Chronic stress impairs performance on hippocampal-mediated spatial tasks and alters hippocampal CA3 dendritic complexity in adult male rats (Conrad, 2006).

 A post-stress recovery period allows for reversal: spatial ability improves or exceeds that of controls and hippocampal dendritic complexity returns to levels of unstressed controls (Hoffman et al., 2011; Ortiz et al., 2014).

 Brain-derived neurotrophic factor (BDNF) is a protein that is required for spatial learning and memory (Radecki et al., 2005) and CA3 dendritic plasticity (Magariños et al., 2011).

 We also recently report that hippocampal BDNF is required for the recovery of spatial ability following the end of chronic stress (Ortiz et al., 2014).

 We previously found that downregulating hippocampal BDNF prevents the recovery of spatial ability following the end of chronic stress (Ortiz et al., 2014): the current study investigates whether hippocampal dendritic architecture also fails to recover and parallels spatial memory ability.

 We hypothesized the hippocampal BDNF mediates the recovery from chronic stress-induced dendritic retraction, which parallels spatial memory deficits.

  The RAWM is a task that requires rodents to have an intact hippocampus to optimally navigate to solve. Cues located outside and around the maze help with this “spatial navigation.”

 A single platform is located at the end of one of the arms and the location remains constant across all trials for a given rat.

  Errors are scored as incorrect entries into non-platformed arms within a given trial.

  shRNA against BDNF will prevent recovery of dendritic complexity: stress-induced CA3 apical dendritic atrophy will persist: Str-Imm = Str-Rec-shRNA. Neurons illustrated are of the SS type.

Stress immediate

Stress recovery

Control Viral Infusion

and surgical rehab

2 weeks

No restraint 42 days

Stress 21 days

Stress 21 days

No restraint 21 days

No restraint 21 days

RAWM testing 3 days

8 trials for 2 days 1 trial day 3

Arrival Euthanize

 Adult male Sprague-Dawley rats (n = 52) underwent stereotaxic surgery to bilaterally infuse an adeno-associated viral vector (AAV) into the hippocampus. AAV encoded for an shRNA directed against BDNF (shRNA) or a “scrambled” shRNA with no known correspondence to rat mRNA (Scr).

 Rats were chronically stressed using wire mesh restraint (6h/d/21d), a procedure known to produce reliable effects on hippocampal morphology and function (McLaughlin, 2007) and then given a 21 day post-stress recovery period (Str-Rec) or not (Str-Imm) and then tested on the radial arm water maze.

  The authors greatly acknowledge Dr. Ernest Terwilliger and Dr. Caroline Bass for designing and supplying the viral vectors used in this study. This research was supported in part by funds from the School of Life Sciences Undergraduate Research (SOLUR) Program through the School of Life Sciences at Arizona State University, Tempe Campus, ASU’s College of Liberal Arts and Sciences, the NIH IMSD program (R25GM099650 to Stuart Newfeld for funding Ortiz), and the National Science Foundation Graduate Research Fellowship Program (DGE-1311230, Ortiz).

  Conrad CD (2006) What is the functional significance of chronic stress-induced CA3 dendritic retraction within the hippocampus? Behav. Cog, Neurosci. Rev., 5, 41-60.

  Hoffman AN, Krigbaum A, Ortiz JB, Mika A, Hutchinson KM, Bimonte-Nelson HA & Conrad CD (2011) Recovery after chronic stress within spatial reference and working memory domains: correspondence with hippocampal morphology. Eur. J. Neurosci., 34, 1023-1030.

  Magariños AM, Li C, Toth JG, Bath KG, Jing D, Lee FS & McEwen BS (2011) Effect of brain-derived neurotrophic factor haploinsufficiency on stress-induced remodeling of hippocampal neurons. Hippocampus, 21, 253-264.

  McLaughlin KJ, Gomez JL, Baran SE & Conrad CD (2007) The effects of chronic stress on hippocampal morphology and function: an evaluation of chronic restraint paradigms. Brain research, 1161, 56-64.

  Ortiz JB, Mathewson CM, Hoffman AN, Hanavan PD, Terwilliger EF & Conrad CD (2014) Hippocampal brain-derived neurotrophic factor mediates recovery from chronic stress-induced spatial reference memory deficits. Eur. J. Neurosci., 40, 3351-3362.

  Radecki, DT, LM Brown, J Martinez & TJ Teyler (2005) BDNF protects against stress-induced impairments in spatial learning and memory and LTP. Hippocampus 15, 246

  After the single retention trial on day 3 of testing, rats were euthanized and brains extracted for Golgi processing.

  Brains were dissected in half, flash frozen in 2-methylbutane, and one hemisphere was processed for Golgi (FD NeuroTechnologies).

  The other hemisphere was placed in a -80°C freezer. Our plans include processing this hemisphere for BDNF expression using ELISA (in progress).

 With this study, we hope to show that recovery from stress-induced hippocampal CA3 dendritic retraction depends on hippocampal BDNF. •  Work is still in progress; approximately five SS and five LS CA3 neurons

have been identified and traced for each rat and 33% have been checked. •  We still have neurons from the CA1 and dentate gyrus to identify and draw. •  We are working on determining the effectiveness of the shRNA in knocking

down hippocampal BDNF using ELISA (in progress).  Uncovering factors, such as BDNF, that are important in mediating plasticity and

facilitating recovery may help identify novel mechanisms that can be targeted to enhance resilience in the face of adversity.

Con-Scr Unstressed Control rats

Str-Imm-Scr Chronically stressed

rats no recovery

Str-Rec-Scr Chronically stressed rats with recovery

Str-Rec-shRNA Chronically stressed rats with

recovery & downregulated hippocampal BDNF

  Golgi staining enables the observation of the entire neuron and its dendritic arbors, yet stains about 1% - 2% of neurons. Consequently, Golgi provides clear, isolated, and fully impregnated neurons.

  We found many individual neurons in the hippocampus that could be viewed and traced using a Camera Lucida drawing tube.

  Two main neuronal types were drawn and checked for dendritic complexity: “short shaft (SS),” with an apical dendrite that bifurcates proximal to the soma (above, right), and “long shaft (LS),” that bifurcates distal from the soma (adjacent, right).

  We predict that shRNA against hippocampal BDNF will prevent stress-induced spatial memory deficits from recovering, so that Str-Rec-shRNA = Str-Imm-Scr.

  Consequently, dendritic arbor complexity will correspond with behavior on RAWM

Background

Predicted Behavioral Results

Acknowledgements & References

Radial Arm Water Maze (RAWM)

Brain Extraction and Processing

Golgi Staining

Predicted Morphological Results

Summary

Purpose and Hypothesis

General Methods

Chronic Variable Stress Effects on Anxiety and Expression of Organic Cation Transporter 3

1.  Amphoux,  A.,  Vialou,  V.,  Drescher,  E.,  Bruss,  M.,  Mannoury  La  Cour,  C.,  Rochat,  C.,  Millan,  M.J.,  Giros,  B.,  Bonisch,  H.,  Gautron,  S.,  2006.  DifferenFal  pharmacological  in  vitro  properFes  of  organic  caFon  transporters  and  regional  distribuFon  in  rat  brain.  Neuropharmacology  50,  941-­‐952.  

2.  Baganz,  N.L.,  Horton,  R.E.,  Calderon,  A.S.,  Owens,  W.A.,  Munn,  J.L.,  WaUs,  L.T.,  Koldzic-­‐Zivanovic,  N.,  Jeske,  N.A.,  Koek,  W.,  Toney,  G.M.,  Daws,  L.C.,  2008.  Organic  caFon  transporter  3:  Keeping  the  brake  on  extracellular  serotonin  in  serotonin-­‐transporter-­‐deficient  mice.  Proceedings  of  the  NaFonal  Academy  of  Sciences  of  the  United  States  of  America  105,  18976-­‐18981.  

3.  Feng,  N.,  Mo,  B.,  Johnson,  P.L.,  Orchinik,  M.,  Lowry,  C.A.,  Renner,  K.J.,  2005.  Local  inhibiFon  of  organic  caFon  transporters  increases  extracellular  serotonin  in  the  medial  hypothalamus.  Brain  research  1063,  69-­‐76.  

4.  Feng,  N.,  Telefont,  M.,  Kelly,  K.  J.,  Orchinik,  M.,  Forster,  G.  L.,  Renner,  K.  J.,  &  Lowry,  C.  A.  (2009).  Local  perfusion  of  corFcosterone  in  the  rat  medial  hypothalamus  potenFates  d-­‐fenfluramine-­‐induced  elevaFons  of  extracellular  5-­‐HT  concentraFons.  Hormones  and  behavior,  56(1),  149-­‐157.  

5.  Gasser,  P.J.,  Lowry,  C.A.,  Orchinik,  M.,  2006.  CorFcosterone-­‐sensiFve  monoamine  transport  in  the  rat  dorsomedial  hypothalamus:  potenFal  role  for  organic  caFon  transporter  3  in  stress-­‐induced  modulaFon  of  monoaminergic  neurotransmission.  The  Journal  of  neuroscience  :  the  official  journal  of  the  Society  for  Neuroscience  26,  8758-­‐8766.  

6.  Daws,  L.C.,  2009.  Unfaithful  neurotransmiUer  transporters:  focus  on  serotonin  uptake  and  implicaFons  for  anFdepressant  efficacy.  Pharmacol  Ther  121,  89-­‐99.  

7.  Marcinkiewcz,  C.a.,  and  D.p.  Devine,  2015.  ModulaFon  of  OCT3  Expression  by  Stress,  and  AnFdepressant-­‐like  AcFvity  of  Decynium-­‐22  in  an  Animal  Model  of  Depression.  Pharmacology  Biochemistry  and  Behavior  33-­‐41.  

8.  Reber,  Stefan  O.,  and  Inga  D.  Neumann,  2008.  Defensive  Behavioral  Strategies  and  Enhanced  State  Anxiety  during  Chronic  Subordinate  Colony  Housing  Are  Accompanied  by  Reduced  Hypothalamic  Vasopressin,  But  Not  Oxytocin,  Expression.  Annals  of  the  New  York  Academy  of  Sciences  184-­‐95.  

•   This  project  was  funded  by  a  grant  awarded  to  MO  from  the  NaFonal  Science  FoundaFon  (NSF  0922085)  and  BarreU  Faculty  Support  Funds.  • This  research  was  supported  in  part  by  funds  from  the  School  of  Life  Sciences  Undergraduate  Research  (SOLUR)  Program  through  the  School  of  Life  Sciences  at  Arizona  State  University,  Tempe  Campus  

•   Although  there  were  no  significant  differences  in  adrenal  weights,  the  reduced  weight  gain  in  the  CVS  group  is  consistent  with  predicFons  that  these  rats  were  chronically  stressed  •   Contrary  to  predicFons,  there  was  no  difference  between  CVS  and  unstressed  rats  in  terms  of  sucrose  preference8  •   Also  contrary  to  predicFons,  CVS  rats  showed  a  non-­‐significant  tendency  for  reduced  latency  in  the  novel  feeding  environment.  The  non-­‐significant  tendency  to  consume  food  in  the  home  environment  suggests  that  CVS  rats  were  not  as  moFvated  or  hungry  as  controls.  •   Preliminary  opFmizaFon  of  fluorescent  in  situ  hybridizaFon  using  mouse  brains  revealed  OCT3  mRNA  labeling  in  key  limbic  regions.  •   Future  experiments  will  compare  OCT3  mRNA  and  protein  expression  in  CVS  vs.  control  rats  •   Although  there  were  no  significant  differences  in  behavioral  tests,  we  are  examining  differences  in  OCT3  expression  between  CVS  and  control  rats  for  potenFal  paUerns  and  correlaFons.  

• Organic  caFon  transporters  (OCT)s  are  a  family  of  transporters  for  monoamines  (serotonin  (5-­‐HT),  dopamine,  and  norepinephrine)  in  the  human  and  murine  brain.1  

•   OCT3  is  believed  to  help  clear  extra-­‐neuronal  monoamines,  thereby  helping  to  terminate  signaling2-­‐5    

• One  known  regulator  of  OCT3  is  corFcosterone  (the  stress  steroid  hormone),  which  can  alter  5-­‐HT  transport,  clearance  and  neurotransmission,  as  well  as  HPA  axis  negaFve  feedback  and  regulaFon5  

•   Altered  expression  of  OCT3  may  play  a  role  in  stress  coping7  

•   Numerous  basic  and  clinical  studies  from  the  last  three  to  four  decades  have  demonstrated  that  imbalances  in  5-­‐HT  are  linked  to  certain  mood  disorders,  such  as  anxiety  and  depression6  

•   In  previous  studies,  chronic  variable  stress  (CVS)  has  produced  anxiety  and  depressive  like  behaviors  as  measured  by  novelty  suppressed  feeding  and  sucrose  preference  tesFng    

•   Since  OCT3  is  involved  in  5-­‐HT  clearance  and  transport,  it  is  likely  that  OCT3  is  influenced  by  chronic  stress    •   We  hypothesized  that  OCT3  is  a  mechanisFc  link  between  stress,  anxiety,  and  depression  •   We  predicted  that  CVS  would  increase  anxiety  and  depressive-­‐like  behaviors  and  increase  expression  of  OCT3  

Chronic  Variable  Stress  Protocol  

*

a)  There  was  no  significant  difference  in  adrenal  weights  between  CVS  and  control  rats  b)  CVS  rats  gained  less  weight  than  control  rats  (p  <  .01)    c)  No  significant  difference  was  found  in  sucrose  preference  and  total  volume  drunk  between  CVS  and  control  rats,  although  CVS  rats  appeared  to  drink  more  sucrose  soluFon  and  total  liquid  volume  overall.  d)  No  significant  difference  was  found  in  latency  to  approach  food  in  bright  arena  or  food  eaten  in  home  cage  between  CVS  and  control  rats,  although  CVS  rats  displayed  a  non-­‐significant  tendency  for  reduced  latency  and  feeding  in  home  cage.  

Cerebral  Aqueduct  400x  Bregma  -­‐3.52  

Dorsal  Hippocampus  CA3  400x  Bregma  -­‐3.52  

•   20  Sprague  Dawley®  rats  were  used  in  this  study,  10  received  unpredictable  CVS  for  14  days  while  10  received  no  stress  (see  CVS  protocol/Fmeline).  •   Following  last  administraFon  of  CVS,  all  rats  were  given  both  sucrose  soluFon  and  tap  water  available  to  them,  and  the  volume  of  liquid  drunk  was  measured.  •   Novelty  suppressed  feeding  tesFng  measured  latency  to  approach  food  when  rats  were  placed  in  a  novel,  well-­‐lit  environment,  as  well  as  subsequent  feeding  in  the  home  cage  (during  the  dark  cycle  when  rats  are  acFve).  •   Following  behavioral  tesFng,  rats  were  euthanized,  adrenals  were  removed  and  weighed,  and  brains  were  extracted  and  flash  frozen.  • RNA  was  reverse  transcribed  and  the  polymerase  chain  reacFon  (PCR,  HotStar  Taq  Qiagen)  was  performed  using  primers  that  contained  the  T7  phage  promoter  sequence.    •   Riboprobes  were  in  vitro  transcribed  using  a  T7  kit  (Ambion)  containing  BioFn-­‐16-­‐labeled  uridine  triphosphate  • For  opFmizaFon  of  in  situ  hybridizaFon,  10  µm  secFons  (Bregma~-­‐3.52)  of  mouse  brains  were  fixed  and  hybridized  with  5µg  of  the  bioFn-­‐labeled  riboprobe  at  80°C  overnight.  The  next  day,  secFons  were  washed  with  increasing  stringency,  and  slides  were  incubated  using  Streptavidin-­‐AlexaFluor  488  conjugate.  •   Following  1  hr  incubaFon  with  the  conjugate  soluFon,  slides  were  incubated  in  1%  Sudan  Black  to  reduce  auto-­‐fluorescence  .  

Piper  Boyll1,  Pooja  R.  Paode1,2,  Emmanuel  Fonseca2,  J.  Bryce  OrFz2,  Joshua  S.  Talboom3,4,  Jeremiah  Molinaro1,  Salma  Kemmou2,  Eshaan  J.  Daas1,2,  Cheryl  D.  Conrad2, and  Miles  Orchinik1  

 1Arizona  State  University,  School  of  Life  Sciences,  Tempe,  AZ  85287,  2Arizona  State  University,  Department  of  Psychology,  Tempe  AZ  85287  3Banner  Sun  Health  Research  InsFtute,  Sun  City,  AZ    85351,  4Arizona  Alzheimer’s  ConsorFum,  Phoenix,  AZ    85014  

a)   b)  

c)  

d)