UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The effects of localized application of oxytocin and vasopressin in the central nervous system Tiberiis, Bruce Edmund 1983

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1983_A1 T53.pdf [ 5.49MB ]
Metadata
JSON: 831-1.0095890.json
JSON-LD: 831-1.0095890-ld.json
RDF/XML (Pretty): 831-1.0095890-rdf.xml
RDF/JSON: 831-1.0095890-rdf.json
Turtle: 831-1.0095890-turtle.txt
N-Triples: 831-1.0095890-rdf-ntriples.txt
Original Record: 831-1.0095890-source.json
Full Text
831-1.0095890-fulltext.txt
Citation
831-1.0095890.ris

Full Text

THE EFFECTS OF LOCALIZED APPLICATION OF OXYTOCIN AND VASOPRESSIN IN THE CENTRAL NERVOUS SYSTEM by BRUCE EDMUND TIBERIIS B . A . , U n i v e r s i t y o f A rkansas , 1966 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Phys io logy ) We accept t h i s t h e s i s as conforming to the requ i red standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1983 © Bruce Edmund T i b e r i i s , 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of PH/SIQ LOG/ The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 2 V A MIL DE-6 (3/81) i i ABSTRACT Immunocytochemical s t ud ies have demonstrated tha t nerve f i b e r s con-t a i n i n g immunoreactive oxy toc in and vasop ress in p r o j e c t to many areas of the c e n t r a l nervous system, i n c l u d i n g the hippocampus and the l a t e r a l septum ( B u i j s , 1980; Sofroniew and We ind l , 1978) . B i o c h e m i c a l , phys i o -l o g i c a l and behav io ra l s t u d i e s of the e f f e c t s of these pept ides on the CNS have i n d i c a t e d tha t they are i nvo l ved in f u n c t i o n s as d i v e r s e as the con t ro l of se ro ton in tu rnover (Auerbach and L i p t o n , 1982) , the r e g u l a -t i o n of body temperature (Kas t ing e t . a l . , 1979) and the r e t e n t i o n of cond i t i oned behav ior (de Wied e t . a l . , 1974; Koob and Bloom, 1982). The presence of immunoreactive vasop ress in ( iAVP) i n the hippocampus of Wis ta r r a t s was conf i rmed by radioimmunoassay. The vasopress in con-ten t of the do rsa l hippocampus was 30.3 ± 7.3 pg iAVP/mg s o l u b l e p r o t e i n and tha t o f the ven t ra l hippocampus was 81.4 + 8.3 pg iAVP/mg s o l u b l e p r o t e i n , wh i l e t i s s u e from the ce reb ra l co r tex con ta ined no de tec tab le v a s o p r e s s i n . That t h i s immunoreac t iv i ty was due to vasop ress in was con-f i rmed by the absence of immunoreac t iv i ty i n hippocampal or c o r t i c a l t i s s u e from B r a t t l e b o r o r a t s , which are g e n e t i c a l l y unable to syn thes i ze v a s o p r e s s i n . Vasopress in a p p l i e d by i on topho res i s was found to i nc rease the a c t i v i t y o f neurones in the l a t e r a l septum and i n the hippocampus of the anes the t i zed r a t . There was no obvious d i f f e r e n c e between the response of spontaneously a c t i v e c e l l s and the response of c e l l s e x c i t e d by cont inuous i on topho res i s o f glutamate or a c e t y l c h o l i n e . Repeated a p p l i c a t i o n o f vasop ress in r e s u l t e d i n a d e c l i n e in the magnitude of i i i the response , but a t l e a s t par t of t h i s d e c l i n e was due to p rog ress i ve blockage of the m i c rop ipe t t e ba r re l r a the r than to t a c h y p h y l a x i s . O x y t o c i n , t es ted on ly i n the septum, was w i thout e f f e c t . When a p p l i e d by super fus ion onto r a t hippocampal s l i c e s , the NHP pept ides were found to i nc rease the a c t i v i t y of 88% of spontaneously a c t i v e c e l l s and to induce a c t i v i t y i n many neurones tha t were not spontaneously a c t i v e . A r g i n i n e v a s o p r e s s i n , l y s i n e v a s o p r e s s i n , a r g i n i n e v a s o t o c i n , and oxy toc i n were found to be of roughly equ i va len t po tency , producing a dose dependent response in the range 10~ 9 - 10~ 6 M. Most c e l l s were t es ted w i th more than one pept ide and were always found to respond e i t h e r to a l l o r to none of them. There was no d e c l i n e in respons iveness when c e l l s were sub jec ted to repeated a p p l i c a t i o n s of p e p t i d e , but cont inuous a p p l i c a -t i o n caused the c e l l s to become unrespons ive . Fo l l ow ing cont inuous a p p l i -c a t i o n o f o x y t o c i n , a c e l l f a i l e d to respond to both oxy toc in and vaso-p r e s s i n , as would be expected i f the two pept ides were a c t i n g on the same r e c e p t o r . The analogues ddOT, ddAVP, and G l y 7 0 T were a l s o a c t i v e , but the oxy toc in fragment PLG had no e f f e c t , and the vasopress in fragment DGAVP was ext remely weak. The response to the pept ides cou ld be b locked by vasopress in a n t a g o n i s t s . The pept ide s e n s i t i v e c e l l s appeared to be pyramidal c e l l s r a t h e r than i n t e r n e u r o n e s , s i nce the pept ide induced a c t i v i t y cou ld be i n h i b i t e d f o r about 200-600 msec by e l e c t r i c a l s t i m u l a t i o n of the st ratum rad ia tum. i v TABLE OF CONTENTS Page A b s t r a c t i i Table o f Contents i v L i s t o f Tables v i L i s t o f F igures v i i Abb rev i a t i ons i x Acknowledgements x i i Chapter I. I n t r oduc t i on 1 A. Endocr ino logy o f the Neurohypophysial System 1 1. The Hypothalamo-neurohypophysial System 1 2 . Endocr ine Ac t i ons o f the Neurohypophysial P e p t i d e s . . . 2 B. The Neurohypophysial Pept ides and the Cen t ra l Nervous System 9 1. Ex t rahypotha lamic P r o j e c t i o n s 9 2 . E f f e c t s on the Cent ra l Nervous System 12 3. The Presen t Study 26 C. Exper imenta l Techniques 28 1. Radioimmunoassay 28 2 . M i c r o i o n t o p h o r e s i s 30 3 . The Hippocampal S l i c e . 3 2 Chapter I I . Radioimmunoassay Experiments 38 A. Methods 38 1. Summary 38 2 . T issue Sample P repa ra t i on 38 3 . Acetone/Pet ro leum E ther E x t r a c t i o n . . . 39 V Page 4 . Radioimmunoassay 40 B. Vasopress in Content o f Rat B r a i n T i ssues 44 1. Resu l t s and A n a l y s i s 44 2 . D i s c u s s i o n 46 Chapter I I I . Neu rophys io l og i ca l Experiments 48 A. Methods 48 1. In_ V i t r o S tud ies 48 2 . In V ivo S tud ies 57 B. I on topho re t i c A p p l i c a t i o n o f Neurohypophysial P e p t i d e s . . . 61 1. Resu l t s and A n a l y s i s 61 2 . D i s c u s s i o n 71 C. In V i t r o Experiments 76 1. Resu l t s and A n a l y s i s 76 2 . D i s c u s s i o n . 93 Chapter IV. O x y t o c i n , Vasopress in and the CNS 106 References .110 v i LIST OF TABLES Table Page I. The Amino A c i d Sequences of the Major Mammalian Neurohypophysial Hormones 3 I I . R e l a t i v e Potency of NHP Pept ides and Analogues on NHP Endocr ine Receptors 6 I I I . Behav io ra l E f f e c t s o f Neurohypophysial Pept ides 19 IV. Vasopress in Content of Rat B ra in T issues 45 V. A c t i v i t y o f Neurohypophysial Pept ides and Analogues in Hippocampal S l i c e s 80 v i i LIST OF FIGURES F igure Page 1. The Anatomy o f the Rat Hippocampal S l i c e 33 2 . P o s i t i o n o f E lec t rodes f o r Recording the Response to S c h a f f e r C o l l a t e r a l S t i m u l a t i o n . . 35 3 . Standard Curve o f an AVP Radioimmunoassay 42 4 . S e r i a l D i l u t i o n o f Hippocampal T i ssue 47 5 . Schemat ic Diagram o f the Incubat ion Chamber 51 6 . Apparatus f o r M a i n t a i n i n g Hippocampal S l i c e s 54 7. Response o f Neurones i n the Hippocampus to Ion tophores is o f AVP 63 8. Response o f Neurones i n the Septum to Ion tophores is o f AVP. . . . '. 65 9 . I n h i b i t i o n o f Neurones i n the Septum by Ion topho re t i c Currents 66 10. Neurones Responding w i th a Combination o f E x c i t a t i o n and I n h i b i t i o n 67 11. Dec l i ne i n Responsiveness During Repeated A p p l i c a t i o n s of AVP 68 12. E x c i t a t i o n Produced Dur ing Ion tophores is o f H + Ions . . . 70 13. Response to Ion tophores is o f AVP In V i t r o 72 14. E f f e c t o f Super fus ion o f AVP on Hippocampal Neurones In V i t r o 77 15. E f f e c t o f LVP, AVT, and OXT on Hippocampal Neurones In_ V i t r o 78 16. E f f e c t o f SST on a Neurone I n s e n s i t i v e to AVP 79 17. F a i l u r e o f a Hippocampal Neurone to Respond to PLG. . . . 82 18. Response o f a Hippocampal Neurone to Low Concent ra -t i o n s o f Oxytoc in 83 19. E f f e c t o f DGAVP on Hippocampal Neurones 85 v i i i F igure Page 20 . Antagonism o f the Response to ddOT by d(CH 2 ) 5 Tyr(Me)AVP 87 21 . Antagonism o f the Response to G l y 7 0 T by d(CH 2 ) . 5 -D-Tyr(Me)VAVP 89 22. Dec l i ne i n Responsiveness o f a C e l l Sub jec ted to Cont inuous Exposure to Oxy toc in 100 i x ABBREVIATIONS Ach A c e t y l c h o l i n e ACTH a d r e n o c o r t i c o t r o p i c AD recep to r media t ing the a n t i d i u r e t i c e f f e c t s of vasop ress in AVP a r g i n i n e vasopress in AVT a r g i n i n e vaso toc in B/F bound/ f ree r a t i o BSA bovine serum albumin °C deg rees . cen t i grade cGMP 3 ' , 5 ' - c y c l i c guanosine monophosphate CNS c e n t r a l nervous system CRF c o r t i c o t r o p i n r e l e a s i n g f a c t o r CSF ce reb rosp ina l f l u i d d(CH 2 ) 5 -D-Tyr (Me)VAVP [1-(B -mercapto-3 ,3-cyc lopentamethy leneprop ion ic a c i d ) , 2 - ( 0 - m e t h y l ) - D - t y r o s i n e , 4 - v a l i n e ] a r g i n i n e -vasopress in d(CH 2 ) 5 Tyr (Me)AVP [ l - (3 -mercap to -3,B -cyc lopen tamethy leneprop ion ic a c i d ) , 2 - ( 0 - m e t h y l ) t y r o s i n e ] a r g i n i n e - v a s o p r e s s i n ddAVP [1 - d e a m i n o - l , 6 - d i c a r b a ] a r g i n i n e - v a s o p r e s s i n dDAVP [1 -deamino-8 -D-a rg in ine ]vasopress in ddOT [1 - d e a m i n o - l , 6 - d i c a r b a ] o x y t o c i n D6AVP [ d e s g l y c i nami d e - 8 - a r g i n i ne]vasopres s i n DGLVP [ d e s g l y c i n a m i d e - 8 - l y s i n e ] v a s o p r e s s i n DPM d ischarges per minute dPTyr(Me)AVP [1 - d e a m i n o p e n i c i l l a m i n e , 2 - ( 0 - m e t h y l ) t y r o s i n e ] a r g i n i n e - v a s o p r e s s i n EDTA disodium dihydrogen e thy lened iam ine te t raace ta te d ihyd ra te X EEG e lec t roencephalogram g grams o r g r a v i t i e s , depending on the contex t G l y 7 0T [ 7 - g l y c i n e ] o x y t o c i n HeDI heterozygous f o r d iabe tes i n s i p i d u s HF recep to r media t ing the r e l e a s e of hemato log ica l f a c t o r by vasop ress in HoDI homozygous f o r d iabe tes i n s i p i d u s iAVP AVP immunoreact iv i ty i c v i n t r a c e r e b r o v e n t r i c u l a r I .D. i nne r d iameter ip i n t r a p e r i t o n e a l IU I n t e rna t i ona l Un i t kg k i log ram kHz k i l o H e r t z KMNCs potassium permanganate LVP l y s i n e vasop ress in M molar MBq MegaBecquerels ME recep to r media t ing the mi l k e j e c t i n g a c t i o n of oxy toc in mg m i l l i g r a m s min minutes ml m i l l i l i t r e s mm m i l l i m e t r e s mM m i l l i m o l a r MSH me lanocy te - s t imu la t i ng hormone Mfi megohms x i nA nanoamperes NHP neurohypophysia l ng nanograms NSB n o n s p e c i f i c b ind ing OT recep to r media t ing the u t e r o t o n i c e f f e c t of oxy toc in OXT oxy toc in OVLT organum vasculosum of the lamina te rmina l i s pg picograms PLG p r o l y l - l e u c y l - g l y c i n a m i d e PVN p a r a v e n t r i c u l a r nucleus sc subcutaneous SCN suprach iasmat ic nucleus SEM standard e r r o r o f the mean SON sup raop t i c nucleus SST somatos ta t in yA microamperes yg micrograms y l m i c r o l i t r e s x i i ACKNOWLEDGEMENTS I wish to thank my two s u p e r v i s o r s , Dr . Nadine Wi lson and Dr. Hugh McLennan, f o r t h e i r suppor t , t h e i r a d v i c e , and t h e i r e x t r a o r d i n a r y p a t i e n c e . Dr . Wi lson was e x c e p t i o n a l l y generous wi th her t i m e , her knowledge o f endoc r i no logy , and her l a b o r a t o r y f a c i l i t i e s ; w i thout her understanding and encouragement, t h i s p r o j e c t would never have been completed. The neurophys io logy exper iments were conducted under the s u p e r v i s i o n of Dr . McLennan, and h i s s u g g e s t i o n s , comments, and guidance proved i n v a l u a b l e . I thank,IJohn Ngsee f o r h i s he lp and f o r h i s common sense , and Dr. Ralph K e e l e r , Dr . John Brown, and Dr . Owen Parkes f o r t h e i r adv i ce and sugges t i ons . A d d i t i o n a l thanks are due to Steve K e h l , Andrew L a r d e r , and Graham C o l l i n g r i d g e f o r answering my many quest ions about neuro-p h y s i o l o g y , arid to Ron Walker f o r t each ing me eve ry th ing I know about h i s t o l o g y . Dr. M. Manning o f the Medica l Co l lege o f Oh io , To ledo , Oh io , k i n d l y prov ided a supply o f vasop ress in ana logues. Th is t h e s i s was typed by Co l l een C o l c l o u g h . F i n a l l y , I thank R ichard S t a f f o r d , C h r i s t i n e N e l s o n , and David C o l l i n s , f o r e v e r y t h i n g . 1 CHAPTER I. INTRODUCTION A. ENDOCRINOLOGY OF THE NEUROHYPOPHYSIAL SYSTEM 1. THE HYPOTHALAMO-NEUROHYPOPHYSIAL SYSTEM The i d e n t i t i e s o f the a c t i v e p r i n c i p l e s o f the p o s t e r i o r p i t u i t a r y were e s t a b l i s h e d when Du Vigneaud (1956) syn thes i zed the pept ides oxy toc in and vasopress in and demonstrated t h a t the chemical and b i o l o g i c a l p r o p e r t i e s o f the s y n t h e t i c pept ides were i d e n t i c a l to those of p u r i f i e d p i t u i t a r y e x t r a c t s . E rns t Scha r re r had proposed as e a r l y as 1928 tha t the hormones were syn thes i zed by neurones i n the hypothalamus r a t h e r than by the c e l l s i n the p o s t e r i o r p i t u i t a r y i t s e l f (Knowles, 1974) , but i t was not u n t i l the 1950's t ha t the concept of neu rosec re t i on became w ide ly accep ted . Th is acceptance was based l a r g e l y on the work o f E rns t and Ber ta Schar re r and Wolfgang Bargmann, who demonstrated h i s t o l o g i c a l l y t ha t c e r t a i n ve r teb ra te and i n v e r t e b r a t e neurones conta ined g ranu la r pro te inaceous ma te r i a l s i m i l a r to the m a t e r i a l t h a t c h a r a c t e r i z e d p r o t e i n s e c r e t i n g c e l l s elsewhere i n the body (Bargmann and S c h a r r e r , 1951). In,mammals, t h i s "neu rosec re to ry " ma te r i a l was found to o r i g i n a t e i n the c e l l bodies o f magnoce l lu la r neurones i n the sup raop t i c and p a r a v e n t r i c u l a r n u c l e i and to be t ranspo r ted v i a the hypothalamo-neurohypophysial t r a c t to nerve te rm ina l s i n the p i t u i t a r y (Bargmann and S c h a r r e r , 1951; Bern and Knowles, 1966; Knowles, 1974) . Release of o x y t o c i n and vasopress in i n t o the bloodstream i nvo l ves d e p o l a r i -z a t i o n o f the nerve te rm ina l s by a c t i o n p o t e n t i a l s o r i g i n a t i n g i n the c e l l bodies ( C r o s s , 1973; 1974) . 2 2 . ENDOCRINE ACTIONS OF THE NEUROHYPOPHYSIAL PEPTIDES The amino a c i d sequences of the mammalian neurohypophysia l (NHP) pept ides are shown in Table I. The p o s t e r i o r p i t u i t a r y of the adu l t mammal con ta ins both oxy toc i n and v a s o p r e s s i n . Oxytoc in (OXT), which i s re l eased by r e f l e x e s o r i g i n a t i n g i n the mammary glands and i n the rep roduc t i ve t r a c t , causes mi l k e j e c t i o n dur ing s u c k l i n g and c o n t r a c t i o n of the uterus dur ing c h i l d b i r t h (Share and Grosvenor , 1974). Vasopress in has both vasopressor and a n t i d i u r e t i c e f f e c t s and i s r e l eased by inc reased plasma osmo la r i t y or decreased blood volume ( S c h r i e r e t . a l . , 1979). Vasopress in has r e c e n t l y been shown to i nc rease plasma l e v e l s of b lood c l o t t i n g f a c t o r V I I I and of plasminogen a c t i v a t o r (Cash e t . a l . , 1978; Cor t e t . a l . , 1981; Mannucc i , 1977). Th is e f f e c t i s thought to be i n d i r e c t , w i th vasop ress in promoting r e l e a s e of a hemato log ica l f a c t o r r e l e a s i n g f a c t o r from the b ra i n or p i t u i t a r y (Cor t e t . a l . , 1981). The vasopress in found in most mammals i s a r g i n i n e vasop ress in (AVP) , but i n the p ig and r e l a t e d spec ies and in some marsup ia ls the p i t u i t a r y con ta ins l y s i n e vasop ress in (LVP) i ns tead of or i n a d d i t i o n to AVP (Chauvet e t . a l . , 1981). When used expe r imen ta l l y i n o ther mammals, such as the r a t and r a b b i t , LVP has q u a l i t a t i v e l y the same e f f e c t s as AVP, a l though i t i s l e s s a c t i v e on a weight bas i s (Berde and Bo issonnas , 1966). A r g i n i n e vaso toc in (AVT), a neurohypophysia l pept ide found i n non-mammalian ve r teb ra tes (Bern and Knowles, 1966; Acher , 1981), i s in te rmed ia te in s t r u c -tu re between oxy toc in and AVP. Vaso toc in b i o a c t i v i t y has been detec ted i n the p i t u i t a r i e s of f e t a l mammals (sheep and s e a l s ) by V i z s o l y i and Perks (1969) and i s probably i nvo l ved in water f l ow across the amn io t i c membrane ( V i z s o l y i and P e r k s , 1974). Vaso toc in may a l s o be present i n 3 Tab le I. The amino a c i d sequences o f the major mammalian neurohypophysia l hormones. 1 2 3 4 5 6 7 8 9 C y s - T y r - I l e - G l n - A s n - C y s - P r o - L e u - G l y N H 2 1 S S - J Oxytoc in (OXT) C y s - T y r - P h e - G l n - A s n - C y s - P r o - A r g - G l y N H 2 L s S J Arg in i ne Vasopress in (AVP) C y s - T y r - P h e - G l n - A s n - C y s - P r o - L y s - G l y N H 2 J S S - J Lys ine Vasopress in (LVP) C y s - T y r - I l e - G l n - A s n - C y s - P r o - A r g - G l y N H 2 J S S _ J A r g i n i n e Vaso toc in (AVT) Note tha t the sequences d i f f e r on ly i n p o s i t i o n 3 ( l i e or Phe) and i n p o s i t i o n 8 (Leu , A r g , or L y s ) . 4 adu l t mammalian b ra i n t i s s u e ; Pavel (1971) has repor ted AVT b i o a c t i v i t y i n bovine p i n e a l s t a l k and subcommissural organ (SCO), and Rosenbloom and F i s h e r (1975) have found AVT immunoreac t iv i t y i n r a b b i t SCO. Vaso-t o c i n i nc reases p r o l a c t i n r e l ease and has an an t igonado t roph ic e f f e c t when i n j e c t e d i n t o r a t s (B lask e t . a l . , 1978; B lask and Vaughan, 1980). Recent work by Pevet e t . a l . (1981) however, i n d i c a t e s tha t the bovine p inea l g land con ta ins no AVT, AVP, or OXT; the A V T - l i k e b i o a c t i v i t y was a t t r i b u t e d i ns tead to an u n i d e n t i f i e d pept ide which i s immunolog ica l l y s i m i l a r to AVT and AVP. The NHP pept ides a r e - s t o r e d i n the p i t u i t a r y i n a s s o c i a t i o n w i th c a r r i e r p r o t e i n s , the neurophysins (Ho l lenberg and Hope, 1968; Rob inson, 1975). I t i s thought t h a t each nonapept ide and i t s cor respond ing neurophysin are fragments o f a common p recurso r molecule (Watson e t . a l . , 1982) . Both oxy toc i n and vasopress in may be i nvo l ved i n the con t ro l of the a n t e r i o r p i t u i t a r y . Vasopress in has been demonstrated by immunocyto-chemis t ry i n the median eminence o f the r a t and monkey (Defend in i and Zimmerman, 1978) and i s present i n h igh concen t ra t i ons i n the hypophysia l po r ta l b lood o f the monkey (Zimmerman e t . a l . , 1973). Vasopress in p ro -motes re l ease o f a d r e n o c o r t i c o t r o p i c hormone (ACTH) from the r a t p i t u i t a r y , probably a c t i n g i n con junc t i on w i th another r e l e a s i n g f a c t o r ( G i l l e s and Lowry, 1979; Lu tz -Bucher e t . a l . , 1980) . An enzyme i n the r a t median eminence c leaves o x y t o c i n to r e l e a s e the carboxy te rmina l t r i p e p t i d e p r o l y l - l e u c y l - g l y c i n a m i d e (PLG, a l s o known as M I F - I ) , which i n h i b i t s the r e l e a s e o f melanocyte s t i m u l a t i n g hormone (MSH) from the in te rmed ia te lobe (Cel l ' s e t . a l . , 1971; Schwartz and W a l t e r , 1974) . 5 NHP Pept ide Receptors There are a t l e a s t 5 d i s t i n c t types o f mammalian NHP pept ide r e c e p t o r . As i n d i c a t e d i n Table I I , the recep to rs are d i s t i n g u i s h a b l e by t h e i r d i f -f e r e n t s p e c i f i c i t i e s f o r the va r ious NHP pept ides and ana logues. The recep to rs med ia t ing the o x y t o c i c (OT) response i n the uterus d i f f e r s l i g h t l y from those med ia t ing the m i l k e j e c t i n g (ME) response i n the mammary g l a n d , but both are more s e n s i t i v e to OXT than to AVP (Berde and Bo issonnas , 1966). In c o n t r a s t , the recep to rs f o r vasopressor (VP) a c t i v i t y , f o r a n t i d i u r e t i c (AD) a c t i v i t y , and f o r hemato log ica l f a c t o r (HF) r e l e a s i n g a c t i v i t y are more s e n s i t i v e to AVP than to OXT (Berde and Bo issonnas , 1966; Cash e t . a l . , 1978). The termino logy used here f o r the recep to rs i s based on the cor respond ing b i o a s s a y s ; the VP recep to r has a l s o been r e f e r r e d to i n the l i t e r a t u r e as the smooth muscle (SM) or Vi r e c e p t o r , the AD recep to r as the V 2 r e c e p t o r , and the HF recep to r as the HF-R or plasminogen a c t i v a t o r (PA) recep to r (PA r e c e p t o r , Cash e t . a l . , 1978; SM, AD, and HF-R r e c e p t o r s , Cor t e t . a l . , 1981; and Vi and V 2 r e c e p t o r s , Muh le tha le r e t . a l . , 1982). There i s ev idence tha t a d d i t i o n a l types o f NHP pept ide recep to r are present i n mammalian t i s s u e s . For example, desg lyc inamide LVP (DGLVP) has been found to produce changes i n cond i t i oned behav ior and i n se ro ton in tu rnover (Bohus, 1974; Ramaekers e t . a l . , 1977). S ince DGLVP has very l i t t l e OT, VP, AD, HF, or c o r t i c o t r o p h i n r e l e a s i n g f a c t o r (CRF) a c t i v i t y (de Wied e t . a l . , 1972; Cash e t . a l . , 1978), i t s e f f e c t s on behav ior and on se ro ton in tu rnover i n d i c a t e the e x i s t e n c e o f a separate type of vaso-p r e s s i n recep to r i n the r a t CNS. A l t u r a (1975) has demonstrated tha t there may be a t l e a s t 3 d i s t i n c t AVP recep to rs i nvo l ved i n v a s o c o n s t r i c -6 Table I I . R e l a t i v e Potency o f NHP Pept ides and Analogues on NHP Endocr ine Recep to rs . PEPTIDE OT ME VP AD HF OXT 100 100 1 1 0 AVP 4 14 100 100 100 LVP 1 13 68 62 ~100 AVT 26 47 61 62 ~ 5 0 dDAVP 0 0 0 >100 >100 DGAVP/DGLVP 0 ? 0 1 0 G ly 7 0T ~200 ~100 0 0 ? dDAla 9 AVP ? 0 0 >100 d(CH 2 ) 5 TyrOMe)AVP ANT I ? ANT I 0 ? d(GH 2 ) 5 DTyr(Me)VAVP ? ? ANT I ANT I ? The a c t i v i t y o f AVP at the AD, VP, and HF recep to rs and of OXT at the OT and ME recep to rs have been a r b i t r a r i l y se t a t 100, and the a c t i v i t i e s o f the o ther pept ides and analogues are expressed as a percentage o f these a c t i v i t i e s . The va lues i n the l i t e r a t u r e are not e n t i r e l y i n agreement (Sawyer e t . a l . , 1981) , and the a c t i v i t i e s g iven here should the re fo re be cons idered approx imat ions . I n a c t i v i t y or very weak a c t i v i t y i s r e p r e -sented by a " 0 " ; " ? " i n d i c a t e s t ha t the i n fo rma t ion i s not a v a i l a b l e ; "ANTI" i n d i c a t e s t h a t the analogue ac ts as an a n t a g o n i s t . Re fe rences : Berde and Bo issonnas , 1966 (OXT, AVP, LVP, AVT) ; Cash e t . a l . , 1978 (HF r e c e p t o r ) ; De Wied e t . a l . , 1972( DGAVP); Cor t e t . a l . , 1981 (dDA la 9 AVP) ; Sawyer e t . a l . , 1981 (dDAVP); Lowbridge e t . a l . , 1977 ( G l y 7 0 T ) : and Kruszynsk i e t . a l . , 1980 and Manning e t . a l . , 1982 ( a n t a g o n i s t s ) . 7 t i o n ; the recep to rs d i f f e r i n t h e i r r e l a t i v e s e n s i t i v i t i e s to P h e 2 , H e 3 , and Orn 8 s u b s t i t u t e d analogues o f AVP and are found i n d i f f e r e n t reg ions o f the r a t c i r c u l a t o r y system ( a o r t a , mesenter ic a r t e r i o l e , mesenter ic v e n u l e ) . In a d d i t i o n , the e f f e c t o f AVT on l u t e i n i z i n g hormone and f o l l i c l e - s t i m u l a t i n g hormone r e l e a s e i n the r a t (B lask e t . a l . , 1978) and on melanogenesis i n the hamster (Logan and Weatherhead, 1981) are mediated by AVT s p e c i f i c r e c e p t o r s . I t i s a l s o p o s s i b l e tha t there are s p e c i f i c recep to rs f o r the NHP pept ide f ragments , such as PLG. These a d d i -t i o n a l c l a s s e s o f recep to rs have not ye t been s tud ied i n d e t a i l and are t h e r e f o r e not i nc luded in Table I I . The B r a t t l e b o r o Rat The study o f the NHP pept ides has been g r e a t l y f a c i l i t a t e d by the d i scove ry t ha t c e r t a i n r a t s o f the B r a t t l e b o r o s t r a i n are incapab le o f s y n t h e s i z i n g AVP. The c o n d i t i o n , c h a r a c t e r i z e d by the i n a b i l i t y to con-cen t ra te u r ine (d iabe tes i n s i p i d u s , D I ) , i s i n h e r i t e d as an autosomal r e c e s s i v e t r a i t ; r a t s homozygous f o r the d i s o r d e r (HoDI r a t s ) are com-p l e t e l y l a c k i n g i n AVP, wh i l e heterozygous (HeDI) r a t s have on l y a p a r t i a l impairment o f AVP syn thes i s ( V a l t i n e t . a l . , 1965) . As would be expec ted , the ch ron i c p o l y u r i a o f the HoDI r a t s r e s u l t s i n a v a r i e t y o f e l e c t r o l y t e and hormonal a b n o r m a l i t i e s , i n c l u d i n g d e h y d r a t i o n , hypoka lemia , hyper ten-s i o n , rena l damage, i nc reased ren in and ang io tens in II r e l e a s e , and a reduced p i t u i t a r y - a d r e n a l response to s t r e s s ( B a i l e y and We iss , 1981) . I n j e c t i o n o f AVP w i l l b lock the water l o s s , but long- te rm t reatment w i th AVP i s needed to r e s t o r e rena l f u n c t i o n to normal . The oxy toc in content o f the p o s t e r i o r p i t u i t a r y i s reduced i n HoDI r a t s , presumably because 8 oxy toc i n i s r e l eased to compensate f o r the d e f i c i e n c y of AVP ( V a l t i n e t . a l . , 1965). In a d d i t i o n , HoDI r a t s have an impai red a b i l i t y to respond to oxy toc in (Goren e t . a l . , 1980; Han i f e t . a l . , 1982) . D i f f e r e n c e s in the g-endorphin content o f b ra i n t i s s u e s (Pi t tman e t . a l . , 1982) , i n the reg iona l d i s t r i b u t i o n o f op ia te recep to rs ( R i g t e r e t . a l . , 1979) , and in se ro ton in and catecholamine metabol ism (Kovacs e t . a l . , 1980) have a l s o been r e p o r t e d . B r a t t l e b o r o r a t s have proved i n v a l u a b l e as a con t ro l i n immunoassay and immunocytochemical s t u d i e s , s i nce any immunoreact iv i ty found i n HoDI r a t s i s o b v i o u s l y not due to the presence of AVP (Vandesande and D i e r i c k x , 1975; B u i j s and Swaab, 1979). The use of HoDI. r a t s as c o n t r o l s i n p h y s i o l o g i c a l and behav io ra l s t u d i e s i s l e s s s t r a i g h t f o r w a r d . I t i s reasonable to assume, o f cou rse , t ha t a response demonstrated i n HoDI r a t s does not depend on AVP - the presence of r ecu r ren t i n h i b i t i o n i n the HoDI r a t , f o r example, has been c i t e d as ev idence tha t the i n h i b i t i o n i s not mediated by AVP (D re i f uss e t . a l . , 1973) - but i t i s more d i f f i c u l t to prove the o p p o s i t e , t ha t a f u n c t i o n found to be impai red i n HoDI r a t s i s normal ly mediated by AVP. Th is i s t rue both because the de fec t i n the HoDI r a t might be an i n d i r e c t e f f e c t o f the AVP d e f i c i e n c y , due, perhaps, to hypokalemia or to the d i s t u rbed adrenal response , and because one cannot be c e r t a i n t h a t the l a c k o f AVP i s the on ly gene t i c de fec t present i n the HoDI r a t . The importance of t h i s po in t i n e v a l u a t i n g the r o l e of AVP i n behav io r (v ide i n f r a ) has been d i scussed more f u l l y by B a i l e y and Weiss (1981) , to whom the reader i s r e f e r r e d f o r a more d e t a i l e d d e s c r i p -t i o n o f the b e h a v i o r a l , m e t a b o l i c , and endocr ine d i f f e r e n c e s between HoDI, HeDI, and normal r a t s . Comparison o f HoDI and normal r a t s i s a l s o c o m p l i -9 cated by the s m a l l e r s i z e and reduced body f a t o f HoDI r a t s , and the r e s u l t i n g d i f f i c u l t y i n c a l c u l a t i n g e q u i v a l e n t dosages (Carey and M i l l e r , 1982; Pi t tman e t . a l . , 1982) . In some types o f exper iments , r a t s t r e a t e d w i th a vasopress in an tagon is t may be more appropr ia te c o n t r o l s than HoDI an ima ls . B. THE NEUROHYPOPHYSIAL PEPTIDES AND THE CENTRAL NERVOUS SYSTEM 1. EXTRAHYPOTHALAMIC PROJECTIONS OF NEURONES CONTAINING NHP PEPTIDES The same s t a i n i n g techn iques t ha t had been used to demonstrate the hypothalamo-neurohypophysia l f i b e r s - the Gomor i , a l d e h y d e - f u c h s i n , and pseudoisocyanine techn iques - a l s o revea led hypothalamic f i b e r s p r o j e c t -ing to o ther areas o f the c e n t r a l nervous system (CNS), i n c l u d i n g the septum, the hippocampus, and the habenular reg ion (Bern and Knowles, 1966; S t e r b a , 1974). These f i b e r s appeared to t e r m i n a t e , not on blood v e s s e l s as i n the p i t u i t a r y , but on o ther neurones. When s t a i n e d w i th a KMn04 o x i d a t i o n technique and viewed under the e l e c t r o n m ic roscope , the t e rm ina l s were found to form synapses not n o t i c e a b l y d i f f e r e n t from the synapses formed by convent iona l neurones ( S t e r b a , 1974; S te rba e t . a l . , 1979) . These s t a i n i n g t e c h n i q u e s , however, were not s p e c i f i c f o r the NHP pept ides (Bern and Knowles, 1966; S t e r b a , 1974; B u i j s , 1980) , and the repor ts d i d not r ece i ve widespread a t t e n t i o n . The development o f immunological techniques f o r s t a i n i n g t i s s u e s (Vandesande, 1979) a l lowed f o r much g rea te r s p e c i f i c i t y and s e n s i t i v i t y . Immunocytochemical s t ud ies i n the r a t and monkey revea led tha t oxy toc in and vasop ress in were syn thes i zed by separate popu la t ions of neurones i n 10 the sup raop t i c nucleus (SON) and the p a r a v e n t r i c u l a r nuc leus (PVN), the AVP c o n t a i n i n g neurones being i n the ven t ra l po r t i on o f the SON and the medial p o r t i o n o f the PVN (Vandesande and D i e r i c k x , 1975) , and by neurones ou ts ide these n u c l e i i n areas as d i s t a n t as the p r e o p t i c n u c l e u s , the t r i a n g u l a r nuc leus of the septum, the s u b s t a n t i a innomina ta , and the zona i n c e r t a (Sofroniew and We ind l , 1981; Antunes and Zimmerman, 1978; K e l l y and Swanson, 1980). In a d d i t i o n , p a r v o c e l l u l a r neurones i n the sup ra -ch iasma t i c nucleus (SCN) were found to con ta in vasop ress in but not oxy toc in (Sofroniew and We ind l , 1978; B u i j s e t . a l . , 1978) . The immunocytochemical s t u d i e s a l s o conf i rmed the ex i s t ence of the ex t rahypotha lamic p r o j e c t i o n s . F i b e r s con ta i n i ng oxy toc i n p r o j e c t c a u d a l l y i n t o the medul la and s p i n a l c o r d , but f i b e r s c o n t a i n i n g vasop ress in are r e l a t i v e l y sparse in these areas (Swanson, 1977; N i l a v e r e t . a l . , 1980; Seybold and E l d e , 1980) . Most o f the f i b e r s i n the r o s t r a l p o r t i o n o f the b r a i n , on the o ther hand, con ta in vasop ress in r a t h e r than oxy toc in ( B u i j s e t . a l . , 1978; B u i j s , 1980) . Termina ls immunoreactive f o r vasop ress i n have been demonstrated by both l i g h t and e l e c t r o n microscopy i n the l a t e r a l habenu la , the l a t e r a l septum, the amygdala, and the hippocampus (Sofroniew and We ind l , 1978; B u i j s e t . a l . , 1978; B u i j s and Swaab, 1979; B u i j s , 1980) . Termina ls c o n t a i n i n g oxy toc in have been found i n the amygdala and the hippocampus but not i n the l a t e r a l septum or the l a t e r a l habenula ( B u i j s and Swaab, 1979) . Accord ing to B u i j s (1980) , there are two routes by which f i b e r s immuno-r e a c t i v e f o r AVP reach the hippocampus: (1) a dorsa l p r o j e c t i o n pass ing v i a the f o r n i x and the f i m b r i a to terminate p r i m a r i l y i n the ven t ra l hippocampus, and (2) a separate ven t ra l p r o j e c t i o n pass ing through the amygdala. The f i b e r s were repor ted to terminate among the a p i c a l and 11 basal dend r i t es o f the pyramidal c e l l s . Sofroniew and Weindl (1981) have conf i rmed the ex i s t ence o f the v e n t r a l pathway, but the f i b e r s appeared to terminate i n the mo lecu la r l a y e r o f the dentate gyrus and in the h i l a r r e g i o n , and they found no evidence of a do rsa l p r o j e c t i o n . The d isc repancy between these two s t u d i e s suggests t h a t the ven t ra l and dorsa l p r o j e c t i o n s do not con ta in the same immunoreactive m a t e r i a l . S ternberger (1982) has argued t h a t the immunoreac t iv i ty de tec ted i n h i s t o l o g i c a l s t u d i e s i s not due to the vasop ress in and o x y t o c i n molecu les themse lves , which are l o s t when the s e c t i o n s are washed, but to l a r g e r fragments o f the p recurso r mo lecu les . I t i s l i k e l y t ha t the a n t i s e r a used by B u i j s (1980) and by Sofroniew and Weindl (1981) d i f f e r i n t h e i r a f f i n i t i e s f o r c e r t a i n of these f ragments . Because of the d i f f i c u l t i e s i nvo l ved i n t r a c i n g i n d i v i d u a l immuno-r e a c t i v e f i b e r s through severa l microscope s e c t i o n s , i t has not always been p o s s i b l e to determine which hypothalamic nucleus i s the o r i g i n of a g iven pathway. Sofroniew and Weindl (1978) , f o r example, a t t r i b u t e d the f i n e f i b e r s i n the l a t e r a l septum and the thalamus to a p r o j e c t i o n from the SCN, but a p r o j e c t i o n from the PVN seems more l i k e l y . Les ions of the SCN r e s u l t i n l o s s o f immunoreac t iv i ty on ly i n the p e r i v e n t r i c u l a r n u c l e u s , the dorsomedial n u c l e u s , and the organum vasculosum of the lamina t e rm in -a l s (OVLT) (Hoorneman and B u i j s , 1982) , and i n j e c t i o n of 3 H-Leuc ine i n t o the SCN r e s u l t s i n very l i t t l e l a b e l l i n g ou ts ide the hypothalamus (Berk and F i n k e l s t e i n , 1981) . A p r o j e c t i o n from the SON i s u n l i k e l y , s i nce e l e c t r i c a l s t i m u l a t i o n of the septum does not cause an t id romic e x c i t a -t i o n o f c e l l s i n the SON (Pou la i n e t . a l . , 1980) . The caudal p r o j e c t i o n to the cord and the medul la probably o r i g i n a t e s i n the PVN, s i nce i t can 12 be abo l i shed by b i l a t e r a l l e s i o n s in t h i s nucleus ( N i l a v e r , 1980) , and s ince i n j e c t i o n o f ho rse rad i sh perox idase i n t o the cord r e s u l t s i n l a b e l l i n g o f neurones i n the PVN (Ono e t . a l . , 1978) . Accord ing to B u i j s (1980) , the AVP and oxy toc i n con ta i n i ng f i b e r s i n the hippocampus appear to be a p r o -j e c t i o n o f the PVN. 2 . EFFECTS OF THE NHP PEPTIDES ON THE CNS The e x i s t e n c e o f nerve te rm ina l s c o n t a i n i n g oxy toc in and vasop ress in i n the CNS (v ide supra) suggests t h a t the pept ides can ac t d i r e c t l y on nerve c e l l s . There i s cons ide rab le ev idence t ha t t h i s i s the case . The b i o c h e m i c a l , p h y s i o l o g i c a l , and behav io ra l s t ud ies to be desc r i bed below demonstrate c l e a r l y t h a t the NHP pept ides can a f f e c t CNS f u n c t i o n , but i t i s hot always c l e a r t ha t the pept ides were a c t i n g d i r e c t l y on recep to rs i n CNS t i s s u e ra the r than on endocr ine recep to rs ou ts ide the CNS. S ince t h i s ques t ion i s r e l e v a n t to a l l the s t u d i e s to be desc r ibed below, the types of ev idence used to d i s t i n g u i s h cen t ra l e f f e c t s from those secondary to changes ou t s i de the CNS w i l l be d i scussed before the s tud ies themselves are p resen ted . That the pept ides ac t d i r e c t l y on CNS t i s s u e i s most apparent i n the i n v i t r o and the i o n t o p h o r e t i c exper iments , i n which a c t i o n s at pe r i phe ra l recep to rs would be i m p o s s i b l e . The e f f e c t s o f smal l (1 ng) q u a n t i t i e s o f pept ide i n j e c t e d i n t o the ce reb ra l v e n t r i c l e s are a l s o un-l i k e l y to i nvo l ve p e r i p h e r a l a c t i v i t y , e s p e c i a l l y i f i t can be shown t ha t much l a r g e r subcutaneous (sc) doses are requ i red to produce the same responses . That subcutaneous i n j e c t i o n s can produce the same response as i n t r a c e r e b r o v e n t r i c u l a r ( i c v ) i n j e c t i o n s i m p l i e s , o f c o u r s e , t ha t 13 the pept ides can c ross the b l o o d - b r a i n b a r r i e r . Whi le t h i s po in t i s s t i l l c o n t r o v e r s i a l , recent work in guinea p igs i n d i c a t e s tha t smal l q u a n t i t i e s o f the NHP pept ides en te r the ce reb rosp ina l f l u i d (CSF) i f the pe r i phe ra l dose i s s u f f i c i e n t l y l a rge (Jones and Rob inson, 1982) . A l t e r n a t i v e l y , the pept ides might be a c t i n g on the c i r c u m v e n t r i c u l a r organs or on the a n t e r i o r p i t u i t a r y , e i t h e r o f which would be a c c e s s i b l e from both the b lood and the CSF (Landgraf e t . a l . , 1979; Van D i j k e t . a l . , 1981) . Exper iments i n which l a r g e r q u a n t i t i e s o f pept ide (100 ng o r more) are i n j e c t e d i n t o the v e n t r i c l e s should be i n t e r p r e t e d w i th c a u t i o n , as p h y s i o l o g i c a l changes not d i r e c t l y r e l a t e d to neuronal a c t i v i t y may o c c u r . For example, i n j e c t i o n o f l a rge doses o f AVP i n t o the v e n t r i c l e s o f monkeys has been repor ted to cause an i nc rease i n the water p e r m e a b i l i t y o f b ra in c a p i l l a r i e s (Ra i ch le and Grubb, 1978). Subcutaneous i n j e c t i o n s o f DGLVP have been found to produce the same changes i n cond i t i oned behav ior as subcutaneous i n j e c t i o n s o f AVP or LVP (Bohus, 1974; de Wied, 1980). S ince DGLVP has l i t t l e endocr ine a c t i v i t y , (v ide supra) i t s e f f e c t on CNS f u n c t i o n cannot be secondary to vasocon-s t r i c t i o n , a n t i d i u r e s i s , o r ACTH r e l e a s e . The f a c t tha t the DGLVP recep to r i s d i s t i n c t from the c l a s s i c a l endocr ine r e c e p t o r s , however, does not n e c e s s a r i l y prove tha t i t i s l o c a t e d i n the CNS. DGAVP has sometimes been used i ns tead of DGLVP. I t i s g e n e r a l l y assumed t ha t DGAVP i s a l s o l a c k i n g in endocr ine a c t i v i t y , a l though except f o r i t s i n a b i l i t y to promote ACTH r e l e a s e (Van Di j k e t . a l . , 1981) , t h i s assumption does not seem to have been t e s t e d . 14 Biochemica l S tud ies Perhaps the best ev idence tha t CNS t i s s u e con ta ins recep to rs f o r the NHP pept ides i s prov ided by the b iochemica l changes tha t can be p ro -duced by the pept ides i n v i t r o . The oxy toc i n fragment PL6, f o r example, has been found to promote cGMP produc t ion i n a m i tochondr ia l and synaptosomal r a t b ra i n p repa ra t i on in v i t r o ( S p i r t e s e t . a l . , 1980) . - 9 - G + AVP, a t concen t ra t i ons o f 10 to 10 M, inc reased the K induced synthe-s i s and re l ease of se ro ton in from r a t hippocampal s l i c e s (Auerbach and L i p t o n , 1982) , and both oxy toc in and AVP (5X10" 7 M) reduced the K + evoked re l ease of 3H-dopamine from ra t s t r i a t a l s l i c e s ( S t a r r , 1982) . Many of the b iochemica l changes observed in v i vo are a l s o thought to be mediated by recep to rs in the CNS, a l though the ev idence i s u s u a l l y l e s s d i r e c t than w i th the in v i t r o s t u d i e s . I n t r ace reb ra l m i c r o i n j e c t i o n s of AVP (50 pg) i n t o c e r t a i n areas o f the r a t b r a i n , i n c l u d i n g the septum and the hippocampus, have been repor ted to a l t e r catecholamine tu rnover (Kovacs e t . a l . , 1979a; 1979b), and subcutaneous i n j e c t i o n s of DGLVP (5 yg) can a f f e c t the se ro ton in content o f the r a t hippocampus (Ramaekers e t . a l . , 1977). A d e f i c i e n c y i n 3 H - c o r t i c o s t e r o n e b ind ing capac i t y has been found in the hippocampus and the a n t e r i o r p i t u i t a r y of HoDI r a t s . Th i s d e f i c i e n c y can be c o r r e c t e d by subcutaneous i n j e c t i o n s of AVP, dDAVP, or DGAVP (1-2 yg d a i l y f o r 1 week) but not by oxy toc in or ACTH^-io (Ve ld ius and de K l o e t , 1982). The mechanism by which vasopress in a f f e c t s the 3 H - c o r t i c o s t e r o n e b ind ing c a p a c i t y and the l o c a t i o n o f the vasopress in recep to r are unknown. 15 P h y s i o l o g i c a l S tud ies Cooper e t . a l . (1979) and Kas t ing e t . a l . (1979) , us ing push -pu l l cannulae i n s e r t e d i n t o the septum of sheep, have repor ted tha t immuno-r e a c t i v e AVP i s r e l eased from the septum dur ing endotox in - induced f e v e r . I n j e c t i o n of AVP v i a the cannula reduced the f e b r i l e response to endotox in wh i le i n j e c t i o n o f an t i -AVP serum or o f a vasop ress in an tagon is t i nc reased the f e v e r , suggest ing tha t endogenous AVP re leased by t e rm ina l s i n the septum has an a n t i - f e b r i l e a c t i o n in sheep. There i s a l s o ev idence tha t endogenous AVP may be i nvo l ved i n the response to endotox in i n r a t s , s i nce systemic i n j e c t i o n of endo tox i n , which normal ly causes a f a l l i n body temperature i n r oden t s , produces the oppos i te r e a c t i o n in HoDI r a t s . A f t e r ch ron i c pret reatment w i th subcutaneous AVP, however, the HoDI r a t s respond normal ly to endotox in (Ngsee e t . a l . , 1980) . C e n t r a l l y admin is te red AVP (1 u g , i c v ) not on ly reduces the body tempera-tu re of r a t s , i t can a l s o cause convu ls ions i f g iven in repeated doses (Kas t ing e t . a l . , 1980). A p o s s i b l e r e l a t i o n between the a n t i - f e b r i l e and the c o n v u l s i v e e f f e c t s o f AVP has been s tud ied by Kas t ing e t . a l . (1981) , who found tha t both HoDI r a t s and Long-Evans r a t s t r ea ted wi th an t i -AVP serum ( i c v ) had h igher t h resho lds f o r hea t - induced convu ls ions than d id unt reated Long-Evans r a t s , r a i s i n g the p o s s i b i l i t y tha t AVP re l eased dur ing f eve r may be p a r t l y r e s p o n s i b l e f o r f e b r i l e c o n v u l s i o n s . Neu rophys io log i ca l S tud ies Pept ide induced a l t e r a t i o n s i n neuronal a c t i v i t y have a l s o been r e -p o r t e d . In the r a b b i t , c e n t r a l l y admin is te red LVP (approx imate ly 0 .2-20 ng , i c v ) i nc reased the m u l t i u n i t d i scharge ra te in the PVN and SON, wh i l e 16 oxy toc in had the oppos i te e f f e c t (Schwarzberg e t . a l . , 1974). I n j e c t i o n o f DGAVP (20 ng , i c v ) caused inc reased e lec t roencepha logram (EEG) the ta f requenc ies in r a t s , wh i le i n j e c t i o n o f an t i -AVP serum caused a decrease (Urban and De Wied, 1978) . Urban(1981) has a l s o r e p o r t e d , however, tha t as l i t t l e as 1 0 - 3 pg o f AVP o r oxy toc in reduced the ampl i tude of t he ta rhythms when i n j e c t e d d i r e c t l y i n t o the septum o f r a t s . The d isc repancy may r e f l e c t the d i f f e r e n c e in dose or i n s i t e o f i n j e c t i o n . I n j e c t i o n of LVP (5-12 ng) d i r e c t l y i n t o the hippocampus o f anes the t i zed r a t s has been repor ted to cause hippocampal spread ing depress ion (Huston and J a k o b a r t l , 1977). A more d i r e c t demonstrat ion o f the e f f e c t o f the NHP pept ides on neuronal a c t i v i t y has been repor ted by N i c o l 1 and Barker (1971) , who found t h a t i on topho res i s o f LVP i n h i b i t e d 80% o f a n t i d r o m i c a l l y i d e n t i f i e d sup raop t i c neurones and e x c i t e d 90% of c o r t i c a l neurones t e s t e d i n the b ra i n of the pen toba rb i t a l anes the t i zed c a t . The authors proposed tha t the AVP r e l e a s e d by axon c o l l a t e r a l s might be r e s p o n s i b l e f o r the r e c u r -ren t i n h i b i t i o n produced by an t i d rom ic s t i m u l a t i o n o f SON neurones. Recurrent i n h i b i t i o n has a l s o been observed i n the r a t SON (D re i f uss and K e l l y , 1972) , but the r o l e o f AVP i n t h i s phenomenon has been d i spu ted by D r e i f u s s e t . a l . (1973) , who demonstrated r ecu r ren t i n h i b i t i o n in the AVP d e f i c i e n t HoDI r a t . Whi le t h i s f i n d i n g would seem to exc lude AVP from c o n s i d e r a t i o n as the agent caus ing the i n h i b i t i o n , the ques t ion has not y e t been comple te ly r e s o l v e d . Leng and Wiersma (1981) , us ing more s o p h i s t i c a t e d t e c h n i q u e s , have r e c e n t l y shown tha t an t id romic s t i m u l a t i o n caused a reduc t i on o f burs t l eng th i n p h a s i c a l l y f i r i n g SON c e l l s i n Long Evans r a t s but not i n HoDI r a t s . I t would be h e l p f u l to determine whether r ecu r ren t i n h i b i t i o n in the SON can be b locked by l o c a l a p p l i c a t i o n o f a 17 vasopress in a n t a g o n i s t . In c o n t r a s t to the study by N i c o l l and Barker (1971) , Moss e t . a l . (1972) found i on topho res i s o f AVP to have r e l a t i v e l y l i t t l e e f f e c t on a n t i d r o m i c a l l y i d e n t i f i e d neurosecre to ry c e l l s i n urethane anes the t i zed r a t s and r a b b i t s ; AVP i n h i b i t e d on ly one o f 6 SON c e l l s t es ted ( spec ies not s p e c i f i e d , one SON c e l l was e x c i t e d ) and i n h i b i t e d on ly 2 o f 16 r a b b i t and 13 r a t PVN c e l l s (no PVN c e l l s were e x c i t e d ) . Un l i ke AVP, oxy toc in e x c i t e d a ma jo r i t y o f a n t i d r o m i c a l l y i d e n t i f i e d PVN c e l l s i n both the r a t and r a b b i t b r a i n , but d id not e x c i t e c e l l s i n the SON, c o r t e x , or tha lamus. The a c t i v i t y o f oxy toc i n i s not con f ined to neurosecre to ry c e l l s , however; i n a study by Mor r i s e t . a l . (1980) , oxy toc in a p p l i e d by i on topho res i s o r p ressure e j e c t i o n onto c e l l s i n the r a t caudal medul la was found to depress neuronal a c t i v i t y i n a l l areas t es ted (dorsa l column n u c l e u s , t r i g e m i n a l nucleus c a u d a l i s , r e t i c u l a r f o r m a t i o n , and in the area o f the nucleus ambiguus). Behav io ra l S tud ies Perhaps the most w ide l y used approach to s tudy ing the c e n t r a l e f f e c t s o f the NHP pept ides has been to determine the behav io ra l response to c e n t r a l or pe r i phe ra l i n j e c t i o n s . Cen t ra l i n j e c t i o n s o f oxy toc in or AVP (100-400 ng , i c v ) were repor ted to induce maternal behavior i n female r a t s , AVP being somewhat l e s s e f f e c t i v e than o x y t o c i n i n t h i s regard (Pedersen e t . a l . , 1982) . Cen t ra l (150-500 ng , i c v ) or sys temic (1500 ng , sc) i n j e c t i o n s o f LVP have been found to have an a n t i n o c i c e p t i v e e f f e c t i n r a t s (Kordower e t . a l . , 1981) , a f i n d i n g c o n s i s t e n t w i th the presence of immunoreactive f i b e r s i n the s u b s t a n t i a g e l a t i n o s a of the s p i n a l cord 18 ( N i l a v e r e t . a l . , 1980) . The l a r g e doses requ i red i n both these s t u d i e s , however, suggest t ha t the e f f e c t s may not be c e n t r a l l y mediated. Pe r i phe ra l i n j e c t i o n s of the o x y t o c i n fragment PLG p o t e n t i a t e the behav io ra l e f f e c t s o f L-Dopa (exc i tement , aggress iveness ) i n both hypophysectomized and i n -t a c t mice ( K a s t i n e t . a l . , 1976) and cause more r a p i d development of morphine t o l e r a n c e i n r a t s (van Ree and De Wied, 1976) . In m ice , c e n t r a l i n j e c t i o n s of the NHP pept ides (AVP, LVP, AVT, or o x y t o c i n ; 10-180 ng , i c v ) caused an i nc rease i n spontaneous f o r a g i n g , s c r a t c h i n g , and squeak-ing behav io r (Delanoy e t . a l . , 1979) . These s tud ies are summarized i n Table I I I . In some l a b o r a t o r i e s , however, c e n t r a l l y admin is te red vasopress in produces convu ls ions or o ther types of severe motor d i s t u r b a n c e . Kruse e t . a l . (1977) found t ha t AVP, AVT, or LVP i n j e c t e d i n t o the l a t e r a l v e n t r i c l e s of r a t s caused b a r r e l r o t a t i o n , p r o s t r a t i o n , and sometimes dea th . The t h r e s h o l d dose f o r the p roduc t ion o f ba r re l r o t a t i o n - a r o t a t i o n around the l o n g i t u d i n a l a x i s which has been desc r ibed by Kas t ing e t . a l . (1980) as a form o f u n i l a t e r a l convu l s i on - was 8 ng f o r AVP and AVT and 1.5 ng f o r LVP. O x y t o c i n , w i th a t h r e s h o l d dose of 1 y g , was ext remely weak i n t h i s t e s t . S i m i l a r r e s u l t s were desc r i bed by Abood e t . a l . (1980) who found tha t i n t r a v e n t r i c u l a r i n j e c t i o n o f 100 ng of AVP produced s e i z u r e s i n r a t s . Oxytoc in (1 yg) not on ly f a i l e d to produce convu ls ions but b locked the c o n v u l s i v e e f f e c t of a subsequent dose of AVP. Whether oxy toc i n s p e c i f i c a l l y an tagon izes AVP or whether i t has a more general a n t i c o n v u l s a n t a c t i o n i n t h i s t e s t i s not c e r t a i n , a l though the f i n d i n g t h a t oxy toc i n ( ~ 6 ng , i c v ) r a p i d l y te rminates glutamate induced s e i z u r e s i n r a b b i t s (Schulz e t . a l . , . 1 9 7 4 ) would support the l a t t e r i n t e r -p r e t a t i o n . Table I I I . Behav iora l E f f e c t s o f Neurohypophysial P e p t i d e s . PEPTIDE DOSE RESPONSE REFERENCE OXT 100-400 ng i c v Maternal Behav ior Pedersen e t . a l . , 1982 AVP ~ 8 ng i c v Ba r re l Ro ta t ion Kruse e t . a l . , 1977 OXT ~1 yg i c v ii " AVP 1 0 - 6 0 ng i c v Foraging (mice) Delanoy e t . a l . , 1979 OXT . 180 ng i c v AVP 100 ng i c v Convuls ions Abood e t . a l . , 1980 OXT 5 yg i c v A n t i c o n v u l s i v e n LVP 150-500 ng i c v A n t i n o c i o c e p t i o n Kordower e t . a l . , 1981 LVP 1500 ng sc II II AVP 1 yg i c v Immobi l i ty Kas t ing e t . a l . , 1980 AVP (3rd dose) 10 ng i c v Convuls ions II AVP 1 ng i c v Memory Retent ion Koob e t . a l . , 1981b AVP 1 yg sc " The behav io ra l response to i n t r a c e r e b r o v e n t r i c u l a r ( i c v ) and subcutaneous (sc) i n j e c t i o n s o f the NHP pept ides i s shown. A l l experiments were performed w i th r a t s except as i n d i c a t e d . In the ^ s t u d i e s by Kruse e t . a l . (1977) and by Delanoy e t . a l . (1979) , the dose l i s t e d f o r AVP a p p l i e s to £ LVP and AVT as w e l l . 20 I t i s not c l e a r why vasop ress in produces such extreme responses i n some l a b o r a t o r i e s but not i n o t h e r s . Kruse e t . a l . (1977) , f o r example, repor ted a 33% m o r t a l i t y i n r a t s g iven as l i t t l e as 40 ng of LVP, wh i le Kordower e t . a l . (1981) mention on ly an a n t i n o c i o c e p t i v e e f f e c t w i th doses as high as 500 ng. The s l i g h t d i f f e r e n c e s in technique between the va r ious s tud ies do not prov ide an obvious exp lana t i on f o r the d i s c r e p a n c y , and there i s no c l e a r ev idence t ha t the response v a r i e s accord ing to the s t r a i n o f r a t or the commercial source of the hormone. The response does, however, depend on the prev ious h i s t o r y of the r a t . Exper iments i n v o l v i n g repeated i n j e c t i o n s of AVP (Burnard e t . a l . , 1982; Kas t i ng e t . a l . , 1980) have shown t ha t the convu l s i ve a c t i o n o f AVP i nvo l ves a s e n s i t i z a t i o n p rocess ; r a t s t r e a t e d w i th an i n i t i a l i n j e c t i o n o f AVP (1 y g , i c v ) e x h i b i t e d on ly minor e f f e c t s such as i m m o b i l i t y , but a second i n j e c t i o n 2 days l a t e r produced f u l l c o n v u l s i o n s , and on the f i f t h day of the exper iment an i n j e c t i o n o f on ly 10 ng was s u f f i c i e n t to produce f u l l c o n v u l s i o n s . HoDI r a t s d i d not d i f f e r from normal ra t s i n t h e i r i n i t i a l response but became s e n s i t i z e d more r a p i d l y . The mechanism o f t h i s s e n s i t i z a t i o n was not determined. Kas t i ng e t . a l . (1980) have noted the s i m i l a r i t y between AVP s e n s i t i z a t i o n and k i n d l i n g , a process by which repeated e l e c t r i c a l o r chemical s t i m u l a t i o n o f c e r t a i n b ra i n a r e a s , p a r t i -c u l a r l y the amygdala, r e s u l t s i n a lowered t h resho ld f o r convu ls ions ( G i r g i s , 1981) . I t i s p o s s i b l e t ha t i n t r a v e n t r i c u l a r AVP s t imu la tes neurones i n the amygdala, an area known to be innerva ted by f i b e r s c o n t a i n -ing immunoreactive AVP ( B u i j s and Swaab, 1979) , and t he re fo re produces k i n d l i n g . I f t h i s i s the c a s e , r a t s s e n s i t i z e d to AVP should a l s o be more s u s c e p t i b l e to s e i z u r e s produced by o ther methods of s t i m u l a t i n g the 21 amygdala, a p o s s i b i l i t y t ha t has not ye t been t e s t e d . A l t e r n a t i v e l y , cen t ra l i n j e c t i o n s o f vasop ress in may cause an i nc rease i n AVP recep to rs i n the b r a i n , thus render ing the r a t s more s e n s i t i v e to AVP but not to o ther convu l s i ve agen ts . A general i nc rease in AVP recep to rs in the b ra in i s u n l i k e l y , s ince the s e n s i t i z e d r a t s are not more respons ive to the hypothermic a c t i o n s o f AVP, but an i nc rease con f ined to a p a r t i c u l a r pathway remains a p o s s i b i l i t y . Whatever the mechanism, one obvious i m p l i -c a t i o n o f the s e n s i t i z a t i o n i s t h a t r a t s used i n behav io ra l exper iments shou ld not be g iven repeated i n j e c t i o n s o f AVP. F a i l u r e to app rec ia te t h i s f a c t may account f o r some of the c o n f l i c t i n g dose response r e l a t i o n -sh ips mentioned above. The recep to rs i nvo l ved i n the p roduc t ion o f s e i z u r e s and r e l a t e d behav io r , u n l i k e the c l a s s i c a l endocr ine recep to rs desc r ibed in Table I I , appear to be more s e n s i t i v e to LVP than to AVP. In the study by Abood e t . a l . (1980) , f o r example, LVP was repor ted to be 3 to 5 t imes more e f f e c t i v e as a convu l s i ve agent than was AVP. Odd ly , removal o f the g lyc inamide res idue by t r y p t i c d i g e s t i o n - to produce DGLVP and DGAVP - dest royed the convu lsan t a c t i v i t y o f LVP but not t h a t o f AVP. I t i s p o s s i b l e how-e v e r , t h a t the convu lsan t a c t i v i t y o f the DGAVP was due to contaminat ion w i th i n t a c t AVP. As noted above, Kruse e t . a l . (1977) a l s o found LVP to be more a c t i v e than AVP i n produc ing ba r re l r o t a t i o n , a l though comparison o f the two pept ides was d i f f i c u l t because o f the "ext remely f l a t " dose response c u r v e s . DGLVP was not e f f e c t i v e ; i n f a c t , Kruse e t . a l . (1977) suggest t ha t DGLVP may have a b l o c k i n g e f f e c t , s ince LVP d id not induce ba r re l r o t a t i o n in r a t s p re t rea ted w i th a h igh dose (5 y g , i c v ) o f DGLVP. There may a l s o be recep to rs i n the b r a i n t ha t respond s p e c i f i c a l l y 22 to AVT. Pavel has repor ted t h a t ex t remely smal l amounts o f AVT ( 1 0 ~ 1 8 g) i n j e c t e d i n t o the t h i r d v e n t r i c l e o f the ca t can induce slow wave s l eep (Pavel e t . a l . , 1977a) and can reduce plasma C o r t i s o l l e v e l s (Pavel e t . a l . , 1977b), p o s s i b l y by a c t i n g on neurones i n the m i d l i n e raphe nuc leus . Oxytoc in and AVP were i n a c t i v e i n t h i s t e s t , even when doses as h igh as 1 0 " 6 pg were used. In view of the q u a n t i t i e s o f AVT i n v o l v e d , the repo r t s shou ld probab ly be viewed wi th c a u t i o n . E f f e c t s on Condi t i nned Behav ior Behav io ra l s t u d i e s have a l s o i m p l i c a t e d the NHP pept ides i n the con t ro l o f memory p r o c e s s e s . P o s t e r i o r lobectomized r a t s are l e s s r e s i s t a n t than normal r a t s to e x t i n c t i o n of a cond i t i oned avoidance response , but t h i s d e f i c i t can be c o r r e c t e d by i n j e c t i o n o f vasop ress in (~ 1 y g , sc) (Burbach and de Wied, 1981). Vasopress in i s a l s o e f f e c t i v e in p reven t ing the amnesia caused by puromycin, C 0 2 , . o r e l e c t r o c o n v u l s i v e shock (F lexner e t . a l . , 1978; Kovcics e t . a l . , 1979a). The e f f e c t i s not con f ined to ra t s w i th a memory d e f e c t ; vasop ress in g iven to normal r a t s w i l l a l s o de lay e x t i n c t i o n o f a c t i v e o r pass ive cond i t i oned avoidance responses (Bohus e t . a l . , 1972; 1978) . Whi le the t im ing o f the t r i a l s and the i n j e c t i o n s v a r i e s i n the d i f f e r e n t exper imen ts , i t appears tha t the vasop ress in i s e f f e c t i v e when i n j e c t e d s h o r t l y before or a f t e r (w i t h i n about one hour) the l e a r n i n g t r i a l or s h o r t l y before the r e t e n t i o n t r i a l but not i f i n j e c t e d a t i n t e r -mediate t imes . S ince vasop ress in i n j e c t e d at the t ime o f the l e a r n i n g t r i a l can i nc rease r e t e n t i o n measured days l a t e r , when the pept ide i s no longer p resen t , the i nc rease has been a t t r i b u t e d to improved c o n s o l i d a -t i o n of memory (de Wied, 1980; K r e j c i e t . a l . , 1979). R i g t e r (1982) has 23 argued t ha t the improved c o n s o l i d a t i o n can not be the r e s u l t o f changes i n a t t e n t i o n , a r o u s a l , o r m o t i v a t i o n , s i nce vasopress in i n j e c t e d immediate ly a f t e r the l e a r n i n g t r i a l i s completed has the same e f f e c t as e a r l i e r i n -j e c t i o n s . The e f f e c t i v e n e s s o f i n j e c t i o n s g iven j u s t p r i o r to the r e t e n -t i o n t r i a l suggests t ha t the pept ide a l s o promotes r e t r i e v a l o f memory (Bohus e t . a l . , 1972; 1978; Kovacs e t . a l . , 1979a). In most l a b o r a t o r i e s the a c t i o n o f oxy toc in i s oppos i te to t ha t o f v a s o p r e s s i n , i n h i b i t i n g both c o n s o l i d a t i o n and r e t r i e v a l of memory (Bohus e t . a l . , 1978) , but i n some exper iments oxy toc in has been found to mimic the memory promoting a c t i o n s o f vasop ress in (van Wimersma Greidanus e t . a l . , 1981) . I n t r a v e n t r i c u l a r i n j e c t i o n s o f an t i -AVP serum i n t e r f e r e w i th memory p r o c e s s e s , wh i le a n t i - o x y t o c i n serum enhances memory, as would be expected i f endogenous pept ides are i nvo l ved i n memory processes (Bohus e t . a l . , 1978; Burbach and de Wied, 1981). The memory de fec t observed i n HoDI r a t s a l s o suggests a r o l e f o r endogenous AVP (de Wied, 1980) . The vasopress in an tagon is t dPTyr(Me)AVP (25 y g , sc) antagonizes the e f f e c t of exogenous AVP on behav io r , and , i n h igher doses (100 ug) hastens e x t i n c t i o n o f con-d i t i o n e d responses in r a t s not t r e a t e d w i th AVP (Le Moal e t . a l . , 1981; Koob e t . a l . , 1981a; Koob e t . a l . , 1981b). As would be expec ted , the oxy toc in an tagon is t N-a - a c e t y l - [ 2 - 0 - m e t h y l t y r o s i n e ] oxy toc in enhances cond i t i oned avoidance behav ior ( K r e j c f e t . a l . , 1981) . The s i t e o f a c t i o n of the pept ides has not been c o n c l u s i v e l y d e t e r -mined, but the e f f e c t on memory i s probably not the r e s u l t o f hormonal ac t i ons a t p e r i p h e r a l s i t e s , s i nce vasopress in i n j e c t e d i n t o the l a t e r a l v e n t r i c l e s i s e f f e c t i v e a t much lower doses (1 ng) than vasopress in i n -j e c t e d s y s t e m i c a l l y (1 ug) (Koob e t . a l . , 1981b), and s ince DGLVP produces 24 the same e f f e c t s as vasop ress in (de Wied e t . a l . , 1972). M i c r o i n j e c -t i ons o f AVP (50 pg) i n t o the hippocampal dentate gy rus , the dorsa l septum, o r the do rsa l raphe* nucleus enhanced "performance i n a pass ive avoidance memory t e s t . Oxytoc in had the oppos i te e f f e c t when i n j e c t e d i n t o the dentate gyrus or the raphe nucleus but mimicked the e f f e c t of AVP i n the septum (Kovcics e t . a l . , 1979a; 1979b). Th i s work suggests t ha t the r e -sponse to oxy toc i n v a r i e s accord ing to the s i t e o f a c t i o n and may e x p l a i n why oxy toc in i s more v a r i a b l e i n i t s a c t i o n s than i s vasop ress in (van Wimersma Greidanus e t . a l . , 1981) . Another exp lana t i on f o r the v a r i a b i l i t y observed w i th oxy toc in i s t ha t the nonapept ides may be e n z y m a t i c a l l y de-graded to a c t i v e f ragments . Accord ing to t h i s v iew, proposed by Burbach and de Wied (1981) , the N-terminal hexapept ide o f vasop ress in promotes c o n s o l i d a t i o n of memory wh i l e the C- te rmina l t r i p e p t i d e promotes r e t r i e v a l . The i n t a c t vasop ress in mo lecu le , as mentioned above, f a c i l i t a t e s both con-s o l i d a t i o n and r e t r i e v a l . Both the C- te rmina l and N- terminal fragments o f oxy toc in f a c i l i t a t e memory processes (Burbach and de Wied, 1981; F lexner e t . a l . , 1978) and thus have an a c t i o n oppos i te to t ha t of the i n t a c t oxy toc in mo lecu le . The response to an i n j e c t i o n o f oxy toc in would t he re fo re depend on the ex ten t to which the molecule i s c leaved i n v i v o . Th i s s i t u a t i o n i s s i m i l a r to t ha t desc r i bed by C e l i s e t . a l . (1971) , i n which the e f f e c t o f oxy toc in on MSH re l ease i s thought to be c o n t r o l l e d by v a r i a t i o n s i n the a c t i v i t y o f p r o t e o l y t i c enzymes i n the median eminence. In c l i n i c a l s t u d i e s , pe r i phe ra l a d m i n i s t r a t i o n of vasopress in has been repor ted to improve memory i n humans (Moeglen e t . a l . , 1979; Weingartner e t . a l . , 1981) . Changes i n mental s t a t e (eupho r i a , w e l l -25 being) and i n EEG a c t i v i t y have a l s o been repor ted (Moeglen e t . a l . , 1979) . The memory enhancing a c t i o n s o f vasop ress in are not an e n t i r e l y con-s i s t e n t f i n d i n g . For example, Bohus e t . a l . (1978) repor ted tha t vaso-p r e s s i n f a c i l i t a t e d memory i n a pass ive avoidance t e s t ( l a tency to en ter a box i n which a shock had been rece i ved 24 hours p r e v i o u s l y ) , but Hos te t te r e t . a l . (1980) were unable to demonstrate any e f f e c t of vasop ress in i n the same t e s t , and R i g t e r (1980) was ab le to demonstrate enhanced avoidance behav ior on l y i n r a t s t ha t had been a l lowed to become f a m i l i a r w i th the apparatus before the l e a r n i n g t e s t . Sahgal e t . a l . (1982) found tha t vasop ress in produced a bimodal response - both the number o f very shor t l a t e n c i e s and the number o f very long l a t e n c i e s were i nc reased by vaso-p r e s s i n . The bimodal response was s a i d to be more c o n s i s t e n t w i th i n -creased a rousa l than w i th improved memory. The memory de fec t a t t r i b u t e d to HoDI r a t s (de Wied, 1980; Burbach and de Wied, 1981) has been quest ioned by some workers . HoDI r a t s were repor ted by B r i t o e t . a l . (1981) to l e a r n more s l ow l y than c o n t r o l s , p o s s i b l y due to g rea te r t i m i d i t y , but to r e t a i n avoidance behav io r l o n g e r , a f i n d i n g not c o n s i s t e n t w i th a s p e c i f i c de fec t i n r e t e n t i o n . S i m i l a r r e s u l t s were repor ted by M i l l e r e t . a l . (1976) , who found t ha t HoDI r a t s had no de fec t i n r e t e n t i o n t ha t cou ld not be accounted f o r by the de lay i n l e a r n i n g , and by Carey and M i l l e r (1982) , who found tha t HoDI r a t s had b e t t e r r e t e n t i o n of pass ive avoidance be-hav io r than d i d Long Evans r a t s . These s t u d i e s do not argue aga ins t a r o l e f o r the NHP pept ides i n behav io r , but they do suggest tha t i t may be premature to a s c r i b e tha t r o l e to a s p e c i f i c e f f e c t on memory p rocesses . 26 3 . The Present Study The s t u d i e s desc r ibed above prov ide compe l l ing ev idence tha t the NHP pept ides are i nvo l ved i n CNS f u n c t i o n , but desp i t e the d i v e r s i t y of these s t u d i e s r e l a t i v e l y l i t t l e i s known about the nature of tha t invo lvement . In some types o f exper imen ts , p a r t i c u l a r l y those i n which the behav io ra l response to cen t ra l or pe r i phe ra l i n j e c t i o n o f pept ide are measured, the repor ted e f f e c t s o f the pept ides are not e n t i r e l y c o n s i s t e n t (Hos te t te r e t . a l . , 1980; Sahgal e t . a l . , 1982) . Th i s i n c o n s i s t e n c y i s not s u r p r i s -i n g , s i nce the ex tens i ve f i b e r p r o j e c t i o n s revea led by immunocytochemistry suggest t ha t the pept ides are a c t i v e i n many areas of the CNS, and there i s ev idence t ha t they may produce opposing responses i n d i f f e r e n t a r e a s . For example, oxy toc in enhances r e t e n t i o n o f cond i t i oned behav ior when i n -j e c t e d i n t o the septum but decreases r e t e n t i o n when i n j e c t e d i n t o the dentate gyrus or the do rsa l raphe nucleus (Kovacs e t . a l . , 1979b). An understanding of the f unc t i ons o f the NHP pept ides i n the CNS w i l l r equ i r e a more d e t a i l e d knowledge of the a c t i o n s of these pept ides a t i n d i v i d u a l t a rge t areas than i s c u r r e n t l y a v a i l a b l e . In p a r t i c u l a r , i t would be use fu l to know what types of NHP pept ide recep to r are present i n each a r e a . Which a r e a s , f o r example, respond to o x y t o c i n , and which respond to DGLVP? Do some areas respond to fragments o f NHP pept ides? I n j e c t i o n of pept ide i n t o the t a rge t a r e a , however, i s not an e n t i r e l y s a t i s f a c t o r y t echn ique , s i nce the pept ide i s not p rope r l y con f ined to the s i t e o f i n j e c t i o n . Kovacs e t . a l . (1982) have shown tha t the m i c r o -i n j e c t i o n o f an t i -AVP serum i n t o the dentate gyrus o f the dorsa l h ippo-campus r e s u l t e d i n the spread of an t i se rum, t raced by an immunoperoxidase techn ique , a long the su r face o f the hippocampus as f a r c a u d a l l y as the 27 ven t ra l hippocampus and r o s t r a l l y to the l a t e r a l septum. Ion tophores is prov ides a much more l o c a l i z e d a p p l i c a t i o n o f a pept ide and a l l ows the response of i n d i v i d u a l neurones to be mon i to red , bu t , u n t i l r e c e n t l y , i on topho res i s o f oxy toc in and vasop ress in had been attempted on ly i n the hypothalamus and the co r tex ( N i c o l l and B a r k e r , 1971; Moss e t . a l . , 1972) . A l t e r n a t i v e l y , the t i s s u e of i n t e r e s t can be i s o l a t e d and s tud ied in v i t r o , bu t , as o f the t ime t ha t the present i n v e s t i g a t i o n was begun, t h i s technique had been used on ly to demonstrate the p resynap t i c a c t i o n s of the NHP pep t i des . The present study was undertaken to determine the e f f e c t o f the NHP pept ides on the a c t i v i t y o f i n d i v i d u a l neurones i n the septum and the h ippo-campus of the r a t . Both o f these areas are known to be innerva ted by immunoreactive f i b e r s ( B u i j s , 1980) . As desc r ibed more f u l l y i n the p r e -v ious s e c t i o n o f t h i s r e p o r t , the septum i s b e l i e v e d to be the s i t e respon-s i b l e f o r the hypothermic ac t i ons of vasop ress in (Kas t i ng e t . a l . , 1979) , and both the septum and the hippocampus have been i m p l i c a t e d i n the be-h a v i o r a l and b iochemica l responses to the NHP p e p t i d e s . The pept ides were a p p l i e d both by i on topho res i s i n the septum and the hippocampus of the i n t a c t r a t and by super fus ion i n the i n v i t r o hippocampus, and the r e -sponse was moni tored by e x t r a c e l l u l a r m i c r o p i p e t t e s . In a d d i t i o n , the AVP content o f the do rsa l and of the ven t ra l po r t i ons o f the hippocampus was measured by radioimmunoassay, s i nce there appears to be a disagreement i n the immunocytochemical repo r t s as to whether f i b e r s con ta i n i ng immuno-r e a c t i v e AVP are found i n both these areas or on ly i n the ven t ra l h ippo-campus ( B u i j s e t . a l . , 1978; B u i j s , 1980; Sof ron iew and W e i n d l , 1981). 28 C. EXPERIMENTAL TECHNIQUES 1. RADIOIMMUNOASSAY The f i r s t radioimmunoassay was developed by Yalow and Berson (1959) , who d i scove red tha t 1 3 1 I - i n s u l i n cou ld be q u a n t i t a t i v e l y d i s p l a c e d from i n s u l i n - b i n d i n g a n t i b o d i e s by the a d d i t i o n o f un labe l l ed i n s u l i n . The technique was found to be g e n e r a l l y a p p l i c a b l e , not j u s t to o ther pept ide hormones, but to v i r t u a l l y any a n t i g e n i c subs tance . The p r i n c i p l e on which the assay i s based i s r e l a t i v e l y s imp le : u n l a b e l l e d an t igen i s a l lowed to compete w i th l a b e l l e d an t igen ( " t r a c e r " ) f o r a l i m i t e d number o f an t ibody b ind ing s i t e s . The amount o f t r a c e r t ha t w i l l b ind to the a n t i -body i s dependent on the amount o f u n l a b e l l e d an t igen which i s added; the g rea te r the amount o f u n l a b e l l e d a n t i g e n , the sma l l e r the p ropo r t i on o f t r a c e r t ha t w i l l be bound. To determine the concen t ra t i on o f an t igen i n the sample, the p ropo r t i on o f t r a c e r bound i n the presence of the sample i s compared to the p ropo r t i on bound i n the presence of known ( "s tandard" ) concen t ra t i ons o f u n l a b e l l e d a n t i g e n . The an t igen may not be the on l y substance i n the sample tha t can a f f e c t the b i n d i n g , however. Ions conta ined i n or added to the sample and chemica ls used to preserve the sample ( h e p a r i n , b a c t e r i o s t a t i c agen ts , and enzyme i n h i b i t o r s ) can i n t e r f e r e w i th the an t i gen -an t i body r e a c t i o n (Yalow and Be rson , 1971a, 19:71b). The k i n e t i c s o f the r e a c t i o n can a l s o be a l t e r e d by t i s s u e or plasma p r o t e i n s which are capable o f b ind ing the l a b e l l e d or u n l a b e l l e d an t igen (Tho re l l and L a r s o n , 1978, p. 169) . De-s t r u c t i o n o f the ant ibody or o f the l a b e l by p r o t e o l y t i c enzymes i n the sample w i l l reduce the b i n d i n g , wh i l e d e s t r u c t i o n o f u n l a b e l l e d an t igen w i l l have the oppos i te e f f e c t (Rober tson , 1977; Yalow and S t r a u s , 1980). 29 In te r fe rence by s a l t s and chemica ls can o f ten be min imized by d i s s o l v i n g the s tandard i n a s o l u t i o n s i m i l a r i n composi t ion to the sample; t h i s i s u s u a l l y f e a s i b l e on ly f o r samples in r e l a t i v e l y s i m p l e , non-prote inaceous s o l u t i o n s such as CSF. I n t e r f e r i n g substances can sometimes be removed by s imple sepa ra t i on techn iques ( d i a l y s i s , chromatography), but i n many cases i t i s necessary to use more e labo ra te p u r i f i c a t i o n procedures . Acetone/pet ro leum e the r e x t r a c t i o n (Rober tson , 1973) , f o r example, i s use-f u l f o r removing p ro te i ns and f a t s from smal l p e p t i d e s . Even i f i n t e r f e r e n c e by un re la ted substances i s e l i m i n a t e d , the measure-ment w i l l be accura te on ly i f the an t igen used as a s tandard i s immuno-l o g i c a l l y i d e n t i c a l to the an t igen present i n the sample (Yalow and Berson , 1971b). In the case o f the pept ide hormones, t h i s i d e n t i t y i s o f ten d i f -f i c u l t to e s t a b l i s h . Spec ies d i f f e r e n c e s i n hormone s t r u c t u r e , he te ro -gene i t y o f hormone forms w i t h i n a s p e c i e s , and the ex i s t ence of hormone fragments and p recu rso rs can compl ica te i n t e r p r e t a t i o n of the exper iment or r e s u l t i n c o n f l i c t i n g repo r t s from d i f f e r e n t l a b o r a t o r i e s (Yalow and Be rson , 1971a). For example, i f the hormone to be measured i s present as par t o f an immuno log ica l l y l e s s a c t i v e p r e c u r s o r , the assay w i l l p ro -duce d e c e p t i v e l y low v a l u e s . Conve rse l y , e r roneous ly high va lues can occur i f the sample con ta ins pept ides immuno log ica l l y s i m i l a r to the hormone in q u e s t i o n . For t h i s reason , i t i s e s s e n t i a l to determine the ex ten t to which the ant iserum w i l l c ross reac t w i th any r e l a t e d pept ides tha t may be present i n the sample. Severa l techn iques have been developed to demonstrate t ha t the immunoreactive ma te r i a l i n the sample i s i d e n t i c a l to the s t anda rd . One can show, f o r example, tha t the sample and the s tandard behave i d e n t i c a l l y i n seve ra l chromatographic systems. I d e n t i t y i s a l s o 30 i m p l i e d , but not proved (Yalow and Be rson , 1971a) , i f s e r i a l d i l u t i o n o f the sample produces a curve p a r a l l e l to or super imposable on the s tandard cu rve . Perhaps the most e f f e c t i v e method o f e s t a b l i s h i n g the i d e n t i t y o f the ma te r i a l i n the sample i s to compare the immunoreact iv i ty o f the sample wi th t ha t o f s i m i l a r t i s s u e obta ined from hormone d e f i c i e n t mutants or from animals s u r g i c a l l y depr ived of the source of the hormone. The cho ice o f techniques w i l l depend on the nature o f the sample, the type o f i n t e r -ference a n t i c i p a t e d , and the m a t e r i a l s a v a i l a b l e . 2 . MICR0I0NT0PH0RESIS M i c r o i o n t o p h o r e s i s has become a we l l accepted method f o r app l y i ng drugs and o ther subs tances , such as amines, amino a c i d s , and p e p t i d e s , i n t o the immediate v i c i n i t y o f the i n d i v i d u a l nerve c e l l s ( K e l l y e t . a l . , 1975) . In the most w ide l y used v a r i a t i o n o f the t echn ique , s o l u t i o n s of the compounds to be tes ted are p laced i n separate b a r r e l s o f a m u l t i b a r r e l m i c r o p i p e t t e , the cen t ra l ba r re l o f which i s used to record the a c t i v i t y o f the c e l l . The compounds are e x p e l l e d from the m ic rop ipe t t e ba r re l by pass ing a cu r ren t o f approp r ia te p o l a r i t y through the b a r r e l . C a t i o n i c molecu les are e x p e l l e d by a p o s i t i v e vo l tage app l i ed to the b a r r e l , wh i l e a n i o n i c molecu les are e x p e l l e d by a negat ive v o l t a g e . D i f f u s i o n of the compound from the m ic rop ipe t t e t i p between a p p l i c a t i o n s i s prevented by the passage of a r e t a i n i n g cu r ren t o f oppos i te p o l a r i t y to the e j e c t i n g c u r r e n t . S ince the r e t a i n i n g cu r ren t draws the compound away from the t i p , there i s a de lay i n re lease of the compound when the e j e c t i n g cu r ren t i s a p p l i e d again (Pu rves , 1979; C la rke e t . a l . , 1973) . Fo l l ow ing t h i s d e l a y , the ra te o f r e l ease i s constant and i s u s u a l l y p ropo r t i ona l to 31 the cu r ren t f l ow (Brad ley and Candy, 1970; Z ieg lgansberger e t . a l . , 1969) . Molecu les w i th a h igh charge/mass r a t i o w i l l , i n g e n e r a l , be e x p e l l e d more r a p i d l y than more neu t ra l mo lecu les . The net charge on the molecule can be maximized by a d j u s t i n g the pH o f the s o l u t i o n , c a t i o n i c molecules being r e l a t i v e l y more p o s i t i v e l y charged a t low pH. Much o f the cu r ren t w i l l , o f c o u r s e , be c a r r i e d by the more mobi le H + and OH" ions ra the r than by the compound of i n t e r e s t . S ince H + ions can themselves a f f e c t neuronal a c t i v i t y (F rede r i ckson e t . a l . , 1971; Gruol e t . a l . , 1980) , i t i s necessary to show tha t the e f f e c t observed dur ing i on topho res i s o f the compound cannot be d u p l i c a t e d by i on topho res i s o f H + or OH" ions from a con t ro l s o l u t i o n . A l t e r n a t i v e l y , one can demonstrate tha t the e f f e c t can be b locked by a s p e c i f i c a n t a g o n i s t . S i m i l a r c o n s i d e r a t i o n s app ly to N a + , C I " , a c e t a t e , or any o ther ions present i n the s o l u t i o n . The re l ease of a compound i s sometimes prevented by blockage of the e l e c t r o d e t i p . Blockage i s i n d i c a t e d by a sharp i nc rease i n the r e s i s -tance o f the b a r r e l , by o s c i l l a t i o n s i n the r e s i s t a n c e , or by e l e c t r i c a l no ise (Z i eg lgansbe rge r , e t . a l . , 1969, 1974) . Z ieg lgansberger e t . a l . (1974) , i n a study o f the re l ease o f l a b e l l e d amino ac i ds and amines from i o n t o p h o r e t i c p i p e t t e s , a t t r i b u t e d the blockage o f the p i p e t t e s to a t t a c h -ment of the compounds to dust and g l ass p a r t i c l e s i n the t i p . The e f f e c t was compared to t ha t of a semipermeable membrane, s i nce cu r ren t cou ld s t i l l be passed through the b locked t i p , but the cu r ren t was c a r r i e d by smal l ions i ns tead o f by the l a b e l l e d compounds. S i m i l a r r e s u l t s were repor ted by Purves (1979) , who found t ha t sudden i nc reases i n r e s i s t a n c e caused a complete c e s s a t i o n o f r e l ease o f a f l u o r e s c e n t dye even though cu r ren t was s t i l l passed . Pep t ides are e s p e c i a l l y l i k e l y to cause blockage 32 o f the p i p e t t e t i p (Palmer e t . a l . , 1980) , presumably because of t h e i r r e l a t i v e l y low charge to weight r a t i o and t h e i r tendency to s t i c k to g l a s s . N a t u r a l l y , e f f e c t s observed dur ing the passage of cu r ren t through a b locked p i p e t t e cannot be a t t r i b u t e d to the drug or pept ide being s t u d i e d . 3 . THE HIPPOCAMPAL SLICE The t ransve rse hippocampal s l i c e , f i r s t developed by Skrede and Westgaard (1971) , has q u i c k l y become one of the most popular neurophys io -l o g i c a l p repa ra t i ons a v a i l a b l e . One reason f o r t h i s p o p u l a r i t y i s the we l l de f i ned anatomical s t r u c t u r e o f the hippocampus. As i l l u s t r a t e d s c h e m a t i c a l l y i n F igu re 1, t h i s s t r u c t u r e i s preserved i n the hippocampal s l i c e . The two major c e l l types are arranged i n l a y e r s , the pyramidal c e l l l a y e r o f the hippocampus proper ( d i v i d e d i n t o reg ions CA1 to CA4) and the granu le c e l l l a y e r o f the dentate gy rus . S ince these l a y e r s are r e a d i l y v i s u a l i z e d under low power m a g n i f i c a t i o n i n v i t r o , the e l e c t r o d e s can be p o s i t i o n e d more e a s i l y and w i th g rea te r accuracy than i s p o s s i b l e w i th s t e r e o t a x i c t echn iques . Interneurones are a l s o present i n the h ippo-campus; these are l oca ted p r i m a r i l y i n or near the pyramidal c e l l l a y e r s (Lee e t . a l . , 1980) , but s ca t t e red c e l l s are found throughout the h ippo-campus (Shepard, 1974; G a l l e t . a l . , 1981). Fur thermore, the major f i b e r p r o j e c t i o n s w i t h i n the hippocampus - the pe r fo ran t path p r o j e c t i o n to the granule c e l l s , the mossy f i b e r pathway from the granu le c e l l s to CA3, and the S c h a f f e r c o l l a t e r a l pathway from CA3 to CAT - are a l l a l i g n e d roughly p a r a l l e l to the p a r a s a g i t t a l plane (L0mo, 1971) . I f the hippocampus i s cu t a long the same plane (F igu re I B ) , the p r o j e c t i o n s w i l l remain i n t a c t i n the s l i c e p r e p a r a t i o n . In the present exper iment the Scha f f e r c o l l a t e r a l 33 S C H A F F E R C O L L A T E R A L C A 1 P Y R A M I D A L C E L L v P Ij LAYEf R ' / S T R A T U M ] RADIATUM P E R F O R A N T PATH C A 3 P Y R A M I D A L C E L L D E N T A T E A R E A G R A N U L E C E L L S E P T U M F I M B R I A S L I C E H I P P O C A M P U S C A U D A L F igure 1. The anatomy o f the r a t hippocampal s l i c e . (A) Schemat ic drawing of the hippocampal s l i c e as i t appears under low power m a g n i f i c a t i o n . The pyramidal c e l l l a y e r ( t r i a n g l e s ) and the granu le c e l l l a y e r ( c i r c l e s ) are i n d i c a t e d , and r e p r e s e n t a t i v e c e l l types have been sketched to i n d i c a t e the o r i e n t a t i o n o f the d e n d r i t e s . Only those p r o j e c t i o n s d i scussed i n the t e x t are shown. (B) Schematic drawing o f the hippocampus as seen from above, to show the o r i e n t a t i o n o f the s l i c e . F igu res adapted from Skrede and Westgaard, 1971; Shepard , 1974; and D ing led ine e t . a l . , 1980. 34 pathway was s t imu la ted by an e l e c t r o d e i n the st ratum rad ia tum, and the response was moni tored w i th an e l e c t r o d e i n the c e l l body l a y e r of area CA1 (F igu re 2 ) . The evoked f i e l d response (F igu re 2B) i nc ludes a broad p o s i t i v e p o t e n t i a l , due to the summed post synap t i c p o t e n t i a l s i n the d e n d r i t e s , and a sharp negat ive popu la t i on sp i ke r e s u l t i n g from the summed a c t i o n p o t e n t i a l s i n the c e l l body l a y e r . The response of a s i n g l e py ra -midal c e l l (F igu re 2C) i s u s u a l l y a s i n g l e a c t i o n p o t e n t i a l f o l l owed by a pe r i od o f i n h i b i t i o n . The c e l l s i n the i n v i t r o p repa ra t i on have a r e l a t i v e l y constant and c o n t r o l l e d envi ronment. S ince there i s , o f cou rse , no blood f l o w , there are no changes in b lood p ressure o r i n the concen t ra t i ons o f hormones or a n e s t h e t i c s i n the b lood . The absence of v a s c u l a r and r e s p i r a t o r y p u l s a -t i o n s has made the s l i c e p a r t i c u l a r l y use fu l f o r i n t r a c e l l u l a r r e c o r d i n g . The e x t r a c e l l u l a r space i s d i r e c t l y a c c e s s i b l e to exper imenta l man ipu la -t i o n , so tha t t r a n s m i t t e r s , hormones, and pharmacolog ica l agents can be added w i thou t the impediment o f a b lood b r a i n b a r r i e r . S p e c i a l techn iques f o r app l y i ng the subs tances , such as i on topho res i s or pressure e j e c t i o n , are requ i red on ly i f the a p p l i c a t i o n i s to be r e s t r i c t e d to a p a r t i c u l a r p o r t i o n o f the s l i c e . The a c c e s s i b i l i t y o f the e x t r a c e l l u l a r space a l s o means t ha t the behav io r o f the c e l l s w i l l be dependent, i n p a r t , on the i o n i c compos i t ion o f the ba th ing medium, the e x c i t a b i l i t y o f the c e l l s being i nc reased by a h igh K + or a low C a + + c o n c e n t r a t i o n . Th i s i s an advantage i n the sense tha t the exper imenter can choose the optimum c o n d i -t i o n s f o r a p a r t i c u l a r type o f exper iment , but i t i s imposs ib le to say what c o n d i t i o n s produce the most "normal " behav ior ( S c h w a r t z k r o i n , 1981) . Most workers f i n d t ha t a medium w i th r e l a t i v e l y h igh concen t ra t i ons of 35 B 1 mV + 50 m s e c 0.5mV 5 0 0 msec F igure 2 . P o s i t i o n o f e l e c t r o d e s f o r r eco rd ing the response to Scha f f e r c o l l a t e r a l s t i m u l a t i o n . (A) Schematic drawing of hippocampal s l i c e , as i n F igu re 1, showing the p o s i t i o n o f the e l e c t r o d e s f o r r eco rd ing the response o f CAT pyramidal neurones to s t i m u l a t i o n of the Scha f f e r c o l l a t e r a l pathway. (B) F i e l d p o t e n t i a l produced by S c h a f f e r c o l l a t e r a l s t i m u l a t i o n , recorded w i th a f i l t e r o f 0 . 1 - 3 kHz. (C) S i n g l e u n i t response to s t i m u l a t i o n o f the same pathway, recorded w i th a f i l t e r o f 1-3 kHz. 36 both K + (5-6 mM) and C a + + (2 .0 mM) produces a more s t a b l e p r e p a r a t i o n , e s p e c i a l l y f o r i n t r a c e l l u l a r r e c o r d i n g , than does a medium con ta i n i ng more p h y s i o l o g i c a l l y " c o r r e c t " c o n c e n t r a t i o n s . Th is b e l i e f has been quest ioned by D ing led ine e t . a l . (1980) , who repo r t t ha t s u i t a b l e r e c o r d -ings can be made w i th a r t i f i c i a l CSF con ta i n i ng 3 .0 -3 .5 mM K + and 1.2 mM Ca . The c h i e f d isadvantage o f the i n v i t r o p repa ra t i on i s t ha t the t i s s u e i s i n e v i t a b l y damaged in c u t t i n g - the axons o f a l l the pyramidal c e l l s and o f some of the granule c e l l s and in terneurones w i l l be seve red , and many c e l l s w i l l have severed dend r i t es as w e l l . I n t r a c e l l u l a r r e c o r d -ings i n d i c a t e , however, t ha t the membrane p r o p e r t i e s of the c e l l s ( r e s t i n g membrane p o t e n t i a l and conductance) are u s u a l l y qu i t e s i m i l a r to those measured i n v i vo (Schwar t zk ro i n , 1977). The l o s s of the a f f e r e n t p r o j e c -t i o n s to the hippocampus w i l l a l t e r the behav ior of the c e l l s i n ways tha t cannot be e a s i l y p r e d i c t e d . In a d d i t i o n , the s l i c e s are sub jec ted to a pe r iod o f anox ia and to cons ide rab le mechanical trauma dur ing d i s s e c t i o n and c u t t i n g . A f u r t h e r d e c ! i n e i n t i s s u e v i a b i l i t y occurs dur ing the course o f the exper iment , a l though t h i s d e t e r i o r a t i o n i s u s u a l l y very g r a d u a l ; s l i c e s have been kept a l i v e as long as 40 hours under s t e r i l e c o n d i t i o n s (Swanson e t . a l . , 1982). The c o n d i t i o n o f the s l i c e can be judged from the e l e c t r o p h y s i o l o g i c a l responses , p a r t i c u l a r l y by the l o s s o f i n h i b i t i o n w i th double pu lse s t i m u l a t i o n and the appearance of m u l t i p l e popu la t i on s p i k e s , and by the v i s u a l appearance of the s l i c e (D ing led ine e t . a l . , 1980; T e y l e r , 1980). While the i n v i t r o hippocampal s l i c e i s an ex t remely use fu l p repara -t i o n , pe rm i t t i ng exper imenta l man ipu la t ions t ha t would not be f e a s i b l e 37 in v i v o , i t should be remembered t ha t even the h e a l t h i e s t s l i c e i s g r o s s l y abnormal compared to the hippocampus o f the consc ious r a t . I t i s probably b e s t , t h e r e f o r e , to i n t e r p r e t the r e s u l t s o f i n v i t r o exper iments as i n d i -c a t i n g t h a t the t i s s u e i s capable of a p a r t i c u l a r response - as e v i d e n c e , f o r example, t ha t a p a r t i c u l a r pathway e x i s t s or tha t a c e l l type has recep to rs f o r a p a r t i c u l a r t r a n s m i t t e r - r a the r than as i n d i c a t i n g tha t the observed response a c t u a l l y occurs i n v i v o . 38 CHAPTER I I . RADIOIMMUNOASSAY EXPERIMENTS A . METHODS 1. SUMMARY As desc r i bed in more d e t a i l below, samples o f b r a i n t i s s u e from e i t h e r male W is ta r r a t s or female HoDI r a t s were homogenized, and the vasopress in was e x t r a c t e d w i th 0.2 M a c e t i c a c i d (Wi lson and Smi th , 1969) . The p ro te i ns were removed by p r e c i p i t a t i o n w i th ace tone , the l i p i d s r e -moved w i th petroleum e t h e r , and the vasop ress in content o f the t i s s u e s was determined by radioimmunoassay. A l l s o l u t i o n s , samples, and con ta ine rs were kept i n i ce throughout the homogeniza t ion , e x t r a c t i o n , and r a d i o -immunoassay procedures . A l l g lassware used f o r t i s s u e samples o r pept ide s o l u t i o n s was s i l i c o n i z e d . 2 . TISSUE SAMPLE PREPARATION Each r a t was anes the t i zed w i th urethane ( 1 . 0 - 1 . 5 g/kg body we igh t , i . p . ) and d e c a p i t a t e d . I t s b ra i n was immediately removed, r i n s e d wi th c o l d s a l i n e , and p laced i n a d d i t i o n a l co l d s a l i n e i n a p e t r i d i s h . The hippocampi were d i s s e c t e d f r ee and d i v i d e d i n t o approx imate ly equal r o s t r a l and caudal p o r t i o n s , and a sample o f ce reb ra l co r tex of s i m i l a r s i z e was removed from the m i d l i n e area above the septum on each s i d e . R igh t and l e f t po r t i ons o f each t i s s u e were combined, r i n s e d w i th s a l i n e , homogenized i n 0.2 M a c e t i c a c i d w i th a T e f l o n coated p e s t l e i n a P o t t e r -Ehlvehjem assembly , and c e n t r i f u g e d a t 15,000 g f o r 30 minutes . The supernatant was l y o p h i l i z e d and s to red a t -20°C. T i ssues from 6 Wis ta r 39 r a t s were combined to form the "pooled hippocampal t i s s u e " and the "pooled c o r t i c a l t i s s u e " r e f e r r e d to i n the " R e s u l t s and A n a l y s i s " s e c -t i o n . The "poo led HoDI co r t ex " c o n s i s t e d o f the e n t i r e ce reb ra l co r tex caudal t o the septum from 3 HoDI r a t s . Normal ly the d i s s e c t i n g d i sh and ins t ruments were r i n s e d between d i s s e c t i o n s . One b ra i n from an HoDI r a t , however, was d e l i b e r a t e l y d i s s e c t e d i n the s a l i n e remain ing from the d i s -s e c t i o n o f a W is ta r r a t b ra in to determine whether vasop ress in l e a k i n g from the severed p i t u i t a r y s t a l k cou ld be contaminat ing the t i s s u e s dur ing d i s s e c t i o n . 3 . ACETONE/PETROLEUM ETHER EXTRACTION Vasopress in was e x t r a c t e d from the l y o p h i l i z e d samples by a m o d i f i -c a t i o n o f the method of Robertson e t . a l . (1973) . Each t i s s u e sample was d i s s o l v e d in 2.0 ml o f 2.0 mM a c e t i c a c i d , and 200 u l was removed f o r p r o t e i n de te rmina t ion (B io rad P r o t e i n Assay K i t I, w i th bovine gamma g l o b u l i n as a s tanda rd ) . Acetone (2 .0 ml) was added to the remaining 1.8 ml to p r e c i p i t a t e the p r o t e i n s , and the sample was mixed on a Vor tex mixer and cen t i f uged (1200 g f o r 50 m inu tes ) . A f t e r the supernatant was decanted and saved , the p e l l e t was resuspended i n 1.0 ml acetone and c e n t r i f u g e d a g a i n , the two supernatants were combined, and the p e l l e t was d i s c a r d e d . L i p i d s were then removed by the a d d i t i o n o f 5.0 ml o f petro leum e t h e r . The two phases were mixed on a Vor tex and a l lowed to separate f o r a t l e a s t one hour before the e ther (upper phase) was a s p i r -ated and d i s c a r d e d . The t h i n f i l m a t the i n t e r f a c e was a l lowed to remain w i th the lower phase. A f t e r the a d d i t i o n o f 100 u l of 0.2 M a c e t i c a c i d , the sample was evaporated to l e s s than i t s o r i g i n a l 2 .0 ml volume under 40 a stream o f n i t rogen gas to remove the acetone and any remaining petroleum e t h e r . The sample remained in the i ce bath dur ing the e v a p o r a t i o n . The f i n a l s o l u t i o n was l y o p h i l i z e d and s to red a t -20°C u n t i l assayed. 4 . RADIOIMMUNOASSAY The vasopress in content o f the b ra i n t i s s u e s was measured by r a d i o -immunoassay. The ant iserum ( "R-20" ) was obta ined from a DI r a t immunized w i th LVP conjugated to bovine serum albumin (BSA) by the carbod i im ide technique (Goodf r iend e t . a l . , 1964) . The ant iserum i s s p e c i f i c f o r v a s o p r e s s i n , a r g i n i n e vasopress in being about tw ice as e f f e c t i v e as l y s i n e v a s o p r e s s i n , and does not c ross reac t s i g n i f i c a n t l y (<1%) w i th o x y t o c i n , AVT, i s o t o c i n , or a n g i o t e n s i n . The vasopress in used as a s tandard i n the assay was a p o s t e r i o r p i t u i t a r y e x t r a c t (2.1 IU/ml) k i n d l y p rov ied by Dr. R . E . Weitzman of Harbor General H o s p i t a l , To r rance , C a l i f o r n i a . The ex -t r a c t was s tandard i zed a g a i n s t s y n t h e t i c AVP (Spectrum L a b o r a t o r i e s ) to express the potency on a weight b a s i s . Iod ina ted AVP was prepared as desc r ibed by Weitzman and F i s h e r (1976) , based on the Chloramine-T technique developed by Greenwood e t . a l . (1963) f o r i o d i n a t i o n o f growth hormone. S y n t h e t i c AVP (Spectrum, 10 yg i n 10 y l o f 0.05 M a c e t i c a c i d ) was mixed w i th 15 y l o f 0.5 M phosphate bu f f e r (pH 7 . 4 ) , 10 y l o f Chloramine-T (1 .0 mg/ml ) , and 15 y l N a 1 2 5 I (Amersham, 37 M B q / y l ) . The r e a c t i o n was a l lowed to proceed f o r 50 seconds a t room temperature and was then stopped by a d d i t i o n o f 100 y l Pentax BSA (25% i n S a l i n e ) . B io -Rad anion exchanger AG1-X10 (200 y l of a 250 mg/ml s u s -pension) was added to remove unreacted i o d i n e , the mix ture was c e n t r i -fuged b r i e f l y , and the supernatant was a p p l i e d to a column o f CM Sephadex 41 C-25 (9x800mm) and e l u t e d w i th 0.6 M Na ace ta te bu f fe r (pH 4.85) a t 4°C. The monoiodinated hormone had a s p e c i f i c a c t i v i t y o f 19-39 MBq per ug o f AVP immunoreac t iv i t y (1150-2250 DPM/pg i A V P ) . A l l assays were performed in t r i p l i c a t e . The reagents were d i s s o l v e d i n 0.15 M phosphate b u f f e r , pH 7 . 2 , a t the concen t ra t i ons i n d i c a t e d . The r e a c t i o n mix ture c o n s i s t e d o f 200 u l of s tandard or sample, 200 y l o f the R-20 ant iserum (at 1:12000 d i l u t i o n ) , and 400 y l o f bu f f e r c o n t a i n i n g 0.1 M EDTA and 1% r a b b i t serum. The n o n s p e c i f i c b ind ing (NSB) was measured i n tubes c o n t a i n i n g 200 y l o f bu f f e r i n p lace of the an t i se rum. The assay was incubated a t 4°C f o r 3 days , 200 y l o f l a b e l l e d hormone (3000 DPM) was added to each t u b e , and the assay was re tu rned to the co ld f o r an a d d i t i o n a l two days . Bound and unbound ( " f r e e " ) f r a c t i o n s were separated by the a d d i t i o n o f 200 y l o f dex t ran-coa ted charcoa l (250 mg of dext ran T-70 and 2.5 g o f charcoa l per 100 ml b u f f e r ) , f o l l owed by c e n t r i -f uga t i on (1200 g f o r 10 m inu tes ) . Each bound f r a c t i o n (supernatant ) was poured i n t o a separate tube and both bound and f r ee f r a c t i o n s were counted on a Nuc lea r -Ch icago S e r i e s 1185 automated gamma counte r . The r a t i o o f bound to f r ee r a d i o a c t i v i t y ( " B / F " ) was c a l c u l a t e d w i th the a i d o f an Amdahl 470 V /8 computer, and a s tandard c u r v e , showing the r e l a t i o n between the B/F r a t i o and the amount o f u n l a b e l ! e d hormone, was p l o t t e d on semi -l o g a r i t h m i c paper (F igu re 3 ) . The s e n s i t i v i t y o f the assay , c a l c u l a t e d as the amount o f hormone requ i red to produce a 20% depress ion i n b i n d i n g , was 0.24 + 0.02 pg (mean + SEM), and the i n t r a - a s s a y c o - e f f i c i e n t o f v a r i a -t i o n a t 1.0 pg was 3.3% (n=4). In p r e l i m i n a r y exper imen ts , the t i s s u e was assayed us ing a guinea b i g an t i se rum, GP-15. Th is an t i se rum, a l s o produced by immunizat ion w i th 42 F igure 3 . Standard curve o f an AVP radioimmunoassay. The assay was prepared as desc r ibed in the t e x t , us ing the R-20 ant iserum and a t o t a l i ncuba t i on time o f 5 days. The n o n - s p e c i f i c b i nd ing was c a l c u l a t e d as the p ropo r t i on o f t o t a l r a d i o a c t i v i t y appear ing i n the "bound" f r a c t i o n o f tubes con ta in i ng 200 u l o f bu f f e r i n s t e a d of ant iserum (equat ion 1 ) . As i n d i c a t e d in equat ion 2 , the NSB was sub t rac ted before the B/F r a t i o s were c a l c u l a t e d . The terms " B " and " F " are the amounts o f r a d i o a c t i v i t y (as DPM) in the bound and f ree f r a c t i o n s , r e s p e c t i v e l y , wh i l e "b " and "f" r e f e r to the cor responding q u a n t i t i e s i n the n o n - s p e c i f i c b ind ing tubes . Values are p l o t t e d as means + SEM, n=3. A V P ( p g / a s s a y tube) 44 LVP coupled to BSA, does not d i s t i n g u i s h between LVP and AVP but shows l e s s than 0.5% c r o s s - r e a c t i o n w i th o x y t o c i n , i s o t o c i n , and a n g i o t e n s i n . The GP-15 ant iserum was a l s o used f o r a v a r i e t y of o ther purposes such as check ing s o l u t i o n s and measuring the amount o f vasop ress in e j e c t e d from i o n t o p h o r e t i c m i c r o p i p e t t e s . The assay was s i m i l a r to the R-20 assay except t ha t the EDTA was o m i t t e d , BSA (25 mg/100 ml) was sometimes s u b s t i t u t e d f o r the r a b b i t serum, and the t o t a l i ncuba t i on per iod was o c c a s i o n a l l y reduced to 3 days. B. VASOPRESSIN CONTENT OF RAT BRAIN TISSUES 1. RESULTS AND ANALYSIS The amounts o f vasop ress in in the hippocampus and the ce reb ra l co r tex o f 9 male Wis ta r r a t s (220-430 g) and 7 female HoDI r a t s (182-250 g) were determined by radioimmunoassay and are summarized i n Table IV. Vaso-p r e s s i n was de tec ted i n a l l 9 o f the ven t ra l hippocampal samples and i n a l l but one o f the 9 do rsa l hippocampal samples from the Wis ta r r a t s . The amount o f vasopress in i n the ven t ra l hippocampus (81.4 + 8 .3 pg iAVP/mg s o l u b l e p ro te i n ) was 2.7 t imes g rea te r than i n the dorsa l hippocampus (30.3 + 7.5 pg iAVP/mg s o l u b l e p r o t e i n ) . In c o n t r a s t to t h i s , none of the c o r t i c a l samples from the W is ta r r a t s con ta ined de tec tab le vasopress in ( < 6 . 2 pg iAVP/mg s o l u b l e p r o t e i n ) . No immunoreacti v i t y was detec ted i n any o f the t i s s u e samples from the HoDI r a t s , con f i rm ing tha t the immuno-r e a c t i v e ma te r i a l i n the W is ta r r a t s was in f a c t AVP. The recovery o f 11.25 pg AVP added to each o f 4 HoDI c o r t i c a l samples and 2 W is ta r c o r t i c a l samples was 45.7% + 4.18% (mean + SEM, n=6), and the va lues i n Table IV have t h e r e f o r e been co r rec ted a p p r o p r i a t e l y . S ince 45 TISSUE n iAVP (pg/mg s o l u b l e p ro te i n ) HoDI V. HIPP. 6 n . d . HoDI D. HIPP. 5 n . d . HoDI CORTEX 2 n . d . WISTAR V. HIPP. 9 81.4 + 8.3 WISTAR D. HIPP. 9 30.3 ± 7.3 WISTAR CORTEX 7 n . d . TABLE IV. VASOPRESSIN CONTENT OF RAT BRAIN TISSUES A l l va lues have been co r rec ted f o r e x t r a c t i o n l o s s as desc r i bed i n the t e x t . The va lues are means + SEM. AVP was not de tec ted ( n .d . ) i n the W is ta r co r tex o r i n the HoDI t i s s u e s . The h ippo-campal t i s s u e was d i v i d e d i n t o ven t ra l (V. HIPP.) and dorsa l (D. HIPP.) p o r t i o n s . Each sample represen ts the t i s s u e from one r a t . the AVP was added to the t i s s u e immediately a f t e r d i s s e c t i o n , the c o r r e c -t i o n f a c t o r should compensate f o r l o s s o f hormone dur ing a l l s tages of t i s s u e p rocess ing and f o r the e f f e c t of n o n s p e c i f i c i n t e r f e r e n c e . To determine whether the vasopress in found i n the Wis ta r hippocampi cou ld have r e s u l t e d from contaminat ion o f the t i s s u e dur ing d i s s e c t i o n (by pept ide r e l e a s e d , f o r example, from the severed p i t u i t a r y s t a l k ) , an HoDI r a t b ra i n ( the "contaminat ion c o n t r o l " ) was d i s s e c t e d in s a l i n e p r e v i o u s l y used f o r d i s s e c t i o n of a Wis ta r r a t b r a i n . The d i s s e c t i o n was d e l i b e r a t e l y performed i n a manner in tended to maximize the chance o f con tamina t ion . The t i s s u e samples from t h i s b ra in d i d not con ta in de tec tab le v a s o p r e s s i n . Th i s f i n d i n g , and the absence of vasopress in i n the Wis ta r c o r t i c a l samples, i n d i c a t e t ha t contaminat ion cou ld not have accounted f o r the vasopress in detected i n the Wis ta r hippocampal t i s s u e s . 46 To a l l ow l a r g e r amounts o f ma te r i a l to be assayed , b ra i n t i s s u e from severa l r a t s was combined ("pooled t i s s u e s " ) . Pooled hippocampal t i s s u e prepared from 6 male W is ta r r a t s (410-480 g) conta ined 236 pg iAVP/mg s o l u b l e p r o t e i n (va lue co r rec ted as desc r i bed above) , 2.9 t imes as much as the s i n g l e ven t ra l hippocampal samples, and pooled c o r t i c a l t i s s u e from the same ra t s conta ined 26.3 pg iAVP/mg s o l u b l e p r o t e i n , even though iAVP was not de tec tab le i n the s i n g l e c o r t i c a l samples. Pooled c o r t i c a l t i s s u e from 3 HoDI r a t s (185-215 g) f a i l e d to y i e l d de tec tab le iAVP. The curve produced by s e r i a l d i l u t i o n o f the pooled t i s s u e was p a r a l l e l to t ha t produced by s e r i a l d i l u t i o n o f the AVP standard (F igu re 4 ) , as would be expected i f the immunoreactive ma te r i a l i n the t i s s u e was immuno log ica l l y i d e n t i c a l to AVP. 2 . DISCUSSION Immunocytochemical s t u d i e s in the r a t ( B u i j s e t . a l . , 1978; B u i j s and Swaab, 1979; B u i j s , 1980; Sof ron iew and W e i n d l , 1981) have demon-s t r a t e d t ha t hypothalamic neurones con ta in i ng vasopress in p r o j e c t to the hippocampus. The present study conf i rms the presence of iAVP in the r a t hippocampus and suggests t h a t the ven t ra l hippocampus con ta ins more o f t h i s pept ide than does the do rsa l p o r t i o n . Th is f i n d i n g i s c o n s i s t e n t w i th the repo r t by B u i j s (1980) t h a t the immunoreactive f i b e r s p ro j ec t p r i m a r i l y to the ven t ra l hippocampus, but i t i s not i n agreement w i th the work o f Sofroniew and Weindl (1981) , who found the do rsa l hippocampus to be devoid o f vasop ress in c o n t a i n i n g f i b e r s . The d i f f e r e n c e between these two immunocytochemical s t ud ies has been d i scussed more f u l l y i n Chapter I o f t h i s t h e s i s . 47 A V P ( p g / a s s a y tube) 10 r 5 0 PROTEIN ( u g / a s s a y tube) F igu re 4 . S e r i a l d i l u t i o n o f hippocampal t i s s u e . The curves produced by s e r i a l d i l u t i o n o f the AVP standard (open c i r c l e s ) and o f the pooled W is ta r hippocampal t i s s u e ( c l osed c i r c l e s ) are shown. The a b s c i s s a f o r the t i s s u e curve i s expressed i n yg s o l u b l e p r o t e i n / a s s a y tube. The r e l a t i o n between the two absc i ssae i s a r b i t r a r y and was chosen to f a c i l i t a t e compar ison. Values are p l o t t e d as means + SEM, n=3. 48 The ce reb ra l c o r t e x , i nc luded i n the study as a c o n t r o l , d i d not con ta in de tec tab le v a s o p r e s s i n . While immunoreactive f i b e r s have been found i n some c o r t i c a l a r e a s , such as the e n t o r h i n a l r e g i o n , the t i s s u e i nc luded i n the present study was taken from the dorsa l su r face o f the c o r t e x , where immunoreactive f i b e r s have not been : repo r ted . George and Jacobowi tz (1975) measured the AVP content o f 32 areas of the Sprague-Dawley r a t b ra i n by radioimmunoassay. They were unable to de tec t any AVP i n the hippocampus or i n any o ther ex t rahypotha lamic t i s s u e , i n c l u d i n g areas such as the habenula and the organ vasculosum o f the lamina te rmina l i s (OVLT) which have been repor ted to con ta in immunoreac-t i v e f i b e r s ( B u i j s e t . a l . , 1978; B u i j s , 1980; Sofroniew and We ind l , 1978) . Th i s f a i l u r e , however, i s probably a t t r i b u t a b l e to the l ack of s e n s i t i v i t y o f t h e i r assay (100 pg/mg p r o t e i n ) . Using a more s e n s i t i v e assay system, Dogterom e t . a l . (1978) found immunoreactive vasop ress in i n a v a r i e t y o f ex t rahypotha lamic areas (hippocampus, c o r t e x , septum, OVLT, amygdala, nuc leus p a r a f a s c i c u l a r i s , cho ro id p l e x u s , and medul la) o f the W is ta r r a t b r a i n . The a n t e r i o r hippocampus con ta ined 58 + 37 pg iAVP/mg dry we igh t , the p o s t e r i o r hippocampus con ta ined 3 0 + 1 1 pg iAVP/mg dry we igh t , and the cor tex con-t a i n e d 4.6 + 1 . 2 pg iAVP/mg dry we igh t . No iAVP was found i n any HoDI t i s s u e s . In t h e i r s t udy , i n which the samples were " d i s s e c t e d or punched out " o f f reeze d r i e d b ra i n s e c t i o n s , the " a n t e r i o r " hippocampus inc luded on ly the a n t e r i o r t i p of the hippocampus and the " p o s t e r i o r " hippocampus extended on l y as f a r c a u d a l l y as plane A2420 (Kb'nig and K l i p p e l , 1967) . Most o f the t i s s u e r e f e r r e d to as " v e n t r a l hippocampus" i n the present study was not i nc luded in t h e i r s tudy . The amount o f vasop ress in detec ted 49 i n the hippocampus i n the present study would correspond to approx imate ly 2 pg iAVP/mg dry we igh t , computed f o r the hippocampus as a who le , and was t he re fo re cons i de rab l y l e s s than repor ted i n t h e i r s tudy . The d i s -crepancy may be p a r t l y the r e s u l t of d i f f e r e n c e s i n d i s s e c t i o n techn ique . In the present s tudy , the f i m b r i a was removed and on ly the vasopress in i n the hippocampus i t s e l f was measured. S ince the immunoreactive f i b e r s run through the f i m b r i a to reach the hippocampus ( B u i j s , 1980) , the con-c e n t r a t i o n o f vasop ress in may be..higher i n the f i m b r i a than i n the h ippo-campus. The repo r t by Dogterom and h i s coworkers does not mention whether the f i m b r i a was removed. In a study by Hawthorn e t . a l . (1980) , the r a t hippocampus was found to con ta in 27 pg iAVP/mg t o t a l p r o t e i n , which was about 4 t imes g rea te r than repor ted i n the present study (approx imate ly 6.7 pg iAVP/mg t o t a l p r o t e i n ) . Vasopress in was a l s o repor ted i n such ex t rahypotha lamic areas as the tha lamus, amygdala, and ce rebe l l um, and smal l amounts were present i n some c o r t i c a l a r e a s . No HoDI c o n t r o l s were used , but t h i n l a y e r chromatography o f the hippocampal t i s s u e suggested tha t the immunoreactive ma te r i a l was in f a c t AVP. As a f u r t h e r check, the curves produced by s e r i a l d i l u t i o n o f 3 o f the b r a i n t i s s u e s (not i n c l u d i n g the hippocampus) were compared to the s tandard c u r v e ; whether the curves were p a r a l l e l , as c la imed by the a u t h o r s , i s deba tab le . The f i n d i n g o f immunoreactive AVP i n the r a t hippocampus supports the immunocytochemical demonstrat ion o f immunoreactive f i b e r s i n the hippocampus. The ev idence would seem to be s u f f i c i e n t to j u s t i f y neuro-p h y s i o l o g i c a l s t u d i e s on the a c t i o n o f AVP and r e l a t e d pept ides on h ippo-campal neurones. Such s t u d i e s form the b a s i s o f the remainder o f t h i s t h e s i s . 50 CHAPTER I I I . NEUROPHYSIOLOGICAL EXPERIMENTS A. METHODS 1 . IN VITRO STUDIES Hippocampal s l i c e s were prepared from e i t h e r male W is ta r r a t s (160-360 g) or female HoDI r a t s (180-190 g ) . The pept ides to be s tud ied were added to the pe r f us i on l i q u i d , and the responses of i n d i v i d u a l neurones were recorded e x t r a c e l l u l a r l y w i th s i n g l e ba r re l g l ass m i c r o -p i p e t t e s . S l i c e P repa ra t i on Each ra t was decap i t a ted and i t s b ra i n was p laced i n oxygenated (95% 0 2 , 5% C0 2 ) Krebs s o l u t i o n i n a p e t r i d i s h . One hippocampus was d i s s e c t e d f r e e , us ing Te f l on coated s p a t u l a s , and cut i n t o 400 urn t r a n s -verse s l i c e s , approx imate ly i n the plane o f the hippocampal l a m e l l a (Skrede and Westgaard, 1971) , on a Mc l lwa in t i s s u e chopper. The s l i c e s were immediate ly t r a n s f e r r e d to a ny lon net and p laced i n a narrow, r e c -t angu la r i ncuba t i on chamber i n a b lock o f a c r y l i c (F igu re 5 ) . The lower su r face o f each s l i c e was in con tac t w i th a t h i n f i l m of oxygenated Krebs s o l u t i o n t ha t f lowed along the bottom o f the chamber a t a cons tant ra te (about 0.5 m l / m i n u t e ) , and the upper su r face was exposed to the humid i -f i e d 0 2 / C 0 2 m i x t u r e . The chamber was conta ined i n a water bath main ta ined a t a cons tant temperature . In most exper iments a temperature of 33°C was chosen as a compromise between the more r a p i d d e t e r i o r a t i o n o f the s l i c e s t ha t occur red at h igher temperatures and the poor respons iveness observed w i th lower tempera tures . 51 F igure 5 . Schematic diagrams o f the i ncuba t i on chamber. A ny lon net i s g lued to an a c r y l i c f rame, which i s then p laced i n a narrow groove i n an a c r y l i c b l o c k . The f low o f the Krebs s o l u t i o n i s i n d i -ca ted by the broad a r rows . The net r e s t s on the bottom of the chamber, and the Krebs s o l u t i o n must t h e r e f o r e f low through the mesh o f the n e t , pass ing a long the unders ide o f the s l i c e . The s o l u t i o n then f lows under the edge o f the frame i n t o a second chamber, where i t i s removed by an a s p i r a t o r . An a c r y l i c d i v i d e r , shown i n cut-away v iew , prevents the a s p i r a t o r from drawing a i r over the s l i c e . R E C O R D I N G 53 The apparatus used f o r most o f the i n v i t r o exper iments i s shown s c h e m a t i c a l l y i n F igu re 6. Oxygenated Krebs s o l u t i o n s to red in the b a r r e l s o f 50 ml p l a s t i c sy r i nges was a l lowed to f low by g r a v i t y through Tygon tub ing ( I . D . 0.06 inch) to a d r i p chamber (a 5 ml p l a s t i c sy r inge) and then through S i l a s t i c tub ing to the reco rd ing chamber. The Tygon tub ing cou ld be c lamped, and s o l u t i o n was a l lowed to f low from on l y one o f the sy r i nges a t a t ime . Pept ides were a p p l i e d by i n t e r r u p t i n g the f low o f con t ro l s o l u t i o n and s imu l taneous ly removing the clamp on the tub ing from a r e s e r v o i r o f s o l u t i o n c o n t a i n i n g the p e p t i d e . The f low ra te cou ld be c o n t r o l l e d by an ad jus tab le clamp below the d r i p chamber. The ra te was moni tored by t im ing the drops i n the d r i p chamber w i th a s t o p w a t c h , p rev ious measurements having e s t a b l i s h e d tha t the volume o f the drops d i d not d i f f e r by more than 10%. The r e l a t i v e he igh ts o f the i n d i v i d u a l sy r inges were ad jus ted as needed to ensure tha t the f low ra te was not a f f e c t e d by changing from one s o l u t i o n to another . The des ign of the apparatus used i n some o f the exper iments was s l i g h t l y d i f f e r e n t from tha t i l l u s t r a t e d i n F igure 6 . There were d i f f e r e n c e s , f o r example, i n the types and s i z e s o f con ta ine rs used f o r the Krebs s o l u t i o n s , i n the des ign o f the frame used to ho ld the ny lon n e t , and i n the method of channe l ing the s o l u t i o n from the net to the a s p i r a t o r . Separate d r i p chambers were sometimes used f o r each source of s o l u t i o n to reduce the l ag time when changing s o l u t i o n s . The separate chambers were j o i n e d by a 5-way connector to a s i n g l e S i l a s t i c tube . A more e labo ra te a r range-ment, i n v o l v i n g 6 sy r i nges and 2 d r i p chambers, was used i n seve ra l e x p e r i -ments to a l l o w pept ides and an tagon is t s to be a p p l i e d s imu l t aneous l y . The Krebs s o l u t i o n conta ined 124 mM N a C l , 5.0 mM KC1, 26.0 mM NaHC0 3 , 54 F igure 6. Apparatus f o r ma in ta in i ng hippocampal s l i c e s i n v i t r o . The e l e c t r o d e i s p o s i t i o n e d i n the s l i c e (not shown), which r e s t s on a ny lon net i n an a c r y l i c chamber (see F igure 5 ) . The chamber i s p laced i n a heated water ba th . The Krebs s o l u t i o n i s a l lowed to f low by g r a v i t y from 50 ml s y r i n g e s . The he igh ts o f the sy r i nges can be i n d i v i d u a l l y ad jus ted to prov ide a uni form f low r a t e . 55 o 2/co 2 K R E B S 5 0 m l S Y R I N G E S C L A M P • 56 1.25 mM NaHPOit, 2.0 mM MgS0 4 , 10.0 mM g l u c o s e , and 2.0 mM C a C l 2 . S o l u t i o n s con ta i n i ng lower concen t ra t i ons of CaCl. 2 (1 .0 or 1.5 mM) were a l s o t e s t e d , and the amount o f NaCl was ad jus ted a c c o r d i n g l y to ma in ta in i s o t o n i c i t y . In some exper iments the pept ides were s imp ly d i s s o l v e d d i r e c t l y i n the Krebs s o l u t i o n . In o t h e r s , the l y o p h i l i z e d pept ides were f i r s t d i s s o l v e d in 20-100 y l of a c i d (1 .0 mM HC1 i n 140 mM N a C l ) . The a c i d s o l u t i o n was then d i l u t e d 500-1000 f o l d w i th Krebs s o l u t i o n and n e u t r a l i z e d w i th 20-100 "ul o f NaOH (1.0 mM NaOH i n 140 mM N a C l ) . A l l con t ro l and pept ide s o l u t i o n s were a c i d i f i e d and n e u t r a l i z e d i n the same way i n any g iven exper iment . The pept ides i nc luded AVP (Sigma, grade V I I I ) , LVP (Sigma, grade I V ) , Oxytoc in (S igma) , ddOT (Beckman), ddAVP (Beckman), PLG (Beckman), and somatos ta t in (Serono) . Dr . M. Manning o f the Medica l Co l l ege o f Oh io , To ledo , Oh io , k i n d l y p rov ided AVP, DGAVP, G l y 7 - 0 T , d (CH 2 ) 5 Tyr (Me)AVP, and d (CH 2 ) 5 -D-Ty r (Me)VAVP. Recording Techniques The response to the pept ides was moni tored by e x t r a c e l l u l a r un i t r e c o r d i n g . S i n g l e b a r r e l g l a s s m i c r o p i p e t t e s f i l l e d w i th 4 M NaCl and having an impedance o f 0 . 5 - 4 . 0 Mfi were lowered i n t o the c e l l body l a y e r w i th a manual ly operated mic roman ipu la to r . Accurate p o s i t i o n i n g of the e l e c t r o d e was f a c i l i t a t e d wi th a s t e r e o s c o p i c m ic roscope. The s i g n a l from the m ic roe lec t rode was passed to a p r e a m p l i f i e r , and the a m p l i f i e d , compensated s i g n a l was then sent to a d i f f e r e n t i a l a m p l i f i e r f o r f u r t h e r a m p l i f i c a t i o n and f i l t e r i n g (0 .1 -3 kHz or 1-3 kHz) and was d i s p l a y e d on an o s c i l l o s c o p e . Severa l combinat ions o f Tek t ron i x a m p l i f i e r s and o s c i l l o -scopes were used. A p la t inum w i r e running the length o f the i ncuba t i on 57 chamber served as the re fe rence e l e c t r o d e . ( S i l v e r w i res were found to t a r n i s h i n the presence of s u l f u r c o n t a i n i n g p e p t i d e s . ) The f i l t e r e d s i g n a l was a l s o conducted to an EKEG Neuroratemeter or a Winston d i s c r i m i n a t o r . Sp ikes above a predetermined ampl i tude a c t i v a t e d a Schmidt t r i g g e r , and the output from the t r i g g e r was d i s p l a y e d s imu l taneous ly w i th the f i l t e r e d s i g n a l on a dual beam storage o s c i l l o s c o p e . The t r i g g e r l e v e l was ad jus ted to prevent i t s being a c t i v a t e d by background n o i s e , and a second output from the t r i g g e r was reco rded , as sp ikes per second, on an E s t e r l i n e - A n g u s char t r e c o r d e r . The c o n d i t i o n o f the s l i c e s was judged by both v i s u a l ( v i s i b i l i t y o f the c e l l l i n e , t e x t u r e o f the su r face ) and e l e c t r o p h y s i o -l o g i c a l c r i t e r i a (Tey le r , 1980). Evoked p o t e n t i a l s were s tud ied by pos i t ion 1 - : ing a b i p o l a r s t i m u l a t i n g e l e c t r o d e ( tw i s ted Nichrome w i re ) i n the Scha f f e r c o l l a t e r a l pathway and app ly ing s t i m u l i o f 1 .0 -1 .5 msec du ra t i on and u s u a l l y o f 10-30 v o l t ampl i tude e i t h e r w i th a D i g i t i m e r D100 t imer and a D i g i t i m e r i s o l a t e d s t i m u l a t o r model DS-2 or w i th a Grass s t i m u l a t o r S4KR and a Grass s t imu lus i s o l a t i o n u n i t SIU5. The evoked p o t e n t i a l s were recorded through the same m ic roe lec t rode used f o r s i n g l e un i t r e c o r d -ing but w i th a low-pass f i l t e r o f 1 Hz. 2 . IN VIVO STUDIES The e f f e c t o f vasopress in and oxy toc i n on neurones in the l a t e r a l septum and the hippocampus was s tud ied i n v i vo i n 24 male a l b i n o r a t s (240-485 g ) . The pept ides were a p p l i e d by m i c r o i o n t o p h o r e s i s , and the response o f the neurones was moni tored by e x t r a c e l l u l a r u n i t r e c o r d i n g . 58 M i c r o p i p e t t e s M u l t i b a r r e l m i c r o p i p e t t e s , c o n s i s t i n g o f 7 fused g l a s s c a p i l l a r y tubes were purchased from Vancouver S c i e n t i f i c G lassb low ing and were drawn to a f i n e t i p on a v e r t i c a l m ic roe lec t rode p u l l e r . The t i p was broken back to 5-12 ym, and the m ic rop ipe t t e was b o i l e d u n t i l a l l b a r r e l s were f i l l e d w i th water . Most o f the water was then withdrawn from each b a r r e l and rep laced e i t h e r w i th 4 M NaCl f o r reco rd ing ( c e n t r a l b a r r e l ) o r w i th the appropr ia te s o l u t i o n (glutamate 0.5 M, pH 8 . 6 ; a c e t y l c h o l i n e bromide 1 M, pH 4 . 9 , o r the p e p t i d e ) . The m i c r o p i p e t t e , w i th i t s t i p immersed i n wa te r , was then r e f r i g e r a t e d f o r a t l e a s t 24 hours to a l l ow the s o l u t i o n s to d i f f u s e i n t o the t i p . The pept ides t es ted were a r g i n i n e vasop ress in (Sigma grade V I I I , Spect rum) , l y s i n e vasopress in (Sigma Grade I V ) , and oxy toc i n (S igma). A l l o f these are s u p p l i e d as a l y o p h i l i z e d powder and were d i s s o l v e d i n d i l u t e a c e t i c a c i d f o r use i n t h i s e x p e r i -ment. Sigma AVP grade V I , which i s s u p p l i e d as a 0.4 mM s o l u t i o n c o n t a i n -ing 0.33 mg NaCl and 0.03 mg NH^Cl per m l , and Sandoz oxy toc in (Syn toc inon-10) which con ta ins ch lo robu tano l (5 mg/ml) , were used l e s s f r e q u e n t l y . A l l o f these pept ides had a pronounced tendency to b lock the b a r r e l s o f the m i c r o p i p e t t e . In an e f f o r t to c i rcumvent t h i s prob lem, the pept ides were t e s t e d a t many d i f f e r e n t concen t ra t i ons (10~ 6 to 5X10 - l f M) in va r ious concen t ra t i ons o f a c e t i c a c i d (10~ 5 to 2X10" 2 M) . The more d i l u t e s o l u -t i o n s con ta ined 10-40 mM NaCl i n a d d i t i o n to the pept ide and a c e t i c a c i d . The a b i l i t y o f the m i c rop ipe t t es to e j e c t AVP was determined by immersing the t i p o f the m i c r o p i p e t t e s and a s i l v e r ground w i re i n t o 1.0 ml s a l i n e , app l y i ng the appropr ia te cu r ren ts (+20 to +60 nA) f o r 30 to 60 minu tes , and measuring the AVP content o f the s a l i n e by radioimmunoassay. The 59 measured ra te o f e j e c t i o n o f AVP was compared to the t h e o r e t i c a l ra te o f r e l e a s e , which was c a l c u l a t e d assuming a charge o f +2 f o r the AVP molecule and a p p o r t i o n i n g the t o t a l cu r ren t accord ing to the r e l a t i v e concen t ra -t i o n s and mo lecu la r weights o f a l l p o s i t i v e l y charged substances p resen t . The ra tes o f e j e c t i o n from each o f 6 f r e s h l y prepared s i n g l e b a r r e l m i c r o -p i p e t t e s con ta i n i ng 3 d i f f e r e n t AVP s o l u t i o n s were 0 .015-2 .0 pg /nA /m in , compared to t h e o r e t i c a l ra tes o f 2 . 0 - 4 . 2 pg /nA/min . The ra tes o f r e l e a s e from each o f 5 b a r r e l s o f 2 m u l t i b a r r e l m i c rop ipe t t es were below the l i m i t o f de tec t i on (< 0.006 pg /nA/min , compared to t h e o r e t i c a l ra tes o f 0 .09 -11 .8 pg /nA /m in ) . These two m ic rop ipe t t es had been p r e v i o u s l y used i n exper iments and had been prone to b l ockage , as desc r i bed i n the "Resu l t s and A n a l y s i s " s e c t i o n . Surgery Each r a t was anes the t i zed w i t h urethane (1.5 g / k g , i p ) , i t s head was shaved, and i t was p laced in a Kopf s t e r e o t a x i c ho lde r w i th the i n c i s o r bar 4.2 mm below the mid-aura l l i n e . The s k i n over the s k u l l was cut a long the m i d l i n e w i th a s c a l p e l , and the s k u l l was c l ea red w i th a p e r i o s t e a l e l e v a t o r and a l lowed to d ry . The s k u l l over the area to be s t u d i e d was opened w i th a denta l d r i l l , the f l a p o f s k u l l was removed wi th f o r c e p s , and the dura was c a r e f u l l y cu t away. P a r t i c u l a r care was taken when removing the s k u l l and dura over the septum to avo id damage to the s u p e r i o r s a g i t t a l s i n u s . B leed ing from bone was c o n t r o l l e d , when necessa ry , w i th bone wax, and b leed ing from smal l v e s s e l s was stopped w i th S t r o l l ' s h y d r o c e l l u l o s e wedges or Upjohn gel foam. The exposed b r a i n t i s s u e was kept mois t w i th warm Locke ' s s o l u t i o n (37°C) . When surgery was comple te , the s k i n was r a i s e d and sutured to an a c r y l i c h o l d e r . 60 The r e s u l t i n g pocket was f i l l e d w i th Locke ' s s o l u t i o n which was then covered w i th a l a y e r o f minera l o i l . Recording and Ion tophores is The equipment and procedures f o r s t i m u l a t i o n and f o r reco rd ing s i n g l e u n i t s and evoked responses were e s s e n t i a l l y the same as those used f o r the i n v i t r o exper imen ts , except as desc r i bed below. The c e n t r a l ba r re l o f the 7 ba r re l m i c rop ipe t t e served as the reco rd ing e l e c t r o d e (0 .4 -2 .0 Ki), and a c o n c e n t r i c s t e e l e l e c t r o d e w i th a t i p sepa ra t i on o f 0.5 mm was used f o r s t i m u l a t i o n . The re fe rence e l e c t r o d e was a w i re c l i p p e d to the edge of the s u r g i c a l i n c i s i o n . Currents f o r i on topho res i s were c o n t r o l l e d w i th potent iometers and were passed through 1000 Mft s e r i e s r e s i s t o r s before being conducted by s i l v e r w i res i n t o the b a r r e l s o f the micro p i p e t t e s . A panel o f l i g h t s i n d i c a t e d the amount o f cu r ren t a c t u a l l y pass -ing through the c i r c u i t and t he re fo re showed whether the b a r r e l was b l o c k e d , Re ta in i ng cu r ren ts o f 5-15 nA and o f oppos i te p o l a r i t y to the e j e c t i n g cu r ren ts prevented d i f f u s i o n from the t i p o f the m i c r o p i p e t t e (Pu rves , 1979; C la rke e t . a l . , 1973; K e l l y e t . a l . , 1975). The reco rd ing e l e c t r o d e was lowered i n t o the b ra i n i n 4 ym steps us ing an e l e c t r o n i c a l l y c o n t r o l l e d m ic rod r i ve mounted on a l a rge m i c r o -man ipu la to r . The coord ina tes f o r e l e c t r o d e placement were determined from an a t l a s o f the r a t b ra i n by Kdnig and K l i p p e l (1967) except t ha t measurements were made from bregma, cor responding to t h e i r plane A7190 ym, i n s t e a d o f from the i n t e r - a u r a l l i n e . The coord ina tes below are given as the d i s t ance i n m i l l i m e t e r s a n t e r i o r ( A ) , p o s t e r i o r ( P ) , o r l a t e r a l ( L ) , to bregma, or ven t ra l (V) to the su r face o f the c o r t e x . Recordings 61 were made i n the l a t e r a l septum i n the area from bregma to 0.9 mm a n t e r i o r to bregma. The l a t e r a l and ven t ra l coo rd ina tes depended on the a n t e r i o r -p o s t e r i o r p o s i t i o n but were t y p i c a l l y i n the range of 0 . 8 - 1 . 2 L and 3 . 0 -5.0 V. The s t i m u l a t i n g e l e c t r o d e was u s u a l l y p laced i n the i p s i l a t e r a l hippocampus a t 3.5 P, 2.5 L, 2.5 V. Recordings were a l s o made i n the h ipp^ campus ( 4 . 0 - 5 . 2 P, 2 . 0 - 3 . 5 L ) , and i n t h i s case the depth o f the e l e c t r o d e was determined by l o c a t i n g the evoked response i n the dentate gyrus (L0mo, 1971a) f o l l o w i n g s t i m u l a t i o n o f the i p s i l a t e r a l en to rh i na l co r tex (8.1 P, 4 .3 L, 2.5 V ) . At the end of the exper iment , an 18 uA anodal c u r -ren t was passed through the s t i m u l a t i n g e l e c t r o d e f o r 20-30 seconds. The b ra in was then f i x e d i n f o rma l i n c o n t a i n i n g 1% potassium f e r r o c y a n i d e , f r o z e n , s e c t i o n e d , and s t a i n e d , and the P russ ian b lue spot marking the p o s i t i o n of the i r o n depos i t was l oca ted under the mic roscope. Less f r e -q u e n t l y , the p o s i t i o n o f the reco rd ing e l e c t r o d e was determined by e j e c t i n g pontamine sky b lue (4% i n 1M NaCl) from one of the ou ter b a r r e l s of the m i c rop ipe t t e (12 uA X 10 m inu tes ) . B. I0NT0PH0RETIC APPLICATION OF NEUROHYPOPHYSIAL PEPTIDES 1. RESULTS AND ANALYSIS The e f f e c t o f neurohypophysia l pept ides a p p l i e d by i on topho res i s to c e l l s i n the l a t e r a l septum or the hippocampus was s tud ied in 24 male Wis ta r r a t s (240-485 g ) . Ion tophores is o f vasop ress in (AVP or LVP) was accompanied by a gradual i nc rease i n the f i r i n g ra te i n 19 of 100 c e l l s i n the l a t e r a l septum and i n 20 of 45 c e l l s i n the pyramidal c e l l l a y e r 62 of the hippocampus (F igures 7 and 8 ) . None of the 4 c e l l s t es ted i n the granu le c e l l l a y e r o f the dentate gyrus were e x c i t e d by v a s o p r e s s i n . There was no obvious d i f f e r e n c e between the e f f e c t of vasop ress in on c e l l s f i r -ing spontaneously and on those e x c i t e d by cont inuous a p p l i c a t i o n o f glutamate or a c e t y l c h o l i n e . Depress ions of spontaneous or drug induced f i r i n g were observed l e s s f r e q u e n t l y than e x c i t a t i o n s and were c h a r a c t e r -i z e d by a sudden onset and o f f s e t (F igure 9 ) . A few c e l l s responded w i th both a sudden i n h i b i t i o n and a gradual e x c i t a t i o n (F igu re 1 0 ) , and sudden e x c i t a t i o n s were observed i n two c e l l s . Ion tophores is of oxy toc i n was attempted on ly i n the septum. E x c i t a -t i o n s were never observed dur ing i on topho res i s o f o x y t o c i n , a l though sudden i n h i b i t i o n s were observed w i th 7 of the 30 c e l l s t e s t e d . Ion tophores is o f e i t h e r pept ide o f ten r e s u l t e d i n gradual b lockage of the m i c r o p i p e t t e ba r re l - t ha t i s , i n an i nc rease i n the r e s i s t a n c e of the ba r re l so tha t on ly smal l cu r ren ts cou ld be passed . Tests on 5 p a r t i a l l y b locked m ic rop ipe t t e b a r r e l s revea led t ha t none cou ld expel de tec tab le AVP ( l i m i t o f d e t e c t i o n was 0.006 pg /nA/min ; see Chapter I I I , s e c t i o n A 2 ) . In c o n t r a s t , AVP r e l e a s e from unblocked m ic rop ipe t t es was in the range 0 .015-2 .0 pg/nA/min (n=6). For reasons d i scussed i n Chapter I, s e c t i o n C2 , one would not expect s i g n i f i c a n t pept ide r e l e a s e from b locked or p a r t i a l l y b locked m i c r o p i p e t t e s , the cu r ren t being c a r r i e d i ns tead by H + or o ther smal l i o n s . M i c r o p i p e t t e b a r r e l s c o n t a i n i n g oxy toc in were b locked much more f r e q u e n t l y than b a r r e l s c o n t a i n i n g the more h i g h l y charged molecules AVP or LVP. Repeated a p p l i c a t i o n s of vasop ress in sometimes r e s u l t e d i n a d e c l i n e i n the respons iveness of the c e l l (F igu re 11 ) . In some cases t h i s d e c l i n e 63 F igure 7. Response o f neurones i n the hippocampus to i on topho res i s o f AVP. In t h i s and a l l subsequent ratemeter r e c o r d s , the f i r i n g ra te i s measured on the o rd ina te i n sp i kes per second and t ime on the a b s c i s s a i n m inu tes . The i o n t o p h o r e t i c cu r ren ts were a p p l i e d f o r the pe r iod o f t ime represented by the h o r i z o n t a l b a r s ; the numbers over the bars i n d i c a t e the magnitude o f the cu r ren t i n nanoamperes. Th is f i g u r e i l l u s t r a t e s the response o f three neurones in the hippocampus to AVP. No te , i n t race B, the lack o f response to i on topho res i s of H + ions from a d i l u t e s o l u t i o n o f a c e t i c a c i d . 65 40 i AVP 80 AVP 50 AVP 30 40 AVP 40 AVP 50 AVP 90 AVP 90 Minutes F igure 8. Response o f neurones i n the septum to i on topho res i s of AVP, The responses o f 3 c e l l s are shown. 66 B LVP 90 AVP 50 AVP 10 AVP 8| AVP 30 OXT 30 10 Minutes F igu re 9 . I n h i b i t i o n o f neurones i n the septum by i o n t o p h o r e t i c c u r r e n t s . The a c t i v i t y o f the neurone represented i n t r ace A was the r e s u l t of con-t inuous a p p l i c a t i o n o f glutamate (25nA) dur ing the pe r iod of the r eco rd i ng , The c e l l i n t race B was spontaneously a c t i v e . 57 0 I , , , : , 6 8 10 Minutes F igure 10. Neurone responding w i th an i n h i b i t i o n fo l l owed by an e x c i t a t i o n . In t r a c e A , glutamate (4 nA) was a p p l i e d c o n t i n u o u s l y ; i n t r a c e B, from the same hippocampal neurone, no glutamate was a p p l i e d . 68 40 AVP 50 AVP 50 T " 4 o m m o AVP 50 AVP 50 Minutes F igu re 11 . Dec l i ne in respons iveness dur ing repeated a p p l i c a t i o n s o f AVP. The responses of a s i n g l e hippocampal neurone to repeated a p p l i c a t i o n s of AVP are shown. The three t r a c e s are cont inuous except as i n d i c a t e d . 69 may have been due to p rog ress i ve b lockage of the m ic rop ipe t t e t i p , as e v i -denced by the i n c r e a s i n g r e s i s t a n c e of the b a r r e l , r a t h e r than to t a c h y p h y l a x i s . The sudden i n h i b i t i o n s , on the o ther hand, were not a l t e r e d by changes i n r e s i s t a n c e and were probably due to n o n - s p e c i f i c cu r ren t e f f e c t s . Cont ro l s o l u t i o n s , c o n s i s t i n g of d i l u t e s o l u t i o n s of a c e t i c a c i d i d e n t i c a l to those used to d i s s o l v e the p e p t i d e s , were t es ted on most c e l l s t ha t responded to v a s o p r e s s i n . Of the 39 c e l l s e x c i t e d by AVP or LVP, 6 were a l s o e x c i t e d by the con t ro l s o l u t i o n (F igure 12 ) . The r e -sponse to the con t ro l s o l u t i o n would seem to suggest t ha t the e x c i t a t i o n s cou ld have been caused by H + ions ra the r than by v a s o p r e s s i n , but t h i s was not the c a s e : the e x c i t a t i o n s were never observed dur ing i on topho res i s o f o x y t o c i n , even though most o f the cu r ren t from the oxy toc in b a r r e l s would be c a r r i e d by H + i o n s . Assay o f the a c e t i c a c i d s o l u t i o n s from two m ic rop ipe t t es revea led tha t the s o l u t i o n s were contaminated w i th AVP ( 3 X 1 0 - 9 M and 5X10" 9 M, compared to 4X10 - I + M AVP i n the vasop ress in b a r r e l ) . Contaminat ion to t h i s ex ten t i s probably unavo idab le ; the m ic ro -p i p e t t e s were s to red i n a r e f r i g e r a t o r w i th the t i p s immersed i n water and were t h e r e f o r e coated w i th a cont inuous f i l m of condensed wa te r , which would a l l o w d i f f u s i o n between the upper ends of the b a r r e l s . Some of the oxy toc in b a r r e l s might a l s o have been contamined by v a s o p r e s s i n . The vasop ress in would not have been e x p e l l e d as r e a d i l y from the oxy toc in b a r r e l s as from the con t ro l b a r r e l s , however, s i nce the oxy toc in b a r r e l s a lmost i n v a r i a b l y e x h i b i t e d some degree of b lockage and would t he re fo re have passed much l e s s pept ide f o r a g iven c u r r e n t . The response to i o n t o p h o r e t i c a p p l i c a t i o n o f vasop ress in was a l s o AVP 80 40 1 H + 80 H + 80 -1— 4 to cu Q. 00 AVP 220 H + 90 40 i F igure 12. 0 Minutes E x c i t a t i o n produced dur ing i on tophores i s o f H + i ons , c o n s i s t e n t l y to both H + and AVP. — The two hippocampal neurones responded o 71 s tud ied in hippocampal s l i c e p repa ra t i ons from 8 Wis ta r r a t s . Gradual e x c i t a t i o n s were observed w i th 5 o f the 14 c e l l s t es ted (F igu re 13 ) . The response d i d not d i f f e r from those repor ted f o r the i n v i vo s t u d i e s . The r e s u l t s presented here i n d i c a t e tha t AVP and LVP can cause r e l a -t i v e l y r a p i d changes i n the a c t i v i t y of neurones i n the septum and the hippocampus. Neurones i n the septum d id not seem to respond to o x y t o c i n , but i t i s p o s s i b l e t ha t the oxy toc in was not e x p e l l e d from the m ic ro -p i p e t t e s i n s u f f i c i e n t q u a n t i t i e s . Oxytoc in was not t es ted in the h ippo-campus. S ince the response to vasop ress in occur red even in v i t r o , i t i s not l i k e l y to have been the r e s u l t o f v a s o c o n s t r i c t i o n . Fur thermore, L a s s o f f and A l t u r a (1980) have repor ted tha t vasop ress in does not cause c o n s t r i c t i o n o f the ce reb ra l m i c r o v a s c u l a t u r e . 2. DISCUSSION Nerve t e rm ina l s c o n t a i n i n g immunoreactive vasop ress in have been demon-s t r a t e d a t both the l i g h t and e l e c t r o n m ic roscop i c l e v e l i n the l a t e r a l septum and the hippocampus of the r a t (Sofroniew and We ind l , 1978; B u i j s and Swaab, 1979; B u i j s , 1980) . The work repor ted here i n d i c a t e s tha t neurones in these areas respond to vasop ress in a p p l i e d by i on topho res i s and t h e r e f o r e suggests t ha t vasop ress in i s , i n f a c t , the a c t i v e p r i n c i p l e , or at l e a s t one of the a c t i v e p r i n c i p l e s , r e l eased by the f i b e r s . That vasop ress in can a f f e c t the f i r i n g ra tes o f neurones i s n o t , i n i t s e l f , new. N i c o l l and Barker (1971) found tha t 80% of neurones i n the ca t sup raop t i c nucleus (SON) were i n h i b i t e d by i on topho res i s of LVP. A few e x c i t a t o r y responses were a l s o observed . The oppos i te response was F igu re 13. Response to i on topho res i s o f AVP i n v i t r o . The e f f e c t o f AVP on two neurones i n the i n v i t r o hippocampus i s i l l u s t r a t e d . The AVP was a p p l i e d by i on topho res i s i n the c e l l body l a y e r o f the CAT r e g i o n . 73 observed i n the c o r t e x , where 90% of neurones were e x c i t e d by LVP. The s e n s i t i v i t y o f c o r t i c a l neurones i s somewhat s u r p r i s i n g , s i nce there i s no ev idence tha t f i b e r s c o n t a i n i n g vasop ress in p r o j e c t to t h i s a r e a . In a s i m i l a r study by Moss e t . a l . (1972) , oxy toc in a p p l i e d by i on topho res i s was found to e x c i t e a ma jo r i t y of neurosecre to ry c e l l s i n the p a r a v e n t r i -c u l a r nucleus (PVN) o f the r a t and r a b b i t . The pept ide had no e f f e c t on non-neurosecre tory c e l l s i n the PVN - t ha t i s , on c e l l s which were not a n t i d r o m i c a l l y invaded f o l l o w i n g s t i m u l a t i o n o f the neura l lobe - or on c e l l s i n the SON, c o r t e x , or tha lamus. Very low concen t ra t i ons of pept ide (10~ 7 M) and weak cu r ren ts (0 .5-30 nA) were used , and the responses were r e l a t i v e l y s m a l l ; the authors repo r t t ha t the use of h igher concen-t r a t i o n s o f o x y t o c i n i n the m i c r o p i p e t t e caused such "massive e x c i t a t i o n " tha t the c e l l s were " l o s t " . Vasopress in (AVP) a p p l i e d i n the same way had l i t t l e e f f e c t on SON (one c e l l e x c i t e d , one i n h i b i t e d , 3 una f fec ted) or PVN c e l l s (2 of 29 PVN c e l l s were i n h i b i t e d ) . The s e n s i t i v i t y of neurosecre to ry c e l l s to neurohypophysia l pep t ides seems to extend even to i n v e r t e b r a t e s p e c i e s . Vasopress in or oxy toc in a p p l i e d e i t h e r i n the bath ing medium ( t h resho ld 1 0 " 9 M) or by i on topho res i s caused long l a s t i n g e x c i t a t i o n o f neurosecre to ry c e l l s o f the s n a i l A p l y s i a c a l i f o r n i c a (Barker e t . a l . , 1975). The neurosecre to ry c e l l s are thought to r e l e a s e a pept ide s i m i l a r to the neurohypophysia l pept ides (Ba rke r , 1976) . As d i scussed more f u l l y i n the i n t r o d u c t i o n to t h i s t h e s i s , however, there has been r e l a t i v e l y l i t t l e d i r e c t ev idence tha t neurones i n pu ta t i ve t a r g e t areas ou t s i de the hypothalamus respond to the NHP p e p t i d e s . Mo r r i s e t . a l . (1980) showed t ha t o x y t o c i n , a p p l i e d by i on topho res i s o r p ressure e j e c t i o n , depressed the a c t i v i t y o f neurones i n the r a t caudal 74 m e d u l l a , but there was no c l o s e c o r r e l a t i o n between the l o c a t i o n of the respons ive c e l l s and the areas where immunoreactive f i b e r s were found . The present study demonstrates tha t the a c t i v i t y of neurones in two areas r o s t r a l to the hypothalamus can be a l t e r e d by l o c a l a p p l i c a t i o n o f v a s o p r e s s i n . Both o f these a r e a s , the septum and the hippocampus, have p r e v i o u s l y been suggested as p o s s i b l e t a r g e t areas of the NHP pep t i des . Workers in Ca lgary (Kas t ing e t . a l . , 1979) , f o r example, have shown tha t vasop ress in has an a n t i p y r e t i c e f f e c t when i n j e c t e d i n t o the septum of sheep. Because of the d i f f e r e n c e i n spec ies and t echn ique , i t i s not c l e a r whether the AVP s e n s i t i v e neurones s tud ied i n the present experiment correspond to those i nvo l ved i n a n t i p y r e s i s . Kova'cs e t . a l . (1979b) have repor ted tha t vasop ress in f a c i l i t a t e d pass i ve avoidance behav ior when i n -j e c t e d i n t o the dorsa l sep ta l n u c l e i o r the medial sep ta l n u c l e i of the r a t . In c o n t r a s t to the present study however, oxy toc in was found to be e q u a l l y e f f e c t i v e . Th is d i sc repancy may r e f l e c t the d i f f e r e n c e i n l o c a -t i o n (dorsa l and medial po r t i ons o f the septum versus the l a t e r a l septum). The demonstrat ion tha t i on topho res i s o f AVP and LVP can a l t e r the a c t i v i t y o f hippocampal neurones would seem to be i n agreement w i th the repor t by Huston e t . a l . (1977) t ha t i n j e c t i o n o f 10 uM LVP i n t o the hippocampus of anes the t i zed r a t s r e s u l t e d i n hippocampal spreading d e p r e s s i o n . The d i f f e r e n c e between the e x c i t a t i o n s observed w i th i on topho res i s and the depress ion t ha t occur red w i th the in t rah ippocampal i n j e c t i o n s do not represen t a c o n f l i c t , s i nce spread ing depress ion can r e s u l t from the mas-s i v e d e p o l a r i z a t i o n induced by an e x c i t a t o r y agent . In the same s tudy , f o r example, su r face a p p l i c a t i o n o f the d e p o l a r i z i n g agent KC1 onto the co r tex caused c o r t i c a l spread ing d e p r e s s i o n . The r o l e of the NHP pept ides 75 i n hippocampal f u n c t i o n w i l l be exp lo red more f u l l y i n the next s e c t i o n of t h i s t h e s i s . Whi le the work repor ted here i n d i c a t e s tha t vasop ress in e x c i t e s neurones in the l a t e r a l septum and the hippocampus of the r a t , i on topho res i s was found to be a l e s s than i d e a l techn ique f o r s tudy ing pept ide induced e x c i t a t i o n s . The problem of contaminat ion of the con t ro l b a r r e l s cou ld probably be so lved by the use of lower concen t ra t i ons of the p e p t i d e s . Most workers seem to use qu i t e h igh concen t ra t i ons of pept ide f o r i o n -t opho res i s (Yarbrough and Haub r i ch , 1978; F e l i x and P h i l l i p s , 1979; Duggan e t . a l . , 1979) , but the work by Moss e t . a l . (1972) and by Barker e t . a l . (1975) , desc r i bed above, i n d i c a t e s tha t much lower concen t ra t i ons would have been e f f e c t i v e . The tendency o f the pept ides to b lock the m ic ro -p i p e t t e ba r re l was a more se r i ous problem. Fur the r study of the pept ides would r equ i r e the use of a n t a g o n i s t s , which are more l i p o p h i l i c than oxy toc i n and vasop ress in and would t he re fo re be even more d i f f i c u l t to expel from the m i c r o p i p e t t e . Palmer e t . a l . (1980) have suggested tha t m ic ropressure e j e c t i o n i s a more s u i t a b l e technique than i on topho res i s f o r the study of p e p t i d e s . A l t e r n a t i v e l y , one can apply the pept ides v i a the bath s o l u t i o n in an i n V i t r o p r e p a r a t i o n . Th is l a t t e r approach was chosen and i s the bas i s f o r the remainder of t h i s r e p o r t . 76 C. IN VITRO EXPERIMENTS 1. RESULTS AND ANALYSIS The NHP pept ides were found to have a pronounced, dose dependent e x c i t a t o r y e f f e c t when a p p l i e d i n concen t ra t i ons of TO" 9 to T O - 6 M onto r a t hippocampal s l i c e s . An example o f the e x c i t a t o r y a c t i o n of 0.5 yM AVP can be seen i n F igu re 14. Not a l l c e l l s responded to the pept ides (see be low) , and many c e l l s t ha t appeared to respond were r e j e c t e d f o r f u r t h e r study f o r t e c h n i c a l reasons such as smal l sp i ke ampl i tude or exces -s i v e background n o i s e . The data below are based on a study of 57 c e l l s t ha t responded unambiguously to repeated a p p l i c a t i o n s o f pep t i de . The e f f e c t s o f the o ther 3 n a t u r a l l y o c c u r r i n g NHP pept ides - LVP, AVT, and oxy toc i n - were q u a l i t a t i v e l y and q u a n t i t a t i v e l y s i m i l a r to tha t of AVP (F igu re 15 ) . Most neurones were t es ted w i th more than one of these p e p t i d e s , and i n a l l cases responded e i t h e r to a l l o f the pept ides t es ted ( "pept ide s e n s i t i v e c e l l s " ) o r to none o f them. No c e l l was found which responded to one NHP pept ide but f a i l e d to respond to ano ther . Th is was not t rue of the pept ide somatos ta t in (SST) : wh i l e one c e l l responded to both SST and o x y t o c i n , two c e l l s s e n s i t i v e to the NHP pept ides f a i l e d to respond to SST, and one c e l l i n s e n s i t i v e to AVP was e x c i t e d by SST (F igure 16 ) . Severa l s y n t h e t i c analogues o f the NHP pept ides were a l s o t es ted (Table IV ) . The oxy toc in fragment PLG was found to be t o t a l l y i n a c t i v e i n t e s t s on 4 c e l l s s e n s i t i v e to oxy toc in and to AVT. The ratemeter record of a c e l l t ha t was una f fec ted by 1 0 " 5 M PLG but which responded to oxy toc in a t concen t ra t i ons as low as 1 0 " 9 M i s shown i n F igu res 17 and 77 a . oo AVP 0.5 uM Minutes F igure 14. E f f e c t o f supe r fus ion o f AVP on hippocampal neurones i n v i t r o . The supe r fus i ng s o l u t i o n was a l lowed to f low around the hippocampal s l i c e s c o n t i n u o u s l y . Dur ing the pe r i od represented by the h o r i z o n t a l ba r , a s o l u t i o n c o n t a i n i n g 0.5 uM AVP was s u b s t i t u t e d f o r the con t ro l s o l u t i o n . Both neurones shown i n t h i s f i g u r e were i n the same s l i c e . LVP 0.4 uM Minutes F igure 15. E f f e c t o f LVP, AVT, and OXT on hippocampal neurones i n v i t r o . The responses i n t r ace A and t r a c e B were from the same c e l l . AVP 0.4 yM SST 0.4 yM 20 4 o Minutes F igure 16. The e f f e c t o f SST on a neurone i n s e n s i t i v e to AVP. Note the d i s c o n t i n u i t y i n the time s c a l e . 80 PEPTIDE ACTION AVP AGONIST LVP AGONIST AVT AGONIST OXT AGONIST PLG INACTIVE DGAVP WEAK AGONIST ddOT AGONIST ddAVP AGONIST Gly 7 OT AGONIST d(CH 2 ) 5 Tyr(Me)AVP ANTAGONIST d(CH 2 ) 5 -D-Tyr (Me)VAVP ANTAGONIST Table V. A c t i v i t y o f Neurohypophysial Pept ides and Analogues i n Hippocampal S l i c e s . 81 18. G l y 7 O T , which i s a c t i v e a t oxy toc in (OT o r ME) recep to rs but not a t vasop ress in (VP or AD) recep to rs (Lowbridge e t . a l . , 1977) , was t e s t e d on 4 pept ide s e n s i t i v e c e l l s and was found to be as e f f e c t i v e as o x y t o c i n . In c o n t r a s t , DGAVP, t es ted on two c e l l s s e n s i t i v e to AVP, produced on ly a very weak e x c i t a t i o n o f one c e l l (F igu re 19) and f a i l e d to e x c i t e the o t h e r . The oxy toc i n analogue desamino -d i ca rba -oxy toc in (ddOT), which i s p ro tec ted aga ins t enzymat ic a t t ack a t the N-terminus and a t the d i s u l f i d e bond, was found to be an e f f e c t i v e agon i s t when t e s t e d on 6 pept ide s e n s i -t i v e c e l l s . The a c t i v i t y o f the analogue was s l i g h t l y g rea te r than t ha t o f o x y t o c i n , sugges t ing tha t oxy toc in may be p a r t i a l l y i n a c t i v a t e d by aminopept idase a c t i v i t y i n the hippocampal s l i c e . The equ i va len t AVP ana logue, ddAVP, was a l s o a c t i v e (1 c e l l ) , but i t s a c t i v i t y was not com-pared w i th tha t o f AVP. The response to a l l o f the NHP pept ides cou ld be b locked by p r i o r a p p l i c a t i o n o f the vasop ress in an tagon is t d (CH 2 ) 5 Tyr (Me)AVP. The s p e c i f i -c i t y o f the an tagon is t i s apparent i n F igu re 20 ; p r i o r a p p l i c a t i o n of an tagon is t a t a concen t ra t i on o f 0.1 uM almost comple te ly b locked the response to 0.4 uM ddOT but had no e f f e c t on the response to 2 mM a c e t y l c h o l i n e . The an tagon is t was a l s o t es ted a g a i n s t AVP, LVP, AVT, and oxy toc in and was e f f e c t i v e aga ins t each of these p e p t i d e s . In some exper iments , the an tagon is t was d i s s o l v e d i n 1% a c e t i c a c i d before being d i l u t e d 5000 f o l d i n Krebs s o l u t i o n . Cont ro l s o l u t i o n s c o n t a i n i n g the same amount o f a c e t i c a c i d had no de tec tab le e f f e c t on the respons iveness o f the c e l l s . A second vasop ress in a n t a g o n i s t , d(CH 2 ) 5 -D-Tyr( .Me)VAVP, was t e s t e d aga ins t AVP, o x y t o c i n , ddOT, and G ly OT. L i k e d (CH 2 ) 5 Tyr (Me)AVP, t h i s an tagon is t c o n s i s t e n t l y b locked the e f f e c t o f the NHP pept ides but 82 Minutes F igure 17. F a i l u r e o f a hippocampal neurone to respond to PLG. The 3 t r a c i n g s are from a s i n g l e c e l l which responded c o n s i s t e n t l y to AVT and to OXT but d i d not respond to PLG. 83 F igure 18. Response o f a hippocampal neurone to low concen t ra t i ons of OXT. Both t r a c i n g s are from the same c e l l shown i n F igure 17, which f a i l e d to respond to 10 uM PLG. The response to 10" 9 M OXT was de layed f o r 12 minu tes . s p i k e s / s e c 85 0.5 mV 1 s e c 50 msec 5 mV + F igu re 19. E f f e c t o f DGAVP on hippocampal neurones. (A) DGAVP was found to produce a weak e x c i t a t i o n o f one c e l l but (B) f a i l e d to e x c i t e a second pept ide s e n s i t i v e c e l l from the same r a t . (C) S t i m u l a t i o n o f the Scha f fe r c o l l a t e r a l pathway r e s u l t e d i n an i n h i b i t i o n o f the Gly 7 OT induced a c t i v i t y o f the c e l l i n t race B. Two sweeps were super imposed. (D) The popu la t i on sp i ke produced by s t i m u l a t i o n o f the same pathway (30 V o l t s , 0.2 msec) . 86 F igure 20. Antagonism o f the response to ddOT by d(CH 2)5Tyr(Me)AVP. The three t r a c i n g s are from the same c e l l , before ( A ) , dur ing ( B ) , and a f t e r (C) exposure to the an tagon is t ("ANTAG"). A p p l i c a t i o n o f the an tagon is t i s i n d i c a t e d by the dot ted l i n e . The an tagon is t was a p p l i e d f o r 10 minutes be fo re the s t a r t o f t r a c e B, and had been d i scon t i nued 20 minutes p r i o r to the s t a r t o f t r ace C. The response to ddOT, but not t ha t to a c e t y l c h o l i n e (Ach ) , was almost comple te ly b locked by the a n t a g o n i s t . In C, the c e l l i s beg inn ing to recover from the e f f e c t s o f the a n t a g o n i s t . 88 not tha t o f a c e t y l c h o l i n e (F igure 21 ) . The two an tagon is ts were t es ted on a t o t a l o f 15 c e l l s and never f a i l e d to b lock the response to any of the p e p t i d e s . The changes i n f i r i n g ra te produced by a p p l i c a t i o n o f the pept ides cannot be a t t r i b u t e d to mechanical d i s tu rbance o f the s l i c e , s i nce changing from one con t ro l s o l u t i o n to another never produced a de tec tab le change i n the f i r i n g ra te o f the c e l l s . S i m i l a r l y , changes i n the f low ra te o f the super fus ing s o l u t i o n were u s u a l l y found to be w i thout e f f e c t , even when the ra te was more than doub led . On one o c c a s i o n , however, a 17% i n -crease i n f l ow ra te was fo l l owed by a s l i g h t e x c i t a t i o n . To ta l c e s s a t i o n of the f low produced a.pronounced e x c i t a t i o n o f one c e l l . Repeated b r i e f a p p l i c a t i o n s o f pept ide by super fus ion d i d not cause pept ide s e n s i t i v e c e l l s to become l e s s r e s p o n s i v e , p rov ided tha t the f i r i n g ra te o f the c e l l was a l lowed to re tu rn to basal l e v e l s between a p p l i c a t i o n s . When sub jec ted to cont inuous a p p l i c a t i o n of pept ide however, the c e l l s became unresponsive f a i r l y q u i c k l y . As can be seen i n F igure 22 , a c e l l rendered unresponsive by cont inuous super fus ion w i th oxy toc in appeared t h e r e a f t e r to be i n s e n s i t i v e to AVP as w e l l , a f i n d i n g tha t suggests tha t both pept ides ac t on the same r e c e p t o r . There were few spontaneously a c t i v e c e l l s i n the s l i c e p r e p a r a t i o n s , but 7 of 8 spontaneously a c t i v e c e l l s (88%) were pept ide s e n s i t i v e . Most o f the pept ide s e n s i t i v e c e l l s i nc luded i n t h i s study had l i t t l e or no spontaneous a c t i v i t y but cou ld be induced to f i r e by super fus ion w i th pept ide - the c e l l s were d i scove red by exposing the s l i c e s to one of the pep t ides and then search ing f o r a c t i v e c e l l s . The pept ide s e n s i t i v e c e l l s were l oca ted i n the pyramidal c e l l l a y e r o f area CA1. S t i m u l a t i o n Gly 7 OT 0.4 uM I Ach 4 mM i « Figure 2 1 . Antagonism o f the response to G ly 7 0T by d (CH 2 ) 5 -D-Tyr (Me)VAVP. The an tagon i s t d ( c H 2 ) 5 -D-Tyr (Me)VAVP ("ANTAG") complete ly b locked the response to G l y 7 0T but not the response to Ach . Both t r aces are from the same c e l l , and the time s c a l e i s cont inuous except as i n d i c a t e d . 90 Figure 22 . Dec l i ne i n respons iveness o f a c e l l sub jec ted to cont inuous exposure to o x y t o c i n . There i s a 14 minute gap between the two t r a c i n g s . The e f f e c t o f a 25 minute a p p l i c a t i o n o f oxy toc i n i s shown (minute 14 to minute 39 on the t r a c i n g s ) ; the response to oxy toc in began to d e c l i n e w i t h i n 5-6 minutes . The s l i c e was then washed w i th c o n t r o l s o l u t i o n f o r 10 minutes (minute 39 to minute 4 9 ) , but the c e l l remained r e l a t i v e l y unresponsive to both o x y t o c i n and AVP. Th is c e l l had p r e v i o u s l y responded c o n s i s t e n t l y to repeated a p p l i c a t i o n s of oxy toc i n and AVP. 92 o f the S c h a f f e r c o l l a t e r a l pathway r e s u l t e d i n i n h i b i t i o n o f the pept ide induced a c t i v i t y o f these c e l l s (F igure 19C) , i n d i c a t i n g tha t they were pyramidal c e l l s r a t h e r than in te rneurones (Lee e t . a l . , 1980) . No pept ide s e n s i t i v e c e l l s were found i n area CA3 or area CA4 or i n the dentate gy rus , but these th ree reg ions were not exp lo red e x t e n s i v e l y . There was no obvious d i f f e r e n c e i n respons iveness between the l e f t and r i g h t h ippo-campi or between the ven t ra l and do rsa l po r t i ons o f the hippocampus. S l i c e p repa ra t i ons from .almost h a l f o f the ra t s s tud ied conta ined no pept ide s e n s i t i v e c e l l s , even though the s l i c e s o f ten appeared to be as hea l thy as the more respons ive s l i c e s when judged by the usual e l e c t r o -p h y s i o l o g i c a l c r i t e r i a (D ing led ine e t . a l . , 1980) . The cause o f t h i s v a r i a t i o n was not determined - the d i f f i c u l t i e s i n de termin ing why some s l i c e s f a i l to "work" have been d i scussed by Schwar tzk ro in (1981) - but i t seems l i k e l y t h a t the l ack o f respons iveness was a c o n d i t i o n o f the s l i c e s themse lves , perhaps the r e s u l t o f trauma dur ing c u t t i n g , ra the r than a p roper ty of the r a t from which the s l i c e s were t aken . In p a r t i c u l a r , the pept ide s e n s i t i v i t y d i d not depend on p r i o r exposure of the r a t to AVP, s i nce s l i c e s prepared from HoDI r a t s (2 female r a t s , 180-190 g) were as respons ive as those from Wis ta r r a t s (41 male r a t s , 160-360 g ) . F u r t h e r -more, one W is ta r r a t was i n j e c t e d w i th AVP (1 y g , i c v ) two days p r i o r to the exper iment and should t he re fo re have been s e n s i t i z e d to the pept ide (Kas t i ng e t . a l . , 1980) , but no respons ive c e l l s were found i n the s l i c e s from t h i s r a t . The response may be temperature dependent, s i nce s l i c e s main ta ined a t room temperature (8 r a t s ) , r a the r than a t 33°C, were i n -s e n s i t i v e to the p e p t i d e s . No pept ide s e n s i t i v e c e l l s were found in s l i c e s main ta ined i n 1.0 mM C a + + (1 r a t ) or i n 1.0 mM C a + + w i th 6.25 mM K + 93 (1 r a t ) , but t h i s f i n d i n g may be c o i n c i d e n t a l , s i nce the s l i c e s i n these two p repara t ions appeared to be i n r e l a t i v e l y poor c o n d i t i o n . S l i c e s t es ted i n 1.5 mM C a + + (1 r a t ) d i d not appear l e s s respons ive than those i n 2.0 mM C a + + (40 r a t s ) . 2. DISCUSSION Comparison o f I on tophore t i c and Super fus ion S tud ies The e x c i t a t i o n of r a t hippocampal neurones by oxy toc i n and vasop ress in a p p l i e d i n v i t r o demonstrates t ha t these pept ides can ac t d i r e c t l y on b ra in t i s s u e . The r e s u l t s c o n f i r m , moreover, t h a t the e x c i t a t o r y responses observed dur ing i on topho res i s o f vasop ress in were due to the pept ide and not to the e j e c t i o n o f H + ions or to n o n s p e c i f i c cu r ren t e f f e c t s . There were minor d i f f e r e n c e s however, between the e f f e c t s observed w i th i o n -tophores i s and those observed w i t h s u p e r f u s i o n : (1) The response was more pronounced when the vasopress in was a p p l i e d by s u p e r f u s i o n . Th is was not an unexpected f i n d i n g , s i nce the amount o f vasop ress in r e l eased from the m i c r o p i p e t t e s was r e l a t i v e l y s m a l l , and s i n c e the pept ide would be l a r g e l y con f ined to the area of the c e l l body. S u p e r f u s i o n , on the o ther hand, would a l l ow the pept ide to reach the d e n d r i t e s , where the f i b e r s immuno-r e a c t i v e f o r vasop ress in are repor ted to synapse ( B u i j s e t . a l . , 1978; B u i j s and Swaab, 1979) . (2) There was a d i f f e r e n c e i n the t ime course o f the response: the f a c t t ha t the a c t i v i t y o f the c e l l s o f ten began to i nc rease w i t h i n seconds when vasop ress in was a p p l i e d by i on topho res i s (F igu re 8) i n d i c a t e s tha t the de lay observed w i th super fus ion was due almost e n t i r e l y to the t ime requ i red f o r the pept ide to reach the c e l l . (3) Repeated i on topho res i s of vasop ress in o f ten r e s u l t e d i n a r e l a t i v e l y 94 r a p i d d e c l i n e i n the magnitude of the response (F igure 1 1 ) , an e f f e c t not observed when the pept ide was a p p l i e d by s u p e r f u s i o n . Th is d i f f e r e n c e was a c h a r a c t e r i s t i c o f the method o f a p p l y i n g the pept ide r a t h e r than the type o f p r e p a r a t i o n , s i nce i on topho res i s r e s u l t e d i n a d e c l i n e both i n v i vo and i n v i t r o , and i t t h e r e f o r e seems l i k e l y t ha t the d e c l i n e was due to p rog ress i ve blockage o f the m i c rop ipe t t e t i p r a t h e r than to t a c h y p h y l a x i s . A l t e r n a t i v e l y , the l o c a l concen t ra t i on o f vasop ress in a t the c e l l body may be h igher dur ing i on topho res i s than dur ing s u p e r f u s i o n , r e s u l t i n g i n a more r a p i d d e c l i n e i n respons i veness . Nature o f the Hippocampal Receptors Con f i rmat ion t ha t the response to the NHP pept ides was mediated by s p e c i f i c recep to rs was found in the a b i l i t y o f two vasopress in an tagon is t s to b lock the response. The an tagon is t d(CH 2 ) 5 Tyr(Me)AVP (Kruszynsk i e t . a l . , 1980) b locks the response to vasop ress in a t the VP (vasopressor ) recep to r but has n e g l i g i b l e a c t i v i t y a t the AD ( a n t i d i u r e t i c ) r e c e p t o r , wh i l e d (CH 2 ) 5 -D-Tyr (Me)VAVP (Manning e t . a l . , 1982) i s a potent an tagon is t a t both recep to rs (Table I I ) . S ince both an tagon is t s were able to b lock the e x c i t a t i o n s i n the hippocampal s l i c e , the responses could not have been mediated by the AD r e c e p t o r . Oxytoc in and G1y 7 0T, which are r e l a t i v e l y i n a c t i v e a t the vasop ress in recep to rs (Berde and Bo i ssonnas , 1966; Lowbridge e t . a l . , 1977; Cash , 1978) , may have been a c t i n g on oxy toc in recep to rs s i m i l a r to those i n the uterus or the mammary g land (the OT and the ME r e c e p t o r s , r e s p e c t i v e l y ) . Th is would not be i n c o n s i s t e n t w i th the a b i l i t y o f the vasop ress in an tagon is t d (CH 2 ) 5 Tyr (Me)AVP to b lock the hippocampal response to o x y t o c i n , s i nce t h i s an tagon is t has a l s o been repor ted to b lock the a c t i o n o f oxy toc i n on the r a t u terus i n v i t r o 95 (Kruszynsk i e t . a l . , 1980) . The e f f e c t o f d (CH 2 ) 5 -D-Tyr (Me)VAVP on OT and ME recep to rs has not been r e p o r t e d . I t seems more l i k e l y , however, t h a t oxy toc in and vasopress in were both a c t i n g on the same popu la t ion o f r e c e p t o r s . Th i s i n t e r p r e t a t i o n i s supported by the f a c t t h a t the hippocampal neurones were never found to d i s t i n g u i s h between the two p e p t i d e s ; c e l l s respons ive to e i t h e r o f these pept ides were always found , when t e s t e d , to be about e q u a l l y respons ive to the o t h e r . The demonstrat ion t h a t cont inuous t reatment w i th oxy toc in rendered a c e l l r e f r a c t o r y to both oxy toc i n and vasop ress in a l s o i n d i c a t e s a common recep to r f o r the two p e p t i d e s . I f t h i s i s the c a s e , the h ippo-campal r ecep to r must be d i f f e r e n t from the endocr ine NHP pept ide recep to rs l i s t e d i n Table I I , a l l o f which are r e l a t i v e l y s e l e c t i v e f o r oxy toc in or v a s o p r e s s i n . There i s a cons ide rab le body of e v i d e n c e , summarized i n Chapter I o f t h i s t h e s i s , t ha t a d d i t i o n a l types o f NHP pept ide recep to r are present i n mammalian t i s s u e s , but d e t a i l e d i n fo rma t ion about these recep to rs i s l a c k i n g . The s p e c i f i c i t y o f the hippocampal recep to r i s s i m i l a r to t ha t o f the NHP pept ide recep to r i n the nervous system of the s n a i l , i n t h a t both recep to rs respond to o x y t o c i n , AVP, LVP, and AVT but not to PLG or DGLVP (Barker e t . a l . , 1975) . There i s a l s o ev idence t ha t the NHP pept ides are conver ted to s m a l l e r fragments by enzymes i n the b r a i n (Burbach e t . a l . , 1980; Burbach and de Wied, 1981) , and t ha t these fragments are r e s p o n s i b l e f o r a t l e a s t some o f the e f f e c t s o f the pept ides (F lexner e t . a l . , 1978; Wal ter e t . a l . , 1975; 1978). Burbach e t . a l . (1980) have repor ted tha t oxy toc in can be c leaved by enzymes found i n a synaptosomal plasma membrane p repara t i on o f r a t l i m b i c b ra i n t i s s u e . Both aminopept idase a c t i v i t y and "C- te rm ina l 96 c l e a v i n g pep t i dase " a c t i v i t y were found, line C- te rmina l p e p t i d a s e , which was present i n on ly smal l amounts, r e l eased g lyc inamide and Leu-GlyNH 2 from o x y t o c i n . I t seems u n l i k e l y , however, t h a t the e x c i t a t i o n s observed i n the present study were caused by a c t i v e f ragments. I f the pept ides were conver ted to a c t i v e fragments by an aminopept idase, f o r example, one would expect the enzyme r e s i s t a n t analogue ddOT to be l e s s a c t i v e than o x y t o c i n ; i n f a c t , the oppos i te was found t o be the c a s e . C leavage .o f the C- termina l p o r t i o n of the pept ides would be e q u a l l y u n l i k e l y to p ro -duce a c t i v e f ragments , s i nce removal o f even a s i n g l e C- te rmina l res idue from AVP r e s u l t e d i n a f ragment , DGAVP, a lmost t o t a l l y l a c k i n g i n a c t i v i t y . Oxytoc in can be s p l i t a t the 6-7 bond by an enzyme present i n the r a t median eminence ( C e l i s e t . a l . , 1971), but not y e t de tec ted i n the h ippo-campus, t o produce t o c i n o i c a c i d (0Ti_ 6) and PLG ( 0T 7 _ 9 ) . T o c i n o i c a c i d was not t e s t e d i n the present s tudy , but PLG was found to be t o t a l l y i n -a c t i v e . Both PLG ( K a s t i n e t . a l . , 1976) and DGAVP (Abood e t . a l . , 1980) a l t e r behav io r i n the i n t a c t r a t , but they must b ind to recep to rs d i f -f e r e n t from those s tud ied i n the present exper iment . T r i t i a t e d PLG i n -j e c t e d i n t o the l a t e r a l v e n t r i c l e o f the r a t has been shown to b ind s e l e c t i v e l y to hippocampal t i s s u e ( P e l l e t i e r e t . a l . , 1975), but the l a b e l was l a r g e l y con f ined to the c e l l body l a y e r , wh i l e the te rm ina l s immuno-r e a c t i v e f o r oxy toc i n and vasopress in are found p r i m a r i l y i n the d e n d r i t i c reg ion ( B u i j s e t . a l . , 1978; B u i j s and Swaab, 1979). E f f e c t o f Somatos ta t in The pept ide somatos ta t in (SST) , a t a concen t ra t i on o f 0.05-0.5 uM, was a l s o found to e x c i t e hippocampal neurones. The f a c t t ha t SST e x c i t e d a somewhat d i f f e r e n t popu la t i on o f neurones from the NHP pept ides conf i rms 97 t ha t SST must have been a c t i n g on a d i f f e r e n t se t o f r e c e p t o r s . Dodd and K e l l y (1978) have repor ted t ha t 3 mM SST a p p l i e d by i o n t o p h o r e s i s , by pressure i n j e c t i o n , o r i n the form o f d r o p l e t s caused r a p i d d e p o l a r i z a t i o n and an i nc rease i n e x c i t a b i l i t y o f CA1 c e l l s i n the r a t hippocampal s l i c e . P i t tman and S i g g i n s (1981) , on the o ther hand, found- t ha t 0 .12 -1 .2 ]M SST added to the bath s o l u t i o n i n h i b i t e d spontaneous a c t i v i t y m the r a t hippocampal s l i c e ; 9 o f 10 pyramidal c e l l s were hype rpo la r i zed by the pep t i de . The d i f f e r e n c e i n response cannot be a t t r i b u t e d to the h igher concen t ra t i on used by Dodd and K e l l y , s i nce e x c i t a t i o n s were observed i n the present study w i th concen t ra t i ons s i m i l a r to those used by Pi t tman and S i g g i n s . I n h i b i t i o n s would not have been de tec tab le i n the present study because o f the lack o f spontaneous a c t i v i t y . R e l a t i o n o f E x c i t a t i o n s to B iochemica l Changes There have been repo r t s t ha t oxy toc in and vasop ress in a f f e c t the r e l e a s e of s e r o t o n i n , dopamine, and norep inephr ine from nerve te rm ina l s i n seve ra l areas of the r a t b r a i n , i n c l u d i n g the hippocampus and the septum (Ramaekers e t . a l . , 1977; Schwarzberg e t . a l . , 1981). Th is response i s probably not i nvo l ved i n the e x c i t a t i o n s observed i n the present e x p e r i -ment, however. The s e r o t o n e r g i c and ca techo l aminerg ic inpu ts are severed i n the s l i c e p r e p a r a t i o n , and the NHP pept ides are repor ted to be incapab le o f i n c r e a s i n g the spontaneous e f f l u x o f these substances from i n a c t i v e t e rm ina l s i n v i t r o . For example, i n a study o f r a t hippocampal s l i c e s by Auerbach and L i p t o n (1982) , AVP d id not a f f e c t spontaneous se ro ton in r e l e a s e , even though the same concen t ra t i on o f AVP ( 1 0 " 9 - 1 0 ~ 6 M) i nc reased the ra te o f K + induced r e l e a s e . Fur thermore, the e f f e c t of AVP on K + i n -98 duced se ro ton in r e l ease was con f ined to the dentate a r e a , the s e r o t o n e r g i c p r o j e c t i o n to CA1 being u n a f f e c t e d . S i m i l a r l y , S t a r r (1982) has repor ted t h a t oxy toc in and vasop ress in a t tenuated the K + induced r e l e a s e but not the spontaneous r e l e a s e o f 3H-dopamine from r a t s t r i a t a l s l i c e s . The a b i l i t y o f AVP to i nc rease the c o r t i c o s t e r o n e b ind ing c a p a c i t y o f hippocampal t i s s u e (Ve ld ius and de K l o e t , 1982) would a l s o seem to be un re la ted to the e x c i t a t i o n o f hippocampal neurones, s i nce the b ind ing c a p a c i t y was a l s o i nc reased by DGAVP, which was i n a c t i v e in the present exper imen ts , and was not i nc reased by o x y t o c i n . E f f e c t of NHP Pept ides on Hippocampal A c t i v i t y Gahwi ler (1978) has s t u d i e d the e f f e c t o f the NHP pept ides on c u l t u r e d neurones from seve ra l areas o f the r a t b r a i n i n c l u d i n g the hippocampus and the hypothalamus. O x y t o c i n , a p p l i e d i n the ba th ing medium a t a con-c e n t r a t i o n o f 1 0 " 5 M, was found to e x c i t e 3 o f 9 hippocampal c e l l s but d i d not a f f e c t c e l l s from the sup raop t i c area or the hypothalamus. The e x c i t a -t i o n o f hippocampal c e l l s by oxy toc in i s i n agreement w i th the present s tudy , a l though the c u l t u r e d c e l l s appear to be l e s s respons ive than those in the hippocampal s l i c e . LVP, a t 1 0 " 7 to 1 0 " 6 M, caused a r a p i d , r e v e r s i b l e i n h i b i t i o n o f the hypothalamic c e l l s bu t , i n c o n t r a s t to the present f i n d i n g s , d i d not a l t e r the a c t i v i t y o f c e l l s from the hippocampus. C e l l s from the cerebe l lum and from the locus coeru leus were a l s o unaf fec ted by the pep t i de . The lack o f e f f e c t o f LVP on c u l t u r e d hippocampal c e l l s i s d i f f i c u l t to e v a l u a t e , however, s ince LVP was not t es ted i n the same concen t ra t i on as o x y t o c i n . SST was found to have no e f f e c t on the c u l t u r e d hippocampal o r hypothalamic c e l l s a t a concen t ra t i on o f 1 0 " 5 M, a l though 99 hippocampal c e l l s were e x c i t e d by 5X10" 7 M SST i n the present s tudy . S ince the present work was begun, the r e s u l t s of a s i m i l a r study of r a t hippocampal s l i c e s have been pub l i shed by workers i n Geneva (Muh le tha le r e t . a l . , 1982; D r e i f u s s and M u h l e t h a l e r , 1981 , 1982a). Vaso-p r e s s i n was found to i nc rease the spontaneous a c t i v i t y of CA1 neurones, and the response cou ld be b locked by the an tagon is t d (CH 2 ) 5 Tyr (Me)AVP. The concen t ra t i ons of pept ide (10~ 8 -10~ 6 M) and the t ime course and magnitude o f the responses were comparable to those repor ted i n t h i s t h e s i s . A l s o i n agreement w i th the present s tudy , repeated a p p l i c a t i o n s of pept ide r e s u l t e d i n l i t t l e or no t achyphy lax i s - whether t achyphy lax i s occur red w i th c o n t i n u -ous a p p l i c a t i o n was not r e p o r t e d ; and HoDI r a t s were found to be as respon-s i v e as normal r a t s . The medium used by Muh le tha le r e t . a l . (1982) had a low C a + + and M g + + content (1 .0 mM C a + + ; 1.4 mM M g + + ) and a high K + content (6.24 mM) compared to the medium used i n the present experiment (2.0 mM C a + + , 2 .0 mM M g + + , and 5.0 mM K + ) . As a r e s u l t , the c e l l s i n t h e i r p repara t ions had a g rea te r spontaneous f i r i n g ra te (mean 13 sp i kes per second) , the a c t i v i t y o c c u r r i n g as i n t e r m i t t e n t b u r s t s . F i f t y - s i x (89%) o f 63 spon-taneous ly a c t i v e c e l l s were repor ted to respond to AVP and LVP. M i ih le tha le r e t . a l . (1982) were p r i m a r i l y concerned w i th the e f f e c t s o f vasop ress in r a t h e r than of o x y t o c i n , and t h e i r i n v e s t i g a t i o n was t h e r e -f o r e con f ined to the ven t ra l hippocampus, the dorsa l hippocampus being r e l a t i v e l y devo id o f vasop ress in c o n t a i n i n g te rm ina l s ( B u i j s e t . a l . , 1978) . Oxy toc in was .a l so t es ted and was found to e x c i t e 5 o f 6 c e l l s , but the e f f e c t o f the an tagon is t on oxy toc in induced e x c i t a t i o n s was not t es ted and no oxy toc in analogues were s t u d i e d . The N-terminal vasop ress in fragment DGAVP and the cor respond ing C- te rmina l fragment g lyc inamide were 100 repor ted to be i n a c t i v e . The weak e x c i t a t i o n s produced by DGAVP i n the present exper iments would probably not have been de tec ted i n t h e i r s tudy because o f the h igh l e v e l o f spontaneous a c t i v i t y . The s p e c i f i c AD recep to r agon i s t DDAVP, which was not t e s t e d i n the present exper iment , was found by Muh le tha le r to be much weaker than AVP o r LVP. The two s t u d i e s are t he re fo re i n agreement t h a t the response was not mediated by the AD r e c e p t o r , and the lack o f response to DDAVP would seem to prec lude the HF recep to r as we l l (Table I I ) . Muh le tha le r e t . a l . (1982) demonstrated tha t the e x c i t a t i o n s produced by the pept ides were not due to a p resynap t i c a c t i o n , s i nce the response was not b locked by p r o p a n o l o l , a t r o p i n e , o r naloxone or by a b o l i s h i n g a l l s y n a p t i c a c t i v i t y w i th 10 mM M g + + . I n t r a c e l l u l a r s t u d i e s (D re i f uss and M u h l e t h a l e r , 1981; MUhle tha le r and D r e i f u s s , 1981) i n d i c a t e d tha t vaso-p r e s s i n caused a s l i g h t d e p o l a r i z a t i o n and a smal l i nc rease i n conductance i n a few o f the e x c i t e d c e l l s . The f a c t t h a t oxy toc in and vasop ress in were found to e x c i t e c e l l s i n the present study does not n e c e s s a r i l y mean tha t the p h y s i o l o g i c a l f u n c t i o n o f these pept ides i s e x c i t a t o r y . As mentioned above i n r e l a t i o n to SST, which e x c i t e s hippocampal neurones i n some l a b o r a t o r i e s and i n -h i b i t s them i n o t h e r s , the nature of the response may depend on the e x p e r i -mental c o n d i t i o n s . In the present exper iment , i n h i b i t i o n s would not have been detec ted because o f the low l e v e l o f spontaneous a c t i v i t y i n the NHP s e n s i t i v e c e l l s , but Muh le tha le r and D re i f uss (1981) repo r t t ha t i n t h e i r i n t r a c e l l u l a r s t u d i e s vasopress in was found to i n h i b i t a lmost as many c e l l s as i t e x c i t e d . Fur thermore, they have repor ted i n a more recent study (D re i f uss and M u h l e t h a l e r , 1982b) t ha t the c e l l s e x c i t e d by oxy toc in 101 and vasop ress in appeared to be i n t e r n e u r o n e s . . Pyramidal c e l l s were e i t h e r i n h i b i t e d or una f fec ted by the p e p t i d e s , and i n t r a c e l l u l a r record ings i n d i c a t e d tha t the i n h i b i t i o n s were the r e s u l t o f e x c i t a t i o n o f i n h i b i t o r y i n te rneu rones . Th is f i n d i n g i s not incompat ib le w i th t h e i r e a r l i e r r epo r t t ha t 89% of spontaneously a c t i v e c e l l s were e x c i t e d by v a s o p r e s s i n , s i nce the i n t e r m i t t e n t b u r s t i n g a c t i v i t y o f these c e l l s suggests t ha t they were probably in te rneurones (see D r e i f u s s and M i i h l e t ha le r , 1982b). I t i s not c l e a r , however, why they found most c e l l s to be e x c i t e d i n t h e i r e a r l i e r i n t r a c e l l u l a r work (D re i f uss and M u h l e t h a l e r , 1981) , s i nce one would expect most o f the impaled c e l l s to be pyramidal c e l l s , and i t i s d i f f i -c u l t to r e c o n c i l e t h e i r f i n d i n g s wi th the present work, i n which the neurones e x c i t e d by the pept ides were c l e a r l y pyramidal c e l l s (F igure 19 ) . The most obvious d i f f e r e n c e i n the techniques used i n the two l a b o r a t o r i e s i s i n the composi t ion o f the medium, which conta ined 2.0 mM C a + + i n the present study and i n some of the i n t r a c e l l u l a r work by Muh le tha le r and D r e i f u s s (1981) , but conta ined 0 . 8 - 1 . 0 mM C a + + i n t h e i r e x t r a c e l l u l a r s t u d i e s (Muh le tha le r and D r e i f u s s , 1981; Muh le tha le r e t . a l . , 1982). I t i s p o s s i b l e t h a t the pyramidal c e l l s are e x c i t e d by vasop ress in when the C a + + concen t ra t i on i s h i g h , but t ha t the in te rneurones are e x c i t e d when the C a + + concen t ra t i on i s low. The low C a + + concen t ra t i on may a l s o be the cause o f the pronounced spontaneous a c t i v i t y o f in terneurones repor ted by D re i f uss and Muh le tha le r (1982b). I f the i n h i b i t i o n o f the pyramidal c e l l s i n the low C a + + medium was caused by the e x c i t a t i o n o f i n t e rneu rones , i t i s p o s s i b l e tha t the e x c i t a -t i o n o f the pyramidal c e l l s i n the present work was secondary to i n h i b i -t i o n o f the i n te rneu rones . A l t e r n a t i v e l y , i t i s p o s s i b l e tha t the pept ides 102 have a d i r e c t e x c i t a t o r y e f f e c t on both the pyramidal c e l l s and the i n t e r -neurones, and t ha t the net e f f e c t on pyramidal c e l l a c t i v i t y i s a balance between the d i r e c t e x c i t a t i o n and the i n h i b i t i o n produced by inc reased in terneurone a c t i v i t y . Moreover, there have been repo r t s tha t the i n t e r -neurones may have both e x c i t a t o r y and i n h i b i t o r y e f f e c t s on pyramidal c e l l s i n v i t r o (Swanson e t . a l . , 1982, page 640 ) . CSF Pept ides and Hippocampal Func t ion I t i s u n l i k e l y t ha t endogenous oxy toc i n and vasopress in i n the CSF would cause e x c i t a t i o n o f neurones i n the CA1 reg ion of the hippocampus, s i nce the lowest concen t ra t i on o f pept ide producing a measurable e x c i t a -t i o n i n the s l i c e p repa ra t i on (10~ 9 M) was about 10-100 f o l d g rea te r than the concen t ra t i on o f the pept ides in the r a t CSF (Dogterom e t . a l . , 1977) . The CSF borne pept ides cou ld produce s i g n i f i c a n t e f f e c t s on l y i f the neurones are more respons ive in v i v o than i n v i t r o o r i f the concen t ra t i on o f the pept ides i n the CSF i s d r a s t i c a l l y i nc reased under some c i r cums tances , such as du r ing f e b r i l e convu ls ions (Kas t i ng e t . a l . , 1980) . The f a c t t ha t the neurones appeared to be unable to d i s t i n g u i s h between oxy toc in and vasopress in would a l s o argue aga ins t a r o l e f o r the CSF borne pep t i des . R e l a t i o n o f Hippocampal E x c i t a t i o n to Behav io ra l Changes There i s cons ide rab le ev idence tha t some o f the behav io ra l changes produced by i n j e c t i o n of oxy toc in or vasopress in may i nvo l ve the h ippo-campus (Kovacs e t . a l . , 1982; Ramaekers e t . a l . , 1977) , and the pept ide s e n s i t i v i t y o f hippocampal neurones demonstrated i n the present i n v i t r o exper iments would be c o n s i s t e n t w i th such a s u g g e s t i o n . I n t r a v e n t r i c u l a r i n j e c t i o n o f 1.0 ng of v a s o p r e s s i n , the dose most f r e q u e n t l y used i n 103 s t u d i e s o f cond i t i oned behav io r , would r e s u l t i n a concen t ra t i on g rea te r than 1 0 " 9 M i n the CSF, and the 100 ng i n j e c t i o n s used to produce c o n v u l -s ions in r a t s (Abood e t . a l . , 1980) would y i e l d a concen t ra t i on i n excess o f 1 0 " 7 M. S ince these concen t ra t i ons are comparable to those found to e x c i t e hippocampal neurones in v i t r o , i t would not be unreasonable to suspect t ha t the response to the c e n t r a l l y admin is te red vasopress in might be the r e s u l t o f pept ide induced e x c i t a t i o n o f hippocampal neurones. An e x c i t a t i o n o f hippocampal neurones c o u l d , f o r example, e x p l a i n the convu l s i ons produced by i c v i n j e c t i o n s of v a s o p r e s s i n . The major d i f -f i c u l t y w i th t h i s proposa l i s t h a t o x y t o c i n , which i s as e f f e c t i v e as vaso -p r e s s i n i n e x c i t i n g hippocampal neurones, has l i t t l e o r no convu lsan t a c t i v i t y (Abood e t . a l . , 1980) . S i m i l a r l y , the a b i l i t y o f vasop ress in to pro long r e t e n t i o n o f cond i t i oned behav ior cannot be exp la i ned by the e x c i t a t i o n o f hippocampal CA1 neurones, s i nce o x y t o c i n , which produces the same e x c i t a t i o n , does not produce the same changes i n cond i t i oned be-h a v i o r . I t seems more l i k e l y , i n f a c t , t ha t the prolonged r e t e n t i o n i n -vo lves the dentate gyrus of the hippocampus ra the r than the CAT r e g i o n . I t has been repor ted t ha t AVP i n j e c t e d i n t o the dentate gyrus f a c i l i t a t e s r e t e n t i o n o f avoidance behav io r , wh i l e oxy toc in has the oppos i te e f f e c t (Kovacs e t . a l . , 1979b), and t ha t an t i -AVP serum, i n j e c t e d i n t o the dentate g y r u s , impa i rs r e t e n t i o n o f avoidance behav ior (Kovcfcs e t . a l . , 1982). I t i s somewhat d i s t u r b i n g however, t h a t the photograph used to i n d i c a t e the cannula placement i n the l a t t e r study shows t ha t the cannula was i n the CA3 reg ion r a t h e r than i n the dentate gy rus . The ant iserum re l eased from the c a n n u l a , t r aced by an immunoperoxidase t echn ique , reached CA3 neurones and p o s s i b l y some CA1 and CA4 neurones and spread through the 104 v e n t r i c l e to the l a t e r a l septum but d i d not reach the granu le c e l l l a y e r o f the den ta te . AVP promotes se ro ton in r e l e a s e from the dentate gyrus but not from the CA1 reg ion (Auerbach and L i p t o n , 1982) , and there i s i n -d i r e c t ev idence t ha t the hippocampal se ro ton in tu rnover i s i nvo l ved i n the memory r e l a t e d ac t i ons of vasop ress in (Ramaekers e t . a l . , 1977) . The enhancement o f c e r t a i n spontaneous behav iors ( h y p e r a c t i v i t y , grooming, and fo rag ing ) i n mice i s qu i t e s i m i l a r i n i t s pept ide s p e c i f i c i t y to the e x c i t a t i o n o f CA1 neurones (Delanoy e t . a l . , 1979) . O x y t o c i n , v a s o p r e s s i n , and vaso toc in were found to be s t rong agon i s t s i n both systems, wh i l e DGAVP and DDAVP were weak or i n a c t i v e . I t i s t he re fo re p o s s i b l e tha t the changes i n spontaneous behav io r r e s u l t , a t l e a s t i n p a r t , from a change i n neuronal a c t i v i t y i n the CA1 reg ion of the h ippo -campus. The s i g n i f i c a n c e of t h i s behav io ra l response i s unknown. Cone!us ions The present s tudy demonstrates t ha t oxy toc in and vasopress in can cause r a p i d and profound changes i n the a c t i v i t y o f neurones i n the CA1 reg ion o f the r a t hippocampus. The nature o f the response , e x c i t a t i o n or i n h i b i t i o n , depends on the exper imenta l c o n d i t i o n s , and i t i s p o s s i b l e t h a t the response o f the pyramidal c e l l s i s secondary to changes i n i n t e r -neurone a c t i v i t y . Determining the r e l a t i o n o f the in v i t r o response to the p h y s i o l o g i c a l ac t i ons o f these pept ides w i l l r e q u i r e f u r t h e r s tudy . There i s apparen t l y more than one type o f NHP pept ide recep to r i n the hippocampus, s i nce the r e c e p t o r ( s ) r e s p o n s i b l e f o r the e x c i t a t i o n o f CA1 c e l l s appear to be d i f f e r e n t from the recep to r which mediates the 105 e f f e c t o f vasop ress in on se ro ton i n re lease i n the dentate gy rus . The PLG b ind ing s i t e present on CA1 neurones ( P e l l e t i e r e t . a l . , 1975) may rep re -sent an a d d i t i o n a l type o f NHP pept ide r e c e p t o r , the f u n c t i o n of which i s unknown. A comparison o f the pept ide s p e c i f i c i t y observed i n the present experiment w i th the s p e c i f i c i t y repor ted i n o ther s t u d i e s suggests t ha t the convu l s i ve and memory-re lated a c t i o n s o f vasop ress in do not d i r e c t l y i nvo l ve the CA1 reg ion and may be r e l a t e d i ns tead to the con t ro l o f s e r o -ton in metabol ism i n the dentate gy rus . The e x c i t a t i o n o f CA1 neurones by the NHP pept ides may be i nvo l ved i n the repor ted e f f e c t s o f these pept ides on spontaneous behav io r i n m ice . 106 CHAPTER IV. OXYTOCIN, VASOPRESSIN AND CNS FUNCTION I t has not been proven t ha t oxy toc in and vasop ress in a re . . invo lved in s y n a p t i c t r a n s m i s s i o n . The c r i t e r i a by which a substance i s e s t a b l i s h e d as a media tor o f s y n a p t i c t r ansm iss i on are the sub jec t o f some debate , s i nce some o f the c r i t e r i a o r i g i n a l l y desc r ibed f o r s t u d i e s o f smal l molecules may not be e n t i r e l y r e l e v a n t to pept ides (Werman, 1972; Bu rns tock , 1976; Barch ias e t . a l . , 1978) , but use fu l ev idence would c e r t a i n l y i nc l ude the demonstrat ion t ha t (1) the substance i s present i n and re l eased from s y n a p t i c t e r m i n a l s , and (2) t ha t the e f f e c t o f the endogenous substance can be i m i t a t e d by exogenous a p p l i c a t i o n and b locked by agents t h a t antagonize the exogenous a p p l i c a t i o n . That o x y t o c i n and vasopress in are present i n nerve te rm ina l s i n many areas o f the CNS has been we l l e s t a b l i s h e d by immunocytochemical and radioimmunoassay s t u d i e s , but r e l ease has been demonstrated on ly i n the septum (Cooper e t . a l . , 1979). I n j e c t i o n of AVP i n t o the septum o f sheep reduces the f e b r i l e response to endo tox in , wh i l e i n j e c t i o n o f a vasop ress in an tagon is t has the oppos i te e f f e c t (Cooper e t . a l . , 1979; Kas t ing e t . a l . , 1979). Th is f i n d i n g suggests t ha t the re l ease o f endogenous AVP may have an a n t i f e b r i l e a c t i o n i n the septum. Ion tophores is o f AVP was found to e x c i t e sep ta l neurones i n the present exper iment , but i t i s not c e r t a i n t ha t the popu la t i on o f neurones e x c i t e d by i on topho res i s o f AVP i s i n v o l v e d in the a n t i f e b r i l e response , and i on topho res i s o f an antago-n i s t was not a t tempted. The a c t i o n s o f oxy toc i n and vasopress in a p p l i e d to the hippocampus have been desc r ibed both i n the present study and i n the work o f Muh le tha le r e t . a l . (1982) , but wh i le the pept ides are known to be present i n nerve te rm ina l s i n the hippocampus ( B u i j s , 1980) , the c i rcumstances under which they are r e l eased and the response 107 to endogenous re l ease are e n t i r e l y unknown. Pi t tman e t . a l . (1981) have shown t h a t c e l l s i n the PVN can be a n t i -d r o m i c a l l y a c t i v a t e d by s t i m u l a t i o n o f the areas known to con ta in immuno-r e a c t i v e t e r m i n a l s , such as the l a t e r a l septum, the amygdala, and the per iaqueduc ta l g ray . I f s t i m u l a t i o n o f the a n t i d r o m i c a l l y i d e n t i f i e d c e l l s cou ld be shown to produce a measurable response i n the t a r g e t a r e a , i t might be p o s s i b l e to demonstrate t ha t the response cou ld be im i t a t ed by exogenous pept ide a p p l i e d , f o r example, by pressure i n j e c t i o n , or tha t i t cou ld be b locked by l o c a l a p p l i c a t i o n o f an a n t a g o n i s t . High f requency s t i m u l a t i o n might be necessary to r e l ease enough pept ide to produce a s i g n i f i c a n t response. I t i s not o b v i o u s , however, what type of response shou ld be measured; one might be able to de tec t a change i n u n i t a c t i v i t y , a m o d i f i c a t i o n o f the response evoked by s t i m u l a t i o n o f a t h i r d a r e a , or a b iochemica l or t r o p h i c change. That the changes produced by the pept ides may be more sub t l e than s imp ly e x c i t a t i o n or i n h i b i t i o n i s i n d i c a t e d i n a repo r t by Chepkova (1981) , who s tud ied the e f f e c t o f LVP on long term p o t e n t i a t i o n (LTP) o f the S c h a f f e r c o l l a t e r a l pathway i n mouse hippocampal s l i c e s . The LVP was added to the bath a t a concen t ra t i on o f 1-5 uM f o r a pe r i od of 30-60 minu tes , and LTP was induced by t e t a n i c s t i m u l a t i o n a p p l i e d dur ing t h i s p e r i o d . The pept ide d i d not i nc rease the i n i t i a l magnitude of the LTP, measured as the change in ampl i tude o f the CA1 popu la t i on s p i k e , and i n most cases d i d not a f f e c t the s i z e o f the popu la t ion sp i ke measured p r i o r to the LTP. The pept ide d i d appear , however, to pro long the du ra t i on o f LTP; t ha t i s , i n s l i c e s t r e a t e d w i th the p e p t i d e , the ampl i tude of the po ten t i a t ed popu la t i on sp ike d e c l i n e d more s l ow ly than i n unt reated 108 s l i c e s . Chepkova noted the s i m i l a r i t y between t h i s response and the r e -por ted a b i l i t y o f vasop ress in to pro long the du ra t i on o f cond i t i oned behav io r . U n f o r t u n a t e l y , the experiment d i d not e s t a b l i s h tha t the a c t i o n o f the pept ide was a c t u a l l y r e l a t e d to LTP, s i nce the popu la t i on sp i ke normal ly d e c l i n e s i n v i t r o even i n unpoten t ia ted s l i c e s . To e s t a b l i s h t ha t vasop ress in pro longs LTP, i t would be necessary to show t h a t . i t does not slow the d e c l i n e o f a second, unpoten t ia ted pathway. I t would a l s o be h e l p f u l to demonstrate t h a t the e f f e c t o f the vasop ress in on LTP cou ld be b locked by a vasop ress in a n t a g o n i s t . Whether the a c t i o n s of oxy toc i n and vasopress in i n the CNS are r e -l a t e d to t h e i r endocr ine ac t i ons i s not known. I f the f i b e r s p r o j e c t i n g to the va r ious po r t i ons o f the CNS are from the same c e l l s tha t p r o j e c t to the p o s t e r i o r p i t u i t a r y - such a p o s s i b i l i t y i s suggested by the b i -p o l a r and m u l t i p o l a r shape o f the c e l l s (Defend in i and Zimmerman, 1978) -then the pept ides w i l l be re l eased i n t o these areas under the same c i r -cumstances under which they are r e l e a s e d i n t o the b l ood . I f t ha t i s the c a s e , the CNS func t i ons must be r e l a t e d to the endocr ine f u n c t i o n s . B u i j s (1980) has po in ted out t h a t many o f the areas innerva ted by f i b e r s con-t a i n i n g v a s o p r e s s i n , such as the OVLT, the p e r i v e n t r i c u l a r n u c l e u s , and the c h o r o i d , are thought to be i nvo l ved i n water ba lance . Termina ls immunoreactive f o r vasop ress in have a l s o been found i n areas concerned w i th c a r d i o v a s c u l a r f u n c t i o n (Sof ron iew and S c h r e l l , 1981) . S i m i l a r l y , the promotion o f maternal behav io r by o x y t o c i n (Pedersen e t . a l . , 1982) would seem to be a reasonable a c t i o n f o r a hormone re l eased dur ing suck-l i n g . On the o ther hand, i f the CNS p r o j e c t i o n s are from a popu la t i on of neurones d i s t i n c t from those p r o j e c t i n g to the p o s t e r i o r p i t u i t a r y , then 109 i t i s p o s s i b l e t ha t the re l ease o f pept ide i n t o synapses i n the CNS i s un re la ted to the endocr ine a c t i o n o f the p e p t i d e s . Whi le t h i s po in t i s s t i l l c o n t r o v e r s i a l , e l e c t r o p h y s i o l o g i c a l exper iments by Pi t tman e t . a l . (1981) i n d i c a t e t ha t the PVN c e l l s p r o j e c t i n g to the l a t e r a l septum, the amygdala, and the per iaqueduc ta l gray do not p r o j e c t to the p i t u i t a r y . S i m i l a r l y , h i s t o l o g i c a l s t ud ies suggest t ha t the PVN c e l l s p r o j e c t i n g to the medul la and the sp ina l cord are separate from those p r o j e c t i n g to the p i t u i t a r y (Swanson and Sawchenko, 1980). I t was suggested i n the i n t r o d u c t i o n to t h i s t h e s i s t ha t an under-s tand ing o f the f unc t i ons o f vasop ress in and oxy toc in i n the CNS would be f a c i l i t a t e d by a more d e t a i l e d knowledge o f the ac t i ons of the pept ides in i n d i v i d u a l t a r g e t a r e a s . In p a r t i c u l a r , d i f f e r e n c e s i n the responses to i n j e c t i o n s of AVP, o x y t o c i n , DGLVP, and PLG suggest t ha t there may be reg iona l d i f f e r e n c e s i n pept ide s p e c i f i c i t y . The i n v i t r o s l i c e p repa ra -t i o n was found i n the present study to be a s a t i s f a c t o r y system f o r i n -v e s t i g a t i n g the e f f e c t s o f the pept ides in the hippocampus and would probably be e q u a l l y use fu l i n s tudy ing o ther a r e a s , such as the septum, the amygdala, the m e d u l l a , the s p i n a l c o r d , and the hypothalamus. The response can be l o c a l i z e d much more a c c u r a t e l y i n v i t r o than i s p o s s i b l e w i th m i c r o i n j e c t i o n t echn iques . In a d d i t i o n , s y n t h e t i c analogues can be screened f o r CNS a c t i v i t y much more r a p i d l y i n v i t r o than in v i v o . 110 REFERENCES Abood, L . G . ; Knapp, R . ; M i t c h e l l , T . ; Booth , H. and Schwab, L. (1980) Chemical requirements o f vasop ress in f o r ba r re l r o t a t i o n convu ls ions and r e v e r s a l by o x y t o c i n . J . N e u r o s c i . Research 5 :191-199. Ache r , R. (1981) E v o l u t i o n o f neuropep t ides . Trends i n Neurosc ience 4 : 225-229. A l t u r a , B. (1975) Dose-response r e l a t i o n s h i p s f o r a r g i n i n e vasop ress in and s y n t h e t i c analogs on three types of r a t b lood v e s s e l s : p o s s i b l e e v i -dence f o r reg iona l d i f f e r e n c e s i n vasop ress in recep to r s i t e s w i t h i n a maml. J . Pharmacol . Exp. Ther . 193:413-423. Antunes, J . L . and Zimmerman, E.A. (1978) The hypothalamic magnoce l lu la r system of the rhesus monkey: an immunocytochemical s tudy . J . Comp. N e u r o l . 181:539-566. Auerbach, S . and L i p t o n , P. (1982) Vasopress in augments d e p o l a r i z a t i o n -induced r e l e a s e and syn thes i s o f se ro ton in i n hippocampal s l i c e s . J .  N e u r o s c i . 2 :477-482. B a i l e y , W.H. and We iss , J . M . (1981) Avoidance c o n d i t i o n i n g and endocr ine f u n c t i o n i n B r a t t l e b o r o r a t s . ln_ J . L . M a r t i n e z , J r . , R.A. Jensen , R .B . Mess ing , H. R i g t e r and J . L . McGaugh ( E d i t o r s ) Endogenous Pept ides and Lea rn -ing and Memory P rocesses , pp. 371-395. Academic P r e s s , New York . Ba rchas , J . D . ; A k i l , H . ; E l l i o t t , G . R . ; Holman, R .B . and Watson, S . (1978) Behav io ra l neurochemis t ry : neuro regu la to rs and behav io ra l s t a t e s . Sc ience 200:964-973. Bargmann, W. and S c h a r r e r , E. (1951) The s i t e of o r i g i n of the hormones of the p o s t e r i o r p i t u i t a r y . Amer. S c i e n t i s t 39:255-259. B a r k e r , J . L . (1976) P e p t i d e s : r o l e s i n neuronal e x c i t a b i l i t y . P h y s i o l . Rev. 56 :435-452. B a r k e r , J . L . ; I f s h i n , M.S. and G a i n e r , H. (1975) S tud ies on b u r s t i n g pacemaker p o t e n t i a l a c t i v i t y i n mol luscan neurons. I I I . E f f e c t s o f hormones. B ra in Res. 84 :501-513 . Berde , B. and Bo i ssonnas , R.A. (1966) Syn the t i c analogues and homologues of the p o s t e r i o r p i t u i t a r y hormones. In G.W. H a r r i s and B.T. Donovan ( E d i t o r s ) The P i t u i t a r y G land , pp. 627R>61. Bu t te rwo r ths , London. Be rk , M.L. and F i n k e l s t e i n , J . A . (1981) An au to rad iog raph i c de te rmina t ion o f the e f f e r e n t p r o j e c t i o n s of the suprach iasmat ic nucleus of the hypo tha la -mus. B ra in Res. 226 :1 -13 . B e r n , H.A. and Knowles, F.G.W. (1966) N e u r o s e c r e t i o n . In L. M a r t i n i and W.F. Ganong ( E d i t o r s ) Neuroendocr ino logy. V o l . 1, pp. 139^"186. Academic P r e s s , New York . I l l B l a s k , D .E. and Vaughan, M.K. (1980) Naloxone i n h i b i t s a r g i n i n e v a s o t o c i n -(AVT) induced p r o l a c t i n r e l e a s e in u re thane -anes the t i zed male r a t s in v i v o . N e u r o s c i . L e t t . 18:181-184. B l a s k , D . E . ; Vaughan, M .K . ; R e i t e r , R . J . and Johnson, L . Y . (1978) In f luence of a r g i n i n e v a s o t o c i n on the es t rogen- induced surge of LH and FSH i n adu l t ova r iec tomized r a t s . L i f e S c i . 23:1035-1040. Bohus, B. (1974) The i n f l u e n c e of p i t u i t a r y pept ides on b ra i n cen te rs con-t r o l l i n g autonomic responses . In D .F . Swaab and J . P . Schade ( E d i t o r s ) I n t e g r a t i v e Hypothalamic A c t i v i t y , pp. 175-183. E l s e v i e r , Amsterdam. Bohus, B . ; Ade r , R. and de Wied, D. (1972) E f f e c t s o f vasop ress in on a c t i v e and pass i ve avoidance behav io r . Hormones and Behav ior 3 :191-197. Bohus, B . ; Kovcics, G . L . and de Wied, D. (1978) O x y t o c i n , v a s o p r e s s i n , and memory: oppos i te e f f e c t s on c o n s o l i d a t i o n and r e t r i e v a l p rocesses . B ra in Res. 157:414-417. B r a d l e y , P . B . and Candy, J . M . (1970) Ion tophore t i c r e l e a s e of a c e t y l c h o l i n e , n o r a d r e n a l i n e , 5-hydroxyt ryptamine and D - l y s e r g i c a c i d d ie thy lamide from m i c r o p i p e t t e s . B r i t . J . Pharmacol . 40 :194-201 . B r i t o , G . P . ; Thomas, G . J . ; G i n g o l d , S . I . and Gash, D.M. (1981) Behav io ra l c h a r a c t e r i s t i c s o f v a s o p r e s s i n - d e f i c i e n t r a t s ( B r a t t l e b o r o s t r a i n ) . B ra in Res. B u l l . 6 : 71 -75 . B u i j s , R.M. (1980) Immunocytochemical demonstrat ion of vasop ress in and o x y t o c i n i n the r a t b ra i n by l i g h t and e l e c t r o n mic roscopy . J . Histbchem. Cytochem. 28:357-360. B u i j s , R .M. and Swaab, D.F . (1979) Immuno-electron m i c r o s c o p i a l demonstra-t i o n of vasop ress in and oxy toc i n synapses in the l i m b i c system of the r a t . C e l l T i s s . Res. 204:355-365. B u i j s , R . M . ; Swaab, D . F . ; Dogterom, J . and van Leeuwen, F.W. (1978) I n t r a -and ex t rahypotha lamic vasop ress in and o x y t o c i n pathways i n the r a t . Cel1  T i s s . Res. 192:423-433. Burbach, J . P . H . and de Wied, D. (1981) Memory e f f e c t s and b ra i n p r o t e o l y s i s o f neurohypophyseal hormones. '_In D.H. S c h l e s i n g e r ( E d i t o r ) Neurophpophyseal  Pep t ide Hormones and Other B i o l o g i c a l l y A c t i v e P e p t i d e s , pp. 69-87 . E l s e v i e r , New York . Burbach, J . P . H . ; de K l o e t , E .R. and de Wied, D. (1980) Oxytoc in b i o t r a n s -fo rmat ion i n the r a t l i m b i c b r a i n : c h a r a c t e r i z a t i o n o f pept idase a c t i v i t i e s and s i g n i f i c a n c e i n the fo rmat ion of oxy toc in f ragments . B ra in Res. 202: 401-414. Bu rna rd , D .M, ; P i t t m a n , Q . J . and V e a l e , W.L. (1982) Vasopress in - i nduced convu ls ions in the B r a t t l e b o r o r a t . Canada Phys io logy 12 :97 . 112 Burns tock , G. (1976) Do some nerve c e l l s r e l e a s e more than one t r a n s -m i t t e r ? Neuroscience 1:239-248. Carey , R . J . and M i l l e r , M. (1982) Absence of l e a r n i n g and memory d e f i c i t s i n the v a s o p r e s s i n - d e f i c i e n t r a t ( B r a t t l e b o r o s t r a i n ) . Behav. B ra in Res. 6 : 1 - 1 3 . Cash , J . D . ; Gader , A . M . A . ; Mu lder , J . L . and C o r t , J . H . (1978) S t r u c t u r e -a c t i v i t y r e l a t i o n s o f the f i b r i n o l y t i c response to vasopress ins i n man. C l i n . S c i . Mo lec . Med. 54:403-409. C e l i s , M . E . ; T a l e i s n i k , S . and W a l t e r , R. (1971) Regu la t i on of fo rmat ion and proposed s t r u c t u r e o f the f a c t o r i n h i b i t i n g the r e l e a s e of melanocyte s t i m u l a t i n g hormone. P r o c . Nat . Acad . S c i . (USA) 68:1428-1433. Chauvet , M .T . ; Hurpet , D . ; Chauvet , J . and Acher , R. (1981) Phylogeny of neurohypophyseal hormones. I n t l . J . Pept ide P r o t e i n Res. 17 :65 -71 . Chepkova, A . N . (1981) E f f e c t o f vasop ress in on c h a r a c t e r i s t i c s o f prolonged p o s t t e t a n i c p o t e n t i a t i o n in s l i c e s of hippocampus. Zhurhal Vysshei NerVoi  Deya T e l n o s t i - I. P. Pavlova 31:427-430. C l a r k e , G . ; H i l l , R .G. and Simmonds, M.A. (1973) M i c r o i o n t o p h o r e t i c r e -lease of drugs from m i c r o p i p e t t e s : use of 2 4 N a as a model. B r i t . J .  Pharmacol . 48 :156-161 . Cooper, K . E . ; K a s t i n g , N.W.; L e d e r i s , K. and V e a l e , W.L. (1979) Evidence suppor t ing a r o l e f o r endogenous vasop ress in in na tu ra l suppress ion of f e v e r i n sheep. J . P h y s i o l . (Lond.) 295 :33-45 . C o r t , J . H . ; F ischman, A . J . ; Dodds, W . J . ; Rand, J . H . and Schwar tz , I .L . (1981) New category o f vasop ress in recep to r i n the c e n t r a l nervous system. I n t l . J . Pep t ide P r o t e i n Res. 17 :14 -22 . C r o s s , B .A. (1973) Un i t responses i n the hypothalamus. In W.F. Ganong and L. M a r t i n i ( E d i t o r s ) F r o n t i e r s i n Neuroendocr ino logy,~T973. pp. 133-171. Oxford Un iv . P r e s s , New York . C r o s s , B.A. (1974) The Neurosecre tory impu lse . ln_ F. Knowles and L. V o l ! r a t h ( E d i t o r s ) Neurosecre t ion - the F i n a l Neuroendocr ine Pathway, pp. 115-128. S p r i n g e r - V e r l a g , New York . D e f e n d i n i , R. and Zimmerman, E.A. (1978) The magnoce l lu la r neurosecre to ry system of the mammalian hypothalamus. ln_ S . R e i c h l i n , R . J . B a l d l e s s a r i n i and J . B . Mar t i n ( E d i t o r s ) The Hypothalamus, pp. 137-154. Raven P r e s s , New York . Delanoy, R . L . ; Dunn, A . J . and W a l t e r , R. (1979) Neurohypophyseal hormones and behav io r : e f f e c t s o f i n t r a c e r e b r o v e n t r i c u l a r l y i n j e c t e d hormone analogues in m ice . L i f e S c i . 24 :651-658. 1 1 3 de Wied, D. (1980) Pept ides and adapt ive behav io r . J j i D. de Wied and P.A. van Keep ( E d i t o r s ) Hormones and the B r a i n , pp. 103-113. U n i v e r s i t y Park P r e s s , B a l t i m o r e . de Wied, D . ; Greven, H .M. ; Lande, S. and W i t t e r , A. (1972) D i s s o c i a t i o n of the behav io ra l and endocr ine e f f e c t s of l y s i n e vasop ress in by t r y p t i c d i g e s t i o n . B r i t . J . Pharmacol . 45 :118-122. de Wied, D . ; Bohus, B. and van Wimersma Gre idanus , T j . B . (1974) The hypothalamo-neurohypophysia l system and the p r e s e r v a t i o n of cond i t i oned avoidance behav ior i n r a t s . J j i D .F . Swaab and J . P . Schade ( E d i t o r s ) I n t e g r a t i v e Hypothalamic A c t i v i t y , pp. 417-428. E l s e v i e r , Amsterdam. D i n g l e d i n e , R . ; Dodd, J . and K e l l y , J . S . (1980). The i n v i t r o b ra in s l i c e as a use fu l neu rophys io l og i ca l p repara t i on f o r i n t r a c e l l u l a r r e c o r d -i n g . J . N e u r o s c i . Methods 2 :323-362. Dodd, J . and K e l l y , J . S . (1978) Is somatos ta t in an e x c i t a t o r y t r a n s m i t t e r i n the hippocampus? Nature 273:674-675. Dogterom, J . ; van Rheenenverberg, C . M . F . ; van Wimersma Gre idanus , T j . B . and Swaab, D.F. (1977) Vasopress in and oxy toc in i n ce reb rosp ina l f l u i d o f r a t s . J . Endocr ino logy 72:74P-75P. Dogterom, J . ; S n i j d e w i n t , F .G.M. and B u i j s , R.M. (1978) T h e . d i s t r i b u t i o n o f vasop ress in and oxy toc i n i n the r a t b r a i n . N e u r o s c i . L e t t . 9 :341-346. D r e i f u s s , J . J . and K e l l y , J . S . (1972) Recur rent i n h i b i t i o n o f a n t i -d r o m i c a l l y i d e n t i f i e d r a t sup raop t i c neurons. J . P h y s i o l . (Lond.) 220: 87-103. D r e i f u s s , J . J . and M u h l e t h a l e r , M. (1981) Vasopress in - i nduced modulat ion o f f i r i n g ra te i n hippocampal s l i c e s . J . P h y s i o l . (Lond.) 318:18P. D r e i f u s s , J . J . and M u h l e t h a l e r , M. (1982a) Vasopress in e x c i t e s neurones i n hippocampal s l i c e s from B r a t t l e b o r o r a t s . J . P h y s i o l . (Lond.) 327: 45P-46P. D r e i f u s s , J . J . and M u h l e t h a l e r , M. (1982b) Neurohypophysial hormones: pr imary e f f e c t on in terneurones i n r a t hippocampal s l i c e s . Communicated to the P h y s i o l o g i c a l S o c i e t y (London), J u l y , 1982. D r e i f u s s , J . J . ; Nordmann, J . J . and V i n c e n t , J . D . (1973) Recurrent i n h i b i t i o n of sup raop t i c neurosecre to ry c e l l s i n homozygous B r a t t l e b o r o r a t s . J . P h y s i o l . (Lond.) 237:25P. Duggan, A . W . ; G r i e r s m i t h , B . T . ; Head ley , P .M. and H a l l , J . G . (1979) Lack o f e f f e c t o f substance P a t s i t e s i n the s u b s t a n t i a g e l a t i n o s a where Met-enkepha l in reduces the t r ansm iss ion of n o c i c e p t i v e impu lses . N e u r o s c i . L e t t . 12:313-317. Du V igneaud, V. (1956) Hormones of the p o s t e r i o r p i t u i t a r y g l and : oxy toc i n and v a s o p r e s s i n . The Harvey Lec tu res 5 0 : 1 - 2 6 . 114 F e l i x , D. and P h i l l i p s , M. I . (1979) I n h i b i t o r y e f f e c t s o f l u t e i n i z i n g hormone r e l e a s i n g hormone (LHRH) on neurones i n the organium vasculosum lamina te rmina l i s (OVLT). B r a i n Res. 169:204-208. F l e x n e r , J . B . ; F l e x n e r , L . B . ; W a l t e r , R. and Hoffman, P . L . (1978) ADH and r e l a t e d p e p t i d e s : e f f e c t o f p re - or p o s t t r a i n i n g t reatment on puromycin amnesia. Pharmacol . Biochem. Behav. 8 : 93 -95 . F r e d e r i c k s o n , R . C . A . ; J o r d a n , L .M . and P h i l l i s , J . (1971) A c t i o n of nor -ad rena l i ne on c o r t i c a l neurons: e f f e c t s o f pH. B r a i n Res. 35:556-560. G a h w i l e r , B . H . (1978) A c t i o n o f pept ide hormones on e l e c t r i c a l a c t i v i t y of c u l t u r e d neurons from d i f f e r e n t b ra i n a r e a s . N e u r o s c i . L e t t . , Supp l . 1:218. G a l l , C ; B recha , N . ; K a r t e n , H . J . and Chang, K. (1981) L o c a l i z a t i o n of e n k e p h a l i n - l i k e immunoreac t iv i t y to i d e n t i f i e d axonal and neuronal popu la -t i o n s of the r a t hippocampus. J . Comp. N e u r o l . 198:335-350. George, J . M . and J a c o b o w i t z , D.M. (1975) L o c a l i z a t i o n o f vasop ress in in d i s c r e t e areas of the r a t hypothalamus. B r a i n Res. 93:363-366. G i l l i e s , G. and Lowry, P . (1979) Cor t i co t rop in 'n r e l e a s i n g f a c t o r may be modulated v a s o p r e s s i n . Nature 278:463-464. G i r g i s , M. (1981) K i n d l i n g as a model f o r l i m b i c e p i l e p s y . Neurosc ience 16:1695-1706. Good f r i end , T . L . ; L e v i n e , L. and Fasman, G.D. (1964) An t i bod ies to b radyk i n i n and a n g i o t e n s i n : a use o f ca rbod i im ides i n immunology. Sc ience 144:1344-1346. Goren, H . J . ; Geonzon, R . M . ; H o l l e n b e r g , M .D . ; L e d e r i s , K. and Morgan, D.0 . (1980) Oxy toc in a c t i o n : l ack o f c o r r e l a t i o n between recep to r number and t i s s u e respons i veness . I^ n D. Oxender, A . Blume, I. Diamond and C . F . Fox ( E d i t o r s ) Membrane T ranspor t and Neuro recep to rs , pp. 235-241. A lan R. L i s s , I n c . , New York . Greenwood, F . C . and Hunter , W.M. (1963) The p repa ra t i on o f 1 3 1 I - l a b e l l e d human growth hormone o f h igh s p e c i f i c a c t i v i t y . Biochem J . 89 :114-123. G r u o l , D . L . ; B a r k e r , J . L . ; L i - Y e n , M .A . ; Macdonald, J . F . and Smi th , T . G . , J r . (1980) Hydrogen ion mimicry o f pept ide a c t i o n s . - _In J . L . Barker and T . G . Smi th , J r . ( E d i t o r s ) The Role o f Pept ides i n Neuronal F u n c t i o n , pp. 301-316. Marcel Dekker, New York . H a n i f , K . ; L e d e r i s , K . ; H o l l e n b e r g , M.D. and Goren, H . J . (1982) I n a b i l i t y o f oxy toc in to a c t i v a t e pyruvate dehydrogenase i n the B r a t t l e b o r o r a t . Sc ience 216:1010-1012. Hawthorn, J . ; Ang , V . T . Y . and J e n k i n s , J . S . (1980) L o c a l i z a t i o n of vasop ress in i n the r a t b r a i n . B r a i n Res. 197 :75 -81 . 115 H o l l e n b e r g , M.D. and Hope, D.B. (1968) The i s o l a t i o n o f the na t i ve hormone b ind ing p ro te i ns from bovine p i t u i t a r y p o s t e r i o r l o b e s . Biochem. 106:557-564. H o s t e t t e r , G . ; Jubb, S . L . and K o z l o w s k i , G . P . (1980) An i n a b i l i t y of subcutaneous vasop ress in to a f f e c t pass ive avoidance behav io r . Neuroendocr ino!ogy 30:174-177. Huston, J . P . and J a k o b a r t l , L. (1977) Evidence f o r s e l e c t i v e s u s c e p t i -b i l i t y o f hippocampus to spread ing depress ion induced by v a s o p r e s s i n . N e u r o s c i . L e t t . 6 :69 -72 . Jones , P.M. and Rob inson , I . C . A . F . (1982) D i f f e r e n t i a l c lea rance of neurophysin and neurohypophysia l pep t ides from the ce reb rosp ina l f l u i d i n consc ious guinea p i g s . Neuroendocr ino!ogy 34 :297-302. K a s t i n , A . J . ; P l o t n i k o f f , N . P . ; S c h a l l y , A . V . and Sandman, C .A . (1976) Endocr ine and CNS e f f e c t s o f hypothalamic pept ides and MSH. Jjn S. Ehrenpre is and I . J . Kopin ( E d i t o r s ) Reviews o f Neurosc ience . V o l . 2 , pp. 111-148. Raven P r e s s , New York . K a s t i n g , N.W.; Cooper, K . E . and V e a l e , W.L. (1979) A n t i p y r e s i s f o l l o w i n g pe r f us i on o f b ra i n s i t e s w i th v a s o p r e s s i n . E x p e r i e n t i a 33:208-209. K a s t i n g , N.W.; V e a l e , W.L. and Cooper, K . E . (1980) Convu ls i ve and hypothermic e f f e c t s o f vasop ress i n i n the b r a i n o f the r a t . Can. J .  P h y s i o l . Pharmacol . 58:316-319. K a s t i n g , N.W.; V e a l e , W . L . ; Cooper, K . E . and L e d e r i s , K. (1981) Vaso-p r e s s i n may mediate f e b r i l e c o n v u l s i o n s . B r a i n Res. 213:327-333. K e l l y , J . and Swanson, L.W. (1980) A d d i t i o n a l f o r e b r a i n reg ions p r o j e c t -ing to the p o s t e r i o r p i t u i t a r y : p r e o p t i c r e g i o n , bed nucleus of the s t r i a te rmina l i s , and zona i n c e r t a . B r a i n Res. 197 :1 -9 . K e l l y , J . S . ; Simmonds, M.A. and St raughan, D.W. (1975) M i c roe lec t r ode techn iques . ln_ P . B . Brad ley ( E d i t o r ) Methods i n B r a i n Research , pp. 333-337. John Wi ley and Sons, Toron to . Knowles, F. (1974) Twenty years o f n e u r o s e c r e t i o n . Jjn F. Knowles and L. Vol 1 ra th ( E d i t o r s ) Neurosecre t ion - the F ina l Neuroendocr ine Pathway, pp. 3 -11 . S p r i n g e r - V e r l a g , New York . Kb'nig, J . F . R . and K l i p p e l , R.A. (1967) The Rat B r a i n . Robert E. K r i e g e r , C o . , Hun t ing ton , N.Y. Koob, G . F . and Bloom, F . E . (1982) Behav io ra l e f f e c t s o f neuropep t ides : endorphins and v a s o p r e s s i n . Ann. Rev. P h y s i o l . 44 :571-582. Koob, G . F . ; E t t e n b e r g , A . ; Le M o a l , M. and Bloom, F . E . (1981a) Vaso-p r e s s i n p o t e n t i a t i o n i n the performance o f a learned a p p e t i t i v e t a s k . Soc . N e u r o s c i . A b s t r . 7 :30 . 116 Koob, G . F . ; Le M o a l , M . ; G a f f o r i , 0 . ; Manning, M . ; Sawyer, W . H . ; R i v i e r , J . and Bloom, F . E . (1981b) A rg i n i ne vasop ress in and a vasop ress in antago-n i s t pep t i de : oppos i te e f f e c t s on e x t i n c t i o n o f a c t i v e avoidance i n r a t s . Regu l . Pep t ides 2 :153-163. Kordower, J . H . ; S i k o r s z k y , V . ; C o r t , J . and Bodnar, R . J . (1981) A n t i -n o c i c e p t i v e e f f e c t s of c e n t r a l and sys temic a d m i n i s t r a t i o n o f l y s i n c e vasop ress in and s t r u c t u r a l ana logues. Soc. N e u r o s c i . A b s t r . 7 :30 . Kovacs, G . L . ; Bohus, B. and V e r s t e e g , D.H.G. (1979a) The e f f e c t s of vasop ress in on memory p rocesses : the r o l e o f noradrenerg ic neu ro t rans -m i s s i o n . Neuroscience 4:1529-1537. Kovcics, G . L . ; Bohus, B . ; V e r s t e e g , D . H . G . ; de K l o e t , E .R . and de Wied, D. (1979b) E f f e c t o f oxy toc i n and vasopress in on memory c o n s o l i d a t i o n : s i t e s o f a c t i o n and ca techo lam ine rg i c c o r r e l a t e s a f t e r l o c a l m i c r o i n j e c -t i o n i n t o l i m b i c - m i d b r a i n s t r u c t u r e s . B r a i n Res. 175:303-314. Kova*cs, G . L . ; Szabo, G . ; Szontagh, L . ; Medve, L . ; Te legdy , G. and L a s z l o , F .A . (1980) H e r e d i t a r y d iabe tes i n s i p i d u s i n r a t s . A l t e r e d ce reb ra l indolamine and catecholamine metabo l ism. Neuroendocr ino logy 31 :189-193. Kovacs , G . L . ; B u i j s , R . M . ; Bohus, B . and van Wimersma Gre idanus , T j . B . (1982) M i c r o i n j e c t i o n o f a r g i n i n e 8 - v a s o p r e s s i n ant iserum i n t o the dorsa l hippocampus a t tenuates pass ive avoidance behav ior i n r a t s . P h y s i o l . Behav. 28 :45 -48 . K r e j c f , I.; Kupkova", B . ; Metys , J . ; B a r t h , T. and J o s t , K. (1979) Vasopress in ana logs : seda t i ve p r o p e r t i e s and pass ive avoidance behav ior i n r a t s . E u r . J . Pharmacol . 56 :347-353. K r e j c f , I.; Kupkova, B. and D labac . A. (1981) Pass i ve avoidance behav io r : oppos i te e f f e c t s o f oxy toc in analogs w i th agon is t and an tagon is t p r o p e r t i e s . R e g u l . Pep t i des 2 :285-291 . K ruse , H . ; van Wimersma Gre idanus , T j . B . and de Wied, D. (1977) Ba r re l r o t a t i o n induced by vasop ress in and r e l a t e d pept ides i n r a t s . Pharmacol . Biochem. Behav. 7 :311-313. K r u s z y n s k i , M . ; Lammek, B . ; Manning, M . ; S e t o , J . ; H a i d e r , J . and Sawyer, W.H. (1980) [ l - ( 3 - m e r c a p t o - 3 , 3 - c y c l o p e n t a m e t h y l e n e p r o p i o n i c a c i d ) , 2 - ( 0 - m e t h y l ) t y r o s i n e ] a r g i n e - v a s o p r e s s i n and [ l - ( g - m e r c a p t o - 8 , 3 - c y d o p e n t a -methy leneprop ion ic a c i d ) ] a r g i n i n e - v a s o p r e s s i n , two h i g h l y potent antago-n i s t s o f the vasopressor response to a r g i n i n e - v a s o p r e s s i n . J . Med. Chem. 23 :364-368. Landgra f , R . ; E rm isch , A. and Heb, J . (1979) I n d i c a t i o n s f o r a b ra in uptake o f l a b e l l e d vasop ress in and o x y t o c i n and the problem of the b l o o d -b r a i n b a r r i e r . Endok r i no log ie 73 :77 -81 . L a s s o f f , S . and A l t u r a , B.M. (1980) Do pi a l te rmina l a r t e r i o l e s respond to l o c a l p e r i v a s c u l a r a p p l i c a t i o n o f the neurohypophyseal hormones, vaso -p r e s s i n and oxy toc in? B ra in Res. 196:266-269. 117 Lee , H . K . ; Dunwiddie, T. and H o f f e r , B. (1980) E l e c t r o p h y s i o l o g i c a l i n t e r a c t i o n s o f enkepha l ins w i th neuronal c i r c u i t r y i n the r a t hippocampus. I I . E f f e c t s on i n te rneu ron e x c i t a b i l i t y . B r a i n Res. 184:331-342. Le M o a l , M . ; Koob, G . F . ; Koda, L . Y . ; Bloom, F . E . ; Manning, M . ; Sawyer, W.H. and R i v i e r , J . (1981) Vasopressor recep to r an tagon i s t prevents behav io ra l e f f e c t s o f v a s o p r e s s i n . Nature 291:491-493. Leng, G. and Wiersma, J . (1981) E f f e c t s o f neura l s t a l k s t i m u l a t i o n on phas ic d ischarge o f sup raop t i c neurones i n B r a t t l e b o r o r a t s devo id o f v a s o p r e s s i n . J . Endocr ino logy 90:211-220. Logan, A . and Weatherhead, B. (1981) E f f e c t s o f a -me lanocy te - s t imu la t i ng hormone and [ 8 - a r g i n i n e ] - v a s o t o c i n upon melanogenesis i n h a i r f o l l i c l e melanocytes vn v i t r o . J . Endocr ino logy 91:501-507. Lj6mo, T . (1971) Pa t te rns o f a c t i v a t i o n i n a monosynapt ic c o r t i c a l pa th -way: the pe r fo ran t path inpu t to the dentate area of the hippocampal f o rma t i on . B r a i n Res. 12 :18 -45 . Lowbr idge, J . ; Manning, M . ; H a l d a r , J . and Sawyer, W.H. (1977) Syn thes is and some pharmacolog ica l p r o p e r t i e s o f [ 4 - t h r e o n i n e , 7 - g l y c i n e ] o x y t o c i n , [1 - ( L -2 -hyd roxy -3 -mercap top rop ion i c a c i d ) , 4 - t h r e o n i n e , 7 - g l y c i n e ] o x y t o c i n (Hydroxy[Thr 1* , G l y 7 ] o x y t o c i n ) , and [ 7 - G l y c i n e ] o x y t o c i n , pept ides w i th h igh o x y t o c i n - a n t i d i u r e t i c s e l e c t i v i t y . J . Med. Chem. 20 :120-123. L u t z - B u c h e r , B . ; Koch , B . ; M i a l h e , C. and B r i a u d , B. (1980) Involvement o f vasop ress in i n c o r t i c o t r o p i n - r e l e a s i n g e f f e c t of hypothalamic median eminence e x t r a c t . Neuroendocr ine!ogy 30:178-182. Manning, M . ; Olma, A . ; K l i s , W.A. and K o l o d z i e j c z y k , A . M . (1982) Design o f more potent an tagon i s t s o f the a n t i d i u r e t i c responses to a r g i n i n e -v a s o p r e s s i n . J . Med. Chem. 25 :45 -50 . Mannucc i , P . M . ; P a r e t i , F . I . ; R u g g e r i , Z . M . and C a p i t a n i o , A. (1977) l -Deamino-8 -D-a rg in ine v a s o p r e s s i n : a new pharmacolog ica l approach to the management o f hemophi l ia and von W i l l e b r a n d ' s d i s e a s e . Lancet 1 (8017) :869-872. M i l l e r , M . ; Ba r randa , E . G . ; Dean, M.C. and B r u s h , F .R . (1976) Does the r a t w i th h e r e d i t a r y hypothalamic d iabe tes i n s i p i d u s have impai red avoidance l e a r n i n g and /o r performance? Pharmacol . Biochem. Behav. V o l . 5 , S u p p l . 1 , pp. 35-40. Moeglen, J . M . ; A u d i b e r t , A . ; T i m s i t - B e r t h i e r , M . ; O l i v e r o s , J . C . and J a n d a l i , M.K. (1979) V a s o p r e s s i n , l e a r n i n g , and memory: p r e l i m i n a r y r e s u l t s i n man. J j i A . P o l l e n " and R.M. MacLeod ( E d i t o r s ) Neuroendocr in- o l ogy : B i o l o g i c a l and C l i n i c a l A s p e c t s , pp. 47 -53 . Academic P r e s s , New York . M o r r i s , R . ; S a l t , T . E . ; So f ron iew, M.V. and H i l l , R .G. (1980) Ac t i ons o f m i c r o i o n t o p h o r e t i c a l l y a p p l i e d o x y t o c i n , and immunohistochemical l o c a l i z a t i o n o f o x y t o c i n , vasopress in and neurophysins i n the r a t caudal medu l l a . N e u r o s c i . L e t t . 18:163-168. 118 Moss, R . L . ; D y b a l l , R . E . J , and C r o s s , B .A. (1972) E x c i t a t i o n o f a n t i -d r o m i c a l l y i d e n t i f i e d neurosecre to ry c e l l s o f the p a r a v e n t r i c u l a r nuc leus by oxy toc in a p p l i e d i o n t o p h o r e t i c a l l y . Exper imenta l Neurology 34 :95-102. M u h l e t h a l e r , M. and D r e i f u s s , J . J . (1981) Vasopress in enhances spontaneous neuronal a c t i v i t y i n hippocampal s l i c e s . Sbc . N e u r o s c i . A b s t r . 7 :430. M u h l e t h a l e r , M . ; D r e i f u s s , J . J . and Gahw i l e r , B . H . (1982) Vasop ress in e x c i t e s hippocampal neurones. Nature 296:749-751. Ngsee, J . ; J e n k i n s , C . J . and W i l s o n , N. (1980) Temperature response to endotox in in B r a t t l e b o r o r a t s . Canada Phys io logy 11:109. N i c o l l , R.A. and B a r k e r , J . L . (1971) The pharmacology o f r ecu r ren t i n h i b i t i o n i n the sup raop t i c neurosec re to ry system. B r a i n Res. 35 :501-511. N i l a v e r , G . ; Zimmerman, E . A . ; W i l k i n s , J . ; M i c h a e l s , J . ; Hoffman, D. and S i l ve rman. . A . (1980) Magnoce l l u l a r hypothalamic p r o j e c t i o n s to the lower b r a i n stem and s p i n a l cord o f the r a t . Neuroendocr ino logy 30:150-158. Ono, T . ; N i s h i n o , H . ; Sasaka , K . ; Muramoto, K . ; Yano, I. and Simpson, A. (1978) P a r a v e n t r i c u l a r nuc leus connect ions to s p i n a l cord and p i t u i t a r y . N e u r o s c i . L e t t . 10 :141-146. Pa lmer , M .R . ; Wuer the le , S . M . and H o f f e r , B . J . (1980) P h y s i c a l and p h y s i o l o g i c a l c h a r a c t e r i s t i c s o f m ic ropressure e j e c t i o n of drugs from m u l t i b a r r e l e d p i p e t t e s . Neuropharmacology 19:931-938. P a v e l , S . (1971) Evidence f o r the ependymal o r i g i n o f a r g i n i n e v a s o t o c i n i n the bovine p inea l g l a n d . Endocr ino logy 89:613-614. P a v e l , S . ; P s a t t a , D. and G o l d s t e i n , R. (1977a) Slow-wave s leep induced i n ca ts by ex t remely small. iamounts o f s y n t h e t i c and p inea l vaso toc in i n j e c t e d i n t o the t h i r d v e n t r i c l e o f the b r a i n . B r a i n Res. B u l l . 2 : 251-254. P a v e l , S . ; C r i s t o v e a n u , A . ; G o l d s t e i n , R. and C a l b , M. (1977b) I n h i b i t i o n o f r e l ease o f c o r t i c o t r o p i n r e l e a s i n g hormone i n ca ts by ex t remely smal l amounts o f vaso toc in i n j e c t e d i n t o the t h i r d v e n t r i c l e o f the b r a i n . Evidence f o r the involvement o f 5 -hyd roxy t r yp tamine -con ta in ing neurons. Endocr ino logy 101:672-678. Pedersen , C . A . ; Asche r , J . A . ; Monroe, Y . L . and Prange, A . J . , J r . (1982) Oxytoc in induces maternal behav ior i n v i r g i n female r a t s . Sc ience 216: 648-650. P e l l e t i e r , G . ; L a b r i e , F . ; K a s t i n , A . J . ; Coy, D. and S c h a l l y , A . V . (1975) Rad ioautograph ic l o c a l i z a t i o n o f r a d i o a c t i v i t y i n r a t b ra i n a f t e r i n t r a -v e n t r i c u l a r or i n t r a c a r o t i d i n j e c t i o n o f 3 H - L - p r o l y l - L - l e u c y l - g l y c i n a m i d e . Pharmacol . Biochem. Behav. 3 :675-679. 119 P£vet , P . ; Neacsu, C ; H o l d e r , F . C . ; R e i n h a r z , A . ; Dogterom, J . ; B u i j s , R . M . ; Guerne, J . M . and V i v i e n - R o e l s , B. (1981) The v a s o t o c i n - 1 i k e b i o l o g i c a l a c t i v i t y p resent i n the bovine p inea l i s due to a compound d i f f e r e n t from v a s o t o c i n . J . Neura l T rans . 51:295-302. P i t t m a n , Q . J . and S i g g i n s , G.R. (1981) Somatosta t in hype rpo la r i zes hippocampal pyramidal c e l l s i n v i t r o . B r a i n Res. 221:402-408. P i t t m a n , Q . J . ; Blume, H.W. and Renaud, L . P . (1981) Connect ions of the hypothalamic p a r a v e n t r i c u l a r nucleus w i th the neurohypophys is , median eminence, amygdala, l a t e r a l septum and midbra in per iaqueducta l g ray : an e l e c t r o p h y s i o l o g i c a l study i n the r a t . B r a i n Res. 215 :15-28 . P i t tman , Q . J . ; Rogers , J . and Bloom, F . E . (1982) A rg i n i ne vasopress in d e f i c i e n t B r a t t l e b o r o r a t s f a i l to develop t o l e rance to the hypothermic e f f e c t s of e t h a n o l . Regu l . Pept ides 4 : 3 3 - 4 1 . P o u l a i n , D .A . ; E l l e n d o r f f , F. and V i n c e n t , J . D . (1980) Septa l connect ions w i th i d e n t i f i e d oxy toc in and vasopress in neurones i n the sup raop t i c nucleus o f the r a t . An e l e c t r o p h y s i o l o g i c a l i n v e s t i g a t i o n . Neuroscience 5: 379-387. Pu rves , R.D. (1979) The phys ics o f i o n t o p h o r e t i c p i p e t t e s . J . N e u r o s c i . Methods 1:165-178. Raemakers, F . ; R i g t e r , H. and Leonard, B . E . (1977) P a r a l l e l changes i n behav ior and hippocampal se ro ton in metabol ism i n r a t s f o l l o w i n g treatment w i th desg lyc inamide l y s i n e v a s o p r e s s i n . B ra in Res. 120:485-492. R a i c h l e , M.E. and Grubb, R . L . , J r . (1978) Regu la t i on o f b r a i n water p e r m e a b i l i t y by c e n t r a l l y - r e l e a s e d v a s o p r e s s i n . B r a i n Res. 143:191-194. R i g t e r , H. (1982) Vasopress in and memory: the i n f l u e n c e of p r i o r exper -ience w i th the t r a i n i n g s i t u a t i o n . Behav. and N e u r o l . B i o l . 34 :337-351. Rober tson , G . L . (1977) The r e g u l a t i o n o f vasop ress in f u n c t i o n i n hea l th and d i s e a s e . Recent P rog . Horm. Res. 33:333-385. Rober tson , G . L . ; Mahr, E . A . ; A t h a r , S . and S i n h a , T . (1973) Development and c l i n i c a l a p p l i c a t i o n o f a new method f o r the radioimmunoassay of a r g i n i n e vasop ress in i n human plasma. J . C l i n . I nves t . 52:2340-2352. Rob inson, A . G . (1975) I s o l a t i o n , a s s a y , and s e c r e t i o n of i n d i v i d u a l human neurophys ins . J . C l i n . I nves t . 55:360-367. Rosenbloom, M. and F i s h e r , D.A. (1975) A r g i n i n e vaso toc in i n the r a b b i t subcommissural o rgan . Endocr ino logy 96:1038-1039. S a h g a l , A . ; K e i t h , A . B . ; Wr igh t , C. and Edwardson, J . A . (1982) F a i l u r e o f vasop ress in to enhance memory i n a pass i ve avoidance task i n r a t s . N e u r o s c i . L e t t . 28 :87 -92 . 120 Sawyer, W.H . ; Grzonka , Z . and Manning, M. (1981) Neurohypophysial pept ides - des ign of t i s s u e - s p e c i f i c agon i s t s and a n t a g o n i s t s . Mo lec . and C e l l E n d o c r i n o l . 22:117-134. S c h r i e r , R.W.; B e r l , T . and Anderson, R . J . (1979) Osmotic and nonosmotic con t ro l o f vasop ress in r e l e a s e . Amer. J . P h y s i o l . 235:F321-332. S c h u l z , H . ; Schwarzberg, H. and Unger, H. (1974) The e f f e c t of i n t r a -v e n t r i c u l a r ^ a p p l i e d oxy toc in on s e i z u r e s of r a b b i t s induced by Na-g lu tamate . In_ A . M i t r o ( E d i t o r ) Ependyma and Neurohormonal R e g u l a t i o n , pp. 269-280. Veda P u b l . , B r a t i s l a v a . Schwar tz , I .L . and W a l t e r , R. (1974) Neurohypophyseal hormones as p recursors of hypophys io t rop i c hormones. I s r e a l J . Med. S c i . 10:1288-1293. S c h w a r t z k r o i n , P .A . (1977) Fur ther c h a r a c t e r i s t i c s o f hippocampal CA1 c e l l s i n v i t r o . B ra in Res. 128 :53-68 . S c h w a r t z k r o i n , P .A . (1981) To s l i c e or not to s l i c e . J j i G.A. Kerkut and H.V. Wheal ( E d i t o r s ) E l e c t r o p h y s i o l o g y o f I so l a t ed Mammalian CNS Prepa ra - t i o n s . pp. 15-50 . Academic P r e s s , New York . Schwarzberg, H . ; S c h u l z , H. and Ungar, H.. (1974) The d i scha rge ra te o f hypothalamic neurones o f r a b b i t s a f t e r i n t r a v e n t r i c u l a r vasop ress in and oxy toc in a p p l i c a t i o n a t va r i ous c o n c e n t r a t i o n s . J j i A . M i t r o ( E d i t o r ) Ependyma and Neurohormonal R e g u l a t i o n , pp. 261-267. Veda P u b l . , B r a t i s l a v a . Schwarzberg, H . ; Kovacs , G . L . ; Szabo, G. and Te legdy , G. (1981) I n t r a -v e n t r i c u l a r a d m i n i s t r a t i o n o f vasop ress in and oxy toc in a f f e c t s the s teady-s t a t e l e v e l s o f s e r o t o n i n , dopamine and norep inephr ine i n r a t b r a i n . E n d o c r i n o l o g i a Exper imental i s 15 :75-78 . S e y b o l d , V. and E l d e , R. (1980) Immunohistochemical s t ud ies o f p e p t i d e r g i c neurons i n the do rsa l horn of the s p i n a l c o r d . J . Histochem. Cytochem. 28:367-370. Share , L. and Grosvenor , C . E . (1974) The neurohypophys is . j_n S .M. McCann ( E d i t o r ) Endocr ine Phys io logy (Phys io logy S e r i e s One, V o l . 5 ) . pp. 1-30. Un iv . Park P r e s s , B a l t i m o r e . Shepard, G.M. (1974) The Synapt i c O rgan i za t i on o f the B r a i n . Oxford U n i v e r s i t y P r e s s , New York . Skrede , K . , J r . and Westgaard, R .H. (1971) The t ransve rse hippocampal s l i c e : a w e l l - d e f i n e d c o r t i c a l s t r u c t u r e main ta ined i n v i t r o . B r a i n Res. 35 :589-593. So f ron iew, M.V. and S c h r e l l , U. (1981) Evidence f o r a d i r e c t p r o j e c t i o n from oxy toc in and vasopress in neurons i n the hypothalamic p a r a v e n t r i c u l a r nucleus to the medul la ob longa ta . N e u r o s c i . L e t t . 22 :211-217. 121 Sof ron iew, M.V. and W e i n d l , A . (1978) P r o j e c t i o n s from the p a r v o c e l l u l a r v a s o p r e s s i n - and neurophys in - c o n t a i n i n g neurons o f the suprach iasmat ic n u c l e u s . Amer. J . Anat . 153:391-430. So f ron iew , M.V. and W e i n d l , A . (1981) Cent ra l nervous system d i s t r i b u t i o n o f v a s o p r e s s i n , o x y t o c i n , and neurophys in . ln_ J . l . M a r t i n e z , J r . , R.A. Jensen , R .B . Mess ing , H. R i g t e r and J . L . McGaugh ( E d i t o r s ) Endogenous  Pept ides and Learn ing and Memory P rocesses , pp. 327-359. Academic P r e s s , New York . : ~ S p i r t e s , M .A . ; Gerber , A . R . ; Wood, K . S . ; S a r t o r , A . O . and C h r i s t e n s e n , C.W. (1980) The e f f e c t s o f MIF-I on i n v i t r o cGMP produc t ion by a p a r t i c u l a t e r a t b ra i n f r a c t i o n . Neuropharmacology 19:687-689. S t a r r , M.S. (1982) In f luence of pept ides on 3H-dopamine r e l e a s e from super fused r a t s t r i a t a l s l i c e s . Neurochem. I n t l . 4 :233-240 . S t e r b a , G. (1974) Ascending neurosecre to ry pathways of the p e p t i d e r g i c t ype , jjn F. Knowles and L. V o l ! r a t h ( E d i t o r s ) Neurosecre t ion - the F i n a l  Neuroendocrine Pathway, pp. 38-47. S p r i n g e r - V e r l a g , New York . S t e r b a , G . ; H o h e i s e l , G . ; Wege l in , R . ; Naumann, W. and Schober , F. (1979) Pept ide c o n t a i n i n g v e s i c l e s w i t h i n the neuro-neuronal synapses. B r a i n Res. 169 :55-64 . S te rnbe rge r , L . A . (1982) T r a n s i t i o n o f immunocytochemistry from an anatomical to a b iochemica l s c i e n c e : e v o l u t i o n o f the concept o f neuro-t ypy . Neurosc ience 7:2937-2947. Swanson, L.W. (1977) Immunohistochemical evidence f o r a neurophys in -con ta i n i ng autonomic pathway a r i s i n g i n the p a r a v e n t r i c u l a r nucleus o f the hypothalamus. B r a i n Res. 128:346-353. Swanson, L.W. and Sawchenko, P . E . (1980) P a r a v e n t r i c u l a r nuc l eus : a s i t e f o r the i n t e g r a t i o n o f neuroendocr ine and autonomic mechanisms. Neuro- endoc r ino logy 31:410-417. Swanson, L .W. ; T e y l e r , T . J . and Thompson, R . F . (1982) Hippocampal l o n g -term p o t e n t i a t i o n : mechanisms and i m p l i c a t i o n s f o r l e a r n i n g . N e u r o s c i . Res. Prog . B u l l . 20 :613-769. T e y l e r , T . J . (1980) B r a i n s l i c e p r e p a r a t i o n : hippocampus. B r a i n Res. B u l l . 5 :391-403. T h o r e l l , J . I . and L a r s o n , S . M . (1978) Radioimmunoassay and Re la ted  Techn iques . C .V . Mosby C o . , S a i n t L o u i s . Urban, I . J . A . (1981) I n t r asep ta l a d m i n i s t r a t i o n o f vasop ress in and oxy toc i n a f f e c t s hippocampal e lec t roencepha logram i n r a t s . Exper imenta l Neurology 74: 131-147. Urban, I . J . A . and de Wied, D. (1978) Neuropept ides : e f f e c t s on pa ra -d o x i c a l s l eep and the ta rhythm i n r a t s . Pharmacol . Biochem. Behav. 8 :51 -59 . 122 V a l t i n , H . ; Sawyer, W.H. and S o k o l , H.W. (1964) Neurohypophyseal p r i n c i p l e s i n r a t s homozygous and heterozygous f o r hypothalamic d iabe tes i n s i p i d u s ( B r a t t l e b o r o s t r a i n ) . Endocr ino logy 77:701-706. Vandesande, F. (1979) A c r i t i c a l rev iew of immunocytochemical methods f o r l i g h t m ic roscopy . J . N e u r o s c i . Methods 1 :3 -23 . Vandesande, F. and D i e r i c k x , K. (1975) I d e n t i f i c a t i o n o f the vasop ress in producing and of the oxy toc i n producing neurons in the hypothalamic magnoce l lu la r neurosecre to ry system of the r a t . C e l l T i s s . Res. 164:153-162. van D i j k , A . M . A . ; Lodew i j ks , H . M . J . M . ; van Ree, J . M . and van Wimersma Gre idanus , T j . B . (1981) I n h i b i t o r y and s t i m u l a t o r y a c t i o n o f vasop ress in on the s e c r e t i o n o f co r t i co t r op in ' n i n r a t s : s t r u c t u r e - a c t i v i t y s tudy . L i f e S c i . 29:1107-1116. van Ree, J . M . and de Wied, D. (1976) P r o l y l - l e u c y l - g l y c i n a m i d e (PLG) f a c i l i t a t e s morphine dependence. L i f e S c i . 19:1331-1340. van Wimersma Gre idanus , T j . B . ; Bohus, B. and de Wied, D. (1981) Vaso-p r e s s i n and oxy toc i n i n l e a r n i n g and memory. J^n J . L . M a r t i n e z , J r . , R.A. Jensen , R .B . Mess ing , H. R i g t e r , and J . L . McGaugh ( E d i t o r s ) Endogenous  Pept ides and Learn ing and Memory P r o c e s s e s , pp. 413-427. Academic P r e s s , New York. V e l d i u s , D. and de K l o e t , E .R . (1982) V a s o p r e s s i n - r e l a t e d pept ides i n -crease the hippocampal c o r t i c o s t e r o n e recep to r c a p a c i t y of d iabe tes i n s i p i d u s ( B r a t t l e b o r o ) r a t s . Endocr ino logy 110:153-157. V i z s o l y i , E. and P e r k s , A . M . (1969) New neurohypophysia l p r i n c i p l e i n f o e t a l mammals. Nature 223:1169-1171. V i z s o l y i , E. and P e r k s , A . M . (1974) The e f f e c t o f a r g i n i n e vaso toc in on the i s o l a t e d amn io t i c membrane of the guinea p i g . Can. J . Z o o ! . 52: 371-386. W a l t e r , R . ; Hoffman, P . L . ; F l e x n e r , J . B . and F l e x n e r , L . B . (1975) Neuro-hypophyseal hormones, analogs, . .and f ragments: t h e i r e f f e c t on puromycin induced amnesia. P roc . Nat . Acad . S c i . (USA) 72:4180-4184. W a l t e r , R . ; van Ree, J . M . and de Wied, D." (1978) M o d i f i c a t i o n of c o n d i -t i oned behav ior o f r a t s by neurohypophyseal hormones and ana logues. P r o c . Nat . Acad . S c i . (USA) 75:2493-2496. Watson, S . J . ; S e i d a h , N.G. and C r e t i e n , M. (1982) The carboxy terminus of the p recu rso r to vasop ress in and neurophys in : immunocytochemistry i n the r a t b r a i n . Sc ience 217:853-855. Weingar tner , H . ; G o l d , P . ; B a l l e n g e r , J . C ; Sma l l be rg , S . A . ; Summers, R . ; Rubinow, D .R . ; P o s t , R.M. and Goodwin, F .K . (1981) E f f e c t s of Vasopress in on human memory f u n c t i o n s . Sc ience 211:601-603. 123 Weitzman, R . E . and F i s h e r , D.A. (1976) Mono and d i i o d i n a t e d a r g i n i n e v a s o p r e s s i n : an t ibody and membrane b ind ing s t u d i e s . P r o c . I n t l . Congress Endoc r i no logy , Hamburg, Germany, 1976. Werman, R. (1972) CNS c e l l u l a r l e v e l : membranes. Ann. Rev. P h y s i o l . 34:337-374. W i l s o n , N. and Sm i th , M. (1969) I s o l a t i o n and amino a c i d sequence of neurohypophyseal hormones of P a c i f i c chinook salmon (Oncorhynchus t schawy tscha) . Gen. Comp. Endocr ino logy 13:412-424. Yalow, R .S . and Berson , S . A . (1959) Assay o f plasma i n s u l i n i n human sub jec ts by immunological methods. Nature (Lond.) 184:1648-1649. Yalow, R . S . and Be rson , S .A . (1971a) Problems o f v a l i d a t i o n o f r a d i o -immunoassays. J j i W.D. Odel l and W.H. Daughaday ( E d i t o r s ) P r i n c i p l e s of  Compet i t ive P r o t e i n - B i n d i n g Assays , pp. 374-400. J . B . L i p p i n c o t t , Toronto . Yalow, R . S . and Be rson , S . A . (1971b) I n t roduc t i on and General Cons ide ra -t i o n s . J j i W.D. Ode l l and W.H. Daughaday ( E d i t o r s ) P r i n c i p l e s of Compet i - t i v e P r o t e i n - B i n d i n g Assays , pp. 1-21. J . B . L i p p i n c o t t , Toron to . Ya low, R . S . and S t r a u s , E. (1980) Problems and p i t f a l l s i n the r a d i o -immunoassay of g a s t r o i n t e s t i n a l hormones. In G . B . J . G lass ( E d i t o r ) G a s t r o i n t e s t i n a l Hormones, pp. 751-767. Raven P r e s s , New York . Yarbrough, G .G . and Haub r i ch , D.R. (1978) Thy ro t rop in r e l e a s i n g hormone (TRH) and MK-771 i n t e r a c t i o n s w i th CNS c h o l i n e r g i c mechanisms. Jjn R.W. R y a l l and J . S . K e l l y ( E d i t o r s ) Ion tophores is and T ransmi t t e r Mechanisms  i n the Mammalian Cent ra l Nervous System, pp. 136-138. E l s e v i e r , Amsterdam. Z i e g l g a n s b e r g e r , W.; He rz , A. and Teschemacher, H. (1969) E l e c t r o p h o r e t i c r e l e a s e o f t r i t i u m - l a b e l l e d g lu tamic a c i d from m ic rop ipe t t es i n v i t r o . B r a i n Res. 15:298-300. Z i e g l g a n s b e r g e r , W.; Sothmann, G. and Herz , A. (1974) Ion tophore t i c r e l ease of substances from m ic rop ipe t t es in v i t r o . Neuropharmacology 13: 417-422. Zimmerman, E . A . ; Carme l , P .W. ; Husa in , M .K . ; F e r i n , M . ; Tannenbaum, M . ; F r a n t z , A . G . and Rob inson , A . G . (1973) Vasopress in and neurophys in : h igh concen t ra t i ons i n monkey hypophyseal po r ta l b l o o d . Sc ience 182: 925-927. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0095890/manifest

Comment

Related Items