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The  role  of  parvalbumin-­‐positive  interneurons  in  rodent  primary  somatosensory  

cortex  during  object  localization  

 

 

 

 

 

 

 

 

 

NEUR  494  Honors  Thesis  

Jason  Strawbridge  

University  of  Southern  California  

Faculty  Advisor:  Andrew  Hires  

May  13th,  2015  

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Abstract    Our  internal  representation  of  the  external  world  is  based  on  patterns  of  neural  activity  in  our  cortex.  These  patterns  are  profoundly  influenced  by  inhibitory  neurons.  The  most  numerous  class  of  inhibitory  neurons  in  the  cortex  express  parvalbumin  (PV+).  The  function  of  these  neurons  during  active  sensation  is  unknown.  We  sought  to  determine  the  role  PV+  interneurons  play  in  the  cortical  processing  of  the  sense  of  touch  in  the  mouse  whisker  system.  We  addressed  how  PV+  neuron  activity  shapes  the  activity  patterns  in  primary  somatosensory  cortex  (S1)  associated  with  whisker-­‐object  contact  and  whisker  motion  during  whisker-­‐mediated  object  localization.  We  recorded  whisker  motion  and  object  contact  with  1ms  precision,  calculated  sensory  input,  and  correlated  these  variables  spiking  patterns  in  S1  neurons.  We  then  optogentically  silenced  PV+  interneuron  activity  in  the  whisker  representation  of  S1  during  object  localization  and  compared  how  the  encoding  of  sensory  and  motor  variables  differed  between  these  two  states.  Finally,  we  linked  these  changes  in  neural  activity  to  shifts  in  perception  of  object  location.  This  revealed  underlying  computational  mechanisms  by  which  object  location  is  perceptually  constructed  from  neural  activity  in  S1.  

Introduction    Sensation,  or  the  initial  stage  in  detection  of  stimulus  properties,  is  a  relatively  well-­‐understood  physiological  process.  Humans,  along  with  many  other  species,  are  able  to  sense  the  external  world  through  a  number  of  biochemical  pathways  activated  by  stimulus-­‐driven  receptors  within  the  cells  of  sensory  organs.  The  human  finger,  for  example,  contains  multiple  classes  of  mechanoreceptors  that  respond  to  varying  degrees  of  touch,  pressure,  and  vibration1.  Action  potentials  produced  by  these  receptors  are  sent  to  the  brain  via  the  central  nervous  system,  where  they  are  processed  in  the  somatosensory  cortex  in  order  to  create  a  recognizable  percept.  Yet  the  actual  process  of  perception,  unlike  sensation,  is  not  clearly  defined.  Decoding  the  nature  of  conscious  perception  is  especially  difficult  in  somatosensory  regions,  where  perceptions  of  sensory  signals  are  built  from  neural  activity  patterns  within  complex  parallel  and  hierarchical  circuits.  Some  fundamental  characteristics  of  processing  within  primary  somatosensory  cortex  (S1)  have  been  identified;  previous  research  within  S1  has  revealed  the  neuronal  connections  underlying  S1  circuits2,3,  and  has  established  a  morphological  and  physiological  understanding  of  the  neurons  that  make  up  these  connections4.  However,  the  functional  organization  of  circuits  underlying  perceptual  processes  in  S1  has  yet  to  be  determined.  One  particularly  significant  obstacle  in  the  way  of  understanding  this  functional  organization  is  a  lack  of  knowledge  regarding  the  effects  of  interneuron  influence  within  layers  of  somatosensory  cortex.  The  construction  of  neural  activity  patterns  is  highly  regulated  by  feed-­‐forward  inhibition  from  GABAergic  interneurons.  Previous  work  has  identified  parvalbumin-­‐positive  (PV+)  interneurons  as  the  primary  source  of  this  inhibition5,6,  and  has  revealed  the  mechanisms  by  which  PV+  interneurons  regulate  excitatory  spiking  rates7.  Although  these  findings  provide  

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valuable  information  about  the  synaptic  activity  of  PV+  interneurons,  a  quantitative  understanding  of  how  these  interneurons  influence  conscious  perception  has  yet  to  be  developed.  We  sought  to  shed  light  on  the  nature  of  touch  perception  by  investigating  the  role  that  PV+  interneurons  play  in  shaping  touch-­‐evoked  neural  activity  patterns.    We  conducted  our  investigation  of  PV+  interneurons  by  performing  electrophysiological  and  behavioral  analysis  in  mice  during  optogenetic  manipulation.  Mice  are  ideal  experimental  subjects  for  the  exploration  of  sensory  processing  mechanisms  because  of  their  cortical  similarities  to  human  models;  many  aspects  of  cortical  circuits  that  process  touch  are  conserved  across  both  humans  and  mice2.  Additionally,  the  nature  of  whisker-­‐based  tactile  mechanisms  allows  precise  measurement  of  sensory  input.  Mice  sweep  their  whiskers  back-­‐and-­‐forth  to  detect,  locate  and  identify  objects8,9,  much  like  humans  use  their  fingers.  Precise  measurement  of  touch  input  can  be  performed  by  tracking  whisker  deformations  with  high-­‐speed  video  recording10.  Furthermore,  the  head-­‐fixed  procedures11,12  and  optogenetic  techniques13-­‐16  previously  established  in  mice  models  mice  offer  a  degree  of  experimental  control  necessary  for  the  manipulation  of  specific  behaviors  and  neuron  subtypes.      In  mice,  whiskers  perform  the  duty  of  active  touch  sensation;  signal  transduction  is  mediated  by  mechanoreceptors  within  the  whisker  follicle17.  Inputs  from  sensory  receptors  within  the  whiskers  ascend  through  the  principal  trigeminal  nucleus  to  the  ventral  posterior  medial  (VPM)  thalamic  nucleus,  where  they  are  then  relayed  to  the  primary  somatosensory  cortex  (S1)18,19.  Neuronal  input  from  each  individual  whisker  is  processed  topographically  in  a  single  “barrel”  within  S120.  Intrinsic  signal  imaging  of  animals  trimmed  to  a  single  whisker  permits  the  identification  of  barrel  circuitry  based  on  whisker  input  and  allows  us  to  pinpoint  the  location  of  neural  processing  within  S1  for  individual  whiskers21.  Furthermore,  this  mapping  enables  precise  targeting  during  optogenetic  manipulation,  so  that  optogenetically-­‐tagged  cell  types  are  only  inhibited  or  activated  within  a  specific  cortical  area22.    Previous  research  within  rodent  primary  somatosensory  cortex  has  revealed  important  information  regarding  the  mechanisms  underlying  general  tactile  processing  within  barrel  circuitry.  The  parallel  and  hierarchical  processing  streams  that  form  the  neural  basis  of  touch  perception  operate  between  multiple  layers  of  cortex  within  S12.  Sensory  input  from  whisker  mechanoreceptors  first  arrives  in  layers  4  (L4)  and  5B  after  ascending  through  the  VPM  of  the  thalamus18,23.  These  signals  ascend  to  L3  and  then  descend  to  the  pyramidal  neurons  of  L5B,  which  serve  as  the  major  output  centers  from  S124-­‐26.  While  the  precise  functions  of  neural  activity  patterns  within  L3  and  5B  have  yet  to  be  determined  conclusively,  functional  circuitry  within  L4  has  been  characterized  in  greater  detail.  In  one  experiment,  optogenetic  stimulation  of  L4  excitatory  neurons  in  S1  using  touch-­‐like  patterns  evoked  illusory  perception  of  object  location22.  Follow-­‐up  research  has  indicated  that  excitatory  cells  within  L4  of  S1  are  actually  able  to  represent  the  time  of  touch  onset,  strength,  and  direction  with  high  precision27;  these  results  suggest  

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that  L4  extracts  a  “when”  and  “what”  touch  signal  from  whisker  input.  Furthermore,  these  studies  have  found  evidence  supporting  the  idea  that  local  feedfoward  inhibition  from  PV+  interneurons  cancels  excitatory  signals  from  the  VPM  generated  by  self-­‐motion;  suggesting  that  PV+  interneuron  activity  allows  cells  in  L4  to  discriminate  the  temporal  and  spatial  nature  of  a  stimulus27.    The  implication  that  PV+  interneuron  inhibition  might  regulate  object  localization23  was  the  basis  of  our  current  investigation.  While  the  results  of  previous  experiments  targeting  excitatory  cells  and  PV+  cells  within  L4  provided  a  clearer  look  at  the  mechanisms  underlying  tactile  perception,  the  conclusions  drawn  from  these  results  had  notable  limitations.  We  hoped  to  expand  upon  the  results  from  earlier  experiments  while  avoiding  these  limitations.  A  key  distinguishing  feature  of  our  current  research  is  the  utilization  of  a  conditional  discrimination  paradigm,  an  adaptation  of  the  tactile  discrimination  paradigm  used  in  previous  experiments18.  In  the  experimental  paradigm  used  during  earlier  research,  mice  were  required  to  determine  the  spatial  location  of  an  object  relative  to  their  current  position18.  However,  sample  object  stimuli  in  these  experiments  were  presented  in  only  two  positions  (go  and  no-­‐go);  mice  may  have  been  able  to  solve  the  task  by  simply  limiting  their  whisking  range  and  subsequently  identifying  the  presence  of  a  pole  within  that  range.  As  such,  there  is  a  possibility  that  these  mice  were  not  discriminating  precise  pole  location  and  were  performing  the  task  without  referencing  an  internal  motor  model.  In  comparison,  mice  performing  a  continuous  discrimination  task  based  on  variable  pole  position  (multiple  go  and  no-­‐go  positions)  are  more  likely  to  use  an  internal  motor  model  that  reflects  the  precise  spatial  location  of  the  pole.  In  a  continuous  discrimination  task,  the  degree  of  motor-­‐referenced  discrimination  can  be  quantified  by  the  difference  in  lick  probability  between  go  trials  and  no-­‐go  trials  when  at  least  one  touch  occurs  during  the  trial.        An  important  goal  of  our  research  was  to  quantify  the  mechanism  by  which  PV+  interneuron  inhibition  influenced  object  perception.  Conclusions  from  previous  investigations  of  excitatory  spiking  in  S1  have  suggested  that  object  location  might  be  encoded  by  either  excitatory  spike  count  or  excitatory  spike  pattern18.  An  encoding  method  based  on  spike  count  implies  that  a  greater  number  of  excitatory  spikes  correspond  to  the  perception  of  closer  object  location  (due  to  a  greater  number  of  object  touches),  and  that  less  spikes  corresponds  to  a  further  location  (less  touches)18.    In  contrast,  an  encoding  method  based  on  spike  pattern  implies  that  specific  patterns  of  neural  activity  correspond  to  specific  object  locations  in  space18.  Our  hypothesis  was  that  mice  would  encode  object  location  during  the  continuous  discrimination  task  by  counting  the  number  of  touch-­‐evoked  spikes  in  S1.  In  order  to  test  this  hypothesis,  we  altered  both  timing  precision  and  number  of  touch-­‐evoked  spikes  in  S1  by  inhibiting  local  PV+  inhibitory  interneurons;  we  believed  that  silencing  these  cells  would  increase  overall  excitatory  activity  within  S1,  leading  to  a  greater  number  of  spikes,  and  shifting  the  animal’s  perception  of  object  location  to  more  proximal  positions.        

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Results    PV+  interneurons  within  somatosensory  cortex  fire  in  response  to  object  contact  and  whisker  movement    Mice  were  trimmed  to  a  single  whisker  (C2)  in  order  to  maintain  precise  stimulus  control,  and  to  ensure  that  sensory  input  during  head-­‐fixed  behavior  was  introduced  at  distinct  and  quantifiable  locations.  Electrophysiological  recordings  were  targeted  to  the  C2  whisker  representation  within  the  barrel  field  via  intrinsic  signal  imaging.  Cell-­‐attached  pipette  recordings  were  made  within  the  C2  barrel  in  an  effort  to  identify  PV+  interneurons.  PV+  interneurons  were  distinguished  by  their  fast  spike-­‐rate  and  distinct  waveforms.        

 Figure  1.  Left:  microcircuit  diagram  of  feed-­‐forward  inhibition  from  PV+  interneurons  onto  excitatory  pyramidal  cells  within  L4  of  primary  somatosensory  cortex;  recording  electrodes  were  targeted  to  PV+  interneurons.  Right:  spike  waveform  used  to  distinguish  whether  cell  recorded  from  was  in  fact  a  PV+  interneuron.    Electrophysiological  recordings  of  spiking  activity  during  behavior  from  identified  PV+  interneurons  were  paired  with  high-­‐speed  whisker  tracking  video  in  order  to  determine  the  relationship  between  PV+  cell  activity  and  sensorimotor  input.  An  increase  in  PV+  interneuron  spike  rate  was  observed  during  whisking  behavior  and  was  found  to  correlate  with  whisking  strength.  Furthermore,  an  increase  in  PV+  interneuron  spike  rate  immediately  following  the  onset  of  touch  indicated  a  touch-­‐aligned  response  from  the  cell.  Touches  with  greater  force  demonstrated  an  enhanced  spiking  response  compared  to  those  with  lesser  force.      

           Figure  2.  Bottom  (left):  PV+  cell  activity  immediately  followed  object-­‐touch.  Bottom  (right):  Spike  rate  of  PV+  cell  increased  with  greater  whisking  strength.  Top:  PV+  cell  spiking  was  greater  when  whiskers  hit  objects  with  higher  impact  force.  

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 Optogenetic  inhibition  of  PV+  interneurons  increases  the  number  of  spikes  from  excitatory  neurons  within  primary  somatosensory  cortex    Cell-­‐attached  pipette  recordings  were  made  from  excitatory  cells  within  the  C2  barrel  of  S1.  Excitatory  cells  were  identified  by  a  short-­‐latency  spiking  response  to  object  touch  and  by  their  recognizable  waveform.  Initial  recordings,  in  the  absence  of  PV+  photoinhibition,  served  as  a  baseline  for  excitatory  cell  activity.  Recordings  were  then  made  from  excitatory  cells  during  PV+  photoinhibition  at  varying  light  intensities  of  5mW,  10mW,  and  20mW.  Selective  transgenic  expression  of  halorhodopsin,  a  light-­‐gated  chloride  ion  channel,  permitted  the  optogenetic  inhibition  of  PV+  interneurons.  Recordings  from  excitatory  neurons  during  photoinhibition  of  PV+  cells  revealed  an  increase  in  excitatory  spike  rate  during  the  illumination  period.  Furthermore,  this  increase  in  excitatory  spiking  that  positively  correlated  with  photoinhibition  intensity;  a  slight  increase  in  spike  rate  compared  to  baseline  and  controls  (0mW)  was  observed  at  5mW  (p<0.002),  and  a  large  increase  in  spike  rate  was  observed  at  20mw  (p<1e-­‐29).      

 

 Figure  3.    Left:  Raster  plot  of  excitatory  spiking  patterns  in  S1  during  photoinhibition  of  PV+  cells  at  various  intensities  and  during  control  trials  (no  photoinhibition).  Right:  Mean  spike  rate  of  excitatory  cells  increases  during  photoinhibition  of  PV+  cell  activity.  Higher  spike  rates  correlate  with  higher  photoinhibition  intensities.      Silencing  the  activity  of  PV+  interneurons  shifts  perception  of  pole  location  in  the  proximal  direction  during  tactile  discrimination    Mice  used  as  experimental  subjects  underwent  roughly  2  weeks  of  behavioral  training  in  order  to  learn  the  continuous  discrimination  task.  In  this  task,  mice  were  required  to  accurately  determine  which  side  of  a  virtual  boundary  a  pole  was  presented  on.  Pole  positions  anterior  to  the  virtual  boundary  represented  no-­‐go  trials,  during  which  the  mice  learned  to  refrain  from  licking.  Conversely,  pole  positions  posterior  to  the  virtual  boundary  represented  go  trials,  during  which  mice  learned  to  lick,  in  order  to  obtain  a  water  reward.  Go  and  no-­‐go  trials  were  

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presented  with  random  but  equal  probability  (50%),  and  pole  position  within  both  go  and  no-­‐go  ranges  varied  randomly  along  the  anterior-­‐posterior  axis.  Accurate  hits  during  go  trials  led  to  water  rewards,  and  false  alarms  during  no-­‐go  trials  led  to  time-­‐outs  (~3  seconds).  Correct  rejections  during  no-­‐go  trials  were  not  rewarded  (no  water  given),  and  misses  during  go  trials  were  not  punished  (no  timeout).      

Figure  4.    Object  localization  task  performed  during  behavioral  experiments.  Left:  during  go-­‐trials,  a  pole  is  presented  in  locations  closer  to  the  animal’s  head;  a  licking  response  indicates  perception  of  closer  pole  location  and  leads  to  a  water  reward.  Right:  during  no-­‐go  trials,  the  pole  is  presented  in  farther  locations;  licking  responses  will  lead  to  false  alarms  and  time-­‐outs.  The  absence  of  a  licking  response  indicates  perception  of  farther  object  location.        Once  subjects  achieved  a  consistent,  high  level  of  accuracy  during  their  training  sessions  (~75%  accuracy  or  higher),  we  manipulated  the  activity  of  

PV+  interneurons  using  photoinhibition  of  the  targeted  S1  area  mapped  during  intrinsic  signal  imaging.  Photoinhibition  was  delivered  during  roughly  20%  of  all  trials  within  each  behavioral  session,  and  occurred  pseudorandomly  among  trials.  Performance  curves  for  photoinhibition-­‐mediated  trials  were  plotted  separately  from  the  baseline  curves  in  order  to  quantify  the  effects  of  PV+  inhibition  on  tactile  discrimination  ability.  The  intensity  of  the  photoinihibiting  pulse  train  remained  fixed  for  the  duration  of  a  single  behavioral  session  but  varied  between  sessions;  laser  power  alternated  between  5mW,  10mW,  and  20mW.  Separate  performance  curves  were  plotted  for  each  mW  value  in  order  to  measure  the  relative  behavioral  differences  between  the  varying  intensities.      Grand  mean  psychometric  performance  curves  plotted  for  control  trials  (no  photoinhibition)  demonstrated  that  the  subject  was  adept  at  performing  the  discrimination  task.  For  trials  in  which  the  pole  was  in  the  most  proximal  positions,  the  probability  of  a  lick  was  relatively  high  (70%).  Lick  probability  decreased  as  the  pole  moved  toward  the  virtual  boundary,  and  remained  relatively  low  for  all  trials  in  which  the  pole  was  in  the  no-­‐go  ranges  (30%).  Grand  mean  psychometric  performance  curves  plotted  during  photoinhibition  trials,  however,  exhibited  a  significantly  different  behavioral  result.  During  photoinhibition  trials,  the  probability  of  a  lick  was  as  high  as  90%  in  the  most  proximal  locations.  This  increase  in  lick  probability  was  seen  throughout  the  continuum,  in  both  go  and  no-­‐go  ranges;  during  no-­‐go  trials,  the  subject’s  probability  of  licking  rose  to  around  60-­‐70%.        

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 Figure  5.  Lick  probability  during  trials  with  various  pole  locations.  During  control  trials  (no  photoinhibition,  black  dots),  lick  probability  was  relatively  high  in  proximal  pole  locations  and  relatively  low  for  distal  pole  locations.  During  PV+  cell  photoinhibition  trials  (orange),  lick  probability  was  higher  relative  to  control  trials  for  all  pole  positions.  

         Discussion    The  results  obtained  from  electrophysiological  recordings  of  PV+  interneurons  within  the  C2  representation  of  the  somatosensory  cortex  demonstrate  the  involvement  of  PV+  cell  activity  in  whisker-­‐mediated  touch  sensation.  Furthermore,  the  recordings  from  excitatory  neurons  during  PV+  cell  inhibition  reveal  the  importance  of  PV+  cell  activity  in  regulating  the  neural  firing  patterns  of  excitatory  neurons  in  S1.  These  results  show  that  PV+  cells  have  the  appropriate  activity  to  regulate  both  the  pattern  and  number  of  touch-­‐evoked  excitatory  spikes  in  S1.      The  results  of  PV+  inhibition  during  tactile  discrimination  behavior  reveal  that  the  silencing  of  PV+  cell  activity,  and  the  increase  in  excitatory  spike  rate  that  follows,  shifts  the  animal’s  perception  of  object  location  to  more  proximal  positions.  This  suggests  that  the  animal  is  encoding  object  location  based  on  a  spike-­‐count  mechanism,  not  a  spike-­‐pattern  mechanism.  If  object  location  were  encoded  by  the  recognition  of  specific  spike-­‐patterns,  then  an  increase  in  overall  spike  rate  (as  a  result  of  PV+  cell  inhibition)  would  disrupt  the  spike  pattern  and  impair  the  animal’s  ability  to  discriminate  pole  location.  In  this  scenario,  we  would  expect  to  see  a  flattening  of  the  psychometric  performance  curve;  discriminatory  ability  would  be  impaired  and  performance  would  trend  toward  chance  levels,  regardless  of  pole  location.  However,  performance  curves  plotted  for  photoinhibition  trials  showed  a  markedly  different  effect:  photoinhibition  increased  lick  probability  among  all  trials,  in  both  go  and  no-­‐go  ranges.  These  results,  instead,  support  our  original  hypothesis  that  object  location  is  encoded  with  spike  count;  an  increase  in  spike  rate  during  PV+  cell  inhibition  leads  to  an  increase  in  lick  probability  because  the  animal  is  counting  the  number  of  S1  spikes  in  order  to  determine  the  precise  spatial  location  of  the  pole.  

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 Figure  6.  Psychometric  performance  curves  generated  from  PV+  cell  photoinhibition  during  object  localization  behavior  (right)  more  closely  support  a  spatial  encoding  mechanism  based  on  spike  count  in  S1  (pink  curve,  right).    Although  our  results  provide  evidence  for  a  spike-­‐count  hypothesis,  these  results  have  a  few  significant  limitations.  First  and  foremost,  the  behavioral  data  gathered  in  our  research  comes  from  only  a  single  mouse  over  the  course  of  5  photoinhibition  sessions.  In  order  to  eliminate  the  possibility  of  subject-­‐to-­‐subject  variability  and  provide  more  conclusive  evidence  for  a  spike-­‐count  hypothesis,  we  need  to  focus  our  efforts  on  obtaining  similar  data  from  additional  trials  across  a  larger  number  of  mice.  Additionally,  future  experiments  should  aim  to  quantify  the  manner  in  which  spike  patterns  are  changed  during  photoinhibition  of  PV+  interneurons;  such  quantification  is  necessary  in  order  to  completely  exclude  the  possibility  that  mice  might  be  using  a  pattern-­‐based  spike  model  for  object  location  encoding.      Finally,  while  these  results  do  provide  evidence  for  a  hypothesized  function  of  PV+  interneurons  within  primary  somatosensory  cortex,  they  are  limited  in  the  sense  that  they  do  not  reveal  information  regarding  PV+  interneuron  function  within  specific  layers  of  the  cortex.  Previous  studies  have  suggested  that  excitatory  neurons  within  somatosensory  regions  play  layer-­‐specific  roles  in  sensory  processing;  future  efforts  to  investigate  the  details  of  PV+  interneuron  function  should  target  individual  layers  in  order  to  quantify  potential  differences  between  PV+  interneuron  functions  throughout  the  processing  circuit.    Methods    Surgery  for  head-­‐fixation    Mice  (~2-­‐6  months  old)  were  deeply  anesthetized  with  2%  (volume  in  oxygen)  isoflurane  and  then  mounted  into  a  stereotaxic  apparatus  on  top  of  a  heated  blanket.  After  mice  were  fixed  into  the  apparatus,  isoflurane  levels  were  lowered  to  1.5%.  The  eyes  were  covered  with  petroleum  jelly,  and  the  scalp  was  cleaned  with  ethanol  

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and  betadine.  Topical  anesthesia  was  achieved  via  the  injection  of  a  0.5%  marcaine  solution  under  the  scalp.  Ketofen  was  injected  subcutaneously  to  prevent  inflammation  and  buprenorphine  (opioid  analgesic)  was  injected  into  the  intraperitoneal  cavity.  A  flap  of  skin  (approximately  1cm)  was  removed  from  the  dorsal  skull,  and  any  leftover  periostium  was  removed.  After  the  skull  was  sterilized  with  cotton  swabs,  bone  was  scraped  with  a  scalpel  to  ensure  proper  bonding  of  glue.  A  thin  layer  of  cyanoacrylic  glue  was  placed  onto  the  skull,  and  the  head  plate  was  placed  directly  on  top  of  the  glue.  Dental  acrylic  was  used  to  cover  any  exposed  glue  and  to  cement  the  head  plate  in  the  correct  position.    Intrinsic  signal  imaging    In  order  to  precisely  target  the  primary  somatosensory  cortex  during  optogenetic  stimulation,  intrinsic  signal  optical  imaging  was  performed  on  each  mouse  prior  to  behavioral  training.  These  images,  obtained  by  capturing  small  activity-­‐based  changes  from  the  illuminated  brain,  enabled  accurate  morphological  mapping  of  the  dorsal  cortex.  Imaging  was  performed  through  the  skull  in  animals  anesthetized  with  isoflurane  (1.5%  volume  in  oxygen).    

                       Figure  7.  Left:  illustration  of  intrinsic  signal  imaging  technique  used  to  target  the  C2  whisker  representation  within  primary  somatosensory  cortex.  Middle:  changes  in  oxygen  content  of  hemoglobin  are  revealed  during  illumination  with  640nm  (red)  light.  Right:  changes  in  blood  volume  allow  imaging  of  arterial  structure  during  illumination  with  525nm  (green)  light.    Water  restriction    In  order  to  produce  the  necessary  motivation  required  for  behavioral  training,  mice  were  put  on  water  restriction;  this  produced  thirst-­‐mediated  motivation  necessary  for  learning  the  task.  Water  restriction,  as  opposed  to  food  restriction,  was  used  as  a  motivator  due  to  the  significant  number  of  health  problems  that  have  been  documented  in  mice  placed  on  food  restriction  in  previous  research.  Water  restriction  typically  began  at  least  three  days  after  surgery,  allowing  the  mice  

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enough  time  to  recover  from  the  operation.  During  water  restriction,  mice  were  given  exactly  1mL  of  water  each  day,  which  was  dispensed  into  bowls  inside  the  cages.  Dry  food  was  available  to  the  mice  in  unlimited  quantity.      Each  day,  while  mice  were  given  water,  each  mouse  was  also  scored  on  a  health  assessment  sheet.  Health  scores  were  determined  by  monitoring  4  different  aspects  of  mouse  behavior  and  appearance.  First,  movement  was  observed  and  monitored  for  abnormal  gait.  Second,  posture  and  grooming  were  assessed  by  taking  note  of  any  hunched  stances  or  ruffled  fur.  Third,  signs  of  eating  and  drinking  were  determined  by  observing  the  number  of  feces  and  urine  traces  in  the  cage.  Fourth,  signs  of  dehydration  were  monitored  by  pinching  the  fur  of  the  mouse  and  looking  for  prolonged  tenting  of  the  fur  upon  its  retraction.  Finally,  each  mouse  was  weighed  in  order  to  determine  whether  or  not  the  rate  of  weight  loss  was  at  an  unhealthy  level.  Each  health  assessment  category  was  given  a  number  score,  with  higher  numbers  indicating  unhealthy  appearances  or  behavior.  Mice  that  received  a  health  score  of  4  or  greater  were  given  an  additional  0.5  mL  of  water  for  the  day.  Mice  that  received  a  health  score  of  6  or  greater  were  removed  from  water  restriction  and  given  access  to  unlimited  water.      Mice  that  obtained  water  rewards  via  behavioral  training  were  allowed  to  continue  their  training  until  they  wanted  to  stop  on  their  own  volition.  Mice  that  received  less  than  0.6  mL  of  water  during  behavioral  training  sessions  were  given  supplemental  water  up  to  0.6  mL.      Head-­‐fixation  and  behavioral  set-­‐up    Mice  perform  tactile  discrimination  via  whisker-­‐mediated  sensation;  head-­‐fixation  of  the  mice  ensured  that  the  resting  position  of  any  given  whisker  was  fixed  at  a  single  value,  enabling  the  quantification  of  whisker  movement.    Prior  to  head-­‐fixation,  mice  were  placed  into  an  acrylic  tube  so  that  movement  of  the  body  was  limited.  Then,  in  order  to  achieve  head-­‐fixation,  the  head  plate  was  inserted  into  stainless  steel  holders  inside  the  behavioral  apparatus.  The  head  plate  was  secured  to  the  holders  by  the  tightening  of  thumbscrews,  which  clamped  the  head  plate  into  place.      The  acrylic  tube  containing  the  mouse  was  coated  in  aluminum  foil  and  linked  to  a  grounding  wire,  in  order  to  provide  an  electrical  connection  between  the  mouse  and  a  lickport  placed  roughly  1mm  away  from  the  mouth.  Extension  of  the  tongue  and  contact  with  the  lickport  effectively  completed  the  electrical  circuit  and  provided  a  signal  to  the  behavioral  software,  indicating  that  a  lick  was  made.      The  MATLAB-­‐based  BControl  software  was  used  for  behavioral  control  and  measurement.  High-­‐frame  video  acquisition  of  whisker  movement  was  obtained  using  StreamPix  software  via  a  Basler  504k  camera  under  940nm  illumination.  

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Precision  tracking  of  whisker  motion  was  achieved  using  the  Janelia  Whisker  Tracker  program.    

 Figure  8.  Head-­‐fixed  behavioral  set-­‐up  for  tactile  discrimination  paradigm.  Behavioral  facilitation,  whisker  imaging,  photoinhibition,  and  electrophysiological  recording  can  all  be  performed  simultaneously  when  using  head-­‐fixed  experimental  paradigms.      Optogenetic  photoinhibition    Optogenetic  tagging  of  specific  cell  types  permitted  neural  manipulation  during  behavior.  PV-­‐Cre  knock-­‐in  mice  were  crossed  to  AI-­‐39  mice  that  expressed  halorhodopsin  in  Cre,  permitting  the  optogenetic  manipulation  of  PV+  interneurons.    

 Photoinhibition  was  remotely  controlled  along  with  behavior  through  the  use  of  BControl  software  on  a  single  computer.  A  separate  computer  was  used  to  run  Ephus  software,  which  triggered  high-­‐speed  video  recording  for  each  trial,  and  aligned  video  recording  with  photoinhibition  pulses.  Light  pulses  were  delivered  to  target  regions  within  S1  of  the  exposed  cortex  by  a  473nm  laser  under  the  control  of  an  acousto-­‐optical  modulator  and  a  real-­‐time  Linux  system.  In  order  to  prevent  mice  

from  responding  to  the  sound  of  the  laser  shutter  opening  and  not  to  the  targeted  photoinhibition  itself,  a  ‘sham  pulse’  of  roughly  0.1mW  was  delivered  during  non-­‐inhibition  trials  onto  the  head  plate  of  the  mouse.                Figure  9.  Selective  illumination  of  mouse  cortex  during  delivery  of  a  4  second  10mW  laser  pulse.  Intrinsic  signal  images  allowed  identification  of  C2  whisker  representation  in  primary  somatosensory  cortex;  photoinhibition  was  targeted  to  the  identified  representation.      

             

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