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Calcium-related signal transduction systems in developing visual cortex Jia, Wei-Guo 1991

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& i  CALCIUM- RELATED SIGNAL TRANSDUCTION SYSTEMS IN DEVELOPING VISUAL CORTEX By Wei-GuoJia B.Sc, Fudan University, 1982 M.Sc. Dalhousie University, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES NEUROSCIENCE PROGRAMME  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA January 1991 ©  Wei Guo Jia, 1991  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood that  copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada  DE-6 (2/88)  C a - R E L A T E D SIGNAL TRANSDUCTION SYSTEMS IN 2 +  DEVELOPING VISUAL CORTEX (Abstract) Neuronal connections in cat visual cortex are highly visual  experience  model  of  this  neural  cortical  postnatal  plasticity.  The  plasticity  development  of  transduction protein  kinase  inositol  1,4,5  C,  investigated  own  time-table  culminate  the  of  The  receptors  the  results  methods show  and laminar  and display  the  In  in  as  this  as of  useful  underlying thesis,  surface  dependent second  and  kinase  messenger  alpha-1 II  develops  various  elements  colocalization during  the  that  are  cell  dependent surface  on subcortical  receptors  and  afferents.  their  second  The  sequence  of  and  environmentally  development  of  the  determined, molecules  for  and with all and  results  suggest  messenger  targets  develop in specific temporal and spatial patterns, which genetically  and  critical  period; and, only at this age, the cortical levels of the receptors kinases  and  targets,  immunocytochemistry  distribution; the  the  signal  receptors  that each receptor  maximal  a  calcium-related  cell  calcium/calmodulin using  serve  type-1 muscarinic  examples  phosphotate  autoradiography. its  as  and thus  unclear.  elements  including  systems  age  biochemical mechanisms  remain  several  systems,  adrenoceptor  were  at early  susceptible to  and  may this  signal  be both specific  transduction  results in a series of modifications in the morphology and physiology of the developing cortex leading to its maturation.  ii  Table of Contents Abstract  11  List of tables  iv  List of figures  v  Chapter one GENERAL INTRODUCTION  Pi  Chapter two p7 DEVELOPMENT OF ALPHA ADRENOCEPTORS AND A COMPARISON WITH M l CHOLINERGIC RECEPTOR Chapter three p24 POSTNATAL ONTOGENY OF PROTEIN KINASE C IN THE VISUAL CORTEX Chapter four p41 CALCIUM/CALMODULIN DEPENDENT KINASE II IN CAT VISUAL CORTEX AND ITS DEVELOPMENT Chapter five p5 3 POSTNATAL DEVELOPMENT OF INOSITOL 1,4,5-TRIPHOSPHATE RECEPTORS: A DISPARITY WITH PROTEIN KINASE C Chapter six GENERAL CONCLUSIONS AND DISCUSSION  p62  Figures  p80  Acknowledgement  p i 27  Biblliography  p i 28  List of Tables Table 1. Operated animals used in study of chapter one  pll  Table 2. Bindings of [ H]prazosin and [ H]rauwolscine in the visual 3  3  cortex of operated animals  pi5.1  Table 3. Sequential development of cell surface receptors  p6 9  Table 4. Morphological and physiological development of cat visual cortex  p72  iv  List of Figures Figure 1  Autoradiograms of [ H]prazosin (ccl)and [ H]rauwolscine 3  3  (a2)binding in developing visual cortex Figure 2  p80  Autoradiograms of [ H]pirenzepine binding for M l 3  cholinergic receptors in developing visual cortex. Figure 3  Development of alpha adrenoceptors and M l cholinoceptors at different depth of visual cortex.  Figure 4  p8 2  Autoradiograms of [ H]prazosin (al) and [ H]rauwolscine 3  3  (oc2) bindings in operated animals. Figure 5  p8 3  Densitometric analysis of the ligand binding data of figure 4.  Figure 6  p8 0  p85 Comparison of development of alpha-1, alpha-2  adrenoceptors and M l cholinoceptors in various cortical laminae. Figure 7  p87  Comparison in laminar distributions of the three receptors in developing visual cortex.  p8 9  Figure. 8 Light micrographs of immunostaining for protein kinase C in area 17 (a) and area 18 (b) of kitten visual cortex  p9 1  Figure 9 Area 17 (top) and area 18 (bottom) of the visual cortex stained with the polyclonal antibodies to PKC at day 10.  p93  Figure 10 High magnification of PKC immunostaining in area 17 of the visual cortex at day 40.  p93  Figure 11 Bundles of fibers with PKC immunoreactivity in upper layer VI of area 17 at day 40.  p9 3  Figure 12 Cytoplasm of a PKC immunoreactive cell from a postnatal day 10 kitten visual cortex.  p95  Figure 13 Electron micrographs of immunoreactive profiles taken from the visual cortex of a postnatal day 30 kitten. p97 Figure 14 Distribution of PKC immunoreactive structures in a v  postnatal day 4 kitten visual cortex.  p9 9  Figure 15 PKC immunoreactivity in the visual cortex surgically isolated at day 14. Figure 16  plOl  Immunostaining of CAM-K II antibody in tissues perfused  with two different protocols  pi03  Figure 17 CAM-K II in cat visual cortex.  p i 05  Figure 18 CAM-K II immunoreactivity in kitten visual cortex (area 17) at various ages.  pi07  Figure 19 High magnification microphotograph taken from an area in upper layer IV of the animal at 24 days of age.  p i 09  Figure 20 CAM-K II immunoreactivity in an animal with an early LGN-lesioned.  p i 09  Figure 21 Cytoplasm of CAM-KII immunoreactive neurons from an adult cat (left) and a postnatal day 14 kitten (right) visual cortex.  pill  Figure 22 Electron micrographs of post-synaptic CAM-K II immunoreactive profiles taken from an adult cat (left) and a postnatal day 4 kitten (right) visual cortex.  p1 1 3  Figure 23 Electron micrographs of presynaptic CAM-KII immunoreactive profiles taken from an adult cat (left) and a postnatal day 4 kitten (right) visual cortex.  p i 15  Figure 24 Charectelization of [ H]IP3 binding in cat visual 3  cortex.  p 117  Figure 25 Color coded optical density of autoradiography of [ H ] I P 3 3  and [ H]PDBu in adjacent sections of the visual cortex and 3  hippocampus in developing kitten brain (left panel).  p i 19  Figure 26 Development of [ H]IP3 and [ H]PDBu binding sites in 3  3  visual cortex.  p 1 21  Figure 27 Autoradiography of [ H]IP3 and [ H]PDBu binding in 3  3  undercut visual cortex (left) and the results of densitometry (right).  pi23 vi  Figure 28 Colocalization of receptors for PI turnover in developing visual cortex. Figure 29  p i 25  Comparison of development of receptors for PI turnover in the visual cortex.  p i 25  vii  Chapter One GENERAL INTRODUCTION During early age of postnatal life, the visual cortex displays a remarkable early  age  synaptic  plasticity 198,199,200. can  cause  connections  Abnormal visual exposure at this  profound and permanent in the cortex ",41,182.  abnormalities  in  Young children with  4  peripheral visual disorders such as cataract, hyperopia or myopia will  develop  reduced visual capabilities  (e.g.  amblyopia) in the  affected eye in adulthood, unless the optical defect is corrected early in life. The  permanent defective  consequences  been widely studied in animal  of abnormal vision have  models .41,182,198,199,200. 40  it has been  well established that kittens reared with one eyelid sutured during the first three months of postnatal life sustain permanent blindness in the sutured eye when it is reopened, although the eye and the retina are normal.  Further, anatomic and physiologic studies indicate  that there are a series of changes in the central parts of the visual system.  In the cerebral visual cortex, most cells become monocularly  driven by stimulation through the normal eye and fewer than 10% of the  cells  respond to  stimulation through the  sutured eye.  In  addition, injection of [ H]proline in one eye, to label the areas on the 3  visual  cortex  remarkable  that receive  shrinkage  the  inputs from  in those areas  of  that eye,  cortex  reveals  representing  a the  deprived eye and a corresponding expansion in the areas receiving input from the non-deprived  eye85,i°9.  These results indicate that  monocular deprivation at an early age causes profound modification in synaptic connections in the visual pathways. around postnatal  week  3 to week  In cat, this age is  13 and this  period of  time  characterized with the highest susceptibility to visual experience in organization of the visual cortex is called "the critical period" "' . 1  1  200  Based on the above good  model  for  connections neuronal  the  which  has  to  various  of  the visual system formation  become  widely  of  has provided a  neuronal  accepted  as  formation  procedure  the  dominance  neurons  synaptic on  and to  adjust  to  of  adapt  their responses  to  changes  1 5 1  .  1 5 2  -  .  1 6 8  activities  monocularly  by  activity  hand,  in  or  the  is  of  a  highly  both  deprived  continually These  may  a blockade  connections  presynaptic  kittens  inhibiting  addition,  strengthened long  depending  term  hippocampus, facilitates  results  indicate  interrupt formation  of presynaptic  that  of  activity  disruption  significance  on activity.  This phenomenon  tetanic  (LTP)  stimulation  of  connections,  response  which  2 0  lasts  i  1  certain  or  of L T P cannot be overemphasized?'  modification  of  synaptic  important  clues  transmission; to  the  in  cortex  1 8 1  . be  or  pathways  an  even  elevated  days.  The  L T P directly reflects  therefore,  mechanisms  the  is best known  presynaptic  results  hours  can  cerebral  n  of  normal  columns  connection  . 86.  and  On  driven by the  synaptic  by  cortical  synapses.  established  potentiation  synaptic  postsynaptic  provide  an  and  either  the  can also prevent the formation of ocular dominance In  dynamic  In the visual cortex, one can prevent ocular  29  postsynaptic other  of  cells .i??.  overactivating  and  basis  This plasticity enables organisms  environments  depending  postsynaptic  as  synaptic  situations.  The  eye  study  plasticity73,177.  themselves new  discoveries,  of  studies  on L T P  synaptic  formation  strengthening. Evidence  new pre-  from the  synapse and  mainly  opening  chemical  substances  receptors,  strengthening  post-synaptic  by  released  or its  study of L T P suggests that the formation of a  which  elements . 21  signals (e.g.  transfer  is based on the interaction  released  This from  interaction  certain ion channels  signal  into  bind  the  to cause cell  2  established  neuronal. terminals.  neurotransmitters) the  is  between  cell  to  their  either  by  The  specific directly  membrane depolarization/  -  hyperpolarization proteins)  which  1  including  signal  relatively  slow  often  coupled  protein  DNA-binding  but l o n g  term  new  synapses  intracellular and  results  signal transduction  corresponding  during In  protein  the d e v e l o p m e n t view  a  systems  of  Since  of  synaptic  of cellular  investigated.  Three  distributed  has  been  be  case  densest is  [ H]  shown  that  IV,  while  the  have  in  been  the f o r m a t i o n o f responses,  second an  visual  drawn  the  layers V  from  are not  these  receptors  (nAChRs)  labeled  studies:  homogeneously  c o r t e x i 69,170,201.  density  and  VI  of binding  For  sites  a p p e a r s to  appear the least d e n s e 3 3 . i n 1  acetylcholinergic receptors  On  the  l a b e l G A B A A receptors i n adult cat, i t  to  III  through  the  systematically  (mAChRs),  3  I  role  for  cortex,  binding o f a specific ligand [ H]quinuclidinylbenzinlate i n layer  the  messengers  important  has b e e n  cortex of  layers  the highest  o f muscarinic  found  gene  signal transduction  receptors  the v i s u a l  muscimol  3  appears i n l a y e r I V 1 7 1 .  in  in  throughout  i n layer  the  conclusions  receptors  instance, u s i n g  play  connections  o f neurotransmitter  Many  may  or  cellular to  as p r o t e i n  2 9  development  1.  of  causes  o f the b r a i n .  the i m p o r t a n c e  development  such  synthesis  receptors  kinases  a group of  usually  i n the c e l l ,  series from  messengers  8  protein  from  second  proteins ,18,44,49,132,157,163.  transcription32,44,49,64,66,69,94,96,99,120,136,196. new  messenger  phosphorylate  messenger  changes  (G-  proteins  second  The  to  kinases  v i a second  phosphorylation,  to  i o n channels33.  some  pathway  GTP-dependent  activating  some  activate specific  proteins  This  by  are  or  systems ^,197 further  or  b u t a n d the l e a s t  the other  (QNB),  dense  binding  hand, n i c o t i n i c a c e t y l c h o l i n e r g i c  [ H]nicotine,  with  the  3  are l o c a t e d  specifically  layer IV150. 2.  The  distributions during  numbers i n visual  of  cortex  development 69,172. 1  neurotransmitter undergo i  n  a  receptors  series  of dynamic  contrast to the l a m i n a r  3  and  their  changes  distribution o f  mAChRs in adult cats, these receptors are concentrated in the middle layers in newborn kittens. are  expressed  disappear  in  in  During development, mAChR binding sites  superficial  layer  and  IV170,17 1,192.  sites  cortex.  Almost two thirds of receptors as  development  (3-adrenergic,  muscarinic,  layers  and  The redistribution  binding such  during postnatal  deeper  is  gradually  of  common  receptor in  visual  studied in our laboratory,  opioid,  C C K and  glutamate  receptors, show a similar form of redistributions. In  all cases, the redistribution occurs during the critical period.  Furthermore, the overall number of each type the peak within that critical period.  of receptors  reaches  After that time, the number of  receptors is either maintained or reduced to adult levels. 3.  Redistribution of receptors is dependent on normal input to the If part of the white matter underneath the visual cortical  cortexi73.  grey  matter  is  surgically  undercut,  the  cortical  neurons  that  are  innervated by those fibers will be isolated from the input of lateral geniculate visual  nucleus ( L G N ) and other regions.  cortex,  some  receptors  In kittens with undercut  do n o t show  normally  occurs during the critical period.  mAChR:  kittens  were  undercut at postnatal  the  redistribution that  One such example day  were labeled with [ H]QNB three weeks later. 3  23,  and  retained their immature pattern, i.e. layer IV is densely heterogeneous  presynaptic  input  distribution, the  dependence  mAChRs  Instead of showing the  adult pattern of binding (all layers, except layer IV), the  The  highly  and especially  labeledi73.  dynamic the  receptors  change,  highest of  of  receptors  in  important roles development In couple  visual in the  cortex  suggest  that  the  activation  development  organization of neuronal connections  play during  of the cortex.  order to induce long term cellular responses, to  its  activity  during the critical period,all of these characteristics receptors  is  second of  messenger  various  systems.  kinases,  which  4  receptors  Normally, this should  be  must  results  in  considered  as  receptors  for second  messengers.  The present  work is mainly  focused on calcium-related second messenger systems, i.e., calciumCa  + +  /calmodulin  dependent  protein  kinase  II  (Ca  /CAM-K  ++  II  system) and phosphoinositide (PI system). The  Ca /CAM-K  II second messenger  + +  mobilized by elevations  in intracellular free calcium level.  neuronal activities can result in increases intracellular  Ca  extracellular  + +  systemU.34.6i.i62.i88 is  in the concentration of  ([Ca ]i) by either activating C a ++  sources  or  mobilizing  Many  influx from  + +  intracellular  Ca  stores.  + +  Increased [Ca ]i activates CAM-K II through an interaction between ++  the kinase and the Ca /calmodulin complex. ++  Another second messenger calcium  is  inositol  system  that is closely  related with  trisphosphate/diacylglycerol  (IP3/DG)  The I P 3 / D G second messenger system is triggered by  14,15,57,143,175.  occupation of several types of receptors, such as M l muscarinic cholinergic receptors, a, adrenergic receptors, 5-HTlc receptors, etc. Stimulation  of  phospholipase  these  C (PLC) via  phosphatidylinositol messengers,  receptors  results  the  G-protein(s)57,79.  4,5-bisphosphate  DG and I P 3 .  in  activation  PLC hydrolyses  (PIP2) to release  The former activates  of  two second  a calcium/lipid-  dependent protein kinase, protein kinase C (PKC) and the latter reacts with its specific intracellular receptors to cause C a from intracellular  storesi6.  Both the C a / C A M - K II and IP3/DG ++  have been widely studied in the brain. are  deeply  neuronal  involved in many plasticity  and  second messenger  neuronal functions,  development  systems  It is accepted that the two (for  Ca  17,23,34,61,64,74,101,102,104,120,125,141,162,196;  ++  especially /CAM-K for  2,32,49,64,66,94,96,115.-118,120,121,123,126.132,136,140,157.163,208).  K  release  + +  in II:  IP3/DG: Both C A M -  II and PKC exist in high concentrations in the brain and are  responsible  for the  phosphorylation of 5  various neuronal  specific  proteins  that  formation  are  or  cytoskeleton  important  modulation.  during Some  development targets  and  include  proteins and the growth associated  synaptic  synapsin  I,  protein (GAP 43).  Both kinases are reported play a role in LTP - 10,120,123.  An increase  1 1  in I P 3 during LTP was also been reported?,25,117. It is possible that the three second messengers, calcium, IP3 and DG  can have  synergistic  interactions  perform various cellular responses. and  IP3 since  hydrolysis  of  they PIP2.  can  be  under certain conditions  to  It is particularly likely for D G  produced simultaneously  from  the  Several groups have reported that there are  additive effects of calcium and activators of PKC to induce full secretory responses in blood platelets and other cells . 13  well  It is also  known that a number of proteins, such as synapsin  I and  microtubule associated protein 2 (MAP 2), are common substrates of both CAM-K II and PKC.  However, little evidence has been collected  to support this speculation in the nervous system. on  To throw a light  the issue, I compared the postnatal ontogeny and localization of  C a / C A M - K II and IP3/DG systems in kitten visual cortex. + +  This is  under the presumption that if the two systems indeed play roles in cortical development  in a synergistic manner, C A M - K  II, the IP3  receptor, and PKC should be labelled at similar locations and show similar ontogenic profiles in developing visual cortex. To  this  aim, immunocytochemistry and autoradiography were  utilized to localize the related molecules in cat visual cortex. investigation  was particularly focused on changes  The  with age in the  distributions and on the comparison among these molecules.  In  order to reveal the relationship between the development of these second  messenger  systems  and  neuronal  activity,  various  manipulations were performed on the visual cortex at different ages and  the effects of these manipulations on expression and distribution  of these molecules were investigated.  6  Chapter Two DEVELOPMENT OF ALPHA ADRENOCEPTORS AND A COMPARISON WITH M l CHOLINERGIC RECEPTOR INTRODUCTION: As  the gates  surface  receptors to PI  coupled  have  receptors  with  phosphates addition,  their  there  agonists + +  Both  considered from  as  the  from  evidence  receptors  cortical locus  (NA)  modulatory  bundles  to v a r i o u s  cortex,  cortical  et  cholinergic  reduced a  Bear  found  that  reported  and  application  o f NA  dominance,  the  cells  i n kitten  and  ACh  systems  a n d ACh  of  that  results  play  cortex roles  65.  fibers  i n the b a s a l  run through cortex.  destruction  dorsal In c a t o f the  results  deprivation,  On  9  C  systems  a n d ACh  innervation  to  kinase  (ACh)  cells  combined  In  responses  protein  NA  and  in  stores24,97.  the c e r e b r a l  plasticity .  in  a  suggesting  the other hand, i t was  synchronized  with  combined  in a long-lasting modification i n  orientation selectivity visual  by  to m o n o c u l a r  stimulation  inositol  fibers  noradrenergic  dominance  visual  These  areas  p h y s i o l o g i c a l response  l o s s o f the o c u l a r  ocular  al  1 1 2  cholinergic  increase  cellular  systems.  that are  o f the  and cholinergic  (LC)  coeruleus  respectively.  visual  many  are m e d i a t e d  telencephalon^, and project  rapid  cell  Stimulation  intracellular  that  noradrenergic  and Ml  studied. a  pathways,  the r e c e p t o r s  receptors  causes  release  is also  o f these  activation49.  widely  transduction  Among  role.  a l adrenergic  most  Ca  signal  a crucial  been  and  stimulation  arise  play  turnover,  receptors  are  of intracellular  and direction  These  in cortical  results plasticity  preference  of  that the  NA  imply in a  cooperative  manner. The visual  postnatal cortex  identification  has and  development been laminar  of  reported  muscarinic  p r e v i o u s l y 170.  distribution  7  receptors  of  in  kitten  i n a d d i t i o n , the  alpha-adrenoceptors  in  adult cat visual cortex were reported by Parkinson et a l . to examine systems  in  the  development  with  receptors  of  the  adrenoceptors  autoradiography  [ H]rauwolscine28, 3  .  In order  the functional contribution of adrenergic and cholinergic  ontogeny of a l and a2 sections  1 4 5  by  respectively.  were labelled  visual was  cortex,  investigated  utilizing  the in  postnatal adjacent  [ H]prazosin 3  and  As a comparison, M l cholinergic  by [ H]pirenzepine 3  animals in the same age groups.  8  in the  visual cortex  of  METHODS AND MATERIALS Animals A total of twenty-one cats of various ages (from postnatal day 1 to adult) were utilized in the present  study.  anesthetized  the  and perfused  through  phosphate buffer (0.1M, pH 7.4) for 1 min.  Animals were deeply  aorta  with  cold  (8°C )  The perfused brains were  rapidly removed and stored at -20°C. Some of animals were subjected to surgical manipulations before the perfusion. facemask  Kittens were anesthetized with helothane to effect via  and placed in stereotaxic  were performed (Table 1).  frame.  Four types of surgery  In the first group {front cut), a 1 mm  thick trench of cortical tissue was aspirated by suction to a depth of 1 cm from the cortical surface  (extending  down to  the  corpus  callosum) and extending from the midline to about 7 mm lateral of the midline on one side of the brain. about  A.P.  modulatory forebrain  12.0  anterior  fibers  and  had  The section was located at the  effect  of  interrupting  arising from brainstem, hypothalamic and basal  sources . 9  aspirated on one side.  In the second group (LGN lesion),  LGN was  In animals of the third group, the unilateral  front cuts were combined with ipsilateral L G N lesion.  In one  additional kitten, the optic tract on the left side was severed at postnatal day 10 and the animal was perfused at day 59.  The  operations were performed at various ages and all operated kittens were given an injection of penicillin G (10,000 IU/kg) and a topical broad spectrum antibiotic is applied to the incision for 5 days post operatively.  Animals were allowed to survive at least two weeks  before sacrifice. process.  The lesion sites were examined during sectioning  Removal of LGN and severing of the dorsal bundles were  confirmed by greatly reduced [ H]nicotine binding in the ipsilateral 3  visual staining  cortexl50 and by a negative result of cholinesterase in the  ipsilateral  cortex 9  posterior  to  the  lesion  (AChE) site , 9  respectively. Autoradiography Thin (12|im) coronal sections of the visual cortex from animals at various ages were cut on a cryostat, and thaw-mounted onto gelatinsubbed slides.  Sections  were incubated for one hour at 4 ° C in  phosphate buffer (PB, 0.1 M, pH 7.7) containing 0.75nM [ H]prazosin 3  (24.4 Ci/mmol) or 0.6nM [ H]rauwolscine (75.0 Ci/mmol, both from 3  NEN) for a l and a2 receptors, respectively.  Nonspecific binding was  less than 15% of the total binding at current concentrations of ligands determined  by  concentration)  adding  phentolamine  (Sigma,  into the incubation media.  10|iM,  The incubations  final were  followed by 3x10 min wash for [ H]prazosin and 3x5 min wash for 3  [ H]rauwolscine in PB at 4°C. For M l sites, sections were incubated in 3  0.02M  Tris  buffer  [ H]pirenzepine 3  (pH  (82.0  7.5)  with  Ci/mmol,  0.01M  Sigma)  for  temperature followed by a 2X3 min wash.  MgCl2 60  and 5 nM  min  at room  Sections were rapidly  dried in a stream of room air and apposed to LKB Ultrofilm for 10 weeks. Quantitative The and  analysis  autoradiography images were captured with a video camera,  input into a computer for densitometry  analysis.  Averaged  optical density (OD) of any given region of cortex was measured and expressed  in pseudocolor after subtracting background.  The OD  values were calibrated into concentration of the bound radioactive ligand using [ H]standards (Amersham). 3  were applied for statistical analysis.  10  Student t-test or A N O V A  Table 1 Operated animals used in study of chapter one Animal  Operation type  Operation age (Postnatal day)  Sacrifice age  RB50  left FC  adult  90 days survival  RB69  right FC  PI 1  P40  RB71  right FC  Pll  P40  RB52  right LGN(-)  adult  14 days survival  RB61  right LGN (-)  adult  60 days survival  RB68  right LGN (-)  Pll  P40  RB79  right LGN (-)  Pll  P90  RB46  right FC/LGN (-)  PI 1  P40  RB47  right FC/LGN (-)  RB78  Left OT(-)  Pll  P40  P10  P59  FC, front cut; LGN(-), LGN lesion; FC/LGN(-), front cut combined with LGN lesion; OT(-), optic tract lesion.  11  Results Postnatal development The  of alpha-1  laminar distribution of  adrenoceptors the  visual cortex varies with age (Fig.l).  alpha-1  adrenoceptors  in  the  At postnatal day 1, binding of  [ H]prazosin in the visual cortex was at a very low level, although a 3  mild density of silver grains could be seen in cortical layer I. In contrast,  high binding was  found in the  subcortical plate.  By  postnatal day 10, cortical layers I and IV were densely labeled by the ligand and the binding in the rest of the laminae remained low. Unlike the binding in layer I, the densely  labelled middle layer  particularly demarcated the visual cortex (areas 17 and 18). postnatal day 1, levels of [ H]prazosin in the subplate  consistently  3  increased until P10.  During this period, the largest proportion of the  binding was located in the subplate. [ H]prazosin  binding increased  3  particularly  in the  After  By postnatal day 30 to 40,  in all cortical laminae  superficial and middle layers  while  showed the least radioactivity among all cortical layers.  (Fig.  3),  layer VI There was  still a strong binding in the subplate although the width was greatly reduced.  The number of binding sites in this zone declined gradually  and finally disappeared in the visual cortex by postnatal day 60 (not shown) but they were still present in some cortical areas, such as the entorhinal cortex, to a lesser extent until adulthood.  Between P40  and P75, there was still a substantial increase in density of binding sites in the cortex.  However, there was not much change in the  laminar distribution pattern of the binding.  The total binding in the  visual cortex started to decline between P75 and P120.  During this  period of time, the greatest reduction occurred in the middle layer while binding in the superficial layers declined less (Fig.3). heterogeneous  reduction  in  the  binding  among  different  This layers  resulted in a new laminar pattern, showing a higher density of silver grains in layers II and III than in layers IV, V and VI.  12  This new  pattern remained into adulthood. Postnatal development Development receptors  in  of the  [ H]prazosin.  of alpha-2 adrenoceptor  [ H]rauwolscine  binding sites representing  3  visual  cortex  somewhat  resembles  that  a2 of  The binding achieved its maximum around the same  3  age as that of [ H]prazosin (P75) and the number of binding sites 3  declined subsequently.  Again, this reduction was most remarkable  in the middle layer and the superficial layers presented the densest binding of the ligand in adulthood. ligands  differs  [ H]rauwolscine 3  in  several  However, binding of the two  aspects.  First  all,  binding  of  in the subplate disappeared by postnatal day 30,  much earlier than that of [ H]prazosin; 3  representing  of  Secondly,  silver  grains  [ H]rauwolscine binding sites appeared in layer IV by 3  P20, about 10 days later than the appearance of [ H]prazosin binding 3  sites in the same layer (Fig. 3);  Thirdly, the maximal binding density  of [ H]rauwolscine during the critical period was 12-fold higher than 3  during the first month while it was less than 3-fold for [ H]prazosin 3  (Fig. 3);  Finally, in adult visual cortex, the binding of [ H]rauwolscine 3  in layer II was 3-fold higher than in layers IV-VI while the binding of [ H]prazosin in the top half of the cortex was only about 50% 3  higher than that in the bottom half (Fig. 3 and Fig. 6). Development of muscarinic type-1 receptors The postnatal development of [ H]pirenzepine binding sites in the 3  cat visual cortex has been reported previously 149. agrees with those results (Fig.2 and 3).  The present work  Briefly, the [ H]pirenzepine 3  binding sites were first apparent in layer IV in the neonatal cortex. The number of binding sites in this layer reached its maximum by P30 and subsequently reduced.  In the superficial layers (II and III)  and deep layers (V and VI), the binding was low in first two weeks, and rapidly increased around P20.  13  The peak in layers I-III was  reached around P40, a week later than that in layer IV. layers  V-VI reached  essentially  a maximum at  about  P40  Binding in  and remained  unchanged thereafter and the density of binding in these  layers then declined to adult level.  As a consequence of a greater  decrease of binding in the middle layers comparing to the other layers,  the adult laminar distribution of M l receptors  was  quite  different from that of young animals, showing heavily labelled layers II, III and VI with relatively lower density of binding in the middle layers, especially layer IV. Effects of inputs deprivation on development of alpha adrenoceptor alpha-1  receptors  (Figures 4, 5 and Table 2a)  L G N lesions  performed at a young age (Pll) resulted in a significant decrease (P<0.05) in binding of [ H]prazosin in the ipsilateral visual cortex 3  when the animals were perfused either at P40 or at P90 (Fig.4a,c and Fig.5a,c).  Although the "front cut" itself showed little effect (Table  2a), the loss in the binding resulting from combination of the front cut and ipsilateral L G N lesion  was  significantly  greater (p<0.01,  ANOVA, in all cortical layers) than that of LGN lesion only.  There  was no alteration in alpha-1 receptors when the LGN lesion performed at adulthood (Fig. 4g, 5g and Table 2a). decline  of  binding in  the  grey  matter,  was  Contrary to the  a striking increase  in  radioactivity was found in the ipsilateral subcortical plate in animals following the early LGN lesion with or without front cut (Fig. 4a,c; Fig. 5a, c and Table 2a).  No effect was seen with the optic tract lesion  (Fig.4e; Fig. 5e and Table 2a). Alpha-2  receptors (Figures 4, 5 and Table 2b)  The effects of an  early L G N lesion or combination of a LGN lesion and front cut on alpha-2 receptors.  receptors  was  more  remarkable  than  that  of  alpha-1  Again, the most striking effects were seen in animals  having the combination of LGN lesion and front cut.  14  Unlike alpha-1  receptors,  the  reduction  in  density  of  silver  homogeneous across the cortical laminae.  grains  was  not  The most affected cortical  layer was layer IV, where the binding of [ H]rauwolscine 3  LGN lesion was about half of the control value.  following  In contrast, there was  no change in layer I when the animal was sacrificed at P40, 4 weeks following  the surgery.  However, this heterogeneous reduction in  different layers was less obvious in a kitten that was perfused at P90, 11 weeks after the operation (Fig. 5 b and d).  In this animal,  although there was still a greater decrease of binding in layer IV, the extent of decline in layer I was similar to that in layers II and III. The same LGN lesion in adult animals did not significantly change the binding density in the cortex (Fig. 5h).  As with alpha-1 receptors,  neither the optic tract lesion  nor a pure front cut had  (Fig. 5f)  significant effect on the number of alpha-2 receptors.  15  Table 2 Binding of [ H]Prazosin and [ H]Rauwolscine in the Visual Cortex of Operated Animals 3  3  a) [ H]prazosin 3  0T(-)  FC  Young LGN (-)  LGN(-)/FC  Adult LGN(-)  Layers I-III  101.9±2.1 (4)  97.1±2.3 (8)  86.3±2.0 (9)  76.2±2.7 (6)  99.9±3.6(8)  n  Layer IV  103±l.l (4)  96.8+2.8 (8)  91.2±3.7 (9)  73.7±3.1 (6)  96.0±2.7(8)  n  Layers V-VI  102±4.3 (4)  97.6±2.4 (8)  93.0±1.6 (9)  77.9±3.7 (6)  102.4±2.7(8)  Layers I-VI  102±2.3 (4)  96.1±2.2 (8)  90.8±2.4 (9)  76.1±3:3 (6)  99.8±2.7(8)  SP  99.0±1.8 (4)  105.0±3.5 (8)  202.8±15.4 (9)  147.1±8.8 (6)  n  n  n  n  n  n  n  n  n  n  b  a  b  a  C  C  c  b  b  n  n  b  b) [ H]rauwolscine 3  OT(-)  FC  Young LGN (-)  LGN(-)/FC  Adult LGN(-)  Layers I-III  107.7±7.7 (4)  106.5±4.2 (8)  74.0+2.6 (9)  74.8+8.l (4)  99.4±3.5 (8)  Layer IV  109.0+4.8 (4)  97.5±3.2 (8)  50.6±2.9 (9)  32.0+3. l (4)  100+_4.8 (8)  Layers V-VI 1 1 0 . 4 ± 4 . 6 ( 4 )  105.5±5.2 (8)  54.3 + 3.4 (9)  58.2+1.9 (4)  106±13 (8)  Layers I-VI  107.6±3.8 (8)  66.9±1.3 (9)  52.1±2.2 (4)  95.5±6.2 (8)  n  n  n  108.2±2.7 (4) n  n  n  n  n  c  C  c  C  a  c  c  C  n  n  n  n  OT(-), optic tract lesion; FC, front cut; LGN(-), LGN lesion; SP, subcortical plate, n, P>0.05; a, P<0.05; b, P<0.005; P<0.0005  DISCUSSION Five alpha adrenoceptors have been cloned, namely three alphal's (la, lb, lc)i30 and two alpha-2's (2a, 2b)i08. was reported in  PI  Most recently, it  alpha-lb but not alpha-la receptor  that  turnoveri27.  was  involved  Prazosin does not distinguish these alpha-1  subtypes, so that the results reported above reflect overall changes of the alpha-l's. affinity  On the other hand, although rauwolscine has equal  to both 2a and 2b  s u b t y p e s 2 7,  the  autoradiograms  of  [ H]rauwolscine shown above probably represent alpha-2a sites since 3  the majority of alpha-2 receptors in the cerebral cortex are believed to  be  alpha-2a2 7.  Both alpha-2 subtypes  are considered to be  negatively coupled with adenylate cyclase via Gi, i.e., activation of either of the two can reduce cellular cAMP levels 7,28. 2  NA and ACh fibers arise from the locus coeruleus (LC)U2 and cells in  the  basal  noradrenaline  telencephalon^,  Fibers  containing  or dopamine-[3-hydroxylase (DBH) have been widely  studied in the cortex. in  respectively.  In rat, NA axons derived from LC are observed  the cerebral cortex at very early developmental stage (El7) and  cover the entire hemisphere endogenous  by  birthH6.  in kitten visual cortex,  monoamines are already measurable at birth and level  of NA increases gradually with age and reaches 50% of its adult level by postnatal week 11-13 . 98  This slow increase in endogenous NA at  early postnatal age bears resemblance to the development of alpha adrenoceptors  observed here.  Interestingly,  while  the  NA level  keeps increase till adulthood, the ligand binding sites for the alpha adrenoceptors increases at early age followed by a dramatical drop in adulthood.  This suggests that both subtypes of alpha receptors are  transiently "over-expressed" at early postnatal ages. Compared  with  alpha receptors,  a relatively  increase in binding sites for [ H]pirenzepine was 3  cortex.  Immunocytochemical 16  early  and  fast  seen in neonatal  visualization  of  cholineacetyltransferase  (ChAT) in both cat 79 and rat visual cortex 1  45  indicated low concentrations of ChAT activity in the first week, with a significant increase during subsequent weeks. unique long fibers  It was reported that  with ChAT immunoreactivity run preferentially  within layers I and IV of kitten visual cortex while, in adult cat, the superficial  layers receive  the  strongest cholinergic  innervation 79. 1  This redistribution of ChAT positive fibers resembles the changes in Ml  receptor binding pattern during development. Despite reportedly low levels of ChAT, the M l receptor-related  intracellular  signal  pathway  seems already  fully  functional  at a  neonatal age since high levels of phosphoinositide turnover can be induced by muscarinic agonists in both  rat6,72  and kitten cortex  4 8  .  Unlike alpha adrenoceptors, the binding of which in adult animals is only slightly higher than that in newborn kittens, the number of M l binding sites in adulthood is four-fold that in newborn kittens (Fig.3 and  Fig.6).  Interestingly,  adult cortex is similar to animals. sites  or even lower  than6.72  that in neonatal  The paradoxical changes in number of receptor binding  and level  indicate  48  the carbachol-stimulated PI turnover in  an  of  PI turnover stimulated  alteration  in efficacy  of  the  by the  receptor may  coupling  between  the  receptor and the second messenger system during development. None  of the  three  receptor binding sites developed  across the six cortical layers. slopes of development common feature  uniformly  This is manifested by difference in  curves for each cortical layer (Fig.3).  of development  shared by the three receptors  A is  that layer IV presented the most binding sites early in life and that the binding densities were reduced in this layer greater than in other layers at late ages. animals  showing  superficial results  145  .  consistent  This developmental pattern resulted in the adult the  cortical  highest  layers,  density  which  is  of  binding  consistent  sites with  in  the  previous  These laminar distributions of the receptors are also with  that  of  NA-containing  17  terminals,  which  are  reportedly  concentrated  cortex93.  In all cortical layers, the a l sites showed a higher level of  expression  at postnatal day 1 and a slower rate of increase  the  a2  or M l sites  in upper  (Fig. 6).  and lower  layers  of  cat visual  In the middle and deep  than did  layers, the  increase in a2 binding sites was delayed at least a month beyond the time  when  were  also  the other two receptors  Differences  started to increase.  noted in the falling phases of the developmental  profiles.  While the number of the two alpha receptors dropped rapidly, the number o f M l receptors declined less and more slowly.  Figure 7 illustrates the typical relative laminations of the three receptors that,  in the visual cortex  in newborn kittens,  completely  separate  from that of alpha adrenoceptors.  distributions of the three  In  adulthood,  laminations  identical.  physiological NA  results  are  colocalization of  Ml  coupled  and a l  showed  to  the  are more  data  cortical  neurons65.  same  PI  or less overlapped. are  consistent  synergistic  from  there  would systems  be  a boost  almost  At later ages,  receptors  are  is  effects  almost  with  the  of exogenous  As both M l and a 1  second  messengers,  of the two further suggests that the synergistic  second messenger system. cholinergic  of  which  the two may result  cortex,  receptors  These  and ACh on single  receptors  It is clear  the distribution of M l receptors  the  completely  at various postnatal ages.  in activation  of this  the  effects  common  These data also predict that, in newborn little  via  cooperation  Ml  and  between  alpha  adrenergic and  receptors  since  the  concentration of the latter is very low. Changes  in  laminar  patterns  development  indicate  that  receptors  different  ages.  transiently in  at  show  receptors  different To  the  cell  binding  populations  wit, cells  in  sites  during  express  the middle  the layer  high levels of the receptors at early ages and cells  the superficial  most  of  layers  in the  in turn  adult.  become  the populations  with the  However, it is also possible that the  18  observed shift in laminar pattern of the binding sites may reflect a translocation  of the  receptors  at subcellular level,  i.e.  terminals  versus cell bodies that are located in different cortical layers.  In  addition, a shift in cell types expressing the receptors can also occur during  development  within the  same  observed in the postnatal ontogeny  cortical layers.  This  was  of beta receptors, which are  expressed mainly by pyramidal cells in early life and by astrocytes and  nonpyramidal  technique  cells  in  adulthood 3. 11  utilized in the present  The autoradiographic  work does not have  sufficient  resolution to detect such changes for alpha adrenoceptors. The most striking transient expression of the receptors was seen in the subcortical plate.  Both ligands for the alpha subtypes heavily  bound to the subplate region at early postnatal ages.  The band of  binding in this region gradually shrunk and disappeared by postnatal day 20 for oc2 receptors and by day 60 for a l . [ H]thymidine 3  birthdating experiments,  Shatz  Based on results of and her  colleagues  reported that, in cat visual cortex, the subplate was formed between embryonic day 40 to 60 and cells in this region finally disappeared by 2 months after birth 66.  Neurons in the subplate contain various  1  neurotransmitters early  and peptides  developmental  existing  ages 65. 1  and receive  synaptic  contacts  at  Although the role of these transiently  neurons in the subplate remains to be elucidated, it is  suggested that these neurons may be transient targets of geniculate afferents  and of callosal  projections  during  early developmental  stages while the axons are waiting to invade the cortical platens. The time course of disappearance of the alpha binding sites in the subplate  coincides  almost  exactly  with that of  subplate neurons,  suggesting that the receptors probably are located on these neurons or on terminals making transient contacts on them, and that these receptors may be involved in the novel functions of the subplate cells.  As alpha-2 receptors disappeared in this region much earlier  than alpha-1 type, the two types of receptors might play distinct 19  roles in the subplate. that either the two  The difference in time courses also indicates are located on separate cell populations on  subplate cells with different life-times or expression of the two types of receptors are regulated by distinct mechanisms. Data from operated animals provided further information about the regulation of the receptors.  Radioactivity of bound [ H]prazosin 3  doubled in the subplate from 29 to 79 days following an early L G N lesion.  Although it is difficult to estimate the exact extent of the  lesion  besides  L G N , it  adrenoceptors in the  should  subplate  be  were  safe  to  say  that  alpha-1  up-regulated by abolition of  subcortical inputs, particularly the geniculocortical input.  This up-  regulatory effect is not likely to be due to blockade of neuronal activity  stimulated  abolished  by  vision  retinogeniculate  since  input,  optic  did  not  tract affect  lesions, the  which binding.  Therefore, expression of alpha-1 receptors in the subplate may be regulated either by direct L G N afferents visual  including its basal non-  activity, or by other factors dependent on L G N activity.  Alternatively, since the density of binding in the subplate on the lesion side resembled that of animals at younger ages, it is possible that these "extra" a l binding sites are associated with a population of subplate cells that would have died under normal condition and that the  increased binding was  a consequence  of delayed cell death  following abolition of thalamic afferents on the lesion side.  This  latter possibility perhaps is less likely since Nissl staining did not show any notable difference between the two hemispheres in total number of subplate cells, a comparison of the number of subplate cells  in the  two  hemispheres  between these two possibilities.  shall be necessary  to distinguish  The fact that binding for a l receptor  sites increased after a L G N lesion strongly indicates that the majority of a l receptors in the subplate are not located on LGN terminals. The majority of a l receptors in the grey matter may also not be 20  associated with LGN terminals  since a L G N lesion only resulted in a  minor reduction (about 10%, p<0.01) of the binding density. this minor reduction was  Even  not seen in adulthood, suggesting that  either this small portion of a l  receptors only transiently exists on  LGN terminals or the subcortical regulatory capability for the cortical al  receptors is age-dependent. Alpha 2 binding sites were more sensitive to L G N lesions than  were alpha l's.  In the same operated animals, much larger portions  of [ H]rauwolscine binding sites were lost, particularly in the middle 3  and deep cortical layers, after LGN lesions.  It should be noted that  the rapid increase of cortical a2 receptors does not start until the end of  the  first  performed  month,  (Pll).  about  two  Abolishing  weeks  after  subcortical  the  operation  inputs  prior  to  was the  development  of receptors might be more effective in blocking the  expression.  Again, attenuated visual activity may be not responsible  for the loss of a2 receptors, since there was no reduction following an unilateral optic tract lesion. dependent,  The loss of a2 receptors was also age-  as adult L G N lesions had no significant  effect.  As  stimulation of a2 receptors results in reduced release of NA in the cortex, it is believed that some of these receptors are located on adrenergic terminals and cause an inhibition of NA release, i.e. serve as  autoreceptorsi76.  However, although the front cut should seven  adrenergic fibers of the dorsal bundle, no marked loss in a2 sites was observed in the operated animals at any age. lesion  did  not  entirely  abolish  This could be that the  adrenergic  fibers  despite  the  substantial loss in AChE staining, an index of the total ACh fibers in the same bundle . 9  Another possible explanation is the number of  autoreceptors is such a small proportion of the receptor number that any change  in this  subpopulation could not be detected by the  technique used here. As there was hemispheres  of  often a slight shrinkage (about operated side, decrease in the 21  10-20%) in the  binding might be  partially  due  to possible  cell  death.  However,  this  may  only  contribute a small portion to the total loss of binding sites since number of cortical cells in operated side did not seem much less than that in the control side as assessed by Nissl staining. The effect of interruption of cortical inputs on development of M l receptors has previously been reported by our group 3. It has been 17  shown  that isolation  afferents  of a portion of the visual cortex  (undercut,  see  next chapter  for  detail)  from all  prevents  the  redistribution of the receptor, resulting in a high density of M l sites remaining in layer IV as seen in younger animals. not observed after adult lesions.  This effect was  Undercut reduced the bindings of  [ H]prazosin and [ H]rauwolscine to a similar extent (data not shown) 3  3  as observed in young L G N lesion animals.  Differences in the effects  of cortical input blockade on development of M l receptor and alpha adrenoceptors in cat visual cortex suggests that expression of these sites is regulated in different ways. The present study showed both similarities and dissimilarities in the  development  adrenoceptors.  of  cortical  Ml  In summary, both  maximally expressed at young ages.  cholinoceptor  alpha and M l  and  alpha  receptors  are  These receptors show similar  changes in their laminar distribution patterns during  development.  Effects of blockade of subcortical input on these receptors are all age-dependent.  On the other hand, timetables of development of the  three receptors are quite different; they are therefore not colocalized at certain ages.  In some regions, e.g.  the subplate, M l and a 1  receptors never co-exist, indicating different roles played by the two. The effects of abolition of subcortical afferents on these receptors are quite different, suggesting that either they have different subcellular locations (e.g. terminals versus cell bodies) or that their expressions are regulated by distinct mechanisms. As both M l and a l  receptors are gates to the same PI second  22  messenger  pathway,  the  differences  in  development  leads  to  a  question of how these unique patterns match the development of the second messenger system to which they are coupled. be addressed in the following chapters.  23  This issue is to  Chapter Three POSTNATAL ONTOGENY OF PROTEIN KINASE C IN THE VISUAL CORTEX INTRODUCTION Binding of ACh or NA to M l or a l  receptors  initiates  the  intracellular signal cascade that results in activation of protein kinase C (PKC) via PI second messenger system.  The PKC family is one of  the kinases that are especially abundant in the brain, particularly in the  cerebral cortex  and hippocampus 1.138. 8  This kinase was first  reported in 1977 as a proteolytically activated kinase by Inoue al. .  ej  Two years later, it was further revealed that this kinase was  9 1  calcium-activated  and  phospholipid! .  The function of the enzyme in intracellular signal  84  its  activation  was  also  dependent  on  transduction became clearer when it was found that activity of the kinase was greatly increased by diacylglycerol, an early product of inositol  phospholipid hydrolysis  surface receptors' . 85  induced  by  stimulation  of  cell  Since then, six isozymes of the kinase (a, (31, [32,  y, e and Q have been isolated ,82,105,160 and two additional mRNAs (8 68  and e')i  42  proteins,  have been found in rat brain. PKC plays  important roles  By phosphorylating various in many cellular processes  including cell differentiation, neurite outgrowth, synapse formation and  receptor expression  and regulationi - ,53,69,90,96,99,1 18,1 19,137. 4  52  The development of PKC has been studied in the brain, and evidence shows that its activity varies with age?0,83,134,180,207. In the present work, the postnatal ontogeny of PKC in the visual cortex  was investigated  with polyclonal antibodies against  PKC . 8 4  The results show that PKC level in kitten visual cortex is not only regulated by developmental age, but is also use-dependent.  24  MATERIALS AND METHOD Animals Twenty-one cats of various ages (from postnatal day 1 to adult) were deeply anesthetized  and perfused through the left ventricle or  aorta with a 4% paraformaldehyde and 0.1% glutaraldehyde solution. The  perfused  brains  were  post-fixed  in  4%  paraformaldehyde  overnight at 4°C. Three cats were surgically manipulated before the perfusion. one  In  14-day-old kitten and a second adult cat, a portion of visual  cortex in one hemisphere was completely isolated from the rest of the brain by three  1 cm-deep scalpel cuts (undercut):  two cuts  extended from the midline to the lateral edge of the marginal gyrus, while the third cut ran parallel to the midline but was directed at a 4 5 ° angle in such a way that the white matter underlying the portion of  visual cortex was completely cut.  In another 14-day-old kitten,  the front cut was performed (see chapter one for detailed surgical description).  Both kittens were sacrificed at day 90.  The adult  undercut animal was allowed to survive for two weeks before it was perfused for immunocytochemical examination. Immunocytochemistry The  tissue was cut into 50 |im sections with a vibratome and  incubated with goat polyclonal antibodies against rat protein kinase C provided by F. Huang84 for periods ranging from overnight to 72 hours at 4°C.  The dilution of the antibodies varied from 1:2,000 to  1:12,000 depending on the age of kittens. antibody  concentration  increased  consistently  required  after  day 40.  for  In general, the minimum optimal  immunoreaction  In animals  that had been  operated at an early age, a 1:8000 dilution of the primary antibodies was  used  in order to increase  between the  operated  region  the contrast  in immunoreactivity  and the control areas. 25  Following  incubation 1:400)  for  with two  biotinylated hours  at  rabbit-anti-goat  room temperature,  antiserum the  (Biocan,  sections  were  processed with the avidin-biotin (ABC) system (Vector) for one hour at room temperature, and the immunoreaction was visualized with 0.01%  3\3'-diaminobenzidine  (DAB) and 0.003% H 0 . 2  2  Adjacent  sections were stained with cresyl violet to identify cortical layers. control  experiments,  normal goat serum was  antisera in adjacent sections.  substituted  for  In the  No immunoreactivity was found in  those sections, indicating that the immunostaining was not due to a cross  reaction  microscopic  of  secondery  observations  antibody.  Sections  used  for  light  were then dehydrated with ethanol and  mounted with DPX. Sections  for electron microscopic observations  were washed in  phosphate buffer (PB, pH7.4), and postfixed for 30-60 min in 1% osmium tetroxide dissolved in 0.1M PB (pH 7.4).  They were washed  again in PB, then dehydrated in ethanol at concentrations of 50% for 5 min, 70% (containing 1% uranyl acetate) for 20 min, 90% and 95% for 5 min each, and 100% (2 x 10 min).  They were then immersed in  propylene oxide (2 x 10 min) and finally embedded on glass slides in Araldite (Durcupan ACM;  Fluka) resin.  from the slides and reembedded.  Portions of interest were cut  Serial ultrathin sections were cut  on an ultramicrotome, mounted on Formvar-coated single-slot grids, and viewed under a Phillips 400 at 40 kv. C  26  RESULTS Light microscopic observations PKC-immunoreactive cells were mainly neurons rather than glia or other supportive cells based on the morphological characteristics of the cells. of  the  During postnatal development of the visual cortex, most  PKC-immunopositive neurons  pyramidal  cells  sometimes  of  found.  in  the  different  sizes,  although  Stained  cells  in  the  grey  matter  were  cells  were  matter  were  stellate white  concentrated in the subplate, especially in a region underneath the crown of the lateral gyrus.  Although the staining intensity varied in  individual cells, this may not be due solely to variations in antibody penetration of cells at different depths of tissue, since cells in the same focal plane also showed observed  variable density  by light microscopy.  Hence the  of staining  when  variation in staining  density was at least partly determined by the level of kinase in each cell.  The pattern of PKC immunoreaction in the visual cortex of  kittens varied with age as follows: Postnatal  day 1-4 (n=5).  In kittens of this age group, both area  17 and area 18 were heavily stained (Fig.8ia,b)).  In area 17 (Fig.8ia)),  the most densely stained cells were concentrated in layers II, III, V and VI. border  In addition, the densely packed cells and fibers at the region  between  layers  I  and  II  were  strongly  immunoreactive.  Cells in this region had small somata with unclear  profiles.  immunoreactive  No  originating from these somata. less remained until day 20.  processes  could  be  This densely-stained  visualized  zone more or  Moving from this region toward layers  II and III, the density of stained cells decreased and the cells in these  layers  dendrites.  showed  clearly-outlined  Layer IV showed  somata  with  less reaction product.  contained lightly-stained neurons and a matrix.  apical  This layer  The latter was also  seen in layers II and VI but not in layers III and V.  27  short  In area 18  (Fig.8 l b ) ) , border  layers II and III were most strongly stained.  of  layer  immunoreaction cells,  I/II,  product  particularly, were densely  many neuronal fibers  numerous  packed  were stained.  At the  cells  with  and, among  these  Many  pyramidal cells were found in layers II, III and V.  densely-stained Compared to the  neurons that were PKC-positive in area 17, immunostained cells in area 18 were larger, and the number of immunoreactive cells in layers II and III was much greater than in the corresponding layers of  area  17.  In both areas, the majority of stained  cells were  pyramidal, while in layer IV of area 17, cells with round cell bodies showed weaker immunoreactivity.  At this age, particularly on day 4,  areas 17 and 18 were also characterized by many large and small puncta that were densely stained and distributed mostly in layers V and VI (Fig.14). Layer I in both areas was pale but in some sections, a few small stained cells were sparsely scattered in this layer.  Postnatal Day 10-20 (n=2). were most densely stained.  In area 17 1-4,  layer IV appeared rather  Cells in this layer presented the least immunoreactivity if any  within the cortex, and the matrix was not stained. cells  layers V and VI  Layers II and III were lightly stained.  Unlike the staining pattern of day clear.  (Fig.82a)),  in  layer  IV of  area  18  (Fig. 8 2 b ) )  As in area 17,  appeared  the  least  immunoreactive, while layer I showed more intense staining than at earlier ages.  There were more neuronal fibers, that were probably  dendrites, exhibiting PKC immunoreactivity in both areas at this age than in one-day-old animals.  Another characteristic of the staining  pattern in the visual cortex at this age was the strong difference in intensity of immunoreactivity between areas 17 and 18 (Fig. 9).  The  reaction in the latter area was much stronger than in the former, both in the number of PKC-immunopositive neurons (especially in layer IV) and in the intensity of staining of individual cells.  This  difference remained to a smaller extent until at least postnatal day  28  90.  As shown in Figure 82a,b), the  restricted  to  the  periphery  of  the  immunoreaction nucleus  and  product to  was  dendrites  (particularly in proximal portion of the dendrites), in both area 17 and  area  18  of  10-day-old  animal.  Both  light  and  electron  microscopic observations (Fig. 12) suggested that the reaction product was concentrated in the perinuclear cytoplasm and dendrites but not in the nuclei. Postnatal  day 30-60 {n-6)  (Fig. 83a,b)). As seen at earlier ages,  cells in layer I V of both area 17 and area 18 in this age group presented  the  This difference  weakest immunoreactivity relative  to other laminae.  became greater after postnatal day 40, as evidenced  by a continual reduction in immunoreactivity in the middle layer which extended to the lower part of layer III and the upper part of layer V at later ages.  By counting number of immunoreactive cells in  randomly chose regions, it was estimated that about 40% to 50% of the neurons, mainly pyramidal cells of various sizes, in superficial (II and  III) and  deeper  immunoreactivity, while staining. higher  ( V and  V I ) layers,  exhibited  strong  a further 20% of the cells showed mild  Compared to the extent of immunoreaction at day 10, a proportion  of  the  most  densely  stained  neurons  concentrated in layers II and III in area 17 at these ages.  were  In area  18, by contrast, the relative intensity of staining in layers II and III decreased  and most of cells with the strongest immunoreactivity  were found in layers V and VI. mild staining.  Layer I in both areas exhibited only  Observations at higher magnification indicated that,  unlike the situation at day 10, the entire cell body and the dendrites were labelled in each PKC-immunopositive cell. Staining in a kitten at day 40 gave rise to the most striking observation among animals in this age group. were clearly stained in area 17.  Long apical dendrites  The dendrites originated from the  29  cells in layer V and bifurcated in layers II,III,and IV of the cortex (Fig. 84a) and Fig. 10 for high magnification).  Another interesting  finding at day 40 is shown in Figure 11 at higher magnification. Many PKC-immunopositive axon-like fibers were found in both area 17 and area 18.  These fibers, with swelling varicosities that were  densely stained by the antibodies, run vertically in bundles between layer III and layer V.  The long apical dendrites and axon-like fibers  were occasionally seen in animals of P30 but not at any other ages. Postnatal Day 90 (n=3)and Adult (n=5). The staining profile of the polyclonal antibodies against PKC at day 90 resembled that of adult cats (Fig. 85a,b,)). 95  No marked difference in immunoreactivity  between area 17 and area 18 was found in animals of these later ages.  In general, the extent of the PKC reaction was much less than  in the younger kittens, and a higher concentration of the primary antibodies was necessary to visualize the stained cells.  The PKC-  positive cells were distributed mainly in layer II, upper layer III, lower layer V and layer VI. immunoreactivity  at  antibody.  cells  Some  Cells in the middle layers showed little  normal  dilution  (1:4000)  of  in these laminae, however,  the  primary  were  weakly  immunoreactive at a dilution of 1:2000, while background staining remained the same.  Most of the stained cells at these ages were  pyramidal,  some  although  nonpyramidal cells  were  also  seen.  Examination by light microscopy indicated that the immunoreaction product in the majority of cells was located on the entire cell body, and probably on the membrane and in the cytoplasm, but not in the nucleus (although a few exceptions were noted). were found in a variety of laminae.  Numerous fibers  The diameters of these fibers  suggested that they were more likely dendrites than axons. supported by electron microscopic examinations (see  Electron microscopic observations  30  below).  This was  Cytoplasm.  Fig. 12 shows  an  electron  micrograph  of  an  immunopositive cell body in layer VI of the visual cortex of a 10day-old kitten labelled with the PKC antibodies.  It should be noted  that, in a young kitten, the brain tissue tended to be much more fragile than in an adult animal. achieve  Consequently, it was very difficult to  excellent preservation of  incubating  time required for the  the  tissue (especially  immunoreaction).  some very useful observations were made.  with  the  Nevertheless,  At the level of the cell  body, end-product could be found throughout the cytoplasm (Fig. 12).  The membranes of cytoplasm, mitochondria, Golgi apparatus,  and endoplasmic 12).  reticulum were immunoreactive (arrowheads, Fig.  This staining pattern of the cell body was present in every age  group studied, and no striking differences  could be found during  development. Dendrites. 13C  and  Positive staining was also found in dendrites (Figs. on  the  microtubules and on the membranes of dendritic mitochondria.  No  obvious dendrites  14D).  The  end-product  changes  in  the  could  be  found  was  distribution of among  seen  the  the  mainly  immunoreactivity in  different  ages  studied.  Occasional synapses which were PKC-negative were found contacting positive  dendrites  (Fig. 13C).  This type  of synapse  was  more  frequently encountered in the 30-60 day age group and in adult animals than in the group of newborn kittens (day 1-4).  This is not  surprising, since the overall number of synaptic contacts is very low in newborn kittens38.  When a positive dendrite received a synaptic  contact (Fig. 13C) the post-synaptic opacity was usually similar to or, in some cases, greater than that for a typical asymmetrical contact. Vesicle-containing  profiles.  During  development,  occasional  vesicle-containing profiles were found to be immunoreactive for PKC. Fig. 13A,B illustrates two positive terminals making synaptic contacts with  unlabelled  postsynaptic  elements.  31  In  such  profiles,  the  immunoreaction product was especially  present  in the  presynaptic terminal,  concentrated on the membranes of the synaptic vesicles.  It is interesting to note that many synapses with a positive terminal had adult features:  Terminals were usually large and contained  many synaptic vesicles;  they sometimes showed perforated synaptic  contacts (Fig 13A) and had a well-defined postsynaptic opacity (Fig 13B).  All synapses  with  immunoreactive  presynaptic  terminals  found at this age (P30) were judged to belong to the asymmetrical category. synaptic  Immunopositive contacts  newborn animals.  were  vesicle-containing  also  found  (but  profiles  much more  making  rarely) in  In these animals, however, it was difficult to  classify these synapses.  Despite extensive searches in older animals  (postnatal day 90 to adult), no PKC-positive terminals were found.  It  thus seems that there is a transient expression of PKC in synaptic terminals  which peaks  around postnatal  day 30-60 in cat visual  cortex. Undetermined  profiles.  Light  microscopic  observations  of  newborn kittens revealed that some large puncta (Fig. 14E) were heavily  labelled.  Electron  microscopic observations  showed  that  these large puncta were profiles that were different from the PKCpositive  dendrites  because they  were much more immunoreactive  for PKC, contained more mitochondria, and emitted branches (Fig. 14 A,B,C). Compare Figure 14A to D for example.  Although these two  profiles were taken from the very same section, the larger profile (Fig 14A) is much more immunoreactive and larger in size, and sends prolongations. Although we could not exclude  the possibility that  these large profiles were some type of mature dendrites, they were more likely immature ones since these profiles were not found in older animals.  The prolongations could also be growing heads of  dendritic growth cones, or be possibly dendritic spines, although no synapses or vesicle-containing profiles were seen in apposition to the ends of these branches.  Alternatively, these profiles could be growth  32  cones from axons.  Further investigations will be needed to identify  these profiles clearly. The presence of animals  (newborn  these elements was  observed  until postnatal group day 20)  microscopy and electron microscopy.  only  in young  under both light  In older animals, neither large  immunoreactive puncta nor profiles with appendages could be seen. Operated animals Figure 15 compares immunoreactivity in control regions with that in the part of the visual cortex that was surgically isolated from other areas of the brain on postnatal day 14 in the animal that was perfused on day 90. strikingly  The extent of reaction in the isolated area was  stronger than in the corresponding area of the control  hemisphere (not shown) and in the areas of the cortex surrounding the isolated zone.  The isolated zone had both a greater number of  PKC-positive cells and a higher density of staining.  Interestingly, this  increase in immunoreactive cells did not occur in layer IV.  Here the  low immunoreactivity level was comparable to that in the control tissue. isolated  Therefore, in spite of the increase in immunoreactivity in the area, the  unchanged.  laminar pattern of  the  staining  In contrast to these findings,  was  basically  no alteration in PKC  immunoreactivity was found in visual cortex that had been similarly isolated  in  adulthood95.  Immunostaining in the animal in which  modulatory inputs were interrupted by a front cut on day 14 also showed no difference  between the  operated  hemisphere  and the  control side (data not shown) when the animal was sacrificed on day 90.  33  DISCUSSION The polyclonal antibodies used in the present study were raised in a goat and against rat brain PKC by Dr. F. Huang at N I H . 84  antibodies  bind  to  both  purified or crude  rat brain  PKC and  incubation of these PKC preparations with the antibodies 100%  inhibition  of  the  enzyme  activity.  The  showed  Furthermore, these  antibodies preferentially inhibit type I and type II PKC isozymes with a lower titer against type III. It has been shown that the activity of PKC in rat brain is low at birth.  Thereafter, it gradually increases and reaches its maximum in  the first few weeks of postnatal Stichel ? 1  In cat visual cortex,  Hfe70.83,190,207.  reported that the activity of protein kinase C peaked at  8  about 5 weeks of age  and maintained this level  into adulthood.  Although the present immunoreaction data are not well suited for quantification of the activity of the kinase, it was noticed that the best immunostaining was obtained in tissues at ages from day 10 to day 40  and the immunoreactivity decreased  at later ages.  decrease in the immunoreactivity was shown by:  The  (1) the decline in  the number of stained neurons and in the intensity of the staining after  postnatal  day 40  immunoreactive  level  in the primary in  the  visual cortex,  hippocampus  cortical areas showed little change;  and other  while  the  associated  (2) the higher concentrations of  the antibodies needed to obtain results in tissue older than 50 days postnatal kittens).  (1:4000 for  adult  animals  vs.  1:10,000  for  younger  Neither treatment with Triton-100-X (0.3 to 1%) nor longer  incubation times (up to 72 hr.) noticeably improved the staining in adult tissue;  (3) with the 1:8000 dilution of the antibodies used for  the undercut animal on day 90, the isolated area of the visual cortex displayed good immunostaining while the immunoreaction in control areas  was  rather  poor  (again  consistent  immunoreactivity in the normal visual cortex  34  with  reduced  at later ages).  In  addition,  the  reduction  in  immunoreactivity  heterogeneous in different laminae, showing the middle layers (especially deeper layers.  at  later  ages  was  a greater decrease in  layer IV) than in the superficial and  Therefore, the reduction in immunoreactivity appears  to indeed reflect a decreased amount of PKC isozymes detectable by our  antibodies in adult visual cortex, although the effect of poorer  penetration of the antibodies in adult tissue cannot be completely ruled out. and  Similar results were observed by Stichel and  Singeri80,  in this laboratory (data not shown) with a monoclonal antibody  purchased from Amersham that appears to be more specific for PKC isozyme III .  Immunoreactivity of this latter monoclonal antibody  195  was found in certain populations of neurons only at younger ages and  decreased later in postnatal life.  It is not clear whether the  decrease in PKC immunoreactivity results from the disappearance of a particular population of cells or from the reduced expression of the kinase in these cells. earlier developmental  However, since cell death occurs mainly at stages, it is  more likely  that intracellular  levels of PKC (at least the isozyme(s) recognized by our antibodies) vary,  or  the  kinase  is  only  transiently  expressed  in  certain  populations of neurons in the postnatal visual cortex. One  of  the  regions  that  transiently  displayed  a PKC  immunoreaction product was the border of layers I/I I, presumable cortical  plate,  where  there  was  a group of cells  morphologically  immature in newborn kittens.  heavily  in neonatal  stained  kittens;  in  this  region  are  the  latest-generated  predominantly destined for layer IIH6.  This region  immunoreactivity was  gradually lost, finally disappearing by day 20. cells  that appeared was then  It is believed that ones,  and  are  Disappearance of this highly  immunoreactive region may result from the migration of the cells in the region. neuronal  PKC has been shown to be important in stimulation of  sprouting80,90  and in regulation of cytoskeletal  processes26.  In accordance with these functions, the high level of expression of 35  PKC in this population of cells in newborn kittens suggests that the kinase may participate in maturation of the neurons in this region. In  another  example  of  transient  appearance  of  PKC  immunoreactivity, the reaction product was seen in the long apical dendrites of some pyramidal cells in layer V only in animals of postnatal  ages four to seven weeks, particularly around day 40.  Interestingly, all89  the same type of cells were observed by Tsujino et  using a monoclonal antibody directed against PKC g-isozyme in  rat neocortex.  It is therefore possible that the PKC immunoreactivity  seen i n these dendrites represents  the g-subtype,  which has been  shown to be expressed slowly and does not reach its maximum level until  3-4  weeks postnatal  disappearance  o f the  in  stained  rat  Furthermore, the  brain70,83.  long apical dendrites  at later ages  suggests that this subtype o f PKC is transiently expressed particular population o f pyramidal cells, isozyme  i n this  o r alternatively, that the  changes its subcellular distribution during development o f  kitten visual cortex. More evidence  for the developmentally-regulated  expression o f  PKC at the subcellular level was provided by the electron microscopic observations.  A PKC immunoreaction product could be localized on  presynaptic terminals i n the cortex i n neonatal animals and was most frequently  encountered  around day 30-40.  It  was  not  found,  however, i n presynaptic terminals o f adult tissue^. On the postsynaptic side, PKC immunoreactivity was localized  i n dendrites,  i n perikarya, and  membranes o f both young and adult animals.  o n the  consistently postsynaptic  The finding o f the  large unclassified puncta with strong immunoreactivity is interesting. Morphologically,  they  resembled  dendrites  rather  than  axonal  terminals, but were characterized by prolongations and microtubules. In  particular, they  appeared only i n young kittens,  denser immunostaining than normal dendrites.  36  and showed  These features imply  that they were possibly growth cones of neurites containing high concentrations of PKC, whose roles in elongation process have been studied elsewhere It is  80,90,135.  unclear why the kinase is only transiently  presynaptic membrane, but persistently and sites  perikarya.  seen in the  found in postsynaptic  sites  The peak of expression of PKC at the presynaptic  (P30-40)  occurred  at  the  same  time  development of synapses in the visual cortex.  as  the  maximum  It has been shown  that the number of synapses increases during the first few weeks, and  peaks during the critical  Meanwhile,  period38.  use-dependent  adjustment of synapses to form ocular dominance columns also takes place at an early developmental stage. 4 weeks of between  age 3.60,87 4  appearance  It is most active at around 3-  and then decreases again.  of  presynaptic  PKC and  The correlation development  or  stabilization of synapses strongly argues that PKC in the presynaptic location  may participate in synapse  developing  visual cortex,  potentiation  (LTP) in the  as  has  formation or modification in  been  suggested for long  hippocampus!  10.1 15,1 22,123.  term  In LTP, the  neurotransmitter that is involved in synaptic modification is mainly glutamate .  Interestingly, all synapses with presynaptically located  37  PKC-immunoreactivity found in the visual cortex of young animals were classified containing  round vesicles  excitatory36, excitatory  as asymmetric.  i t can  On the assumption that terminals  and making  contacts  asymmetrical  be suggested that PKC-positive synapses use  neurotransmitter  within the cortex.  Furthermore, the  that the end-product is concentrated on the membranes vesicles  are  agrees to the  note that  synapsin  an fact  o f synaptic  I, a synaptic  vesicle  associated protein, is one of the substrates of PKC and implies that PKC  may  participate in neurotransmitter release during synaptic  formation in developing visual cortex.  A similar function for PKC has  been reported in LTP studies of the hippocampusi 9 . J  unlike the kinase  in  hippocampus  37  and  However,  cerebellum, where  a  high  plasticity remains and PKC has been reported to be present on both pre-  and  post-synaptic  membranes  in  adulthood  1  the  06,203,  presynaptically located PKC in the primary visual cortex appears to be  diminished once  the  use-dependent  synaptic  organization  is  established. It is interesting that the transient presynaptic location of PKC in the developing visual cortex is temporally correlated with transient expression  of  GAP-43-like  immunoreactivity in this  structure . 12  Since GAP-43 is known to be a substrate for P K C ^ . i s e , it would be interesting and important to determine if they are localized within the same specific terminal sites.  Double-label electron microscopic  studies would be required to answer this question. The argument that PKC is involved in use-dependent organization  is  more  undercut experiments.  directly  supported  by  the  synaptic  results  of  the  These results indicate that the number of  immunopositive cells in layers II, III, V and VI of the isolated area was much greater than in the control area for the animal that had been undercut at postnatal day 14, but not for the animal operated in adulthood. the  isolated  animals.  It was clear that the level of the immunoreactivity in area at day 90 closely  resembled  that in younger  Therefore, in this portion of the visual cortex, the decrease  in immunoreactivity of PKC that normally occurs during postnatal development was interrupted by the isolation at an early age.  This  suggests that neuronal activity from subcortical and/or other cortical areas is responsible for the decline in the level of PKC in the normal cat visual cortex.  This effect of afferent activity seems to be absent  in adulthood, at least as measured two weeks after the undercut . 95  The level of the kinase in the cortex may well be regulated by the input at an early postnatal developmental  stage, i.e.,  the critical  period, when synaptic organization in the visual cortex is most plastic and most susceptible to visual experience. 38  I hypothesize that PKC  may be involved in synaptic formation in developing visual cortex and, as a consequence  of establishment of the synaptic connections,  continuing neuronal activity then diminishes the level of the kinase. It is interesting to note that regulation of muscarinic receptors by input activity also appears to occur.  As mentioned earlier, [ H ] Q N B 3  binding in the isolated kitten visual cortex showed a laminar pattern that  resembles  that  of  immature animals.  This  will  be  fully  discussed later in Chapter 6. What component(s) in the external input to the visual cortex are responsible for regulation of the PKC level?  The undercut procedure  isolated the cortical area from two types of input, one from callosal afferents inputs  and from other cortical areas including neuromodulatory  of the forebrain, and the other from the thalamic nuclei,  primarily the lateral geniculate nucleus.  Since no effect was found in  the contralateral visual cortex (in which the callosal projection from the  operated  hemisphere  should  also  be  abolished  in  the  corresponding area of the unoperated cortex), the callosal fibers are unlikely to be involved in the regulatory effects of the undercut.  The  input from the forebrain also does not appears to be crucial, since the "front cut" severed  the  modulatory neuronal pathways  from the  forebrain early in life yet no alteration in PKC immunoreactivity was seen.  Hence, the component most likely to be  responsible is the  geniculate input. If the geniculate  activity directly regulated the level of PKC  detected by the current antibodies in the developing visual cortex, the layer most affected by the undercut would have been layer IV, the major termination lamina of the geniculate input.  As shown in  Fig. 15, there was no obvious difference in this layer between the isolated zone and the control areas, although the immunoreactivity in other laminae was strikingly higher and resembled the level found in younger animals.  Thus, it is more likely that the geniculate input  indirectly regulates the level of PKC in the visual cortex. 39  The lack of  geniculate input may result in reduced neuronal activity in layer IV of the isolated area, which alters the intrinsic neuronal activities of those superficial and deeper layers which are innervated by the cells in layer IV.  As a consequence of this change in activity, the decrease  of PKC in these laminae (which occurs in normal tissue) is blocked, and a high level of the kinase is maintained. PKC level could be regulated at various stages from the gene expression to the enzyme degradation.  An experimental study using  in situ hybridization with cDNAs for PKC mRNAs to detect the level of expression of the kinase in the developing visual cortex would be valuable. The results of immunocytochemistry from the animals operated as both young  and adults  also suggests that PKC labelled  by our  antibodies is localized in neurons of the visual cortex but not on the terminals of subcortical inputs or association fibers.  This is indicated  by the increase or lack of change in PKC immunoreactivity after undercutting of the visual cortex.  Although further  investigations  are necessary, the increase in the level of PKC immunoreactivity in the area isolated from neuronal activities of other CNS structures at early ages further suggests that the change developing experience  visual cortex  is  strongly  in PKC level  influenced  by the  of the animal, and is not determined solely  factors.  40  in the  postnatal by genetic  Chapter Four CALCIUM/CALMODULIN DEPENDENT KINASE II IN CAT VISUAL CORTEX AND ITS DEVELOPMENT INTRODUCTION PKC  is not the only kinase which undergoes  cortical development.  changes  during  Ca /calmodulin-dependent kinase II  (CAM-K  II) represents another important calcium dependent kinase.  This is  2+  an oligomeric enzyme consisting of subunits of various was first purified from rat brain in 1983 . 11  of  CAM-K  II,  the  a , f3/(3', y, and 5  it  Since then, five subunits subunits  have  been  Among them, the a and pVp" subunits are  isolated34,i32,i62,i88.  expressed  sizes6i.i04.  primarily in brain!88 in great abundance.  It is estimated  that the kinase represents 0.3% of total protein in brainii and is especially concentrated in postsynaptic densities, where it comprises up to 30-50% of the total protein 101.103. CAM-K II has spectrum of substrates and  fairly broad  a  including synapsin I, tyrosine hydroxylase,  m icrotu b u1 e-ass ociate d  protein  (MAP-  Accordingly, the kinase has been  2)34,102,104,132,162,164,183,194,196.  suggested to play important roles in many neuronal functions, such as  regulation  of  neurotransmitter  catecholamine release,  and  synthesis,  facilitation  strengthening  of  of  synaptic  transmission74,l02,i20,i22.  Considering the abundance of CAM-K II in the brain, its calciumdependent  activity,  and the  suggested important role  plays in neuronal plasticity in the  hippocampus3,i9,i20,i22,i35,  might expect that CAM-K II would be of  the visual cortex as an effector  signal  cascades.  postnatal  ontogeny  It  is  therefore  the kinase one  involved in the development  of calcium-related intracellular interesting  to  understand  of the kinase in kitten visual cortex  the  and to  compare its development with that of PKC and other elements in  41  calcium  dependent  signal  pathways.  Here,  a  monoclonal  against the oc-subunit of C A M - K  II  and  the  of  was  studied at both the light and electron microscopic levels.  postnatal  development  42  antibody  was utilized to localize the kinase immunoreactivity  of  the  kinase  METHODS AND MATERIALS Animals Fifteen kittens of various ages (postnatal day 1, day 4, day 14, day 15, day 24, day 30, day 40, day 90 and adult) were used in the experiments. completely  In one kitten, the lateral geniculate nucleus (LGN) was removed  thalamus at day 14.  by  an  extensive  lesion  of  the  ipsilateral  The kitten was allowed to survive for another  11 weeks before it was sacrificed at postnatal day 90. In  most cases, the brain tissue was prepared as described in  chapter two. were  In addition, one  perfused  with  propyl)carbodimide  500  ml  (Sigma)  14-day-old and two adult animals of  2%  l-ethyl-3-(3-dimethylamino-  and  2%  paraformaldehyde  followed  by another 500 ml of buffered (pH 7.4)  aldehyde  (EDC-PFA).  glutaraldehyde  into  in  PB  4% paraform-  We chose this method since adding 0.1% 4%  immunoreaction with the  paraformaldehyde antibody.  resulted  in  reduced  Compared with conventional  PFA perfusion, the EDC-PFA perfusion did not change the laminar pattern of the immunostaining (Fig. 16), tended to allow more cellular staining,  and  gave  better  preservation  of  the  ultrastructure.  Therefore, tissue prepared by this procedure was used for electron microscopy. Immunocytochemistry The monoclonal antibody against the cc-subunit of C A M kinase II was generously provided by Dr. M . Kennedy . 51  into 50 \im  sections  with  a vibratome  The tissue was cut  and incubated  with  the  antibody (1/500) and 4% normal horse serum in PB at 4°C overnight. The  sections were then processed with conventional procedures for  both light and electron microscopic, observations (see No  chapter two).  immunoreactivity was found in these sections when the primary  antibody was omitted during the process.  43  44  RESULTS CAM-K  II  representative positive  in  adult  section  neurons  cat  visual  illustrating  in  cat  cortex(n=4):  the  visual  Fig. 17a is a  distribution of cortex.  CAM-K  Neurons  II  with  immunoreactivity for CAM-K II were found in all cortical laminae in adult cat.  In particular, a group of nonpyramidal cells in lower layer  IV were strikingly stained (Fig. 17b) while some lightly labelled cells could be seen in upper layer IV and layer V.  Cells in layers II and  VI  In layers III and V,  also presented strong immunoreactivity.  there were a few large pyramidal cells with weak immunoreaction product. higher  In general, the incidence of nonpyramidal cells appeared among  pyramidal cells. The  the  total  population  of  labelled  neurons  than  No immunoreactive glia-like cells were encountered.  immunoreaction product was mainly present in cell bodies, and  dendrites.  Axons were not seen under light microscopic observation  although axonal terminals were found at the electron microscopic level (Fig. 22). CAM-K II in developing visual cortex Postnatal day 1-4 (n=3)(Fig. CAM-K  II-immunopositive  18a-b):  By postnatal day 4, strong  cells were found in layers V and VI,  although numerous lightly stained cells were also found in upper layers of the visual cortex.  Pyramidal cells in layer V probably are  the earliest cells expressing CAM-K II immunoreactivity since in one of the two animals studied at day 4, large pyramidal cells in layer V were more distinctly stained than other neurons (Fig. 18a) and this pattern somehow Neuropil  resembles  and matrix  in  that seen in a PI kitten (not the  middle  layers  also  showed  shown). strong  immunoreactivity, while the most superficial lamina (layer I) was rather pale at day 4.  Many bipolar-like cells in the white matter  were also found to be immunopositive. 45  Most of these cells were  present in the region closely adjacent to layer IV. Postnatal sizes  day 14-24 (n=3) (Fig. 18c,d): Many neurons of various  and morphologies  showed  animals of this age group.  CAM-K  II immunoreactivity in  These neurons were located through  layers II to VI with particular concentration in layers II to IV.  Many  pyramidal cells in layer III and V presented immunoreactivity, while non-pyramidal cells in layer II and IV were also stained. densely  Numerous  labelled particles of various sizes were seen in the visual  cortex  of this age group, especially  Fig. 19).  around day 24 (fig. 18d and  Most of these particles were found in the superficial and  middle cortical layers but not in the deep layers.  Under higher  magnification (Fig. 19), these particles appeared to be terminals of fibers, possibly dendritic growth cones and/or axonal terminals.  This  conclusion was supported by electron microscopic observations (data not shown). Postnatal day 30-40 (n=3) (Fig. 18e): Unlike the staining pattern observed at earlier ages, cells in layers V and VI appeared much less strongly IV/V,  immunoreactive at this age.  Along the border of layer  large pyramidal cells were occasionally positive for CAM-K II.  However, the immunoreaction in these cells was much weaker than that in cells of other layers. CAM-K small  Many large pyramidal cells with strong  II immunoreaction product were seen in layer III, while  non-pyramidal cells in lower  layer IV were also  densely  stained. Postnatal day 90 (n=l) (Fig.l8f):  The most surprising finding in  this animal was a reappearance of many immunopositive neurons in the deeper layers that were barely stained in animals at postnatal days 30-40.  These neurons were also seen in control hemisphere of  the operated animal (perfused at P90) and adult animals. the  intensity  of  the  immunostaining  Ill/upper layer IV and layer V. was most densely labelled.  was  weak  in  However,  lower  layer  A group of cells in deeper layer IV  This laminar pattern was almost identical  46  to that of adult animals. Operated  animal (n=l)  (Fig. 20): In the animal in which an  ipsilateral L G N lesion had been performed at 14 days of age and which  was  allowed  to  survive  until  immunoreactivity in the visual cortex between the two hemispheres. pattern  resembled  the  day  showed  90, striking  On the control side,  normal  adult  CAM-K  pattern,  II  differences the staining with  most  immunopositive neurons concentrated in layers II, deeper IV, and VI.  On the operated side, the number of cells that presented C A M - K  II immunoreaction product was approximately double that of the control cortex. least  However, upper layer IV and layer V still showed the  immunoreactivity,  suggesting  that  the  laminar  pattern  appropriate to the animal's age was maintained. Electron microscopic observations. Cytoplasm.  Figure  21 shows  electron  micrographs  of  immunopositive cell bodies taken from layer VI of the visual cortex of an adult cat ( A ) and of a 4-day-oId kitten (B) labelled with the CAM-K II antibody.  In the cell body, end-product could be found  throughout the cytoplasm but not on plasmic membrane. membranes  of  mitochondria, Golgi  apparatus,  and  While the endoplasmic  reticulum were immunoreactive, no obvious immunoreactivity could be found inside these cell organelles. The nucleoplasm also presented some degree of immunoreactivity. This staining pattern of the cell body  was  present  in every  age  group studied,  and no striking  differences could be found during development. Dendrites.  Positive staining was also found in dendrites at the  different ages studied.  In adult tissue (Fig. 22), the end-product was  seen mainly adjacent to the synaptic contact. It formed a dense and wide aggregate, which was many times larger than the non-labelled post-synaptic opacity. In addition, the kinase was localized along the 47  dendritic  microtubules  as  seen  on  transverse  (Fig.  22A)  and  longitudinal (Fig. 22B) sections of immunopositive dendrites. Dendrites  from young tissue showed  a different  pattern. The  postsynaptic density was not particularly labelled and the kinase did not seem to concentrate on the microtubules. In fact, the kinase was rather uniformly distributed in the dendritic trunk. Vesicle-containing profiles.  In the adult cortex, many vesicle-  containing profiles were found to be immunoreactive for C A M - K II. Figure 23a illustrates a positive terminal making synaptic with unlabelled postsynaptic cortex. of  contacts  elements in layer III of the visual  The end product was concentrated mainly on the membranes  the synaptic vesicles and mitochondria.  Adult immunopositive  terminals were usually large and contained many synaptic vesicles. When  the  visualising contacts,  synaptic the i.e.,  contact  synaptic the  was  cleft,  cut at an angle we  postsynaptic  found  appropriate for  primarily  membrane  had  a  asymmetrical well-defined  postsynaptic opacity (Fig 23a). Immunopositive  vesicle-containing  profiles  contacts were also found in younger animals.  making  synaptic  The kinase was always  present on the synaptic vesicle membranes. It is difficult to evaluate quantitatively  the  number  of  immunopositive  synapses  during  development but, since the number of positive varicosities is higher in the day  20-40  age group as indicated by both electron and light  microscopic observations, it is probably that the number of CAM-K II vesicle-containing profiles is much higher at these ages. Growth cones. At very young ages (day varicosities  1 and day 4 ) , large  were also found in the subplate region and layer VI.  Electron microscopic observations were profiles  which  contained  showed many  that these large puncta  vacuoles  and mitochondria  (Fig.23b). Ultrathin serial sections showed that many of them had a bulbous ending attached to a long tail. For these reasons, we believe that these profiles are neuronal growth cones. 4Q  DISCUSSION The  monoclonal antibody used in the study showed high affinity  to the a subunit of rat brain CAM-K II (50 kD) and recognizes a single protein band of the same molecular weight in crude rat brain homogenates^^.  Our results show that, in cat visual cortex, this  antibody gives rise to a specific immunoreaction of  and that intensity  staining seems to depend on concentrations of the antibody in a  range  of  1/2000  to  1/50  dilution.  The  pattern  of  immunoreactivity in the visual cortex varies with development. kinase  appears  developmental  to  be  highly  expressed  stages and to decline  at  thereafter,  early  the The  postnatal  at least in some  subpopulations of cortical cells, especially the pyramidal neurons in layer V. Subcellularly,  immunoreactivity  locations  that are closely  vitro.  It  has a  been  I,  facilitate  neurotransmitter  the  kinase  was  related with the functions  suggested  synapsin  of  synaptic  that  the  kinase  vesicle-associated release? . 4  found in  revealed / n  phosphorylates  phosphoproteini i , to  This is consistent with its  localization in presynaptic terminals and, especially, on membranes of synaptic vesicles. high rate  n.i6i  gelation.  The kinase can also phosphorylate MAP 2 at a  to suppress microtubule assembly and actin filament  This results in regulation of the neuronal cytoskeleton,  which can affect neurite outgrowth and modulate the shape of both dendrites  and  immunoreaction microtubules  dendritic product agree  spines of  with  the this  51. kinase possible  Our findings was  that  the  concentrated  on  function.  immunoreactivity was also found in the post-synaptic opacity.  Strong This  is consistent with the abundant evidence that CAM-K II is a major postsynaptic The  density protein 101,102.  pattern of immunoreactivity in adult cat visual cortex  similar to that found in monkey with the same antibody?5.  49  is  in the  monkey  visual cortex,  small nonpyramidal cells with  CAM-K  II  immunoreactivity were found in layers II,III, VI and low layer IV( IVCb) and few cells were found in layer V.  It is interesting to note  that, in cat visual cortex, cells in lower layer IV present strong immunoreactivity while this layer contains moderately-stained in  normal monkey  enucleation,  cells  visual of  this  cortex.  Particularly, after  sub-lamina that  were  cells  unilateral  driven  by  the  deprived eye showed an increased immunoreactivity? . 5  With a polyclonal antibody  62  or monoclonal antibodies  against  both a and P subunits , a different distribution of immunoreaction 144  product was found in rat visual cortex.  In these cases, the most  prominently stained neurons were pyramidal cells in layer V and the least prominently labelled were the cells in lower layer IV. distinct  distributions of immunoreaction product with the  The  various  antibodies may reflect differential expression of various subunits in different cell populations.  As the a and P subunits are present in a  4:1 ratio in rat forebrain and a 1:4 ratio in cerebellum . , it is 125  129  possible that the two subunits may also present in different cortical cell populations with different ratios.  Thus, cells that contain the  kinase, but with a high degree of p subunit composition, might show only weak staining or be undetectable with the antibody utilized in the  present  experiments.  Thus,  the  low  levels of  CAM-K  II-  immunoactivity found in pyramidal cells of adult cat visual cortex could be due to a decline in the ratio of a to p subunits in this cell type or to a lower overall level of the kinase. The  different  different  patterns  of  immunostaining  for  CAM-K  II  at  ages strongly imply that the kinase is involved in the  cortical development.  As many pyramidal cells in deep layers were  densely stained at early ages, and the staining of these neurons was reduced at a later stage of the critical period, the kinase may play a specific role in the development of this cell type. kinase  In addition, the  immunoreactivity was highly concentrated in growth cone50  like terminals at early postnatal ages.  The labelling in fact reached  its height at the peak of the critical period for cortical plasticity, suggesting  that  CAM-K  II  is  involved  synaptogenesis or the activity dependent  in  neurite  synapse  outgrowth,  elimination that  occurs at this time. It is interesting that PKC, another Ca -dependent protein kinase, ++  has  an  age-dependent,  but  different  subcellular  and  distribution in the developing cat visual cortex95,i80. in  laminar  I have shown  the previous chapter that PKC is mainly located in superficial  (II/III) and deep (V/VI) cortical layers but not in the middle layers (IV)  of adult cat visual cortex.  postsynaptic  In adult animals,  PKC was found at  locations only, and it was particularly concentrated on  cytoplasmic  membranes  immunoreactivity  was  of  cell  bodies,  rather weak.  where  However,  similarities in the distribution of the two kinases.  CAM-K  there  are  II  some  In young animals,  when PKC is transiently found at presynaptic locations, it is also concentrated on synaptic vesicles involved with making asymmetric synapses, just as is CAM-K  II.  These similarities  suggest that  presynaptically-located PKC and CAM-K II may both be involved in excitatory  neurotransmitter release.  Since  PKC was  presynaptically in visual cortical neurons near the  only found  height  of the  critical period (postnatal day 20-40) while presynaptic CAM-K II was found in both young and adult animals, the presynaptic PKC may play some special role in synaptic formation during the peak of synaptogenesis and CAM-K II may be involved in a more general way with neuronal plasticity in the cortex. As  early abolition of LGN input results  in menifest  increased  levels of both PKC (chapter 2) and CAM-K II immunoreactivities in the developing cortex, it is unlikely that these kinases are associated directly  with  the  thalamic  afferent  terminals,  probably both localized in intracortical neurons.  51  rather  they  are  Furthermore, these  results  indicate  that the  levels of  immunoreactivity of  the  two  kinases  during development  input.  Interestingly, the increase in C A M - K II immunoreactivity is  are down-regulated by the subcortical  seen in all cortical layers except layer V, implying that the level of the kinase in different layers is regulated by different factors.  As  mentioned earlier, the dependence of CAM-K II down-regulation on subcortical input has also been observed in monkey visual cortex . 75  The  elevation of CAM-K II immunoreactivity may reflect an increase  in level of the kinase, implying that the expression of the kinase is regulated  by  the  subcortical  activity.  Alternatively, since  the  antibody binds twice as well to the phosphorylated a subunit as to the  unphosphorylated  form *, so the increase in immunoreactivity 5  may reflect an increased ratio of phosphorylation/dephosphorylation of  the  kinase  in the  layers,  regulated by the LGN input.  suggesting  that this  ratio may be  To distinguish these two regulatory  mechanisms, measuring mRNA levels of the a subunit in control and lesioned hemispheres will be valuable.  52  Chapter Five POSTNATAL DEVELOPMENT OF INOSITOL 1,4,5-TRISPHOSPHATE RECEPTORS: A DISPARITY WITH PROTEIN KINASE C INTRODUCTION Calcium  dependent  protein  intracellular calcium levels.  kinases  are  activated  by  elevated  Intracellular calcium concentrations can  be raised by either Ca -influx from extracellular space or  Ca -  ++  release from intracellular pools. is  inositol  specific  One type of such intracellular stores  1,4,5-trisphosphate (IP3)-sensitive.  breakdown  of  interaction.  IP3 is generated by  phosphatidylinositol-4,5-bisphosphate  phospholipase  + +  C (PLC) is  This receptor-stimulated  activated  (PIP2) when a  by  receptor/ligand  phosphoinositide  first reported by Hokin and Hokin in 1955 . 78  turnover was  Based on accumulating  evidence from a variety of tissues, Berridge and Irvine suggested IP3 as a second messenger that links between receptor stimulation and increased  intracellular calcium^ .  This hypothesis has been widely  accepted.  Intracellular application of IP3 induces a calcium activated  5  potassium current in neuroblastoma cells raphe nucleus . 59  Aplysia  bag  IP3-induced C a  cells,  + +  and neurons of the dorsal  release was directly evidenced in  showed  which  77  a  long-lasting  intracellular calcium following IP3 injection . 55  IP3  have  been  cerebella63.  purified  and  cloned  from  Recently, rat  1 2 8  elevation  of  receptors  for  and  The molecular weight of the receptor protein  260 kDa and it possibly contains transmembrane regions  mouse is  about  that may  form the calcium channel intrinsic to the receptori .  Stimulation of  the  flux  28  purified IP3 receptors  (IP3R)  reconstituted lipid vesicles . 54  in  Purkinje cells,  receptor.  which  can cause C a  + +  through  The localization of IP3Rs is best known  have  Immunocytochemical  the  highest concentration  studies using an antibody  53  of  the  against  IP3R  showed  that the  endoplasmic surfaced can  receptors  reticulum  either  degradated  further (IP4)  tetrakisphosphate  second  various  types  Since  The  16,  pathway  acting  areas  between  PKC  is  the  of  glutamate  In  .  IP3  an  of  smooth  inositol-1,3,4,5-  into  or  inositol-5-  by  recently  reported  Ca  extracellular  to + +  act  as  influx in  To  visual  visual critical  act s y n e r g i s t i c a l l y  [ H]phorbol-12,13-dibutyrate 3  developing  kitten  visual  cortex  PI  3  (areas  54  in 17  is  enriched  in  some previous of  populations  it  turnover  has  3-11  were  during  18)  the  and  used  a  critical  IP3R  whether  PKC  to  [ H]IP3  and  localize  IP3  3  sections and  by  of  development  adjacent  also been  stimulated  development,  and  receptors  e s p e c i a l l y between  turnover  ([ H]PDBu)  contention,  suggesting  certain  whether  PI  activity  was  described  its p e a k  in c o r t i c a l  respectively,  ,  periodi34,i80.  to determine  may  PKC,  cortex,  reaches  glutamate-sensitive  2 0 6  been  cortex, the  receptor  attempt  receptors  in  this  PKC  that  the  of the PIP2  cellular  messenger  et al. reported  has  facilitate  limbs  regulate  second  expressed  cat the  to  IP3  and  transduction  may  demonstrate  two  As  of  and  (DG)  signal  elevation  other  IP3  that  receptors  bifurcating  calcium  the  them.  during  subtype  and  a  each  transiently  that, i n kitten  matches  to  lack  developing  postnatal  4 8  been  regulating  with  which  reported  period  in  Worley  rat brain,  independence  in  the  Generated IP3  nucleuses.  it is b e l i e v e d that the two  colocalization  in  weeks  DG,  cooperate  In  neurons  has  it leads  by  necessary.  chapters,  IP4  synergisticallyi36.  certain  of  inositol-1,4,5-trisphosphate-3-kinase  IP3-stimulated  a c t i v a t i o n o f PKC  of  the  other  o f PIP2 generates d i a c y l g l y c e r o l  breakdown  Since  evidence  possibly  inositol-1,4-phosphate  messenger  simultaneously  by  near  portions  o f cells3°,50,56,159.  the  pathway.  and  special  phosphorylated  by  into  p h o s p h a t a s e s 16,57. another  l o c a t e d on  membranes  structures, p a r t i c u l a r l y  be  signal  are  of  the  hippocampus.  MATERIALS AND METHODS Fourteen cats of various ages (from postnatal day 1 to adult) were used in the present experiment.  For characterization of  binding,  17 and 18)  the  visual cortex  (areas  was  [ H]IP3 3  dissected and  homogenized in 20 volumes of ice-cold buffer (50 mM Tris-HCl, pH 8.5, 1 mM EDTA).  Tissue was pelleted by centrifugation (10,000 g for  5 min.), and resuspended in 50 volumes of the buffer.  Saturation  binding assays were run by incubation with [ H]IP3 (NEN, 3  specific  activity: 17 Ci/mmol) at concentrations of 0.7 to 50 nM in 1 ml of the same buffer for 20 min. at 4°C, and were stopped by centrifugation (10,000 g for 1 min.). To evaluate non-specific unlabelled  binding, 10 mM of  IP3 (Sigma) was added to the incubation medium. The  pellets were washed in 1 ml of the buffer and resuspended in 2 ml of Formula 963 (NEN) for counting of radioactivity in a Beckman LS 2800 liquid scintillation counter. Brain  tissue for autoradiography was  chapter one.  prepared as described in  For the IP3 receptor, sections were incubated with 20  nM of [ H]IP3 in Tris buffer (50 mM Tris-HCl, pH 8.5, 1 mM EDTA) for 3  20 min. at 4°C, followed by 2x30 s wash in the same buffer. [ H]PDBu 3  binding, adjacent  For  sections were incubated with 20 nM  [ H]PDBu (NEN, specific activity: 13.2 Ci/mmol) in Tris-HCl, pH 7.4 for 3  6 hours, .a.t 4 ° C 3 4 followed by 3x10 min. washes in the same buffer. 1  Sections were rapidly dried in a stream of room air and apposed to LKB Ultrofilm for 7 weeks for [ H]IP3and four days for [ H ] P D B u . 3  The  autoradiograms  3  were analysed  described in chapter one.  with computer densitometry  In both cases, non-specific  as  binding was  less than 15% of the total binding as determined by adding 10 u.M of unlabelled  IP3 or unlabelled PDBu (both  incubation media.  55  from Sigma)  into  the  RESULTS The kinetic characteristics of [ H]IP3 binding are shown in figure 3  24.  Binding reached saturation above a concentration of 40 nM. An  Eadie-Hofstee plot of the saturation data indicates that [ H]IP3 binds 3  to a single class of sites with a Kd of 15.13+.1.28 nM and a Bmax of 2.38 +0.13  pmol/mg protein.  This value is compatible with previous  data from rat cerebella . 205  In newborn kittens, a low density of [ H]IP3 binding (Fig.25, 3  26)  was found in the superficial layers of the cortex. In general, however, few binding sites for [ H]IP3were observed until about postnatal day 3  20.  Between 20-30 days of age, the beginning of the critical period,  the density of [ H]IP3 binding sites rapidly increases in the visual 3  cortex  and other areas  Around  of the brain, including the hippocampus.  the peak of the critical period (P30-40), the superficial  cortical layers and layer IV were densely labelled, while layers V and VI showed the lowest density of silver grains.  The density of  the binding in the cortex was reduced in adulthood. Unlike IP3 receptors, the [ H]PDBu-binding site, labelling PKC, was 3  highly  expressed  in  newborn  kitten  distribution in early development  cortex  and  altered  its  to arrive at its mature cortical  laminar pattern by postnatal day 30-40.  The binding for [ H ] P D B u 3  increased during the critical period and then reduced in adulthood (Fig.25, 26).  Strikingly, the laminar distribution of [ H]PDBu binding 3  sites was quite different and actually somewhat complimentary to that of IP3 receptors in both developing and adult visual cortex.  The  lowest [ H]PDBu binding sites in animals older than P40 were found 3  in layer IV, the site of the highest density of IP3 receptors.  Similarly,  the density of [ H]IP3 binding was low in the superficial and deeper 3  layers where strong [ H]PDBu binding was present. 3  Development of both [ H]IP3 and [ H]PDBu binding in the visual 3  3  cortex were analysed quantitatively with computerized densitometry  56  (Fig. 26).  The binding for [ H]PDBu increased within first 3-4 weeks 3  of life whereas the increase in [ H ] I P 3 binding did not occur until 3  postnatal day 20. However, binding for both of the ligands peaked in the middle of the critical period and then declined in adulthood. Expression of [ H]IP3 binding sites in the visual cortex at postnatal 3  day 30-40 is about 9-fold that in newborn kittens, while binding of [ H ] P D B u only increased 1.6-fold from PI to the peak of critical 3  period.  In adult visual cortex, the total binding was reduced by _20%  for [ H]PDBu and by _45% for [ H]IP3, compared to their maximal 3  3  levels at P30-P40. In the hippocampus, the binding patterns of [ H]IP3 and [ H]PDBu 3  were also distinct and largely complementary.  3  Cresyl violet staining  showed that IP3-associated binding was predominantly localized in pyramidal cell regions while [ H]PDBu binding showed a bi-laminar 3  pattern in CA1 and CA2 (Fig. 25), concentrating in both pyramidal cell layers and in the dendritic arbor zone. seen in the dentate gyrus.  An extreme example was  At postnatal day 40, the IP3  receptors  were concentrated in the granule cell layer of the dentate gyrus.  In  contrast, [ H]PDBu-associated grains were almost absent in this zone 3  but instead strongly labelled the dendritic region of the granule cells. Developmentally,  IP3 receptors in the hippocampus also show a  different profile from that of PKC.  The [ H]IP3 binding was apparent 3  around postnatal day 30-40 in CA1, CA2 and the dentate but not in CA3.  The density  increased  of silver grains in the latter region gradually  subsequently.  By  comparison,  substantial  densities  of  [ H]PDBu binding were found in CA1-CA2 areas in kittens as young 3  as PI. Binding levels of [ H ] I P 3 and [ H]PDBu also 3  3  appeared to be  regulated by different factors in developing visual cortex (Fig. 27). In agreement with PKC immunocytochemical results, the binding for [ H]PDBu was elevated 3  in a visual cortical region that had been  surgically isolated from its neuronal connections at postnatal day 24, 57  or in the effect  hemisphere ipsilateral  was  However,  only no  observed  matter  when  in the  to a removal animals  of the L G N ,  operated  manipulations  at  were  a  3  58  young  done,  could be found on [ H]IP3 binding in the operated animals.  and this no  age. effect  DISCUSSION These results show that, although expressions of both IP3R.S and PKC in the visual cortex peak during the critical period, there are striking disparities. PKC  differ from each other in both location  profiles. The  and developmental  Moreover, in the visual cortex at least, they are regulated  by different  mechanisms.  mismatched  revealed ways:  In both visual cortex and hippocampus, IP3R and  locations  of  and [ H]PDBu  binding  3  [ H]IP3 3  by autoradiography can be explained  in two  alternative  IP3Rs and PKC may be in different populations of neuronal  cells; or, alternatively, they may be present in distinct subcellular locations, such as somata versus processes, in the same cells. visual cortex,  In the  the first possibility seems more likely, the staining  pattern of PKC immunoreaction in adult cat visual cortex described in chapter two is very similar to [ H]PDBu binding pattern and PKC 3  immunopositive cells were found principally in the superficial and deep layers but not in layer IV (see chapter two).  It is still possible,  however, that a PKC-negative somata in layer IV may have PKCpositive processes in superficial or deep layers.  The argument that  IP3Rs and PKC may be located in different populations of cells is also supported by the report from Worley et al.206 who showed that, in the external plexiform layer of the olfactory bulb and substantial gelatinosa, the density of [ H]PDBu binding sites is 100-fold higher 3  than that of [ H]IP3206. 3  possibility  However, in the hippocampus, the second  may be more likely, that the  two  second  messenger  receptors  are in the  same cells but are at different intracellular  locations.  [ H]PDBu labelled both somata regions and dendritic zones 3  in both. CA1-2 and the dentate gyrus of the adult hippocampus, while [ H]IP3 only bound to somata regions. 3  The [ H]PDBu-labelled PKC in 3  the dendritic zone is probably localized in the dendrites  of the  pyramidal neurons of CA1-2 and dentate granule cells but not in  59  axonal  terminals  of  input  immunocytochemical unpublished and  by  data  observations  indicating  quinolinic  This  fibers.  acid  in  labelling  cat  in cells,  Therefore,  portion  IP3Rs  are c o l o c a l i z e d i n somata o f h i p p o c a m p a l  two  i n the d e n d r i t e s ,  second  the  glutamatergic  PI  lateral  and  with  that  coincidences  imply  determining  the  in layer  information after  indeed  plays  period,  this  The with  a  IV; 2) peak  PKC  between  they  both  is  the  be  in  selective IV  process  may  these  in  acid  mainly  o f IP3  receptors  also  profile  of  formation  occur  elements  between  cortical  carrying  remains  probably  IP3Rs These  period.  visual  i n the cortex  are p o s t s y n a p t i c a l l y  PI  receptor-mediated  the s y n a p t i c  should  are  terminals  binding  receptors  i f ibotenic  IP3RS  receptor-regulated  formation  the IP3  be the  o n e o f the c r u c i a l  glutamatergic As  to IV,  o f the c r i t i c a l  be  the v i s u a l  layer  1)  glutamate  roles i n  believed  development  synaptic  and  that  cortical  significant:  the general  IP3  is  the c r i t i c a l  play  Considering  nucleus  at the height  that  role  during  in cortical  turnover  the c r i t i c a l  neurons  but not  terminals. l o w number  the r e l a t i v e  important  i n early  messenger cortex.  mismatch  a  PKC a n d  neurons, o n l y  that  terminates  may  isolation,  Hence,  in L G N  deplete  both  during  function.  geniculate  the LGN.  from  cortical  located.  while  peak  o f ibotenate-sensitive  turnover48_both  neurons  terminals)  which  PKC  indicating  mainly  i n layer  and of  cortical  coincidences  concentrated agrees  cortex,  o f normal  from  following  IP3RS  o f both  i n the v i s u a l  development input  a  than  (my  m e s s e n g e r targets at the s u b c e l l u l a r l e v e l .  Expression period  suggesting  by  hippocampus  l e s i o n s i n rat h i p p o c a m p u s ,  o f PDBu-binding204.  evidenced  rather  major  concentrated  is  system  high  level  cortical  transduction  o f PKC  pathways  suggests  development.  is not yet fully  Meanwhile, PKC  i n neonatal  may  at these  selectively ages  60  since  contrasted  IP3  is n o t as  that  Possibly,  functional  kittens,  the PIP2  i n newborn  act within PKC  other  second kitten signal  activity: can  be  regulated  by  many  The  c a s c a d e 137. some DG  neuronal  factors  fact that  cells  different that  sites  hydrolysis  cholinergic  brain  activated  on the c e l l  raises by  may  the p o s s i b i l i t y  two  populations  There  surface.  of phospholipid  receptors  acid  IP3Rs are not w e l l c o l o c a l i z e d w i t h PKC i n  i n adult  are a s y n c h r o n o u s l y  DG53, such as the a r a c h i d o n i c  beside  is some  following  not always  o f receptors  at  evidence indicating  stimulation  cause  I P 3 and  that  of  muscarinic  a c t i v a t i o n o f PKC  in  rat  hippocampus208. I 21a  should and  inverse  (data  the  important  o u t that  the c i n g u l a t e  colocalized discover  point  in some  gyrus,  not s h o w n ) .  circumstances  second  messenger  other  IP3  receptors  Further under system  relationship.  61  cortical  areas,  and  investigations  which exist  the in  two  such  PKC  as  were  area well  are n e c e s s a r y limbs  synchrony  of or  to this  i n an  Chapter Six GENERAL CONCLUSIONS AND DISCUSSION  In the present work, two Ca -related second ++  messenger  systems,  C a / C A M - K II and I P 3 / D G , were studied in developing visual cortex. 2 +  This  study  allows  two dimensions. (first  comparisons  of development  and localization in  First, those features of some cell surface receptors  messengers), M l and a l , can be compared with those of the  second messenger elements, C A M - K II, P K C and IP3Rs. already done profiles and  to some extent in previous chapters, the  and localizations  the  second  of  messenger  the  various  elements  Second, as development  neurotransmitter  can  be  compared  receptors one  with  another. Based  on such comparable analyses  of the  results  described in  previous chapters, the following conclusions can be drawn: 1) In adult visual cortex, although the neurotransmitter receptors,  al  and  M l are colocalized in superficial and deep layers, localization of  the  second  messenger receptors, P K C and IP3Rs, do not show good  coincidence, especially in layer IV. 2) The elements in recognized signal transduction pathways develop  synchronously in the cortex but are expressed  do not  in a specific  sequence. 3)  The development  afferent  of  neurotransmitter receptors  neuronal activity.  In addition, the  is  regulated  development  of  by  some  second messenger elements, such as P K C and C A M - K II, is also usedependent. 4)  While  intracortical  neurotransmitter cells  and  on  receptors the  can  be  located  within  terminals  of  extracortical  both  afferent  fibers, all three second messenger elements, P K C , IP3R and C A M - K II, are mainly localized in intrinsic cortical cells.  62  GENERAL DISCUSSION Technical 1.  considerations  Immunocytochemistry:  The  antibodies used in the investigation of PKC and C A M - K II  were provided by other laboratories and they against rat brain antigens.  were all originally  Since PKC and CAM-K II have never been  purified from cat brain, it was impossible to determine the affinities of  these antibodies to cat brain antigens although western blots of  the antibodies to cat brain homogenate might be helpful to roughly test the specificity of these antibodies.  However, treatment of brain  tissue for immunocytochemistry, such as perfusion and fixation, may have great effects on the affinity and specificity of an antibody to its antigen.  The two  antibodies  used  in  the  present  study  were  relatively insensitive to a PB buffer containing 4% paraformaldehyde. However, PKC polyclonal antibodies were more tolerant to perfusion of  4%  paraformaldehyde/0.1%  glutaraldehyde  while  the  immunoreaction of CAM-K II antibody was weakened by the same concentration of glutaraldehyde in cats (but not in kittens). to  obtain  maximal  ultrastructure  for  preservation  electron  of  both  microscopic  the  antigen  observation,  perfusion was used as described in chapter four.  In order and  EDC-PFA  Although EDC-PFA  perfusion did not alter lamination of the immunostaining of C A M - K II, possible changes in immunoreaction at ultrastructural level must be considered especially when the results were compared with that of  PKC immunocytochemistry.  Fortunately, there  is  not much  likelihood that this was the case since the results of C A M - K II immuno-electron microscopy with EDC-PFA did not of  some  kittens  glutaraldehyde.  perfused  In general,  with any  4%  differ from that  paraformaldehyde/0.1%  fixation  procedure may cause  translocation of some antigens and in addition, DAB reaction may nonspecifically organelles,  coat postsynaptic  caution  membrane and some intracellular  should be taken  63  to  interpret  the  manifested  locations of immunoreaction product. 2. Autoradiography a) Characterization and incubation condition For  economic  reason,  full  binding  characterization  of  some  radioactive ligands [ H]prazosin, [ H]rauwolscine, and [ H]pirenzepine 3  3  3  were not performed in homogenized cat visual cortex.  However, the  concentration of each tritiated ligand was determined in preliminary experiments  to obtain maximal ratio of specific/nonspecific  for autoradiography.  binding  In addition, incubation time for each ligand  was chosen, based on a time course obtained from these preliminary experiments, to ensure that binding reaches equilibrium. since  these preliminary experiments  However,  were normally conducted in  adult tissue, the chosen incubation conditions may not be optimal for tissues at different ages.  This variable should be taken account when  one compares autoradiograms of ligand binding in visual cortex at different ages. 3.Sample size and quantitative analysis Because of limited source of kittens, there were often not enough animals were available at each age, especially autoradiography. qualitative present  Small sample size may not seriously  conclusions  study  for experiments  of developmental  because  1) previous  affect  of the  changes drawn from the  experience  has  shown  that  variation of laminar distribution of a given ligand binding is usually small between different individual animals at same ages, 2) at some key stages of development, period)  and  P90  (end  such as P30-40 (peak of the critical of  the  critical  period),  results  of  autoradiograms in normal animals were similar to control side of operated animals at same ages, bring the total number of animals in each age group up to 3-4 animals. Major  caution  autoradiograms  should  with  be  taken  densitometry. 64  for  quantitative  Absolute  analysis  of  bound radioactivity  levels, therefore optical densities, do vary among individual animals, among  different  sections  from  the  same  experiments performed at different times.  animals  and  among  Thus, the developmental  profiles (e.g., Fig.3, 6, etc.) taken from a few animals at each age should only reflect tendencies of development of given binding sites. In addition, values of bound radioactivity did not directly indicate total number of binding sites since concentrations of ligands used were below saturation. As the isotope used in the study was H , a weak (3 ray source, the 3  factor of quenching also has to be taken into consideration.  It has  been well established that the white matter absorbs more p* rays emitted by  3  H than the grey matter, due to its greater density.  Particularly, as quenching is dependent on the degree of myelination which increases during maturation of the cerebral cortex.  Although  the time course of myelination process in cat visual cortex is not clear, the manifested decreases in binding of ligands at late ages could be partially due to an increased quenching of the radioactivity. Myelination caused quenching may be also partly responsible for apparent  reduction  of  radioactivity  in  layer  IV  during  late  development since this layer shows the heaviest myelination in adult visual cortex. estimated and  by  Therefore, the magnitudes of decreased binding sites densitometry  meanwhile,  the  measurement  increased  binding  could  be  density  overestimated during  early  development could be underestimated because of increased quench effect.  However, it is unlikely that all the decreased densities of  silver grains in the visual cortex at late ages were due to quench effect.  This is supported by several peaces of evidence: 1) densities  of silver grains for [ H]rauwolscine in hippocampus and entorhinal 3  cortex were actually increased in adult animals; 2) binding assay of [ H]IP3 3  with homogenized visual cortex also showed a decreased  Bmax in an adult animal compared with that in a P30 kitten (data not  shown); 3) in the case of [ H]PDBu binding, immunoreactivity of the 3  65  PKC antibodies also reduced at late ages and laminar pattern of PKC immunoreactive cells resembled that of [ H]PDBu binding, especially 3  in adult animals.  Finally, as all the ligands used in the present study  were tritium-labelled and they were applied on the same tissue, the quench factor should be the same, which allows the comparisons Explanations and speculations 1. Why mismatch? As shown in figure 28, the laminar distribution of IP3Rs  differs  from that of other three elements involved in PI turnover, in both neonatal and adult animals.  In the neonatal cortex, while both PKC  and M l receptors are colocalized in the middle layers, the level of IP3Rs  is  very  low  (Fig.  28  and  chapter  4),  concentrations being instead in superficial layers.  with  the  main  In adulthood, the  IP3Rs are found mainly in the middle cortical layer while the highest densities of PKC and the two surface receptors M l and a l are in the superficial layers. It is not uncommon that there are mismatches  in locations of  molecules that are expected to be functionally coupled in the central nervous  system? . 6  One may argue that the mismatches in locations  of IP3R and M l / a l receptors may not be surprising, as PIP2 turnover can be triggered by activation of a number of other receptors as well as  Ml  and  al39,47,57,72,lll,124,191.  If this were the case, however, a logical deduction would be that most phospholipid related cell surface receptors would be located in the middle cortical layer where IP3 receptors are concentrated and the M l / a l receptors would only be exceptions.  Although the laminar  distributions of many receptors are still unknown, receptors such as 5-HTic,  5-HT2 and CCK, that are known to be also linked to PI  turnover have  been  studied in our group and found to be only  transiently expressed in layer IV at young ages, low  66  concentrations  in the middle layer are found in adulthood. understand, on this  It is also difficult to  interpretation, why PKC, which may have a  broader spectrum for its endogenous activators 5 3 , is colocalized with Ml/al The  receptors but not with IP3R in adult visual cortex. question becomes more puzzling with the evidence that high  levels of inositol phosphates can be induced by stimulation of M l receptor in newborn kitten cortex48 and rat pups6 although we have IP3R sites at that age.  found few receptors  are  not  there?  stimulation-generated  Why is IP3 generated  One possibility  is  that  when  the  its  receptor  IP3 does not always act as a second messenger  but simply as a precursor of inositol-1,3,4,5-phosphate certain circumstances (such as in newborn cortex).  (IP4) under  This hypothesis is  based on an emerging understanding of the metabolic pathway of IP3.  It can be either rapidly metabolized to inositol-1,4-phosphate or  further phosphorylated to IP48,16,46,88,89,92. shows  that  Accumulating evidence  IP4 may also play a role in Ca  transduction as another intracellular C a  2 +  2+  -related  modulator.  Indeed, several  groups have reported a specific binding site for IP4 22,187. microsomes,  IP4 stimulates  independent  manner50.  intracellularly lasts  injected  30-60si59.  intracellular  signal in adrenal  calcium release in an I P 3 receptorin  identified  neurons  of  Aplysia,  IP4 causes an inward cation current which  These  results  suggest  that  an  IP4 may regulate excitability of neurons.  elevation  of  It is thus  possible that, in newborn visual cortex, or in some cell populations of adult brain, when the IP3Rs is not fully expressed, IP4 as a metabolic product of IP3 plays a major role.  In the neonatal cortex, stimulation  of M l receptors would result in increased IP3, which would then be rapidly transformed to IP4 to regulate the general excitability of the cells by altering ionic currents across the plasma membrane.  To test  this hypothesis, an investigation of the postnatal development of the IP4  receptor  and inositol-1,4,5-trisphosphate  3-kinase,  that phosphorylates IP3 to IP4, would be desirable.  67  the  enzyme  An alternative explanation is that some cell surface receptors are coupled with other phospholipids instead of PIP2.  In platelets and  vascular  inositol  smooth  muscle,  hormone  stimulated  lipid  metabolism may use PIP2 at the beginning and then switch over to hydrolyse  phosphatidylinositol  (PI)  or  phosphatidylinositol-4-  phosphate (PIP), which release DG and either inositol or inositol-1phosphate  (IP 1), but not I P 3 . 1 4  This pathway  therefore  towards the DG/PKC limb and away from the I P 3 / C a second messenger system. al  2+  biases  branch of this  As mentioned in chapter 4, both M l and  receptors may also couple to phospholipase D or phospholipase The former hydrolyses phosphatidylcholine to produce D G  A25,114.  without IP3 and the latter generates arachidonic acid. substances are activators of PKC On  Both of these  31,158.  the other hand, it should be pointed out that, despite  the  difference in the overall distributions, a certain proportion of IP3R s are  still colocalized with PKC and M l / a l  cortex.  receptors in the visual  The extent of the overlap is maximal during the critical  period (Fig. 28).  The I P 3 / C a  + +  branch of the PI turnover therefore  probably still takes part in the signal transduction in developing cortical  cells  to some  extent and may be particularly important  during the critical period.  2. Sequential programmed development of cortex. A major cause of mismatches discussed above is the asynchrony in  ontogeny  targets  in  of the receptors and their normal second the  developing  cortex.  Figure  29  messenger  compares  the  development of the four elements of the PI turnover system ( M l , a l , PKC, IP3R) in the cortex.  In superficial layers, PKC and a l  receptors  show relative high levels of expression at birth while M l and I P 3 receptors display a delay of 10-20 days before onset of increases.  In  the middle layer, there is a striking difference in the levels of PKC  68  and  IP3R in first 2-3 weeks.  The PKC level is above 80% of its  maximal at this age, while only 10 to 20 percent of IP3Rs have been However, in the next ten days, the number of IP3R  expressed.  dramatically increases time as PKC.  and reaches the maximal level at the same  As for cell surface receptors, the number of M l  receptors increases more rapidly than that of the a l receptors in the first two weeks and arrives at its peak at least three weeks earlier than alpha-1 receptor. development  of the  Unlike the superficial and middle layers,  four receptors  in the  deep layers  is fairly  synchronous, although PKC still shows the highest level at birth and peaks earlier than the others.  It is apparent, therefore, that both cell  surface and intracellular receptors develop at their own specific pace even though they work within the same signal transduction pathway. In  Table 3, several neurotransmitter receptors studied in this group  are listed to show the sequence at which their expressions peak in layer IV of the developing visual cortex.  T a b l e 3 Sequential development of cell surface receptors  Receptors  5-HTia M2  Ageofmaximal P w 4 expression in layer IV  Ml/pl/p2  Pw5 Pw6  5-HTic/2 a 1/2  G A B A A nAChR  PwlO  Pwl4  Pwll  Adult  What does this sequence mean to the maturation of the cortex? Selective formation of synapses in the visual cortex during cortical development  is  a complicated  process.  Besides  direct  visual  stimulation via the geniculate input, many other systems have been suggested to be involved, including spontaneous  activity of cortical  neurons, activity of extraocular muscles and the ascending reticular activating mediated  system, by  corresponding  etc.(review released  receptors  see60).  As these inputs are mainly  neurotransmitters and second  considered as effectors of these inputs. 69  messenger  or  modulators,  systems  can  be  Considering that subcortical  structures sequential  normally  mature  development  in  advance  of different  may, at least partly, reflect  of  the  cortex* ?, 4  the  types of cell surface  receptors  the sequence in which the  different  systems influence the developing cortex.  For example, in first two  weeks of life, M l receptors may be actively involved in the early development of cortex since at this age, cholinergic terminals show high levels of acetylcholine - , 45  and  the  muscarinic-related  148  PI  M l receptors are highly expressed turnover  is  already  functional . 48  Meanwhile, alpha adrenoceptors in the subplate at this stage may be important for interaction cells i .  fibers  and  subplate  Furthermore, since PKC levels are much higher than those of  6 5  IP3R's  between growing-in  in neonatal  stimulated  cortex,  the  intracellular signal  early  and adrenergic  transduction may be biased  DG/PKC pathway and IP3R related C a role at this stage.  cholinergic  + +  toward  release may not play a crucial  However, intracellular calcium levels may still be  elevated by stimulation of M l / a l  receptors since the accelerated PI  turnover can generate IP4 from IP3 to increase C a  + +  influx.  Voltage-  dependent calcium channels labelled with [ H]PN-200 are also highly 3  expressed in layer IV at this early stage . 42  The increased calcium  level may further activate CAM-K II which is highly expressed by the second week in layer IV. How the  is the sequential development of these receptors related with  morphological  cortex?  and physiological  Table 4 summarizes  visual cortex.  development  some developmental  of  the  visual  events in the  The postnatal development of the visual cortex can be  defined by two major stages, pre-critical and critical periods. the pre-critical period (postnatal  During  week 1-2 (Pwl-Pw2)), the cortical  lamination is still undergoing final  organization, axons from both  extracortical sources and intracortical neurons are still growing, and dendrites  of pyramidal cells in layers  developed . 139  II, III and V are poorly  At this stage, the development of the visual cortex is 70  ^  probably mainly controlled by genetic factors (natural eye opening is around P8 in kittens) although spontaneous activity of the cortex is also important.  The cortex at this age is undergoing a preparation for  the critical period when selective synaptic connection, modification and  stabilization  occur.  At  this  time,  consistent  with  the  morphological development, both PKC and CAM-K II, the final targets of  calcium-related signal  transduction pathways,  growth cone-like terminals and dendrites (see  are enriched in  chapter II and III).  High immunoreactivity of PKC is also present in the cortical plate in which cells are still migrating to form superficial layers at this early stage of development. During the critical period (Pw3-Pwl2), the number of synapses in the visual cortex increases dramatically in the first few weeks and declines by Pwl038,202. segregating  to  Meanwhile, afferent terminals from L G N are  form specific  according to activity driven  by  connections  with  cortical  either of the two  neurons  eyes85,86,i09.  in  addition, intracortical neuronal circuit is also built up to form specific "patches".  As consequences of synapse  formation and neuronal  circuit rewiring, cells show more specific response to their preferred visual stimuli and adult-like physiological properties of the visual cortex  emerge.  At this developmental  stage, the external visual  environment has the greatest influence on the cortex.  This influence  is imposed upon the visual cortex through several pathways only  retino-geniculo-cortical  activating  pathway)  various  sy Stems -67,109,151,152,167,168,174,181. 9  71  in the  nervous  system  (not by  neurotransmitter  Table 4 Morphological and physiological development of cat visual cortex Developmental stage  Pre-critical period (Postnatal week 1-2)  Critical period (Pw3-12)  Laminar formation  Layer V-VI: matured Layer IV: formed (by PW1) Layer ll-lll: forming (PW1-PW4) subplate: thick  all layers are matured subplate disappears by Pw9  Synaptogenesis low density of synapses in grey matter (7.5% of adulthood at PD1), appearance of dendritic spines (PD7-10), L G N projection  axons continue to develop and the terminals are segregated to form terminals form a continous band ocular dominance columns in layer IV  Physiological properties conduction velocity: orientation selectivity: receptive field: direction selectivity: ocular dominance: velocity selectivity:  References:38,io9,i47  highest by Pw6 then declines  slow present poor poor poor low speed  increases rapidly in Pw2-6 increase of high-selective cells starting to develop by Pw3 matured by Pw4 formed by Pw6-8 increase of high speed -responding cells during Pw4  i  In accordance with the extensive involvement of multiple systems in cortical development, most cell surface receptors are maximally expressed during the critical period.  In addition, second messenger  receptors are also present at maximal concentrations in the cortex (Fig. 29). densest  For instance, it is during this stage that IP3 shows its  binding  intracellular  to  calcium  receptors,  suggesting  pools  be  may  that the  actively  IP3-sensitive  involved.  More  importantly, distributions of  various receptors show the  overlap at this age (Fig.28).  Furthermore, PKC and C A M - K II are  72  maximal  concentrated vesicles  in both pre- and postsynaptic  and  cytoskeletal  elements  in  membranes,  terminals.  synaptic  Presumably,  phosphorylation of the corresponding proteins plays important roles in  regulating  neurotransmitter  release,  postsynaptic  dendritic spine and synapse modification, etc.  However, within the  critical period, the time at which receptors achieve expression varies.  responses,  their maximal  M l and 5-HTia receptors reach their peaks about  one month earlier than a 1/2 adrenoceptors and 5-HTic/2's (Table 3). It seems that different receptors may play their roles in turn during the  critical  expressions. expressing  period It  is  by  sequential  then  culmination  reasonable  to  of  suggest  their maximal that  the  early  receptors, such as M l , may be particularly involved in  induction of the plasticity and those late expressed may be more important for maintenance or turning off of the plasticity or for final tuning of neuronal circuits. It is interesting to notice that several receptors studied are first maximally expressed in layer IV and the densities of these receptors are reduced later in this layer which often receptor densities in the adult cortex. both  immunocytochemical 192  although,  as  mentioned  in  presents the lowest  This argument is supported by  and  "technical  autoradiographic  evidence  consideration", ' factor  of  quenching caused by increased myelination at late ages can not be ignored for autoradiographic results.  Layer IV bears unique features  of cortical development also in other aspects. retino-geniculo  As the gateway of the  pathway to the visual cortex, it shows the highest  concentration of the geniculate input as defined by both physiological and anatomical evidence^,86,109.  And, most interestingly, it displays  the most narrow temporal window for influence of visual experience on its synaptic formation.  In other words, the plasticity in layer IV  is only displayed around Pw3-7 and after which this layer loses its susceptibility layers,  to environment influence  in contrast,  especially  the  73  within a few  superficial  weeks; other  ones maintain their  plasticity to a certain extent in  This transiently  aduithood58,i3i.  displayed plasticity in layer IV seems to correlate with transient expression o f a number of receptors in the layer, further implicating the  involvement  of  these receptors  in cortical plasticity  during  development. 3. What decides the development of the receptors? As suggested above, cell surface receptors and second messenger targets develop in specific temporal and spatial patterns and this specific  sequence  of  development  of  the  molecules  for  signal  transduction may result in a series of modifications of morphology and physiology in the developing cortex and lead to maturation of the cortex.  The next obvious question is how this complex sequence  is governed?  What factor(s) decides this sequence?  Results from surgically manipulated animals in the present work throw some light on this question.  Manipulations performed in the  present work were designed to abolish two main types of inputs: i) modulatory afferents from the basal forebrain and hindbrain, and ii) subcortical afferents from the thalamus.  As shown in the previous  chapters, concentrations of both the neurotransmitter receptors and protein  kinases  subcortical  are  inputs,  more  or less  particularly  the  affected  by  removal  L G N afferents,  abolition of modulatory inputs (by the front cut). removal of either type of input had no effect in  adult  but  of  the  not by  In addition, cats.  For the  neurotransmitter receptors studied here and from previous work of our  groups,  isolation of the cortex from the subcortical inputs in  young animals mainly results in either reduction in numbers of binding  sites  process  of  or blockade  the  receptors.  of  the  normal  Unlike  the  laminar redistribution cell  surface  receptors,  concentrations of the two kinases, P K C and C A M - K II, are increased when the cortex is isolated from subcortical inputs.  74  Furthermore,  these  increases  are  not  homogenous  across  cortical  laminae,  indicating that the manipulations have selective effects on certain populations of neurons. These results strongly indicate that:  1) cortical levels of many  receptors (both cell surface and intracellular ones) are at least partly regulated  by extracortical activity  and are not solely  genetically  determined; 2) the effects are heterogeneously imposed on specific populations of cortical cells; and 3) the influence of input activity is age-dependent. Although the reduction in numbers of some receptors, nicotinic  ACh receptors  150  such as  and probably certain population of a 2  adrenoceptors, can occur simply because these receptors are located on the terminals of subcortical afferents surgery, activity  it  is  almost  completely  which degenerate after the  unknown  regulates cellular levels of  other  how  the  receptors  and  However, it is likely that the effects of deafferentation reciprocal  interactions  between  the  cell  surface  subcortical kinases.  are due to  receptors  and  kinases. It has been showed that phosphorylation of cell surface receptors is an important process to induce down-regulation of the receptors . 71  PKC has been found to be involved in down-regulation of both aadrenergic  and  muscarinic  Although immunocytochemistry  cholinergic  receptors35,96,ioo,i07,i54.  and autoradiography utilized in the  present work do not necessarily reflect activity of the kinases, an increased PKC level suggests the possibility of an extensive downregulation  of  deafferentation.  cell  surface  receptors  by  P K C following  the  On the other hand, little is known about metabolism  of PKC or CAM-K II and how extracellular signals affects cellular levels of the kinases (most work only measures changes in activity). However, some evidence shows that a second messenger may play a role in influencing the turnover of its target kinase.  In several non-  neural cell lines, prolonged high levels of cAMP results in enhanced 75  degradation  of  the  catalytic  subunit  of  the cAMP-dependent  kinase 153.  This phenomenon seems similar to the case of cell surface  receptors,  which  are  down-regulated  by  stimulation and up-regulated by denervation.  prolonged  agonist  It is thus conceivable  that metabolism of PKC is up-regulated by a possibly declining DG level following the changes in M l or a l receptors as a consequence of reduced cortical activity.  Similarly, CAM-K II turnover rate may be  regulated by intracellular calcium levels,  which may also decline  after isolation of the cortex from subcortical inputs. apparent correlation between development  As there is no  of the kinases  and any  particular receptor, it is likely that changes in the kinase levels are due to the combined effect of alterations in many receptors. Regardless of the mechanisms involved, the increases in the levels of the kinases after cortical deafferentation indicate that, in normal young kitten cortex, the kinase levels are tonically down-regulated by  subcortical inputs.  substrates  This suggests that many proteins that are  for the kinases  may tend to be in a dephosphorylated  state in highly activated cortical regions.  This is supported by the  finding that MAP 2, a substrate of PKC, CAM-K dependent  kinase (PKA),  II and cAMP-  is mostly phosphorylated in dark-reared  kittens and, once the kittens are exposed to light, the MAP 2 becomes dephosphorylated and can then be easily phosphorylated by PKA in vitro . 4  Interestingly,  dark-rearing  does  not  have  any  phosphorylation status of MAP 2 in adult cats . 4  effect  on  Similarly, cortical  levels of PKC are not regulated by deafferentation in adulthood. similar  age-dependence  adrenoceptors  and  other  is  also cell  seen  in  surface  the  regulation  receptors 3. 17  of  A  alpha  This age-  dependent feature in activity-regulation means that the metabolism of  these receptors  and kinases  are most  environment influences in immature cortex. 76  susceptible  to external  4.  What is the significance of this temporal plasticity in metabolism of the elements involved in signal transduction? of  the  metabolism  of cell  surface  receptors  The susceptibility  and coupled  second  messenger systems (and perhaps also plasma ion channel proteins), in my point of view, is an essential part of the biochemical basis underlying the plasticity manifested age.  in the visual cortex  at young  In other words, the so-called "critical period" for the visual  cortex  is  mainly  determined  by  the  period  of  time  when  the  metabolism of these molecules is most susceptible to influence of the external world.  Each receptor molecule (including ion channels) on  the cytoplasmic membrane can be thought of as an interface of the cell with the outside world.  Second messengers and their target  kinases work like a multi-channel-amplifier within the cell. channel represents of  the  amplifier,  a second messenger system. which  controls  Each  The overall output  intracellular reactions  causing  modifications in morphology and physiology of the cell in response to external stimulation, is determined by transfer functions the  channels  quantitative  and the and  overall  qualitative  input from the  combination  of  of each of  interfaces.  different  The  types  of  receptors and their spatial distribution on the cell surface constitutes a specific  conformation of the input to the amplifier.  Apparently,  any conformational change in the overall input (such as alteration in density of any particular type of receptor), or any modification in the transfer function (such as elevation in cellular level of any particular kinase) will alter the output of the amplifier, resulting in a change of the cellular response to a given stimulation. the  conformation  particularly,  the  and  transfer  alterations  functions  are  dependent  Since, as shown above, vary on  with the  age  and,  extracellular  environment, the output of the amplifier changes as a function of both age and environmental influence.  77  This leads to morphological  and physiological modifications of the cell during development and causes the manifestation of cortical susceptibility to the environment. It is  also predictable  receptors  that,  if the  conformation  and the transfer functions  of  cell  surface  of second messenger systems  are no longer dependent on external environment, as shown in adult cortex, the cortical cells will lose a great deal of plasticity in response to changes of environment. This is exactly the case in the adult visual cortex. However, alterations in the conformation and transfer functions are not solely dependent on the external environment.  The numbers  and distributions of some cell surface receptors, such as a l one),  are not or only slightly affected  activity.  by alterations  of cortical  In addition, IP3 receptors, as an element in our amplifier,  also show no change after such manipulations. output  (chapter  of the  signal transduction pathways  Hence, the overall  are undoubtedly  also  controlled by genetic factors. In summary, the present study chose some elements involved in calcium-dependent  signal  transduction  significance  of  signal  transduction  development  by  investigating  distribution  and  influence  the of  signal  membrane development study.  transduction  phospholipases,  in  postnatal  examine plasticity  the and  ontogenesis, cortical afferents  on  the  Of course, many other elements  pathways, play  to  cortical  extracortical  development of these molecules. within  pathways  such  equally  as  G proteins  important roles  of the cortex and they were not investigated  in  and the  in this  The results obtained here indicate that elements of signal  transduction, even though within the same pathway, do not develop synchronously and that the development  of these elements in the  visual cortex is regulated by both environmental and genetic factors. As a consequence of these developmental asynchronies, conformation of the interfaces,  which are composed of different 78  types of cell  surface vary  receptors,  with  age.  transduction cortex,  and  Regulated  pathways,  alter  total  their  output by  behavior  with  age.  pathways  most  susceptible  certain developmental "critical  the  As  and the to  second  total  individual cells,  environment is  of  as  their output the  messenger  output well  as  of  these  signal  the  entire  visual  responses  to  of  transduction  signal  environmental  79  external  influence  stages, the visual cortex is most plastic  period".  systems  at  at this  FIGURES: Figure 1,  Autoradiograms of [ H]prazosin (al) and [ H]rauwolscine 3  3  (a2) binding in developing visual cortex.  The animal age (postnatal  days) is given in the middle of each pair of adjacent sections.  Note  that the binding of both ligands demarcate the visual cortex (areas 17 and 18) from the subadjacent  cingulate cortex and area 19  laterally in kittens between 10 and 40 days of age.  The binding  sites are concentrated in subplate/white matter at neonatal ages (P0-P10).  The numbers beside the scale represent color coding of  optical densities. Scale bar=6 mm Figure 2,  Autoradiograms of [ H]pirenzepine binding for M l 3  muscarinic cholinergic receptors in visual cortex of kittens of various ages.  Conventions are the same as in Figure 1.  See text for a detailed  description. O.D., optical density. Scale bar=2 mm  80  Figure 3,  Development of alpha adrenoceptors and M l  cholinoceptors at different depths of the visual cortex. autoradiograms  The  in figure 1 and 2 were analysed using quantitative  densitometry and the optical density was calibrated to radioactivity of bound ligands with tritiated standards.  The cortical layers were  determined by comparing the autoradiograms stained with Cresyl violet.  to the same sections  Each point in the curves represents an  average of the measurements in at least four sections.  alpha-1 receptors  | I  1 PSO  POO  P7S  P120  Adult  Postnatal Day  alpha-2 receptors  I  1  i  P30  POO  P75  P120  Adult  Postnatal Day  1  I  M1 receptors  120 100 -  ao 60 -  Layer I III Layer IV Layer V VI P10  P20  P40  POO  Postnatal Day  82  P1?0  Adull  Figure 4,  Autoradiograms of [ H]prazosin 3  (alpha-1)  [ H]rauwolscine (alpha-2) binding in operated animals, 3  lesion combined with front cut at P14 with 4-weeks d, L G N lesion at P l l  with 10-weeks survival;  lesion at P10 with 7-weeks survival; adulthood with 10-weeks survival.  and a and b,  survival; c and  e and f, optic tract  g and h, L G N lesion in O.D., optical density  83  LGN  84  Figure 5, 4.  Densitometric analysis of the ligand binding data of figure  The optical densities were calibrated as described in figure  Note that, after early L G N  lesion and combined LGN/front cut  surgeries, the reduction in the radioactivity  is uniform in all  layers on the operated side except for subplate (labelled as SP) receptors (a and c). for a 2  3.  It is however  heterogeneous across the  receptors (b and d) with the greatest decrease in layer  85  cortical for a 1 layers IV.  86  Figure 6,  Comparison of development of alpha-1, alpha-2  adrenoceptors and M l cholinoceptors in various cortical laminae. Data are expressed as percentages of the maximal binding for each ligand in sections of the visual cortex at various ages.  It is clear that  the maximal binding in superficial layers for M l receptors is achieved around P60, which is later than in the middle and deep layers (around P40).  The numbers of the two alpha adrenoceptor  sites achieve their maxima in all laminae at the same age (around P75).  Meanwhile, in the superficial layers, development of all the  three ligand binding sites shows two stages in their rising phases with various latencies in first few weeks followed by a rapid increase.  However, in the middle and deep layers, only a2 receptor  sites show a 30-day latency in their development.  Each point  represents an average of measurements from at least four sections.  87  Layers I-III !  T  1  1  1  1  1  1  1  1  •  1  10 20 30 40 60 75 120 Adult Postnatal day  Layer IV  1  10 20 30 40 60 75 120 Adult Postnatal day  Layers V-VI  T  1  1  1  1 —  1  1  1  1  1  1  10 20 30 40 60 75 120 Adult Postnatal day  82  Figure 7,  Comparison of laminar distribution of the three receptors  in developing visual cortex.  In order to fit the binding density of the  three ligands into the same scale of the ordinate, radioactivity of each of the bound ligands is normalized as a ratio of the maximal value in the sections at each age.  Note that, except for postnatal day  1, the distribution of M l overlaps that of alpha receptors although the identical laminar patterns of M l and a l receptors are only seen in adulthood.  The two alpha receptors are colocalized in most layers.  However, at P30, despite a colocalization in the middle layer, the binding of [ H]prazosin and [ H]rauwolscine peaked in very different 3  3  layers (the outmost for a2 and the deepest for a l ) .  89  90  Figs. 8:  Light micrographs of immunostaining for protein kinase C in  area 17  (a)  and area 18 (b)  Cortical layers cells . 1 0  were determined by examining the morphology of the  Notice the pronounced decrease in immunoreactivity in both  areas during development. layer IV  of kitten visual cortex at various ages.  The immunostaining was  and then at later ages in other layers.  description.  1), postnatal day 1; 2), P10;  Scale bars=100 u m .  91  reduced first  in  See text for detailed  3) P 20; 4) P 40;  5) P  90.  5a-  4b ',"•'•«.'  5b  • • V . '  II/III  i  \  '  -.  / •  v. \  4  IV  ?2.2  • » ^ « «  Figure 9: Area 17 (top) and area 18 (bottom) of the visual cortex stained with the polyclonal antibodies at day 10.  The overall  immunoreactivity in area 18 is remarkably stronger than area 17. The laminar patterns, however, are similar in both areas. horizontally-oriented cells can be seen in the white matter.  Many Scale  bar=200 u.m. Figure 10: High magnification of immunostaining in area 17 of the visual cortex at day 40. densely stained.  Pyramidal cells in layers II, III and V were  Stained long apical dendrites originate from  pyramidal cells in layer V, bifurcate and terminate in various superficial cortical layers.  Arrows show such a dendrite bifurcating  in layer II/III and terminating in layer II; black arrow heads show another one bifurcating at the border of layers III/IV and terminating in layer III.  Empty arrow heads show some faintly  stained fibers with varicosities in layers IV, V and VI but not in the superficial layers.  Scale bar=100 (im.  Figure 11: Bundles of fibers with PKC immunoreactivity in upper layer VI of area 17 at day 40.  These fibers possess numerous  varicosities (arrows) which show high levels of PKC immunoreactivity.  Both pyramidal and nonpyramidal cells can be  seen to be PKC positive.  Scale bar=20 pm.  93  Figure day  12: Cytoplasm of a P K C  10 kitten  immunoreactive cell from a postnatal  visual cortex. Note that the end-product can be found  throughout the cytoplasm and that the membranes of the organelles  are  immunoreactive.  that the  cytoplasmic  staining  is more  membranes  obvious  Another feature are  between the cytoplasm and  membranes. This cell body development,  No  staining  stained (arrowheads).  nucleoplasm, possibly because of the better age studied.  of the  This of  observed at  striking differences were found during  m, mitochondria; er, endoplasmic reticulum.  95  is  the  preservation  staining pattern was  1 pm  cell  these every  postnatal Scale  bar=  Figure 13: Electron micrographs of immunoreactive profiles taken from the visual cortex of a postnatal day 30 kitten. A) An immunoreactive vesicle-containing profile (+) makes a "perforated" synaptic contact (arrows) with an immunonegative dendritic spine (-). This synapse is classified as asymmetric because of the  presence of the postsynaptic opacity. Note that another  immunonegative vesicle-containing profile (-) is in close apposition to the same postsynaptic target. Scale bar= 0.20 um B ) An immunopositive vesicle-containing profile (+) makes a synaptic contact (arrows) on a small immunonegative dendritic profile (-). Note also the well-defined postsynaptic opacity. Scale bar= 0.20 pm C) An immunoreactive dendritic profile (+) which receives a synaptic contact (arrows) from a immunonegative vesicles-containing profile (-). Note that microtubules, plasma membranes and portions of the mitochondria membranes are stained (arrowheads). Scale bar= 0.20 pm  97  Figure  14: Distribution of P K C  immunoreactive structures in a  postnatal day 4 kitten visual cortex. A , B , C ) Electron micrographs from serial sections through a structure that was highly immunoreactive (+) This  profile  for the antibody against  contains mitochondria (small  arrows)  PKC.  and microtubules  but does not have any synaptic vesicles and does not make any synaptic  contacts. The  structure  sends prolongations (large  arrows)  through the neuropil. These profiles could be found only in very young animals. Scale bars for all three pictures= 1pm D ) Electron micrograph of a profile lightly immunoreactive (+) PKC.  for  This micrograph has been taken from the same ultrathin section  as that shown in A). Note the immunoreaction is weaker  than the  profile shown in A) and that this profile is smaller in size. Because of the presence of the microtubules and of mitochondria, I  classified  this profile as a dendrite. Scale bar= 0.25 p m E)  H i g h power light micrograph of layer V in the visual cortex of a  postnatal day 4 kitten.  A few large pyramidal cells in layer V  shown  in the middle part of the picture are PKC-positive. Note the highly immunoreactive  puncta marked with arrowheads.  99  Scale bar=15  pm  /oo  Figure 15: PKC immunoreactivity in the visual cortex surgically isolated at day 14.  The immunoreaction was processed after  perfusion at postnatal day 90.  Square area in figure a) is shown as  figure b) with high magnification.  Immunoreactivity in layers II/III  and V/VI is much higher in isolated region (right side of b) than corresponding layers in the neighboring control areas and in the contralateral unoperated hemisphere (not shown).  Notice that the  staining in layer IV is not much different between the isolated region and control areas.  In a), Scale bar=300 pm; in b), Scale bar=200 pm.  101  !°2  Figure  16  Immunostaining of C A M - K II  antibody in tissues perfused  with two different protocols, E D C - P F A (upper panel) and P F A (lower panel).  Immunoreaction with E D A - P F A  stronger and more cells were stained.  103  only  perfusion tends to be  Scale bar=350 p m .  Figure 17, CAM-K II in cat visual cortex,  a) Neurons with strong  immunoreactivity are concentrated in layers II, III, VI and lower layer IV. The  Many dendritic fibers are present at the top of layer II.  strong, but diffuse immunostaining in layer I may partly  represent an edge artifact.  The strongest CAM-K II  immunoreactivity is present in nonpyramidal cells in lower layer IV. These cells compose a thin lamina with a thickness of 3 or 4 celllayers.  Scale bar= 200 pm; b) High magnification of nonpyramidal  cells in lower layer IV. Scale bar= 70 pm.  105  Fig  18, CAM-K II immunoreactivity in kitten visual cortex (area 17)  at various ages.  The numbers at upper right corner represent the  age at perfusion,  a) the pattern of the day 1-4 age group, in which  cells in superficial laminae show unclear outlines and the pyramidal cells in layer V are stained with the antibody.  Scale bar= 50 pm; b)  another pattern in the same group, in which the morphology of the cells in the superficial layers seems more mature.  Some large  pyramidal cells in layer V are still densely stained.  In general, the  immunoreaction in tissues of this age group is relatively weaker than that at later ages.  Scale bar= 50pm;  c) day 14: Many neurons show  strong immunoreactivity in both somata and fibers.  These neurons,  both pyramidal and nonpyramidal and of various sizes, are distributed over all cortical layers with the highest density in layers II-IV.  d) day 24: Many immunopositive neurons with numerous  densely stained particles can be seen at this age. concentrated in layers II-IV.  These particles are  Note that both large cells in upper  layer IV and small cells in lower layer IV are stained.  Large  pyramidal cells in layer V are still strongly immunoreactive, while staining in cells of layer VI has started to fade,  e) day 40: While  numerous neurons in the superficial layers are CAM-K II immunopositive, cells in layers V and VI show weak immunoreactivity.  Note that the staining of some large pyramidal  cells in layer III is weaker than that of nonpyramidal cells and small cells in lower layer IV are less densely stained than the large ones in upper layer IV.  f) day 90: The strong immunopositive cells are  localized in three bands, namely layer II/III, lower layer IV and layer VI. Scale bars=100 pm  107  Figure 19, High magnification photomicrograph taken from an area in upper layer IV of the animal at 24 days of age. are densely stained.  Numerous puncta  Many puncta are shown to be expanded  terminals of weakly stained fibers (thin arrows). presumably growth cones. approaching cell bodies.  They are  Thick arrows show some puncta Scale bar= 100pm  Figure 20, CAM-K II immunoreactivity in an animal with an early LGN lesion,  a) the control hemisphere, b) the operated hemisphere.  Note that there are more immunopositive cells in all layers but layer V on the operated side.  Scale bar=100pm  109  I vo  Figure 2 1 : Cytoplasm of CAM-KII immunoreactive neurons from an adult cat (left) and a postnatal day 4 kitten (right) visual cortex. Note that the end-product can be found throughout the cytoplasm and that the membranes of the endoplasmic reticulum (er) and of the mitochondria (m) are immunoreactive in both adult and young animals. However, no obvious immunoreactivity can be found inside these cell organelles or on the cell membranes, m, mitochondria; er, endoplasmic reticulum. Scale bar= 1 pm  111  M2  Figure 22: Electron micrographs of post-synaptic CAM-K II immunoreactive profiles taken from an adult cat (A and B) and a postnatal day 4 kitten (C) visual cortex. A: An immunonegative vesicle-containing profile makes a synaptic contact ( upper large arrow) with an immunopositive dendritic shaft. The end-product forms a dense and wide aggregate adjacent to the post-synaptic opacity: Compare the size of the post-synaptic opacity of immunonegative dendrites (lower large arrows). Microtubules are also immunoreactive (small arrows). Scale bar= 0.50 pm B: An immunopositive dendrite(*) cut transversely.  Note that the  immunoreactivity is concentrated in microtubules (small arrows) and in the membranes of the mitochondrion (arrowheads).  Scale bar=0.5  pm. C: An immunonegative vesicle-containing profile makes a synaptic contact (arrow) on an immunopositive dendritic profile. Note that the immunoreaction end-product in the  profiles is more uniformly  distributed than in A. Scale bar= 0.50 pm  113  Figure 23: Electron micrographs of presynaptic CAM-K II immunoreactive profiles taken from an adult cat (A) and a postnatal day 4 kitten (B) visual cortex. A: A vesicle-containing profile immunoreactive for CAM-K II (+) makes a synaptic contact (large arrow) on an immunonegative postsynaptic element. The synapse is classified as asymmetrical because the postsynaptic membrane has a well-defined postsynaptic opacity. The  end product is concentrated mainly on the membranes of the  synaptic vesicles (small arrows) and mitochondria. Note the small immunonegative vesicle-containing profile(-) located close to the CAM-KII terminal. Scale bar= 0.25 pm B: A large immunopositive nerve growth cone was found in the subplate region and in the layer VI. It contains many vacuoles and has a bulbous ending attached to a tail containing microfilaments. Scale bar= 0.5 pm.  115  lib  Figure 24, Saturation  Characterization of [ H ] I P 3 binding in cat visual cortex. 3  curve of the binding with Eadie-Hofstee plot (insert).  point represents  an average  of three  117  measurements.  Each  4  8000 -\  pH]IP(nM) 3  118  Figure 25,  Color coded optical density of autoradiography of [ H ] I P 3 3  and [ H ] P D B u binding in adjacent sections of the visual cortex and 3  hippocampus in developing kitten brain (left panel).  Optical density  of the area between the two lines was analysed for radioactivity, after calibration with H-standards (Amersham).3  shown in right panel.  T h e results are  In both the visual cortex and hippocampus,  binding of tritiated IP3 and P D B u (nCi/mg protein) peak at different laminar locations.  A t P40, the binding of [ H ] P D B u peaks in dendritic 3  regions (arrows) of the dentate gyrus and that of [ H ] I P 3 peaks in the 3  layer of granule cell somata. D, dentate gyrus.  119  O . D . , optical density.  I'liinmii  I'IIIII'I  t'liii-iiiii,  120  h'niii'i  Figure 26,  Development of [ H]IP3 and [ H]PDBu binding sites in  visual cortex. layers.  3  3  A) Comparison of the binding among different cortical  Development of IP3Rs shows a delay at early ages when  [ H]PDBu binding is increasing, although at different rates in the 3  various cortical layers. in developing cortex.  B) Comparison of binding of the two ligands Data are expressed as the percentages of the  maximal binding at all ages studied.  High levels of [ H]PDBu binding 3  sites exist at age PI while few IP3R sites are present until postnatal day 20.  PKC reaches its maximal levels earlier than IP3R, except in  layer IV where they peak at the same age.  121  122  Figure 27  Autoradiography of [ H]IP3 and [ H]PDBu binding in 3  3  undercut visual cortex (left) and the results of densitometry  (right).  The isolated zones are indicated by arrows. The binding of [ H]IP3 3  shows little difference between the undercut zone and the control hemisphere  (1.873±0.026 nCi/mg and 1.897+0.091 nCi/mg protein,  respectively), while the binding for [ H]PDBu in the isolated region 3  (211.092+12.044 nCi/mg) is more than twice (t=8.548, p<0.0001) that of the control side (105.237+2.879 nCi/mg).  123  O.I).  124  Figure 28,  Colocalization of receptors for PI turnover in developing  visual cortex.  The density of each ligand in cortical layers is  expressed as a percentage of the maximal binding density across all cortical laminae.  Notice the distributions of a l and IP3R completely  differ from that of M l and PKC  in newborn cortex and a high degree  of overlap are seen during the critical period.  In adults, despite the  colocalization of M l , a l and PKC, lamination of IP3R is still distinct. Figure 29,  Comparison of development of receptors for PI turnover  in the visual cortex.  Data for each autoradiographic are expressed in  percentages of the maximal binding density among animals with various ages. measurements.  Each point represents an average of at least four See text for details.  125  Postnatal Day 1  Mill  IV  V  VI  SP  Cortical Layer Postnatal D a y 30-40  60  40 4  20 J II  III  IV  V  VI  C o r t i c a l Layer  Adult  <rt NAR MIAChH PKC IPjR  I  II  III  IV  V  C o r t i c a l Layer  L a y e r s I - III  '  10 20 30 40 60 75 120 Postnatal Day  L a y e r IV  1  10  20 30 40 60 75 120 Postnatal Day  L a y e r s V - VI  1  10 20 30 40 60 75 120 Adult Postnatal Day  126  ACKN0WLELX3EMENT I  thank Dr. F. Huang and Dr. M . Kennedy for providing the P K C  CAM Dr. M .  K  II  antibodies.  I  also gratefully  acknowledge my  and  supervisor,  Cynader, for his many helpful and stimulating suggestions and  for the lesioned animals operated by him. 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