ABSTRACT         Title  of  Dissertation:       INFLUENCE  OF  ACUTE  AND  CHRONIC  EXERCISE   ON  MARKERS  OF  HIPPOCAMPAL  PLASTICITY         Andrew  Carmen  Venezia,  Doctor  of  Philosophy,  2016       Dissertation  Directed  by:   Professor  Stephen  M.  Roth     Department  of  Kinesiology         Exercise   and   physical   activity   are   lifestyle   behaviors   associated   with   enriched   mental  health.    Understanding   the  mechanisms  by  which  exercise  and  physical   activity   improve   mental   health   may   provide   insight   for   novel   therapeutic   approaches   for  numerous  mental  health  disorders.  This  dissertation   reports   the   findings   from   three   studies   investigating   the   influence   of   acute   and   chronic   exercise   on   behavioral   and  mechanistic  markers   of   hippocampal   plasticity   and   delves   into   the   potential   role   of   noradrenergic   signaling   in   the   hippocampal   adaptations   with   exercise.     The   first   study   assessed   the   effects   of   long-­term   voluntary   wheel   running   on   hippocampal   expression   of   plasticity-­associated   genes   and   proteins   in   adult  male   and   female  C57BL/6J  mice,   highlighting   sex   differences  in  the  adaptations  to  long-­term  voluntary  wheel  running.    The  second   study   examined   the   influence   of   acute   exercise   intensity   on   AMPA   receptor   phosphorylation,   a   mechanism   essential   for   hippocampal   plasticity,   plasticity-­ associated   gene   expression,   spatial   learning   and   memory,   and   anxiety-­like   behavior.     The   unexpected   finding   that   acute   exercise   increased   anxiety-­like       behavior  encouraged  investigation  into  the  role  of  central  noradrenergic  signaling   in   acute   exercise-­induced   anxiety.     The   third   study   determined   how   previous   exposure  to  voluntary  wheel  running  modulates  the  response  to  an  acute  bout  of   exercise,  focusing  primarily  on  transcription  of  the  important  plasticity-­promoting   gene,   brain-­derived   neurotrophic   factor.   Using   a   pharmacological   approach   to   compromise   the   locus   coeruleus   noradrenergic   system,   a   system   that   is   implicated   in  age-­related  mental  health  disorders  such  as  Alzheimer’s  Disease,   the   third   study   also   investigated   the   influence   and   interaction   of   the   noradrenergic  system  and  acute  exercise  on  expression  of  multiple  brain-­derived   neurotrophic   factor   transcripts.     Together,   this   dissertation   reports   the   findings   from   a   series   of   experiments   that   explored   similarities,   differences,   and   interactions   between   the   effects   of   acute   and   chronic   exercise   on   markers   of   hippocampal  plasticity  and  behavior.    Further,  this  work  provides  insight  into  the   role  of  the  noradrenergic  system  in  exercise-­induced  hippocampal  plasticity.             Influence  of  Acute  and  Chronic  Exercise  on  Markers  of  Hippocampal  Plasticity           by       Andrew  Carmen  Venezia           Dissertation  submitted  to  the  Faculty  of  the  Graduate  School  of  The     University  of  Maryland,  College  Park  in  partial  fulfillment  of     the  requirements  for  the  degree  of     Doctor  of  Philosophy   2016                               Advisory  Committee:         Professor  Stephen  M  Roth,  Chair       Professor  Elizabeth  Quinlan       Professor  Erica  Glasper     Professor  J.  Carson  Smith       Professor  Matthew  Roesch,  Dean’s  Representative                             ©  Copyright  by     Andrew  Carmen  Venezia   2016       ii   Table  of  Contents     Table  of  Contents  ................................................................................................  ii   List  of  Figures  .....................................................................................................  iv   List  of  Abbreviations  ...........................................................................................  v   Chapter  1.  Introduction  &  Specific  Aims  ...........................................................  1     Specific  Aim  1  ........................................................................................................  2     Specific  Aim  2  ........................................................................................................  5     Specific  Aim  3  ........................................................................................................  5     Specific  Aim  4  ........................................................................................................  6     Specific  Aim  5  ........................................................................................................  7   Chapter  2.  Review  of  Literature  .........................................................................  9     Introduction  ............................................................................................................  9     Physical  Activity  is  Beneficial  for  Healthy  Cognitive  Aging  ..................................  12     Physical  Activity/Exercise  Training  Increases  Brain  Volume  ...............................  14     Physical  Activity  and  Structural  Adaptations  in  Rodents  .....................................  16   Brain-­Derived  Neurotrophic  Factor  is  Essential  for  Structural  and  Functional   Hippocampal  Plasticity  .........................................................................................  17     Exercise  Training/Fitness  and  Peripheral  BDNF  Levels  ......................................  22   Physical  Activity  Increases  Hippocampal  Bdnf  in  Rodents  ..................................  23   Sex  Differences  in  Exercise-­Induced  BDNF  Expression  .....................................  26   Transcription  of  the  BDNF  Gene  .........................................................................  28   Acute  Exercise  and  Memory  ................................................................................  31   Acute  Exercise  Increases  Peripheral  BDNF  in  Humans  ......................................  34   Acute  Exercise  and  Hippocampal  Bdnf  in  Rodents  .............................................  35   Catecholamines  and  Exercise  –  The  Link  Between  the  Periphery  and  Improved   Memory  ................................................................................................................  37   Peripheral  Epinephrine  Increases  Central  Norepinephrine  Release  ...................  40   Central  Norepinephrine  Levels  and  Exercise  ......................................................  42   Norepinephrine  and  Plasticity  ..............................................................................  43   Noradrenergic  System  and  Exercise  Training-­Induced  Plasticity  and  Bdnf   Expression  ...........................................................................................................  45   Acute  Exercise  and  Anxiety  .................................................................................  47   Training  Influences  Catecholamine  Response  to  Acute  Exercise  .......................  49   Training  Influences  BDNF  Response  to  Acute  Exercise  .....................................  51   Summary  .............................................................................................................  53   Chapter  3.  Sex-­Dependent  and  Independent  Effects  of  Long-­Term  Voluntary   Wheel  Running  on  Bdnf  mRNA  and  Protein  Expression  ...............................  54     Abstract  ................................................................................................................  55     Introduction  ..........................................................................................................  57       iii     Methods  ...............................................................................................................  60     Results  .................................................................................................................  63     Figures  .................................................................................................................  65     Supplemental  Table  1  ..........................................................................................  79     Discussion  ...........................................................................................................  80     Competing  Interest,  Funding,  Authors’  Contributions  ..........................................  88   Chapter   4.   Acute   Forced   Exercise   Increases   Expression   of   Bdnf   IV   and   Induces  Anxiety  Like  Behavior  in  C57BL/6J  Mice  ..........................................  89     Introduction  ..........................................................................................................  90     Methods  ...............................................................................................................  94     Results  ...............................................................................................................  101     Figures  ...............................................................................................................  105     Discussion  .........................................................................................................  123   Chapter   5.  Bdnf   Transcription   is  Differentially  Regulated  by  DSP-­4,  Acute   Forced  Treadmill  Exercise,  and  Voluntary  Wheel  Running  ........................  137     Introduction  ........................................................................................................  138     Methods  .............................................................................................................  141     Results  ...............................................................................................................  146     Figures  ...............................................................................................................  149     Discussion  .........................................................................................................  159   Chapter  6.  Summary,  Conclusions,  Limitations,  and  Future  Directions  ....  167     Overall  Summary  ...............................................................................................  167   Limitations  and  Considerations  ..........................................................................  171   Future  Directions  ...............................................................................................  174   Appendix  A.  Primer  Sequences  .....................................................................  177   Appendix  B.  Venezia  A.C.  et  al.  (2015)  ..........................................................  178   Appendix  C.  Curriculum  Vitae  ........................................................................  203   Bibliography  .....................................................................................................  212               iv   List  of  Figures   Figure  1.  Average  daily  running  distance  for  male  and  female  C57Bl/6J  mice  ..  67   Figure  2.  Male  and  female  body  mass  ...............................................................  68   Figure  3.  Five  months  of  voluntary  wheel  running  increases  Bdnf  transcription  in   a  transcript  and  sex-­dependent  manner  ..............................................................  69   Figure   4.   Five   months   of   voluntary   wheel   running   does   not   influence   hippocampal  GluR1,  NR2A,  NR2B,  and  NR2B/NR2A  mRNA  expression  ..........  72   Figure   5.   Five   months   of   voluntary   wheel   running   does   not   influence   hippocampal  total  Bdnf  protein  levels  ..................................................................  75   Figure  6.  Five  months  of  voluntary  wheel  running  significantly  increases  mature   Bdnf  protein  levels  in  the  male  hippocampus  ......................................................  77   Figure   7.   Intraperitoneal   injection   of   epinephrine   induces   GluR1   Ser845   phosphorylation  in  the  hippocampus  .................................................................  110   Figure  8.  Acute  exercise  does  not  influence  GluR1  phosphorylation  in  the  mouse   hippocampus  .....................................................................................................  111   Figure   9.  Acute  exercise  does  not   influence  glutamate   receptor  subunit  mRNA   expression  in  the  mouse  hippocampus  .............................................................  113   Figure   10.  High-­intensity  exercise   increases   transcript-­specific  Bdnf  expression ...........................................................................................................................  114   Figure   11.  Acute  exercise  does  not   influence   time  spent  with  objects  or  object   placement  memory  ............................................................................................  115   Figure   12.   High-­intensity   acute   exercise   reduces   exploratory   behavior   in   the   novel  object  placement  task  ..............................................................................  116   Figure  13.  Open  Field  Task  0-­5  Minutes.  High   intensity  acute  exercise   induces   anxiety-­like  behavior  in  the  open  field  task  during  the  first  five  minutes  of  the  task ...........................................................................................................................  117   Figure  14.  Open  Field  Task  5-­10  Minutes.  High-­intensity  acute  exercise  induces   anxiety-­like  behavior   in   the  open   field   task  during   the  second   five-­minute  block   (5-­10  min)  of  the  task  ........................................................................................  119   Figure   15.   Open   Field   Task   10-­15   Minutes.   High-­intensity   acute   exercise   induces   anxiety-­like   behavior   in   the   open   field   task   during   the   third   five-­minute   block  (10-­15  min)  of  the  task  .............................................................................  121   Figure   16.   Chronic   exercise   but   not   acute   exercise   increases   GluR1   Ser845   phosphorylation  and   total  GluR1  protein  expression   in   the  mouse  hippocampus ...........................................................................................................................  152   Figure   17.   Acute   and   chronic   exercise   do   not   influence   glutamate   receptor   subunit  mRNA  expression  .................................................................................  153   Figure   18.  Acute  and   chronic   exercise   increase   total  Bdnf   and  Bdnf   IV  mRNA   expression  .........................................................................................................  154   Figure   19.   Acute   exercise   and   DSP-­4   lesioning   influence   Bdnf   transcript   expression  .........................................................................................................  155   Figure  20.  b2-­adrenergic  receptor  mRNA  expression  is  not  influenced  by  DSP-­4   treatment  or  acute  exercise  ...............................................................................  158         v   List  of  Abbreviations       b2AR:  b2-­adrenergic  receptor;;  official  gene  symbol:  Adrb2   ActB:  Actin,  beta   ACTH:  Adrenocorticotropin  Hormone   AMPA:  α-­amino-­3-­hydroxyl-­5-­methyl-­4-­isoxazole-­propionate   AMPAR:  AMPA  receptor   AUC:  Area  under  the  curve   BCA:  Bicinchoninic  acid   BDNF:  Brain-­derived  neurotrophic  factor   BDNF:  Protein  in  humans  *     BDNF:  Gene  in  humans*     Bdnf:  Protein  in  rodents*     Bdnf:  Gene  or  mRNA  in  rodents*   CAMKII:  Calcium/calmodulin-­dependent  protein  kinase  II     CNS:  Central  nervous  system   CREB:  cAMP  response  element  binding  protein   DSP-­4:    N-­(2-­Chloroethyl)-­N-­ethyl-­2-­bromobenzylamine  hydrochloride   ERK:  Extracellular  regulated  MAP  kinase   fMRI:  Functional  magnetic  resonance  imaging   Gapdh:  Glyceraldehyde-­3-­phosphate  dehydrogenase   GluR1:  Glutamate  ionotropic  receptor  AMPA  type  subunit  1;;  Official  gene   symbol:  Gria1   HPA:  Hypothalamus-­pituitary-­adrenal   IGF-­1:  Insulin  like  growth  factor  1   IP:  Intraperitoneal     IPGTT:  Intraperitoneal  glucose  tolerance  testing   KO:  Genetic  knockout   LC:  Locus  coeruleus   LTP:  Long-­term  potentiation   mGluR:  Metabotropic  glutamate  receptor   MHPG:  3-­methoxy-­4-­hydroxyphenylglycol   mRNA:  Messenger  ribonucleic  acid   NA:  Noradrenergic   NMDA:  N-­methyl-­d-­aspartate   NMDAR:  NMDA  receptor   NOP:  Novel  object  placement   NR2A:  glutamate  receptor,  ionotropic,  NMDA2A;;  official  gene  symbol:  Grin2a   NR2B:  Glutamate  receptor,  ionotropic,  NMDA2B;;  official  gene  symbol:  Grin2b   NT4:  Neurotrophin  4   NTS:  Nucleus  of  the  solitary  tract   PGC-­1a:  Peroxisome  proliferator-­activated  receptor  γ  coactivator  1  alpha;;  official   gene  symbol:  Ppargc1a   PKA:  Protein  kinase  A   PTSD:  Post-­traumatic  stress  disorder       vi   PVN:  Paraventricular  nucleus  of  the  hypothalamus   qPCR:  Quantitative  polymerase  chain  reaction   SDS:  Sodium  dodecyl  sulfate   Ser831:  Serine  831   Ser845:  Serine  845   tPa:  Tissue  plasminogen  activator;;  official  gene  symbol:  Plat   TrkB:  Neurotrophic  receptor  tyrosine  kinase  2;;  official  gene  symbol:  NTRK2     UMD:  University  of  Maryland   VO2max:  Maximal  oxygen  consumption         *This  is  the  format  for  all  official  gene  and  protein  names  in  the  document.     Capitalization  may  vary  when  common  abbreviations  are  used  instead  of  official   gene  symbols  (e.g.  GluR1,  NR2A,  NR2B,  TrkB).    Genes  and  mRNAs  are   italicized  throughout  the  document.             1   Chapter  1.  Introduction  and  Specific  Aims   Physical   activity   promotes   brain   health   by   inducing   adaptations   that   increase   hippocampal   volume,   improve   hippocampal-­dependent   learning   and   memory,   decrease   anxiety   and   depression,   and   lower   the   risk   of   cognitive   impairment  and  dementia  later  in  life.    The  overall  aim  of  this  dissertation  project   is   to   identify   potential   mechanisms   mediating   these   favorable   adaptions   to   exercise.    The   literature  reporting  the  cognitive  benefits  of  exercise  training  and   chronic   physical   activity   in   humans   and   rodents   is   vast.   Non-­human   studies   examining   the   mechanisms   of   plasticity   induced   by   chronic   exercise   have   primarily  focused  on  “short-­term”  chronic  exercise  exposures  (<  one  month)  that   may   reflect   adaptations   in   response   to   the   novelty   of   the   activity.     In   rodents,   voluntary   running   activity   decreases   over   time,   so   utilizing   short-­term   exercise   studies  may   favor  plasticity  by  capturing   the  novelty  of   running  and  high  wheel   activity.     Further,   studies   typically   use   only   one   sex   in   the   sample   population,   which   has   limited   our   understanding   of   any   potential   sex   differences   in   the   response   to   exercise   training.     Chapter   Three   of   this   dissertation   contains   a   manuscript  published  in  Physiology  and  Behavior  in  2015  that  reports  a  series  of   experiments  that  tested  the  hypotheses  of  Specific  Aim  1.       Aim   #1:    Determine   the   effect   of   five  months   of   voluntary  wheel   exposure   on   hippocampal  mRNA  expression  of  plasticity-­associated  genes  in  adult  male  and   female  mice.           2   Hypothesis   #1:     Due   to   the   gradual   reduction   in   voluntary  wheel   activity   over   time,  plasticity-­associated  gene  expression  will   not  differ  between  exercise  and   sedentary  mice.   Exploratory   Hypothesis   #1:   Any   observed   differences   in   mRNA   expression   between  exercise  and  sedentary  mice  will  be  sex-­dependent.   The  purpose  of  this  investigation  was  to  determine  the  influence  of  long-­term  (20   weeks)   voluntary   wheel   running   on   expression   of   genes   known   to   play   critical   roles  in  exercise-­induced  enhancement  of  brain  health  in  rodents.    Moreover,  our   sample   consisted   of   both   male   and   female   C57BL/6J   mice,   providing   the   opportunity  to  investigate  sex  differences  in  the  response  to  long-­term  voluntary   wheel   running.     We   observed   that   long-­term   wheel   running   increased   the   expression  of  a  critical  neurotrophin  for  brain  health  and  plasticity,  brain-­derived   neurotrophic   factor   (Bdnf),   in  a   transcript  and  sex  specific  pattern.  Bdnf  protein   analysis  concurred  with  the  mRNA  data,  demonstrating  that   long-­term  voluntary   wheel  running  increases  Bdnf  mRNA  and  mature  protein  only  in  male  mice.       In  contrast   to   the   large  number  of  studies  examining   the  mechanisms  of   chronic   exercise   training,   research   examining   the   effects   of   acute   bouts   of   exercise   on   brain   health   has   been   limited   to   correlational   investigations   of   peripheral   markers   in   human   participants   or   investigations   in   rodents   using   multiple-­day   “acute”  exercise   interventions   instead  of   true  single  acute  bouts  of   exercise.    The  mechanisms  mediating  the  effects  of  chronic  exercise  training  are   likely  due  to  accumulated  changes  induced  by  repeated  bouts  of  acute  exercise,       3   and   characterizing   the   immediate   benefits   of   acute   exercise   is   essential   to   understanding   and   optimizing   exercise   training   and   future   “exercise-­like”   interventions   for   hippocampal   plasticity.       Bdnf   is   known   to   be   essential   for   exercise-­induced   improvements   in   memory   but   how   expression   of   Bdnf   in   the   brain   is   influenced   by   truly   acute   bouts   of   exercise   is   currently   unknown.     Understanding   the   temporal  dynamics  of  Bdnf  expression   in   response   to  acute   exercise  offers  the  opportunity  to  optimize  acute  exercise  prescription  to  enhance   brain  plasticity.    In  addition,  in  vitro  and  ex  vivo  exposure  of  hippocampal  neurons   to  hormones  and  neurotrophins  known   to  be  upregulated  during  acute  exercise   regulates  phosphorylation  and  trafficking  of  the  GluR1  subunit  of  the  AMPA-­type   glutamate   receptor   (AMPAR),   but   the   ability   for   exercise   itself   to   initiate   these   pathways   in   the   hippocampus   has   not   been   studied.   Trafficking   of   the   AMPA-­ type   ionotropic  glutamate   receptor   (AMPAR)   to  and   from  the  synapse   is  crucial   for   synaptic   plasticity   at   excitatory   synapses.     Activity-­dependent   receptor   trafficking   is  mediated   by   phosphorylation   of   the  GluR1   subunit   of   the  AMPAR   and  can  be  engaged  by  physiologically  arousing  stimuli   (e.g.  emotional   stimuli,   catecholamines),  which   in   turn   reduces   the   threshold   for   long-­term  potentiation   (LTP)  and  enhances  learning  and  memory.    Exercise  is  a  non-­invasive,  practical   stimulus   with   the   potential   to   engage   this   critical   molecular   mechanism   to   enhance   synaptic   plasticity   and   learning.   Short-­term   exercise   training   also   reduces   the   threshold   for   LTP,   a   mechanism   of   activity-­dependent   synaptic   plasticity   known   to   contribute   to   many   forms   of   memory   formation,   but   the   pathway   that   links   exercise   to   enhanced   synaptic   plasticity   is   not   understood.         4   Exercise   activates   the   “fight   or   flight”   response,   which   is   characterized   by   an   elevation   in   peripheral   and   central   catecholamines.    Peripheral   catecholamines   (epinephrine   and   norepinephrine)   activate   the   central   noradrenergic   system   causing  the  release  of  norepinephrine  in  the  brain.    Norepinephrine  signaling  is  a   potent   memory   enhancer   and   strongly   influences   plasticity   in   multiple   brain   regions,   including   the   hippocampus.     The   influence   of   exercise   on   peripheral   epinephrine   and   central   norepinephrine  might   explain   how  exercise,   an   activity   that   primarily   influences   peripheral   tissues,   has   such   a   strong   effect   on   hippocampal   plasticity.     However,   catecholamine   signaling   and   central   noradrenergic   activation   are   also   associated   with   anxiety   and   anxiety-­like   behavior.    If  acute  exercise  is  to  be  used  as  a  non-­pharmacological  approach  to   enhance  memory  and  mental   health,   understanding   the  behavioral   response   is   essential,   especially   in   rodents,   which   are   commonly   used   to   investigate   how   exercise   influences   brain   plasticity   and   behavior.     Chapter   Four   contains   a   manuscript   that   reports   the   experiments   undertaken   to   test   the   hypotheses   of   Specific   Aims   2   and   3.     I   utilized   a   truly   acute   bout   of   treadmill   exercise   by   familiarizing   mice   to   the   treadmill   without   any   running   activity.     Mice   were   exposed   to   behavioral   testing   or   sacrificed   immediately   after   the   acute   bout   of   treadmill  exercise.   Aim  #2:    Determine  the  effect  of  acute  exercise  and  exercise  intensity  on  GluR1   phosphorylation,  the  expression  of  specific  plasticity-­associated  genes,  and  novel   object  location  memory  in  three-­month  old  C57BL/6J  mice.         5   Hypothesis  #2:  Acute  treadmill  exercise  will  increase  GluR1  phosphorylation  at   sites   critical   for  GluR1  membrane   trafficking   in   an   intensity-­dependent  manner,   with  greater  phosphorylation  in  response  to  high-­  compared  to  moderate-­intensity   running.    Acute  exercise  will  also  increase  plasticity-­associated  gene  expression   and  novel  object  location  memory  performance  in  an  intensity-­dependent  manner.   Aim   #3:     Determine   if   acute   high-­intensity   exercise   increases   anxiety-­like   behavior  in  the  open  field  task  and  if  this  behavioral  phenotype  is  attenuated  with   pre-­treatment  with  the  selective  noradrenergic  neurotoxin  DSP-­4.   Hypothesis   #3:    An  acute  bout  of  high-­intensity  exercise  will   increase  anxiety-­ like  behavior  in  the  open  field  task.   Exploratory  Hypothesis  #3.  The  exercise-­induced  anxiety-­like  behavior  will  be   attenuated  in  mice  treated  with  DSP-­4.   We   observed   that   acute   forced   treadmill   exercise   does   not   influence   GluR1   phosphorylation   of   the   AMPAR   but   does   increase   expression   of   the   rapidly   transcribed   Bdnf   transcript   IV.   Bdnf   is   a   complex   gene   producing   up   to   22   possible  transcripts.    These  and  previously  reported  data  support  the  conclusion   that  Bdnf   IV   is   sensitive   to   numerous   stimuli,   including  exercise,   and   is   rapidly   transcribed.     I   also   observed   that   acute   exercise   did   not   improve  memory   in   a   one-­trial  spatial  memory  task.    The  lack  of  improvement  was  likely  due  to  anxiety-­ like   behavior   during   the   task,   as   I   observed   anxiety-­like   behavior   following   exercise   in  a   locomotor-­dependent  anxiety   task   following  exercise   that  was  not   rescued  by   lesioning  central  noradrenergic  signaling.    This  behavior  might  have       6   been  due  to  other  signaling  factors  (e.g.  dopaminergic,  glucocorticoid,  amygdalar   activity,  etc.)  or  fatigue  following  exercise.     Though   this   dissertation   sought   to   address   how   a   truly   acute   bout   of   exercise   influences   markers   of   hippocampal   plasticity,   it   is   important   to   understand   acute   exercise   in   the   context   of   regular   physical   activity.     Physical   activity  is  important  for  physical  and  mental  health,  and  it  is  therefore  encouraged   that   individuals   perform   regular   physical   activity   or   purposeful   exercise.     So,   if   acute  exercise   is   to  be  effectively  utilized   to  enhance  brain  health  and  memory   capability,  it  must  be  understood  how  acute  and  chronic  exercise  interact  and  the   mediating   mechanisms   of   a   potential   interaction.     Chapter   Five   contains   a   manuscript   describing   the   approach,   results,   and   interpretation   of   a   series   of   experiments  designed  to  test  the  hypotheses  for  Specific  Aims  4  and  5.       Aim  #4:  Determine  if  chronic  exercise  influences  the  effect  of  acute  exercise  on   GluR1   protein   phosphorylation   and   mRNA   expression   of   plasticity-­associated   genes.   Hypothesis  #4:  Because  chronic  exercise  will  increase  baseline  levels  of  GluR1   phosphorylation   and   plasticity-­associated   gene   expression,   chronic   voluntary   wheel   running  will  minimize   the   response   to  acute  exercise.    This  will  minimize   the   absolute   difference   in   the   level   of   GluR1   phosphorylation   and   plasticity-­ associated   gene   expression   in   mice   that   received   chronic   exercise   +   acute   exercise   versus   acute   exercise   alone.  However,   the   relative   response   to   acute   exercise  will  be  lower  in  chronically  exercise-­trained  mice.       7   Aim   #5:     Determine   the   influence   of   acute   exercise   and   locus   coeruleus   noradrenergic  signaling  on  specific  Bdnf  transcript  expression.       Hypothesis  #5:    Acute  exercise-­induced  expression  of  Bdnf  IV  will  be  dependent   on   noradrenergic   signaling   and   will   be   attenuated   by   DSP-­4.     Acute   exercise-­ induced   expression   of   total  Bdnf  mRNA  will   persist   following   DSP-­4   treatment   due  to  increased  expression  of  other  Bdnf  transcripts.       Mice   were   housed   for   one   month   with   either   freely   rotating   or   locked   running  wheels   before   exposure   to   an   acute   bout   of   forced   treadmill   exercise.     Though   previous   research   suggests   enhanced   capacity   to   secrete   catecholamines  following  exercise  training,  animals  and  humans  show  a  reduced   stress  response  to  psychological  and  physical  stress  following  exercise  training.     We   observed   that   both   acute   and   chronic   exercise   increased   Bdnf   mRNA   expression  but  chronic  exercise  blunted  the  influence  of  acute  exercise  on  Bdnf   IV  mRNA  expression.    We  then  investigated  if  noradrenergic  signaling  influences   transcription   of   multiple   Bdnf   transcripts   and   observed   that   compromising   noradrenergic  signaling  reduced  Bdnf  IV  mRNA  expression  but  this  was  rescued   with   acute   exercise,   contrary   to   our   original   hypothesis   that   compromising   noradrenergic  signaling  would  attenuate   the  acute  exercise-­induced   increase   in   this   transcript.    Though  compromising  noradrenergic   signaling   reduced  Bdnf   IV   expression,   it   increased   Bdnf   VI   expression,   demonstrating   the   remarkable   complexity   of   Bdnf   gene   transcription   and   the   influence   of   noradrenergic   signaling.       8   This   dissertation   begins   with   a   review   of   literature   (Chapter   Two)   examining  the  influence  of  acute  and  chronic  exercise  on  mental  health  and  the   structural  and   functional  brain  plasticity  observed   in  humans  and   rodents.    The   review   of   literature   is   followed   by   three   chapters   in  manuscript   form   (Chapters   Three,  Four,  and  Five)  that  describe  the  methodological  approaches,  results,  and   interpretations  of  the  dissertation  experiments.    Finally,  Chapter  Six  summarizes   the  findings  of  all   three  studies  and  discusses   implications  and  future  directions   for  this  area  of  research.             9   Chapter  2.    Review  of  Literature   Introduction     Mental   health   disorders   are   an   increasing   health   burden   in   the   United   States   and   around   the   world.     Disorders   such   as   anxiety,   depression,   post-­ traumatic   stress   disorder   (PTSD),   and   age-­related   dementias   (e.g.   Alzheimer’s   disease)  are  debilitating  conditions   that  not  only   impact   the  suffering   individual,   but  also  family,  friends,  and  the  economy.  The  National  Institute  of  Mental  Health   claims   that  6.7%  of  U.S.  adults  experienced  an  episode  of  major  depression   in   2014   (Center   for   Behavioral   Health   Statistics   Quality,   2015)   and   others   have   reported  lifetime  prevalence  of  28.8%  and  6.8%  for  anxiety  disorders  and  PTSD,   respectively   (Kessler   et   al.,   2005).     Moreover,   in   the   2014-­2015   Alzheimer’s   Progress  Report,   the  National   Institute   of   Aging   stated   that   roughly   five  million   Americans   are   currently   living   with   Alzheimer’s   disease,   and   with   the   growing   aging   population   and   lack   of   current   therapies,   this   number   is   expected   to   increase  (National  Institute  on  Aging,  2015).    These  mental  health  disorders  span   the  age  spectrum  and  negatively  impact  the  lives  of  millions  of  young  and  aged   individuals.    An  abundance  of  research  suggests  that  exercise  is  a  potential  non-­ invasive   therapeutic   technique   that   could   be   used   to   treat   and   defend   against   anxiety,  depression,  PTSD,  and  age-­related  dementias.    Research  is  still  needed   to  understand   the  most  effective  exercise   regimens,   the  mechanisms  of  action,   and   sensitive   brain   regions   mediating   the   beneficial   adaptations   to   best   treat   and/or   prevent   these   debilitating   conditions.     Exercise   may   serve   as   a   stand-­     10   alone   treatment   or   be   used   in   combination   with   medications   and   cognitive   therapies.     Moreover,   in   addition   to   improving   the   mental   health   of   patient   populations,   exercise   has   the   potential   to   improve   cognitive   function   in   healthy   individuals.     Effective   exercise   strategies   may   be   used   to   accelerate   and   strengthen   learning   in   students   of   all   ages   and   improve   training   practices   in   corporate,  military,  and  government  agencies.     Though   multiple   brain   regions   influence   and/or   are   affected   by   mental   health  disorders,  the  hippocampal  formation  is  linked  to  the  development  and/or   progression   of   many   disorders   affecting   Americans   today   such   as   anxiety,   depression,   PTSD,   schizophrenia,   and   age-­related   dementias   (Pajonk   et   al.,   2010;;  Small  et  al.,  2011).    Both  structural  and   functional  changes  coincide  with   disease   progression,   but   remarkably,   this   structure   is   also   highly   sensitive   to   physical   activity   and   exercise   training   (Voss   et   al.,   2013),   highlighting   the   therapeutic   potential   of   exercise   to   treat   these   mental   health   disorders.     The   hippocampus   is   a   brain   structure   important   for   the   formation   of   episodic   and   spatial  memories  and  plays  an  important  role  in  emotional  regulation,  containing   a  high  content  of  stress  hormone  receptors  (Osborne  et  al.,  2015).     It  mediates   these  cognitive  functions  via  a  unique  gradient  along  the  longitudinal  axis  of  the   structure.     Using   lesioning   techniques   in   rodents   and   fMRI   in   humans,   researchers   have   identified   that   the   dorsal   (posterior   in   humans)   region   is   particularly   involved   in   spatial   navigation  while   the   ventral   (anterior   in   humans)   region  is  principally  involved  in  emotional  responses.    This  functional  delineation   along   the   longitudinal   axis   is   consistent   with   the   cortical   and   subcortical       11   connections  found  along  the  longitudinal  axis  (Strange  et  al.,  2014).    For  example,   the  fear  and  emotional  center  of  the  brain,  the  amygdala,  and  the  infralimbic  and   prelimbic   cortices   (cingulate   areas   involved   in   emotional   regulation)   are   more   connected  with  the  ventral  hippocampus  while  information  from  the  visual  cortex   projects   primarily   to   the  dorsal/posterior   hippocampus   (Amaral  &  Witter,   1989).     Further  support  for  a  longitudinal  gradient  of  function  comes  from  identification  of   unique  transcriptional  profiles  along  the  longitudinal  axis  (Thompson  et  al.,  2008;;   Dong  et  al.,  2009).     Importantly,  exercise   influences  both  the  dorsal  and  ventral   hippocampus  (Schoenfeld  et  al.,  2013).     In  addition   to   the   longitudinal  axis,   the   hippocampus  contains  unique  subfields  (dentate  gyrus  à  CA3  à  CA1)  that  are   uniquely  sensitive  to  disease  and  environmental  stimuli  (McGaugh  &  Roozendaal,   2002;;   Small   et   al.,   2011).     Exercise   training   leads   to   structural   adaptations   throughout  the  hippocampal  subfields  (Eadie  et  al.,  2005;;  Stranahan  et  al.,  2007;;   Lin  et  al.,  2012)  and  can  serve  as  an  intervention  to   improve  the  functional  and   structural   integrity   of   this   brain   region   that   is   so   critical   for   cognitive   and   emotional  health.     This  review  of  literature  will  provide  a  detailed  analysis  of  the  research  in   the  field  of  physical  activity/exercise  training  on  brain  health  and  function.     I  will   focus   on   how   exercise   influences   the   human   and   rodent   hippocampus,   highlighting   structural   and   functional   adaptions   and   the  mechanisms  mediating   these  effects.    Consistent  with   the  order  of  experiments   in   the  dissertation,   this   literature  review  will  begin  with  a  review  of  studies  examining  the  use  of  chronic   exercise  to  influence  brain  health,  then  will  transition  to  studies  of  acute  exercise       12   (or  acute  psychological  stress  as  a  surrogate  for  acute  exercise),  and  will   finish   with   a   review   of   literature   reporting   potential   interactions   between   acute   and   chronic  exercise.       Physical  Activity  is  Beneficial  for  Healthy  Cognitive  Aging   Exercise   is   an   excellent   environmental   stimulus   for   maintaining   brain   health  into  old  age.    Large-­scale  epidemiological  studies  have  shown  that  higher   levels   of   chronic   physical   activity   are   associated  with   reduced   risk   of   cognitive   decline   and   age-­related   dementias   such   as   Alzheimer’s   disease   (Laurin  et   al.,   2001;;  Yaffe  et  al.,   2001;;  Weuve  et  al.,   2004;;  Rovio  et  al.,   2005;;  Larson,  2006;;   Angevaren  et  al.,  2008;;  Middleton,  2011;;  Buchman  et  al.,  2012).    Though  many   of  the  epidemiological  studies  used  very  general  methods  of  physical  activity  (e.g.   self-­reported  physical  activity)  and/or  cognitive  functioning  assessment  (e.g.  Mini-­ Mental  State  Exam),  they  offered  powerful  evidence  for  the  benefit  of  exercise  on   brain   health   and   provided   the   stimulus   for   more   systematic   investigations   of   exercise   and   brain   health/plasticity   that   have   followed.     Larson   et   al.   (2006)   investigated  the  relationship  between  self-­reported  physical  activity  and  rates  of   dementia   over   an   average   of   6.2   years   in   2,581   cognitively   intact   men   and   women   aged   65   years   and   older.     They   found   that   individuals   who   exercised   three  or  more  times  per  week  had  32%  reduced  odds  of  dementia.  Similarly,  in  a   group  of  16,466  women  aged  70  years  and  older,  researchers  found  that  women   who   participated   in   the   greatest   amount   of   leisure   time   physical   activity   (assessed   via   biennial   questionnaires)   were   20%   less   likely   to   be   cognitively       13   impaired  when  compared  to  women  who  participated  in  the  least  physical  activity   (Weuve  et   al.,   2004).     Consistent   with   these   relatively   short   follow-­up   periods,   another  investigation  reported  that  individuals  who  participated  in  physical  activity   two   or   more   times   per   week   had   52%   lower   odds   of   dementia   21   years   later   (Rovio   et   al.,   2005).     These   earlier   studies   that   used   self-­reported   physical   activity  have  been  supported  by  more  advanced  measures  of  energy  expenditure   and  activity,  such  as  doubly  labeled  water,  indirect  calorimetry  (Middleton,  2011),   and   actigraphy   (Buchman   et   al.,   2012).     Though   not   all   investigations   have   reported   beneficial   effects   of   self-­reported   physical   activity   on   the   risk   of   developing   dementia   (Rovio   et   al.,   2007),   the   literature   provides   exceedingly   strong   evidence   to   support   that   chronic   physical   activity   is   an   effective   way   to   reduce  cognitive  decline  and  age-­related  cognitive  impairments.    It  is  important  to   note   that   these   longitudinal   studies   have   demonstrated   that   physical   activity   performed  in  early  or  mid-­life  predicts  cognitive  functioning  later  in  life  (Sofi  et  al.,   2010).    Remarkably,  an  investigation  by  Middleton  et  al.  (2010)  showed  that  self-­ reported   physical   activity   during   the   teenage   years   was   most   predictive   of   cognitive  aging   in  elderly  women.    Women  who  were  physically   inactive  during   their  teenage  years  could  reduce  their  risk  of  cognitive  impairment  by  becoming   active  during  their  30s  and  50s  but  there  was  no  additional  benefit  of  becoming   active  in  their  30s  and  50s  if  the  subjects  were  already  active  during  the  teenage   years   (Middleton  et  al.,  2010).  Taken   together,   the   literature  on  cognitive  aging   informs   the   importance   of   understanding   the  most   effective   exercise   strategies   that   can  be  performed   throughout   the   lifespan   to  maintain  brain  health   into  old       14   age.     The   structural   adaptations   occurring   in   the   human   and   rodent   brain  with   exercise   training   and   the  mechanisms   responsible   have   received   considerable   attention   from   researchers,   as   these   adaptations   likely   mediate   the   cognitive   benefit   of   regular   physical   activity   and   risk   of   age-­related   cognitive   impairment   and  dementias.   Physical  Activity/Exercise  Training  Increases  Brain  Volume     The  maintenance  of  cognitive   function  with  physical  activity   is  potentially   due  to  the  remarkable  preservation  of  brain  tissue  volume  observed  in  physically   active   and/or   highly   fit   adults   compared   to   sedentary   adults   (Colcombe   et   al.,   2003;;   Erickson   et   al.,   2009;;   2010).     This   is   important   as   the   aging   process   is   associated  with   a   loss   of   gray   and  white  matter   tissue   volume   (Resnick  et   al.,   2003)  and  a  decrease   in  hippocampal  volume  (Ylikoski  et  al.,  2000),  which  has   been  reported  to  occur  more  rapidly  than  in  other  cortical  areas  (Jernigan  et  al.,   2001).     Intervention  studies  have  provided  strong  support   for   the  association  of   physical   activity   and   brain   tissue   volume   by   demonstrating   that   tissue   volume   actually  increases  with  physical  activity.    Colcombe  et  al.  (2006)  reported  that  six   months  of  aerobic  exercise   training   increased  gray  and  white  matter   volume   in   individuals   aged   60-­79   years.   These   researchers   did   not   investigate   the   association  between  increased  tissue  volume  and  cognitive  function.    Erickson  et   al.   (2011)  showed   that  one  year  of  structured  aerobic  exercise   training   in  older   adults   increased   the  size  of   the  human  anterior  hippocampus  by  approximately   2%.     Importantly,   control   subjects   in   this   study   showed   a   reduction   in       15   hippocampal  volume  of  approximately  1.4%  over  the  same  12-­month  intervention   period.    This  shows  remarkable  structural  plasticity  in  the  adult  brain  as  exercise   not   only   prevented   age-­related   tissue   loss   but   also   increased   tissue   volume.     While  the  aerobic  exercise  group  in  this  study  did  not  perform  better  on  a  spatial   memory  task  compared  to   the  stretching  control  group,  cardiorespiratory   fitness   and   changes   in   hippocampal   volume   were   associated   with   spatial   memory   performance  before  and  after  the  intervention  (Erickson  et  al.,  2011).    Increased   hippocampal  volume  has  also  been  reported  with  six  months  of  aerobic  exercise   training   in   women   aged   70-­80   years   with   suspected  mild   cognitive   impairment   (Brinke  et  al.,  2015).       The   aged   population   has   received   the   most   attention   in   the   field   of   physical   activity   and   brain   health   since   there   is   generally   greater   variability   in   cognitive   function   in   this  group  compared  to  young  adults  who  have  higher  and   more  stable  cognitive  function  (Voss  et  al.,  2011).    The  limited  research  in  young   and  middle-­aged  adults  has  provided  conflicting  evidence   for   the  structural  and   functional   benefits   of   regular   physical   activity   or   exercise   training   in   these   age   groups  (Prakash  et  al.,  2015);;  however,  Pereira  et  al.   (2007)  showed  that   three   months  of  aerobic  exercise  training   increased  cerebral  blood  volume  (which  the   authors  suggest  is  an  imaging  “correlate”  of  neurogenesis)   in  the  hippocampi  of   adults   aged   21-­45   years   (mean   age   33   yrs).     The   authors   also   reported   an   association  between  cardiorespiratory   fitness  and   learning  on  a  verbal  memory   task  and  a   trend   for   improved  delayed   recall   after  exercise   training.  This   study   shows   that   structural   and   functional   adaptations   to   exercise   training   are   not       16   limited   to   the   old.    Overall,   the   literature   reveals   that   the   human   hippocampus   displays  remarkable  structural  plasticity  in  response  to  regular  physical  activity  in   both  young  and  old  adults.    Research  in  rodents  has  furthered  our  understanding   of  these  structural  changes  and  the  critical  mechanisms  of  structural  plasticity.   Physical  Activity  and  Structural  Adaptations  in  Rodents     Exercise   induces   structural   adaptations   in   the   rodent   hippocampus.     Rodent  research  has  definitively  demonstrated  that  physical  activity  is  effective  in   stimulating  adult  neurogenesis  in  the  dentate  gyrus  of  the  hippocampus  (Voss  et   al.,  2013).    In  addition  to  increasing  the  number  of  neurons  in  the  dentate  gyrus,   physical   activity   increases   dendritic   branching   and   synaptogenesis   throughout   the  hippocampus  (Eadie  et  al.,  2005;;  Redila  &  Christie,  2006;;  Stranahan  et  al.,   2007;;   Lin   et   al.,   2012).     Lin   et   al.   (2012)   showed   that   four   weeks   (5   d/wk)   of   forced   treadmill   running  or  voluntary  wheel   running   increased   the  dendritic   field   and   spine   density   in   the   CA3   region   of   the   rat   hippocampus.   Another   study   showed   that   two   months   of   voluntary   wheel   running   increased   dendritic   spine   density  in  granule  cells  of  the  dentate  gyrus  and  pyramidal  cells  in  CA1  and  layer   III  of  the  entorhinal  cortex  (Stranahan  et  al.,  2007).    These  structural  adaptations   are   most   likely   responsible   for   the   observed   increase   in   hippocampal   size   following   an   aerobic   exercise   intervention   (Pereira   et   al.,   2007)   and   are   consistent   with   the   numerous   functional   and   behavioral   adaptions   observed   following  exercise  training  and  physical  activity  such  as  enhanced  hippocampal-­ dependent   learning   and   memory   (Gomez-­Pinilla   &   Hillman,   2013;;   Voss   et   al.,       17   2013),   hypothalamus-­pituitary-­adrenal   (HPA)   axis   regulation   (Stranahan   et   al.,   2008),   and   reduced   anxiety-­   and   depression-­like   behavior   (Sciolino   &  Holmes,   2012;;   Holmes,   2014).     These   hippocampal   adaptations   demonstrate   the   sensitivity  of  this  brain  region  to  exercise  training  and  physical  activity,  consistent   with   adaptations   observed   in   human   studies.     Moreover,   these   structural   adaptations  are  due  to  numerous  signaling  mechanisms  that  occur  with  physical   activity.     Thoroughly   understanding   these   signaling   mechanisms   will   greatly   enhance  the  ability  to  maintain  and  improve  brain  health  with  exercise  and  other   “exercise-­like”  interventions.    Brain  derived  neurotrophic  factor  (BDNF)  has  been   identified  as  a  critical  neurochemical  for  normal  brain  health,  structural  plasticity,   behavioral  adaptations,  and  exercise  induced-­enhancement  of  brain  health.    The   importance  of  BDNF   in  normal   brain  health  and  exercise   induced  hippocampal   plasticity  will  be  discussed  in  detail  over  the  next  several  sections  of  this  review.       Brain-­Derived   Neurotrophic   Factor   Is   Essential   for   Structural   and   Functional  Hippocampal  Plasticity     Research   has   identified   a   number   of   potential   mechanisms   that   might   mediate   the   effect   of   chronic   exercise   on   hippocampal   structure   and   function.     Elevated  expression  (mRNA  and/or  protein)  and  downstream  signaling  of  BDNF   has   been   identified   as   a   primary   exercise-­induced   regulator   of   functional   and   structural  plasticity.    BDNF  is  a  critical  factor  in  the  maintenance  of  optimal  brain   health   and   plays   an   integral   role   in   functional   and   structural   plasticity   in   the   hippocampus  throughout  the  lifespan.    It  is  a  remarkable  signaling  protein  of  the       18   neurotrophin  family,  a  group  of  structurally  related  proteins  that  are  important  for   neural   growth   and   development   (Poo,   2001).     BDNF   is   important   for   structural   adaptations   such   as   hippocampal   neurogenesis   (Lee  et   al.,   2002;;  Rossi  et   al.,   2006;;   Sairanen,   2005;;   Scharfman   et   al.,   2005;;   Taliaz   et   al.,   2009)   and   dendritic/synaptic   development     (Alonso   et   al.,   2004;;   Bergami   et   al.,   2008;;   Bohlen   und   Halbach   et   al.,   2008),   as   well   as   functional   adaptations   at   the   synaptic  (Korte  et  al.,  1995;;  Figurov  et  al.,  1996;;  Korte  et  al.,  1996;;  Patterson  et   al.,  1996;;  Kang  et  al.,  1997;;  Ma  et  al.,  1998;;  Chen  et  al.,  1999;;  Zakharenko  et  al.,   2003)  and  behavioral   levels  (Linnarsson  et  al.,  1997;;  Ma  et  al.,  1998;;  Mu  et  al.,   1999;;  Mizuno  et  al.,  2000;;  Alonso  et  al.,  2002;;  Heldt  et  al.,  2007;;  Bekinschtein  et   al.,   2008).     Using   RNA   interference   (Taliaz   et   al.,   2009)   and   genetic   manipulations   to   knockout   (KO)   or   knockdown  Bdnf   (Rossi   et   al.,   2006)   or   its   receptor  (TrkB)  (Bergami  et  al.,  2008),  researchers  have  demonstrated  that  Bdnf   signaling   is   critical   for   adult   neurogenesis   to   occur   under   normal   conditions   (Taliaz  et  al.,  2009)  or  following  enriched  housing  conditions  (Rossi  et  al.,  2006).     Importantly,   though   TrkB   also   binds   neurotrophin-­4   (NT4),   another   member   of   the   neurotrophin   family,   Rossi   et   al.   (2006)   demonstrated   that   Nt4   KO   mice   showed  normal  neurogenesis  following  environmental  enrichment,  whereas  Bdnf   heterozygous   knockdown   mice   had   significantly   reduced   hippocampal   neurogenesis.       Remarkably,  neurogenesis  can  be  induced  by  exogenous  delivery  of  Bdnf   (Scharfman  et  al.,  2005).    Bergami  et  al.  (2008)  used  a  TrkB  inducible  KO  mouse   model   to   demonstrate   that   TrkB   signaling   (and   presumably   Bdnf   signaling)   is       19   necessary   for   development   of   newborn   neurons   in   adult  mice.    TrkB   KO  mice   also  showed  reduced  dendritic  and  spine  growth,  which   is  consistent  with  other   studies   that   have   reported   that   Bdnf-­TrkB   signaling   is   necessary   for   dendritic   spine  density  in  the  hippocampus  (Alonso  et  al.,  2004;;  Bohlen  und  Halbach  et  al.,   2008).     In   fact,   exogenous   Bdnf   delivery   increases   dendritic   spine   density   in   mature   hippocampal   neurons   in   culture   (Tyler   &   Pozzo-­Miller,   2001;;   Ji   et   al.,   2010).    The  newborn  neurons   from  TrkB  KO  mice   in   the  Bergami  et  al.   (2008)   investigation  also  displayed  reduced  capacity  for  long-­term  potentiation  (LTP),  a   cellular   model   for   memory.   This   is   consistent   with   other   studies   that   have   demonstrated   that  Bdnf-­TrkB  signaling   is  critical   for   inducing  synaptic  plasticity.     Korte   et   al.   (1995)   deleted   the   Bdnf   coding   sequence   in   mice   to   create   heterozygous   knockdown   and   homozygous   KO   mice   and   observed   impaired   long-­term  potentiation  (LTP)  in  CA3-­CA1  synapses.    Only  approximately  30%  of   slices  from  knockdown  and  KO  mice  displayed  LTP  following  tetanus  stimulation   compared   to   approximately   87%   from  wild   type  mice   showing   successful   LTP.     Of  note,  when  LTP  did  occur  in  the  mutant  mice,  it  displayed  lower  amplitude  and   a   rapid   decline   in   amplitude,   suggesting   that   maintenance   of   LTP   was   also   significantly   impaired  in  these  mice.    Other   investigations  have  shown  that  Bdnf   is   important   for   both   the   early   and   late   phases   of   LTP   (Figurov   et   al.,   1996;;   Patterson  et  al.,  1996;;  Kang  et  al.,  1997).    Korte  et  al.  (1996)  later  demonstrated   that  re-­expressing  Bdnf  in  CA1  neurons  (ex  vivo)  with  an  adenovirus  rescued  the   impaired   LTP   observed   in   the   Bdnf-­mutant   mice.     Other   studies   have   used   various  approaches  to  block  Bdnf  availability  (Ma  et  al.,  1998;;  Chen  et  al.,  1999)       20   or   signaling   (Figurov   et   al.,   1996;;   Minichiello   et   al.,   2002)   to   demonstrate   its   importance  in  LTP.    Notably,  the  impaired  LTP  observed  in  slices  from  Bdnf  KO   animals  can  be  rescued  with  recombinant  Bdnf    (Patterson  et  al.,  1996).       In  light  of  the  evidence  of   impaired  neurogenesis,  compromised  neuronal   structure,  and  reduced  plasticity  following  treatments  that  reduce  Bdnf  availability   or  signaling,  it   is  no  surprise  that  these  treatments  also  have  deleterious  effects   on  memory  (Linnarsson  et  al.,  1997;;  Ma  et  al.,  1998;;  Mu  et  al.,  1999;;  Mizuno  et   al.,   2000;;   Alonso   et   al.,   2002;;   Heldt   et   al.,   2007;;   Bekinschtein   et   al.,   2008).     Reducing   Bdnf   availability   impairs   learning   and/or   memory   in   the   novel   object   recognition  task  (Heldt  et  al.,  2007),  Morris  water  maze  (Linnarsson  et  al.,  1997;;   Mu  et  al.,  1999),  non-­swimming  maze  tasks  (Mizuno  et  al.,  2000),  and  one-­trial   avoidance   tasks   (Ma   et   al.,   1998;;   Alonso   et   al.,   2002).     It   is   important   to   recognize  that  learning  itself  is  associated  with  increased  expression  of  Bdnf  (Ma   et  al.,  1998;;  Mizuno  et  al.,  2000;;  Alonso  et  al.,  2002;;  Bekinschtein  et  al.,  2008;;   Lubin  et  al.,  2008),  and  remarkably,  Bdnf  availability   is   important  for  both  short-­   and   long-­term   memory   formation   and   persistence.     Bekinschtein   et   al.   (2008)   investigated   the   role   of   Bdnf   in   long-­term   memory   formation/persistence   and   observed  that  Bdnf  was  necessary  and  sufficient  for  protein  synthesis-­dependent   long-­term   memory   persistence.   Infusion   of   anisomycin,   a   protein   synthesis   inhibitor,   into   the   hippocampus   12   hours   after   a   one-­trial   inhibitory   avoidance   paradigm   reduced   memory   performance   seven   days   but   not   two   days   after   training,   suggesting   an   impairment   of   long-­term   memory   retention   but   not   formation.     Infusion   of   a   recombinant   Bdnf   protein   15   minutes   after   the       21   anisomycin   infusion   rescued   memory   persistence.     Further,   a   weak   inhibitory   avoidance   training   paradigm   that   was   unable   to   induce   Bdnf   expression   and   long-­term  memory   persistence   resulted   in   long-­term  memory   persistence  when   coupled  with  infusion  of  a  recombinant  Bdnf.    Alonso  et  al.  (2002)  used  an  anti-­ Bdnf  antibody   infused   into  CA1  of   the  dorsal  hippocampus   to  demonstrate   that   Bdnf   is   essential   for   short-­term   and   long-­term   memory   performance   in   an   inhibitory  avoidance  task.    When  the  anti-­Bdnf  antibody  was  infused  15  minutes   prior   to   the   training   trial,   performance   was   impaired   1.5   and   24   hours   later.     Interestingly,  when   the  antibody  was  delivered  one  or   four   hours   after   training,   performance  24  hours  later  was  impaired  but  not  when  it  was  delivered  at  0  or  6   hours   post-­training.     Infusion   of   recombinant   Bdnf   15   minutes   before   or   immediately  after  training  improved  short-­term  memory  (1.5  hrs),  while  infusion  at   1   or   4   hours   post   training   improved   long-­term   memory   (24   hrs).     These   data   suggest  that  there  are  critical  windows  in  time  in  which  Bdnf  expression  is  crucial   for  the  formation  of  short-­  and  long-­term  memory.           Research  on  BDNF  has  provided  profound  evidence  of  the  significance  of   this  neurotrophin  in  the  maintenance  and/or  enhancement  of  brain  health.    Aging,   obesity,   diabetes,   and   numerous   mental   health   disorders   such   as   depression,   anxiety,   and   Alzheimer’s   disease   are   associated   with   reduced   BDNF   levels,   which   may   contribute   to   memory   impairments   (Marosi   &   Mattson,   2014).     Exercise   training   is   an  effective  way  of   increasing  BDNF  expression   in   healthy   and  pathological  populations   (Cotman  &  Berchtold,  2002;;  Cotman  et  al.,  2007).     Moreover,   BDNF   is   essential   for   many   of   the   beneficial   adaptations   observed       22   following   exercise   training;;   however,   many   questions   about   exercise   and   Bdnf   expression   still   remain.   For   example,   what   are   the   most   effective   exercise   strategies  to  increase  BDNF  expression  and  what  signaling  mechanisms  induced   by  exercise  stimulate  BDNF  expression?    Though  research  has  established  that   maintaining  a  certain  level  of  BDNF  expression  is  important  for  brain  health,  the   studies  by  Bekinschtein  et  al.   (2008)  and  Rossi  et  al.   (2006)   reveal   that  certain   “windows  of  opportunity”  exist  for  BDNF  expression  to  influence  memory.    Once   questions   such   as   these   are   adequately   addressed,   exercise   training   can   be   tailored  to  most  effectively  increase  BDNF  expression  to  enhance  brain  health  in   healthy  and  pathological  populations.       Exercise  Training/Fitness  and  Peripheral  BDNF  Levels   Generally,  it  is  believed  that  exercise  training  is  associated  with  increased   circulating   BDNF,   though   research   in   healthy   adults   has   provided   inconclusive   evidence  with  some  studies  showing  higher  basal  BDNF  concentrations  following   training  or  in  highly  fit   individuals  (Zoladz  et  al.,  2008;;  Erickson  et  al.,  2011),  no   difference  (Castellano  &  White,  2008;;  Schiffer  et  al.,  2009;;  Flöel  et  al.,  2010),  or   even  lower  BDNF  in  trained  or  highly  fit   individuals  (Chan  et  al.,  2008;;  Nofuji  et   al.,  2008;;  Currie  et  al.,  2009;;  Babaei  et  al.,  2014).    These  discordant  findings  are   potentially  due  to  differences  in  sample  sizes,  training  status  of  the  participants,   types  of  exercise  training  protocols,  or  whether  BDNF  was  measured  in  serum  or   plasma  (Knaepen  et  al.,  2010).    Moreover,  the  biological  importance  of  circulating   BDNF   levels   for   hippocampal   health   is   not   understood.     Erickson   et   al.   (2011)       23   demonstrated   that   the   increase   in  hippocampal   volume   following   six  months  of   aerobic   exercise   training   was   associated   with   increased   serum   BDNF.     An   association   between   peripheral   BDNF   levels   and   anterior   hippocampal   volume   was  also  observed  by  Wagner  et  al.   (2015);;  however,   they  actually  observed  a   reduction   in   hippocampal   volume   following   a   six-­week   exercise   training   intervention   that   improved   fitness.    Higher   levels  of  BDNF  were  still   associated   with   greater   hippocampal   volume   even   though   training   was   associated   with   reduced   hippocampal   volume.     It   is   curious   that   both   fitness   and   BDNF   are   associated  with  hippocampal  volumes,  yet  numerous  studies  indicate  that  higher   fit  individuals  have  lower  peripheral  BDNF  levels.    This  questions  the  usefulness   of   using   peripheral   BDNF   levels   to   infer   adaptations   occurring   in   the   hippocampus.     Currently,   it   is   necessary   to   use   animals   to   characterize   the   influence  of  exercise  on  hippocampal  Bdnf  and   the  mediating  mechanisms  and   associated  adaptations.     Physical  Activity  Increases  Hippocampal  Bdnf  in  Rodents   In  the  rodent  hippocampus,  Bdnf  protein  and  mRNA  are  elevated  following   both  short-­term   (≤  seven  days;;  Neeper  et  al.,  1995;;  1996;;  Molteni  et  al.,  2002;;   Vaynman   et   al.,   2003;;   Berchtold   et   al.,   2005;;   Huang   et   al.,   2005;;   Ding   et   al.,   2011;;  Sartori  et  al.,  2011)  and  longer  exercise  exposures  (>  seven  days;;  Molteni   et  al.,  2002;;  Farmer  et  al.,  2004;;  Berchtold  et  al.,  2005;;  Liu  et  al.,  2009;;  Berchtold   et  al.,  2010;;  Ding  et  al.,  2011;;  Kobilo  et  al.,  2011;;  Sartori  et  al.,  2011;;  Marlatt  et   al.,   2012;;   Wrann   et   al.,   2013).     Importantly,   studies   show   that   both   forced       24   treadmill  exercise  and  voluntary  wheel  running  increase  hippocampal  expression   of  Bdnf  (Liu  et  al.,  2009;;  Alomari  et  al.,  2013).    It  is  well  established  that  exposure   to  physical  activity  in  rodents  improves  spatial  (Fordyce  &  Farrar,  1991;;  Vaynman   et   al.,   2004;;   Gomez-­Pinilla   et   al.,   2008;;   Liu   et   al.,   2009;;   Creer   et   al.,   2010;;   Intlekofer  et  al.,  2013)  and  non-­spatial  (O'Callaghan  et  al.,  2007;;  Liu  et  al.,  2008;;   2009)   memory;;   however,   blocking   Bdnf-­TrkB   signaling   (Vaynman   et   al.,   2004;;   Gomez-­Pinilla   et   al.,   2008;;   Intlekofer   et   al.,   2013)   prevents   such   beneficial   adaptations  to  exercise  training,  including  improved  spatial  memory  and  elevated   expression  of  plasticity  associated  markers.    Using  a  recombinant  human  TrkB-­ IgG  chimera  to  block  Bdnf  action,  Vaynman  et  al.  (2004)  demonstrated  that  Bdnf   signaling  was  necessary  for  the  improvement  in  Morris  water  maze  performance   afforded  by  one  week  of  voluntary  wheel  running.    Importantly,  the  chimera  alone   did   not   impair   task   performance;;   it   merely   attenuated   the   beneficial   effect   of   voluntary   exercise.   The   chimera   also   blocked   the   exercise-­induced   increase   in   CREB   phosphorylation   and  mRNA   expression   of  Bdnf,  TrkB,  Synapsin   1,   and   Creb.    This  group  later  replicated  the  finding  that  one  week  of  voluntary  exercise   improved  performance  on  the  Morris  water  maze  and  that  this  was  blocked  using   the  TrkB-­IgG  chimera.    They  further  extended  the  findings  of  their  previous  report   to   show   that   blocking   Bdnf   signaling   attenuated   the   metabolic   transcriptional   profile   induced   by   one   week   of   voluntary   running   (Gomez-­Pinilla   et   al.,   2008).     Lin  et   al.   (2012)   reported   that   injecting  a  TrkB   inhibitor,  K252A,   into   the  dorsal   hippocampus   prevented   the   exercise   facilitation   of   contextual   fear   learning,   a   hippocampal-­dependent   fear   learning   task.     Unfortunately,   K252A   might   block       25   activity  of  other  important  kinases,  such  as  CAMKII,  and  therefore,  it  is  difficult  to   attribute   this   finding   solely   to   Bdnf-­TrkB   signaling.     Other   researchers   have   attempted   to   mimic   the   effects   of   exercise   using   intracerebral   injections   of   a   human  recombinant  Bdnf  and  observed  a  similar  improvement  in  spatial  learning   between  mice  exposed   to  seven  days  of   forced   treadmill   running  and   the  mice   injected   with   the   recombinant   Bdnf   (Griffin   et   al.,   2009;;   Bechara   et   al.,   2014).     These   mimicking   studies   only   demonstrate   that   the   effects   of   exercise   and   infusions  of  recombinant  Bdnf  have  similar  effects  on  spatial  learning  but  do  not   provide   evidence   that  Bdnf   is  mediating   the   effect   of   exercise.     The  mimicking   investigations   would   be   stronger   if   the   recombinant   Bdnf   infusion   rescued   compromised   exercise-­induced   facilitation   of   learning   by   a   manipulation   that   reduces  endogenous  Bdnf  availability.    Other  mRNA  and  protein  targets  such  as   tissue-­type   plasminogen   activator   (tPa),   insulin   like   growth   factor   (Igf-­1),   and   peroxisome  proliferator-­activated  receptor  γ  coactivator  1  alpha  (Pgc-­1a)  have  all   been   shown   to   be   critical   for   exercise-­induced   adaptations   in   the   rodent   hippocampus  and  remarkably,  each  of  these  proteins  is  linked  to  Bdnf  signaling   or  expression  (Vaynman  et  al.,  2004;;  Ding  et  al.,  2006;;  2011;;  Voss  et  al.,  2013;;   Wrann  et  al.,  2013).       An  interesting  caveat  to  the  research  examining  exercise  training  and  Bdnf   expression   in   rodents   is   that   the  majority   of   research   has   focused   on   exercise   exposures  from  seven  to  28  days.    In  fact,  “chronic”  or  “long-­term”  exercise  has   not  been  adequately  defined  in  the  literature.    Importantly,  wheel  running  volume   declines  over   time  (Richter  et  al.,  2014;;  Venezia  et  al.,  2015)  and   it   is  possible       26   that   the   high   levels   of   running   and   novelty   of   the   new   environment   upon   introduction  of   a   running  wheel   stimulate  Bdnf  expression   to  a   level   that   is   not   observed   following   prolonged   exposure   to   a   wheel.     However,   there   is   some   evidence   of   elevated   Bdnf   expression   with   longer   exercise   exposures.     Ninety   days   of   wheel   running   was   effective   at   increasing   hippocampal   Bdnf   protein   expression  in  rats  (Berchtold  et  al.,  2005).    Eight  months  of  wheel  (Marlatt  et  al.,   2012)  and   treadmill   (O'Callaghan  et  al.,   2007)   running  have   resulted   in  greater   Bdnf  expression  in  older  animals;;  however,  aging  is  associated  with  a  decrease   in   Bdnf   expression   (O'Callaghan   et   al.,   2009)   and   the   benefits   of   long-­term   exercise  training  on  Bdnf  expression  in  these  investigations  may  be  a  rescue  of   compromised   Bdnf   expression   due   to   aging   in   lifelong   sedentary   conditions.     Whether  similar  benefits  would  be  observed  in  young  adult  rodents  is  not  known.       Sex  Differences  in  Exercise  Induced  BDNF  Expression     Research   in   both   humans   and   animals   suggests   a   complex   relationship   between  sex  hormones  and  Bdnf  expression  (Carbone  &  Handa,  2013;;  Pluchino   et   al.,   2013).     The   menstrual   cycle   influences   peripheral   levels   of   BDNF   and   women  with   normal   ovulatory   cycles   have   higher   levels   of   BDNF   compared   to   amenorrhoeic   or   postmenopausal  women   (Begliuomini  et   al.,   2007).    Hormone   therapy   used   for   male-­to-­female   transsexuals   is   associated   with   reduced   peripheral   BDNF   (Fuss   et   al.,   2015).     Even   diurnal   fluctuations   in   peripheral   BDNF  differ  between  sexes,  with  men  experiencing  greater  variation  throughout   the  day  (Piccinni  et  al.,  2008;;  Choi  et  al.,  2011).    Concerning  exercise  and  BDNF       27   in   humans,   a   recent   meta-­analysis   reported   that   effect   sizes   were   generally   smaller   in   studies   that   included   females   (Szuhany   et   al.,   2015)   and   in   a   systematic   review,   Knaepen   et   al.   (2010)   suggest   that   sex   might   be   a   contributing   factor   to   differences   observed   between   studies   reporting   the   influence  of  exercise  on  peripheral  BDNF.       A  limited  number  of  studies  have  utilized  both  male  and  female  animals  in   investigations   on   the   influence   of   exercise   on   hippocampal   Bdnf   expression.     Berchtold   et   al.   (2001)   reported   that   five   days   of   wheel   exposure   increased   hippocampal  Bdnf  mRNA  and  protein  expression  in  female  rats;;  however,  in  rats   that  were  ovariectomized  seven  weeks  before  exercise  exposure,  exercise  was   unable   to   increase   hippocampal   Bdnf   mRNA   or   protein.     Ovariectomy   itself   reduced  hippocampal  Bdnf  mRNA  and  protein.    Gallego  et  al.  (2015)  reported  an   increase  in  hippocampal  Bdnf  mRNA  and  protein  expression  following  21  days  of   voluntary  wheel  exposure  in  adolescent  male  and  female  mice  while  Titterness  et   al.   (2011)   reported  no  differences   in  Bdnf  protein   in  male  or   female  adolescent   rats.     However,   Titterness   et   al.   (2011)   also   reported   that   exercise   facilitated   synaptic  plasticity  (i.e.  LTP)  only   in  adolescent  males,  which   is  surprising   in   the   absence   of   increased   Bdnf   expression.     Understanding   sex   differences   in   response   to  environmental   factors  and   therapeutic   treatments   is   very   important   for  optimizing  brain  health  (Cahill,  2006)  and  much  remains  unknown  about  how   sex  and  exercise  interact  to  enhance  critical  signaling  factors  such  as  BDNF.    For   example,  as  males  generally  display  diurnal  variation  in  BDNF  levels,  potentially   there  is  an  optimal  time  to  participate  in  exercise  activities  to  effectively  increase       28   BDNF   levels.    Moreover,   in   females,   exercise   participation   could   be   tailored   to   the  menstrual  cycle  or  hormone  replacement  therapies.         Transcription  of  the  BDNF  Gene     Age,   sex,   and   exercise   type,   duration,   and   intensity   all   influence   BDNF   availability  through  intricately  controlled  transcription  of  the  highly  complex  BDNF   gene.    Total  Bdnf  mRNA  expression  is  the  result  of  the  combined  expression  of   multiple  Bdnf  transcripts.    The  Bdnf  gene  was  originally  characterized  as  having   four   non-­coding   exons   (I,   II,   III,   IV)   that   alternatively   splice   to   one   3’   protein   coding  exon  (V).    However,  in  2007,  the  gene  was  re-­characterized  and  found  to   have   eight   non-­coding   exons   [I,   II,   III   (new),   IV   (previously   III),   V   (new),   VI   (previously   IV),  VII   (new),  VIII   (new)]  with   individual  promoters   that  all   splice   to   one  3’  protein  coding  exon  (exon  IX;;  previously  V)  (Aid  et  al.,  2007).    In  addition,   the  gene  has  two  3’  polyadenylation  sites  (Timmusk  et  al.,  1993;;  Aid  et  al.,  2007).     This  complexity  results  in  as  many  as  22  possible  transcripts  (Zheng  et  al.,  2012).   Interestingly,  all  Bdnf  transcripts  are  translated  into  the  same  protein  (proBDNF),   which   is   then   cleaved   to   produce   the   mature   plasticity-­associated   protein.     Though  it  is  not  fully  understood  why  so  many  transcripts  are  needed  to  produce   the   same   protein,   the   transcripts   have   different   subcellular   localization   and   transport  capabilities  (Baj  et  al.,  2011;;  2013)  and  the  large  number  of  transcripts   offers   tight   temporal   and   regional   control   of   transcription,   mRNA   survival,   and   distribution.     For   example,   Baj   et   al.   (2013)   reported   a   unique   spatial   code   of   Bdnf   transcripts   throughout   the   hippocampal   subfields   under   rest,  with  Bdnf  VI       29   being   the  primary   transcript   found   in   the  dendrites  of  all  hippocampal  subfields;;   however,   after   pilocarpine-­induced   neuronal   activity,  Bdnf   transcripts   IV   and  VI   were  highly  present  in  hippocampal  dendrites.    Bdnf  IV  was  originally  believed  to   be  restricted  to  the  soma  even  with  neuronal  activity  (Chiaruttini  et  al.,  2008)  but   the  more   recent   study   by   Baj   et   al.   (2013)   demonstrated   that   this   transcript   is   transported   to   the  dendrites   following  high  neuronal  activity.    Bdnf   transcript   IV   (driven   by   promoter   IV)   has   been   the   target   of  much   of   the   research   on  Bdnf   transcription.     Though   Bdnf   is   considered   to   be   an   activity-­regulated   gene,   promoter   IV-­driven  Bdnf   transcription   is  especially   sensitive   to  neuronal   activity   (Tao   et   al.,   1998;;   2002;;   Martinowich   et   al.,   2003).     Intense   investigation   of   transcriptional   regulation   of   exon   IV   has   revealed   this   transcript   to   be   calcium   sensitive,   containing   at   least   three   calcium-­responsive   elements   around   the   transcriptional  initiation  site  of  exon  IV  (Zheng  et  al.,  2011;;  2012).    While  in  vitro   work   demonstrates   a   clear   association   between   neuronal   activity   and  Bdnf   IV   transcription  (Tao  et  al.,  1998;;  Martinowich  et  al.,  2003),  environmental  stimuli  in   vivo  result  in  additional  Bdnf  transcript-­specific  transcription.    Using  a  contextual   fear   conditioning   paradigm   in   rats,   Lubin   et   al.   (2008)   demonstrated   that   hippocampal   total   Bdnf   mRNA   (exon   IX)   was   elevated   after   both   context   exposure  alone  (no  shock)  and  associative  fear  conditioning,  though  the  elevated   expression   of   total  Bdnf   was  mediated   by   different   exon-­containing   transcripts.     Following   context   exposure   alone,   the   elevated   total   Bdnf  mRNA   was   due   to   elevated  Bdnf  I  and  VI  mRNA,  while  after  associative  fear  conditioning  it  was  due   to   elevated   Bdnf   IV   expression.     Acute   immobilization   stress   differentially       30   influences   transcription   of   specific   transcripts   depending   on   the   length   of   immobilization.    Two  hours  of  immobilization  resulted  in  reduced  total  Bdnf  (exon   IX),  Bdnf  I,  and  Bdnf  IV  compared  to  non-­stress  controls  (Fuchikami  et  al.,  2008).     An   earlier   study   by   Marmigere   et   al.   (2003)   demonstrated   that   transcripts   containing   exons   I,   II,   and   IV   (III   in   the   paper)   were   elevated   after   short   exposures   to   immobilization  stress   (15  –  60  minutes).    Bdnf   IV  expression  was   elevated  at  15  minutes,  which  suggests  a  very  rapid  stimulus-­induced  expression   of  this  transcript,  though  it  reached  peak  expression  at  60  minutes.    Transcripts  I   and  II,  were  not  yet  elevated  at  15  minutes  but  also  reached  peak  expression  at   60  minutes.    Transcript  VI  expression  was  unchanged  at  60  minutes  and   lower   than   controls   at   180   minutes   (Marmigère   et   al.,   2003).     Concerning   exercise   training  and   transcript-­specific  Bdnf  expression,   three  weeks  of  voluntary  wheel   running   in   C57BL/6J   mice   was   sufficient   to   elevate   mRNA   for   total   Bdnf   and   exons   I   and   IV   in   the   hippocampus   but   did   not   increase   exon   VI   expression   (Intlekofer  et   al.,   2013).     These   changes   in  Bdnf   transcription  were   associated   with   cognitive   benefits   in   an   object   location   task   that   were   blocked   by   hippocampal  infusion  of  a  short-­interfering  RNA  for  Bdnf.    A  study  by  Zajac  et  al.   (2009)   reported   that   eight   weeks   of   wheel   running   increased   hippocampal   expression  of  exons  I,  II,  III,  IV,  and  VI  in  females  and  exons  I,  II,  and  III  in  males.     Baj  et  al.  (2012)  reported  an  increase  in  total  Bdnf  following  28  days  of  voluntary   wheel  running  in  the  somata  and  apical  dendrites  of  CA3.    There  was  no  effect  of   exercise  on  Bdnf  IV  expression  but  there  was  an  increase  in  CA1  and  CA3  Bdnf   VI  expression.     In  addition,  Bdnf  exon  IV   in   the  rodent  hippocampus  undergoes       31   epigenetic   modifications   with   seven   days   of   running   wheel   exposure,   which   suggested  enhanced  transcriptional  capabilities  (Gómez-­Pinilla  et  al.,  2010).       These   studies   demonstrate   that   control   of   BDNF   transcription   is   highly   complex,   with   different   environmental   stimuli   inducing   unique   transcriptional   profiles   in   a   region-­,   time-­,   and   sex-­dependent   manner.     Compromised  BDNF   expression  influences  a  number  of  mental  health  disorders,  so  understanding  the   BDNF  transcripts  most  influenced  by  these  disorders  and  affected  by  common  or   novel   treatment   interventions  will  greatly  enhance  approaches  to   treating  at-­risk   or   suffering   individuals.     It   is   strongly   supported   that   regular   chronic   physical   activity  is  beneficial  for  overall  health,  though  to  truly  capitalize  on  the  benefits  of   exercise  as  a  non-­invasive  therapeutic  strategy  for  mental  health  disorders,   it   is   critical   to   understand   the   adaptations   occurring   in   response   to   acute   bouts   of   exercise.    A  thorough  understanding  of  the  immediate  and  delayed  adaptions  to   acute   exercise   can   identify   exercise   strategies   to   be   used   most   effectively   to   enhance  mental  health.    For  example,  to  improve  cognitive  function  in  cognitively   impaired  older  adults  or  Alzheimer’s  disease  patients,   there  may  be  an  optimal   approach  of  combining  acute  exercise  bouts  with  memory  training,  or  an  optimal   intensity   of   acute   exercise   combined   with   a   pharmacological   approach.     Answering  these  types  of  questions  will  require  thorough  investigations  of  acute   exercise  and  the  adaptations  and  mediating  mechanisms.   Acute  Exercise  and  Memory       32   The   impact   of   acute   bouts   of   physical   activity   on   the   brain   is   less   understood  than  chronic  physical  activity  or  exercise  training.    This  may  be  due   to   the   lack   of   acute   exercise   studies   in   rodents   to   identify   mechanisms   and   structural   adaptations.     It   is   important   to   remember   that   the   mechanisms   mediating  the  effects  of  chronic  physical  activity  or  exercise  training  are  likely  the   result  of  accumulated  repeated  bouts  of  acute  exercise.     Indeed,  acute  bouts  of   physical  activity  have  been  reported  to  improve  cognitive  performance  in  humans,   though   differences   in   exercise   protocols,   types   of   cognitive   measures,   and   whether   cognitive   testing   took   place   before,   during,   or   after   exercise  make   the   data   difficult   to   interpret   (Brisswalter   et   al.,   2002;;   Tomporowski,   2003;;   Lambourne  &  Tomporowski,  2010;;  Chang  et  al.,  2012;;  Roig  et  al.,  2013).    Much   of   the   research   to   date   has   focused   on   acute   exercise-­induced   arousal   and   cognitive  processing  during   the  acute  bout  of  exercise,  or   focused  on  cognitive   tasks  designed  to  assess  executive  functioning.    However,  recent  meta-­analyses   have  provided   support   for   an  association  between  acute  exercise  and  memory   performance,  especially  when  the  cognitive  task   is  performed  after   the  exercise   (Lambourne  &  Tomporowski,  2010;;  Chang  et  al.,  2012;;  Roig  et  al.,  2013).    Using   a   meta-­analytic   approach,   Lambourne   and   Tomporowski   (2010)   explored   the   influence  of  acute  exercise   timing   relative   to  cognitive   task  performance.    They   determined   that   cognitive   task   performance   was   impaired   when   the   task   was   performed   during   the   first   20   minutes   of   acute   exercise.     However,   task   performance   was   facilitated   when   the   task   was   performed   after   the   first   20   minutes  of  exercise  initiation.    They  also  found  an  improvement  in  cognitive  task       33   performance   when   the   task   was   performed   after   the   acute   bout   of   exercise.     Interestingly,   they   reported   larger  effects   for  memory  performance  compared   to   processing  and/or  reaction  time  following  an  acute  bout  of  exercise.    Concerning   memory,   Coles   and   Tomporowski   (2008)   reported   that   40  minutes   of   exercise   improved   long-­term  memory   on   a   delayed   free   recall   test   in   a   group   of   young   adults.    Individuals  exercised  for  40  minutes  (30  minutes  at  60%  VO2max;;  5  min-­ warm   and   5  min   cool-­down)   prior   to   the   encoding   phase   of   the  memory   task.     Interestingly,   the   benefit   of   acute   exercise   was   only   observed   for   long-­term   memory,  with   no  benefit   observed   for   short-­term  memory.    A   study   by   Labban   and  Etnier   (2011)   tested   the   effect   of   acute   exercise   on   long-­term  memory   by   presenting   subjects   with   two   paragraphs   after   a   bout   of   cycling   exercise   and   reported   that   these   subjects   recalled   significantly   more   than   non-­exercise   controls  after  a  35-­minute  delay.    The  subjects  that  performed  exercise  after  the   learning  phase  of  the  memory  task  did  not  recall  more  than  non-­exercise  controls,   demonstrating   the   benefit   of   exercise   prior   to   learning.   In   contrast,   exercise   following   learning  has  also  been  shown  to  be  effective  at  enhancing  memory   in   certain  populations  (Segal  et  al.,  2012).    Segal  et  al.  (2012)  showed  that  an  acute   bout  of  exercise  after  picture  viewing  enhanced  memory  for  emotional  images  in   older   adults   with   or   without   mild   cognitive   impairment.     These   studies   on   the   benefits   of   acute   exercise   on   memory   are   supported   by   a   more   recent   meta-­ analysis  by  Roig  et  al.  (2013),  which  reported  that  acute  cardiovascular  exercise   is   associated  with   small   to  moderate  effects   on  memory  with   the   larger   effects   (moderate   to   large)  observed   for   long-­term  memory   (compared   to  short-­term  or       34   working   memory).   More   research   is   certainly   needed   to   understand   the   effectiveness  of  acute  exercise  on  cognitive   function.    Factors  such  as   intensity   of  exercise  (Winter  et  al.,  2007)  might  play  a  role  and  certain  populations  might   benefit  more   from  acute  exercise.  For  example,  populations  at   risk  or   suffering   from  mental  health  disorders,  which  may  be  associated  with  compromised  BDNF   levels   and   other   important   signaling   factors,   may   be   particularly   sensitive   to   properly  prescribed  acute  exercise.     Acute  Exercise  Increases  Peripheral  BDNF  in  Humans     The   mechanisms   mediating   the   beneficial   effects   of   acute   exercise   on   memory  performance  in  humans  are  not  fully  understood.    Based  on  the  findings   of   chronic   exercise   training   studies   in   rodents   and   humans,   researchers   have   attempted   to  use  correlational  analyses   to  support   the  hypothesis   that  elevated   peripheral   BDNF   is   the   mechanism   mediating   the   cognitive   enhancement   afforded  by  acute  exercise   (Piepmeier  &  Etnier,  2015).     Indeed,  acute  exercise   increases   peripheral   BDNF   levels   in   humans   (Vega   et   al.,   2006;;   Ferris   et   al.,   2007;;  Winter  et   al.,   2007;;   Rasmussen  et   al.,   2009;;  Griffin  et   al.,   2011)   and   in   some   studies,   the   increases   are   associated   with   improved   cognitive   task   performance   (Ferris  et   al.,   2007;;  Winter  et   al.,   2007;;  Griffin  et   al.,   2011).     For   example,   Ferris   et   al.   (2007)   showed   that   serum  BDNF  was   elevated   after   an   acute   bout   of   exercise   at   an   intensity   above   the   ventilatory   threshold,   but   this   effect  did  not  persist   if   the  acute  exercise  was  performed  at  an   intensity  below   the  ventilatory  threshold.    The  high-­intensity  acute  exercise  was  associated  with       35   better   performance   on   the   Stroop   Color   and   Word   Test,   a   task   of   executive   functioning.    Winter   et   al.   (2007)   reported   that   faster   vocabulary   learning   after   high-­intensity   running   was   associated   with   increased   levels   of   BDNF.     It   is   important  to  recognize  that  exercise  increases  levels  of  many  circulating  factors   and   using   correlational   analyses   with   selected   factors   like   BDNF   should   be   interpreted   with   caution.     It   is   difficult   to   link   the   elevation   of   peripheral   BDNF   levels   with   improved   cognitive   task   performance   since   70-­80%   of   circulating   BDNF   is   released   from   the   brain   into   systemic   circulation   and   therefore   is   no   longer   in   the   vicinity   of   the   brain   regions   associated   with   enhanced   cognitive   performance   (Rasmussen   et   al.,   2009).   More   studies   and   a   more   thorough   understanding  of  the  benefits  of  elevated  peripheral  BDNF  levels  for  brain  health   are  necessary.    Animal  models  provide  an  effective  way  to  understand  how  acute   physical   activity   influences   expression   of   plasticity-­associated   genes   in   the   hippocampus.   Acute  Exercise  and  Hippocampal  Bdnf  in  Rodents     How  a  single  bout  of  acute  exercise  influences  hippocampal  Bdnf  levels  is   not   fully   understood.     Obstacles   to   interpreting   the   acute   exercise   and   hippocampal   Bdnf   literature   include   differences   in   exercise   exposures   and   various  acclimation  protocols   that  might   induce  adaptations   independent   of   the   acute   exercise   or  may   interact  with   the   acute   bout   of   exercise.    Goekint   et   al.   (2012)   found  no  effect  of  60  minutes  of   treadmill   running  on  Bdnf  protein   in   the   rat   hippocampus   immediately   following   or   two   hours   after   an   exercise   bout.         36   However,  Oliff  et  al.  (1998)  reported  that  both  six  hours  and  12  hours  of  voluntary   wheel  running  increased  Bdnf  mRNA  in  the  rat  hippocampus.    Importantly,  in  this   study   the   rats   were   housed   in   cages   with   voluntary   running   wheels   for   three   nights   to   acclimate   the   rats   to   the   environment.     This   “training”   period   was   followed  by  10  days  of  no  wheel  exposure  before  the  acute  bout  of  exercise  (six   or   12   hours   of   housing   with   a   running   wheel).     The   authors   reported   that   six   hours  of  running  significantly   increased  Bdnf  mRNA  in  CA1,  CA3,  and  the  hilus   region  of  the  hippocampus.    The  six  and  12  hour  runs  also  significantly  increased   Bdnf  exon  I  expression  in  the  dentate  gyrus,  CA3,  and  hilus.    Exon  II  expression   was  increased  in  CA1  after  12  hours  of  running.    Exon  IV  (exon  III  in  the  paper)   was   not   elevated   following   acute   exercise;;   however,   this   finding   is   difficult   to   interpret   because   researchers   reported   that   rats   that   underwent   three   days   of   “training”  followed  by  10  days  of  no-­wheel  exposure  had  elevated  Bdnf  mRNA  in   CA1  and  elevated  BDNF  IV  in  all  hippocampal  regions  examined.    This  finding  is   important  because  it  highlights  the  need  to  examine  truly  acute  bouts  of  exercise   since   even   10   days   of   sedentary   conditions   may   not   negate   the   effects   of   previous  physical   activity.    Rasmussen  et  al.   (2009)   reported   that   two  hours  of   treadmill   running   to  exhaustion   increased  Bdnf  mRNA   in   the  hippocampus  and   cortex   at   two   hours   post-­running   but   not   immediately   post-­running.     Similar   to   Goekint   et   al.   (2012)   and  Oliff   et   al.   (1998),   the  mice   in   the  Rasmussen   et   al.   (2009)   investigation   underwent   a   familiarization   protocol   that   involved   exercise   training   followed   by   a   “wash   out”   prior   to   the   acute   bout   of   exercise.     These   studies  suggest  that  Bdnf  expression  can  be  induced  by  acute  bouts  of  exercise       37   but  also  highlight  the  need  to  understand  the  adaptations  to  truly  acute  bouts  of   exercise.     The   evidence   for   rapid   induction   of   Bdnf   with   other   environmental   stimuli,   such   as   immobilization   stress   (Marmigère   et   al.,   2003)   suggests   that   exercise   may   also   be   able   to   induce   rapid   increases   in   Bdnf   expression.     Elevated   Bdnf   mRNA   expression   following   acute   exercise   may   prime   the   hippocampus   for   learning   by   providing   the   mRNA   to   be   translated   during   encoding  and  consolidation  and  signal  for  architectural  adaptations  necessary  for   the  formation  and  persistence  of  memory.         Research   in   humans   and   rodents   demonstrates   that   acute   exercise   can   influence  central  and  peripheral  BDNF  levels,   though  many   important  questions   still   remain.  For  example,  what   is   the   importance  of  peripheral  BDNF  and  what   exercise-­induced   mechanisms   are   stimulating   the   production   and   release   of   central  BDNF?    Potentially,   circulating   factors   released   in   response   to  exercise   activate   signaling   pathways   to   increase   expression   of   BDNF.     Catecholamines   are   a   promising   target   as   they   are   influenced   by   acute   exercise,   strongly   influence  memory  and  plasticity,  and  regulate  BDNF  expression.   Catecholamines   and   Exercise   –   The   Link   Between   the   Periphery   and   Improved  Memory?     Research   in   stress   and   emotion   has   identified   arousal   and   subsequent   activation   of   the   noradrenergic   (NA)   system   as   a   critical   and   potent   memory   enhancer   (McGaugh,   2013).   Both   peripheral   levels   of   the   catecholamine   epinephrine  and  central  levels  of  the  neurotransmitter  norepinephrine  (which  are       38   strongly   correlated   and   discussed   below)   are   associated   with   strength   of   memories.    Flashbulb  memories,  which  are  very  vivid  and  long-­lasting  memories   of  significant  and  arousing  public  events   (Brown  &  Kulik,  1977),  and  PTSD  are   good  examples  of  the  effect  of  emotion  and  adrenergic  enhancement  of  memory.   Remarkably,   both   the   β-­adrenergic   receptor   blocker   propranolol   and  morphine   (which   blocks   norepinephrine   release)   decrease   the   risk   of   developing   PTSD   when  provided  shortly  after  a  traumatic  event  (Vaiva  et  al.,  2003;;  Holbrook  et  al.,   2010).  Moreover,  compromised  memory  with  b-­blockers  has  also  been  reported   in   human   investigations   beyond   those   concerning   PTSD   (Chamberlain   &   Robbins,   2013).     For   example,   using   propranolol,   Cahill   et   al.   (1994)   reported   that  b-­adrenergic  receptors  were  necessary  for  enhanced  memory  of  emotionally   charged   stories   relative   to   neutral   stories.     In   humans,   both   direct   epinephrine   infusions   (Cahill   &   Alkire,   2003)   and   cold   pressor   stress   (Cahill,   2003),   a   treatment  that  stimulates  the  release  of  peripheral  epinephrine,  improve  memory.   Segal  and  Cahill  (2009)  found  a  significant  correlation  between  levels  of  salivary   α-­amylase  (a  marker  of  central  NA  activation)  after  viewing  a  series  of  images  of   varying  emotional  grade  and  memory  recall  of  those  emotional  images  one  week   later.    In  rodents,  b-­adrenergic  receptor  blockers,  such  as  propranolol,  have  been   shown   to   block   spatial   and   contextual   fear   memories   when   infused   into   the   hippocampus   (Ji,  Wang,  et   al.,   2003;;   Ji,   Zhang,  et   al.,   2003),  while   infusion   of   norepinephrine   into   the   hippocampus   and   peripheral   injections   of   epinephrine   improve   inhibitory   avoidance   and   contextual   fear   memory,   respectively     (Izquierdo  et  al.,  1998;;  Hu  et  al.,  2007).    It  is  beyond  the  scope  of  this  review  to       39   comprehensively  review  all  of  the  research  on  the  memory  enhancing  effects  of   NA   stimulation.     Investigations   of   the   effects   of   NA   stimulation   on   encoding,   consolidation,  and  retrieval  of  memories  spans  decades  and  numerous  excellent   reviews   have   been   written   on   the   topic   (for   examples   see   McGaugh   &   Roozendaal,  2002;;  Kensinger,  2009;;  Chamberlain  &  Robbins,  2013;;  McGaugh,   2013;;   O'Dell   et   al.,   2015;;   Osborne   et   al.,   2015).     Though  much   research   has   investigated   the  memory-­enhancing  effects  of  stress  and  catecholamines,  more   research  is  needed  to  provide  useful  strategies  to  utilize  the  memory-­enhancing   effects   of   stress   and   catecholamines.     Potentially,   exercise   can   be   used   as   a   non-­invasive   and   non-­traumatic   environmental   stimulus   to   activate   the   NA   system   to   enhance   memories   similar   to   the   enhanced   memory   observed   with   psychological  stress.     Circulating   epinephrine   might   be   the   signaling   factor   between   the   periphery  and  the  hippocampus  during  exercise  that  mediates  the  enhancement   of  memory  and  hippocampal  plasticity.     In  a  2002   review  of   literature  on  acute   bouts  of  physical  activity  and  cognitive  performance,  it  was  suggested  that  for  an   acute  bout  of  exercise  to  impact  cognition,  the  exercise  must  be  intense  enough   to  increase  circulating  catecholamines  (Brisswalter  et  al.,  2002).  Indeed,  exercise   of   sufficient   duration   and   intensity   increases   peripheral   catecholamines,   a   necessary   response   for   cardiovascular  and  metabolic  adjustments   to   the  acute   exercise  (Tipton,  2006;;  Zouhal  et  al.,  2008).    Exercise  of  short  duration  and  high   intensity,   or   long   duration   and   low   intensity   is   sufficient   to   significantly   elevate   peripheral   levels   of   catecholamines   (Zouhal   et   al.,   2008).   This   is   due   to   an       40   increase  in  catecholamine  secretion  and  potentially  a  reduction  in  catecholamine   clearance  (Zouhal  et  al.,  2008).  Previous  research  has  tried  to  identify  if  exercise-­ induced   arousal   improves   cognition,   though   many   studies   have   employed   cognitive  tests  that  measure  simple  cognitive  domains  such  as  reaction  time  and   employed   the   cognitive   task   during   exercise   (McMorris   et   al.,   2008;;   2009).     Potentially,  the  cognitive  resources  required  during  some  types  of  exercise  (e.g.   treadmill   exercise)   may   impair   performance   during   exercise   (Lambourne   &   Tomporowski,   2010)   and   more   research   is   needed   specifically   examining   memory,  which   is   not   comparable   to   tasks   assessing   reaction   time.    However,   there  is  evidence  of  a  relationship  between  acute  exercise-­induced  elevations  of   epinephrine  and  memory  performance  (Winter  et  al.,  2007).    An  investigation  by   Winter   et   al.   (2007)   reported   that   high-­intensity   running   increased   peripheral   levels   of   epinephrine   and   memory   performance   on   a   word   recall   task   at   one   week  and  eight  months  after  learning.    The  improved  memory  performance  was   positively   correlated   with   epinephrine   levels.   Though   epinephrine   does   not   directly   influence   hippocampal   plasticity   and   improve   memory,   peripheral   epinephrine   has   direct   influences   on   central   levels   of   the   plasticity-­promoting   neurotransmitter,  norepinephrine.       Peripheral  Epinephrine  Increases  Central  Norepinephrine  Release   Unlike   cortisol   and   other   circulating   factors,   epinephrine   does   not   cross   the   blood-­brain  barrier.  Instead,  epinephrine  mediates  its  effects  through  activation  of   β-­adrenergic   receptors   on   vagal   afferents   that   terminate   on   brainstem   NA   cell       41   groups  in  the  nucleus  of  the  solitary  tract  (NTS)  (McGaugh  &  Roozendaal,  2002).   The   NTS   stimulates   release   of   norepinephrine   from   the   locus   coeruleus   (LC),   which   has   extensive   innervations   to   numerous   brain   regions,   including   the   hippocampus   (Osborne   et   al.,   2015).     Miyashita   and   Williams   (2004)   demonstrated   that   an   intraperitoneal   (IP)   injection   of   saline   had   no   effect   on   hippocampal  extracellular  levels  of  norepinephrine,  however  following  a  single  IP   injection   of   epinephrine   there   was   a   significant   increase   in   hippocampal   norepinephrine.     This   finding   demonstrates   that   a   stimulus   that   increases   peripheral   levels  of  epinephrine  (e.g.  acute  exercise)  can   increase  hippocampal   levels  of  norepinephrine.    Remarkably,  stimulation  of  the  ascending  vagus  nerve   (which   increases  central  norepinephrine)  post-­learning   improves  memory   (Clark   et   al.,   1998),   consistent   with   the   effects   of   epinephrine   infusions   (McGaugh,   2013).   In   addition,   blocking   NTS   activity   with   lidocaine   blocks   the   effects   of   peripheral   epinephrine   on  memory   consolidation   (Williams   &  McGaugh,   1993).     These   investigations   demonstrate   that   peripheral   epinephrine   stimulates   the   ascending  vagus  nerve-­LC-­NA  system  and  exerts   its  memory  enhancement  by   increasing   levels   of   central   norepinephrine.     It   is   important   to   note   that   glucocorticoids  are  also  known  to  be  important  for  the  memory  enhancing  effects   of  acute  psychological  stress  (McGaugh  &  Roozendaal,  2002)  but  it  is  likely  that   glucocorticoids   and   catecholamines   work   in   concert   to   influence   memory   and   plasticity   (Osborne   et   al.,   2015).     In   fact,   blocking   NA   signaling   prevents   the   memory   enhancing   effects   of   glucocorticoids   (Quirarte   &   Roozendaal,   1997;;   Roozendaal   &   Nguyen,   1999).     Roozendaal   and   Nguyen   (1999)   blocked   NA       42   signaling   in   the   amygdala   using   the   β -­adrenergic   blocker   atenolol   and   attenuated   the  memory-­enhancing  effects  of  direct   infusions  of  a  glucocorticoid   agonist  into  the  dorsal  hippocampus.   Central  Norepinephrine  Levels  and  Exercise   In   addition   to   an   increase   in   peripheral   catecholamines,   rodent   research   suggests  that  central  norepinephrine  levels  are  elevated  during  exercise  (Pagliari   &   Peyrin,   1995a;;   1995b).     Pagliari   et   al.   (1995a)   found   that   acute   treadmill   exercise   increased   circulating   epinephrine,   brain   norepinephrine,   and   the   norepinephrine  metabolite  MHPG   in  an  exercise  duration-­dependent  manner   in   the   rat   cortex.     They   observed   that   the   elevation   in   peripheral   epinephrine   preceded  the  increase  in  central  norepinephrine,  which  is  consistent  with  the  idea   that   exercise-­induced   release  of   epinephrine   stimulates   the  exercise-­stimulated   release  of   central   norepinephrine.  However,   the   literature   reporting  an  effect  of   acute   exercise   on   hippocampal   levels   of   norepinephrine   are   not   conclusive.     Goekint   et   al.   (2012)   found   no   effect   of   one   hour   of   treadmill   running   on   hippocampal   norepinephrine   in   rats,   using   microanalysis   during   and   after   the   running.     Dunn   et   al.   (1996)   reported   that   chronic   treadmill   running   increased   basal  hippocampal  levels  of  the  extraneuronal  norepinephrine  metabolite  MHPG,   suggesting  release  and  subsequent  breakdown  of  norepinephrine,  but  no  effect   on   hippocampal   norepinephrine   levels.     Interpretation   of   research   measuring   norepinephrine   levels   in   the   hippocampus   following   exercise   is   difficult,   as   it   is   not   clear   which   measurement   technique   (extra-­synaptic,   tissue   content,       43   metabolites)  most   accurately   reflects  biologically   relevant   norepinephrine   in   the   hippocampus.   Research   in   humans   supports   that   acute   exercise   increases   central   noradrenergic   activation.     Measuring   salivary   α-­amylase   is   an   effective   way  to  predict  central  NA  activation  (Chatterton  et  al.,  1996)  and  research  shows   that  levels  of  salivary  α-­amylase  increase  with  exercise  (Chatterton  et  al.,  1996;;   Allgrove   et   al.,   2008;;   Segal   et   al.,   2012).     Segal   et   al.   (2012)   reported   that   exercise-­induced   elevation   in   salivary   α-­amylase   was   correlated   with   performance  on  a  memory  recall  trial.    These  researchers  had  older  adults  view  a   series   of   images   of   varying   emotional   charge   followed   by   a   six-­minute   bout   of   exercise   at   70%   VO2max,   which   was   sufficient   to   increase   salivary   α-­amylase   and  improve  memory  for  emotional  images.       Norepinephrine  and  Plasticity   Norepinephrine   is   important   for   normal   brain   function   and   a   potent   memory  enhancer.     In  addition   to  activation  of   the  ERK  pathway   (O'Dell  et  al.,   2015)   and   influencing   glucose  metabolism   (Osborne  et   al.,   2015)   in   the   brain,   norepinephrine   induces   plasticity   through   glutamate   receptor   phosphorylation   and  CREB-­mediated  transcription.  Norepinephrine  is  a  NA  neurotransmitter  that   is   synthesized   and   released   from   the   LC.   The   LC   projects   to   numerous   brain   regions   including   the   amygdala,   frontal   cortex,   and   hippocampus.     The   β-­ adrenergic   receptor   is   a   G-­coupled   protein   receptor   that   induces   numerous   downstream  signaling  cascades  (through  cyclic  AMP)  including  posttranslational   modifications   to   key   plasticity-­mediating   molecules   and   CREB-­mediated       44   transcription   (Gelinas,   2005;;   McGaugh,   2013).   Glutamate   is   the   major   neurotransmitter  of   the  central  nervous  system  (CNS),  and  glutamate   receptors   (AMPA,  NMDA,  mGluR)  mediate   the  majority  of   synaptic  communication   in   the   mammalian  brain.  The  AMPA  type  glutamate  receptor  consists  of  multiple  GluR   subunits   (GluR1-­4)   and   is   critical   for   synaptic   communication   and   plasticity   (Santos   et   al.,   2009).   The   adult   mammalian   brain   contains   heterotetramers   composed  of  GluR1/GluR2  and  GluR2/GluR3  combinations  (Santos  et  al.,  2009).     Phosphorylation  of  specific  sites  (Ser831  and  Ser845)  on  GluR1  is  critical  for  the   synaptic  activity-­induced  extra-­synaptic  insertion  of  the  receptor  and  subsequent   migration   to   the   synapse   (Huganir   &   Nicoll,   2013).     Extra-­synaptic   insertion   lowers   the   threshold   for   LTP,  a   cellular   representation  of   learning  and  memory   (Oh   et   al.,   2006).   Hu   et   al.   (2007)   showed   that   norepinephrine   exposure   to   hippocampal  neurons  in  culture  increased  phosphorylation  of  Ser845  and  Ser831   and  reduced  the  threshold  for  LTP  (Hu  et  al.,  2007).    IP  injections  of  epinephrine   in   the   mouse   increased   phosphorylation   of   Ser845   (the   PKA   dependent   site;;   downstream  of  β2-­adrenergic   receptor)  on  GluR1  and  reduced  the   threshold   for   learning   (Hu   et   al.,   2007).     The   effect   of   norepinephrine   on   AMPA   receptor   phosphorylation   is  dependent  on   the  β-­adrenergic   receptor,  as  exposure   to   fox   urine   induced   Ser845   phosphorylation,   but   this   effect   was   blocked   by   pretreatment  with  the  β-­adrenergic  receptor  blocker  propranolol  (Hu  et  al.,  2007).     Importantly,   when   the   authors   used   a  mutant  mouse   with   a   knock-­in  mutation   that   prevented   phosphorylation   of   Ser845   and   Ser831,   IP   injections   of   epinephrine   no   longer   reduced   the   threshold   for   learning.     An   investigation   by       45   Chai  et  al.   (2014)  showed   that   the  enhancement  of  extinction  memory  afforded   by  infusions  of  norepinephrine  into  the  dorsal  hippocampus  was  associated  with   increased   membrane   GluR1,   Ser845   phosphorylation,   and   CREB   phosphorylation.    Enhanced  learning/memory  and  a  lower  threshold  for  LTP  and   learning  have  also  been  observed  following  short-­term  exercise  training  (Farmer   et  al.,  2004;;   Intlekofer  et  al.,  2013).  Understanding   the  mechanisms  and  stimuli   that   cause   phosphorylation   and  membrane   insertion   of   the  GluR1   subunit   is   a   focus  of   intense   investigation  and  exercise-­induced  norepinephrine   release   is  a   potential  mechanism  to  induce  this  favorable  adaptation.    Moreover,  β-­adrenergic   signaling  plays  a  crucial  role  in  acute  physical-­stress  induced  transcription  in  the   rodent   hippocampus   (Roszkowski  et   al.,   2016)   and  might   be  a   critical   factor   in   exercise  induced-­Bdnf  expression.       Noradrenergic   System   and   Exercise   Training-­Induced   Plasticity   and   Bdnf   Expression   The  beneficial  adaptations  in  the  hippocampus  following  exercise  training   are  blocked  by  peripheral  administration  of  propranolol  (Ivy  et  al.,  2003)  or  DSP-­4   (Garcia   et   al.,   2003),   a   LC-­NA-­selective   neurotoxin.     Based   on   the   similarities   between   the   effects   of   antidepressant   treatment   and   physical   activity   on   the   hippocampus   (e.g.   increased  Bdnf,   neurogenesis),   researchers   suggested   that,   like   antidepressants,   which   increase   and   prolong   extrasynaptic   monoamine   levels,  exercise  might  mediate  its  effects  through  elevated  norepinephrine.    Ivy  et   al.   (2003)   demonstrated   that   three   days   of   housing   with   a   voluntary   running       46   wheel  increased  hippocampal  Bdnf  mRNA  levels  but  this  was  blocked  by  nightly   IP  injections  of  propranolol  over  those  three  days.    Garcia  et  al.  (2003)  reported   that   one   week   of   voluntary   wheel   exposure   increased   total   Bdnf   mRNA   expression   in   the   rat   hippocampus   and   this  was   attenuated   in   rats   pre-­treated   with   DSP-­4   one   week   prior   to   wheel   exposure.     The   researchers   also   investigated  the  effect  of  exercise  and  DSP-­4  on  exon-­specific  Bdnf  transcription.     Exon  I  expression  was  elevated  with  one  week  of  exercise  in  the  dentate  gyrus   and   DSP-­4   significantly   attenuated   this.     Exon   II   expression   was   actually   increased   in  CA1,  CA2,  and  CA3  with   combined  exercise  and  DSP-­4.     In   fact,   exercise   mice   injected   with   saline   had   significantly   less   exon   II   expression   compared   to   exercise   mice   injected   with   DSP-­4   and   there   was   no   effect   of   exercise  alone  on  exon  II  mRNA  expression.    Similar  results  were  observed  for   exon  IV  (exon  III   in  the  paper),  with  DSP-­4  actually  increasing  levels  of  exon  IV   expression  and  no  effect  of  exercise  alone.    The  discrepancy  between  total  Bdnf   mRNA  and  transcript-­specific  expression  is  curious  and  deserves  more  research.     This  could  be  due   to   the  unusual  properties  of  DSP-­4,  which   lesions  NA  nerve   terminals   and   drastically   reduces   tissue   content   of   norepinephrine   in   the   hippocampus,   but   has   also   been   shown   to   increase   levels   of   extrasynaptic   norepinephrine  (Ross  &  Stenfors,  2014).    To  support  the  effect  of  norepinephrine   on  Bdnf,  Chen  et  al.   (2007)  showed  that  norepinephrine   increased  Bdnf  protein   expression  and  signaling  in  hippocampal  culture.    Baj  et  al.  (2012)  reported  that   incubating   neurons  with   norepinephrine   increases   Bdnf  mRNA   targeting   to   the   dendrites   and   this   was   mediated   primarily   by   Bdnf   exon   VI.   These   data       47   demonstrate   an   important   relationship   between   exercise,   the   NA   system,   and   Bdnf,  however  the  relationship  between  acute  exercise  and  central  NA-­mediated   enhancement  of  hippocampal  plasticity  and  Bdnf  expression  is  unknown.   Acute  Exercise  and  Anxiety   Norepinephrine   is   strongly   associated   with   the   pathogenesis   of   anxiety.     Interestingly,   norepinephrine   is   associated   with   both   anxiolytic   and   anxiogenic   behavioral   responses   (Goddard  et   al.,   2010).     In   response   to  acute   stress,  NA   neurons  in  the  LC  and/or  signals  from  the  limbic  system  stimulate  the  HPA  axis,   resulting   in   release   of   corticotropin-­releasing   hormone   from   the   paraventricular   nucleus   of   the   hypothalamus   (PVN),   which   then   stimulates   the   production   and   secretion  of  adrenocorticotropin  hormone  (ACTH)  from  the  pituitary  gland.    Upon   release  of  ACTH,  corticosterone  is  released  from  the  adrenal  cortex  (Goddard  et   al.,   2010).     In   addition   to   the   aforementioned   HPA   axis,   stress   signals   arising   from   the   limbic   system   or   hypothalamus   stimulate   the   LC   to   release   norepinephrine   centrally   and   further   stimulates   the   release   of   norepinephrine   from  the  sympathetic  nervous  system  and  norepinephrine  and  epinephrine  from   the   adrenal   medulla   (Sothmann   et   al.,   1996).     These   adaptations   to   stress   –   whether   psychological   or   physical   –   are   important   for   the   “fight   or   flight”   response   ,   though   in   rodents,   avoidance   in   the   form   of   freezing   is   a   common   response  to  stressful  situations  (Goddard  et  al.,  2010)  and  is  frequently  used  as   a   measure   of   anxiety   and   fear   in   rodent   behavioral   paradigms.     Though   high   levels  of  catecholamines  can  result  in  strong  memory  enhancement,  as  seen  with       48   flashbulb  memories,   it   is   reasonable   to  assume   that  high-­intensity  exercise   that   increases   catecholamine   levels   may   induce   anxiety   and   be   detrimental   to   cognitive   task   performance.    Numerous   investigations   have   been   conducted   to   determine  the  influence  of  chronic  exercise  on  anxiety  in  humans  and  anxiety-­like   behavior   in   rodents,   but   the   results   have  been   inconclusive.     In   rodents,   some   investigations   report   anxiolytic   effects   while   others   report   anxiogenic   effects   of   exercise  (Sciolino  &  Holmes,  2012).    The  effects  of  acute  exercise  and  exercise-­ induced  stress  hormones  on  anxiety-­like  behavior  are  not  understood,  especially   in   animal   models   where   the   effects   of   exercise   on   anxiety   have   been   almost   exclusively   studied   in   the   context   of   chronic   exercise   training   and   response   to   stressful  situations.       Two  meta-­analyses,  one  examining  research  pre-­1991  (Petruzzello  et  al.,   1991)  and  one  examining   research  since  1991   (Ensari  et  al.,  2015)  have  been   conducted   and   support   that   acute   exercise   has   small   but   significant   beneficial   effects   on   state   anxiety   in   humans.     However,   exercise   that   exceeds   the   anaerobic  or  ventilatory  threshold  has  been  associated  with  reduced  affect  during   exercise  (Bixby  et  al.,  2001;;  Ekkekakis  et  al.,  2008;;  Lind  et  al.,  2008)  and  other   research  provides  evidence  of   increased   feelings  of   stress  after  acute  exercise   (Hopkins  et   al.,   2012).     Even   though   exercise   in   humans   rarely   results   in   high   levels  of  anxiety  or   increased  rates  of  panic  attacks  (O'connor  et  al.,  2000),   the   reduced   affect   observed   in   humans  may   present   itself   as   increased   anxiety   in   rodents   since   the   acute   high-­intensity   exercise   is   uncontrollable,   novel,   and   results   in   increased   stress   hormone   levels.     Duman   et   al.   (2008)   reported       49   increased   anxiety-­like   behaviors   following   three   weeks   of   voluntary   wheel   running   if   the  behavioral   task   took  place   the  morning  after   a   night   of   voluntary   running   wheel   access.     If   the   task   was   performed   24   hours   after   cessation   of   voluntary   running,   the   animal   displayed   reduced   anxiety-­like   behavior.     Other   studies   have   also   reported   anxiogenic   effects   of   chronic   voluntary   exercise   in   rodents  (Fuss  et  al.,  2009;;  2010;;  Onksen  et  al.,  2012)  and  Dishman  et  al.  (1996)   reported   that  eight  weeks  of   treadmill   running   reduced  activity   in   the  open   field   task   (anxiety-­like   behavior)   24   hours   after   the   last   running   exposure   but   eight   weeks   of   voluntary   wheel   running   increased   activity   in   the   open   field   task   (anxiolytic-­like  behavior).       The   influence   of   acute   exercise   on   animal   behavior   is   important   to   understand   since   animal   models   are   commonly   used   to   examine   the   effectiveness  of  treatment  strategies.    Acute  exercise  is  a  promising  approach  to   treat  conditions  such  as  PTSD  (Powers  et  al.,  2015),  but  a  potential  anxiety-­like   effect  observed   in  animals  might   interfere  with   the   interpretation  of  mechanistic   and   behavioral   findings   in   rodents.     For   this   reason,   it   is   important   to   fully   characterize   the   behavioral   response   to   acute   exercise   in   animal   models   to   determine   the   most   effective   way   to   use   these   models   to   understand   the   application  of  acute  exercise  for  therapies  and  cognitive  training.       Training  Influences  Catecholamine  Response  to  Acute  Exercise     Exercise   training   in  humans   is  associated  with  a   reduced  catecholamine   response   to   the   same   absolute   exercise   intensity   but   a   higher   capacity   to       50   increase   catecholamine   release   at   maximal   exercise   intensities   (Kjaer,   1992;;   Zouhal  et  al.,  2008).    Similarly,  in  rodents,  exercise  training  is  associated  with  an   increased   capacity   for   peripheral   epinephrine   release   (Zouhal   et   al.,   2008);;   however,   the   available   literature   in   rodents   suggests   that   previous   exposure   to   exercise  reduces  the  central  NA  response  to  both  homotypic  (e.g.  exercise)  and   heterotypic   physical   or   psychological   stressors   (e.g.   immobilization   stress).     Concerning  running,  Pagliari  and  Peyrin  (1995b)  reported  that  mice  trained  for  12   days   to   run   for   one   hour   had   significantly   increased   cortical   norepinephrine   during  a  two  hour  run  compared  to  mice  trained  for  12  days  to  run  for  two  hours.     Dishman  et  al.   (1997)   reported   that   rats  housed  with  a  voluntary   running  wheel   for   9-­12   weeks   before   exposure   to   uncontrollable   foot   shock   and   an   escape   shuttle  box  task  had  61%  higher  levels  of  norepinephrine  in  the  LC  compared  to   sedentary   controls.     In   mice   that   experienced   controllable   footshock,   LC   norepinephrine  concentrations  were  49%  higher  than  sedentary  controls.    These   data   indicate   that  wheel   running  protects  against  depletion  of  norepinephrine   in   the  LC.    The  protection  of  LC-­  norepinephrine  depletion  with  exercise  was  also   confirmed   in   an   investigation   that   utilized   six  weeks  of   forced   treadmill   running   prior   to   exposure   to   immobilization   stress   or   treadmill   running   (Dishman  et   al.,   2000).    The  blunting  of  norepinephrine  depletion  with  stress  was  observed  in  the   LC   and   brain   regions   innervated   by   the   LC   including   the   amygdala   and   the   hippocampus.   After   40   minutes   of   a   scrambled   footshock,   rats   housed   with   a   voluntary   running   wheel   for   4-­5   weeks   had   significantly   lower   levels   of   norepinephrine   in  the  frontal  cortex  compared  to  rats  housed  in  standard  cages       51   (Soares  et  al.,  1999).    The  authors  suggested   this  demonstrated  a   reduction   in   the   stress-­induced   release   of   norepinephrine   following   housing   with   a   running   wheel.    Not  only  is  norepinephrine  release  and  depletion  influenced  by  voluntary   wheel  and   forced   treadmill   running  but   stress-­induced  activity   in   the  LC   is  also   reduced.     Rats   housed   with   running   wheels   for   six   weeks   have   significantly   reduced  c-­Fos  expression  in  the  LC  following  inescapable  tail  shocks  compared   to  mice  housed  with   locked  wheels   (Greenwood  et  al.,  2003).    Taken   together,   these  studies  provide  strong  evidence   that  exercise   training   reduces   the  stress   response  in  rodents  to  both  homotypic  and  heterotypic  stressors.    Knowing  that   norepinephrine  and  β-­adrenergic  signaling  (Garcia  et  al.,  2003;;   Ivy  et  al.,  2003)   are   important   for   Bdnf   expression,   this   reduction   in   norepinephrine   release   following   exercise   training   provides   the   potential   for   a   reduction   in   acute   exercise-­induced  Bdnf  expression  following  exercise  training.   Training  Influences  Bdnf  Response  to  Acute  Exercise     Cardiovascular  fitness  or  a  previous  history  of  physical  activity   influences   the  peripheral  BDNF  response  to  acute  exercise  (Knaepen  et  al.,  2010).    Griffin   et   al.   (2011)   reported   that   an   acute   bout   of   exercise   to   exhaustion   increased   serum   BDNF   immediately   after   the   exercise   bout   but   this   was   attenuated   in   subjects  who  underwent  three  or  five  weeks  of  aerobic  exercise  training.    Wagner   et   al.   (2015)   reported   a   similar   blunting   of   the   peripheral   BDNF   response   to   exhaustive   acute   exercise   following   eight   weeks   of   exercise   training.     It   is   important   to   note   that   the   acute   exercise   protocols   in   both   studies   were   to       52   exhaustion  before  and  after  training,  so  the  effects  of  the  acute  exercise  cannot   be  attributed  to  reduced  relative  exercise  intensity.    Not  all  studies  have  reported   a   blunting   of   acute   exercise-­induced   BDNF   response,   however.   Seifert   et   al.   (2010)   reported   an   increase   in   BDNF   release   from   the   human   brain   (brachial   artery   –   jugular   vein   difference)   with   acute   exercise   of   varying   intensities   and   reported   no   effect   of   three   months   of   aerobic   training   on   acute   exercise   stimulated  release.     In  contrast  to  what  has  been  observed  peripherally  in  humans,  research  in   the  rodent  hippocampus  suggests   that  previous  exposure   to  exercise   increases   the   ability   to   increase   Bdnf   levels   with   an   additional   exposure   to   exercise.     Berchtold  et  al.  (2005)  exposed  rats  to  a  voluntary  running  wheel  either  daily  or   every   other   day   over   a   28   day   period;;   both   of   these   protocols   increased  Bdnf   protein   in   the   hippocampus.     After   a   period   of   seven   or   14   days   of   no   wheel   running,   two  days  of   voluntary  wheel   running   increased  Bdnf  expression   in   the   hippocampus   only   in   mice   that   were   previously   exposed   to   voluntary   wheel   running.     An   interesting   finding   from   this   investigation   was   that   even   though   intermittent   running   every   other   day  was   as   effective   at   increasing  Bdnf   in   the   hippocampus  as  daily  running,  Bdnf  protein  returned  to  baseline  by  day  three  of   sedentary  conditions  in  the  intermittent  group  but  remained  elevated  for  a  week   in   the   daily   running   group.     This   demonstrates   that   while   various   exercise   exposures  may  result  in  similar  elevations  in  Bdnf,  the  lasting  benefits  may  differ   depending   on   the   stimulus.     Concerning   transcriptional   regulation   of   Bdnf,   Gomez-­Pinilla   et   al.   (2010)   reported   that   one  week   of   voluntary  wheel   running       53   reduced   methylation   and   increased   mRNA   expression   of   Bdnf   IV.     Reduced   methylation  of  this  transcript  suggests  increased  transcriptional  capability,  though   how  long  this  reduced  methylation  persists  is  unknown.    These  studies  in  rodents   suggest   that   previous   exposure   to   voluntary  wheel   running   augments   the  Bdnf   response   to   voluntary   wheel   running.   It   is   not   known   if   previous   exposure   to   voluntary   wheel   running   will   augment   the   Bdnf   response   to   forced   treadmill   exercise,  given  that  the  physical  and  psychological  stress  associated  with  forced   treadmill  running  is  different  from  voluntary  wheel  running.   Summary   Much   research   has   been   done   over   the   past   30   years   to   understand   how   exercise   influences   the   human   and   rodent   brain.     Though   much   has   been   discovered,   many   questions   remain   unanswered.     Only   with   rigorous   investigations   utilizing   animal   models   and   unique   exercise   approaches   can   exploration   into   the   mechanisms   mediating   the   favorable   hippocampal   adaptations  to  acute  and  chronic  exercise  be  truly  accomplished.    Though  animal   models   are   currently   necessary   for   this,   careful   consideration   must   be   taken   when   translating   exercise   and   behavioral   observations   to   human   application.     This  dissertation  project  contains  a  series  of   investigations  designed  to  address   many  of  the  questions  presented  in  the  review  of  literature  and  provides  exciting   new  information  on  the  influence  of  both  acute  and  chronic  exercise  on  markers   of  hippocampal  plasticity  and  animal  behavior.         54   Chapter  3.     Aim   #1:    Determine   the   effect   of   five  months   of   voluntary  wheel   exposure   on   hippocampal  mRNA  expression  of  plasticity-­associated  genes  in  adult  male  and   female  mice.       Published  Manuscript     Physiology  &  Behavior  156  (2016)  8-­15     Title:    Sex-­Dependent  and   Independent  Effects  of  Long-­Term  Voluntary  Wheel   Running  on  Bdnf  mRNA  and  Protein  Expression   Authors:   Andrew   C.   Venezia1,2,   Lisa   M.   Guth1,   Ryan   M.   Sapp1,   Espen   E.   Spangenburg1,  Stephen  M.  Roth1,2   Department   of   Kinesiology,   School   of   Public   Health,   University   of   Maryland,   College  Park,  MD1   Neuroscience  and  Cognitive  Science  Program,  University   of  Maryland,  College   Park,  MD2             55   Abstract   The   beneficial   effects   of   physical   activity   on   brain   health   (synaptogenesis,   neurogenesis,   enhanced   synaptic   plasticity,   improved   learning   and   memory)   appear   to   be   mediated   through   changes   in   region-­specific   expression   of   neurotrophins,   transcription   factors,   and   postsynaptic   receptors,   though   investigations   of   sex   differences   in   response   to   long-­term   voluntary   wheel   running  are  limited.    Purpose:  To  examine  the  effect  of  five  months  of  voluntary   wheel   running  on  hippocampal  mRNA  and  protein  expression  of   factors   critical   for  exercise-­induced  structural  and  functional  plasticity  in  male  and  female  adult   mice.   Methods:   At   8   weeks   of   age,   male   and   female   C57BL/6   mice   were   individually   housed  with   (PA;;   n=20;;   10  male)   or  without   (SED;;   n=20;;   10  male)   access  to  a  computer  monitored  voluntary  running  wheel.    At  28  weeks,  all  mice   were   sacrificed   and   hippocampi   removed.   Total   RNA   was   isolated   from   the   hippocampus   and   expression   of   total   Bdnf,   Bdnf   transcript   IV,   tPA,   Pgc-­1a,   GluR1,  NR2A,  and  NR2B  were  assessed  with  quantitative  RT-­PCR  and  total  and   mature  Bdnf  protein  were  assessed  with  ELISA.  Results:  We  found  significantly   higher  Bdnf   IV  mRNA   expression   in   PA  males   (p=0.03)   and   females   (p=0.03)   compared   to   SED   animals.     Total   Bdnf   mRNA   expression   was   significantly   greater   in   PA   males   compared   to   SED   males   (p=0.01),   but   there   was   no   difference   in   females.     Similarly,   we   observed   significantly   higher  mature   Bdnf   protein   in   PA   males   compared   to   SED   males   (p=0.04),   but   not   in   females.     Conclusion:  These  findings  indicate  that  the  impact  of  long-­term  voluntary  wheel   running  on   transcriptional  and  post-­translational   regulation  of  Bdnf  may  be  sex-­     56   dependent,   though   the   activity-­dependent   Bdnf   IV   transcript   is   sensitive   to   exercise  independent  of  sex.       Key   Words:     Brain-­Derived   Neurotrophic   Factor;;   Sex-­Differences;;   Exercise;;   Physical  Activity;;  Hippocampus       57   Introduction     Chronic  exercise  training  and  physical  activity  have  remarkable  effects  on   the  human  and  rodent  hippocampus  (Voss  et  al.,  2013).    Structural  adaptations   observed   in   the   hippocampus   in   response   to   exercise   training   and   physical   activity   include  synaptogenesis,  dendritic  arborization,  and  neurogenesis   (Eadie   et   al.,   2005;;   Redila   &   Christie,   2006;;   Stranahan   et   al.,   2007;;   Lin   et   al.,   2012)   while   functional  adaptations   include  enhanced   learning  and  memory,   increased   amplitude   of   long-­term   potentiation   (LTP),   and   reduced   threshold   for   LTP   (van   Praag,  Christie,  et  al.,  1999;;  Kida  et  al.,  2002;;  Farmer  et  al.,  2004;;  Titterness  et   al.,   2011).     Moreover,   hippocampal   neurons   show   increased   mitochondrial   biogenesis  in  response  to  chronic  exercise  training  (Steiner  et  al.,  2011).    These   structural   and   functional   adaptations   are   believed   to   be   the   result   of   increased   expression   of   important   neurotrophins,   transcription   factors,   and   postsynaptic   receptors.           Elevated  expression  (mRNA  and/or  protein)  and  downstream  signaling  of   brain-­derived   neurotrophic   factor   (BDNF)   has   been   identified   as   a   primary   exercise-­induced   regulator   of   functional   and   structural   plasticity   since   blocking   Bdnf  expression  or   signaling  attenuates   improvement   in   learning,  memory,  and   expression   of   genes   important   for   synaptic   plasticity   in   the   hippocampus   following   exercise   training   (Vaynman   et   al.,   2004;;   Gomez-­Pinilla   et   al.,   2008).     Circulating  BDNF   is   elevated   in   humans   following   acute   exercise   and   exercise   training  (Gold  et  al.,  2003;;  Vega  et  al.,  2006;;  Ferris  et  al.,  2007;;  Erickson  et  al.,   2011).   In   the   rodent   hippocampus,   Bdnf   protein   and   mRNA   are   elevated       58   following  brief  exercise  exposures  (≤  7  days)  (Neeper  et  al.,  1995;;  1996;;  Molteni   et   al.,   2002;;  Vaynman  et   al.,   2003;;  Berchtold  et   al.,   2005;;  Huang  et   al.,   2005;;   Ding  et  al.,  2011;;  Sartori  et  al.,  2011)  and   longer  exercise  exposures  (>7  days)   (Molteni  et  al.,  2002;;  Farmer  et  al.,  2004;;  Berchtold  et  al.,  2005;;  Liu  et  al.,  2009;;   Berchtold  et  al.,  2010;;  Ding  et  al.,  2011;;  Kobilo  et  al.,  2011;;  Sartori  et  al.,  2011;;   Marlatt   et   al.,   2012;;  Wrann   et   al.,   2013;;   Darlington   et   al.,   2014).     Importantly,   most   studies   in   rodents   that   attempt   to   address   mechanisms   mediating   the   cognitive  enhancing  effects  of  chronic  exercise  have  focused  on  exercise  training   ranging   from  ~7   days   to   3  months   and   how   longer   voluntary  wheel   exposures   influence  mRNA  expression  of  plasticity-­associated  genes  is  not  fully  understood.     When  mice   are   exposed   to   a   voluntary   running  wheel,   activity   decreases   over   time   (Richter   et   al.,   2014;;   Venezia   et   al.,   2015)   and   focusing   on   short-­term   chronic  exercise  favors  plasticity  by  highlighting  the  response  to   the  high  wheel   activity   and   the   novelty   of   activity.   This   might   present   a   biased   view   of   the   benefits   of   chronic   exercise   training   on   plasticity-­associated   gene   and   protein   expression.    However,  Marlatt  et  al.  (2012)  showed  that  eight  months  of  voluntary   wheel   running   increased   Bdnf   protein   expression   in   17-­month   old   female   C57BL/6J  mice   that   began   running  at   nine  months  of   age.    This   suggests   that   long-­term  voluntary  exercise  maintains  elevated  Bdnf  expression  that  is  normally   observed  following  short-­term  exercise  exposure.     It   is  not  fully  understood  how   long-­term   voluntary   wheel   exposure   influences   young-­adult   male   and   female   hippocampal  plasticity-­associated  gene  and  protein  expression.           59     Importantly,  the  Bdnf  gene  is  highly  complex,  containing  eight  non-­coding   exons  with  individual  promoters  that  all  splice  to  one  3’  protein  coding  exon  (exon   IX).     Though  Bdnf   is   considered  an  activity-­regulated   gene,   promoter   IV-­driven   Bdnf   transcription   is   especially   sensitive   to   neuronal   activity   (Tao   et   al.,   1998;;   2002)   and   environmental   stimuli   (Lubin   et   al.,   2008;;   Intlekofer   et   al.,   2013).     Interestingly,  all  Bdnf  transcripts  are  translated  into  the  same  protein  (proBDNF),   which   is   then  cleaved   to  produce   the  mature  plasticity-­associated  protein.     It   is   not  fully  understood  how  long-­term  voluntary  exercise  influences  the  transcription   and  post-­translational  processing  of  Bdnf.     The  hippocampus  is  a  sexually  dimorphic  structure  (Madeira  &  Lieberman,   1995)   and   environmental   stimuli   result   in   sex-­dependent   hippocampal   adaptations   (Cahill,  2006).     In   fact,  many  stimuli  will   result   in  similar  behavioral   responses  in  males  and  females,  but  the  mechanisms  by  which  these  responses   are   mediated   may   be   different   between   the   sexes   (Cahill,   2006).     Exercise   results  in  beneficial  adaptations  to  the  hippocampus  in  both  males  (Farmer  et  al.,   2004)  and   females   (van  Praag,  Christie,  et  al.,   1999),   though   it   is  not   known   if   exercise   is   stimulating   the   same   signaling   pathways   in   both   sexes.     Further,   research   in   adolescent   rats   supports   that   sex   differences   exist   in   the   hippocampal  response  to  exercise  (Titterness  et  al.,  2011).    The  purpose  of  the   present  investigation  was  to  examine  how  five  months  of  voluntary  wheel  running   influences  hippocampal  mRNA  and  protein  expression  in  adult  male  and  female   C57BL/6J   mice.     We   hypothesized   that   long-­term   chronic   voluntary   wheel   running   would   have   small   or   no   effects   on   hippocampal   mRNA   expression   of       60   plasticity-­associated  genes  due  to  reduced  wheel  running  over  time  and  that  any   observed  differences  in  mRNA  expression  would  be  sex-­dependent.    We  focused   our  investigation  on  Bdnf  mRNA  and  protein  as  well  as  other  genes  important  for   the   effect   of   exercise   on   structural   and   functional   plasticity,   mitochondrial   biogenesis,  and  synaptic  transmission.   Methods     Animals   and  Voluntary  Wheel  Running:  Male   and   female  C57BL/6J  mice  were   used   in   this   investigation.    All  animals  were  cared  for  by  University  of  Maryland   veterinary   staff   and   kept   on   12hr   light/12hr   dark   cycle   and   provided   standard   rodent  chow  ad  libitum.    All  protocols  were  IACUC  approved.    At  eight  weeks  of   age,  male   and   female   C57Bl/6J  mice   were   individually   housed   with   (n=20;;   10   male)   or   without   (n=20;;   10   male)   continuous   access   to   a   computer-­monitored   voluntary   running   wheel   (Lafayette   Instruments,   Lafayette   IN).     Mice   were   sacrificed  at  28  weeks  of  age.       Tissue   Collection   &   Processing:     All   mice   were   exposed   to   intraperitoneal   glucose   tolerance   testing   (IPGTT)  24  hours  before  sacrifice.    Mice  were   fasted   (ad   libitum   water   access)   for   6   hours   prior   to   IPGTT.   Baseline   blood   glucose   measurements  were  made  and   then  each  mouse  was   injected   intraperitoneally   with  2.0  mg  of  D-­glucose  (Sigma-­Aldrich,  St.  Louis,  MO)  per  gram  of  body  mass.   Blood  glucose  was  measured  15,  30,  60,  90,  and  120  minutes  after  injection  in  all   animals.    All  blood  glucose  measurements  were  made  on  blood  removed  from  a   single  tail  snip.  Following  the  glucose  tolerance  test  animals  were  returned  to  ad       61   libitum   food   and   water   access.   On   the   day   of   sacrifice,   total   body   mass   of   anesthetized   mice   was   recorded   and   mice   underwent   euthanasia   by   exsanguination   followed   by   removal   of   the   heart   under   isoflurane   anesthesia.   The  hippocampus  was  isolated,  halved,  and  immediately  frozen  in  liquid  nitrogen.       Gene  Expression:    Prior  to  nucleic  acid  isolation,  hippocampi  were  homogenized   in   TRIzol   reagent   (Life   Technologies,   Grand   Island,   NY,   USA)   using   a   glass   Dounce   homogenizer.     Total   RNA   was   isolated   with   TRIzol   reagent   following   manufacturers   instructions   and   quantified   via   spectrophotometry.     Reverse   transcription  was  performed  with  1  μg  of  total  RNA  with  the  High-­Capacity  cDNA   RT   kit   (Life   Technologies).     Real-­time   quantitative   PCR   (qPCR)   was   used   to   assess   mRNA   expression   of   total   Bdnf   (exon   IX);;   Bdnf   exon   IV   (Bdnf   IV);;   peroxisome  proliferator-­activated  receptor  γ  coactivator  1  alpha  (Pgc-­1α);;   tissue   plasminogen   activator   (tPa);;   glutamate   receptor,   ionotropic,   AMPA   1   (GluR1);;   glutamate  receptor,  ionotropic,  NMDA2A  (NR2A),  glutamate  receptor,  ionotropic,   NMDA2B   (NR2B);;   and   glyceraldehyde-­3-­phosphate   dehydrogenase   (Gapdh;;   expression   control;;   primer   sequences   listed   in   Supplemental   Table   1).     Primer:probe   assays   were   purchased   pre-­made   (Pgc-­1α,   tPa,   GluR1,   NR2A,   NR2B,  Gapdh)  or  designed  (Bdnf   IX,  Bdnf   IV)   for   the  mRNA  sequence  of  each   gene  using  Integrated  DNA  Technologies’  PrimeTime  qPCR  Assay  designer.    All   primer  pairs  except  Bdnf  total  spanned  exons  to  prevent  amplification  of  genomic   DNA.   Because  Bdnf   total   is   represented   by   amplification   of   only   exon   IX,   this   primer  pair   could  not   span  exons.    Efficiency   for  each  primer:probe  assay  was   determined  prior  to  use.         62   Bdnf  Protein:  Total  and  mature  Bdnf  protein  levels  were  measured  using  the  E-­ Max  Bdnf  ELISA  kit  (Promega,  WI,  USA)  according  to  manufacturer’s  instructions.   Tissues  were  homogenized  on   ice   in   lysis  buffer   [137mM  NaCl,  20mM  Tris-­HCl   (pH   8.0),   1%   NP40,   10%   glycerol,   0.5mM   sodium   vanadate,   and   protease   inhibitor   cocktail   (complete   mini   EDTA-­free   protease   inhibitors,   Roche,   1   tablet/10ml)].   Homogenized   samples   were   diluted   in   two   volumes   DPBS   containing   calcium   and   magnesium   (Life   Technologies,   NY,   USA)   and   centrifuged  for  3  min.  at  13,500  rpm  at  4oC  (Berchtold  et  al.,  2005).  Supernatant   was  collected  and  total  protein  concentration  determined  by  a  Bicinchoninic  acid   (BCA)   assay   following   manufacturer’s   instructions   (Pierce   Biotechnology,   IL,   USA).     Samples   were   then   diluted   in   1x   block   and   sample   buffer.   For   determination  of  total  Bdnf,  samples  were  acidified  with  1N  HCl  for  15  minutes  to   pH  2-­3  and  neutralized  with  1N  NaOH  to  pH  7-­8.    The  standard  curve  produced   from  Bdnf  standard  dilutions  produced  an  R-­value  of  >0.99.   Statistics:  T-­tests  were  used  to  test  for  differences  in  body  mass  and  IPGTT  area   under   the   curve   (AUC).    Running  wheel   activity  was   analyzed  with   a   repeated   measures  ANOVA.    Protein  and  mRNA  data  were  analyzed  by  two-­way  ANOVA   (exercise  x  sex)  and  pre-­planned  LSD  post  hoc  contrasts  to  compare  exercise  vs.   sedentary  within  sexes  and  male  vs.  female  within  exercise  conditions.    A  p≤0.05   was  considered  statistically  significant.           63   Results   Wheel   Running:    Wheel   running   data   are   shown   in   Figure   1.     The   repeated   measures  ANOVA  revealed  a  significant  effect  of   time   (F(3,12)=14.80;;  p=0.0002)   and  a  tendency  for  an  interaction  between  sex  and  time  (F(3,12)=3.22;;  p=0.06)  for   average  distance  ran  per  24  hours.  Females  ran  significantly  more  during  week  1   than   males   (t(10.6)=2.24;;   p<0.05);;   however,   during   week   20,   males   ran   significantly  more  than  females  (t(16)=-­2.94;;  p<0.01).       Body   Mass   and   GTTs:     Female   runners   weighed   significantly   less   than   their   sedentary  counterparts  after   five  months  of  running  wheel  exposure  (t(18)=-­2.37;;   p=0.03;;  Fig.   2).     There  was  a   tendency   for  male   runners   to  weigh   significantly   less  than  sedentary  males  (t(17)=-­1.92;;  p=0.07;;  Fig.  2).    There  was  no  significant   effect   of  wheel   running   on   blood  glucose   response   (AUC)   to   an   IP   injection   of   glucose  (data  not  shown).   Gene  Expression:    Gene  expression  data   are   shown   in  Figures   3   and  4.     The   two-­way  ANOVA  revealed  a  main  effect  of  exercise  (F(1,33)=10.89;;  p=0.002)  but   no  main  effect  of  sex  or  an  exercise  by  sex  interaction  on  Bdnf  IV  (Fig.  3).  Five   months   of   voluntary   wheel   running   led   to   significantly   greater   Bdnf   IV   gene   expression   compared   to   sedentary   living   conditions   (Fig.   3a)   and   this   effect   remained  when  sexes  were  separated  in  the  analysis  (males:  t(16)=2.41,  p=0.03,   Fig.  3b;;   females:   t(17)=2.32,  p=0.03,  Fig.  3c).    The   two-­way  ANOVA   revealed  a   significant   interaction   between   exercise   and   sex   for   total   Bdnf   mRNA   (F(1,33)=4.98;;  p=0.03).  There  was  no  main  effect  of  exercise  or  sex  on  total  Bdnf       64   mRNA   levels.  Post-­hoc  analysis   revealed   that  when   the  sexes  were  separated,   exercise  males  had  significantly  higher  total  Bdnf  mRNA  expression  compared  to   sedentary   males   (t(16)=2.76;;   p=0.01,   Fig.   3b)   and   this   was   not   observed   in   females   (Fig.   3c).   There   was   no   significant   effect   of   five   months   of   voluntary   wheel  running  or  sex  on  Pgc-­1a,  tPa,  or  glutamate  receptor  subunit  expression  in   either  sex  (Figs.  3  and  4).   Bdnf  Protein:    The  two-­way  ANOVA  revealed  no  significant  effects  of  exercise  or   sex   or   an   interaction   between   exercise   condition   and   sex   on   total   (Fig.   5)   or   mature  (Fig.  6)  Bdnf  protein.    However,  based  on  our  mRNA  data,  we  analyzed   by   sex   and   found   that  mature  Bdnf   levels  were   significantly   higher   in   exercise   males   compared   to   sedentary  males   (t(15)=2.31,   p=0.04,   Fig.   6b),   an   effect   not   observed   in   females.  Moreover,  sedentary  males  had  significantly   lower  mature   Bdnf  protein  compared  to  sedentary  females  (t(16)=2.25,  p=0.04,  Fig.  6b).             65   Figure   1.     Average   daily   running   distance   for   male   and   female   C57Bl/6J   mice.     Females   ran   significantly   more   per   day   during   week   one   (p=0.03)   and   significantly  less  per  day  during  week  twenty  (p<0.01)  compared  to  males.    Error   bars  represent  SEM   Figure   2.     Male   and   female   body   mass.     Females   with   access   to   voluntary   running   wheels   had   significantly   lower   body   mass   compared   to   sedentary   females   (p=0.03).     There   was   a   tendency   for   males   with   access   to   voluntary   running  wheels  to  have  lower  body  mass  compared  to  sedentary  males  (p=0.07).     *p<0.05;;  #p<0.1.  Error  bars  represent  SEM.   Figure   3.     Five   months   of   voluntary   wheel   running   increases   Bdnf   transcription   in   a   transcript   and   sex-­dependent   manner.     Target   mRNA   expression   is  presented  as  ddCt  relative   to  Gapdh.  A)  qPCR  analysis   indicated   that   five  months  of  wheel  exposure   increased  Bdnf   IV   in   the  combined  sample   (p=0.004)   but   did   not   influence  Bdnf   total,  Pgc1a,   or   tPa   expression.     B)   Five   months   of  wheel   exposure   increased  Bdnf   IV   (p=0.03)   and   total  Bdnf   (p=0.01)   expression   but   had   no   effect   on   Pgc1a   or   tPa   expression   in   males.     C)   Five   months  of  wheel   exposure   increased  Bdnf   IV   (p=0.03)   expression  but   not   total   Bdnf,  Pgc1a,  or  tPa  in  females.    Error  bars  represent  SEM.       Figure   4.     Five   months   of   voluntary   wheel   running   does   not   influence   hippocampal   GluR1,   NR2A,   NR2B,   and   NR2B/NR2A   mRNA   expression.     Target  mRNA  expression  is  presented  as  ddCt  relative  to  Gapdh.  qPCR  analysis   indicated  that  there  was  no  significant  effect  of  chronic  wheel  exposure  on  GluR1,       66   NR2A,  or  NR2B  mRNA  expression   in  males,   females,  or   the  combined  sample.     Error  bars  represent  SEM.       Figure   5.     Five   months   of   voluntary   wheel   running   does   not   influence   hippocampal   total   Bdnf   protein   levels.     A)   Total   Bdnf   protein   in   combined   sample  of  males  and  females.    Data  presented  as  percent  of  sedentary  controls.   Five  months  of  wheel  exposure  did  not  influence  total  Bdnf  protein  expression.  B)   Five   months   of   wheel   exposure   did   not   significantly   affect   total   Bdnf   protein   expression  in  males  or  females.  Error  bars  represent  SEM.       Figure  6.    Five  months  of  voluntary  wheel   running  significantly   increases   mature  Bdnf  protein  levels  in  the  male  hippocampus.    A)  Mature  Bdnf  protein   in   combined   sample   of   males   and   females.     Data   presented   as   percent   of   sedentary  controls.  Five  months  of  wheel  exposure  did  not  influence  mature  Bdnf   protein  expression.    B)  Five  months  of  wheel   exposure   increased  mature  Bdnf   protein   expression   in  males   (p=0.04)   but   had   no   effect   in   females.     Sedentary   females   had   significantly   higher   mature   Bdnf   protein   expression   compared   to   sedentary  males  (p=0.04).      Error  bars  represent  SEM.               67   Figure  1.         * * 0 2000 4000 6000 8000 10000 12000 Week 1 Week 5 Week 10 Week 20 M et er s/ 24 h rs Male Female     68   Figure  2.     # * 0 10 20 30 40 Male Female B od y M as s (g ) Sedentary Runners     69   Figure  3.     * 0 0.5 1 1.5 2 Bdnf Bdnf IV Pgc1a tPa R el at iv e m R N A Ex pr es si on (2 -Δ Δ C t ) Sedentary Running Wheel A     70     * * 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 Bdnf Bdnf IV Pgc1a tPa R el at iv e m R N A Ex pr es si on (2 -Δ Δ C t ) Males B     71     * 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 Bdnf Bdnf IV Pgc1a tPa R el at iv e m R N A Ex pr es si on (2 -Δ Δ C t ) Females C     72   Figure  4.     0 0.5 1 1.5 2 GluR1 NR2A NR2B NR2B/NR2A R el at iv e m R N A Ex pr es si on (2 -Δ Δ C t ) Sedentary Running Wheel A     73     0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 GluR1 NR2A NR2B NR2B/NR2A R el at iv e m R N A Ex pr es si on (2 -Δ Δ C t ) Males B     74         0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 GluR1 NR2A NR2B NR2B/NR2A R el at iv e m R N A Ex pr es si on (2 -Δ Δ C t ) Females C     75   Figure  5.     0 50 100 150 200 Sedentary Running wheel To ta l B dn f P ro te in (% o f S ed en ta ry C on tr ol ) A     76         0 20 40 60 80 Male Female To ta l B dn f P ro te in (p g/ m l o f s am pl e) Sedentary Running Wheel B     77   Figure  6.     0 50 100 150 200 Sedentary Running wheel M at ur e B dn f P ro te in (% o f S ed en ta ry C on tr ol ) A     78           * * 0 10 20 30 40 50 Male Female M at ur e B dn f P ro te in (p g/ m l o f s am pl e) Sedentary Running Wheel B     79   Supplemental  Table  1.  Primer  sequences  for  genes  of  interest.       mRNA  Target   Primer  Sequences     Bdnf  total   Primer  1:  5’  –  CCATAAGGACGCGGACTTGTAC  -­3’   Primer  2:  5’  –  AGACATGTTTGCGGCATCCAGG  -­3’     Bdnf  IV   Primer  1:  5’-­  CAGAGCAGCTGCCTTGATGTT  -­3’   Primer  2:  5’-­  GCCTTGTCCGTGGACGTTTA  -­3’     Pgc-­1a   Primer  1:  5’  –  GGTGTCTGTAGTGGCTTGATTC  -­3’   Primer  2:  5’  –  GTTCCCGATCACCATATTCCA  -­3’     tPa  (Plat)   Primer  1:  5’  –  CAACCAAGACCTCCACGA  -­3’   Primer  2:  5’  –  CACATCCTTCTGCCCACA  -­3’   NR2A  (Grin2a)   Primer  1:  5’  –  TGCTCATCACCTCATTCTTCT  C  –  3’  Primer  2:  5’  –  GATTGACCTCGCTCTGCTC  –  3’   NR2B  (Grin2b)   Primer  1:  5’  –  CACAAACATCATCACCCACAC  -­3’  Primer  2:  5’  –  TTGACTTCTCTGTGCCCTTC  –  3’   GLUR1  (Gria1)   Primer  1:  5’  –  TGGCGAGGATGTAGTGGTA  –  3’  Primer  2:  5’  –  AAGAAAAAGGAGAGGCTGGTG  –  3’   Gapdh   Primer  1:  5’  –  AATGGTGAAGGTCGGTGTG  –  3’  Primer  2:  5’  –  GTGGAGTCATACTGGAACATGTAG  –  3’             80   Discussion     We   found   a   sex-­   and   transcript-­dependent   effect   of   long-­term   voluntary   wheel   running   on  Bdnf   transcription.     Five   months   of   voluntary   wheel   running   increased  Bdnf  IV  gene  expression  but  had  no  effect  on  total  Bdnf  expression  in   the  combined  sample   (male  &   female).     Interestingly,  when  males  and   females   were  separated  for  analysis,  we  observed  an   increase   in  Bdnf   IV   in  both  males   and   females  and  an   increase   in   total  Bdnf   in  males  only.    Moreover,  we   found   that   five   months   of   voluntary   wheel   running   increased   mature   Bdnf   protein   in   males  but  had  no  effect  in  females,  which  is  consistent  with  total  Bdnf  mRNA  and   provides  strong  evidence  for  sex-­dependent  effects  of  long  term  exercise  training   on  Bdnf  expression  and  processing.    These  are  interesting  observations  because   voluntary  wheel  running  enhances  hippocampal  plasticity  in  both  males  (Farmer   et  al.,  2004)  and  females  (van  Praag,  Christie,  et  al.,  1999).         Numerous  studies  have  demonstrated  that  both  brief  and  longer  exercise   training   exposures   increase   hippocampal   Bdnf   mRNA   and   protein   expression   (Neeper  et  al.,  1996;;  Molteni  et  al.,  2002;;  Vaynman  et  al.,  2003;;  2004;;  Berchtold   et   al.,   2005;;   2010;;  Ding  et   al.,   2011;;   Sartori  et   al.,   2011).     In   addition,   human   studies  have  demonstrated  that  peripheral  BDNF  levels  are  elevated  with  aerobic   exercise   training   (Szuhany   et   al.,   2015).     However,   we   report   here   that   five   months  of  voluntary  wheel  running  increases  total  Bdnf  mRNA  and  mature  Bdnf   protein  only  in  male  mice.    When  males  and  females  were  combined  for  analysis,   there  were   no   significant   effects   of   exercise   on  Bdnf  mRNA   or  mature   protein       81   expression.    The  majority  of   research   to  date  has  primarily  used  only  males  or   only   females,  making  our   results  difficult   to  compare   to   the   literature.  However,   Gallego  et  al.   (2015)     reported   that  21  days  of  voluntary   running  wheel  access   increased  Bdnf  protein  and  mRNA  expression  in  the  hippocampus  of  both  male   and  female  adolescent  C57Bl/6J  mice.    Both  age  and  duration  of  wheel  exposure   have   been   reported   to   influence   Bdnf   expression   (Adlard   et   al.,   2005),   which   might  explain  the  difference  between  the  results  reported  in  Gallego  et  al.  (2015)   and   this   investigation.    A   recent  meta-­analysis   concluded   that  exercise   training   increases  peripheral  levels  of  BDNF  in  humans,  though  effect  sizes  were  smaller   for  studies  that  included  females  in  the  sample  (Szuhany  et  al.,  2015).    Titterness   et  al.  (2011)  reported  sex  differences  in  hippocampal  LTP  following  two  weeks  of   voluntary  wheel  running   in  adolescent  rats,   though  there  were  no  differences   in   Bdnf   protein   expression   in   either   males   or   females.     Other   research   has   demonstrated   that   voluntary   wheel   running   does   increase   Bdnf   mRNA   expression   in   females   but   the   expression   is   dependent   on   sex   hormones   (Berchtold   et   al.,   2001).     There   is   strong   evidence   that   sex   hormones   are   important  regulators  of  Bdnf  expression  (Carbone  &  Handa,  2013;;  Pluchino  et  al.,   2013).     In   humans,   plasma   BDNF   fluctuates   during   the   menstrual   cycle   and   women   who   experience   normal   ovulatory   cycles   have   higher   plasma   BDNF   compared  to  amenorrhoeic  or  postmenopausal  women  (Begliuomini  et  al.,  2007).     Further,  in  male-­to-­female  transsexuals,  12-­months  of  hormone  therapy  results  in   reduced   serum   BDNF   (Fuss   et   al.,   2015).   These   studies   suggest   a   complex   relationship  between  sex  hormones  and  BDNF  in  humans.  Further  research  with       82   long-­term  exercise   training   in   ovariectomized  mice   is   necessary.    Potentially,   a   non-­Bdnf  pathway  plays  a  more  important  role  in  exercise-­induced  hippocampal   plasticity   in   females  compared   to  males,  whereas  males  may   rely  more  heavily   on  Bdnf-­mediated  plasticity.           There   is   evidence   of   differential   hippocampal   Bdnf   expression   between   males  and  females  following  acute  and  chronic  stress  (Lin  et  al.,  2009).    Females   have   a   higher   prevalence   of  mental   disorders   such   as   clinical   depression   and   post-­traumatic   stress   disorder,   though   animal   research   suggests   that   chronic   stress  leads  to  more  structural  damage  to  the  male  hippocampus  (Cahill,  2006).     The   literature   suggesting   sex   differences   in   hippocampal   adaptations   to   stress   offers  another  potential  explanation  for  the  findings  reported  here.    The  animals   in  this  investigation  underwent  glucose  tolerance  testing  one-­day  prior  to  sacrifice.     This  was  done   to   determine   if   any  whole  body  metabolic   adaptations  occurred   following   five  months  of  voluntary  wheel   running.    The   IPGTT  was  novel   to   the   rodents   and   required   handling   and   a   tail   snip   and,   though   we   took   every   precaution   to   minimize   the   stress   response,   the   procedure   was   undoubtedly   novel  and  presented  an  opportunity  for  stress.    Lin  et  al.  (2009)  reported  that  in   response  to  an  acute  foot  shock,  female  rats  responded  with  greater  Bdnf  protein   expression  in  the  dentate  gyrus  whereas  stressed  and  control  male  rats  showed   no   difference   in   Bdnf   expression.     Moreover,   rodents   that   are   chronically   exercised  have  a  lower  stress  response  to  stressful  stimuli  (Dishman  et  al.,  1997;;   1998;;  Greenwood  et  al.,  2003;;  2005)  and  therefore  Bdnf  expression  in  sedentary   females  may  have  been  greater  compared  to  exercise  females  in  response  to  the       83   IPGTT   stress,   masking   any   observable   effect   of   the   chronic   physical   activity.     Though  there  was  no  difference  between  sedentary  male  and  sedentary  female   total  Bdnf  mRNA  expression,   there  was  a   significant   difference   in  mature  Bdnf   protein   and   a   tendency   for   a   difference   in   total   Bdnf   protein   (p=0.08)   between   sedentary  males  and  sedentary  females.         Interestingly,  Bdnf   IV  mRNA  was  greater   in  exercised  mice  compared   to   sedentary  mice,  and  this  effect  remained  when  analyzing  sexes  separately.    Bdnf   IV  mRNA  expression  is  stimulated  with  neural  activity  (Martinowich  et  al.,  2003),   exercise   (Gómez-­Pinilla  et  al.,  2010;;   Intlekofer  et  al.,  2013),  and  other  external   stimuli  (Lubin,  2011).    Remarkably,  Bdnf  IV  promoter  methylation  is  reduced  with   fear  learning  (Lubin,  2011)  and  short-­term  exercise  (Gómez-­Pinilla  et  al.,  2010),   and   decreased   promoter   methylation   suggests   greater   transcriptional   activity   (Martinowich  et  al.,  2003).    This  is  a  potential  mechanism  mediating  the  effects  of   long-­term  exercise  training  on  Bdnf  IV  transcription.    The  finding  that  both  males   and  females  had  increased  expression  of  Bdnf  IV,  though  only  males  had  higher   total   Bdnf   suggests   that   exercise   stimulates   sex-­specific   up-­   and/or   down-­ regulation  of  transcript-­specific  Bdnf  gene  expression.         The  elevation  in  Bdnf  protein  in  males  was  limited  to  mature  Bdnf  with  no   difference  in  total  Bdnf,  suggesting  that  five  months  of  voluntary  wheel  exposure   selectively  increases  expression  of  the  mature  plasticity-­promoting  Bdnf  isoform.     Sartori  et  al.  (2011)  reported  that  28  days  of  voluntary  wheel  running  selectively   increased   mature   Bdnf   with   no   difference   in   the   immature   proBdnf   in   male       84   C57/Bl6  mice.    In  contrast,  Ding  et  al.  (2011)  reported  that  seven  days  of  wheel   exposure   increased   both   mature   and   proBdnf   in   the   rat   hippocampus.     Differences   in  animal  model  and  exercise  duration   likely  explain   the  differences   between  our  findings  and  those  of  Ding  and  colleagues  (2011).     Curiously,  five  months  of  voluntary  wheel  running  did  not  impact  the  other   mRNA   targets   measured   in   the   present   study.   tPa   and   Pgc-­1a   mRNA   are   reportedly  increased  with  voluntary  wheel  running  (Sartori  et  al.,  2011;;  Steiner  et   al.,  2011),  an  effect  we  did  not  observe.     tPa  has  been  shown   to   influence   the   beneficial   effects   of   exercise   on   hippocampal   function   and   is   known   to   be   an   important  enzyme   in   the  cleavage  of  apoptotic  proBdnf   to  generate   the  mature   and  plasticity-­promoting  mature  Bdnf   (Pawlak  et  al.,  2005;;  Sartori  et  al.,  2011).       Interestingly,   Sartori   et   al.   (2011)   also   used   C57Bl/6J   mice   and   qPCR   to   demonstrate   that   voluntary   wheel   running   increases   tPa   expression   in   the   hippocampus.     Animals   in   the   Sartori   et   al.   (2011)   investigation   were   only   provided  access  to  a  voluntary  running  wheel  for  28  days.    Longer  exposure  to  a   voluntary  running  wheel  may  result  in  a  return  to  control  levels  of  tPa.    Pgc-­1a  is   a   co-­transcription   factor   that   regulates   mitochondrial   biogenesis   and   when   co-­ expressed  with  other   tissue-­  and   temporal-­specific   transcription   factors,  Pgc-­1α   stimulates   the   transcription   of   genes   necessary   for   mitochondrial   biogenesis   (Finck,   2006).     Mitochondrial   biogenesis   in   the   rodent   hippocampus   has   been   observed   following   exercise   training   (Steiner   et   al.,   2011)   and   we   recently   reported  that  in  utero  exercise  exposure  increases  Pgc-­1α  expression  in  offspring   hippocampus   (Venezia  et  al.,  2015)   (Appendix  B).     In   the  current   investigation,       85   we   observed   no   effect   of   long-­term   wheel   running   on   Pgc-­1α.     Steiner   et   al.   (2011)   reported   an   increase   in   hippocampal   Pgc-­1α   following   an   eight-­week   treadmill   exercise   protocol.     Importantly,   forced   and   voluntary   exercise   are   distinct   forms   of   exercise,   generally   associated   with   different   levels   of   stress   hormones  (Yanagita  et  al.,  2007;;  Hayes  et  al.,  2008;;  Liu  et  al.,  2009;;  Ke  et  al.,   2011),   and   volume   and   intensity   of   exercise   (Hayes   et   al.,   2008;;   Leasure   &   Jones,   2008).     Indeed,   voluntary   and   forced   exercise   induce   both   similar   and   distinct  structural  and  functional  adaptations  to  the  rodent  brain  (Burghardt  et  al.,   2004;;  Ploughman  et  al.,  2005;;  Hayes  et  al.,  2008;;  Leasure  &  Jones,  2008;;  Liu  et   al.,  2009;;  Toscano-­Silva  et  al.,  2010;;  Ke  et  al.,  2011;;  Kinni  et  al.,  2011),  which   might   explain   the  discrepancy  between  our  data  and  Steiner  et   al   (2011).    We   also   observed   no   influence   of   chronic   wheel   running   on   glutamate   receptor   subunit  expression.    Short-­term  exposure  to  a  voluntary  running  wheel  increases   mRNA   expression   of   the  NR2B   subunit   of   the   NMDA   glutamate   receptor   and   higher   expression   of   this   subunit   is   associated   with   a   more   plastic   synapse   (Molteni   et   al.,   2002;;   Farmer   et   al.,   2004).     There   is   limited   and   inconsistent   (increases,  decreases,  no  effect)  data  on  the  influence  of  exercise  on  GluR1  and   NR2A  subunit  mRNA  expression  (Molteni  et  al.,  2002;;  Dietrich  et  al.,  2005;;  Ni  et   al.,  2009;;  Real  et  al.,  2010).    Potentially,  due  to  the  long  duration  of  running  and   the   steady   decline   in   wheel   activity   over   the   course   of   the   five   months,   the   stimulus   was   not   intense   enough   to   maintain   elevated   mRNA   expression   of   plasticity-­associated   genes.     Further,   differences   in   mRNA   expression   of   plasticity-­associated  genes  may  have  been  observed   if  we   investigated  specific       86   hippocampal   subfields   (dentate   gyrus,   CA1,   and   CA3)   instead   of   whole   hippocampal   homogenates.     Hippocampal   subfields   contain   specific   cell   types   and   varying   levels   of   sensitivity   and   adaptations   to   stimuli   including   exercise   (Andersen   et   al.,   2006;;   Voss   et   al.,   2013).   Future   investigations   should   utilize   additional  methods  of  mRNA  detection  such  as  in  situ  hybridization.       A  limitation  of  our  investigation  was  that  the  control  group  was  not  housed   with   a   locked   running   wheel.     The   differences   observed   in   Bdnf   mRNA   and   protein   expression   were   potentially   due   to   enriched   housing   or   the   combined   effects  of  enriched  housing  and  running.    Sartori  et  al.   (2011)  observed  greater   mature  Bdnf   protein   in  mice   housed  with   a   locked   running  wheel   compared   to   mice  housed  in  standard  cages  without  a  wheel,  demonstrating  that  the  presence   of  a  wheel  can   influence  Bdnf  expression   independent  of   running.     Importantly,   our  data  still  demonstrate  that   long-­term  housing  with  a  freely  rotating  voluntary   running   wheel   influences   Bdnf   expression   and   processing   differently   between   sexes.   Summary.     The   present   data   suggest   that   long-­term   voluntary   exercise   has   limited   and   sex-­dependent   effects   on   hippocampal   mRNA   and   Bdnf   protein   expression.   Due   to   the   limited   effects   of   long-­term   voluntary   wheel   running   observed  in  our  investigation,  we  speculate  that  voluntary  wheel  running  volume   and/or  intensity  might  need  to  be  maintained  or  even  manipulated  (i.e.,  increase   intensity)  to  most  effectively  maintain  hippocampal  plasticity-­associated  gene  and   protein  expression.    This  might  explain  why  we  saw  a  greater  benefit  of  exercise       87   in  males,  which  maintained  their   running  volume  better   than  females.  Voluntary   wheel  running  is  a  good  model  for  unstructured  leisure-­time  physical  activity  but   might  not  be  the  best  model   for  exercise  training,  which   is  generally  associated   with  structured  frequency,  intensity,  and  duration  components.    Forced  exercise,   though   associated   with   elevated   stress   hormones   (Ploughman   et   al.,   2005;;   Yanagita  et  al.,  2007;;  Hayes  et  al.,  2008;;  Liu  et  al.,  2009;;  Ke  et  al.,  2011),  might   be   an   alternative   to   enhance   brain   health   and   plasticity   (Hayes   et   al.,   2008;;   Leasure  &  Jones,  2008;;  Liu  et  al.,  2009;;  Toscano-­Silva  et  al.,  2010;;  Kinni  et  al.,   2011;;  Lin  et  al.,  2012)  in  the  long-­term  if  declining  voluntary  running  volume  and   intensity   is   limiting   plasticity,   which   is   yet   to   be   determined.     Future   research   should   examine   the   effectiveness   of   long-­term   forced   exercise   due   to   the   high   levels  of  stress  associated  with  this  model  of  exercise.           88   Competing  interest   None  declared   Funding   This  work  was   supported   by  NIH   grant  HD062868,   The  College   of  Health   and   Human   Performance   Public   Health   Research   Seed   Money   Program   award   (S.M.R.   and   E.E.S.),   NIH   T32   AG000268   (A.C.V.   and   L.M.G.),   and   NIH   F31   MH103951-­01A1  (A.C.V.)   Authors’  contributions.   ACV,  LMG,  EES,  and  SMR  designed  the  study;;  ACV,  LMG,  and  RMS  collected   the  data;;  data  analysis,  preparation  of   figures,  and  drafting   the  manuscript  was   done   by   ACV;;   ACV,   LMG,   RMS,   EES,   and   SMR   edited   and   revised   this   manuscript;;  and  all  authors  approved  the  final  version.             89   Chapter  4.       Aim  #2.    Determine  the  effect  of  acute  exercise  and  exercise  intensity  on  GluR1   phosphorylation,  the  expression  of  specific  plasticity-­associated  genes,  and  novel   object  location  memory  in  three-­month  old  C57BL/6J  mice.     Aim   #3:     Determine   if   acute   high-­intensity   exercise   increases   anxiety-­like   behavior  in  the  open  field  task  and  if  this  behavioral  phenotype  is  attenuated  with   pre-­treatment  with  the  selective  noradrenergic  neurotoxin  DSP-­4.   Title:     Acute   Forced   Exercise   Increases   Expression   of   Bdnf   IV   and   Induces   Anxiety-­Like  Behavior  in  C57BL/6J  Mice.   Authors:  Andrew  C.  Venezia1,2,  Molly  M.  Hyer2,3,  Erica  R.  Glasper1,3,  Stephen  M.   Roth1,2,  Elizabeth  Quinlan2,4   Department   of   Kinesiology,   School   of   Public   Health,   University   of   Maryland,   College  Park,  MD1   Neuroscience  and  Cognitive  Science  Program,  University   of  Maryland,  College   Park,  MD2   Department  of  Psychology,  University  of  Maryland,  College  Park,  MD3   Department  of  Biology,  University  of  Maryland,  College  Park,  MD4         90   Introduction       Exercise  is  an  effective  way  to  maintain  and  improve  brain  health,  and  research   in  humans  and  rodents  indicates  that  the  hippocampus,  a  brain  region  important   for   memory   formation   and   the   response   to   emotional   and   stressful   situations   (Moser  &  Moser,  1998;;  Strange  et  al.,  2014),  is  particularly  sensitive  to  physical   activity   and   exercise   (Voss   et   al.,   2013).     Adaptations   observed   in   the   hippocampus  with  exercise  training  include  neurogenesis  (van  Praag,  Christie,  et   al.,   1999;;   van   Praag,   Kempermann,   et   al.,   1999),   dendritic   arborization   (Stranahan  et  al.,  2007;;  Lin  et  al.,  2012),  and  increased  amplitude  and  reduced   threshold  of  long  term  potentiation  (LTP;;  van  Praag,  Christie,  et  al.,  1999;;  Farmer   et   al.,   2004),   a   long-­lasting   enhancement   of   synaptic   strength   and   a   leading   candidate   for   the  molecular  mechanism  of  memory   formation   (Malenka,   1999).     Though  numerous  studies  have  investigated  the  mechanisms  that  mediate  these   beneficial   adaptations   in   response   to   exercise   training,   the   mechanisms   that   directly   mediate   enhanced   synaptic   efficacy   following   exercise   have   not   been   identified.     Moreover,   the   molecular   events   that   occur   in   response   to   a   single   bout  of  acute  exercise  have  not  been  thoroughly  investigated.    Chronic  exercise,   which  has  been  the  focus  of  the  majority  of  research  to  date,  is  the  accumulation   of  individual  acute  bouts  of  exercise,  each  of  which  can  be  optimized  to  enhance   the  benefits  of  chronic  exercise  training  on  brain  health.   The  enhancement  of  synaptic  transmission  mediating  LTP  is  a  result  of  an   increase  in  the  number  of  synaptic  AMPA  receptors  (AMPARs)  that  mediate  the       91   majority   of   the   response   to   synaptically-­released   glutamate   (Huganir   &   Nicoll,   2013).    AMPARs  are  heterotetramers   composed  of  GluR1,  GluR2,  GluR3,   and   GluR4  subunits,  with   the  adult  brain  primarily  containing  AMPARs  composed  of   GluR1/GluR2   and   GluR2/GluR3   subunit   combinations.     The   threshold   for   LTP   can   be   reduced   by   phosphorylation   of   the  C-­terminal   tail   of   the  GluR1   subunit   and   subsequent   insertion   of   GluR1   containing   AMPARs   into   the   perisynaptic   membrane,   making   this   receptor   more   available   for   subsequent   migration   and   “capture”  by  the  synapse  (Kessels  &  Malinow,  2009).    Phosphorylation  of  serine   831  (Ser831)  on  GluR1  by  CAMKII  increases  the  frequency  of  synaptic  “capture”   of  AMPARs  and  ion  channel  open  probability  (Kessels  &  Malinow,  2009;;  Huganir   &  Nicoll,  2013),  while  phosphorylation  of  serine  845  (Ser845)  by  PKA  increases   perisynaptic   insertion   and   decreases   AMPAR   internalization   (Oh   et   al.,   2006;;   Santos  et  al.,  2009).     In   fact,  mutations   that  prevent  phosphorylation  of  Ser831   and   Ser845   compromise   synaptic   plasticity   (Lee   et   al.,   2003)   and   prevent   the   lowered  threshold  for  LTP  and  learning  triggered  by  exogenous  catecholamines   (Hu  et  al.,  2007).    Moreover,  mimicking  phosphorylation  with  knock-­in  mutations   reduces   the   threshold   for   LTP   and   occludes   the   effects   of   exogenous   norepinephrine   (Makino   et   al.,   2011).     Understanding   the   mechanisms   and   stimuli  that  cause  phosphorylation  and  membrane  insertion  of  GluR1  is  a  focus  of   intense   investigation   and   exercise   has   the   potential   to   be   a   practical,   non-­ invasive   strategy   to   achieve   this   molecular   event.     In   fact,   following   cortical   infarction,   exercise   training   can   induce   Ser845   phosphorylation   in   the   rodent   cortex  (Mizutani  et  al.,  2015),  indicating  that  physical  activity  has  the  potential  to       92   induce   this   adaptation.    Whether   or   not   acute   exercise   can   achieve   this   in   the   healthy  hippocampus  is  unknown.  The  reduced  threshold  for  LTP  (Farmer  et  al.,   2004)  and  learning  (Intlekofer  et  al.,  2013)  observed  following  exercise  training  in   healthy  rodents  may  be  mediated  by  phosphorylation  of  important  sites  on  the  C-­ terminal  tail  of  GluR1;;  however,  this  has  not  been  thoroughly  investigated  in  the   rodent   hippocampus.     Acute   exposure   to   psychological   stress   or   peripheral   injections   of   epinephrine   increase   phosphorylation   of   Ser845   on   GluR1   and   reduce  the   threshold   for  LTP  and   learning  (Hu  et  al.,  2007).     Importantly,  acute   bouts   of   exercise   also   increase   release   of   peripheral   epinephrine   and   central   norepinephrine   (Pagliari   &   Peyrin,   1995a;;   Zouhal   et   al.,   2008).     Like   acute   exposure   to   psychological   stress,   a   single   acute   bout   of   exercise   has   the   potential  to  influence  the  phosphorylation  status  of  this  important  AMPA  receptor   subunit   and   might   be   the   mechanism   by   which   acute   bouts   of   exercise   strengthen  memory  formation  (Lambourne  &  Tomporowski,  2010;;  Intlekofer  et  al.,   2013;;  Roig  et  al.,  2013).   Brain-­derived  neurotrophic   factor   (Bdnf)   is  a  critical  protein   for   functional   and  structural  hippocampal  plasticity  and   is  upregulated  with  chronic  and  short-­ term   exercise   training   (Vivar   et   al.,   2012;;   Voss   et   al.,   2013).     Though   the   importance   of   Bdnf   is   well   known   (Park   &   Poo,   2013),   the   extent   to   which   individual   bouts   of   exercise   influence   Bdnf   expression   is   not   understood.     Research   investigating   “acute”  exercise   in   rodents  generally   follows  a  period  of   treadmill  running  familiarization  or  is  a  voluntary  running  wheel  exposure  lasting   from  several   hours   to  multiple   days.    Bdnf   expression   is   important   for   different       93   stages   of   memory   formation   (Bekinschtein   et   al.,   2014)   so   understanding   the   temporal   dynamics   of   Bdnf   expression   and   the   stimuli   that   upregulate   its   expression  is  important  to  most  effectively  utilize  exercise  or  exercise-­like  stimuli   to  enhance  memory.    In  addition,  transcription  of  other  plasticity-­associate  genes,   such  as  glutamate  receptor  subunits,  may  also  be   influenced  by  acute  bouts  of   exercise   and   identifying   these   genes   could   shed   light   on   the  mechanisms   that   lead  to  memory  enhancement  following  acute  and  chronic  exercise.   Though   research   in   humans   suggests   acute   exercise   can   improve   memory  (Lambourne  &  Tomporowski,  2010;;  Roig  et  al.,  2013),  this  has  not  been   adequately  investigated  in  rodents.    Since  rodents  are  commonly  used  as  models   to  understand   the  mechanisms   that  drive  behavioral  adaptations,   it   is   important   to   understand   the   behavioral   modifications   occurring   in   response   to   acute   exercise  if  this  model  is  to  be  used  appropriately.    We  investigated  the  influence   of  a  single  bout  of  acute  treadmill  exercise  on  GluR1  phosphorylation,  plasticity-­ associated  gene  expression,  and  performance  on  a  one-­trial  spatial  memory  task   and  a   locomotor-­dependent   anxiety   test.     Further,   because  exercise   stimulates   the   release  of  central  and  peripheral  catecholamines   (Pagliari  &  Peyrin,  1995a;;   Chatterton  et  al.,  1996;;  Zouhal  et  al.,  2008),  which  are  known  to  influence  anxiety   like   behavior   (Goddard   et   al.,   2010),   we   also   wanted   to   understand   how   noradrenergic  signaling  influences  animal  behavior.  To  explore  this  relationship,   we  utilized  a  pharmacological  approach  to  compromise  the  central  noradrenergic   system  prior  to  acute  exercise  and  assessment  of  anxiety-­like  behavior.       94   Methods   Mouse  model.  Three-­month  old  male  C57BL/6J  mice  (Jackson  Laboratories  (Bar   Harbor,   ME,   USA)   were   used   in   this   investigation.     This   mouse   strain   is   commonly  used  to  study  the  impact  of  exercise  on  brain  phenotypes  and  in  our   lab  displays  avid   treadmill   running  activity  and  normal  physiological  adaptations   to   exercise   (e.g.   improved   glucose   metabolism,   lower   body   mass,   enhanced   markers  of  oxidative  capacity,  etc.;;  Ludlow  et  al.,   2012;;  Guth  et  al.,   2013).    All   mice  were  group  housed  and  cared  for  by  UMD  veterinary  staff.    Mice  were  kept   on  a  12hr  light/12hr  dark  cycle  and  provided  standard  rodent  chow.    All  protocols   were  approved  by  the  University  Institutional  Animal  Care  and  Use  Committee.       Overview  and  Treadmill  Protocol:    To  address  the  question  of  how  acute  exercise   influences  markers  of  hippocampal  plasticity,  mice  were  randomly  separated  into   three   groups:   1)   treadmill   without   exercise   (CON;;   n=12);;   2)  moderate-­intensity   acute   treadmill  exercise   (MOD;;  n=12);;  3)  high-­intensity  acute   treadmill  exercise   (HI;;  n=12).  For  three  days  leading  up  to  the  experiment,  mice  were  placed  on  the   stationary  treadmill   for  five  minutes  per  day,  during  which  the  electrical  stimulus   grid   at   the   end   of   the   treadmill   belt   was   activated,   so   that   all   mice   were   familiarized   with   the   stimulus   and   treadmill-­testing   environment.   During   active   treadmill  running,  the  stimulus  grid  provides  a  weak  foot  shock,  which  causes  an   involuntary  muscle  contraction  that  encourages  running.  Tactile  stimulation  to  the   tail  was  used  to  encourage  mice  to  run  prior  to  touching  the  stimulus  grid,  which   reduced  the  number  of  stimulus  grid  touches  (unpublished  observation).    On  day       95   four,  MOD  and  HI  group  mice  were  placed  on   the   treadmill,  one  at  a   time,  and   the   acute   bout   of   exercise   was   initiated.   Each  mouse   underwent   a   six-­minute   warm  up,  where  the  first  minute  was  a  no-­exercise  treadmill  exposure;;  thereafter   the  treadmill  belt  began  to  move  at  5  m/min,  increasing  1  m/min  every  minute  for   five  minutes.    The  treadmill  speed  was  then  incrementally  increased  to  the  group-­ appropriate   speed   and   the   mouse   ran   for   30   minutes   at   this   pace.     In   our   laboratory,  this  warm  up  is  an  effective  method  to  encourage  mice  to  run  on  the   treadmill  without  multiple  acclimation  trials.  The  MOD  group  ran  for  30  minutes  at   12  m/min  at  0%  grade;;  this  stimulus  has  been  reported  to  be  ~75%  of  VO2max  in   adult  C57BL/6J  mice  (Schefer  &  Talan,  1996).    The  HI  group  ran  for  30  minutes   at  a  speed  ranging  from  15-­17  m/min  at  0%  grade,  depending  on  running  ability;;   this   speed   has   been   reported   to   be   ~80%   of   VO2max   in   adult   C57BL/6J   mice   (Schefer  &  Talan,  1996).    Mice  in  the  CON  group  were  placed  on  the  stationary   treadmill   for  36  minutes  with   the  electrical  stimulus  grid  activated.     Immediately   following   the   treadmill   bout,   mice   were   sacrificed   for   gene   expression   and   biochemical  analysis.     Tissue  Processing:  Mice  used  for  mRNA  and  protein  analysis  were  sacrificed  by   decapitation   under   isoflurane   anesthesia   immediately   following   the   acute   exercise  bout.  The  hippocampus  was  halved,  isolated,  and  immediately  frozen  in   liquid  nitrogen.    For  western  blot  analysis,  the  hippocampus  was  sonicated  in  1%   SDS,   boiled   for   10   minutes   (Hu   et   al.,   2007),   and   stored   at   -­80°C.     Protein   concentration   was   determined   by   spectrophotometry   using   the   BCA   Protein   Assay   (Pierce®,   Rockford,   IL).     RNA   was   isolated   from   the   remaining   half       96   hippocampus.     Samples   were   homogenized   in   a   glass   Dounce   tissue   homogenizer   followed  by  RNA   isolation  using  TRI  Reagent   (Life  Technologies,   Grand   Island,   NY,   USA).     RNA   quantity   and   purity   were   assessed   by   UV   spectroscopy.       Western  Blotting:    Twenty-­five  μg  of  protein  was  loaded  onto  polyacrylamide  gels   and   electrophoresed,   followed   by   transfer   to   nitrocellulose   membranes   and   immunoblotting.   Nitrocellulose   membranes   were   incubated   with   anti-­phospho-­ GluR1  (Ser845;;  Millipore  or  Ser831;;  Millipore,  Billerica,  MA)  antibodies,  stripped   in   a   glycine-­  HCl   stripping   solution,   and   re-­probed  with   an  anti-­GluR1  antibody   (Millipore).     Although   the   short   time   between   the   initiation   of   the   exercise   bout   and  sacrifice  (30  minutes)  should  prevent  any  changes  in  total  GluR1  translation,   we  also  blotted  for  total  GluR1  protein  followed  by  stripping  and  re-­probing  for  the   neuronal   nuclear   marker   NeuN   (Millipore).     Appropriate   fluorescent   secondary   antibodies   were   used   for   detection.   A   Typhoon   scanner   (GE   Healthcare   UK   Limited,  Buckinghamshire,  England)  was  used   to  digitize   the   fluorescent  signal.     Levels  of  phosphorylation,  expressed  as   the   ratio  of  phospho-­GluR1  divided  by   total  GluR1  intensity  from  the  same  lane,  were  used  for  statistical  analysis.         To   confirm   that   peripheral   epinephrine   increases   hippocampal   GluR1   Ser845  phosphorylation,  a  subsample  of  mice  were  injected  with  saline  (10ml/kg;;   n=3)  or  epinephrine  (0.5mg/kg  at  10  ml/kg;;  n=4)  and  sacrificed  15  minutes  post-­ injection.    Hippocampi  were  sonicated   in   lysis  buffer   [50  mM  Tris-­HCl   (pH  7.4),       97   150   mM   NaCl,   1%   NP-­40,   protease   inhibitor   cocktail]   and   immunoblotted   as   described  above.     Gene  Expression:    One  μg  of  total  RNA  was  reverse  transcribed  into  cDNA  using   the  High  Capacity  cDNA  Reverse  Transcription  Kit   (Applied  Biosystems,  Foster   City,   CA).     Real-­time   quantitative   PCR   (qPCR)   was   used   to   assess   mRNA   expression  of   total  brain  derived  neurotrophic   factor   (Bdnf;;  exon   IX),  Bdnf  exon   IV  (Bdnf  IV),  GluR1,  NR2A,  NR2B,  Gapdh,  and  ActB  (Gapdh  &  ActB;;  expression   controls;;   primer   sequences   listed   in   Appendix   A).     Primer:probe   assays   were   purchased   pre-­made   [GluR1   (Gria1),   NR2A   (Grin2a),   NR2B   (Grin2b),   Gapdh,   ActB]  or  designed  (Bdnf  IX,  Bdnf  IV)  for  the  mRNA  sequence  of  each  gene  using   Integrated   DNA   Technologies’   PrimeTime   qPCR   Assay   designer.     All   primer   pairs  except  Bdnf  total  spanned  exons  to  prevent  amplification  of  genomic  DNA.   Because  Bdnf   total   is   represented  by   amplification  of   only   exon   IX,   this   primer   pair   could   not   span   exons.     Efficiency   for   each   primer:probe   assay   was   determined  prior  to  use.    qPCR  data  were  normalized  to  the  geometric  mean  of   Gapdh   and   ActB   using   the   -­ΔΔCt   method   (Vandesompele   et   al.,   2002;;   Schmittgen   &   Livak,   2008)   and   expressed   as   fold   induction   (2-­ΔΔCt)   of   mRNA   expression  compared  to  the  control  group  (1.0-­fold  induction).     Object  Location  Memory:  A  subset  of  three-­month  old  male  C57BL/6J  mice  were   tested  on  the  object  location  memory  task  immediately  following  the  acute  bout  of   exercise   or   no-­exercise   treadmill   exposure.     The   treadmill   familiarization   approach  was  the  same  as  described  above.    Mice  in  the  treadmill  control  group       98   (n=15)  sat  on   the  stationary   treadmill   for  36  minutes  while  mice   in   the  exercise   group   (n=15)   ran  on   the   treadmill  at  15   to  17  m/min   for  30  minutes   following  a   six-­minute  warm-­up.    The  procedures   for   the  object   location   task  were  adapted   from  Barker  and  Warburton  (Barker  &  Warburton,  2011).    Prior  to  test  day,  mice   were  exposed   to   the   testing  apparatus   (43x43x21.5  cm  open   field  box)   for   five   minutes/day   over   two   consecutive   days   to   acclimate   them   to   the   behavioral   procedure.    On  the  test  day,   immediately   following  the  treadmill  exposure,  mice   were  placed  in  the  testing  apparatus  for  the  familiarization  phase  of  the  task  and   allowed  to  explore  the  box  and  two  identical  objects  (small,  Duplo  blocks)  for  five   minutes   and   then   returned   to   their   home   cages   for   a   fifteen-­minute   inter-­trial   interval.    Following  this   interval,  mice  were  returned  to  the  testing  apparatus  for   the  test  phase  when  they  were  presented  with  one  of  the  objects  from  the  initial   exposure  phase  and  a  third  object  that  was  identical  to  the  initial  exposure  phase   objects,  though  it  was  moved  to  a  different  location  of  the  box.    Mice  explored  the   box   and   the   two   objects   for   five  minutes.     The   left/right   position   of   the  moved   object  was   counterbalanced  between  mice.    Behavior   during   the   familiarization   and   test   phases   was   monitored   using   EthoVision   XT   11   Behavioral   Tracking   Software   (Noldus,   Leesburg,   VA),   which   provides   automatic   tracking,   analysis,   and  storage  of  animal  activity  and  behavior.  Object   interaction   (time  spent  with   sample   objects   and   number   of   interactions),   latency   to   approach   objects,   and   total  distance  moved  were  recorded  for  each  mouse.   Open   Field   Behavior   Task:     An   additional   subset   of   three-­month   old   male   C57BL/6J  mice  were  used   to  assess  anxiety-­like  behavior  and   the   influence  of       99   noradrenergic   signaling   on   behavior   following   acute   exercise.     Mice   were   separated  into  four  groups:  1)  Stationary  Treadmill  –  Saline  (CON-­SAL;;  n=9);;  2)   Stationary   Treadmill   –   DSP-­4   (CON-­DSP4;;   n=10);;   Treadmill   Exercise   –   Saline   (EX-­SAL;;  n=8);;  Treadmill  Exercise  –  DSP-­4   (EX-­DSP4;;  n=9).    Mice  underwent   the  same   treadmill   familiarization  and  running  as  described  above  and  ran  at  a   treadmill   speed   between   15   and   17   m/min   depending   on   running   ability.     Immediately  after   the  acute  bout  of  exercise  or  no-­exercise   treadmill   exposure,   performance   on   the   open   field   task   was   assessed.   Immediately   following   the   treadmill   exposure,  mice  were   placed   in   the   testing   apparatus   (43x43x21.5   cm   field  box)  and  allowed  to  explore  for  15  minutes.  Behavior  was  monitored  using   the  EthoVision  XT  11  Behavioral  Tracking  Software.    Total  distance  moved,  time   spent   in  central  and  peripheral   zones,  and   time  spent  grooming  were   recorded   and  separated  into  five  minute  blocks  (0-­5  minutes,  5-­10  minutes,  10-­15  minutes).   N-­(2-­chloroethyl)-­N-­ethyl-­2-­bromobenzylamine   (DSP-­4):     DSP-­4   is   a   selective   neurotoxin   that  specifically   lesions   the   locus  coeruleus   (LC)  noradrenergic   (NA)   system.   DSP-­4   dramatically   reduces   tissue   levels   of   norepinephrine   in   regions   innervated  by  the  LC,  such  as  the  hippocampus  (Ross,  1976;;  Ögren  et  al.,  1980;;   Jonsson  et  al.,  1981;;  Archer  et  al.,  1982;;  Anisman  et  al.,  1984;;  Zahniser  et  al.,   1986;;  Bennett  et  al.,  1990;;  Scullion  et  al.,  2009;;  Szot  et  al.,  2010),  though  leaves   non-­LC   NA   neurons   and   serotonergic   and   dopaminergic   systems   essentially   unaffected   (Ross   &   Stenfors,   2014).     This   chemical   is   commonly   used   to   irreversibly  disrupt  central  NA  signaling  because  it  easily  crosses  the  blood  brain   barrier   and   therefore   can   be   injected   systemically   (Ross   &   Stenfors,   2014).         100   Importantly,  DSP-­4  treatment  does  not  alter  running  activity  in  rodents  (Garcia  et   al.,  2003).  DSP-­4  (Sigma  Aldrich)  was  prepared   in  0.9%  saline  and  a  single  50   mg/kg  dose  was  delivered  by   IP   injection   in  a  volume  of  10  ml/kg;;   this  dose   is   frequently  used   in  both   rats  and  mice  and   is  effective   in  depleting  hippocampal   norepinephrine   (Ross   &   Stenfors,   2014).     Control   mice   received   a   single   IP   injection   of   0.9%   saline.     Solutions   were   prepared   for   five   animals   and   any   remaining  solution  was  discarded.    Injections  were  delivered  within  15  minutes  of   solution   preparation   and   was   kept   out   of   the   light.   Animals   received   injections   seven   days   prior   to   treadmill   familiarization   (10   days   prior   to   experimental   treadmill   day).     This   dose   of   DSP-­4   and   interval   between   injection   and   task   performance   results   in   >90%   reduction   in   hippocampal   norepinephrine   in   C57BL/6J  mice  (Scullion  et  al.,  2009).   Statistical   Analysis:   To   determine   differences   in   GluR1   protein   expression/   phosphorylation   and   mRNA   expression   between   groups   we   used   a   one-­way   analysis   of   variance   with   Tukey’s   post   hoc   comparisons   when   appropriate   (p<0.05  considered  statistically  significant).  Object  location  memory  performance   was   analyzed   with   a   repeated   measures   ANOVA   and   Sidak’s   multiple   comparison  test  when  appropriate.    Open  field  behavior  data  was  analyzed  using   a   two-­way  ANOVA  (treadmill  exposure  x  drug   treatment)  and  Tukey’s  post  hoc   comparisons  when  appropriate  (p<0.05  considered  statistically  significant).           101   Results   GluR1   phosphorylation:     We   were   able   to   confirm   that   an   IP   injection   of   epinephrine   was   sufficient   to   induce   phosphorylation   of   Ser845   (t(5)=   3.048;;   p=0.03)   but   did   not   influence   Ser831   phosphorylation   or   GluR1   protein   expression   (Fig.   7).     In   contrast,   an   acute   bout   of   high-­   or   moderate-­intensity   treadmill  running  did  not  influence  phosphorylation  of  Ser845  or  Ser831  (Fig.  8).       Glutamate  Receptor  mRNA:  There  was  no  effect  of  moderate-­  or  high-­intensity   acute  exercise  on  GluR1,  NR2A,  or  NR2B  mRNA  expression  (Fig.  9).     Bdnf   mRNA:     There   was   an   intensity-­dependent   significant   effect   of   acute   treadmill  exercise  on  Bdnf   IV  expression   (F(2,  32)=3.79;;  p=0.03;;  Fig.  10A).    High   intensity   treadmill   exercise   resulted   in   higher   mRNA   expression   compared   to   controls  (adjusted  p=0.03).    There  was  no  significant  effect  of  acute  exercise  on   total  Bdnf  mRNA  expression  (Fig.  10B).     Object  Location  Task:    There  were  no  main  effects  or  interaction  effect  of  acute   exercise  or  phase  of  the  task  on  total  time  spent  exploring  the  two  objects  (Fig.   11A).    During  the  test  phase,  there  was  no  significant  difference  between  controls   and  exercisers   in   time  spent  with   the  newly  moved  object  relative  to   time  spent   with  both  objects  (Fig.  11B).    There  was  a  main  effect  of  acute  exercise  on  total   distance  moved  (F(1,28)=14.06;;  p=0.008)  but  no  main  effect  of  test  phase.    There   was  a  tendency  for  an  interaction  effect  between  acute  exercise  and  test  phase   on   total  distance  moved  (F(1,28)=3.684;;  p=0.07).    Mice  exposed   to  high-­intensity   treadmill   running   moved   significantly   less   (total   distance   traveled)   during   the       102   familiarization  phase  compared  to  treadmill  controls  (Fig.  12A;;  p=0.0003).    There   was  a  main  effect  of  acute  exercise  on  number  of   interactions  with   the  objects   (F(1,28)=4.553;;   p=0.04)   and   an   interaction   effect   (F(1,28)=6.938;;   p=0.01)   but   no   main   effect   of   test   phase.     The   treadmill   exercisers   interacted   with   the   two   objects   significantly   less   frequently   than   treadmill   controls   during   the   familiarization  phase  (Fig.  12B;;  p=0.003).       Open  Field  Task:     0-­5  minutes:  During   the   first   five  minutes  of   the  open   field   task  we  observed  a   main   effect   of   exercise   (F(1,32)   =   33.09;;   p   <0.0001)   and   an   interaction   between   drug  and  exercise  (F(1,32)  =  4.25;;  p=0.048)  but  no  main  effect  of  drug  (Fig.  13A).     Post   hoc   analysis   revealed   that   EX-­SAL   mice   had   significantly   lower   total   distance   moved   during   the   first   five   minutes   compared   to   CON-­SAL   mice   (adjusted   p   <0.0001)   (Fig   13A).     There   was   no   significant   difference   between   CON-­DSP4  and  EX-­DSP4,  though  EX-­DSP4  had  significantly  less  total  distance   traveled  than  CON-­SAL  (adjusted  p=0.003)  (Fig.  13A).  We  also  observed  a  main   effect  of  exercise  on  time  spent  grooming  during  the  first  five  minutes  in  the  open   field   task   (F(1,32)   =   35.52;;   p<0.0001)   but   no   main   effect   of   the   drug   or   an   interaction   effect   (Fig   13B).     Post   hoc   analysis   revealed   that   both   EX-­SAL   (adjusted   p=0.0007)   and  EX-­DSP4   (adjusted   p=0.002)   spent   significantly  more   time  grooming  compared  to  CON-­SAL  mice  (Fig.  13B).    Further,  EX-­DSP4  mice   spent   significantly   more   time   grooming   compared   to   CON-­DSP4   (adjusted   p=0.002;;  Fig.  13B).    There  was  no  main  effect  of  exercise  or  drug  or  interaction       103   effect  for  number  of  entries  into  the  center  of  the  testing  arena  (Fig.  13C)  or  the   amount  of  time  spent  in  the  center  of  the  arena  (Fig.  13D).           5-­10  minutes:     During   the   second   five-­minute   block   of   the   open   field   task,   we   observed  a  main  effect  of  exercise  (F(1,32)  =13.32;;  p=0.0009)  but  no  main  effect  of   drug  or  interaction  effect  on  total  distance  moved  (Fig.  14A).    Post  hoc  analysis   revealed   significantly   less   distance   traveled   in   EX-­SAL   (adjusted   p=0.03)   and   EX-­DSP4  (adjusted  p=0.02)  compared  to  CON-­SAL  mice  (Fig.  14A).    There  was   no  significant  difference  between  CON-­DSP4  and  EX-­DSP4  mice.    There  was  a   main  effect  of  exercise   (F(1,31)  =  28.31;;  p<0.0001)  on   total   time  spent  grooming   but   no   main   effect   of   drug   or   interaction   effect   during   the   second   five   minute   block   (Fig.   14B).     Post   hoc   analysis   revealed   that   EX-­DSP4   mice   spent   significantly  more  time  grooming  than  CON-­SAL  (adjusted  p=0.0006)  and  CON-­ DSP4   (adjusted   p=0.0001)   mice   (Fig.   14B).     There   was   no   main   effect   of   exercise  or  drug  or  an  interaction  effect  for  frequency  of  entries  (Fig.  14C)  or  time   spent  (Fig.  14D)  in  the  center  of  the  testing  arena.     10-­15   minutes:     During   the   final   five-­minute   block   of   the   open   field   task,   we   observed  a  main  effect  of  exercise  (F(1,32)  =  10.55;;  p=0.003)  and  a  main  effect  of   the  drug  (F(1,32)  =  5.091;;  p=0.03)  but  no  interaction  between  exercise  and  drug  for   total   distance   traveled   (Fig.   15A).     Post   hoc   analysis   revealed   that   EX-­SAL   (adjusted   p=0.006)   and   EX-­DSP4   (adjusted   p=0.003)   had   significantly   less   distance  traveled  compared  to  CON-­SAL  (Fig  15A).    Moreover,  CON-­DSP4  had   significantly  less  distance  traveled  compared  to  CON-­SAL  (adjusted  p=0.02;;  Fig       104   15A).    We  observed  a  main  effect  of  exercise  on   time  spent  grooming  (F(1,31)  =   14.95;;  p=0.0005)  but  no  main  effect  of  drug  or   interaction   (Fig  15B).    Post  hoc   analysis   revealed   that   EX-­DSP4   mice   spent   significantly   more   time   grooming   than  CON-­SAL  (adjusted  p=0.01)  and  CON-­DSP4  (adjusted  p=0.002)  mice  (Fig   15B).    We  observed  a  main  effect  of  exercise  in  the  frequency  of  entries  (F(1,32)  =   6.869;;  p=0.01)  and  time  spent  (F(1,32)  =  5.393;;  p=0.03)  in  the  center  of  the  testing   arena  (Fig  15C).   Running  performance:    We  observed  no  significant  difference  between  EX-­SAL   and   EX-­DSP4   in   running   performance,   indicated   by   number   of   stimulus   grid   touches.    However,  we  did  observe  a  significant  negative  correlation  between  the   total  number  of  stimulus  grid  touches  and  distance  traveled  during  the  first   five-­ minute  block  of   the  open  field   task  (p=0.005).    Better   running  performance  was   associated  with  higher  activity   in   the  open   field   task  during   the   first   five-­minute   block.    This  correlation  was  no  longer  observed  during  the  second  and  third  five-­ minute  blocks.    There  was  no  correlation  between  running  performance  and  time   spent  self-­grooming  or  entries  into  the  center  of  the  testing  arena  during  any  five-­ minute  time  block.           105   Figure   7.   Intraperitoneal   injection   of   epinephrine   induces   GluR1   Ser845   phosphorylation   in   the   hippocampus.    A)  Blots   for  p-­Ser845,  p-­Ser831,  and   total  GluR1,  15  minutes  following  saline  (n=3)  or  epinephrine  (n=4)  IP  injections.     B)  IP  injection  of  epinephrine  increased  the  ratio  of  Ser845  phosphorylation  over   GluR1  protein  expression   (p=0.03).     There  was  no   influence  of   epinephrine  on   the  ratio  of  Ser831  phosphorylation  over  GluR1  or  the  ratio  of  GluR1  over  NeuN.   Error  bars  represent  SEM.  *  indicates  p<0.05   Figure  8.  Acute  exercise  does  not   influence  GluR1  phosphorylation   in   the   mouse  hippocampus.    Mice  were  exposed  to  30  minutes  (after  6  minute  warm   up)  of  moderate-­intensity  (12m/min;;  n=11),  high-­intensity  (15-­17m/min;;  n=11),  or   no   exercise   treadmill   exposure   (n=11).     Mice   were   sacrificed   and   hippocampi   isolated  immediately  after  treadmill  exposure.    Whole  hippocampal  homogenates   were  sonicated  and  boiled  in  1%  SDS.    There  was  no  significant  effect  of  acute   exercise  on  GluR1  phosphorylation.    Error  bars  represent  SEM.       Figure   9.  Acute   exercise   does   not   influence   glutamate   receptor   subunit   mRNA   expression   in   the   mouse   hippocampus.     Mice   were   exposed   to   30   minutes   (after   6  minute  warm  up)  of  moderate-­intensity   (12m/min;;   n=11),   high-­ intensity   (15-­17m/min;;   n=12),   or   no   exercise   treadmill   exposure   (n=12)   Mice   were  sacrificed  and  hippocampi  immediately  isolated.    mRNA  was  isolated  from   whole   hippocampal   homogenates   in   Trizol.     Target   mRNA   expression   is   presented  as  2-­ΔΔCt   relative   to   the  geometric  mean  of  ActB   and  Gapdh.     qPCR       106   analysis  indicated  that  there  was  no  significant  effect  of  acute  exercise  on  GluR1,   NR2A,  or  NR2B  mRNA  expression.    Error  bars  represent  SEM.       Figure   10.   High-­intensity   exercise   increases   transcript-­specific   Bdnf   expression.     Mice   were   exposed   to   30   minutes   (after   6   minute   warm   up)   of   moderate-­intensity   (12m/min;;   n=12),   high-­intensity   (15-­18   m/min;;   n=12),   or   no   exercise   treadmill   (n=12).     Mice   were   sacrificed   and   hippocampi   immediately   isolated.    mRNA  was   isolated   from  whole   hippocampal   homogenates   in   Trizol.     Target  mRNA  expression  is  presented  as  2-­ΔΔCt  relative  to  the  geometric  mean  of   ActB  and  Gapdh.    A)  A  significant  effect  of  exercise  on  Bdnf  IV  mRNA  expression   was   observed   (F(2,   32)=3.79;;   p=0.03),   after   an   acute   bout   of   high-­intensity   exercise  relative  to  controls  (p=0.03).    B)  We  found  no  effect  of  exercise  on  total   Bdnf  mRNA  expression.    Error  bars  represent  SEM.  *  indicates  p<0.05   Figure   11.  Acute   exercise   does   not   influence   time   spent   with   objects   or   object   location   memory.     Mice   were   exposed   to   30   minutes   (after   6   minute   warm  up)   to  high-­intensity   (15-­17  m/min;;  n=15)  or  no  exercise   treadmill   (n=15)   and   immediately  underwent   the  novel  object  placement   task.    A)  There  was  no   main   effect   of   acute   exercise,   test   phase,   or   interaction   in   the   time   spent   exploring   the   two  objects.    B)  There  was  no  effect  of  acute  exercise  on  %  time   spent  exploring  the  moved  object  relative  to  the  time  spent  exploring  both  objects.     Error  bars  represent  SEM.               Figure   12.  High-­intensity   acute   exercise   reduces   exploratory   behavior   in   the   object   location   task.     Mice   were   exposed   to   30  minutes   (after   6  minute       107   warm  up)   to  high   intensity   (15-­17  m/min;;  n=15)  or  no  exercise   treadmill   (n=15)   and  immediately  underwent  the  novel  object  location  task.    A)  There  was  a  main   effect   of   acute   exercise   on   total   distance   traveled   (F(1,28)=14.06;;   p=0.008)   with   mice  exposed  to  high  intensity  treadmill  running  having  significantly  less  distance   traveled   during   the   familiarization   phase   compared   to   treadmill   controls   (p=0.0003).    B)  There  was  a  main  effect  of  acute  exercise  (F(1,28)=4.553;;  p=0.04)   and  an  interaction  between  acute  exercise  and  test  phase  (F(1,28)=6.938;;  p=0.01)       in   number   of   interactions   with   the   objects.   Mice   exposed   to   high-­intensity   treadmill   running  had   significantly   fewer   interactions   (#  of   interactions)  with   the   objects  during  the  familiarization  phase  relative  to  the  treadmill  controls  (p=0.003).     Error  bars  represent  SEM.    *  indicates  significantly  different  from  control  (p<0.05)               Figure   13.     Open   Field   Task   0-­5   Minutes.   High   intensity   acute   exercise   induces   anxiety-­like   behavior   in   the   open   field   task   during   the   first   five   minutes  of  the  task.    Saline-­injected  and  DSP-­4-­injected  mice  were  exposed  to   30  minutes  (after  6  minute  warm  up)  of  high  intensity  (15-­17  m/min;;  EX-­SAL  n=8,   EX-­DSP4   n=9)   or   no   exercise   treadmill   exposure   (CON-­SAL   n=9,   CON-­DSP4   n=10)  and  immediately  underwent  the  open  field  task.  A)  Mice  exposed  to  high-­ intensity   treadmill   running   had   significantly   less   activity   measured   as   total   distance  traveled  (F(1,32)  =  33.09;;  p<0.0001).    x’s  indicate  mice  with  ³  15  stimulus   grid   touches.     B)   Mice   exposed   to   high-­intensity   treadmill   running   spent   significantly  more  time  self-­grooming  compared  to  no  exercise  treadmill  controls   (F(1,32)  =  35.52;;  p<0.0001).    C)  There  was  no  significant  effect  of  exercise  or  drug   on   number   of   entries   into   the   center   of   the   testing   arena.   D)   There   was   no       108   significant   effect   of   exercise   or   drug   on   time   spent   in   the   center   of   the   testing   arena.  Error   bars   represent  SEM.     *   indicates   significantly   different   from  CON-­ SAL  (p<0.05).    $  indicates  significantly  different  from  CON-­DSP4  (p<0.05)   Figure   14.   Open   Field   Task   5-­10   Minutes.   High-­intensity   acute   exercise   induces  anxiety-­like  behavior  in  the  open  field  task  during  the  second  five-­ minute  block  (5-­10  min)  of  the  task.    Saline-­injected  and  DSP-­4-­injected  mice   were   exposed   to   30  minutes   (after   6  minute  warm   up)   of   high-­intensity   (15-­17   m/min;;   EX-­SAL   n=8,   EX-­DSP4   n=9)   or   no   exercise   treadmill   exposure   (CON-­ SAL  n=9,  CON-­DSP4  n=10)  and   immediately  underwent   the  open  field   task.  A)   Mice   exposed   to   high-­intensity   treadmill   running   had   significantly   less   activity   measured  as  total  distance  traveled  (F(1,32)  =13.32;;  p=0.0009).    x’s  indicate  mice   with   ³   15   stimulus   grid   touches.     B)   Mice   exposed   to   high-­intensity   treadmill   running   spent   significantly   more   time   self-­grooming   compared   to   no-­exercise   treadmill  controls  (F(1,31)  =  28.31;;  p<0.0001).    C)  There  was  no  significant  effect   of  exercise  or  drug  on  number  of  entries  into  the  center  of  the  testing  arena.  D)   There  was  no  significant  effect  of  exercise  or  drug  on  time  spent  in  the  center  of   the   testing   arena.   Error   bars   represent   SEM.     *   indicates   significantly   different   from   CON-­SAL   (p<0.05).     $   indicates   significantly   different   from   CON-­DSP4   (p<0.05)   Figure   15.  Open   Field   Task   10-­15   Minutes.  High-­intensity   acute   exercise   induces   anxiety-­like   behavior   in   the   open   field   task   during   the   third   five-­ minute  block  (10-­15  min)  of  the  task.    Saline-­injected  and  DSP-­4-­injected  mice       109   were   exposed   to   30  minutes   (after   6  minute  warm   up)   of   high-­intensity   (15-­17   m/min;;   EX-­SAL   n=8,   EX-­DSP4   n=9)   or   no   exercise   treadmill   exposure   (CON-­ SAL  n=9,  CON-­DSP4  n=10)  and   immediately  underwent   the  open  field   task.  A)   Mice   exposed   to   high-­intensity   treadmill   running   had   significantly   less   activity   measured  as  total  distance  traveled  (F(1,32)  =  10.55;;  p=0.003).    Mice  injected  with   DSP-­4  had  significantly  less  activity  measured  as  total  distance  traveled  (F(1,32)  =   5.091;;   p=0.03).     x’s   indicate   mice   with   ³   15   stimulus   grid   touches.     B)   Mice   exposed   to   high-­intensity   treadmill   running   spent   significantly   more   time   self-­ grooming  compared  to  no-­exercise  treadmill  controls  (F(1,31)  =  14.95;;  p=0.0005).     C)  Mice  exposed  to  high-­intensity  treadmill  running  had  significantly  fewer  entries   into   the   center   of   the   testing   arena   compared   to   no-­exercise   treadmill   controls   (F(1,32)   =   6.869;;   p=0.01).     D)   Mice   exposed   to   high-­intensity   treadmill   running   spent   significantly   less   time   in   the  center  of   the   testing  arena  compared   to  no-­ exercise  treadmill  controls  (F(1,32)  =  5.393;;  p=0.03).    Error  bars  represent  SEM.    *   indicates  significantly  different  from  CON-­SAL  (p<0.05).    $   indicates  significantly   different  from  CON-­DSP4  (p<0.05)             110   Figure  7.                   Saline Epinephrine Saline Epinephrine Saline Epinephrine 0.0 0.5 1.0 1.5 Ser845/GluR1 Ser831/GluR1 GluR1/NeuN * O .D . % C on tro l A   B   Epinephrine  Saline       111   Figure  8.                 112             113   Figure  9.       Control Moderate High 0.5 1.0 1.5 R el at iv e m R N A e xp re ss io n (2 -Δ ΔC t ) GluR1 Control Moderate High 0.5 1.0 1.5 R el at iv e m R N A e xp re ss io n (2 -Δ ΔC t ) NR2A Control Moderate High 0.5 1.0 1.5 R el at iv e m R N A e xp re ss io n (2 -Δ ΔC t ) NR2B A B C     114   Figure  10.       Control Moderate High 0.5 1.0 1.5 R el at iv e m R N A e xp re ss io n (2 -Δ ΔC t ) Bdnf IV * A B Control Moderate High 0.5 1.0 1.5 R el at iv e m R N A e xp re ss io n (2 -Δ ΔC t ) Bdnf     115   Figure  11.       0 10 20 30 40 50 Se co nd s Total Time Spent Exploring Either Object Control Exercise A Familiarization Test Control Exercise 0 20 40 60 80 100 Pe rc en t Discrimination Ratio (Time Spent with New Object/Total Time Spent with Both Objects) B     116   Figure  12.           0 1000 2000 3000 cm Total Distance Moved Control Exercise * Familiarization Test A 0 10 20 30 40 50 # of In te ra ct io ns Number of Interactions With Objects * Familiarization Test B     117   Figure  13         Activity (0-5 min) Saline DSP4 Saline DSP4 0 1000 2000 3000 D is ta nc e (c m ) Control Exercise * * Saline DSP4 Saline DSP4 0 20 40 60 80 100 120 140 Se co nd s Grooming (0-5 min) Control Exercise * * $ A B     118         Saline DSP4 Saline DSP4 0 10 20 30 # of E nt rie s Frequency in Center (0-5 min) Control Exercise Saline DSP4 Saline DSP4 0 20 40 60 % o f T ot al T im e Time in Center (0-5 min) Control Exercise C D     119   Figure  14.         Activity (5-10 min) Saline DSP4 Saline DSP4 0 500 1000 1500 2000 2500 D is ta nc e (c m ) * * ControlExercise Saline DSP4 Saline DSP4 0 20 40 60 80 100 120 140 Se co nd s Grooming (5-10 min) Control Exercise* $p=0.055 A B     120         Saline DSP4 Saline DSP4 0 5 10 15 20 25 # of E nt rie s Frequency in Center (5-10 min) Control Exercise Saline DSP4 Saline DSP4 0 10 20 30 40 % o f T ot al T im e Time in Center (5-10 min) Control Exercise C D     121   Figure  15.         Activity (10-15) Saline DSP4 Saline DSP4 0 1000 2000 3000 D is ta nc e (c m ) Control Exercise* ** Saline DSP4 Saline DSP4 0 20 40 60 80 100 120 140 Se co nd s Grooming (10-15 min) Control Exercise * $ A B     122         Saline DSP4 Saline DSP4 0 5 10 15 20 25 # of E nt rie s Control Exercise * Frequency in Center (10-15 min) Saline DSP4 Saline DSP4 0 10 20 30 40 % o f T ot al T im e Time in Center (10-15 min) Control Exercise* C D     123   Discussion   We  found  that  a  single  30-­minute  bout  of  high  intensity  treadmill  exercise   was   sufficient   to   increase   mRNA   expression   of   Bdnf   transcript   IV;;   however,   unlike  acute  psychological  stress  (Hu  et  al.,  2007)  or  peripheral  catecholamines,   one  30-­minute  bout  of  acute  exercise  did  not  influence  the  phosphorylation  status   of   the  GluR1  subunit   of   the  AMPAR.    To  determine   if   acute  exercise   improves   memory   performance   in   mice,   an   effect   observed   in   humans   (Lambourne   &   Tomporowski,  2010;;  Roig  et  al.,  2013),  we  exposed  mice   to  a   low  stress,  one-­ trial  memory  task  immediately  following  the  acute  bout  of  exercise.    We  observed   that  mice   exposed   to   the   acute   bout   of   exercise   spent   less   time   exploring   the   environment  and   interacting  with  the  objects  to  be  remembered.    Therefore,  we   tested   if  mice   exposed   to   acute   exercise   displayed   anxiety-­like   behavior   in   the   open  field  task  and  if  the  behavior  associated  with  acute  exercise  was  influenced   by   central   noradrenergic   signaling.    We   found   that   acute   exercise   significantly   reduced   locomotor  activity  and  significantly   increased   time  spent  self-­grooming,   common   indicators  of  an  anxious  phenotype.    Moreover,  our  data  suggest   that   LC-­noradrenergic  signaling  may  influence  behavior  following  treadmill  exposure.   We   were   able   to   replicate   previous   findings   that   an   IP   injection   of   epinephrine  increases  phosphorylation  of  Ser845  of  GluR1  but  did  not  influence   phosphorylation  of  Ser831   (Hu  et   al.,   2007).    We  hypothesized   that,   like  acute   psychological   stress   and   peripheral   injections   of   epinephrine   (Hu  et   al.,   2007),   acute   forced   treadmill   exercise   would   increase   phosphorylation   of   GluR1   at       124   Ser845   in   the   hippocampus,   potentially   via   the   release   of   catecholamines   and   central   noradrenergic   signaling.   However,   30   minutes   of   high-­   or   moderate-­ intensity  acute  exercise  was  insufficient  to  induce  Ser845  phosphorylation.    While   there   is   evidence   of   short-­term   physical   activity   increasing   phosphorylation   of   Ser845  in  the  rodent  cortex  (Mizutani  et  al.,  2015),  this  has  not  been  investigated   following  a  single  bout  of  acute  exercise.    Our  data  suggest   that  a  single  acute   bout  of  exercise  does  not  induce  phosphorylation  of  Ser845  on  GluR1.   Potentially,  we  did  not  observe  an  effect  of   the  acute  exercise  on  GluR1   phosphorylation   because   30   minutes   of   exercise   was   not   sufficient   to   elevate   peripheral   epinephrine   or   central   norepinephrine.     Pagliari   and   Peyrin   (1995a)   found   that   cortical   norepinephrine   did   not   increase   in   response   to   treadmill   running  until  after  ~40  minutes  of  exercise   in   the  rat  while  Goekint  et  al.   (2012)   observed   no   influence   of   60   minutes   of   treadmill   running   on   extracellular   hippocampal   norepinephrine.     Interestingly,   Goekint   et   al.   (2012)   observed   a   significant  effect  of  exercise  on  dopamine  release,  another  neuromodulator   that   can  increase  GluR1  Ser845  phosphorylation  via  PKA  signaling  (Carr  et  al.,  2010).   In  contrast  to  these  experiments,  Dishman  et  al.  (2000)  reported  that  15  minutes   of   treadmill   running  or   immobilization  stress  decreased  norepinephrine   levels   in   the   LC   and   hippocampus,   likely   through   release   and   metabolism   of   the   neurotransmitter.  Potentially,  longer  more  exhausting  exercise  would  result  in  an   increase  in  GluR1  phosphorylation.    Alternatively,  it  is  possible  that  30  minutes  of   exercise  was  sufficient   to  elevate  central  norepinephrine,   though  sacrificing   the   mice   immediately   after   the   bout   of   exercise   did   not   provide   enough   time   for       125   intracellular   signaling   necessary   for   phosphorylation   of   GluR1.     In   our   epinephrine   injection   experiment,   we   waited   15   minutes   post-­injection   before   sacrifice,  similar  to  the  procedure  of  Hu  et  al.  (2007).    It  is  possible  that  we  would   have  observed  an  effect   if  we  waited   longer  between   the  cessation  of  exercise   and  sacrifice  of  the  animals.    Exposure  to  the  treadmill  environment  alone  may  have  been  sufficient  to   elevate   Ser845   phosphorylation,   which  might   have  masked   the   effect   of   acute   exercise.    The  three  days  of  acclimation  to  the  treadmill  environment  and  the  36-­ minute  exposure   to   the   treadmill  even   in   the  stationary  controls   likely  produced   novelty-­induced  arousal  which  may  have  activated  the  noradrenergic  system  and   stimulated  hippocampal  b2  adrenergic  receptors  (King  &  Williams,  2009).    Indeed,   novelty   exploration   increases   neuronal   activity   in   the   LC   and   release   of   norepinephrine   in   the  hippocampus   (Sara  et  al.,  1994).    Without  a  cage-­control   group   that   did   not   undergo   the   acclimation   or   experimental   day   treadmill   exposure,   we   are   unable   to   determine   if   treadmill   exposure   alone   increased   phosphorylation  of  the  GluR1  subunit  of  the  AMPA  receptor.       In   addition   to   Ser845,   it   is   curious   that   we   did   not   observe   an   effect   of   exercise   on   Ser831   phosphorylation.     Though   we   hypothesized   that   elevated   noradrenergic  signaling  due  to  the  intense  forced  exercise  would  induce  Ser845   phosphorylation,   Ser831   is   phosphorylated   by   CAMKII   (Barria   et   al.,   1997),   a   kinase  that  has  been  linked  to  exercise-­induced  hippocampal  plasticity  (Voss  et   al.,  2013).    Once  again,   it   is  possible  that  exposure  to  the  novel  environment  of       126   the   treadmill   even   in   controls   was   sufficient   to   elevate   CAMKII   activity   and   subsequent   phosphorylation   of   Ser831,   occluding   any   additional   benefit   of   the   exercise  exposure.         There   was   no   effect   of   acute   exercise   on   mRNA   expression   of  GluR1,   NR2A,  or  NR2B.    It  is  likely  that  the  short  time  between  the  start  of  exercise  and   sacrifice  was  insufficient  for  activity-­induced  transcription  of  glutamate  receptors.     The   literature   reporting   the   effects   of   exercise   training   on   glutamate   receptor   expression  is  inconsistent.    Previous  research  has  reported  an  increase  in  NR2B   mRNA   expression   following   short-­term   exposure   to   a   voluntary   running   wheel   (Molteni  et  al.,  2002;;  Farmer  et  al.,  2004).    Higher  expression  of  NR2B  relative  to   NR2A   is  associated  with  a  more  plastic  synapse  (Tang  et  al.,  1999;;  Cao  et  al.,   2007),   making   this   a   potential   mechanism   for   exercise-­induced   plasticity   and   reduced  threshold  for  LTP  and  learning.  Molteni  et  al.  (2002)  reported  that  three   days   of   voluntary   running,   a   protocol   generally   considered   acute   exercise,   increased  both  NR2B,  NR2A,  and  to  a  much  lesser  extent,  GluR1  expression  in   the   rat   hippocampus.    NR2A   remained   significantly   different   than   controls   after   seven  days  of  wheel   running  but  was  no   longer  significantly  different  after  one-­ month  of  exposure.    Ni  et  al.  (2009)  found  that  GluR1  mRNA  expression  was  not   influenced  by  six  days  of  daily  treadmill  running  in  healthy  Sprague-­Dawley  rats.   Our  data  demonstrate  that  an  acute  bout  of  treadmill  exercise  does  not  stimulate   rapid  transcription  of  glutamate  receptor  subunits  in  the  mouse  hippocampus  and   multi-­day   exposures   or   voluntary  wheel   running  may   be   necessary   to   increase   mRNA  expression  of  these  subunits.       127   Brain-­Derived   Neurotrophic   Factor.    We   observed   a   significant   effect   of   acute   exercise  and  acute  exercise  intensity  on  Bdnf  IV  mRNA  expression  but  observed   no   effect   on   total   Bdnf.     Numerous   studies   have   demonstrated   that   exercise   increases   protein   and   mRNA   expression   of   Bdnf   in   the   rodent   hippocampus   (Neeper  et  al.,  1996;;  Molteni  et  al.,  2002;;  Vaynman  et  al.,  2003;;  2004;;  Berchtold   et  al.,  2005;;  Ding  et  al.,  2011;;  Sartori  et  al.,  2011;;  Venezia  et  al.,  2016).    To  our   knowledge,  this  is  the  first  investigation  to  report  that  one  acute  bout  of  exercise   can   increase  expression  of  Bdnf  IV.    The  effect  was  only  observed  in  mice  that   were   exposed   to   high-­intensity   treadmill   running,   which   likely   caused   high   neuronal   activity   in   the   hippocampus   that   resulted   in   rapid   transcription   of   the   activity-­dependent   Bdnf   transcript   IV.     Bdnf   is   important   for   hippocampal   neurogenesis  (Lee  et  al.,  2002;;  Sairanen,  2005;;  Scharfman  et  al.,  2005;;  Rossi  et   al.,  2006;;  Taliaz  et  al.,  2009),  synaptic  plasticity  (Korte  et  al.,  1995;;  Figurov  et  al.,   1996;;   Korte   et   al.,   1996;;   Patterson   et   al.,   1996;;   Kang   et   al.,   1997;;   Ma   et   al.,   1998;;  Chen  et  al.,  1999;;  Zakharenko  et  al.,  2003),  and  memory  (Linnarsson  et  al.,   1997;;  Ma  et  al.,  1998;;  Mu  et  al.,  1999;;  Mizuno  et  al.,  2000;;  Alonso  et  al.,  2002;;   Heldt   et   al.,   2007;;   Bekinschtein   et   al.,   2008),   all   of   which   are   associated   with   exercise  training  (Cotman  et  al.,  2007;;  Vivar  et  al.,  2012;;  Voss  et  al.,  2013).     In   fact,  blocking  Bdnf  action  with  an  antibody  that  binds  to  its  receptor  prevents  the   exercise-­induced   improvement   in   spatial   memory   and   expression   of   plasticity-­ associated   genes   (Vaynman   et   al.,   2004).   The   finding   that   an   acute   bout   of   exercise   of   only   30   minutes   influenced   transcription   of   the   Bdnf   gene   is       128   interesting  and  suggests   that   the  process  of  hippocampal  plasticity  begins  with   short  exposures  to  exercise,  albeit  forced  and  highly  stressful  in  our  investigation.   Acute   exercise   caused   an   increase   in   Bdnf   IV   but   not   total   Bdnf   expression,  suggesting  that  Bdnf  IV  transcription  is  rapidly  initiated  in  response  to   exercise,   similar   to   an   immediate   early   gene,   while   the   other  Bdnf   transcripts   have   a   slower   pattern   of   transcription.    Bdnf   IV   is   highly   sensitive   to   neuronal   activity   (Tao   et   al.,   1998;;   2002;;   Martinowich   et   al.,   2003)   and   hippocampal   expression  increases  in  response  acute  immobilization  stress  (Marmigère  et  al.,   2003),   fear  conditioning   (Lubin  et  al.,  2008),  and  exercise   training   (Zajac  et  al.,   2009;;   Intlekofer  et  al.,   2013).     It   is   important   to  note   that   our  exercise  protocol   was  highly  stressful  as   it  was  unpredictable  and  uncontrollable  making  it  similar   in   certain   ways   to   both   fear   conditioning   and   immobilization   stress.     In   a   fear   conditioning   task,   context   exposure   alone   increased   total   Bdnf   expression   through  increased  expression  of  Bdnf  I  and  VI,  though  following  associative  fear   conditioning,  the  increase  in  total  Bdnf  transcription  was  mediated  by  an  increase   in  Bdnf  IV  transcription  (Lubin  et  al.,  2008).    Marmigere  et  al.  (2003)  reported  that   15   and   60  minutes   of   acute   immobilization   stress   increased  Bdnf   IV   (III   in   the   paper)   in   the   rat   hippocampus,   demonstrating   a   rapid   transcription   of  Bdnf   IV,   which  was  more  rapid  than  total  Bdnf  mRNA  and  other  Bdnf  transcripts  that  were   not   influenced   by   immobilization   stress   until   60   minutes.     This   presents   a   possible  explanation  for  why  we  observed  significantly  increased  Bdnf  IV  mRNA   after  30  minutes  but  no  change  in  total  Bdnf  at  this  time  point.           129   Though   forced   treadmill   exercise   is   stressful   because   it   is   unpredictable   and   uncontrollable,   both  moderate-­intensity   and   high-­intensity   treadmill   running   were   similarly   uncontrollable   and   unpredictable   and   we   only   observed   a   difference   in  Bdnf   IV  expression   in   the  high-­intensity  group.    This   indicates   that   signaling   processes   unique   to   the   high-­intensity   exercise   are   stimulating   transcription   of   Bdnf   IV.     Concerning   the   influence   of   acute   exercise   on   Bdnf   expression,   research  has  been   inconsistent   in  both   the   research  approach  and   reported   findings.   Oliff   et   al.   (1998)   reported   that   six   hours   of   voluntary   wheel   running  increased  Bdnf  mRNA  in  the  rat  hippocampus  (hilus,  CA1,  and  CA3)  but   had  no  effect  on  Bdnf  IV  expression.    Importantly,  these  investigators  acclimated   mice  to  the  running  wheel  for  three  nights  followed  by  a  10-­day  washout  period.   Though  they  observed  no  difference  in  Bdnf  IV  mRNA  expression  after  six  or  12   hours  of  voluntary  wheel  running,  they  found  that  mice  that  underwent  the  three   days   of   wheel   acclimation   but   no   acute   wheel   exposure   10   days   later   had   significantly   elevated  Bdnf   IV   in   all   hippocampal   regions  examined  and  greater   total  Bdnf  in  CA1.    This  is  interesting  and  demonstrates  the  lasting  influence  that   acclimation  protocols  can  have  on  hippocampal  Bdnf  expression.  Rasmussen  et   al.  (2009)  reported  significantly  greater  Bdnf  mRNA  in  the  hippocampus  two  and   six  hours  post-­treadmill  running  to  exhaustion  but  not  immediately  after  exercise.     Similar   to   Oliff   et   al.   (1998),   Rasmussen   et   al.   (2009)   used   an   acclimation   protocol  that  included  running  on  the  treadmill  for  multiple  days  before  the  acute   treadmill   running.    These  current  data  and  previously  published   research  show   that  Bdnf  IV  transcription  is  sensitive  to  acute  exercise  and  is  rapidly  transcribed       130   upon  exposure.    Potentially,   longer  acute  exercise  exposures  are  necessary   to   significantly  increase  total  Bdnf  expression.     The  novel  object   location   task   is  a  hippocampal-­dependent  memory   task   (Barker   &   Warburton,   2011).     We   hypothesized   that   an   acute   bout   of   high-­ intensity  exercise  would   lower   the   threshold   for  memory   formation  and   improve   memory  performance  as  has  been  shown  with  epinephrine  injections  (Hu  et  al.,   2007)  and  three  weeks  of  voluntary  wheel  running  (Intlekofer  et  al.,  2013).  In  the   current   investigation,   mice   exposed   to   high-­intensity   acute   exercise   showed   significantly   less  exploratory  behavior   (frequency  of   interaction  with  objects  and   significantly   less   distance   moved)   during   the   familiarization   phase   of   the   task   compared  to  treadmill  controls.    Therefore,   the  mice  were  not  actively  exploring   the   novel   objects   that   were   to   be   remembered.     This   behavior   suggested   an   anxious   phenotype   in   the   mice   exposed   to   treadmill   running   (Crawley,   1985).     The   relationship   between   exercise   and   anxiety   in   rodents   is   complex   and   the   literature  supports  both  anxiolytic  and  anxiogenic  effects  of  exercise  (for  review,   see  Sciolino  &  Holmes,  2012).    Potentially,  elevated  stress  hormones  induced  by   acute   exercise,   which   we   thought   would   improve   memory,   contributed   to   the   anxiety-­like   behavior   but   returned   to   baseline   by   the   test   phase,   when   we   no   longer  observed  reduced  exploratory  behavior.    Another  potential  explanation  is   that  the  mice  were  simply  fatigued  following  the  bout  of  treadmill  exercise.    This   is  unlikely  because   the   treadmill  exercise  was  not  exhaustive,   though  even   low   levels  of  fatigue  could  have  contributed  to  the  observed  behavior.  The  behavioral   profile   observed   in   the   object   location   task   following   acute   exercise   was       131   consistent  with  observations  in  the  open  field  task,  where  exercised  mice  showed   significantly   less   activity   during   the   task   and   spent   significantly  more   time   self-­ grooming.    We  also  observed  reduced  entries  and  total  time  spent  in  the  center   of   the   testing   arena   in   exercised   mice   during   the   final   five   minute   time   block.     This   is   a   hallmark  measure   of   anxiety   behavior   in   the   open   field   task   (Prut   &   Belzung,  2003).    Self-­grooming  is  a  complex  behavior  that  can  be  interpreted  as   a   marker   of   anxiety   since   stressful   situations   and   high   levels   of   anxiety   can   increase  grooming  behavior  (for  review,  see  Kalueff  et  al.,  2015).  Moreover,  this   behavior  can  be  reduced  with  the  use  of  benzodiazepines  (Kalueff  et  al.,  2015).     However,  grooming  behavior  is  mediated  by  many  brain  regions/circuits  and  can   be  influenced  by  numerous  pharmacological  manipulations  (Kalueff  et  al.,  2015),   so   interpretation   of   this   behavior   following   forced   exercise   is   difficult.     It   could   represent  reduced  vigilance  and  more  internally  directed  behavior  (Sothmann  et   al.,   1996).     Taken   together,   these   data   suggest   that   acute   exercise   stimulates   behaviors   that   may   be   interpreted   as   anxiogenic;;   however,   more   research   is   needed  to  determine   if   this   is   truly  anxiety-­like  behavior  or  an  effect  of  exercise   on  motivation  or  fatigue.   The  majority  of  published  research  on  exercise  and  anxiety  has  reported   anxiolytic  effects  of  exercise  (Sciolino  &  Holmes,  2012);;  however,  a  few  studies   have  reported  anxiogenic  effects  (Fuss  et  al.,  2009;;  2010;;  Onksen  et  al.,  2012).     The   investigations   of   exercise   and   anxiety   have   focused   on   chronic   voluntary   wheel  running  and  the  anxiogenic  mechanism  identified,  increased  neurogenesis,   is   unlikely   to   explain   our   findings   since   one   30-­minute   bout   of   exercise   is   not       132   sufficient   to   generate   new   functioning   neurons   (Van   der   Borght   et   al.,   2009).   Salam  et  al.   (2009)  provided  C57Bl/6J  mice  with  access   to  a  voluntary   running   wheel  for  two  weeks  prior  to  exposure  to  the  open  field  task  and  found  conflicting   results.    They  reported   that   running-­exposed  mice  spent  significantly  more   time   in   the   center   of   the   testing   box   and   entered   the   center   more   frequently   than   sedentary  mice.    These  behaviors  are   indicative  of   less  anxiety;;  however,   they   also  observed  significantly   less  activity  and  more  grooming  behavior   in  runners,   which   is   indicative   of   higher   levels   of   anxiety,   similar   to   what   we   observed.   Interestingly,  Duman  et  al.   (2008)   reported   that   three  weeks  of  voluntary  wheel   running  in  C57Bl/6J  mice  increased  anxiety-­like  behavior  (reduced  activity)  in  the   open   field   task   if   the   task   was   initiated   the   morning   after   a   night   of   voluntary   wheel  running.    In  contrast,  they  observed  anxiolytic-­like  behavior  if  the  task  was   initiated  24  hours  after  the  last  exposure  to  the  voluntary  running  wheel.    These   data  suggest  a  transient  anxiogenic  effect  of  exercise.         Potentially,   our   observed   behavior   resulted   from   elevated   release   of   adrenal  stress  hormones  and  central  noradrenergic  signaling,  which  we  predicted   would  improve  learning  and  memory.    The  reduced  locomotor  behavior  observed   in   the  object   location  and  open  field   tasks   is  similar   to   the  home  cage  behavior   we  observed  following  an   IP   injection  of  epinephrine  (unpublished  observation).     Administration   of   selective   norepinephrine   reuptake   inhibitors   used   as   antidepressants   (e.g.   reboxetine)   have   been   shown   to   be   initially   anxiogenic   (Inoue   et   al.,   2006),   though   become   anxiolytic   after   chronic   administration   by   reducing   stress-­induced   cortical   norepinephrine   release   (Dazzi   et   al.,   2003).         133   Potentially,   acute   and   chronic   exercise   function   similarly   to   acute   and   chronic   treatment   with   selective   norepinephrine   reuptake   inhibitors   by   increasing   extrasynaptic  norepinephrine,  which  is  acutely  anxiogenic  but  becomes  anxiolytic   with  chronic  exposure.     Indeed   the  role  of  norepinephrine   in  anxiety   is  complex   and  is  associated  with  both  anxiolytic  and  anxiogenic  behavior  depending  on  the   type   of   acute   stress   stimulating   the   norepinephrine   release   (for   review,   see   Goddard   et   al.,   2010).     We   hypothesized   that   the   anxiogenic-­like   behavior   observed   following   acute   exercise  would   be   attenuated  with   pre-­treatment  with   the  selective  neurotoxin  for  the  LC-­  noradrenergic  system,  DSP-­4.    The  influence   of   DSP-­4   on   behavior   in   the   open   field   task   was   small.   In   contrast   to   our   hypothesis,  mice  exposed  to  exercise  and   injected  with  DSP-­4  had  significantly   reduced  activity  compared  to  control  mice  injected  with  saline;;  however,  they  did   not   have   significantly   reduced   activity   compared   to   control   mice   injected   with   DSP-­4  at  any  time  point.    This  suggests  that  DSP-­4  attenuates  the  effect  of  acute   exercise   on   exploratory   behavior   but   does   not   rescue   the   effect   of   exercise   completely.     Further,  mice   injected  with  DSP-­4   and   exposed   to   exercise   spent   significantly   more   time   self-­grooming   compared   to   both   saline   and   DSP-­4   injected   treadmill   control  mice,  and  DSP-­4  did  not  attenuate   the  effect  of  acute   exercise  on   frequency  of  entries  or   time  spent   in   the  center  of   the  arena   in   the   last   five   minutes   of   the   open   field   task.     These   data   indicate   that   DSP-­4   was   ineffective  at  preventing  the  anxiety-­like  effect  of  acute  forced  treadmill  exercise,   though  it  did  attenuate  the  effect  of  acute  exercise  on  activity  during  the  task.       134   DSP-­4   treatment   has   been   shown   to   influence   open   field   behavior   by   reducing  overall  activity  but  this  can  be  attenuated  with  chronic  mild  stress  (Harro   et  al.,  1999).    We  did  not  observe  a  significant  reduction  in  activity  in  animals  that   received  DSP-­4   alone,   potentially   due   to   the  mild   stressful   nature   of   the   novel   treadmill   environment.   An   inverted-­U   effect   of   LC-­derived   norepinephrine   may   exist,   with   both   low   and   high   levels   resulting   in   reduced   exploratory   activity.   Potentially   other   stress   hormones   (e.g.   corticosterone)   and   amygdalar   activity,   independent  of  LC  innervation,  were  sufficient  to  cause  the  behavioral  profile  that   we  observed.    Moreover,  high   levels  of  exogenous  catecholamines  can  bypass   the   LC   to   exert   their   behavioral   effects.     Bennett   et   al.   (1990)   reported   that     peripheral   injections   of   epinephrine   following   DSP-­4   treatment   can   attenuate   impairments   in   active   avoidance   induced   by   DSP-­4.     This   indicates   that   high   levels   of   peripheral   catecholamines   can   bypass   the   LC   noradrenergic   system,   and  our  exercise  protocol  may  have  been  psychologically  and  physically  stressful   enough  to  accomplish  this.     The   major   limitation   of   this   investigation   was   the   exclusion   of   a   no-­ treadmill  control  group.    We  were  primarily   interested  in  the  influence  of   forced-­   exercise,  not  the  influence  of  the  novel  treadmill  environment.    We  hypothesized   that  the  novel  environment  alone  may  induce  plasticity  and  designed  our  study  to   observe   the  effects  of   the   treadmill  exercise  beyond   the  effects  of  novelty.    We   did  not  envision  an  occlusion  of  plasticity  markers  by   the  novelty;;  however,  not   having  a  home-­cage  control  prevents  us  from  being  able  to  determine  if  our  lack   of  observed  effect  on  GluR1  phosphorylation  and  total  Bdnf  was  due  to  the  novel       135   environment   masking   the   effects   of   acute   exercise.   Another   limitation   to   this   investigation   is   the   absence   of   a   clear   understanding   of   the   tissue   and   extracellular   content   of   norepinephrine   in   response   to   exercise   and   DSP-­4.     There   is   enough   evidence   to   support   that   DSP-­4   reduces   tissue   content   of   norepinephrine  (Ross,  1976;;  Ögren  et  al.,  1980;;  Jonsson  et  al.,  1981;;  Archer  et   al.,   1982;;   Anisman   et   al.,   1984;;   Zahniser   et   al.,   1986;;   Bennett   et   al.,   1990;;   Scullion   et   al.,   2009;;   Szot   et   al.,   2010),   though   the   possibility   of   increased   extracellular   content   of   norepinephrine   (Ross   &   Stenfors,   2014)   following   treatment  allows  for  uncertainty.       Summary:    The  data  presented  here  indicate  that  a  single  acute  bout  of  exercise   does  not  influence  GluR1  phosphorylation  but  does  stimulate  the  transcription  of   the  important  plasticity-­promoting  gene,  Bdnf,   in  a  transcript-­dependent  manner.     Further,   our   data   suggest   that   following   acute   exercise,   locomotor   and   exploratory   behavior   are   reduced   and   associated   with   an   increase   in   self-­ grooming  behavior.    Importantly,  these  data  suggest  that  tasks  with  low  intrinsic   motivation   and   dependent   on   locomotor   and   exploratory   behavior   should   be   avoided  when  testing  for  memory  or  anxiety  following  acute  exercise  exposures.     Exercise  does  not  increase  anxiety  or  incidents  of  anxiety  attacks  in  humans  and   generally   appears   to  be  anxiolytic   in   rodents   (O'connor  et  al.,   2000;;  Sciolino  &   Holmes,  2012;;  Ensari  et  al.,  2015),  so  we  are  hesitant  to  conclude  that  the  acute   bout  of  exercise   is  actually  anxiogenic.     Instead,   it   results   in  behaviors   that  can   be   interpreted  as  an  anxious  phenotype.    Careful  consideration  should  be  used       136   when   selecting   the   appropriate   behavioral   task   following   acute   exercise   exposures.         137   Chapter  5.     Aim  #4:  Determine  if  chronic  exercise  influences  the  effect  of  acute  exercise  on   GluR1   protein   phosphorylation   and   mRNA   expression   of   plasticity-­associated   genes.   Aim   #5:     Determine   the   influence   of   acute   exercise   and   locus   coeruleus   noradrenergic  signaling  on  specific  Bdnf  transcript  expression.       Title:     Bdnf   Transcription   is   Differentially   Regulated   by   DSP-­4,   Acute   Forced   Treadmill  Exercise,  and  Voluntary  Wheel  Running   Authors:  Andrew  C.  Venezia1,2,  Elizabeth  Quinlan2,  Stephen  M.  Roth1,2   Department   of   Kinesiology,   School   of   Public   Health,   University   of   Maryland,   College  Park,  MD1   Neuroscience  and  Cognitive  Science  Program,  University   of  Maryland,  College   Park,  MD2           138   Introduction   A  history  of  physical  activity  or  exercise  training  can  alter  the  human  and   animal   response   to   an   acute   bout   of   psychological   or   physical   stress.   This   modulation  of  the  response  to  acute  physical  stress  by  previous  physical  activity   or  exercise   training  has   implications   for   the  application  of  acute  exercise  as  an   effective   intervention   to   improve   memory   and   mental   health.     Brain-­derived   neurotrophic   factor   (BDNF),   a   neurotrophin   critical   for   exercise-­induced   brain   plasticity  (Vaynman  et  al.,  2004),   is   influenced  by  previous  physical  activity  and   fitness  (Knaepen  et  al.,  2010).    In  humans,  acute  exercise-­induced  elevations  of   peripheral   BDNF   may   be   reduced   after   exercise   training   (Griffin   et   al.,   2011;;   Wagner  et  al.,  2015),  while  research  in  rodents  suggests  that  short-­term  exercise   training   increases  hippocampal  Bdnf  expression  to  a  greater  magnitude   in  mice   previously  exposed  to  physical  activity  (Berchtold  et  al.,  2005).  In  addition  to  Bdnf,   catecholamines   [epinephrine   (adrenaline)   and   norepinephrine   (noradrenaline)]   are   neuromodulators   important   for   mental   health   and   memory   performance   (McGaugh   &   Roozendaal,   2002).     Acute   elevations   in   catecholamines   are   associated   with   improved   memory   performance   and   enhanced   plasticity   (McGaugh,   2013;;   O'Dell   et   al.,   2015),   while   loss   of   normal   noradrenergic   signaling   is  associated  with  age-­related  memory  disorders  such  as  Alzheimer’s   disease   (Szot   et   al.,   2006)     Moreover,   normal   catecholamine   signaling   is   necessary  for  exercise-­induced  elevations  in  Bdnf  (Garcia  et  al.,  2003;;  Ivy  et  al.,   2003).     In   response   to   acute   exercise   of   sufficient   intensity   and   duration,   catecholamine   levels   increase   to   mediate   cardiovascular   and   metabolic       139   adaptations   to   physical   stress   (Tipton,   2006).     Importantly,   catecholamine   responses  to  acute  exercise  are  influenced  by  exercise  training  and  fitness  (Kjaer,   1998;;   Zouhal   et   al.,   2008).   In   trained   humans,   circulating   catecholamines   are   lower   at   a   given   absolute   exercise   intensity   but   higher   at   the   same   relative   or   maximal   exercise   intensity   compared   to   untrained   individuals   (Dishman   &   Jackson,   2000;;   Zouhal   et   al.,   2008).     Similarly,   rodents   exposed   to   exercise   training   have   a   greater   capacity   for   catecholamine   release   as   indicated   by   increased  adrenal  weight  and  adrenaline  content  (Zouhal  et  al.,  2008),  but  may   have   a   reduced   catecholamine   response   to   acute   physical   or   psychological   stress.     Indeed,   after   voluntary   wheel   running,   rodents   exposed   to   acute   psychological  or  physical  stress  have  a  lower  stress  response  as  determined  by   reduced   stress-­induced   peripheral   and   central   catecholamine   release/depletion   (Dishman  et  al.,  1997;;  2000;;  Greenwood  et  al.,  2003)  or  hypothalamic-­pituitary-­ adrenal  (HPA)  axis  activation  (Dishman  et  al.,  1998).     We  have  previously  investigated  (Chapter  4)  the  impact  of  acute  exercise   in   lifelong  sedentary  animals  on  expression  of  Bdnf  mRNA  and  phosphorylation   of   the   GluR1   subunit   of   the   AMPA-­type   glutamate   receptor   (AMPAR).     The   importance  of  Bdnf  expression  in  hippocampal  plasticity   is  well  supported  in  the   literature   (for   review,   see  Park   &  Poo,   2013)   and   our   data   demonstrate   that   a   single   30-­minute   acute   bout   of   high-­intensity   forced   exercise   rapidly   increases   expression  of  Bdnf  transcript  IV  in  the  mouse  hippocampus.    Transcription  of  the   Bdnf  gene   is   highly   complex,   producing   up   to   22   possible   transcripts   from   the   nine-­exon  gene.    Of   these,  Bdnf   transcript   IV  appears   to  be  highly   sensitive   to       140   physical  activity  (Gómez-­Pinilla  et  al.,  2010;;  Intlekofer  et  al.,  2013;;  Venezia  et  al.,   2016).       We   hypothesized   that   acute   exercise  would   increase   phosphorylation   of   Ser845   on   the   Glur1   subunit   of   the   AMPAR   due   to   an   elevation   in   peripheral   catecholamines.     Application   of   exogenous   catecholamines   increases   phosphorylation   of   Ser845   (chapter   4)   and   this   is   associated   with   a   reduced   threshold   for   long-­term   potentiation   (LTP)   and   enhanced   learning   (Hu   et   al.,   2007),  making   it   a   desirable   adaptation.     However,   in   contrast   to   our   previous   hypothesis,  we  did  not  observe  a  change  in  Ser845  phosphorylation  in  response   to  acute  exercise  (chapter  4),  potentially  due  to  the  short  duration  of  exercise  and   the   short   time   between   the   start   of   exercise   and   sacrifice   of   the   animal.     Importantly,   all   experiments   in   the   previous   investigation   were   carried   out   in   lifelong   sedentary   animals.    While   this   design   is   optimal   for   understanding   the   impact   of   a   truly   acute   bout   of   exercise,   it   is   not   ideal   for   human   application.     Exercise  should  be  performed   regularly  and  consistently,  and   therefore   if  acute   exercise   is   to   be   used   as   a   therapeutic   or   cognitive   aid,   it   is   necessary   to   understand   the   response   to   acute   exercise   in   the   context   of   regular   voluntary   exercise.   Our   previous   data   suggest   that   an   acute   bout   of   exercise   may   be   effective  at   enhancing  plasticity  and   it   is  essential   to  understand   if   these  same   effects  are  observed  following  exercise  training.       Noradrenergic   signaling   plays   an   essential   role   in   hippocampal   Bdnf   expression  (Garcia  et  al.,  2003;;  Ivy  et  al.,  2003;;  Chen  et  al.,  2007;;  Akhavan  et  al.,       141   2008).    When  noradrenergic  signaling  is  blocked,  the  effect  of  exercise  on  Bdnf   transcription  is  attenuated  (Garcia  et  al.,  2003;;  Ivy  et  al.,  2003)  and  remarkably,   this   extends   to   in   utero   exercise   exposure   (Akhavan   et   al.,   2008).     Russo-­ Neustadt  et  al.  (2004)  showed  that  two  days  of  voluntary  exercise  or  reboxetine   (selective   norepinephrine   reuptake   inhibitor)   both   increased   total  Bdnf   levels   in   the   rodent   hippocampus.     Exercise   and   reboxetine   also   had   unique   effects   on   expression   of   specific  Bdnf   transcripts.     Both   treatments   alone   increased  Bdnf   exon  II,  and  when  reboxetine  and  voluntary  exercise  were  combined  there  was  a   robust  increase  in  Bdnf  transcripts  I,  II,  and  IV  (referred  to  as  transcript  III  in  the   paper)  in  some  or  all  hippocampal  regions  examined.    These  data  demonstrate  a   relationship   between   exercise,   norepinephrine,   and   Bdnf   transcription.     In   the   present   study,   we   investigated   the   influence   of   one   month   of   voluntary   wheel   running   on   the   response   to   an   acute   bout   of   forced   treadmill   exercise.     We   investigated   Ser845   phosphorylation   of   GluR1,   glutamate   receptor   subunit   mRNA   expression,   and   Bdnf   mRNA   expression.     Moreover,   due   to   the   relationship   between   norepinephrine   and  Bdnf   expression,   we   investigated   the   influence   of   acute   exercise   and   DSP-­4-­induced   noradrenergic   lesioning   on   transcript-­specific  Bdnf  expression.       Methods     Mouse  model  and  overview.  Male  C57BL/6J  (Jackson  Laboratories,  Bar  Harbor,   ME,  USA)  mice  were  used,  as   they  are  commonly  used   to  study   the   impact  of   exercise  on  brain  phenotypes  and  in  our  lab  display  avid  treadmill  running  activity       142   and   normal   physiological   responses   to   exercise   training   (improved   glucose   metabolism,   lower   body   mass,   increased   markers   of   oxidative   capacity,   etc.;;   Ludlow   et   al.,   2012;;   Guth   et   al.,   2013).     All   mice   were   cared   for   by   UMD   veterinary   staff   and   kept   on   12hr   light/12hr   dark   cycle   and   provided   standard   rodent  chow.    All  protocols  were  approved  by  the  University   Institutional  Animal   Care   and   Use   Committee.     We   performed   two   separate   experiments   to   understand  how  previous  physical  activity  and  noradrenergic  signaling  influence   markers  of  plasticity.     In   the   first  experiment,  mice  were  housed  with  or  without   access   to   a   freely   moving   voluntary   running   wheel   for   one   month   before   exposure   to   an   acute   bout   of   exercise.     In   the   second   experiment,  mice  were   injected  with  a  locus  coeruleus-­noradrenergic  specific  neurotoxin  (DSP-­4)  prior  to   the  acute  bout  of  exercise.       Overview   of   Acute   Exercise   after   One   Month   of   Voluntary   Wheel   Running   Experiment:   To   determine   if   one   month   of   voluntary   wheel   running   influences   acute   exercise-­induced   markers   of   hippocampal   plasticity,   two-­month-­old   C57BL/6J   mice   were   separated   into   voluntary   running   and   sedentary   groups.     Voluntary   running   mice   were   individually   housed   in   cages   containing   freely   moving   running  wheels   and   sedentary  mice  were   individually   housed   in   cages   containing  locked  running  wheels.    These  housing  conditions  were  maintained  for   one  month  before  acute  exercise  exposure.    Mice  were  randomly  assigned  to  six   groups:   1)   sedentary-­no   acute   exercise   (CON;;   n=10);;   2)   chronically   active-­no   acute   exercise   (PA-­CON;;   n=9);;   3)   sedentary-­moderate-­intensity   acute   exercise   (MOD;;  n=10);;  4)  chronically  active-­moderate-­intensity  acute  exercise  (PA-­MOD;;       143   n=10);;   5)   sedentary-­high-­intensity   acute   exercise   (HI;;   n=10);;   6)   chronically   active-­high-­intensity  acute  exercise  (PA-­HI;;  n=10).    For  three  days  leading  up  to   the  experiment,  mice  were  placed  on  the  stationary  treadmill  for  five  minutes  per   day,  during  which  the  electrical  stimulus  grid  at  the  end  of  the  treadmill  belt  was   activated  to  familiarize  them  with  the  stimulus  and  treadmill-­testing  environment.   Therefore,   they   had   no   previous   treadmill   running   prior   to   the   acute   bout,   only   exposure   to   the   treadmill   environment   to   prevent   a   response   to   a   novel   environment  on  the  experimental  day.  On  day  4,  all  exercise  group  mice  (MOD,   PA-­MOD,  HI,   and  PA-­HI)  were  placed  on   the   treadmill,   one  at   a   time,   and   the   acute  bout  of  exercise  was  initiated.  Each  mouse  underwent  a  six-­minute  warm   up,  where   the   first  minute  was  a  no-­exercise   treadmill   exposure;;   thereafter   the   treadmill  belt  began  to  move  at  5  m/min,  increasing  1  m/min  every  minute  for  five   minutes.     The   treadmill   speed  was   then   incrementally   increased   to   the   group-­ appropriate   speed   and   the  mouse   ran   for  45  minutes   at   this   pace.     The  MOD   group  ran  for  45  minutes  at  12  m/min  at  0%  grade  and  the  HI  group  ran  for  45   minutes  at  a  speed  of  18  m/min  at  0%  grade.    Tactile  stimulation  to  the  tail  was   used  to  encourage  mice  to  run  prior  to  touching  the  stimulus  grid,  which  reduces   the   number   of   stimulus   grid   touches   (unpublished   observation).     Following   the   acute  bout  of  exercise,  both  groups   remained  on   the  stationary   treadmill   for  an   additional  15  minutes  prior  to  sacrifice.  Animals  in  the  CON  groups  were  placed   on   the   stationary   treadmill   for   66   minutes   with   the   electrical   stimulus   grid   activated.         144   Overview  for  DSP-­4  Lesioning  of  Locus  Coeruleus  and  Treadmill  Exercise.  Three   month   old  male  C57Bl/6J  mice  were   separated   into   four   groups:   1)   Stationary   Treadmill   –   Saline   (CON-­SAL;;   n=9);;   2)   Stationary   Treadmill   –   DSP-­4   (CON-­ DSP4;;   n=9);;   Treadmill   Exercise   –   Saline   (EX-­SAL;;   n=7);;   Treadmill   Exercise   –   DSP-­4  (EX-­DSP4;;  n=9).  Mice  were  housed  in  standard  cages  with  2-­3  mice  per   cage.     Due   to   fighting,   certain   mice   needed   to   be   separated   and   individually   housed.     Individually   housed   mice   are   identified   in   the   data   figures.     Mice   underwent   the  same  three-­day  treadmill   familiarization  protocol  and  exercise  as   described  above.    Mice  ran  at  a  speed  between  12  and  18  m/min  depending  on   running  ability.   N-­(2-­chloroethyl)-­N-­ethyl-­2-­bromobenzylamine   (DSP-­4):     DSP-­4   (Sigma   Aldrich)  was  prepared  in  0.9%  saline  and  a  single  50  mg/kg  dose  was  delivered   by  IP  injection  in  a  volume  of  10  ml/kg;;  this  dose  is  frequently  used  in  both  rats   and   mice   and   is   effective   in   depleting   hippocampal   norepinephrine   (Ross   &   Stenfors,   2014).     Control   mice   received   a   single   IP   injection   of   0.9%   saline.     Solutions   were   prepared   for   five   animals   and   any   remaining   solution   was   discarded.     Injections  were   delivered  within   15  minutes   of   solution   preparation   and   were   kept   out   of   the   light.   Mice   received   injections   seven   days   prior   to   treadmill  familiarization  (10  days  prior  to  experimental  treadmill  day).       Western  Blotting:    Twenty-­five  μg  of  protein  was  loaded  onto  polyacrylamide  gels   and   electrophoresed,   followed   by   transfer   to   nitrocellulose   membranes   and   immunoblotting.   Nitrocellulose   membranes   were   incubated   with   anti-­phospho-­     145   GluR1   (Ser845;;   Millipore,   Billerica,   MA)   antibody,   stripped   in   a   glycine   HCl   stripping   solution   and   re-­probed   with   anti-­Glur1   antibody   (Millipore).     Because   one  month  of  wheel  running  could  potentially  increase  Glur1  protein  expression,   we   also   blotted   for   Glur1   protein   then   stripped   the   membrane   and   blotted   for   Gapdh   (Millipore).    Appropriate   fluorescent  secondary  antibodies  were  used   for   detection.  A  Typhoon  scanner  (Amersham  Biosciences)  was  used  to  digitize  the   fluorescent  signal.       Gene  Expression:    One  μg  of  total  RNA  was  reverse  transcribed  into  cDNA  using   the  High  Capacity  cDNA  Reverse  Transcription  Kit   (Applied  Biosystems,  Foster   City,   CA).     Real-­time   quantitative   PCR   (qPCR)   was   used   to   assess   mRNA   expression  of  GluR1  (Gria1),  NR2B  (Grin2b),  NR2A  (Grin2a),  Bdnf  (exon  IX),  and   Bdnf  IV  for  the  wheel  running  and  acute  exercise  experiment.    qPCR  was  used  to   assess  mRNA  expression  of  b2-­adrenergic  receptor  (b2AR),  Bdnf  I,  Bdnf  II,  Bdnf   III,  Bdnf  IV,  Bdnf  VI,  and  total  Bdnf  (exon  IX)  for  the  DSP-­4-­lesioning  experiment.     Gapdh,  and  ActB  served  as  expression  controls  for  both  experiments.  All  primer   sequences  are  listed  in  Appendix  A.    Primer:probe  assays  were  purchased  pre-­ made   (Gapdh,  ActB,  NR2A,  NR2B,  GluR1,  b2AR)  or   designed   (Bdnf   I,  Bdnf   II,   Bdnf  III,  Bdnf  IV,  Bdnf  VI,  total  Bdnf)  for  the  mRNA  sequence  of  each  gene  using   Integrated  DNA  Technologies’  PrimeTime  qPCR  Assay  designer   and  efficiency   tested  prior  to  use.    All  primer  pairs  except  Bdnf  total  and  b2AR  spanned  exons  to   prevent   amplification   of   genomic   DNA.     Because  Bdnf   total   is   represented   by   amplification  of  only  exon  IX,  this  primer  pair  could  not  span  exons.    b2AR  is  an       146   intron-­less  gene.    qPCR  data  were  normalized  to  the  geometric  mean  of  Gapdh   and  ActB   using   the   -­ΔΔCt   method   (Vandesompele   et   al.,   2002;;   Schmittgen   &   Livak,   2008)   and   expressed   as   fold   induction   (2-­ΔΔCt)   of   mRNA   expression   compared  to  the  control  group  (1.0-­fold  induction).       Statistical  Analysis.    Protein  and  mRNA  data  were  analyzed  by  two-­way  ANOVA   (acute  exercise  intensity  x  running  wheel  or  acute  exercise  x  drug  treatment)  and   Tukey’s  post  hoc  comparisons  when  appropriate  (p<0.05  considered  statistically   significant).       Results   Acute  Exercise  after  One  Month  of  Voluntary  Wheel  Running     GluR1.    We   observed   a   main   effect   of   the   running   wheel   on   GluR1   protein   expression   (F(1,53)=5.383;;   p=0.02)   and   Ser845   phosphorylation   (F(1,53)=4.287;;   p=0.04)   (Fig.   16).    Mice   housed  with   a   voluntary   running  wheel   for   one  month   had   significantly   higher   GluR1   protein   expression   and   Ser845   phosphorylation   compared  to  mice  housed  with  a  locked  wheel.    There  was  no  significant  effect  of   running  wheel  on  the  ratio  of  phosphorylated  Ser845  over  total  GluR1  expression.     There  was  no  significant  effect  of  acute  exercise  on  GluR1  expression  or  Ser845   phosphorylation.       Glutamate  Receptor  mRNA  expression:    We  observed   no   significant   effects   of   the   running   wheel   or   acute   exercise   on   GluR1,   NR2B,   or   NR2A   mRNA   expression  (Fig.  17).       147   Total   Bdnf   mRNA   expression.     There   was   a  main   effect   of   the   running   wheel   (F(1,52)=8.621;;   p=0.005)   and   a   main   effect   of   acute   exercise   (F(2,52)=3.372;;   p=0.04)  on  total  Bdnf  mRNA  expression  but  no  running  wheel  and  acute  exercise   interaction  (Fig.  18A).  MOD  (adjusted  p=0.005),  HI  (adjusted  p=0.02),  and  PA-­HI   (adjusted   p=0.006)   had   significantly   higher  Bdnf   mRNA   expression   than   CON   mice.     There   was   no   significant   difference   in   total   Bdnf   mRNA   expression   between  mice  exposed  to  moderate  and  high   intensity   treadmill   running  and  no   differences  observed  within  acute  exercise  groups  (i.e.  MOD  vs  PA-­MOD;;  HI  vs   PA-­HI).   Bdnf   IV   mRNA   expression.     There   was   a   main   effect   of   the   running   wheel   (F(1,52)=14.59;;   p=0.0004),   a   main   effect   of   acute   exercise   (F(2,52)=17.41;;   p<0.0001),   and   a   running   wheel   by   acute   exercise   interaction   (F(2,52)=5.209;;   p=0.009)   for  Bdnf   IV  mRNA  expression  (Fig.  18B).    Post  hoc  analysis   revealed   that  MOD  (adjusted  p<0.0001)  and  HI  (adjusted  p<0.0001)  mice  had  significantly   greater   Bdnf   IV   mRNA   expression   compared   to   CON   mice.     MOD   mice   had   significantly   higher   Bdnf   IV   mRNA   expression   compared   to   PA-­MOD   mice   (adjusted   p=0.002).     There   was   a   tendency   for   PA-­HI   to   have   higher  Bdnf   IV   expression  than  CON  mice  (p=0.07).       Running  Performance.    Within  exercise  groups,  there  was  a  main  effect  of  acute   exercise   (F(1,35)=19.05;;   p=0.0001),   but   no   main   effect   of   the   running   wheel   or   interaction  effect  for  stimulus  pad  touches  (data  not  shown).    HI  intensity  runners   had   significantly   more   stimulus   pad   touches   than   MOD   intensity   runners   but       148   there  were   no   differences  within   acute   exercise   intensity   groups   (MOD   vs  PA-­ MOD  or  HI  vs  PA-­HI).     DSP-­4-­Lesioning  of  Locus  Coeruleus  and  Treadmill  Exercise   Total   Bdnf   (Fig   19A):     We   observed   a   main   effect   of   treadmill   exercise   (F(1,30)=6.111;;   p=0.02)   but   no   effect   of   the   drug   treatment   or   an   interaction   between  treadmill  exercise  and  drug  on  total  Bdnf  mRNA  expression.    Post  hoc   analysis   revealed   a   significant   difference   between   CON-­DSP4   and   EX-­DSP4   (adjusted  p=0.03).   We   observed   no   effects   of   treadmill   exercise   or   drug   treatment   on  Bdnf   I   (Fig   19B),  Bdnf  II  (Fig  19C),  or  Bdnf  III  (Fig  19D)  mRNA  expression.   Bdnf  IV  (Fig  19E):  We  observed  a  main  effect  of  treadmill  exercise  (F(1,30)=12.92;;   p=0.001),   a   main   effect   of   drug   treatment   (F(1,30)=5.106;;   p=0.03),   but   no   interaction   between   treadmill   exercise   and   drug   treatment   on   Bdnf   IV   mRNA   expression.    Post  hoc  analysis   revealed  a  significant  difference  between  CON-­ DSP4  and  EX-­DSP4  (adjusted  p=0.02).   Bdnf  VI   (Fig  19F):    We  observed  a  main  effect  of  drug   treatment   (F(1,30)=17.69;;   p=0.0002)   but   no   effect   of   treadmill   exercise   or   interaction   on  Bdnf   VI   mRNA   expression.    Post  hoc  analysis  revealed  a  significant  difference  between  EX-­SAL   and  EX-­DSP4  (adjusted  p=0.006).   We  observed  no  effects  of  treadmill  exercise  or  drug  treatment  on  b2AR  (Fig  20)   mRNA  expression.       149   Figure  16.    Chronic  exercise  but  not  acute  exercise  increases  GluR1  Ser845   phosphorylation  and  total  GluR1  protein  expression  in  the  mouse   hippocampus.    Mice  were  housed  in  cages  with  either  locked  or  freely  rotating   running  wheels  for  one  month  and  exposed  to  45  minutes  (after  6-­minute  warm   up)  of  moderate-­intensity  (12m/min;;  n=20;;  10  runners  10  sedentary),  high-­ intensity  (18m/min;;  n=20;;  10  runners  10  sedentary),  or  no  exercise  treadmill   exposure  (n=20;;  10  runners  10  sedentary)  followed  by  15  minutes  of  rest  on  the   treadmill.    Mice  were  sacrificed  and  hippocampi  isolated  immediately  after  the   15-­minute  rest.    Mice  housed  with  voluntary  running  wheels  had  significantly   more  Ser845  phosphorylation  compared  to  sedentary  mice  (F(1,53)=4.287;;   p=0.04).    Mice  housed  with  voluntary  running  wheels  also  had  significantly  more   GluR1  expression  compared  to  sedentary  mice  (F(1,53)=5.383;;  p=0.02).    There   was  no  effect  of  voluntary  wheel  running  on  the  ratio  of  pSer845  over  GluR1.     There  was  no  effect  of  acute  exercise  on  GluR1  expression  or  phosphorylation.     Error  bars  represent  SEM.    *  denotes  significance  at  p<0.05.   Figure  17.    Acute  and  chronic  exercise  do  not  influence  glutamate  receptor   subunit  mRNA  expression.  Mice  were  housed  in  cages  with  either  locked  or   freely  rotating  running  wheels  for  one  month  and  exposed  to  45  minutes  (after  6-­ minute  warm  up)  of  moderate-­intensity  (12m/min;;  n=20;;  10  runners  10   sedentary),  high-­intensity  (18m/min;;  n=20;;  10  runners  10  sedentary),  or  no   exercise  treadmill  exposure  (n=20;;  10  runners  10  sedentary)  followed  by  15   minutes  of  rest  on  the  treadmill.    Mice  were  sacrificed  and  hippocampi  isolated   immediately  after  the  15  minute  rest.    Target  mRNA  expression  is  presented  as       150   2-­ΔΔCt  relative  to  the  geometric  mean  of  ActB  and  Gapdh.    There  was  no  effect  of   acute  exercise  or  running  wheel  on  GluR1  (A),  NR2B  (B),  or  NR2A  (C).   Figure  18.  Acute  and  chronic  exercise  increase  total  Bdnf  and  Bdnf  IV   mRNA  expression    Mice  were  housed  in  cages  with  either  locked  or  freely   rotating  running  wheels  for  one  month  and  exposed  to  45  minutes  (after  6-­minute   warm  up)  of  moderate-­intensity  (12m/min;;  n=20;;  10  runners  10  sedentary),  high-­ intensity  (18m/min;;  n=20;;  10  runners  10  sedentary),  or  no  exercise  treadmill   exposure  (n=20;;  10  runners  10  sedentary)  followed  by  15  minutes  of  rest  on  the   treadmill.    Target  mRNA  expression  is  presented  as  2-­ΔΔCt  relative  to  the   geometric  mean  of  ActB  and  Gapdh.    A)  Total  Bdnf:  There  was  a  main  effect  of   running  wheel  (F(1,52)=8.621;;  p=0.005)  and  a  main  effect  of  acute  exercise   (F(2,52)=3.372;;  p=0.04)  but  no  wheel  running  x  acute  exercise  interaction.    B)  Bdnf   IV.  There  was  a  main  effect  of  running  wheel    (F(1,52)=14.59;;  p=0.0004),  a  main   effect  of  acute  exercise  (F(2,52)=17.41;;  p<0.0001),  and  a  wheel  running  x  acute   exercise  interaction  (F(2,52)=5.209;;  p=0.009).    Error  bars  represent  SEM.    *   denotes  significantly  different  than  CON  (p<0.05).  $  denotes  significant  difference   from  CON  housing  within  acute  exercise  groups  (p<0.05).   Figure  19.    Acute  exercise  and  DSP-­4  lesioning  influence  Bdnf  transcript   expression.      Mice  were  injected  with  either  saline  or  DSP-­4  ten  days  prior  to   exposure  to  45  minutes  (after  6-­minute  warm  up)  of  treadmill  exercise  (n=16;;  7   saline  9  DSP-­4)  or  no  exercise  treadmill  exposure  (n=18;;  9  saline  9  DSP-­4)   followed  by  15  minutes  of  rest  on  the  treadmill.    Target  mRNA  expression  is       151   presented  as  2-­ΔΔCt  relative  to  the  geometric  mean  of  ActB  and  Gapdh.    A)  Total   Bdnf:  There  was  a  main  effect  of  treadmill  exercise  (F(1,30)=6.111;;  p=0.02)  but  no   effect  of  the  drug  treatment  or  treadmill  x  drug  interaction.  B)  Bdnf  I:  There  was   no  influence  of  exercise  or  drug  on  Bdnf  I  mRNA  expression.    C)  Bdnf  II:  There   was  no  influence  of  exercise  or  drug  on  Bdnf  II  mRNA  expression.  D)  Bdnf  III:   There  was  no  influence  of  exercise  or  drug  on  Bdnf  III  mRNA  expression.    E)   Bdnf  IV:    There  was  a  main  effect  of  treadmill  exercise  (F(1,30)=12.92;;  p=0.001),  a   main  effect  of  drug  treatment  (F(1,30)=5.106;;  p=0.03),  but  no  treadmill  x  drug   interaction.    F)  Bdnf  VI:    There  was  a  main  effect  of  drug  treatment  (F(1,30)=17.69;;   p=0.0002)  but  no  effect  of  treadmill  exercise  or  treadmill  x  drug  interaction.  *   indicates  significantly  different  (p<0.05).    x’s  represent  data  points  of  animals  that   were  individually  housed  due  to  fighting  with  cage  mates.   Figure  20.    b2-­adrenergic  receptor  mRNA  expression  is  not  influenced  by   DSP-­4  treatment  or  acute  exercise.      Mice  were  injected  with  either  saline  or   DSP-­4  ten  days  prior  to  exposure  to  45  minutes  (after  6  minute  warm  up)  of   treadmill  exercise  (n=16;;  7  saline  9  DSP-­4)  or  no  exercise  treadmill  exposure   (n=18;;  9  saline  9  DSP-­4)  followed  by  15  minutes  of  rest  on  the  treadmill.    Target   mRNA  expression  is  presented  as  2-­ΔΔCt  relative  to  the  geometric  mean  of  ActB   and  Gapdh.    There  was  no  effect  of  acute  exercise  or  DSP-­4  on  b2AR  mRNA   expression           152   Figure  16.                 153   Figure  17.     Sedentary Wheel 0.0 0.5 1.0 1.5 Re la tiv e m RN A ex pr es si on (2 -Δ ΔC t ) GluR1 Control Moderate High Sedentary Wheel 0.0 0.5 1.0 1.5 Re la tiv e m RN A ex pr es si on (2 -Δ ΔC t ) NR2B Sedentary Wheel 0.0 0.5 1.0 1.5 Re la tiv e m RN A ex pr es si on (2 -Δ ΔC t ) NR2A A B C     154   Figure  18         Sedentary Wheel 0.5 1.0 1.5 Re la tiv e m RN A ex pr es si on (2 -Δ ΔC t ) Bdnf Control Moderate High * * ** Sedentary Wheel 0.5 1.0 1.5 2.0 Re la tiv e m RN A ex pr es si on (2 -Δ ΔC t ) Bdnf IV * * $ A B     155   Figure  19         Saline DSP-4 0.5 1.0 1.5 Re la tiv e m RN A ex pr es si on (2 - Δ Δ Ct ) Total Bdnf Control Exercise * A B Saline DSP-4 0.5 1.0 1.5 Re la tiv e m RN A ex pr es si on (2 - Δ Δ Ct ) Bdnf I     156         C D Saline DSP-4 0.5 1.0 1.5 Re la tiv e m RN A ex pr es si on (2 - Δ Δ Ct ) Bdnf II Saline DSP-4 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Re la tiv e m RN A ex pr es si on (2 - Δ Δ Ct ) Bdnf III     157       Saline DSP-4 0.5 1.0 1.5 2.0 Re la tiv e m RN A ex pr es si on (2 - Δ Δ Ct ) Bdnf IV * * Saline DSP-4 0.0 0.5 1.0 1.5 2.0 2.5 Re la tiv e m RN A ex pr es si on (2 - Δ Δ Ct ) * $ Bdnf VI * E F     158   Figure  20         Saline DSP-4 0.0 0.5 1.0 1.5 2.0 Re la tiv e m RN A ex pr es si on (2 - Δ Δ Ct ) β2 Adrenergic Receptor Control Exercise     159   Discussion   We   observed   that   one   month   of   voluntary   wheel   running   increased   expression   of   GluR1   protein   and   phosphorylation   at   Ser845.     Moreover,   we   confirmed  our  previous  findings  (Chapter  4)   that  acute  forced  treadmill  exercise   increased   Bdnf   IV  mRNA   expression,   though   remarkably,   this   effect   of   acute   exercise   was   attenuated   by   one-­month   of   voluntary   wheel   running.     We   also   observed  that  both  one  month  of  chronic  voluntary  wheel  running  and  45  minutes   of   acute   exercise   at   either   high   or  moderate   intensity   increased   expression   of   total  Bdnf  mRNA.    We  hypothesized  that  the  blunting  of  Bdnf  IV  expression  after   one  month  of  voluntary  wheel  running  was  due  to  reduced  noradrenergic  activity   after   exercise,   so   we   lesioned   the   locus   coeruleus-­noradrenergic   system   with   DSP-­4  prior   to   the   acute   exercise.    Contrary   to   our   hypothesis,   acute   exercise   increased  Bdnf  IV  and  total  Bdnf   in  DSP-­4  treated  mice.    Additionally,  we  found   that  DSP-­4  increased  expression  of  Bdnf  transcript  VI.   Glutamate   Receptors:    We   observed   a   significant   increase   in   phospho-­ Ser845   following   one   month   of   voluntary   wheel   running.     An   increase   in   phosphorylated  Ser845  is  indicative  of  more  membrane-­inserted  AMPA  receptors   and  is  believed  to  reduce  the  threshold  for  synaptic  plasticity  and  learning  (Hu  et   al.,  2007;;  Makino  et  al.,  2011).    We  also  observed  a  significant  increase  in  total   GluR1  protein  expression.    Mizutani  et  al.   (2015)   found  higher   levels  of  Ser845   phosphorylation  in  a  model  of  cortical   infarction  following  one  week  of  voluntary   wheel   running   but   observed   no   effect   on   total   GluR1   protein   expression.         160   Differences  in  the  brain  region,   length  of  wheel  running  exposure,  and  health  of   the   tissue   may   account   for   the   differences   between   our   investigation   and   Mizutani  et  al.  (2015).    Indeed,  Dietrich  et  al.  (2005)  reported  that  four  weeks  of   voluntary  wheel  running  (the  same  duration  of  wheel  exposure  as  in  the  current   investigation)   increased   protein   levels   of   GluR1   in   the   mouse   cortical   post   synaptic   density,   suggesting   high   levels   of   Ser845   phosphorylation.     In   the   current  investigation,  the  increase  in  GluR1  protein  was  of  similar  magnitude  with   the   elevation   in  Ser845   phosphorylation   and   therefore  we   did   not   observe   any   differences   in   the   ratio  of   phospho-­Ser845  over   total  GluR1.    Upon  exogenous   catecholamine  delivery  or  psychological  stress  there  is  an  increase  in  the  ratio  of   Ser845   to   total  GluR1   [Chapter  4,   (Hu  et  al.,  2007)].    We  believe  our  observed   response  to  be  a  favorable  adaptation  to  chronic  exercise  because  maintaining  a   low   ratio   of   phospho-­Ser845   to   GluR1,   while   increasing   both   phospho-­Ser845   and   GluR1   protein   may   result   in   more   membrane-­inserted   AMPARs   and   also   more  available  GluR1  containing  AMPARs  to  be  phosphorylated  and  trafficked  to   the  synapse  in  response  to  an  appropriate  stimulus.     We   did   not   observe   a   significant   effect   of   acute   exercise   on   Ser845   phosphorylation,  which   is  consistent  with  our  previous   investigation   (Chapter  4)   that  examined  mice  sacrificed  immediately  after  exposure  to  30  minutes  of  acute   exercise.    Potentially,  the  acute  bout  of  exercise  was  not  sufficient  in  intensity  or   duration  to  lead  to  a  widespread  elevation  of  norepinephrine  [or  dopamine,  which   also   has   the   potential   to   phosphorylate  Ser845   (Price  et   al.,   1999)]   throughout   the   hippocampus.     Moreover,   we   used   whole   hippocampal   homogenates.         161   Potentially,   acute   forced   treadmill   running   influences   the   hippocampus   in   a   region-­specific  manner.  Numerous  studies  suggest  that  the  dorsal  hippocampus   is   important   for   spatial   memory   and   the   ventral   hippocampus   is   important   for   anxiety-­like  behaviors  (Bannerman  et  al.,  2004)  and  running  has  been  shown  to   affect   the   rodent   ventral   and  dorsal   hippocampus  differently   (Schoenfeld  et   al.,   2013).   Therefore,   it   is   possible   that   the   effects   of   our   acute   bout   of   forced   exercise  may  differ  along  the  longitudinal  axis  of  the  hippocampus.     The  increase  in  GluR1  protein  with  one  month  of  voluntary  wheel  running   was   not   consistent   with   our   qPCR   findings   of   GluR1   mRNA,   which   was   not   different   from   sedentary   controls.    We   also   did   not   observe   an   effect   of  wheel   running   or   acute   exercise   on   NMDA   receptor   subunits.     Molteni   et   al.   (2002)   reported  that  three  days  of  voluntary  wheel  running  increased  NR2B  and  NR2A   mRNA  expression  in  the  rat  hippocampus,  though  these  were  no  longer  different   than   controls   after   28   days   of   wheel   running.     Potentially,   we   would   have   observed  an  effect  on  glutamate   receptor   subunit   expression  at  an  earlier   time   point.     The   observation   that   neither   chronic   exercise   nor   45   minutes   of   acute   exercise   increased  expression  of  glutamate   receptor  subunit  mRNA  expression   is  consistent  with  our  previous   investigations  (Chapters  3  and  4;;  Venezia  et  al.,   2016).         Bdnf   transcription:    We  observed  a  significant  effect  of  voluntary  wheel   running   and  acute  exercise  on  total  Bdnf  expression.    Both  one  month  of  voluntary  wheel   running   and   acute   treadmill   running   increased   expression   of   total  Bdnf   mRNA       162   with   no   interaction   between   the   exercise   stimuli.     It   is   well   supported   in   the   literature  that  one  month  of  voluntary  wheel  running  increases  hippocampal  Bdnf   mRNA   (for   review,   see   Cotman   et   al.,   2007;;   Voss   et   al.,   2013),   however   the   influence  of  one  bout  of  acute  exercise  has  not  been  thoroughly  investigated.    In   contrast   to   the   results   observed   immediately   following   30   minutes   of   acute   exercise  in  our  previous  investigation  (Chapter  4),  45  minutes  of  running  followed   by  15  minutes  of  rest  resulted  in  an  intensity-­independent   increase  in  total  Bdnf   mRNA.     This   suggests   that   30  minutes   of   acute   exercise  was   not   sufficient   in   duration   and   that   either   45  minutes   of   exercise   or   a   60-­minute   delay   from   the   initiation  of  exercise  to  sacrifice  is  required.    Interestingly,  both  acute  and  chronic   exercise   increased  Bdnf   transcription   to   similar   levels  with  no  additive  effect   of   the   two.     This   indicates   that   one   bout   of   acute   exercise   is   equally   effective   at   increasing  this  important  neurotrophin  as  one  month  of  voluntary  wheel  running.     This  finding  requires  further  investigation  to  determine  if  the  effects  on  total  Bdnf   mRNA  expression  are  more  persistent   following  chronic  exercise   than   following   an  acute  bout  of  exercise.    Berchtold  et  al.  (2005)  reported  that  even  though  both   daily   and   intermittent   (every   other   day)   exposure   to   voluntary   wheel   running   increased  Bdnf  protein  expression  to  similar  levels,  daily  wheel  running  resulted   in  more  persistent  elevation  in  Bdnf  levels.         Both   acute   and   chronic   exercise   significantly   influenced   Bdnf   IV   expression;;  however,  in  contrast  to  total  Bdnf,  there  was  an  interaction  between   voluntary  wheel  running  and  acute  exercise.    Moderate-­  and  high-­intensity  acute   exercise   increased  Bdnf   IV   mRNA   only   in  mice   housed   with   a   locked   running       163   wheel.  A  history  of  physical  activity  has  previously  been  shown  to  influence  Bdnf   protein  expression  in  response  to  short-­term  exercise  exposure.    Berchtold  et  al.   (2005)  showed  that  two  days  of  wheel  running  increased  Bdnf  protein  expression   only  in  mice  that  were  previously  exposed  to  14  days  of  wheel  running.  Further,   Gomez-­Pinilla   et   al.   (2010)   reported   that   one  week   of   voluntary  wheel   running   decreased  Bdnf   IV   promoter  methylation.     A   reduction   in   promoter  methylation   suggests   greater   transcriptional   capacity   following   a   short   exposure   to   activity.     These   two  studies  suggest   that  a  previous  history  of  physical  activity   increases   the  capacity  to  induce  Bdnf  transcription;;  however,  we  saw  a  blunting  of  Bdnf  IV   expression   after   one  month   of   voluntary   wheel   running.     This   suggests   that   a   signaling   mechanism   upstream   of   Bdnf   IV   transcription   is   downregulated   following   voluntary   wheel   running.     We   hypothesized   that   the   modulated   signaling  mechanism  is  exercise-­induced  noradrenergic  signaling.   Noradrenergic   signaling   is   an   important   regulator   of   Bdnf   expression.     Application   of   norepinephrine   to   neurons   in   culture   or   injection   of   exogenous   norepinephrine  in  vivo  can  increase  expression  of  Bdnf  (Chen  et  al.,  2007;;  Mello-­ Carpes   et   al.,   2016),   similar   to   what   is   observed   following   treatment   with   antidepressants,  including  norepinephrine  reuptake  inhibitors  such  as  reboxetine   (Russo-­Neustadt  et  al.,  2004;;  Larsen  et  al.,  2008;;  Musazzi  et  al.,  2009;;  Baj  et  al.,   2012).     Exercise-­induced   Bdnf   transcription   is   dependent   on   b-­adrenergic   receptor  (Ivy  et  al.,  2003)  and  normal  LC-­noradrenergic  signaling  (Garcia  et  al.,   2003).     If   either   of   these   are   compromised,   exercise   no   longer   increases  Bdnf   expression   (Garcia   et   al.,   2003;;   Ivy   et   al.,   2003).     Further,   though   exercise       164   training   is   associated   with   an   increased   capacity   for   catecholamine   release   (Zouhal  et  al.,  2008),  a  previous  history  of  physical  activity   is  associated  with  a   reduced  stress   response   (Dishman  et  al.,  1997;;  1998;;  2000;;  Greenwood  et  al.,   2003)  For  example,  norepinephrine  release/depletion  in  the  LC  and  hippocampus   in  response  to  treadmill  or  immobilization  stress  is  reduced  following  six  weeks  of   treadmill   training   (Dishman   et   al.,   2000).     This   led   us   to   hypothesize   that   the   blunting   of  Bdnf   IV   expression   following   one  month   of   voluntary  wheel   running   was  due  to  reduced  acute  exercise-­induced  noradrenergic  signaling  after  wheel   exposure.    Our   analysis   revealed   that   acute   exercise   increased   total  Bdnf   and   Bdnf  IV  only  in  DSP-­4  treated  mice  (though  there  was  a  main  effect  of  exercise).   This   is   interesting  since  Garcia  et  al.   (2003)  reported  that  DSP-­4  prevented  the   voluntary   wheel   running-­induced   increase   in   total  Bdnf   expression   but   did   not   decrease  Bdnf  expression   in  sedentary  controls.    This   is   in  agreement  with   the   effects   of   propranolol,   a   bAR-­antagonist,   on   exercise   induced  Bdnf  expression   (Ivy  et   al.,   2003).    We   speculate   that   the   treadmill   environment   alone   induced   Bdnf   and   Bdnf   IV   expression   through   noradrenergic   signaling   and   DSP-­4   attenuated   this;;   however,   acute   high-­intensity   exercise   was   sufficient   to   overcome   the   reduction   in   LC-­derived   norepinephrine,   potentially   through   LC-­ norepinephrine   independent   pathways   such   as   strong   synaptic   activity   at   glutamatergic  synapses  or  serotonergic  signaling.    Indeed,  DSP-­4  is  reported  to   leave   serotonergic   signaling   intact,   and   serotonin   signaling   may   also   be   important  for  exercise-­induced  Bdnf  expression  (Ivy  et  al.,  2003;;  Russo-­Neustadt   et  al.,  2004).       165   Interestingly,   in   the   Garcia   et   al.   (2003)   investigation,   one   week   of   voluntary  exercise  did  not  increase  Bdnf  IV  but  DSP-­4  and  DSP-­4  plus  one  week   of   exercise   did   increase  Bdnf   IV.     Again,   this   increase   with   DSP-­4   alone   and   DSP-­4   plus   exercise   relative   to   saline   controls   is   inconsistent  with   our   findings   and  might  be  explained  by  our  mice  being  exposed  to  the  treadmill   immediately   before   sacrifice   and   differences   between   one-­week   of   voluntary   wheel   running   and  an  acute  bout  of  forced  exercise.  We  also  observed  an  increase  in  Bdnf  VI   expression   with   DSP-­4   treatment.     Interestingly,   Russo-­Neustadt   et   al.   (2004)   reported  that  Bdnf  VI  was  reduced  following  one  week  of  reboxetine  or  combined   reboxetine  and  exercise.    Reboxetine  is  a  norepinephrine  reuptake  inhibitor  and   should   increase   norepinephrine   availability,   especially   when   combined   with   exercise,  whereas  DSP-­4  should  reduce  norepinephrine  availability.    Therefore,  it   appears  that  high   levels  of  norepinephrine  reduce  Bdnf  VI  expression  while   low   levels   increase   Bdnf   VI   expression.     Understanding   why   DSP-­4   plus   acute   exercise  in  the  current  investigation  increased  Bdnf  VI  more  than  exercise  alone   requires  further  investigation.         We  did  not  observe  an  effect  of  acute  exercise  or  DSP-­4  on  Bdnf  I,  II,  or  III.     This  is  interesting  since  Bdnf  I  has  been  shown  to  be  influenced  by  exercise  (Oliff   et   al.,   1998;;  Garcia  et   al.,   2003;;  Russo-­Neustadt  et   al.,   2004;;   Intlekofer  et   al.,   2013),  while  Bdnf   II   expression   is   increased  by  norepinephrine,  norepinephrine   reuptake  inhibitors,  and  exercise  (Russo-­Neustadt  et  al.,  2004;;  Zajac  et  al.,  2009;;   Musazzi  et  al.,   2014).  Garcia  et  al.   (2003)   reported   that  one  week  of   voluntary   wheel  running  increased  expression  of  Bdnf  I,  which  was  attenuated  with  DSP-­4,       166   and  Bdnf  II,  which  was  potentiated  with  DSP-­4.  We  did  not  observe  an  effect  of   either   DSP-­4   or   acute   exercise   on   Bdnf   I   or   Bdnf   II.     Bdnf   I   is   sensitive   to   exposure  to  novel  contexts  (Lubin  et  al.,  2008),  so  even  though  Bdnf  I  is  sensitive   to   acute   exercise   (Oliff   et   al.,   1998)   and   stress   (Marmigère   et   al.,   2003),   the   treadmill  environment  alone  may  have  been  sufficient  to  increase  expression  and   mask  the  effects  of  exercise.    It  is  possible  that  multiple  days  of  exercise  (Zajac   et   al.,   2009)   or   very   long   acute   exercise   protocols   (Oliff   et   al.,   1998)   are   necessary  to  increase  expression  of  Bdnf  II  and  III.   Summary:     Our   data   support   that   one   truly   acute   bout   of   exercise   increases   expression  of   total  Bdnf   and  Bdnf   transcript   IV;;   however,   previous  exposure   to   voluntary   wheel   running   may   reduce   the   effectiveness   of   acute   exercise   to   increase  Bdnf   IV  expression.    Our  data   further  demonstrate   that  acute  exercise   has   the   capacity   to   increase   Bdnf   transcription   even   with   compromised   noradrenergic   signaling,   which   has   implications   for   the   treatment   of   disorders   associated  with  reduced  noradrenergic  signaling  and  LC  neurodegeneration  such   as  Alzheimer’s  disease  (Szot  et  al.,  2006).    We  also  report  that  increased  Ser845   phosphorylation  and  expression  of  GluR1  protein  may  be  a  potential  mechanism   by   which   exercise   training   increases   hippocampal   plasticity,   though   these   adaptations  are  not  induced  by  a  single  acute  bout  of  exercise.         167   Chapter  6.    Summary,  Conclusions,  Limitations,  and  Future  Directions   Overall   Summary:     The   overall   aim   of   this   dissertation   research   was   to   explore   the  mechanisms  by  which   exercise   influences   brain   health,   specifically   focusing  on   the  hippocampus,  a  brain   region  critical   for  memory  and  emotional   health.     The   dissertation   reports   the   findings   from   three   investigations,   each   exploring  the   influence  of  physical  activity  on  markers  of  hippocampal  plasticity,   through   three   unique   approaches.     The   dissertation   provides   a   comprehensive   look  at   the   influence  of  different  exercise  exposures,   from  a  single  exposure  of   acute   forced   exercise   to   long-­term   voluntary   exercise.     The   first   investigation   explored   how   long-­term   physical   activity   influences   markers   of   hippocampal   plasticity,   which   has   implications   for   understanding   why   people   who   are   physically  active   throughout   their   lifetime  maintain  brain  health   into  old  age.     In   addition  to  being  uniquely  long  in  comparison  to  the  majority  of  existing  published   research  (i.e.  20  weeks  vs.  a  typical  range  of  seven  to  28  days),  this  investigation   was  also  unique  because  it  included  both  male  and  female  mice.    We  observed   sex   differences   in   the   influence   of   long-­term   exercise   exposure,   with   exercise-­ induced   increases   in  Bdnf  protein  and  mRNA  expression  only  detected   in  male   mice   (Chapter   3,   Figs.   3   &   6).   Further   research   is   required   to   uncover   the   mechanisms  associated  with   the  observed   sex  differences.    We  speculate   that   sex  differences  in  exercise-­induced  Bdnf  expression  were  due  to  sex  hormones   or  differences  observed   in   running  behavior,  since  males   ran  significantly  more   during   the   last   week   of   wheel   exposure   and   maintained   their   running   levels   throughout   the   exposure   more   than   females   (Chapter   3,   Fig.   1).     This   is       168   noteworthy   since   most   studies   use   shorter   chronic   exercise   protocols   and   capitalize  on  the  high  wheel  activity  that  is  observed  during  the  first  few  weeks  of   wheel   exposure.     Treadmill   running  might   be   a  more   appropriate   approach   for   investigating  how  long-­term  exercise  influences  brain  health  in  rodents  since  the   volume  and  intensity  of  exercise  can  be  manipulated  and  maintained.       Though   we   observed   sex   differences   in   total   Bdnf   mRNA   and   protein   expression  in  response  to  five-­months  of  voluntary  wheel  running,  Bdnf  transcript   IV  expression  was   increased   in  both  sexes  (Chapter  3,  Fig.  3).    Bdnf   IV  mRNA   content   increased   following  30  minutes  of  high-­intensity   (Chapter  4,  Fig.  10)  or   45  minutes   of   high-­   or   moderate-­intensity   forced   treadmill   running   (Chapter   5,   Figs  18&19)  and  was  supported  in  three  separate  experiments,  providing  strong   evidence  that  acute  exercise  promotes  plasticity  by  increasing  expression  of  this   rapidly   transcribed   Bdnf   transcript.     Acute   exercise   also   increased   total   Bdnf   expression  when  performed  for  45  minutes  but  not  for  30  minutes  (Chapters  4  &   5,  Figs.  10,  18,  &  19).    Interestingly,  while  five  months  of  voluntary  wheel  running   and  one  bout  of  acute  forced  exercise  increased  Bdnf  IV  expression,  one  month   of  voluntary  wheel   running  was  not  sufficient   to   increase  expression  of  Bdnf   IV   and  actually  prevented  the  acute  exercise-­induced  transcription  (Chapter  5,  Fig.   18).     The   blunting   effect   of   one   month   of   voluntary   wheel   running   was   not   observed   in   total  Bdnf   (Chapter  5,  Fig.  18).    Lesioning   the   locus  coeruleus  with   DSP-­4  also  reduced  Bdnf  IV  expression,  but  remarkably,  this  was  rescued  by  an   acute   bout   of   forced   exercise   (Chapter   5,   Fig.   19)     The   rescue   of   Bdnf   IV   expression   with   acute   exercise   has   important   implications   for   exercise   in   the       169   aged   population   and   individuals   suffering   from   Alzheimer’s   Disease   who   experience  neurodegeneration  of  the  locus  coeruleus  (Chalermpalanupap  et  al.,   2013).     These   results   demonstrate   that   exercise   can   increase  Bdnf   expression   even  when  challenged  by  a  compromised  noradrenergic  system.       Though  acute  exercise  increases  expression  of  Bdnf,  a  neurotrophin  that   is   necessary   for   hippocampal   plasticity   and   memory,   we   were   unable   to   determine  if  this  increase  is  associated  with  improved  memory.    Mice  exposed  to   a  memory  task  immediately  after  acute  forced  treadmill  running  failed  to  perform   the   task  due   to   significantly   reduced  exploratory   behavior   (Chapter   4,  Fig.   12).     We  tested  whether  this  reduction  in  exploratory  behavior  would  also  be  observed   in   a   traditional   anxiety   task   that   utilizes   a   similar   testing   environment   as   our   memory   task.    Once   again,   acute   forced   treadmill   running   reduced   exploratory   behavior   (Chapter   4,   Figures   13,   14,   &   15).     Interestingly,   lesioning   the   noradrenergic  system  partially  attenuated   this  effect.    Mice   injected  with  DSP-­4   and   exercised   did   not   show   reduced   exploratory   behavior   compared   to   mice   injected  with  DSP-­4  and  exposed  to  the  stationary  treadmill.    However,  they  did   have  significantly  less  activity  than  saline  injected  controls  and  therefore  it  is  not   possible   to   say   that   lesioning   the   LC   rescued   this   behavioral   phenotype.     Interestingly,  exercised  mice  also  spent  significantly  more  time  self-­grooming  and   this  was  not  attenuated  by  DSP-­4  (Chapter  4,  Figures  13,  14,  &  15).    The  finding   that  acute  exercise  increases  anxious  behavior   in  mice  and  prevents  them  from   performing  exploratory-­based  memory  tasks  highlights  the  obstacles  faced  when   trying   to  determine   the  effectiveness  of  acute  exercise  at   improving  memory  or       170   reducing   anxiety.     Non-­locomotor   dependent   tasks   that   do   not   rely   heavily   on   exploratory   behavior   (e.g.   virtual   reality)   and/or   contain   a   high   level   of   intrinsic   motivation  should  be  strongly  considered  when  selecting  behavioral   tasks  to  be   performed  after  exercise  exposures.    A  potential  option   for  a  memory   task   is  a   one-­trial  spatial   learning  task  that  relies  on  sexual  drive  of  male  mice  to   identify   the   location   of   previously   present   female   mice   (Meier   et   al.,   2010;;   Fellini   &   Morellini,  2013)     Another  consistent   finding   in  all   three  studies   is   that  exercise  has   limited   or  no  effects  on  hippocampal  mRNA  expression  of  glutamate  receptor  subunits.     The  data  indicate  no  changes  in  GluR1,  NR2A,  or  NR2B  mRNA  expression  after   one   (Chapter   5,   Fig.   17)   or   five   (Chapter   1,   Fig.   4)  months   of   voluntary  wheel   running,   or   30   (Chapter   4,   Fig.   9)   or   45   (Chapter   5,   Fig.   17)  minutes   of   acute   forced  exercise.    Potentially,  these  mRNAs  are  expressed  at  such  high  levels  in   the  hippocampus  that  changes  with  exercise  are  difficult   to  detect,  especially   in   whole   hippocampal   homogenates.     One  month   of   voluntary   wheel   running   did   increase   the   expression   of   GluR1   protein   and   its   phosphorylation   at   Ser845   (Chapter  5,  Fig.  16),  which  is  a  favorable  adaptation  and  should  be  investigated   further.     For   example,   by   keeping   the   ratio   of   phosphorylated   Ser845   to   total   GluR1   low  but   increasing  absolute   values  of   each,  potentially   the   synapse  has   increased   synaptically   inserted   AMPARs,   yet   in   response   to   the   appropriate   stimulus   (e.g.   IP   injection   of   epinephrine),   rapid   phosphorylation   of   available   GluR1   at   Ser845   can   increase   peri-­synaptic   insertion   of   GluR1-­containing   AMPARs.     In   contrast   to   our   hypothesis,   one   bout   of   acute   exercise   did   not       171   induce   phosphorylation   of   Ser845   and   this   was   confirmed   by   two   experiments   (Chapters  4  &  5).       Overall,   this   dissertation   provides   strong   and   convincing   evidence   that   both   acute   and   chronic   exercise   increase   expression   of   Bdnf   in   the   mouse   hippocampus.    Though  the  literature  supports  that  short-­term  (seven  to  28  days)   exercise   increases  Bdnf   transcription   in   the   hippocampus,   the   effectiveness   of   one  bout  of  exercise  and/or  long-­term  wheel  running  (>90  days)  in  adult  rodents   was  previously  unknown.    This  dissertation  also  provides  evidence  that  exercise   has   little   or   no   effect   on   glutamate   receptor   expression   or   phosphorylation   of   GluR1,   though   the   effects   may   be   subtle   and   difficult   to   detect   with   the   methodological  approaches  utilized  in  these  investigations.    It  also  provides  “food   for   thought”   concerning   the   selection   and   timing   of   behavioral   tasks   when   exploring  behavioral  adaptations  to  exercise.       Limitations   and   Considerations:     We   did   not   have   the   capability   to   comprehensively  examine  the   influence  of  DSP-­4  treatment   in  our  experiments.     There   are   some   inconsistencies   reported   in   the   literature   on   the   influence   of   DSP-­4   on   both   behavior   and   extracellular   norepinephrine.     Though   DSP-­4   dramatically  reduces  tissue  levels  of  norepinephrine  in  regions  innervated  by  the   LC   such  as   the   hippocampus   (Ross,   1976;;  Ögren  et   al.,   1980;;   Jonsson  et   al.,   1981;;  Archer  et  al.,  1982;;  Anisman  et  al.,  1984;;  Zahniser  et  al.,  1986;;  Bennett  et   al.,   1990;;   Scullion   et   al.,   2009;;   Szot   et   al.,   2010),   increased   extracellular   norepinephrine   in   the   frontal   cortex   (Kask   et   al.,   1997;;   Hughes   &   Stanford,       172   1998a;;   1998b)   and   elevated   b-­adrenergic   receptor   expression   in   the   hippocampus   (Zahniser   et   al.,   1986)   have   also   been   reported.   The   increased   extracellular   norepinephrine   in   brain   regions   innervated   by   the   LC,   despite   dramatic   reductions   in   dopamine   beta   hydroxylase   (rate-­limiting   enzyme   in   norepinephrine   synthesis)   and   tissue   content   of   norepinephrine,   are   potentially   due   to   non-­LC   noradrenergic   neurons   releasing   norepinephrine   into   the   extracellular  space  and  the  inability  of  LC  terminals  to  take  up  the  norepinephrine   in   the   surrounding   region   (Ross  &  Stenfors,   2014).    DSP-­4   exerts   its   effect   by   irreversibly  disrupting  the  norepinephrine  transporter  (Ross  &  Stenfors,  2014).    In   contrast  to  reports  of  elevated  extracellular  norepinephrine  after  DSP-­4  treatment,   others   have   shown   that   corticotropin-­releasing   factor   (CRF)   -­stimulated   norepinephrine   release   from   the   LC   is   greatly   reduced   in   rats   pre-­treated   with   DSP-­4  (Zhang  et  al.,  1998;;  Palamarchouk  et  al.,  2000).    Together,  the  research   on   DSP-­4   suggests   that   there   may   be   an   elevation   in   basal   extracellular   norepinephrine,   though   stress   induced   norepinephrine   release   in   the   hippocampus  is  attenuated  with  pre-­treatment  of  DSP-­4.    There  was  no  change   in   b-­adrenergic   receptor   expression   in   our   sample   but   we   did   not   have   the   capability   to   comprehensively   explore   tissue   content   and  extracellular   levels   of   norepinephrine   in   these   investigations.     Previous   research   on   DSP-­4,   which   spans  decades,   combined  with  our  data  suggest   that   the  drug  was  effective  at   lesioning  the  LC  in  our  animals.       173   Another  potential  limitation  of  our  investigation  was  the  housing  conditions   of   the   animals.     The   C57BL/6J   mouse   model   was   selected   due   to   its   generalizability   and   common   use   in   exercise   studies.     However,   there   were   issues  with   in-­cage   fighting  when  mice  were  group  housed  and   therefore  some   animals  had  to  be  individually  caged.    Social  isolation  can  influence  hippocampal   plasticity  (Stranahan  et  al.,  2006)  but   it   is   important   to  recognize  that  C57BL/6J   male  mice  can  be  highly  aggressive  (Roubertoux  et  al.,  2005)  and  therefore  more   research  is  needed  in  this  mouse  strain  to  understand  the  influence  of  individual   and   group   housing.     It   is   not   clear   which   would   be   more   detrimental   to   hippocampal  plasticity,  being  aggressively  attacked  or  being  individually  housed.   Our  mRNA   data   do   not   indicate   a   clear   benefit   or   adverse   effect   of   individual   versus   group   housing   in   these  mice   and   no   individually   housed   animals   were   used  in  the  behavioral  tasks.   Another   factor   to   consider   was   that   all   treadmill   running   and   behavioral   testing  occurred  during  the  light  phase  of  the  light-­dark  cycle.    Mice  are  nocturnal   and   perform   the  majority   of   their   running   during   the   dark   cycle.     Having  mice   perform  treadmill  running  and  behavioral  testing  during  the  light  phase  may  have   added  an  additional  level  of  stress  to  the  exercise  that  would  have  been  avoided   if  the  tasks  were  performed  during  the  dark  phase.    Time  of  day  (Hopkins  &  Bucci,   2010)   and   phase   of   light   cycle   (Huynh  et   al.,   2011)   can   influence   behavior   on   anxiety  tasks  and  performing  anxiety  tasks  during  the  light  cycle  can  even  mask   the   anxiogenic   effect   of   chronic   stress   (Huynh   et   al.,   2011);;   however,   we   observed   significant   differences   in   exploratory   behavior   between   exercise   and       174   sedentary  mice,   suggesting   that   if   the   light   cycle   influenced   the   data,   exercise   modulated   this  effect.    The   interaction  of  exercise  and   light-­dark  cycle   requires   further  investigation.     Future   Directions:     Many   questions   were   generated   upon   examining   and   interpreting   the   data   from   this   dissertation   project.     Even   though   there   is   little   evidence   to   suggest   that   acute   exercise   induces   anxiety   in   humans,   mice   are   commonly  used  as  a  model  for  exercise  research,  so  understanding  the  influence   of  acute  exercise  on  anxiety-­like  behavior  is  necessary.    Future  research  should   approach   acute   exercise-­induced   anxiety   with   a   battery   of   anxiety   tests,   potentially   in   both   the   light   and   dark   phases.     To   examine   the   effects   of   truly   acute   bouts   of   exercise   and   avoid   a   training   effect,   these   investigations   will   require   a   large   sample   of   animals   where   each   undergoes   a   session   of   acute   exercise   followed   by   a   behavioral   task.     It   remains   to   be   determined   if   acute   exercise   will   induce   anxiety   in   non-­exploratory   behavior-­dependent   tasks   and   whether  motivation  or  fatigue  is  a  contributing  factor  to  the  anxiety-­like  behavior   we  observed.       Future   research   should   also   attempt   to   address   the   fate   of   the   acute   exercise-­induced   Bdnf   IV   mRNA   expression.     Experiments   designed   to   determine  whether  Bdnf   transcript   IV   is   also   rapidly   translated  or   is   shuttled   to   dendrites  for  later  translation  will  be  valuable  for  understanding  the  importance  of   the  rapid  transcription  of  Bdnf  IV.    In  addition,  the  observations  in  this  dissertation   indicate   that  both  acute  and  chronic  exercise   increase  expression  of   total  Bdnf       175   mRNA   to   a   similar   magnitude,   suggesting   no   additional   benefit   of   multiple   voluntary  wheel  running  sessions  compared  to  one  bout  of  treadmill  exercise.    A   time   course   experiment   to   identify   the   lasting   effects   of   acute   and   chronic   exercise  on  Bdnf  expression  is  warranted.       The   importance   of   this   research   lies   in   its   translatability   to   therapeutic   human  exercise  interventions.  The  finding  in  this  dissertation  that  an  acute  bout   of   exercise   can   increase   transcription   of   Bdnf   even   when   challenged   with   compromised   LC   noradrenergic   signaling   has   important   implications.     Norepinephrine   is   important   for   brain   health   and   plays   an   important   role   in   defending   against   Alzheimer’s   Disease   through   Bdnf-­related   signaling   mechanisms   (Counts   &   Mufson,   2010;;   Liu   et   al.,   2015).     However,   though   norepinephrine   is   important   for  reducing  Alzheimer’s  Disease  pathology,   the  LC   experiences   early   and   rapid   neurodegeneration   in   Alzheimer’s   Disease   (Chalermpalanupap  et   al.,   2013).    More   research   is   needed   to   understand   the   ability   of   acute   high-­intensity   exercise   to   increase   Bdnf   expression   after   LC   lesioning.     Use   of  β -­adrenergic   blockers   such   as   propranolol   will   identify   if   remaining   norepinephrine   from   non-­LC   sources   is   influencing   this   effect.     It   is   possible   that   overcoming   compromised   LC   function   with   acute   exercise   is   intensity   dependent,   which   will   have   important   implications   for   translatability.     This   is   an   extremely   important   area   of   research   and   understanding   the   mechanisms  to  defend  against  and  treat  diseases  such  as  Alzheimer’s  Disease   remains  a   top  priority.     This   dissertation  provides  evidence   that   acute  exercise       176   might   be   an   effective   non-­invasive   therapeutic   technique   to   enhance   brain   plasticity   and   may   be   robust   enough   to   influence   brain   plasticity   even   with   a   compromised  noradrenergic  system.           177   Appendix  A   mRNA  Target   Primer  Sequences     Bdnf  total   Primer  1:  5’  –  CCATAAGGACGCGGACTTGTAC  -­3’   Primer  2:  5’  –  AGACATGTTTGCGGCATCCAGG  -­3’   Bdnf  I   Primer  1:  5’-­  GACACATTACCTTCCTGCATCT  -­3’  Primer  2:  5’-­  GGATGGTCATCACTCTTCTCAC  -­3’   Bdnf  II   Primer  1:  5’-­  GCCTTCATGCAACCGAAGTA  -­3’  Primer  2:  5’-­  GTGGTGTAAGCCGCAAAGA  -­3’   Bdnf  III   Primer  1:  5’-­  GCCTTCATGCAACCGAAGTA  -­3’  Primer  2:  5’-­  GGGCCGGATGCTTCATT  -­3’   Bdnf  IV   Primer  1:  5’-­  CAGAGCAGCTGCCTTGATGTT  -­3’  Primer  2:  5’-­  GCCTTGTCCGTGGACGTTTA  -­3’   Bdnf  VI   Primer  1:  5’-­  GCTGGCTGTCGCACGGTTCCCAGT  -­3’  Primer  2:  5’-­  GAAGTGTACAAGTCCGCGTCCTTA  -­3’   ß-­AR   Primer  1:  5’-­  GCCAGATACAATCCATACCATCA  -­3’  Primer  2:  5’-­  TCGCTATGTTGCTATCACATCG  -­3’     Pgc-­1a   Primer  1:  5’  –  GGTGTCTGTAGTGGCTTGATTC  -­3’   Primer  2:  5’  –  GTTCCCGATCACCATATTCCA  -­3’     tPa  (Plat)   Primer  1:  5’  –  CAACCAAGACCTCCACGA  -­3’   Primer  2:  5’  –  CACATCCTTCTGCCCACA  -­3’   ActB   Primer  1:  5’-­  GAC  TCA  TCG  TAC  TCC  TGC  TTG  –  3’  Primer  2:  5’  –  GAT  TAC  TGC  TCT  GGC  TCC  TAG  –  3’   NR2A  (Grin2a)   Primer  1:  5’  –  TGC  TCA  TCA  CCT  CAT  TCT  TCT  C  –  3’  Primer  2:  5’  –  GAT  TGA  CCT  CGC  TCT  GCT  C  –  3’   NR2B  (Grin2b)   Primer  1:  5’  –  CAC  AAA  CAT  CAT  CAC  CCA  CAC  -­3’  Primer  2:  5’  –  TTG  ACT  TCT  CTG  TGC  CCT  TC  –  3’   GLUR1  (Gria1)   Primer  1:  5’  –  TGG  CGA  GGA  TGT  AGT  GGT  A  –  3’  Primer  2:  5’  –  AAG  AAA  AAG  GAG  AGG  CTG  GTG  –  3’   Gapdh   Primer  1:  5’  –  AAT  GGT  GAA  GGT  CGG  TGT  G  –  3’  Primer  2:  5’  –  GTG  GAG  TCA  TAC  TGG  AAC  ATG  TAG  –  3’             178   Appendix  B.  Neuroreport  (2015)  vol.  26  (8)  pp.  467-­472   Title:   Lifelong   Parental   Voluntary   Wheel   Running   Increases   Offspring   Hippocampal  Pgc-­1α  mRNA  Expression  But  Not  Mitochondrial  Content  or  Bdnf   Expression.   Andrew  C.  Venezia1,2,  Lisa  M.  Guth1,  Espen  E.  Spangenburg1,  and  Stephen  M.   Roth1,2   1   Department   of   Kinesiology,   School   of   Public   Health,   University   of   Maryland,   College  Park,  MD  20742,  USA   2  Neuroscience  and  Cognitive  Sciences  Program,  University  of  Maryland,  College   Park,  Maryland  20742,  USA             179   Abstract   When   exercise   is   initiated   during   pregnancy,   offspring   of   physically   active   mothers  have  higher  hippocampal  expression  of  brain  derived  neurotrophic  factor   (Bdnf)   and   other   plasticity   and   mitochondrial-­associated   genes,   resulting   in   hippocampal   structural   and   functional   adaptations.     In   the   present   study,   we   examined  the  effects  of  lifelong  parental  voluntary  wheel  running  (before,  during,   and   after   pregnancy)   on   offspring   hippocampal   mRNA   expression   of   genes   implicated   in   the   exercise-­induced   improvement   of   cognitive   function.   C57BL/6   mice   were   individually   housed   at   8   weeks   of   age   with   (EX;;   n=20)   or   without   (SED;;  n=20)  access  to  a  computer-­monitored  voluntary  running  wheel  (VRW)  for   12   weeks   prior   to   breeding.   EX   breeders   maintained   access   to   the   VRW   throughout   breeding,   pregnancy,   and   lactation.    Male   offspring  were   housed   in   sedentary  cages,  regardless  of  parental  group,  and  were  sacrificed  at  8  (n=18)  or   28  weeks  (n=19).    PCR  was  used  to  assess  mRNA  expression  of  several  genes   and  mitochondrial  content  (ratio  of  mitochondrial  to  nuclear  DNA)  in  hippocampal   homogenates.     We   found   significantly   higher   peroxisome   proliferator-­activated   receptor   γ   coactivator   1   alpha   (Pgc-­1α)   mRNA   expression   in   EX   offspring   compared   to  SED  offspring  at  8  wks   (p=0.04),   though   the  effect  was  no   longer   present   at   28   wks.     There   was   no   difference   in   mitochondrial   content   or   expression  of  Bdnf  or  any  other  mRNA  targets  between  offspring  at  8  or  28  wks.   In   contrast   to   exercise   initiated   during   pregnancy,   parental   voluntary   physical   activity  initiated  early  in  life  and  maintained  throughout  pregnancy  has  little  effect       180   on   offspring   mRNA   expression   of   genes   implicated   in   exercise-­induced   hippocampal  plasticity.     Key  words   Brain   derived   neurotrophic   factor;;   BDNF;;   PGC-­1;;   Exercise;;   Pregnancy;;   Offspring;;  Hippocampus           181   Introduction       Physical  exercise  during  pregnancy   improves  health-­related  outcomes   in   both  mother  and  offspring  [1-­3].    In  the  pregnant  mother,  exercise  can  reduce  the   risk  of  gestational  diabetes,  preeclampsia,  and  excessive  gestational  weight  gain,   conditions   associated   with   negative   health   outcomes   in   the   offspring   [2].     Remarkably,  exercise  during  pregnancy  also  impacts  offspring  brain  health  both   in   early   postnatal   development   and   adulthood   [4-­11].     In   humans,   in   utero   exercise   exposure   is   associated   with   greater   cognitive   performance   in   early   postnatal  development   [10]  and  higher   intelligence  scores  at  5  years  of  age   [9]   relative  to  children  of  mothers  that  did  not  exercise  during  pregnancy.    Numerous   studies   in   rodents  show  that   in  utero  exercise  exposure   increases  hippocampal   expression   of   brain   derived   neurotrophic   factor   (Bdnf)   [4;;   5;;   7;;   12];;   cell   proliferation  and  neuron  differentiation  [5;;  6];;  increased  mitochondrial  content  and   expression  of  genes  associated  with  mitochondrial  biogenesis  [13];;  and  improved   performance  on  spatial  [4;;  8]  and  non-­spatial  memory  tasks  [5;;  7;;  14].    In  addition   to   the   influence   of   maternal   exercise,   long-­term   paternal   forced   exercise   enhances   male   offspring   neurotrophin   expression   and   spatial   learning   and   memory  performance   [15],   suggesting   transgenerational   inheritance  of  exercise   effects   beyond   direct   in   utero   exposure.     There   is   remarkable   consistency   between  the  effects  of  in  utero  and  adult  exercise  exposure  on  the  hippocampus.     For   example,   adult   exercise   exposure   increases   Bdnf   protein   and   mRNA   expression,   enhances   neurogenesis   and   cell   survival,   increases   mitochondrial   content,   and   improves   learning   and   memory   [reviewed   in   16;;   17].   Further,       182   exercise   lowers   the   risk   of   Alzheimer’s   disease   (AD)   in   humans   and   reduces   pathology   after   disease   onset   in   transgenic   animals   [18].     Similar   results   are   observed  following  in  utero  exercise  exposure  in  AD  transgenic  mice  [19].         To   examine   the   impact   of   parental   exercise   on   offspring   hippocampal   phenotype,   researchers   have   primarily   initiated   maternal   exercise   during   pregnancy,  rather  than  prior  to  gestation.  Exercise  during  pregnancy  is  useful  for   highlighting   the   specific   effect   of   in   utero   exercise   exposure;;   however,   though   beginning  exercise  during  pregnancy  is  recommended  [2],  only  a  low  percentage   of  women   report  being  more  active  during  pregnancy   than  before   [20].    As   the   physical   changes   that   occur   during   pregnancy   favor   a   sedentary   lifestyle,   it   is   more   likely   that  women  who   exercise   regularly  will   continue   to   exercise   during   pregnancy,   while   women   who   are   sedentary   prior   to   pregnancy   will   remain   sedentary.  Thus,  examining  the  impact  of  exercise  prior  to  and  during  pregnancy   on  offspring  phenotypes  is   important  to  understand  the  effectiveness  of   in  utero   exercise  exposure   for  enhancing  brain  health.    For   this   reason  we   investigated   the   influence   of   lifelong   parental   exercise   on   offspring   hippocampal   gene   expression  and  mitochondrial   copy  number  at   two  different  offspring  ages.  Our   gene   targets   were   specifically   selected   based   on   previous   literature   reporting   sensitivity   to   adult   exercise   training   and/or   in   utero   exercise   exposure   and   we   hypothesized  that  mRNA  for  Bdnf  (and  related  processing  and  signaling  markers),   growth   factors,   the   mitochondrial   biogenesis   regulator   peroxisome   proliferator-­ activated   receptor   γ   coactivator   1   α   (Pgc-­1α),   and   synaptic   markers   would   be   elevated  in  offspring  of  lifelong  physically  active  parents.           183   Methods   Animals  and  Experimental  Design     All   animal   procedures   were   performed   in   accordance   with   the   National   Institutes   of   Health   guidelines   and   were   approved   by   the   Institutional   Animal   Care  and  Use  Committee  at   the  University  of  Maryland.    This   investigation  was   part   of   a   larger   investigation   designed   to   examine   whole   body   and   tissue   (skeletal   muscle,   white   adipose,   liver)   metabolic   phenotypes   in   multiple   generations  of   offspring  of   exercised  vs.   sedentary  parents.    The  availability   of   these  mice  offered  a  unique  opportunity  to  test  an  equally  important  yet  unrelated   hypothesis   that   lifelong  parental  physical  activity   increases  plasticity  associated   mRNA  expression  in  offspring  hippocampus.     Twenty   male   and   twenty   virgin   female   eight-­week-­old   C57BL/6J   mice   (Jackson   Laboratories,   Bar   Harbor,   ME,   USA)   were   randomly   separated   into   individual   cages   with   (F0   EX)   or   without   (F0   SED)   access   to   a   computer   monitored   running   wheel   (Lafayette   Instruments,   Lafayette   IN).     Though   individual  housing  may  influence  behavior  and  hippocampal  plasticity  it  does  not   influence   rodent   running   behavior   [21]   and   is   consistent   with   previous   investigations   of   maternal   exercise   [4-­8].   After   12   weeks   of   voluntary   wheel   running,  males  were  randomly  housed  with  females  from  like  groups  (1  male  and   1  female  per  cage;;  EX  with  EX  and  SED  with  SED).    During  the  breeding  period,   both   males   and   females   in   the   EX   mating   group   maintained   running   wheel   access;;   however,   running   activity   could   not   be   monitored   during   this   period.         184   Males  were  removed  after  pregnancy  was  visually  confirmed  by  vaginal  plug  or   after  2  weeks  of  pairing.    EX   females  maintained   running  wheel  access  during   pregnancy  and   lactation.     Two  F0  EX  breeding  pairs   did   not   produce   viable  F1   offspring.  The  resulting  offspring  made  up  the  F1  generation.    Average  litter  size   for  F1  offspring  was  6.1  ±  0.6  EX  and  6.4  ±  0.5  SED  offspring/litter;;  there  was  no   significant  difference  in  litter  size  between  groups.  Litters  with  8  or  fewer  offspring   were   included   for   analysis.   Only   male   offspring   are   presented   due   to   fewer   female  mice  available  from  exercised  parents  compared  to  sedentary  parents.  F1   males  were  weaned  at  21  days  of  age,  group-­housed  in  standard  cages  without   running  wheel  access  and  were  sacrificed  at  8  (n=18)  or  28  weeks  (n=19).    The   animals   sacrificed   at   28   weeks   were   bred   at   8   weeks   of   age   and   individually   housed  thereafter  until  sacrifice.  No  more  than  3  offspring  per  litter  were  studied   per   age   group.    A   standard   diet   (Purina  Mills   LLC,   St.   Louis,  MO,  USA;;  RMH   3000;;  60%  carbohydrate,  14%  fat,  and  26%  protein)  and  water  were  provided  ad   libitum  to  animals  of  all  experimental  groups.   Tissue  Collection  &  Processing   Twenty-­four  hours  prior   to  sacrifice,  all  F1  mice  were  exposed   to   intraperitoneal   glucose  tolerance  testing.    This  procedure  was  performed  to  address  the  overall   hypothesis   of   the   investigation,   though   the   data   will   not   be   discussed   in   this   report.    Euthanasia  by  exsanguination  by  cardiac  puncture   followed  by   removal   of   the  heart  was  performed  under   isoflurane  anesthesia.  The  hippocampus  was   isolated,  halved,  and  immediately  frozen  in  liquid  nitrogen.    Prior  to  nucleic  acid       185   isolation,   hippocampi   were   homogenized   using   a   glass   Dounce   homogenizer.     Total  RNA  was  isolated  with  TRIzol  reagent  (Life  Technologies,  Grand  Island,  NY,   USA)  following  manufacturers  instructions  and  quantified  via  spectrophotometry.     Reverse   transcription   was   performed   with   1   μg   of   total   RNA   with   the   High-­ Capacity   cDNA  RT   kit   (Life   Technologies).     Following  RNA   isolation  DNA  was   isolated  from  TRIzol  reagent  and  quantified  via  spectrophotometry.       Gene  Expression   Real-­time  quantitative  PCR  was  used  to  assess  mRNA  expression  of  total  brain   derived   neurotrophic   factor   (Bdnf;;   exon   IX),   Bdnf   exon   IV   (Bdnf   IV),   Pgc-­1α,   tissue   plasminogen   activator   (tPa),   and   glyceraldehyde-­3-­phosphate   dehydrogenase  (Gapdh;;  expression  control).    Primer  and  probe  sequences  were   purchased  pre-­made  (Pgc-­1α,  tPa,  Gapdh)  or  designed  (Bdnf  IX,  Bdnf  IV)  for  the   mRNA  sequence  of  each  gene  using   Integrated  DNA  Technologies’  PrimeTime   qPCR  Assay   designer.     Primer   sequences   are   listed   in  Supplemental   Table   1.     Bdnf   IV,   Pgc-­1α,   tPa,   and   Gapdh   primer   pairs   spanned   exons   to   prevent   amplification  of  genomic  DNA.  Because  Bdnf  total  is  represented  by  amplification   of  exon  IX,  the  protein  coding  exon  that  is  present  in  each  transcript,  this  primer   pair   could   not   span   exons.     Efficiency   for   each   primer:probe   assay   was   determined  prior  to  use.    RT-­PCR  was  used  to  measure  the  expression  of  insulin   like  growth  factor  1  (Igf1),  vascular  endothelial  growth  factor  (Vegf),  neurotrophic   tyrosine   kinase   receptor   type   2   (TrkB),   calpactin   (p11),   synapsin   1   (Syn1),   synaptobrevin   (Vamp2),   synaptotagmin   1   (Syt1),   and   synaptophysin   (Syp).         186   Gapdh   was   used   as   an   expression   control   for   RT-­PCR.     All   RT-­PCR   primers   were  designed  to  span  exons.       Mitochondrial  Copy  Number   DNA  was  subjected  to  real-­time  quantitative  PCR  and  comparison  of  β-­actin  and   cytochrome   b   amplification   was   used   to   determine   the   relative   amounts   of   nuclear  and  mtDNA,   respectively.    These  primer:probe  assays  were  purchased   pre-­made   from  Integrated  DNA  Technologies  and  efficiency   tested  prior   to  use.     Primer  sequences  are  listed  in  Supplemental  Table  1.   Statistical  Analysis   Unpaired   t-­tests  were  used   to  compare  gene  expression  between  EX  and  SED   groups   within   age   using   SAS   version   4.2.     One-­tailed   t-­tests   were   used   to   examine  Bdnf  mRNA  expression,  Pgc-­1α  mRNA  expression,  and  mitochondrial   copy  number.    Two-­tailed  t-­tests  were  used  to  examine  all  other  mRNA  targets.   Significance  was  achieved  at  p<0.05.   Results   F0  Wheel  Activity     Running   data   for   the   F0   breeders   are   shown   in   Figure   1.     Peak   running   was   achieved  during  week  4  (6689  meters/24hrs)   for  males  and  week  2   for   females   (7209  meters/24hrs).    In  males  there  was  a  steady  decline  in  running  activity  until   the   final   week,   when   the   lowest   running   distances   were   recorded   (1505       187   meters/24hrs).     In   females,   lowest  activity  was  recorded  during  pregnancy   (347   meters/24hrs).     Running   activity   increased   to   pre-­pregnancy   levels   following   pregnancy.       F1  Eight-­Week  Offspring  Outcomes   Hippocampal  gene  expression  data  for  F1  8-­week  offspring  are  shown  in  Figure  2.     Eight-­week   old   offspring   of   EX   parents   had   significantly   higher  Pgc-­1α   mRNA   expression   compared   to   offspring   of   SED   parents   (p=0.04,   Fig.   2a).     We   observed  no  significant  differences  between  offspring  of  EX  and  SED  parents  in   any  other  targets  measured  (Fig.  2b).    There  was  no  effect  of  parental  exercise   on   offspring  Gapdh   expression   (confirmed  with   both   qPCR   and   gel-­based  RT-­ PCR).     We   also   observed   no   differences   between   offspring   of   EX   and   SED   parents  in  mitochondrial  copy  number  (Figure  3).     F1  Twenty-­Eight  Week  Offspring  Outcomes   Hippocampal  gene  expression  data  for  F1  28  week  are  shown  in  Figure  4.    The   difference  in  Pgc-­1α  mRNA  expression  observed  in  8-­week  old  animals  was  no   longer   present   in   the   28-­week   old   offspring.    We   observed   no   differences   any   mRNA  target  between  28-­week  old  offspring  of  EX  and  SED  parents.  There  was   no   effect   of   parental   exercise   on   offspring  Gapdh   expression   (confirmed   with   both   qPCR   and   gel-­based   RT-­PCR).     We   observed   no   differences   in   mitochondrial   copy   number   between   28-­week-­old   offspring   of   EX   and   SED   parents  (Fig.  3).           188   Discussion     We   report   here   that   parental   exercise   training   prior   to,   during,   and   after   (lactation)  gestation  results  in  greater  hippocampal  Pgc-­1α  gene  expression  at  8   weeks  of  age  in  male  EX  offspring  that  returns  to  baseline  by  28  weeks  of  age.     This  change  in  Pgc-­1α  expression  was  not  accompanied  by  higher  mitochondrial   copy   number,   as   might   be   expected   based   on   the   known   role   of   Pgc-­1α   in   mitochondrial   biogenesis   [22;;   23].   In   contrast   to   previous   studies   initiating   exercise  during  pregnancy,  we  observed  no  significant  difference  in  Bdnf  mRNA   expression  between  offspring  of  EX  and  SED  parents  at  8  or  28  weeks  of  age.         Maternal   exercise   beginning   during   pregnancy   has   numerous   health   benefits   to  offspring.     In   the  offspring  hippocampus,  maternal  exercise  (whether   swimming,   treadmill   running,   or   voluntary   wheel   running)   beginning   during   pregnancy  leads  to  changes  in  Bdnf  mRNA  and  protein  expression  [4;;  5;;  7;;  12],   enhanced  learning  and  memory  performance  [4;;  5;;  7;;  8;;  14],  neurogenesis  [5;;  6],     mitochondrial   biogenesis,   and   mRNA   expression   of   genes   associated   with   mitochondrial   biogenesis   and   oxidative   metabolism   [13].     We   observed   that   maternal   and   paternal   exercise   beginning   early   in   life   and   continuing   through   mating,   gestation,   and   lactation   resulted   in   no   difference   in   male   offspring   hippocampal  mRNA  expression  of  any  of  our  targets  with  the  exception  of  Pgc-­ 1α.    Pgc-­1α  was  elevated  at  8  weeks  in  offspring  of  EX  parents,  though  returned   to  baseline  by  28  weeks.    Pgc-­1α  is  a  co-­transcription  factor  that  is  considered  a   regulator  of  mitochondrial  biogenesis.    When  co-­expressed  with  other  tissue-­  and       189   temporal-­specific   transcription   factors,   Pgc-­1α   stimulates   the   transcription   of   genes   necessary   for   mitochondrial   biogenesis   [24].     Pgc-­1α   expression   is   induced  in  many  tissues,  including  the  brain,  in  response  to  physical  exertion  [17;;   23].     Using   three   different   exercise   durations,   Park   et   al.   [13]   showed   that   offspring   of   treadmill-­exercised   mothers   had   more   hippocampal   mitochondria,   determined   by   measuring   mitochondrial   DNA   relative   to   nuclear   DNA,   and   greater   levels   of   Pgc-­1α   protein   expression.     This   effect   was   observed   in   offspring  of  mothers  who  exercised  the  longest  of  three  exercise  durations.    Even   though  we  observed  a  significant  effect  of  parental  exercise  on  offspring  Pgc-­1α   expression   at   8   weeks,   we   did   not   observe   an   accompanying   increase   in   mitochondrial   content   at   8  or   28  weeks.    Potentially,  we  missed   the  window  of   observation  for  a  change  in  mitochondrial  content  that  took  place  between  8  and   28  weeks  before  returning  to  baseline.       Based   on   previous   literature,   we   hypothesized   that   parental   physical   activity   prior   to,   during,   and   after   gestation   would   result   in   an   increase   in   neurotrophin  and  growth  factor  mRNA  expression,  however  this  hypothesis  was   not   supported.     A   number   of   studies   have   reported   an   effect   of   maternal   and   paternal   exercise   on   offspring   hippocampal  Bdnf   expression,   though   how   long   this  effect  persists  still   remains   to  be  determined.    Parnpiansil  et  al.   [4]  showed   that  treadmill  exercise  in  pregnant  rats  resulted  in  greater  offspring  hippocampal   Bdnf  mRNA  expression  at  post-­natal   (PN)  day  0   (PN0),  no  difference  at  PN14,   and  significantly   lower  Bdnf  expression  at  PN28.     In  contrast,  Kim  et  al.   [7]  and   Lee  et   al.   [5]   showed   that  Bdnf  mRNA  expression  was  elevated   in  offspring  of       190   treadmill-­   and  swim-­trained  mothers,   respectively,   at   29  days  after   birth.    Even   though  these  studies  reported  significantly  greater  Bdnf  expression  in  offspring  of   exercise  mothers   compared   to   offspring   of   sedentary  mothers   one  month   after   birth,   our   youngest   time   point   was   two  months   of   age  which  may   explain  why   even   in   our   youngest   group   of   offspring,   we   observed   no   differences   in   neurotrophin  (Bdnf)  or  growth  factor  (Igf1  and  Vegf)  mRNA  expression.  Yin  et  al.   [15]  reported  enhanced  Bdnf  expression  (mRNA  and  protein)  and  spatial  memory   performance   in   ~one-­month   old   male   offspring   of   treadmill   exercised   male   C57BL/6J   mice.     Again,   this   is   much   younger   than   our   youngest   group,   suggesting   the   influence   of   parental   exercise   is   relatively   short-­lived.     It   is   important   to  note   that   the  offspring   in   these  studies,   and   in  our   study,  were  all   sedentary   and   it   is   possible   that   offspring   of   physically   active   parents   would   maintain  the  benefits  of  parental  physical  activity  if  they  themselves  are  provided   access  to  exercise.       Though  we  did  not  observe  differences   in   synaptic   (Syn1,  Vamp2,  Syt1,   Syp),   neurotrophin   (Bdnf,   Bdnf   IV),   neurotrophin   receptor   (TrkB),   posttranslational   processor   (tPa,   p11),   or   growth   factor   (Vegf,   Igf1)   mRNA   expression,  structural  adaptations  may  have  occurred  that  were  not  represented   by  differences   in  mRNA  expression.    Bick-­Sander  et  al   [6]   reported  differences   between   offspring   of   exercise   (beginning   during   pregnancy)   and   sedentary   mothers   in  neuron  survival  and   total  granule  cell   numbers  at  PN36,  despite  no   significant  differences   in  mRNA  expression  of  Vegf,  Fgf-­2,  or   Igf1.    These  data   suggest  lasting  structural  changes  that  are  not  mirrored  by  lasting  differences  in       191   mRNA   expression.   Future   studies   are   needed   to   determine   if   lifelong   parental   physical  activity  influences  hippocampal  structure  and  function  in  offspring  in  the   absence  of  differences  in  mRNA  expression.     In   addition   to   the   age   at   which   the   offspring   were   examined,   another   difference   between   our   study   and   previous   studies   is   the   timing   and   mode   of   exercise  exposure.    We  provided  mice  with  a  running  wheel  for  12  weeks  prior  to   mating  and  pregnancy.    Previous  studies  have  used  forced  exercise  modes  such   as  swimming  or  treadmill  running,  which  add  additional  stress,  but  also  have  the   benefit   of   controlled   exercise   duration   (swimming   and   treadmill)   and   intensity   (treadmill).     Studies   that   have   used   voluntary   wheel   running   have   initiated   exercise   during   pregnancy.     In   addition   to   the   influence   of   novelty/enrichment,   mice   are   very   active   in   the   first   few   weeks   of   wheel   exposure,   with   declining   activity   thereafter   (Fig   1).     Compared   to   Bick-­Sander   et   al.   [6],   who   used   the   same  strain  of  mice  as  our  study,  our  pregnant  mice   ran  much   less.    Between   days  5  and  10  of  pregnancy,  mice   in   the  Bick-­Sander  study   ran  between  2500   and   3000   meters/day,   while   in   our   study,   pregnant   mice   ran   less   than   700   meters/day   (see   Fig   1).     The   dependence   of   the   exercise   volume   is   also   supported  by   investigations  using   forced  exercise.  Park  et  al.   [13]   reported   that   offspring   of   the   most   active   pregnant   mice   were   significantly   different   than   controls  when  hippocampal  mitochondrial  content,  mitochondrial  enzyme  activity,   and  Pgc-­1α  protein  expression  were  assessed.           192     A   limitation  of   this   investigation  was   the  exposure  of  offspring   to  glucose   tolerance   testing,   which  may   have   added   additional   stress   to   the   animals   and   influenced   hippocampal   gene   expression.   Weekly   handling   of   our   mice   to   prepare   them   for   the   stress   of   the   GTT   likely   reduced   the   stress   response,   though  we   cannot   rule   out   an   influence   of   the   procedure   on   gene   expression.   Another   limitation   of   our   study   is   that  we   did   not   observe   and   record  maternal   care,  which  can  strongly  influence  hippocampal  development  and  plasticity  [25].   Though  not  a  limitation  of  our  study,  it  is  possible  that  providing  offspring  access   to   a   voluntary   running   wheel   may   expose   an   exercise-­induced   adaptation   to   parental  exercise,  which  provides   these  mice  with  a  plasticity  advantage  above   offspring  of  sedentary  parents.    This   is  a  question   that  should  be  addressed   in   future  investigations.       We   believe   that   our   data,   in   the   light   of   previously   reported   findings,   suggest  a  high  volume  and/or  intensity  of  physical  activity  during  pregnancy  must   be  maintained   to  observe   the  beneficial   effects  of  maternal   physical   activity   on   hippocampal   plasticity-­associated   gene   expression   and   mitochondrial   copy   number.    The  steady  decrease  in  voluntary  wheel  running  observed  over  the  12   weeks  prior   to  mating,   in   both   parents,   and  during   pregnancy   in  mothers,  may   have   prevented   the   strong   effect   observed   in   other   investigations.     Treadmill   exercise  may  be  more  effective  at  maintaining  intensity  and  volume  prior  to  and   during  pregnancy.               193   Competing  interest   None  declared   Funding   This  work  was   supported   by  NIH   grant  HD062868,   The  College   of  Health   and   Human   Performance   Public   Health   Research   Seed   Money   Program   award   (S.M.R.  and  E.E.S.),  and  NIH  T32  AG000268  (A.C.V  and  L.M.G.).   Authors’  contributions.   ACV,   LMG,   EES,   and   SMR   designed   the   study;;   ACV   and   LMG   collected   the   data;;  data  analysis,  preparation  of  figures,  and  drafting  the  manuscript  was  done   by  ACV;;  ACV,  LMG,  EES,  and  SMR  edited  and  revised  this  manuscript;;  and  all   authors  approved  the  final  version.             194   References  Cited   1.   Clapp  JF  III.  Exercise  during  pregnancy:  a  clinical  update.  Clinics  in  sports   medicine  2000;;   2.   Committee  OPAGA.  U.S.  Department  of  Health  and  Human  Services  and   Physical  Activity  Guidelines  Advisory  Committee.  2008;;  1–683.   3.   Downs  DS,  Chasan-­Taber  L,  Evenson  KR,  Leiferman  J,  Yeo  S.  Physical   Activity  and  Pregnancy.  Res  Q  Exerc  Sport  2012;;  83  (4):  485–502.   4.   Parnpiansil  P.  Exercise  during  pregnancy  increases  hippocampal  brain-­ derived  neurotrophic  factor 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 self-­reported  physical  activity  and   sedentary  behaviors  among  pregnant  women:  NHANES  1999–2006.   Preventive  Medicine  2010;;  50  (3):  123–128.   21.   Stranahan  AM,  Khalil  D,  Gould  E.  Social  isolation  delays  the  positive   effects  of  running  on  adult  neurogenesis.  Nat.  Neurosci.  2006;;  9  (4):  526– 533.   22.   Scarpulla  RC,  Vega  RB,  Kelly  DP.  Transcriptional  integration  of   mitochondrial  biogenesis.  Trends  in  Endocrinology  &  Metabolism  2012;;       196   23.   Arany  Z.  PGC-­1  coactivators  and  skeletal  muscle  adaptations  in  health  and   disease.  Current  Opinion  in  Genetics  &  Development  2008;;  18  (5):  426– 434.   24.   Finck  BN.  PGC-­1  coactivators:  inducible  regulators  of  energy  metabolism   in  health  and  disease.  Journal  of  Clinical  Investigation  2006;;  116  (3):  615– 622.   25.   van  Hasselt  FN,  Cornelisse  S,  Yuan  Zhang  T,  Meaney  MJ,  Velzing  EH,   Krugers  HJ,  et  al.  Adult  hippocampal  glucocorticoid  receptor  expression   and  dentate  synaptic  plasticity  correlate  with  maternal  care  received  by   individuals  early  in  life.  Hippocampus  2011;;  22  (2):  255–266.                 197   Figure  1.    Average  running  activity  of  mice.    Data  are  shown  as  average  distance   run  over  24  hours  per  week.    Running  data  were  not  collected  during  mating  due   to   the   presence   of   two   mice   in   the   cage   and   the   inability   to   determine   which   mouse  was  using  the  running  wheel.    The  litters  were  delivered  between  23  and   24  weeks  on  the  timeline.    There  was  a  six-­day  span  between  first  and  last  litter,   thus   pre-­weaned   mice   had   access   to   the   wheel.    We   cannot   rule   out   the   possibility   that   pre-­weaned  mice  are   contributing   to   recorded  wheel   revolutions   during  week  27.     Figure  2  (A-­B).    Whole  hippocampal  homogenate  mRNA  levels  in  F1  8  week  old   male  offspring  of  exercise  (n=10)  and  sedentary  (n=8)  parents.    Bars  represent   mean  (±SEM)  mRNA  expression  relative  to  Gapdh  mRNA  expression.    *  denotes   significance  at  p<0.05.   Figure  3.    Mitochondrial  DNA  content  in  F1  8  wk  and  28wk  old  male  offspring  of   exercise  and  sedentary  parents.    There  were  no  significant  differences  in  CytB   mitochondrial  DNA  content  relative  to  ActB  DNA  content  between  offspring  of   exercise  and  sedentary  parents  at  8  weeks  or  28  weeks  of  age.    Results  are   means  ±SEM.       Figure   4   (A-­B).    Whole  hippocampal  homogenate  mRNA   levels   in  F1  28  week   old   male   offspring   of   exercise   (n=9)   and   sedentary   (n=10)   parents.     Bars   represent  mean  (±SEM)  mRNA  expression  relative  to  Gapdh  mRNA  expression.     *  denotes  significance  at  p<0.05.   Supplemental   Table   1.     Primer   sequences   for   genes   of   interest.         198   Figure  1.                     0.0 1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 8 9 10 11 12 13 14 15 16 17 18 19 22 23 24 25 26 27 M et er s/ 24 h rs Age (weeks) Male Female     199   Figure  2.                 * 0 0.5 1 1.5 2 Bdnf Bdnf IV Pgc1a tPa Re la tiv e m R N A ex pr es si on (2 -Δ Δ C t )! F1!8wk! Exercise Offspring Sedentary Offspring 0 0.5 1 1.5 2 Igf 1 Ve gf p1 1 Trk B Sy p Sy t1 Va mp 2 Sy n1 R el at iv e m R N A ex pr es si on (A U )! A B     200       Figure  3.         0 0.5 1 1.5 2 8wk 28wk C yt B re la tiv e to A ct B (2 -Δ Δ C t ) Exercise Offspring Sedentary Offspring     201   Figure  4.         0 0.5 1 1.5 2 Bdnf Bdnf IV Pgc1a tPa Re la tiv e m R N A ex pr es si on (2 -Δ Δ C t )! F1!28wk! Exercise Offspring Sedentary Offspring 0 0.5 1 1.5 2 Igf 1 Ve gf p1 1 Trk B Sy p Sy t1 Va mp 2 Sy n1 R el at iv e m R N A ex pr es si on (A U )! A B     202   Supplemental  Table  1.     mRNA  Target   Primer  Sequences     Bdnf  total   Primer  1:  5’  –  CCATAAGGACGCGGACTTGTAC  -­3’   Primer  2:  5’  –  AGACATGTTTGCGGCATCCAGG  -­3’     Bdnf  IV   Primer  1:  5’-­  CAGAGCAGCTGCCTTGATGTT  -­3’   Primer  2:  5’-­  GCCTTGTCCGTGGACGTTTA  -­3’     Pgc-­1a   Primer  1:  5’  –  GGTGTCTGTAGTGGCTTGATTC  -­3’   Primer  2:  5’  –  GTTCCCGATCACCATATTCCA  -­3’     tPa  (Plat)   Primer  1:  5’  –  CAACCAAGACCTCCACGA  -­3’   Primer  2:  5’  –  CACATCCTTCTGCCCACA  -­3’   Syt1   Forward:  5’-­  CGG  TCC  TCG  CTC  CAG  TTT  CCC  –  3’  Reverse:  5’  –  CGG  GAC  GGC  AAG  GGC  AAT  GT  –  3’   Syp   Forward:  5’  –  GCC  GAC  TGG  GCT  GTT  CCG  AC  –  3’  Reverse:  5’  –  CCC  CCA  GCC  ACC  AGC  TGA  TTC  –  3’   Vamp2   Forward:  5’  –  CAG  GCC  CAG  GTG  GAT  GAG  GTG  GT  -­3’  Reverse:  5’  –  CTG  GAG  GGC  ATC  TGC  ACG  GTC  –  3’   TrkB   Forward:  5’  –  GCA  TGA  AAG  GCC  CAG  CTT  CGG  T  –  3’  Reverse:  5’  –  GGG  ACC  GCC  CTC  CGA  AGA  AGA  –  3’   Vegf   Forward:  5’  –  GCC  GAG  CTC  ATG  GAC  GGG  TG  –  3’  Reverse:  5’  –  GGT  GCA  GCC  TGG  GAC  CAC  TTG  –  3’   Gapdh   Forward:  5’  –  TCA  AGC  TCA  TTT  CCT  GGT  ATG  ACA  –  3’  Reverse:  5’  –  TCT  TGC  TCA  GTG  TCC  TTG  CT  -­3’   Syn1   Forward:  5’  –  GCC  AAT  GGT  GGA  TTC  TCT  GT  -­3’  Reverse:  5’  –  CAG  CAC  AAA  GTC  TGG  CTT  CA  -­3’   Igf1   Forward:  5’  –  TGG  ATG  CTC  TTC  AGT  TCG  TG  –  3’  Reverse:  5’  –  GCA  ACA  CTC  ATC  CAC  AAT  GC  –  3’   p11   Forward:  5’  –  GCG  TAC  AAA  GAC  CGC  CGG  TC  -­  3’  Reverse:  5’  –  GTC  GAA  ACC  TGG  GCC  CCG  AAG  -­3’    β-­actin   Primer  1:  5’  –  CTTGATCTTCATGGTGCTAGGAG  -­3’  Primer  2:  5’  –  CGTTGACATCCGTAAAGACCT  -­3’   Cytochrome  B   Primer  1:  5’  –  TATTCCTTCATGTCGGACGA  -­3’  Primer  2:  5’  –  AAATGCTGTGGCTATGACTG  -­3’           203   Appendix  C   Andrew  Carmen  Venezia   Avenezia@umd.edu   University  of  Maryland   School  of  Public  Health   College  Park,  MD  20742   (607)  765-­7897     Education     January  2011  to  present   Ph.D.  Neuroscience  and  Cognitive  Science   University  of  Maryland   Department  of  Kinesiology   Mentors:  Dr.  Stephen  Roth  (Kinesiology)  &  Dr.  Elizabeth  Quinlan  (Biology)   Dissertation  title:    Effects  of  Acute  and  Chronic  Exercise  on  Markers  of   Hippocampal  Plasticity  and  Behavior       August  2009  to  December  2010  (transferred  to  Neuroscience  and  Cognitive   Science  Program)   Ph.D.  Program  in  Kinesiology   University  of  Maryland   Mentor:  Dr.  Stephen  Roth     January  2008  to  May  2009   M.S.  Exercise  Science   Bloomsburg  University   Master’s  thesis  mentor:  Dr.  Eric  Rawson   Title:  Physical  Activity  “Dose”  and  Cognitive  Processing  in  Older  Adults.     August  2003  to  December  2007   B.S.  Exercise  Science   Bloomsburg  University     Publications     Refereed  Research  Articles:     1.   Venezia,  A.C.,  Guth,  L.M.,  Sapp  R.  M.,  Spangenburg,  E.E.,  Roth,  S.M.   Sex-­dependent  and  independent  effects  of  long-­term  voluntary  wheel   running  on  hippocampal  Bdnf  expression.  Submitted  to  Physiology  and   Behavior.         204   2.   Venezia  A.C.,  Guth  L.M.,  Spangenburg  E.E.,  Roth  S.M.  (2015).  Lifelong   Parental  Voluntary  Wheel  Running  Increases  Offspring  Hippocampal  Pgc-­1α   mRNA  Expression  But  Not  Mitochondrial  Content  or  Bdnf  Expression.   Neuroreport  26  (8):  467-­72.     3.   Guth  L.M.,  Ludlow  A.T.,  Witkowski  S.,  Lima  L.,  Marshall  M.R.,  Venezia   A.C.,  Xiao  T.,  Lee  M.L.,  Spangenburg  E.E.,  Roth  S.M.  (2013).  Sex-­ Specific  Effects  of  Exercise  Ancestry  on  Metabolic,  Morphological,  and   Gene  Expression  Phenotypes  in  Multiple  Generations  of  Mouse  Offspring.   Exp  Physiol  98  (10):  1469–1484.     Invited  Reviews:     1.   Venezia  A.C.,  Roth  S.M.  (2015).  Recent  research  in  the  genetics  of   exercise  training  adaptation  Medicine  and  Sport  Science  -­  'Genetics  and   Sports',  2nd  revised  and  extended  edition.    In  Press.     2.   Rawson  E.S.,  Venezia  A.C.  (2011).    Use  of  Creatine  in  the  Elderly  and   Evidence  for  Effects  on  Cognitive  Function  in  Young  and  Old.    Amino   Acids  40(5):1349-­62.       In  Preparation:     1.   Venezia,  A.C.,  Quinlan,  E.M.,  Roth  S.M.    The  Influence  of  Acute  and   Chronic  Exercise  on  Hippocampal  GluR1  and  Bdnf  Transcript  Expression.     In  preparation  for  Genes,  Brain  and  Behavior.     2.   Venezia,  A.C.,  Hyer,  M.M.,  Glasper  E.R.,  Roth  S.M.,  Quinlan,  E.M.  The   Effect  of  One  Bout  of  Acute  Exercise  on  AMPA  Receptor  Phosphorylation,   Bdnf  Expression,  and  Behavior.  In  preparation  for  Genes,  Brain  and   Behavior.     3.   Guth  L.M.,  Venezia  A.C.,  Marini  M.P.,  Beltran,  E.P.,  Spangenburg,  E.E.,   Roth  S.M.  Effects  of  Exercise  Ancestry  on  Metabolic,  Morphological,  and   Gene  Expression  Phenotypes  in  Multiple  Generations  of  Mature  Mouse   Offspring.    In  preparation  for  Experimental  Physiology.   Presentations     Invited  Presentations:     1.   Venezia,  A.C.    Acute  Exercise  May  Induce  Hippocampal  Plasticity…Or   Anxiety.    NACS  Research  Day,  University  of  Maryland         205   2.   Panel  Discussion  with:  Clevenger  S.,  Venezia,  A.C.,  Patel  P.  Science,   Social  Responsibility,  and  Crisis.  8th  Annual  Physical  Cultural  Studies   Graduate  Student  Conference:  “Bodies,  Science  and  Technology,”   University  of  Maryland     Research  Presentations:     1.   Steele,  C.N.,  Fradkin,  A.J.,  Andreacci,  J.L.,  Venezia,  A.C.,  Rawson,  E.S.   Effects  of  Age  and  Sex  on  Muscle  Function  During  Isovelocity   Contractions.    Slide  presentation.    Annual  Meeting  of  the  Mid-­Atlantic   Chapter  of  the  American  College  of  Sports  Medicine,  October  2014,   Harrisburg,  PA.     2.   Venezia,  A.C.,  Guth,  L.M.,  Spangenburg,  E.E.,  Roth,  S.M.  Sex-­dependent   and  independent  effects  of  long-­term  voluntary  wheel  running  on   hippocampal  gene  expression.    Poster  presentation,  American  College  of   Sports  Medicine  Annual  Meeting.  June  2014,  Orlando,  FL.     3.   Kayes,  M.  K.,  Venezia,  A.C.,  Sprenger,  A.M.,  Roth,  S.M.,  Dougherty,   M.R.,  Bolger,  D.J.,  Hatfield,  B.D.,  Variability  in  Learning  in  Adults   Explained  by  Cardiovascular  Fitness,  Physical  Activity,  and  APOE   Genotype.  Poster  presentation,  American  College  of  Sports  Medicine   Annual  Meeting.  June  2014,  Orlando,  FL.     4.   Venezia,  A.C.,  Guth,  L.M.,  Marini,  M.P.,  Smith,  J.C.,  Spangenburg,  E.E.,   Roth,  S.M.  Impact  of  Parental  Voluntary  Wheel  Running  on  Offspring   Hippocampal  Gene  Expression  in  C57BL/6  Mice.  Poster  presentation,   Neuroscience  2012,  October  2012,  New  Orleans,  LA.         5.   Venezia,  A.C.,  Guth,  L.M.,  Marini,  M.P.,  Beltran,  E.P.,  Spangenburg,  E.E.,   Roth,  S.M.  Effects  of  Parental  Physical  Activity  on  Hippocampal  Gene   Expression  in  C57BL/6  Mice.    Poster  presentation,  American  College  of   Sports  Medicine  Annual  Meeting.  June  2012,  San  Francisco,  CA.     6.   Guth,  L.M.,  Venezia,  A.C.,  Marini,  M.P.,  Beltran,  E.P.,  Spangenburg,  E.E.,   Roth,  S.M.  Effects  of  Physical  Activity  Ancestry  on  Aspects  of  Body   Composition  and  Glucose  Tolerance  in  Mice.    Poster  presentation,   American  College  of  Sports  Medicine  Annual  Meeting.  June  2012,  San   Francisco,  CA.     7.   Marini,  M.P.,  Guth,  L.M.,  Venezia,  A.C.,  Beltran,  E.P.,  Spangenburg,  E.E.,   Roth,  S.M.  Effects  of  Chronic  Exercise  on  DNA  Methyltransferase   Expression  in  Mouse  Testes.    Poster  presentation,  American  College  of   Sports  Medicine  Annual  Meeting.  June  2012,  San  Francisco,  CA.           206   8.   Rawson,  E.S.,  Venezia,  A.C.,  Still,  C.D.  No  Adverse  Effects  Associated   with  Low-­Dose  Longer-­Duration  Creatine  Supplementation  in  Older   Adults.    Poster  presentation,  American  College  of  Sports  Medicine  Annual   Meeting.    May  2012,  San  Francisco,  CA.     9.   Guth  L.M.,  Ludlow  A.T.,  Witkowski  S.,  Marshall  M.R.,  Lima  L.,  Venezia   A.C.,  Xiao  T,  Lee  M-­L.T.,  Spangenburg  E.E.,  and  Roth  S.M.  Exercise   ancestry  decreases  lipogenesis-­related  gene  expression  in  skeletal   muscle  of  male  offspring.  Oral  and  poster  presentation,  Experimental   Biology  2011,  March  2011,  Washington,  D.C.  FASEB  J  25:862.3.       10.  Venezia,  A.C.,  Ludlow,  A.T.,  Witkowski,  S.,  Marshall,  M.R.,  Spangenburg,   E.E.,  Roth,  S.M.,  Effect  of  one  year  of  voluntary  wheel  running  on   transcript  specific  hippocampus  Bdnf  gene  expression.  Poster   presentation,  ACSM’s  Conference  on  Integrative  Physiology  of  Exercise,   September  2010,  Miami  Beach,  FL.     11.  Guth  L.M.,  Ludlow  A.T.,  Witkowski  S.,  Marshall  M.R.,  Lima  L.,  Perret  K.,   Caffes  N.,  Venezia  A.C.,  Spangenburg  E.E.,  and  Roth  S.M.   Transgenerational  effects  of  physical  activity  ancestry  on  mouse  body   composition,  glucose  metabolism,  and  gene  expression.  Poster   presentation,  ACSM’s  Conference  on  Integrative  Physiology  of  Exercise,   September  2010,  Miami  Beach,  FL.     12.  Venezia,  A.C.,  Ludlow,  A.T.,  Witkowski,  S.,  Marshall,  M.R.,  Spangenburg,   E.E.,  Roth,  S.M.,  Effect  of  one  year  of  voluntary  wheel  running  on   transcript  specific  hippocampus  Bdnf  gene  expression.    Slide   Presentation,  Department  of  Kinesiology  Annual  Exercise  Physiology   Retreat,  August  2010,  Beltsville,  MD       13.  Venezia,  A.C.,  Smoliga,  J.,  Still,  C.D.,  Rawson,  E.S.    Are  Older  Adults   Less  Physically  Active  than  Young  Adults?  Presented  updated  data  as  a   free  communication  slide  presentation  at  the  Annual  Meeting  of  the  Mid-­ Atlantic  Chapter  of  the  American  College  of  Sports  Medicine,  November   2009,  Harrisburg,  PA.     14.  Venezia,  A.C.,  Wierzbicki,  J.S.,  Reitmeyer,  D.,  Shick,  A.,  Still  C.D.,   Rawson,  E.S.  Physical  Activity  and  Cognitive  Processing  in  Older  Men   and  Women.  Poster  presentation,  American  College  of  Sports  Medicine   National  Conference,  May  2009,  Seattle,  WA.       15.  Venezia,  A.C.,  Wierzbicki  J.  S.,  Reitmeyer  D,  Schick  A,  Rawson  E.S.,   Physical  Activity  and  Cognitive  Processing  in  Older  Men  and  Women.     Free  communication  slide  presentation,  Annual  Meeting  of  the  Mid-­Atlantic       207   Chapter  of  the  American  College  of  Sports  Medicine,  November  2008,   Harrisburg,  PA.       16.  Wierzbicki,  J.S.,  Reitmeyer,  D.,  Shick,  A.,  Venezia,  A.C.,  Still,  C.D.,   Rawson,  E.S.  Are  Older  Adults  Less  Physically  Active  than  Young  Adults?   Presented  for  Jackie  Wierzbicki  as  a  free  communication  slide   presentation,  Annual  Meeting  of  the  Mid-­Atlantic  Chapter  of  the  American   College  of  Sports  Medicine,  November  2008,  Harrisburg,  PA.       Chaired  Sessions:     1.   Venezia,  A.C.  (Chair)  Genetics  of  Physical  Activity,  Exercise  Training,   and  Sport  Performance.    MARC-­ACSM  Annual  Meeting,  Harrisburg,   PA,  2015     2.   Venezia,  A.C.  (Chair)  Mid-­Atlantic  Regional  Chapter  of  American   College  of  Sports  Medicine  Student  College  Bowl.    MARC-­ACSM   Annual  Meeting,  Harrisburg,  PA,  2014     3.   Venezia,  A.C.  (Chair)  Meet  the  Experts.    MARC-­ACSM  Annual   Meeting,  Harrisburg,  PA,  2014       4.   Venezia,  A.C.  (Chair)  Mid-­Atlantic  Regional  Chapter  of  American   College  of  Sports  Medicine  Student  College  Bowl.  MARC-­ACSM   Annual  Meeting,  Harrisburg,  PA,  2013     5.   Venezia,  A.C.  (Chair)  Meet  the  Experts.    MARC-­ACSM  Annual   Meeting,  Harrisburg,  PA,  2013       Research  Support       August  2014  –  January  2016   National  Institute  of  Mental  Health  of  the  National  Institutes  of  Health,  National   Research  Service  Award.    Individual  Pre-­Doctoral  Fellowship  ($56,306).    Title:     Acute  Exercise  and  Hippocampal  Plasticity.     June  2014  –  2015   University  of  Maryland,  College  Park  Department  of  Kinesiology.  Graduate   Research  Initiative  Program  Grant  ($2,500).    Title:    Acute  Exercise,   Catecholamines,  and  Hippocampal  Plasticity.     December  2012  –  2013   University  of  Maryland,  College  Park  Department  of  Kinesiology.  Graduate   Research  Initiative  Program  Grant  ($2,500).    Title:    Acute  Exercise  and  AMPA   Receptor  GluR1  Subunit  Phosphorylation.       208     February  2010  –  2011   University  of  Maryland,  College  Park  Department  of  Kinesiology  Graduate   Research  Initiative  Program  Grant  ($2,500).    Title:  Effect  of  One  Year  of   Voluntary  Wheel  Running  on  DNA  Methylation  in  the  Hippocampus.       Awards  and  Honors     Spring  2009   Bloomsburg  University  Bill  Sproule  Award:    Senior  Exercise  Science  Major  with   the  highest     GPA.           Teaching  Experience     Instructor:     University  of  Maryland,  College  Park     Spring  2016     SPHL498F  Social,  Political,  &  Ethical  Issues  in  Public  Health  Instructor     University  of  Maryland,  Shady  Grove     Fall  2015     •   KNES360  Physiology  of  Exercise  (Co-­Instructor)     Teaching  Assistant:     University  of  Maryland  College  Park     Semester     Course   Fall  2009   KNES360  -­  Physiology  of  Exercise  Laboratory  Instructor  (TA)   KNES360  -­  Physiology  of  Exercise  Laboratory  Instructor  (TA)   KNES132  -­  Beginning  Badminton   KNES160  -­  Beginning  Volleyball   Spring  2010   KNES360  -­  Physiology  of  Exercise  Laboratory  Instructor  (Lead  TA)   KNES360  -­  Physiology  of  Exercise  Laboratory  Instructor  (Lead  TA)   KNES100  -­  Intermediate  Basketball     Summer   2010   KNES157  -­  Beginning  Weight  Training   KNES157  -­  Intermediate  Weight  Training       209   Fall  2010   KNES360  -­  Physiology  of  Exercise  Laboratory  Instructor  (Lead  TA)   KNES360  -­  Physiology  of  Exercise  Laboratory  Instructor  (Lead  TA)   KNES100  -­  Intermediate  Basketball   Fall  2011   KNES360  –  Physiology  of  Exercise  Laboratory  Instructor  (Lead  TA)   KNES360  –  Physiology  of  Exercise  Laboratory  Instructor  (Lead  TA)   Following  the  fall  2011  semester  I  restructured  the  weekly  laboratory  assignments  for   future  semesters.    These  changes  remain  the  format  for  the  course.       Academic  Research  Positions     University  of  Maryland       January  2012  to  Present   •   National  Institute  of  Health  Pre-­doctoral  Research  Trainee     January  2011  to  August  2011   •   Research  assistant:  Dr.  Stephen  Roth’s  NIH  Funded  “Role  of  Maternal   Exercise  Environment  on  Transgenerational  Offspring  Health.”     Bloomsburg  University     January  2008  to  July  2009   •   Research  assistant:  Dr.  Eric  Rawson’s  NIH  Funded  “Creatine   Supplementation  and  Cognitive  Function  in  the  Elderly.”       Job  Experience     February  2009  to  May  2009   Intern,  Geisinger  Core  Genomics  Laboratory,  Danville,  PA.       September  2006  to  June  2009   Fitness  Aide,  Geisinger  Fitness  Center,  Danville,  PA.         June  2007  to  July  2007   Intern,  Geisinger  Medical  Center  Stress  Laboratory  and  Cardiac  Rehabilitation   Program,  Danville,  PA.       June  2006  to  August  2006   Trainer,  Edge  Fitness  and  Martial  Arts,  Towanda,  PA.             210   Professional  Memberships   American  College  of  Sports  Medicine     Mentoring     August  2013  to  July  2014   Matthew  Ballew  –  Senior  Kinesiology/Pre-­medicine  Student     August  2012  to  May  2013   Rhea  Ramakrishnan  –  Sophomore  Biology  Student     August  2011  to  May  2012   Kelly  Protzko  –  Senior  Kinesiology  Honors  Student.    Mentored  through  senior   thesis  project.     Service     University  of  Maryland     Department  of  Kinesiology:     October  2014  to  May  2015   Student  Representative  on  Kinesiology  Department  Graduate  Committee       August  2012  to  August  2013   Kinesiology  Department  Graduate  Student  Council     School  of  Public  Health:     August  2013  to  August  2014   School  of  Public  Health  Senate  Executive  Committee  (elected  position)     Neuroscience  and  Cognitive  Science  Program:     August  2013  to  present   Neuroscience  and  Cognitive  Science  Recruitment  Fest  Committee     Bloomsburg  University     September  2008  to  May  2009   Bloomsburg  University  Student  Grievance  Board     American  College  of  Sports  Medicine     May  2014  to  Present       211   American  College  of  Sports  Medicine  Student  Affairs  Committee     May  2013  to  June  2015   Student  Representative  on  Mid-­Atlantic  Regional  Chapter  of  American  College  of   Sports  Medicine  Executive  Board             212   Bibliography:   Adlard,  P.A.,  Perreau,  V.M.,  &  Cotman,  C.W.  (2005)  The  exercise-­induced   expression  of  BDNF  within  the  hippocampus  varies  across  life-­span.   Neurobiology  of  Aging,  26,  511–520.   Aid,  T.,  Kazantseva,  A.,  Piirsoo,  M.,  Palm,  K.,  &  Timmusk,  T.  (2007)  Mouse  and   rat  BDNF  gene  structure  and  expression  revisited.  Journal  of  Neuroscience   Research,  85,  525–535.   Akhavan,  M.M.,  Emami-­Abarghoie,  M.,  Safari,  M.,  Sadighi-­Moghaddam,  B.,   Vafaei,  A.A.,  Bandegi,  A.R.,  &  Rashidy-­Pour,  A.  (2008)  Serotonergic  and   noradrenergic  lesions  suppress  the  enhancing  effect  of  maternal  exercise   during  pregnancy  on  learning  and  memory  in 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