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Studies involving somatostatin systems in the rodent central nervous system Radke, James Melvin 1987

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STUDIES INVOLVING SOMATOSTATIN SYSTEMS I N THE RODENT CENTRAL NERVOUS SYSTEM. by JAMES MELVIN B.Sc.(hon.) U n i v e r s i t y  RADKE of B r i t i s h  C o l u m b i a , 1985  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE  STUDIES  (Neuroscience)  We  accept to  this  thesis  the required  as c o n f o r m i n g standards  THE UNIVERSITY OF B R I T I S H COLUMBIA April,  1987  c ) J a m e s M e l v i n Radke, 1987  In  presenting  degree  this  at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department publication  this or of  and study.  this  his  or  her  Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6(3/81)  that the  representatives.  may be It  thesis for financial gain shall not  permission.  requirements  I further agree  thesis for scholarly purposes by  the  for  an  advanced  Library shall make it  that permission  for extensive  granted  head  is  by the  understood be  that  allowed without  of  my  copying  or  my written  ii ABSTRACT  Somatostatin Several being  q u i t e heterogenous with  little  i n the  how  the  they  functional  other  and  relatively  striatal  interact with  high  the b r a i n .  distribution  Presently,  somatostatin  other  inputs  and  chemical  very  systems i n  t r a n s m i t t e r systems.  m e n t a l and  of  somatostatin  s t u d i e s have e s t a b l i s h e d t h a t t h e important  dopaminergic  f o r locomotor  activity  T h e s e s y s t e m s have a l s o b e e n i m p l i c a t e d i n s e v e r a l neural diseases  Parkinson s 1  In the somatostatin  somatostatin  such  as  schizophrenia, depression,  first  experiment,  i n t e r a c t i o n s between dopamine  s y s t e m s were e x a m i n e d u s i n g p a r a d i g m s  levels  amphetamine- and reinforcing  t o dopamine a g o n i s t s .  by  the drug  o f amphetamine.  i n t e r a c t i o n by  a dopamine a n t a g o n i s t , on term treatment  found  to  apomorphine-mediated motor b e h a v i o u r s aspects  The  second of the  examining the e f f e c t s  central  somatostatin  with h a l o p e r i d o l decreased  of attenuate but  not  experiment dopamine-  of h a l o p e r i d o l ,  levels.  striatal  and  involving  Depletion  c y s t e a m i n e was  to f u r t h e r c h a r a c t e r i z e the nature  somatostatin  and  disease.  b e h a v i o u r a l responses  attempted  interactions  to  systems.  to the b a s a l g a n g l i a are  reward.  as  concentrations  systems.  f u n c t i o n s of  neurotransmitter  Previous  the  throughout  f o l l o w i n g r e p o r t i s a summary o f e x p e r i m e n t s u n d e r t a k e n  assess  and  limbic  i s known a b o u t t h e  t h e b r a i n and  with  found  s t u d i e s have e s t a b l i s h e d i t s a n a t o m i c a l  appearing  The  i s a neuropeptide  Short  somatostatin  iii levels.  Long term  to a l t e r  somatostatin levels  Since  treatment  appear  the e f f e c t s  the e f f e c t s results  experiment  lesions indicate  dependent i n c r e a s e i n n i g r a l striatal  or c o r t i c a l  disagreement  studied  t o examine t h e  a n i m a l model o f P a r k i n s o n ' s d i s e a s e and examine  of dopaminergic  of this  t o be n o r m a l i n  o f MPTP p o i s o n i n g i n m i c e on  s o m a t o s t a t i n l e v e l s was a l s o  accuracy of t h i s  failed  i n t h e caudate-putamen.  somatostatin levels  Parkinsonian brains, central  ( 8 months) w i t h h a l o p e r i d o l  on s o m a t o s t a t i n l e v e l s . that  MPTP c a u s e s  a dose  somatostatin l e v e l s without  levels.  These r e s u l t s  The  altering  are i n p a r t i a l  w i t h r e s u l t s o b t a i n e d from both  post-mortem  P a r k i n s o n i a n b r a i n s a n d p r i m a t e s g i v e n MPTP, t h e r e b y q u e s t i o n i n g t h e a c c u r a c y o f t h i s mouse m o d e l o f P a r k i n s o n ' s d i s e a s e . The  final  experiment  examined t h e e f f e c t s  anticonvulsant-antidepressant somatostatin responsible  levels  carbamazepine  i n the r a t .  Although  f o r the therapeutic e f f e c t s  unknown, p r e v i o u s s t u d i e s have s u g g e s t e d treatment  of both manic-depression  with the a b i l i t y  o f t h i s drug  of the  on c e n t r a l t h e c h e m i c a l mechanisms  o f carbamazepine that  i t s efficacy  t o reduce  t h e abnormal s o m a t o s t a t i n  observed  i n these d i s e a s e s .  In this  acute,  chronic,  nor withdrawal  c h r o n i c treatment  rats.  were f o u n d  from  to alter  The l a c k o f e f f e c t s  i n the  a n d e p i l e p s y may be a s s o c i a t e d  levels  carbamazepine  are  the levels  of carbamazepine  experiment,  neither  with  of somatostatin i n on b a s a l  iv somatostatin susceptible  l e v e l s may  these  recent observations  results  cells  are  the i n t e r a c t i o n s  somatostatin  levels  n e r v o u s s y s t e m and p r o v i d e  and f u n c t i o n s o f s o m a t o s t a t i n  n o r m a l and a b n o r m a l  situations.  are d i s c u s s e d i n the context  of abnormal  diseases of the c e n t r a l  the  somatostatin  t o carbamazepine o n l y under p a t h o l o g i c a l  Together,  into  indicate  brain.  in some  of  several insight  systems i n  V  TABLE OF CONTENTS  Abstract  i i  Table of contents List  v  of f i g u r e s  v i i  Acknowledgements  ix  INTRODUCTION  1  Somatostatin  i n the basal ganglia  1  Dopamine-somatostatin i n t e r a c t i o n s  4  Somatostatin  5  i n neural disease  Rationale  6  EXPERIMENTS 1. The e f f e c t s  o f c y s t e a m i n e on s o m a t o s t a t i n  immunoreactivity  and on d o p a m i n e - m e d i a t e d  behaviours.  Introduction  8  Methods  10  RIA  14  methods  Results  16  Discussion  24  2. The e f f e c t s treatment  o f s h o r t and l o n g t e r m h a l o p e r i d o l  on s o m a t o s t a t i n  immunoreactivity.  Introduction  31  Methods  32  Results  34  Discussion  35  vi 3. The e f f e c t s o f MPTP p o i s o n i n g on immunoreactivity  somatostatin  i n t h e mouse.  Introduction  39  Methods  40  Results  41  Discussion  42  4. The e f f e c t s o f c a r b a m a z e p i n e (DMI) on s o m a t o s t a t i n  and d e s m e t h y l i m i p r a m i n e  immunoreactivity.  Introduction  47  Methods  48  Results  50  Discussion  50  GENERAL  DISCUSSION  Dopamine-somatostatin Somatostatin  interactions  i n neuro-psychiatric  The f u n c t i o n o f s o m a t o s t a t i n  diseases  55 57 61  REFERENCES  62  APPENDIX  88  vii LIST  OF  Figure  FIGURES  1:  Effects  of  4 hour p r e t r e a t m e n t  cysteamine locomotor Figure  2:  Effects  on  with  amphetamine-induced  activity  18  of 7 hour p r e t r e a t m e n t  cysteamine  on  striatal  with  somatostatin  immunoreactivity Figure  3:  Effects  o f 12  cysteamine  on  19  hour p r e t r e a t m e n t  with  apomorphine-induced  stereotypy Figure  4:  Effects  21  of  13 h o u r p r e t r e a t m e n t  cysteamine  on  striatal  with  somatostatin  immunoreactivity Figure  5:  Effects  22  of cysteamine  pretreatment  on  amphetamine-induced p l a c e p r e f e r e n c e Figure  6:  Effects on  F i g u r e 7:  of repeated  striatal  Effects  cysteamine  somatostatin  25  administration  i m m u n o r e a c t i v i t y . . . 26  of s h o r t term h a l o p e r i d o l  a d m i n i s t r a t i o n on  central  somatostatin  immunoreactivity Figure  8:  Effects  of  l o n g term  Figure  9:  l o n g term  35 and  withdrawal  following  h a l o p e r i d o l a d m i n i s t r a t i o n on  central  somatostatin  Effects  o f MPTP p o i s o n i n g on  catecholamine  levels  immunoreactivity  i n mice  36  striatal 43  viii Figure  10: E f f e c t s  o f MPTP p o i s o n i n g on  somatostatin Figure  11: E f f e c t s  12: E f f e c t s  somatostatin  o f DMI  somatostatin Figure  13: An example curve  immunoreactivity  44  of carbamazepine a d m i n i s t r a t i o n  on c e n t r a l Figure  central  i m m u n o r e a c t i v i t y . . . . 51  a d m i n i s t r a t i o n on  central  immunoreactivity  of a radioimmunoassay  52 standard 89  ix  ACKNOWLEDGEMENTS This  t h e s i s was w r i t t e n b y me, b u t t h e c o l l e c t i o n and  interpretation would l i k e Fibiger  o f d a t a was p e r f o r m e d  t o thank  b y numerous p e o p l e .  D r . A. J . MacLennan and D r . H. C.  f o r p r o v i d i n g me w i t h t h e t i s s u e  haloperidol  experiment.  Also,  samples f o r t h e  the catecholamine  w i t h t h e MPTP s t u d y was p e r f o r m e d  b y P a u l Cumming.  a s s i s t a n c e o f D r . M. T. M a r t i n - I v e r s o n was a p p r e c i a t e d when I was t r y i n g behavioural  studies  Finally, appreciation Vincent. sciencific unnoticed.  t o understand  a r e supposed  I would l i k e  His help i n trying  The  greatly what  these  t o mean.  t o e x p r e s s my  f o r the guidance  analysis  deepest  p r o v i d e d by Dr. S t e v e n t o t e a c h me t o p e r f o r m  r e s e a r c h and t o w r i t e a b o u t  i t h a s n o t gone  I  1  INTRODUCTION  Somatostatin  i n the Basal  Somatostatin active peptide this  peptide  somatotropin referred  was  Ganglia  first  recognized  by B r a z e a u and  as a  physiologically  his collegues  from ovine hypothalami  and  (1973) who  found  ( g r o w t h hormone) r e l e a s e ; t h u s  t o as  SRIF  (somatotropin  i t inhibited  somatostatin  release inhibiting  S u b s e q u e n t s t u d i e s have r e v e a l e d t h a t s o m a t o s t a t i n various endocrine throughout al.,  cells,  e t a l . , 1975;  J o h a n s s o n e t a l . , 1984; 1985).  Within  concentration  i s found  h y p o t h a l a m u s and  the  intermediate  levels  undetectable  amounts i n t h e c e r e b e l l u m  somatostatin present has  the  14  two  cleaved  It  appears t h a t somatostatin-14  prosomatostatin trypsin-like  olfactory  a 28  i s 116  peptide d i r e c t l y  conversion  be  is  low  active mainly  amino a c i d  peptide  somatostatin  (Epelbaum,  gene  the  (Gomez e t a l . , 1 9 8 5 ) , o r by (see review  and  1986).  c l e a v e d from e i t h e r  from somatostatin-28  or  bulb.  amino a c i d s l o n g  amino a c i d p r o s o m a t o s t a t i n can  et a l . ,  somatostatin-14  C h a r a c t e r i z a t i o n of the  is  (Beal et  c o r t e x , and  which c o n t a i n s  revealed that preprosomatostatin  and  system,  physiologically  amino a c i d p e p t i d e , b u t  i t s structure.  t o a 92  and  in  i n high  In the b r a i n somatostatin  a l s o b e e n shown t o e x i s t ,  within has  as  at l e a s t  Vincent  limbic  i n t h e b a s a l g a n g l i a and  peptides.  i s present  e t a l . , 1981;  K o b a y a s h i e t a l . , 1977;  T h e r e a p p e a r s t o be  factor).  nervous system  Finley  the b r a i n s o m a t o s t a t i n i n the  i s often  i n the p e r i p h e r a l nervous system,  v a r i o u s r e g i o n s of the c e n t r a l  1986a; B r o w n s t e i n  isolated  by  a  2 Epelbaum, 1 9 8 6 ) . different may  be  Studies  have s u g g e s t e d t h e s e two  d i s t r i b u t i o n s i n the  due  to d i f f e r e n t  processing  brain  regions  peptides  ( P a t e l e t a l . , 1981)  having  which  d i f f e r e n t methods  somatostatin-28 to somatostatin-14  (Zingg  have  of  and  Patel,  1983). Within relatively  the high  rat basal  concentrations  c a u d a t e - p u t a m e n , and  low  v e n t r a l tegmental area 1975;  ganglia,  i n the  levels  quite  Lesion (nucleus  low  studies  i n the i n the  neurons.  however o t h e r  Beal  afferents  to the  t h a l a m u s and somatostatin  Early studies  Terry,  of  a m y g d a l a do  the  from the not  et  suggested the  ( B e a l and  Martin,  the  striatal  i s present  in  hypothalamus  as  (Beal et a l . ,  l e s i o n s of  substantia  striatal  the  nigra,  levels  of  striatal  neurons, reduce  kainic the  1983).  s t u d i e s have a l s o d e m o n s t r a t e d  ( F i n l e y e t a l . , 1981;  a l . , 1985).  this  In c o n t r a s t ,  striatal  the  ( P a l k o v i t s e t a l . , 1980),  cortex,  reduce the  the  (1983;  region.  neurons d i s p l a y i n g s o m a t o s t a t i n striatum  and  caudate-putamen,  Furthermore,  ( B e a l e t a l . , 1985).  somatostatin  presence of  Vincent  his collegues  to support  1980).  striatum  Immunohistochemical  within  the  somatostatin  l e s i o n s , which d e s t r o y  levels  and  dorsal-lateral  s t u d i e s have f a i l e d  C r o w l e y and  in and  nigra  r a t i n d i c a t e t h a t much o f  a s o u r c e f o r some s t r i a t a l  acid  substantia  accumbens + c a u d a t e - p u t a m e n ) s o m a t o s t a t i n  intrinsic  1985;  n u c l e u s accumbens  i n the  1986a) have a l s o shown t h a t w i t h i n are  i s found  ( B e a l e t a l . , 1986a; B r o w n s t e i n e t a l . ,  K o b a y a s h i e t a l . , 1977).  levels  somatostatin  the  immunoreactivity  Johansson e t a l . ,  U l t r a s t r u c t u r a l studies  indicate that  1984;  3 these  cells  1982;  Takagi  et  are medium-sized a s p i n y e t a l . , 1983;  a l . , 1982a).  cytoplasm  Vincent  These c e l l s  neurons and  have an  (DiFiglia  Johansson,  indented  1983;  of  a l s o b e e n shown t o c o n t a i n an  s t r i a t a l n e u r o n s has  diaphorase  enzyme a c t i v i t y  selectively  stained  that allows  particular  these  cells  ( V i n c e n t e t a l . , 1983a).  This  characteristic  of  these  cells  allows  differentiated  from the  other  major s t r i a t a l  population,  the  cholinergic cells,  them t o be  Immunohistochemical somatostatin  neuropeptide Y supported  by  peptides  radioimmunoassay  have a s i m i l a r  somatostatin their  asymmetric 1983).  to  be  latter  easily  (Vincent  distribution  that  striatum also  i n the  but  do  e t a l . , 1983b). some o f  the  contain This  s t u d i e s showing t h a t t h e s e  cells  synaptic  contacts  s t u d i e s of  striatum  symmetrical neurons  i n p u t on  (DiFiglia  is  two  (Beal  and  At p r e s e n t ,  somatostatin  the  containing  synaptic connections  dendrites,  and  Aronin,  synapses w i t h  (DiFiglia  the  i n d i c a t e t h a t these  Somatostatin-immunoreactive  primarily  1983).  NADPH-  1986a).  striatal  striatal  population  ( V i n c e n t e t a l . , 1982a; 1982b; 1 9 8 3 a ) .  Immunohistochemical  most o f  i n the  rich  rough  also aspiny  s t u d i e s have a l s o r e v e a l e d  containing cells  a  interneuron  which are  d i s p l a y NADPH-diaphorase a c t i v i t y  Martin,  and  c o n t a i n i n g w e l l d e v e l o p e d G o l g i a p p a r a t u s and Furthermore, t h i s  Aronin,  Vincent  nucleus,  endoplasmic reticulum.  not  and  and  interneurons  1982;  dendrites  t r a n s m i t t e r s of  receive  v i a b o t h symmetric  terminals  Aronin,  neurons  of  1982; the  Takagi  et a l . ,  appear t o and  spines  Takagi cells  make c o n t a c t  and  form of  other  et a l . ,  with  which  i s unknown,  but  4 may  include  the  medium s p i n y  major output neurons of  the  neurons which are  b e l i e v e d t o be  the  striatum.  Somatostatin-dopamine interactions The the  two  main a s c e n d i n g dopaminergic  nigrostriatal  substantia  tract  n i g r a , and  to the  rat  the mesolimbic p r o j e c t i o n to the the  arising  area.  v e n t r a l tegmental  connections  Dopamine t e r m i n a l s symmetric  and  t h e y make a r e  medial p r e f r o n t a l  i n the  asymmetric  forming axo-dendritic,  cortex  The  poorly  the  understood.  s t r i a t u m have b e e n shown t o make b o t h contacts  with  a wide v a r i e t y of  a x o - s p i n o u s , and  ( A r l u i s o n e t a l . , 1984; 1981).  still  the  nucleus  A l t h o u g h t h e s e systems have been e x t e n s i v e l y s t u d i e d , synaptic  are  caudate-putamen o r i g i n a t i n g i n  accumbens, o l f a c t o r y t u b e r c l e , and from the  systems i n the  axo-somatic  F r e u n d e t a l . , 1984;  neurochemical i d e n t i t i e s  of  cells,  contacts  P i c k e l et a l . ,  these postsynaptic  cells  a r e m o s t l y unknown. T h e r e i s some e v i d e n c e s u p p o r t i n g o f c e n t r a l dopamine n e u r o n s may  be  the  view t h a t  i n f l u e n c e d by  somatostatin  ( B e a l and increase  Martin,  the  Reisine,  r e l e a s e of  1983;  turnover  S t a r r , 1982)  tritiated  of  striatal  and  the  dopamine.  spontaneous  K -evoked +  (Starr,  dopamine f r o m r a t s t r i a t a l  results  have a l s o b e e n o b s e r v e d  i n vivo  nucleus  (Chesselet  1983).  and  Reisine,  and  1984a; 1986b) i n f u s i o n s o f  Somatostatin a l s o appears to i n c r e a s e and  activity  somatostatin.  B o t h i n t r a v e n t r i c u l a r ( G a r c i a - S e v i l l a e t a l . , 1978) intrastriatal  the  i n the  slices. cat  (Chesselet 1982) Similar  caudate  5 Some o f through  the  cyclic  somatostatin  effects  AMP.  Intraventricular  cyclase  can  activity  mediated  a d m i n i s t r a t i o n of  p r o d u c e s an e l e v a t i o n i n s t r i a t a l  ( H e r c h l e t a l . , 1977). somatostatin  o f s o m a t o s t a t i n m i g h t be  cyclic  AMP  levels  More r e c e n t s t u d i e s have i n d i c a t e d  inhibit  the  s t i m u l a t i o n of  p r o d u c e d by dopamine  striatal  (Chneiweiss  that  adenylate  e t a l . , 1985;  Moser e t a l . , 1986) . The striatal  dopamine s y s t e m a l s o a p p e a r s t o be somatostatin  administration of  disorders. treatment diabetes  regard,  i n the  s t r i a t u m ( B e a l and  the  chronic  Martin,  levels  1984c).  i n neural disease  T h e r e has somatostatin  In t h i s  of n e u r o l e p t i c s produces a r e d u c t i o n i n the  somatostatin  Somatostatin  neurons.  able t o modulate  been c o n s i d e r a b l e i n t e r e s t  i n t h e p e r i p h e r y and For  ( G e r i c h , 1 9 7 6 ) , and  f u n c t i o n s of  i t s impications i n  example s o m a t o s t a t i n  of p a n c r e a t i c endocrine  i n the  analogs  tumours  acromegaly  endocrine  are being  tested for  (Wood e t a l . , 1 9 8 5 ) ,  (Kingsnorth et a l . ,  1986). Recently, shown t o be Huntington's striatal cells  somatostatin  altered  i n the b r a i n have a l s o been  i n several neuronal  disorders.  d i s e a s e , w h i c h i s c h a r a c t e r i z e d by  tissue,  t h e r e a p p e a r s t o be  (Dawborn e t a l . , 1985;  striatal  levels  somatostatin  F e r r a n t e e t a l . , 1985)  p o s t mortem b r a i n s o f H u n t i n g t o n ' s B e a l e t a l . , 1984;  severe  a s p a r i n g of  concentrations being  In loss  of  somatostatin resulting  in  greatly elevated i n  p a t i e n t s ( A r o n i n e t a l . , 1983;  N e m e r o f f e t a l . , 1983;  Sagar e t a l . , 1984).  6 In c o n t r a s t , o t h e r d i s o r d e r s such and  Javoy-Agid,  and  schizophrenia  striatal  1985;  ( N e m e r o f f e t a l . , 1983)  Most c l i n i c a l  Included fluid et  E p e l b a u m e t a l . , 1983;  somatostatin  somatostatin  and  i n the  (CSF)  to  have p r o v i d e d  some i n t e r e s t i n g  results.  of diseases  somatostatin  (Agren  levels  are: Parkinson's  disease  and  Rubinow e t a l . , 1985; syndromes  As  somatostatin  (Gomez e t a l . , 1986a; disease  Lundqvist,  G e r n e r and  Rubinow, 1 9 8 6 ) , and  clinical levels  of  addition,  setting,  T h a i e t a l . , 1985).  altered  affectively fluphenazine  CSF  CSF  l e v e l s when t h e  rats  somatostatin  i l l p a t i e n t s on treatment  levels  (Rubinow e t a l . , 1984;  ( H i g u c h i e t a l . , 1986).  z i m e l i d i n e (ZMI),  With regard  b e e n shown t o l o w e r  are abnormal, 1985;  In  have b e e n r e p o r t e d i n  carbamazepine,  has  1984c).  (Gattez e t a l . , 1986).  (Rubinow, 1 9 8 6 ) .  drug  levels  -28  Martin,  striatal  n e u r o l e p t i c s have a l s o b e e n shown t o a l t e r  carbamazepine, t h i s  humans  somatostatin  the  and  ( B e a l and  Yamada,  In  somatostatin-14  rat  1986b;  patients with  p r e v i o u s l y mentioned, n e u r o l e p t i c s reduce i n the  (Cramer  (Cramer e t a l . ,  1984;  (Cramer e t a l . , 1985;  levels  normal  study  disease  D r u g r e g i m e n s have a l s o b e e n shown t o a l t e r levels.  1984)  showing abnormal c e r e b r o s p i n a l  e t a l . , 1986), H u n t i n g t o n ' s  dystonic  a p p e a r t o have  lumbar p u n c t u r e s  list  (Agid  Rinne e t a l . ,  s t u d i e s use  1981), d e p r e s s i o n 1982;  disease  levels.  a l . , 1985), A l z h e i m e r ' s  Raskind  as P a r k i n s o n ' s  i n both  or  to  somatostatin  affectively i l l  Rubinow, 1986)  and  i n kindled  7  The  purpose of the  f o l l o w i n g s e t o f e x p e r i m e n t s was  to  examine f u r t h e r t h e p o s s i b l e i n t e r a c t i o n s between dopamine somatostatin involve was  i n the b a s a l g a n g l i a .  a b e h a v i o u r a l approach to study  f o l l o w e d by  two  of the  the  MPTP on  somatostatin  (DMI)  on  the  examined t o d e t e r m i n e i f t h e s e e f f e c t s by  levels  of  clinically  altering  experiments  interaction.  the e f f e c t s levels.  a n t i d e p r e s s a n t s carbamazepine  desmethylimipramine  some o f t h e i r  initial  experiments i n v o l v i n g  d r u g s h a l o p e r i d o l and effects  The  and  This  of  the  Finally,  the  and  somatostatin important  somatostatin  were  drugs  systems.  have  8  EXPERIMENT 1.  THE EFFECTS OF CYSTEAMINE ON SOMATOSTATIN IMMUNOREACTIVITY AND ON DOPAMINE MEDIATED BEHAVIOURS.  INTRODUCTION Very l i t t l e  i s presently  known a b o u t t h e f u n c t i o n o f  somatostatin  i n various  somatostatin  i n t o t h e v e n t r i c l e s have been  field  activity  (Havlicek  Rezek e t a l . , 1976; behaviours  motor  (Vecsei  i n t o the striatum  impairment  established  t e s t s which  the  have  P l o t n i k o f f e t a l . , 1982; stereotypic  Direct  a l s o been  i n f u s i o n s of  shown t o  l o c o m o t i o n and  while higher  studies  influence  producing  doses  produce  some o f t h e i r  i n v o l v i n g t h e dopamine  functions.  a p p e a r t o be d e p e n d e n t  behaviours,  Two  common  behavioural  upon dopamine s y s t e m s  stereotypy  and  locomotor  s u c h as s n i f f i n g ,  chewing,  systems  are  activity. and gnawing  r a t , a p p e a r t o be m e d i a t e d v i a d o p a m i n e r g i c r e c e p t o r s  caudate-putamen,  while  locomotor a c t i v i t y  The m e s o l i m b i c dopamine s y s t e m has r e w a r d mechanisms u s i n g  electrical  self  stimulation  a l s o been  the  ( K e l l y e t a l . , 1975). shown t o be  d i f f e r e n t paradigms, (Corbett  in  i n the  appears t o i n v o l v e  m e s o l i m b i c pathway t o t h e n u c l e u s accumbens  in  open  (Rezek e t a l . , 1 9 7 7 ) .  dopamine a g o n i s t - i n d u c e d Stereotypic  shown t o a l t e r  et al.,1982).  and b i t i n g ,  Numerous b e h a v i o u r a l have  of  and e l e c t r i c a l s e l f - s t i m u l a t i o n  low d o s e s i n c r e a s i n g  scratching  Infusions  e t a l . , 1983a; 1 9 8 4 ) ,  (Rezek e t a l . , 1 9 7 6 ) ,  behaviour, with stereotypic  of the b r a i n .  e t a l . , 1976;  Vecsei  of h y p o t h a l a m i c s i t e s somatostatin  regions  and W i s e ,  involved  including: 1980),  conditioned  9 reinforcers  ( T a y l o r and R o b b i n s ,  conditioning Phillips,  ( S p y r a k i e t a l . , 1983; s e e r e v i e w  1986).  paradigm suggest locomotor  that the mesolimbic  e t a l . , 1983; 1985; M i t h a n i e t a l . , 1986; Radke e t  temporarily deplete the l e v e l s approximately  one week  Millard  1982;  Szabo a n d R e i c h l i n ,  affect  of somatostatin  ( B e a l and M a r t i n ,  1981).  1984b; Brown e t a l . ,  Cysteamine does n o t appear t o  i n c l u d i n g v a s o p r e s s i n , enkephalin, VIP,  B e a l , 1987) and s i n c e p r o l a c t i n  i s present  central  understand  (Chattha  i n v e r y low  somatostatin  may  systems.  b e t t e r t h e i n t e r a c t i o n s between dopamine and  somatostatin  i n the basal ganglia, the e f f e c t s  somatostatin  b y c y s t e a m i n e on a m p h e t a m i n e - i n d u c e d  activity,  Y  i n t h e b r a i n (Fuxe e t a l . , 1 9 7 7 ) , c y s t e a m i n e  used t o study To  appears  and p r o l a c t i n  ( P a l k o v i t s e t a l . , 1982) and n e u r o p e p t i d e  concentrations be  f o r studying  e t a l . , 1982; P a l k o v i t s e t a l . , 1982; S a g a r e t a l . ,  other peptides,  CCK, LHRH  tool  systems i s the a m i n o t h i o l cysteamine which  1983;  and  ( D i S c a l a e t a l . , 1985;  1987a).  somatostatin  for  preference  mediated reward and  A possible u s e f u l pharmacological  to  preference  b y F i b i g e r and  Recent s t u d i e s u s i n g the p l a c e  mechanisms may be d i f f e r e n t i a t e d  Martin-Iverson al.,  1 9 8 4 ) , and p l a c e  of reduction of locomotor  a p o m o r p h i n e - i n d u c e d s t e r e o t y p y , and a m p h e t a m i n e - i n d u c e d  place preference  c o n d i t i o n i n g were e x a m i n e d .  10  METHODS AND MATERIALS  Subjects M a l e Long  Evans  rats  were u s e d a n d h o u s e d  (Charles Rivers, weighing  4-5 p e r c a g e  under  a 12 h l i g h t  - 20:00) w i t h a d l i b i t u m  a c c e s s t o f o o d and water.  left  f o rat least  i n their  home c a g e s  l a b o r a t o r y b e f o r e e x p e r i m e n t s were  275-350 g  1 week a f t e r  cycle  (08:00  A l l rats arrival  were  atthe  conducted.  Drugs Cysteamine dissolved (s.c.)  (p-mercaptoethylamine  i n 0.9% s a l i n e  hydrochloride,  (100 mg/ml) a n d i n j e c t e d  dissolved  was  subcutaneously  (1 m l / k g ) .  D-amphetamine s u l p h a t e ( S m i t h K l i n e  (i.p.)  Sigma) was  i n saline  (1 m l / k g ) .  dissolved  (1.5 mg/ml) a n d i n j e c t e d  intraperitoneally  Apomorphine h y d r o c h l o r i d e  (Sigma)(0.5mg/ml)  i n 0.9% s a l i n e w i t h a s c o r b a t e (0.3 mg/ml) added a s  an a n t i o x i d a n t . on i c e d u r i n g  a n d F r e n c h ) was  This  s o l u t i o n was k e p t p r o t e c t e d  the i n j e c t i o n procedures.  from  l i g h t and  R a t s were i n j e c t e d  with  1 ml/kg, s . c . .  Procedure Experiment  la.  cm) a c t i v i t y beams.  Locomotor  cages  a c t i v i t y was m e a s u r e d i n c i r c u l a r (61  (BRS/LVE),  t r a n s e c t e d by 6 i n f r a r e d  photocell  Photobeam i n t e r r u p t i o n s were r e c o r d e d a n d a n a l y z e d w i t h a  NOVA IV/X m i n i c o m p u t e r  ( D a t a G e n e r a l ) e q u i p p e d w i t h a Manx  interface  (GC C o n t r o l s ) .  and s o f t w a r e  R a t s were i n j e c t e d  with  11 cysteamine  or s a l i n e  (n= 11 p e r g r o u p ) 4 h o u r s p r i o r t o  amphetamine  injections.  injections,  r a t s were p l a c e d i n t o  and  a c t i v i t y was  was  then  were k i l l e d striata  recorded  administered,  additional  One h o u r p r i o r  3 hours.  the locomotor  f o r 1 hour  recorded  A t t h e end o f locomotor  by c e r v i c a l d i s l o c a t i o n ,  Experiment  S t e r e o t y p y was  lb.  and a c l e a r  (p. 14).  observed  while  r a t s were i n s t e e l tilted  P l e x i g l a s d o o r on one s i d e .  injected with cages.  T h e y were t h e n  (12 h o u r s a f t e r  Rats  t h a t had  (n=19) o r  An o b s e r v e r behaviour  s c a l e of K e l l y points:  blind  to the treatment  d u r i n g 30 s e c o n d  e t a l . , (1976).  0 = a s l e e p o r immobile;  of the r a t s  periods every  1 = active;  2 = active  continuous  i n one l o c a t i o n ;  o r gnawing; 6 = c o n t i n u o u s immediately  10 m i n u t e s f r o m  5-  rating  T h i s s c a l e has t h e f o l l o w i n g  3 = a c t i v e with continuous  sniffing  rated  using the 7-point  bursts of s n i f f i n g ;  licking  pretreatment)  a p o m o r p h i n e , and r e p l a c e d i n t o t h e s t e r e o t y p y  65 m i n u t e s p o s t - a p o m o r p h i n e i n j e c t i o n ,  killed  mirror  (n=20) were g i v e n 1 h o u r e x p o s u r e t o t h e s t e r e o t y p y c a g e s  (habituation).  their  the rats  f o r somatostatin  b e e n p r e t r e a t e d 11 h o u r s p r e v i o u s l y w i t h c y s t e a m i n e saline  Amphetamine  f o r an  testing  (35 x 40 x 18 cm) w i t h a s t e e l mesh f l o o r ,  underneath  cages,  t h e b r a i n s were removed, t h e  d i s s e c t e d o u t on i c e and a s s a y e d as d e s c r i b e d l a t e r  activity  (habituation).  and a c t i v i t y was  immunoreactivity  cages  t o amphetamine  sniffing;  4 =  5 = s n i f f i n g with bursts of  licking  f o l l o w i n g the l a s t  with  o r gnawing.  Rats  observation period,  were  their  b r a i n s were removed, t h e s t r i a t a were d i s s e c t e d and t h e l e v e l s o f  12 somatostatin described  later  Experiment conducted into  immunoreactivity (p.  lc. with  c o n d i t i o n i n g and  f o u r s h u t t l e boxes  (80 x 25 x 36  c o m p a r t m e n t s was  (34 x 25  cm),  c l o s e d by  distinctive  connected  guillotine  Translucent Plexiglas  i l l u m i n a t i o n of the balanced  on  box  t h a t the  crossings  interior  a fulcrum; time  c o u l d be  the  and  lids  cm),  doors.  i n the allowed  on  for  r a t s were p l a c e d i n one  of the  side) with  the doors  time  the  number o f c r o s s i n g s f r o m one  recorded two  over  and  consecutive  immediately  procedure  was  the  2,  4,  an  an  immediately  was  two  spent  the  of  equipment.  During  Phase 1  compartments i n each  compartment  compartment t o t h e was  (start  other  conducted  on  i n j e c t i o n of cysteamine or v e h i c l e . f o r each of injected  e a c h day  and  4 days  f o r 30 minutes.  injections  of  (Day  1,  3,  p l a c e d w i t h i n the  8 ) , r a t s were c o n f i n e d t o t h e after  compartment  i n j e c t i o n o f amphetamine o r v e h i c l e and  conducted  same t i m e 6 and  (grid  E a c h box  r a t s were c o n f i n e d t o t h e n o n - s t a r t  Phase 2 ) ; e a c h r a t was at  of f l o o r  days.  after  hours a f t e r  (solid  t h e number  a 15 m i n u t e p e r i o d . T h i s p r o c e d u r e  I n P h a s e 2,  14  the  2  diffuse  electromechanical  (Pretest),  open, and  (8 x 8 x  of a r a t ' s weight t i l t i n g  each s i d e ,  recorded with  was  Each of the  type  T h i s experiment c o n s i s t e d of three phases.  and  as  each d i v i d e d  a tunnel  of the compartments.  shifting  spent  by  testing  i n the c o l o u r of the w a l l s  brown o r b l a c k w i t h w h i t e s t r i p e s )  such  radioimmunoassay  Place preference  w h i c h c o u l d be  o r mesh).  by  14).  2 compartments  6 cm)  determined  saline;  On  This 5 and  7 of  compartment  a l t e r n a t e days  start  12-  (Day  compartment  t h e r e were no  injections  13 made 12-14 h o u r s p r i o r t o t h e s e i n j e c t i o n s . assigned  t o one o f 4 t r e a t m e n t g r o u p s  AMPHETAMINE, first the  drug  second  refers  refers  killed, the  Immediately  their brains  i m m e d i a t e l y b e f o r e compartment  a single  15 m i n u t e  test of place  a s i n P h a s e 1. No i n j e c t i o n s  after  t h i s 15 m i n u t e  preceded  t e s t , the rats  removed, and t h e s t r i a t a  dissected  were  o u t and  l e v e l s o f s o m a t o s t a t i n i m m u n o r e a c t i v i t y d e t e r m i n e d by  radioimmunoassay  Statistical  with  pretreatment;  n = 10 p e r g r o u p ) .  p r e f e r e n c e was c o n d u c t e d , test.  (SALINE + SALINE, SALINE +  t o t h e 12-14 h o u r  t o the treatment  D u r i n g Phase 3 ( T e s t ) ,  this  randomly  CYSTEAMINE + SALINE, CYSTEAMINE + AMPHETAMINE; t h e  i n each p a i r  confinement;  R a t s were  as d e s c r i b e d  later  (p.  14).  Analysis  L o c o m o t o r a c t i v i t y was s u b j e c t e d t o a n a l y s i s  of variance  2 factors:  and 1 r e p e a t e d  factor  1 between f a c t o r  ( b l o c k s o f 10 m i n ) .  made f o l l o w i n g  (drug treatment)  Individual  t h e method o f W i n e r  planned comparisons  (1971).  were  Peak, t o t a l and  m e d i a n s t e r e o t y p y s c o r e s were a n a l y s e d s e p a r a t e l y w i t h one-way analysis  of variance.  analysis  o f v a r i a n c e w i t h 2 between f a c t o r s  and  P l a c e p r e f e r e n c e d a t a was s u b j e c t e d t o  amphetamine X s a l i n e )  test).  Planned comparisons  r e s u l t s were a l s o  and one w i t h i n  factor  were c o n d u c t e d .  subjected to analysis  (cysteamine X  saline  (second p r e t e s t  Biochemical assay  of variance.  X  14  Biochemical  Assays  Tissue Preparation. R a t s were k i l l e d removed, and NAS  by c e r v i c a l d i s l o c a t i o n ,  the s t r i a t a ,  i n c l u d i n g b o t h t h e caudate-putarr.en  ( b i l a t e r a l ) were d i s s e c t e d  r a d i o i m m u n o a s s a y was acetic  acid  and  centrifuged  o u t on  weighed, b o i l e d  then sonicated.  a t 4°C  f o r 20 min  Samples were s t o r e d  t h e b r a i n s were  ice.  Tissue for  f o r 10 m i n u t e s  The  s a m p l e s were  a t 10,000 rpm,  a t -70°C u n t i l  and  and  i n 2.0  N  then  lyophilized.  assayed.  Radioimmunoassay. Somatostatin-like  i m m u n o r e a c t i v i t y was  r a d i o i m m u n o a s s a y w h i c h compared t i s s u e somatostatin-like  activity  concentrations of s y n t h e t i c The  buffer  sodium b a r b i t a l , 0.25  mM  mM  The  generously  and  28  a monoclonal  prepared (Ab  i n equimolar  and  0.1%  stock  BSA  Brown, MRC  s o l u t i o n was  azide/BSA  solution  B.  a stock solution,  a t a 1:1  dilution  further and  and  diluted  stored  buffer.  fresh  100  500  weekly.  recognizes both  The  antibody  was  Regulatory Peptide  C.  Antibody  solutions sodium a z i d e  f r o z e n a t -70°C. fold On  mM  chloride,  and  c o n t a i n i n g 0.1%  kept  a t 4°C.  sodium  (BSA)  3) t h a t  ratios.  Group, D e p a r t m e n t o f P h y s i o l o g y , U. from  mM  serum a l b u m i n  and was  s u p p l i e d by Dr J . C.  were p r e p a r e d  by v a r y i n g  f o r t h e a s s a y c o n t a i n e d 24  bovine  (pH 7 . 4 ) ,  a n t i b o d y was  s o m a t o s t a t i n 14  somatostatin i n assay  s o d i u m a c e t a t e , 43.6  t h i m e r o s a l , 0.5%  KlU/ml a p r o t i n i n  c o n c e n t r a t i o n s of  t o a s t a n d a r d c u r v e formed  s o l u t i o n used 3.4  measured u s i n g a  i n a 0.1%  t h e day  The  sodium  of the  assay  15 t h i s was  f u r t h e r d i l u t e d i n c o l d assay b u f f e r  concentration  of  the  c h l o r a m i n e T method  day  0.1%  of  an  assay, the  trifluoroacetic  cartridge, ranging  was  washes.  The  purified  acetonitrile  wash.  solution  to obtain  CPM/  100  ul.  The  synthetic  -70°C i n 10u.g 40u.M BSA,  40%  500  On  the  labelled  day  of  final On  per  the  the  the  of  third  somatostatin  day, was  0.1%  per  of  1.0  p e r f o r m e d by  (C^g  ml 28%  adding  stored  aliquot  in  was  0.975  to  triplicate.  i n assay b u f f e r the  standard  f r e e and  curve  f o r 3 days bound  dextran-T70  (Norit-neutral; Fisher) The  to  in duplicate.  incubated  hormone f r e e p l a s m a .  at  aprotinin  analyzed  the  2500  lactose,  a range of  range of  of  in  gradient  140mM  analyzed  and  On  column  approximately  t u b e was  separation  (Pharmacia)-coated charcoal phosphate b u f f e r w i t h  ml  pak  from the  a s s a y , an  t u b e and  sensitivity  0.4  dissolved  a c i d , and  to obtain  K.  -20°C.  ( P e n i s u l a L a b s ) was  ED80 = 70-100 pg)  volume o f  at  Dr.  diluted in buffer  s a m p l e s were d i l u t e d a p p r o p r i a t e l y  (ED20 = 5.0-10.0 pg;  4°C.  l a b e l was  a c i d , lOOmM f o r m i c  d i l u t e d i n assay b u f f e r  results within  The  steps  obtained  concentration  picograms somatostatin  obtain  at  purified  i n 2%  by  step  aliquots in a solution containing  lOmM c i t r i c  Tissue  acetonitrile  was  somatostatin-14  (80,000 K l U / l i t e r ) . serially  an  acetonitrile  a final  was  p u r i f i e d w i t h a sep  somatostatin  The  stored  l a b e l l e d somatostatin  a c i d and  to  generously supplied U.B.C.., and  Waters Assoc.) u s i n g  f r o m 20%  (^^1)  ( P e n i s u l a Labs.) i o d i n a t e d  Kwok, D e p a r t m e n t o f P h y s i o l o g y , the  final  1:500,000.  Tyrosine-l-somatostatin via  to a  in  0.5%  t u b e s were  16  centrifuged containing  f o r 30 m i n u t e s  a t 3,000 RPM,  t h e bound s o m a t o s t a t i n , was  radioactivity  i n the c h a r c o a l p e l l e t  s o m a t o s t a t i n was  and  the supernatant,  removed.  The  containing  c o u n t e d on a gamma c o u n t e r  4) e q u i p p e d w i t h P C - D a t a A c q u i s i t i o n  the f r e e  labelled  (Packard, M u l t i  and A n a l y s i s  System  Prias  (PC-  DAAS) t o a n a l y z e t h e d a t a . R e s u l t s were e x p r e s s e d as p i c o g r a m s milligram  t i s s u e wet  of somatostatin per  weight.  RESULTS Experiment l a . Cysteamine testing 30 min  injections  significantly  individual  min  interaction  10 min  habituation  group  interaction  not  significant. and  10 min  less  < 1.0  first  significant  p < 0.01),  and  The  activity  10  of the 2  the remaining b l o c k s  for a l l 3 blocks).  stimulation  t r e a t e d group  the cysteamine  pretreated  of locomotor a c t i v i t y (Figure  Planned comparisons  than  1 ) , w i t h the main  ( F ( l , 1 6 ) = 7.38,  between t r e a t m e n t and  the t h i r d  the  b l o c k : F ( l , 1 6 ) = 83.15, p <  p < 0.05).  treatment s i g n i f i c a n t  The  first  ( F ( 5 , 8 0 ) = 3.64,  amphetamine i n j e c t i o n ,  seen i n the s a l i n e cysteamine  1 ) , as i n d i c a t e d by a  s i g n i f i c a n t l y during  (F(l,16)  exhibited  locomotor  b l o c k : F ( l , 1 6 ) = 23.11, p < 0.001; t h i r d  d i d not d i f f e r  Following  of  (first  b l o c k : F ( l , 1 6 ) = 5.21,  groups of  (Figure  comparisons  0.001; s e c o n d  to  reduced locomotor a c t i v i t y d u r i n g  of h a b i t u a t i o n  treatment X time  g i v e n 3 hours p r i o r  time  was effect  p < 0.025).  ( b l o c k s o f 10 min)  revealed  that during  through t o the n i n t h time b l o c k s the  the  was  17 cysteamine controls 8.25,  p r e t r e a t e d group d i f f e r e d  significantly  from t h e  ( F ( l , 1 6 ) = 14.76, 10.47, 21.08, 17.94, 15.01,  7.68, r e s p e c t i v e l y , Examination  p < 0.025 i n a l l c a s e s ) .  of the s t r i a t a l  two g r o u p s o f r a t s  somatostatin levels  revealed a s i g n i f i c a n t  6.69, p < 0.02) i n t h e c y s t e a m i n e compared w i t h t h e c o n t r o l g r o u p These r e s u l t s  indicate  systemic  injections  striatal  somatostatin levels  hyperactivity.  14.92,  that  group  f o r these  reduction (F(l,20) =  (38.12 ± 1.20 pg/mg)  (49.40 ± 2.54 pg/mg) ( F i g u r e 2 ) . 4 hour p r e t r e a t m e n t  o f 100 mg/kg c y s t e a m i n e , a n d amphetamine  reduced induced  with both  18  900 -  habituation  amph  20  40  60  80  100  120  140  160  180  TIME (min)  FIGURE 1.  E f f e c t o f 4 hour p r e t r e a t m e n t w i t h s a l i n e  cysteamine  (100 mg/kg, SC) on amphetamine  locomotor a c t i v i t y .  (1 ml/kg) o r  (1.5 mg/kg, I P ) i n d u c e d  D a t a r e p r e s e n t mean v a l u e s o f beam  i n t e r u p t i o n s p e r 10 m i n u t e b l o c k f o r e a c h g r o u p . * p < 0.05 compared activity.  to saline  group's t e n minute  block  19  FIGURE 2.  Effect  o f 7 hour p r e t r e a t m e n t w i t h s a l i n e  cysteamine  (100 mg/kg, SC) on s t r i a t a l  accumbens) s o m a t o s t a t i n l e v e l s The  tissue  obtained i n this  following.the values  f o r each  (caudate-putamen  a s m e a s u r e d by  immediately  D a t a r e p r e s e n t s mean ± SEM  group.  * p < 0.05 compared t o s a l i n e  + nucleus  radioimmunoassay.  s t u d y were f r o m r a t s  locomotor t e s t i n g .  (1 ml/kg) o r  group.  20  Experiment l b . As c a n be o b s e r v e d i n F i g u r e with  cysteamine  exhibited  12 h o u r s p r i o r  significant  stereotypy,  relative  3, t h e g r o u p  but  and t h e t o t a l  reductions  i n apomorphine-induced  to the s a l i n e  p r e t r e a t e d group.  although  ( F ( l , 3 7 ) = 2.13, p > 0 . 1 ) . T h u s ,  apomorphine produced  stereotypy  was o f a s i g n i f i c a n t l y cysteamine  somatostatin  levels  decreased  shorter  h a l f that of c o n t r o l s  significantly  were n o t that  (peak e f f e c t ) o f  duration. experiment (saline:  had s t r i a t a l  59.43 ± 8.61  30.38 ± 3.35 pg/mg, n = 8; F ( l , 1 5 )  p < 0.025) ( F i g u r e 4 ) .  pretreatment  scores,  r a t s as i n c o n t r o l s , t h e  treated rats i n this  pg/mg, n = 9; c y s t e a m i n e :  scores  i t appears  a s i m i l a r degree  i n cysteamine p r e t r e a t e d  6.28,  ( F ( l , 3 7 ) = 4.25, p <  i n t h e peak s t e r e o t y p y  stereotypy  The  This  ( F ( l , 3 7 ) = 4.81, p < 0.05) s t e r e o t y p y  apparent reductions  significant  pretreated  t o apomorphine i n j e c t i o n s  r e d u c t i o n was a p p a r e n t i n b o t h t h e m e d i a n 0.05)  of rats  Thus,  systemic  cysteamine  depleted  striatal  somatostatin  apomorphine induced  stereotypy.  and  =  21  PEAK  FIGURE 3.  Effect  or cysteamine induced  MEDIAN  TOTAL  o f 12 h o u r p r e t r e a t m e n t  with  (100 mg/kg, SC) on a p o m o r p h i n e  stereotypy.  intervals,  '  Total  = sum a l l 7 s c o r e s  and peak = t h e h i g h e s t  r e p r e s e n t mean ± SEM f o r e a c h  single  (1 ml/kg)  (0.5 mg/kg, SC) taken  a t t e n minute  score obtained.  group.  * p < 0.05 compared t o t h e s a l i n e  saline  group.  Data  22  8 0  7 0  -  I SAL  FIGURE 4. or  Effect  cysteamine  i CYST  o f 13 h o u r p r e t r e a t m e n t w i t h s a l i n e  (100 mg/kg, SC) on s t r i a t a l  n u c l e u s accumbens) s o m a t o s t a t i n l e v e l s radioimmunoassay. rats  The t i s s u e  immediately f o l l o w i n g  (caudate-putamen  s t u d y were  the stereotypy t e s t i n g .  * p < 0.05 compared t o s a l i n e  +  as measured by  obtained i n this  r e p r e s e n t s mean ± SEM v a l u e s f o r e a c h  (1 ml/kg)  group.  group.  Data  from  23  Experiment l c . The induced  i n f l u e n c e of cysteamine pretreatment place  variance  preferences  revealed  c y s t e a m i n e and pretest  the  t h a t t h e r e was  amphetamine on  to t e s t  significant  i s depicted no  the  ( F ( l , 3 0 ) = 0.62,  effect  of  i n Figure  interaction  last  pretest  comparisons exhibited  p  compartment  > 0.1),  Thus, r e p e a t e d on  conditioned  due  the  than those  to the  preference  the  i n the  of  between  p  < 0.1).  t h a t t h e r e was time spent  test  day,  in  relative  groups  saline  treated no  ( F ( l , 3 0 ) = 0.16, without  p  >  0.1).  significant  preferences.  p r o d u c e d a s u b s t a n t i a l (65.2%)  i n both s a l i n e p  and  amphetamine  < 0.005) ( F i g u r e  6).  i n s a l i n e p r e - t r e a t e d r a t s were o b s e r v e d from r a t s i n the  extensive  handling  experiment r e l a t i v e  experiments, or t o the  the  to the  influence  other  experiments;  r a t s r e c e i v e d i n the l o c o m o t o r and  Amphetamine t r e a t m e n t s had  somatostatin  levels  no  significant  v e h i c l e p r e t r e a t e d group  to  this  may  place  stereotypy  (possibly stressful)  injections.  i n the  a  Planned  T h e r e was  was  from  conditioned  < 0.025), t h e  ( F ( l , 1 6 ) = 34.4,  levels  but  < 0.05).  cysteamine pretreatment  groups  Analysis  amphetamine-treated  interaction evident  somatostatin  Somatostatin  be  p  amphetamine c o n d i t i o n e d p l a c e  d e p l e t i o n of  higher  p  ( F ( l , 3 0 ) = 0.54,  Cysteamine pretreatment  be  5.12,  i n time spent  ( F ( l , 3 0 ) = 5.63,  cysteamine X t e s t  effect  (F(l,30) =  increase  groups d i d not  5.  amphetamine, i n c r e a s i n g t h e  indicated that while  an  amphetamine-  change i n p l a c e p r e f e r e n c e  a m p h e t a m i n e - a s s o c i a t e d compartment on  to the  on  of  repeated  effect  (F(l,16)  on =  24 1.27,  p > 0.1),  treatments  nor d i d i t i n t e r a c t  ( F ( 1 , 1 6 ) = 2.58,  p >  s i g n i f i c a n t l y with  cysteamine  0.1).  DISCUSSION  The  present results  indicate  that  d e p l e t i o n s produced with cysteamine a t t e n u a t i o n o f t h e motor e f f e c t s al.  (1984) have p r e v i o u s l y  injections  of cysteamine  reduces spontaneous  s t r i a t a l somatostatin  are associated with  o f dopamine a g o n i s t s .  reported  that  after  locomotor a c t i v i t y .  during habituation  cysteamine.  I n agreement w i t h  also  That t h i s  (Figure  1).  this,  reduced  Amphetamine  a t t e n u a t e d by t h i s  treatment 4  latter  is a  attenuation  result  o f a m o d u l a t i o n o f t h e a c t i o n s o f c e n t r a l dopamine a c t i v i t y , not merely a r e f l e c t i o n  et  4 hours b e f o r e b e h a v i o u r a l t e s t i n g  i n d u c e d m o t o r s t i m u l a t i o n was hours  Vecsei  intraventricular  cysteamine g i v e n subcutaneously 3 hours b e f o r e t e s t i n g locomotor a c t i v i t y  an  of lower b a s a l  activity  and  levels, i s  s u p p o r t e d by a s t u d y p e r f o r m e d  i n c o l l a b o r a t i o n with Drs. M a r t i n -  I v e r s o n and V i n c e n t .  study  1986)  rats  receiving  with cysteamine activity induced  In t h i s 11 h o u r ,  i n s t e a d of 3 hour,  (100 mg/kg, s . c . )  pretreatment  showed no change i n h a b i t u a t i o n  b u t d i d show a s i g n i f i c a n t locomotor a c t i v i t y .  (Martin-Iverson et a l . ,  reduction  Furthermore,  i n amphetamine-  intra-accumbens  25  o n us Q  tn  o UJ  z g Q Z  o o UJ  sal+sal  FIGURE 5.  Effect  or cysteamine  sal+amp  sal+cys  o f 12 h o u r p r e t r e a t m e n t  with saline  (100 mg/kg, SC) d u r i n g c o n d i t i o n i n g ,  p l a c e p r e f e r e n c e w i t h s a l i n e o r amphetamine Data  cys+amp  r e p r e s e n t mean v a l u e s ± SEM o f t i m e  s h u t t l e b o x compartment b e f o r e  (1 ml/kg)  on c o n d i t i o n e d  (1.5 mg/kg, I P ) .  spent by each  ( p r e t e s t ) and a f t e r  group i n a  (test) the  conditioning. * indicates  the t e s t  compared t o t h e p r e t e s t  t i m e s were s i g n i f i c a n t l y  t i m e s , p < 0.05.  different  2 6  180 170 -  sal+sal  FIGURE 6.  Effect  sal-i-amp  of four injections  mg/kg, SC) g i v e n o v e r e i g h t d a y s injections,  on t h e s t r i a t a l  radioimmunoassay. immediately  Data  of s a l i n e  T i s s u e samples  levels  were o b t a i n e d  test  o r c y s t e a m i n e (100  to saline  o f no  as measured by from  session of place  r e p r e s e n t mean v a l u e s  * p < 0.05 c o m p a r e d  cys+amp  f o l l o w e d by 3 days  somatostatin  f o l l o w i n g the f i n a l  conditioning.  sal+cys  groups.  rats preference  ± SEM f o r e a c h  group.  27 cysteamine induced  i n j e c t i o n s were a l s o f o u n d  locomotor  a c t i v i t y without  ( M a r t i n - I v e r s o n e t a l . , 1986). somatostatin  mediated  locomotor  habituation after  11  similar  hour, but  not  open f i e l d levels et  results 24  a l . , 1986).  stressful  after  with  ( B e a l and  Martin,  the  1984b;  but  not  initial  4  reduce  somatostatin Martin-Iverson  reflect  a  changes i n  somatostatin,  apomorphine  (Figure 3).  attenuates As  result  and  is  this  cysteamine, the  stereotypy  apomorphine i s a suggests  central  striatum.  that  Martin  direct  somatostatin  levels  finding  (Beal  that  and et a l . ,  In c o n t r a s t w i t h  that somatostatin  to  cysteamine  1984b; M a r t i n - I v e r s o n  suggestion.  the o b s e r v a t i o n s  The  a d m i n i s t r a t i o n of  dopamine o r dopamine m e t a b o l i t e  Martin,1984a; Beal  induced  o f dopamine a c t i v i t y p o s t - s y n a p t i c  t e r m i n a l s i n the  n e i t h e r p e r i p h e r a l nor  consistent with  amphetamine  p e r i p h e r a l a d m i n i s t r a t i o n of  striatal  dopamine-releasing  are  cysteamine to  e a r l y change i n a c t i v i t y may  modulate the e f f e c t s  results  reduction i n  even though cysteamine reduces  dopamine r e c e p t o r a g o n i s t , t h i s  altered  intra-accumbens  levels.  which reduces  may  or  that  by V e c s e i e t a l . (1984) s h o w i n g  associated with  activity,  p r o d u c e d by  indicate  3 hour p r e t r e a t m e n t  In a d d i t i o n to cysteamine a l t e r i n g locomotor  habituation activity  ( M a r t i n - I v e r s o n e t al.,1986) i s  observed  hours  The  factor  somatostatin  72  amphetamine-  a r e d u c t i o n i n dopamine  observed  hour, pretreatment  activity,  f o r over  The  ( F i g u r e 1)  hour p r e t r e a t m e n t to the  systemic  correlate with  activity.  activity  altering  These r e s u l t s  r e d u c t i o n s by e i t h e r  cysteamine i n j e c t i o n s  to attenuate  1986)  these  infusions alter  28  striatal  dopamine t u r n o v e r  et.al.,1978)  and  ( B e a l and M a r t i n , 1 9 8 4 a ;  Garcia-Sevilla  r e l e a s e ( C h e s s e l e t and R e i s i n e , 1 9 8 3 ) ,  and t h a t  h a l o p e r i d o l c a n b l o c k t h e motor e f f e c t s  of somatostatin  et  that i n addition to  a l . , 1983b).  somatostatin  These s t u d i e s suggests  (Vecsei  a c t i n g p o s t s y n a p t i c t o dopamine t e r m i n a l s , t h e r e may  a l s o be a f e e d b a c k mechanism t o t h e dopamine t e r m i n a l s when striatal  somatostatin  cells  are active.  While cysteamine treatment  attenuated  dopamine a g o n i s t s , t h e r e w a r d i n g  t h e motor e f f e c t s o f  a c t i o n o f amphetamine, a s  r e v e a l e d by t h e c o n d i t i o n e d p l a c e p r e f e r e n c e p r o c e d u r e , reduced.  This finding  motor and r e i n f o r c i n g  supports  h a d no e f f e c t  amphetamine somatostatin on  (DiScala et.al.,1985;  Martin-Iverson  on p l a c e p r e f e r e n c e  (DiScali cells  (SL 76002) a t t e n u a t e d  et.al.,1985)  locomotor  For activity,  c o n d i t i o n i n g i n d u c e d by  suggesting  GABA a n d  i n t h e b a s a l g a n g l i a may have o p p o s i t e  t h e motor s t i m u l a n t p r o p e r t i e s o f dopaminergic  neither  that the  M i t h a n i e t a l . , 1987; Radke e t a l . , 1 9 8 7 a ) .  example, a GABA a g o n i s t but  p r e v i o u s work s u g g e s t i n g  a c t i o n s o f dopamine a g o n i s t s a r e  independent o f each other et.al.,1985;  was n o t  a p p e a r s t o be e s s e n t i a l  actions  drugs, but  f o r the rewarding  p r o p e r t i e s of  amphetamine. While both and  reinforcement  1986; be  dopamine-mediated a c t i v i t y  ( K e l l y e t a l . , 1977)  ( C o r b e t t a n d W i s e , 1984; F i b i g e r  S p y r a k i e t a l . , 1983; T a y l o r a n d R o b b i n s ,  d e p e n d e n t on t h e m e s o l i m b i c  differentiation  of these  dopamine t r a c t ,  two b e h a v i o u r s  by d r u g  and P h i l l i p s ,  1984) a p p e a r t o the observed manipulations  29 indicates  f u r t h e r work on  this  a s p e c t o f dopamine f u n c t i o n i s  essential. It  i s interesting  d e p l e t e d more t h a n  65%  cysteamine  treatments.  depletions  of c e n t r a l  of  cysteamine  et.al.,1975; and  t o note  i n the p r e s e n t Previous  results  has  but  not  cell  M a r t i n 1984b; Brown  Sagar e t . a l . , 1 9 8 2 ;  the  somatostatin  i s supported  bodies.  Strikant be  i n only  by  immunohistochemical  study,  nucleus  and  the c e n t r a l  cysteamine  s o m a t o s t a t i n but  3 for detailed  effect  on  somatostatin-28 bodies  has  more  somatostatin-14.  a useful pharmacological  a c t i o n s of somatostatin. been used  been used  mostly  levels  agent  Prior  with  to  to study p e r i p h e r a l  to study c e n t r a l  seizures (Higuchi et.al.,1983;  somatostatin see  experiment  discussion).  i s apparent  s o m a t o s t a t i n may  has  i t has  changes i n k i n d l i n g  accumbens t e r m i n a l  t h e t e r m i n a l s h a v e more t o be  depleted  A s t u d y by B a k h i t e t a l . , (1983)  t o have no  Cysteamine appears which to study  from  somatostatin pool i n c e l l  somatostatin-28,  not  injections  s u s c e p t i b l e t o the a c t i o n s of  immunoreactivity  shown c y s t e a m i n e  It  single  t h a t s o m a t o s t a t i n may  compartmentalized;  This hypothesis  suggesting the  this  by  that  ( M a r t i n - I v e r s o n e t al.,1986) i n which cysteamine  somatostatin fields,  ( B e a l and  T h i s suggests  c e r t a i n compartments b e i n g  repeated  r e p o r t s have a l s o n o t e d  P a l k o v i t s et.al.,1982;  differentially  never  study even w i t h  s o m a t o s t a t i n produced  are not complete  Patel,1984).  cysteamine.  t h a t s o m a t o s t a t i n was  from  the p r e s e n t  play a role  the r e i n f o r c i n g  study using cysteamine,  i n modulating  t h e motor e f f e c t s ,  a c t i o n s , o f dopamine a g o n i s t s .  that but  Furthermore,  30  it  is likely  that t h i s modulation  dopamine-releasing depletions  reduce  occurs postsynaptic to the  terminals i n the striatum, s i n c e t h e motor e f f e c t s  of both  indirect  somatostatin and  a c t i n g dopamine r e c e p t o r a g o n i s t s , w h i l e n o t a p p e a r i n g dopamine m e t a b o l i s m . striatal  To i n v e s t i g a t e  somatostatin c e l l s  afferents,  the e f f e c t s  l e v e l s was  investigated  further  direct  to a l t e r  the p o s s i b i l i t y of  b e i n g u n d e r t h e i n f l u e n c e o f dopamine  o f c h r o n i c h a l o p e r i d o l on (experiment 2 ) .  somatostatin  31  EXPERIMENT 2 .  THE EFFECTS OF HALOPERIDOL ON SOMATOSTATIN IMMUNOREACTIVITY.  INTRODUCTION As  mentioned i n the d i s c u s s i o n o f experiment  growing evidence t h a t striatum  somatostatin  neurons i n t h e  have t h e i r m a j o r p h y s i o l o g i c a l a c t i o n s  dopamine t e r m i n a l s .  Therefore  effects  prescribed  affect  containing  of c l i n i c a l l y dopaminergic  postsynaptic  i t was o f i n t e r e s t  s y s t e m s , on s o m a t o s t a t i n  dopamine  have e x a m i n e d t h e e f f e c t s  t r e a t m e n t on n o n - d o p a m i n e r g i c s y s t e m s , GABA  t o examine t h e  levels.  w h i c h i s commonly u s e d a s an a n t i s c h i z o p h r e n i c studies  to  d r u g s , w h i c h have b e e n shown t o  important drug i s h a l o p e r i d o l , a d i r e c t  Several  1, t h e r e i s  One  antagonist,  drug. of neuroleptic  i n c l u d i n g those  containing  (Gunne a n d HaggstrOm, 1 9 8 3 ) , s u b s t a n c e P (Hanson e t a l . ,  1980), n e u r o t e n s i n 1983), o p i o i d s (Beal  (Govoni e t a l . , 1980), c h o l e c y s t o k i n i n  (Hong e t a l . , , 1 9 7 8 ) , a s w e l l  and M a r t i n ,  1984c).  These s t u d i e s  as  (Frey,  somatostatin  are useful f o r a better  u n d e r s t a n d i n g o f dopamine i n t e r a c t i o n s w i t h o t h e r  transmitter  s y s t e m s , b u t t h e y a l l s h a r e a common p r o b l e m , t h e l e n g t h o f treatment. for of  In the c l i n i c a l  s e v e r a l months o r y e a r s , a short  long  duration,  term treatment  insight  lasting  setting,  neuroleptic  as w e l l  lasts  whereas t h e s e s t u d i e s  are generally  three  Examination of  o r f o u r weeks.  i n animals would t h e r e f o r e  i n t o t h e n e u r o n a l mechanisms i n v o l v e d  treatment  treatment  provide  a better  i n maintenance of  as i n t h e development o f t h e d y s k i n e t i c  32 movements o f t e n and  associated  with  long  term n e u r o l e p t i c  treatment  withdrawal. The  purpose of the f o l l o w i n g  s e t o f e x p e r i m e n t s was t o  examine a n d compare t h e e f f e c t s o f h a l o p e r i d o l three  weeks  levels  (short  term) and e i g h t months  of somatostatin  immunoreactivity  Furthermore, the e f f e c t of withdrawal after  long  term a d m i n i s t r a t i o n  (long  treatment  f o r both  term) on t h e  i n the r a t b r a i n .  (2 months) o f h a l o p e r i d o l  were a l s o  examined.  METHODS AND MATERIALS  Subjects. Male W i s t a r u n d e r a 12 h o u r chow p r o v i d e d  rats  (Woodlyn) were g r o u p h o u s e d 4 p e r c a g e  light/dark cycle with  ad l i b i t u m .  standard  For the long  laboratory r a t  term s t u d i e s  r a t s began t h e experiment a t a young age, w h i l e  (8 m o n t h s ) ,  i n the short  term  e x p e r i m e n t , a g e d r a t s were u s e d t o c o n t r o l f o r t h e e f f e c t s o f a g i n g on n e u r o c h e m i s t r y .  Drugs. Haloperidol solution  b a s e was d i s s o l v e d  (10 mg/ml) a n d s t o r e d  s o l u t i o n s were made m o n t h l y .  i n a 0.5% l a c t i c  a t 4°C i n t h e d a r k . Aliquots  d i l u t e d w i t h d i s t i l l e d water and g i v e n water. rats  B a s e d on a p i l o t  of the stock  acid Fresh  stock  s o l u t i o n were  t o t h e animals as d r i n k i n g  s t u d y p e r f o r m e d b y A. J . MacLennan, t h e  consumed 1.3-1.5 mg o f h a l o p e r i d o l / k g / d a y .  33  Procedure. This (short  term t r e a t m e n t ) ,  supplied vehicle rats  study consisted  in their solution  received  by  d r i n k i n g water (n=12).  b e t w e e n 1:00  sample d o r s a l olfactory  the  to the  tubercle,  boiling  1,  substantia  the  immunoreactivity  long  treatment),  or  pm.  (n=8).  term treatment)  vehicle  Their  (n=6), by  brains  of  nigra, cut  and on  the  cold  0.1  N HCl  N acetic acid.  c o l d 0.1  N HCl  was  followed  cervical  compared t o p r e p a r i n g  cortical  cortex,  ventral  tegmental  microtome,  described  in  used i n s t e a d  Preliminary  d i d not  rats  were removed  a freezing  f o r r a d i o i m m u n o a s s a y as  except that  use  term  medial p r e f r o n t a l  from s e c t i o n s  s a m p l e s i n 2.0  revealed  (long  (n=10) o r v e h i c l e  (n=8)  3:00  striatum,  samples p r o c e s s e d  experiment  2b  n u c l e u s accumbens, c a u d a t e - p u t a m e n , a  a r e a were d i s s e c t e d and  haloperidol  a n i m a l s were s a c r i f i c e d  and  2a  (1.3-1.5 mg/kg/day) (n=12) o r  haloperidol  The  In experiment  3 weeks o f  In experiment  haloperidol  2 months o f w a t e r .  samples of  experiments.  (withdrawal f o l l o w i n g  8 months o f  dislocation and  2c  three  r a t s were g i v e n  8 months o f  In experiment received  of  effect  of  studies somatostatin  tissue with  2.0  N  acetic  acid.  Statistics. D a t a was  e x p r e s s e d as  i m m u n o r e a c t i v i t y per values and  f o r each b r a i n  analysis  individual  picograms of  milligram region  within  between t r e a t m e n t  brain  region  was  t i s s u e wet  and  somatostatin weight.  e a c h g r o u p were  Mean  S.E.M)  calculated  c o n t r o l groups f o r  performed using  (±  Student's  each t-test.  34  RESULTS As  shown i n F i g u r e  7,  3 week t r e a t m e n t w i t h  haloperidol C  significantly accumbens  reduced somatostatin  (t(22)=2.55; p  <0.005), and  the  l e v e l s i n the  <0.025) c a u d a t e - p u t a m e n  v e n t r a l tegmental area  Long t e r m t r e a t m e n t however o n l y in  the  other  n u c l e u s accumbens regions  controls  8).  being  Following  h a l o p e r i d o l treatment  f r o m c o n t r o l l e v e l s i n any  of  (t(22)=2.34; p  <0.05) w i t h  significantly  the  levels regions  p  <0.05). levels  levels in a l l  d i f f e r e n t from  w i t h d r a w a l from  somatostatin  (t(22)=3.46;  reduced somatostatin  (t(16)=2.2; p  examined not  (Figure  nucleus  long  term  were n o t d i f f e r e n t examined  (Figure  8).  DISCUSSION The  e f f i c a c y of  c o r r e l a t e d w i t h the dopamine r e c e p t o r s effects  of  theory  antagonistic (Creese et  antipsychotics  animal studies insight  neuroleptic  potency of  a l . , 1976).  require  be  these drugs The  therapeutic  mechanisms i n v o l v e d  continuous h a l o p e r i d o l  plausible  W h i t e and  explanation,  neuroleptics  can  Wang, 1 9 8 3 ) .  recent  studies  and  have begun t o p r o v i d e i n t h i s delay. treatment  A  Although t h i s have shown  a f f e c t s e v e r a l non-dopaminergic  some  popular  results in a  d e v e l o p m e n t o f d e p o l a r i z a t i o n i n a c t i v a t i o n o f dopamine (Bunney, 1984;  on  s e v e r a l days t o d e v e l o p ,  involving neuroleptics  i n t o the i s that  treatment appears to  cells  is a  that systems  slow  35  300  -r  280 260 x:  CTX  FIGURE 7 . ( 22  ) or  central  E f f e c t of  NAS OT  short  haloperidol  ( £3  NAS  term  mPFC  (1.3-1.5  )  The  sample d o r s a l  = nucleus  brain  regions  to the  OT  VTA  mg/kg/day) on as measured  the  examined i n c l u d e :  striatum,  C-P  tegmental area.  SN  = substantia  = caudate  Data represents  nigra,  mean v a l u e s  group. < 0.05  compared t o c o n t r o l  group.  and  ± SEM  vehicle l e v e l s of  by CTX  VTA for  =  putamen,  accumbens s e p t i , mPFC = m e d i a l p r e f r o n t a l  = olfactory tubercle,  *.p  SN  ( 3 weeks) t r e a t m e n t w i t h  somatostatin immunoreactivity  radioimmunoassay. cortical  C-P  cortex, =  ventral  each  36  JZ  v  cn  \  E cn cI  o o  mPFC  FIGURE 8. ( J72  E f f e c t of  ) or  long  haloperidol  (£51)  of w i t h d r a w a l from long ( ggg  haloperidol  ) on  immunoreactivity regions  C-P  (6* months) t r e a t m e n t w i t h  (1.3-1.5 mg/kg/day) and  term treatment w i t h v e h i c l e  the  l e v e l s of  as m e a s u r e d by  examined i n c l u d e :  striatum,  term  CTX  nigra,  represents  mean v a l u e s < 0.05  and  VTA  OT  ( ^  The  sample d o r s a l  brain to  = olfactory tubercle,  f o r each  compared t o a p p r o p r i a t e  effects ) or  = n u c l e u s accumbens  = v e n t r a l tegmental area.  + SEM  the  vehicle  somatostatin  radioimmunoassay.  = cortical  mPFC = m e d i a l p r e f r o n t a l c o r t e x ,  * p  central  = c a u d a t e putamen, NAS  substantia  VTA  Data  group. control  group.  the septi, SN  =  37 including  t h o s e c o n t a i n i n g GABA  substance  P  (Hanson e t a l . ,  (Hong e t a l . ,  1978),  cholecystokinin  (CCK)  c h a n g e s may  a l s o be  chronic  the neurochemical  the l e v e l s The  1987c).  showed t h a t  s h o r t term  significantly  reduced  term h a l o p e r i d o l  understanding  A slight  tegmental  treatment.  s o m a t o s t a t i n neurons  administration  interact  treatment  of  gave r a t s  on  i n the  decrease  a r e a was  also  These r e s u l t s 1,  found  after  support the  t h a t dopamine  the a c t i v a t i o n  of s o m a t o s t a t i n neurons,  enhanced a f t e r  haloperidol  d e c r e a s e d dopamine a c t i v i t y ,  treatment  idea and  may  which i n t u r n  somatostatin that  short  Therefore a  decrease  p o s s i b i l i t y w o u l d be  the  i n somatostatin  i n dopamine r e c e p t o r a c t i v a t i o n by h a l o p e r i d o l  Another  i n the  caudate-putamen  i n the b a s a l g a n g l i a .  e x p l a i n the r e d u c t i o n i n s t r i a t a l  rats  haloperidol  decrease  is  of  treatment,  T h i s agrees with  (1984c) who  i n the d i s c u s s i o n of experiment  observed.  of  somatostatin levels  found decreases  accumbens.  i n the v e n t r a l  oral  caudate-putamen.  f o r 3 weeks and  the nucleus  could  These  the  changes o c c u r r i n g d u r i n g n e u r o l e p t i c  o b s e r v a t i o n s o f B e a l and M a r t i n  stated  and  i n the s i d e e f f e c t s  To p r o v i d e a more c o m p l e t e  n u c l e u s accumbens and  levels  1987c),  o f s o m a t o s t a t i n were e x a m i n e d .  with haloperidol  and  and  opioids  1984c),  i n the delayed onset of  of v a r i o u s lengths of h a l o p e r i d o l  results  injections  Radke e t a l . , Radke e t a l . ,  of neuroleptics,  treatment.  the e f f e c t s  1980;  important  1983),  Radke e t a l . , 1 9 8 7 b ) ,  ( F r e y , 1983;  (Govoni e t a l . ,  effects  1080;  s o m a t o s t a t i n ( B e a l and M a r t i n ,  neurotensin  clinical  (Gunne and HaggstrOm,  levels  somatostatin release  t o compensate f o r t h e  thereby r e d u c i n g the  measurable  38 levels  of s o m a t o s t a t i n .  very speculative h a l o p e r i d o l on  and more d e t a i l e d  mechanisms i n v o l v e d contrast  are r e q u i r e d  ventral  t o the s h o r t term  suggest  study,  understand  the  levels.  i n the  treatment  to control  the a l t e r a t i o n s  i n dopamine r e c e p t o r b l o c k a d e .  In  but not a f t e r  l o n g term  summary, t h e e f f e c t s  on s o m a t o s t a t i n l e v e l s previous  studies.  treatment  which are a l t e r e d  i n the  levels  following  f o l l o w i n g withdrawal  treatment  significant  provide  further  haloperidol  with  be  long term  treatment  l o n g term  of the b r a i n evidence  important  from  remained  l o n g term  r e g i o n s examined.  f o r an  reduced.  haloperidol  These  were  results  i n t e r a c t i o n o f dopamine w i t h and  suggest  i n the c l i n i c a l  neuroleptic  with  haloperidol  changes i n s o m a t o s t a t i n l e v e l s  s o m a t o s t a t i n i n the b a s a l g a n g l i a , s y s t e m s may  s h o r t term  r e t u r n to c o n t r o l values except i n  Furthermore,  i n any  P  treatment.  of s h o r t term  In c o n t r a s t ,  time  substance  after  t h e n u c l e u s accumbens where s o m a t o s t a t i n l e v e l s  detected  over  i n the b a s a l g a n g l i a are c o n s i s t e n t  these a l t e r e d  no  These  Similar  c h a n g e s have a l s o b e e n o b s e r v e d w i t h n e u r o t e n s i n and  treatment  and  levels.  s o m a t o s t a t i n c o n t a i n i n g neurons  (Radke e t a l . , 1987b: 1 9 8 7 c ) ,  (8  nucleus  c a u d a t e - p u t a m e n , b u t n o t t h e n u c l e u s accumbens, a d a p t to  of  or  i n the caudate-putamen  a r e a were s i m i l a r that  this is  of the e f f e c t s  l o n g term  somatostatin levels  Somatostatin levels  tegmental  findings  to f u l l y  i n reducing somatostatin  months) o n l y d e c r e a s e d accumbens.  examination  that  s o m a t o s t a t i n s y n t h e s i s (mRNA l e v e l s )  somatostatin release  In  I t s h o u l d be m e n t i o n e d  treatment.  that  somatostatin  side effects  associated  39  EXPERIMENT 3.  THE E F F E C T S OF MPTP ON IMMUNOREACTIVITY  SOMATOSTATIN  I N THE MOUSE.  INTRODUCTION L e s i o n s o f dopamine t r a c t s b y 6-hydroxydopamine have b e e n shown t o a l t e r GABA, a c e t y l c h o l i n e 1983),  (Kim, 1 9 7 3 ) ,  and s u b s t a n c e  not appear  several transmitter  to alter  and M a r t i n , 1 9 8 3 ) .  in striatal  term h a l o p e r i d o l  treatment  finding  treatment  observations levels 1983;  i n the r a t (Beal  c o n f l i c t s with the following  involving  long  3 ) , as w e l l  ( A g i d and J a v o y - A g i d ,  e t a l . , 1984).  short  experiment  term  as w i t h  i n P a r k i n s o n ' s d i s e a s e where s t r i a t a l  a r e normal Rinne  (Chang e t a l . ,  ( B e a l and M a r t i n , 1984c;  (experiment  including  6-OHDA l e s i o n s do  somatostatin levels  but i s c o n s i s t e n t with studies  neuroleptic  be  cholecystokinin  somatostatin levels  This latter  changes o b s e r v e d  3),  systems,  P (Hanson e t a l . , 1 9 8 1 ) . striatal  (6-OHDA)  somatostatin  1985; E p e l b a u m e t a l . ,  This suggests  that  6-OHDA l e s i o n s  may  a u s e f u l model f o r P a r k i n s o n ' s d i s e a s e , w i t h r e s p e c t t o non-  dopaminergic  a s p e c t s o f t h i s d i s e a s e and t h e i r  implications f o r  treatment. Recently,  t h e n e u r o t o x i n MPTP  (1-methyl-4-phenyl-1,2,3,6-  t e t r a h y d r o p y r i d i n e ) h a s b e e n shown t o c a u s e P a r k i n s o n - l i k e symptoms i n humans  ( L a n g s t o n e t a l . , 1983) and h a s b e e n u s e d  model o f t h i s  d i s e a s e i n both primates  mice  e t a l . , 1984; H e i k i l l a  (Hallman  b e e n shown t o a f f e c t various neuropeptides  (Burns e t a l . ,  et al.,  s e v e r a l non-dopaminergic  1983). systems,  ( A l l e n e t a l . , 1986; Zamir  as a  1983) and MPTP h a s including  et al.,  1984).  40 Since  several  non-dopaminergic  systems a r e a l t e r e d i n  P a r k i n s o n ' s d i s e a s e ( A g i d and J a v o y - A g i d , 1983; et  Rinne  e t a l . , 1984),  al.,1983;  poisoning  Hanson e t a l . , 1 9 8 1 ;  two  found  drugs The  a s w i t h 6-OHDA l e s i o n s  Kim e t a l . ,  ( A l l e n e t a l . , 1986; Zamir  of t h e nondopaminergic those  as w e l l  1985; E p e l b a u m e t a l . ,  et al.,  (Chang  1973) a n d MPTP 1984),  a  comparison  c h a n g e s a f t e r MPTP a n d 6-OHDA l e s i o n s  with  i n P a r k i n s o n ' s d i s e a s e may e s t a b l i s h w h i c h o f t h e s e  produces purpose  a more a c c u r a t e model o f P a r k i n s o n ' s d i s e a s e .  of the following  e f f e c t s o f MPTP a d m i n i s t r a t i o n  experiment  i n C57 m i c e o n t h e l e v e l s o f  somatostatin immunoreactivity t o determine a c c u r a t e model o f t h e n e u r o c h e m i c a l Parkinsonism.  This study a l s o  was t o examine t h e  i f MPTP p r o d u c e s a n  changes o b s e r v e d i n  further  examined t h e i n t e r a c t i o n s  between dopamine a n d s o m a t o s t a t i n i n t h e b r a i n .  METHODS AND MATERIALS Subjects. M a l e C57 m i c e group  housed  (Charles Rivers) weighing  20-25 grams were  (5-6/cage) i n p o l y p r o p y l e n e cages  and m a i n t a i n e d  w i t h i n a fume h o o d f o r t h e d u r a t i o n o f t h e e x p e r i m e n t .  Food and  w a t e r were p r o v i d e d a d l i b i t u m .  Drugs. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) ( R e s e a r c h B i o c h e m i c a l s I n c . ) was d i s s o l v e d  i n saline at  c o n c e n t r a t i o n s o f 40 mg/ml, a n d 50 mg/ml a n d i n j e c t e d i . p . , (l.Oml/lOOg).  41  Procedure. C57 m a l e m i c e were r a n d o m l y a s s i g n e d a c o n t r o l group  (n=7) r e c e i v i n g a d a i l y  ml/100 g) f o r two d a y s , a s e c o n d g r o u p  (n=7) r e c e i v i n g two  o f 40 mg/kg MPTP, a n d a t h i r d  injections  o f 50 mg/kg MPTP.  injection,  a n i m a l s were s a c r i f i c e d  the  cortex,  dissected  (n=6) g i v e n two days a f t e r  the l a s t  by d e c a p i t a t i o n and samples o f  c a u d a t e - p u t a m e n , a n d t h e s u b s t a n t i a n i g r a were  on i c e , a n d p r e p a r e d  i n experiment  1.  were p r o c e s s e d  f o r radioimmunoassay  as d e s c r i b e d  I n a d d i t i o n , samples o f t h e caudate-putamen  f o r catecholamine  performance l i q u i d detection  groups;  i n j e c t i o n o f s a l i n e (1.0  injections  Fourteen  t o one o f t h r e e  chromatography  levels  using  (HPLC) w i t h  reverse  phase  high  electrochemical  (Cumming e t a l . , 1 9 8 6 ) .  RESULTS As  shown i n F i g u r e  reductions  in striatal  9, MPTP p o i s o n i n g dopamine l e v e l s .  resulted i n significant F o r t h e 2x40 mg/kg  g r o u p , t h e r e d u c t i o n was 75 %, a n d t h e r e d u c t i o n w i t h mg/kg g r o u p was 77 %. homovanillic  acid  There  (HVA),  serotonin  Figure somatostatin  acid  b u t no c h a n g e s were o b s e r v e d i n  levels.  10 i l l u s t r a t e s levels  in striatal  3,4-dihydroxy-phenyl-acetic  (DOPAC), a n d n o r e p i n e p h r i n e , striatal  were a l s o d e c r e a s e s  t h e 2x50  the e f f e c t s  i n various  immunoreactivity  i n the cortex  MPTP p o i s o n i n g .  Instead,  o f MPTP p o i s o n i n g  brain regions.  on t h e  Somatostatin  a n d s t r i a t u m was n o t c h a n g e d b y  t h e r e was a s i g n i f i c a n t  increase i n  42 somatostatin  levels  i n the s u b s t a n t i a n i g r a  d o s e d e p e n d e n t , w i t h t h e 2x40 g r o u p of  56  % from c o n t r o l v a l u e s  2x50 g r o u p  %  appeared  to  be  having a s i g n i f i c a n t increase  ( t ( 1 2 ) = 3.33,  an i n c r e a s e o f 72  that  (t(ll)  p < 0.025),  = 3.48,  and  the  p < 0.025).  DISCUSSION  Recent brain  observations of a l t e r e d  tissues  Huntington's  chorea  ( A r o n i n e t a l . , 1983; Sagar  ( A g i d and  Javoy-Agid,  al.,1984)  have i l l u s t r a t e d To d e v e l o p  neurochemical  1985;  e t al.,1984)  Beal et a l . , and  the importance  Rinne  Agid,  an a c c u r a t e a n i m a l m o d e l o f a d i s e a s e , t h e be  secondary  t o the  neurochemical  changes found  levels  1983;  are reduced  Rinne  1985;  Epelbaum e t a l . ,  Rinne  In c o n t r a s t with these f i n d i n g s , poisoning  to increase n i g r a l  catecholamines c o r t e x and  decreased.  ( A g i d and  i n the f r o n t a l c o r t e x  1983;  our  JavoyHowever,  and  ( A g i d and  Javoy-  et al.,1984). s t u d y has  somatostatin levels  The  changes i n  et al.,1984).  h i p p o c a m p u s o f demented P a r k i n s o n i a n p a t i e n t s Agid,  with  i n the d i s e a s e .  have b e e n o b s e r v e d  Epelbaum e t a l . ,  somatostatin  believed  d y s f u n c t i o n , s h o u l d a l s o be c o n s i s t e n t  somatostatin levels 1985;  et  of neuropeptides i n  I n p o s t - m o r t e m s t u d i e s o f P a r k i n s o n i a n b r a i n s no nigral  1984;  Parkinson's disease  E p e l b a u m e t a l . , 1983;  a l t e r a t i o n s w h i c h may  major neurochemical the observed  i n post-mortem  f r o m A l z h e i m e r ' s d i s e a s e ( D a v i e s e t a l . , 1980),  N e m e r o f f e t a l . , 1983;  pathology.  peptide levels  shown MPTP  as t h e l e v e l s  l e v e l s of s o m a t o s t a t i n i n the  s t r i a t u m were u n c h a n g e d .  of  43  200 190 180 170 160  FIGURE  -r  -  9.  Effects  of saline  d o s e s o f 2 x 40 mg/kg striatal  catecholamine  expressed  i n t h e mouse.  of control,  g r o u p s b e i n g , dopamine  dihydroxyphenylacetic acid  homovanillic  acid  (Q  ) o r MPTP a t  ) and 2 x 50 mg/kg, I P (  levels  as a p r e c e n t a g e  the c o n t r o l weight,  (  ( 2 x 1 ml/lOOg)  )  o  The r e s u l t s a r e  with the absolute values of  (DA) = 11,145  ± 324 n g / g wet  (DOPAC) = 1,323 ± 59 n g / g ,  (HVA) = 1,812 ± 137 n g / g , n o r e p i n e p h r i n e  337 ± 69 n g / g , s e r o t o n i n (5-HT) = 480 ± 15 n g / g . * p < 0.05 compared t o c o n t r o l  n  group.  (NE) =  44  100  -r  90 -  CTX  FIGURE 10.  Effects  of s a l i n e  d o s e s o f 2 x 40 mg/kg levels  of central  radioimmunoassay. sample  SN  C - P  ( 2 x 1 ml/lOOg)  ( JZ2 >  o  r  M  P  T  P  a  t  ( £\] ) and 2 x 50 mg/kg, SC ( jggj ) on t h e  somatostatin  immunoreactivity  B r a i n r e g i o n s examined  a s m e a s u r e d by  i n c l u d e ; CTX =  cortical  d o r s a l t o t h e s t r i a t u m , C-P = c a u d a t e putamen, SN =  substantia nigra.  D a t a r e p r e s e n t s mean v a l u e s  group. * p < 0.05 compared t o c o n t r o l  group.  ± SEM f o r e a c h  45 These r e s u l t s  are i n p a r t i a l  studies  i n primates.  (1984),  found  levels  Both A l l e n e t al.(1986)  MPTP t o have no e f f e c t  MPTP  and Zamir e t a l .  on n i g r a l  somatostatin  i n primates.  The using  disagreement with previous  present  r e s u l t s w i t h MPTP a r e s i m i l a r  6-OHDA, a n o t h e r  disease. levels  proposed animal  studies  model f o r P a r k i n s o n ' s  6-OHDA l e s i o n s do n o t a f f e c t  striatal  i n t h e r a t ( B e a l and M a r t i n , 1 9 8 3 ) .  however d i d n o t measure n i g r a l  to earlier  somatostatin  B e a l and M a r t i n  somatostatin  levels  study,  s o i t i s p r e s e n t l y unknown w h e t h e r n i g r a l  levels  a r e i n c r e a s e d as w e l l ,  f o l l o w i n g 6-OHDA  (1983)  i n their  somatostatin  lesions.  A p o s s i b l e e x p l a n a t i o n f o r t h e d i s c r e p a n c y between t h e results MPTP  of t h i s  nigral  s y s t e m s i n t h e mouse.  somatostatin  the primate  quite  given  levels  As shown i n F i g u r e 10,  i n t h e mouse were f a i r l y  and r a t , t h e l e v e l s  high,  the  whereas  i n the substantia nigra are  l o w ( s e e F i g u r e 7, e x p e r i m e n t 2 ) . The  levels  reason  why h a l o p e r i d o l d o e s a f f e c t  i n t h e r a t (experiment  n e i t h e r MPTP nor  animals  ( A l l e n e t a l . , 1986; Z a m i r e t a l . , 1984) i s t h e anatomy o f  somatostatin  in  experiment and s t u d i e s i n o t h e r  2; B e a l a n d M a r t i n ,  somatostatin  1984c) b u t  ( F i g u r e 10; A l l e n e t a l . , 1984; Z a m i r e t a l . ,  6-OHDA l e s i o n s  somatostatin  striatal  ( B e a l and M a r t i n ,  levels  1983) a l t e r  striatal  i s p r e s e n t l y unknown b u t may i n d i c a t e  c h a n g e s i n dopamine r e c e p t o r s , b u t n o t dopamine l e v e l s , greater  i n f l u e n c e on s o m a t o s t a t i n  1986)  that  have a  neurons.  I n summary, m i c e g i v e n MPTP show i n c r e a s e s i n n i g r a l somatostatin  levels.  These o b s e r v a t i o n s  are not consistent with  46  either  MPTP s t u d i e s i n p r i m a t e s ,  mortem P a r k i n s o n ' s  brains.  does s u p p o r t  earlier  striatal  these  somatostatin  somatostatin transmitter  levels  or with  f i n d i n g s from  However, t h e p r e s e n t  investigation  s t u d i e s i n s h o w i n g no r e d u c t i o n s i n  levels.  The c h a n g e s o b s e r v e d  f o l l o w i n g MPTP may r e f l e c t  interactions  post-  i n different  i n nigral  differencesi n  species. Further  work  involving  the interactions  different  s p e c i e s may h e l p i n t h e i n t e r p r e t a t i o n o f t h e s e  results.  Finally,  these  o f dopamine a n d s o m a t o s t a t i n i n  results  expand t h e l i s t  catecholaminergic  t r a n s m i t t e r s a f f e c t e d b y MPTP.  u s i n g MPTP s h o u l d  recognize  actions  of this  toxin.  these  o f nonFurther  work  c h a n g e s when e x a m i n i n g t h e  47  EXPERIMENT 4,  THE EFFECTS OF CARBAMAZEPINE AND  DESMETHYLIMIPRAMINE (DMI)  ON SOMATOSTATIN  IMMUNOREACTIVITY.  INTRODUCTION As  shown i n e x p e r i m e n t s  dopaminergic clinical al.,  have b e e n shown t o a l t e r  affect  and  2,  levels  important  manic-depression, by  Post  is  levels One  e t a l . , 1983  unique not  has  and  to t h i s drug  Human s t u d i e s have a l s o  appear t o a l t e r  carbamazepine treatment  of  the  c a r b a m a z e p i n e has central  a l t e r e d by  interest useful  and  illness,  also  i t i s of other  is  i n the  Rubinow, 1 9 8 6 ) .  treatment  e p i l e p s y (see Clinically,  CSF  somatostatin  an o b s e r v a t i o n w h i c h  low  somatostatin  levels  (DMI)  somatostatin  its clinical  somatostatin  and  (Rubinow,  shown a c o r r e l a t i o n between  the dexamethasone s u p p r e s s i o n t e s t  i n f l u e n c e on  In  during  s i n c e desmethylimipramine  1986).  not  levels  and  b e e n shown t o l o w e r  do  or  c a n be  b e e n shown t o be  lithium  from  drug  of p a t i e n t s with a f f e c t i v e  fairly  somatostatin  p a r o x y s m a l p a i n d i s o r d e r s , and  carbamazepine treatment levels  levels.  i n the r a t b r a i n ,  drugs.  c a r b a m a z e p i n e w h i c h has  reviews  somatostatin  n e u r o l e p t i c s were shown t o  i n t e r e s t whether s o m a t o s t a t i n  of  disrupt  n e u r o l e p t i c s ( G a t t a z e t a l . , 1986),  human CSF  In experiment  somatostatin  clinically  drugs which  a n t i d e p r e s s a n t s , a n t i c o n v u l s a n t s (Rubinow e t  Rubinow, 1986)  treatment.  3,  systems appear t o a l t e r  setting  1984;  2 and  levels with  (Rubinow, 1 9 8 6 ) .  efficacy via a  both  escape Whether  direct  s y s t e m s i s p r e s e n t l y unknown.  48 The due  a n t i c o n v u l s a n t p r o p e r t i e s o f c a r b a m a z e p i n e m i g h t a l s o be  to i t s interactions with c e n t r a l  Cortical  somatostatin  epilepsy  ( H i g u c h i e t a l . , 1984;  patients with  levels  intractable  Recently,  Higuchi  decreases  somatostatin  brain of  areas  this peptide.  somatostatin  (Rubinow  levels  K a t o e t a l . , 1 9 8 3 ) , and i n  shown t h a t  i n kindled rats,  carbamazepine but only i n the  1986;  a r e a b n o r m a l , as i n k i n d l e d  Kato e t a l . , 1983), o r Rubinow,  not  was  i n the r a t .  s t u d y was  a l s o examined  appear t o a l t e r  CSF  The  affective  t o determine i f acute basal  a d m i n i s t r a t i o n of acute  as a c o n t r o l g r o u p s i n c e DMI  somatostatin  rats  1986).  or c h r o n i c a d m i n i s t r a t i o n of carbamazepine a l t e r s  c h r o n i c DMI  levels  t h a t carbamazepine might reduce  only i f they  e t a l . , 1984;  levels  of  e p i l e p s y (Nadi e t a l . , 1986).  The p u r p o s e o f t h e p r e s e n t  somatostatin  models  s e i z u r e - a s s o c i a t e d e l e v a t i o n s i n the  ( H i g u c h i e t a l . , 1984; illness  1986;  T h i s suggests  levels  systems.  are i n c r e a s e d i n animal  e t a l . (1986) have  showing  somatostatin  levels  and  does  i n patients with  affective disorders.  METHODS AND MATERIALS Subj e c t s . Male Long Evans were g r o u p h o u s e d with  rats  ( C h a r l e s R i v e r ) w e i g h i n g 250-300 grams  4 p e r cage under  f o o d and w a t e r p r o v i d e d  ad  a 12 h o u r l i g h t / d a r k c y c l e  libitum.  49  Drugs. Carbamazepine  (Sigma) was d i s s o l v e d  (20 mg/ml) a n d i n j e c t e d hydrochloride  i n 70% g l y c e r o l  i . p . (1.0 m l / k g ) .  (DMI) ( M e r r e l  (5.0 mg/ml) a n d i n j e c t e d  solution  Desmethylimipramine  Dow) was d i s s o l v e d  indistilled  water  i . p . (1.0 m l / k g ) .  Procedure. Experiment  4a.  R a t s were r a n d o m l y a s s i g n e d t o one o f f o u r  groups;  1) c o n t r o l  vehicle  ( 7 0 % g l y c e r o l , i . p . ) f o r 12 c o n s e c u t i v e d a y s , 2) a c u t e  group  (n=7) g i v e n  group  (n=8) g i v e n  11 d a y s o f v e h i c l e  by one i n j e c t i o n o f c a r b a m a z e p i n e chronic  group  2 d a i l y injections of  (n=8) r e c e i v i n g  mg/kg), 4) c h r o n i c  injections  their  12 d a y s o f c a r b a m a z e p i n e  withdrawal group  sacrificed.  last  (n=8) r e c e i v i n g  were d i s s e c t e d prepared  groups;  from s e c t i o n s  4b.  1) c o n t r o l  group  DMI  sample  A l l rats  n u c l e u s accumbens,  dorsal  as d e s c r i b e d  (n=9) r e c e i v i n g  (dB^O, i . p . ) ,  to the striatum microtome and  i n e x p e r i m e n t 1.  3) c h r o n i c  12 h o u r s p r i o r  accumbens, c a u d a t e - p u t a m e n ,  f o l l o w e d b y 2 d a y s o f DMI  group  A l l rats  to being  14 d a y s o f two d a i l y  2) a c u t e g r o u p (n=9)  solution  (2 x 5.0 mg/kg, i . p . ) .  injection  injections.  c u t on a f r e e z i n g  12 d a y s o f v e h i c l e  (2 x 5.0 mg/mg, i . p . ) , of  10 d a y s o f  R a t s were r a n d o m l y a s s i g n e d t o one o f t h r e e  injections of vehicle receiving  hypothalamus,  and a c o r t i c a l  f o r radioimmunoassay  Experiment  (2 x 20  i n j e c t i o n 30 m i n u t e s p r i o r t o b e i n g  The h i p p o c a m p u s ,  caudate-putamen,  followed  (20 mg/kg) on d a y 12, 3)  carbamazepine f o l l o w e d by 2 days o f v e h i c l e received  (2/day)  (n=7) r e c e i v i n g received  their  14 d a y s last  s a c r i f i c e d and t h e n u c l e u s  and a c o r t e x  sample  dorsal  tothe  50  s t r i a t u m d i s s e c t e d f r o m s e c t i o n s c u t on a f r e e z i n g m i c r o t o m e and prepared  f o r radioimmunoassay,  a s d e s c r i b e d i n e x p e r i m e n t 1.  RESULTS As  shown i n F i g u r e 11, n e i t h e r a c u t e ,  c h r o n i c , nor  withdrawal f o l l o w i n g c h r o n i c a d m i n i s t r a t i o n of carbamazepine significantly brain  the l e v e l s  r e g i o n s examined,  values. did  changed  Similarly,  not a l t e r  of somatostatin  i n any o f t h e  when compared t o c o n t r o l g r o u p mean  acute,  somatostatin  a s w e l l a s c h r o n i c DMI a d m i n i s t r a t i o n levels  i n any b r a i n r e g i o n  examined  (Figure 12).  DISCUSSION The levels  lack of e f f e c t  o f c a r b a m a z e p i n e on b a s a l  ( F i g u r e 11) w o u l d  agree with  the r e s u l t s  somatostatin  of Higuchi e ta l .  (1986) who showed t h a t i n r e g i o n s o f t h e b r a i n w i t h somatostatin the  levels  somatostatin  treatment.  after  kindling  normal  ( i e . hypothalamus,  striatum)  l e v e l s were u n a f f e c t e d b y c a r b a m a z e p i n e  However, H i g u c h i  e t a l . (1986) f o u n d  kindling-induced elevation i n c o r t i c a l  that the  s o m a t o s t a t i n was  by c a r b a m a z e p i n e u s i n g a s i m i l a r  dosage.  c a r b a m a z e p i n e was w i t h o u t  on b a s a l c o r t i c a l  effect  In the present  reduced study,  somatostatin  51-  N Acc  FIGURE 11. E f f e c t and  withdrawal  carbamazepine somatostatin  C - P  HIPPOC  o f v e h i c l e ( [73  following chronic  ) , acute  (Q  ( £j^J ) t r e a t m e n t  (2 x 25 mg/kg, I P ) on t h e l e v e l s immunoreactivity  ), c h r o n i c with  of c e n t r a l  as measured by radioimmunoassay.  B r a i n r e g i o n s examined i n c l u d e ; N Acc = n u c l e u s C-P = c a u d a t e putamen, CTX = c o r t i c a l  accumbens  sample d o r s a l t o t h e  s t r i a t u m , HIPPOC = h i p p o c a m p u s , HT = h y p o t h a l a m u s . r e p r e s e n t s mean v a l u e s  (ggl)  ± SEM o f e a c h  group.  Data  septi,  ,  52  200 190 180 -  CTX  FIGURE 12. (£22)  DMI  Effects treatment  immunoreactivity  of s a l i n e on  = caudate  ( (7/1  = cortical  putamen, NAS  r e p r e s e n t s mean v a l u e s  ) , acute  l e v e l s of c e n t r a l  as m e a s u r e d by  e x a m i n e d i n c l u d e ; CTX C-P  the  C-P  ( [£J  ) , and  Brain regions  sample d o r s a l t o t h e  of each  accumbens s e p t i . group.  chronic  somatostatin  radioimmunoassay.  = nucleus  + SEM  NAS  striatum, Data  53 levels.  Recently,  N a g a k i e t a l . (1985) f o u n d  a t a d o s e o f 50 mg/kg d a i l y alter  central  slight (200  somatostatin  decreases  carbamazepine  (acute and c h r o n i c treatment) levels,  i n somatostatin  mg/kg) i n t h e c o r t e x  that  however t h e y d i d o b s e r v e  levels with  (acute)  d i d not  a very high  a n d hippocampus  very  dose  (chronic  treatment). C a r b a m a z e p i n e h a s been shown t o a f f e c t catecholamines al.,  (Kowalik  n u c l e o t i d e s , a n d GABA  (Post e t a l . 1983).  i s p o s s i b l e that the carbamazepine-induced  somatostatin  levels  observed  (1986) a r e t h e r e s u l t systems.  by H i g u c h i  e t a l . (1986) a n d Rubinow  coexist i n various regions, including  carbamazepine i n k i n d l e d r a t s  levels  the cortex  o f GABA a r e i n c r e a s e d  ( H i g u c h i e t a l . 1986).  i t i s possible that increases i n c o r t i c a l  associated with  other  m i g h t be GABA, s i n c e GABA a n d  (Hendry e t a l . 1984), and c o r t i c a l  Therefore  Thus,  reductions i n  o f c a r b a m a z e p i n e ' s a c t i o n s on t h e s e  A prime candidate  somatostatin  by  e t a l . , 1985; P u r d y e t  1987; W a l d m e i e r e t a l . 1 9 8 4 ) , a c e t y l c h o l i n e , t h y r o i d  hormones, c y c l i c it  e t a l . 1984; P o s t  central  the s u s c e p t i b i l i t y  of somatostatin  GABA may be cells to  carbamazepine's a c t i o n s . With regard effect  t o DMI, Rubinow  on CSF s o m a t o s t a t i n  In agreement w i t h alterations  this,  i n central  by M a r t i n - I v e r s o n  i n affectively  ill  patients.  r a t s g i v e n DMI d i d n o t show a n y somatostatin  levels  (Figure 12).  A  study  e t a l . (1983) h a s shown DMI t o e n h a n c e dopamine  mediated behaviours, B a s e d on r e s u l t s  levels  (1986) h a s shown DMI t o have no  p o s s i b l y b y a n o n - d o p a m i n e r g i c mechanism.  from experiment  1, s h o w i n g c y s t e a m i n e t o  54 a t t e n u a t e dopamine m e d i a t e d b e h a v i o u r s , DMI  a possible  t o e n h a n c e dopamine m e d i a t e d b e h a v i o u r s  increasing  striatal  somatostatin  experiment  i n d i c a t e DMI  dopamine-mediated behaviours Further  analysis  transmitters  involving  w o u l d be  activity.  i s probably  not  The  having  t h e e f f e c t s o f DMI  1985), s u b s t a n c e  P  (Nemeroff,  may  1986)  l e v e l s i n the  supports  or  DMI  do  on  the  clinical  mechanisms by w h i c h DMI For both  mechanism o f a c t i o n  lack  on  other  i t s e f f e c t s on  drugs,  is clearly  further  problem.  appear t o a l t e r T h i s suggests  abnormal  fails  on  that  somatostatin somatostatin  to e s t a b l i s h  dopamine-mediated  work t o e s t a b l i s h  warranted.  such  neurotensin  o f e f f e c t o f DMI  o b s e r v a t i o n s but has  this  (DiScali et a l . ,  not  rat brain.  a c t i n d i r e c t l y t o reduce  Furthermore,  behaviours.  i t s effects  p r o v i d e some a n s w e r s t o t h i s  summary, c a r b a m a z e p i n e and  c a r b a m a z e p i n e may  levels  ( K e l l e y e t a l . , 1979)  somatostatin  levels.  r e s u l t s of  known t o a f f e c t d o p a m i n e - m e d i a t e d b e h a v i o u r s , (Radke e t a l . , 1 9 8 7 a ) , GABA  basal  by-  v i a a s o m a t o s t a t i n mechanism.  as a c e t y l c h o l i n e  In  mechanism f o r  the  the  55  GENERAL DISCUSSION  Dopamine-somatostatin i n t e r a c t i o n s  The  effects  of  somatostatin  dopamine-mediated behaviours al.,  1986)  suggests  role  i n modulating  the  occurs  Furthermore,  hyperactivity)  and  of both  1986). alter  that  and  infusions into ( B e a l and  somatostatin  R e i s i n e , 1983;  somatostatin supported  can by  t h a t h a l o p e r i d o l can ventricular  infusions  this  block  Starr,  somatostatin  appear to a l t e r  by  the  observations  dopamine  the  striatum  Martin,  1982)  and  K -induced +  suggesting  terminals.  This i s  Since  has  (Starr,  slices,  dopamine a c t i v i t y ,  i t i s possible that somatostatin  do  basal  showing  i n c r e a s e i n motor a c t i v i t y  infusions.  et a l . ,  1984a; G a r c i a -  by V e c s e i e t a l . (1983b)  the  reduce  stereotypy)  enhances both  a c t on d o p a m i n e r g i c a study  terminals i n  cysteamine  striatal  -^-dopamine r e l e a s e f r o m r a t s t r i a t a l  further  not,  and  a c t i o n s , of  1984a; 1984b; M a r t i n - I v e r s o n  dopamine t u r n o v e r  e t a l . , 1978)  (Chesselet 1982)  appear t o a l t e r  Martin,  play a  (amphetamine-induced  (apomorphine-induced  However, s o m a t o s t a t i n  Sevilla  that  d e p l e t i o n s by  et  n e u r o n s may  reinforcing  This i s f u r t h e r supported  ( B e a l and  striatal  the  i t is likely  indirect  direct  c y s t e a m i n e does not  metabolism  not  on  1; M a r t i n - I v e r s o n  postsynaptic to dopamine-releasing  dopamine a g o n i s t s . that  cysteamine  somatostatin  the motor, but  striatum, since somatostatin  t h e motor e f f e c t s  by  (experiment  that s t r i a t a l  dopamine a g o n i s t s . modulation  d e p l e t i o n by  caused  somatostatin but  both  cysteamine  presynaptic  does and  56  postsynaptic presynaptic increased  i n t e r a c t i o n s with somatostatin  somatostatin  dopamine t e r m i n a l s .  a c t i o n s may  activity  be  B a s e d on  the  of  case.  effects  i n somatostatin  interpretation The  control values interesting  after  long of  appears to  s t a t e d by  d y s k i n e s i a may  be  the  GABA n e u r o n s .  be  reduced.  These blockade  s y n t h e s i s or  an  s t u d i e s i n v o l v i n g the  r e l e a s e and/or s y n t h e s i s  levels  of  a more p r e c i s e  i n the  observations  caudate-putamen  of  long  to  term n e u r o l e p t i c  tardive dyskinesia  d y s k i n e s i a has  Gerlach,  stimulate  Another theory  striatal  are  Future the  the  (Casey  and  b e e n s u g g e s t e d t o be  due  s u p e r s e n s i t i v y and/or c h o l i n e r g i c  ( C a s e y and  appear t o enhance o r  affect  term n e u r o l e p t i c t r e a t m e n t i s  the  Tardive  to e i t h e r dopaminergic hypofunction  that  results.  associated with  1984).  had  h a l o p e r i d o l f o r 3 weeks  levels  provide  somatostatin  in light  treatment being Gerlach,  release.  s t r i a t u m may  of these  r e t u r n of  should This  i n somatostatin  o f d o p a m i n e r g i c d r u g s on i n the  expected  i n t e r p r e t e d as dopamine r e c e p t o r  either a reduction  somatostatin  neurons.  treated with  somatostatin  c o u l d be  alter  i f somatostatin  i t w o u l d be  somatostatin  striatal  expected  dopaminergic a c t i v i t y  When a n i m a l s a r e  observations  increase  striatal  during  terminals.  observations  striatal  (Figure 7),  causing  dopamine  these  d i s r u p t i o n of activity  on  only e f f e c t i v e  s i n c e cysteamine does not  dopamine m e t a b o l i s m , w h i c h w o u l d be a tonic effect  The  1984), s i n c e a n t i c h o l i n e r g i c s  tardive dyskinesia  F i b i g e r and  result The  Lloyd  i n humans.  (1984) i s t h a t  of n e u r o l e p t i c - i n d u c e d  damage  tardive to  proposed s y n e r g i s t i c r e l a t i o n s h i p  57 b e t w e e n dopamine and  somatostatin  i n the  striatum i s opposite  the b e l i e v e d i n t e r a c t i o n of dopaminergic acetylcholine  (McGeer e t a l . , 1961;  ( D i S c a l i e t a l . , 1985) somatostatin  systems.  to control  term treatment d y s k i n e s i a by  may  be  levels  related  i n c r e a s i n g the  neurons w i t h  Therefore  i m b a l a n c e between  i n Neuro-psychiatric disease  Alzheimer's  al., and  1985)  d u r i n g s h o r t term  i n t r o d u c t i o n , CSF  (Cramer e t a l . , 1 9 8 1 ) , and  disease, depression  (Agren  1985).  mentally al.,  One is  CSF  Rubinow, 1986)  somatostatin In regard  that  levels  CSF  are unclear  somatostatin  1984;  Rubinow, 1 9 8 6 ) ,  Gerner  and Thai  et  a r e a l s o a l t e r e d when  interpretation.  i s an  (Cramer e t  carbamazepine  (Rubinow e t  or n e u r o l e p t i c s (Gattez e t a l . , 1986).  to a f f e c t i v e  somatostatin  reduced  of  e t a l . , 1986),  Lundqvist,  p r o b l e m w i t h measurements o f p e p t i d e  the d i f f i c u l t y  CSF  somatostatin  levels  including  (Cramer e t a l . , 1985;  i l l p a t i e n t s are t r e a t e d with  1984;  somatostatin  Parkinson's  and  Rubinow e t a l . , 1985;  p a t i e n t s w i t h d y s t o n i c syndromes al.,  treatment.  abnormal i n s e v e r a l n e u r a l d i s e a s e s ,  Yamada, 1982;  long  neurotransmitter  (Gomez e t a l . , 1986a; 1986b; R a s i n d  Huntington's  r e t u r n of  t o the development of t a r d i v e  Somatostatin  a p p e a r t o be  the observed  i n the caudate-putamen a f t e r  observed  i n the  striatal  Radke e t a l . , 1987a) o r GABA  systems, not  As m e n t i o n e d  to  At p r e s e n t  (Sorenson illness,  important levels  levels  i n the  CSF  the o r i g i n s  of  e t a l . , 1981).  there i s growing  element  evidence  i n this disease.  in affectively  i l l humans  The has  58 been c o r r e l a t e d w i t h the al.,  1984)  1984).  and  levels  reduced  c o r r e l a t e d w i t h escape from (Rubinow, 1 9 8 6 ) .  ill  humans  kindled  CSF  escape from  not  abnormal s o m a t o s t a t i n  'basal"  somatostatin  ACTH s e c r e t i o n  levels  has  the b a s a l l e v e l s  are abnormal  affectively  Rubinow, 1986)  and  a l s o been c o r r e l a t e d  to  These  t o have i t s e f f e c t  reducing  on  s i n c e s o m a t o s t a t i n has  (Brown e t a l . , 1984;  of experiment  i s important  suggests  1986;  i n both  t h a t c a r b a m a z e p i n e c o u l d be  Richardson  Litvin  the been  shown  et a l . ,  e t a l . , 1983). 4 indicate  t h a t carbamazepine  of s o m a t o s t a t i n  i n d i s c u s s i n g these  carbamazepine appears levels  levels  t h e dexamethasone s u p p r e s s i o n t e s t .  results  are  been  R e i s i n e , 1985;  alter  levels  T r e a t m e n t w i t h c a r b a m a z e p i n e , w h i c h has  dexamethasone s u p p r e s s i o n t e s t  The  Lundqvist,  test  ( H i g u c h i e t a l . , 1986)  hypothalamic  1986;  and  (Rubinow e t  t h e dexamethasone s u p p r e s s i o n  o b s e r v a t i o n s would suggest  to i n h i b i t  (Agren  somatostatin  (Rubinow e t a l . , 1984;  rats  noradrenaline  noradrenaline metabolites  Furthermore,  shown t o l o w e r  of both  to reduce  i n the r a t .  results  somatostatin  ( H i g u c h i e t a l . , 1986;  t h a t carbamazepine i s not having  The  does term  because  l e v e l s when  these  Rubinow, 1 9 8 6 ) . a direct  effect  This on  somatostatin. C a r b a m a z e p i n e has systems,  including  nucleotides et  al.,  1985;  GABA, a c e t y l c h o l i n e ,  (see review  a l . , 1985)  and  b e e n shown t o a f f e c t  by  Post  Carbamazepine's e f f e c t s  on  neurochemical  vasopressin,  cyclic  e t a l . , 1983), substance  catecholamines  P u r d y e t a l . , 1977;  other  (Kowalik  e t a l . , 1984;  P  (Jones  Post  et  Waldmeier e t a l . , 1984).  s o m a t o s t a t i n may  i n v o l v e one  of  these  59 other  t r a n s m i t t e r s y s t e m s s i n c e s e v e r a l o f them have b e e n shown  to a f f e c t 1979;  hypothalamic  Richardson  somatostatin  e t a l . , 1980;  Another p o s s i b i l i t y , o f c a r b a m a z e p i n e on by  to double  somatostatin  when a n i m a l s  carbamazepine does not its  ability  t o lower  including  Two  the  of  In Huntington's  al., et  1985)  and  while  could  illness  somatostatin  or  lesions  other  the  somatostatin  experiments to spare  striatal  results  (experiment  stress,  kindling.  levels  are  report,  are  a s p a r i n g of  (Dawbarn e t a l . , 1985; levels  i n the  striatal  cells  a u s e f u l model of t h i s  report  t o d r u g s when u n d e r  epilepsy.  cells  striatal  acid  B a s e d on this  cells  1986;  i n v o l v e changes i n the  that are p e r t i n e n t to t h i s  Recently,  killing  providing  may  levels,  (Higuchi et a l . ,  Ferrante  are e l e v a t e d  B e a l e t a l . , 1984a; N e m e r o f f e t a l . , 1983;  1984).  quinolinic  basal somatostatin  d i s e a s e t h e r e a p p e a r s t o be  somatostatin  a l . , 1986;  al.,  Since  involved i n affective  d i s e a s e and  study  are under s t r e s s .  d i s o r d e r s i n which c e n t r a l  Huntington's  A  somatostatin  Rubinow, 1986)  altered,  effect  shown h y p o t h a l a m i c  appear t o a l t e r  somatostatin  stress  significantly  striatal  neurons d u r i n g s t r e s s .  abnormal s o m a t o s t a t i n  Rubinow e t a l . , 1984; susceptibility  Sheppard e t a l . , 1979).  however, w o u l d i n v o l v e a d i r e c t  A r a n c i b i a e t a l . (1984) has  release  r e l e a s e (Epelbaum e t a l . ,  (Aronin Sagar  r a t have shown somatostatin  neurons  (Beal e t a l . , 1986c),  thus  disease.  of the b e h a v i o u r a l study presented  1), the  et  increase i n somatostatin  c o n t r i b u t e t o the motor problems seen i n  in  levels  Huntington's  et  60 disease.  Treatment  re-establishing diseased  with cysteamine  a balanced neurochemical  striatum.  In f a c t ,  e v a l u a t e d as a p o s s i b l e Unfortunately,  failed  the treatment  to alter  future  still  useful  recently  failed  1986). to r e l i e v e the  This  treatment  of somatostatin suggesting Cysteamine,  i n Huntington's  that  or similar  patients i n  epilepsy,  cortical  epileptic  somatostatin levels  tissue  involving  a n i m a l models o f  show an i n c r e a s e i n c o r t i c a l  decrease  I n t h e animal models, cysteamine both  somatostatin levels  a l . , 1984; H i g u c h i e t a l . ,  somatostatin  somatostatin  Carbamazepine a l s o seizures  reduces  i n kindled  suggesting  rats  only cysteamine  reduces  carbamazepine  in  kindled  in  kindling  a c t i v i t y (Assouline  1983) s u g g e s t i n g t h a t factor  the increased  i n epilepsy.  t h e i n c r e a s e d s o m a t o s t a t i n l e v e l s and  (Higuchi et a l . ,  somatostatin i s involved  S i n c e both carbamazepine  1986),  i n epileptic  and c y s t e a m i n e  further behaviours.  attenuate k i n d l i n g , but  basal somatostatin levels,  i s acting  indirectly  to decrease  this  cysteamine  inhibits  seizures.  suggests  somatostatin  animals, but somatostatin i s a very important since  et a l . ,  h a s b e e n shown t o  and s e i z u r e  l e v e l s may be an i m p o r t a n t  appear  (Nadi e t a l . , 1986).  ( H i g u c h i e t a l . , 1983; H i g u c h i e t a l . , 1986; K a t o  1983).  that  used  of p a t i e n t s .  i s c o n s i s t e n t with experiments  e p i l e p s y which a l s o  et  has been  w i t h i n the  studies.  t o be i n c r e a s e d i n f o c a l  levels  environment  u s e d was t o o low.  prove  In i n t r a c t a b l e  This  group  t h e r e f o r e be u s e f u l i n  (Shults e t a l . ,  regimen  CSF l e v e l s  the dose o f cysteamine drugs, might  cysteamine  treatment  motor a b n o r m a l i t i e s i n t h i s also  might  Future  factor studies  61  involving  somatostatin  i n kindling  e x p e r i m e n t s may p r o v i d e  better understanding  of the c l i n i c a l  in  treatment.  The  e p i l e p s y and i t s  importance of somatostatin  f u n c t i o n s o f s o m a t o s t a t i n systems  The  functions of somatostatin  are o b v i o u s l y d i f f e r e n t striatum, and  somatostatin  systems i n t h e b r a i n  fordifferent  brain regions.  may be i m p o r t a n t  tardive dyskinesia.  i n Huntington's  i n epilepsy.  disease  I n the hypothalamus,  s o m a t o s t a t i n may c o n t r i b u t e t o t h e h o r m o n a l i m b a l a n c e occurring  In the  I n t h e hippocampus and c o r t e x ,  s o m a t o s t a t i n may be a f a c t o r  be  a  i n mental i l l n e s s .  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The  Somatostatin  radioimmunoassay  standard curve developed  somostostatin  i m m u n o r e a c t i v i t y i n t i s s u e was d i s c u s s e d i n t h e  methods s e c t i o n description  t o measure t h e l e v e l s o f  of experiment  of t h i s procedure  a t y p i c a l standard cureve  1 (p.14).  F o r a more  an i l l u s t r a t i o n  complete  of the assay  and o f  i s shown b e l o w .  P r o t o c o l f o r s o m a t o s t a t i n RIA Buffer (u.1)  AB (ul)  200 300 100  100 100  Tracer (ul)  Standard (lOOul)  CPM (mean)  %Bound  51.4  0.987pg 1.975pg 3.950pg 7.825pg 15.62pg 31.25pg 62.50pg 125.Opg 250.Opg 500.Opg  1215 2500 1277 1415 1443 1532 1698 1953 2266 2416 2446 2442  2207 2012  11.7 19.5  100  Samples #101(0.82mg) #201(0.66mg)  SOM-IR (pg/mg)  48, 43, 42, 38, 32, 21, 9, 3, 2, 2,  65.0 54.0  89  Q Z ZD O  m  7.8  500  SOMATOSTATIN  F i g u r e 13.  An example o f a s t a n d a r d c u r v e u s e d  somatostatin immunoreactivity increasing is  (pg)  i n tissues.  l e v e l s of synthetic  the percent of synthetic  w h i c h c a n be c a l c u l a t e d  The X - a x i s r e p r e s e n t s  somatostatin-14  somatostatin-14  x  iQO.  used.  The  Y-axis  bound t o t h e a n t i b o d y  u s i n g t h e f o r m u l a : %B =  s t a n d a r d o r sample c o u n t s  t o measure  t  o  t  a  l  counts  

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