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The systems neurobiology of autism spectrum disorders (ASD) : a histological and functional approach Cairns, James Maxwell 2015

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	  	  	  THE	  SYSTEMS	  NEUROBIOLOGY	  OF	  AUTISM	  SPECTRUM	  DISORDERS	  (ASD):	  A	  HISTOLOGICAL	  AND	  FUNCTIONAL	  APPROACH	  	  	  	  by	  	  	  James	  Maxwell	  Cairns	  	  	  	  B.Sc.,	  The	  University	  of	  British	  Columbia,	  2012	  	  	  	  A	  THESIS	  SUBMITTED	  IN	  PARTIAL	  FULFILLMENT	  OF	  THE	  REQUIREMENTS	  FOR	  THE	  DEGREE	  OF	  	  	  MASTER	  OF	  SCIENCE	  	  in	  	  THE	  FACULTY	  OF	  GRADUATE	  AND	  POSTDOCTORAL	  STUDIES	  	  (Neuroscience)	  	  	  THE	  UNIVERSITY	  OF	  BRITISH	  COLUMBIA	  	  (Vancouver)	  	  March	  2015	  	  	  © James	  Maxwell	  Cairns,	  2015	  	  	  	  	  	  	  	  	   ii	  Abstract	  	  	  Objective:	  To	  explore	  the	  relationship	  between	  cerebellar	  pathology	  and	  changes	  in	  neuronal	  activity	  in	  mouse	  models	  of	  autism-­‐like	  phenotypes.	  	  Methods:	  We	  used	   the	   rotarod	   test	   as	   a	  measure	  of	   sensorimotor	   function	   in	  our	  mice	  and	  as	   a	  means	   to	   trigger	  neuronal	   activation.	   Following	  behavioural	   testing	  we	  obtained	  brain	  tissue	  from	  our	  ASD-­‐like	  mouse	  models	  and	  used	  histology	  and	  microscopy	  to	  examine	  the	  expression	  of	  cFos	  (a	  reporter	  of	  neuronal	  activity)	  and	  several	   other	   structural	   and	   functional	   markers	   to	   evaluate	   cerebellar	   pathology.	  Finally,	   we	   looked	   at	   differences	   in	   the	   morphology,	   distribution	   and	   number	   of	  cerebellar	   glia	   in	   our	   ASD-­‐like	   mouse	   models	   to	   determine	   if	   reactive	   gliosis	  contributes	  to	  further	  cerebellar	  pathology	  in	  adult	  mice.	  	  Results:	   Compared	   to	   wildtype	   littermates,	   Lc/+	   mutant	   mice	   performed	  significantly	   worse	   on	   the	   rotarod	   assay	   of	   sensorimotor	   function	   (p<0.0001).	   In	  addition,	   Lc/+	   mutants	   have	   significantly	   increased	   neuronal	   activity	   in	   the	  cerebellum	   and	   cortex	   at	   rest	   and	   following	   cerebellar	   rotarod	   activation	   as	  compared	   to	  wildtype	   littermates	   (p<0.05	   for	  each	  group).	  Lurcher	   chimeras	  with	  the	   severest	   cerebellar	  pathology	  have	   increased	  neuronal	   activity	   in	   the	  GCL	  and	  CN	  neurons,	  but	  decreased	  neural	  activity	  in	  inhibitory	  PCs	  and	  they	  have	  increased	  numbers	  of	  activated	  microglia	  and	  Bergmann	  glia	  in	  the	  cerebellar	  cortex.	  Fmr1	  KO	  mice	   have	   a	   slight	   decrease	   in	   PC	   numbers	   as	   compared	   to	   Fmr1	   wildtypes	  	   iii	  (p=0.0862	  n.s)	  with	  alterations	  in	  neuronal	  activity	  at	  rest	  in	  the	  cerebellar	  GCL	  and	  cortex.	  	  Conclusion:	   Variable	   cerebellar	   pathology	   seen	   in	   human	   cases	   of	   autism	   and	   in	  ASD-­‐like	   mouse	   models	   in	   the	   form	   of	   neuron	   loss,	   microgliosis	   and	   astrogliosis	  leads	   to	   changes	   in	   excitatory	   and	   inhibitory	   activity	   in	   surviving	   cerebellar	  neurons.	  Structural	  and	  functional	  changes	  documented	  in	  Lc/+	  mutants,	  chimeras	  and	  Fmr1	  KO	  mice	  revealed	  neuroanatomical	  abnormalities	  and	  functional	  changes	  in	   cerebellar	   neural	   circuits	   which	   may	   lead	   to	   a	   better	   understanding	   of	   the	  neurobiological	   changes	   occurring	   in	   the	   cerebellum	   that	   contribute	   to	   ASD-­‐like	  phenotypes.	   By	   identifying	   cerebellar	   neurons	   and	   glia	   that	   are	   involved	   in	  pathological	   processes	   in	   mouse	   models	   of	   neurodevelopmental	   disorders,	   it	   is	  hoped	   that	   these	   results	   will	   provide	   fresh	   insights	   into	   neurobiological	   changes	  underlying	  ASD-­‐like	  phenotypes.	  	  	  	  	  	  	  	  	  	  	  	  	  	   iv	  Preface	  	   Histological,	  pathological,	  morphological	  and	  functional	  data	  for	  Lc/+	  mutant,	  Lurcher	  chimera,	  Fmr1	  KO	  mice	  and	  wildtype	  littermates	  was	  collected	  at	  the	  Centre	  for	  Molecular	  Medicine	  and	  Therapeutics	  (CMMT),	  BC	  Children’s	  Hospital	  Campus,	  UBC,	   Vancouver,	   British	   Columbia,	   Canada	   between	   2011	   and	   2014.	   Behavioural	  testing	   of	   Lc/+	   mutants	   and	   wildtype	   littermates	   took	   place	   at	   UBC/CMMT	   and	  behavioural	   testing	   of	   Lurcher	   chimeras	   was	   conducted	   at	   the	   Department	   of	  Psychology,	   University	   of	   Memphis,	   Memphis,	   Tennessee,	   USA.	   Methodologies	  involving	  the	  breeding	  and	  use	  of	  mice	  in	  experiments	  were	  reviewed	  and	  approved	  by	   the	  UBC	  Animal	   Care	  Committee	   according	   to	   ethical	   standards	   and	   guidelines	  established	  by	   the	  CCAC.	   (UBC/CMMT	  Goldowitz	  Lab-­‐Breeding	  Protocol	  A12-­‐0190	  and	   UBC/CMMT	  Goldowitz	   Lab-­‐Developmental	   Analysis	   of	   Axonal	   Pathways	   A13-­‐0177).	  	  	   Some	   experimental	   findings	   presented	   in	   this	   thesis	   were	   submitted	   in	  abstract	   form	   and	   as	   a	   poster	   presentation	   at	   the	   2014	   Canadian	   Association	   of	  Neuroscience	   (CAN)	   Meeting	   in	   Montréal,	   Quebec.	   I,	   James	   Cairns,	   was	   the	   lead	  author	   of	   the	   abstract	   and	   poster	   and	   presented	   the	   research	   poster	   at	   the	   2014	  CAN	   meeting.	   Studies	   presented	   in	   this	   thesis	   have	   not	   been	   submitted	   for	  publication	  at	  the	  time	  of	  thesis	  submission.	  	  	   v	  	   I,	   James	  Cairns,	  was	  the	  primary	  investigator	  on	  the	  project,	  responsible	  for	  brain	   tissue	   preparation,	   embedding,	   sectioning,	   histological	   and	   morphological	  analyses,	  microscopy,	  data	  analysis,	  statistical	  analysis	  and	  manuscript	  composition.	  Dr.	   Daniel	   Goldowitz	   and	   CMMT	   research	   associates	   Joanna	   Yeung,	   Fernando	  Villegas,	   Dr.	   Doug	   Swanson	   and	   Peter	   Zhang	   were	   involved	   in	   Lurcher	   chimera	  mouse	   generation	   and	   aided	   in	   the	   writing	   of	   experimental	   and	   animal	   care	  protocols.	   Dr.	   Price	   Dickson	   and	  Dr.	   Guy	  Mittleman	   at	   the	   University	   of	  Memphis	  aided	  in	  the	  collection	  and	  analysis	  of	  behavioural	  data	   for	  Lurcher	  chimeric	  mice.	  Dr.	   Daniel	   Goldowitz	   was	   the	   supervisory	   author	   and	  mentor	   on	   the	   project	   and	  provided	  guidance	  on	  study	  design	  and	  thesis	  revisions.	  Anna	  Sinova,	  Ronny	  Chan	  and	   Praneetha	   Potluri	   in	   the	   Goldowitz	   lab	   assisted	   with	   tissue	   preparation,	   cell	  counts	  and	  immunostaining	  for	  some	  brain	  tissue	  analyzed	  in	  the	  project.	  	  	  	  	  	  	  	  	  	  	  	  	   vi	  Table	  of	  Contents	  	  Abstract……………………………………………....…………………………………………………………	  ii	  Preface………………………………………………………………....……………………………………….	  iv	  Table	  of	  Contents…………………………………………………...……………………………………..	  vi	  List	  of	  Figures............................................................................................................................x	  List	  of	  Abbreviations…………………………………..………………………………………………..xiv	  Acknowledgements……………………...…………………………………………………………….xviii	  Dedication…………………………………..………………………………………………………………..xix	  Chapter	  1-­‐Introduction	  and	  background……………………………………………………..1	  	   1.1-­‐Autism	  Spectrum	  Disorders	  (ASD)	  and	  the	  cerebellum………...……………...1	  	   1.2-­‐Fragile	  X	  Syndrome	  (FXS)	  and	  the	  cerebellum………………...…………………..4	  	   1.3-­‐Activated	  microglia	  and	  astroglia	  seen	  in	  cases	  of	  ASD…………………...……6	  1.4-­‐Experimental	  mouse	  models	  of	  Autism	  Spectrum	  Disorders	  (ASD)…….11	  1.4.1-­‐Lurcher	  mutant	  (Lc/+)	  mouse……………………………………………..11	  1.4.2-­‐Lurcher	  chimeric	  mice………………………………………………………..13	  1.4.3-­‐Fragile	  X	  Mental	  Retardation	  (Fmr1	  KO)	  mice……………………...17	  	  	  	  1.5-­‐The	  use	  of	  cFos	  as	  an	  indirect	  marker	  of	  neuronal	  firing	  to	  study	   	  changes	  in	  neural	  activity	  in	  mouse	  models	  of	  ASD-­‐like	  phenotypes……......19	  	  1.6-­‐Overview	  of	  the	  study……………………………………………………………………...21	  	   	   1.6.1-­‐Aims	  and	  purpose……………………………………………………………....21	  	   1.6.2-­‐Hypotheses………………………………………………………………………...22	  	   1.6.3-­‐Challenges	  and	  further	  considerations……..……………………….....22	  	   vii	  Chapter	  2-­‐Materials	  and	  methods……………………………………………………………….24	  	   2.1-­‐Behavioural	  testing	  of	  Lc/+	  mutants,	  controls	  and	  Lurcher	  chimeras..24	  	   	   2.1.1-­‐Rotarod	  testing	  of	  Lurcher	  mutant	  mice	  and	  wildtype	  mice...24	  	   2.1.2-­‐Rotarod	  testing	  of	  Lurcher	  chimera	  mice……………………………25	  2.2-­‐Preparation	  of	  brain	  tissue	  for	  immunohistochemical	  analysis……...….26	  2.3-­‐Immunohistochemical	  staining	  and	  analysis……………..……………………...27	  	   2.3.1-­‐Purkinje	  cell	  staining	  and	  visualization…………….………………..27	  	   2.3.2-­‐cFos	  immunostaining	  and	  analysis………….…………………………28	  	   2.3.3-­‐Immunofluorescence	  for	  cFos,	  Calbindin,	  Iba1	  and	  GFAP.…...29	  2.4-­‐Analysis	  and	  quantification	  of	  histological	  and	  functional	  data….……30	  	   2.4.1-­‐Analysis	  and	  quantification	  of	  Purkinje	  cells………….…………..30	  	  	   2.4.2-­‐cFos	  analysis	  and	  quantification…………….………………………….31	  2.5-­‐Statistical	  analysis…………………………………………………………………………31	  Chapter	  3-­‐Experiments	  and	  results……………………….…………………………………..34	  3.1-­‐Lurcher	  mutants	  and	  wildtype	  (+/+)	  littermates……………………………..34	  3.1.1-­‐Cerebellar	  pathology	  in	  Lc/+	  mutants	  as	  compared	  to	  +/+	   	  controls……………………………………………………………………………………34	  	  	  	  	  	  	  	  	  3.1.2-­‐Lc/+	  mutants	  exhibit	  increased	  cFos	  expression	  under	   	  basal	  conditions	  and	  following	  rotarod	  cerebellar	  activation……...35	  	  3.2-­‐Lurcher	  chimeras…………………………………………………………………………..45	  	   3.2.1-­‐Lurcher	  chimeras	  exhibit	  variable	  cerebellar	  pathology……..45	  	  	  	  	  	  3.2.2-­‐cFos	  staining	  in	  cerebellar	  granule	  cells	  is	  inversely	   	  correlated	  with	  the	  number	  of	  surviving	  PCs	  in	  Lc	  chimeras……….48	  	  	   viii	  	  	  	  3.2.3-­‐cFos	  staining	  in	  cerebellar	  Purkinje	  cells	  is	  positively	   	  correlated	  with	  the	  number	  of	  surviving	  PCs	  in	  Lc	  chimeras………...52	  	  	  	  	  3.2.4-­‐cFos	  staining	  in	  cerebellar	  nuclei	  (CN)	  neurons	  is	   	  	  	  	  inversely	  correlated	  with	  the	  number	  of	  surviving	  PCs	  and	  the	  	   	  number	  of	  cFos	  positive	  PCs	  in	  Lc	  chimeras……….………………………...60	  	  	  	  	  3.2.5-­‐Changes	  in	  the	  number,	  morphology	  and	  distribution	  of	   	  	  	  	  	  cerebellar	  glia	  following	  Purkinje	  cell	  death	  in	  Lc	  mutants	  and	  	   	  chimeras………………………..……………………………………………………………66	  	  3.3-­‐Fmr1	  KO	  mice……………………………………………………………….………………....77	  	  	  	  	  3.3.1-­‐Trend	  of	  decreased	  PC	  numbers	  and	  altered	  PC	  morphology	   	  in	  Fmr1	  KO	  mice	  as	  compared	  to	  Fmr1	  wildtype	  mice………….……….77	  	  3.3.2-­‐Altered	  baseline	  cFos	  expression	  in	  Fmr1	  KO	  mice……………...80	  Chapter	  4-­‐Discussion………………………………………………………………………………….88	  	  4.1-­‐Elevated	  cFos	  staining	  in	  Lc/+	  mutant	  mice	  suggests	  that	  Lc/+	  mutants	   	  	  	  	  	  have	  increased	  neural	  activity	  in	  the	  cerebellum	  and	  connected	  cortical	  	   	  regions	  due	  to	  the	  developmental	  loss	  of	  inhibitory	  cerebellar	  PCs..………..88	  	  	  	  	  4.2-­‐Increased	  cFos	  staining	  in	  cerebellar	  GCs	  of	  Lurcher	  chimeras	  with	   	  	  	  	  the	  highest	  degree	  of	  cerebellar	  pathology	  suggests	  that	  there	  is	  a	  shift	  in	   	  the	  neural	  activity	  of	  surviving	  pre-­‐synaptic	  GCs	  in	  response	  to	  PC	  death..94	  	  4.3-­‐Higher	  numbers	  of	  cFos	  positive	  PCs	  in	  wildtype-­‐like	  Lurcher	  chimeras	   	  represents	  increased	  inhibition	  of	  post-­‐synaptic	  CN	  neurons….......................97	  	  	  	  4.4-­‐Cerebellar	  nuclei	  neuronal	  activity	  is	  inversely	  correlated	  with	  the	   	  	  	  amount	  of	  inhibition	  received	  from	  cerebellar	  PCs	  indicating	  that	  a	  loss	  	   	  	   ix	  of	  inhibitory	  PCs	  leads	  to	  a	  shift	  in	  the	  efferent	  output	  of	  the	  cerebellum…101	  	  	  4.5-­‐Cerebellar	  glial	  cells	  undergo	  changes	  in	  structure	  and	  function	   	  	  following	  cell	  death	  and	  neurodegeneration	  in	  mouse	  models	  of	  ASD-­‐like	  	   	  phenotypes…………………………………………………………………………………………...103	  	  4.6-­‐Fmr1	  KO	  mice	  exhibit	  altered	  resting	  state	  cFos	  expression	  in	  the	  cerebellum	  and	  cortex	  suggesting	  that	  changes	  in	  cerebellar	  and	  cortical	  activity	  are	  common	  features	  associated	  with	  FXS	  and	  other	  forms	  of	  	  ASD...............................................................................................................................................110	  4.7-­‐ASD	  as	  a	  neurodevelopmental	  disorder	  caused	  by	  an	  imbalance	  	  in	  excitatory	  and	  inhibitory	  synaptic	  transmission	  in	  the	  CNS…..............…….113	  4.8-­‐Future	  directions	  and	  conclusions……………………….…………………………...118	  	  References……………………………………………………………………………………………….....120	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	   x	  List	  of	  Figures	  Figure	  1	   Abercrombie	  correction	  formula	  for	  PC	  counts...........................................	  ............................................................................................	  33	  Figure	  2	   Cerebellum	  of	  a	  wildtype	  (+/+)	  mouse	  stained	  for	  Calbindin-­‐D28K...........................................................................................................................................34	  Figure	  3	   Performance	  on	  the	  rotarod	  test	  of	  sensorimotor	  function…………..37	  Figure	  4	   Average	  density	  of	  cFos	  positive	  cells	  in	  wildtype	  (+/+)	  vs.	  Lc/+	  mutant	  animals	  rotarod	  activation	  and	  baseline	  levels……………………....…….................	  ...................................................................................	  38	  Figure	  5	   Average	  density	  of	  cFos	  staining	  in	  the	  cerebellum	  of	  wildtype	  mice	  as	  compared	  to	  Lc/+	  mutant	  mice…………………………………………………………………………...	  ...........................................	  39	  Figure	  6	   Average	  density	  of	  cFos	  staining	  in	  the	  orbitofrontal	  cortex	  of	  wildtype	  mice	  as	  compared	  to	  Lc/+	  mutant	  mice…………………………………………………	  ............................................................................	  40	  Figure	  7	   Average	  density	  of	  cFos	  staining	  in	  the	  posterior	  cortex	  of	  wildtype	  mice	  as	  compared	  to	  Lc/+	  mutant	  mice……………………………………………………………….	  ..........................................................	  41	  Figure	  8	   Average	  density	  of	  cFos	  staining	  in	  the	  somatomotor	  cortex	  of	  wildtype	  mice	  as	  compared	  to	  Lc/+	  mutant	  mice…………………………………………………	  ............................................................................	  42	  Figure	  9	   Micrographs	  showing	  cFos	  activation	  in	  rotarod-­‐activated	  Lc/+	  mutants	  and	  wildtype	  (+/+)	  mice………………………………………………………………………..	  ...............................................	  43	  Figure	  10	   Micrographs	  showing	  basal	  cFos	  activation	  in	  Lc/+	  mutants	  and	  wildtype	  (+/+)	  mice……………………………………………………………………………………………	  ......................	  44	  	   xi	  Figure	  11	   Bar	  graph	  showing	  PC	  counts	  in	  individual	  Lc	  chimeras,	  gross	  cerebellar	  pathology	  in	  Lc	  chimeras	  and	  GCL	  morphology	  in	  Lc	  chimeras…………….	  ................................................................................................................	  48	  Figure	  12	   Relationship	  between	  the	  number	  of	  surviving	  PCs	  and	  the	  average	  cFos	  staining	  density	  in	  the	  GCL	  of	  the	  cerebellum	  of	  Lc	  chimeras..………………………	  ..................................................................................................	  50	  Figure	  13	   Differences	  in	  the	  size	  and	  area	  of	  the	  cerebellar	  GCL	  and	  differences	  in	  cFos	  staining	  in	  the	  cerebellar	  GCL	  in	  Lc	  chimeras…………………………………………...	  ..........................................................................	  51	  Figure	  14	   Relationship	  between	  the	  average	  number	  of	  cFos	  positive	  Purkinje	  cells/section	  and	  %	  chimerism	  in	  individual	  Lc	  chimeras…………………………................	  .................................................................................	  53	  Figure	  15	   Average	  number	  of	  cFos	  positive	  PCs/section	  in	  17	  Lc	  chimeras	  with	  variable	  cerebellar	  pathology……………………………………………………………………………..	  ..............................	  54	  Figure	  16	   Photomicrographs	  showing	  cFos	  positive	  PCs	  in	  the	  cerebellum	  of	  two	  different	  Lc	  chimeras	  along	  with	  cFos	  staining	  in	  the	  GCL....................................................	  .........................................................................................	  55	  Figure	  17	   Photomicrographs	  showing	  co-­‐localization	  of	  Calbindin-­‐D28K	  and	  cFos	  in	  cerebellar	  Purkinje	  cells	  of	  Lc	  chimeras………………………………………......……….	  ...............................................................	  59	  Figure	  18	   Light	  photomicrographs	  of	  DAB	  staining	  showing	  the	  difference	  in	  the	  density	  and	  number	  of	  cFos	  positive	  CN	  neurons	  in	  individual	  Lc	  chimeras………….	  ....................................................................................................................	  61	  Figure	  19	   Immunofluorescence	  staining	  showing	  differences	  in	  the	  density	  and	  number	  of	  cFos	  positive	  CN	  neurons	  in	  individual	  Lc	  chimeras…………………………….	  ............................................................................................	  62	  	   xii	  Figure	  20	   Bar	  graph	  showing	  the	  average	  number	  of	  cFos	  positive	  CN	  neurons	  in	  Lc	  chimeras……………………………………………………………………………………………………….	  63	  Figure	  21	   Photomicrographs	  demonstrating	  Tbr1	  immunofluorescence	  in	  CN	  neurons	  of	  a	  Lc/+	  and	  wildtype	  mouse………………………………………………………………..	  .....................................................	  65	  Figure	  22	   Photomicrographs	  demonstrating	  differences	  in	  cerebellar	  microglial	  morphology	  in	  a	  Lc/+	  and	  wildtype	  mouse………………………………………………………….	  .............................................................	  67	  Figure	  23	   Photomicrographs	  showing	  differences	  in	  the	  distribution	  and	  morphology	  of	  microglia	  in	  the	  cerebellar	  cortex	  of	  individual	  Lc	  chimeras………….	  ....................................................................................................................	  69	  Figure	  24	   Bar	  graph	  showing	  the	  average	  number	  of	  Iba1	  positive	  microglia	  in	  the	  cerebellar	  cortex	  of	  19	  Lc	  chimeras……………………………………………………………….	  .................................................	  70	  Figure	  25	   Iba1	  and	  GFAP	  staining	  of	  microglia	  and	  cerebellar	  astroglia	  in	  a	  Lc/+	  mutant	  and	  wildtype	  mouse……………………………………………………………………………….	  ..................................	  73	  Figure	  26	   Immunofluorescence	  showing	  GFAP,	  Iba1	  and	  DAPI	  staining	  in	  the	  cerebellar	  cortex	  of	  3	  different	  Lc	  chimeras	  at	  10x	  magnification…………………………	  ........................................................................................	  75	  Figure	  27	   Immunofluorescence	  showing	  GFAP,	  Iba1	  and	  DAPI	  staining	  in	  the	  cerebellar	  cortex	  of	  3	  different	  Lc	  chimeras	  at	  20x	  magnification………………………....	  .......................................................................................	  76	  Figure	  28	   Average	  Number	  of	  Purkinje	  cells	  in	  Fmr1	  KO	  and	  wildtype	  mice	  and	  appearance	  of	  cerebellar	  PCs	  as	  shown	  by	  Calbindin-­‐D28K	  staining…………………….	  .........................................................................................................	  78	  	   xiii	  Figure	  29	   Cresyl	  violet	  counterstaining	  of	  Fmr1	  wildtype	  and	  Fmr1	  KO	  mouse	  brain	  tissue………………………………………………………………………………………………………..	  .....	  79	  Figure	  30	   Overview	  of	  brain	  regions	  analyzed	  when	  looking	  at	  cFos	  staining	  in	  Lc/+	  mutant	  and	  Fmr1	  KO	  mouse	  models…………………………………………………………....	  .........................................................	  80	  Figure	  31	   Bar	  graphs	  showing	  the	  average	  number	  of	  cFos	  positive	  cells	  in	  four	  different	  brain	  regions	  under	  basal	  conditions	  in	  Fmr1	  KO	  and	  wildtype	  mice…...............	  .........................................................................................................................	  82	  Figure	  32	   Photomicrographs	  of	  cFos	  staining	  in	  the	  cerebellar	  GCL	  of	  a	  Fmr1	  KO	  and	  Fmr1	  wildtype	  mouse…………………………………………………………………………………..	  ..............................	  83	  Figure	  33	   Photomicrographs	  of	  cFos	  staining	  in	  the	  orbitofrontal	  cortex	  of	  a	  Fmr1	  KO	  and	  Fmr1	  wildtype	  mouse…………………………………………………………………....	  ................................................	  83	  Figure	  34	   Photomicrographs	  of	  cFos	  staining	  in	  the	  posterior	  cortex	  of	  a	  Fmr1	  KO	  and	  Fmr1	  wildtype	  mouse……………………………………………………………………………..	  .....................................	  84	  Figure	  35	   Photomicrographs	  of	  cFos	  staining	  in	  the	  somatomotor	  cortex	  of	  a	  Fmr1	  KO	  and	  Fmr1	  wildtype	  mouse…………………………………………………………………….	  ................................................	  84	  Figure	  36	   Scatterplots	  showing	  the	  relationship	  between	  the	  average	  number	  of	  PCs	  and	  basal	  cFos	  staining	  in	  Fmr1	  wildtype	  versus	  Fmr1	  KO	  mice……………………..	  ..............................................................................................................	  86	  	  	  	  	  	   xiv	  List	  of	  Abbreviations	  	  ABC:	  Avidin-­‐Biotin	  Complex	  reaction	  ACC:	  Anterior	  Cingulate	  Cortex	  ADI-­‐R:	  Autism	  Diagnostic	  Interview-­‐Revised	  	  ADOS-­‐G:	  Autism	  Diagnostic	  Observation	  Schedule-­‐Generic	  AMPA:	  α-­‐Amino-­‐3-­‐hydroxy-­‐5-­‐methyl-­‐4-­‐isoxazolepropionic	  acid	  ANOVA:	  Analysis	  of	  Variance	  statistical	  test	  AP:	  Action	  Potential	  ASD:	  Autism	  Spectrum	  Disorder	  BSA:	  Bovine	  Serum	  Albumin	  BTBR:	  Black	  and	  Tan	  BRachyury	  mouse	  	  CA:	  Cornu	  Ammonis	  (of	  the	  hippocampus)	  cFos:	  FBJ	  murine	  osteosarcoma	  viral	  oncogene	  homolog	  CN:	  Cerebellar	  Nuclei	  CNS:	  Central	  Nervous	  System	  CV:	  Cresyl	  Violet	  staining	  DAB:	  Diaminobenzidene	  	  DAPI:	  4',6-­‐diamidino-­‐2-­‐phenylindole	  Dlx1:	  distal-­‐less	  homeobox	  1	  EM:	  Electron	  Microscopy	  Fmr1:	  Fragile	  X	  Mental	  Retardation	  1	  gene	  FMRP:	  Fragile	  X	  Mental	  Retardation	  Protein	  	   xv	  FXS:	  Fragile	  X	  Syndrome	  GABA:	  γ-­‐Aminobutyric	  acid	  GABAAR:	  ionotropic	  GABAA	  receptor	  GABABR:	  	  metabotropic	  GABAB	  receptor	  	  GABRB3:	  Gamma-­‐aminobutyric	  acid	  receptor	  subunit	  beta-­‐3	  GAD:	  Glutamate	  Decarboxylase	  (GAD65	  and	  GAD67	  isoforms)	  GC:	  Granule	  Cells	  GCL:	  Granule	  Cell	  Layer	  GFAP:	  Glial	  Fibrillary	  Acidic	  Protein	  G.O.F:	  Gain	  of	  Function	  mutation	  Grid2	  aka	  GluRδ2:	  Glutamate	  receptor,	  ionotropic,	  delta	  2	  HLA:	  Human	  Leukocyte	  Antigen	  HPA:	  Hypothalamic-­‐Pituitary	  Axis	  Hz:	  Hertz	  	  IAHP:	  After-­‐hyperpolarization	  current	  Iba1:	  ionized	  calcium-­‐binding	  adapter	  molecule	  1	  IFN-­‐γ:	  Interferon-­‐γ	  IHC:	  Immunohistochemistry	  IL-­‐6:	  Interleukin-­‐6	  iNOS:	  inducible	  Nitric	  Oxide	  Synthase	  ION:	  Inferior	  Olivary	  Nucleus	  IQ:	  Intelligence	  Quotient	  KO:	  Knockout	  mouse	  	   xvi	  Lc/+:	  Lurcher	  mouse	  L.O.F:	  Loss	  of	  Function	  mutation	  LTP:	  Long	  Term	  Potentiation	  M1	  Microglia:	  pro-­‐inflammatory	  subtype	  of	  microglia	  M2	  Microglia:	  anti-­‐inflammatory	  or	  neuro-­‐protective	  subtype	  of	  microglia	  mEPSCs:	  miniature	  Excitatory	  Post-­‐synaptic	  Currents	  mIPSCs:	  miniature	  Inhibitory	  Post-­‐synaptic	  Currents	  ML:	  Molecular	  Layer	  MRI:	  Magnetic	  Resonance	  Imaging	  mRNA:	  messenger	  RNA	  NGS:	  Normal	  Goat	  Serum	  NKCC:	  Na-­‐K-­‐Cl	  co-­‐transporter-­‐1	  NMDA:	  N-­‐methyl-­‐D-­‐Aspartate	  Nna1:	  ATP/GTP	  binding	  protein-­‐1	  NO:	  	  Nitric	  Oxide	  O.C.T.:	  Optimal	  Cutting	  Temperature	  compound	  for	  embedding	  tissue	  P:	  Postnatal	  day	  	  PBS:	  Phosphate	  buffered	  saline	  PBS-­‐T:	  Phosphate	  buffered	  saline	  with	  Triton	  PC:	  Purkinje	  cell	  PCD:	  Purkinje	  Cell	  Degeneration	  mouse	  PCL:	  Purkinje	  cell	  Layer	  PDD-­‐NOS:	  Pervasive	  developmental	  disorder	  not	  otherwise	  specified	  	   xvii	  PET:	  Positron	  Emission	  Tomography	  PFA:	  Paraformaldehyde	  PVN:	  Paraventricular	  Nucleus	  of	  the	  Hypothalamus	  RNA:	  Ribonucleic	  Acid	  rpm:	  rotations	  per	  minute	  Scn1a: sodium	  channel,	  voltage-­‐gated,	  type	  I,	  alpha	  subunit	  sIPSCs:	  spontaneous	  Inhibitory	  Post-­‐synaptic	  Currents	  SK:	  small	  conductance	  calcium-­‐activated	  potassium	  channels	  SPECT:	  single	  photon	  emission	  computed	  tomography	  TNF-­‐α :	  Tumour	  Necrosis	  Factor-­‐α	  WT:	  wildtype	  mouse	  	  	  	  	  	  	  	  	  	  	  	  	  	   xviii	  Acknowledgements	  	   I	   would	   like	   to	   thank	   my	   supervisor	   Dr.	   Daniel	   Goldowitz	   for	   his	   ongoing	  support	  and	  guidance	  throughout	  my	  thesis	  project.	   	   I	  would	  also	  like	  to	  thank	  my	  committee	  members	  Dr.	  Tim	  Murphy,	  Dr.	  Anthony	  Bailey	  and	  Dr.	  Ruth	  Grunau	   for	  their	   feedback	   and	   advice	   on	   this	   research	   project.	   Thank	   you	   to	   the	   previous	  graduate	  student	  Ms.	  Anna	  Sinova	  in	  the	  Goldowitz	  lab	  for	  getting	  me	  started	  on	  the	  project	   and	   Mr.	   Ronny	   Chan	   and	   Ms.	   Praneetha	   Potluri	   for	   their	   assistance	   with	  sectioning	   and	   immunostaining	   of	   brain	   tissue.	   Finally,	   I	   would	   like	   to	   extend	   a	  special	  thank	  you	  to	  the	  lab	  members	  and	  technicians	  in	  the	  Goldowitz	  lab	  for	  their	  expertise	  and	  knowledge	  in	  the	  generation	  and	  production	  of	  chimeric	  mice.	  	   I	  would	  also	  like	  to	  say	  a	  warm	  thank	  you	  to	  members	  of	  the	  Goldowitz	  Lab	  for	   their	   friendship	   and	   ongoing	   support	   throughout	   my	   time	   in	   the	   lab.	   Your	  patience,	  guidance	  and	  support	  were	  invaluable	  in	  the	  development	  and	  writing	  of	  this	  thesis.	  	  	  	  	  	  	  	  	  	   xix	  	  	  	  Dedication	  	   To	  my	  family	  and	  friends,	  this	  thesis	  is	  dedicated	  to	  you.	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	   1	  Chapter	  1-­‐Introduction	  and	  background	  	  1.1-­‐Autism	  Spectrum	  Disorders	  (ASD)	  and	  the	  cerebellum	  	  	   Autism	  and	  Autism	  Spectrum	  Disorders	  (ASD)	  are	  a	  heterogeneous	  group	  of	  disorders,	   with	   the	   severity	   of	   symptoms	   varying	   widely	   between	   individuals.	  Autism	   can	   be	   characterized	   by	   symptoms	   such	   as	   impaired	   and	   disordered	  language,	   decreased	   social	   interactions,	   extreme	   fixation	   on	   certain	   objects	   or	  activities	  and	  impaired	  fine	  and	  gross	  motor	  movements	  [1,2].	  Clinically,	  ASD	  can	  be	  separated	   into	   3	   categories:	   autistic	   disorder,	   Asperger’s	   disorder	   and	   pervasive	  developmental	   disorder	   not	   otherwise	   specified	   (PDD-­‐NOS)	   [3].	   Those	   diagnosed	  with	  Asperger’s	   disorder	   have	   normal	   or	   above	   average	   cognitive	   skills	   and	  don’t	  have	   language	  deficits;	   those	  diagnosed	  with	  PDD-­‐NOS	  have	   social	  deficits,	  but	  do	  not	   meet	   the	   full	   diagnostic	   criteria	   for	   autistic	   disorder	   [3].	   In	   addition,	   a	  disproportionate	  number	  of	  people	  diagnosed	  with	  autism	  are	  male,	  with	  a	  ratio	  of	  4	  males:	  1	  female	  being	  diagnosed	  with	  some	  form	  of	  ASD	  [3].	  	  ASDs	  are	  diagnosed	  using	   behavioural	   measures	   and	   diagnostic	   tests	   such	   as	   the	   Autism	   Diagnostic	  Interview-­‐Revised	  (ADI-­‐R)	  and	  the	  Autism	  Diagnostic	  Observation	  Schedule-­‐Generic	  (ADOS-­‐G)	   [4].	   However,	   to	   date	   there	   are	   no	   consistently	   used	   biological	   tests	   or	  clinically	  relevant	  biomarkers	  available	  to	  accurately	  diagnose	  ASD.	  	  	   Many	  studies	  have	  found	  that	  altered	  structural	  and	  functional	  connectivity	  between	  adjacent	  and	  distant	  brain	  regions	  may	  play	  a	  role	  in	  the	  neuropathology	  of	  ASD.	   For	   example,	   it	   is	   now	   known	   that	   there	   are	   structural	   and	   functional	  abnormalities	   in	   the	   cerebellum	   and	   connected	   brain	   regions	   in	   both	   people	  	   2	  diagnosed	  with	  ASD	  and	  also	  in	  mouse	  models	  that	  display	  autistic-­‐like	  behavioural	  phenotypes	  and	  neuropathology	  [5].	  Examples	  of	  abnormalities	   include:	  decreased	  numbers	  of	  neurons	   in	   the	   cerebellum,	   altered	  migration	  patterns	  of	  neurons	  and	  abnormal	   synaptogenesis	   between	   neurons	   [6].	   In	   humans	   diagnosed	   with	   ASD	  common	  neuroanatomical	  changes	  include	  variable	  loss	  of	  cerebellar	  Purkinje	  Cells	  (PCs),	   abnormal	   clustering	  of	  neurons	   in	   the	   inferior	  olivary	  nucleus	   (ION)	  and	   in	  some	  patients,	  decreased	  thickness	  of	  the	  cortical	  grey	  matter	  as	  the	  patients	  aged	  [7].	  	  	   Neuroimaging	   studies	   of	   ASD	   patients	   have	   discovered	   abnormalities	   in	  lobules	   VI	   and	  VII	   of	   the	   cerebellar	   vermis	   [8].	   This	   is	   significant	   because	   studies	  have	   found	   that	   the	  posterior	   vermis	   of	   the	   cerebellum	  has	   a	   functional	   link	  with	  cortical	   brain	   regions	   that	   are	   involved	   in	   the	   interpretation	   and	   production	   of	  language	   [9].	   In	   clinical	   studies	   evaluating	   patients	   with	   acquired	   lesions	   to	   the	  posterior	  vermis	  of	  the	  cerebellum	  (lesions	  similar	  to	  damage	  seen	  in	  ASD	  patients)	  deficits	  in	  language,	  cognition	  and	  executive	  functions	  were	  also	  observed	  [10].	  This	  provides	   evidence	   that,	   through	   its	   connections	   with	   cortical	   regions,	   the	  cerebellum	   plays	   an	   important	   role	   in	   modulating	   higher	   order	   brain	   functions,	  including	  cognition	  and	  language.	  	  	   In	  addition	  to	  the	  neuroanatomical	  and	  behavioural	  studies	  of	  ASD,	  the	  field	  of	  neurogenetics	  has	  also	  provided	  valuable	  information	  regarding	  the	  etiology	  and	  pathological	   features	   of	   ASD	   and	   related	   neurodevelopmental	   disorders.	   ASD	  	   3	  heritability	  estimates	  from	  numerous	  studies	  are	  quite	  high,	  ranging	  from	  37-­‐90%,	  which	  suggests	  that	  the	  genetic	  contribution	  to	  individual	  cases	  of	  ASD	  is	  significant	  [11,12].	  Multiple	  studies	  have	  also	  demonstrated	  that	  ASD	  appears	  to	  be	  a	  polygenic	  neurodevelopmental	   disorder,	  with	   different	   types	   of	   genetic	   variants	   at	   genomic	  loci	   each	   contributing	   a	   small	   risk	   to	   developing	   the	   disorder	   [13].	   Several	  previously	   identified	   genetic	   variants	   that	   have	   been	   linked	   to	   ASD	   or	   related	  disorders	  also	  show	  overlap	   in	   their	  effects	  on	  cellular	   function	  and	  cell	   signaling.	  Mutations	   linked	   with	   autism	   commonly	   affect	   neural	   processes	   including	  synaptogenesis,	   neural	   signaling	   and	   neuronal	   migration	   in	   brain	   regions	  responsible	  for	  integrating	  neural	  impulses	  from	  multiple	  cortical	  association	  areas	  (including	  the	  prefrontal	  cortex,	  temporal	  association	  areas	  and	  the	  cerebellum)	  [7].	  Specifically,	  mutations	  in	  synaptic	  proteins	  such	  as	  Neurexin-­‐1,	  -­‐2,	  and	  Neuroligin-­‐3	  (which	  are	  important	  for	  the	  stabilization	  of	  synapses	  and	  trans-­‐synaptic	  signaling)	  have	  been	  discovered	  in	  a	  subset	  of	  ASD	  patients	  [14,15].	  	  	   Taken	   together,	   neuroanatomical,	   behavioural,	   neuroimaging	   and	   genetic	  studies	   of	   the	   etiology	   and	   neuropathology	   of	   ASD	   highlight	   the	   heterogeneity	   of	  symptoms	   and	   the	  wide	   variety	   of	   neuropathology	   that	   can	   be	   seen	   in	   individual	  cases	   of	   autism.	   Experimental	   evidence	   also	   points	   to	   the	   importance	   of	   the	  cerebellum	   and	   abnormalities	   in	   cerebellar	   structure	   and	   function	   in	   the	  pathophysiology	  of	  ASD.	  It	  is	  for	  these	  reasons	  that	  reliable	  experimental	  models	  of	  ASD	   are	   needed	   to	   study	   the	   underlying	   structural	   and	   functional	   changes	   in	   the	  cerebellum	  and	  other	  brain	  regions	  implicated	  in	  the	  pathogenesis	  of	  autism.	  	   4	  1.2-­‐Fragile	  X	  Syndrome	  (FXS)	  and	  the	  cerebellum	  Fragile	   X	   Syndrome	   is	   a	   neurodevelopmental	   disorder	   characterized	   by	  expansion	  of	  a	  CGG	  trinucleotide	  repeat	  in	  the	  FMR1	  gene,	  which	  is	  located	  on	  the	  X	  chromosome	  [143].	  Clinically,	  Fragile	  X	  syndrome	  (FXS)	  is	  characterized	  by	  mental	  retardation,	   learning	   difficulties,	   macro-­‐orchidism,	   cognitive	   disability,	   impaired	  socialization	   and	   communication	   skills,	   repetitive	   behaviours,	   seizures	   and	  hyperactivity	   [27,144].	   FXS	   affects	   ~1:4500	   males	   and	   ~1:9000	   females	   and	  interestingly	  30%	  of	  people	  diagnosed	  with	  FXS	  are	  also	  diagnosed	  with	  some	  form	  of	   ASD	   [7,28].	   FXS	   is	   one	   of	   the	   most	   common	   monogenic	   neurodevelopmental	  disorders	   associated	   with	   autistic	   symptoms	   and	   since	   it	   shares	   many	   clinical	  features	   with	   cases	   of	   ASD	   has	   become	   a	   popular	   research	   area	   to	   study	   the	  neurogenetic	  bases	  of	  ASD	  and	  related	  neurodevelopmental	  disorders	  [145].	  	  	   Fragile	  X	  Mental	  Retardation	  Protein	  (FMRP)	  (the	  gene	  product	  of	  the	  FMR1	  gene)	  plays	  an	   important	   role	   in	   the	   regulation	  of	  numerous	   synaptic	  mRNAs	  and	  recent	  evidence	  suggests	  that	  it	  may	  play	  an	  important	  role	  in	  the	  neuronal	  activity-­‐dependent	   transport	  of	  mRNAs	  encoding	   synaptic	  proteins	   to	   specific	   synapses	   in	  the	   CNS	   [33].	   This	   has	   led	   researchers	   to	   hypothesize	   that	   FXS	   is	   a	  neurodevelopmental	   disorder	   characterized	   by	   an	   imbalance	   in	   excitatory	   and	  inhibitory	  synaptic	  transmission.	  Many	  recent	  experiments	  studying	  FXS	  in	  humans	  have	  found	  that	  patients	  diagnosed	  with	  the	  disorder	  have	  an	  imbalance	  in	  cortical	  excitation	  and	  inhibition	  [108].	  Evidence	  to	  support	  this	  has	  come	  from	  the	  finding	  that	   20-­‐25%	   of	   FXS	   patients	   suffer	   from	   some	   form	   of	   epilepsy,	   which	   is	  	   5	  characterized	  by	  suppressed	  inhibitory	  cortical	  synaptic	  transmission	  [143].	  In	  post	  mortem	  studies	  of	  brain	   tissue	   from	  FXS	  patients	   the	  most	  consistent	  pathological	  findings	   have	   been	   increased	   numbers	   of	   immature	   dendritic	   spines	   on	   cortical	  pyramidal	   neurons,	   which	   provides	   evidence	   that	   changes	   in	   cortical	   neuron	  structure	   and	   function	   could	   contribute	   to	  many	   of	   the	   behavioural	   and	   cognitive	  abnormalities	   seen	   in	   FXS	   [146].	   However,	   to	   date	   very	   few	   experiments	   have	  studied	  the	  neuropathological	  and	  functional	  changes	  in	  the	  cerebellum	  of	  patients	  diagnosed	  with	   FXS,	   which	   is	   odd	   because	   cerebellar	   pathology	   has	   been	   seen	   in	  post	   mortem	   and	   neuroimaging	   studies	   of	   patients	   diagnosed	   with	   FXS	   and	   in	  patients	  diagnosed	  with	  both	  FXS	  and	  ASD	  [8].	  	  	   Evidence	  to	  support	  the	  hypothesis	  that	  cerebellar	  pathology	  contributes	  to	  ASD-­‐like	  behavioural	   symptoms	   in	   cases	  of	  FXS	  has	   come	   from	  both	  post	  mortem	  and	   imaging	  studies	  of	  FXS	  patients.	   In	  a	  post	  mortem	  study	  of	  3	  males	  diagnosed	  with	   FXS,	   histopathological	   changes	   were	   observed	   in	   the	   midline	   vermis	   and	  postero-­‐lateral	   cerebellar	   cortex	   of	   all	   3	   subjects	   [147].	   The	   most	   common	  pathological	   findings	  in	  the	  cerebellar	  cortex	  (identified	  by	  hematoxylin,	  eosin	  and	  calbindin	  staining),	  included	  patchy	  loss	  of	  PCs	  with	  a	  loss	  of	  up	  to	  40%	  of	  cerebellar	  PCs	   in	   vermal	   and	   lateral	   cerebellar	   lobules	   compared	  with	   healthy	   age	  matched	  controls	  [147].	  In	  addition	  to	  decreased	  PC	  number	  and	  density	  in	  certain	  regions	  of	  the	  cerebellum	  researchers	  also	   found	  abnormal	  clustering	  of	  surviving	  PCs	   in	   the	  internal	  granular	  layer	  (IGL)	  of	  the	  cerebellum	  and	  decreased	  dendritic	  arborization	  of	   surviving	   PCs	   [147].	   These	   findings	   are	   consistent	   with	   neuroanatomical	  	   6	  abnormalities	  and	  changes	  in	  PC	  morphology	  seen	  in	  idiopathic	  cases	  of	  ASD	  and	  in	  mouse	  models	  of	  ASD	  including	  the	  Lc/+	  mutant	  and	  chimeric	  mouse	  [7,17,22].	  	  	  	   In	  addition	  to	  post	  mortem	  studies	  evaluating	  cerebellar	  pathology	   in	  cases	  of	  FXS,	  several	  neuroimaging	  studies	  have	  been	  conducted	  to	  look	  at	  changes	  in	  the	  structure	  of	  the	  cerebellum	  in	  the	  living	  brains	  of	  patients	  with	  FXS.	  Structural	  MRI	  studies	  have	  shown	  that	  FXS	  patients	  have	  smaller	  cerebellar	  volumes	  as	  compared	  to	  healthy	  age	  matched	  controls,	  particularly	  in	  the	  posterior	  lobe	  of	  the	  cerebellum	  [148].	   In	   more	   detailed	   structural	   MRI	   studies	   researchers	   recruited	   84	   children	  diagnosed	  with	  FXS	  and	  72	  age-­‐matched	  controls	  (subjects	  ranged	  from	  1	  year	  old	  to	   23	   years	   of	   age)	   and	   they	   found	   that	   the	   size	   of	   the	   posterior	   lobe	   of	   the	  cerebellar	   vermis	   was	   correlated	   with	   levels	   of	   FMRP	   in	   the	   cerebellum	   [149].	  Neuroimaging	   studies	   have	   also	   found	   that	   lobules	   VI	   and	   VII	   of	   the	   cerebellar	  vermis	   undergo	   atrophy	   in	   patients	   diagnosed	   with	   FXS	   [149].	   Taken	   together,	  neuroanatomical	   and	   neuroimaging	   findings	   in	   humans	   suggest	   that	   changes	   in	  cerebellar	  neuron	  structure	  and	  function	  as	  a	  result	  of	  loss	  of	  function	  mutations	  in	  the	  FMR1	  gene	   could	   produce	  ASD-­‐like	   symptoms	   in	   patients	   diagnosed	  with	   FXS	  and	  related	  neurodevelopmental	  disorders.	  	  1.3-­‐Activated	  microglia	  and	  astroglia	  seen	  in	  cases	  of	  ASD	  Microglia	  are	  specialized	  glial	  cells	  of	  mesenchymal	  origin	  and	  they	  play	  an	  important	   role	   in	   inflammatory	   and	   immune	   responses	   following	   traumatic	   brain	  injury,	   infection	   or	   in	   cases	   of	   neurodegenerative	   disease	   such	   as	   Alzheimer’s	  	   7	  disease	   and	  multiple	   sclerosis	   [80].	  Microglia	   are	   related	   to	  peripheral	  monocytes	  and	  upon	  activation	  will	  rapidly	  change	  their	  cell	  morphology	  to	  display	  thicker	  cell	  bodies	   and	   cell	   processes	   and	   like	   macrophages,	   microglia	   possess	   phagocytic	  properties	   that	   allow	   them	   to	   engulf	   cellular	   debris	   and	   microbes	   following	  activation	  [81].	  Resting	  or	  “ramified”	  microglia	  have	  thin,	  spindly	  cell	  processes	  and	  less	   conspicuous	   cell	   bodies,	   but	   with	   activation	   they	   become	   fatter	   and	   more	  amoeboid-­‐like	   in	   appearance	   when	   viewed	   underneath	   the	   microscope	   [81].	  Microglial	   activation	   leads	   to	   changes	   in	   microglia	   shape,	   microglia	   distribution	  within	   the	   CNS,	   changes	   in	   gene	   expression	   and	   changes	   in	   cell	   behaviour	   [82].	  Activated	  microglia	  can	  respond	  to	  gradients	  of	  chemokines	  and	  migrate	  to	  the	  site	  of	   injury	   within	   the	   CNS	   and	   once	   at	   the	   site	   of	   injury	   microglia	   can	   themselves	  release	   chemokines	   and	   cytokines	   to	   recruit	   other	   effector	   immune	   cells	   to	  participate	  in	  the	  immune	  or	  inflammatory	  response	  [81].	  	  	   It	   is	   also	   important	   to	   note	   that	   microglia	   can	   have	   multiple	   different	  phenotypes	   upon	   activation.	   In	   the	   past	   few	   years	  microglia	   have	   been	   separated	  into	   two	   distinct	   functional	   subsets	   based	   on	   differential	   gene	   expression,	  expression	  of	  cell	  surface	  markers,	  production	  of	  different	  subsets	  of	  cytokines	  and	  chemokines	   and	   differences	   in	   functional	   properties	   [83,84].	   M1	   microglia	   are	  involved	  in	  pro-­‐inflammatory	  responses	  within	  the	  CNS	  and	  they	  secrete	  cytokines	  such	   as	   TNF-­‐α	   and	   IFN-­‐γ	   that	   promote	   inflammation	   [84].	   Alternatively,	   M2	  microglia	   are	   involved	   in	   neuroprotective	   and	   regenerative	   responses	   within	   the	  CNS	   and	   they	   secrete	   anti-­‐inflammatory	   cytokines	   and	   neurotrophic	   factors	   [85].	  	   8	  There	  are	  probably	  many	  different	   subtypes	  of	  microglia	  within	   these	   two	  groups	  based	   on	   functional	   and	   structural	   properties,	   but	   it	   is	  most	   important	   to	   realize	  that	  microglia	  have	  numerous	  and	  sometimes	  opposing	  functions	  within	  the	  CNS.	  	  In	  human	  cases	  of	  ASD	  there	  is	  an	  increasing	  body	  of	  evidence	  that	  suggests	  abnormal	   immune	  responses	  may	  contribute	  to	  pathophysiological	  changes	  within	  the	  developing	  CNS	  and	   lead	   to	   the	  development	  of	  ASD	   in	  a	  subset	  of	   individuals	  [86].	  Previous	  studies	  in	  humans	  have	  found	  that	  a	  subset	  of	  people	  diagnosed	  with	  ASD	  have	  elevated	  levels	  of	  pro-­‐inflammatory	  cytokines	  (such	  as	  IL-­‐6	  and	  TNF-­‐α)	  in	  their	   systemic	   circulation	   and	   in	   post-­‐mortem	   brain	   tissue	   [87].	   In	   addition,	  molecular	   genetics	   experiments	   have	   identified	   several	   putative	  ASD-­‐linked	   genes	  that	   have	   several	   immune	   system	   functions	   including	   genes	   from	   the	   HLA	   family	  and	   complement	   cascade	   [88,89].	   Finally,	  more	   detailed	   post-­‐mortem	   histological	  studies	  of	  brain	   tissue	   from	  children	  and	  adults	  diagnosed	  with	  ASD	  were	   carried	  out	  to	  evaluate	  microglia	  and	  astroglia	  morphology	  and	  the	  activation	  state	  of	  these	  neuroglial	  cells	  in	  multiple	  brain	  regions	  [90].	  	  	   In	   a	   paper	   published	   by	   Vargas	   et	   al.	   researchers	   found	   marked	   diffuse	  activation	  of	  astroglia	  and	  microglia	   in	  several	  cortical	  brain	  regions,	  but	  they	  also	  found	  significant	  neuroglial	  activation	  in	  the	  cerebellum	  and	  subcortical	  brain	  areas	  of	   post-­‐mortem	   tissue	   from	   individuals	   diagnosed	   with	   ASD	   [90].	   More	   detailed	  immunohistological	   analysis	   of	   microglial	   activation	   in	   autopsies	   of	   individuals	  diagnosed	  with	  ASD	   also	   found	   increased	   numbers	   of	   Iba1	   positive	  microglia	   and	  	   9	  peripheral	   macrophages	   in	   the	   dorsolateral	   prefrontal	   cortex,	   which	   is	   another	  brain	   region	   previously	   implicated	   in	   cases	   of	   autism	   and	   other	  neurodevelopmental	  disorders	  [91].	  	   Following	  neurodegeneration	  in	  various	  brain	  regions	  resident	  astroglia	  also	  become	  activated,	  which	  is	  reflected	  by	  an	  increase	  in	  the	  thickness	  of	  cell	  processes	  and	  an	  increase	  in	  the	  expression	  of	  cytoskeleton-­‐associated	  intermediate	  filaments	  such	   as	   GFAP	   and	   vimentin	   [39,40].	   These	   structural	   and	   functional	   changes	   in	  astroglia	  represent	  astrogliosis	  and	  are	  associated	  with	  pathological	  changes	  in	  the	  CNS	  and	  have	  been	  documented	  in	  numerous	  human	  cases	  of	  ASD.	  In	  post-­‐mortem	  studies	  of	  brain	  tissue	  from	  people	  diagnosed	  with	  ASD	  it	  was	  found	  that	  there	  are	  reduced	   numbers	   of	   PCs	   in	   the	   cerebellum	   (ranging	   from	   50-­‐95%	   PC	   loss	   as	  compared	  to	  healthy	  controls	  in	  certain	  regions	  of	  the	  cerebellum)	  and	  that	  PC	  loss	  in	  some	  cases	  is	  accompanied	  by	  gliosis	  [103,104].	  In	  addition	  it	  has	  been	  previously	  documented	   that	   a	   subset	   of	   people	   diagnosed	   with	   ASD	   have	   a	   decrease	   in	   the	  density	  of	  GCs	  in	  the	  cerebellum	  and	  that	  some	  individuals	  have	  changes	  in	  the	  size	  and	  morphology	   of	   CN	   neurons,	  which	   are	   neuroanatomical	   changes	   also	   seen	   in	  mouse	  models	  of	  ASD	  [105].	  In	  a	  number	  of	  cases	  it	  was	  also	  noted	  that	  a	  decrease	  in	  PC	  number	  within	   the	  cerebellum	  was	  accompanied	  by	  a	  significant	   increase	   in	  GFAP	  staining	  of	  Bergmann	  glia	   in	   the	  ML	  of	   the	  cerebellum	  [104].	   	  These	  specific	  neuropathological	  changes	  in	  the	  cerebella	  of	  ASD	  patients	  are	  only	  seen	  in	  a	  subset	  of	   ASD	   cases	   and	   again	   reflect	   the	   heterogeneity	   in	   the	   presentation	   of	  	   10	  neurobehavioural	   and	   neuroanatomical	   phenotypes	   in	   different	  neurodevelopmental	  disorders.	  	   More	   recently,	   researchers	   in	   Japan	   used	   a	   combination	   of	   structural	  Magnetic	   Resonance	   Imaging	   (MRI)	   and	   Positron	   Emission	   Tomography	   (PET)	   to	  look	  at	  the	  differences	  in	  the	  number	  and	  distribution	  of	  activated	  microglia	  in	  brain	  regions	  of	  20	  people	  diagnosed	  with	  ASD	  versus	  20	  healthy	  controls	  [92].	  Authors	  of	  the	   study	   injected	   subjects	   with	   a	   radiotracer	   that	   specifically	   binds	   to	   activated	  microglia	   and	   then	   utilized	   PET	   imaging	   to	   determine	   the	   amount	   of	   radiotracer	  binding	  in	  various	  brain	  regions	  [92].	  They	  found	  that	  there	  was	  increased	  binding	  of	  the	  radiolabelled	  substance	  to	  activated	  microglia	  in	  the	  cerebellum,	  orbitofrontal	  cortex,	  anterior	  cingulate	  cortex,	  midbrain	  and	  pons	  in	  subjects	  with	  ASD	  compared	  to	   healthy	   controls	   [92].	   These	   results	   are	   some	   of	   the	   first	   to	   look	   at	   the	  distribution	   and	   level	   of	   activation	   of	   microglia	   in	   the	   living	   human	   brain	   and	  supports	  the	  notion	  that	  microglia	  may	  play	  a	  bigger	  role	  in	  the	  pathophysiological	  mechanisms	  underlying	  or	  contributing	   to	  symptoms	  of	  ASD.	   In	  addition,	  multiple	  mouse	  models	  of	  ASD-­‐like	  phenotypes	  also	  exhibit	  increased	  activation	  of	  microglia	  and	  astroglia	   in	   the	  cerebellum	  and	  other	  brain	  regions	  with	  pathological	   changes	  seen	  in	  human	  cases	  of	  autism.	   	  	   	  	  	  	  	   11	  1.4-­‐Experimental	  mouse	  models	  of	  Autism	  Spectrum	  Disorders	  (ASD)	  1.4.1-­‐Lurcher	  mutant	  (Lc/+)	  mouse	  Over	  the	  years,	  many	  mouse	  models	  have	  been	  developed	  and	  used	  to	  study	  the	  neural	  and	  biological	  correlates	  of	  ASD-­‐like	  phenotypes.	  One	  mouse	  model	  used	  to	   study	   autistic-­‐like	   behavioural	   and	   neuroanatomical	   phenotypes	   is	   the	   Lurcher	  mutant	   (Lc/+)	   mouse.	   The	   Lurcher	   mutant	   mouse	   is	   characterized	   by	   a	   gain	   of	  function	  mutation	  in	  the	  Grid2	  gene	  (GluRδ2	  ionotropic	  receptor),	  which	  induces	  a	  constitutive	   leak	   current	   through	   the	   GluRδ2	   receptor	   triggering	   increased	  depolarization	  of	  neurons	  [16].	  In	  the	  mouse	  brain	  Grid2	  is	  expressed	  at	  high	  levels	  in	  cerebellar	  Purkinje	  cells	  (PCs),	  which	  are	  the	  major	  output	  of	  the	  cerebellum	  [16].	  Sustained	   and	   prolonged	   depolarization	   of	   cerebellar	   PCs	   leads	   to	   excitotoxic	   cell	  death	   of	   the	  majority	   of	   PCs	   in	   the	   first	   2-­‐3	  weeks	   of	   life	   [16].	   In	   addition	   to	   the	  primary	   loss	   of	   PCs	   there	   is	   also	   substantial	   secondary	   loss	   of	   cerebellar	   granule	  cells	   (GCs)	   and	   inferior	   olivary	   neurons	   (IONs),	   which	   are	   the	   primary	   afferent	  fibres	  that	  synapse	  onto	  PCs	  [17].	  This	  makes	  the	  Lurcher	  mutant	  mouse	  a	  valuable	  model	   in	   which	   to	   study	   the	   effects	   of	   targeted	   neuronal	   cell	   death	   and	   the	  responses	  of	  pre-­‐synaptic	  afferent	  neurons	  following	  the	  loss	  of	  their	  post-­‐synaptic	  targets.	  	  	   The	   secondary	   loss	   of	   cerebellar	  GCs	   and	   inferior	   olivary	  neurons	   starts	   to	  occur	  at	  postnatal	  day	  8	  (P8)	   in	  Lc/+	  mutants	  and	  along	  with	  the	  complete	   loss	  of	  PCs	  leads	  to	  drastic	  changes	  in	  cerebellar	  gross	  anatomy,	  histology	  and	  physiology	  [17].	   Interestingly,	   the	  primary	  post-­‐synaptic	  targets	  of	  PCs,	  cerebellar	  nuclei	  (CN)	  	   12	  neurons,	   do	   not	   degenerate	   and	   the	   numbers	   of	   CN	   neurons	   are	   comparable	  between	   Lc/+	   mutants	   and	   wildtype	   (+/+)	   mice	   [17].	   The	   considerable	  morphological	  and	  functional	  changes	  in	  the	  Lurcher	  cerebellum	  create	  behavioural	  deficits	  which	  resemble	  some	  ASD-­‐like	  behavioural	  phenotypes,	  such	  as	  behavioural	  inflexibility,	   impaired	   fine	  and	  gross	  motor	  movements	  and	  decreased	  exploratory	  behaviours	   when	   mice	   are	   placed	   in	   a	   novel	   environment	   [139,140].	   The	  behavioural	   abnormalities	   observed	   in	   Lurcher	   mutants	   model	   some	   core	   ASD	  behavioural	   symptoms	   in	   humans	   including:	   restricted	   interests	   and	   behaviours,	  repetitive	   behaviours	   and	   impaired	   fine	   and	   gross	  motor	   activity	   [1,2].	   For	   these	  reasons,	   Lc/+	   mutant	   mice	   provide	   a	   valuable	   tool	   with	   which	   to	   study	   the	  neuropathological	  and	  behavioural	  correlates	  of	  ASD-­‐like	  phenotypes.	  	  	   Firstly,	  Lurcher	  mutants	  have	  impaired	  fine	  and	  gross	  motor	  movements	  due	  to	  extensive	  cerebellar	  pathology	  such	  as	  PC,	  GC	  and	  ION	  neuron	  death.	  On	  climbing	  tasks	  and	  dynamic	  equilibrium	  measures	  of	  motor	  function	  and	  coordination,	  Lc/+	  mutants	   perform	   significantly	   worse	   than	   their	   wildtype	   littermate	   counterparts	  [18].	  Lurcher	  mutants	  also	  exhibit	  deficits	  in	  the	  exploration	  of	  novel	  environments	  when	  compared	  to	  wildtype	  controls	  as	  measured	  by	  a	  decreased	  frequency	  of	  nose	  poking	   into	  different	  holes	   [140].	  This	  behavioural	  phenotype	   is	   thought	   to	  model	  restricted	   interests	   and	   behaviours	   as	   seen	   in	   human	   cases	   of	   ASD	   when	  encountering	  novel	  objects	  and	  environments	  and	  is	  proposed	  to	  be	  due	  to	  reduced	  motivation	   to	   explore	   novel	   situations	   and	   deficits	   in	   spatial	   perception	   [141].	   In	  addition,	   Lurcher	   mutants	   have	   deficits	   in	   spatial	   learning	   and	   memory	   as	  	   13	  demonstrated	  by	  their	  poor	  performance	  on	  the	  Morris	  water	  maze	  when	  compared	  to	  wildtype	  controls	  [19].	  Finally,	  Lurcher	  mutants	  also	  appear	  to	  have	  differences	  in	   anxiety-­‐related	   behaviour	   as	   compared	   to	   wildtype	   mice	   because	   they	   spend	  more	  time	  in	  the	  open	  areas	  of	  a	  radial	  arm	  maze	  [20].	  	  	  The	   differences	   in	   anxiety-­‐related	   behaviours	   and	   changes	   in	   the	   stress	  response	   in	   Lurcher	   mutants	   may	   be	   explained	   in	   part	   by	   the	   finding	   that	   the	  cerebellum	   has	   reciprocal	   connections	   with	   the	   hypothalamus	   and	   brain	   regions	  involved	  in	  mediating	  the	  Hypothalamic-­‐Pituitary	  Axis	  (HPA)	  stress	  response	  [21].	  Using	   viral	   vector	   tracing	   techniques,	   researchers	   were	   able	   to	   demonstrate	   that	  there	  are	  monosynaptic	  neural	  connections	  between	  the	  CN	  and	  the	  Paraventricular	  Nucleus	  (PVN)	  of	  the	  hypothalamus,	  suggesting	  that	  the	  cerebellum	  may	  be	  able	  to	  modulate	  HPA	  axis	  activity	  in	  response	  to	  stressful	  stimuli	  [21].	  	  1.4.2-­‐Lurcher	  chimeric	  mice	  A	   chimera	   is	   an	   organism	   that	   is	   composed	   of	   two	   or	  more	   populations	   of	  genetically	   distinct	   cells	   [23,152].	   Mouse	   chimeras	   can	   be	   composed	   of	   both	  wildtype	   and	  mutant	   cell	   types	   and	   they	   have	   been	   used	   extensively	   to	   study	   the	  roles	  of	  genes	   in	   tissue	  specific	  development	  and	   in	  numerous	  cell	   lineage	  studies	  [23,153,154].	  Lurcher	  chimeras	  were	  originally	  developed	  to	  study	  the	  site	  of	  gene	  action	  of	   the	  Lurcher	  mutation	  and	  when	   it	  was	  discovered	   that	   a	  Grid2	  mutation	  leads	   to	   the	   intrinsic	  death	  of	   cerebellar	  PCs,	   researchers	  were	  able	   to	   look	  at	   the	  effect	  of	  variable	  PC	  death	  on	  afferent	  GCs	  and	  IONs	  during	  the	  development	  of	  the	  	   14	  cerebellum	  [17,35].	  The	  Lurcher	  chimera	  mouse	  is	  composed	  of	  a	  variable	  number	  of	  cells	  derived	  from	  either	  a	  wildtype	  (+/+)	  embryo	  or	  Lc/+	  mutant	  embryo,	  which	  means	   that	   the	  number	  of	   cerebellar	  PCs	  derived	   from	  either	   genotype	   can	   range	  from	  ~1	  to	  99%	  [17,35].	  The	  more	  PCs	  that	  are	  derived	  from	  the	  mutant	  embryo	  the	  higher	   the	   %	   of	   PC	   death	   in	   each	   chimera	   [17,35].	   Each	   Lurcher	   chimera	   has	  variable	  loss	  of	  cerebellar	  PCs	  (ranging	  from	  ~1%	  to	  99%	  PC	  death)	  and	  as	  a	  result	  also	  has	  variable	  secondary	  loss	  of	  cerebellar	  GCs	  and	  inferior	  olivary	  neurons	  [22].	  These	  features	  of	  the	  Lurcher	  chimera	  mouse	  are	  valuable	  because	  it	  allowed	  us	  to	  study	   the	   relationship	   between	   variable	   cerebellar	   pathology	   in	   individual	   mice	  with	   a	   continuum	  of	   changes	   in	   the	   structure	   and	   function	  of	   surviving	   cerebellar	  neurons	  and	  glia.	  	   To	   create	   a	   Lurcher	   chimera,	   embryos	   from	   both	   a	   Lurcher	   mutant	   and	  wildtype	   mouse	   are	   incubated	   together	   overnight	   where	   they	   fuse	   to	   form	   a	  blastocyst	  [17].	  The	  chimeric	  blastocyst	  is	  then	  taken	  and	  implanted	  into	  a	  pseudo-­‐pregnant	  female	  mouse,	  where	  the	  embryo	  develops	  to	  full-­‐term	  and	  is	  delivered	  as	  a	   healthy	  mouse	   pup	   [17].	  When	   generating	   Lc	   chimeras	  we	   expected	   to	   produce	  mice	  of	  different	  genotypes	  at	  a	  specific	  ratio.	  One	  quarter	  of	  our	  chimeric	  mice	  are	  expected	  to	  be	  +/+↔+/+	  wildtype	  mice,	  half	  are	  expected	  to	  be	  +/+↔Lc/+	  chimeras	  and	   one	   quarter	   of	   the	   mice	   are	   expected	   to	   be	   Lc/+↔Lc/+	   mutants	   with	   no	  surviving	  PCs.	  In	  a	  pioneering	  study	  by	  Wetts	  and	  Herrup,	  independent	  cell	  markers	  for	  PCs,	  GCs	  and	  inferior	  olivary	  neurons	  were	  used	  to	  determine	  if	  mutant-­‐derived	  cells	   could	   be	   rescued	   by	   cell	   interactions	   with	   wildtype	   neurons	   within	   the	  	   15	  cerebellum	   of	   a	   Lurcher	   chimera	   [22].	   The	   researchers	   discovered	   that	   the	   Lc/+	  fraction	   of	   PCs	   still	   died	   with	   interactions	   from	   wildtype	   neurons,	   but	   that	   Lc/+	  derived	  GCs	  and	  IONs	  could	  be	  rescued	  within	  the	  chimera,	  suggesting	  that	  PC	  death	  in	  the	  Lurcher	  mutant	  is	  intrinsic	  and	  a	  direct	  result	  of	  the	  Grid2	  mutation	  [22].	  	  In	  addition	  to	  having	  variable	  loss	  of	  cerebellar	  PCs,	  GCs	  and	  olivary	  neurons,	  there	   is	   increasing	  evidence	   that	  Lurcher	   chimeras	  also	  exhibit	   variability	   in	  ASD-­‐like	  behavioural	  phenotypes.	  This	  is	  significant	  because	  it	  will	  allow	  researchers	  to	  study	  the	  relationship	  between	  targeted	  neuronal	  cell	  death	  in	  the	  cerebellum	  and	  a	  spectrum	   of	   behavioural	   phenotypes	   that	   model	   ASD-­‐like	   behaviour.	   Lurcher	  mutants	   and	   chimeras	   have	   been	   used	   extensively	   to	   study	   sensorimotor	  dysfunction	  with	   varying	   degrees	   of	   cerebellar	   pathology,	   but	   very	   little	   research	  has	   been	   conducted	   to	   look	   at	   deficits	   in	   higher	   order	   brain	   functions	   such	   as	  cognition	  and	  attention	  [17].	  	   Recently,	  Lurcher	  chimeras	  have	  been	  tested	  on	  tasks	  that	  look	  at	  both	  lower	  and	   higher	   order	   behavioural	   flexibility	   as	   a	   model	   of	   cognition	   and	   executive	  functions	   and	   how	   PC	   function	   relates	   to	   performance	   on	   these	   behavioural	  measures.	  Behavioural	  flexibility	  is	  defined	  as	  a	  type	  of	  executive	  function,	  which	  is	  the	   ability	   to	   adapt	   one’s	   behaviour	   to	   changing	   tasks	   and	   environments	   [142].	  	  Previous	   studies	   in	   humans	   have	   shown	   deficits	   in	   higher	   order	   behavioural	  flexibility	   in	   ASD	   patients	   with	   IQ	   and	   severity	   of	   ASD	   symptoms	   varying	   widely	  between	   individuals	   [24].	   Using	   behavioural	   approaches	   that	   have	   been	  modified	  	   16	  for	  testing	  the	  same	  parameters	  in	  Lurcher	  chimeras,	  it	  is	  also	  possible	  to	  test	  both	  lower	  and	  higher	  order	  behavioural	  flexibility	  and	  the	  role	  of	  PCs	  in	  producing	  and	  modifying	  these	  behaviours.	  For	  example,	  it	  was	  found	  that	  Lurcher	  chimeras	  with	  fewer	  surviving	  PCs	  had	  deficits	  on	  a	  serial	  reversal-­‐learning	  task	  (which	  measures	  lower	   order	   behavioural	   flexibility)	   suggesting	   that	   the	   cerebellum	   plays	   an	  important	  role	   in	  attention	  and	  cognition	   in	  tasks	  assessing	  higher	  brain	  functions	  [25].	  	  	  	   In	  human	  ASD	  cases,	  it	  is	  becoming	  increasingly	  clear	  that	  a	  subset	  of	  autistic	  patients	   also	   have	   deficits	   in	   executive	   functions	   that	   require	   the	   integration	   of	  neural	   inputs	   from	  many	  cortical	  and	  subcortical	  structures	  [150,151].	   In	  humans,	  the	  seat	  of	  executive	  functions	  resides	  in	  the	  prefrontal	  cortex,	  which	  is	  involved	  in	  attention,	   cognition,	   decision-­‐making	   and	   personality	   among	   many	   other	   higher	  order	   brain	   functions	   [150,151].	  Using	   behavioural	   tests	   that	  were	  modified	   from	  assays	  used	  to	  assess	  executive	  functions	  in	  humans,	  it	  is	  now	  also	  possible	  to	  study	  related	   brain	   functions	   such	   as	   attention	   and	   cognition	   in	   Lurcher	   chimeras.	   For	  example,	   the	   attentional	   set-­‐shifting	   task	   requires	   mice	   to	   decide	   between	   two	  dimensions	   during	   the	   behavioural	   test	   to	   obtain	   a	   food	   pellet	   as	   a	   reward	   [26].	  Success	   on	   this	   test	   relies	   on	   intact	   frontal	   lobe	   functions	   in	  mice	   and	  behaviours	  assessed	  with	  this	  task	  may	  be	  similar	  to	  executive	  functions	  in	  humans	  [26].	  	   	  	  	   17	  1.4.3-­‐Fragile	  X	  Mental	  Retardation	  (Fmr1	  KO)	  mice	  	   FXS	   is	   caused	   by	   loss	   of	   function	   mutations	   in	   the	   Fragile	   X	   Mental	  Retardation	  Protein	  (FMRP)	  or	  by	  transcriptional	  repression	  of	  the	  FMR1	  gene	  [27].	  FXS	   is	  characterized	  histologically	  by	  changes	   in	  the	  number,	  size	  and	  structure	  of	  dendritic	   spines	   found	   on	   cortical	   neurons,	   which	   has	   led	   many	   researchers	   to	  suggest	  that	  changes	  in	  dendritic	  spine	  morphology	  may	  lead	  to	  changes	  in	  synaptic	  function	  producing	  the	  cognitive	  and	  learning	  deficits	  seen	  in	  FXS	  patients	  [29].	  To	  further	   study	   the	   genetic	   and	  molecular	   mechanisms	   underlying	   FXS,	   Oostra	   and	  Willems	  created	  a	  mouse	  model	  of	  FXS	  that	  models	  many	  Fragile	  X-­‐like	  phenotypes	  seen	  in	  humans	  [27].	  As	  in	  humans	  Fmr1	  KO	  mice	  also	  display	  an	  overall	  increase	  in	  the	  number	  and	   length	  of	  cortical	  dendritic	  spines	  providing	  evidence	  that	  altered	  cortical	  synaptic	  plasticity	  may	  also	  produce	  many	  of	  the	  behavioural	  deficits	  seen	  in	  the	  Fmr1	  KO	  mouse	  model	  [29].	  	  	   With	   the	  development	  of	  Fmr1	  KO	  mice,	   researchers	  are	  now	  able	   to	  study	  the	   neurobiological	   correlates	   and	   changes	   in	   brain	   structure	   and	   function	   that	  underlies	   FXS-­‐like	   symptoms.	   Fmr1	   KO	   mice	   are	   characterized	   by	   hyperactivity,	  increased	  incidence	  of	  seizures	  and	  macro-­‐orchidism,	  which	  are	  similar	  features	  to	  those	   seen	   in	   human	   cases	   of	   FXS	   [30].	   Fmr1	   KO	   mice	   also	   exhibit	   deficits	   on	  behavioural	   tasks	   that	   assess	   associative	   learning	   [31].	   In	   a	   classical	   eye-­‐blinking	  conditioning	   paradigm	   mediated	   by	   activity	   in	   the	   cerebellum,	   Fmr1	   KO	   mice	  performed	   significantly	   worse	   than	   wildtype	   controls,	   indicating	   that	   changes	   in	  cerebellar	  synaptic	  plasticity	  may	  also	  contribute	  to	  features	  of	  FXS	  [31].	  	   18	  Finally,	   in	  addition	  to	  neuroanatomical	  and	  behavioural	  studies	  of	  the	  Fmr1	  KO	  mouse	   recent	   research	  has	   focused	   on	   the	   genetic	   and	  molecular	  mechanisms	  underlying	   FXS.	   FMRP	   can	   be	   found	   in	   many	   different	   neurons	   in	   the	   Central	  Nervous	  System	  (CNS)	  and	   is	   frequently	   found	   in	  dendritic	   spines	  associated	  with	  cytosolic	   polyribosomes	   [32].	   It	   was	   discovered	   that	   FMRP	   is	   an	   RNA-­‐binding	  protein	  and	  is	  incorporated	  into	  ribonucleoprotein	  complexes	  where	  it	  functions	  as	  a	   translational	   repressor	   during	   protein	   synthesis	   [32].	   With	   loss	   of	   function	  mutations	   in	   FMRP	   one	   would	   expect	   to	   see	   increased	   expression	   of	   synaptic	  proteins	   targeted	   by	   FMRP	   and	   in	   Fmr1	  KO	  mice	   scientists	   found	   both	   increased	  rates	   of	   synaptic	   protein	   synthesis	   and	   markedly	   increased	   levels	   of	   synaptic	  proteins	   [32].	   Increased	   expression	   of	   specific	   synaptic	   proteins	   in	  Fmr1	  KO	  mice	  may	  lead	  to	  alterations	  in	  synaptic	  plasticity	  and	  mediate	  changes	  in	  dendritic	  spine	  morphology	  as	  seen	  in	  cortical	  and	  cerebellar	  neurons.	  	   Fmr1	  KO	  mice	  provide	  a	  useful	  model	  to	  study	  both	  FXS	  and	  ASD	  because	  of	  overlapping	   neuroanatomical	   abnormalities	   and	   behavioural	   phenotypes	   seen	   in	  both	   of	   these	   neurodevelopmental	   disorders.	   For	   example,	   in	   humans	   diagnosed	  with	  both	  FXS	  and	  ASD	  it	  was	  found	  that	  there	  are	  anatomical	  abnormalities	  seen	  in	  the	  vermis	  lobules	  VI	  and	  VII	  of	  the	  cerebellum,	  not	  observed	  in	  patients	  diagnosed	  with	   FXS	   alone	   [8].	   More	   recently,	   it	   was	   discovered	   that	   FMRP	   targets	   many	  putative	  ASD-­‐linked	  genes	  such	  as	  Neuroligin-­‐3	  and	  Neurexin-­‐1,	  which	  are	   located	  at	  numerous	  synapses	  in	  the	  CNS	  [33].	  Together,	  Lurcher	  mutant,	  Lurcher	  chimera	  and	   Fmr1	   KO	   mice	   provide	   useful	   tools	   with	   which	   to	   study	   the	   relationship	  	   19	  between	   histological	   and	   functional	   changes	   in	   the	   cerebellum	   with	   the	   ASD-­‐like	  behavioural	  phenotypes	  observed	  in	  these	  mouse	  models.	  	  1.5-­‐The	  use	  of	  cFos	  as	  an	  indirect	  marker	  of	  neuronal	  firing	  to	  study	  changes	  in	  neural	  activity	  in	  mouse	  models	  of	  ASD-­‐like	  phenotypes	  	  	  	   cFos	  is	  a	  nuclear	  proto-­‐oncogene	  that	  has	  previously	  been	  used	  as	  an	  indirect	  marker	   of	   neural	   activity	   for	  mapping	  poly-­‐synaptic	   neural	   pathways	   in	   the	  brain	  and	  spinal	   cord	   [42].	   cFos	  was	   initially	  used	  by	  Dragunow	  and	  colleagues	   to	   trace	  neural	   pathways	   involved	   in	   the	   generation	   and	   propagation	   of	   epileptic	   seizures	  following	  electrical	  stimulation	  of	  the	  amygdala	  in	  rats	  [43].	  Since	  cFos	  is	  expressed	  at	   low	   levels	   in	  adult	  neurons,	  but	  at	  significantly	  higher	   levels	   following	  different	  types	  of	  neuronal	  stimulation	  it	  is	  a	  useful	  functional	  marker	  for	  measuring	  changes	  in	  neural	  activity	  in	  different	  brain	  regions	  [41].	  	  	   	  In	   human	   cases	   of	   autism	   there	   is	   evidence	   to	   suggest	   that	   core	   autistic	  behaviours	   are	   caused	   by	   an	   imbalance	   between	   excitatory	   and	   inhibitory	  neurotransmission	   in	  different	  brain	   regions	   [155].	  Work	  using	  Scn1a	  and	  Shank3	  mice	   has	   also	   shown	   that	   these	   mice	   have	   an	   increased	   ratio	   of	   excitatory	   to	  inhibitory	   neurotransmission,	   which	   may	   cause	   ASD-­‐like	   behavioural	   phenotypes	  seen	   in	   these	  mouse	  models	   [156,157].	   Recently,	   imaging	   studies	   have	   suggested	  that	   altered	   connectivity	   between	   the	   cerebellum,	   subcortical	   and	   cortical	   brain	  regions	  may	  also	  play	  a	  role	  in	  the	  production	  of	  autistic	  behaviours.	  Using	  Diffusion	  Tensor	   Imaging	   (DTI)	   Japanese	   researchers	   found	   that	   autistic	   subjects	   had	  decreased	  fractional	  anisotropy	  (FA)	  and	  axial	  diffusivity	  (AD)	  measurements	  in	  the	  	   20	  superior	  cerebellar	  peduncle	  as	  compared	   to	  control	  subjects	   [158].	  Decreased	  FA	  and	  AD	  measures	  in	  the	  superior	  cerebellar	  peduncles	  of	  autistic	  subjects	  were	  also	  correlated	  with	  their	  performance	  on	  motor	  tasks	  suggesting	  that	  altered	  cerebellar	  connectivity	  and	  function	  is	  linked	  to	  motor	  impairments	  in	  autistic	  subjects	  [158].	  	  	  Mouse	   models	   of	   autism-­‐like	   phenotypes	   also	   display	   neuropathological	  changes	   in	   the	   cerebellum	  with	   corresponding	   changes	   in	   cerebellar	   connectivity	  and	   function.	   Previous	   studies	   using	   the	   Lc/+	   mutant	   mouse	   have	   shown	   that	  cerebellar	   pathology	   (like	   developmental	   PC	   death)	   leads	   to	   altered	   connectivity	  between	  the	  dentate	  nuclei	  (DN)	  of	  the	  cerebellum	  and	  connected	  brain	  regions	  like	  the	   medial	   prefrontal	   cortex	   (mPFC)	   [159].	   In	   Lc/+	   mutants	   there	   is	   decreased	  dopamine	  release	  in	  the	  mPFC	  following	  electrical	  stimulation	  of	  the	  DN,	  suggesting	  that	   cerebellar	   pathology	   associated	  with	   ASD	   leads	   to	   changes	   in	   neural	   activity	  within	   distinct	   cerebello-­‐cortical	   circuits	   [159].	   Developmental	   cerebellar	  abnormalities	  appear	  to	  be	  fairly	  common	  in	  cases	  of	  autism	  and	  in	  models	  of	  ASD-­‐like	   phenotypes,	   suggesting	   that	   structural	   and	   functional	   reorganization	   of	  cerebellar	   neural	   circuits	   with	   corresponding	   changes	   in	   neuronal	   activity	  contributes	   to	   core	   ASD-­‐like	   phenotypes	   such	   as	   stereotyped	   and	   repetitive	  behaviours	  [25,159].	  	  Since	   Lc/+	   mutants	   and	   chimeras	   exhibit	   developmental	   PC	   loss	   and	  cerebellar	   pathology	   similar	   to	   human	   ASD	   cases,	   we	   were	   interested	   in	  determining	  if	  there	  are	  corresponding	  changes	  in	  neural	  activity	  that	  are	  related	  to	  	   21	  the	  severity	  of	  cerebellar	  pathology.	  We	  chose	  to	  use	  the	  rotarod	  test	  as	  a	  measure	  of	   sensorimotor	   activity	   and	   because	   it	   triggers	   neuronal	   activation	   in	   the	  cerebellum	  and	  connected	  brain	  regions	  [45].	  By	  measuring	  cFos	  expression	  at	  rest	  and	   corresponding	   increases	   in	   cFos	   expression	   following	   rotarod	   activation,	   we	  were	  able	  to	  use	  cFos	  as	  an	  indirect	  marker	  of	  neural	  activity	  and	  compare	  changes	  in	  neuronal	  activation	  with	  the	  degree	  of	  cerebellar	  pathology	  in	  our	  mouse	  models	  of	  ASD-­‐like	  phenotypes.	  	  	  1.6-­‐Overview	  of	  the	  study	  	   1.6.1-­‐Aims	  and	  purpose	  Autism	   Spectrum	   Disorders	   (ASD)	   and	   related	   neurodevelopmental	  disorders	  are	  heterogeneous	   in	   their	   clinical	   appearance	  and	   in	  neuropathological	  changes	   that	   occur	   in	   the	   brains	   of	   autistic	   individuals.	   There	   are	   currently	   no	  widely	  used	  biological	  assays	  available	  to	  aid	  in	  the	  diagnosis	  of	  ASD	  and	  a	  clearer	  understanding	   of	   the	   pathological	   and	   neurobiological	   changes	   in	   the	   cerebellum	  and	  other	  brain	  regions	  implicated	  in	  the	  pathogenesis	  of	  autism	  may	  lead	  to	  better	  diagnostic	  criteria	  for	  the	  treatment	  of	  ASD.	  The	  current	  study	  uses	  mouse	  models	  of	  autism-­‐like	  phenotypes	   to	   investigate	  neurobiological	  and	   functional	  changes	   in	  the	   cerebellum	   to	   better	   understand	   the	   corresponding	   neurobiological	   and	  neurodevelopmental	   mechanisms	   that	   contribute	   to	   human	   cases	   of	   autism.	   A	  primary	  aim	  of	  the	  study	  is	  to	  document	  pathological	  changes	  in	  cerebellar	  neurons	  and	  glial	  cells	  and	  to	  explore	  the	  relationship	  between	  cerebellar	  pathology	  seen	  in	  mouse	  models	  of	  ASD-­‐like	  phenotypes	  and	  cerebellar	  abnormalities	  seen	  in	  human	  	   22	  cases	  of	  autism.	  Another	  primary	  aim	  of	  the	  study	  is	  to	  determine	  if	  the	  variable	  loss	  of	  Purkinje	  cells	  leads	  to	  compensatory	  or	  pathological	  changes	  in	  neural	  activity	  in	  surviving	   cerebellar	   neurons	   in	   mouse	   models	   exhibiting	   variable	   cerebellar	  pathology	  and	  ASD-­‐like	  behavioural	  phenotypes.	  	  	   1.6.2-­‐Hypotheses	  I)	   Purkinje	   cell	   death	   is	   one	   of	   the	   most	   common	   neuroanatomical	  abnormalities	   seen	   in	   the	  brains	  of	   autistic	   individuals	   and	  we	  predict	   that	  Lc/+	  mutants,	  Lurcher	  chimeras	  and	  Fmr1	  KO	  mice	  that	  exhibit	  PC	  loss	  also	  display	   correlated	   changes	   in	   cerebellar	   intrinsic	   and	   extrinsic	   neuronal	  activity	  using	  cFos	  as	  an	  indirect	  reporter	  of	  neural	  activity.	  II)	  Changes	  in	  neuroglia	  structure	  and	  function	  have	  been	  well	  documented	  in	   the	   brains	   of	   some	   autistic	   individuals	   and	   since	   Lc/+	   mutants	   and	  chimeras	   display	   cerebellar	   pathology	   which	   reflects	   cerebellar	  abnormalities	  seen	  in	  human	  cases	  of	  ASD,	  we	  predict	  that	  there	  are	  similar	  changes	  in	  neuroglial	  structure	  and	  function	  in	  our	  mouse	  models	  of	  autism-­‐like	  phenotypes.	  	  1.6.3-­‐Challenges	  and	  further	  considerations	  This	   study	   faces	   a	   challenge	   in	   the	   application	   of	   neuroanatomical	   and	  functional	  findings	  from	  our	  ASD-­‐like	  mouse	  models	  to	  human	  cases	  of	  autism.	  Since	  ASD	  is	  clinically	  heterogeneous	  with	  variability	   in	  the	  presentation	  and	  severity	  of	  behavioural	  symptoms	  seen	  in	  individual	  patients,	  it	  is	  important	  to	  note	  that	  not	  all	  	   23	  cases	  of	  autism	  are	  characterized	  by	  neuroanatomical	  and	  functional	  abnormalities	  in	  the	  cerebellum.	  Some	  cases	  of	  autism	  are	  characterized	  by	  morphological	  changes	  in	  cortical	  brain	  regions	  with	  no	  obvious	  cerebellar	  pathology	  and	  yet	  other	  studies	  find	   no	   gross	   morphological	   changes	   in	   the	   cerebellum	   or	   cerebral	   cortices.	   Our	  chosen	   mouse	   models	   are	   characterized	   by	   olivo-­‐cerebellar	   degeneration	   and	  neuropathological	  changes	  in	  the	  cerebellar	  cortex	  to	  reflect	  similar	  cerebellar	  and	  brainstem	   abnormalities	   documented	   in	   human	   cases	   of	   ASD	   and	   related	  neurodevelopmental	  disorders.	  Therefore,	  this	  thesis	  will	  focus	  on	  neuroanatomical	  and	  associated	  functional	  changes	  in	  the	  cerebellum	  seen	  in	  both	  mouse	  models	  and	  human	  cases	  of	  neurodevelopmental	  disorders.	  Many	  other	  mouse	  models	  of	  ASD-­‐like	  phenotypes	  are	  available	   to	   study	   the	  neurobiological	   changes	   seen	   in	  autism	  and	  we	  will	   reference	   these	   studies	   for	   the	  purpose	   of	   discussion	   in	   applying	   our	  results	  and	  findings	  to	  human	  cases	  of	  autism.	  	  	  	  	  	  	  	  	  	  	  	   24	  Chapter	  2-­‐Materials	  and	  methods	  2.1-­‐Behavioural	  testing	  of	  Lc/+	  mutants,	  controls	  and	  Lurcher	  chimeras	  2.1.1-­‐Rotarod	  testing	  of	  Lurcher	  mutant	  mice	  and	  wildtype	  (+/+)	  mice	  	   Lurcher	   mutant	   and	   wildtype	   littermates	   were	   first	   tested	   on	   the	   rotarod	  assay	  of	   sensorimotor	   function	   to	  measure	  cerebellar	   function	   in	  Lurcher	  mutants	  as	  compared	  to	  wildtype	  controls.	  The	  rotarod	  test	  was	  also	  chosen	  as	  a	  behavioural	  paradigm	   because	   it	   triggers	   cerebellar	   activation	   and	   allowed	   us	   to	   analyze	  changes	   in	   the	   expression	   of	   the	   cFos	   protein	   in	   various	   populations	   of	   neurons,	  which	   acts	   as	   an	   indirect	   reporter	   of	   changes	   in	   neural	   activity	   following	  behavioural	   testing	   [34].	   During	   the	   rotarod	   test	   mice	   walked	   on	   an	   elevated,	  rotating	  bar	  and	  during	  the	  course	  of	  testing	  the	  speed	  of	  the	  rotarod	  was	  increased	  from	  4	  rotations	  per	  minute	  (rpm)	  to	  40	  rpm.	  All	  wildtype	  control	  mice	  were	  tested	  first	  with	  each	  individual	  animal	  being	  tested	  on	  the	  rotarod	  for	  a	  maximum	  time	  of	  300	  seconds.	  Once	  rotarod	  testing	  had	  been	  completed	  for	  all	  the	  wildtype	  mice	  the	  average	  time	  spent	  on	  the	  rotarod	  was	  calculated	  for	  all	  the	  control	  animals	  tested.	  The	  average	  amount	  of	   time	   that	   the	  wildtype	  animals	  could	  stay	  balanced	  on	   the	  rotarod	   bar	   became	   the	   target	   time	   that	   the	   Lurcher	  mutants	   had	   to	   reach	  while	  being	  tested	  on	  the	  rotarod.	  	  	   Following	  testing	  of	  the	  wildtype	  animals,	  Lc/+	  mutants	  were	  placed	  on	  the	  rotarod	   and	   each	   animal	   was	   required	   to	   reach	   the	   target	   time	   (which	   was	   the	  average	  time	  spent	  on	  the	  rotarod	  by	  all	  tested	  wildtype	  mice).	  If	  one	  of	  the	  Lurcher	  mutant	  mice	  fell	  off	  the	  rotarod	  it	  was	  consistently	  placed	  back	  on	  the	  rotarod	  bar	  	   25	  until	  the	  criteria	  time	  had	  been	  reached.	  The	  number	  of	  falls	  and	  number	  of	  sessions	  on	  the	  rotarod	  bar	  were	  recorded	  for	  each	  Lurcher	  mutant	  and	  the	  duration	  of	  each	  session	  on	  the	  rotarod	  bar	  before	  the	  target	  time	  was	  reached	  was	  summed	  for	  each	  mouse.	  Following	  this	  it	  was	  then	  possible	  to	  calculate	  the	  average	  performance	  of	  each	  Lc/+	  mutant	   on	   the	   rotarod	   test	   as	   a	  measure	   of	   sensorimotor	   function	   and	  compare	   the	   performance	   of	   the	   wildtype	   mice	   as	   a	   group	   against	   the	   Lurcher	  mutant	  mice	  as	  a	  group	  on	  a	  test	  of	  cerebellar	  function.	  	  	  2.1.2-­‐Rotarod	  testing	  of	  Lurcher	  chimera	  mice	  	   Like	  Lurcher	  mutant	  and	  wildtype	  controls,	  Lurcher	  chimera	  mice	  were	  also	  tested	   on	   the	   rotarod	   task	   of	   sensorimotor	   function	   to	   trigger	   neural	   firing	   in	  cerebellar	  neurons	  and	  cFos	  expression	  in	  activated	  neurons	  was	  used	  as	  a	  reporter	  of	   neural	   activity.	   The	   same	  behavioural	   protocol	   as	   outlined	   above	  was	   followed	  except	   that	   all	   Lurcher	   chimeras	   spent	   90	   seconds	   on	   the	   rotarod.	   If	   the	   Lurcher	  chimera	   being	   tested	   had	   cerebellar	   ataxia	   and	   kept	   falling	   off	   the	   rotarod	   the	  experimenter	   placed	   the	   chimera	   back	   on	   the	   rotarod	  until	   the	   90	   seconds	   target	  time	   had	   been	   reached.	   This	   was	   done	   so	   that	   all	   Lurcher	   chimeras	   received	   the	  exact	  same	  testing	  measures	  on	  the	  behavioural	  paradigm	  and	  to	  ensure	  that	  each	  animal	  had	   the	   same	   level	   of	   cerebellar	   activation.	  By	   ensuring	   that	   each	   chimera	  received	   a	   constant	   level	   of	   cerebellar	   activation	  we	  were	   able	   to	   simultaneously	  compare	  and	  contrast	  the	  severity	  of	  cerebellar	  pathology	  and	  changes	  in	  neuronal	  function	   between	   individual	   Lurcher	   chimeras.	   Following	   rotarod	   testing	   Lurcher	  mutants,	  Lurcher	  chimeras	  and	  wildtype	  animals	  received	  an	  Avertin	  overdose	  90	  	   26	  minutes	  after	  cerebellar	  activation	  and	  were	  subsequently	  trans-­‐cardially	  perfused	  with	  1x	  PBS	  followed	  by	  4%	  Paraformaldehyde	  (PFA).	  90	  minutes	  was	  a	  sufficient	  time	  period	  for	  translation	  and	  synthesis	  of	  new	  proteins	  to	  occur	  and	  allowed	  us	  to	  look	  at	  the	  differences	  in	  cFos	  protein	  expression	  in	  different	  groups	  of	  neurons	  as	  a	  measure	  of	  changes	  in	  neural	  activity	  in	  response	  to	  rotarod	  activation.	  	  2.2-­‐Preparation	  of	  brain	  tissue	  for	  immunohistochemical	  analysis	  Following	  behavioural	  testing	  we	  were	  interested	  in	  assessing	  structural	  and	  functional	   changes	   in	   the	  cerebellum	  and	  cortical	   regions	  of	  brain	   tissue	   from	  our	  ASD-­‐like	  mouse	  models.	   To	   prepare	   brain	   tissue	   for	   downstream	   histological	   and	  neuroanatomical	  analysis	  we	  perfused,	  embedded	  and	  sectioned	  brain	   tissue	   from	  our	   3	  ASD-­‐like	  mouse	  models.	   Lurcher	   chimeras,	   Lurcher	  mutants,	  Fmr1	  KO	  mice	  and	  wildtype	   littermates	  were	   trans-­‐cardially	   perfused	   and	  brain	   tissue	  was	   fixed	  with	   room	   temperature	   PBS	   followed	   by	   4%	   Paraformaldehyde	   (PFA)	   solution.	  Brains	  were	  immediately	  dissected	  and	  removed	  from	  the	  skull	  and	  cut	  in	  half	  mid-­‐sagittally	  at	  the	  corpus	  callosum.	  Brain	  tissue	  was	  then	  post-­‐fixed	  in	  PFA	  overnight	  and	  transferred	  to	  PBS	  with	  sodium	  azides	  for	  storage	  at	  4	  Celsius.	  Two	  days	  before	  sectioning,	   brain	   tissue	   was	   placed	   in	   30%	   sucrose	   plus	   sodium	   azides	   (for	  preservation	  of	  brain	   tissue)	  until	   the	  brains	  had	  sunk	   to	  bottom	  of	   the	  glass	   jars.	  Finally,	   brains	  were	   embedded	   in	   O.C.T.	   compound	   using	   isopentane	   and	   dry	   ice.	  The	   embedded	   brain	   tissue	   was	   then	   stored	   in	   the	   -­‐80	   Celsius	   freezer	   for	  downstream	  experiments	  and	  analysis.	  	  	   27	  	   Next,	   embedded	  brain	   tissue	  was	   sectioned	   into	  para-­‐sagittal	   slices	  using	  a	  cryostat.	  The	  left	  halves	  of	  the	  brain	  tissue	  were	  used	  for	  Calbindin-­‐D28K	  staining	  to	  visualize	  the	  loss	  of	  cerebellar	  PCs	  and	  the	  right	  halves	  of	  the	  brain	  tissue	  were	  used	  for	  cFos	  staining	  and	  analysis.	  Tissue	  being	  stained	  for	  Calbindin-­‐D28K	  (to	  visualize	  and	   quantify	   cerebellar	   PCs)	   was	   sectioned	   at	   25µm	   and	   mounted	   directly	   onto	  Fisher	   Superfrost-­‐plus	   slides.	   Brain	   tissue	   being	   used	   for	   cFos	   analysis	   was	  sectioned	   at	   40µm	   and	   tissue	   sections	   were	   placed	   into	   cell	   culture	   well	   plates	  containing	  PBS	  and	  sodium	  azides	  for	  free-­‐floating	  immunohistochemistry	  (IHC).	  	  2.3-­‐Immunohistochemical	  staining	  and	  analysis	  2.3.1-­‐Purkinje	  cell	  staining	  and	  visualization	  Next,	  we	  were	  interested	  in	  quantifying	  cerebellar	  pathology	  in	  brain	  tissue	  by	   calculating	   the	   number	   of	   surviving	   PCs	   in	   each	   ASD-­‐like	   mouse.	   To	   quantify	  cerebellar	   pathology,	   sections	   were	   examined	   and	   visualized	   by	  immunohistochemistry	   and/or	   cresyl	   violet	   staining.	   For	   immunohistochemical	  analysis,	   sections	   were	   rinsed	   with	   PBS	   and	   then	   incubated	   with	   blocking	   buffer	  containing	  bovine	  serum	  albumin	  and	  normal	  goat	  serum	  for	  20	  minutes.	  Sections	  were	   then	   incubated	   overnight	   with	  mouse	  monoclonal	   anti-­‐Calbindin	   antibodies	  (1:1000	  dilution;	  Abcam,	  Cambridge,	  MA,	  USA).	  Sections	  were	  incubated	  with	  anti-­‐mouse	  secondary	  antibody	  (1:200	  dilution;	  Vector	  Labs,	  Burlingame,	  CA,	  USA)	  for	  an	  hour	   following	  3	  washes	   in	  PBS-­‐T.	   Immunoreactivity	  was	   then	  visualized	  by	  using	  an	   ABC	   reaction	   (Vectastain	   kit;	   Vector	   Labs,	   Burlingame,	   CA,	   USA)	   and	  diaminobenzidine	   (DAB)	   (Sigma-­‐Aldrich,	   St.	   Louis,	   MO,	   USA).	   Slides	   were	   also	  	   28	  counterstained	  with	  cresyl	  violet	  to	  visualize	  GCL	  morphology	  and	  dehydrated	  using	  ascending	   alcohol	   concentrations	   prior	   to	   being	   cleared	   with	   xylenes.	   Glass	  coverslips	  were	  then	  applied	  with	  Permount	  solution.	  	   2.3.2-­‐cFos	  immunostaining	  and	  analysis	  To	   observe	   and	   quantify	   changes	   in	   neuronal	   activity	   in	   cerebellar	   and	  cortical	   neurons	   following	   rotarod	   activation	   and	   at	   rest,	   we	   utilized	   cFos	  expression	  in	  neurons	  as	  an	  indirect	  marker	  of	  increased	  neural	  activity	  [34].	  Free-­‐floating	  sections	  were	  selected	  and	  we	  performed	  immunohistochemistry	  to	  identify	  and	  quantify	  cFos	  activation	   in	  the	  cerebellum,	  orbital	  cortex,	  somatomotor	  cortex	  and	  posterior	  cortex	  in	  each	  brain.	  Sections	  used	  for	  immunohistochemical	  analysis	  were	  washed	   in	   PBS	   and	   incubated	  with	   blocking	   buffer	   (containing	  Normal	  Goat	  Serum	   (NGS)	   and	  30%	  Bovine	   Serum	  Albumin	   (BSA))	   prior	   to	   being	   incubated	   in	  polyclonal	   anti-­‐cFos	   antibodies	   at	   4	   Celsius	   overnight	   (1:500	   dilution,	   Santa	   Cruz	  Biotechnology,	  Dallas,	  Texas,	  USA).	  Sections	  were	  then	  rinsed	  three	  times	  in	  PBS-­‐T	  and	  incubated	  for	  1	  hour	  in	  anti-­‐rabbit	  secondary	  antibodies	  (1:200	  dilution;	  Vector	  Labs,	   Burlingame,	   CA,	   USA).	   cFos	   immunoreactivity	   was	   visualized	   using	   the	  VectaStain	   ABC	   reaction	   (Vectastain	   kit;	   Vector	   Labs,	   Burlingame,	   CA,	   USA)	   and	  diaminobenzidine	  (DAB,	  Sigma-­‐Aldrich,	  St.	  Louis,	  MO,	  USA).	   	  Free-­‐floating	  sections	  were	   then	  mounted	  onto	  slides	  and	  allowed	   to	  adhere	   to	   the	  slides	  overnight	   in	  a	  humid	  incubation	  chamber.	  Sections	  were	  then	  dehydrated	  in	  ethanol	  of	  increasing	  concentrations	  and	  cleared	  in	  xylenes	  prior	  to	  being	  coverslipped.	  Analysis	  of	  cFos	  positive	   cells	   was	   performed	   using	   a	   light	   transmission	   microscope	   and	   cFos	  	   29	  staining	  (as	  a	  marker	  for	  neural	  activation)	  was	  quantified	  using	  ImageJ64	  software	  (NIH	   ImageJ	  Software)	  and	  allowed	   for	  analysis	  of	  differences	   in	  neural	  activation	  between	  wildtype	  animals	  and	  autistic-­‐like	  phenotype	  animals.	  	  2.3.3-­‐Immunofluorescence	  for	  cFos,	  Calbindin,	  Iba1	  and	  GFAP	  We	   performed	   immunofluorescence	   to	   visualize	   co-­‐localization	   of	   cFos	  (reporter	   of	   neural	   activity)	   and	   Calbindin-­‐D28K	   (structural	   marker	   of	   PCs)	   to	  observe	  the	  amount	  of	  inhibitory	  cerebellar	  cortical	  outflow	  to	  CN	  neurons.	  We	  then	  used	   Iba1	   immunostaining	   to	   quantify	   and	   study	   morphological	   changes	   in	  cerebellar	  microglia	  and	  GFAP	  as	  a	  marker	  for	  activated	  Bergmann	  glia	  in	  the	  ML	  of	  the	  cerebellum	  to	  study	  pathological	  changes	  in	  glial	  cells	  in	  the	  cerebellum	  that	  are	  associated	   with	   developmental	   neuronal	   degeneration.	   (As	   a	   positive	   control	   to	  ensure	   that	   the	   GFAP	   staining	   protocol	   had	   worked	   we	   imaged	   numerous	   GFAP	  positive	  astrocytes	  in	  the	  grey	  matter	  of	  the	  hippocampus	  in	  the	  same	  sections	  used	  to	  analyze	  cerebellar	  GFAP	  staining).	  	  	  Brain	  tissue	  sections	  already	  mounted	  on	  glass	  slides	  were	  selected	  and	  we	  performed	   immunofluorescence	   to	   visualize	   the	   co-­‐localization	   of	   cFos	   and	  Calbindin-­‐D28K	   positive	   PCs,	   Iba1	   positive	  microglia	   in	   the	   cerebellum	   and	   GFAP	  positive	  astrocytes	  and	  Bergmann	  glia	  in	  the	  cerebellar	  cortex.	  Slides	  were	  washed	  3	  times	   in	   PBS	   and	   then	   incubated	   for	   30	   minutes	   in	   a	   humid	   chamber	   at	   room	  temperature	   in	   blocking	   buffer	   containing	   NGS	   and	   30%	   BSA.	   Next,	   slides	   were	  incubated	   in	   various	   10	  antibodies	   overnight	   in	   a	   humid	   incubation	   chamber	   at	   4	  	   30	  Celsius	   (1.	   Mouse	   monoclonal	   Anti-­‐Calbindin	   Abs,	   1:1000	   dilution,	   Abcam,	  Cambridge,	  MA,	  USA,	  2.	  Rabbit	  polyclonal	  Anti-­‐cFos	  Abs,	  1:500	  dilution,	  Santa	  Cruz	  Biotechnology,	   Dallas,	   Texas,	   USA,	   3.	   Mouse	   monoclonal	   Anti-­‐GFAP	   Abs,	   1:200	  dilution,	   Cell	   Signaling	   Technology,	   Danvers,	   Massachusetts,	   USA,	   4.	   Rabbit	  polyclonal	  Iba1	  Abs,	  1:500	  dilution,	  Wako,	  Osaka,	   Japan).	  The	  next	  day	  slides	  were	  again	  washed	  3	  times	  in	  PBS	  and	  then	  incubated	  for	  1	  hour	  in	  a	  humid	  incubation	  chamber	  with	   two	  20	  antibodies	  and	  DAPI	  (Goat	  Anti-­‐Mouse	  Alexa	  594	  Abs	  (Red),	  Goat	  Anti-­‐Rabbit	  Alexa	  488	  Abs	  (Green)	  or	  Goat	  Anti-­‐Mouse	  Alexa	  488	  Abs	  (Green),	  Goat	   Anti-­‐Rabbit	   Alexa	   594	   Abs	   (Red),	   Life	   Technologies,	   Burlington,	   Ontario,	  Canada).	  Slides	  were	  then	  washed	  3	  times	  in	  the	  dark	  with	  PBS	  and	  then	  coverslips	  were	  applied	  using	  FluoroSave.	  Slides	  were	  then	  stored	  in	  the	  dark	  at	  4	  Celsius	  for	  downstream	  analysis.	  	  2.4-­‐Analysis	  and	  quantification	  of	  histological	  and	  functional	  data	  2.4.1-­‐Visualization	  and	  quantification	  of	  Purkinje	  cells	  	  	   For	  Purkinje	  cell	  counts,	  five	  sections	  spaced	  20	  sections	  apart	  were	  used	  for	  quantification	  of	  PCs	  in	  each	  mouse.	  Purkinje	  cell	  counts	  were	  done	  manually	  using	  a	   standard	   brightfield	   light	   microscope	   equipped	   with	   a	   10x	   eyepiece	   and	   a	   25x	  objective	  lens.	  Purkinje	  cells	  were	  only	  counted	  if	  the	  nucleus	  was	  visible,	  which	  was	  easily	   identifiable	   since	   it	   appeared	   light	   against	   the	  dark	   surrounding	   cytoplasm.	  The	  total	  number	  of	  Purkinje	  cells	  was	  then	  estimated	  for	  the	  entire	  cerebellum	  and	  the	  Abercrombie	  correction	  (See	  Figure	  1)	  was	  performed	  to	  account	  for	  split	  nuclei,	  section	  thickness	  and	  the	  average	  diameter	  of	  each	  PC.	  In	  addition,	  pictures	  of	  PCs	  	   31	  were	   taken	   at	   5x,	   10x	   and	   20x	   magnification	   to	   visualize	   the	   structure	   and	  morphology	  of	  individual	  PCs.	  	  2.4.2-­‐cFos	  analysis	  and	  quantification	  cFos	  positive	  cells	  were	  visualized	  and	  quantified	  using	  a	  light	  transmission	  microscope	  equipped	  with	  a	  camera.	  We	  took	  pictures	  of	  different	  brain	  regions	  at	  20x	  magnification	  and	  then	  quantified	  the	  cFos	  stained	  neuron	  cell	  bodies	  using	  the	  ImageJ64	  program	  (NIH	  ImageJ	  Software).	  We	  measured	  the	  total	  area	  of	  each	  brain	  region	  in	  mm2	  and	  counted	  the	  number	  of	  cFos	  positive	  neurons	  (number	  of	  circular	  particles)	   in	   different	   brain	   regions	   to	   obtain	   the	   density	   of	   cFos	   positive	   cells	   in	  each	  brain	  region	  (cFos	  positive	  cells/mm2).	  Finally,	  we	  averaged	  the	  density	  of	  cFos	  positive	  cells	   in	  a	  particular	  brain	   region	   from	  all	   the	  brains	  stained	  and	  analyzed	  and	   looked	   for	   differences	   in	   the	   number	   and	   density	   of	   cFos	   stained	   cells	   (as	   a	  functional	  measure	  of	   changes	   in	  neural	   activity)	   between	  our	   autistic-­‐like	  mouse	  models.	  	  2.5-­‐Statistical	  analysis	  	   After	   averaging	   PC	   numbers	   from	   5	   representative	   sections	   of	   each	   brain	  stained	  with	  Calbindin-­‐D28K,	  we	  used	  the	  Abercrombie	  correction	  to	  obtain	  a	  total	  PC	  count	  for	  the	  entire	  cerebellum	  of	  each	  mouse	  which	  was	  used	  as	  a	  quantifiable	  measure	  of	  cerebellar	  pathology	  in	  the	  3	  ASD-­‐like	  mouse	  models.	  After	  applying	  the	  Abercrombie	   correction	   to	   obtain	   a	   corrected	   total	   number	   of	   PCs	   for	   each	  cerebellum	   we	   used	   an	   unpaired	   t-­‐test	   to	   determine	   if	   there	   was	   a	   statistically	  	   32	  significant	  difference	   in	   cerebellar	  pathology	  between	  different	   groups	  of	   autistic-­‐like	  mouse	  models.	  	  	  	   After	   calculating	   average	   cFos	   cell	   counts	   and	   cFos	   staining	   density	   in	  different	   brain	   regions	   for	   each	  mouse,	  we	   calculated	   the	   average	   density	   of	   cFos	  staining	  for	  each	  brain	  region	  (cFos	  positive	  cells/mm2)	  in	  each	  autistic-­‐like	  mouse	  model.	   We	   then	   used	   an	   unpaired	   t-­‐test	   between	   different	   groups	   of	   mice	   to	  determine	   if	   the	   differences	   in	   cFos	   immunostaining	   in	   our	   mouse	   models	   were	  statistically	  significant.	  It	  is	  important	  to	  note	  that	  while	  cresyl	  violet	  (CV)	  staining	  also	  labels	  cell	  nuclei,	  it	  is	  not	  possible	  to	  quantify	  and	  analyze	  CV	  stained	  cells	  in	  the	  cerebellar	   granule	   cell	   layer	   (GCL)	   as	   the	   GCs	   are	   packed	   too	   closely	   together	   to	  effectively	   count	  manually	   using	   a	   light	  microscope	   or	   using	   a	   computer	   program	  such	  as	  ImageJ64	  (NIH	  ImageJ	  Software).	  	   For	   analyzing	   the	   density	   of	   cFos	   positive	   cells	   in	   the	   GCL	   and	   CN	   of	   the	  cerebellum	   and	   the	   density	   of	   Iba1	   positive	   microglia	   in	   the	   cerebellar	   cortex	   of	  Lurcher	  chimeras,	  we	  assigned	  Lurcher	  chimeras	  to	  3	  experimental	  groups	  based	  on	  the	   quantification	   of	   surviving	   PCs	   in	   the	   left	   hemi-­‐cerebellum	  of	   each	   animal.	   PC	  counts	   acted	   as	   a	   reliable	   and	   replicable	   measure	   of	   cerebellar	   pathology	   in	   our	  mouse	  models	  of	  olivo-­‐cerebellar	  neurodegeneration.	  For	  statistical	  analysis	  of	  the	  3	  groups	  of	  Lurcher	  chimeras	  we	  used	  a	  multiple	  comparisons	  of	   the	  mean	  analysis	  using	  a	  one-­‐way	  ANOVA.	  	  	   33	  Finally,	  we	  used	  ImageJ64	  (NIH	  ImageJ	  Software)	  to	  perform	  cell	  counts	  for	  immunofluorescence	   staining	   for	   cFos	   positive	   cells,	   Calbindin	   positive	   PCs,	   Iba1	  positive	   microglia	   and	   GFAP	   positive	   astroglia.	   We	   took	   pictures	   of	   different	   cell	  types	  at	  10x	  and	  20x	  magnification	  using	  an	  immunofluorescence	  light	  microscope	  equipped	   with	   a	   camera.	   Different	   cell	   types	   were	   counted	   in	   20x	   magnification	  images	  of	  various	  brain	  regions	  using	  an	  automated	  cell	  counting	  program	  included	  as	  an	  application	  with	  ImageJ64	  (NIH	  ImageJ	  Software).	  Cell	  counts	  from	  individual	  sections	  were	  averaged	  for	  each	  brain	  analyzed	  and	  we	  obtained	  cell	  counts	  for	  cFos	  positive	   neurons	   and	   Iba1	   positive	   microglia	   in	   brain	   regions	   of	   interest.	   The	  average	  number	  of	  cells/mm2	  for	  different	  cell	  types	  were	  calculated	  by	  measuring	  the	   area	   of	   analysis	   in	   each	   photomicrograph	   and	   dividing	   the	   number	   of	   cells	   in	  each	  picture	  by	  the	  area	  of	  analysis.	  Figure	   1.	   Abercrombie	   Correction	   formula	   to	   account	   for	   Purkinje	   cell	   diameter,	  section	   thickness	   and	   split	   Purkinje	   cell	   nuclei	   in	   estimating	   the	   total	   number	   of	  Purkinje	  cells	  in	  a	  whole	  cerebellum.	  	  	   34	  Chapter	  3-­‐Experiments	  and	  results	  	  3.1-­‐Lurcher	  mutants	  (Lc/+)	  and	  wildtype	  (+/+)	  littermates	  3.1.1-­‐Cerebellar	  pathology	  in	  Lc/+	  mutants	  as	  compared	  to	  +/+	  controls	  	   As	   observed	   in	   previous	   studies,	   Lurcher	   mutants	   lose	   100%	   of	   their	  cerebellar	  PCs	  as	  compared	  to	  the	  wildtype	  animals	  [16].	  However,	  we	  were	  able	  to	  calculate	  an	  average	  number	  of	  PCs	  in	  the	  Lurcher	  wildtype	  cerebellum	  by	  taking	  5	  representative	  sections	  from	  each	  wildtype	  mouse	  analyzed	  to	  act	  as	  controls	  for	  PC	  counts	   when	   looking	   at	   PC	   counts	   in	   Lurcher	   chimeras	   (See	   Figure	   2).	   Using	   the	  Abercrombie	  Correction	  we	   calculated	   that	   the	  average	  wildtype	  hemi-­‐cerebellum	  contains	  49,237	  +/-­‐	  2,230	  PCs	  and	  an	  average	  wildtype	  whole	  cerebellum	  contains	  98,474	  +/-­‐	  4,460	  PCs.	  	  	  	  	  Figure	  2.	  Cerebellum	  of	  a	  wildtype	  (+/+)	  control	  mouse	  stained	  for	  Calbindin-­‐D28K	  at	   5x	  magnification	   (left)	   and	   at	   20x	  magnification	   (right)	   showing	  numerous	  PCs	  and	  their	  complex	  dendritic	  arbors	  at	  higher	  magnification.	  	  	  	   	  	   35	  3.1.2-­‐Lc/+	   mutants	   exhibit	   increased	   cFos	   expression	   under	   basal	  conditions	  and	  following	  rotarod	  cerebellar	  activation	  	  To	  study	  differences	  in	  neuronal	  activity	  between	  Lc/+	  mutants	  and	  wildtype	  littermates	  we	  quantified	  the	  density	  of	  cFos	  positive	  cells	  in	  different	  brain	  regions.	  cFos	  is	  expressed	  at	  higher	  levels	  in	  neurons	  following	  bursts	  of	  APs	  and	  therefore	  acts	  as	  a	  reporter	  of	  recent	  neuronal	  activity	  [34].	  We	  selected	  para-­‐sagittal	   tissue	  sections	   from	  Lurcher	  mutant	  and	  wildtype	   littermates	   that	  were	  stained	   for	  cFos	  and	   Lurcher	   mutant	   and	   wildtype	   mice	   were	   separated	   into	   2	   different	  experimental	   groups.	   Half	   of	   the	   animals	   received	   rotarod	   activation	   (a	   dynamic	  equilibrium	   task	   which	   measures	   sensorimotor	   function	   mediated	   by	   the	  cerebellum	  [34])	  and	  half	  of	  the	  animals	  received	  no	  rotarod	  activation	  representing	  a	   resting	   or	   basal	   state	   of	   neural	   activity.	  With	   extensive	   PC	   death	   (>90%	   of	   PCs	  dying)	   we	   saw	   a	   significant	   decrease	   in	   the	   amount	   of	   time	   that	   Lurcher	  chimeras/mutants	   could	   stay	   on	   the	   rotarod	   as	   compared	   to	  wildtype	   (+/+)	  mice	  (See	  Figure	  3),	  which	  suggests	  that	  performance	  on	  the	  rotarod	  test	  is	  related	  to	  the	  extent	   of	   cerebellar	   pathology	   (particularly	   related	   to	   the	   loss	   of	   cerebellar	   PCs).	  Brain	   tissue	   was	   harvested	   90	   minutes	   after	   any	   behavioural	   testing	   and	   tissue	  slices	  were	  stained	  using	  IHC	  to	  look	  for	  cFos	  immunoreactivity	  in	  4	  different	  brain	  regions:	   the	   cerebellum,	   orbitofrontal	   cortex,	   posterior	   cortex	   and	   somatomotor	  cortex.	  	  	   We	  found	  that	  those	  animals	  (Lurcher	  mutants	  and	  wildtype	  littermates)	  that	  received	  rotarod	  activation	  (active	  state)	  had	  significantly	  higher	  expression	  of	  cFos	  positive	   cells	   in	   all	   brain	   regions	   that	  were	   analyzed	  as	   compared	   to	   animals	   that	  	   36	  received	   no	   rotarod	   activation	   (resting	   state).	   Within	   both	   groups	   that	   received	  rotarod	  activation	  (active	  state)	  or	  no	  rotarod	  activation	  (basal	  state),	  Lc/+	  mutants	  had	   significantly	   higher	   cFos	   expression	   in	   the	   cerebellum,	   orbitofrontal	   cortex,	  posterior	   cortex	   and	   somatomotor	   cortex	   as	   compared	   to	   wildtype	   animals.	  Expression	   of	   cFos	   was	   highest	   in	   the	   cerebellum	   for	   both	   Lurcher	   mutants	   and	  wildtype	   mice	   following	   rotarod	   activation	   and	   was	   lower	   in	   all	   other	   cortical	  regions	  analyzed	  (See	  Figures	  4-­‐10	  for	  graphs	  and	  photomicrographs).	  	  	  	  	  	  	  	   37	  	  	  Figure	   3.	   Performance	   on	   the	   rotarod	   test	   of	   sensorimotor	   function	   in	   wildtype	  mice	   (n=7)	   (blue)	   and	   in	  Lc/+	  mutant	  mice	   (n=7)	   (red).	   The	   top	   graph	   shows	   the	  performance	   of	   individual	   animals	   and	   the	   bottom	   graph	   shows	   the	   average	  performance	  of	  wildtype	  mice	  versus	  Lurcher	  mutants	  (p<0.0001).	  Wildtype (+/+) AWildtype (+/+) BWildtype (+/+) CWildtype (+/+) GWildtype (+/+) HWildtype (+/+) IWildtype (+/+) JLc/+ Mutant DLc/+ Mutant ELc/+ Mutant FLc/+ Mutant KLc/+ Mutant LLc/+ Mutant MLc/+ Mutant N0100200300400Rotarod Time for  Wildtype (+/+) and Lc/+ Mutant MiceAnimal IDAverage Rotarod Time (seconds)Wildtype (+/+) Mice Lc/+ Mutant Mice0100200300Animal GenotypeRotarod Time (seconds)Average Rotarod Time (in seconds) in WT (+/+) vs. Lc/+ Mutant Mice****	   38	  	  	  	  Figure	  4.	  Graphs	  showing	  the	  average	  density	  (cells/mm2)	  of	  cFos	  positive	  cells	  in	  the	  cerebellum	  (red),	  orbitofrontal	  (blue),	  posterior	  (green)	  and	  somatomotor	  cortex	  (yellow)	  of	  Lc/+	  mutants	  (n=5	  for	  each	  group)	  versus	  wildtype	  (WT)	  mice	  (n=5	  for	  each	  group).	  Top	  graph	  shows	  cFos	  staining	  from	  rotarod-­‐activated	  animals	  and	  the	  bottom	  graph	  shows	  cFos	  staining	  from	  animals	  that	  received	  no	  rotarod	  activation	  (basal	   state)	   (p<0.05	   for	   all	   groups	   in	   both	   basal	   cFos	   staining	   and	   active	   cFos	  staining	  conditions).	  +/+ cFos Active CerebellumLc/+ Mutant cFos Active Cerebellum+/+ cFos Active Orbitofrontal CortexLc/+ Mutant cFos Active Orbitofrontal Cortex+/+ cFos Active Posterior CortexLc/+ Mutant cFos Active Posterior Cortex+/+ cFos Active Somatomotor CortexLc/+ Mutant cFos Active Somatomotor Cortex010002000300040005000Average Density of cFos Positive Cells in Wildtype (+/+) vs. Lc/+ Mutant Animals Rotarod ActivationDensity of cFos Positive Cells(cells/mm2) ******** **** ****Animal Groups+/+ cFos Basal CerebellumLc/+ Mutant cFos Basal Cerebellum+/+ cFos Basal Orbitofrontal CortexLc/+ Mutant cFos Basal Orbitofrontal Cortex +/+ cFos Basal Posterior CortexLc/+ Mutant cFos Basal Posterior Cortex+/+ cFos Basal Somatomotor CortexLc/+ Mutant cFos Basal Somatomotor Cortex0100020003000Average Density of cFos Positive Cells in Wildtype (+/+) Mice vs. Lurcher Mutants (Lc/+) Basal ActivationDensity of cFos Positive Cells(cells/mm2) Animal Groups******* *	   39	  	  	  	  Figure	   5.	   The	   average	   density	   of	   cFos	   staining	   (cFos	   positive	   cells/mm2)	   in	   the	  cerebellum	  of	  +/+	  (n=5	  mice)	  and	  Lc/+	  (n=5	  mice)	  animals.	  The	  top	  panel	  shows	  the	  average	   density	   of	   cFos	   staining	   in	   rotarod-­‐activated	   mice	   (p<0.0001)	   and	   the	  bottom	   panel	   shows	   the	   average	   density	   of	   cFos	   staining	   under	   resting	   (basal)	  conditions	  (p<0.0005).	  	  Wildtype (+/+) Mice Lc/+ Mutant Mice050100150200250Animal StrainAverage Number of cFos Positive Cells (cells/mm2)(expressed as % of wildtype)Average Density of cFos Positive Cells in the Cerebellum Rotarod Activated cFos Levels****Wildtype (+/+) Mice Lc/+ Mutant Mice050100150200250Animal StrainAverage Number of cFos Positive Cells (cells/mm2)(expressed as % of wildtype)Average Density of cFos Positive Cells in the Cerebellum Basal cFos Levels***	   40	  	  	  	  Figure	   6.	   The	   average	   density	   of	   cFos	   staining	   (cFos	   positive	   cells/mm2)	   in	   the	  orbitofrontal	  cortex	  of	  +/+	   (n=5	  mice)	  and	  Lc/+	  (n=5	  mice)	  animals.	  The	  top	  panel	  shows	   the	   average	   density	   of	   cFos	   staining	   in	   rotarod-­‐activated	  mice	   (p<0.0001)	  and	   the	   bottom	   panel	   shows	   the	   average	   density	   of	   cFos	   staining	   under	   resting	  (basal)	  conditions	  (p<0.05).	   	  Wildtype (+/+) Mice Lc/+ Mutant Mice0100200300Animal StrainAverage Number of cFos Positive Cells (cells/mm2)(expressed as % of wildtype)Average Density of cFos Positive Cells in Orbitofrontal Cortex Rotarod Activated cFos Levels****Wildtype (+/+) Mice Lc/+ Mutant Mice050100150200250Animal StrainAverage Number of cFos Positive Cells (cells/mm2)(expressed as % of wildtype)Average Density of cFos Positive Cells in Orbitofrontal Cortex Basal cFos Levels*	   41	  	  	  	  Figure	   7.	   The	   average	   density	   of	   cFos	   staining	   (cFos	   positive	   cells/mm2)	   in	   the	  posterior	   cortex	   of	   +/+	   (n=5	   mice)	   and	   Lc/+	   (n=5	   mice)	   animals.	   The	   top	   panel	  shows	   the	   average	   density	   of	   cFos	   staining	   in	   rotarod-­‐activated	  mice	   (p<0.0001)	  and	   the	   bottom	   panel	   shows	   the	   average	   density	   of	   cFos	   staining	   under	   resting	  (basal)	  conditions	  (p<0.0005).	  Wildtype (+/+) Mice Lc/+ Mutant Mice0100200300Animal StrainAverage Number of cFos Positive Cells (cells/mm2)(expressed as % of wildtype)Average Density of cFos Positive Cells in Posterior Cortex Rotarod Activated cFos Levels****Wildtype (+/+) Mice Lc/+ Mutant Mice0100200300400Animal StrainAverage Number of cFos Positive Cells (cells/mm2)(expressed as % of wildtype)Average Density of cFos Positive Cells in Posterior Cortex Basal cFos Levels***	   42	  	  	  	  Figure	   8.	   The	   average	   density	   of	   cFos	   staining	   (cFos	   positive	   cells/mm2)	   in	   the	  somatomotor	  cortex	  of	  +/+	  (n=5	  mice)	  and	  Lc/+	  (n=5	  mice)	  animals.	  The	  top	  panel	  shows	   the	   average	   density	   of	   cFos	   staining	   in	   rotarod-­‐activated	  mice	   (p<0.0001)	  and	   the	   bottom	   panel	   shows	   the	   average	   density	   of	   cFos	   staining	   under	   resting	  (basal)	  conditions	  (p<0.05).	   	  	  Wildtype (+/+) Mice Lc/+ Mutant Mice050100150200250Animal StrainAverage Number of cFos Positive Cells (cells/mm2)(expressed as % of wildtype)Average Density of cFos Positive Cells in Somatomotor Cortex Rotarod Activated cFos Levels****Wildtype (+/+) Mice Lc/+ Mutant Mice0100200300Animal StrainAverage Number of cFos Positive Cells (cells/mm2)(expressed as % of wildtype)Average Density of cFos Positive Cells in Somatomotor Cortex Basal cFos Levels*	   43	  	  	  	  Figure	   9.	  Micrographs	   showing	   cFos	   activation	   in	   rotarod-­‐activated	  Lc/+	  mutants	  (top	  panels)	  and	  wildtype	  mice	  (bottom	  panels).	  Pictures	  taken	  at	  20x	  magnification.	  	   44	  	  	  	  Figure	  10.	  Micrographs	  showing	  basal	  cFos	  activation	  in	  Lc/+	  mutants	  (top	  panels)	  and	  wildtype	  littermates	  (bottom	  panels).	  Pictures	  are	  taken	  at	  20x	  magnification.	  	  	   45	  3.2-­‐Lurcher	  chimeras	  	   3.2.1-­‐Lc	  chimeras	  exhibit	  variable	  cerebellar	  pathology	  	  	   To	   date	   we	   have	   tested	   39	   Lurcher	   chimeras	   to	   study	   differences	   in	  cerebellar	  pathology,	  ASD-­‐like	  behavioural	  phenotypes	  on	  paradigms	  testing	  higher	  order	  brain	  functions	  (such	  as	  behavioural	  flexibility)	  and	  differences	  in	  cerebellar	  neuronal	  activity.	  We	  separated	  Lurcher	  chimeras	  into	  5	  experimental	  groups	  based	  on	  the	  number	  of	  surviving	  PCs	  as	  a	  measure	  of	  cerebellar	  pathology.	  As	  expected	  some	   of	   the	   Lc	   chimeras	   generated	   had	   no	   surviving	   PCs	   and	   resembled	   Lc/+	  mutants.	   A	   second	   group	   of	   mice	   consisted	   of	   ataxic	   Lc	   chimeras	   with	   very	   few	  surviving	  PCs	  (>0-­‐3,000	  PCs	  in	  the	  left	  hemi-­‐cerebellum),	  the	  third	  group	  of	  Lurcher	  chimeras	   had	   an	   intermediate	   number	   of	   surviving	   PCs	   (>3,000-­‐20,000	   surviving	  PCs	   in	   the	   left	   hemi-­‐cerebellum)	   with	   no	   ataxia	   and	   the	   fourth	   group	   of	   mice	  consisted	  of	  Lurcher	  chimeras	  with	  >20,000-­‐32,000	  surviving	  PCs	  in	  the	  left	  hemi-­‐cerebellum.	   The	   fifth	   group	   of	   mice	   resembled	   wildtype	   animals	   with	   a	   large	  cerebellum	   and	   large	   number	   of	   surviving	   PCs	   (>32,000	   PCs	   in	   the	   left	   hemi-­‐cerebellum).	   Mice	   were	   treated	   as	   Lurcher	   wildtypes	   if	   they	   had	   a	   comparable	  number	  of	  PCs	  as	  seen	  in	  wildtype	  mice	  assessed	  in	  Section	  3.1.1	  (Established	  using	  a	  95%	  confidence	  interval	  of	  cerebellar	  PC	  counts	  in	  Lc	  chimeras).	  Lurcher	  chimeras	  underwent	  behavioural	   testing	  before	  brain	   tissue	  was	  harvested	   for	  downstream	  analysis.	  The	  left	  side	  of	  each	  Lurcher	  chimera	  brain	  was	  stained	  for	  Calbindin-­‐D28K	  to	   analyze	   differences	   in	   cerebellar	   PC	   death	   and	   some	   brains	   were	   also	  counterstained	  with	   cresyl	   violet	   (CV)	   to	   determine	   if	   there	   were	   any	   qualitative	  differences	  in	  the	  morphology	  of	  the	  GCL.	  	   46	  Each	  Lurcher	  chimera	  analyzed	  had	  a	  different	  number	  of	  surviving	  PCs	  and	  the	  size	  and	  morphology	  of	  the	  cerebellum	  varied	  widely	  from	  animal	  to	  animal.	  The	  Lurcher	  chimera	  with	  the	  highest	  number	  of	  surviving	  PCs	  had	  31,641	  surviving	  PCs	  in	   the	   left	   hemi-­‐cerebellum	   and	   the	   Lurcher	   chimera	   with	   the	   lowest	   number	   of	  surviving	  PCs	  had	  19	  PCs	   in	   the	   left	   hemi-­‐cerebellum	   (See	  Figure	  11).	   In	   previous	  studies	  using	  Lurcher	   chimeras	   it	  was	   found	   that	   those	   chimeras	  with	   fewer	   than	  10%	  surviving	  PCs	  (as	  compared	  to	  wildtype	  numbers	  of	  PCs)	  had	  cerebellar	  ataxia	  as	  seen	  in	  Lc/+	  mutants	  [35].	  Our	  study	  confirmed	  these	  findings	  as	  chimeras	  with	  <10%	   surviving	   PCs	   had	   cerebellar	   ataxia	   and	   had	   a	   cerebellum	   with	   similar	  appearance	  and	  morphology	  to	  the	  cerebellum	  of	  Lc/+	  mutants,	  characterized	  by	  its	  small	  size	  and	  drastic	  loss	  of	  cerebellar	  neurons.	  Finally,	  we	  found	  extensive	  losses	  of	  cerebellar	  GCs	  (as	  shown	  by	  CV	  staining)	  in	  those	  chimeras	  with	  the	  fewest	  PCs.	  	  	  	  	  	  	  	  	   47	  	  	  	   48	  	  Figure	  11.	  Bar	  graph	  showing	  the	  variability	  in	  the	  number	  of	  surviving	  PCs	  in	  the	  left	   hemi-­‐cerebellum	   of	   Lurcher	   chimeras	   analyzed	   in	   2013-­‐2014	   (top	   panel).	  Photomicrographs	   showing	   variable	   cerebellar	   pathology	   in	   3	   different	   Lurcher	  chimeras	  (middle	  panel).	  Photomicrographs	  showing	  changes	  in	  the	  appearance	  and	  morphology	   of	   the	   granule	   cell	   layer	   (GCL)	   in	   a	   wildtype	   animal	   and	   3	   different	  Lurcher	  chimeras	  (bottom	  panel).	  	  	   3.2.2-­‐cFos	   staining	   in	   cerebellar	   granule	   cells	   is	   inversely	   correlated	  with	  the	  number	  of	  surviving	  PCs	  in	  Lc	  chimeras	  	  Next,	  we	  were	   interested	   in	   determining	   if	   there	   is	   a	   relationship	   between	  the	  degree	  of	  cerebellar	  pathology	  (death	  of	  PCs	  and	  GCs)	  seen	  in	  Lurcher	  chimeras	  and	  neuronal	  activity	  in	  surviving	  pre-­‐synaptic	  cerebellar	  granule	  cells.	  As	  described	  above	   we	   separated	   the	   Lurcher	   chimeras	   into	   5	   experimental	   groups	   based	   on	  cerebellar	  PC	  counts	  as	  a	  quantifiable	  measure	  of	  cerebellar	  pathology.	  We	  used	  IHC	  to	  look	  at	  the	  density	  of	  cFos	  staining	  (cFos	  positive	  cells/mm2)	  in	  the	  right	  side	  of	  the	   brains	   of	   the	   same	   Lurcher	   chimeras	   used	   for	   Calbindin-­‐D28K	   staining	   to	  explore	   the	   relationship	   between	   the	   number	   of	   surviving	   PCs	   and	   changes	   in	  	   49	  neuronal	   activity	   in	   response	   to	   PC	   death.	   We	   found	   that	   there	   is	   an	   inverse	  relationship	  between	  the	  numbers	  of	  surviving	  PCs	  and	  the	  density	  of	  cFos	  staining	  in	  the	  cerebellar	  GCL,	  suggesting	  that	  those	  Lurcher	  chimeras	  with	  fewer	  surviving	  PCs	  have	   increased	  neuronal	  activity	   in	  surviving	  pre-­‐synaptic	  GCs	  (See	  Figures	  12	  and	  13).	  	  	   	  	  	  	  	  	  	  	  	  	  	  	  	  	  	   	  	   50	  	  	  	  	  Figure	  12.	  Relationship	  between	  the	  number	  of	  surviving	  PCs	  and	  the	  average	  cFos	  staining	   density	   (cFos	   positive	   cells/mm2)	   in	   the	  GCL	   of	   the	   cerebellum.	  Note	   the	  inverse	  relationship	  between	  the	  number	  of	  surviving	  PCs	  and	  cFos	  staining	  density	  in	  the	  GCL	  of	  the	  cerebellum.	  Lurcher	  chimeras	  with	  the	  fewest	  PCs	  have	  the	  highest	  neuronal	   activity	   in	   the	   GCL	   as	   shown	   by	   cFos	   staining.	   There	   is	   no	   significant	  difference	  in	  the	  density	  of	  cFos	  staining	  in	  the	  GCL	  between	  Lurcher	  wildtype	  (+/+)	  animals	   and	   wildtype-­‐like	   Lurcher	   chimeras	   or	   between	   Lc/+	   mutants	   and	   Lc/+	  mutant-­‐like	  chimeras,	  but	  there	  is	  a	  statistically	  significant	  difference	  in	  the	  density	  of	   cFos	   staining	   in	   the	   GCL	   between	   wildtype-­‐like	   chimeras	   (Group	   4),	   chimeras	  with	   intermediate	   numbers	   of	   surviving	   PCs	   (Group	   3)	   and	   ataxic	   chimeras	   with	  <10%	  wildtype	  numbers	  of	  PCs	  (Group	  2).	  	  Lurcher Wildtype (+/+) (n=4)Wildtype-like Lurcher chimeras (n=12)Intermediate chimeras (n=7)Lc/+ mutant-like chimeras (n=9)Lc/+ mutants (n=7)010002000300040005000Average Number of cFos Positive Cells/mm2 of GCL in the Cerebellum of Lurcher ChimerasLurcher chimeras Grouped by Number of Surviving PCsAverage Number of cFos Positive Cells (cells/mm2)*********p<0.001p<0.001p<0.05	   51	  	  	  	  	  Figure	  13.	  Differences	  in	  the	  size	  and	  area	  of	  the	  cerebellar	  GCL	  between	  an	  ataxic	  Lurcher	   chimera	   and	   a	   wildtype-­‐like	   chimera	   (top	   panels).	   Differences	   in	   cFos	  expression	  in	  the	  cerebellar	  GCL	  in	  3	  different	  Lurcher	  chimeras	  (bottom	  panels).	  	   52	  3.2.3-­‐cFos	   staining	   in	   cerebellar	  Purkinje	   cells	   is	  positively	   correlated	  with	  the	  number	  of	  surviving	  PCs	  in	  Lc	  chimeras	  	  In	   addition	   to	   analyzing	   the	   density	   of	   cFos	   staining	   in	   the	   GCL	   in	   the	  cerebellum	  of	  Lurcher	  chimeras,	  we	  also	  assessed	  the	  number	  of	  cFos	  positive	  PCs	  in	  17	  Lurcher	  chimeras	  to	  determine	  if	  there	  are	  differences	  in	  PC	  neural	  activity	  in	  chimeras	   with	   differing	   numbers	   of	   surviving	   PCs.	   We	   found	   that	   those	   Lurcher	  chimeras	  with	  the	  highest	  number	  of	  surviving	  PCs	  also	  had	  the	  most	  cFos	  positive	  PCs	  in	  the	  cerebellum	  and	  the	  lowest	  density	  of	  cFos	  staining	  in	  the	  cerebellar	  GCL	  (See	   Figures	   14,	   15	   and	   16).	   Our	   finding	   that	   the	   number	   of	   cFos	   positive	   PCs	   is	  positively	   correlated	  with	   the	   number	   of	   surviving	   PCs	   and	   negatively	   correlated	  with	  the	  density	  of	  cFos	  staining	  in	  the	  cerebellar	  GCL	  suggests	  that	  there	  is	  a	  loss	  of	  inhibition	  from	  the	  cerebellar	  cortex	  in	  Lurcher	  chimeras	  with	  increasing	  cerebellar	  pathology	  and	  neuron	  loss.	  	  	  It	  must	  be	  noted	  that	  while	  Lc/+	  mutant-­‐like	  chimeras	  had	  no	  cFos	  positive	  PCs,	   they	   did	   have	   small	   numbers	   of	   surviving	   Calbindin-­‐D28K	   positive	   PCs	  confirming	  that	  these	  animals	  are	  true	  chimeras.	  However,	  in	  those	  ataxic	  chimeras	  with	   a	   few	   surviving	   Calbindin-­‐D28K	   positive	   PCs	  we	   do	   not	   see	   cFos	   expression	  within	   the	  cell	  bodies	  of	  PCs	  and	  PC	  dendritic	   trees	  are	  stunted	  with	   less	  complex	  branching	  patterns	  as	  compared	  to	  PCs	  seen	  in	  wildtype	  animals.	  Finally,	  increased	  cFos	  staining	  in	  the	  cerebellar	  GCL	  of	  Lurcher	  chimeras	  also	  supports	  the	  idea	  that	  the	  loss	  of	  a	  population	  of	  post-­‐synaptic	  target	  neurons	  leads	  to	  intrinsic	  changes	  in	  the	   firing	   of	   pre-­‐synaptic	   neurons.	   Although	   the	   exact	   mechanisms	   underlying	  changes	   in	   neuronal	   excitability	   following	   post-­‐synaptic	   cell	   death	   are	   not	   fully	  	   53	  understood,	   a	   recent	   study	   suggests	   that	   GABAergic	   interneuron	   death	   in	   the	  hippocampus	  can	  lead	  to	  changes	  in	  both	  LTP	  induction	  and	  intrinsic	  firing	  patterns	  of	  surviving	  hippocampal	  neurons	  [36].	  	  	  	  Figure	   14.	   Relationship	   between	   the	   average	   number	   of	   cFos	   positive	   Purkinje	  cells/section	   and	   %	   chimerism	   in	   individual	   Lurcher	   chimeras	   (%	   chimerism	   is	  determined	   by	   the	   total	   number	   of	   surviving	   wildtype	   PCs	   in	   the	   left	   hemi-­‐cerebellum	  of	  each	  Lurcher	  chimera).	  There	  is	  a	  strong	  positive	  correlation	  between	  %	  chimerism	  and	  the	  average	  number	  of	  cFos	  positive	  PCs/section	  (as	  a	  marker	  of	  neuronal	   activity)	   in	   Lurcher	   chimeras.	   Those	   chimeras	  with	   the	   fewest	   surviving	  PCs	   also	   have	   the	   fewest	   cFos	   positive	   PCs/section	   suggesting	   that	   these	   animals	  have	   the	   lowest	   levels	   of	   neural	   activity	   in	   the	   inhibitory	   PCL.	   Each	   dot	   in	   the	  scatterplot	   represents	   a	   single	   Lurcher	   chimera	   and	   the	   correlation	   coefficient	  r=0.9531.	  	  	  	  0 20 40 60 80 1000200400600% Chimerism (Based on Total Numbers of PCs in the Left Hemi-Cerebellum)Average Number of cFos Positive Purkinje Cells/Section Relationship Between the Average Number of cFos Positive Purkinje Cells/Section and % Chimerism in Lurcher chimerasr=0.9531	   54	  	  	  	  Figure	   15.	   Average	   number	   of	   cFos	   positive	   PCs/section	   in	   17	   Lurcher	   chimeras	  with	  variable	  cerebellar	  pathology.	  The	  graph	  above	  shows	  the	  average	  number	  of	  cFos	  positive	  PCs	  in	  3	  different	  groups	  of	  Lurcher	  chimeras	  based	  on	  quantification	  of	  surviving	  PCs	  in	  the	  cerebellum	  as	  a	  measure	  of	  cerebellar	  pathology.	  The	  average	  number	   of	   cFos	   positive	   PCs	   in	   the	   cerebellum	   of	   Lurcher	   chimeras	   is	   inversely	  correlated	  with	   the	   average	   density	   of	   cFos	   positive	   GCs.	   Chimeras	   shown	   in	   red	  have	  >20,000-­‐32,000	  PCs	  in	  the	  left	  hemi-­‐cerebellum,	  chimeras	  shown	  in	  green	  have	  intermediate	   cerebellar	   pathology	   with	   >3,000-­‐20,000	   surviving	   PCs	   in	   the	   left	  hemi-­‐cerebellum	  and	  chimeras	  with	  no	  cFos	  positive	  PCs	  have	  >0-­‐3,000	  surviving	  PCs	   in	   the	   left	   hemi-­‐cerebellum	   as	   shown	   by	   Calbindin-­‐D28K	   staining.	   Although	  Lc/+	   mutant-­‐like	   chimeras	   have	   no	   cFos	   positive	   PCs,	   they	   do	   have	   surviving	  Calbindin-­‐D28K	   positive	   PCs	   confirming	   that	   these	   animals	   are	   chimeras	   (p<0.01	  between	   the	  means	  of	  Group	  2	   and	  Group	  3	   and	  p<0.0001	  between	   the	  means	  of	  Group	  2	  and	  Group	  4	  and	  between	  the	  means	  of	  Group	  3	  and	  Group	  4).	  	  	  	  	  	  	   	  Group #2 >0-3,000 PCs (n=6)Group #3 >3,000-20,000 PCs (n=3)Group #4 >20,000-32,000 PCs (n=8)0200400600Average Number of cFos Positive Purkinje Cells/Section in the Cerebellum of Lurcher chimeras Lurcher chimeras grouped by Number of Surviving PCsAverage Number of cFos Positive Purkinje Cells**********	   55	  	  	  	  	  	  	  	  	  	  	  	  Figure	   16.	  Photomicrographs	  showing	  cFos	  positive	  PCs	   in	   the	  cerebellum	  of	   two	  different	  Lurcher	  chimeras	  along	  with	  cFos	  staining	  in	  the	  GCL.	  The	  number	  of	  cFos	  positive	  PCs	  is	  positively	  correlated	  with	  the	  number	  of	  surviving	  PCs	  in	  Lc	  chimeras	  and	  inversely	  correlated	  with	  the	  density	  of	  cFos	  staining	  (cells/mm2	  of	  GCL)	  in	  the	  GCL	  of	  the	  cerebellum.	  	  	   	  	  	  	  	   56	  The	   strong	   positive	   correlation	   between	   %	   chimerism	   (defined	   as	   total	  number	  of	  surviving	  wildtype	  PCs	  in	  the	  left-­‐hemi	  cerebellum)	  in	  individual	  Lurcher	  chimeras	  and	  the	  number	  of	  cFos	  positive	  PCs/section	  suggests	  that	  with	  increasing	  cerebellar	  pathology	  there	  is	  decreased	  inhibition	  from	  PC	  outputs	  in	  the	  cerebellar	  cortex	  to	  CN	  neurons.	  Lurcher	  mutant-­‐like	  chimeras	  have	  the	   lowest	  %	  chimerism	  with	   the	   fewest	   surviving	   PCs	   in	   the	   left	   hemi-­‐cerebellum	   and	   the	   fewest	   cFos	  positive	  PCs/section	  as	  a	  quantitative	  measure	  of	   the	  overall	  number	  of	   inhibitory	  PC	   efferents	   to	   CN	   neurons.	   The	   strong	   positive	   correlation	   between	   total	   PC	  number	   in	   the	   left	   hemi-­‐cerebellum	   and	   the	   average	   number	   of	   cFos	   positive	  PCs/section	   in	   the	  right	  hemi-­‐cerebellum	  follows	  a	   linear	  relationship	  and	  as	   total	  PC	  numbers	  decrease	  so	  does	  the	  average	  number	  of	  cFos	  positive	  PCs.	  To	  confirm	  that	  PCs	  were	  expressing	  cFos	  we	  used	  double	  immunofluorescence	  to	  visualize	  PCs	  expressing	  the	  structural	  marker	  Calbindin-­‐D28K	  and	  the	  functional	  marker	  cFos.	  	  We	   utilized	   indirect	   immunofluorescence	   to	   examine	   co-­‐localization	   of	  Calbindin-­‐D28K	  (a	  structural	  marker	  expressed	  at	  high	  levels	  in	  cerebellar	  PCs)	  and	  cFos	   (a	   functional	   marker	   expressed	   at	   higher	   levels	   following	   recent	   neural	  activity)	   to	   determine	   if	   there	   is	   a	   shift	   in	   the	   amount	   of	   inhibitory	   cerebellar	  outflow	  in	  Lurcher	  chimeras	  with	   increasing	  cerebellar	  pathology.	   In	  wildtype-­‐like	  chimeras	   with	   higher	   numbers	   of	   surviving	   PCs	   we	   found	   large	   numbers	   of	   PCs	  expressing	  Calbindin-­‐D28K	  and	  cFos	  suggesting	  robust	  inhibitory	  activity	  within	  the	  PCL.	  In	  Lurcher	  chimeras	  with	  intermediate	  numbers	  of	  surviving	  PCs	  we	  found	  less	  intense	  co-­‐localization	  of	  Calbindin-­‐D28K	  and	  cFos	  and	  also	  observed	  that	  surviving	  	   57	  cerebellar	   PCs	   had	   smaller	   and	   less	   complex	   dendritic	   trees	   as	   compared	   to	  wildtype	  controls	  and	  wildtype-­‐like	  Lurcher	  chimeras	   (data	  not	  shown).	  Finally,	   in	  ataxic	  Lurcher	  chimeras	  with	  fewer	  surviving	  PCs	  we	  observed	  no	  co-­‐localization	  of	  cFos	  and	  Calbindin-­‐D28K,	  confirming	  that	  these	  animals	  have	  almost	  no	  inhibitory	  PC	  projections	   to	   cerebellar	  nuclei	   (CN)	  neurons	  deep	  within	   the	   cerebellar	  white	  matter.	   These	   experiments	   demonstrate	   that	   there	   is	   a	   shift	   in	   the	   balance	   of	  excitatory/inhibitory	   neural	   activity	   within	   the	   cerebellar	   cortex	   following	  developmental	  PC	  death	  (See	  Figure	  17).	  	  	  	  	  	  	   58	  	  	  	   59	  	  	  	  Figure	  17.	  Photomicrographs	  showing	  co-­‐localization	  of	  Calbindin-­‐D28K	  and	  cFos	  in	   cerebellar	   PCs	   demonstrating	   decreased	   inhibitory	   PC	   neural	   activity	   in	   those	  Lurcher	   chimeras	   with	   increasing	   severity	   of	   cerebellar	   pathology.	   The	   top	   panel	  shows	  high	  levels	  of	  cFos	  and	  Calbindin	  co-­‐localization	  in	  a	  Lurcher	  chimera	  with	  a	  large	   number	   of	   surviving	   PCs,	   the	  middle	  panel	   shows	   lower	   levels	   of	   cFos	   and	  Calbindin	   co-­‐localization	   in	   a	   Lurcher	   chimera	   with	   intermediate	   numbers	   of	  surviving	  PCs	  and	  the	  bottom	  panel	  shows	  no	  co-­‐localization	  of	  cFos	  and	  Calbindin	  in	   an	   ataxic	   chimera	  with	   few	   surviving	   PCs.	  DAPI	   staining	   in	  blue	   shows	   the	   cell	  nuclei	  of	  cerebellar	  GCs.	  	  	   	  	  	  	  	   	  	   60	  3.2.4-­‐cFos	   staining	   in	   cerebellar	   nuclei	   (CN)	   neurons	   is	   inversely	  correlated	  with	   the	   number	   of	   surviving	   PCs	   and	   the	   number	   of	   cFos	  positive	  PCs	  in	  Lc	  chimeras	  	  Next,	   to	   explore	   the	   downstream	   effects	   of	   losing	   inhibitory	   cerebellar	   PC	  outputs	  we	  analyzed	  the	  density	  of	  cFos	  staining	  in	  Cerebellar	  Nuclei	  (CN)	  neurons.	  CN	  neurons	  receive	  direct	  synaptic	  connections	  from	  axons	  of	  cerebellar	  PCs	  and	  we	  predict	  that	  there	  should	  be	  corresponding	  changes	   in	  the	  density	  of	  cFos	  positive	  cells	  in	  the	  CN	  depending	  on	  the	  amount	  of	  inhibition	  that	  CN	  neurons	  receive	  from	  PCs.	  When	  we	  looked	  at	  the	  density	  of	  cFos	  staining	  within	  the	  CN,	  we	  found	  that	  the	  number	   of	   cFos	   positive	   CN	   neurons/mm2	   was	   inversely	   correlated	   with	   the	  number	  of	  surviving	  PCs	   in	  the	  Lurcher	  chimeras	  and	  qualitatively	  those	  chimeras	  with	   the	   lowest	   level	   of	   co-­‐localization	   of	   Calbindin-­‐D28K	   and	   cFos	   had	   an	  appreciably	  higher	  number	  of	  cFos	  positive	  CN	  neurons	   indicating	  higher	   levels	  of	  neural	  activity	  within	  the	  CN	  (See	  Figures	  18,	  19	  and	  20).	  Conversely,	  those	  animals	  with	  the	  highest	  number	  of	  surviving	  cerebellar	  PCs	  and	  increased	  inhibition	  of	  CN	  neurons	  had	  the	  lowest	  density	  of	  cFos	  positive	  CN	  neurons	  indicating	  lower	  levels	  of	  neural	  activity	  within	  the	  CN.	  	  	  	  	  	  	   61	  	  	  	  Figure	   18.	   Light	  photomicrographs	  of	  DAB	  staining	   showing	   the	  difference	   in	   the	  density	   and	   number	   of	   cFos	   positive	   CN	   neurons	   in	   individual	   Lurcher	   chimeras.	  The	   top	   panels	   show	   photomicrographs	   of	   cFos	   positive	   CN	   neurons	   in	   Lurcher	  chimeras	   at	   10x	   magnification.	   Lurcher	   chimera	   I	   has	   the	   highest	   number	   of	  surviving	  PCs	  and	  the	  lowest	  level	  of	  cFos	  staining	  in	  the	  CN,	  Lurcher	  chimera	  L	  has	  an	  intermediate	  number	  of	  surviving	  PCs	  and	  an	  intermediate	  level	  of	  cFos	  staining	  in	  CN	  neurons	  and	  Lurcher	  chimera	  G	  (ataxic)	  has	  few	  surviving	  PCs	  and	  the	  highest	  density	  of	  cFos	  staining	  in	  CN	  neurons.	  The	  bottom	  panels	  show	  cFos	  staining	  in	  CN	  neurons	   at	   20x	   magnification.	   Lurcher	   chimera	   I	   has	   the	   highest	   number	   of	  surviving	  PCs	  and	  has	  the	  lowest	  density	  of	  cFos	  staining	  in	  the	  CN,	  Lurcher	  chimera	  L	  has	  an	  intermediate	  number	  of	  surviving	  PCs	  and	  an	  intermediate	  number	  of	  cFos	  positive	  neurons	  in	  the	  CN	  and	  Lurcher	  chimera	  Q	  (ataxic)	  has	  the	  fewest	  surviving	  PCs	  in	  the	  cerebellum	  and	  has	  the	  highest	  density	  of	  cFos	  staining	  in	  the	  CN.	  	  	   62	  	  	  Figure	   19.	   Immunofluorescence	   staining	   showing	   differences	   in	   the	   density	   and	  number	  of	  cFos	  positive	  CN	  neurons	  in	  individual	  Lurcher	  chimeras.	  The	  top	  panel	  shows	   photomicrographs	   of	   cFos	   positive	   CN	   neurons	   (shown	   in	   green).	   Lurcher	  chimera	   A	   has	   the	   highest	   number	   of	   surviving	   PCs	   and	   the	   lowest	   level	   of	   cFos	  staining	  in	  the	  CN,	  Lurcher	  chimera	  E	  has	  an	  intermediate	  number	  of	  surviving	  PCs	  and	   an	   intermediate	   level	   of	   cFos	   staining	   in	   CN	   neurons	   and	   Lurcher	   chimera	  D	  (ataxic)	  has	  few	  surviving	  PCs	  and	  the	  highest	  level	  of	  cFos	  staining	  in	  CN	  neurons.	  The	  bottom	  panel	   shows	   the	   same	   relationship	   except	   in	   these	   photomicrographs	  cFos	   is	   shown	   in	   red,	   Calbindin-­‐D28K	   is	   shown	   in	  green	  and	  DAPI	   staining	   of	   cell	  nuclei	   is	   shown	   in	   blue.	   The	   number	   of	   cFos	   positive	   CN	   neurons	   is	   inversely	  correlated	   with	   the	   number	   of	   surviving	   inhibitory	   PCs	   and	   the	   extent	   of	   co-­‐localization	  of	  cFos	  and	  Calbindin-­‐D28K	  in	  the	  PCL.	  	   63	  	  	  	  	  	   	  	  	  	  Figure	   20.	   Bar	   graph	   showing	   the	   average	   number	   of	   cFos	   positive	   CN	   neurons	  (cells/mm2)	  in	  Lurcher	  chimeras.	  The	  blue	  bar	  indicates	  that	  the	  Lurcher	  chimeras	  were	   ataxic	   and	   had	   >0-­‐3,000	   PCs	   in	   the	   left	   hemi-­‐cerebellum,	   the	   green	   bar	  indicates	   that	   the	   Lurcher	   chimeras	   had	   intermediate	   cerebellar	   pathology	   with	  >3,000-­‐20,000	   PCs	   in	   the	   left	   hemi-­‐cerebellum	   and	   the	   red	  bar	   indicates	   that	   the	  Lurcher	  chimera	  had	  a	  wildtype-­‐like	  cerebellum	  with	  >20,000-­‐32,000	  PCs	  in	  the	  left	  hemi-­‐cerebellum.	   Neural	   activation	   as	   shown	   by	   cFos	   in	   CN	   neurons	   is	   inversely	  correlated	  with	  the	  number	  of	  surviving	  inhibitory	  PCs	  in	  the	  cerebellum	  of	  Lurcher	  chimeras	  and	  inversely	  correlated	  with	  the	  number	  of	  cFos	  positive	  PCs	  in	  Lurcher	  chimeras	  (p<0.0001	  between	  the	  mean	  of	  each	  group).	  	  	  	  	  Group #2 >0-3,000 PCs (n=4)Group #3 >3,000-20,000 PCs (n=4)Group #4 >20,000-32,000 PCs (n=5)0100200300400500Lurcher Chimeras Grouped by Cerebellar PathologyAverage Number of cFos Positive Cells(cells/mm2)Average Number of cFos Positive Cerebellar Nuclei (CN) Neurons (cells/mm2)************	   64	  We	   confirmed	   that	   cFos	   positive	   cells	   were	   indeed	   CN	   neurons	   by	   taking	  serial	   sections	   of	   Lurcher	   chimera	   brain	   tissue	   and	   used	   immunofluorescence	   to	  visualize	  Tbr1	  and	  Calbindin-­‐D28K	  expression	  in	  cerebellar	  neurons	  within	  sections	  adjacent	   to	   brain	   tissue	   used	   for	   cFos	   and	   Calbindin-­‐D28K	   immunofluorescence.	  Tbr1	   is	   a	   transcription	   factor	   that	   is	  widely	   expressed	   in	  developing	  CNS	  neurons	  and	   in	   the	   adult	  mammalian	   brain	   it	   is	   also	   strongly	   expressed	   in	   a	   subset	   of	   CN	  neurons	  [78].	  In	  previous	  histological	  studies	  of	  the	  CN	  it	  was	  found	  that	  Tbr1	  is	  co-­‐expressed	  in	  CN	  neurons	  that	  also	  express	  parvalbumin,	  which	  is	  a	  calcium-­‐binding	  protein	  expressed	  at	  high	   levels	   in	   the	  CN	   [79].	   In	  our	  experiments	  we	   found	   that	  Tbr1	   is	   expressed	   in	   CN	  neurons	   of	   Lurcher	  mutants	   and	   chimeras	   and	   using	   the	  Allen	   Brain	   Atlas	   as	   a	   cross-­‐reference,	   we	   confirmed	   that	   the	   cFos	   positive	   CN	  neurons	   identified	   in	   our	   double	   immunofluorescence	   experiments	   were	   at	   the	  correct	  anatomical	  location	  within	  the	  cerebellum	  (See	  Figure	  21).	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	   65	  	  	  	  Figure	   21.	   Photomicrographs	   demonstrating	   Tbr1	   immunofluorescence	   in	   CN	  neurons	   of	   a	  wildtype	   control	  mouse	   top	  panel	  and	   a	  Lc/+	  mutant	  mouse	  bottom	  panel	  showing	  the	  morphology	  and	  anatomical	  location	  of	  CN	  neurons	  deep	  within	  the	  cerebellar	  white	  matter.	  The	  faint	  red	  Calbindin-­‐D28K	  staining	  surrounding	  the	  green	  Tbr1	  positive	  CN	  neurons	   in	  the	  Lurcher	  wildtype	  mouse	  may	  represent	  the	  axons	  of	  PCs	  before	  they	  form	  synaptic	  connections	  with	  their	  CN	  neuron	  targets.	  	   	  	   66	  3.2.5-­‐Changes	  in	  the	  number,	  morphology	  and	  distribution	  of	  cerebellar	  glia	  following	  Purkinje	  cell	  death	  in	  Lc	  mutants	  and	  chimeras	  	   In	   addition	   to	   neuronal	   cell	   death	   and	   changes	   in	   neuronal	   activity	   in	  surviving	   PCs,	   GCs	   and	   CN	   neurons,	   we	   have	   also	   noticed	   a	   change	   in	   the	  morphology	  and	  distribution	  of	  glial	  cells	  within	  the	  cerebellum	  of	  Lc/+	  mutants	  and	  chimeras.	   In	   Lc/+	   mutants	   and	   ataxic	   Lurcher	   chimeras	   (with	   a	   mutant-­‐like	  cerebellum),	  we	  observed	  an	  increase	  in	  the	  number	  of	  microglia	  invading	  the	  grey	  matter	   of	   the	   cerebellar	   cortex	   and	   have	   also	   seen	   a	   significant	   difference	   in	   the	  morphology	  of	  microglia	  relative	  to	  the	  extent	  of	  cerebellar	  pathology	  in	  individual	  mice.	  In	  wildtype	  mice	  microglia	  have	  thin	  and	  spindly	  processes	  and	  non-­‐descript	  cell	  bodies,	  but	   in	  the	  Lc/+	  mutant	  cerebellum	  microglia	  have	  thicker,	  well-­‐formed	  processes	  with	  a	  much	  fatter	  and	  prominent	  cell	  body	  (See	  Figure	  22).	  This	  change	  in	  microglial	  cell	  morphology	  likely	  reflects	  a	  change	  in	  microglial	  cell	  function	  and	  in	   the	  Lc/+	  mutant	   cerebellum	   appears	   to	   be	  well	   localized	   to	   the	   site	   of	   original	  cerebellar	  PC	  and	  GC	  death	  in	  the	  grey	  matter	  of	  the	  cerebellar	  cortex.	  	  The	   same	   changes	   in	   the	   distribution	   and	  morphology	   of	  microglia	   can	   be	  observed	   in	   the	   cerebellum	   of	   individual	   Lurcher	   chimeras	   (See	   Figure	   23)	   and	  appears	   to	  be	  well	   correlated	  with	   the	   extent	  of	   cerebellar	  pathology,	  particularly	  the	  extent	  of	  primary	  PC	  death	  and	  secondary	  loss	  of	  cerebellar	  GCs.	  In	  those	  ataxic	  Lurcher	   chimeras	   with	   the	   fewest	   surviving	   PCs	   and	   GCs	   we	   observed	   increased	  numbers	  of	  microglia	  invading	  the	  cerebellar	  cortical	  grey	  matter	  and	  we	  also	  saw	  microglia	  with	  altered	  cell	  morphology	  with	  thicker	  cell	  processes	  and	  fatter,	  more	  prominent	   cell	   bodies.	   In	   Lurcher	   chimeras	  with	   the	   highest	   number	   of	   surviving	  	   67	  PCs	  there	  are	  fewer	  microglia	  localized	  to	  the	  cerebellar	  cortical	  grey	  matter	  and	  the	  microglia	   have	   thinner,	   skinnier	   processes	  with	   less	   prominent	   cell	   bodies.	   These	  morphological	   differences	   may	   reflect	   a	   change	   in	   the	   activation	   state	   of	   the	  microglia	   in	   response	   to	   cerebellar	   neuropathology,	   but	   further	   experiments	   are	  necessary	  to	  determine	  the	  exact	  functional	  and	  activation	  state	  of	  these	  microglia.	  	  	  	  Figure	   22.	   Photomicrographs	   demonstrating	   differences	   in	   cerebellar	   microglial	  morphology	  in	  a	  wildtype	  littermate	  (left)	  and	  a	  Lc/+	  mutant	  mouse	  (right).	   In	  the	  wildtype	   mouse	   microglia	   have	   thinner	   and	   skinnier	   cell	   processes	   and	   in	   the	  Lurcher	  mutant	  mouse	   the	  microglia	  have	   thicker,	  more	  prominent	   cell	   processes	  and	   a	   more	   pronounced	   cell	   body	   indicative	   of	   a	   change	   in	   cell	   function	   and	  activation	  state.	  	  	   68	  	  	  	   69	  	  	  Figure	   23.	   Photomicrographs	   showing	   differences	   in	   the	   distribution	   and	  morphology	   of	   microglia	   in	   the	   cerebellar	   cortex	   of	   individual	   Lurcher	   chimeras.	  The	  top	  photomicrograph	  shows	  a	  few	  spindly	  microglia	  in	  a	  wildtype-­‐like	  Lurcher	  chimera	  with	  a	  large	  number	  of	  surviving	  PCs.	  The	  middle	  photomicrograph	  shows	  a	  larger	  number	  of	  microglia	  with	  more	  prominent	  cell	  processes	  and	  cell	  bodies	   in	  the	  cerebellar	  cortex	  of	  a	  Lurcher	  chimera	  with	  more	  extensive	  PC	  loss.	  The	  bottom	  photomicrograph	   shows	   a	   large	   number	   of	   microglia	   with	   well-­‐developed	   cell	  processes	  in	  the	  cerebellar	  cortex	  of	  an	  ataxic	  Lurcher	  chimera	  with	  >90%	  PC	  loss.	  	   	  	  	  	  	  	  	  	  	  	  	  	   70	  	  	  	  	  	  	  	  	  	  Figure	  24.	  Bar	  graph	  showing	  the	  average	  number	  of	  Iba1	  positive	  microglia	  in	  the	  cerebellar	   cortex	   of	   19	   Lurcher	   chimeras.	   The	  blue	  bar	   indicates	   that	   the	   Lurcher	  chimeras	  were	  ataxic	  and	  had	  >0-­‐3,000	  PCs	   in	   the	   left	  hemi-­‐cerebellum,	   the	  green	  bar	  indicates	  that	  the	  Lurcher	  chimeras	  had	  intermediate	  cerebellar	  pathology	  with	  >3,000-­‐20,000	   PCs	   in	   the	   left	   hemi-­‐cerebellum	   and	   the	   red	  bar	   indicates	   that	   the	  Lurcher	   chimeras	   had	   a	  wildtype-­‐like	   cerebellum	  with	   >20,000-­‐32,000	  PCs	   in	   the	  left	   hemi-­‐cerebellum	   (p<0.0001	   between	   the	  means	   of	   Group	   2	   and	   Group	   3	   and	  Group	  2	  and	  Group	  4	  and	  p<0.01	  between	  the	  means	  of	  Group	  3	  and	  Group	  4).	  	  	  	  	  	   	  Group #2 >0-3,000 PCs (n=9)Group #3 >3,000-20,000 PCs (n=4)Group #4 >20,000-32,000 PCs (n=6)0100200300400500Density of Microglia (cells/mm2) in the Cerebellar CortexLurcher Chimeras Grouped by Cerebellar PathologyAverage Number of Iba1 Positive Cells(cells/mm2)**********	   71	  As	   shown	   above	   the	   morphology	   and	   distribution	   of	   microglia	   within	   the	  cerebellar	   cortex	   varies	   between	   individual	   Lurcher	   chimeras.	   Increased	  microgliosis	   appears	   to	   be	   coupled	   with	   the	   severity	   of	   cerebellar	   pathology	   and	  with	   increased	   death	   of	   PCs	   and	   GCs	   in	   the	   cerebellar	   cortex,	   we	   see	   increased	  numbers	  of	  microglia	  with	  prominent	  cell	  processes	  and	  cell	  bodies	  in	  the	  cerebellar	  grey	  matter	  (See	  Figure	  24).	   Interestingly,	  all	  of	  the	  Lurcher	  mutants	  and	  chimeras	  evaluated	   in	   these	   experiments	   were	   adults	   (P60+)	   and	   months	   after	   the	   initial	  degeneration	   of	   cerebellar	   PCs	   and	   secondary	   loss	   of	   GCs	   those	   animals	  with	   the	  most	   severe	   cerebellar	   pathology	   had	   the	   most	   pronounced	   changes	   in	   the	  morphology	  and	  distribution	  of	  cerebellar	  microglia.	  Future	  experiments	  need	  to	  be	  conducted	  to	  determine	  the	  activation	  state	  of	  these	  microglia	  in	  individual	  Lurcher	  chimeras	   and	   to	   determine	   if	   these	   microglia	   are	   playing	   a	   neuro-­‐protective	   or	  neuro-­‐degenerative	  role	  in	  the	  cerebellum	  of	  these	  ASD-­‐like	  mouse	  models.	  	  	  	   Finally,	   we	   explored	   the	   qualitative	   differences	   in	   the	   distribution	   and	  morphology	   of	   glial	   cells	   located	   in	   the	   cerebellar	   cortex	   of	   Lurcher	  mutants	   and	  chimeras.	  We	   specifically	   looked	  at	   the	  morphology	  and	  distribution	  of	  Bergmann	  glia,	   which	   are	   a	   type	   of	   radial	   astroglia	   located	   in	   the	   molecular	   layer	   of	   the	  cerebellum	   [37].	   In	   previous	   studies	   looking	   at	   GFAP	   expression	   in	   cerebellar	  astroglia,	   researchers	   found	   that	   in	   control	   mice	   (wildtype	   animals)	   GFAP	  expression	  in	  Bergmann	  glia	  was	   lower	  than	  GFAP	  expression	  in	  all	  other	  types	  of	  astroglia	   found	   in	   the	   cerebellar	   cortex	   [37].	   In	   many	   studies	   it	   has	   been	   well	  documented	  that	  astrogliosis	  (an	  increase	  in	  the	  number	  of	  reactive	  astrocytes)	  is	  a	  	   72	  common	   pathological	   hallmark	   seen	   after	   traumatic	   brain	   injury,	   CNS	   infection,	  stroke	   and	   during	   the	   course	   of	   neurodegenerative	   diseases	   [38].	   Histologically,	  astrogliosis	   is	   characterized	   by	   an	   increase	   in	   the	   size	   of	   astroglial	   processes	   and	  upregulation	  of	   the	  astroglial	   intermediate	   filaments	  Glial	  Fibrillary	  Acidic	  Protein	  (GFAP)	  and	  vimentin	   [39,40].	  We	  chose	   to	  use	   immunofluorescence	   to	   look	  at	   the	  morphology	  of	  astroglia	  in	  the	  cerebellar	  cortex	  to	  see	  if	  there	  is	  astrogliosis	  in	  the	  cerebellum	   of	   Lurcher	  mutants	   and	   chimeras	   following	   the	   neurodegeneration	   of	  PCs	  and	  GCs	  in	  the	  first	  2-­‐3	  weeks	  of	  life.	  	  	   We	   first	   analyzed	   GFAP	   expression	   in	   Lc/+	   mutant	   mice	   and	   wildtype	  littermates	   to	   see	   if	   there	   was	   an	   appreciable	   difference	   in	   the	   appearance	   or	  morphology	   of	   cerebellar	   astroglia	   between	   the	   two	   groups	   of	   animals.	  We	   found	  that	  Bergmann	  glia	  in	  Lurcher	  mutants	  have	  markedly	  increased	  GFAP	  expression	  in	  the	   molecular	   layer	   of	   the	   cerebellum	   as	   compared	   to	   wildtype	   littermates	   (See	  Figure	  25).	  This	  increase	  in	  GFAP	  expression	  in	  Bergmann	  glia	  within	  the	  cerebellar	  cortex	   is	   consistent	   with	   previous	   descriptions	   of	   astrogliosis	   following	  neurodegeneration	   in	  other	  brain	   regions	   [39,40].	   In	  wildtype	  mice,	   expression	  of	  GFAP	   was	   significantly	   lower	   in	   the	   cerebellar	   cortex	   and	   in	   some	   brains	   GFAP	  expression	  could	  not	  be	  detected	  in	  the	  molecular	  layer	  of	  the	  cerebellum	  at	  10x	  and	  20x	  magnification.	  	   73	  	  	  Figure	  25.	  GFAP,	   Iba1	  and	  DAPI	   immunofluorescence	   in	  the	  cerebellar	  cortex	  of	  a	  wildtype	  control	  mouse	  (lower	  panels)	  and	  a	  Lurcher	  mutant	  mouse	  (upper	  panels)	  showing	  increased	  numbers	  of	  microglia	  and	  increased	  intensity	  of	  GFAP	  staining	  in	  the	   molecular	   layer	   of	   the	   cerebellar	   cortex	   of	   Lurcher	   mutant	   mice.	   Increased	  intensity	   of	   GFAP	   staining	   in	   Lurcher	   mutant	   mice	   is	   consistent	   with	   reactive	  astrogliosis	   following	   the	  degeneration	  of	   cerebellar	  neurons.	  Brain	   tissue	   is	   from	  adult	  (P60+)	  mice.	  	  	  	   We	  also	  analyzed	  the	  appearance	  and	  morphology	  of	  cerebellar	  astroglia	   in	  Lurcher	   chimera	  mice	   to	   determine	   if	   there	  was	   variability	   in	   the	  morphology	   of	  astroglia	   in	   the	   cerebellar	   cortex	   corresponding	   to	   the	   severity	   of	   cerebellar	  pathology	  in	  individual	  chimeras.	  In	  wildtype-­‐like	  chimeras	  with	  higher	  numbers	  of	  surviving	   PCs	   and	  GCs,	  we	   saw	  no	   increase	   in	   GFAP	   expression	   in	   Bergmann	   glia	  within	   the	   molecular	   layer.	   In	   Lurcher	   chimeras	   with	   a	   significant	   loss	   of	   PCs	  (without	   cerebellar	   ataxia),	  we	  observed	   a	   large	   increase	   in	   the	   intensity	   of	  GFAP	  staining	  of	  Bergmann	  glia	  in	  the	  molecular	  layer	  of	  the	  cerebellum.	  In	  ataxic	  Lurcher	  	   74	  chimeras	  with	  >90%	  PC	  death	  we	  observed	  an	  even	  larger	  increase	  in	  the	  intensity	  of	  GFAP	  staining	  of	  Bergmann	  glia	  in	  the	  molecular	  layer	  (See	  Figure	  26	  and	  27).	  	   The	  increased	  intensity	  of	  GFAP	  staining	  and	  the	  changes	  in	  the	  morphology	  of	  Bergmann	  glia	  in	  the	  molecular	  layer	  of	  the	  cerebellum	  are	  again	  consistent	  with	  astrogliosis	  following	  the	  initial	  degeneration	  of	  cerebellar	  PCs	  and	  GCs	  in	  the	  first	  2-­‐3	   weeks	   of	   life	   and	   appears	   to	   be	   correlated	   with	   the	   severity	   of	   cerebellar	  pathology	  in	  individual	  Lurcher	  chimeras.	  Finally,	  we	  have	  seen	  significant	  numbers	  of	  GFAP	  positive	  Bergmann	  glia	  and	  Iba1	  positive	  microglia	  within	  the	  same	  regions	  of	  the	  cerebellar	  cortex	  of	  Lurcher	  mutant	  and	  Lurcher	  chimeric	  mice	  with	  extensive	  cerebellar	   pathology.	   In	   ataxic	   Lurcher	  mutants	   and	   chimeras	   there	   are	   increased	  numbers	   of	   microglia	   and	   increased	   intensity	   in	   the	   staining	   of	   GFAP	   positive	  Bergmann	   glia	   in	   the	   same	   regions	   of	   the	   cerebellar	   cortex	   suggesting	   that	   the	  degree	  of	  reactive	  gliosis	  is	  related	  to	  the	  degree	  of	  cerebellar	  damage	  and	  extent	  of	  cerebellar	  neuron	  death.	  	  	   75	  	  	  Figure	   26.	   Immunofluorescence	   showing	   GFAP,	   Iba1	   and	   DAPI	   staining	   in	   the	  cerebellar	   cortex	   of	   3	   different	   Lurcher	   chimeras	   at	   10x	   magnification.	   Lurcher	  chimera	   J	   (top	   left	  panel)	   is	   a	   wildtype-­‐like	   chimera	   and	   has	   low	   levels	   of	   GFAP	  staining	   in	   the	  molecular	   layer	  of	   the	  cerebellum	  and	  has	  only	  a	   few	  Iba1	  positive	  microglia	  present	   in	  the	  cerebellar	  cortex.	  Lurcher	  chimera	  E	  (top	  right	  panel)	  has	  considerably	  higher	  numbers	  of	  GFAP	  positive	  Bergmann	  glia	  in	  the	  molecular	  layer	  and	  there	  are	  several	  more	  Iba1	  positive	  microglia	  localized	  in	  the	  cerebellar	  cortex.	  Lurcher	  chimera	  D	  with	  the	  highest	  degree	  of	  cerebellar	  pathology	  (bottom	  panel)	  has	   intense	   GFAP	   staining	   of	   Bergmann	   glia	   in	   the	  molecular	   layer	   and	   there	   are	  numerous	   microglia	   localized	   to	   the	   cerebellar	   cortex.	   The	   overlap	   of	   astrocytes	  expressing	   high	   levels	   of	   GFAP	   and	   numerous	   Iba1	   positive	   microglia	   in	   the	  cerebellar	   cortex	   corresponds	   with	   the	   number	   of	   surviving	   PCs	   and	   degree	   of	  cerebellar	  pathology	  in	  individual	  Lurcher	  chimeras.	  In	  those	  animals	  with	  the	  most	  severe	  cerebellar	  pathology	  there	  is	  extensive	  evidence	  of	  reactive	  gliosis,	  which	  is	  a	  pathological	  feature	  seen	  in	  many	  neurological	  and	  neurodegenerative	  disorders.	  	  	  	  	   76	  	  	  Figure	   27.	   Immunofluorescence	   showing	   GFAP,	   Iba1	   and	   DAPI	   staining	   in	   the	  cerebellar	   cortex	   of	   3	   different	   Lurcher	   chimeras	   at	   20x	   magnification.	   Lurcher	  chimera	   F	   (top	   left	   panel)	   is	   a	   wildtype-­‐like	   chimera	   and	   has	   no	   evident	   GFAP	  staining	   in	   the	  molecular	   layer	  of	   the	  cerebellum	  and	  has	  only	  a	   few	  Iba1	  positive	  microglia	  present	   in	  the	  cerebellar	  cortex.	  Lurcher	  chimera	  E	  (top	  right	  panel)	  has	  considerably	  higher	  numbers	  of	  GFAP	  positive	  Bergmann	  glia	  in	  the	  molecular	  layer	  and	  there	  are	  several	  more	  Iba1	  positive	  microglia	  localized	  to	  the	  cerebellar	  cortex.	  Lurcher	  chimera	  D	  with	  the	  highest	  degree	  of	  cerebellar	  pathology	  (bottom	  panel)	  has	   intense	   GFAP	   staining	   of	   Bergmann	   glia	   in	   the	  molecular	   layer	   and	   there	   are	  numerous	   microglia	   localized	   to	   the	   cerebellar	   cortex.	   The	   overlap	   of	   astrocytes	  expressing	   high	   levels	   of	   GFAP	   and	   numerous	   Iba1	   positive	   microglia	   in	   the	  cerebellar	   cortex	   corresponds	   with	   the	   number	   of	   surviving	   PCs	   and	   cerebellar	  pathology	   in	   individual	   Lurcher	   chimeras.	   In	   those	   animals	   with	   the	  most	   severe	  cerebellar	   pathology	   there	   is	   extensive	   evidence	   of	   reactive	   gliosis,	   which	   is	   a	  pathological	  feature	  seen	  in	  many	  neurological	  and	  neurodegenerative	  disorders.	  	  	  	  	  	  	  	   77	  3.3-­‐Fmr1	  KO	  mice	  3.3.1-­‐Trend	   of	   decreased	   PC	   numbers	   and	   altered	   PC	   morphology	   in	  Fmr1	  KO	  mice	  as	  compared	  to	  Fmr1	  wildtype	  mice	  	   Based	   on	   evidence	   that	   there	   are	   cerebellar	   abnormalities	   in	   humans	  diagnosed	   with	   both	   ASD	   and	   FXS	   and	   that	   Fmr1	  KO	  mice	   have	   altered	   synaptic	  plasticity	   in	   the	   cerebellum,	   we	   wanted	   to	   explore	   the	   relationship	   between	  cerebellar	  pathology	  and	  changes	  in	  neuronal	  activity	  in	  Fmr1	  KO	  mice	  [8,32].	  First,	  we	   stained	   brain	   tissue	   from	   6	   Fmr1	  KO	   mice	   and	   5	   Fmr1	  wildtype	   mice	   using	  monoclonal	  antibodies	  against	  Calbindin-­‐D28K	  to	  determine	  if	  there	  is	  cerebellar	  PC	  loss	   in	  Fmr1	  KO	  mice	  (similar	  to	  PC	   loss	  seen	   in	  the	  brains	  of	  Lurcher	  mutant	  and	  chimeric	  mice).	  We	  found	  that	   there	   is	  an	  average	  decrease	  of	  ~15%	  in	  cerebellar	  PC	  numbers	   in	  Fmr1	  KO	  mice	  as	  compared	  to	  Fmr1	  wildtype	  mice	  (See	  Figure	  28),	  but	   this	  difference	   in	  PC	  number	  was	  not	   statistically	   significant	  between	   the	   two	  groups	  (p=0.0862).	  	  	   PC	   loss	   in	   Fmr1	  KO	   mice	   was	   extremely	   variable	   and	   cell	   loss	   was	   more	  prominent	   in	  certain	   lobules	  of	   the	  cerebellum	  as	  compared	  to	  others.	   In	  addition,	  the	  distribution	  of	  PCs	  within	  the	  cerebellum	  was	  heterogeneous	  between	  different	  Fmr1	  KO	  mice,	  with	  no	  obvious	  or	  consistent	  pattern	  of	  cell	  loss	  in	  different	  regions	  of	   the	   cerebellum.	   The	  more	   diffuse	   and	  heterogeneous	   histopathological	   changes	  seen	   in	   the	  Fmr1	  KO	  mouse	  cerebellum	  as	  compared	   to	   the	  more	  widespread	  and	  homogeneous	   cerebellar	   pathology	   seen	   in	   Lurcher	   mutants	   and	   chimeric	   mice	  could	  be	  a	  result	  of	  the	  numerous	  synaptic	  proteins	  targeted	  by	  FMRP.	  With	  a	  loss	  of	  function	  mutation	  in	  the	  FMRP	  protein,	  there	  could	  be	  numerous	  subtle	  changes	  in	  	   78	  neuronal	  structure	  and	  function	  because	  of	  changes	  in	  the	  expression	  of	  numerous	  synaptic	  proteins	  [32].	  	  	  Figure	   28.	   Bar	   graph	   showing	   the	   average	   number	   of	   PCs	   in	   the	   left	   hemi-­‐cerebellum	   of	   Fmr1	   KO	   mice	   and	   Fmr1	   wildtype	   mice	   (top	   panel).	   There	   is	   an	  average	   loss	   of	   ~15%	   of	   cerebellar	   PCs	   in	   Fmr1	  KO	   mice	   as	   compared	   to	   Fmr1	  wildtype	  mice	   (p=0.0862	   n.s.)	   Photomicrographs	   of	   cerebellar	   PCs	   at	   5x	   and	   20x	  magnification	  (bottom	  panel)	  showing	  the	  difference	  in	  the	  number	  and	  morphology	  of	  PCs	  between	  Fmr1	  KO	  mice	  and	  Fmr1	  wildtype	  mice.	  	  Fmr1 WT (n=5) Fmr1 KO (n=6)0200004000060000Average Number of Purkinje Cells in the Cerebellum of Fmr1 MiceAnimal GenotypeAverage Number of Purkinje Cells p=0.0862 n.s.	   79	  Next,	  we	  counterstained	  the	  same	  brain	  tissue	  with	  CV	  to	  determine	  if	  there	  was	   a	   difference	   in	   the	  morphology	   and	   thickness	   of	   the	   cerebellar	   GCL	   between	  Fmr1	   KO	  mice	   and	  Fmr1	  wildtype	  mice.	  We	   found	   that	   there	  were	   no	   qualitative	  differences	  in	  the	  appearance	  or	  morphology	  of	  the	  GCL	  (See	  Figure	  29),	  suggesting	  that	   there	   is	   not	   significant	   cell	   death	   of	   cerebellar	  GCs	   in	   response	   to	   the	   loss	   of	  their	  post-­‐synaptic	  PC	  targets	  as	  seen	   in	  Lurcher	  mutants	  and	  chimeras.	  However,	  we	   found	   that	   PCs	   in	   Fmr1	   KO	  mice	   appeared	   to	   have	   smaller	   and	   less	   complex	  dendritic	   trees	   when	   compared	   to	   PCs	   seen	   in	   Fmr1	  wildtype	   mice,	   which	   have	  dendritic	   trees	   with	   numerous	   complex	   branches.	   This	   observed	   change	   in	   the	  morphology	   of	   cerebellar	   PCs	   in	   Fmr1	   KO	  mice	   could	   reflect	   changes	   in	   synaptic	  plasticity	  in	  cerebellar	  neurons	  such	  as	  GCs	  and	  PCs.	  	  Figure	   29.	   Cresyl	   Violet	   counterstaining	   of	   Fmr1	  wildtype	   and	   Fmr1	   KO	   mouse	  brain	  tissue	  showing	  no	  significant	  differences	  in	  the	  morphology	  of	  the	  GCL.	  	   80	  3.3.2-­‐Altered	  baseline	  cFos	  expression	  in	  Fmr1	  KO	  mice	  Finally,	  we	  examined	  changes	  in	  cFos	  expression	  in	  neurons	  as	  a	  reporter	  of	  neural	   activity	   in	   the	   cerebellar	   GCL,	   orbitofrontal	   cortex,	   posterior	   cortex	   and	  somatomotor	  cortex	  (See	  Figure	  30).	  We	  again	  used	  cFos	  as	  an	  immunohistological	  marker	  of	  neuronal	  activity	  to	  evaluate	  differences	  in	  basal	  neural	  activity	  between	  Fmr1	   KO	   mice	   and	   Fmr1	   wildtype	   mice.	   We	   found	   that	   there	   were	   decreased	  numbers	   of	   cFos	   positive	   cells	   in	   the	   cerebellar	   GCL,	   posterior	   cortex	   and	  somatomotor	   cortex	   of	   Fmr1	   KO	   mice	   as	   compared	   to	   Fmr1	   wildtype	   mice.	  Interestingly,	  we	  found	  increased	  levels	  of	  cFos	  staining	  in	  the	  orbitofrontal	  cortex	  of	  Fmr1	  KO	  mice	  as	  compared	  to	  Fmr1	  wildtype	  mice	  (See	  Figures	  31-­‐35),	  suggesting	  that	  under	  resting	  conditions	  there	  is	   increased	  neural	  activity	  in	  the	  orbitofrontal	  cortex	  of	  Fmr1	  K.O.	  mice	  as	  compared	  to	  Fmr1	  wildtype	  animals.	  	  Figure	   30.	   Overview	   of	   brain	   regions	   analyzed	   when	   looking	   at	   cFos	   staining	   in	  Lurcher	  mutant	  and	  Fmr1	  KO	  mouse	  models.	  	   81	  	  	  Fmr1 WT (n=4) Fmr1 KO (n=6)050100150Average Number of cFos Positive Cells in the Somatomotor Cortex (% of Controls)Animal GenotypeNumber of cFos Positive Cells (% of Controls)p<0.001***Fmr1 WT (n=4) Fmr1 KO (n=6)050100150200Average Number of cFos Positive Cells in the Orbital Cortex (% of Controls)Animal GenotypeNumber of cFos Positive Cells (% of Controls)p<0.05*	   82	  	  	  Figure	   31.	   Bar	   graphs	   showing	   the	   average	   number	   of	   cFos	   positive	   cells	   (as	   a	  measure	  of	  neural	  activation)	  in	  four	  different	  brain	  regions	  under	  basal	  conditions.	  There	   was	   decreased	   cFos	   expression	   in	   the	   cerebellar	   GCL	   (p<0.001),	   posterior	  cortex	  (p<0.005)	  and	  somatomotor	  cortex	  (p<0.001)	  in	  Fmr1	  KO	  mice	  as	  compared	  to	   wildtype	   littermates.	   However,	   we	   found	   increased	   cFos	   activation	   in	   the	  orbitofrontal	  cortex	  (p<0.05)	  of	  Fmr1	  KO	  mice	  as	  compared	  to	  Fmr1	  wildtypes.	  The	  average	  number	  of	  cFos	  positive	  cells	  in	  Fmr1	  KO	  mice	  are	  expressed	  as	  a	  %	  of	  Fmr1	  wildtype	  controls.	  Fmr1 WT (n=4) Fmr1 KO (n=6)050100150Average Number of cFos Positive Cells in the Posterior Cortex (% of Controls)Animal GenotypeNumber of cFos Positive Cells (% of Controls)p<0.005**Fmr1 WT (n=4) Fmr1 KO (n=6)050100150Average Number of cFos Positive Cells in the Cerebellar GCL (% of Controls)Animal GenotypeNumber of cFos Positive Cells (% of Controls)p<0.001***	   83	  	  	  Figure	  32.	  Photomicrograph	  showing	  increased	  cFos	  staining	  in	  the	  cerebellar	  GCL	  of	  a	  Fmr1	  WT	  mouse	  left	  panel	  as	  compared	  to	  an	  Fmr1	  KO	  mouse	  right	  panel.	  	  	  	  	  	  	  Figure	   33.	  Photomicrograph	  showing	  decreased	  cFos	  staining	   in	   the	  orbitofrontal	  cortex	  of	  a	  Fmr1	  WT	  mouse	  left	  panel	  as	  compared	  to	  a	  Fmr1	  KO	  mouse	  right	  panel.	  	  	  	  	   84	  	  	  Figure	   34.	   Photomicrograph	   showing	   increased	   cFos	   staining	   in	   the	   posterior	  cortex	  of	  a	  Fmr1	  WT	  mouse	  left	  panel	  as	  compared	  to	  a	  Fmr1	  KO	  mouse	  right	  panel.	  	  	  	  	  	  	  Figure	   35.	  Photomicrograph	  showing	   increased	  cFos	  staining	   in	   the	  somatomotor	  cortex	  of	  a	  Fmr1	  WT	  mouse	  left	  panel	  as	  compared	  to	  a	  Fmr1	  KO	  mouse	  right	  panel.	  	  	  	  	  	  	  	  	  	   85	  	  	  	  	  30000 40000 50000 60000 70000050100150Relationship between the Average Number of Purkinje Cells and the Number of cFos Positive Cells in the Somatomotor Cortex of Fmr1 KO miceAverage Number of Purkinje CellsNumber of cFos Positive Cells (% of Controls)Fmr1 WT (n=4)Fmr1 KO (n=6)30000 40000 50000 60000 70000050100150200250Relationship between the Average Number of Purkinje Cells and the Number of cFos Positive Cells in the Orbitofrontal Cortex of Fmr1 KO miceAverage Number of Purkinje CellsNumber of cFos Positive Cells (% of Controls)Fmr1 WT (n=4)Fmr1 KO (n=6)	   86	  	  	  	  Figure	   36.	   Scatterplots	   showing	   the	   relationship	   between	   the	   average	   number	   of	  surviving	   PCs	   and	   basal	   cFos	   staining	   in	   the	   somatomotor	   cortex,	   orbitofrontal	  cortex,	  posterior	  cortex	  and	  cerebellar	  GCL	  in	  Fmr1	  wildtype	  versus	  Fmr1	  KO	  mice.	  The	   plots	   indicate	   that	   there	   is	   no	   significant	   correlation	   between	   average	   PC	  numbers	   and	   the	   density	   of	   cFos	   staining	   in	   any	   of	   the	   analyzed	   brain	   regions	   in	  Fmr1	  KO	   and	  Fmr1	  wildtype	  mice,	   but	   that	   there	   is	   a	   significant	   difference	   in	   the	  average	  density	  of	  cFos	  staining	   in	  the	  4	  brain	  regions	  analyzed	  between	  Fmr1	  KO	  and	  Fmr1	  wildtype	  mice.	  30000 40000 50000 60000 70000050100150Relationship between the Average Number of Purkinje Cells and the Number of cFos Positive Cells in the Posterior Cortex of Fmr1 KO miceAverage Number of Purkinje CellsNumber of cFos Positive Cells (% of Controls)Fmr1 WT (n=4)Fmr1 KO (n=6)30000 40000 50000 60000 70000050100150Relationship between the Average Number of Purkinje Cells and the Number of cFos Positive Cells in the Cerebellar GCL of Fmr1 KO miceAverage Number of Purkinje CellsNumber of cFos Positive Cells (% of Controls)Fmr1 WT (n=4)Fmr1 KO (n=6)	   87	  	   Finally,	  we	   compared	   the	   relationship	   between	   the	   average	  number	   of	   PCs	  and	   the	   average	   density	   of	   cFos	   staining	   in	   Fmr1	  KO	   and	   Fmr1	   wildtype	  mice	   to	  determine	  if	  there	  is	  a	  correlation	  between	  total	  PC	  number	  and	  cFos	  staining	  in	  the	  cerebellar	   GCL	   and	   cortical	   brain	   regions.	  We	   found	   that	   there	  was	   no	   significant	  relationship	   between	   the	   average	   total	   number	   of	   PCs	   and	   the	   average	   density	   of	  cFos	  staining	  in	  the	  somatomotor	  cortex,	  orbitofrontal	  cortex,	  posterior	  cortex	  and	  cerebellar	  GCL	  under	  baseline	  conditions	  in	  Fmr1	  KO	  and	  Fmr1	  wildtype	  mice	  (See	  Figure	  36).	  This	   suggests	   that	  PC	  number	   is	  not	   tightly	   correlated	  with	  changes	   in	  neural	   activity	   as	   reported	   by	   cFos	   staining	   in	   Fmr1	   KO	   mice	   and	   wildtype	  littermates.	   However,	   we	   did	   see	   a	   significant	   difference	   in	   the	   density	   of	   cFos	  staining	  between	  Fmr1	  KO	  and	  Fmr1	  wildtype	  mice	  suggesting	  that	  null	  mice	  have	  altered	   neuronal	   activity	   at	   rest	   in	   the	   cerebellar	   GCL	   and	   cortical	   regions	   that	   is	  linked	  to	  changes	  in	  synaptic	  protein	  expression	  with	  loss	  of	  function	  mutations	  in	  the	  Fmr1	  gene	  [32].	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	   88	  Chapter	  4-­‐Discussion	  	  4.1-­‐Elevated	   cFos	   staining	   in	   Lc/+	   mutant	  mice	   suggests	   that	   Lc/+	  mutants	  have	  increased	  neural	  activity	  in	  the	  cerebellum	  and	  connected	  cortical	  brain	  regions	  due	  to	  the	  developmental	  loss	  of	  inhibitory	  cerebellar	  PCs	  	   In	  previous	  studies	  using	  cFos	  as	  a	  marker	  of	  metabolic	  activity	   in	  neurons	  for	  the	  purposes	  of	  poly-­‐synaptic	  pathway	  tracing	  researchers	  have	  found	  low,	  but	  measurable	  expression	  of	  cFos	  mRNA	  in	  adult	  neurons	  and	  cFos	  positive	  neurons	  in	  the	   neocortex	   and	   limbic	   system	   of	   control	   rat	   brains	   [41,42].	   In	   our	   first	  experiments	   looking	   at	   basal	   cFos	   activation	   in	   the	   cerebellum	   and	   other	   cortical	  regions	   in	   Lc/+	  mutant	   and	  wildtype	  mice,	   we	   also	   found	   relatively	   low	   levels	   of	  cFos	  expression	   in	  all	  brain	   regions	  analyzed	  with	   the	  highest	  basal	   levels	  of	   cFos	  seen	  in	  Lc/+	  mutants.	  Therefore,	  it	  was	  first	  important	  to	  establish	  controls	  to	  look	  at	   basal	   cFos	   staining	   in	   our	   Lc/+	   mutants	   and	   wildtype	   animals	   to	   look	   at	   the	  specific	   changes	   in	   cFos	   staining	   following	   activation	   of	   the	   cerebellum	   and	  connected	   brain	   regions	   during	   rotarod	   activation	   (a	   behavioural	   test	   specifically	  used	   to	   assess	   sensorimotor	   function).	   Previous	   studies	   using	   unilateral	   electrical	  stimulation	   of	   the	   rat	   amygdala	   have	   shown	   specific	   and	   rapid	   increases	   in	   cFos	  expression	   levels,	   which	   is	   consistent	   with	   our	   findings	   that	   the	   use	   of	   a	   specific	  behavioural	  task	  like	  the	  rotarod	  test	  can	  reliably	   induce	  increases	  in	  cFos	  protein	  levels	  in	  adult	  neurons	  [43].	  	  	   After	   rotarod	   activation	   we	   found	   that	   Lc/+	  mutant	   mice	   had	   significantly	  higher	   cFos	   expression	   not	   only	   in	   the	   cerebellum,	   but	   also	   in	   the	   orbitofrontal,	  somatomotor	   and	   posterior	   cortices	   which	   suggested	   that	   Lurcher	   mutants	   had	  	   89	  increased	  firing	  of	  action	  potentials	  in	  these	  brain	  regions	  as	  compared	  to	  wildtype	  littermates.	   Previous	   neuroanatomical	   studies	   of	   the	   Lurcher	  mutant	  mouse	   have	  reported	  the	  complete	  absence	  of	  cerebellar	  PCs	  and	  secondary	  death	  of	  up	  to	  90%	  of	  cerebellar	  GCs	  and	  70%	  of	  IONs	  by	  postnatal	  day	  90	  (P90),	  resulting	  in	  the	  loss	  of	  the	   sole	   efferent	   cerebellar	   cortical	   pathway	   to	   the	   brainstem,	   thalamus	   and	  cerebral	  cortex	  [44].	  However,	  despite	  the	  death	  of	  all	  PCs	  and	  the	  majority	  of	  pre-­‐synaptic	  GCs	  there	  is	  not	  significant	  death	  of	  CN	  neurons	  in	  the	  deep	  grey	  matter	  of	  the	  cerebellum	  [45].	  The	  relative	  preservation	  of	  CN	  neurons	  within	  the	  cerebellum	  despite	   extensive	   cerebellar	   cortical	   pathology	   suggests	   that	   some	   functional	  cerebellar	   outflow	   to	   the	   rest	   of	   the	   CNS	   is	   intact	   in	   the	   form	   of	   CN	   projection	  neurons	   [45].	  The	  observed	  preservation	  of	  CN	  neurons	   in	  Lc/+	  mutants	  may	  also	  explain	  why	  Lurcher	  mice	  have	  increased	  excitability	  within	  the	  CNS	  suggesting	  that	  the	  loss	  of	  GABAergic	  PCs	  leads	  to	  a	  shift	  in	  the	  balance	  of	  excitation	  and	  inhibition	  in	  cerebellar	  efferent	  pathways	  projecting	  to	  subcortical	  and	  cortical	  brain	  regions	  [46].	  	   Consistent	   with	   these	   findings,	   elevated	   levels	   of	   cFos	   under	   both	   basal	  (resting)	  conditions	  and	  after	  rotarod	  activation	  in	  Lc/+	  mutant	  mice	  suggests	  that	  increased	  metabolic	  activity	  within	  cFos	  positive	  neurons	  can	  be	  used	  as	  an	  indirect	  measure	  of	  increased	  neural	  activity	  within	  the	  cerebellum	  and	  cerebral	  cortices.	  In	  addition,	   since	   cerebellar	  PCs	  are	   the	   start	  of	   cerebellar	  outflow	   to	   the	   rest	  of	   the	  CNS,	   the	   complete	   loss	   of	   the	   GABAergic	   PC	   population	   should	   lead	   to	   decreased	  inhibition	  of	   surviving	  CN	  neurons	  and	   lead	   to	   a	   shift	   in	   the	  neural	   activity	  of	   the	  	   90	  heterogeneous	   population	   of	   surviving	   post-­‐synaptic	   excitatory	   (glutamatergic)	  projection	  neurons	  and	  inhibitory	  (glycinergic	  and	  GABAergic)	  CN	  neurons	  [47,48].	  	  	   The	   cerebellum	  has	   numerous	   indirect	   connections	  with	   different	   regions	   of	  the	   cerebral	   cortex	   via	   the	   brainstem	   and	   thalamus	   through	   poly-­‐synaptic	   neural	  circuits.	  One	  of	   the	  most	   important	  cerebello-­‐cortical	  pathways	   in	  mammals	   is	   the	  dentato-­‐rubro-­‐thalamo-­‐cortical	   tract	   [161].	   CN	   neurons	   in	   the	   dentate	   nucleus	  project	  to	  the	  contralateral	  red	  nucleus,	  then	  neurons	  from	  the	  red	  nucleus	  project	  to	   ventro-­‐lateral	   and	   ventro-­‐anterior	   nuclei	   of	   the	   thalamus	   and	   thalamo-­‐cortical	  neurons	   project	   to	   the	   motor	   cortex	   to	   modulate	   excitatory	   motor	   output	   [161].	  Since	  PCs	   are	   the	   sole	   cerebellar	   cortical	   outputs	   to	   the	  CN	  neurons	   it	   is	   believed	  that	  varying	   inhibitory	  activity	   in	  PCs	   is	  responsible	   for	  modulating	  cortical	  motor	  output	   through	   cerebello-­‐cortical	   pathways	   [161].	   The	   modification	   of	   excitatory	  cortical	  motor	  output	  by	  the	  cerebellum	  plays	  an	  important	  role	  in	  regulating	  motor	  activity	   and	   providing	   feedback	   to	   the	  motor	   cortex	   during	   fine,	   coordinated	   and	  skilled	  movements	   and	  may	   explain	   why	   some	   patients	   with	   ASD	   and	   associated	  cerebellar	  pathology	  have	  impairments	  in	  fine	  and	  gross	  motor	  tasks	  [161].	  	  	   In	   addition	   to	   its	   role	   of	   modulating	   motor	   activity,	   there	   is	   increasing	  evidence	   to	   suggest	   that	   through	   cerebello-­‐cortical	   connections	   the	   cerebellum	   is	  also	   able	   to	   modulate	   neural	   activity	   in	   higher	   brain	   regions	   like	   the	   prefrontal	  cortex.	  There	  are	  two	  major	  poly-­‐synaptic	  pathways	  from	  CN	  neurons	  in	  the	  dentate	  nucleus	  to	  the	  mPFC	  and	  through	  these	  pathways	  the	  cerebellum	  is	  able	  to	  modulate	  	   91	  dopamine	  release	  in	  the	  forebrain	  [162].	  The	  first	  pathway	  starts	  with	  glutamatergic	  projection	  neurons	  in	  the	  dentate	  nucleus,	  which	  indirectly	  modulate	  the	  activity	  of	  dopaminergic	  neurons	   in	   the	   ventral	   tegmental	   area	   (VTA)	   that	   in	   turn	  project	   to	  the	   mPFC	   where	   dopamine	   is	   released	   [163].	   The	   second	   pathway	   starts	   with	  glutamatergic	  neurons	   in	  the	  dentate	  nucleus	  that	  project	   to	  the	  medio-­‐dorsal	  and	  ventro-­‐lateral	  nuclei	  of	   the	   thalamus,	  which	   in	   turn	  send	  glutamatergic	  outputs	   to	  the	  mPFC	  to	  modulate	  dopamine	  release	  from	  dopaminergic	  mesocortical	  neurons	  [164].	   In	   Lc/+	   mutant	   mice	   that	   lose	   almost	   all	   of	   their	   cerebellar	   PCs	   there	   is	  decreased	  dopamine	  release	  in	  the	  mPFC	  following	  electrical	  stimulation	  of	  the	  CN	  neurons	  in	  the	  dentate	  nucleus	  [159].	  	  This	  suggests	  that	  PCs	  may	  also	  play	  a	  role	  in	  indirectly	   modulating	   neural	   activity	   in	   brain	   regions	   underlying	   higher	   brain	  functions	  such	  as	  decision-­‐making	  [25].	  	  	   In	   Lc/+	   mutant	   mice	   we	   found	   increased	   density	   of	   cFos	   staining	   in	   the	  somatomotor,	  posterior	  and	  orbitofrontal	  cortices	  under	  both	  basal	  conditions	  and	  following	  rotarod	  activation,	  suggesting	  that	  Lurcher	  mutants	  have	  increased	  neural	  activity	   in	   these	   brain	   regions.	   This	   data	   suggests	   that	   following	   the	   loss	   of	  inhibitory	  PC	  outputs	  to	  CN	  neurons	  there	  is	  an	  increase	  in	  excitatory	  output	  from	  CN	  projection	  neurons,	  which	  in	  turn	  leads	  to	  an	  increase	  in	  the	  activation	  of	  cortical	  neurons	   through	   multiple	   indirect	   cerebello-­‐cortical	   pathways.	   Although	   we	   can	  quantify	   differences	   in	   neural	   activity	   using	   cFos	   as	   an	   indirect	  marker	   of	   neural	  activation,	   it	   remains	   to	   be	   determined	   which	   cerebello-­‐cortical	   circuits	   exhibit	  changes	   in	   structure	   and	   function	   to	   produce	   the	   changes	   in	   neural	   activity	  	   92	  observed	  in	  the	  cerebral	  cortex.	  However,	  data	  from	  this	  study	  and	  others	  looking	  at	  the	  role	  of	  cerebellar	  modulation	  of	  neural	  activity	  suggests	  that	  the	  cerebellum	  can	  influence	  neural	  activity	  in	  a	  wide	  variety	  of	  neural	  circuits	  within	  the	  CNS.	  	   	  	   To	  understand	  how	  the	  loss	  of	  inhibitory	  neurons	  like	  PCs	  can	  lead	  to	  a	  shift	  in	  the	  neural	  activity	  of	  post-­‐synaptic	  neurons	  researchers	  have	  developed	  transgenic	  mouse	  models	  to	  study	  molecular	  and	  biophysical	  changes	  in	  ion	  channels	  that	  leads	  to	  altered	  neuronal	  firing.	  To	  look	  at	  the	  mechanisms	  underlying	  altered	  CN	  neuron	  activity	  scientists	   inhibited	   the	  activity	  of	   the	  small	   conductance	  calcium-­‐activated	  (SK)	   class	   of	   potassium	   channels	   by	   expressing	   a	   transgene	   that	   is	   a	   dominant	  inhibitory	  construct	  of	  the	  SK	  channel	  isoform	  seen	  in	  CN	  neurons	  [49].	  It	  was	  found	  that	   suppressing	   the	   activity	   of	   SK	   channels	   led	   to	   abolition	   of	   the	   after-­‐hyperpolarization	   current	   (IAHP)	   in	   CN	   neurons	   and	   caused	   increases	   in	   the	  spontaneous	   firing	   rate	   of	   CN	   neurons,	   which	   are	   both	   electrophysiological	  measures	  indicative	  of	  increased	  neuronal	  excitability	  [49].	  In	  addition	  to	  changes	  in	  the	   molecular	   and	   biophysical	   properties	   of	   neuronal	   ion	   channels	   an	   emerging	  body	  of	  research	  suggests	  that	  altered	  connectivity	  between	  neurons	  can	  also	  lead	  to	  changes	  in	  neuronal	  excitability	  and	  firing	  of	  APs	  in	  neural	  circuits.	  	  	   	  Altered	   connectivity	   between	   cerebellar	   and	   cortical	   neurons	   in	   Lc/+	  mutants	   is	   reflected	   by	   an	   increase	   in	   the	   cFos	   staining	   density	   of	   neurons	   in	   the	  cerebellum	   and	   cortical	   brain	   regions	   at	   rest	   or	   following	   rotarod	   activation.	   To	  understand	  how	  altered	  cerebellar	  connectivity	  may	  lead	  to	  global	  changes	   in	  CNS	  	   93	  excitability	  researchers	  have	  studied	  the	  electrical	  properties	  of	  cerebellar	  neurons	  in	   vitro	   and	   in	   vivo	   using	   mouse	   models.	   Electrophysiological	   studies	   have	   found	  that	  CN	  neurons	  are	  able	  to	  fire	  spontaneously	  at	  relatively	  high	  frequencies	  in	  the	  absence	  of	  other	  synaptic	  inputs	  [50].	  In	  vitro	  recordings	  of	  mammalian	  CN	  neurons	  found	   a	   spontaneous	   firing	   rate	   of	   10-­‐50	   Hz	   in	   the	   absence	   of	   other	   synaptic	  connections	   [51].	   However,	   in	   animal	   models	   the	   activity	   of	   CN	   neurons	   and	  modulation	  of	  CN	  neuronal	  activity	  through	  synaptic	  inputs	  is	  much	  more	  complex.	  Previous	  histological	  studies	  of	  CN	  neurons	  have	  found	  that	  the	  CN	  are	  composed	  of	  a	  heterogeneous	  population	  of	  larger	  glutamatergic	  neurons	  and	  smaller	  glycinergic	  and	   GABAergic	   neurons,	   with	   all	   cell	   types	   receiving	   synaptic	   input	   from	   several	  inhibitory	  PCs	  [52,74].	  CN	  neurons	  also	  receive	  numerous	  excitatory	  synaptic	  inputs	  from	  pontine	  mossy	   fibres	  and	  climbing	   fibres	   from	  the	   inferior	  olivary	  nucleus	   in	  the	   medulla,	   but	   mossy	   fibres	   also	   excite	   GABAergic	   PCs	   indirectly	   through	  cerebellar	  GCs	  [53].	  	   	  The	  numerous	  excitatory	  and	  inhibitory	  synaptic	  connections	  made	  with	  CN	  neurons	   are	   altered	   significantly	   in	   Lurcher	  mutant	   and	   chimeric	  mice	   due	   to	   the	  extensive	  loss	  of	  inhibitory	  PCs	  [54].	  The	  loss	  of	  CN	  neuron	  inhibition	  following	  PC	  degeneration	  leads	  to	  compensatory	  changes	  in	  the	  neural	  activity	  of	  surviving	  CN	  neurons	  in	  order	  to	  maintain	  a	  minimal	  level	  of	  cerebellar	  outflow	  to	  the	  rest	  of	  the	  CNS	   [54].	   Altered	   cerebellar	   outflow	   between	   CN	   neurons	   and	   cortical	   neurons	  through	  poly-­‐synaptic	  cerebello-­‐cortical	  circuits	  may	  explain	  how	  developmental	  PC	  	   94	  death	  can	  lead	  to	  altered	  neuronal	  activity,	  as	  reflected	  by	  increased	  cFos	  staining	  in	  the	  orbitofrontal,	  somatomotor	  and	  posterior	  cortices	  of	  Lc/+	  mutant	  mice.	  	  Besides	   studying	   cFos	   activation	   in	   Lurcher	   mutants	   (which	   have	  homogeneous	   cerebellar	  pathology	  with	   consistent	  morphological	   and	  histological	  abnormalities	  between	  individual	  animals),	  we	  also	  wanted	  to	  evaluate	  changes	   in	  the	   structure	  and	   function	  of	   cerebellar	  neurons	   in	  a	  mouse	  model	   that	  had	  more	  variable	  pathological	  and	  histological	  changes	  in	  the	  cerebellum.	  Lurcher	  chimeras	  (that	   have	   a	   cerebellum	   of	   variable	   size	   and	   variable	   loss	   of	   cerebellar	   neurons)	  provided	   a	   valuable	  model	  with	   a	   spectrum	   of	   cerebellar	   pathology	   and	   ASD-­‐like	  behavioural	   phenotypes	   to	   study	   the	   neurobiological	   correlates	   of	   ASD-­‐like	  phenotypes	  in	  a	  mouse	  model	  [22].	  	  4.2-­‐Increased	   cFos	   staining	   in	   cerebellar	   GCs	   of	   Lurcher	   chimeras	   with	   the	  highest	   degree	   of	   cerebellar	   pathology	   suggests	   that	   there	   is	   a	   shift	   in	   the	  neural	  activity	  of	  surviving	  pre-­‐synaptic	  GCs	  in	  response	  to	  PC	  death	  	  	   Granule	   cells	   are	   small,	   excitatory	   glutamatergic	   neurons	   found	   in	   the	  innermost	   layer	   of	   the	   cerebellar	   cortex	   and	   they	   receive	   excitatory	   pre-­‐synaptic	  input	  from	  pontine	  mossy	  fibres	  [55].	  The	  axons	  of	  granule	  cells	  bifurcate	  into	  two	  parallel	   fibres	   before	   making	   contact	   with	   their	   post-­‐synaptic	   target	   cells,	  GABAergic	   PCs	   [55].	   Each	   PC	   receives	   up	   to	   200,000	   synaptic	   inputs	   from	   pre-­‐synaptic	  GCs	  and	  this	  massive	  number	  of	  neural	  inputs	  is	  filtered	  and	  integrated	  to	  produce	  a	  single	  unified	  neural	  output,	  which	  is	  conveyed	  along	  the	  axon	  of	  the	  PC	  [56].	  In	  Lurcher	  chimeras	  where	  there	  is	  variable	  primary	  loss	  of	  inhibitory	  PCs	  and	  	   95	  secondary	  loss	  of	  GCs	  and	  IONs,	  we	  determined	  that	  there	  is	  a	  change	  in	  the	  density	  of	   cFos	   staining	   in	   surviving	   GCs	   that	   is	   inversely	   correlated	  with	   the	   number	   of	  surviving	  PCs	  in	  the	  cerebellum	  of	  Lurcher	  chimeras.	  	   With	   increasing	   PC	   death	   in	   Lurcher	   chimeras	   we	   saw	   corresponding	  increases	  in	  the	  staining	  density	  of	  cFos	  within	  pre-­‐synaptic	  GCs.	  This	  suggests	  that	  even	  with	  the	  loss	  of	  their	  post-­‐synaptic	  target	  neurons	  surviving	  cerebellar	  GCs	  are	  able	   to	   increase	   their	   neural	   activity,	   perhaps	   to	  maintain	   a	   threshold	   of	   synaptic	  transmission	   within	   the	   cerebellar	   cortex.	   Previous	   studies	   have	   also	   reported	  changes	  in	  the	  intrinsic	  firing	  rate	  of	  neurons	  and	  changes	  in	  neuronal	  excitability	  in	  response	   to	   the	   death	   of	   post-­‐synaptic	   target	   neurons	   [57].	   Following	   prolonged	  changes	  in	  the	  neural	  activity	  of	  a	  population	  of	  neurons	  researchers	  have	  observed	  both	  pre-­‐synaptic	  and	  post-­‐synaptic	  changes	  in	  synaptic	  transmission	  and	  neuronal	  excitability	   [58].	   Researchers	   have	   dubbed	   this	   phenomenon	   as	   homeostatic	  plasticity,	  with	  neurons	  being	  able	  to	  change	  their	  excitability	  to	  maintain	  a	  balance	  of	  excitatory	  and	  inhibitory	  synaptic	  transmission	  in	  order	  to	  maintain	  homeostasis	  in	   neural	   functioning	   [59].	   When	   populations	   of	   neurons	   are	   no	   longer	   able	   to	  maintain	  a	  balance	  in	  synaptic	  transmission	  in	  response	  to	  pathological	  changes	  in	  the	   brain,	   an	   imbalance	   in	   excitation	   and	   inhibition	   occurs	   which	   is	   thought	   to	  manifest	   in	   the	   form	   of	   neurological	   disorders	   such	   as	   epilepsy,	   ASD	   and	  schizophrenia	  [60].	  	  	   96	  An	  example	  of	  how	   the	  death	  of	   a	  population	  of	  post-­‐synaptic	  neurons	   can	  lead	   to	   changes	   in	   neural	   activity	   of	   surviving	   pre-­‐synaptic	   neurons	   has	   been	  demonstrated	   in	   the	   mouse	   hippocampus.	   Researchers	   generated	   distal-­‐less	  homeobox	  1	  (Dlx1	  -­‐/-­‐)	  mice,	  which	  exhibit	  postnatal	  loss	  of	  a	  group	  of	  hippocampal	  inhibitory	  interneurons	  at	  around	  30	  days	  after	  birth	  [61].	  In	  response	  to	  the	  loss	  of	  the	   hippocampal	   interneurons	   Dlx1	   -­‐/-­‐	   mice	   show	   decreased	   inhibitory	   synaptic	  transmission	  and	  go	  on	  to	  develop	  epilepsy,	  which	  reflects	  a	  global	  increase	  in	  CNS	  excitability	   [61].	   In	   addition	   to	   pathophysiological	   changes	   in	   neural	   excitability,	  researchers	   also	   documented	   compensatory	   changes	   in	   a	   population	   of	   excitatory	  CA1	   hippocampal	   neurons,	   which	   exhibited	   a	   decrease	   in	   intrinsic	   excitability	  shown	   by	   a	   decrease	   in	   mEPSCs	   (mini	   Excitatory	   Post-­‐Synaptic	   Currents)	   and	   a	  decreased	   ratio	   of	   AMPA/NMDA	   receptors	   [36].	   This	   highlights	   that	   with	   the	  selective	  loss	  of	  a	  population	  of	  neurons	  one	  may	  observe	  both	  pathological	  changes	  in	  neuronal	  excitability	  and	  compensatory	  changes	  in	  neuronal	  activity	  in	  surviving	  neurons	  to	  maintain	  a	  balance	  of	  synaptic	  transmission.	  	  	   Similar	   to	  Dlx1	   -­‐/-­‐	  mice,	   Lurcher	  mutants	   and	   chimeras	   lose	   inhibitory	  PCs	  shortly	   after	   birth	  with	   subsequent	   loss	   of	   excitatory	   cerebellar	  GCs	   and	   climbing	  fibres.	  The	  postnatal	  neurodegenerative	  changes	  in	  the	  cerebellar	  cortex	  also	  lead	  to	  changes	  in	  the	  balance	  of	  excitation	  and	  inhibition	  within	  the	  cerebellum.	  With	  the	  loss	  of	   cerebellar	  PCs	   in	  Lurcher	   chimeras	   there	  were	   changes	   in	   cFos	   expression	  not	  only	   in	  pre-­‐synaptic	  GCs,	  but	  also	  changes	   in	  cFos	  expression	  within	  surviving	  PCs	  and	  changes	  in	  the	  density	  of	  cFos	  staining	  within	  CN	  neurons.	  While	  cFos	  is	  a	  	   97	  useful	   functional	  marker	  to	  detect	  changes	   in	  neural	  activity	  within	  populations	  of	  neurons,	  it	  cannot	  be	  used	  to	  determine	  whether	  these	  changes	  in	  neural	  firing	  are	  pathological	  alterations	  in	  neuronal	  excitability	  or	  compensatory	  changes	  in	  neural	  firing	  to	  maintain	  a	  balance	  in	  excitatory	  and	  inhibitory	  synaptic	  transmission.	  It	  is	  likely	   that	   differences	   in	   cFos	   expression	   in	   different	   populations	   of	   cerebellar	  neurons	  reflect	  both	  pathological	  and	  homeostatic	  changes	  in	  neuronal	  excitability	  as	   has	   been	   previously	   documented	   in	   hippocampal	   neurons	   using	   the	   Dlx1	   -­‐/-­‐	  mouse	  [36].	  	  4.3-­‐Higher	   numbers	   of	   cFos	   positive	   PCs	   in	   wildtype-­‐like	   Lurcher	   chimeras	  represents	  increased	  inhibition	  of	  post-­‐synaptic	  CN	  neurons	  	  	   In	   addition	   to	   seeing	   an	   increase	   in	   the	   density	   of	   cFos	   staining	   in	   the	  cerebellar	  GCL,	  we	  also	  observed	  differences	  in	  the	  number	  of	  cFos	  positive	  PCs	  in	  Lurcher	  chimeras	  with	  variable	  cerebellar	  pathology.	  Those	  Lurcher	  chimeras	  with	  higher	   numbers	   of	   surviving	   PCs	   also	   had	   a	   higher	   number	   of	   cFos	   positive	   PCs,	  reflecting	  an	  increased	  number	  of	  active	  PCs	  following	  rotarod	  activation.	  When	  we	  looked	  at	   individual	  Lurcher	  chimeras	  we	  observed	  a	  wide	  range	  of	  differences	   in	  neural	  activity	   in	  PC	  outputs	  as	   shown	  by	  quantification	  of	   cFos	  expression	   in	   the	  Purkinje	   cell	   layer	   (PCL).	   Those	   chimeras	  with	  wildtype-­‐like	   cerebellums	   and	   the	  lowest	   measures	   of	   cerebellar	   pathology	   (determined	   by	   PC	   counts	   and	   GCL	  morphology)	  had	  the	  highest	  levels	  of	  inhibitory	  PC	  activity	  in	  the	  PCL,	  as	  reflected	  by	   higher	   total	   numbers	   of	   cFos	   positive	   PCs.	   Lurcher	   chimeras	   with	   more	   cFos	  positive	   PCs	   also	   had	   sparser	   and	   less	   dense	   cFos	   staining	   in	   the	  GCL,	   suggesting	  	   98	  that	  the	  synchronous	  activity	  of	  a	  group	  of	  pre-­‐synaptic	  GCs	  leads	  to	  patterned	  post-­‐synaptic	  PC	  inhibitory	  outflow	  [50].	  	   In	   electrophysiological	   studies	   of	   PC	   activity	   it	   was	   discovered	   that	   under	  basal	  conditions	  PCs	  are	  spontaneously	  active	  and	  can	  fire	  at	  a	  rate	  of	  50	  Hz	  leading	  to	  significant	  inhibition	  of	  CN	  neurons	  at	  rest	  [62].	  In	  wildtype	  mice	  PCs	  outnumber	  post-­‐synaptic	  CN	  neurons	  by	  a	  factor	  of	  11:1	  and	  during	  a	  behavioural	  task	  leading	  to	  cerebellar	  activation	  synchronized	  patterns	  of	  action	  potential	  (AP)	  spikes	  in	  PCs	  exert	   powerful	   inhibitory	   control	   over	   the	   activity	   of	   CN	   neurons	   [63,64].	  Experimental	   studies	   of	   PC	   activity	   have	   also	   shown	   that	   the	   coordinated	   neural	  activity	   of	   a	   population	  of	   PCs	  determines	   the	   amount	   of	   inhibition	   and	   the	   firing	  rate	   of	   CN	   neurons	   during	   a	   cerebellar	   task	   [65].	   This	   idea	   is	   consistent	  with	   the	  finding	   of	   increased	   numbers	   of	   cFos	   positive	   PCs	   in	   Lurcher	   chimeras	   with	   a	  wildtype-­‐like	   cerebellum	   because	   after	   90	   seconds	   of	   rotarod	   activation	   we	   see	  increased	   expression	   of	   cFos	   (as	   an	   indirect	  marker	   of	   increased	   neural	   activity)	  within	  a	  large	  population	  of	  inhibitory	  PCs.	  Finally,	  we	  have	  also	  seen	  that	  Lurcher	  chimeras	   with	   the	   most	   cFos	   positive	   PCs	   (confirmed	   by	   double	  immunofluorescence	  labeling	  of	  PCs	  with	  Calbindin-­‐D28K	  and	  cFos)	  have	  the	  fewest	  cFos	   positive	   CN	   neurons	   (CN	   neuron	   identity	   confirmed	   by	   Tbr1	   staining)	  supporting	  the	   idea	  that	  neural	  activity	   in	  CN	  neurons	   is	   inversely	  correlated	  with	  the	  amount	  of	  activity	  in	  inhibitory	  PCs	  during	  cerebellar	  activation	  [66].	  	  	   99	  	   In	   functional	   studies	   looking	   at	   the	   synchrony	   of	   PC	   burst	   firing	   it	   was	  determined	   that	   PCs	   exhibit	   a	   higher	   degree	   of	   synchronous	   AP	   firing	   during	   a	  cerebellar	   task	   [67].	   In	   an	   experiment	   looking	   at	   synchronous	   firing	   of	   complex	  spikes	  in	  a	  population	  of	  rat	  PCs,	  it	  was	  discovered	  that	  there	  is	  prolonged	  inhibition	  of	  CN	  neurons	   in	  response	   to	  coordinated	  complex	  spike	  activity	   in	   inhibitory	  PCs	  [68].	  These	   functional	   studies	  highlight	   the	  complexity	  of	   synaptic	   transmission	   in	  cerebellar	  PCs	  and	  their	  control	  of	  neural	  activity	  in	  post-­‐synaptic	  CN	  neurons,	  but	  they	   also	   demonstrate	   that	   with	   the	   loss	   of	   cerebellar	   cortical	   inhibition	   and	  increasing	  cerebellar	  pathology	  there	  is	  a	  shift	  in	  the	  structure	  and	  function	  of	  post-­‐synaptic	   neurons.	   It	  must	   be	   noted	   that	  while	   cFos	   expression	   cannot	   be	   used	   to	  determine	  the	  timing	  or	  kinetics	  of	  neural	  activity	  within	  neurons,	  it	  does	  allow	  one	  to	  look	  at	  changes	  in	  cerebellar	  function	  and	  pathology	  simultaneously	  in	  the	  same	  brain	  tissue	  specimens.	  	   Our	   cFos	   and	   Calbindin-­‐D28K	   co-­‐localization	   experiments	   allowed	   us	   to	  visualize	  an	  increase	  in	  cFos	  expression	  in	  Calbindin-­‐D28K	  positive	  PCs	  following	  90	  seconds	   of	   rotarod	   activation	   of	   the	   cerebellum.	   This	   was	   important	   because	   it	  allowed	  us	  to	  confirm	  the	  specific	  identity	  of	  cerebellar	  neurons	  expressing	  cFos	  and	  to	  standardize	  our	  cFos	   labeling	  and	   imaging	  protocol	   for	  cerebellar	  PCs.	  Previous	  studies	  have	  also	  used	  the	  immediate	  early	  gene	  Fos	  as	  a	  marker	  of	  neural	  activation	  in	  combination	  with	  other	  structural	  cell	  markers	  such	  as	  Parvalbumin	  and	  various	  isoforms	   of	   Calbindin	   to	   look	   at	   correlated	   changes	   in	   cell	   activation	   in	   different	  populations	   of	   cerebellar	   neurons	   [69].	   Utilizing	   immunofluorescence	   techniques	  	   100	  researchers	  were	  able	  look	  at	  changes	  in	  cFos	  expression	  in	  different	  populations	  of	  wildtype	  rat	  cerebellar	  neurons	  using	  antibodies	  against	  various	  structural	  markers	  (such	  as	  Calbindin	  to	   identify	  cerebellar	  PCs)	  [70].	  Following	  electrical	  stimulation	  of	  the	  inferior	  cerebellar	  peduncle	  to	  activate	  both	  mossy	  fibres	  and	  climbing	  fibre	  afferents,	  researchers	  saw	  an	  increase	  in	  cFos	  expression	  in	  both	  excitatory	  granule	  cells	  and	  their	  post-­‐synaptic	  PC	  targets	  [70].	  	  Next,	   immunofluorescence	   co-­‐localization	   experiments	   of	   cFos	   and	  Calbindin-­‐D28K	   allowed	   us	   to	   visualize	   the	   subcellular	   localization	   of	   the	   cFos	  protein	   within	   PCs.	   We	   found	   cFos	   expression	   to	   be	   highest	   in	   the	   cytoplasm,	  expressed	   at	   lower	   levels	   in	   the	   PC	   dendritic	   tree	   and	   very	   low	   to	   no	   expression	  within	  the	  nucleus	  of	  PCs	  (which	  appears	  dark	  against	  the	  surrounding	  cytoplasm	  in	  our	  staining	  protocols).	  This	  finding	  is	  significant	  because	  it	  tells	  us	  that	  our	  staining	  for	  cFos	  is	  not	  only	  specific	  to	  populations	  of	  cerebellar	  neurons,	  but	  also	  specific	  for	  a	  particular	  subcellular	   location	  within	  these	  neurons.	  Past	  studies	  using	  cFos	  as	  a	  marker	   of	   neural	   activation	   have	   found	   that	   many	   different	   physiological	   stimuli	  such	   as	   neuropharmacological	   agonists,	   behavioural	   testing	   paradigms	   and	   direct	  electrical	   stimulation	   of	   a	   brain	   region	   can	   reliably	   induce	   cFos	   expression	   in	  discrete	   populations	   of	   neurons,	   which	   is	   particularly	   useful	   for	   mapping	  connectivity	   between	   different	   brain	   regions	   [71,72].	   Others	   have	   found	   that	  electrical	  stimulation	  of	   the	   inferior	  cerebellar	  peduncle	   leads	  to	  specific	   increases	  in	   cFos	   expression	  within	   the	   cytoplasm	   and	   proximal	   dendrites	   of	   PCs,	   which	   is	  similar	   to	   the	   distribution	   of	   cFos	   seen	  within	   PCs	   of	   Lurcher	   chimeras	   following	  	   101	  rotarod	   testing	   [70].	   These	   co-­‐localization	   experiments	   allowed	  us	   to	   validate	   our	  cFos	  staining	  protocol	  as	  a	  viable	  method	  for	  looking	  at	  an	  indirect	  marker	  of	  neural	  activity	  in	  the	  cerebellum	  and	  connected	  brain	  regions.	  	  After	  quantifying	  cFos	  expression	  in	  both	  cerebellar	  GCs	  and	  PCs	  in	  Lurcher	  chimeras,	  we	  were	  also	  interested	  in	  examining	  coincident	  expression	  of	  cFos	  in	  CN	  neurons	   using	   immunohistochemical	   techniques.	   Studying	   cFos	   expression	   in	   CN	  neurons,	  PCs	  and	  GCs	  allowed	  us	  to	  evaluate	  structural	  and	  functional	  changes	  in	  a	  cerebellar	  neural	  circuit	  as	  they	  relate	  to	  variable	  neuropathological	  changes	  in	  the	  cerebellum	   of	   Lurcher	   chimeric	   mice.	   Since	   cFos	   is	   expressed	   in	   many	   types	   of	  neurons	  during	  the	  development	  of	  the	  nervous	  system,	  but	  only	  at	  low	  levels	  under	  basal	   conditions	   in	   adult	   neurons,	   cerebellar	   activation	   induces	   rapid,	   detectable	  changes	   in	   gene	   expression	   of	  Fos	  within	   subsets	   of	   cerebellar	   neurons	   [73].	   This	  allowed	   us	   to	   quantify	   changes	   in	   cFos	   protein	   expression	   in	   neurons	   reflecting	  changes	   in	   excitatory	   and	   inhibitory	   neurotransmission	   within	   the	   cerebellum	   of	  individual	  Lurcher	  chimeras	  [73].	  	  4.4-­‐Cerebellar	   nuclei	   neuronal	   activity	   is	   inversely	   correlated	   with	   the	  amount	   of	   inhibition	   received	   from	   cerebellar	   PCs	   indicating	   that	   a	   loss	   of	  inhibitory	  PCs	  leads	  to	  a	  shift	  in	  the	  efferent	  output	  of	  the	  cerebellum	  	  	   The	  CN	  are	  found	  deep	  within	  the	  cerebellum	  embedded	  within	  white	  matter	  tracts	   and	   are	   composed	   of	   a	   heterogeneous	   population	   of	   neurons	  with	   variable	  morphologies	   [50].	   There	   are	   two	  major	  populations	   of	   neurons	   found	  within	   the	  CN;	   larger	   diameter	   glutamatergic	   neurons	   that	   project	   to	   cortical	   regions	   and	  	   102	  smaller	   inhibitory	  (glycinergic	  and	  GABAergic)	  neurons	  that	  project	  to	  the	  inferior	  olive	  in	  the	  medulla	  to	  provide	  negative	  feedback	  to	  IONs	  that	  in	  turn	  project	  to	  the	  cerebellar	   cortex	  as	   climbing	   fibre	  afferents	   [74].	  All	   cerebellar	  outflow	  except	   for	  vestibular	  information	  passes	  through	  the	  CN	  and	  with	  the	  early	  loss	  of	  PCs	  (which	  represents	  the	  start	  of	  all	  cerebellar	  outflow)	  CN	  neurons	  lose	  significant	  inhibitory	  inputs	  that	  has	  been	  shown	  to	  lead	  to	  both	  pathological	  and	  compensatory	  changes	  in	  remaining	  CN	  synapses	  [50].	  	  	   Previous	   studies	   conducted	   in	   Lc/+	   mutant	   mice	   have	   confirmed	   the	  importance	   of	   CN	   neurons	   in	   regulating	   the	   balance	   of	   inhibitory	   and	   excitatory	  synaptic	   transmission	   in	   cerebellar	   outflow	   using	   both	   histological	   and	  electrophysiological	   approaches	   [75,76,77].	   In	   the	   absence	   of	   PCs	   there	   are	  significant	   compensatory	   changes	   in	   the	   CN	   neurons	   to	   maintain	   a	   threshold	   of	  cerebellar	  outflow	  to	   the	  rest	  of	   the	  CNS	  [75].	  Using	  histological	   techniques	   it	  was	  discovered	   that	   the	   number	   of	   GABAergic	   and	   glycinergic	   neurons	  within	   the	   CN	  increases	   and	   that	   inhibitory	   synaptic	   nerve	   terminals	   increase	   in	   size	   to	  compensate	  for	  the	  loss	  of	  PC	  inhibitory	  inputs	  to	  CN	  neurons	  [75].	  From	  postnatal	  day	  8	   (P8)	   to	  postnatal	  day	  22	   (P22)	   (during	   the	  period	  of	  PC	  death)	   researchers	  found	  that	  there	  are	  also	  increases	  in	  the	  size	  of	  both	  miniature	  IPSCs	  (mIPSCs)	  and	  spontaneous	   IPSCs	   (sIPSCs),	   which	   reflect	   compensatory	   increases	   in	   inhibitory	  synaptic	  transmission	  to	  make	  up	  for	  the	  loss	  of	  GABAergic	  PCs	  [76].	  Finally,	  at	  the	  molecular	   level	   it	   was	   determined	   that	   there	   is	   increased	   aggregation	   of	   GABAA	  receptors	  (GABAARs)	  and	  gephyrin	  (a	  protein	  that	  frequently	  clusters	  with	  GABAARs	  	   103	  at	   inhibitory	   synapses)	   in	  CN	  neurons	  of	  Lc/+	  mutants,	  which	   again	   suggests	   that	  there	   are	   numerous	   compensatory	   mechanisms	   in	   post-­‐synaptic	   CN	   neurons	   to	  maintain	  a	  minimal	  level	  of	  cerebellar	  function	  [77].	  	  The	   same	   pathological	   and	   compensatory	   changes	   in	   cerebellar	   cortex	  structure	  and	  function	  can	  be	  observed	  in	  Lurcher	  chimeras,	  but	  to	  varying	  degrees	  in	  individual	  chimeras	  depending	  on	  the	  severity	  of	  cerebellar	  pathology.	  Using	  PC	  counts	   as	   a	   quantitative	   measure	   of	   cerebellar	   pathology	   in	   individual	   Lurcher	  chimeras,	   we	   found	   that	   the	   density	   of	   cFos	   positive	   CN	   neurons	   is	   inversely	  correlated	  with	  not	   only	   the	   total	   number	  of	   surviving	  PCs	   in	   the	   cerebellum,	  but	  also	   inversely	   correlated	  with	   the	   number	   of	   cFos	   positive	   cells	   in	   the	   PCL.	  Using	  cFos	  staining	   in	   the	  PCL	  as	  a	  measure	  of	   inhibitory	  PC	  outflow	  to	  CN	  neurons	  our	  findings	  support	  previous	  anatomical	  and	  neurophysiological	  studies	  that	  find	  there	  is	   a	   compensatory	   increase	   in	   neural	   activity	   within	   CN	   neurons	   following	  pathological	  events	  in	  the	  cerebellar	  cortex	  [75,76,77].	  	  4.5-­‐Cerebellar	  glial	  cells	  undergo	  changes	  in	  structure	  and	  function	  following	  cell	  death	  and	  neurodegeneration	  in	  mouse	  models	  of	  ASD-­‐like	  phenotypes	  	   We	  have	  seen	  that	  there	  are	  changes	  in	  cFos	  expression	  in	  the	  GCL,	  PCL	  and	  CN	   neurons	   of	   Lurcher	   chimeras	   in	   response	   to	   cerebellar	   neuron	   death	   and	  neurodegeneration	  within	  the	  cerebellum.	  These	  structural	  and	  functional	  changes	  within	   cerebellar	   neural	   circuits	   also	   lead	   to	   drastic	   changes	   in	   the	   number,	  distribution	   and	   morphology	   of	   cerebellar	   glial	   cells.	   In	   Lurcher	   mutants	   and	  chimeras	   there	   are	   noticeable	   differences	   in	   the	   morphology	   and	   distribution	   of	  	   104	  Iba1	  positive	  microglia	   and	   the	  expression	  of	  GFAP	  within	  Bergmann	  glial	   cells	   in	  the	  molecular	  layer	  (ML)	  is	  drastically	  altered	  in	  Lc/+	  mutant-­‐like	  chimeras	  versus	  wildtype-­‐like	  chimeras.	  In	  both	  human	  cases	  of	  ASD	  and	  in	  animal	  models	  of	  autism-­‐like	   phenotypes	   it	   is	   becoming	   increasingly	   clear	   that	   glial	   cells	  may	   play	   a	  much	  bigger	   role	   in	   the	   neuropathological	  mechanisms	   underlying	   neurodevelopmental	  disorders	  than	  previously	  thought.	  	  In	  addition	  to	  human	  studies	  looking	  at	  microglial	  activation	  in	  the	  brains	  of	  patients	  diagnosed	  with	  ASD,	   several	   studies	  have	  also	   looked	  at	   the	   activation	  of	  neuroglial	   cells	   in	   the	   cerebellum	   of	   ASD-­‐like	   mouse	   models.	   The	   Purkinje	   Cell	  Degeneration	  (PCD)	  mouse	  loses	  PCs	  starting	  in	  the	  third	  to	  fourth	  week	  of	  life	  and	  PCD	  mice	  exhibit	  increased	  microglia	  proliferation	  and	  activation	  corresponding	  to	  the	  onset	  of	  cerebellar	  neuron	  death	  [93].	  The	  PCD	  mouse	  model	  is	  characterized	  by	  an	   autosomal	   recessive	   mutation	   in	   the	   nna1	   gene,	   which	   leads	   to	   the	   death	   of	  various	  neuronal	  populations	  in	  the	  CNS	  at	  different	  time	  points	  [94].	  Cerebellar	  PCs	  are	  the	  first	  discrete	  neuronal	  population	  to	  undergo	  cell	  death	  between	  three	  and	  four	   weeks	   after	   birth	   and	   like	   Lc/+	  mutant	   mice,	   PCD	   mice	   develop	   cerebellar	  ataxia	  due	  to	  the	  selective	  loss	  of	  cerebellar	  PCs	  [95].	  PCD	  and	  Lurcher	  mutant	  mice	  both	   exhibit	   developmental	   PC	   death	  with	   pathology	   in	   the	   cerebellar	   cortex	   and	  provide	   valuable	   insights	   into	   the	   features	   of	   neuroglial	   activation	   in	   the	   CNS	  following	  the	  loss	  of	  specific	  populations	  of	  cerebellar	  neurons.	  	  	   105	  In	  one	  study	  scientists	  followed	  the	  morphology,	  distribution	  and	  activation	  state	  of	  microglia	  over	  various	  postnatal	  time	  points	  that	  corresponded	  to	  the	  onset	  and	   course	   of	   PC	   death	   in	   PCD	  mice	   [93].	   At	   P15	  microglia	   observed	   by	   confocal	  microscopy	   in	  the	  cerebellum	  of	  a	  PCD	  mouse	  had	  thin,	  spindly	  cell	  processes	  and	  smaller	  cell	  bodies	  with	  a	  similar	  distribution	  within	  the	  cerebellum	  as	  seen	  in	  age	  matched	  wildtype	  control	  mice	  [93].	  However,	  following	  the	  onset	  of	  PC	  death	  in	  the	  cerebellum	   of	   PCD	   mice	   there	   was	   a	   rapid	   change	   in	   the	   distribution	   and	  morphology	   of	   Iba1	   positive	   cells,	   with	   microglia	   possessing	   thicker	   and	   more	  prominent	  cell	  bodies	  and	  cell	  processes	  and	  more	  microglia	  migrating	   to	   the	  PCL	  and	   molecular	   layer	   (ML)	   as	   compared	   to	   control	   animals	   [93].	   Finally,	   the	  researchers	   observed	   that	   by	   P25	   activated	   microglia	   were	   almost	   exclusively	  localized	  to	  the	  PCL	  and	  using	  electron	  microscopy	  (EM)	  determined	  that	  activated	  microglia	  were	  engulfing	   the	  degenerating	  PCs	   [93].	   In	  PCD	  mice	   it	  was	  clear	   that	  reactive	  gliosis	  occurred	  following	  the	  initial	  death	  of	  PCs	  in	  the	  cerebellum,	  but	   it	  remains	  to	  be	  determined	  whether	  further	  neurodegeneration	  and	  cell	  death	  occurs	  following	  microglial	  activation	  into	  adulthood	  in	  these	  mice.	  	  In	   Lurcher	   mutant	   and	   chimeric	   mice	   we	   also	   observed	   differences	   in	   the	  morphology,	  distribution	  and	  numbers	  of	  Iba1	  positive	  microglia	  in	  the	  cerebellum.	  In	   adult	   Lc/+	  mutants	   there	   were	   increased	   numbers	   of	   microglia	   located	   in	   the	  cerebellar	   cortical	   grey	   matter	   and	   those	   microglia	   in	   the	   cerebellar	   cortex	   had	  enlarged	  cell	  bodies	  with	  shorter	  and	  thicker	  cell	  processes	   indicative	  of	  activated	  microglia	  when	  compared	   to	  microglia	  observed	   in	  wildtype	   littermates.	  Wildtype	  	   106	  animals	  had	  fewer	  microglia	   localized	  to	  the	  cerebellar	  cortex	  and	  the	  microglia	  in	  wildtype	   animals	   had	   longer,	   thinner	   cell	   processes	  with	   a	   non-­‐descript	   cell	   body	  indicative	   of	   resting	   microglia.	   These	   morphological	   and	   anatomical	   changes	   of	  microglia	   in	  Lurcher	  mutants	  are	  consistent	  with	  previous	  descriptions	  of	  changes	  in	   microglial	   structure	   and	   function	   following	   CNS	   injury	   and	   neurodegeneration	  [81].	  	   In	  Lurcher	   chimeras	   that	  have	  more	  variability	   in	   cerebellar	  pathology	  and	  loss	   of	   cerebellar	  neurons	  we	  observed	   a	   spectrum	  of	   changes	   in	   the	  morphology	  and	  distribution	  of	   Iba1	  positive	  microglia.	  For	  example,	   in	  Lurcher	  chimeras	  with	  an	  intermediate	  number	  of	  surviving	  PCs	  we	  found	  microglia	  that	  had	  enlarged	  cell	  bodies	   and	   processes,	   but	   not	   to	   the	   same	   extent	   as	   a	   fully	   activated	   phagocytic	  microglia	  seen	  in	  Lc/+	  mutants	  or	  PCD	  mice	  [93].	  Lurcher	  chimeras	  also	  have	  more	  subtle	   differences	   in	   the	   distribution	   and	   number	   of	   microglia	   in	   the	   cerebellar	  cortex	   than	   Lurcher	   mutant	   or	   PCD	   mice	   [93].	   Indeed,	   we	   saw	   an	   intermediate	  number	  of	  microglia	  localized	  to	  the	  cerebellar	  cortex	  in	  Lurcher	  chimeras	  with	  an	  intermediate-­‐sized	   cerebellum	   and	   an	   intermediate	   number	   of	   surviving	   PCs	   as	  compared	  to	  wildtype-­‐like	  chimeras	  and	  ataxic	  chimeras.	  	   By	   studying	   differences	   in	  microglial	   activation	   and	  microglial	   distribution	  within	   the	   cerebellum	   of	   individual	   chimeras	   we	   were	   able	   to	   develop	   a	   more	  complete	  understanding	  of	  structural	  and	  morphological	  changes	   in	  neuroglia	  that	  appear	   to	   be	   along	   a	   continuum	   in	   the	   Lurcher	   chimera	   mouse	   model.	   Having	  	   107	  variability	  in	  the	  severity	  of	  reactive	  gliosis	  in	  the	  cerebellum	  of	  individual	  Lurcher	  chimeras	  also	  supports	   the	  notion	   that	   there	  could	  be	  many	  subtypes	  of	  microglia	  with	   different	   functions	   in	   the	   CNS	   [84].	   Finally,	   it	   is	   interesting	   to	   note	   that	   the	  observed	  morphological	  and	  anatomical	  changes	  in	  microglia	  persisted	  for	  months	  after	  the	  initial	  degeneration	  of	  PCs	  and	  the	  secondary	  loss	  of	  GCs	  and	  IONs	  in	  Lc/+	  mutants	  and	  chimeras	   [17].	  Histological	  evidence	  suggests	   that	  microglia	   localized	  to	   the	   cerebellar	   cortex	   in	   Lurcher	  mutants	   and	   chimeras	  with	   the	  most	   extreme	  pathology	   remain	   in	   an	   activated	   state	   and	   don’t	   return	   to	   a	   resting	   or	   quiescent	  state	  after	  the	  death	  of	  PCs	  and	  GCs	  [81,82].	  Therefore,	  future	  experiments	  must	  use	  appropriate	   markers	   to	   determine	   the	   activation	   or	   functional	   state	   of	   these	  cerebellar	   microglia	   because	   prolonged	   microglial	   activation	   can	   lead	   to	   further	  neurobiological	   deficits	   in	   the	  CNS	   as	   reflected	  by	   changes	   in	  behaviour	   in	  mouse	  models	  and	  human	  cases	  of	  neurodevelopmental	  disorders.	  	  	   In	   previous	   studies	   researchers	   documented	   increased	   expression	   of	   pro-­‐inflammatory	   markers	   such	   as	   IL-­‐6	   in	   the	   cerebellum	   of	   PCD	   mice,	   which	   is	  indicative	  of	  a	  pro-­‐inflammatory	  microglial	  response	  that	  is	  associated	  with	  a	  more	  severe	   form	  of	   reactive	  gliosis	   [93,96].	   In	  addition	   to	   increased	  expression	  of	  pro-­‐inflammatory	   cytokines	   and	   chemokines	  microglia	   also	   express	   elevated	   levels	   of	  the	   enzyme	   inducible	   Nitric	   Oxide	   Synthase	   (iNOS)	   under	   pro-­‐inflammatory	  conditions	  in	  the	  CNS	  [97].	  iNOS	  is	  the	  enzyme	  responsible	  for	  creating	  nitric	  oxide	  (NO)	  in	  macrophages	  and	  microglia	  in	  order	  to	  degrade	  phagocytosed	  pathogens	  or	  cellular	  debris,	  but	  in	  conditions	  of	  prolonged	  and	  chronic	  inflammation	  can	  lead	  to	  	   108	  further	  tissue	  damage	  and	  cell	  death	  [98].	  Therefore	  in	  future	  experiments	  looking	  at	  the	  activation	  state	  of	  microglia	  in	  the	  cerebellum	  of	  Lurcher	  chimeras	  we	  could	  evaluate	   the	  degree	  of	   co-­‐localization	  of	   Iba1	   and	   iNOS	   in	  microglia,	  which	  would	  indicate	  whether	  microglia	   located	   in	   the	   cerebellar	   cortex	   are	   involved	   in	   a	   pro-­‐inflammatory	  or	  anti-­‐inflammatory	  response.	  	  	   In	   addition	   to	   quantifying	   microglia	   and	   studying	   their	   morphology	   and	  distribution	   within	   the	   cerebellar	   cortex	   we	   also	   analyzed	   the	   qualitative	  appearance	   and	   distribution	   of	   Bergmann	   glia	   within	   the	   ML	   of	   the	   cerebellum.	  Bergmann	   glial	   cells	   are	   a	   type	   of	   protoplasmic	   unipolar	   astrocyte	  with	   their	   cell	  bodies	  located	  close	  to	  PCs	  and	  their	  radial	  processes	  extending	  through	  the	  ML	  to	  wrap	  around	  numerous	  synapses	  located	  on	  the	  PC	  dendritic	  tree	  [99,100].	  During	  neural	  development	  Bergmann	  glial	  cells	  are	  tightly	  associated	  with	  cerebellar	  GCs	  and	  play	  an	  important	  role	  in	  the	  glial	  cell-­‐mediated	  migration	  of	  GCs	  [101].	  In	  the	  adult	   brain	   Bergmann	   glia	   are	   intertwined	   with	   the	   cell	   bodies	   and	   dendrites	   of	  cerebellar	   PCs	   and	   appear	   to	   participate	   in	   important	   protective	   and	   supportive	  functions	  at	  PC	  synapses	  [102].	  	  Since	  Bergmann	  glia	  are	  closely	  associated	  with	  PCs	  in	  the	  adult	  cerebellum	  we	  hypothesized	  that	  there	  are	  changes	  in	  the	  morphology	  and	  expression	  of	  glial	  cell	  markers	  like	  GFAP	  following	  the	  degeneration	  of	  PCs	  in	  Lurcher	  mutants	  and	  chimeras.	  	   In	   a	   recent	   study	   looking	   at	   changes	   in	   glia	   structure	   and	   function	   in	  Lc/+	  mutant	   mice	   it	   was	   reported	   that	   gliosis	   in	   the	   cerebellum	   was	   abnormally	  	   109	  prolonged	  and	  that	  structural	  and	   functional	  changes	   in	  cerebellar	  astroglia	  began	  following	   degeneration	   of	   cerebellar	   neurons	   and	   persisted	   for	   days	   to	   months	  [106].	  The	  authors	  also	  found	  significantly	  increased	  numbers	  of	  activated	  microglia	  in	   the	   vicinity	   of	   the	   PCL	   at	   P13	   following	   the	   onset	   of	   PC	   death	   and	   cerebellar	  microgliosis	   lasted	   for	  months	   after	   the	   initial	   degeneration	  of	   cerebellar	  PCs	   and	  GCs,	   suggesting	   that	   chronic	   activation	   of	   cerebellar	   glial	   cells	   may	   play	   an	  important	   role	   in	  promoting	   further	   cerebellar	   damage	   [106].	   Finally,	   the	   authors	  discovered	  significantly	  elevated	  levels	  of	  GFAP	  mRNA	  in	  cerebellar	  astroglia	  of	  Lc/+	  mutants	  between	  P22	  and	  P25	  (following	  the	  majority	  of	  PC	  degeneration)	  and	  also	  between	  P36	  and	  P110	  (following	  further	  PC	  death	  and	  secondary	  loss	  of	  cerebellar	  GCs)	   as	   compared	   to	   low	   GFAP	   mRNA	   expression	   at	   the	   same	   time	   points	   in	  wildtype	  control	  mice	  [106,107].	  	  	   These	   results	   are	   consistent	  with	   our	   observed	   changes	   in	   the	   distribution	  and	  morphology	  of	  microglia	  and	  Bergmann	  glia	  in	  the	  cerebellar	  cortex	  of	  the	  Lc/+	  mutant	   and	   chimeric	   mice.	   However,	   in	   individual	   Lurcher	   chimeras	   we	   see	   a	  broader	  distribution	  of	   changes	   in	   the	   structure	   and	  distribution	  of	  microglia	   and	  astroglia.	   For	   example,	   in	   Lurcher	   chimeras	   with	   an	   intermediate	   degree	   of	  cerebellar	   pathology	   we	   see	   more	   diffuse	   microglial	   activation	   throughout	   the	  cerebellum	  and	  we	   see	  moderate,	   patchy	   increases	   in	  GFAP	   staining	  of	  Bergmann	  glia	  in	  the	  ML	  of	  the	  cerebellar	  cortex.	  These	  observations	  suggest	  that	  activation	  of	  glial	  cells	  within	  the	  cerebellum	  of	  Lurcher	  chimeras	  is	  variable	  and	  that	  the	  severity	  of	  cerebellar	  gliosis	  is	  dependent	  on	  the	  extent	  of	  initial	  PC	  and	  GC	  degeneration	  in	  	   110	  the	  first	  few	  weeks	  of	  life.	  In	  addition,	  histopathological	  findings	  in	  one	  study	  of	  post	  mortem	  human	  brain	  tissue	   from	  5	  out	  of	  6	  subjects	  came	  from	  adult	  males	  (over	  the	   age	   of	   20)	   diagnosed	   with	   ASD	   [104].	   Similar	   to	   human	   studies	   evaluating	  pathological	  changes	  in	  the	  cerebella	  of	  autistic	  individuals	  our	  Lurcher	  mutant	  and	  chimeric	   mice	   were	   all	   adults	   (P60	   or	   older)	   and	   exhibited	   long	   lasting	   and	  prolonged	   changes	   in	   the	  morphology	   and	   distribution	   of	   cerebellar	   glial	   cells.	   In	  conjunction	   with	   data	   from	   previous	   studies	   our	   results	   support	   the	   idea	   that	  cerebellar	   gliosis	   occurs	   as	   a	   result	   of	   the	  degeneration	  of	   discrete	  populations	  of	  cerebellar	  neurons	  and	   that	  astrogliosis	  and	  microgliosis	   responses	  are	  prolonged	  in	  Lurcher	  mutant	  and	  chimeric	  mice	  [99,104,106].	  	  4.6-­‐Fmr1	   KO	   mice	   exhibit	   altered	   resting	   state	   cFos	   expression	   in	   the	  cerebellum	   and	   cortex	   suggesting	   that	   changes	   in	   cerebellar	   and	   cortical	  activity	  are	  common	  features	  associated	  with	  FXS	  and	  other	  forms	  of	  ASD	  	  	   Fragile	   X	   Syndrome	   (FXS)	   is	   a	   monogenic	   neurodevelopmental	   disorder	  characterized	   by	   various	   mutations	   in	   the	   FMR1	   gene,	   which	   is	   located	   on	   the	   X	  chromosome	   [131].	   The	   gene	   product	   FMRP	   is	   involved	   in	   the	   translation	   of	  multiple	  proteins	  found	  in	  CNS	  neurons	  and	  with	  loss	  of	  function	  mutations	  can	  lead	  to	   the	   altered	   expression	   of	   multiple	   proteins	   involved	   in	   synaptic	   transmission,	  synaptic	  adhesion	  and	  cell	  signaling	  cascades	  [125].	   In	  Fmr1	  KO	  mice	  it	  was	  found	  that	   complete	   knockout	   of	   the	   FMRP	   protein	   leads	   to	   altered	   inhibitory	   and	  excitatory	   synaptic	   transmission	   in	   multiple	   cortical	   brain	   regions	   [132,133].	  Evidence	  to	  support	  this	  finding	  has	  found	  both	  increased	  and	  decreased	  neuronal	  excitability	   in	   different	   brain	   regions.	   Some	   studies	   have	   found	   decreased	  	   111	  GABAergic	   neurotransmission	   in	   cortical	   brain	   regions	   resulting	   in	   increased	  excitatory	   synaptic	   transmission	   in	   cortical	   neural	   networks	   [134].	   Researchers	  have	   found	   decreased	   levels	   of	   cortical	   GABAA	  subunits,	   decreased	   expression	   of	  GAD	   mRNA	   in	   both	   cerebellar	   and	   cortical	   neurons	   and	   decreased	   levels	   of	   the	  scaffolding	  protein	  gephyrin	   (which	   is	   responsible	   for	   the	  clustering	  of	  GABAA	  and	  glycinergic	  receptors	  at	  the	  post-­‐synaptic	  membrane)	  in	  the	  CNS	  of	  Fmr1	  KO	  mice,	  which	  suggests	  that	  these	  mice	  have	  decreased	  inhibition	  from	  GABAergic	  neurons	  leading	  to	   increased	  neural	  network	  excitability	  [135,136,137].	  On	  the	  other	  hand,	  electrophysiology	   experiments	   have	   documented	   increased	   basal	   inhibitory	  synaptic	  currents	  in	  brain	  regions	  like	  the	  striatum	  suggesting	  that	  under	  different	  conditions	  and	  in	  various	  brain	  regions	  Fmr1	  KO	  mice	  have	  both	  altered	  excitatory	  and	  inhibitory	  synaptic	  transmission	  as	  compared	  to	  wildtype	  controls	  [138].	  	   We	   found	   interesting	   results	  when	  we	  examined	  a	   cohort	  of	  Fmr1	  KO	  mice	  versus	  Fmr1	  wildtype	  littermates.	  Under	  resting	  conditions	  we	  found	  that	  Fmr1	  KO	  mice	   had	   significantly	   decreased	   cFos	   expression	   (used	   as	   a	   reporter	   of	   neural	  activity)	   in	   the	   cerebellar	   GCL,	   posterior	   cortex	   and	   somatomotor	   cortex	   as	  compared	  to	  Fmr1	  wildtypes.	  However,	  we	  documented	  increased	  cFos	  expression	  in	   the	   orbitofrontal	   cortex	   of	   Fmr1	   KO	   mice	   versus	   Fmr1	   wildtype	   mice,	   which	  suggests	   that	   even	  under	   basal	   conditions	   both	   excitatory	   and	   inhibitory	   synaptic	  transmission	  can	  be	  altered	   in	   the	  CNS	  of	  Fmr1	  KO	  mice.	  Finally,	   it	  must	  be	  noted	  that	   cFos	   expression	   in	   cortical	   neurons	   at	   rest	   could	   represent	   increased	   neural	  activity	  in	  populations	  of	  excitatory	  pyramidal	  neurons	  and/or	  increased	  activity	  in	  	   112	  GABAergic	   cortical	   interneurons.	   This	   is	   because	   cFos	   labels	   the	   cell	   bodies	   of	   all	  subtypes	   of	   recently	   active	   neurons	   and	   therefore	   it	   is	   difficult	   to	   discern	   the	  identity	  of	  cFos	  labeled	  cortical	  neurons	  without	  further	  morphological,	  histological	  or	  functional	  analysis	  [34].	  	  Anatomical	   evidence	   for	  altered	   synaptic	   transmission	   in	   the	   cerebellum	  of	  Fmr1	   KO	   mice	   has	   come	   from	   morphological	   analyses	   of	   PCs.	   We	   observed	   a	  relatively	   small	   decrease	   in	   the	   numbers	   of	   PCs	   and	   decreased	   complexity	   of	   PC	  dendritic	  branches	  in	  Fmr1	  KO	  mice	  versus	  wildtype	  littermates.	  We	  also	  observed	  variable	  and	  heterogeneous	  losses	  of	  PCs	  in	  the	  cerebella	  of	  some	  knockouts	  versus	  controls,	   but	   there	   was	   no	   obvious	   or	   consistent	   pattern	   of	   PC	   loss	   in	   specific	  cerebellar	   lobules	   or	   regions	   of	   the	   cerebellar	   cortex	   between	   individual	   mice.	  Though	   the	  difference	   in	  PC	  number	  was	  not	   significantly	  different	  between	  Fmr1	  KO	  mice	  and	  wildtype	  littermates	  as	  seen	  in	  Lc/+	  mutants	  and	  chimeric	  mice,	  there	  was	   a	   trend	   of	   decreased	   PC	   numbers	   and	   cerebellar	   abnormalities	   in	   knockout	  mice	   as	   compared	   to	   wildtype	   littermates.	   These	   results	   suggest	   that	   cerebellar	  pathology	   in	   the	   form	   of	   variable	   PC	   loss	   and	   subsequent	   changes	   in	   cerebellar	  neuron	   structure	   and	   function	  may	   be	   a	   common	   pathological	   process	   associated	  with	  autism	  and	  related	  neurodevelopmental	  disorders	  such	  as	  FXS.	  	  	  	   Finally,	  our	  data	  reveals	  that	  there	  is	  no	  significant	  correlation	  between	  the	  total	  number	  of	  PCs	  and	  the	  average	  density	  of	  cFos	  staining	  as	  a	  marker	  of	  baseline	  neural	  activity	  in	  the	  cerebellum	  and	  cerebral	  cortices	  of	  Fmr1	  mice.	  This	  indicates	  	   113	  that	  changes	   in	  neural	  activity	   in	  the	  GCL	  of	   the	  cerebellum	  and	  cortical	  regions	   is	  not	   tightly	   associated	  with	   total	   PC	   numbers	   as	  we	   have	   seen	   previously	   in	   Lc/+	  mutants	  and	  chimeras.	  However,	   there	  are	  significant	  differences	   in	   the	  density	  of	  cFos	  staining	  in	  the	  cerebellar	  GCL	  and	  cerebral	  cortices	  of	  Fmr1	  KO	  mice	  indicating	  that	  there	  are	  underlying	  changes	  in	  cerebellar	  and	  cortical	  neural	  circuits	  as	  seen	  in	  Lurcher	   mutant	   and	   chimeric	   mice.	   The	   observed	   changes	   in	   neural	   activity	   in	  cerebellar	   and	  cortical	  neurons	  of	  Fmr1	  mice	   suggests	   that	   altered	   connectivity	   in	  neural	  pathways	  may	  be	  a	  common	  mechanism	  underlying	  ASD-­‐like	  phenotypes	  in	  many	   different	   mouse	   models	   of	   neurodevelopmental	   disorders	   [159].	   However,	  differences	   in	   the	  patterns	  of	  cFos	  staining	  between	  Lurcher	  mutant	  and	  Fmr1	  KO	  mice	   highlights	   that	   changes	   in	   neural	   activity	   and	   connectivity	   can	   vary	   widely	  between	   mouse	   models	   and	   that	   changes	   in	   neural	   structure	   and	   function	   are	  heterogeneous	  in	  different	  models	  of	  ASD-­‐like	  phenotypes.	  	  4.7-­‐ASD	   as	   a	   neurodevelopmental	   disorder	   caused	   by	   an	   imbalance	   in	  excitatory	  and	  inhibitory	  synaptic	  transmission	  in	  the	  CNS	  	  	   In	  recent	  years	  many	  papers	  have	  been	  published	  reporting	  a	   link	  between	  dysfunction	   in	   GABAergic	   transmission	   in	   ASD	   and	   related	   neurodevelopmental	  disorders.	  Our	  studies	  in	  Lurcher	  mutant,	  Lurcher	  chimera	  and	  Fmr1	  KO	  mice	  have	  also	   shown	   that	   there	   are	   imbalances	   in	   inhibitory	   and	   excitatory	  neurotransmission	   within	   the	   cerebellum	   with	   increasing	   cerebellar	   pathology	  leading	   to	   changes	   in	   neural	   activity	   markers	   in	   surviving	   cerebellar	   neurons.	  Studies	  looking	  at	  the	  molecular	  and	  genetic	  basis	  of	  ASD	  in	  humans	  have	  found	  that	  one	  of	   the	  most	   frequently	   reported	   chromosomal	   abnormalities	   in	  ASD	  cases	  are	  	   114	  microduplications	   and	   microdeletions	   of	   segments	   of	   chromosome	   15q11-­‐q13	  [108].	   Interestingly,	   this	   region	   on	   the	   long	   arm	   of	   chromosome	   15	   contains	  numerous	   genes	   that	   encode	   GABAA	   subunits	   [108].	   Microduplications	   of	  chromosome	   15q11-­‐q13	   tend	   to	   be	   more	   frequently	   associated	   with	   ASD	   than	  microdeletions	  and	  microduplications	   in	   this	  region	  are	  more	  commonly	   inherited	  from	   the	  maternal	   chromosome	  suggesting	   that	   these	   chromosomal	  abnormalities	  represent	  a	  form	  of	  genomic	  imprinting	  [109,110].	  Finally,	  in	  cases	  of	  chromosome	  15q11-­‐q13	   microduplications	   some	   patients	   also	   exhibit	   epilepsy	   which	   is	   a	  neurological	   disorder	   also	   characterized	   by	   increased	   neuronal	   excitability	   in	   the	  CNS	  [111].	  It	  is	  important	  to	  note	  that	  only	  a	  small	  subset	  of	  people	  diagnosed	  with	  ASD	  have	  associated	  deletions/duplications	  in	  chromosome	  15	  segments	  containing	  genes	  encoding	  for	  GABAA	  subunits	  and	  studies	  have	  estimated	  that	  the	  prevalence	  for	  chromosome	  15q11-­‐q13	  microduplications	  in	  ASD	  patients	  is	  between	  0.5%	  and	  3%	  [112].	  	  	   In	  addition	   to	  genetic	  studies	  of	  mutations	  and	  chromosomal	  abnormalities	  associated	  with	  autism,	  post-­‐mortem	  studies	  of	  human	  brain	  tissue	  have	  also	  found	  altered	  expression	  of	  mRNA	  and	  proteins	  involved	  in	  GABAergic	  transmission	  in	  the	  brains	   of	   people	   diagnosed	   with	   ASD.	   Previous	   studies	   have	   found	   reduced	  expression	  of	   the	   two	  major	   isoforms	  of	  Glutamate	  Decarboxylase	   (GAD)	   (enzyme	  involved	   in	   GABA	   synthesis	   from	   glutamate),	   GAD65	  and	   GAD67	   in	   the	   cerebellum	  and	   also	   found	   decreased	   GAD67	   mRNA	   levels	   in	   GABAergic	   cerebellar	   PCs	  [113,114].	  Decreased	  levels	  of	  GABAA	  subunits	  have	  also	  been	  found	  in	  ASD	  patient	  	   115	  brain	   tissue	   with	   decreased	   expression	   of	   the	   GABRB3	   subunit	   in	   the	   cerebellar	  vermis	  [115].	  With	  decreased	  expression	  of	  mRNA	  and	  proteins	  that	  are	  involved	  in	  GABAergic	   transmission	   other	   studies	   have	   found	   a	   corresponding	   increase	   in	  synaptic	   proteins	   involved	   in	   excitatory	   glutamatergic	   transmission	   in	   the	   same	  brain	   regions	   [116].	   Increased	   levels	   of	   AMPA	   receptors	   (which	   are	   involved	   in	  basal	  synaptic	   transmission	  at	  glutamatergic	  synapses)	  and	  elevated	  expression	  of	  glutamate	   transporters	   have	   been	   documented	   in	   the	   cerebellum	   and	   other	   brain	  regions	  in	  brain	  tissue	  derived	  from	  ASD	  patients	  [116].	  	  	   In	   our	   Lurcher	   chimera	   mice	   we	   also	   observed	   numerous	   changes	   in	   the	  excitability	  of	  cerebellar	  neurons	  following	  PC	  death.	  With	  increasing	  pathology	  we	  saw	  increased	  neural	  activation	  as	  shown	  by	  cFos	  staining	  in	  surviving	  GCs	  and	  CN	  neurons	   and	   decreased	   neural	   activation	   in	   surviving	   inhibitory	   PCs.	   In	   addition,	  with	  increasing	  pathological	  changes	  in	  the	  cerebellum	  we	  saw	  that	  individual	  PCs	  have	   shorter	   dendritic	   trees	  with	   less	   complex	   branching	   patterns	  with	  which	   to	  form	   synapses	   with	   pre-­‐synaptic	   GCs	   and	   climbing	   fibres.	   Changes	   in	   neuron	  structure	  and	  function	  were	  also	  coupled	  with	  pathological	  changes	  in	  microglia	  and	  astroglia	   within	   the	   cerebellum	   and	   a	   change	   in	   the	   morphology	   and	   function	   of	  resident	   neuroglia	   can	   also	   contribute	   to	   imbalances	   in	   excitatory	   and	   inhibitory	  synaptic	  transmission	  in	  the	  CNS.	  Deficits	  in	  GABAergic	  transmission	  in	  humans	  and	  mouse	  models	  of	  ASD-­‐like	  phenotypes	  leads	  to	  both	  pathological	  and	  compensatory	  changes	   in	   cerebellar	  neuron	   structure	  and	   function	  and	  also	  provides	  a	  potential	  target	  in	  the	  CNS	  that	  may	  be	  useful	  for	  the	  development	  of	  therapeutics.	  	   116	  Evidence	  for	  developing	  therapeutic	  agents	  targeting	  multiple	  proteins	  found	  at	  GABAergic	  synapses	  has	  come	  from	  pre-­‐clinical	  studies	  involving	  animal	  models	  of	  ASD,	   in	  vitro	  post	  mortem	  studies	  of	  human	  brain	   tissue	  and	  imaging	  studies	  of	  ASD	   patients.	   One	   recent	   study	   found	   that	   ASD-­‐linked	   genes	   involved	   in	   synaptic	  transmission	  in	  the	  CNS	  are	  expressed	  at	  high	  levels	   in	  GABAergic	   interneurons	  in	  multiple	   brain	   regions	   [117].	   Other	   studies	   have	   reported	   a	   reduction	   in	   both	  ionotropic	   GABAA	   receptors	   and	   metabotropic	   GABAB	   receptors	   in	   the	   anterior	  cingulate	  cortex	  (ACC)	  in	  autistic	  brains	  versus	  healthy	  controls,	  which	  is	  significant	  because	  the	  ACC	  has	  previously	  been	  implicated	  in	  neuronal	  dysfunction	  underlying	  ASD	  symptoms	  [118,119].	  	  With	   the	   advent	   of	   improved	   neuroimaging	   techniques	   researchers	   used	  single	  photon	  emission	  computed	  tomography	  (SPECT)	  and	  the	  tracer	  123I-­‐iomazenil	  (which	  is	  a	  GABAA	  receptor	  binding	  benzodiazepine	  ligand)	  to	  determine	  that	  there	  is	  reduced	  binding	  of	  the	  GABAA	  binding	  ligand	  to	  GABAA	  receptors	  in	  the	  medial	  and	  superior	   frontal	   cortex	   of	   autistic	   patients	   [120].	   Finally,	   a	   recent	   study	   using	   the	  radioactive	   tracer	   [11C]	   Ro15-­‐4513	   (which	   specifically	   binds	   to	   α5	   subunits	   of	  GABAA	   receptors)	   and	   PET	   imaging	   found	   that	   there	   is	   significantly	   decreased	  expression	   of	   the	   α5	   GABAA	   subunits	   in	   the	   limbic	   regions	   of	   the	   brain	   of	   ASD	  patients	  as	  compared	  to	  control	  subjects	  [121].	  	  Current	   pharmacological	   agents	   used	   to	   treat	   ASD	   symptoms	   include	  anticonvulsant	  drugs	  because	  up	  to	  30%	  of	  people	  diagnosed	  with	  ASD	  also	  suffer	  	   117	  from	  some	  form	  of	  epilepsy	  [122].	  Some	  anticonvulsant	  drugs	  also	  have	  anxiolytic	  effects	   and	   it	   has	   been	   proposed	   that	   they	   inhibit	   the	   degradation	   or	   reuptake	   of	  GABA	  from	  the	  synapse	  or	  promote	  an	  increase	  in	  the	  synthesis	  of	  GABA	  in	  the	  pre-­‐synaptic	   nerve	   terminal	   suggesting	   that	   anticonvulsant	   drugs	   can	   potentiate	  GABAergic	   transmission	   and	   be	   useful	   in	   the	   treatment	   of	   ASD	   [123,124].	   In	   pre-­‐clinical	   animal	   models	   of	   ASD	   and	   related	   neurodevelopmental	   disorders	   GABA	  agonists	  have	  been	  tested	  for	  their	  efficacy	  in	  normalizing	  GABAergic	  transmission	  in	  animal	  models	  with	  social	  and	  cognitive	  deficits	  that	  resemble	  ASD-­‐like	  behaviour	  [125].	  Studies	  using	  the	  BTBR	  and	  Scn1a+/-­‐	  mouse	  models	  of	  autism	  (both	  of	  which	  have	   defects	   in	   GABAA	  mediated	   synaptic	   transmission	   and	   ASD-­‐like	   behavioural	  phenotypes)	   found	   that	   treatment	   with	   low	   doses	   of	   benzodiazepines	   and	   other	  positive	   allosteric	  modulators	  of	  GABAA	  receptor-­‐mediated	   transmission	   improved	  cognitive	  and	  social	  behavioural	  deficits	  in	  these	  mice	  [126,127].	  	  	   New	   novel	   agents	   for	   the	   treatment	   of	   ASD	   behavioural	   deficits	   include	   a	  selective	   inhibitor	   of	   the	   NKCC1	   chloride	   transporter	   (which	   transports	   chloride	  ions	  into	  neurons)	  called	  bumetanide	  [128].	  In	  clinical	  trials,	  autistic	  children	  were	  treated	   with	   bumetanide	   for	   3	   months,	   which	   led	   to	   improvements	   in	   autistic	  behaviours,	   facial	   emotional	   learning	   and	   increased	   brain	   activation	   in	   regions	  responsible	  for	  social	  and	  emotional	  processing	  [129,130].	  Taken	  together,	  this	  data	  suggests	  that	  identifying	  structural	  and	  functional	  changes	  in	  GABAergic	  neurons	  in	  ASD	   and	   related	   neurodevelopmental	   disorders	   will	   aid	   in	   the	   identification	   of	  	   118	  putative	   therapeutic	   targets	   and	   focus	   the	   development	   of	   novel	   pharmacological	  agents	  (such	  as	  bumetanide)	  to	  alleviate	  ASD	  behavioural	  deficits.	  	  4.8-­‐Future	  directions	  and	  conclusions	  	   In	   order	   to	   fully	   understand	   the	   role	   of	   cerebellar	   pathology	   in	   our	  mouse	  models	  of	  ASD-­‐like	  phenotypes	  future	  studies	  should	  be	  conducted	  as	  a	  follow-­‐up	  to	  this	  thesis:	  I)	  Follow	  up	  studies	  using	  appropriate	  markers	  to	  determine	  structural	  and	  functional	  changes	  in	  CN	  neurons	  to	  determine	  the	  extent	  of	  plasticity	  in	  CN	  neurons	  following	  PC	  death	   in	  Lurcher	  chimeras	  and	  related	  mouse	  models	  of	  cerebellar	  neurodegeneration.	  II)	   Experiments	   using	   pro-­‐inflammatory	   and	   anti-­‐inflammatory	  markers	   to	  determine	   the	   activation	   state	   of	   cerebellar	  microglia	   and	   astroglia	   and	   to	  determine	  the	  functional	  role	  of	  neuroglia	  in	  the	  adult	  cerebellum	  following	  neuronal	  degeneration	  in	  mouse	  models	  of	  neurodevelopmental	  disorders.	  III)	   Further	   studies	   looking	   at	   the	   role	   of	   structural	   and	   functional	  reorganization	   of	   cerebello-­‐cortical	   circuits	   in	   producing	   changes	   in	   neural	  activity	   underlying	   ASD-­‐like	   behaviours	   in	   mouse	   models	   of	   ASD-­‐like	  phenotypes.	  	  The	  results	  from	  this	  thesis	  provide	  fresh	  insights	  into	  neuroanatomical	  and	  histological	  changes	  in	  cerebellar	  neurons	  and	  neuroglia	  and	  how	  changes	  in	  neuron	  and	   glia	   structure	   and	   function	   relate	   to	   cerebellar	  pathology	   in	  mouse	  models	   of	  	   119	  ASD-­‐like	  phenotypes.	  Furthermore,	  by	  using	  Lurcher	  chimeras	  as	  a	  model	  of	  ASD-­‐like	   phenotypes	   the	   current	   findings	   demonstrate	   a	   quantitative	   relationship	  between	   pathological	   changes	   in	   the	   cerebellum	   and	   altered	   neuronal	   activity	   as	  reflected	   by	   changes	   in	   cFos	   (a	   reporter	   of	   neuronal	   activity)	   within	   cerebellar	  neurons.	  These	  results	  are	  valuable	  because	  Lurcher	  chimeras	  demonstrate	  a	  wide	  range	   of	   ASD-­‐like	   neurobiological	   and	   behavioural	   phenotypes,	   which	   more	  accurately	  models	  human	  cases	  of	  ASD	  that	  are	  characterized	  by	  variable	  cerebellar	  pathology.	  Although	  Lc/+	  mutants,	  Lurcher	  chimeras	  and	  Fmr1	  KO	  mice	  can	  only	  be	  used	  to	  model	  a	  subset	  of	  neurobiological	  and	  behavioural	  ASD-­‐like	  phenotypes,	  this	  thesis	  adds	  to	  our	  understanding	  of	  how	  changes	  in	  cerebellar	  neurons	  and	  glia	  may	  contribute	   to	   the	   development	   of	  ASD	   and	   related	  neurodevelopmental	   disorders.	  These	  mouse	  models	  also	  provide	  insights	  into	  how	  changes	  in	  cerebellar	  structure	  and	  function	  during	  normal	  brain	  development	  (such	  as	  physiological	  PC	  death)	  are	  involved	  in	  the	  formation	  and	  remodeling	  of	  cerebellar	  neural	  circuits	  [160].	  	  	   	  	  	  	  	   	  	  	  	   	  	  	  	  	   120	 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