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Macro-glial specialization in the brain Thompson, Sharleen Grace 1986-12-31

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MACRO-GLIAL SPECIALIZATION IN THE BRAIN BY  SHARLEEN GRACE THOMPSON  B.  SC. The U n i v e r s i t y  A THESIS THE  SUBMITTED  of British  Columbia,  IN PARTIAL FULFILMENT  REQUIREMENTS MASTER  FOR  OF  THE  DEGREE  1976  OF  OF  SCIENCE  in  THE  DEPARTMENT OF  We  FACULTY  GRADUATE  PSYCHIATRY,  as  the required  UNIVERSITY  OF  OCTOBER  (c)Sharleen  Grace  STUDIES  D I V I S I O N OF  accept thesis to  THE  OF  NEUROSCIENCES  conforming standard  BRITISH  COLUMBIA  198 6  Thompson,  1986  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree a t the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may  be granted by the head o f  department o r by h i s or her r e p r e s e n t a t i v e s .  my  It is  understood t h a t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be allowed without my  permission.  Department o f The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  Date  written  THESIS This  thesis  examines  specialization. the  It starts  development  each  of  ideas  of  cells  glial  heterogeneity.  the  cells from  and  how  various both  The  between  different  The  results  indirect  of  to  as  both  numbers  from  experiment,  proline  demonstrated glial  cells  experiment  or  and the  glial  evidence  for  development  i n primary  in  cultures  similar  anatomical  of  be  distinct  i n other  alternate interpretation  of  thiamine  cell  been  and  of  direct  or  presented. the  cellular  results  show  not a l l  regional differences in  of  In  concept on  In  a  small  the  the  formation  dehydrogenase  i n only  a l l data  staining.  glutamate  types.  deficiency in rat brain.  i i  but  1-pyrroline  heterogeneity  an  The  oligodendrocytes  present  are  a n a l y s i s of  in rat brain.  are  provide  heterogeneity  alternate route  shown t o not  of  heterogeneity  morpholgically  cell  ornithine via  and  more  types  d i f f e r e n c e s have  oligodendrocytes  there  an  biochemical are  hemosiderin  and  and  how  in  different  provided  lines  of  demonstrating  increase  experiments which  i s an  oligodendrocytes density  description  spectacular recent  cell  for glial  localization  an  cell  types.  three  experiment  localization primary  of  cells,  for biochemical  that  cell  evidence  first  second  most  These  cells  historical  glial  technique  in different  among  for glial  morphologically  each  regions.  an  allowed  i n c r e a s i n g evidence in vivo,  found  The  about  various  glial  evidence  with  t e c h n o l o g i c a l advance  understanding  is  the  ABSTRACT  is  subset  of  third  i s used  to  biochemical  provide effects  The  c o n c l u s i o n summarizes the c o n t r i b u t i o n of the  experiments t o the a l r e a d y s t r o n g evidence heterogeneity  and  heterogeneity  r a t h e r than homogeneity c o u l d a f f e c t  the  neurosciences.  suggests  for g l i a l  ways t h a t assumptions of  glial research  TABLE OF CONTENTS T i t l e Page Thesis Abstract Table o f Contents L i s t of Tables L i s t of Figures Table o f A b b r e v i a t i o n s D e f i n i t i o n o f G l i a C e l l Types  i i i iv v vi v i i viii-xii  Introduction H i s t o r y o f Development o f Today's Ideas on S t r u c t u r e and F u n c t i o n Function of G l i a G l i a and N e u r o t r a n s m i t t e r s New Techniques E n a b l i n g Advances i n Understanding G l i a (a) T i s s u e C u l t u r e (b) Freeze F r a c t u r e Techniques (c) Markers i) Fibrous Protein ii) Glutamine Synthetase i i i ) Carbonic Anhydrase iv) Other Markers G l i a Heterogeneity-Morphological Developmental D i f f e r e n c e s - a source of Heterogeneity Heterogeneity i n Tissue Cultures (A) Developmental Changes i n C u l t u r e (B) C u l t u r e C o n d i t i o n s , Development and H e t e r o g e n e i t y (C) C e l l Development and D i f f e r e n t a t i o n i n Response t o I n j u r y (D) H e t e r o g e n e i t y Between D i f f e r e n t G l i a Not E x p l a i n e d By Development or C u l t u r a l Conditions H e t e r o g e n e i t y Between and W i t h i n G l i a C e l l L i n e s : D i f f e r e n t Areas L a b e l l i n g In V i v o D i f f e r e n c e s i n G l i a l C e l l s from D i f f e r e n t Areas o f t h e B r a i n  1 1 5 11 21 21 21 25 25 29 30 31 34 44 49 49 53 61 63 65 77  Summary o f Evidence f o r Biochemical Differences i n Glia  86  Experimental R a t i o n a l e and A b s t r a c t Experiment 1 Experiment 2 Experiment 3  88 91 117 127  Conclusion Acknowledgements References  143 144 145  - iv -  LIST  OF  Table  I  Minor  astrocyte  Table  II  Oligodendrocyte  Table  III  Effects cell  TABLES  cell  marker  and m y e l i n  of culture  markers  Pg.  3 2-33  Pg.  35-36  Pg.  59-60  c o n d i t i o n s on  characteristics  Table  IV  Comparative  values  o f glutamate  uptake  Pg.  70  Table  V  Comparative  values  of glutamine  uptake  Pg.  72  Table  VI  Comparative  values  of high  Pg.  73  affinity  GABA u p t a k e Table  VII  Iron  staining  Table  VIII  Enzyme  levels  deficient  i n r a t brain i n control,  and r e c o v e r e d  - v -  by  area  Pg.  115-116  thiamine rats  Pg.  136  LIST  Fig. 1  OF  FIGURES  Microscopic pictures  of iron  staining  in r a t brain. Fig. 2  Pg. 104,  Photographs o f whole s a g i t t a l  sections  of i r o n s t a i n i n g i n r a t b r a i n . Fig. 3  Pg. 108  Schematic diagram o f F i g . 2 showing c e l l u l a r types by area  Fig. 4  106  Pg. 110  H a l f photographs and h a l f schematic drawings o f c o r o n a l s e c t i o n o f iron staining i n r a t brain.  Fig. 5  Schematic r e p r e s e n t a t i o n  Pg. 112,  114  of the  c o n v e r s i o n o f p r o l i n e and o r n i t h i n e t o glutamate and GABA. Fig. 6  1-Pyrroline  dehydrogenase s t a i n i n g o f  Bergmann g l i a i n c e r e b e l l u m Fig. 7  1-Pyrroline  o f dentate gyrus.  Pg. 12 6  P r o l i n e oxidase s t a i n i n g o f Bergmann g l i a i n cerebellum  Fig. 9  Pg. 12 6  dehydrogenase s t a i n i n g  of a s t r o c y t e s Fig. 8  Pg. 124  Pg. 12 6  GABA-T s t a i n i n g i n thiamine deficient rat  - vi -  Pg. 142  TABLE OF ABBREVIATIONS FULL WORD ABBREVIATION 1ST PG. USED ACh 15 Acetylcholine Acetylcholinesterase AChE 15 Adenosine T r i p h o s p h a t e ATP 79 Adenosine-5-Triphosphatase ATPase 10 Calcium Ca++ 12 COMT Catechol-O-Methyl T r a n s f e r a s e 15 CNS 6 C e n t r a l Nervous System CAT Choline Acetyltransferase 89 CAMP C y c l i c Adenine Monophosphate 18 Diaminobenzaldehyde DAB 89 D i b u t y r y l C y c l i c Adenine Monophosphate dBcAMP 17 Dopamine DA 11 Electroencephalogram EEG 89 Gamma-Aminobutyric A c i d GABA 9 Gamma-Aminobutyric A c i d Transaminase GABA-T 15 G l i a l F i b r i l l a r y Acidic Protein GFAP 25 Glutamic A c i d Decarboxylase GAD 89 Glutamine Synthetase GS 16 Histamine Type I Receptor 18 HI Histamine Type I I Receptor H2 18 Magnesium Mg++ 76 Maximum V e l o c i t y o f R e a c t i o n Vmax 12 M i c h a e l i s Constant ( C o n c e n t r a t i o n of Substate a t 1/2 Vmax) KM 70 MAO Monoamine Oxidase 15 NAD 117 N i a c i n e Adenine D i n u c l e o t i d e NA Noradrenaline 11 117 O r n i t h i n e Oxo-Acid Aminotransferase OrnT 8 Potassium K+ 127 PT Pyrithiamine 1-Pyrroline-5-Carboxylate P5C 89 P y r r o l i n e - 5 - C a r b o x y l a t e Dehydrogenase Pro 89 1 - P y r r o l i n e Dehydrogenase PDH 89 Serotonin 5HT 11 Na+ 11 Sodium 127 TD Thiamine D e f i c i e n t TTP 128 Thiamine T r i p h o s p h a t e TCA CYCLE 17 T r i c a r b o x y l i c Acid Cycle  - vii -  DEFINITIONS OF GLIAL CELL TYPES OLIGODENDROCYTES  A c l a s s of g l i a c e l l  f i r s t s t a i n e d and  seen by G o l g i a f t e r he invented h i s s i l v e r s t a i n i n g technique. considerable  There i s  morphological  h e t e r o g e n e i t y w i t h i n t h i s group.  They  are t r a d i t i o n a l l y c l a s s i f i e d by where they a r e l o c a t e d and how they a s s o c i a t e w i t h o t h e r c e l l s o r by t h e i r n u c l e a r and c y t o p l a s t i c  ASTROCYTES  densities.  The second major c l a s s o f g l i a l They a r e l a r g e r than  cells.  oligodendrocytes,  have p a l e s t a i n i n g n u c l e i and e l e c t r o n l i g h t cytoplasm.  C a j a l , the f i r s t to  d e s c r i b e them d i v i d e d them i n t o two s u b c l a s s e s : f i b r o u s and p r o t o p l a s m i c , based on t h e presence  o r number o f  f i b e r s w i t h i n t h e c e l l body.  Those  d e s c r i b e d by C a j a l a r e now c o n s i d e r e d as £-ASTROCYTE  OOastrocytes.  May be i n t e r m e d i a t e type between an OC-astrocyte and l i g h t oligodendrocyte.  - viii -  MICROGLIA  A c l a s s of g l i a o r i g i n a l l y d e f i n e d by the s i l v e r carbonate method of Del Rio Hortega.  T h e i r o r i g i n and  c l a s s i f i c a t i o n i s highly controversial.  They a r e not d i s c u s s e d  i n t h i s paper. DISTINCT SUB-CLASSES OF GLIA BERGMANN GLIA  Also called Golgi E p i t h e l i a l  Cells;  they a r e g l i a l c e l l s w i t h c e l l  bodies  l o c a t e d i n o r j u s t below the P u r k i n j e cell  l a y e r o f the cerebellum  having  radiating fibers  and  extending  upward through t o t h e o u t e r s u r f a c e of the molecular  layer.  They share many  of the development and  biochemical  c h a r a c t e r i s t i c s o f g l i a but a l s o have many d i f f e r e n c e s . MULLER GLIA CELLS  A glia cell  i n the r e t i n a o f the eye.  Though not the o n l y g l i a c e l l eye,  i n the  they have been e x t e n s i v e l y  s t u d i e d and have c o n s i d e r a b l e  overlap  of c h a r a c t e r i s t i c s w i t h g l i a of the c e n t r a l nervous system.  - ix -  RADIAL GLIA CELLS  A developmental stage o f many g l i a c e l l s where t h e c e l l body has long arms extending p e r p e n d i c u l a r l y outer surface.  t o some  These r a d i a t i n g  fibers  may a s s i s t i n g u i d i n g o t h e r c e l l s t o their correct  place during  development.  In some areas the r a d i a l  form may p e r s i s t u n t i l adulthood.  The  Bergmann g l i a may be an example of this.  PITUICYTES  P i t u i c y t e s are g l i a l c e l l s i n the neurohypophysis.  They have many  c h a r a c t e r i s t i c of central EPENDYMAL CELLS  glia.  These g l i a - l i k e c e l l s l i n e t h e ventricular and  system w i t h i n  the brain  c e n t r a l canal of the s p i n a l  They may have s p e c i a l  cord.  function i n  b l o o d b r a i n b a r r i e r , and p r o d u c t i o n of cerebrospinal  fluid.  They have many  c h a r a c t e r i s t i c s o f g l i a c e l l s and may evolve from r a d i a l g l i a .  TANYCYTES  Specialized  g l i a with  processes that  radiating  l i n e the v e n t r i c l e ,  p a r t i c u l a r l y the t h i r d v e n t r i c l e . They have many g l i a  - x -  characteristics.  ENTERIC GLIA  G l i a - l i k e c e l l s of the e n t e r i c nervous system t h a t are more l i k e the c e n t r a l g l i a than the  peripheral  Schwann c e l l s SCHWANN CELLS  C e l l s of the p e r i p h e r a l nervous system t h a t wrap the p e r i p h e r a l nerves with l a y e r s of t h e i r e x t e r n a l membrane t o i n s u l a t e nerves from each In s p e c i a l cases, eye,  other.  such as those i n the  they grow w i t h the o p t i c nerve  i n t o the b r a i n and centrally.  are  located  Occasionally,  as i n the  Schwann c e l l s of the o l f a c t o r y nerve, they have p r o p e r t i e s s i m i l a r t o the c e n t r a l g l i a .  RESEARCH CELL TYPES GLIA CELL LINES  Permanent c e l l c u l t u r e s maintained i n l a b o r a t o r i e s and by t r a n s f o r m a t i o n or c h e m i c a l s .  originally  created  by c e r t a i n v i r u s e s  They are b e l i e v e d to  models of b r a i n tumors and  thus to  c e r t a i n c h a r a c t e r i s t i c s of  various  types of b r a i n tumors, and  are  e x t e n s i v e l y used i n r e s e a r c h  because  the l i n e s are s t a b l e and  be  purchased.  can  They have w e l l  defined  be  c h a r a c t e r i s t i c s which cannot be assumed t o be l i k e those o f untransformed some normal retained.  g l i a c e l l s i n v i v o but  c h a r a c t e r i s t i c s have been Some l i n e s , such as C - l or  C-6, may have c h a r a c t e r i s t i c s of gliomas, w h i l e o t h e r s may neural  PRIMARY CULTURES  resemble  tumors o r astrocytomas.  Cultures  recently derived  from  fetal  or neonatal b r a i n and c u l t u r e d f o r s h o r t p e r i o d s o f time.  During t h i s  time they develop through  several  changes o f morphology and b i o c h e m i c a l c h a r a c t e r i s t i c s t h a t can be manipulated by c u l t u r e  conditions.  They a r e thus u s e f u l i n t r y i n g t o understand t h e c h a r a c t e r i s t i c s o f glia.  Since c u l t u r e conditions are  never i d e n t i c a l t o those i n v i v o , many i n v i v o c h a r a c t e r i s t i c s never develop.  - xi i -  Introduction  T h i s t h e s i s examines g l i a l c e l l of  l i t e r a t u r e f o r evidence  g l i a l h e t e r o g e n e i t y and then p r e s e n t s my  specific glial  s t a i n i n g and the e f f e c t s of  m a n i p u l a t i o n on subsets of g l i a .  The  results  on  experimental  r e s u l t s show  c o n s i d e r a b l e evidence f o r m a c r o g l i a l h e t e r o g e n e i t y , both on a r e g i o n a l and a c e l l u l a r b a s i s . u n r e l a t e d procedures,  My  r e s e a r c h shows, i n two  t h a t o n l y c e r t a i n subsets of g l i a l  cell  are s t a i n e d , f u r t h e r s u p p o r t i n g the evidence f o r b i o c h e m i c a l d i f f e r e n c e s between g l i a l  cells.  The c u r r e n t r e s u l t s  t h a t g e n e r a l i z a t i o n from one g l i a l  system t o another  suggest i s no  longer v a l i d . If g l i a l  c e l l h e t e r o g e n i t y e x i s t s , why  l a t e i n coming, and why acceptance  of t h i s  i s the evidence  so  i s t h e r e tremendous r e s i s t a n c e t o the  idea?  Much o f our understanding  of g l i a l c e l l  on work done v e r y e a r l y i n t h i s c e n t u r y .  f u n c t i o n i s based  The  early  assumptions were so w e l l accepted t h a t more r e c e n t r e s u l t s have l a r g e l y been i g n o r e d by n e u r o s c i e n t i s t s .  Basic  n e u r o s c i e n c e t e x t s s t i l l do not devote more than a s m a l l amount o f space t o g l i a ,  g i v i n g l i t t l e more than a simple  d e s c r i p t i o n o f the b a s i c types and perhaps an h i s t o r i c a l on t h e i r  note  function. History of  Development  A l o o k a t the h i s t o r i c a l work done on g l i a i n t r o d u c e the t o p i c of g l i a l c e l l  -  1 -  will  s t r u c t u r a l and  serve t o  functional  heterogeneity.  The  e a r l y r e s e a r c h e r s f a c e d a number of  problems which l e d t o assumptions t h a t formed b i a s e s which prevent  now  the acceptance of some of the f i n d i n g s of  heterogeneity. Virchow i n 1846  was  n e u r o g l i a i n the b r a i n .  the f i r s t t o mention the e x i s t e n c e of He thought t h a t , s i n c e neurons d i d  not appear t o occupy a l l the space i n the b r a i n , t h e r e must be something h o l d i n g the neurons t o g e t h e r ; t h i s he c a l l e d  the  nerve g l u e , or n e u r o g l i a and the German word came t o be adopted.  He d i d not see t h a t the " g l u e " was  composed of c e l l s  because the e a r l y c e l l p r e s e r v a t i o n techniques  were crude and  n e u r o g l i a were the f i r s t c e l l s t o s w e l l and d i s i n t e g r a t e , which made them d i f f i c u l t t o see under l i g h t H i s t o r i c a l l y the main reason  f o r c o n c e n t r a t i o n on neurons  the d i f f i c u l t y i n s t u d y i n g g l i a .  The  was  f a c t t h a t the spaces  between the nerves were occupied by c e l l s now f i r s t observed  microscopes.  called glia  was  by G o l g i (1879) a f t e r he i n v e n t e d the G o l g i  s i l v e r s t a i n i n g method f o r those g l i a l c e l l s now  called  oligodendrocytes. In 1913 found  C a j a l i n v e n t e d the g o l d sublimate method which he  s t a i n e d another  type of non-neural  thus a l l o w i n g the d i f f e r e n t i a t i n g of two oligodendrodcytes,  cell,  the a s t r o c y t e ,  types of c e l l s ,  s t a i n e d b e s t by the G o l g i technique,  the and  the a s t r o c y t e . In 1919  d e l Rio Hortega invented the s i l v e r  carbonate  method which s t a i n e d m i c r o g l i a , the t h i r d major type of g l i a l cell.  Although  t h e r e were advances i n the understanding  the development of these c e l l s ,  -  of  t h e r e were no major a d d i t i o n s  2  -  to  the  understanding  separated, specific  the  of  electron  markers  d i s c u s s i o n of  have  research  surrounded  by  to  that  about  Oligodendrocytes have  been  where  are  come  are  was  cells  on  I have  not  and  cell  included  thesis  m i c r o g l i a and  first  because  they  I  are  controversy.  of  understood  the  brain  to  be  mass  two  l o c a t e d and  methods. how  they  small  (Varon,  in several different by  were  invented  microglia in this  now  20%  classified  they  the  considerable  comprise  until  available.  present  Oligodendrocytes  types  microscope  became  further no  cell  The  oval  cells  1978).  morphologies first  method  associate with  and is  by  other  cells. Oligodendrocytes  occur  intrafasicular  glial,  with  myelinated  may  groups appear  of as  association a  wide  range  Maxwell,  where  independent  with of  1968).  i n rows  fibers.  or  satellite  On  the  as  and  cytoplasmic  basis  these  and  medium  dark  oligodendrocytes,  dark, the  with  stages  (Mori  concurrent  density of  increasing  Leblond  of  Mori  developmental  the  organelles,  and  development  parallels  product,  the  of  In  cells  i n the  reductions  rough  size  The  oligodendrocytes,  3  -  of  are  to  have and  microscopic them  to  into  be  from and  light,  progessing light  to  increase  reticula  in  volume,  and  processes.  final  they  (Caley  i n cytoplasmic  number  myelination.  -  seem  endoplasmic  i n the  found  densities  classified which  matter, in close  are  electron  called  associated  grey  Leblond,1970)  reductions  reduction  mature  (1970)  and  nucleus,  complexity  are  Oligodendrocytes  densities, and  matter,  processes  nerve  neurons. nuclear  their  i n white  Golgi Their  developmental  c h a r a c t e r i z e d by  a  small  electron-dense  scant  and  dense  apparatus, lamellar  bodies and  microtubules  larger  than  electron  no  are 20  and  group  of  to  second  25%  h i g h l y developed  accumulate  protoplasmic  types,  of  with  astrocytes  are  noted  to  on  divided  have  et  a l . 1962),  cells  astrocyte characteristics.  following In the  the  when  glial  early  years  neuroglia. because  that  opposed  to  the view  their  Marinesco clearing glial  fibrous  fibrous  organized protoplasmic  variety  i s being  and  and  mature  and  These  glial  anoxic  into  s e v e r a l t h e o r i e s were  neurons  1896  a wide  heterogeneity  nourished  suggested  also  with  and  of  will  other  be  discussed  examined  in  a  section.  function of  were  are  under  well  are  nuclei  processes  microscope,  do  fully  and  glia.  tissue,  morphology  extensive,  (Palay  c l a s s e s of  staining  them  location,  astrocytes  more  cisternae  intracellular  brain  granules  electron  There  of  numberous  glycogen  the  filaments not.  Golgi  containing  major  1978)  have  based  Now,  the  have p a l e  originally  function.  with  reticula  processes  (Varon,  cytoplasm,  Cajal  eccentrically,  gliofilaments. the  conditions.  cytoplasmic  a  endoplasmic  oligodendrocytes,  light  filaments  with  rough  small  but  comprise  of  often positioned  frequently associated with a  Astrocytes They  cytoplasm  stacks  membranes,  nucleus,  Golgi he  observed  capillaries.  f u n c t i o n was suggested  of  cells  dying as  (1894)  to  that  In  give  they  neurons.  a  had  His,  4  -  forward  thought  that  that  they  1885  Weigart  had  structural a  f o r the  was  as  to  they  end  feet  (1895) support.  histolytic  i n 1887,  p r o v i d i n g guidance  -  put  role  the  growth  in  first of  In  the  to  nerve was  fibers during  the  first  to  embryological  speculate  detoxification  filter  suggested  that  they  compounds  secreted  nutritional dendrites their he for  s t i l l the  served  (1958)  function  between  blood  and  that  insulate of  glial  he  plants,  neurons.  nerve  Cajal  cells  and  ultrastructural  but  now  the  currently  assigned to  understand  to  glia  understand  that  glia  fiber  rejected  also  Golgi's  that rejected  f o r neurons packing  thought  electron  since  material  that  bundles.  intact until  the  split  they  These  Kory  et  al.  microscope  research.  the  understood by  he  (1913)  remained  also  believe  support  f i b e r s and  function  and  b a s i c a l l y the  Function  We  and  biochemical were  not  (19 07)  a  chemically  Lugaro  did  Lugaro  as  brain  and  endings.  to  they  isolated glial  stimulated  remove  because  roots  "noble"  to  nerve  in providing  more  concepts  like  thought  to  their  hypothesis  were  role  on  served by  development.  many o f  of  Glia  functions  functions the  their  glia  include  earlier  heterogeneity  of  are  complex,  some o f  researchers. basic  those In  functions  order  must  be  understood. The (1895), though  concept i s no the  s t r u c t u r a l support,  longer  s e r i o u s l y thought  historical  without  much  gliosis  has  neurons  as  mechanical  of  fact  elaboration. formed  they  are  scar more  disruption.  as  proposed  of  as  i s frequently In  fact,  tissue,  glia  suspectible They  do,  -  5  are  -  when  perhaps ischemia  however,  Weigart  function  mentioned  except  to  a  by  perform  in  even  texts  extensive softer  than  and several  structural fibers  functions.  i n the  brain  c e l l , membranes, myelin.  Glia  synchronous  and  The with  the  appear  most the  most  of  starts  at  4 months  age  i t starts  2;  This  serves  impulses  to  along  strengthening the  the  also  provide  have  nerve  membranes  rate of  rapid  to  fiber  over  of  a  warp  a l l outer  of  and  of  t i l l  about  grows  the  distally.  electrical  white  woof  and  matrix  dendrites  the  their  may  matter  like  surfaces of  connections  (CNS)  myelination  of  feet  between  their  structural  In  T h e i r end  connections  some t y p e s  conduction  fibers.  provide  and  some  of  and  humans,  body  nerve  is  continues  provides  bundles.  time  myelination  cell  saltatory and  system  the  In  the  layers  proliferation  delicate  do  sheaths  and  prior  neuronal  nerves  these  many t y p e s  just  wrap  many  nervous  g e s t a t i o n and  the  oligodendrocytes  with  do  the  speed  of  with  oligodendrocytes.  at  the  cord  central  rapid  differentiation about  o l i g o d e n d r o g l i a do  spinal  forming  first  myelination.  The  CNS.  They  plasma  provide  structural  support.  the  Mariesco's  original  removal  dying  absorb to  be  This at  the  by not  damaged  increase  mean t h a t  in size and  damaged  area.  from  thus  the  the  When and  They  glia  a  also  overlying  of  histolytic  be  correct  neurons,  that  invade  are  repairs  change  form  may  dying  macrophages  sites.  fibrous  brain  neurons  debris of  done does  of  proposal  not are  but  the  wall  scar  most  this  of  of  they  that  tissue  o f f the  at  the  cells.  may seems  damage. activity  proliferate,  they  damaged  leptomeningeal  - 6 -  glia  area  needed  for  in that  involved i n the  s t r u c t u r e so  tough  activity  are  core  area In  of  more of  the  the  fact  i t  was  this  scar tissue  structural  support  Cajal's fibers are  that  role  idea that  and bundles  the nerves  synaptic  mosaics  of small  similar  parcelling  terminals.  astrocytic  a  as t o prevent  (Lasansky,  specializations alterations  (Wolff  Lugaro's compared  non-continuous at  Golgi  areas  role  brain  (1894)  around  by  glial into A  cell  s u r f a c e s i n such  i n a haphazard  manner  membrane  continuous  dynamic  1978) a s i f a c t i v e l y  must  the astrocytic  go t h r o u g h they  o f incoming originally  They  involved  their  may  thought  to include several  involved i n neuronal  cell  end f e e t  but, because non-occluding  have  c a n be  the f i r s t  may  believed most i n and  opportunity  chemicals.  and n u t r i t i o n  (those  filter  a r e no l o n g e r  capillaries  has  be  neuronal  h a v e many  that  support  oligodendrocytes  other  of synaptic  of a detoxification  biochemical evolved  but the  o f t e n i n t e r v e n e between  barrier.  junctions,  the selection  each  Not only  up t h e n e u r o p i l  clusters  isolating  concept  a barrier  chemicals  today.  nerve  procedure.  t o t h e minor  provide  around  and Guldner,  (1907)  i n the blood  from  break  o f impulses  These  and i s o l a t e  containing a synaptic field.  processes  flow  supported  separated  a n d seem t o h a v e  the isolation  coming  highly  each  groups,  1971).  of a  f o r increased efficiency  occurs  Astrocytic  concept  to insulate  processes  regions  or neuronal  to  serve  i s s t i l l  types  play  glia  terminals are also Thin  in  us t h e o r i g i n a l  for glia.  myelinated  cells.  way  gave  different  nutrition:  7 -  the glia  provided  f o r t h e neurons.  bodies  -  that  This  concepts.  lying Freide,  near  Satellite  long  (1966)  idea  axons)  thought  may of  them  as  auxiliary  Astrocytic  end  substances  inward  brain  mass.  few  days  means the  are  but  metabolic  cell  Hertz lines  metabolic  the  to  that  enzymes,  although than  are  utilization white that can  matter the  be  be  that  nutritional  the  Energy  are  proportion  the  The  astrocytes total  Oligodendrocytes  have  astrocytes  (Pevsner,  uptake  by  increased  by  glia  and  2  survive or  rate are  brain a  Cummins  indicating  (K+) a  8  high  metabolic  environment.  -  or  -  This  maintaining  substance. glia  function, be  quite on  in  glial low  some t y p e s  Glia  have  is  the  reductive  of  protein a  protein  al.  (White  and  oxygen of  the  (1979)  radioactive metabolism  potassium  will  1975).  making  higher  et  brain  glia  in  a  oxidoreductase  consume much  1979). of  have in  the  erroneously  1982).  lower  the  than  of  given  also  oligodendrocytes. that  only  known t o  metabolism  and  of  e a r l y work done  (Hertz,  enzymes,  on  of  have  factors  involved  t i s s u e had  neurons  do  Jacobson,  i s now  neurons.  centre  surrounded  be  a  glia  assumptions  astrocytes  neurotransmitters, their  scar  of  suggests  1981).  and  cells  showed  oxidative  considerable Hertz,  glia  for glia.  of  synthesis  of  glial  majority  enzymes  not  classical  (1978)  rates  comparable  may  the  growth  of  transport  neurons  has  f a c t o r ( s ) must  rate  and  that  at  and  nerve  axons  i n the  Cultured  (Ebendal  the  glia  neurons  Medium  this  to  and  unless  growth  some s o l u b l e  high.  cases  glia  for  involved  metabolisms.  neurite  neurons  be  units  neurons  added.  Contrary the  to  many  without  extracts support  f e e t may  In  complimentary  metabolic  levels  oxygen showed markers  of  responsiveness  to  in  Glia of  may  not only  interact with  the i o n conductance  associated  with  and r e c e p t o r  neurons.  potential  that  7 0 - 9 0 mV,  and t h a t  according  t o the Nernst  may  have  varies  to of  0  and  kainic  reported  culture,  acid  acid  (GABA),  i n culture.  to depolarize  But substances  abolished  the depolarization  Kimelberg, receptors the  1984).  e f f l u x o f K+  that  amino  from  K+  D-  and  t h e amino lying  of glial  cells  the glial  cells  current.  c a n be thought  L-aspartate  acids  have  been  i n the vicinity K+  of  conductance  (Bowman a n d may  n o t have  but depolarized This  they  i n the presence  reversibly block  t h e neurons.  Thus  previously  depolarize  being  levels  astrocytes  L-glutamate,  acids,  membrane  o f neurons,  1979).  a property  In vivo  Therefore  f o r these  that  some  traditionally  of extracellular  a l l astrocytes  neurons.  that  (Pevzner,  They  have  a resting  the external  showed  t o neurons.  -aminobutyric  than  equation  (1984)  i n primary  be e x c l u s i v e  with  have  i n the production  Bowman a n d K i m e l b e r g depolarized  higher  b u t may  properties  Astrocytes  i s slightly  some r o l e  neurons  because o f  subject  i s s t i l l  controversial. Lugaro and of  catabolize many  glia the  (1907)  was  substances  substances  and understand functioning Glia  function  also  the f i r s t  that  the uptake.  K+  to clear  released  from  a r e removed  such  actions  that  nerves.  from  glia Now  we  the synaptic  t o be o f major  remove know  cleft  by  importance i n  brain.  seem  to control  as a f i n e tuning  of  to postulate  e x t r a c e l l u l a r K+  mechanism  I t c a n b e shown t h a t  the excess  that  leaks  -  9  levels.  a f t e r t h e neurons glia  could  out o f neurons  -  take  They do most  up  enough  b u t whether  they  a c t u a l l y do  neurons  causes  eventually  sheets  Glial  cells  Pollen, they  an  act  a  high  to  irregular  bodies  by  K+,  (Franck 1978, as  et  and  The  buffer ideally  demonstrated excess  a  because that  ouabain  an  active  not  cells,  increase level;  i n the  the  large  a  Third,  of  to  other  and  neurons  that  K+  and  content  an  because  and  the  1978,  i s as  i n K+ K+  diffusion; energy  as  increase  uptake  into  is further level  10  -  are  sensitive  homostasis  released this  K+  this  is has  i t has in  is  now  been  been  shown  respiration in  3-4  increased  and  from  removed  the high  or  weeks  astrocytes  beyond  K+  Grossman,  astroglia,  cells  may  1982).  excess  in astrocytes  -  a c t i v a t e d by  Hertz,  the  process  adenosine-5  ATPase  bulk-prepared  e x t r a c e l l u l a r K+ K+  uptake  e x t r a c e l l u l a r K+;  cultured  that  have  that  transient  i f the  This  for glia  requires  glial  into  role  through  microdissected  than  K+.  role  and  Schoffeniels,  (Walz  Second,  to  intense  excitable,  This  First,  build-up  astrocytes  and  to  leads  .  K+  for this  The  of  (Trachtenberg  suited  glia  a l . , 1978).  probably  1982)  of  is specifically  Grisar  cells,  (Hertz,  zone  diffusion  spread  many p r o c e s s e s .  K+  cultured  the  1978).  have  of  and  (Prince,  the  electrically  accepted.  surrounding  which  are  prerequisites.  unanimously  fire  from  selectively  et  to  released  r e s t i n g membrane p o t e n t i a l , a r e  ATPase  leads  to  minimize  a  are  with  Prince  three  that  as  (ATPase)  concept  neurons  by  and  a l . , 1978,  neuronal  has  K+  K+  i n e x t r a c e l l u l a r K+  neurons  active transport  triphosphatase  i n question.  dams r e s t r i c t i n g  Glia  permeable  be  as  acting  1970).  have  the  remove  thus  is s t i l l  increase  causes  glial  regions,  this  by  old  i s more an  resting membrane  potentials  u n e q u i v o c a l l y show t h a t  accumulate  large  that  a s t r o c y t e s have  cultures. glial  This  i n both  primary  cultures  exchange  uptake  inhibit;  this  and  by oubain, mechanism  mechanism  because  anhydrase,  take  take  cells  up  i s probably  evidence  A.  also  seem  a sodium  exists.  (Na+)  Although  does n o t  on c a r b o n i c  an i n h i b i t o r  of carbonic  uptake  into  and  abolished.  oubain  cells  t o b e i n v o l v e d i n many  function.  substances  some  As Lugaro  (1907)  r e l e a s e d by neurons.  into  of these  A c t i v e uptake  cultures  line)  dependant  neurons.  (Hertz  i n glial  of several cell  They  suggested, They  may  glia actively  a r e n o t always are capable  of  of  glia.  n e u r o t r a n s m i t t e r s has been  populations.  o f a s t r o c y t e s , Schousboe  up n o r a d r e n a l i n e  that  aspects of  n e u r o t r a n s m i t t e r s and t h e r e i s  o f r e c e p t o r s o n some t y p e s  demonstrated  cell  by  and N e u r o t r a n s m i t t e r s  t o t h e uptake  catabolizing  active  i s inhibited  that  which  up n e u r o t r a n s m i t t e r s by mechanisms  identical  take  exist  of  1982).  neurotransmitter may  uptake  studied glial  some p o t a s s i u m  Glia  Glial  must  showed  neurons i n  i t i s not completely  acetazolamide,  inhibits  Chaban,  ( a much  this  (1982)  criteria  c a t a l y z e d b y t h e Na+,K+-ATPase  another  anhydrase  than  the third  showed  are able to  and Chaban  rates higher  also  cells  cells  of astrocytes, indicating  i s reduced  Therefore  Hertz  satisfies  They C-6  o f K+.  uptake  thus  transport.  ouabain  -K+  amount  these  (1978)  (NA), dopamine  -  11 -  Using showed  primary that  glia  (DA), and s e r o t o n i n  can  (5HT) .  Hertz  dependent (1984a)  manner  neurons.  A high  Dennison, GABA  i s greater found  maximum  velocity  (1981)  been  also  or less  (Vmax)  than  into  cells into  into  of  rather  glia  than  has  been  1971, Matus and  exhibit  that  this  neurons.  into  a Vmax  uptake  eta l .  into on t h e  culture, neurons  that  and  Schousboe  o t h e r s and found  comparable  was  glial  based  (Ca++) o r l o w K+. o f many  uptake  than  i n primary  higher than  eta l .  Balcar  intense  calculated,  i n astrocytes  glial  Hansson  over whether  less  (1978)  t h e work  1981).  showed  glia  by calcium  astrocytes  eta l .  shown t o b e t a k e n up b y  be 2 t o 6 times  reviewed  seem t o  by s l i c e s  and Lungdahl,  and K e l l y ,  Schousboe  be increased  cultured brain  could  acids  of glycine  (Hokfelt  eta l .  also  and W i l k i n  astroglial  i s controversy  t h e uptake  whereas  rate  could  o f amino  a n d L a r s o n e t a l . (1980)  GABA  Hansson  f o r DA.  e t a l . (1982)  uptake  1976, C u r r i e  There  a n d K+.  i n a n energy-  1976).  has repeatedly  neurons,  the  affinity  occurred  neurotransmitters  the uptake  1971, Henn,  dependent.  (1982)  Na+  data  acid  Levi  demonstrated  (Henn,  (1984b)  both  this  was p r e d o m i n a n t l y i n t o  repeatedly  cells  that  that  this  o f t h e amino  showed  cerebellum  of  requiring  t a k e n up by g l i a .  (1982)  Na+  showed  d i d not confirm  Several be  (1982)  t o that  that found i n  slices.  L-Glutamate transport  a n d D- o r L - a s p a r t a t e  systems.  D-aspartate  into  High  glia  affinity  has been  uptake  repeatedly  autoradiographically  (Hokfeld  Kelly,  1975, McLennan,  1974, L a s h e r ,  -  appear  t o share  common  o f glutamate o r demonstrated  and L j u n g d a h l , 1972, Schon  12  -  1976, C u r r i e  and  and  Kelly,  1981). K+  The g l i a l  and i s both  uptake  energy  requires  t h e p r e s e n c e o f b o t h Na+ a n d  and temperature  dependent  (Schousboe,  1978) . Uptake cultured Weiler  o f glutamate has also  cell  lines  et al.,  astrocytes  1979).  al.,  1974, Henn  et  al.,  1970),  and et  al.,  1978).  including  retinal  Neal,  Muller  astrocytes  (Bruun  (1978)  uptake  astrocyte  with  release  into  cell  from  may  neurons  be t h e i r  some o f t h e o t h e r  glial  enough  the sole  to provide  transmitter  uptake  neutral  amino  acids  closely  related  that  fuel  cell  other  Iversen,  high  this  Houser,  than that f o r  enough  rate We  lines.  t o keep  pace  1979).  In  i s probably not know  that  phenomenom materials,  affinity  because and  uptake  into  glia.  B. A l t h o u g h t h e u p t a k e demonstrated,  there  not  to that  identical  of neurotransmitters  i s evidence that which  some  13  -  has  of this  occurs i n neurons  -  In  so that the  (Hertz,  glycine  no h i g h  cell  enough  source.  t h a n GABA  White  (Schousboe  and  or glial  i s not a general  have  Pfeiffer  t h e g l u t a m a t e Vmax f o r  source  lines  fuel  materials,  1977,  (Snodgrass and  be h i g h  and a l s o  i n glia  et al.,  was much h i g h e r  i t may  only  (Faivre-Baumann  and E h i n g e r , 1974,  sensory ganglia  lines  1974,  i n primary culture  showed  many  demonstrated i n  1979, a n d B a l c a r  i n primary culture  some  glutamate  cells  into  et al.,  centrifugation  astrocytomas  Hertz e t a l . ,  Schousboe  been  1974, B a l c a r  1976) a n d a s t r o c y t e s  1977b,  glutamate  I t has also  et al.,  demonstrated  1971, Henn  prepared by g r a d i e n t  et  1974),  (Hamberger,  been  been  uptake i s  i n either  character The  or  uptake  instance, (Hertz,  o f monoamines  i s a t lower  rates  into than  1982) b u t t h e u p t a k e  (Schousboe, lines  quantity.  1978a).  show h i g h  neurons  affinity  (Edwards  The  can also  and M a r t i n  exchange,  L-glutamic brain  and  a large  of glial  number  similar i n rate  cell  t o those of  found  different.  that  acid  For  example, 1  4-acetamido-4 -  stilbene,  an i n h i b i t o r  acid  uptake  by c u l t u r e d  glioma  cell  line  but d i d not a f f e c t synaptosomal  glutamate  tranport  systems  differ  of  of and r a t  uptake.  between  neurons  glia.  glial  glutamate  e t a l . (1982)  specificity  di-valent  cations.  a n d Ca++,  external  glutamate  was  found  whereas  inhibited.  Uptake  cell.  glutamate  that  more  susceptible  of both  o n mono- o r  i s dependant  Furthermore,  by g r a n u l e c e l l s  o f glutamate  i n contrast  i n terms  on b o t h  t o changes i n  astrocyte  uptake  t o be n o n - c o m p e t i t i v e l y i n h i b i t e d by  uptake  Glial  neuronal and  o f dependency  neuronal uptake  w a s c o u p l e d t o 1 Na+  granule  A  Only  concentrations.  D-aspartate  cortex  and i n terms  and i s t h e r e f o r e  ionic  report  carriers exhibit differences  substrate  of  i s higher  be q u i t e  (1983)  Ramaharo-Brandro  Na+  cultures  was a p o t e n t a n d s e l e c t i v e i n h i b i t o r  astrocytes  Therefore  neuronal  o f L-glutamate  uptakes  isothiocyano-2,2'-disulfonic anion  into  cultures, f o r  e t a l . , 1979).  mechanism  Waniewski  However  primary  cells  similar difference  i n astrocytes  ion i n contrast exhibited  t o neurons between  -  was  n o K+  (Drejer  competitively  from  to 2 f o r the induced release  of  e t a l . , 1982).  neuronal and g l i a l  14 -  prefrontal  uptake can  be  shown  f o r GABA u p t a k e .  -alanine neurons  f o r GABA u p t a k e  systems  Glial  release  from  that  into be  found  cells  o r neurons,  (Vernadakis  and  glial  enzymes,  and cases  has n o t been  cells  activity  can  (C-6)  has been  f o r GABA,  stain  (Schousboe however,  activities  (COMT) t h a n  demonstrated  and Sellstrom,  demonstrated  (AChE)  and S e l l s t r o m ,  (Silberstein et a l . ,  e t a l . , 1978),  brain  enzymes.  shown  i n several  brain. glial  to inactivate  1972). GABA  f o r GABA-T  e t a l . , 1972). than  1983) a n d l o w e r  15 -  i n whole  the ability  i n glia.  i s lower  (MAO)  1983) a n d  found  transaminase Bulk prepared (Sellstrom  a s do a s t r o c y t e s  -  of the  monoamine o x i d a s e  suggests they have  astrocytes  cultures,  (Hansson  catabolic  lines of glial  transferase  has been  Tardy  neonatal  t h e t w o GABA  1980).  d e g r a d a t i v e enzyme  cultured  1977,  clonal  o f COMT a n d MAO  catecholamines  (GABA-T),  but not  t o b e d i f f e r e n t i n some  possess  e t a l . , 1976, Hansson  l i n e s which  The  that  blocker  some n e u r o t r a n s m i t t e r s  acetylcholinesterase  catabolizing  presence  cell  i n glia  i s a  Thus  showed  different.  possess higher s p e c i f i c  catchol-O-methyl The  neurons.  (Ach) u p t a k e  and Arnold,  also  monoamine  derivative  release  can also  i n certain  Glia  o f GABA u p t a k e  c a n be demonstrated  acetylcholine  glia  (Hazama  can also  (1978)  o f neurons.  Glia  Although  into  and Dick  are biochemically  cells  this  C.  blocker  and a cyclohexaneamine  specific uptake  i s a specific  Kelly  from  activity i n  i n cerebral than  et a l . ,  cultured  GABA-T  glia  hemispheres  i n neurons  (Kelly  and  Dick, The  GABA In  1978).  glutamate  and  fact  from  eventually  other  glutamine  p l a y s a major  found  only  which  into  glutamine  The  This  (Schousboe  GS  of  et  glutamate to  activity  (Nicklas the  1 week  and  Browning,  In  o l d ones cyclic  generally  are  other  dehydrogenase  neurons  fire,  i t does  and  glutamate  a  i s converted i s  then  to  controversial.  i n the  brain  in vivo  1979)  to  and  the  of astrocytes  consistent  with  concept  relative this  the  i s to  a  activity with  the  not  t o whole  is a  large  late  of in  maturing  of  the  GS,  cultures  increase i n response (dBcAMP)  found  brain  late  i n 3 week  that  extent  have  function  matures  mono-phosphate  cause  from  cultures  but  GS  has  i n primary  cells  1978)  is  glutamate  reconverted  highly  which  age  the than  to is  maturation.  glutamate-metabolizing and  years  glutamate  and  glia.  enzyme  This glutamine  synthesis i s faster  to  in  Some r e s e a r c h e r s , h o w e v e r ,  High  adenine  thought  GS.  of  glutamate,  i n the  i n astrocytes,  accordance  but  of  neurons  where  neurons  enzyme  in glial  cells.  of  of  Martinez-Hermandez,  accumulated  glutamine  di-butyl  Two  this  degradative  one  when t h e  breakdown  catabolized  number  activity  and  a l . , 1980)  low  glial  of  of  a  remains GS  be  the  chemistry  glia,  by  glutamine.  development. rate  of  (Norenberg  activity  converted  up  by  the  pools,  help  schema  restriction  astrocytes  For  i n the  taken  (GS),  i n two  the  also  i n the  i s released  with  t o be  glutamate.  any  exist  smaller pool  released  high  to  glutamate  second  role  i n astrocytes.  thought  can  synthetase  which  been  sources  generated  enzymes,  glutamate  oxaloacetate transferase,  -  16  -  which  convert  glutamate  astrocytic 1980a).  cultures  This  astrocytes  not  that be  high  activities  suggests  that  glutamate  be and  the  converted thus  by  developing  to  to  studies  of  cultures of of  glutamate  for  such  fate  mouse  also  of  (Schousboe  to an  in  acid  (TCA) This  glutamate  cycle would  loop  would  a l t e r n a t i v e route  r a d i o a c t i v e glutamate  never  indicating  in  et a l . ,  accumulated  astrocytes  glutamine  present  substrate.  glutamine  the  are  tricarboxylic  a metabolic  Support  radioactivity  precursor  be  glutamate  completed.  supplied  the  at  may  constituents mean  t o °\ - k e t o g l u t a r a t e ,  the  (Potter  exceeded  other  et  was in  a l . , 1982);  that  of  metabolic  i t s  routes  must  exist. Possible  roles  neurotransmitters However,  Lentzen  specific  enzyme  glial  cells  have and  the  neurotransmitter,  D.  There  possess For in  the  Muller  on  loss  cells  Henn  and  astrocytes  yet  both  (1983)  and  ability  to  dispose  been w i d e l y  Palendker  degrade  of  the  by  using  products,  enkephalin,  aminopeptidase  peptide  investigated.  showed,  examining  to  i s considerable  retina,  selective  by  in helping  and  that  a  peptide  membrane  bound  A.  receptor  example,  not  inhibitors  have  enkephalinase  for glia  or  selectively Memo e t of  DA  carry Henn  that  binding  a l .  and  these (1980)  are  evidence sites  that  for various  destroying (1981)  5HT  some g l i a  were  binding  the able  sites,  can  neurochemicals.  Muller to  glia  show  cells  a  suggesting  that  receptors. showed  linked to  -  dopamine  adenylate  17  -  binding cyclase  sites and  on  stimulate  cAMP  formation,  Astrocytes binding al.,  prepared  that  1984),  their  is  and  of  true  action sites  to  to  are  et  antipsychotic effectiveness  of  their  effectiveness  closely related to  not  c l o s e l y coupled  (1984)  and 2  showed  spinal (H2)  cord  that had  caused  cAMP  in  drugs  many  and  a  response A  number  1181  regulating  (Hansson  in  i s also  with  the to  be  antipsychotics; the  et  bulk  said  this  antipsychotic  dopamine  binding  3'-5'cyclic  adenine  A  reviewed glioma to  NA  (Clark  system  and  the  cell or  line,  (Perkins  cells  on  noted  et  -  (HI)  and  while  -  interactions  have  responses by  receptors similar  Hertz  this  to  and  topic. their  (Gilman  levels  and  of  and  these the have  of  Nirenberg,  human g l i o m a  Both  components  a l . , 1971)  18  may  i n the  1971).  known  cells  increase  isoproterenol was  1  agonist  glia  article  data  Perkins,  a l l the  of  mediated  recent  type  from  hyperpolarizations.  glial  show r e c e p t o r  similar increase  possess  that  HI  cultures  depolarizations  instances  indicate  (1984)  cells,  then  other  i n neurons.  1971).  must  H2  which  Richardson C-6  a  are  seen  1981)  astrocytes  The  agonist,  those  dopamine  i s correlated  histamine  receptors.  impromidine,  the  with  show  drugs  where  the  drugs.  in blocking  as  preparations  mainly  for  drugs  astrocytic cultures  produced  drugs  (Hertz,  the  thiazolethylamine  with  i n dopamine  dopamine  neuronal  al.  type  There  antipsychotic  (cAMP).  brainstem  histamine  by  rich  by  potency  in  seems more  Hosli rat  The  cAMP  monophosphate  blocked  displace  with  which  areas  antipsychotic  correlated  not  i s blocked  from  be  cells.  formation well  can  ability  prepared  which  cell  line types  cAMP receptors  for  adrenergic  astrocytes profiles  drugs.  However, a d r e n e r g i c  and neurons  (Bender  Chronic regulation  of astrocytic  decreased  accumulation with  drugs  receptors  1984) a s o n n e u r o n s .  exposure  cell  can cause  on g l i a  (Hertz  F o r example,  lines  o f cAMP  ^-agonist  pharmacological  1984).  to adrenergic  of adrenergic  drugs  show d i f f e r e n t  and H e r t z ,  exposure  Richardson,  some  may  receptors i n  a  down  and  chronic  t o isoproterenol leads  and a decreased  properties  (Hertz  response  and  to a to  Richardson,  1983) . Various Richardson, bound  antidepressant  1983) a n d i m i p r a m i n e  t o o r taken  because  up by  ^-adrenergic  might  to exist  and an  and  Richardson,  C-6  and astrocytoma  (Hertz  Henn  demonstrated  binding  sites  with  inhibit  production  o f /3-adrenergic  i s also  are  inhibited  (Hertz ligands to  by a l l groups  or antipsychotic  e t a l . , 1982b).  to astrocytes.  dissociates  These  drugs  b u t n o t by a n x i o l y t i c  i n 1980 d e m o n s t r a t e d  diazepam,  such  be  t h e Ot - a n d  of antidepressants  The b i n d i n g lines  might  drugs.  on a s t r o c y t e s .  since  and  7  with  s t i m u l a t i o n o f cAMP  cell  (Hertz  e t a l . , 1983), a r e  of these  interaction  1983) .  antidepressants  drugs  sites  as doxepin  (Whitaker  interact  i s evident  isoproterenol-induced  such  astrocytes but this  nature  also  receptor  .-adrenoreceptors  of  intact  of the lipophilic  Antidepressants  known  drugs,  with less  sites  another rapidly  (Shoemaker  This  binding  of the  selective  benzodiazepine, from  -  binding  19  -  c a n be  better  R05-4864, b e c a u s e i t  astrocytes than  e t a l . , 1983).  benzodiazepine,  from  neuronal  Hertz diazepam may  be  and  Mruerji  binding  on  primary  displaced  by  other  concentrations through  the  of  but of  uptake  not the  may  one  predicted glia  to  be  be  Thus similar  also  occurs This  to  selective  of  true  on  be  more  Our  understanding  heterogeneity over  A) T i s s u e  decade pure,  of  specific  Diazepam  by  high  drugs  may  be  acting  the  various  In  on  this et  different  this  was  was  into  later  a l . , 1982). may  be  profile,  the  relevant  rate.  and  It  astrocytes  some c a s e s ,  clinically  metabolic  GABA u p t a k e  and  (Meldrum  a  manifestation  action.  inhibit  the  astrocytes  into astrocytes  drugs  have  in  in vitro  anticonvulsants, THPO  and  i n normal  which  drug  s t i m u l a t i o n of  or  be  drug-glia  than  the  interaction.  techniques  The  of  be  anti-convulsant  neurons,  New  developed  or  slices  reduction  for astrocytes. may  amount  in culture.  these  GABA u p t a k e  f o r the  effects  drug-neuron  in brain  non-barbituates  those  interaction  Thus  might  their  effective  the  astocytes  potassium-induced  inhibit  basis  might  found  suppress  which  that  large  benzodiazepines  barbituate-induced  be  a  receptors.  i n neurons.  Barbituates  showed  barbituates.  same  Barbituates oxygen  (1980)  only the  e n a b l i n g advances i n understanding  glia  came a b o u t past  decade  and  how  because or  they of  exhibit  new  techniques  so.  Cultures  understanding because  of  glia  of  homogeneous  of  recent  glia  has  progressed  advances  samples.  i n techniques  These  -  20  i n the  are  -  now  for  routinely  last separating prepared,  using  gradient  c e n t r i f u g a t i o n of  astrocytes  prepared  tissue  normal  cell  are  types  and  by  gradient  astrocytes  debris  and  tissue  culture.  centrifugation  but  their  may  be  The  from  fresh  contaminated  functional  by  integrity  other  may  be  impaired. Astrocytes established  cell  which  do  fresh  tissue  select  not  for  immature  5%  cultures  so  to  The have  heterogeneity  evidence  of  Freeze  texture  the  types,  They  are  on  type  cells  and  cultures,  by  primary  procedures usually  must  to  occur  be  particularly  from  during being  less  functionally  selected,  heterogeneity  which  prepared  homogeneous,  believed  the  transformed  are  quite  types:  most  that  about  frequently  these  regional  between v a r i o u s  cell  types.  Techniques fracture  This  views new  technique of  cell  which  allows  surfaces  development  has  more  than  led  to  some  heterogeneity. is a  technique  fractured; platinium fracture  microscopy.  structural  of  microscope  fracture  with  are  treated  are are  freeze  glial  replicated of  and  available.  mechanically  electron  a  general  differentiation  Fracturing  electron  previously  glia  differences  i s now  two  that  types.  glia  added  of  are  knowledge  or  Freeze  detailed  that  cultures  in vivo  techniques  There  true  their  Primary  are  cultures  certain cell  astrocytes.  and  line  non-specific,  similar  B)  culture  represent  brain  culture. than  in  and  lines  This  information.  the  It  i n which  fractured  carbon, upon  -  21  which  are  by  the transmission  several  existence  frozen  is  reveals  yields  show t h e  -  surface  examination  examination can  cells  and  types  of  organization  of the filaments  show d i f f e r e n c e s existence on  cell  within the  of repeating  membranes. cellular  same t y p e The  those  i n junction  parts,  cytoplasmic structure  glial  types  and astrocytes  intra-membranous  particles.  optic  nerve  surrounding 10  nm  filaments.  orthogonal  arrays  of  6 nm, w h i c h  or  inner  to  that  in  pericapillary  membrane  a n d among  cells of  c a n be d i s t i n g u i s h e d o f both  from  10 nm.  have  differing  examined  most  T h e membranes o f  nodes  o f these  faces  of pits  a centre  faces.  with  pattern  astrocytes  t o centre  that  c a n be used  by  protoplasmic  of particles i s similar  a s t r o c y t i c membranes  showed  contains  periodicity  t o p a r t i c l e s on t h e i r The density  i n adult  processes  are characterized  a s t r o c y t i c and s u b p i a l (1984)  of Ranvier  had a s t r o c y t i c  The e x t e r n a l  i n periparenchymal  junctions  function  heterogeneity  on t h e b a s i s  The cytoplasm  corresponds  Waxman a n d B l a c k gap  (1984)  and found  them.  c a n show  1982).  oligodendrocytes  rat  and reveal t h e  o f unknown  types,  and  a n d t h e c h a r a c t e r i s t i c membrane  and Mugnaini,  Waxman a n d B l a c k  confirm  areas.  processes  cell  filaments (Massa  cell  different  cells,  o f bumps  technique  among  astrocytic cell  o f other  between  patterns  This  from  i n t h e cytoplasm,  and l e s s  than  astroglial  membranes.  the orthogonal  arrays and  to identify  these  astrocytic  processes. Anders arrays  and Brightman,  (1979)  of p a r t i c l e s increase  on  i n rats.  They  also  an  increased  number  showed  i n number  showed  reactive  that from  22 -  orthogonal  embryonic  astrocytes  of particles but that  -  these  they  d a y 20  n o t only had  were  also  rearranged seen  to  (Wujek  Landis  and  But  and  Landis  whether  are  1981,  and  Reese C-6  glioma  modified  (1984)  cholesterol  in  not  did  cell  p a r t i c l e s on  contact and  not  with  that  astrocytic  non-neuronal  Brightman,  a l l glia  under  the  find  line.  C-6  found  that  It  have  1979,  orthogonal i s not  them  culture  filipin,  i n the  of  membranes,  had  or  clear that  a  conditions,  than  membrane  membrane  areas  cholesterol  less  less  cholesterol  both  are  found  in perivascular  specialization that  the  they  occur  that  they  are  proteins.  He  associated  of  arrays  involved  therefore  may  In  the  may  will  where in  orthogonal cultures  develop  on  a  they  surfaces  -  23  a  Gotow  barrier to  appear  -  that  lamina these  Such  transport  suggests  only  basal  areas  Na+,K+-ATPase,  transport  arrays  the  the  structural in  forming  on  filipin.  membrane  active  produces  orthogonal  the  that  and  in vivo.  be  acting  either  or  the  This  astrocytes  specifically  found  with  processes.  the  orthogonal  cells.  from  that  e f f e c t on  means  a l k a l i n e phosphatase  which  by  contacting  This  i s somehow p r o t e c t e d  contain  ependymal  areas.  contain  chemical  less  a s t r o c y t i c membranes  other  a  membranes  array-crowded  and  of  Anders  (1981)  characteristic disruption  also  to  them.  Gotow  on  compared  1981).  means t h a t  line,  structure  arrays  where t h e y Reier,  i n the  this  have  orthogonal  Reese,  assemblies  glioma  of  increase  tissue  ordered  astrocytes.  number  membranes  a  more h i g h l y  i n normal The  not  a  in on  exposed  normally  regional suggested  function, i s less,  as  or  cholesterol astrocytes  a l l the to  and  and  surfaces  large  extracellular The  freeze  various  types  astrocytes between form  fracture of  and  Saint  junctions.  between  junctions Marie  techniques compound  to  eye  had  junctions  a  (gap  arrays.  of  different  layer.  communication;  This of  type  the  C) A  tight  septate  impedance; patterns  and  functions:  material;  tight  Glial  patterns  shapes  the  Cells  and  physical  various  gap  junctions  f i r m but  desmosomes  thus  imply  of  w o r k may  -  that  -  types  be  layer types  and  the  of  septate  the  to  the  layers  these  of  of  the  had  features the  contacts  as  other may  have  intracellular  intercellular  well  of  occlusion  cells  not  do  r e t i n a of  equivalent  each  flexible  the  i n the  three  be  -  but  fracture  relationships to  The  between  astrocytes  freeze  d e n s i t i e s of  junctions  -  of  define  1982).  junctions,  cells  occur  i n each  of  d e s m o s o m e s w h i c h may  characteristic  cells  used  pattern  to  oligodendrocytes  heterogeneity  fly.  junctions,  orthogonal  of  house  used  and  Mugnami,  (1983)  glia  be  junctions  adjacent  and  characteristic  and  as  but  Carlson  the  also  oligodendrocytes  describe of  can  Gap  (Massa  and  junctions)  well  studies  oligodendrocytes,  tight  retina  spaces.  adhesion  or  adhesion.  have  extended  of e x t r a c e l l u l a r tissue The  differing i n the  differing  functions.  future  to  glia  CNS.  Markers v a r i e t y of  distinctions  markers  have  been  found  to  be  made b e t w e e n  and  classifications  of  glia  These  cells.  -  24  -  that  within  the  markers  allow various have  provided  a  wealth can  of information  be used  previously identify glial  to identify  subsets  cell  other  have  clues  been  emphasize  the g l i a l  use  o f some m a r k e r s  Some that  to  as t o heterogeneity good  reviews  1982) b u t t h e y  heterogeneity  for defining  markers  were n o t  c a n be used  several  1981, S c h a c h n e r ,  not  types  markers  and t o p r o v i d e There  (Roots,  heterogeneity.  as g l i a ,  so c l a s s i f i e d ,  function.  markers  on g l i a l  of  glial  generally  revealed  purity  of  do  but rather the  of cultures  or similar  purposes. 1) F i b r o u s a)  Glial  proteins  fibrillary  Astrocytes glial  are  specifically  composed  glial  from  protein  reliably  identified  the electron  stains  o f t h e most  fibrillary  isolated and  under  astrocytes  acidic  a r e most  filaments  method  of  acidic  multiple  these  protein  microscope.  fibers  studied  of glial (GFAP).  gliotic  areas  readily  stained  by immunohistochemistry.  may  have Duffy  cells the  of the filaments  some  function  et a l .  i n culture  astrocytes  (1982)  had abundant  round  or polyhedral  parallel  looked  a t GFAP  GFAP  astrocytomas  As processes  i n human  t h e GFAP was  arrays.  -  25  -  markers, originally  and c a n be  t h e shape  GFAP  They  i n astrocytes  and processes,  developed,  gold  e t a l . , 1972)  of  that  astrocytes.  astrocytoma  a r e l a t i o n s h i p between  i n body  of  I t i s the principle  a n d t h e l o c a t i o n o f t h e GFAP.  cells  perinuclear.  (Uyeda  develop  i n maintaining  and found  specific  e t a l . , 1972),  that  1913).  GFAP was  CNS  constituent  The C a j a l  (Cajal,  s c l e r o s i s plaques (Bignami  by t h e presence  t h e shape o f  Spindle  shaped  whereas i n largely  extended  i n dense  There  may  also  be a r e l a t i o n s h i p between m o t i l i t y and  GFAP.  Stellate cells  arrays  o f GFAP  without  these  retracting Salm very  parallel  (1982)  This  into  Suess  r a t p i t u i c y t e s , which are  i s found  in  the fibrous  It  i s also  that  GFAP  mainly  subtype  found  under  glial  cell  cells  nervous  system  A injury  GFAP  permanent  and precedes  cultured  astrocytes  maturation which  increase  and  factor  causes  the pituicytes  where  (Jessen  related  there  astrocytes,  1974), t h e  percentage  of glia  e t a l . , 1984). a l l  are  astrocytes.  to  and Dahl,  o f whether  of the  I t i s denser  i n protoplasmic  i n  Kennedy  astrocytes  answered.  i n GFAP  content  s t a i n i n g c a n be  (Lim e t a l . , 1977),  maturation.  t o be  astrocytes.  types  other  staining i n  capsule  a s t r o g l i o s i s (Bignami GFAP  a s do  after transplant  and a small  the question  has y e t t o be  that  (Bignami  peripheral  that  found  but i s also  glia  noted  p o s i t i v e GFAP  i n mature  i n several  radial  n o t have  the kidney  enteric  contain  extending  filaments,  does  GFAP p o s i t i v e e v e n  glia,  (1982)  glial  (1981)  influences.  the  cells,  that  and P l i s k a  no  Bergmann  spindle  showed  to give  to a region  including  while  constantly  means  pituitary  GFAP  rigid  parallel  were  filaments  strongly  neural  extensive  arrays  p o s i t i v e , do n o t have  organized  remained  were more  with  processes.  astrocytes.  cells.  fibers,  et al.  GFAP  i n culture,  I n some  expressed  t r a n s i e n t l y ; i n humans  ependymal  cells  b e t w e e n week  cell  26  increased  types  i t i s only  -  follows  and Dahl,  brain  13 a n d f u l l  -  rapidly  1975). by  glia  extracts  or  i t may  only  dBcAMP be  expressed i n  term,  In  and i s a l s o  transiently  i n tanycytes  Heterogeneity al.  (1982)  Schwann  cells  CNS.  found  non-myelinated  may  be  been  shown t o  molecular  weight  with  staining  b)  and  of  are  two  Filaments  of  the  et  of  mainly in  number  GFAP w i t h i n  of  the  subtypes  have  central  those  of  that  various the  impurities  the  chemically  same  glia.  The  producing  proteins, related  found  i n some g l i a  within the  to  vimentin to  cell. and  examine  wounding  Stab  there  were  and  (Pixley  the  with  these  older rats,  normally only  no  the  problems antibody  cause.  wide  spaces  open  of  but are  these  and  different constituents is  vimentin.  in a  double  filaments  DeVellis,  only  i n newborn  with  a  gradual  cortical  at  the  edge  that vimentin  spaces  disappear.  and  is lost  rats  and  switch  areas  of  occurs when  in  1984).  at  vimentin-positive cells  occurred  hypothesis  and  astrocytic  w o u n d s w e r e made t o  Vimentin  led to  One  to  GFAP w e r e u s e d  stained for vimentin  between.  with  and  i n 2 0 day  contact  Dahl  subset  increased  composed  mainly  other  experiment  development  This  are  are  systems  labeling  region.  a  were  be  cross-reactivity  Antibodies  when  These  and  i n p e r i p h e r a l and  GFAP, w h i c h  fiber  GFAP  cells.  Vimentin  There from  the  nerve.  heterogeneity  proteins although  itself  of  stained only  axons,  different  GFAP  i n subsets  degeneration.  also  GFAP h a s  1981).  antiGFAP  in rat sciatic  Wallerian  There  be  noticed that  surrounding during  can  (Roots,  the  for  in a in  time the  wound.  when t h e r e such  contact  A l l vimentin-positive cells  -  27  -  is  seem  to  have  eg.  at  l e a s t one  ependymal  cells,  and  portion  cells,  radial  of  tanycytes,  glial.  The  cells  correlates with  volume.  Cells  in culture,  would edge  develop explain  of In  the  adult  fibroblasts,  vimentin  was  and  found  GFAP  and  and  of  of  was  In  CSF,  Muller  vimentin  This  only  in  cells  observed blood  fibers,  at  the  in  vessels, rat  day  11,  the  ventricular  Schnitzer  together  hypothesis  boundary.  embryonic  culture  occurring  fibers,  of  tissue  large At  with  i s much e x t r a c e l l u l a r  only  in radial  vessels.  glial  origin.  fluid  relatively  only  contact  in extracellular  vimentin  the  vimentin  vimentin  loss  there  astrocytes.  observed  blood  to  in  et  i n the  al.  (1981)  cells  which  f i b r o b l a s t morphology.  c)  Desmin  Dahl fiber  and  Bignami  (1982)  showed  system  in glia  cells.  I t was  astrocytes  of  comparison  with  in  of  the  regardless  close  cells  Bergmann  where  appearance  rat,  cells,  cells,  have  the  wound,  the  ependymal  vimentin  cell  disappearance  positive  fluid,  the  brain  and  brain GFAP  spinal  and  spinal  showed cord  not  desmin  uniformly  cord  that  but  that  and  both  a  third  distributed in  in Muller  were  i n the  forms  cells.  similarly  f i b e r s of  A  localized  Muller  cells.  2)  Glutamine  sythetase  GS  is a  astrocyte  major  marker which  catalyses  the  reaction: glutamate It  may  thus  be  +  ammonia involved  +  ATP  >  Glutamine  i n glutamate  -  28  -  +  metabolism  ADP and  +  Pi ammonia  detoxification.  Martinez-Hernandez  immunohistochemical exclusively the  retina.  Muller  a  the  cells,  primary  glia  et  al.  of  activity. location  of  latter  may  localization  tissue, of  be  of  and such  1979),  does  astrocytes.  (Norenberg,  hippocumpus  -  of  GS;  this  enzyme  very  GS  low  glia  as  the  (Norenberg  and  GS,  Gliotic  may  explain  lead  the  There  of  the scar  to  buildup  epileptogenic are  i n t e n s i t y of layers  in  Since  contain  particularly  -  ammonia.  had  do  1983).  a  (1978)  this  i n the  29  just  Browning  astrocytes.  1979).  feet  ended  previously.  but  molecular  are  end  and  compartment  variations The  which  implicates  could  have  in  vascular  activity  discussed  which  in  Kennedy,  staining  glia  contain  of  fibrous  1979,  line  of  brain  and  cell  GFAP,  cells  d i s t r i b u t i o n supports  Nicklas  (Norenberg,  staining  and  as  not  glutamate tissue  to  marker  regional  cerebellum  high  located  rat  barrier against  But  contain  significant in  a  glutamate  better  however,  ammonia  nature  a  GS  is  i t s location  of  perikaryon,  This  astrocytoma  not  studies  used  Muller  confirmed  described  found  Its  do  and  astrocytic processes  C-6  glia  brain  GS  protoplasmic  glia,  cultures.  Marinex-Hernandez, primitive  also  providing  small  (1977)  Marinez-Hernandez,  the  the  (1978)  surface.  (1977b)  astrocyte  that  and  al.  show t h a t  i n the  i n both  and  ependymal  to  microscope  Bergmann  for  Schousboe  Lam  (1983)  limitans  the  function  found  (Norenberg  glia  beneath  and  Electron  Norenberg  ependymal  cells  localization  astrocytes 1982).  glial  Sarthy  cells.  revealed  in  over  techniques  et  GS  the  heavily  stained  3)  Carbonic  Carbonic carbonic as  anhydrase  anhydrase  acid,  hydrates  an e s t e r a s e .  pH,  secretory  the  brain  considered  be  also  I t thus  activities  oligodendrocytes  et  cells  C-6 al.  have  cells (1982)  anhydrase  a n d movement  marker  (Ghandour  r a t cerebral  be be o l i g o d e n d r o c y t e s exists  i n several  sequences  stain  distinct  might  be a s t r o c y t e  However, and  stain  subsets  i n culture f o r CA-2  above  isoenzymes  of glia.  specific cDbAMP  caused  as i n t e n s e l y  -  30  -  as  cells  have  Carbonic different  distinct.  ( C a - 1 a n d CA-2) thought  that  oligodendrocyte  astrocytes  as  of primary  layer.  that  I t was  a n d CA-2  Kimelberg  f o r carbonic  as w e l l  this  isoenzymes  e t a l . , 1978b),  1973).  method  hemispheres,  Rousell  cultures of  (Kimelberg  and a r e immunologically  t o two o f t h e s e  known t o  1978) a n d  i n the monolayer  believed  Antibodies  b u t i s now  Primary  the histochemical  acts  I t used t o  e t a l . , 1979, 1980,  anhydrase  astrocytes  and  I t develops i n  specific  from  acid  of ions.  a n d Lam,  t o form  i n the regulation of  proliferation.  cultures  amino  to alcohols,  do n o t ( D e V e l l i s and B r o o k e r , found  a n d H2O  as g l i a l  e t a l . , 1978).  carbonic  stained  anhydrase  groups  be i n v o l v e d  (Sarthy  as well  a l . , 1979, Mandel  astrocytes but  may  an a s t r o c y t e  on M u l l e r  r e v e r s i b l y CO2  aldehyde  a t t h e same t i m e  be  et  combines  to  may CA-1 specific.  differentiate  oligodendrocytes.  4)  Other  There stain  with  of these There  astrocyte as  a r e a number  astroglia  reports many  markers  well  predominantly.  very  little  recent  specific  that  Many  that  a r e known  specificity  been  Table  substantiated  of markers  a n d now  have  of these  confirmation.  or poorly  a r e a number  o r whose  o f markers  i s now  - 31 -  used  t o be  reported  are I  to  isolated  summarizes  findings. t o be  on o t h e r  considered cell  highly controversial.  types  TABLE I : MINOR ASTROCYTE CELL MARKERS MARKER  TYPE OF CELL  AUTHORS  Non-neuronal enolase *  A s t r o c y t e s cerebellum i n c l u d i n g Bergmann g l i a & cytoplasmic processes  Langley & Ghandour (1982)  OL-2 - G l y c o p r o t e i n  A s t r o c y t e s ; astrocytomas not glioblastomas  Langley et a l .  Tamm-horsefall glycoprotein  Ependymal c e l l s & a s t r o c y t e processes Zalc et a l . of Bergmann f i b e r s o r a s t r o c y t i c f e e t i n c o n t a c t with blood v e s s e l s o r meninges  Sulfogalactosyl ceramide (SGC) **  11  Zalc et a l .  (1982)  (1984)  (1984)  Ml - a n t i g e n  Distinguishes sub-cerebellar astrocytes some but not a l l GFAP+ c e l l s  C-l  Only processes o f Bergmann g l i a & M u l l e r Sommer e t a l . (1981) c e l l s , and ependymal c e l l s , but not other a s t r o c y t e s except i n e a r l y p o s t n a t a l a s t r o c y t e s o f white matter P u r k i n j e c e l l l a y e r and r a d i a l l y o r i e n t e d s t r u c t u r e s o f t e l e n c e p h a l o n , pons, p i t u i t a r y and r e t i n a  - antigen  IgG - RAN 2 # .  A s t r o c y t e p r e c u r s o r c e l l s , ependymal c e l l s , Muller c e l l s leptomeningeal c e l l s  * C a t a l y z e s o x i d a t i o n o f p h o s p h o g l y c e r i c a c i d t o phosphopyruvate ** C l " p o s s i b l y i n v o l v e d i n C l t r a n s p o r t # IgC made by antibody s e c r e t i n g hybridomas and d e f i n e d by antibody  Lagenaur• e ta l .  (1980)  B a r t l e t t e t a l . (1981)  TABLE  I  (continued):  MARKER  TYPE  THY  1  Only  THY  1  Two  Antigen  S  A2B5  100 P r o t e i n  *  SSEA-1 G l y c o l i p i d antigen  N11N1 M o n o c l o n a l antibody  308 M o n o c l o n a l antibody * ** #  #  **  #  subtypes are also cell  MINOR A S T R O C Y T E C E L L  MARKERS  OF C E L L  AUTHORS  of astrocytes that glactocerebroside+  S c h n i t z e r and Schachner (1981)  lines  Kemshead  et a l .  (1982)  Immature a s t r o c y t e s ; o l i g o d e n d r o c y t e s neurons; has considerable r e g i o n a l , d e v e l o p m e n t a l and s p e c i e s v a r i a t i o n  Schnitzer & (1982)  (controversial) specific astrocyte m a r k e r OR i n o l i g o d e n d r o c y t e s , e n d o t h e l i a l c e l l s and neurons a l s o  Gandour e t a l . (1981a) Hyden e t a l . (1980) H a n s s o n e t a l . (1976)  Subtypes o f astrocytes. I n e a r l y mouse cerebellum only i n external granular l a y e r and molecular l a y e r ; l a t e r only small areas i n molecular area  Lagenaur Lagenaur  Human f e t a l b r a i n c u l t u r e s a n d p r i m a t e s p i n a l c o r d ; s u b t y p e s 8 0 - 9 0 % GFAP+ i n some c e l l s o n l y  Dickson  et a l .  (1983)  D i f f e r e n c e s found between and GFAP- a s t r o c y t e s  Dickson  et a l .  (1983)  GFAP+  May b e i n v o l v e d i n b i n d i n g Ca++ & m o v e m e n t o f m o n o v a l e n t O r i g i n a l l y f o u n d on s u r f a c e o f F9 t e t r a c a r c i n o m a cell O r i g i n a l l y r a i s e d t o human n e u r o b l a s t o m a  cations  o r GABA  Schachner  et a l . et a l .  (1982a) (1982b)  transport  As  c a n be seen  astrocytes with  from  are only  specific  t h e development  evidence these  forglial  markers  systems  may  More  came  Thus,  considerable  research  f o r future  that  markers.  on each o f  classification  Since  of g l i a l  o f markers  indicative With  fracture,  understanding diversity  diversity  that  their  been  research  considered  will  use these  found  to start  subtypes  based  on  and p h y s i c a l c e l l  are not only  tools  functional  but are also  subsets  f o r staining,  and e l e c t r o n microscopy,  of the morphological  be c o r r e l a t e d  found.  -  34  -  direct  t h e wide of glia i s  using  there  diversity  to  heterogeneity  heterogeity of glial  i n techniques  and  types.  s t r u c t u r e and b i o c h e m i s t r y ,  c a n be  data  y e t t o be  into  can sometimes  markers  I I summarizes  stainable i n various  of great  advances  o r have  Future  species,  some m a r k e r s  indicators variety  areas,  a l s o have  Table  could  markers  oligodendrocytes  between  myelin  specific.  oligodendrocyte  classify  this  a n d CNS  on markers  oligodendrocyte  freeze  markers f o r  of astrocytes.  technology  be t h e b a s i s  minor  f o rastrocytes.  available  seen  of these  f o r subsets  heterogeneity.  a r e more o r l e s s  other  I , most  o f marker  Oligodendrocytes that  Table  came  of glia  cells. markers, a  new  a n d how  t o the biochemical  TABLE I I :  MARKERS FOR OLIGODENDROCYTES AND MYELIN  MARKER Galactocerebroside  Myelin  basic protein  Myelin  basic protein  M y e l i n s p e c i f i c PNS e p i t h e l i a l c e l l of Early  *  P r o t e i n WI  Myelin  &  W2  + CNS oligodendrocytes v e n t r i c l e s and c h o r o i d  oligodendrocytes  Cultures  Anti-proteolipid antiserum Wolfgram  AUTHORS  LOCATION  Central  of  and  myelin  galactocerebroside +  sheaths  myelin  Raff  et  a l .  (1978)  plexus  sheaths  cells  Sternberger et a l . (1978) H a r t m a n e t al. (1979) R o u s s e l & Nussbaum(1981) Bhat  et  a l .  and a c t i v e l y m y e l i n a t i n g oligodendrocytes  Agrawal  &  Labourdette (1979)  oligodendrocytes  (1981)  & Hartman (1979) et a l .  2',3'-Cyclic nucleotide 3 ' - p h o s p h o h y d r o l a s e **  I n n e r & o u t e r most s h e a t h myelin Species s p e c i f i c , not i n f i s h ; I t s f u n c t i o n may o n l y be c o i n c i d e n t a l l y related to myelin Not i n o l d o l i g o d e n d r o c y t e c u l t u r e s  Szuchet and Stefansson  01—>04  Oligodendrocytes of early postnatal cerebellum, cerebrum, s p i n a l cord, o p t i c nerve & r e t i n a : 0 1 & 0 2 , a n d 03 & 04 o c c u r i n d i f f e r e n t developmental times i n d i f f e r e n t areas  Sommer & S c h a c h n e r (1981)  * ** #  #  Antigen defined Catalyses h y d r o l y s i s of 2 ' , 3 ' - c y c l i c nucleosides to the major component Wolfgram p r o t e i n F o u r m o n o c l o n a l a n t i b o d i e s f r o m mouse myeloma immunized w i t h white matter from corpus callosum  2•-nucleotides;  Schachner  (1980)  (1982)  TABLE I I ( c o n t i n u e d ) : MARKERS FOR OLIGODENDROCYTES AND MYELIN MARKER  LOCATION  MAG  Succinic  dehydrogenase  Butyryl Cholinesterase  *  Antimyelin antiserum  AUTHORS  O l i g o d e n d r o c y t e s , Schwann c e l l s and c e r t a i n areas of periaxonal region of the central and p e r i p h e r a l m y e l i n sheath  Sternberger (1979)  Oligodendrocytes  Mossakowski(1962)  and  astrocytes  Cavangh & Thompson (1954) , O e h m i c h e n (1980)  More a c t i v e i n o l i g o d e n d r o c y t e s than i n other g l i a M y e l i n a t e d f i b e r s , medium and l i g h t o l i g o d e n d r o c y t e s ; n o t d a r k (mature) astrocytes, Golgi epithelium cells, B e r g m a n n f i b e r s a n d some s u b e p e n d y m a l  et a l .  Roussel & (1983)  Nussbaum  cells  P-2 Myelin specific protein  R a b b i t CNS m y e l i n : m o r e i n c a u d a l a r e a s Highest i n s p i n a l cord, lowest i n f r o n t a l c o r t e x ; o n l y i n l a r g e r diameter axons  Trapp e t a l . (1983)  Glycerol-3-phosphate  01igodendrocytes  DeVellis et a l . (1978)  TU-01  Hajos & Rostomian Cerebellar g l i a c e l l s ; only i n microtubules; smooth endoplasmic reticulum outer mitochondrial (1984) membrane; r i b o s o m a l r o s e t t e s  * **  **  Non-specific cholinesterase Anti-tubulin antibody  G l i a l Heterogeneity  Given of  the  old tools  development the  discussed.  The  heterogeneity  most  would  cells.  already  been  given  further  here  as  the  function.  there  environments,  or  the  Most  appearance  microscope, The may  sometimes  be  not  are  method  intermediate  to  types,  The  be  to  has  be  developed  in  morphology  expression  filaments by  et  has  a l .  a  -  37  into  they  Palay  be of  have  are  reflecting chemical  which  have  usable  mature  et  astrocytes  by  electron a l .  (1962).  oligodendrocytes  s t a i n e d by their  the  silver  cytoplasms  described  protoplasmic  -  types  natural  types  ultrastructural  normal  cell  i n the  (1982)  and  forms  of  a s t r o c y t e s and can  and  p h y s i c a l or  catagorized  the  fibrous  whether  local  chemistry  the  be  types  morphology  transitional  described  Reyners  into  seen  so  between  will  astroglial  Both  that  cell  a variation  c a t a g o r i z e d as  between  cell  i t , can  differences  glial  morphological  been  s t i l l  that  improvements  consider  visual  of  difficult.  similar.  radiosensitive  be  distinct  originally  be  the  know  to  location,  cytoplasmic  distinction  carbonate must  as  of  to  adaptions  plasticity.  subgroups.  on  reversible  have  way  recent  types.  based  stages,  described  now  divided astrocytes  cell  a  and  i n t r o d u c t i o n but  i t remains  distinctive  we  first  appears  other  but  as  discription  glia  developmental  been  tools  discuss  i n the  Several  astrocytic  to  brief  types,  discribed  really  be  originally  protoplasmic  been  A  obvious  classical  Cajal  new  heterogeneity,  between  within  of  - Morphological  a  highly  characteristics a s t r o c y t e and  the  light and  oligodendrocyte.  may  be  replacing  a  oligodendrocytes as  alpha-astrocytes,  as  normal  shaped  endoplasmic  reticulum,  are  never  v e s s e l s but,  perivascular cells.  areas,  Koenig  continuum reactive  of  and  found  and  be  are  absence near  This  called, cover  by  i t s  of  the  morphology,  lack  outer  membranes  The  of  f r e q u e n t l y found  a l s o noted between  of  gliofilaments.  frequently satellites  forms  of  d i s t i n g u i s h e d from  of  the  numbers  capable  cells.  lysosomal  (1963)  transitional  cell,  ribosomal  m i c r o g l i a , are  Barron  that  to  the in  nerve  there  is  a  oligodendrocytes  and  been  into  astrocytes.  Oligodendrocyte  morphology  Hortega  long  was  the  Rio  differences  i n oligodendrocytes.  oligodendrocytes latter  cells  to  one  another.  in  terms  of  and  features  the  axons  being  Both and  of  their  subclassified  which them  perineuronal  of  these  shape  of  the  the  cells  i n rows w i t h  with  are  many  variability  i n the  manner  i n which  but  little  branching.  -  Each  38  -  bodies,  Most 1:  close  and He  number size  are  emanate  show  from  abuts  and  small  that  a  of  thus  perineuronal  these  process  soma  i n the  number  processes they  the  heterogeneous  associated.  type  fine  were  i n the  4 prototypes.  oligodendrocytes  describe  interfasicular,  cells  were  to  classified  and  and  sub-divided  first  subclasses  processes  into  fjim i n d i a m e t e r  exhibit  He  frequently aligned  size  with  perivasular  into  (1928)  has  sub-types.  15-20  can  denser  total  like  glial  microglial  coarser  and  in significant  astrocytes are  nucleus,  processes,  ^-astrocytes  or  i t is called,  irregularly  blood  i s present  multipotential reserve  beta-astrocyte,  cellular  It  cells great  the nerve  cell  fiber.  They  are  medulla.  Most  they  fewer  20  have  to  40^ra  processes matter. large  4  either and  similar  these and  cell  wide  Maxwell,  of  1968,  i s between  of  developmental Many  probable  recent  traditionally  highly  axons.  by  their  most  1 or  2.  Type  elongated  These  sub-types  being  dense  t h e r e f o r e have  for oligodendrocytes i s  and  classify  two  cytoplasmic  Leblond them  I t i s not  the  white  are  types  subtypes  the  stain.  Mori  the  in  from  1970) . into  much  classification density  They  light,  k n o w n how  increasing  densities used medium  of  an  systems. is a  It  function  maturity. data  suggest  thought  classical  Ependymal  that  than  n u c l e a r and  to  range  associated with  carbonate,  scheme  oligodendrocytes.  highly  in  These  They  have  to  silver  for this  range  and  1,  2:  i n which  only  cells  and type  distinguished  numerous  directly  body.  are  bi-polar  bi-polar  attached  is  the  less  classification  there  and  type  manner  processes.  mono- o r are  than  i n the  cell,  large  density differences  dark  processes  the  cerebellum,  oligodendrocytes are  differ  deposition of  the  and  overlap  of  as  biochemistry  on  (Caley  but  mono- o r  the  Another based  few  are  throughout  thicker  from  They  similar  cerebrum,  oligodendrocytes are  found  axons.  show  3  and  large  bodies  and  i n diameter,  Type  frequently  i n the  interfasicular  come o u t  size  are  found  glia  cells  of and  as  that  a wide  being  glia  usually  variety  share  resemble  contain vimentin  and  of  cells  not  characteristics  astrocytes.  resemble  astrocytes  morphology. Pituicytes  of  the  neurohypophysis  -  39  -  are  GFAP-positive  and  retain  this  characteristic  e v e n when no  longer  under  neural  influence. Muller being  glia,  They  are  both  GS  The  cells  though  and  carbonic  anhydrase the  like  glia  specialized  of  of  the  fiber  nerve  cells.  the  of  at  retina  from  .  They  form In  the  and  Hopkins,  that  not  homogeneously  nerve  fiber  but  i n the  run  perpendicular  Barber the  o p t i c nerve to  parallel  glia  and  (1982)  -  found nerves  40  -  the  based  the  on  found  are  with  axons  of  layer  the more  the of  morphology, cells astrocytes the  layers and  He  also  processes  might  ganglionic that  align  axons.  these  are  a  ganglion  with  day.  retina  Muller  i n the  the  entire  throughout  to  bundles,  f o r the  vomeronasal  have  with  16th  plexiform  (1980)  only  the  the  the  besides  distributed  specialized  o l f a c t o r y and  are,  Bussow  axon  of to  outer  there  chicks, during  on  fasiculate  and  1981).  profile  processes  fibrous astrocytes  the  Lindsay  seem  basal  that  processes run  cells  retina.  increases  only  throughout  types  1981).  layers that  saw  considered  cell  have  they  but  cells  cells  Muller  inner  (Boycott  retina,  glia  Muller  their  Macaque monkeys  cells,  as  contain  Muscona,  e a r l y embryonic  as  septi  and  i n developmental  are  and  other  the  ( L i n s e r and  of  that  two  of  i n the  amacrine  least  are  glia  In  areas  retina  ganglionic  only  vimentin-positive,  showed  to  the  and  1981)  other  the  recognized  markedly  function.  thickness  been  not  specificity  (1980)  long  is in a l l retina  disappearance  Bussow not  differ  have  are  anhydrase  Muscona,  development final  retina  they  carbonic  enzymes  (Linser  the  GFAP-positive, and  two  of  that  be  cell  axons.  Schwann  cells  closely  of  related  to  central  than  to  they  react  a s t r o c y t e s and  Schwann to  cells  other  antibodies to  filament  protein  from  the  periphery  into  brain,  their  of  glial  are  from  GFAP  human b r a i n .  into  the  of  parts of  both  CNS  traditionally  peripheral  cells  the the  and  along  called  periphery  a  These the  myenteric  49k  nerve  as  develops  Schwann  different  from  true peripheral  in  not  tongues  of  cytoplasm  separate  them  into  membrane  surrounding  which  axons  branch  bundles.  They  individual  however,  also  the  have  cells,  and  because  of  also Schwann  individually between  i t  cells  morphologically  ensheath  glial grow  are,  do  dalton which  They  they  because  glia,  origin.  that  plexus  but  extend  axons  no  cells  and  basement  contain  no  collagen. Tanycytes oriented  the  stage  development remain  Bergmann  that  third than  into  glia  of  et  the  They,  cerbrospinal  fluid,  cell  processes for  most  and  astrocytic  the  other  some  ventricles,  are  GFAP-positive so  at  an  throughout  et  a l . , 1981).  regard  they  similar  dentate  glia  radially  the  DeVietry  i n which  with  are  cells  gyrus,  in contact  and  They  like  of  the  normal  with  are vimentin-positive.  are  extending  of  cells  continue  cerebellum,  or  around  They  a l . , 1981,  like  glia,  called,  bodies  glia  glia  astrocytes.  sometimes  parts of  adulthood,  hippocampal  Bergmann  line  glia-like  ventricle.  are  (Basco  GFAP+  cortex,  specialized  processes  especially earlier  are  the  Golgi  epithelial  specialized  glia  Purkinje cells  thoughout  the  markers.  They  -  41  cells of and  the  -  they  layer.  from  are  cerebellum  radially  molecular evolve  as  with  oriented They  fibers  in  stain the  molecular that  layer  transform  cells  to  of into  their  in  and  found  regular  (1978)  different profile They  and  enzymes  the  and  Bergmann Hatten early were  et  was  are  with  larger,  more  neurons  There  were  associate Time  lapse  arms  of  also with  one  of  enzyme  low  the  pentose  had  resembled arms  in  There  were  to  Bergmann-like  two  types  the  and  but  glia  a  time  the  other  dozen  or  layer.  which  did  not  studied.  migration not  that  the  granular  the  in  neurons  glia,  extensive  astrocytes  shunt.  i t clustered  during  cycle  cerebellum.  three  of  activity  d i f f e r e n t types  galactocerebroside-positive  revealed  acid  i n the of  astrocytes  low  dehydrogenase.  cells  i n which  than  of  phosphate  Bergmann  c e r e b e l l a r neurons  but  citric  glia  two  glial  glucose-6-phosphate  are  only  granule  activities  activities  succinate  glial  type  resembled  photography  the  no  1979)  Bergmann  reductase,  cerebellum.  shorter  and  the  studied  i t and  had  of  not  mouse  GFAP p o s i t i v e :  associated  glia  i n those  (1984)  postnatal  and  a l . ,  after guiding  studied  high  TPNH-tetrazolium  glia  al.  found  et  1971).  dehydrogenase,  Bergmann  high  (Fulop  cells  Andersen  l a c t a t e dehydrogenase,  Therefore,  glial  (Rakic,  L-glutamate  dehydrogenase  cerebellum  position  astrocytes.  nonspecific  of  and a  early  Bergmann  final  Contestabile cells  the  along  the  stellate  astrocytes. Radial  glial  researchers. glia, and  are  cells  They  related  tanycytes  development  been  transform to  which  and  have  have  described  into  astrocytes, a l l contain  42  a  number  distinguishable Bergmann  GFAP a t  s i m i l a r morphology.  -  by  -  glia,  The  types  Muller  some p o i n t  of  in  immature  of cells  their radial  glia  are  classified  filaments (Rakic,  and  1972),  but  developmental  they  that  occur  glia  have  (Rakic,  been  1972,  shown t o Cajal,  a s t r o c y t e s pass  also  develop  from  and  and  1929,  through  ependymal  astroblasts,  Rakic,  Schmechel a and as  the  radial  and  between  a l . , 1983).  mature  Radial  astrocytes  Rakic,  glial  be  describe  in  between  1979),  phase.  subependymal will  both  glia  1979)  et  and  radial  of  cytoplasm  (1929)  noted  into  bundles  into  in radial  (Choi  transform  the  in their  Cajal  been  oligodendrocytes  most  glioblasts  forms have  (Schmechel  of  develop  changes  astrocytes and  also  and  glia  glia  may  glycogen  (1977)  Transitional  radial  of  because  Akers  cortex. and  astrocytic  accumulation  oligodendrocytes. the  as  They  layers  discussed  and  by  may  way  i n the  of  next  section.  and  Using  GFA  to  Rakic  (1980)  developing  showed  evolution of  they  fanned  pial  s u r f a c e where  transition  out  follow the  from  reticular  they  glia  are  guidelines  that  neurons  i n the  and  the  late a  of  very  stages  primary Choi  but  role and  use  developing  a l . (1981)  correlate  end  Levitt  radial  glial  cells  subreticular  zones  to  feet  traditionally  Woodhams e t time  had  and  i n monkey,  that  stayed  until  as  the  the  to astrocytes.  Radial  However,  the  brain  first well. not  to  grow  mouse  appearance A  clear  in cortical  plate found  -  of  43  to  stages.  the development.  hippocampus,  cortical GFAP+  be  during  and  association  early  (1980)  along  cortex  noticed that  i n the  Lapham  supposed  plate  radial  formation  glia  i s evident This  argues  did in  the  against  formation. two  -  types  of  radial  not  glial  cells  i n t h e d e v e l o p i n g c e r e b e l l u m o f t h e human  weeks, ones of  which  i s earlier  extended  from  closely  rat  cell  Seress  (1980)  brain.  Until  walls  were  examined  seen  development  ones  from  The l a t t e r ,  the radial  and f o r t h  i n the wall  The  lower walls  below t h e  a t 20  weeks,  glia.  d a y 10 r a d i a l  i n different  thought.  at 9  area t o the vascular  t h e upper  to the pia.  Bergmann  of the third  morphology  layer,  layer  resembled  previously  the ventricular  the intermediate  Purkinje  than  fetus  glial  glial  cells  cells  were  ventricles,  regions.  of third  After  of postnatal seen  i nthe  and had very d a y 10, o n l y  venticle,  showing  of tanycytes.  variable tanycytes  the postnatal ¥  Developmental D i f f e r e n c e s - A Source Of H e t e r o g e n e i t y  Glia  cells  histories. complete  can have v e r y d i f f e r e n t  Understanding t h e development  and has undergone  the  Golgi  all  non-neuronal  epithelial first the  staining  cells  postulated  neural  cells  tube,  many  cells,  that  germinal c e l l s  was  the first  rather  t h a n mesenchymal ependyma  which  t o prove  just  which  i n origin  do.  germinal c e l l s  produce  away  from  to differentiate  producing  producing. cells  Schaper  a bipotential  44  -  columnar  H i s (1889)  were  and d i f f e r e n t i a t e  as neurons  -  tube.  a r e neuron  glial  that  the layer  from  of  of germinal c e l l s i n  are glial that  using  the precursors arose  of the neural  the theory of 2 types  not  I n 1888 G o l g i ,  the spongioblasts,  i n the walls  (1891)  i s s t i l l  changes.  technique, proposed  and s p o n g i o b l a s t s  primative  developmental  into  cell glial  Lenhosseck epithelial from t h e  (1897) that  argued  migrated  or neuronal  cells.  I t was  not  studies  of  spinal  the  transitional the  origin  noted an  cells  of the  three  cell  unidentified  was  later  silver  until  cord  from  (1909-1911),  of the  neuroectoderm types:  third  elucidated  with this  chick  neuroectoderm was  neurons  type by  appeared  to  early  work  adequate  f o r d e v e l o p m e n t a l work.  method  of  selectively  silver  stains  were  not  oligodendrocytes  and  undifferentiated  cell  some o f cells  the  membrane cells then  had  and  lost  alternate  were  were  known  as  p r o c e s s e s and  attachments  route than  methods  but  noted  external  limiting These  astrocytes  This  cells  that  neuroepithelial  subpial  bipotential  the  stained  (1932)  from  to blood vessels.  from  well  not  spongioblasts.  became  The  for  Penfield  supportive  the  sublimate  astrocytes  derived  used  The  gold  and  type  was  processes extending to the  their  formed  cells  third  staining  o f t h e s e two  precursors.  Cajal  microglia.  completely selective  neither  ependymal  which  stained  This  as  that  astrocytes  ( 1 9 1 9 ) who  the  really  Cajal  first,  them  i s that  neuroglia  established.  later.  identify  classical  found  t o mature  firmly  appeared  in his  embryo,  d e l Rio Hortega  c a r b o n a t e method  problem  Cajal  was  for  thus  which an  glial  development. Until ependymal macroglia.  the cell  Using and  was  The  neuroectoderm glioblasts  development  thymidine  sacrificed  thought  current  after  give  of the t o be  to both  injected a t day  25,  glia  of  after  birth  glial  45  and  i n rats  from The  oligodendrocytes. from  genesis i n the  -  the  the  develop  formation declines.  astrocytes  -  microscope,  the precursor  theory i s that  neuroblast  rise  electron  day  1-21,  somatosensory  cortex from  was  observed  (Ichikawa  an  inside to  outside  The  glioblast  development,  His's  original  theory  after  90  by  cell of  years  types  the  neural of  (stage  are  tube  and  can  and  give  glioblast, astrocyte. early  early of  mature  to  (1976) corpus  small He  callosum  glioblast,  proposed  glioblast  oligodendrocyte  the  >medium  of  challenged these  are  phases the  (stage  Out  the  I).  neuroblasts  to  an  glioblasts  evolved  end,  and  different  mice.  a l .  from upon  They  ependymal  >  oligodendrocyte  or  neuroectoderm injury.  are  of  the  and  f o l l o w i n g sequence glioblast  stage  (1981)  types  glioblast,  two  development  Fujuita et  large  >large  doubt.  into oligodendrocytes  four of  weeks. in  that  comes  fibrous astrocytes observed  occured  in different  that  of  two  was  cells  production  resting microglia  rise  showed  stage  cells  production  and  cells  matrix  It  is s t i l l  same c e l l  resting microglia.  that  i n the  only  i s the  migrate  Sturrock glia  the  When n e u r o b l a s t  which  concluded  the  who  1982).  first  however,  precursor  post-mitotic  which  astrocytes  In  Hirate, i n the  (1980),  but  i s composed  II).  manner  two  Fujita  nothing  develop  begins  cells,  of  mitotic cycle.  these  III  and  immature  early  young  of  development:  light >dark  oligodendrocyte early >mature Skoff  glioblast  >small  >young  astrocyte  astrocyte (1980)  disputed  only  after  neurogenesis.  glia  exist  to  now  gliobast  guide  i n question  the  i n the  the He  concept cites  neurons earlier  -  to  the  -  gliogenesis  observation  their  stages  46  that  place,  (Woodhams  that  though et  occurs radial this  is  a l . , 1981).  He  proposed  from  that  a s t r o b l a s t s and  Astrocytes and,  once  can  proteins  formed,  of  populations have  shown  during and  systems.  to  to  stage. The  (Dahl  et  al.  appear 2  i t was  (Hajos  et  basic  a l . , do  and  suggesting  1981)  not.  acidic  successive  changes  of  subpopulations  et  a l . , 1981,  at  and  from  that  This  a  2  of  to  vimentin  astrocytes  Shaw e t  gliogenesis  may  be  the  the  a l . , 1981,  that  three  16,  cell  on  in  Yen  types  different  some of  postnatal  day  and  than  type  through  glia  different  researchers  which  are  at  10.  type  In  2  1.  be  traced  glioblast  alternate gliogenesis grossly  Type  oligodendrocytes  type  going  glial  times.  a s t r o b l a s t s can  different  of  exist  oligodendrocytes  without  f o r these  populations  day  o p t i c nerve  for  to  at  astrocytes  different  i s evidence  explanation  appear  embryonic  type  seem  describe  that  ventricular cells  in  brain.  theories  of  observe  heterogeneous  in  their  development. These source  of  in  successive  Polyclonal antibodies  (1984)  shown t h a t  came  showed  different  both  noticed  profiles  o p t i c nerve  day  astrocytes Others  types.  developmental  i n the  culture  acidic,  existance  Raff  postnatal  and  glioblasts.  oligodendrocytes  stained  development  glia  development  d i v i d e but (1979)  not  originate  1981).  astrocytes  back  a l .  devleopment  Other  1  of  oligodendrocytes  oligodendroblasts,  can  basic  the  Field,  cells  et  during  ratio  and  divide during  Keenikova  the  astrocytes  different  developmental  confusion  in  heterogeneity.  Not  profiles  i n t e r p r e t i n g the  only  are  there  -  47  -  may  be  literature  different  a  major on  patterns  glia of  gliogenesis  but there  are different  biochemical  abilities  within these  phenomena develop  develop  late.  stimulatory ontogenic (Moonen  stages  glutamate,  brain  (Grisar,  same a g e levels  cultured glial  i n vivo  astrocytes  development like the  this  means  biochemical may  a  i n very  late  into  into  maturation  from  immature  animals  The development  into  a s i t i s much  1974).  than  of  the time  these  amino  i n adult  metabolic  period  when  acids  The i n c r e a s e d  cultured astrocytes occurs  e t a l , 1976, H e r t z  i sthe  aspartate,  late,  carbon  intensity at the  e t a l . , 1979).  Also  (Hertz  may  that  e t a l . , 1978).  evolve  along  with  Therefore  some  the relative  late  compartmentalization.  c a u t i o n must  differences  be used  reported  Observations  when  forglia  be due t o t h e development  interpreting as  some  stage  of the cells  glial  cells  i n the research. Glycogen  developing changes  storage  r a t brain  that  occur.  GS  a t t h e same a g e a n d t h e same o c c u r s i n  of metabolic  hetergeneity used  uptake  differences  only  some  t o have  develops  and B a l a z s ,  (Schousboe  rise  glucose  1970).  of glucose  glutamate  i s noted  biochemical  coincides well with  (Patel  whereas  which  i n brains  (Van den Berg,  intensifies of  of differing  and glutamine  conversion  biochemical  1979) o r i n o l d e r a s t r o c y t e c u l t u r e s  of radioactive  compartmentation  Some  as vimentin,  potassium  o f development of  1977)  example  pronounced  cells.  o n t h e Na+,K+-ATPase  and Franck,  incorporation  such  F o r example  effect  Another  less  early,  patterns  changes w i t h i n r a d i a l i s another Such  example  storage  -  48  first  -  of  of  developmental  appears  on  embryonic  day  14  i n the choroid plexus  midbrain These  and medullary  cells  retained  development storage adult  levels  glycogen In  radial  glia  also  Glycogen  as an energy  work,  phosphatases,  Colmant  (1965)  cells  of  1981).  throughout  showed  storage  This might  source  glial  and B i e s o l d ,  capacity  by p o s t n a t a l day 21.  other  some then  glycogen decreased  indicate  i n perinatal  to  that  metabolism.  noticed increases i n acid  DPNH- a n d T P N H - t e t r a z o l i u m r e d u c t a s e s ,  dehydrogenase,  ^-naphthol  (Bruckner  developed.  i s used  succinate  raphe  the highest  but other  as they  and i n t h e r a d i a l  esterase  5'nucleotidase, phosphamidase,  and  i n oligodendrocytes during postnatal  development. Lagenaur Ml  e t a l . (1980)  to distinguish  Staining adulthood  appeared  10 a n d l a s t e d  but  n o t a l l GFAP+ Neurons  cessation  only  the antibody  they  designated  o f a s t r o c y t e s i n mouse matter  glia  cerebellum.  a t day 7 and l a s t e d  and i n t h e g r a n u l a r  a short time.  In culture  until  layer  on  i t i s i n some  cells. a r e committed  of division  the cells  with,  i n white  and g l i a  differentiation to  subgroups  b u t i n Bergmann  day  used  occurs  and blood  b u t i n most throughout  to cell of these  might  biochemical  variability.  -  49  -  prior  schemes  t h e parenchyma,  vessels they  allowing f o rthe local  lines  the thus  eventually climate to  to final close  interact induce  Heterogeneity A)  Developmental  i n tissue cultures  changes  i n culture  Primary  cultures of glia  provide  information  on developmental  questions  Primary  c u l t u r e s are thought  situation  and develop  characteristics. that  oligodendrocytes  specializations experimental be  carried  glial  such  et a l .  as t i g h t  Several  culture.  These  and  many  heterogeneity.  f o r instance,  junctions.  redevelop Cultures  and developmental  ages  showed membrane  allow  observations  have monitored  f o r changes  a r e b e l i e v e d t o mimic  the i n vivo  i n vivo  B3,f even  researchers  cultures of various  pertinant  closely  (1983),  designated  manipulations  out.  t o mimic  or redevelop  Massa  some  with  to  primary  time i n  developmental  changes i n  vivo. Fedoroff in  et a l .  (1984a)  cultures, originally  from  three  precursor cells  are They  cells  that  contains cells  days  or glioblasts  free  o f t h e next  flat  have  stage  intercellular  microfilaments  The e a r l i e s t are closely Their  each  other  junctions  and c o n t a i n  singly  production  scanty  dispersed  which  bundles  apposed  associated  astroblasts contain  astrocyte epithelial cytoplasm  to a variable  The p r o a s t r o b l a s t s g r a d u a l l y  also  longitudinally  a similar  comes t o a n e n d , t h e t h i r d  -  50  -  degree.  with intermediate  differentiate  morphology  of intermediate  The  lineage, proastroblasts,  filaments.  have  astrocytes  b u t few m i c r o f i l a m e n t s .  of astrocyte  from  newborn mouse  a t low d e n s i t y ,  junctions.  ribosomes  and separate  have  plated  t o f o u r weeks.  rarely  many  examined  fibers. stage  except When begins  that  into they  neuroblast which i s  the  production  o f a s t r o b l a s t s a n d ependymal  migrate  andmature  resting  microglia.  defined  processes  that the  the  route  only  fibrous  astrocytes  general  origin  o f heterogeneity  stage which  have  study  observation  would  is not o f the These  ofdefinition  could  well showed  astrocytes  of astrocytes.  how l a c k  orcellular  This  t o fibrous  the  illustrates  o r astrocytes and  perikarya.  glia  These  as t o  lead t o i nr e a l i t y be  stages.  Marker  changes  research.  also  Schousboe  time  occur  eta l .  i na s t r o c y t e  whole  S100  radial  I tsupports  conditions  different  old  from  also  observations  with  The mature  or subventricular  observations culture  olgodendrocytes  andd i s t i n c t  route.  ventricular  into  cells.  forebrain.  a n d compound (1980)  cultures  i s synthesized  mainly  showed  t o exceed  Labourdette  the  difficulties i n  that the  andMarks  GFAP  level  (1975)  after differentiation  increased i n 4 week  showed  that  i n t h e C-6  line. Changes Schousboe cultures  i nenzymes  etal.  of astrocytes  stimulatory  the  two  i nc u l t u r e  drops  lactate to  three  first  from  in vivo  t o the  week b u t  level.  changes  GABA-T  -  enzymes  o f mice  occur  Lactate above  from  back  51 -  in  primary  or rats.  i nc u l t u r e but t h e  until  4 weeks.  dehydrogenase  that  o f adult  The iso-enzyme immature  i na s t r o c y t e  increases  development.  various  cortex  not  changes.  adult  then  did  during  a t 2-3 w e e k s  to a level  dehydrogenase weeks.  the  i t s peak  e f f e c t s o f K+  parallels weeks  occur  (1980) m o n i t o r e d  Na+,KH—ATPase r e a c h e d  then  also  t o levels  peaks a t  brain,  pattern o f  t o mature  cultures  This  drops  from one  i n the  comparable t o  those but  i n neonatal  found  to  cultures.  uptake  increase  COMT a n d  astrocyte Levi  mouse b r a i n .  MAO  cultures and  was  culture.  toward  (1983)  restricted Therefore  phenomenon  or  in vivo  increased  (Hansson  Ciotti  Carbonic  to  and  to  showed  subset  low  in  primary  1983).  GABA b u t  stellate  was  in differentiated  time  Sellstrom,  mature  a  levels  with  GABA t r a n s p o r t  i s due  anhydrease  not  D-aspartate  astrocytes  in  is a differentiated  in  culture  that  become  prominent. Meller embryonic protein types were  and  Waelsch  brain  were  were  used  which  myelin where  year.  to  identify and  92%  glial  monitored:  GFAP+,  protein+.  the  studied  cultures  Anti-GFAP  either myelin  were  basic as  a  observed  GFAP- and  cells  for  (1984)  basic and  The  cell flat  cells  myelin  types.  from  basic Four  cell  epitheloid cells  protein+  or  -,  oligodendrocytes  astrocytes  oligodendrocytes  and  of  that  astroglial which  originate  were  continuously  d i f f e r e n t i a t e every  20  to  3 0  days. Vernadakis newborn had  and  both  and  adult  and  hand,  cultures  increased  and  these  astrogliosis  astrocyte  and  both from  these  oligodendrocytes  Other  mice  oligodendrocytes  markers, in  Mangoura  seen  people  found and  decreased i n aging  cultures.  shown Wilkin  those  astrocytes, in  mice  only  compared  that  increased adult  for  have  (1983)  a  as  the  days,  i n number.  from  from  newborn  determined  culture.  only  few  cultures  On  the  mice by  other  astrocytes  whereas  This  the  parallels  the  brain. a et  -  variety al.  52  -  of  (1983)  cell  types  examined  in  primary  primary  cultures  made  positive  and were  class  was  from  cerebellar  o f two d i s t i n c t  stellate  i n shape w i t h  processes  while  polygonal  or elongated.  and  therefore  aspartate stellate  but only  longer  change  i n shape  Non-stellate this  not a  a true  true  cells  may  indicate  that  but  other  o f neurons  B)  Effect  Some  such  attempted found  that  more  controlled  only  1% +  up The  of culture but  possibly  undergoing  increases  ability stellate  suggesting  function.  to divide  a  c-AMP i s  morphology  The f a c t  that  but stellate  of committal  of  i s not  d i d not astrocytes,  to  or presence  of particular  factors.  of culture  They  which  of plating,  may b e  t o study  thymidine  interactions.  GABA u p t a k e ,  as state  a t time  i n culture  took  a r e two d i f f e r e n t t y p e s  conditions  of the variability  differences  c-AMP,  continued these  factors  differentiation  t h e 12 d a y s  of biochemical  cells  either  d i d s h o w a w e a k GABA u p t a k e  stages.  One fine  being  u p GABA.  o f d i f f e r e n t i a t i o n and t h a t  indicator  types.  incorporate  took  to cell  GFAP  distributed  Both  cultures,  cell  d i d not increase agent  could  cells  over  were  i n shape,  of division.  density  that  radially  both  the stellate  following  non-stellate  types  capable  i n lower  at later  morphology,  They  which  morphological  was v a r i e d  disappeared  lasted  lost  the other  were  cells  astrocytes  i n cell  mediums.  t h i s by using  a chemically cultures  on c e l l type  Morrison  that  were  f o r f i b r o n e c t i n , a marker  -  may b e d u e t o  slight  and D e V e l l i s  (1983)  a chemically  defined  53 -  development  defined  medium p r o d u c e d  95% a s t r o c y t e s f o rmeningeal  medium. p u r e r and  (GFAP+) a n d or  endothelial  cells.  differentiated at  least  and  cells  positive  some b i o c h e m i c a l  Not the  The  Raff  et  Type  fibroblast-like,  monoclonal pituitary  antibody extract  i n grey  matter.  bound  tetanus  toxin  could of  be  converted  dBcAMP,  were  to  pituitary  or  extract  properties  of  2.  In  GFAP b y  culture  in  If  changes  in culture  done  on  how  which  et  influence  cultures. animals  A  and  meningeal  in vivo  culture  Trimmer  were  not  gain  were  the  bovine  also  morphology, by Type  i n the  1  presence  especially  specific  cultures  which  are  factor.  extracts,  the  by  stimulated  growth  or  most  in  binding of  induced  C u l t u r e c o n d i t i o n s can  the to  type  2  express  thus  morphology.  understanding of  neuron-like  brain  cells  conditions.  changes  understanding  a  factor  of  toxin  and  neonatal  GFAP-  induce  morphology,  tetanus  in  GFAP+.  growth  or  d i d not  from  being  n e u r o n - l i k e morphology  but  developed  bind  epidermal  medium,  cells  both  types  divide  and  serum-free  type  indicating  factors  stimulated to  2 had  A2B5,  extract  GS,  d e s c r i b e two  d i d not  Type  and  and  differentiating  matter,  epidermal  found  pituitary  of white  A2B5,  or  to  a l . (1983)  in culture  are  S-100P  differentiation.  astrocytes  bovine  f o r both  a l l a s t r o c y t e s respond  s a m e way.  1  were m o r p h o l o g i c a l l y  c o n d i t i o n s can the cell  mechanism  induce  c o u l d g i v e us  differences.  conditions influence  a l . (1984) the  decrease  explored  cellular of  fibroblasts  of  the  a l l caused  -  density,  54  an  -  in  an  Much work h a s  been  cultures.  culture  composition  plating  supplementation  the  changes  of  conditions  cerebral  cortical  i n c r e a s e d age  cortical  cultures  increase i n  of  with  fibronectin  the  staining,  and  a  decrease  i n GFAP,  receptors  and  a  decrease  in  express  both  Goldman high  types and  density  reached  higher  densities  less  several  a c t i n and  increase  in cell  GFAP and  retained  of  these  forms  cells  with  take a  narrow  al.  callosum, approach  without  resembles  the  to  and  the  really  this  dBcAMP  develop at  using  that  et  cell  did  polygonal,  thin  whereas not  stained  for  relative in  Both  the  of  spindle-shaped  astroglial  astrocytes  cells  change  conditions  can  al.  found  (1983) forms  vimentin  spontaneously  large  T h u s much o f the  where  the  from  shape  the  as  than  a  cell  of  the  the  there  the  the  cell  -  the normal  layer  of  cells  Both  first. are  in  lower  astrocyte.  responding  conditions.  55  exist within  Such  but  contain cells  special confluent  heterogeneity  same c e l l  -  top  developing  i n t e r f a c e between  r e s u l t of  on  i s smaller  stimulated  with  the  medium. the  filaments,  cytoskeletal proteins.  dBcAMP w h i c h  vimentin,  conditions  (1982),  Fedoroff  cells;  seem  and  r e s u l t s were m i r r o r e d  from  at  small  GFAP+  densities  jS2.  plated  had  were  40%  cytoskeletal actin  These  develop  in culture  precursor  and  and  confluency.  same c u l t u r e .  GFA  normally  processes.  showed  Differences  cultures  to  adrenergic  astrocytes  that  and  amounts o f  these  time  few et  of  flat  fil  quickly,  initial  were  cells  60%  intermediate  low  filaments.  synthesis  Lindsey  cells  large  that  processes  more  at  number,  intermediate  corpus  long  were p l a t e d  rates  with  showed  contained  to  sites,  (1984)  and  that  Astroglial  binding  i n £ 2  increase  Chiu  perikarya  those  of  an  that to  layer  exists  differing  is  Culture cells.  A  subset  inability  to  polylysine were  98%  remain  plate  coated  so  in  high  If  plastic  plates.  at  that  and  myelin  exist  select  were  highly  H2SO4  that  can  selected but  by  high  into  of  of  their  only  on as  differentiated l e v e l s of  sulfides,  associated  presence  subsets  oligodendrocytes  i s shown by of  for  plates,  were  are  basic  b i p o t e n t i a l or to  to  culture  +,  mimics  conditions  postulated  used  These  This  i . e . , the  culture  looking  on  incorporation  metabolism  glycoproteins  be  oligodendrocytes  culture.  myelinogenesis,  been  of  can  galactocerbroside  activity, lipid  differences  they and  CNPase  and  a  with  myelin  associated  protein. change  cells,  multipotential in vivo  and  perhaps  cells.  have  we  are  They  been  have  demonstrated  in  cultures. Raff  et  a l .  differentiates free  medium  serum.  (1984) into  and  an  an  astrocyte  contains  becomes  an  and  GFAP as  vimentin  astrocyte  oligodendrocyte.  progenitor  with  was  a  a  loses  used  marker  as  that  for  which  in  fetal  marker  a  serum  calf  for  astrocytes.  i t retains  i f i t becomes  commitment  cell  i f cultured  i f cultured  filaments  and  The  a  oligodendrocyte  Galactocerebrosidase  oligodendrocytes cell  describe  The  i f i t  v  an  is reversible for  1  to  2  days. Noble cells to  in  of  Murray  optic  divide  soluble  and  by  nerves  the  factors  progenitor  (1984) of  neonatal  presence from  cells  such and  found  of  the  same o r  rats.  purified  astrocytes,  56  -  These  type  1  similar  were  stimulated  astrocytes  producing  oligodendrocytes.  -  very  Noble  a  large and  or number  Murray  speculated optic type to  that  nerve  this  the cells  They  e t a l . (1981)  form  type  A  found  are  morphologically different.  that  and develop  A  cells  equivalent  immature  into  Since  epithelial-like  come m a i n l y  type  these  postnatal cells type  devleopment  decrease.  Another (1982)  who  there  that  treatment  with  type  B.  dBcAMP.  are also  primary  treatment  increase i n type  cell  lines  express  one o r t h e o t h e r  this  finding  may  Pruss respond while  As  colony-forming between  that  found  growth  oligodendrocytes will  not i n primary  causes  or i n adult  parallels  that that  factor only  cultures.  cell  57  A t o type  cells  to that  rats.  B  after express  of true  seen  Since  enzymes,  with  most but not  induced  occurring i n vivo. astrocytes i n culture and Schwann c e l l  do s o i n suspended  -  Hertz  30% e x p r e s s i o n o f  of these  T h i s may  -  type  untreated  be an example  e t a l . (1982)  to fibroblast  shown b y Yu a n d  B i s similar  age o f t h e c u l t u r e  differentiation  that  probably  astrocyte cultures  increasing  both,  propose  interactions  was  A t 31 d a y s ,  A b u t dBcAMP  This  t h e number o f  t h e p r o p o r t i o n o f MAO  i n mouse b r a i n  type  t o dBcAMP  t h e s u b v e n t r i c u l a r zone.  of interaction  decreased  mainly  which  conditions.  example found  from  occurs,  Thus  and c u l t u r e  cells  from  C cultures  respond  are astrocyte progenitor cells,  to the pale  identical  (1983).  t h e same w a y s a s a s t r o c y t e s d o , t h e a u t h o r s  type  but  of antigens  colonies i n culture  s u b v e n t r i c u l a r zone  of the  2 astrocytes but not  et a l .  the  in  be t h e s o u r c e  had a p r o f i l e  r e p o r t e d by R a f f  Juurlink that  may  o l i g o d e n d r o c y t e s and type  1 astrocytes.  cells  subpopulation  be r e l a t e d  mitogen cultures  to the  astrocytes Since in  vivo  ability  the content  serum  changes  differentiation, same p r o g e n i t o r changes  with cells  after  injury.  o f serum  change  may  be what  well  different cells  with  age o f t h e animal,  controls  glial  o r i g i n a t i n g from t h e  a t d i f f e r e n t times  dictated  by the  i n serum.  Table changes  to divide  III lists  of various  some  of the effects  substances  -  i n primary  i n the culture  58  -  cultures  medium.  of  TABLE I I I : MEDIUM CHANGES dcAMP  PROBABLE MECHANISM Through cAMP  EFFECTS OF CULTURE CONDITIONS ON CELL CHARACTERISTICS CELL TYPE  OBSERVED  CHANGES  AUTHORS  P r i m a r i l y F l a t e p i t h e l o i d - l i k e c e l l s change Astrocyte t o larger stellate-type that Cultures resemble astrocytes Increase Actin Actin  GFAP a n d  organized  Microtubules organized into extending into processes Level Levels  o f most o f GS  et a l . Hansson and (1983)  Vimentin  increases less  Fedoroff (1984b); Ronnbeck  enzymes  bundles  increase  Ciesielski-Treska e t a l . (1984) Ciesielski-Treska e t a l . (1982b) Ciesielski-Treska e t a l . (1982a) Schousboe e t a l . (1980a)  decreased  Proportion decreased  W h i t e Se H e r t z (1981) Kimelberg e ta l . (1978a) Hansson & S e l l s t r o m (1983) o f MAO T y p e A t o T y p e B Yu & H e r t z (1982) as i n o l d e r c e l l c u l t u r e s o r i n v i v o w i t h age.  Astrocytes intensely  t o s t a i n f o r CA-II as as oligodendrocytes  Change i n p a t t e r n o f p r o t e i n s y n t h e s i s Increased e f f e c t of potassium s t i m u l a t i o n o n Na+,K+-ATPase MAO a n d COMT i n c r e a s e d  D e c r e a s e d GABA u p t a k e w i t h Vmax a n d K a f f e c t e d  both  Kimelberg (1982)  et a l ,  Hansson e t a l . (1984b)  TABLE MEDIUM CHANGES dcAMP  I I I (continued):  PROBABLE MECHANISM Probably through AMP  EFFECTS  CELL TYPE C-6 Primary astroglial C-6  Horse  Serum  Fetal serum  calf removal  OF  CULTURE CONDITIONS  OBSERVED [S  CELL CHARACTERISTICS  CHANGES  100] p r o t e i n  Increased  ON  AUTHORS  increased  aspartate  Binding pattern of becomes c o n f l u e n t  aminotransferase  concavalin-A  A s t r o c y t e c u l t u r e s from non-serum c o n t a i n i n g medium b e g i n t o e x p r e s s Primary astrocyte cultures  COMT a n d MAO  Prostaglandin PGE1  Through cAMP  G l i a maturation factor Cytosine arabinoside  Fischer et a l . GFAP (1982)  Schousboe e t a l , (1980a)  increased  Hansson & S e l l s t r o m (1983) I n c r e a s e d N a + , K + , A T P a s e a n d GS Schousboe e t a l . (1980) I n c r e a s e d GABA-T a n d a s p a r t a t e a m i n o Tardy e t a l . t r a n s f e r a s e ; same m o r p h o l o g i c a l changes (1981) a s w i t h GFAP  Surface G l i o b l a s t s A s t r o g l i a l receptor  Mitotic inhibitor  Tabuchi e t a l , (1981)  R e t r a c t i o n o f c e l l soma; e x t e n s i o n Hansson & Ronnback of c e l l processes; decreased 3H v a l i n e (1983) incorporation into protein; decreased t o t a l soluable protein; decreased protein s e c r e t i o n ( a l l returned by s o l u b l e b r a i n extracts) Increased glutamate dehydrogenase a n d GABA-T ( a s i n a g e d m i c e )  Hydrocortisone  Tabuchi e t a l . (1981) T a r d y e t a l (1981)  maturation  Lim Ito  (1977) e t a l . (1982)  Cerebellar Astroglial maturation neuronal cultures  Leu  e t a l . (1983)  C)  Cell  development  to  location In  kind  The  depending  on of  of  the  most  earlier  as  1980). even  of  differentiation  response of  to  occurs  some e x t e n t  injury,  the  age  of  i n response  is  i n response  enzymes  to  injury  animal  to  in size become  than  1980).  The  and  f o r those  of  the  (Friede,  increases are  the  1966,  in  reactive active,  or  citric  permanent  and  increase  involved in glycolysis  dehydrogenase  enzyme  in old  (Oehmichen,  enzymes  shunt  succinic The  to  variable,  the  a s t r o c y t e s i n c r e a s e i n number,  f o r those  occurs  the  acid  as  hexose  cycle,  Oehmichen,  as  they  persist  scars.  Morphological ischemic  injury  found  occur  consisted  of  cytoplasm  and  activity.  response  A l l o x i d o r e d u c t i v e enzymes become more  other  to  in  injury.  processes  monophosphate such  cell  type  the  astrocytes. do  of  type  general  number  differentiation  injury  Another injury.  and  changes  were  examined  (Petito  w i t h i n 4 0 mins.  expansion  The  i n astrocytes i n response  rough  and  ER,  number  after  Babiak,  injury.  i n c r e a s e d number  suggesting  of  and  astrocytic  nuclei  found  only  1982)  These of  increased  to and  changes  mitochondria,  metabolic  also  increased  very  early. Murabe morphology  et  i n response  hippocampus. Polynuclear the  a l . (1981)  neurons.  They  to  first  that  kainic  a c i d - i n d u c e d damage  swelled,  a s t r o c y t e s extended  61  then  filaments  processes  A s t r o c y t e s appeared  -  a s t r o c y t e s changed  t o have  -  in  the  developed.  i n areas  vacated  phagocytic  by  activity.  Primary c u l t u r e s d e r i v e d from k a i n i c a c i d l e s i o n e d r a t striatum lead to 2 morphologically (Van A l s t y n e e t a l . , 1983). of l a r g e f l a t c e l l s with processes  distinguishable c e l l  They were mainly  types  (95%) composed  i l l d e f i n e d j u n c t i o n s and no  cellular  but 5% o f the c e l l s were s m a l l w i t h p r o c e s s e s .  Upon  treatment w i t h dBcAMP, the l a r g e immature c e l l s t r a n s f o r m the s m a l l e r type.  to  These newly d e r i v e d s m a l l e r c e l l s e x h i b i t  c e l l - s p e c i f i c markers ( g a l a c t o c e r b r o s i d e on 10% and GFAP on 80%),  p l u s some f e t a l c h a r a c t e r i s t i c s .  Therefore  the l a r g e r  c e l l s were g l i o b l a s t s t h a t were i n the k a i n i c a c i d damaged tissue. Freide  (1966) showed t h a t the o x i d o r e d u c t i v e  oligodendrocytes  enzymes i n  are more a c t i v e than i n r e s t i n g a s t r o c y t e s  but they i n c r e a s e t o above the o l i g o d e n d r o c y t i c l e v e l i n reactive astrocytes. Although o l i g o d e n d r o c y t e s and  t h e i r oxidoreductase  response t o trauma  do not p r o l i f e r a t e , they do grow  enzymes do become more a c t i v e i n  (Ibrahim  e t a l . , 1974).  Colmant  (1965)  n o t i c e d i n c r e a s e s i n a c i d phosphatases, DPNH- and TPNHt e t r a z o l i u m reductases,  s u c c i n a t e dehydrogenase,  5 ' n u c l e o t i d a s e , phosphamidase, a n d / 5 - n a p h t h o l e s t e r a s e . too much damage occurs,  the o l i g o d e n d r o c y t e s  If  will die.  Even w i t h knowing t h a t t h e r e a r e s e v e r a l sources  of  v a r i a t i o n between c e l l s t h a t can e x p l a i n much observed heterogeneity,  there i s s t i l l  t o be due t o these v a r i a b l e s .  heterogeneity C e l l types  t h a t does not seen  i n v i v o and i n  c u l t u r e a r e found t o have a number of b i o c h e m i c a l  - 62 -  differences  t h a t remain unexplained.  I f there are biochemical differences  i n g l i a they must subserve  some d i f f e r e n c e s i n f u n c t i o n .  D) H e t e r o g e n e i t y between d i f f e r e n t g l i a not e x p l a i n e d by development o r c u l t u r e c o n d i t i o n s D i f f e r e n t g l i a l systems even have d i f f e r e n t d e n s i t i e s depending on t h e i r o r i g i n  cellular  (Henn, 1980), and  u l t r a s t r u c t u r a l h e t e r o g e n e i t y has been d e s c r i b e d (Mori and Lebond, 1970). Schachner e t a l . (1977) found t h a t GFAP was l o c a t e d i n v a r i a b l e p l a c e s i n a s t r o c y t e s o f t h e mouse c e r e b e l l u m .  The  l a b e l was found i n c e l l s around t h e g l o m e r u l a r complexes i n the g r a n u l a r l a y e r , i n r a d i a l f i b e r s i n t h e m o l e c u l a r i n t h e sheath surrounding P u r k i n j e c e l l s ,  layer,  and i n a s t r o c y t i c  end f e e t impinging on meninges and b l o o d v e s s e l s ; i n white matter c e l l bodies t h e r e was d i f f u s e c y t o p l a s m i c l a b e l and elongated s t r i n g s of l a b e l . Mize e t a l . (1981) observed t r i t i a t e d GABA l a b e l l i n g i n the s u p e r i o r c o l l i c u l u s o f c a t s and noted t h a t dark o l i g o d e n d r o c y t e s and a s t r o c y t e s accumulated  GABA  moderately,  w h i l e l i g h t o l i g o d e n d r o c y t e s and m i c r o g l i a d i d not.  The dark  o l i g o d e n d r o c y t e s wrap around p r e s y n a p t i c t e r m i n a l s and are t h e r e f o r e l i k e l y candidates f o r t h e removal o f GABA. and H o s l i  Hosli  (1978) observed t h i s b a r r i e r f u n c t i o n working i n the  cultured g l i a l c e l l s of dorsal root ganglia.  I n mixed  c u l t u r e s t h e g l i a , not t h e neurons, would take up GABA, but, i f t h e neurons were i s o l a t e d they took GABA up b e t t e r than the glial cells.  T h i s was i n t e r p r e t e d as meaning t h a t t h e g l i a l  -  63 -  cells  normally  prevents Levi  t h e uptake et al.  astroglial enriched  cells  were  that  cells by  population cell  the ability  acid  neurotransmitters.  stellate  mostly  but, during  and they  were  astrocytes.  Early  f o r GABA, o f GABA  et al.  noted  3H-D-aspartate GFAP-containing  i n substantial  astrocytes  amounts  o f other  shapes  the stellate from  one  o f t h e I C 5 0 s f o r GABA u p t a k e i n h i b i t o r s  indicated  that  astrocytes to a glial  t h e GABA t r a n s p o r t  d i d n o t have transport  lose  their  even  though  i n neuron-enriched  ability  t o take  the stellate (1966)  noted  activity.  cytochrome  morphology that  matched  i n the culture.  that  as c u l t u r e s  astrocytes grow  cells,  activity  -  64 -  may  older,  i s maintained.  not a l l oligodendrocytes  Satellite  oxidase  present  present  generally  but instead  cultures  u p GABA  systems  the features  system  noticed  marked  Levi  of the  examinations and  They  enzyme  The  l a b e l i n g was v a r i a b l e  Autoradiographic  to  12 d a y s , t h e  and, even w i t h i n  of the i n h i b i t o r y interneurons  same  cells  t o be 70-80%  a s shown b y  by  of undifferentiated  larger.  3H-GABA w a s a c c u m u l a t e d  expressed  o f these  the next  increased  that  Freide  which  interneuron-  the undifferentiated  the extent  consistently  composed  astrocytes  into  t o another.  attributed  amino  lightly  determinations  in  with  accumulated,  the stellate only  t h e morphology  cultures  present  but that  stained  correlated  cells,  autoradiography,  a barrier  neurons.  originally  aspartate  forming  cerebellar,  of stellate  astrocytes  zone,  i n post-natal  putative  GFAP-containing number  into  (1983)  primary  accumulate cultures  a c t as a b u f f e r  which  f o r example,  have t h e have  a  i s not observed i n  other  oligodendrocytes.  Szuchet  and  designated culture,  Yim  B3,f,  but  had  which  cultures  the  may  been  shown  were  observed  rate  was  was  There  interspecies  a  few  systems.  i n young  rat  totally  astrocytes  in  weak t o  strong  experiments  were done  is a  very  cell  between  are  studied.  They  extent  resemble  to  several  relatively  compared  differences.  These  chick  ouabain  a  true  i n the  within  body  of  normal  easy  to  glia. they  cell  maintain  These  are  for  long  65  where  1978). 1978d).  variation  laboratory.  cell  the  lines  on  differences  research  C-6  lines are  is  (Benda,  not  1968).  frequently believed  established  periods  relatively  laboratories.  -  species  was  The  to  cell  some lines  readily available,  c h a r a c t e r i s t i c s are  between  (Hertz,  research  but  1979).  astrocytes,  rate  same  have  uptake  (Latzovits,  glial  produced  glial  potassium  (Kimelberg,  of  lines,  first of  of  show c h a r a c t e r i s t i c s t h a t  advantages:  their  cell  many t y p e s  be  and  extensive  line  astrocyte  much h i g h e r  seems t o  glial  there  a  observation  i t s problems.  be  from  rates  cultures  had  without The  Low  i n h i b i t e d by  different glial  because  homogeneous  they  in various  astrocytes  i n primary  between  have  line  glycoprotein  varied  reported  to  almost  the  differences  Heterogeneity  Now  associated  staining that  due  higher  last  since  oligodendrocyte  morphologically  be in  Mouse b r a i n This  was  an  cells.  Some o f  it  found  anti-myelin  galactocerebrosial between  (1984)  However,  -  of  time  and,  stable,  they  because  they  can were  originally  transformed  characteristics brain. the  They  may  metabolic  be  quite  low,  rates  for glia  glial  uptake  types  but  of  similar  not  glia  without  useful  tools  quite  early  of  that  to  can  glia  cell  that  They  be  made  corroborative  o r may  even  which  from  normal example,  believed glial  e r r o n o u s l y low have  glia.  in  For  early  may  have  cell metabolic  characteristics have  a  neuronal  high  (Edwards  glial  to  i s transported into  Such  evidence.  for preliminary  on  glutamate  of  found  originally  i n neurons  cultures not  was  cell  their  different.  gave  lines  seen  glia  work done  example,  glial  or viruses,  like  cells  tissue  For  i n primary  extrapolation  be  (Hertz, 1978b).  o r more  number  entirely  glial  on  scar  characteristics. large  of  based  and  two  not  chemicals  actually  rate  lines  of  are  by  They  affinity  et  a l . ,  1979)  mean  that  findings  cell  lines  remain,  r e s e a r c h because  of  a  to  normal  however,  their  ease  of  of markers  or  use. Glial other be  cell  charcteristics  easily  are  not  of  are  (1981) lines  glial  used  found than  Wilson  neuronal  share  from  always GI  more  clear. and  new  because  glia.  They  can  sometimes  cell  lines  but  these  Specific  G2  but  ^-glutamyl  (Stallcup  not  glial  antigens and  because  they  neuronal lines.  Cohn, are  tumor  Shine  such  as  1976) on  lines  the and  et a l .  transpeptidase i n g l i a l  cell  ones.  a l . (1981) cell  neuronal  lines  in defining  glia  with  s u r f a c e markers  tumor  i n neural et  d e f i n e d as  they  1974),  considered g l i a l  thus  and  are  (Schachner,  surface  are  distinguished  distinctions NS-1  lines  lines.  worked He  extensively  used  -  66  antisera  -  to  define  against  glial  pseudoneuronal relationship other.  For  and  the  classic  example,  the  N4  cell  Pseudoneuronal  and  of  similar  pseudoneuronal found and  to  be  NG2.  of  other  GFAP.  showed MG  no  showed  et  because  after  and  permanent  as  f o r GFAP  subpopulation of  The  that basic  thrived  lines  line  (Kimelberg,  i s lower  cerebellum  may  be  in  than  activity  uptake which  than  the  of  were NG1  cell  lines.  differed  from  i n expression  cells.  reverse  hand,  be  material  glial  cultures  line  Osborn  and  U251 et a l .  glial  cell  i n response  changes  cells  the  called  human g l i a l  because  or  in  to  the  because  containing different  of  i n primary other  been  and  a  found  cultures  there  genetic  than  67  i n most  of  cell  t o have  cultures  classic  -  functioning  glioma  lower  primary  show a  other  glial  lines  by  culture.  and  have  and  each  the  lines  cultures  i n transformed cells  1974)  1977) , f o r e x a m p l e ,  potassium  i t d i d not  the  t h e r e must  GFAP+  primary  biochemical level  cell  Na+,KH—ATPase  98%  by  antigens  doublings but MG  between  related  cell  glial  biopsied  between  This difference  material  that  astrocyte  U333CG/343  difference  marker  possess  that  primary  On  the  lines.  further  a l . (1981)  found  and  neuronal  were  a l . concluded  define  expressed  pseudoglial  both  seven  3%  7/10  neuronal  from  to  lines  channels.  and  GFAP+  seemed  a  K+  normal  lines  was  et  lines  by  lines  Cultures of  the  genetic  and  and  cell  l i n k a g e s between  and  found  dBcAMP.  Na+  lines  a n t i g e n was  neuronal  related  developmental  each  lines  cell  Wilson  Osborn  cell  between  pseudoneuronal  finding  pseudoglial  The  (Hertz,  Nernstian slope of  C-6  lower  glial  Nernstian slope  -  glia.  lines a  glioma  cells  for  from  (Sugaya  et  the  al.,  1979).  NN  cells  are  found  stimulation  (Ciesielski-Treska,  Glioma  lines  cell  bulk-separated systems cells  cultures  the  Their  glycolytic  of  C-6  et  cells  and  cell  than  al.,  are  1976)  C-6  and  most  and  Hutchison,  brain, even  Evidence as  did  not  properties  to  such  GS  than  et  Not a l l  primary  astrocyte  of  glucose  et  are  found  a l . , 1978b),  in  but  t o have  This  different  i n C-6  enzyme  lines  t o dBucAMP  lines of  as  form  A  et  (Haber contain  1982).  metabolism  i n primary  MAO  forms:  culture  (Hertz,  of glutamate  of  (Murphy  i n two  only  i n primary  levels higher  cells  exists  contain  astrocytes  exposure  i t i s not  1973).  activities  activity  o f ATP  astroglia  Astrocytes possess  cell  into  higher  enriched i n  Brooker,  cell  to their  C-6  a l . , 1978).  and  and  than  f o r example  incorporation  K+  cultures.  content  a l . , 1975).  related  show e v i d e n c e as  lipid  however;  f o r compartmentation  strong i n g l i a l seem  et  Kimelberg  but  after  lower  seems t o be  other g l i a l  especially  not  be  higher.  1976)  primary  rate  proportions of types.  activities  both,  lines  than  t o m a i n t a i n a h i g h e r amount  (Passonneau  (DeVellis  1976)  level,  higher  o x y g e n may  a l . , 1979,  Various MAO  the  anhydrase  responsive to  (Norton  lower  ability  rate  Carbonic  in  a  shown by  absence  (Roussel  at  less  a much  a higher glycolytic  as  lactate.  have  astroglia  function  have  also  t o be  is  cultures.  C-6  compartmentalization i n  but  bulk  isolated  glial  cells  did. High  affinity  uptake  autoradiographically al.,  1974,  Henn  et  of  glutamate  in glial  a l . , 1974,  -  cell  lines  Balcar  68  -  has  et  been  demonstrated  (Faivre-Baumann a l . , 1977,  et  Pfeiffer  et  al.,  1976),  1974). to  Na+  system Calcuim  This  may  than  i n C-6  be d i f f e r e n t  cultures (Balcar  cells  Table uptake  uptake  are generally  f o r uptake  centrifugation,  into  that  1975).  uptake  into  primary  o r t h e NN into  glial C-6  1974).  t h e Vmax v a l u e s f o r g l u t a m a t e  higher f o r primary astrocytes  or glial  uptake  cultures.  but i s f o r uptake  et a l . ,  sensitive  The  e t a l . , 1977b)  e t a l . , 1977)  Iversen,  t o be  i n primary  f o r glutamate  (Faive-Bauman  and  shown  (Henn,  that  (Schousboe  IV i n d i c a t e s  (Snodgrass  has been  cells  from  i s not required  line  glioma  astrocytoma  glutamate  stimulation  astrocytes cell  including  cell  -  cultures  prepared by lines.  69  -  of  gradient  astrocytes  Table  IV:  Comparative  Values  (from Cell  of  Hertz,  Km(^M)  i n primary  "  II  II  "  "  "  "  "  culture  11  A  50  5.9  B  30-90  11  "  "  66 cells  11  12-19  D  0.4  E F  14  "  C  0.4-0.6  15  "  3-7.5  10-20  glioma  glioma  Reference  0.8  C-6  NN  Vmax*  22 0  II  11  Uptake  1979)  Description  Astrocytes  Glutamate  0.07  G  0.02-0.03  H  MGM-LM g l i o m a  cells  20  0.3  I  138  cells  65  0.14  J  astrocytes  12  F  12  K  MG  Bulk  glioma  prepared 11  "  »  "  "  "  Bulk  prepared  cerebellar  10  0.06  L  a s t r o c y t e s 15  0.2  M  21  3.5  N  Retina * ( pi m o l / m i n Reference 1978b, =  C  per  codes =  al.,  et  1976,  Weiler and  et  Neal,  Hamberger, 1974,  S  =  A  Hertz  Faivre-Bauman  Balcar  g  protein) =  Schousboe  et et  a l . , 1974,  = Walum  a l . , 1979, 1976, 1971,  0  = Q  Hutchison  et  a l . , 1979b,  a l . , 1977, J  2  =  H  =  F  a l . , 1977b, D  =  =  Henn  Balcar et  and  Weiler,  M  LeCampell  =  et  Lasher,  1975,  et  a l . , 1974,  -  70  et  and  K  =  -  = =  et a l . ,  Hauser,  I  =  Henn,  Shank,  a l . , 1979, R  Hertz  1978,  a l . , 1974,  a l . , 1978,  T  =  B a l c a r and  1978,  Schousboe  B  P  G  1978, =  Schrier Schousboe  N  Henn and et  =  Stewart  1976,  L =  E  et  = White  and  Thompson, a l . , 1977a.  Baetge variety for  et  of  a l . (1979)  glial  glutamate.  Two  high  glutamate  only  moderately  occurred uptake They  rates  had  ways  glutamate that  the  glioma bulk  preparation of  but  glial  and  were  the  same.  differing  the cell  line  to  i n NN,  normal  BE11, B15  uptake, Even  though seem  Km's  cell  the  line. and  cultures  He  some of  their to  vary.  in  between  cells on  had  however,  glutamate  o f Na+  very  Bill,  t o Na+  f o r NN  wide  rates  had  and  d i d not  i n C-6  primary  uptake  coupled  (12 0 mM  influence  examined the  glutamine  kinetic  to preparation but the  mechanism  a  and  18  mM  uptake  of  also  found  other  astrocytes  cells.  also  Again  and  two,  lines.  Ca++-dependent  not  B28  another  cultures  and  r e s e a r c h on  some g l u t a m a t e  was  found  from  was  (1979)  preparations.  and  the  different  lines,  basic  Km  primary  uptake  Hertz  average  (1978b)  found  other c e l l  the  the  differed  prepared  rates  the  cultures)  lines,  cell  specificity  and  and  primary  of  reviewed  and  high uptake;  same  Schousboe  in  glial  varied,  the  lines  lines  uptake  i n most  identical  cell  cell  also  lines  as  -  into  constants varied  the  glioma  indicated  71  uptake  -  line  was  i n Table  various  from about V.  the  or  Table  V: C o m p a r a t i v e  values  different Cell  Astrocytes  "  MG  "  glioma  cell  glial  cell  affinity (1981)  uptake  that  C-6  high  affinity  Henn  0.16  L  3 3 00  5.0  0  150  0.2  D  490  2.9  J  between  (1975)  t h e work  exhibit  h a d a much uptake.  than  that  (1979)  comparable from  72  -  a high  Schousboe that  to that  found i n  the cerebellum but though  reviewed concluded  of astrocytes  -  t o have  sensitive.  capacity  (Table VI) and s i m i l a r l y  lower  and tranformed  others and found  derived  lower  glia  C-6 c e l l s  w a s Na+  a Vmax  Hertz  normal  found  o f many  and i n neurons  cells  literature  varies  Reference  ( f o r r e f e r e n c e s s e e T a b l e IV)  f o r GABA w h i c h  astrocytes  slices  C-6 w a s  also  lines.  compared  cultured brain  uptake  preparations  63 0  line protein  into  Vmax*  "  * ( a m o l / m i n p e r 1 0 0 mg  GABA  culture  uptake  Km(^M) astrocytes  i n primary  " D138  glial  Type  Bulk-prepared  of glutamine  i t was  s t i l l  much o f t h e the capacity  i n culture.  of  Table  VI:  of Cell Bulk  GABA  astrocytes  cerebellar  glioma  "  "  "  " cerebral  "  "  * |Jmol/min  g  wet  have  1977)  of mice  Different Pilkington  et  spontaneous 10  of  cell  References  K 0.0001-0.0002  Q  32  0.002  R  0.22  0.0001  S  nm  vary  even  of  45  0.040  B  prepared  glia  lower  activity  age  (Nicklas  can  also  showed  related cell  3  nm  to  that  C-6  line.  cells  i n morphology,  patterns  b i o c h e m i s t r y such  characteristics  can  be  in a  73  even  between  accumulation  -  1978).  derived number and  a  from  and  that  given  lots.  plate  colony  by  the  of  single  manipulated  -  i n the  However, and  in  differences.  lines  degree  patterns of as  structural  to  and  Browning,  cell  IV)  compared  found  microtubules  the  Table  (Nicklas  than  differed  morphologically within  noted  GABA-T  and  show  that  234  differences of  T  bulk  astrocytoma  the  0.035  ( f o r r e f e r e n c e s see  f i l a m e n t s and  differentiation  40  of  lines  were  K  activity  much  murine  differences  These  Vmax*  low  a l . (1982)  these  (1978)  weight  similar  of  can  preparations  0.6 0.29  a very  and  ratio  line  uptake  P  astrocytes  a s t r o c y t e s and  Browning,  a  glia  affinity  0.27  11  per  also  cultured  brain  high  50  Cultured  C-6  various glial  "  C-6  of  Km(>JM)  "  Cultured  into  Values  Type  prepared  "  Comparative  Benda  display  formation, of  cell  and  S-lOOp.  serums,  plating  density the  and  ability  1970).  to  High  increase (Logan, effect be  other  in  l e v e l s of amino  of  are  For  came o u t  of  cerebella differed short  fibrous  resemble  and  one  no  of  M2  and  which  Golgi  of of  1  was  and long  culture  and  They  and 2  to  i t has  be  the  conditions  mouse  a l l GFAP+  thought  small  somata,  Type  They 3 had and  to  with  were large  were  but  several  monoclonal  cells.  of  clones  bound  These  to  spontaneous  somata,  were  thin.  and  more  matches  were  small  and  time  has  morphologicially.  were h e t e r o d i p l o i d ,  over  changes  8-day p o s t n a t a l  had  astrocytes.  selective  astrocytic cell  had  Type  a  a l . , 1981).  appears  1984).  Type  M3,  et  medium  et a l . ,  and  same c u l t u r e  types  epithelial  processes,  in  the  astrocytes.  filamentous  stable  there  Pressac,  cells  factor that  (Kato  cultures  out  (bovine)  tumor  were p s e u d o d i p l o i d  BSP-3,  resemble  somata,  distinct  established  processes,  proceses,  all  three  (Alliot  antibodies  C-6  biochemically  morphologically.  resemble  to  differ  (Pfeiffer  morphological  time  exactly  serum-less  bring  factor  gliogenesis  under  which  example,  on  s i t u a t i o n s where  differentiation lines  than  serum  and  maturation  in  is lost  calf  critical  postnatal  There  cell  a  example,  S-lOOp  uptake  cells  within  For  fetal  acid  Glia  normal  present  period  accumulate  1976). on  factors.  two  thought flat  thought  to  c h a r a c t e r i s t i c s were  thus  represent  true  differentiation. Cell  lines  biochemistry. than  do  can  differ  For  example,  neurons,  many n e u r o n s  but  and  has  higher  from  an  each  C-6  has  other  in  higher  Na+,K+ p u m p i n g  than  other  -  74  glial  -  basic  l e v e l s of action  cell  S-lOOp  similar  lines.  to  Another  example  was  y-glutamyl the  found  activation  AChE  of  of  substances, It  Cell  has  a  lines  cells  can  such  another  putative created  derived  from  simian  high  as  cell  C-6  did  of  while  the  of  that  acid  They  clones, or  dependent  glycine  other  conditions  the  d e t o x i f i c a t i o n of  and  and  NN  with  have  line  Phillips  the  cell  carefully  a  high  i n one  K55,  transformed  percentage  Pessac,  C-6  glutamine and  capacity  the  culture  while They  the  also  differently  Pessac,  astrocytoma  of  contain  1983),  in alanine.  used  or  et a l .  clone,  cultures high  of  1983).  and  primary  for taurine  uptake  lower. found  lines  class.  The  differences  which  but,  c o n t r o l l e d , some  75  -  were  amino  conditions  -  well.  1980).  transformation,  and  high  (1977) cell  as  f r o m mouse c e r e b e l l a r  (Cambier  that  was  tissue culture  were  a  were h i g h  types  various  A1B1.  Cambier  astrocytes  clones  found  Arnold,  in  cell  systems  particularly  that  (Cambier  cell  glial  i n human  transmitter.  found  clones  (1978a)  i n the  on  in  spontaneous  levels in different  correlated  membranes,  certain clonal lines  are  derived  by  astrocytes  Drummond acid  that  astrocytic cell  Schousboe cultures  involved  transmitter  i n only  that  be  lowest  (Vernadakis  exist  oligodendrocyte-like  than  and  showed  to  i n the  glycine-enriched  virus-4 0  that  across  and  They  i s thought  in various  amino  a  (1981).  mouse c e r e b e l l a r a s t r o c y t e  amounts  noted  acids  found  virus-40.  astrocytic by  be  clones  (1983)  al.  which  i n C-6  differ  cell  simian  et  tremendous v a r i a t i o n between  i s highest  Certain  by  amino  biopeptides  activity  glial  Shine  transpeptidase,  transport  lines.  by  acid i f  not  in  amino  well  l e v e l s were these  statistically  s i g n i f i c a n t d i f f e r e n c e s were s t i l l  found.  found t o be p a r t i c u l a r l y h i g h i n both C-6 Glutamate  levels i n various c e l l  GABA l e v e l s were and B92  lines.  l i n e s v a r i e d between 50.8  158 nmol/mg p r o t e i n and glutamine l e v e l s from 0.8 nmol/mg p r o t e i n .  glial  to  to  107  S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s were  a l s o observed f o r a s p a r t a t e , p r o l i n e , g l y c i n e , v a l i n e , c y s t a t h i o n i n e , i s o l e u c i n e , and  alanine,  leucine.  The uptake of amino a c i d s by these d i f f e r e n t c l o n e s does not n e c e s s a r i l y v a r y i n the same way  as the l e v e l s .  Schier  and Thompson (1974) examined uptake of p u t a t i v e n e u r o t r a n s m i t t e r s by t h r e e c u l t u r e d g l i a l c e l l cell  lines.  The  l i n e s e x h i b i t e d s i m i l a r r a p i d uptake o f glutamate and  Na+  dependent uptake of GABA, as w e l l as pyridoxal-dependent GABA s y n t h e s i s and e x c r e t i o n .  T a u r i n e uptake o c c u r r e d i n a l l  t h r e e , w i t h each showing a f a s t s a t u r a b l e component and a slow n o n - s a t u r a b l e component which v a r i e d i n magnitude between the cell  lines.  There was  one c e l l  l i n e which c o u l d m a i n t a i n a  high concentration gradient of taurine. from c y s t e i n e was Glial cell  S y n t h e s i s of t a u r i n e  o n l y found i n one of these l i n e s .  l i n e s may  a l s o respond d i f f e r e n t l y t o drugs.  Elkouby e t a l . (1982) found t h a t two g l i a l c e l l astrocytoma and C-6  glioma, responded  C-6  i n response t o these hormones.  dexametiasone, C-6  cells  NN  d i f f e r e n t l y t o the  hormones h y d r o c o r t i s o n e and t h y r o x i n e . Ca++,-Mg++ ATPase i n c r e a s e d i n the NN  lines,  The a c t i v i t y of  l i n e but decreased i n  Another  drug,  can be used t o induce GS i n o n l y a subset of  (Holbrook e t a l . ,  1981).  B i g n e r e t a l . (1981) examined v a r i o u s c h a r a c t e r i s t i c s of  - 76 -  f i f t e e n l i n e s of human c e l l s t r a d i t i o n a l l y thought of as being gliomas.  They showed a wide v a r i e t y of human l e u k o c y t e  a n t i g e n phenotypes.  A l l but two,  which were from a b l a c k  p a t i e n t , had type B glucose-6-phosphate isoenzymes. mice, two GFAP+.  dehydrogenase  Only f o u r c o u l d be t r a n s p l a n t e d i n t o  of which grew and then r e g r e s s e d .  Thus each l i n e had a unique  athymic  Only two were  profile.  C e l l l i n e r e s e a r c h t h e r e f o r e shows a v a r i e t y of types of heterogeneity.  The s i g n i f i c a n c e i s unknown but t h e r e are a  number of sources of v a r i a n c e t h a t may differences.  F i r s t , b e i n g transformed c e l l s , they may  e x p r e s s i n g some new may  e x p l a i n some of the  genotype.  be  Second, the transformed  cell  be e x p r e s s i n g d i f f e r e n t p a r t s of the genotype than i s  n o r m a l l y expressed by the p a r e n t c e l l r e s u l t i n g i n a mixing of characteristics.  T h i r d , these v a r i o u s g l i a l types may  be  d e r i v e d from d i f f e r e n t types of p a r e n t c e l l s and r e t a i n the differences.  D i f f e r e n t subtypes of the p r o g e n i t o r c e l l s  be r e l a t e d t o v a r i a b l e s we have a l r e a d y d i s c u s s e d or to  may  perhaps  the areas of the b r a i n from which the c e l l came.  D i f f e r e n c e s i n g l i a l c e l l s from d i f f e r e n t areas o f the b r a i n There has been a wide v a r i e t y o f r e s e a r c h t h a t has shown regional heterogeneity i n g l i a l c e l l s . generated by people who  Much of the data were  d i d not s e t out t o show d i f f e r e n c e s  between r e g i o n s or are minor o b s e r v a t i o n s i n a paper another t o p i c .  There are p r o b a b l y many more examples b u r i e d  i n the l i t e r a t u r e . in  on  Such d i f f e r e n c e s have not been emphasized  i n d i c e s t o the l i t e r a t u r e because i t was  -  77  -  not u n t i l  recently  t h a t an i n t e r e s t i n t h i s s u b j e c t developed . I t has been known f o r a long time t h a t t h e morphology o f glial  c e l l s v a r i e s between d i f f e r e n t areas o f t h e b r a i n .  Examples i n c l u d e g l i a l c e l l s t h a t a r e s p e c i a l i z e d enough t o have s p e c i f i c names, such as Bergmann g l i a , whose v a r i a n c e i n morphology has been p r e v i o u s l y d i s c r i b e d . are areas o f b r a i n where g l i a l c e l l s appear d i f f e r e n t but have not been g i v e n  In a d d i t i o n ,  there  morphologically  s p e c i f i c names.  Astrocytes  o f t h e hippocampus, f o r example, have a c h a r a c t e r i s t i c shape t h a t i s d i f f e r e n t from t h a t seen i n o t h e r a r e a s . Astrocytes  a r e known t o have s e v e r a l d i f f e r e n t types o f  GFAP o f d i f f e r e n t m o l e c u l a r weights and d i f f e r e n t s o l u b i l i t i e s i n water, w i t h those o f h i g h m o l e c u l a r weights b e i n g the l e a s t water s o l u b l e  (Eng, 1982).  These forms a r e unevenly  d i s t r i b u t e d i n t h e b r a i n even though they a r e a l l c a r r i e d on the same gene (Gheuens e t a l . , 1984). i n f l u e n c e t h e shape o f a s t r o c y t e s , explanation  S i n c e GFAP i s known t o  t h i s might be p a r t o f an  f o r some o f t h e shape d i f f e r e n c e s .  GFAP v a r i e s not only i n s t r u c t u r e but i n i t s schedule o f appearance d u r i n g development. GFAP i n o l f a c t o r y bulbs, during  f o r e b r a i n and c e r e b e l l u m o f r a t s  development, u s i n g a double antibody radioimmunoassay.  Each b r a i n r e g i o n for  Weir e t a l . (1984) measured  GFAP.  showed a d i f f e r e n t p a t t e r n o f development  A t b i r t h , GFAP p r o t e i n i n the o l f a c t o r y bulb was 85  times t h a t i n f o r e b r a i n , and 485 times t h a t i n c e r e b e l l u m . The  increase  i n GFAP corresponded w i t h m a t u r a t i o n more than  proliferation.  The p a t t e r n o f i n c r e a s e  i n GS a c t i v i t y was  s i m i l a r t o t h a t o f GFAP i n the f o r e b r a i n and o l f a c t o r y bulbs  - 78 -  but d i f f e r e d markedly i n the cerebellum.  In t h e cerebellum  the maximum i n c r e a s e i n GFAP o c c u r r e d a f t e r t h e peak o f a s t r o g l i a l p r o l i f e r a t i o n and 1 week b e f o r e maximum of  acquisition  GS and S-100 p r o t e i n . The d i s t r i b u t i o n o f a s t r o g l i a l c o n t a c t s on t h e s u r f a c e of  neurons v a r i e s g r e a t l y among b r a i n areas as w e l l as among d i f f e r e n t types o f neurons (Guldner and W o l f f , 1973, P e t e r s and Palay, 1965, and Wolff, 1965).  Neurons and synapses may  even be wrapped d i f f e r e n t l y by s e v e r a l l a y e r s o f g l i a l lamellae  (Guldner and Wolff, 1973, Palay, 1966, Specek, 1968,  and S z e n t a g o t h a i , for  1970).  Palay and Chan-Palay  (1974) showed,  example, t h a t P u r k i n j e c e l l s a r e l a r g e l y covered by  Bergmann g l i a  i n c o n t r a s t t o c e r e b e l l a r i n t e r n e u r o n s which a r e  not wrapped. W o l f f and Guldner  (1978) found t h a t e l e c t r i c a l s t i m u l a t i o n  produced s w e l l i n g o f a s t r o c y t i c processes  i n t h e neocortex.  S i n c e t h i s e x p e r i m e n t a l l y produced f e a t u r e o f c o r t i c a l a s t r o c y t e s e x i s t s normally it  i s suggested  i n c e r t a i n other a s t r o g l i a l  cells  t h a t v a r i a t i o n s o f t h e s t r u c t u r e and  arrangement o f a s t r o g l i a l processes between d i f f e r e n t b r a i n r e g i o n s may r e f l e c t neuronal a c t i v i t i e s . some evidence  There i s , however,  ( d i s c u s s e d below) t h a t these c h a r a c t e r i s t i c s are  not j u s t responses  t o neuronal  i n f l u e n c e s but a r e s t a b l e  c h a r a c t e r i s t i c s o f the g l i a i n v a r i o u s r e g i o n s . There have been numerous o b s e r v a t i o n s o f d i f f e r e n c e s i n number o f g l i a l c e l l s i n v a r i o u s b r a i n a r e a s . Leglond  S z e l i g o and  (1977) not o n l y found d i f f e r e n c e s i n numbers but a l s o  showed t h a t h a n d l i n g o r e n r i c h e d environments caused  - 79 -  increases  in  the  numbers  of  a s t r o c y t e s and  oligodendrocytes  layers  of  the  not  certain the  corpus  in  the  (1980)  densities resting  location. found  Others  For  glycogen  and  CNS  areas,  i n the  that the  a  such  as  different  1972,  the  variability glial  observed  functional  tendency  Penar,  depending  cells  of  has  on been  accumulate  Oehmichen, from  activity  activity  to  in  area  1980).  to  area  developing  of  rat  brain  1981).  glycogen  in various  to  astrocytes are  and  Biesold,  area  and  t h a t have  storage areas  incorporation of  from  that  strong phosphorylase  in radial  can  concentration  found  areas  confirmed  only  neuronal  i n other  is quantitatively  example,  storage  (Bruckner  varies  i n the  (Mossakowski  have  Not  reported  state  i n those  glycogen  and  only  callosum.  Oehmichen varying  cortex  in  be  but  seen  the  precursors  area.  Higher  s u p r a o p t i c and  in  different  ratio  into  of  glia  vs.  glycoconjugates  incorporation levels  arcuate  nucleus  and  were  lowest  in  cerebellum. Other  indices  of  uptake  varies  widely  (1980)  showed  that  only  to  This  enzyme  from  metabolism area  the  i s normally  triphosphate  white  of  specialized DeVellis  the  white  can  area. BB  (ATP)  also  Thompson  matter  of  human  cells  regenerating  matter  Glucose  et a l .  was  localized  cerebrum.  t h a t have  capabilities  contractile would  vary.  isoenzyme  associated with  involved i n transport or  astrocytes  to  creatine kinase  astrocytes of  adenosine cells  glial  appear  systems. to  high such  Therefore  have  functions. et  a l . (1967)  found  -  80  that  -  there  as  i s a regional  difference  i n the  dehydrogenase  with  levels  of  not  explained  be  external  the  Hussain  on  their  such  i s unknown b u t  astrocytes  of  other  and  of  large  the  visual  (Bjorklund  et  benzilate  (Divac  Hansson from  et  various  with  blocked  a  pronounced  in cells  areas  rich by  of  haloperidol,  the  the  non-dopamine  this  ability  reason  not  cells  of  have  functions for  been  to  this  found  other  or  on  between  of  various cortex  catcholamines for  quinuclidinyl  1978). that  astroglial  showed  of  Such cells  stem.  antagonists  parts  -  the  increase  increase from  cAMP  was  after  could  of  be  most  the  striatum  and  Astrocytes  prepared  from  that  can  be  chloropromazine,  a l , 1984b).  81  cultures  increased  binding  antipsychotic  -  from  prefrontal  ligand  show d o p a m i n e  et  various  isolated  apomorphine;  containing  (Hansson  glia  in  i n membrane b i n d i n g  brain  brain  dopamine  from  The  can  glia.  layers  enzyme  i n content  showed  subpopulation  and  internal  higher  This  Muller  differences  antagonist.  i n dopamine the  found  and  (1984a)  from  for  that  This  a muscarinic  dopamine  in a  on  Braestrup,  dopamine  least  blocked  and  regions  incubation by  been  and  al.  the  AMP.  Differences  a l . , 1978),  diazepam  showed  i n d i c e s between  have  naloxone,  tables  enzyme has  literature  brain.  cortex  the  as  hemispheres.  time  not  showing  areas.  transmitter-related regions  but  phosphate  brainstem  cerebral  membranes.  monophosphates  is a  and  (1982)  layers  difference  There  glucose  developmental  retinal  1  of  cerebellum  then  by  and  5 -nucleotidase hydrolyse  the  induction  Kreutzberg the  inducability  drugs, the This  but  brain means  astrocytes do  not  that  have there  must be s p e c i a l i z e d c e l l s i n dopamine-rich areas and t h a t t h e s e c h a r a c t e r i s t i c s o f such s p e c i a l i z e d g l i a a r e s t a b l e i n c u l t u r e where they are n o t under neuronal i n f l u e n c e . Hansson (1984) measured the a c t i v i t i e s o f both MAO and COMT i n primary a s t r o g l i a l c u l t u r e s from newborn r a t b r a i n c u l t i v a t e d from s i x d i f f e r e n t r e g i o n s and i n b r a i n homogenates from t h e s e same r e g i o n s .  The areas compared were t h e c e r e b r a l  c o r t e x , s t r i a t u m , hippocampus, b r a i n stem, and cerebellum. MAO a c t i v i t y was h i g h e r i n the c u l t u r e s from the s t r i a t u m than i n those from the o t h e r b r a i n r e g i o n s .  S t r i a t a l homogenates  showed the same t r e n d which c o n f l i c t s w i t h the r e s u l t s o f Hazama e t a l . (1976) who found no d i f f e r e n c e s i n t h e homogenates.  COMT a c t i v i t y was t h e same i n n e o n a t a l c u l t u r e s  and a d u l t b r a i n homogenates and a l s o showed r e g i o n a l differences.  The lowest a c t i v i t y was found i n the b r a i n stem,  w i t h h i g h e r l e v e l s i n the c o r t e x , s t r i a t u m and c e r e b e l l u m and the  h i g h e s t i n the hippocampus. Henn and Henn (1980) found t h a t g l i a from the caudate had  a much h i g h e r number o f h a l o p e r i d o l b i n d i n g s i t e s and more dopamine s e n s i t i v e a d e n y l a t e c y c l a s e than those from o t h e r brain regions.  Even so, the b i n d i n g s i t e s a r e l o c a t e d on only  a f r a c t i o n o f the a s t r o g l i a l c e l l s o f the caudate. A v e r y s u r p r i s i n g f i n d i n g was t h a t by D e n i s - D o n i n i e t a l . (1984) who showed t h a t d i f f e r e n t g l i a l p o p u l a t i o n s a f f e c t t h e morphology  o f mouse mesencephalic dopaminergic neurons.  Glial  monolayers  c u l t u r e d from the s t r i a t a l o r the mesencephalic  r e g i o n o f t h e embryonic b r a i n were used t o grow dopaminergic neurons  from the mesencephalon.  On mesencephalic g l i a l  - 82 -  cells  the m a j o r i t y  o f t h e dopamine neurons developed a g r e a t number  of h i g h l y branched and v a r i c o s e n e u r i t e s , whereas on s t r i a t a l g l i a they o n l y e x h i b i t e d one long, t h i n , l i n e a r n e u r i t e . morphology o f t h e u n d e r l y i n g  The  g l i a was not v e r y d i f f e r e n t but  they were n o t e q u a l l y s t a i n e d w i t h GFAP, thus showing some heterogeneity  i n the l e v e l of expression  of g l i a l  filament.  Thus t h e c l a s s i c assumption o f g l i a o n l y responding t o t h e i r neuronal environment i s a c t u a l l y found t o be r e v e r s e d . Goldlefter  (1976) found t h a t t h e p e r i v e n t r i c u l a r g l i a o f  the hypothalamus were p o s i t i v e w i t h gonadotrophin o r t o Gomori's s t a i n and such s t a i n i n g i n c r e a s e d dopamine.  on treatment with  Thus t h e g l i a o f t h i s area respond t o  neurotransmitter  and t o a hormone produced by s u r r o u n d i n g  cells. Schousboe (1978b) found t h a t h i g h a f f i n i t y uptake o f GABA occurred  i n p e r i p h e r a l g a n g l i a , r a t r e t i n a , glioma c e l l  s p i n a l cord explant cells  c u l t u r e s , and primary c u l t u r e s o f g l i a l  from t h e cerebellum  areas o f t h e b r a i n . the cerebellum  lines,  and cerebrum b u t n o t from  other  I t was o n l y i n g l i a l c e l l c u l t u r e s from  and cerebrum t h a t t h e l e v e l o f uptake was  comparable t o t h a t i n b r a i n s l i c e s .  T h i s a s t r o c y t i c uptake  was d i f f e r e n t from t h e neuronal system s i n c e i t was s e l e c t i v e l y i n h i b i t e d by |3-proline but not by two s e l e c t i v e i n h i b i t o r s o f neuronal GABA uptake. G l i a l c e l l s may a l s o v a r y neurotransmitters.  i n t h e i r response t o  K r n j e v i c and Schwartz (1967) f i r s t showed  t h a t GABA a p p l i e d i o n t o p h o r e t i c a l l y caused d e p o l a r i z a t i o n o f some, b u t n o t a l l , g l i a l c e l l s  i n the cortex.  - 83 -  They c o u l d not,  however, r u l e out the p o s s i b i l i t y t h a t the depended on p r o x i m i t y r e l e a s e d K+  that,  selectivity  t o GABA-depolarized neurons which  i n turn, depolarized  the nearby g l i a .  GABA-T, the d e g r a d a t i v e enzyme f o r GABA, showed no regional differences  (Hansson, 1984)  the c e r e b r a l c o r t e x ,  striatum,  i n primary c u l t u r e s from  hippocampus, b r a i n stem,  and  c e r e b e l l u m of newborn r a t b r a i n . Glutamate i n d i c e s have a l s o been measured and vary r e g i o n a l l y .  Autoradiographical  s t u d i e s o f glutamate or  D-aspartate h i g h a f f i n i t y uptake ( C u r r i e and showed e x t e n s i v e Bergmann g l i a ,  found t o  Kelly,  1981)  uptake i n t o c e r e b e l l a r g l i a , e s p e c i a l l y  and  t h a t t h i s decreased a f t e r t r a n s e c t i o n  certain projections.  of  T h i s i m p l i e d t h a t these d i f f e r e n c e s  are  a r e s u l t of i n f l u e n c e s of neurons on g l i a , not the s t a b l e c h a r a c t e r i s t i c s that other research  was  indicated.  glia  They a l s o  noted o t h e r d i f f e r e n c e s i n glutamate uptake from d i f f e r e n t regions.  Hansson (1983) a l s o used autoradiography t o show  r e g i o n a l d i f f e r e n c e s i n uptake. t o a l e s s e r extent, a s p a r t a t e ,  She was  taken up  c u l t u r e s from the c e r e b r a l c o r t e x , and,  found t h a t glutamate, readily in  hippocampus, and  striatum  t o a l e s s e r extent, i n c u l t u r e s from the brainstem  cerebellum.  and  and  T h i s i s evidence f o r s t a b l e g l i a l d i f f e r e n c e s i n  glutamate uptake.  V a l i n e , an amino a c i d which i s  mostly i n t o p r o t e i n , was  used as an i n t e r n a l c o n t r o l and  found t o be accumulated t o the same extent i n the primary c u l t u r e s .  The  was  various  Schousboe (1978a) showed a range i n v a l u e s  i n glutamate uptake by a s t r o c y t e s b r a i n regions.  incorporated  c u l t u r e d from d i f f e r e n t  Vmax ranged from 8 nmol./min/mg c e l l  - 84  -  p r o t e i n i n c e l l s from whole cerebrum t o 60 nmol./min/mg i n c e l l s c u l t u r e d from c e r e b r a l c o r t e x , w i t h Km M t o 50 J A M .  Schousboe and Divac  v a r y i n g from 22 0^  (1979) f u r t h e r showed t h a t  the glutamate uptake i n primary a s t r o c y t e c u l t u r e s from neonatal  mice a f t e r t h r e e weeks i n c u l t u r e was  greater i n  c e l l s o r i g i n a t i n g from the p r e f r o n t a l c o r t e x and  neostriatum  than i n those o r i g i n a t i n g from the o c c i p i t a l c o r t e x  or  cerebellum.  the  These r e s u l t s g e n e r a l l y c o r r e l a t e w i t h  synaptosomal uptake of glutamate i n these r e g i o n s and t h a t t h i s g l i a l c h a r a c t e r i s t i c was  indicate  stable f o r at l e a s t  three  weeks i n c u l t u r e without neuronal i n f l u e n c e s . D r e j e r e t a l . (1982) d i d a s i m i l a r experiment and the f o l l o w i n g Vmax v a l u e s 13.9,  - 5.8  neostriatum and not Km  neostriatum  nmol/min/mg c e l l p r o t e i n .  minor d i f f e r e n c e s i n Km  not  f o r astrocytes: p r e f r o n t a l cortex  o c c i p i t a l c o r t e x - 11.4,  cerebellum  where i t was  found  - 27.3,  -  and  There were o n l y  between r e g i o n s except i n the s l i g h t l y higher.  D i f f e r e n c e s i n Vmax  mean t h a t t h e r e are d i f f e r e n c e s i n the number but  i n the p r o p e r t i e s of the t r a n s p o r t s i t e s .  Again the  authors noted the apparent r e l a t i o n s h i p between the r e g i o n a l a b i l i t y o f g l i a t o accumulate glutamate and number of glutaminergic  terminals.  G l y c i n e i s an important i n h i b i t o r y t r a n s m i t t e r a t the s p i n a l l e v e l but not i n the f o r e b r a i n . gradient-separated  a s t r o c y t e s from s p i n a l cord, but not  from f r o n t a l c o r t e x , (Henn, 1980).  I t has been found t h a t those  show a h i g h a f f i n i t y uptake of g l y c i n e  Others have confirmed t h a t the d i s t r i b u t i o n of  g l i a l t r a n s p o r t systems f o r g l y c i n e f o l l o w s the same  - 85  -  d i s t r i b u t i o n as g l y c i n e  ( H o k f e l t and Lungdahl, 1971, Matus and  Dennison, 1971) . The and  conclusions  o f these o b s e r v a t i o n s  on GABA, glutamate,  g l y c i n e i s t h a t t h e r e a r e probably d i f f e r e n c e s i n the  numbers o f uptake s i t e s i n g l i a l c e l l s i n v a r i o u s regions  and t h a t these a r e s t a b l e i n c u l t u r e .  brain  Moreover, the  g l i a l uptake seems t o c o r r e l a t e t o some e x t e n t w i t h the r e g i o n a l d e n s i t y o f t h e amino a c i d boutons.  Schousboe e t a l .  (1980b) suggested t h a t t h i s g l i a l h e t e r o g e n e i t y  must be taken  i n t o account i n t h e i n t e r p r e t a t i o n o f neurochemical changes r e s u l t i n g from s p e c i f i c neuronal d e g r e n e r a t i o n s . the e f f e c t s o f g l i o s i s a f t e r k a i n i c a c i d l e s i o n s  F o r example, could  s e r i o u s l y a f f e c t i n t e r p r e t a t i o n o f b i o c h e m i c a l changes.  Summary o f evidence f o r b i o c h e m i c a l d i f f e r e n t i a t i o n i n g l i a The  evidences f o r r e g i o n a l b i o c h e m i c a l d i f f e r e n c e s i n g l i a  or c u l t u r e d g l i a i s thus q u i t e s t r o n g .  Some d i f f e r e n c e s may  be because o f d i r e c t e f f e c t s o f surrounding neurons b u t some are s t a b l e i n c u l t u r e a f t e r t h e e f f e c t o f t h e neurons i s no longer  there.  These s t a b l e d i f f e r e n c e s may be i n t e g r a l p a r t s  o f t h e g e n e t i c makeup o f these g l i a o r may be i n i t i a t e d a t some c r i t i c a l  developmental p o i n t by i t s environment.  Questions o f t h i s nature have n o t y e t been answered. evidence t h a t r e v e r s e al.  e f f e c t s may be o p e r a t i v e .  There i s  Paterson et  (1977) showed t h a t g l i a l c e l l s r e l e a s e some f a c t o r t h a t  i n f l u e n c e s t h e amount o f n e u r o t r a n s m i t t e r sympathetically conditioned  synthesized  by  d e r i v e d neurons e i t h e r by c o - c u l t u r e d o r  medium.  They a l s o found t h a t C-6 and sympathetic  - 86 -  s a t e l l i t e c e l l s both i n f l u e n c e growth and development o f c h o l i n e r g i c synapses and ACh s y n t h e s i s . There i s a l s o some evidence o f s p e c i e s v a r i a t i o n s .  There  are, f o r example, c o n s i d e r a b l e d i f f e r e n c e s i n t h e r a t e o f potassium uptake i n a s t r o c y t e s c u l t u r e d from c h i c k , r a t o r mouse b r a i n .  Thus f u t u r e r e s e a r c h must be extremely c a r e f u l  i n t r a n s f e r i n g experiments from one s p e c i e s t o another. I f these b i o c h e m i c a l  d i f f e r e n c e s between g l i a o f d i f f e r e n t  areas and s p e c i e s stand t h e t e s t o f time, then t h e d i f f e r e n c e must be e x p l o r e d  f u r t h e r and  considered  on-going neurochemical r e s e a r c h .  i n much o f t h e  In experimental  c a u s i n g damage l e a d i n g t o g l i o s i s ,  conditions  some o f t h e b i o c h e m i c a l  changes w i l l undoubtedly be found t o be due t o g l i a l  changes.  Research on many d i s e a s e s may have t o c o n s i d e r g l i a as being p o s s i b l y involved i n the e t i o l o g y .  There a r e a l r e a d y  f i n d i n g s i n some d i s e a s e s t h a t p o i n t t o t h i s . Carter  research  F o r example,  (1981) observed t h a t GS a c t i v i t y was reduced i n  Huntington's d i s e a s e  i n some areas where i t c o u l d not be  accounted f o r by c e l l l o s s .  I t has a l s o been observed t h a t  thiamine d e f i c i e n t models o f Wernicke-Korsakoff's syndrome produce damage f i r s t brain  (Collins,  i n g l i a l c e l l s o f c e r t a i n areas o f t h e  1968; C o l l i n s and Converse, 1970).  Research aimed a t i d e n t i f y i n g d i f f e r e n c e s i n g l i a has y i e l d e d much.  But t h e r e i s a l s o i n the v a s t l i t e r a t u r e on  s t a i n i n g o f b r a i n c e l l s many c o i n c i d e n t a l r e p o r t s o f s t a i n i n g o f subsets  of g l i a ;  such r e p o r t s tend t o be b u r i e d i n t h e  g e n e r a l i z e d l i t e r a t u r e because i n t e r e s t i n g l i a has been so l i t t l e compared t o t h e i n t e r e s t i n neurons.  - 87 -  EXPERIMENTAL  R A T I O N A L E AND  ABSTRACT  I have used two u n r e l a t e d s t a i n i n g procedures t h a t  stain  predominantly g l i a l c e l l s , but not a l l g l i a l c e l l s , o n l y s u b s e t s o f them.  I have a l s o looked a t a model o f  Wernicke-Korsakoff's syndrome t h a t demonstrates  that the  d i s e a s e may damage t h e g l i a l c e l l s o f o n l y some areas and b e f o r e neuronal damage occurs i n these a r e a s . In  Experiment  1, hemosiderin, a form o f i r o n , was examined  i n t h e b r a i n s o f r a t s u s i n g a P r u s s i a n Blue f o l l o w e d by d i a m i n o b e n z i d i n e (DAB) procedure.  The areas o f t h e b r a i n  c o n t a i n i n g t h e v a r i o u s types o f c e l l u l a r and n o n - c e l l u l a r s t a i n i n g were mapped.  I r o n was found t o be predominantly  l o c a t e d i n o r on o l i g o d e n d r o c y t e s , but not i n a l l areas as t h e r e was a d i s t i n c t r e g i o n a l p a t t e r n o f s t a i n i n g . a l s o some s t a i n i n g i n neurons, of  There was  ependymal c e l l s and a s t r o c y t e s  s p e c i f i c and r e s t r i c t e d areas, and v a r i o u s l e v e l s o f  background  staining.  t e r m i n a l boutons processes.  The background  s t a i n i n g i s probably  on u n s t a i n e d c e l l s o r neuronal o r g l i a l  The r e s u l t s a r e compared t o t h e known anatomy o f  s e v e r a l n e u r o t r a n s m i t t e r systems.  S i g n i f i c a n t o v e r l a p o f the  l o c a t i o n o f i r o n s t a i n i n g was noted w i t h GABA, dopamine, endorphins and e n k e p h a l i n s . In Gelder  Experiment  2, a m o d i f i c a t i o n o f t h e method o f Van  (1965) f o r h i s t o c h e m i c a l s t a i n i n g o f GABA-T c o n t a i n i n g  c e l l s was used t o s t a i n c e l l s c o n t a i n i n g some enzymes c a t a l y z i n g a p o s s i b l e a l t e r n a t i v e route f o r glutamate p r o d u c t i o n i n b r a i n : from p r o l i n e o r o r n i t h i n e which i s  -  88  -  o x i d i z e d t o glutamate v i a l - p y r r o l i n e - 5 - c a r b o x y l a t e (P5C) 1 - p y r r o l i n e dehydrogenase (EC 1.5.1.12;PDH).  PDH  has  by  been  demonstrated i n s e v e r a l b a c t e r i a and mammalian systems (Fig.  5) and,  in g l i a l  i n our experiment, was  found t o be e x c l u s i v e l y  c e l l s such as the Bergmann g l i a of the cerebellum  a s t r o c y t e s of the hippocampus.  P5C  by the a c t i o n of p r o l i n e oxidase reductase,  EC  1.5.1.2,PrO).  can be  formed from p r o l i n e  (pyrroline-5-carboxylate  T h i s enzyme was  also localized  e x c l u s i v e l y i n g l i a l c e l l s but the s t a i n i n g was distinct.  and  much l e s s  Both of these experiments p r o v i d e a d d i t i o n a l  evidence o f g l i a l  cell  specialization.  Experiment 3 o n l y p o s t u l a t e s g l i a l  involvement i n thiamine  d e f i c i e n c y as the technique does not a l l o w f o r c e l l u l a r histochemistry. i n h i b i t o r , was  Pyrithiamine,  a thiamine phosphokinase  fed t o r a t s on a t h i a m i n e - d e f i c i e n t d i e t t o  c r e a t e an animal model o f Wernicke's encephalopathy. of weight l o s s , a t a x i a , and  l o s s of r i g h t i n g r e f l e x were  produced i n r a t s i n t e n days. s a c r i f i c e d and  Symptoms  At t h i s time some r a t s were  the r e s t o f the r a t s were r e t u r n e d t o a normal  d i e t , t o be s a c r i f i c e d o n l y when t h e i r weight had t h e i r pre-experimental  level.  Rats used f o r  returned  to  biochemical  measurements were s a c r i f i c e d by c e r v i c a l f r a c t u r e , t h e b r a i n s d i s s e c t e d i n t o e i g h t r e g i o n s , and g l u t a m i c (GAD)  and  c h o l i n e a c e t y l t r a n s f e r a s e (CAT)  the b r a i n homogenates. h i s t o l o g i c a l observation  a c i d decarboxylase a c t i v i t y measured on  Other r a t s were p e r f u s e d  for  of GABA-T, by a m o d i f i c a t i o n of a  -  89  -  method o f Van G e l d e r  (1965).  GAD a c t i v i t y was found t o be  s i g n i f i c a n t l y reduced i n symptomatic r a t s i n t h e thalamus > c e r e b e l l u m > pons/medulla  > and m i d - b r a i n .  GABA-T s t a i n i n g  was found t o be s i m i l a r l y reduced, w i t h g r e a t e s t l o s s e s thalamus > i n f e r i o r c o l l i c u l u s > pons > and medulla. a c t i v i t y was n o t s i g n i f i c a n t l y a l t e r e d Upon r e t u r n  CAT areas.  t o a normal d i e t , r e c o v e r y o f GAD was s i g n i f i c a n t  o n l y i n t h e thalamus,  w h i l e GABA-T s t a i n i n g r e c o v e r e d a t l e a s t  p a r t i a l l y i n a l l areas a f f e c t e d . i n terms o f g l i a l assumptions  i n any b r a i n  i n the  These r e s u l t s a r e d i s c u s s e d  s p e c i f i c i t y and e f f e c t s t h e s e new  might have on t h e i n t e r p r e t a t i o n o f t h e r e s u l t s .  - 90 -  EXPERIMENT  1  There have been few s t u d i e s examining t h e c e l l u l a r d i s t r i b u t i o n o f i r o n i n b r a i n but i r o n may have an important r o l e i n t h e b r a i n , and may be i n v o l v e d i n s e v e r a l d i s e a s e processes.  The r o l e o f i r o n i n the CNS i s not y e t understood  but low d i e t a r y i r o n i s known t o have a number o f e f f e c t s on b r a i n f u n c t i o n , i n c l u d i n g e f f e c t s on t h e electroencephalogram (EEG)  (Tucker, 1982), and d i s t u r b a n c e s i n c i r c a d i a n rhythm,  t h e r m o r e g u l a t i o n , motor a c t i v i t y decreased  (Youdim e t a l . , 1981) and  a t t e n t i v e n e s s (Youdim e t a l . ,  1980).  The mechanism  of p r o d u c t i o n o f symptoms i n i r o n d e f i c i e n t y i s thought a t l e a s t p a r t l y through  t o be  n e u r o t r a n s m i t t e r s although t h e reduced  c a p a c i t y o f t h e b l o o d t o c a r r y oxygen may have an i n d i r e c t e f f e c t on t h e b r a i n .  Chronic, s l i g h t l y e l e v a t e d l e v e l s o f  i r o n have been shown t o be t o x i c t o both ACh and GABA neurons (Swainman, 1984).  I r o n d e p o s i t s can a l s o occur i n c e r t a i n  d i s e a s e s such as i n H a l l e r v o r d e n - S p a t z Huntington's  d i s e a s e o r Parkinson's  (Bronson,  disease  1980),  (Swainman, 1981).  Non-heme i r o n e x i s t s i n two forms i n t h e b r a i n : f e r r i t i n i s i r o n h e l d i n s t o r a g e by a p r o t e i n forming g l o b u l e s i n lysosomes i n some p a r t s o f t h e b r a i n , and hemosiderin, i s f e r r i c hydroxide  g r a n u l e s d e p o s i t e d more e v e n l y i n c e l l  b o d i e s and p r o c e s s e s . i n the b r a i n .  which  Hemosiderin i s probably t h e form a c t i v e  Hemosiderin r e l e a s e s f e r r i c i r o n on exposure t o  hydrogen c h l o r i d e and potassium the f e r r i c i r o n producing  f e r r o c y n a n i d e can r e a c t with  f e r r i c ferrocyanide (Prussian Blue).  T h i s c l a s s i c P e r l ' s r e a c t i o n can be i n t e n s i f i e d u s i n g a  - 91 -  procedure  o f Nguyen-Legros e t a l (1980).  Diaminobenzidine i s  added t o t h e P r u s s i a n Blue, a l l o w i n g t h e P r u s s i a n Blue t o a c t as a c a t a l y s t f o r t h e o x i d a t i o n o f DAB by hydrogen p e r o x i d e forming an i n t e n s e brown d e p o s i t where t h e i r o n i s . the procedure we used t o s t a i n f o r i r o n .  This i s  We d i d a d e t a i l e d  map and a n a l y s i s o f both c e l l u l a r and n o n - c e l l u l a r i r o n which a l l o w e d c o r r e l a t i o n o f i r o n w i t h known n e u r o t r a n s m i t t e r anatomy.  METHOD  A method s i m i l a r t o t h a t o f Nguyen-Legros e t a l . (1980) was used.  Two s o l u t i o n s were made up j u s t b e f o r e experimental  procedures were s t a r t e d .  S o l u t i o n A: 4% hydrogen c h l o r i d e .  S o l u t i o n B: 4% f e r r o c y a n i d e . B r a i n s t h a t had been p e r f u s e d w i t h phosphate b u f f e r e d s a l i n e f o l l o w e d by 4% formaldehyde/4%  g l u t e r a l d e h y d e , and  s t o r e d f o r a t l e a s t t h r e e days i n t h e same f i x a t i v e s , were used. S l i c e s were c u t on a c r y o s t a t a t 50  and r e a c t e d f o r  twenty minutes i n a 50% mixture o f s o l u t i o n s A and B. r e a c t i o n proceeded not b l u e o r green.  I f the  c o r r e c t l y , t h e s o l u t i o n should be y e l l o w The s l i c e s were then washed i n 0.1 M  phosphate b u f f e r , pH 7 . 4 , f o r 3 t o 5 min. While t h e s e c t i o n s were washing, t h e DAB reagent was made up.  2 0 mg DAB was mixed i n t o 100 ml t r i s b u f f e r pH. 7.6, and  2 drops o f hydrogen p e r o x i d e  (30%) a r e added.  The DAB  (3.3'-diaminobenzidine t e t r a h y d r o c h l o r i d e monohydrate 97%) was o b t a i n e d from  Aldrich.  - 92 -  The s e c t i o n s were p l a c e d i n t h i s r e a c t i o n mixture f o r 1 0 min.  i n t h e dark, then taken out and washed, mounted,  dehydrated  and c o v e r s l i p p e d .  (The darkness o f t h e s t a i n  depends on t h e amount o f H2O2 and t h e time i n t h i s  reaction  mixture). RESULTS S e v e r a l types o f s t a i n i n g were seen.  There were v a r i o u s  l e v e l s o f background s t a i n i n g without c l e a r  cellular  morphology p r e s e n t ( F i g . l a & b ) , areas o f h i g h background with d e f i n i t e c e l l u l a r s t a i n i n g  ( F i g . I c & d ) , and areas with  v e r y low background s t a i n i n g but w i t h d e f i n i t e staining  cellular  ( F i g . Ie & f ) as w e l l as g r a d a t i o n s i n between.  There were a l s o areas o f neuronal s t a i n i n g astrocytic staining  ( F i g . l g ) and o f  ( F i g . l h ) . There were g r a d a t i o n s i n the  background s t a i n i n g o f v a r i o u s areas such t h a t i t was a matter of judgement t o d e c i d e which areas were t o be c a l l e d h i g h , medium, low o r no background s t a i n i n g .  I f e e l that the  background s t a i n i n g i s probably a combination c e l l u l a r p r o c e s s e s and nerve  endings.  F i g . 2 shows photographs  of s a g i t t a l s e c t i o n s of i r o n  s t a i n e d s e c t i o n s showing t h e d e n s i t y o f s t a i n . maps c o r r e s p o n d i n g t o the photographs individual cellular staining  F i g . 3 gives  showing where t h e r e was  ( c i r c l e s ) , o r h i g h o r medium  l e v e l s o f background s t a i n i n g  (dots) o r both.  c o r o n a l s e c t i o n s and t h e c o r r e s p o n d i n g maps. judgemental  of s t a i n i n g of  F i g . 4 presents Because o f the  nature o f the mapping, t h e photographs  may be  u s e f u l i n p r o v i d i n g more d e t a i l e d i n f o r m a t i o n as t o the  -  93  -  density  o f t h e background s t a i n i n g than t h e maps and t a b l e can  p r o v i d e , b u t t h e photographs must be used c a u t i o u s l y regard since  i n this  some dark areas may j u s t r e f l e c t a h i g h  of t h e c e l l u l a r s t a i n i n g .  A l l areas c o n t a i n i n g  density  cellular  s t a i n i n g a r e marked on t h e schematic maps and a r e judged t o be nonambiguous. Most o f t h e c e l l u l a r s t a i n i n g i s thought t o be o f o l i g o d e n d r o c y t e s but t h e r e a r e i s o l a t e d i n d i v i d u a l c e l l s are p r o b a b l y neuronal  (Fig. l g ) .  There were a l s o  that  limited  areas i n t h e o l f a c t o r y b u l b and o l f a c t o r y t r a c t t h a t had what appeared t o be s t a i n i n g o f f i b r o u s background area  a s t r o c y t e s on a low  ( F i g . Ih) and o t h e r areas i n t h e o l f a c t o r y  b u l b t h a t had what appeared t o be a mixture o f s t a i n e d  fibrous  a s t r o c y t e s and neurons o r o l i g o d e n d r o c y t e s i n a h i g h background a r e a . and  There were a l s o r e g i o n s i n t h e area postrema  around t h e v e n t r i c l e s where t h e s t a i n i n g appeared t o be  predominantly i n e p i t h e l i a l  cells.  T a b l e V I I I summarizes t h e areas showing v a r i o u s types o f staining. DISCUSSION  The  most i n t e r e s t i n g o b s e r v a t i o n t h a t  r e s u l t s i s that g l i a l c e l l areas o f t h e b r a i n .  can be made from our  s t a i n i n g i s not t h e same i n a l l  T h i s uneven d i s t r i b u t i o n o f s t a i n e d  glial  c e l l s tends t o support f u r t h e r o t h e r o b s e r v a t i o n s o f g l i a l cell  specializtion.  T h i s must i n d i c a t e t h a t g l i a l  c e l l s are  b i o c h e m i c a l l y d i f f e r e n t i n t h e i r i r o n metabolism and i n iron-related  functions,  whatever they may be.  - 94 -  The f u n c t i o n of  i r o n i s not understood  i n the b r a i n , but our o b s e r v a t i o n s  regional heterogeneity  i n i r o n d e n s i t y and c e l l u l a r  may  of  location  be c o r r e l a t e d w i t h other i n f o r m a t i o n i n an attempt t o  a s s e s s i n which t r a n s m i t t e r systems i r o n - r i c h g l i a l c e l l s  may  be i n v o l v e d . There are numerous t h e o r i e s proposed as t o how i n v o l v e d i n the b r a i n . Turnbull blue the g l i a l  1  E a r l y i r o n l o c a l i z a t i o n studies using  (Spatz, 1922,  D i e z e l , 1954)  l o c a l i z e d iron to  c e l l s of the globus p a l l i d u s , and the s u b s t a n t i a  n i g r a , and, and L u y s  iron i s  t o a l e s s e r extent, the r e d n u c l e u s ,  s t r i a t e body  body, a l l s t r u c t u r e s o f the e x t r a p y r a m i d a l  system.  Spatz noted t h a t i r o n d e p o s i t s o c c u r r e d i n d i s e a s e s i n v o l v i n g the e x t r a p y r a m i d a l Spatz's  system such as Parkinson's,  and Huntington's d i s e a s e s .  observations  i t was  Based on  proposed t h a t i r o n may  Hallervorden these  be i n v o l v e d i n  dopamine metabolism because o f the known importance of dopamine i n the e x t r a p y r a m i d a l  system.  i n c l u d e d o b s e r v a t i o n s t h a t low  i r o n caused a  hypothermic e f f e c t of D-amphetamine and induced  s t e r e o t y p i c behaviour  Supporting  tryptophan  reduced  i n c r e a s e d apomorphine  (Youdim e t a l . , 1981).  e f f e c t s are mediated by dopamine systems. t h a t i r o n may  evidence  I t was  Both  postulated  f u n c t i o n as a c o f a c t o r f o r t y r o s i n e and  hydroxylases  (Youdim e t a l . , 1984)  or may  be  i n v o l v e d i n dopamine r e c e p t o r f u n c t i o n s (Youdim e t a l . , 1980). My except My  f i n d i n g s are s i m i l a r t o those  i n Spatz's  e a r l y work  t h e r e i s no s t a i n i n g i n the subthalamus (Luys body).  f i n d i n g s do show some c o r r e l a t i o n w i t h dopamine  d i s t r i b u t i o n but t h e r e are areas h i g h i n dopamine t h a t do  - 95  -  not  have s p e c i f i c i r o n s t a i n i n g and areas o f i r o n s t a i n i n g where t h e r e a r e no known dopamine t r a c t s , p r o j e c t i o n s o r c e l l bodies.  One o f t h e major dopamine pathways i s t h a t from the  zona compacta o f t h e s u b s t a n t i a n i g r a and c e l l s j u s t medial t o i t t o t h e caudate, putamen, globus p a l l i d u s ,  olfactory  t u b e r c l e , nucleus accumbens, and l a t e r a l amygdala nucleus and f r o n t a l cortex.  In my f i n d i n g s t h e s u b s t a n t i a n i g r a has  s t a i n e d g l i a l c e l l s , as do t h e caudate-putamen, globus p a l l i d u s , amygdala and o l f a c t o r y t u b e r c l e and a l l these areas have h i g h o r medium background  s t a i n i n g as w e l l .  But I f i n d  no i r o n s t a i n i n g i n c e l l s medial t o t h e s u b s t a n t i a n i g r a , and o n l y medium background  s t a i n i n g i n t h e nucleus accumbens.  Small branches o f t h i s dopamine system a r e supposed to  t h e f r o n t a l c o r t e x , a n t e r i o r c o r t e x , and septum.  t o ascend I f i n d no  s t a i n i n g i n any p a r t o f t h e c o r t e x although t h e r e i s a uniform low background  level.  There i s , however, medium  background  s t a i n i n g i n some s e p t a l areas as w e l l as neuronal s t a i n i n g i n the l a t e r a l septum. There a r e o t h e r dopamine pathways such as t h e one from the a r c u a t e nucleus o f t h e hypothalamus t o t h e median eminence. My f i n d i n g s show the a r c u a t e nucleus has o l i g o d e n d r o c y t e s t a i n i n g on a medium background.  I d i d not s t a i n  sections  c o n t a i n i n g t h e median eminence but H i l l and S w i t z e r (1984) found a h i g h c o n c e n t r a t i o n o f i r o n s t a i n e d ependymal c e l l s i n that region. There a r e c e l l s i n t h e medial d o r s a l nucleus o f t h e hypothalamus t h a t a r e thought t o be dopaminergic to  t h e thalamus  and zona i n c e r t a .  - 96 -  that project  I f i n d t h a t t h e medial  d o r s a l nucleus o f t h e hypothalamus has s t a i n e d o l i g o d e n d r o c y t e s w i t h a h i g h background, t h e thalamus has medium s t a i n i n g and some areas w i t h p o s i t i v e o l i g o d e n d r o c y t e s , and t h e zona i n c e r t a has s t a i n e d o l i g o d e n d r o c y t e s b u t no background s t a i n i n g . There a r e a l s o dopamine i n t e r n e u r o n s i n t h e hypothalamus, b r a i n stem and o l f a c t o r y b u l b .  These a r e a l l areas t h a t  c o n t a i n some background s t a i n i n g w i t h s t a i n e d c e l l s i n the o l f a c t o r y bulb. Thus a l l areas o f dopamine c e l l b o d i e s except t h e area medial t o t h e s u b s t a n t i a n i g r a a l s o c o n t a i n s t a i n e d o l i g o d e n d r o c y t e s but i n t h e dopamine t e r m i n a l areas t h e r e i s e v e r y t h i n g from p o s i t i v e s t a i n i n g o f v a r i o u s c e l l types t o no cell  s t a i n i n g , and a range from low t o h i g h i n background  staining.  I t may be r e l e v a n t t h a t t h e dopaminergic  areas  which show t h e l e a s t i r o n s t a i n i n g a r e g e n e r a l l y those o f t h e A10 system  i n which dopamine and c h o l e c y s t o k i n i n a r e  colocalized. involvement  My evidence i s somewhat s u p p o r t i v e o f i r o n i n dopamine metabolism,  particularly  around  n o n - p e p t i d e r g i c dopamine c e l l b o d i e s , b u t t h e l a c k o f a t o t a l match means t h a t i r o n does not e x i s t e x c l u s i v e l y i n a s s o c i a t i o n w i t h dopamine. Other r e s e a r c h e r s have t r i e d t o c o r r e l a t e d i s t r i b u t i o n w i t h GABA neuroanatomy.  iron  Glutamate-binding  p r o t e i n i s known t o c o n t a i n i r o n and i s r e q u i r e d f o r GABA regeneration  (Michaelis et a l . ,  1982).  F r a n c o i s e t a l . (1981)  observed t h a t t h e GABA s t r i a t o - o r p a l l i d o - n i g r a l and c e r e b e l l a r c o r t i c a l pathways o v e r l a p s i g n i f i c a n t l y with  - 97 -  iron  distribution.  They a l s o noted t h a t areas h i g h i n GAD such as  i n t h e s u p e r i o r c o l l i c u l u s and t h e nucleus i n t e r p e d u n c u l a r i s , were a l s o h i g h i n i r o n  ( F r a n c o i s e t a l . , 1981.).  My  o b s e r v a t i o n s c o n f i r m t h e i r f i n d i n g s o f i r o n i n a l l these except  areas  t h e c o r t e x , where we f i n d o n l y low t o medium background  and no c e l l u l a r s t a i n i n g , and t h e c e r e b e l l a r c o r t e x , where t h e r e i s o n l y medium background s t a i n i n g .  Hill  and S w i t z e r  (1984) d i d a study s i m i l a r t o mine u s i n g t h e same technique and  found  s i m i l a r but not i d e n t i c a l r e s u l t s and concluded  h i g h i r o n c o n c e n t r a t i o n s i n g l i a overlapped  that  most s i g n i f i c a n t l y  w i t h areas h i g h i n GAD and GABA; these areas i n c l u d e d t h e v e n t r a l p a l l i d u m , globus p a l l i d u s , s u b s t a n t i a n i g r a pars r e t i c u l a t a , and c e r e b e l l a r n u c l e i .  They p o i n t e d out t h a t  i n j e c t i o n s o f GABA i n t o t h e globus p a l l i d u s l e d t o r e d u c t i o n s in and  i r o n i n t h e i p s i l a t e r a l v e n t r a l p a l l i d u m , globus substantia nigra ( H i l l ,  1984).  pallidus  They thought, however,  t h a t t h e d i s t r i b u t i o n o f i r o n i n d i c a t e d i t was not e x c l u s i v e l y r e l a t e d t o GABA but might be i n v o l v e d i n o t h e r n e u r o t r a n s m i t t e r systems such as enkephalins. My r e s u l t s do not support t h e involvement as s t r o n g l y as do those o f H i l l to  and S w i t z e r .  o f i r o n i n GABA GABA i s thought  be t h e t r a n s m i t t e r o f t h e P u r k i n j e c e l l s o f t h e cerebellum  which p r o j e c t t o the c e r e b e l l a r n u c l e i and o f t h e c e r e b e l l a r basket  c e l l s , G o l g i c e l l s and t h e s t e l l a t e c e l l s , which are  a l l wholly contained  i n t h e c e r e b e l l a r grey matter.  However,  my r e s u l t s do not show any c e l l u l a r s t a i n i n g i n t h e c e r e b e l l a r c o r t e x and o n l y a medium amount of background s t a i n i n g . Although  I do f i n d s i g n i f i c a n t o l i g o d e n d r o c y t e and p o s s i b l y  - 98 -  neuronal s t a i n i n g i n t h e c e r e b e l l a r n u c l e i .  Overall,  does n o t p r o v i d e s t r o n g evidence f o r t h e involvement i n c e r e b e l l a r GABA systems.  this of iron  The p a t t e r n o f hippocampal  s t a i n i n g i s c o n s i s t e n t w i t h i r o n ' s involvement  i n GABA  p r o c e s s e s i n t h a t nucleus as t h e o n l y area o f s t a i n i n g i s a narrow band o f medium background s t a i n i n g around t h e middle of hippocampal  l a y e r s where t h e r e a r e basket c e l l s which a r e  GABAergic.  The h i g h l e v e l s o f s t a i n e d c e l l s and background i n  the globus p a l l i d u s and t h e p a r s r e t i c u l a t a o f t h e s u b s t a n t i a n i g r a a r e c o n s i s t e n t w i t h t h e w e l l e s t a b l i s h e d GABAergic p r o j e c t i o n s between these two s t r u c t u r e s .  GABA i s a l s o t h e  n e u r o t r a n s m i t t e r o f i n t e r n e u r o n s o f t h e o l f a c t o r y bulb which might be c o n s i s t e n t w i t h t h e o b s e r v a t i o n s o f c e l l u l a r for iron i n that region.  staining  However, GABA i s so u b i q u i t o u s i n  b r a i n t h a t i f a l l GABA systems were a s s o c i a t e d w i t h  iron-rich  g l i a o r o t h e r s t r u c t u r e , one would expect a f a r more even d i s t r i b u t i o n o f i r o n than found i n t h i s o r p r e v i o u s s t u d i e s . There i s l i t t l e c o r r e l a t i o n between areas o f h i g h [3H]-GABA uptake  ( I v e r s e n and Schon, 1973), and h i g h i r o n  staining  areas, except t h a t t h e s u b s t a n t i a n i g r a i s h i g h i n both.  Thus  my r e s u l t s o n l y g i v e l i m i t e d support t o i r o n involvement i n GABA metabolism  i n some areas o f b r a i n w i t h these areas  i n c l u d i n g those i n which H i l l showed r e d u c t i o n s i n i r o n pallidal  after  i n j e c t i o n o f GABA.  S e v e r a l r e s e a r c h e r s have suggested a c o n n e c t i o n between 5HT  and i r o n .  I t was observed t h a t  iron-deficient  synaptosomes take up l e s s 5HT than normal synaptosomes and, when i r o n i s r e t u r n e d t o t h e d i e t , uptake  - 99 -  i n c r e a s e d (Kaladhar  and Rao, 1982).  T h i s phenomena extended  to offspring of iron  d e f i c i e n t mothers (Kaladher and Rao, 1983).  These authors  suggest an i r o n dependent s e r o t o n i n b i n d i n g p r o t e i n o r some o t h e r involvement  o f i r o n i n v e s i c u l a r s t o r a g e o f 5HT.  Tamir  e t a l . (1976) noted t h a t t h e b i n d i n g o f s e r o t o n i n by s e r o t o n i n b i n d i n g p r o t e i n was enhanced by Fe2+.  Most 5HT neurons a r e  l o c a t e d i n t h e raphe o r r e t i c u l a r system and p r o j e c t t o the n e o s t r i a t u m , c o r t e x , thalamus, hippocampus, p r e o p t i c nucleus, s e p t a l n u c l e i o r pons.  cerebellum,  Our r e s u l t s show  p o s i t i v e c e l l s o r , a t l e a s t , medium background s t a i n i n g i n a l l the above areas except t h e c o r t e x , b u t a g a i n t h e r e i s no c o n s i s t e n t s t a i n i n g p a t t e r n d i f f e r e n t i a t i n g areas o f p r o j e c t i o n and c e l l b o d i e s .  The ependymal c e l l s l i n i n g t h e  t h i r d v e n t r i c l e a r e s e r o t o n e r g i c and s t a i n h e a v i l y f o r i r o n which might be i n t e r p r e t e d as some support f o r t h e involvement o f i r o n i n 5HT systems. There i s a s t r i k i n g o v e r l a p o f i r o n d i s t r i b u t i o n w i t h some a s p e c t s o f e n k e p h a l i n neuroanatomy.  There a r e both  iron  s t a i n i n g and e n k e p h a l i n c e l l s i n t h e l a t e r a l septum, bed nucleus o f t h e s t r i a t e r m i n a l i s , s t r i a t u m , hypothalamus, amygdala, s u b s t a n t i a n i g r a , medial v e s t i b u l a r nucleus,  nucleus  o f t h e s p i n a l t r a c t o f t h e t r i g e m i n a l , and t h e p e r i a q u a d u c t a l gray, although i n t h e bed nucleus o f t h e s t r i a t e r m i n a l i s and the s p i n a l t r a c t o f t h e t r i g e m i n a l the s t a i n i n g i s only a medium background s t a i n i n g . P-Endorphins and r e l a t e d substances  a l s o have a s i m i l a r  and e x t e n s i v e o v e r l a p p i n g p a t t e r n w i t h i r o n Our evidence  distribution.  i n d i c a t e s t h a t , i f i r o n i s i n v o l v e d i n any  - 100 -  s p e c i f i c n e u r o t r a n s m i t t e r system, i t i s not simple way.  I t may  systems, or be processes.  Our  be  i n v o l v e d i n two  one  or more t r a n s m i t t e r  i n v o l v e d i n some other, as y e t evidence does not  t h e o r i e s p r e v i o u s l y advanced but support any  involved i n a  unhypothesized,  e l i m i n a t e any  of  the  n e i t h e r does i t w h o l l y  of them e i t h e r .  A r e c e n t study  (Y. Noda unpublished) examined the  effects  of a 20 month normal, i r o n d e f i c i e n t , or i r o n abundant d i e t t h r e e enzymes: CAT,  GAD,  r e s u l t s showed t h a t  GAD  inversely brain  and and,  r e l a t e d t o the  r e g i o n s examined.  thought t h a t  tyrosine  i r o n must be  t o some e x t e n t , TH  amount of i r o n i n the CAT  essential  e a r l i e r , and  i n the  do  activity  is  diet in a l l  f o r both GABAergic  concluded t h a t  might r e s u l t i n d e g e n e r a t i o n of the be harmful and  The  a c t i v i t i e s were u n a f f e c t e d .  c a t c h o l a m i n e r g i c systems but  can  h y d r o x y l a s e (TH).  excessive  neurons.  on  Noda  and iron  Iron deposits  occur i n some d i s e a s e s as mentioned  same s t r u c t u r e s  that  are normally h i g h i n  iron.  High l e v e l s of i r o n may  t o the  g e n e r a t i o n of oxygen f r e e r a d i c a l s , OH  mediated c o u p l i n g of O2 and  be harmful because i t can  H2O2/  the  1  due  lead  t o the  iron  so c a l l e d Haber-Weiss  reaction. The  presence of i r o n i n subsets of g l i a l  suggest t h a t :  (1) the  i r o n i s necessary f o r some f u n c t i o n  t h e s e p a r t i c u l a r g l i a l c e l l s ; or c e r t a i n types of neurons, and are  c e l l s might  the  (2) the  of  iron i s essential  g l i a l c e l l s s u r r o u n d i n g them  e i t h e r s u p p l y i n g i r o n t o these neurons or scavenging i t  from the The  e x t r a c e l l u l a r space around them. most s i g n i f i c a n t f i n d i n g of t h i s r e s e a r c h i s  -  101  for  -  the  e x t e n s i v e l o c a l i z a t i o n of hemosiderin t o g l i a and the that t h i s l o c a l i z a t i o n i s different  -  102  -  fact  i n various b r a i n regions.  F i g u r e 1: V a r i o u s Types o f S t a i n i n g F i g . IA F i g . IB F i g . IC  F i g . ID  f o r I r o n i n Rat B r a i n  M i d b r a i n areas showing s e v e r a l d e n s i t i e s o f background s t a i n i n g without any i n d i v i d u a l l y stained c e l l s . C a l i b r a t i o n b a r = 1000 /um. Band o f medium s t a i n i n g w i t h no c e l l s above the pyramidal c e l l l a y e r o f t h e hippocampus. C a l i b r a t i o n b a r = 300 jam. Area i n t h e globus p a l l i d u s w i t h moderately heavy background s t a i n i n g and c l e a r l y s t a i n e d c e l l s , probably oligodendrocytes. C a l i b r a t i o n b a r = 300 fKm. Strands o f l i g h t and dark background s t a i n i n g w i t h h e a v i l y s t a i n e d o l i g o d e n d r o c y t e s among dark s t r a n d s i n t h e s t r i a t u m . C a l i b r a t i o n b a r = 300 p i r n .  - 103 -  - 104 -  Fig.  IE  Fig.  IF  Fig.  1G  Fig.  IH  Several stained oligodendrocytes i n a l i g h t l y s t a i n e d area of the s t r i a t u m . C a l i b r a t i o n bar = 300^on. I n t e r f a s i c u l a r oligodendrocytes against l i g h t background s t a i n i n g of corpus c a l l o s u m . C a l i b r a t i o n bar = 300 /^m. L i g h t l y s t a i n e d neurons on a l i g h t background i n l a t e r a l septum. C a l i b r a t i o n bar = 300 /im. Area i n o l f a c t o r y b u l b w i t h l i g h t background s t a i n i n g showing probable a s t r o c y t i c staining. C a l i b r a t i o n bar = 300 ijim.  - 105  -  - 106 -  Fig. 2  Photographs o f s a g i t t a l s e c t i o n s o f whole r a t b r a i n . F i g . 2a 0.5mm o f the m i d l i n e . F i g . 2b, 1.2 mm o f the m i d l i n e . F i g . 2c, 2.9 mm o f f the midline. C a l i b r a t i o n b a r = 1 mm.  - 107 -  -  108  -  Fig. 3  Schematic diagrams of f i g u r e 2. Circles i n d i c a t e area of c e l l u l a r s t a i n i n g , and dots i n d i c a t e h i g h background s t a i n i n g . C a l i b r a t i o n bar = 1 mm. (See Table VII f o r a b b r e v i a t i o n s . )  - 109  -  - 110 -  Fig. 4  H a l f photographs and h a l f schematic drawing of coronal sections of r a t b r a i n . F i g . 4a, 3.2 mm a n t e r i o r t o bregma. F i g . 4b, 1.4 mm a n t e r i o r t o bregma. F i g . 4c, 0.6 mm a n t e r i o r t o bregma. F i g . 4d, 2.0 mm p o s t e r i o r t o bregma. C a l i b r a t i o n b a r = 1mm. (See T a b l e V I I f o r a b b r e v i a t i o n  - Ill-  explanations)  - He!  Fig. 4  (Continued) H a l f photographs and h a l f schematic drawing o f c o r o n a l s e c t i o n s o f rat brain. F i g . 4 e, 4.0 mm p o s t e r i o r t o bregma. F i g . 4 f , 6.0 mm p o s t e r i o r t o bregma. F i g . 4g, 8.8 mm p o s t e r i o r t o bregma. F i g . 4h, 11.4 mm p o s t e r i o r t o bregma. C i r c l e s i n d i c a t e areas o f c e l l u l a r s t a i n i n g and dots o f h i g h background s t a i n i n g . C a l i b r a t i o n b a r = 1 mm. (See  Table V I I f o r a b b r e v i a t i o n  - 113 -  explanations)  -  114  TABLE V I I : IRON STAINING IN VARIOUS AREAS OP THE BRAIN T a b l e V I I summarizes the type o f s t a i n i n g i n v a r i o u s structures. A l l s t r u c t u r e s not mentioned have no c e l l s and low or no background s t a i n i n g . H, M, L - h i g h , medium, low background s t a i n i n g , 0 - o l i g o d e n d r o c y t e s , N - neurons, A - astrocytes,E - e p i t h e l i a l c e l l s . Brain Structure Symbol Background Cell ACB none Nucleus Accumbens S e p t i M ACE 0 C e n t r a l Amygdala M AHA M 0, N? A n t e r i o r Hypothalamic Area none L a t e r a l Amygdaloid Nucleus AL M AOB M 0, N? A c c e s s o r y O l f a c t o r y Bulb Area Postrema AP H E? A r c u a t e Nucleus o f Hypothalamus ARH M 0 Bed Nucleus o f the A n t e r i o r BCA M 0 Commissure Bed Nucleus o f S t r i a T e r m i n a l i s BST M none A n t e r i o r Commissure CA none S t r i n g s Corpus Callosum CC none S t r i n g s C e r e b e l l a r Grey CG M none Inferior Colliculus CIF 0 M Caudate Putamen CPUH i n S t r i n g s 0 Superior C o l l i c u l u s CS 0 M CSC 0 Commissure o f the S u p e r i o r none Colliculus none L a t e r a l Cuneate Nucleus CUL M D e c u s s a t i o n s o f M e d i a l Lemniscus DLM M 0 D o r s a l M e d i a l Nucleus of DMH H 0 the Hypothalamus D o r s a l Raphe DR M none Endopeduncular Nucleus EP L 0 E x t e r n a l P l e x i f o r m Layer EPL L 0, A of O l f a c t o r y Bulb FX M i n s t r a n d s non< Fornix G none G e n i c u l a t e Body M 0, N? Globus P a l l i d u s GP H 0? GR Nucleus G r a c i l i s L 0 Hypothalamus ( a l l o t h e r areas) H M Habenular Nucleus HN H none none HP M Hippocampus CA 3 Islands of C a l l e j a IC H none IGL M 0?, A I n f e r i o r O l f a c t o r y Bulb IP H 0 I n t e r p e d u n c u l a r Nucleus Locus Coeruleus LC M 0 none LHA M L a t e r a l Hypothalmic Area LL H 0 L a t e r a l Lemniscus 0 LLD M D o r s a l Nucleus o f the L a t e r a l Lemniscus LM L 0 M e d i a l Lemniscus LS N M L a t e r a l S e p t a l Nucleus LT M 0 L a t e r a l Nucleus of Thalamus MFB M 0 M e d i a l F o r e b r a i n Bundle  -  115  Types  TABLE  VII  Brain Structure L a t e r a l Mammillary Nucleus M e d i a l Mammillary Nucleus Medial Septal Nucleus N u c l e u s Accumbens Cochlear Nucleus Dentate Nucleus F a s t i g i a l Nucleus I n t e r p o s i t u s Nucleus of Cerebellum Prepositus Nucleus P o s t e r i o r N u c l e u s o f Thalamus Red N u c l e u s Nucleus of Spinal Tract of the Trigeminal Nerve L a t e r a l V e s t i b u l a r Nucleus Medial V e s t i b u l a r Nucleus Superior V e s t i b u l a r Nucleus S p i n a l V e s t i b u l a r Nucleus I n f e r i o r O l i v a r y Nucleus Optic Tract Pons P o s t e r i o r Hypothalamus P r e t e c t a l Area L a t e r a l Preoptic Area P e r i v e n t r i c u l a r Grey P a r a v e n t r i c u l a r Hypothalamus R e t i c u l a r Formation Rhomboid N u c l e u s o f T h a l a m u s R e t i c u l a r N u c l e u s o f Thalamus Suprachiasmic Nucleus S t r i a M e d u l l a r i s Thalami Substantia Nigra Supraoptic Nucleus of the Hypothalamus S o l i t a r y Nucleus S u p e r i o r O l i v a r y Complex Thalamus ( a l l o t h e r areas) Intermediate Olfactory Tract Nucleus T r i a n g u l a r i s S e p t i Olfactory Tubercle V e n t r a l N u c l e u s o f Thalamus V e r t r o m e d i a l Hypothalamus V e n t r a l Tegmental Nucleus Zona I n c e r t a O l f a c t o r y Nerve Vermian Lobule N u c l e u s o f t h e T h i r d Nerve F a c i a l Nerve Hypoglossal Nucleus  -  (continued)  Symbol ML MM MS NA NC ND NF NI  Cell none  0 none none none  0 0,N?  0 0 0 0  NPH NPT NR NTST  M M L M  NVL NVM NVS NVSP OL OT P PH PRT POA PVG PVH RF RH RT SC SM SN SO  M M M M M none H L M H L M none M M M H H M  SOL SOC T TO I TS TUO VE VMH VTN AI I  none M M 0 M none A? none none H M 0 M 0 none M none M none 0 H 0 , N?, A? none M L 0? M 0 none M  Cl  III VII XII  116  Background H M M M M M M M  -  none  0 0 none none  0 0 none none  0 0 0 none  0 N 0 , N? N? none  0 N?  EXPERIMENT 2 1-Pyrroline  dehydrogenase  (EC 1.5.1.12; PDH) has been  shown i n s e v e r a l b a c t e r i a l and mammalian systems t o be a key enzyme i n t h e pathways from o r n i t h i n e and p r o l i n e t o glutamate (Figure 5).  Ornithine  i s converted t o glutamic a c i d  semialdehyde by o r n i t h i n e ^-transaminase ( O r n i t h i n e - oxo-acid a m i n o t r a n s f e r a s e , EC 2.6.1.13, OrnT) and t h e semialdehyde i s i n e q u i l i b r i u m w i t h P5C which can a l s o be formed from p r o l i n e by t h e a c t i o n o f PrO. (Roberts, 1982). neurotransmitter precursor  The P5C i s o x i d i z e d by PDH t o glutamate  Glutamate i s an important p u t a t i v e i n i t s own r i g h t and i s a l s o an immediate  o f GABA.  Most b r a i n glutamate i s formed from  g l u c o s e through t h e t r i c a r b o x y l i c a c i d c y c l e but t h e r o u t e from o r n i t h i n e o r p r o l i n e o f f e r s a p o s s i b l e a l t e r n a t i v e f o r a s m a l l glutamate p o o l . neurotransmitter  Proline i s also a possible  which has been shown, when i n j e c t e d , t o end  up as GABA i n g l i a l c e l l s  (Van den Berg, 1970).  One o f t h e enzymes, PDH, has been p u r i f i e d from beef l i v e r and i s a m i t o c h o n d r i a l adenine d i n u c l e o t i d e has moderate  enzyme t h a t r e q u i r e s n i c o t i n a m i d e (NAD) ( S t r e c k e r ,  a c t i v i t y i n brain  1971).  (Kawabata  although n o t f u l l y c h a r a c t e r i z e d ,  The other, PrO,  e t a l . , 1980);  i t appears t o be a membrane  bound enzyme which a l s o uses NAD (Boggess e t a l . , 1978). S i n c e both enzymes f u n c t i o n i n t h e presence o f NAD, we thought they might be h i s t o c h e m i c a l l y  l o c a l i z e d by v a r i a t i o n s o f the  t e c h n i q u e developed f o r GABA-T by Van G e l d e r (1965). G e l d e r used t h e NADH produced d u r i n g  - 117 -  Van  t h e metabolism o f GABA t o  reduce n i t r o b l u e t e t r a z o l i u m t o t h e dye formazan which stayed i n t h e c e l l s c o n t a i n i n g t h e GABA-T.  Modifications of t h i s  technique w i t h P5C o r L - p r o l i n e as a s u b s t r a t e were t r i e d as a means o f demonstrating  t h e h i s t o c h e m i c a l l o c a l i z a t i o n of PDH  and PrO i n b r a i n .  METHOD  P5C was prepared  from i t s p r e c u r s o r  ( s u p p l i e d by  Calbiochem o f La J o l l a , C a l i f o r n i a ) a c c o r d i n g t o t h e manufacturer's d i r e c t i o n s : 1 gm o f t h e p r e c u r s o r i s d i s s o l v e d i n 33 ml o f 6N HC1 and brought t o 100 C f o r 45 min. was p u r i f i e d on a 150 x 3 0ml  The P5C  column o f Dowex 50, 8%  c r o s s l i n k e d , mesh 50-100 H+, u s i n g t h e procedures  of Strecker  (1960).  The P5C was e l u t e d and a p o r t i o n o f each f r a c t i o n was  analyzed  f o r P5C by r e a c t i o n with some o-aminobenzaldehyde and  measurement o f t h e absorbance a t 440 nm.  The samples showing  presence o f P5C were combined and l y o p h i l i z e d . Male W i s t a r r a t s weighing 250-350 gm o b t a i n e d Canadian Breeding  L a b o r a t o r i e s were p e r f u s e d  from  intracardially  w i t h 150 ml o f i c e c o l d 0.1M phosphate b u f f e r e d s a l i n e (pH 7.4) f o l l o w e d by 2% g l u t e r a l d e h y d e / 2 %  paraformaldehyde.  S e c t i o n s were c u t on a vibratome and c o l l e c t e d i n 0.1M phosphate b u f f e r .  The f r e e f l o a t i n g s e c t i o n s were s t a i n e d f o r  PDH by p r e i n c u b a t i n g them f o r 2 0 min. a t 37°C i n t h e dark i n 5 ml t r i s h y d r o g e n - c h l o r i d e  0.1M  (pH 8.6), p l u s 0.5 ml NAD+  (10 mg/ml), p l u s 1.5 ml o f s o l u t i o n c o n t a i n i n g 144 mg/ml NaCl, 2 0 mg/ml MgCl and 1 mg/ml KCN.  A f t e r the p r e i n c u b a t i o n , 10 mg  of n i t r o b l u e t e t r a z o l i u m were mixed i n 0.25 ml dimethyl  - 118 -  s u l f o x i d e which was added w i t h 0.25 ml d i s t i l l e d f o l l o w e d by 0.2 ml o f phenazine m e t h o s u l f a t e  water,  (2 mg/ml) and  0.1-0.5 ml o f 150 mg/ml of P5C.  The i n c u b a t i o n was  at  The r e a c t i o n was stopped by  37°C i n t h e dark f o r 45 min.  t r a n s f e r t o a phosphate b u f f e r .  The s e c t i o n s were mounted on  g e l a t i n coated s l i d e s , d r i e d a t l e a s t o v e r n i g h t , xylene and c o v e r s l i p p e d distilled The  water u n l e s s  continued  i n Permamount.  dehydrated i n  A l l s o l u t i o n s were i n  otherwise s p e c i f i e d .  procedure f o r t h e L - p r o l i n e oxidase s t a i n i n g was  i d e n t i c a l except t h a t 0.1-0.5 ml o f 250 mg/ml o f commercially a v a i l a b l e L - p r o l i n e was s u b s t i t u t e d from t h e P5C. C o n t r o l s were done without L - p r o l i n e o r P5C. RESULTS A l l concentrations  o f P5C gave some background s t a i n i n g ,  but t h e r e was a much d a r k e r s p e c i f i c s t a i n i n g of c e r t a i n types of c e l l s .  T h i s was most e v i d e n t  high proportion  i n t h e cerebellum  o f Bergmann type a s t r o c y t e s were d a r k l y and  d i s t i n c t l y stained.  Although t h e r e was a h i g h background  l e v e l i n the granular morphology was e v i d e n t  l a y e r o f t h e cerebellum, no c e l l u l a r i n t h a t l a y e r and t h e s t a i n i n g d e n s i t y  never approached h a l f o f t h a t i n t h e Bergmann g l i a l Figure  cells.  6a shows t h e s t a i n e d Bergmann g l i a l c e l l b o d i e s i n the  Purkinje c e l l  l a y e r and t h e i r f i b e r - l i k e p r o j e c t i o n s i n t o the  molecular l a y e r .  F i g u r e 5b shows t h i s s t a i n i n g i s c o n s i s t e n t  throughout t h e c e r e b e l l a r s e c t i o n s . and  where a  The next most c o n s i s t e n t  c l e a r f i n d i n g was i n the pyramidal c e l l  d e n t a t e gyrus of t h e hippocampus.  - 119 -  l a y e r of the  Here t h e s t a i n i n g was  light  but a d i s t i n c t band o f s t a i n e d hippocampal a s t r o c y t e s c o u l d be distinguished  ( F i g . 7 ) . The o n l y other c l e a r l y s t a i n e d c e l l s  were o c c a s i o n a l a s t r o c y t e - l i k e c e l l s o f t h e corpus  callosum  and o t h e r prominent white t r a c t s . PrO  s t a i n i n g was much l e s s d i s t i n c t and was l i m i t e d t o the  Bergmann g l i a l c e l l s  (Fig. 8).  S e c t i o n s s t a i n e d without  e i t h e r L - p r o l i n e o r P5C showed no  c e l l u l a r s t a i n i n g and o n l y a f a i n t p i n k background s t a i n i n g . DISCUSSION  Our m o d i f i c a t i o n s o f t h e Van Gelder technique  f o r the  h i s t o c h e m i s t r y o f GABA transaminase gave some i n d i c a t i o n o f the probable  l o c a l i z a t i o n o f PDH and PrO.  In both cases  there  was some n o n - s p e c i f i c background s t a i n i n g , b u t i n n e i t h e r case was i t h i g h enough t o i n t e r f e r e w i t h interpretation of specific c e l l probably  loci  staining.  The technique  be used s i m i l a r l y f o r t h e h i s t o c h e m i c a l  o f o t h e r NAD r e q u i r i n g enzymes. all  microscopic  localization  I t may not, however, r e v e a l  o f such enzymes and i s probably  quantitative analysis.  could  not s u i t a b l e f o r  Thus, f o r example, no c e l l u l a r  s t a i n i n g f o r PDH was seen i n t h e c o r t e x although  appreciable,  i f r e l a t i v e l y low, a c t i v i t y was found i n t h a t r e g i o n on biochemical  assay  t h a t t h e technique  (Thompson e t a l . , 1985).  I t i s possible  only gives c l e a r s t a i n i n g of c e l l s  c o n t a i n i n g PDH a t a c t i v i t i e s t h a t approach t h e l e v e l s i n the c e r e b e l l a r Bergmann g l i a l c e l l s .  Increasing the substrate  c o n c e n t r a t i o n d i d not r e s u l t i n s p e c i f i c s t a i n i n g o f more c e l l types.  - 120 -  The s t a i n i n g f o r p r o l i n e was l e s s d i s t i n c t than t h a t f o r PDH and may, i n f a c t , be due t o PDH s i n c e t h e product o f PrO i s P5C which c o u l d be a c t e d on by PDH t o produce glutamate and f u r t h e r NADH ( F i g . 5 ) . T h i s p o s s i b i l i t y g a i n s some support from t h e f a c t t h a t t h e o n l y d e f i n i t e c e l l  s t a i n i n g f o r PrO was  seen i n t h e c e r e b e l l u m although r e g i o n a l d i s t r i b u t i o n data f o r PrO suggest t h e h i g h e s t a c t i v i t i e s a r e i n t h e m i d b r a i n and b r a i n stem (Thompson e t a l . ,  1985).  The most c o n s i s t e n t h i s t o c h e m i c a l f i n d i n g i s t h a t o n l y certain g l i a l  c e l l p o p u l a t i o n s were s t a i n e d .  I t can be argued  t h a t Bergmann g l i a l c e l l s a r e a s p e c i a l type o f g l i a l  c e l l but  the a s t r o c y t e s o f t h e hippocampus, although m o r p h o l o g i c a l l y d i f f e r e n t t o a s t r o c y t e s i n the r e s t of the b r a i n , are not r e c o g n i z e d as a s p e c i f i c subtype.  T h i s f i n d i n g thus supports  a growing body o f evidence o f g l i a l c e l l  specialization.  It  i s tempting t o s p e c u l a t e t h a t t h e chemical s p e c i a l i z a t i o n o f g l i a i s t o p r o v i d e m a t e r i a l s important t o t h e neurons vicinity.  I t i s t r u e t h a t t h e densest s t a i n i n g  c e l l s and o f a s t r o c y t e s i n t h e hippocampal  i n the  (of Bergmann  dentate gyrus) i s  i n r e g i o n s where important glutamate t r a c t s as w e l l as GABA i n t e r n e u r o n s a r e expected.  On t h e o t h e r hand, t h e r e i s  c o n s i d e r a b l e evidence t h a t many c o r t i c o f u g a l and c o r t i c a l commissural  t r a c t s a l s o use glutamate as a t r a n s m i t t e r and  t h a t many c o r t i c a l i n t e r n e u r o n s a r e GABAergic,  but a s t r o c y t e s  s t a i n i n g f o r PDH o r PrO were not seen i n t h e c o r t i c a l  grey  matter. Our r e s e a r c h may be p e r t i n e n t t o t h e study o f a number of f a m i l i a l c o n d i t i o n s i s which t h e p e r i p h e r a l metabolism o f  - 121 -  p r o l i n e or o r n i t h i n e i s known t o be a f f e c t e d . h y p e r p r o l i n e m i a s t h e r e i s a d e f i c i e n c y o f PrO (Haysaka e t a l . , 1974).  1982)  and of PDH  In the i n type 1  i n type I I ( V a l l e e t a l . ,  A v a r i e t y of n e u r o l o g i c a l symptoms, i n c l u d i n g  EEG  a b n o r m a l i t i e s , c o n v u l s i o n s , and mental d e f i c i e n c y , have been r e p o r t e d i n such cases but the f a c t t h a t many are asymptomatic suggests t h e r e i s not a c a u s a l r e l a t i o n s h i p Pavone, 1976).  ( M o l i c c a and  N e v e r t h e l e s s , i t would be o f i n t e r e s t t o study  b r a i n r e g i o n l e v e l s of these enzymes i n p o s t mortem t i s s u e from such cases as compared w i t h c o n t r o l s .  The  same might be  t r u e o f cases o f h y p e r o r n i t h i n e m i a which have been r e p o r t e d t o show atrophy o f o c u l a r t i s s u e  (Haysaka e t a l . ,  1982); s i n c e  OrnT l e v e l s are normally much h i g h e r i n the r e t i n a than i n brain  (Rao and C o t l i e r , 1984), the former may  d r a s t i c changes i f t h i s enzyme i s d e f i c i e n t .  - 122  -  show the most  Fig.  5  Schematic representation p r o l i n e and o r n i t h i n e t o  -  123 -  of the conversions of g l u t a m a t e and GABA.  H C  C H -  2  H N~C H 2  2  2  N H2  C H ^QQQ|_J  2  ORNITHINE  C H - C O O H  H C  CH C H _  2  H ORNITHINE ^-TRANSAMINASE  PROLIN  /  NAD PROLINE  OXIDASE  NADH  - C H  H,C-  2  ~  C H - C O O H  H C  HC-CH -CH -CH-COOH 2  2  NHP Y R R O L ! N E - 5- C A R B O X Y L A T E /  GLUTAMIC  ACID  SEMIALDEHYDE  I - PYRROLINE DEHYDROGENASE NADH  N  A  °  HOOC-CH.-CH,-CH-COOH NH-  G AD  HOOC-CH -CH -CH NH 2  GLUTAMIC  ACID  -  2  GABA 124  -  2  2  Fig.  6  PDH s t a i n i n g i n c e r e b e l l u m (A) Bergmann g l i a l c e l l s showing f i b e r s p r o j e c t i n g up t o t h e c e r e b e l l a r molecular layer. C e l l b o d i e s are l o o s e l y arranged around t h e P u r k i n j e (P) c e l l layer. C a l i b r a t i o n b a r = 100 M m . (B) Same a t lower m a g n i f i c a t i o n . C a l i b r a t i o n b a r = 300 jum.  Fig.  7  PDH s t a i n e d a s t r o c y t e s i n l a y e r o f dentate gyrus of hippocampus. C a l i b r a t i o n b a r = 300 Min.  Fig.  8  PrO s t a i n i n g o f Bergmann g l i a l c e l l s o f c e r e b e l l u m . C a l i b r a t i o n b a r = 100 pirn.  - 125 -  EXPERIMENT 3  Thiamine d e f i c i e n c y l e a d i n g t o  Wernicke-Korsakoff's  syndrome occurs among s e v e r a l p o p u l a t i o n s o f Western  people,  most commonly among a l c o h o l i c s , but a l s o i n people on d i a l y s i s , people with i n t e s t i n a l a b s o r p t i o n d i s e a s e s ( S a s s a r i s e t a l . , 1983), and t h e e l d e r l y deficiency  (Iber e t a l . , 1982).  Thiamine  (TD) can a l s o l e a d t o b e r i b e r i , a p o l y n e u r i t i s  which can occur w i t h c o n g e s t i v e h e a r t  failure.  Werniche's encephalopathy i s a n e u r o l o g i c a l d i s o r d e r with symptoms o f c o n f u s i o n , d i s t u r b a n c e s i n o c u l a r m o t i l i t y , p u p i l l a r y a l t e r a t i o n s , nystagmus, and a t a x i a w i t h  tremors.  I t s symptoms a r e b e l i e v e d t o be t h e d i r e c t r e s u l t o f a biochemical  l e s i o n which can l a r g e l y be r e v e r s e d by thiamine  administration. K o r s a k o f f ' s syndrome i s c h a r a c t e r i z e d by impaired f o r r e c e n t events  and p o l y n e u r i t i s .  I t occurs f r e q u e n t l y with  Wernicke's but does not r e v e r s e w i t h thiamine thiamine  memory  therapy.  Its  r e s i s t a n t symptoms may be the r e s u l t o f s t r u c t u r a l  damage because o f repeated  o r l o n g term thiamine d e f i c i e n c y .  In humans t h e s t r u c t u r a l damage o f K o r s a k o f f ' s syndrome o c c u r s as hemorrhagic l e s i o n s i n the mammillary b o d i e s , p e r i v e n t r i c u l a r r e g i o n s o f t h e thalamus and hypothalamus, p e r i a q u a d u c t a l r e g i o n s o f t h e midbrain  and f l o o r o f the f o u r t h  v e r t r i c l e and i n p a r t s o f t h e cerebellum. pathology  Wernicke's  i s i n s i m i l a r structures i f i t i s present.  We used t h e p y r i t h i a m i n e animal model i n which r a t s are put on a thiamine  d e f i c i e n t d i e t and g i v e n p y r i t h i a m i n e (PT),  - 127 -  an a n t a g o n i s t of thiamine phosphokinase, the enzyme which c o n v e r t s thiamine t o thiamine pyrophosphate.  Using  this  model, symptoms of weight l o s t , a t a x i a , and l o s s o f r i g h t i n g r e f l e x occur i n about 10 days and death  i n 14 days.  produces l e s i o n s i n the l a t e r a l v e s t i b u l a r n u c l e u s , the f o u r t h v e n t r i c l e , mammillary b o d i e s , thalamus, o l i v e , and cerebellum; these are thus s i m i l a r but i d e n t i c a l t o the human p a t t e r n s seen i n  PT f l o o r of inferior  not  Wernicke-Korsakoff's  syndrome. Understanding  the nature of the e a r l y b i o c h e m i c a l  has been the g o a l of much r e s e a r c h s i n c e 1936,  lesions  when P e t e r s  proposed the b i o c h e m i c a l l e s i o n theory t o e x p l a i n the n e u r o l o g i c a l e f f e c t s of thiamine d e f i c i e n c y . t h e o r y was two  Peters' o r i g i n a l  t h a t the b i o c h e m i c a l l e s i o n when found must e x p l a i n  o b s e r v a t i o n s , the s e l e c t i v e v u l n e r a b i l i t y of c e r t a i n  s t r u c t u r e s i n the b r a i n , and the r e v e r s i b i l i t y upon with thiamine. important  treatment  E x p l a i n i n g these o b s e r v a t i o n s remains  i n current research.  The enzymes f o r which thiamine t r i p h o s p h a t e  (TTP)  is a  co-enzyme, as w e l l as s e v e r a l enzymes a s o c i a t e d w i t h v a r i o u s n e u r o t r a n s m i t t e r s , have been examined by p r e v i o u s authors the r e s u l t s do not e x p l a i n f u l l y the nature of the biochemical l e s i o n .  In t h i s experiment we  s y n t h e t i c enzymes f o r GABA and ACh for  but  initial  examined the  and the d e g r a d a t i v e enzyme  GABA f o r t h e i r p o s s i b l e r o l e i n the i n i t i a l  biochemical  lesion. ACh  i s one of the n e u r o t r a n s m i t t e r s p r e v i o u s l y s t u d i e d i n  thiamine d e f i c i e n c y .  A decrease  - 128  i n the TTP-dependent enzyme,  -  p y r u v a t e dehydrogenase,  which i s e s s e n t i a l f o r the p r o d u c t i o n  of a c e t y l - C o A and t h e r e f o r e of ACh, t o reduced s y n t h e s i s of ACh c o n c e n t r a t i o n s of ACh.  would t h e o r e t i c a l l y  lead  and t h e r e f o r e reduced  Decreased s y n t h e s i s of ACh has i n f a c t  been observed, but, although t h e r e were e a r l i e r r e p o r t s (Hamel et a l . ,  1980)  of decreased ACh  r e p o r t s do not c o n f i r m t h i s et a l . ,  1977).  the b r a i n was  c o n c e n t r a t i o n s , most r e c e n t  (Reynolds and B l a s s , 1975,  The d i f f e r e n c e may  1981).  l i e i n the speed a t which  f i x e d and the r e s u l t a n t e x t e n t t o which the  m e t a b o l i c a l l y a c t i v e p o o l s of ACh al.,  Vorhees  are measured ( B a r c l a y e t  S i n c e some o f the p o o l s are o f l i t t l e  functional  v a l u e , t u r n o v e r i s thought t o be a b e t t e r index of f u n c t i o n a l change (Cheney e t a l . ,  1977).  Decreased t u r n o v e r of ACh  been observed even i n the presence of adequate c h o l i n e and CAT  (Thornber e t a l . ,  1980).  has  l e v e l s of  I t i s postulated  t h a t the decrease i n pyruvate dehydrogenase i n v i v o i n t h i a m i n e d e f i c i e n t animals i s not enough t o e x p l a i n a l l the r e d u c t i o n i n ACh L o c k e t t , 1962, al.,  1978)  synthesis.  The l e v e l s of CAT  Heinrich et a l . ,  1973,  (Bhatgat and  Reddy, 1982,  Sacchi et  and the a c t i v i t i e s of c h o l i n e s t e r a s e are r e p o r t e d l y  not decreased  (Takats e t a l . ,  a c t i v i t i e s o f CAT  1981).  We  examined the r e g i o n a l  i n c o n t r o l s , a f t e r the appearance  of  symptoms of thiamine d e f i c i e n c y , and a f t e r r e c o v e r y t o original  weight.  GABA has been found t o be decreased i n the whole b r a i n , pons/medulla,  midbrain, c o r t e x and c e r e b e l l u m p r i o r t o  n e u r o l o g i c a l symptoms i n r a t s on p y r i t h i a m i n e (Butterworth e t al.,  1979,  Butterworth, 1982a).  - 129  These f i n d i n g s have not been  -  confirmed by o t h e r r e s e a r c h e r s ( P l a i t a k i s e t a l . , h i g h a f f i n i t y uptake ( P l a i t a k i s , 1982).  1979).  GABA  i s not a f f e c t e d i n any b r a i n areas We  examined both GAD  and GABA-T i n  s p e c i f i c r e g i o n s of the b r a i n a t the peak of the p y r i t h i a m i n e induced symptoms and a f t e r r e t u r n t o o r i g i n a l weight  on a  normal d i e t . I f the l e s i o n i s fundamentally b i o c h e m i c a l i n nature, then t h e r e s h o u l d be a t l e a s t some r e c o v e r y when thiamine i s r e t u r n e d t o the d i e t . e i t h e r GABA o r ACh  There have been no s t u d i e s done on  enzymes t o see i f these change s e l e c t i v e l y  and i f they r e c o v e r upon r e i n t r o d u c t i o n o f thiamine t o the diet.  I f there i s a biochemical l e s i o n i n v o l v i n g a p a r t i c u l a r  enzyme, and the thiamine d e f i c i e n c y i s stopped j u s t b e f o r e the onset o f symptoms, t h e r e should be t o t a l r e c o v e r y o f  enzymatic  f u n c t i o n ; but i f the c r i t i c a l time t o stop i s p a s t , t h e r e  may  be r e s i d u a l damage due t o prolonged b i o c h e m i c a l d i s r u p t i o n of the c e l l w i t h some consequent  c e l l death.  Recovery  of  b i o c h e m i c a l f u n c t i o n might be e x p l a i n e d i n y e t another  way.  I f l e s i o n s are i n i t i a t e d i n the g l i a , the r e c o v e r a b i l i t y of the e a r l y l e s i o n s may to  be because g l i a l c e l l s have the c a p a c i t y  proliferate. The  glial  i n i t i a l anatomical l e s i o n appears t o occur f i r s t i n  c e l l s i n the areas known t o be most a f f e c t e d by  d e f i c i e n c y such as the l a t e r a l v e s t i b u l a r nucleus 1967).  thiamine  (Collins,  These e a r l y l e s i o n s c o n s i s t of s w e l l i n g o f both  c e l l s and the myelin sheath  (Robertson e t a l . ,  i n v o l v e a s t r o c y t e s more than o t h e r c e l l types Kanabe, 1978).  C o l l i n s and Converse  - 130  -  1968)  and  glial may  (Watanabe and  (1970) noted a l s o t h a t  the Bergmann g l i a l of  f i b e r s a s s o c i a t e d w i t h d e g e n e r a t i n g neurons  t h e c e r e b e l l a r m o l e c u l a r l a y e r were t h e f i r s t t o accumulate  glycogen i n thiamine d e f i c i e n c y . S i n c e g l i a appear t o be t h e f i r s t  s t r u c t u r e s t o change and  s i n c e they do n o t change e q u a l l y i n a l l areas t h e r e may be fundamental glia.  d i f f e r e n c e s i n thiamine dependence o f v a r i o u s  T h i s experiment  concepts o f g l i a l  e x e m p l i f i e s a type o f r e s e a r c h where  h e t e r o g e n e i t y may be r e l e v a n t t o t h e  i n t e r p r e t a t i o n of the data. METHOD  Male W i s t e r r a t s from Canadian  Breeding Farms, weighing  300+12 gms, were g i v e n f r e e access t o water and commercially a v a i l a b l e thiamine d e f i c i e n t d i e t from N u t r i t i o n a l B i o c h e m i c a l s and were i n j e c t e d i n t r a p e r i t o n e a l l y w i t h 0.5 mg/kg o f p y r i t h i a m i n e d a i l y .  The r a t s were housed  i n d i v i d u a l l y i n rooms w i t h o t h e r rodents on a 12 hour on, 12 hour o f f l i g h t schedule.  A l l r a t s were weighed d a i l y and  g r o s s b e h a v i o u r a l changes were noted.  When r a t s  exhibited  s i g n s o f a t a x i a and l o s s o f r i g h t i n g r e f l e x , u s u a l l y on day 10 or  11, they were e i t h e r s a c r i f i c e d f o r immediate use o r were  put on t o a normal d i e t and g i v e n a few shots o f thiamine hydrochloride  (0.5 mg/kg i n t r a p e r i t o n e a l l y ) .  These r a t s were  kept u n t i l they had r e a t t a i n e d t h e i r o r i g i n a l weights, upon which time they had r e g a i n e d t h e i r r i g h t i n g r e f l e x and had l o s t most o f t h e i r a t a x i a .  T h i s was u s u a l l y w i t h i n two weeks.  Rats f o r b i o c h e m i c a l s t u d i e s were s a c r i f i c e d by c e r v i c a l fracture.  The b r a i n s were immediately  - 131 -  removed and d i s s e c t e d  i n t o e i g h t r e g i o n s : cerebellum, pons/medulla, n e o s t r i a t u m , m i d b r a i n , hypothalamus,  thalamus, hippocampus, and c o r t e x .  Each t i s s u e sample was homogenized i n 0.3 ml o r 10 volumes (whichever was l e s s ) o f c o l d 0.25 M s u c r o s e . P o r t i o n s o f t h e homogenate were used f o r d e t e r m i n a t i o n of e i t h e r CAT o r GAD by methods d e s c r i b e d below. CAT was measured by a m o d i f i c a t i o n o f t h e method o f F. Fonnum (1969).  60-180 mg o f t i s s u e was a c t i v a t e d by treatment  w i t h T r i t o n X-100 and then i n c u b a t e d w i t h acetyl-coenzyme A l a b e l l e d w i t h [C14].  The [14C] a c e t y l c h o l i n e was absorbed  onto an i o n exchange column (IG50) and e l u t e d w i t h 3 ml o f 4N acetic acid.  R a d i o a c t i v i t y i n t h e e l u a n t was counted.  GAD a c t i v i t y was determined by a m o d i f i c a t i o n o f t h e method o f Lupien e t a l . (1968).  L-(1-[14C]}-Glutamic a c i d i s  i n c u b a t e d w i t h t i s s u e homogenates i n t h e presence o f p y r r d o x a l phosphase,  and t h e [14C02] produced i s t r a p p e d on hyamine  h y d r o x i d e soaked paper, and t h e r a d i o a c t i v i t y  counted.  Separate r a t s , s a c r i f i c e d by p e r f u s i o n under deep b a r b i t u a t e a n e s t h e s i a , were used f o r t h e GABA-T h i s t o c h e m i s t r y which was done by a method a Van G e l d e r (1965) m o d i f i e d as follows.  Rats a n a e t h e s i z e d w i t h sodium p e n t a b a r b i t a l and  p e r f u s e d i n t r a c a r d i a l l y w i t h 150 ml i c e c o l d 0.1M  phosphate  b u f f e r , pH 7.4, had t h e i r b r a i n s removed, kept i n 0.1M phosphate b u f f e r , s e c t i o n e d a t 50 /Um on an Oxford  Vibratome  and s t a i n e d f o r GABA-T by p r e i n c u b a t i n g f r e e f l o a t i n g  sections  i n t h e dark f o r 2 0 min. i n a r e a c t i o n mixture c o n t a i n i n g 5.0 ml t r i s HCl 0.1M, 0.2 ml o f 250 mg/ml a l p h a - k e t o g l u t a r a t e , 1.5 ml o f a s o l u t i o n c o n t a i n i n g 144 mg/ml NaCl, 2 0 mg/ml MgCl2,  - 132 -  and 1 mg/ml KCN,  and 0.5ml 10 mg/ml NAD  p r e i n c u b a t i o n , 10 mg 2.5  a t pH 8.6.  of n i t r o b l u e t e t r a z o l i u m d i s s o l v e d i n  ml dimethyl s u l f o x i d e and 2.5 ml water, 9.5  phenazine  m e t h o s u l f a t e and 0.2  t o the p r e - i n c u b a t i o n medium. 45 min.  a t 37°C.  A f t e r the  ml o f lmg/5ml  ml of 250 mg/ml GABA are added The  s e c t i o n s are incubated f o r  The r e a c t i o n i s stopped w i t h the t r a n s f e r of  these s e c t i o n s t o 0.1M  phosphate b u f f e r .  The  s e c t i o n s are  mounted on g e l a t i n coated s l i d e s , a i r d r i e d , dehydrated  in  xylene, and c o v e r s l i p p e d w i t h Permamount.  RESULTS  In symptomatic thiamine d e f i c i e n t .treated r a t s GAD decreased  (TD), p y r i t h i a m i n e  a c t i v i t i e s were found t o be  significantly  i n f o u r areas o f the b r a i n : the thalamus >  c e r e b e l l u m > pons/medulla > midbrain  (see T a b l e V I I I A ) .  body weight had r e t u r n e d t o p r e - e x p e r i m e n t a l s i g n i f i c a n t r e c o v e r y of GAD GABA-T s t a i n i n g was  There was  l e v e l s , there  was  a c t i v i t y except i n the thalamus.  most d r a m a t i c a l l y reduced  thalamus (see F i g . 9a, 9b), and next i n the colliculus.  After  i n the  inferior  some l o s s i n the pons and medulla,  but  no change i n o t h e r areas of the b r a i n i n c l u d i n g the cerebellum.  A f t e r r e t u r n t o a normal d i e t , t h e r e i s a t l e a s t  p a r t i a l r e c o v e r y of s t a i n i n g i n a l l areas (see F i g . 9b, There was b r a i n area  affected  9c). no s i g n i f i c a n t change i n CAT  (see Table V I I I B ) .  - 133  -  a c t i v i t y i n any  DISCUSSION  Our f i n d i n g o f a s p e c i f i c l o s s o f t h e two GABA r e l a t e d enzymes, GAD and GABA-T, i n s e v e r a l b r a i n areas i s compatible w i t h t h e f i n d i n g s o f s e v e r a l o t h e r workers. w i t h t h e r e d u c t i o n o f GABA i n whole b r a i n Gubler e t a l . ,  I t i s compatible  (Gaitonde, 1975,  1974) i n both PT and TD r a t s , and w i t h t h e  f i n d i n g s o f reduced GABA c o n c e n t r a t i o n s i n PT r a t s i n t h e cerebellum  (Butterworth e t a l . ,  1978, Butterworth, 1982a,  Butterworth, 1982b), medulla/pons (Butterworth e t a l . , and m i d b r a i n  (Butterworth, 1982b).  1978),  We d i d not, however,  observe a decrease i n GABA-T o r GAD i n c e r e b r a l c o r t e x as Butterworth  (1982b) d i d .  Our f i n d i n g s a r e n o t compatible with  those o f P l a i t a k i s e t a l . (1979) who found no change i n c e r e b e l l u m o r pons/medulla  i n pyrithiamine treated rats.  The  t h a l a m i c changes i n GABA we observed had not been r e p o r t e d elsewhere,  but t h e p e r i v e n t r i c u l a r r e g i o n o f t h e thalamus,  where t h e r e i s a h i g h d e n s i t y o f presumptive (Nagai e t a l . ,  GABAergic neurons  1983), i s an area which, l i k e t h e c e r e b e l l u m  and m i d b r a i n , have n o t a b l e h i s t o p a t h o l o g y i n K o r s a k o f f ' s syndrome.  The f a c t t h a t GAD remained  reduced  of r a t s p u t on a normal d i e t w i t h thiamine  i n t h e thalamus  supplementation  suggests some s t r u c t u r a l damage t o GABAergic systems i n t h i s area. I t i s i n t e r e s t i n g t h a t t h e thalamus,  which has t h e l a r g e s t  e f f e c t s o f thiamine d e f i c i e n c y on GAD and GABA-T, i s a l s o the r e g i o n showing t h e l a r g e s t drops i n GAD and GABA on aging (McGeer and McGeer, 1982).  The i n t r a c y t o p l a s m i c i n c l u s i o n s  - 134 -  found i n t h e thalamus  i n t h i a m i n e - d e f i c i e n t mice have been  s a i d t o be m o r p h o l o g i c a l l y i n d i s t i n g u i s h a b l e from those i n aged mice  (Aikawa e t a l . ,  1983).  TABLE V I I I : Enzyme L e v e l s i n C o n t r o l , T h i a m i n e - D e f i c i e n t and Recovered Rats ( moles/hr-100 mg p r o t e i n ; Mean+S.D.; number r a t s i n parentheses). B r a i n Area  Controls  (7)  Thiamine Def.(7)  A. Glutamic A c i d Decarboxylase Cerebellum 19. 39 + 1. 78 12. 14 + 1. 65# Pons/Medulla 13. 24 + 1. 21  Recovered (5)  17. 64 + 3. 25  11. 38 + 0. 60**  12 .92 + 1. 83  Neostriatum  15. 11 + 1. 06  12. 12 + 5. 60  14. 98 + 1. 24  Midbrain  19. 46 + 0. 95  13. 11 + 5. 21*  17. 74 + 3. 02  12. 13 + 3. 33  14. 26 + 2. 98  Hypothalamus 16. 28 + 1. 52 Thalamus  21. 34 + 1. 88  10. 02 + 2. 30#  14. 91 + 2. 99*  Hippocampus  13 .56 + 2. 15  13. 95 + 0. 97  13. 47 + 0. 85  Cortex  15. 56 + 2. 94  12 .67 + 4. 29  13. 92 + 2. 84  1. 77 + 0. 26  1. 50 + 0. 22  1. 63 + 0. 24  Pons/Medulla 17. 90 + 2. 13  20. 32 + 2. 15  19. 74 + 1. 93  Neostriatum  33 .06 + 5. 49  26. 93 + 2. 76  34. 32 + 3. 27  Midbrain  12. 08 + 1. 21  11. 54 + 1. 40  12. 09 + 1. 02  6. 19 + 0. 48  6. 93 + 0. 64  6. 37 + 0. 35  Thalamus  10. 54 + 1. 59  11. 08 + 1. 69  10. 91 + 0. 99  Hippocampus  11. 21 + 1. 34  10. 12 + 1. 35  11. 37 + 0. 85  Cortex  12. 76 + 3. 65  10. 90 + 2. 05  12 .93 + 2. 64  Choline Acetyltransferase Cerebellum  Hypothalamus  #p<0.001,  **p<0.001, *p<0.02 f o r comparison w i t h c o n t r o l s .  - 135 -  The  l a c k o f change i n CAT d u r i n g thiamine d e f i c i e n c y i s  c o n s i s t e n t w i t h p r e v i o u s r e p o r t s (Bhatgat and L o c k e t t , 1962, Heinrich et a l . ,  1973, Reddy, 1982, S a c c h i e t a l . , 1978,  Thornber  1980).  et a l . ,  T h i s , combined w i t h t h e f a c t  AChE i s a l s o u n a f f e c t e d i n thiamine d e f i c i e n c y 1982,  Takata e t a l . ,  that  (Gibson e t a l . ,  1981) and t h a t a decreased t u r n o v e r o f  a c e t y l c h o l i n e i s observed even i n animals showing normal l e v e l s o f CAT ( S a c c h i e t a l . ,  1978, Thornber  et a l . ,  1980), i s  c o n s i s t e n t w i t h t h e b e l i e f t h a t t h e amount o f enzyme i s not n o r m a l l y r a t e c o n t r o l l i n g and t h a t f a c t o r s such as decreased a v a i l a b i l i t y o f a c e t y l coenzyme A (Vorhees e t a l . ,  1978) o r an  i n h i b i t o r y e f f e c t o f thiamine d e f i c i e n c y on a c e t y l c h o l i n e release  (Dunant and Eder, 1983, Eder e t a l . ,  important  1976) may p l a y  roles.  A h y p o t h e s i s as t o t h e mechanism o f t h e l o s s e s i n GAD and GABA-T must take i n t o account t h e r e g i o n a l observed.  specificity  A s p e c i f i c l o s s o f GAD i s assumed t o be because o f  d e s t r u c t i o n o f GABAergic synaptosomes (Butterworth, 1982a) and perhaps  o f t h e GABA neurons themselves.  I f i t i s assumed t h a t  o n l y neurons a r e i n v o l v e d , i t i s hard t o see why GABA neurons are n o t d e s t r o y e d e q u a l l y i n a l l a r e a s .  Why f o r i n s t a n c e i s  GAD n o t s i g n i f i c a n t l y reduced i n t h e n e o s t r i a t u m o r hippocampus where t h e r e a r e h i g h c o n c e n t r a t i o n s o f GABA neurons o r i n t e r n e u r o n s ?  I t has been suggested t h a t t h e most  a f f e c t e d areas a r e those w i t h h i g h t u r n o v e r r a t e s o f thiamine and h i g h o x i d a t i v e metabolism one  which i s dependent, f o r a t l e a s t  s t e p , on thiamine as a c o f a c t o r  - 136 -  (Dreyfus, 1976).  The  c e r e b e l l u m i s one such area  (Ritchie et a l . ,  1980, 1984).  Decreased a c t i v i t y o f p y r u v a t e dehydrogenase, which i s dependent on t h i a m i n e t r i p h o s p h a t e as a coenzyme, would, f o r example,  l e a d t o decreased i n c o r p o r a t i o n o f g l u c o s e i n t o amino  a c i d s and keto a c i d s o f both t h e TCA c y c l e and GABA shunt (Butterworth e t a l . ,  1978, Butterworth, 1982a).  GAD a c t i v i t y ,  however, i s not known t o be a f f e c t e d by p r e c u r s o r availability. Another h y p o t h e s i s i n v o l v i n g o n l y neurons i s some interneuronal reaction.  F o r example, t h e changes i n  c e r e b e l l a r GAD might be secondary t o changes i n 5HT system which may i n n e r v a t e GABA neurons. suggested  (Chan-Palay e t a l . ,  Such a s i t u a t i o n has been  1977) i n t h e l o s s o f  s e r o t o n e r g i c mossy f i b e r s i n c o n t a c t w i t h t h e c e r e b e l l a r P u r k i n j e c e l l s which a r e GABAergic Onodera e t a l . , al.,  (Chan-Palay e t a l . , 1977,  1981, P l a i t a k i s e t a l . ,  1978b, P l a i t a k i s e t a l . ,  1979).  1978a, P l a i t a k i s e t  Thus  serotonergic  changes might l e a d t o GABA changes and GAD changes and these s e r o t o n e r g i c neurons a r e known t o be s u s c e p t i b l e t o thiamine deficiency  (Plaitakis et a l . ,  1978a).  This hypothesis could  presumably be t e s t e d by examining GAD l e v e l s i n r a t s where l e s i o n s o f t h e s e r o t o n e r g i c neurons have been produced by o t h e r means such as 5,7-dihydroxytryttamine. The r e c o v e r y o f GAD i n our r e s u l t s may i n d i c a t e : b i o c h e m i c a l r e v e r s a l o f changes which reduced t h e a c t i v i t y of GAD, regrowth o f GABAergic synaptosomes c o n t a i n i n g GAD, or, GAD i s a c t u a l l y s e n s i t i v e t o p r e c u r s o r a v a i l a b i l i t y , the recovery of precursors.  GABA-T l o s s and r e c o v e r y c o u l d be  - 137 -  if  i n d i c a t i v e o f l o s s and r e c o v e r y o f synaptosomes which c o n t a i n GABA-T t o r e g u l a t e GABA l e v e l s p r e s y n a p t i c a l l y . The  r e g i o n a l s p e c i f i c i t y and p a r t i a l r e v e r s i b i l i t y o f t h e  changes i n GABAergic systems analyzed under t h i s s c e n a r i o would i n d i c a t e a p o s s i b l e r o l e o f thiamine neurons o r a f f e r e n t s t o such neurons;  i n e i t h e r GABA  t h e r e i s no good  e x p l a n a t i o n why a l l GABA neurons a r e not a f f e c t e d . If g l i a l  c e l l h e t e r o g e n e i t y i s taken i n t o account and  combined w i t h t h e o b s e r v a t i o n s t h a t t h e f i r s t changes t h a t occur i n t h e thiamine d e f i c i e n c y models a r e i n g l i a l  cells,  then t h e i n t e r p r e t a t i o n o f our o b s e r v a t i o n s c o u l d be q u i t e different. The  r e c o v e r y upon r e t u r n o f thiamine,  a part of the  d e f i n i t i o n o f a b i o c h e m i c a l l e s i o n as d e f i n e d by P e t e r s , c o u l d depend upon p r o l i f e r a t i o n o f t h e remaining  glial  c e l l s i n the  damaged areas, o r o f g l i a l c e l l s from surrounding areas t o r e s t o r e normal g l i a l neurons.  f a c t o r s needed t o support t h e GABAergic  S i n c e GABA-T i s i n g l i a as w e l l as neurons,  glial  p r o l i f e r a t i o n might h e l p t o e x p l a i n t h e r e c o v e r i e s i n GABA-T. The  second p a r t o f P e t e r s ' d e f i n i t i o n o f t h e b i o c h e m i c a l  n a t u r e o f thiamine d e f i c i e n c y , t h e s e l e c t i v e v u l n e r a b i l i t y o f c e r t a i n r e g i o n s , may not be due t o r e g i o n a l d i f f e r e n c e s i n neurons b u t be due t o g l i a l h e t e r o g e n e i t y . that only g l i a l  I t has been shown  c e l l s o f c e r t a i n areas show e a r l y  d e f i c i e n c y changes.  thiamine  A l l t h e areas where GABA-T l o s s and  r e c o v e r y were noted a r e areas where g l i a l c e l l damage occurs e a r l y ; an anomaly i s t h e cerebellum where t h e r e a r e e a r l y glial  changes but no GABA-T l o s s e s .  - 138 -  T h i s area i s a l s o the  o n l y one i n which GAD l o s s e s and r e c o v e r y do not seem t o p a r a l l e l GABA-T changes.  T h i s may be because any l o s s o f  neuronal GABA-T i s concealed by GABA-T a c t i v i t y i n t h e Bergmann g l i a which seem t o c o n t a i n u n u s u a l l y h i g h c o n c e n t r a t i o n s o f t h i s enzyme which i s found i n both GABAergic neurons and g l i a i s the g l i a l to  (Nagai e t a l . ,  1983).  In the cerebellum i t  c e l l s of the molecular l a y e r that are the f i r s t  change. T h e r e f o r e t h e i n i t i a l b i o c h e m i c a l l e s i o n may be i n  sub-types  of g l i a l  c e l l s leading to regional g l i a l  cell  loss  which i n t u r n causes changes i n neurons o f t h e s u r r o u n d i n g area. As reviewed of  i n t h e main body o f t h i s t h e s i s c e r t a i n types  g l i a appear t o have t h e a b i l i t y t o take up and m e t a b o l i z e  glutamate  and t o form t h e glutamine r e q u i r e d as a GABA  p r e c u r s o r by GABAergic neurons.  Changes i n these s y m b i o t i c  g l i a might l e a d t o changes i n t h e a c t i v i t y o f GABA Butterworth  neurons.  (1982a) suggests t h a t t h e types o f g l i a l c e l l s may  be important i n d e t e r m i n i n g t h e s e l e c t i v e v u l n e r a b i l i t y o f c e r t a i n areas and notes t h a t g l i a l  cell  l i n e s a r e more  s u s c e p t i b l e t o thiamine d e f i c i e n c y than a r e neuronal Thiamine  pyrophosphatase  lines.  a c t i v i t y was found t o be v e r y h i g h i n  the plasma membrane o f m i c r o g l i a , and o l i g o d e n d r o c y t e s and a s t r o c y t e s a l s o had s i g n i f i c a n t s t a i n i n g i n t h e G o l g i apparatus  (Murabe and Sano, 1981) so an a s s o c i a t i o n between  thiamine and g l i a has been made. Other people have a l s o suggested key r o l e s f o r g l i a i n thiamine d e f i c i e n c y .  Butterworth  (1982a) suggested  - 139 -  that  s e l e c t i v e changes i n g l i a l c e l l  i n t e g r i t y may e x p l a i n GABA  changes i n t h e l a t e r a l v e s t i b u l a r n u c l e u s . t h a t t h e observed  He a l s o p o s t u l a t e d  enhanced glutamate uptake i n e a r l y  thiamine  d e f i c i e n c y may be e x p l a i n e d by t h e p r o l i f e r a t i o n o f g l i a l c e l l s t h a t occurs i n damaged areas. In c o n c l u s i o n , i f g l i a l c e l l h e t e r o g e n e i t y i s assumed, the GABA enzyme changes we have observed may be t h e d i r e c t o r i n d i r e c t r e s u l t o f t h e e a r l y changes i n a subtype o f g l i a l c e l l s . T h i s then serves t o i l l u s t r a t e an example o f t h e types or r e s e a r c h where concepts  o f g l i a l h e t e r o g e n e i t y may be  relevant t o the i n t e r p r e t a t i o n of the r e s u l t s .  -  140 -  F i g u r e 9.  Sagittal  sections of r a t brains  m i d l i n e ) s t a i n e d f o r GABA-T. Thiamine-deficient; th,  (at 2.5 mm  from  A, C o n t r o l ; B,  C, Recovered,  thalamus; p, pons; i c , i n f e r i o r  - 141  -  colliculus.  - 142 -  CONCLUSION  I have i n t h i s t h e s i s reviewed t h e data on many morphologically the b r a i n .  d e f i n e d types o f g l i a o r g l i a l - l i k e c e l l s i n  These c e l l types have v a r i a b l e marker s t a i n i n g ,  vary biochemically,  have d i f f e r e n t develpment p r o f i l e s , and  respond d i f f e r e n t l y t o d i f f e r e n t c u l t u r e c o n d i t i o n and t o injury.  C u l t u r e work shows even more v a r i a b i l i t y .  There are  d i f f e r e n c e s not only between c e l l l i n e s but between primary c u l t u r e s from d i f f e r e n t areas o f t h e b r a i n i n c e l l s t h a t are morphologically  similar.  My experiments have added t o t h i s p i c t u r e .  Experiment 1  showed t h a t g l i a can s t a i n f o r i r o n w i t h a d i s t i n c t p a t t e r n o f d e n s i t y and types o f c e l l s t a i n i n g . one  more example o f r e g i o n a l h e t e r o g e n e i t y .  regional  This i s j u s t  Experiment 2  showed PDH, an enzyme only r e c e n t l y known t o e x i s t , can be s t a i n e d f o r i n a s e l e c t e d few g l i a l c e l l s .  T h i s would  t h e o r e t i c a l l y i n d i c a t e t h a t an a l t e r n a t e r o u t e o f glutamate synthesis  e x i s t s i n these few s e l e c t e d g l i a l  cells.  Experiment 3 i l l u s t r a t e s how assumptions on t h e e x i s t e n c e o f g l i a l heterogeneity  may shed a d i f f e r e n t l i g h t on t h e  i n t e r p r e t a t i o n of research  data.  There remains much r e s e a r c h heterogeneity.  t o be done on g l i a l  I foresee that i t i s h i g h l y probable that a  complimentary map o f s p e c i f i c g l i a f u n c t i o n s w i l l be c r e a t e d w i t h a complexity t h a t may approach t h a t now emerging f o r neurons.  - 143 -  ACKNOWLEDGEMENTS I would l i k e t o thank a l l the members o f the U.B.C. D i v i s i o n o f N e u r o l o g i c a l Research who  were a l l v e r y h e l p f u l ,  e s p e c i a l l y my a d v i s o r Dr. E d i e McGeer whose warmth and g e n e r o s i t y meant a tremendous  amount t o me.  I would a l s o  like  t o thank the Huntington's Disease S o c i e t y and t h e graduate student summer fund which supported me husband who  f i n a n c i a l l y , and  my  typed the document.  The work on the i r o n experiment was M e d i c a l Research C o u n c i l o f Canada.  supported by the  Dr. Y. Noda, a s c i e n t i s t  from the Chugai Research L a b o r a t o r i e s , Tokyo, Japan,  greatly  a s s i s t e d me w i t h the i r o n r e s e a r c h , and was the co-author of a paper submitted t o J.Neurochem. t h a t came out o f t h i s work. would l i k e t o thank Dr. T.W.  I  McBride f o r t h e use o f h i s  microscope. The p y r r o l i n e dehydrogenase  experiments would not have  been done w i t h o u t the o r i g i n a l s u g g e s t i o n from P e t e r Wong, who a l s o c o l l a b o r a t e d w i t h a paper p u b l i s h e d i n J.Neurochem. was  I  supported by t h e G a r f i e l d - W e s t e r n Foundation and M.R.C. of  Canada i n t h i s work. The thiamine experiment was  supported by the M.R.C. o f  Canada and r e q u i r e d the t e c h n i c a l a s s i s t a n c e o f Mrs. Singh.  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