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Light and electron microscopic autoradiographic investigation of the septo-dentate pathway in rat brain Rose, Ann Marie 1976

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LIGHT AND ELECTRON MICROSCOPIC AUTORADIOGRAPHIC INVESTIGATION OF THE SEPTO-DENTATE PATHWAY IN RAT BRAIN by ANN MARIE ROSE B.A., U n i v e r s i t y of Saskatchewan, 1970 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES INSTITUTE OF NEUROLOGICAL SCIENCES DEPARTMENT OF PSYCHIATRY SCHOOL OF MEDICINE we accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1976 (c) Ann"Marie Rose, 1976 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree l y ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th i s thes is for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is fo r f i nanc ia l gain sha l l not be allowed without my writ ten permiss ion. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date ABSTRACT T h i s study was undertaken to r e s o l v e the c o n f l i c t be-tween two e x i s t i n g s t u d i e s r e g a r d i n g the l a y e r i n the dentate gyrus where the s e p t a l f i b e r s t e r m i n a t e . T h i s was accomplished by i n j e c t i n g r a d i o a c t i v e l y l a b e l l e d amino a c i d s i n t o the me-d i a l septum where they were i n c o r p o r a t e d i n t o p r o t e i n by the c e l l bodies and t r a n s p o r t e d to the nerve t e r m i n a l s . Auto-r a d i o g r a p h i c g r a i n counts showed l a b e l l i n g was moderately heavy i n the subgranular zone of the dentate gyrus, whereas l a b e l l i n g was scant i n the molecular l a y e r . These f i n d i n g s support the work of Raisman et a l . , (1965), but i n d i c a t e the degeneration seen by Mosko, et a l . , (1973) i n the molecular l a y e r may have been a r t e f a c t u a l . The u l t r a s t r u c t u r e of the s e p t a l t e r m i n a l s i n the subgranular zone of the dentate gyrus was d e s c r i b e d . These t e r m i n a l s formed asymmetrical synapses onto d e n d r i t e s and s p i n e s , and contained c l e a r round v e s i c l e s . They resembled c h o l i n e r g i c nerve t e r m i n a l s d e s c r i b e d i n other r e g i o n s of the b r a i n . These f i n d i n g s agree with accumulating evidence that the septo-dentate pathway i s c h o l i n e r g i c . i i i . TABLE OF CONTENTS Page No. TJNTRODUCT ION 1 I . Anatomy of the hippocampal region 4 A. C y t o a r c h i t e c t u r a l arrangement 4 B. A f f e r e n t Connections to the Dentate Gyrus 6 C. I n t r i n s i c Connections of the Dentate Gyrus 8 D. E f f e r e n t Connections from the Dentate Gyrus 9 I I . Synaptic O r g a n i z a t i o n of the Dentate Gyrus .... 12 A. Laminar Arrangement 12 B. U l t r a s t r u c t u r e of Synaptic Terminals 13 C. Synaptic A c t i v i t y i n the Dentate Gyrus ... 15 I I I . The Septo-dentate pathway 17 A. The Septum 17 B. C h o l i n e r g i c nature of the Septo-dentate Pathway IB C. I n v e s t i g a t i o n of the S e p t a l Terminals i n the Dentate Gyrus 19 METHODS 21 I. AxDnal Transport to Hippocampal Region 23 I I . Light Microscopic Autoradiography of Septal Terminals 26 I I I . E l e c t r o n Microscopic Autoradiography of Septal Terminals 28 i v . Page No. RESULTS 30 I. Axonal Transport to Hippocampal Region 31 II. D i s t r i b u t i o n of Label in the Dentate Gyrus ... 34 I I I . Ultrastructure of the Septal Terminals ........ 36 DISCUSSION 41 BIBLIOGRAPHY 48 PLATES 56 LIST OF TABLES Page No. TABLE I Time Course of a x o n a l l y t r a n s p o r t e d l a b e l l e d p r o t e i n s a f t e r i n j e c t i o n 3 of H - l e u c i n e i n t o medial septum 32 TABLE I I D i s t r i b u t i o n of s i l v e r g r a i n s i n 3 dentate gyrus a f t e r H - l e u c i n e i n j e c t i o n i n t o medial septum 39 TABLE I I I D i s t r i b u t i o n of s i l v e r g r a i n s i n the synapses of the dentate gyrus 40 v i . LIST OF FIGURES AND ILLUSTRATIONS Page No. FIGURE l a H o r i z o n t a l s e c t i o n of hippocampal region 10 FIGURE l b The l a y e r s of the Dentate Gyrus 10 FIGURE Ic C i r c u i t r y of the Dentate Gyrus 11 FIGURE 2a H o r i z o n t a l s e c t i o n through i n j e c t i o n s i t e ....... 57 FIGURE 2b Transported s i l v e r g r a i n s i n f i m b r i a 57 FIGURE 2c D i s t r i b u t i o n of s i l v e r g r a i n s i n the dentate gyrus 57 FIGURE 3 Histogram of g r a i n counts i n dentate gyrus and c o n t r o l areas 58 FIGURE 4 Comparison of l a b e l l i n g i n dentate gyrus and e n t o r h i n a l cortex 59 FIGURE 5 E l e c t r o n micrograph of neur o p i l e of dentate gyrus showing l a b e l l e d synapse "en passage". 59 FIGURE 6 Comparison of l a b e l l e d synapses i n dentate gyrus 60 ACKNOWLEDGEMENT I would l i k e t o thank my Supervisor, C h r i s F i b i g e r , f o r h i s support and c a r e f u l research guidance; Toshi H a t t o r i f o r h i s patient advice, and Jim M i l l e r f o r h i s c r i t i c a l d i s c u s s i o n . I am g r a t e f u l to the e x c e l l e n t comments and encourage-ment of V i c Bourne, Joanne Susuki, S t e l l a Atmadja, Raja Rosenbluth, and L a s z l o Veto. A s p e c i a l thanks to W. Maxwell Cowan f o r h i s i n t e r e s t and suggestions. Frontisp Pyramidal c e l l s in area CA1 c e l l bodies and p a r a l l e l dendrit by dense neuropil. iece of rat hippocampus. Pyramidal i c trunks can be seen surrounded - 1 -The i d e n t i f i c a t i o n of synapses of v a r i o u s f u n c t i o n a l types and t h e i r l o c a t i o n on the nerve c e l l s r e p r e s e n t s an im-portant problem i n the study of s y n a p t i c a c t i v i t i e s i n the c e n t r a l nervous system. The p o s i t i o n of the incoming synapse on the neuron w i l l i n f l u e n c e the amount of change i n membrane p o t e n t i a l that can be produced i n the e f f e c t o r neuron. A change i n membrane p o t e n t i a l w i l l have a g r e a t e r e f f e c t , i f f o r example, i t occurs at the membrane of the c e l l body than i f i t occurs at the membrane of a d i s t a l branch. T h i s i s because the a c t i o n p o t e n t i a l generated by a c e l l a r i s e s i n the axon h i l l o c k (Katz, 1966). In g e n e r a l , e l e c t r i c a l i n -formation at the d e n d r i t i c extremes spreads p a s s i v e l y t o the c e l l body ( E c c l e s , 1964). T h i s i n v o l v e s a l o s s of s t r e n g t h of the s i g n a l and a delay i n time. T h i s s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p of s y n a p t i c o r g a n i z a t i o n i s t h e r e f o r e fundamental to understanding of the o r g a n i z a t i o n of the b r a i n . The dentate gyrus, as an area of the hippocampal r e g i o n , o f f e r s a unique o p p o r t u n i t y to d e a l with such problems f o r the f o l l o w i n g reasons. Not only have the important a f f e r e n t pathways to t h i s part of the b r a i n been d e s c r i b e d with regard to t h e i r course and t h e i r s i t e of t e r m i n a t i o n ( C a j a l , 1911; B l a c k s t a d , 1956, 1958; B l a c k s t a d , et a l . , 1970; Raisman, et a l . , 1965); but a l s o there i s a l a y e r e d arrangement of these i n p u t s to t h i s s t r u c t u r e . Knowledge of t h i s l a y e r e d arrange-ment makes i t p o s s i b l e t o study inputs to r e s t r i c t e d i d e n t i f i e d - 2 -parts of the granule c e l l , the major c e l l type of the dentate gyrus (Lorento de N6, 1934). An important and well described input to the hi l u s of the dentate gyrus i s from the medial septal nuclei (Crosby, 1917/ ^ " ; Andersen, et a l . , 1961: Shute & Lewis, 1963: Raisman et a l . , 1965). The septum makes connections with both the hippocampus and the dentate gyrus (Raisman, 1965); the dentate gyrus, i n turn, contacts the hippocampal c e l l s (Cajal, 1911). Although t h i s places the dentate gyrus in a prime position to punctuate the septal input to the hippocampus, the functional importance of thi s arrange-ment i s not understood. The anatomical position of the hippocampal region makes i t a key structure of the limbic system. Connections to the hypothalamus through the septum indicate a close involve-ment in such functions as endocrine control and the expression of emotional states (Shepherd, 1974). Several sensory inputs, v i s u a l , auditory, and somatic t r a v e l to the hippocampus (Shepherd, 1974) and connections through the septum have been implicated in physiological arousal (Green & Arduini, 1954). As well, the hippocampus probably plays a r o l e in memory and learning (Shepherd, 1974). How i t does so i s undoubtedly re-lated to the structure-function relationships of the synaptic organization of the hippocampus and dentate gyrus. Although the septo-dentate pathway has been extensively studied (Crosby, 1917; Daitz & Powell, 1954; Raisman, 1966; Shute & Lewis, 1961; Andersen, 1961-/; Mosko, et a l . , 1973), there exist some unresolved questions about i t s exact mode Df - 3 -termination (Raisman, et a l . , 1965: Mosko, et a l . , 1973). This thesis is directed tovjards c lar ifying the precise d i s -tribution of septal nerve terminals in the dentate gyrus and describing their ultrastructural characteristics . - 4 -I. Anatomy of the hippocampal r e g i o n . A. C y t o a r c h i t e c t u r a l arrangement. The hippocampus i s a s i x - l a y e r e d p a l e o c o r t i c a l s t r u c t u r e of the l i m b i c system. S i t u a t e d i n the temporal lobe of mam-malian b r a i n , i t c u r l s from the s e p t a l n u c l e i c a u d a l l y , l a t -e r a l l y and v e n t r a l l y toward the temporal l o b e . The names Ammonshorn (sheep's horn) and hippocampus (sea horse) d e s c r i b e the f o l d i n g of t h i s C-shaped c y l i n d e r . The open face of an-other C-shaped c y l i n d e r , the dentate gyrus, abuts the lower l i p of the hippocampus. E x c e l l e n t three dimensional recon-s t r u c t i o n s of t h i s r a t h e r complex anatomical arrangement are presented by Hjorth-Simonsen, 1972; Hjorth-Simonsen, et a l . , 1972;1975. F i g u r e l a d i a g r a m a t i c a l l y i l l u s t r a t e s the appearance of the hippocampal r e g i o n i n a h o r i z o n t a l s e c t i o n . H o r i z o n t a l s e c t i o n s are used i n an a t o m i c a l s t u d i e s of t h i s r e g i o n because they c l e a r l y show the s i x c o r t i c a l l a y e r s of the hippocampus. In F i g u r e l a these are i d e n t i f i e d by Roman numerals. Layer IV c o n t a i n s the c e l l bodies of the major c e l l type, the pyramidal c e l l . These c e l l s a re l a r g e (20-50 p. i n diameter), pyramid shaped, and g i v e o f f both a p i c a l and b a s i l a r d e n d r i t e s . The b a s i l a r d e n d r i t e s c o n s t i t u t e l a y e r V. The mass of a p i c a l d e n d r i t e s from the pyramidal c e l l s c o n s t i t u t e l a y e r I I I , and t h e i r major d e n d r i t i c trunks form the stratum radiatum, and stratum lacunosum ( l a y e r s I I I and I I ) . Layer I, stratum mol-e c u l a r e i s made up of the s u p e r f i c i a l d e n d r i t i c branches, which border on the hippocampal f i s s u r e . The axons of the pyramidal - 5 -c e l l s form a myelinated l a y e r , the a l v e u s , l a y e r V I . In add-i t i o n to pyramidal c e l l s other s m a l l neurons with short axons are p r e s e n t . These have been e l e g a n t l y i l l u s t r a t e d by C a j a l , (1911) and Lorento de Ntf (1934). A l s o shown i n F i g u r e l a a r e the s u b d i v i s i o n s of the h i p -pocampal r e g i o n : Cornu ammon 1 (CA1), CA2, CA3, CA4 and the dentate gyrus, o f t e n r e f e r r e d to i n the l i t e r a t u r e as the f a s c i a dentata (F.D.) The arrows i n F i g u r e l a show the boun-d a r i e s between C A l , CA2, and CA3. These d i v i s i o n s were est a b -l i s h e d by Lorento de No (1934) i n s i l v e r s t a i n e d m a t e r i a l on the b a s i s of s i z e and appearance of the pyramidal c e l l s . CA3 pyramids are l a r g e r than CAl pyramids. The pyramids of CA4 are s i t u a t e d i n the h i l u s of the dentate gyrus and have a mod-i f i e d form. Here the d i s c r e t e l a y e r i n g p a t t e r n s observed i n other hippocampal a r e a s i s l o s t and the d e n d r i t e s of CA4 modified pyramids extend r a d i a l l y . B l a c k s t a d (1956) adopted the terminology of C a j a l (1911) who c a l l e d the area occupied by CAl & CA2, r e g i o s u p e r i o r , and the area of CA3 and CA4, r e g i o i n f e r i o r . Both of these t e r m i n o l o g i e s a r e commonly found i n l i t e r a t u r e on the hippocampal r e g i o n . The dentate gyrus i s b a s i c a l l y a s i m p l i f i c a t i o n of the hippocampal form. It has t h r e e l a y e r s superimposed on each o t h e r . These have been i n d i c a t e d i n F i g u r e l b . S e v e r a l (6-8) t i g h t l y packed l a y e r s of the major c e l l type, the granule c e l l , make up the g r a n u l a r l a y e r (GRL). Granule c e l l s a re smaller (10 u ) , rounder and more t i g h t l y packed than pyramidal c e l l s . Instead of a major d e n d r i t i c trunk, they send m u l t i p l e a p i c a l - 6 -d e n d r i t i c branches up from the c e l l body (compare F i g u r e l b to f r o n t i s p i e c e ) , and they have no b a s a l d e n d r i t e (Lorento de No, 1934). The d e n d r i t i c branches of the granule c e l l s reach s u p e r f i c i a l l y t o f i l l the molecular l a y e r (MOL). A l s o i n t h i s l a y e r are two types of neurons, d i s p l a c e d g r a n u l e c e l l s and c e l l s with short axons. The subgranular zone c o n t a i n s numerous c e l l types and is* r e f e r r e d to as the polymorphic l a y e r (POL). Immediately below the g r a n u l a r l a y e r two c e l l types a r e to be found? those with axons t e r m i n a t i n g i n the g r a n u l a r l a y e r and c e l l s which send t h e i r axons t o the a l v e u s . Next i s a l a y e r of c e l l s with axons ascending i n t o the molecular l a y e r of the dentate gyrus, and c e l l s with s h o r t axons which terminate i n the polymorphic l a y e r , as w e l l as c e l l s with axons d e s t i n e d f o r the a l v e u s . F i n a l l y a l a y e r of s p i n d l e shaped c e l l s , p o s s i b l y part of CA4 which send t h e i r axons to the a l v e u s ( C a j a l , 1911: Laatsch & Cowan, 1966). The d i v i s i o n between the polymorphic l a y e r of the dentate gyrus and the d e n d r i t i c f i e l d of area CA4 of the hippocampus has a n a t o m i c a l l y no c h a r a c t e r i s t i c a n a t o mical marker. B. A f f e r e n t s Connections t o the Dentate Gyrus. F i b e r s t o the dentate gyrus a r r i v e e i t h e r through the h i l u s of the dentate or from a c r o s s the hippocampal f i s s u r e . ( C a j a l , 1911 and Lorento de No, 1934). The main input to the d e n t a t e gyrus i s through the p e r f o r a n t path. The c e l l bodies of t h i s input l i e i n the l a t e r a l e n t o r h i n a l c o r t e x (A.E.) of - 7 -the same hemisphere, and t h e i r axons pass through ( p e r f o r a t e ) i n t e r v e n i n g areas (the subiculum) before reaching the hippo-campus and c r o s s i n g the hippocampal f i s s u r e t o terminate i n the outer t w o - t h i r d s of the molecular l a y e r of the dentate g y r u s . (Lorento de No, 1934). C a j a l (1911) b e l i e v e d some f i b e r s i n the p e r f o r a n t path were i s s u e d c o n t r a l a t e r a l l y . The p o s s i b i l i t y of a cros s e d pathway from the e n t o r h i n a l area to the dentate gyrus has r e c e n t l y been i n v e s t i g a t e d by H j o r t h -Simonsen & Zimmer (1975). Using degeneration techniques they d e s c r i b e a weak p r o j e c t i o n t o the r o s t r a l dentate gyrus ( s e p t a l p o l e ) . Other f i b e r s a f f e r e n t to the dentate gyrus a r r i v e through t h e h i l u s from the fimbria.. These i n c l u d e f i b e r s from the c o n t r a l a t e r a l hippocampus, the midbrain, and the septum. Commissural f i b e r s from area C M c r o s s i n the v e n t r a l hippo-campal commissure, t r a v e l through the f i m b r i a t o terminate on the d e n d r i t i c branches of the granule c e l l s i n the inner o n e - t h i r d of the molecular l a y e r ( B l a c k s t a d , 1956: Raisman, et a l . , 1965). Re c e n t l y , anatomical mapping of pathways by a u t o r a d i o g r a p h i c techniques (Cowan, 1972) has allowed f o r the i d e n t i f i c a t i o n of midbrain n u c l e i , the raphe nucleus and the lo c u s coeruleus, as an a d d i t i o n a l source of input t o the den-I t a t e gyrus (Conrad, et a l . , 1974: Soegal, et a l . , 1973). These a f f e r e n t s terminate i n the polymorphic l a y e r . A p r e v i o u s l y undescribed source of a f f e r e n t f i b e r s from the suprama mmilary nucleus was observed by Sj^egal & Landis (1974) a f t e r HKP i n -j e c t i o n i n t o the dentate a r e a . F u r t h e r s t u d i e s w i l l be nec-e s s a r y to confirm t h i s . - 8 -L e s i o n s i n the medial s e p t a l r e g i o n have been shown t o cause degeneration i n the subgranular zone of the dentate gyrus. (Raisman et a l . , 1965; Raisman, 1966; Mosko et a l . , 1973) and i n a narrow band s u p e r f i c i a l to the g r a n u l e c e l l s (Mosko, et a l . , 1973). The exact l o c a t i o n of the s e p t a l input t o the dentate gyrus cannot be c l e a r l y deduced from these e x i s t i n g d egeneration s t u d i e s . The present work was undertaken to r e s o l v e t h i s ambiguity. C. I n t r i n s i c Connections of the Dentate Gyrus Short i n t r i n s i c connections from the i p s i l a t e r a l hippo-campus are known from s t u d i e s using s i l v e r s t a i n s ( C a j a l , 1911; Lorento de No, 1934). One of these, an i p s i l a t e r a l pathway from CA3 & 4 p r o j e c t s t o the molecular l a y e r of the dentate gyrus (Zimmer, 1971). Other i n t r i n s i c a s s o c i a t i o n f i b e r s from CAl have been mentioned but not s y s t e m a t i c a l l y i n v e s t i g a t e d (Raisman, et a l . , 1965; Laatsch & Cowan, 1966/ffj;orth-Simonsen & Jeune, 1971). - 9 -D. E f f e r e n t Connections from the Dentate Gyrus The only known output from the dentate gyrus i s through the axons of the g r a n u l e c e l l s , the mossy f i b e r s ( C a j a l , 1911). Along these axons are l a r g e s w e l l i n g s and the axons end i n bulbous t e r m i n a l s on the d e n d r i t i c p o r t i o n s j u s t s u p e r i o r to the pyramidal c e l l b o d i es of ar e a s CA3 and CA4 ( r e g i o i n f e r i o r ) . B e f o r e e n t e r i n g the hippocampus, the axons g i v e o f f s e v e r a l c o l l a t e r a l s w i t h i n the dentate gyrus. The nature of t h i s mossy f i b e r p r o j e c t i o n has been e x p e r i m e n t a l l y s t u d i e d by B l a c k s t a d et a l . , (1970). They found that each l e v e l of the dentate gyrus p r o j e c t s t o no more than a narrow segment {-*500 ju) of the hippocampus. These segments are o r i e n t e d t r a n s v e r s e to the l o n g i t u d i n a l a x i s of the hippocampus. The s e p t a l p o l e of the dentate gyrus c o n t a i n s many granule c e l l s , but no mossy f i b e r s . There are, however, many mossy f i b e r s at the temporal p o l e of the dentate g y r u s . Thus, f i b e r s from the s e p t a l p o r t i o n of the dentate gyrus must course i n a temporal d i r e c t i o n b e f o r e they t e r m i n a t e . T h i s may mean that the mossy f i b e r s connect t h e dentate gyrus t o d i f f e r e n t septo-tempora1 l e v e l s of the r e g i o i n f e r i o r of the hippocampus ( B l a c k s t a d , et a l . , (1970). - 10 -F i g u r e l a H o r i z o n t a l s e c t i o n through hippocampal r e g i o n i l l u s t r a t i n g the a r e a s CA1, CA2, CA3, CM, and the dentate gyrus (F.D.), the s i x l a y e r s of the hippocampus, and the f l m b r i a l input t o t h i s r e g i o n , F. F i g u r e l b Enla r g e d drawing of r e c t a n g u l a r area i n 1A. I l l u s t r a t e d here i s the v e r t i c a l l a y e r i n g of dentate a f f e r e n t f i b e r s onto the d e n d r i t i c t r e e of a g r a n u l e c e l l , and to the polymorphic l a y e r of t h e dentate gyrus (POL). - 1 0 a -- 11 -F i g u r e l c Schematic drawing of a g r a n u l e c e l l showing th e c i r c u i t r y of one segment t r a n s v e r s e to the l o n g i t u d i n a l a x i s of the dentate gyrus. Broken l i n e d e p i c t s d i s p u t e d i n p u t . - l l a P e r f o r a n t p a t h - 12 -I I S y n a p t i c O r g a n i z a t i o n of the Dentate Gyrus A. Laminar Arrangement The hippocampal r e g i o n along with the dentate gyrus expresses a laminar o r g a n i z a t i o n i n t h r e e planes ( F i g u r e s l a , l b , l c ) . In the h o r i z o n t a l plane, t h e r e a r e c l e a r l y d i f f e r e n t r e g i o n s : CAl, CA2, CA3, CA4, and the dentate gyrus (Figure l a ) . These regions are d e f i n e d a c c o r d i n g to the appearance of the major c e l l types as d i s c u s s e d i n S e c t i o n IA. The r e g i o n s a l s o d i f f e r i n the l a y e r i n g of i n p u t s . For example, i n CA3 there i s an a d d i t i o n a l l a y e r , stratum lucidum, j u s t s u p e r f i c i a l to the pyramidal c e l l b o d i es ( l a y e r IV) where the mossy f i b e r s pass. Layer I I , stratum lacunosum, i s more prominent i n CAl and CA2 because i t c o n t a i n s i n t r i n s i c a s s o c i a t i o n f i b e r s from CA3 & CA4 (the S c h a f f e r ' s c o l l a t e r a l s ) . In a v e r t i c a l a x i s t h e r e i s a l a y e r i n g of inputs t o the granule c e l l . The source of these i n p u t s has been d i s c u s s e d i n S e c t i o n IB and the l a y e r i n g of t h e i r nerve t e r m i n a l s i s shown i n F i g u r e l b . The s i g n i f i c a n c e of t h i s l a y e r i n g may be that the incoming s i g n a l s are segregated; they make s y n a p t i c c o n t a c t s a t d i f f e r e n t l e v e l s on the d e n d r i t i c t r e e and soma of the granule c e l l . Along the l o n g i t u d i n a l plane t h e r e i s a segmental arrange-ment. The hippocampal formation i s arranged i n a s e r i e s of p a r a l l e l l a m e l l a e with regard t o i n t r i n s i c and output f i b e r s (Andersen, Bland, Dudar, 1973). These l a m e l l a e are o r i e n t e d t r a n s v e r s e t o the l o n g i t u d i n a l (septo-tempora 1) a x i s of the hippocampal f o r m a t i o n . The b a s i c c i r c u i t r y of one of these l a m e l l a e i s diagrammed i n F i g u r e l c . The e x t e n s i v e axonal - 13 -r a m i f i c a t i o n of s h o r t axon basket c e l l s (B) onto g r a n u l e c e l l d e n d r i t e s ( C a j a l , 1911), and the mossy f i b e r system a r e o r i e n -t e d i n p a r a l l e l s t r i p s a c r o s s the l o n g i t u d i n a l a x i s . T h i s laminar arrangement f a c i l i t a t e s the a n a l y s i s of the s y n a p t i c o r g a n i z a t i o n of t h i s r e g i o n . B. U l t r a s t r u c t u r e of S y n a p t i c T e r m i n a l s Synapses i n the c e r e b r a l cortex can be c l a s s i f i e d on the b a s i s of the d i s t r i b u t i o n of dense m a t e r i a l a s s o c i a t e d with t h e pre and p o s t s y n a p t i c membranes (Gray, 1959). Gray's Type I synapses have an asymmetrical appearance and make c o n t a c t s onto s m a l l d e n d r i t e s and d e n d r i t i c s p i n e s . Gray's Type I I a r e symmetrical i n appearance and occur on the d e n d r i t i c trunks and soma of neurons. Type I I have a somewhat narrower (200 A) s y n a p t i c c l e f t than Type I (300 A ) . In many p a r t s of the b r a i n Type I synapses are a s s o c i a t e d with l a r g e s p h e r i c a l v e s i c l e s i n the p r e s y n a p t i c element and Type I I with s m a l l e r e l l i p s o i d a l or f l a t t e n e d v e s i c l e s (Uchizone, 1965 i n Shepherd, 1974). The d i s t i n c t i o n between round and f l a t v e s i c l e s i s a f f e c t e d by f i x a t i o n procedures (Robertson, 1970: T i s d a l e & Nakajima, 1976). However, t h i s does not n e c e s s a r i l y e x p l a i n d i f f e r e n c e s which appear i n v e s i c l e shape i n the same p r e p a r a t i o n . Bodian (1970) examined v a r i a b l e s of aldehyde f i x a t i o n and found some v e s i c l e s f l a t t e n e d , while others were r e s i s t a n t to f l a t t e n i n g i r r e s p e c -t i v e of v a r i a t i o n i n washing procedure. It may be that mem-branes of c e r t a i n v e s i c l e s a r e more s u s c e p t i b l e to a l t e r a t i o n i n f i x a t i o n b u f f e r s (Bodian, 1970). S i z e of v e s i c l e does not - 14 -appear to be a f f e c t e d by d i f f e r e n t f i x a t i o n procedures ( T i s d a l e & Nakajima, 1976). I s o l a t i o n of these v e s i c l e s has r e v e a l e d t h e i r c l o s e a s s o c i a t i o n with t r a n s m i t t e r substance ( E y z a g u i r r e & K u f f l e r , 1955 i n Pappas & Purpura, 1972). For example, e l e c t r o n l u c e n t v e s i c l e s (300 - 500 & i n diameter) are commonly r e l a t e d t o a c e t y l c h o l i n e content and l a r g e r electron-opaque s y n a p t i c v e s i c l e s (700 - 1000 & i n diameter) to monoamines such as n o r a d r e n a l i n and s e r o t o n i n (Bloom, 1970). Synapses observed by e l e c t r o n microscopy i n the outer mol-e c u l a r l a y e r of the dentate gyrus, are mainly asymmetrical onto spines, although a few are on main d e n d r i t i c s h a f t s (Laatsch & Cowan, 1966). T h i s i s the d e n d r i t i c r e g i o n where the p e r f o r a n t path and p o s s i b l y a s s o c i a t i o n f i b e r s from CAl terminate (Raisman, et a l . , 1965). In the inner t h i r d of the molecular l a y e r , t hree types of synapses are found (Laatsch & Cowan, 1966). One that has a wide asymmetrical t h i c k e n i n g ? another makes a symmetrical contact onto d e n d r i t e s . The t h i r d makes c o n t a c t s only onto s m a l l s p i n e s p r o j e c t i n g from the main d e n d r i t e and v a r i e s i n the type of membrane t h i c k e n i n g . In t h i s r e g i o n t h e commissural f i b e r s from the c o n t r a l a t e r a l area CA4 ( G o t t l i e b & Cowan, 1973), and a s s o c i a t i o n f i b e r s from the i p s i l a t e r a l CA4 (Zimmer, 1971) te r m i n a t e . A p o s s i b l e p r o j e c t i o n from the septum t o t h i s area has been reported by some (Mosko, et a l . , 1973) but not by ot h e r s (Raisman, 1965: Cowan, 1975). - 15 -C. Synaptic A c t i v i t y i n the Dentate Gyrus. In g e n e r a l input t o the dentate gyrus i s e x c i t a t o r y . A v o l l e y i n the p e r f o r a n t path e l i c i t s an EPSP-IPSP ( e x c i t a t o r y post s y n a p t i c p o t e n t i a l - i n h i b i t o r y post s y n a p t i c p o t e n t i a l ) sequence (Andersen, et a l . , 1966). T h i s i s a c h a r a c t e r i s t i c e x c i t a t o r y - i n h i b i t o r y sequence i n the b r a i n which can be ex-p l a i n e d i n the f o l l o w i n g way. A d e p o l a r i z a t i o n (EPSP) and a c t i o n p o t e n t i a l i s produced by the e x c i t a t o r y a f f e r e n t s t i m -u l u s . The f i r i n g of the g r a n u l e c e l l e x c i t e s the basket c e l l s through mossy f i b e r c o l l a t e r a l s . The f i r i n g of basket c e l l s , i n t u r n , produces i n h i b i t i o n (IPSP) of the g r a n u l e c e l l . It has been concluded from u n i t r e c o r d i n g s deep t o the granule l a y e r that t h e r e i s a feedback c i r c u i t from granule c e l l axon c o l l a t e r a l s , through basket c e l l s , back onto g r a n u l e c e l l s (Andersen, 1966,). The i n h i b i t o r y neuron i s not d i r e c t l y a c c e s s i b l e t o the p e r f o r a n t path input as the d e n d r i t e s of t h e basket c e l l s do not extend to the outer r e g i o n of the molecular l a y e r . S t i m u l a t i o n of the c o n t r a l a t e r a l hippocampal area CA3 produces a high amplitude short l a t e n c y response i n the g r a n u l e c e l l s (Andersen et a l . , 196i; ). T h i s e x c i t a t i o n of the g r a n u l e c e l l s by the commissural input i s mediated by e x t e n s i v e syn-a p t i c t e r m i n a l s i n the inner t h i r d of the molecular l a y e r ( G o t t l i e b & Cowan, 1973). S t i m u l a t i o n of the medial septum a c t i v a t e s c e l l s i n two zones of the dentate gyrus, a p p a r e n t l y corresponding to the two blades of the g r a n u l e l a y e r , as w e l l as c e l l s l y i n g i n the - 16 -h i l u s . T h i s a c t i v a t i o n i s f a i r l y widespread along the septo-temporal a x i s (Andersen, 1 9 6 1 ) . The dentate response to s e p t a l s t i m u l a t i o n has a longer l a t e n c y than t o c o n t r a l a t e r a l GA3 s t i m u l a t i o n . It i s not c l e a r that the response i n the dentate gyrus which was d e s c r i b e d by Andersen i s a response to input from t h e c h o l i n e r g i c c e l l b odies i n the septum which p r o j e c t v i a the f i m b r i a (see S e c t i o n I I I B), s i n c e s e c t i o n i n g the f i m b r i a d i d not a b o l i s h the response. The response measured by Andersen supports both Raisman's and Mosko's f i n d i n g s . C e l l s i n the h i l u s of the dentate gyrus respond to s e p t a l s t i m u l a t i o n and t h i s agrees with both a u t h o r s ' f i n d i n g s of t e r m i n a l degeneration i n t h i s a r e a . However s i n c e Andersen has not i d e n t i f i e d the exact l a y e r he i s r e c o r d i n g from, nor the c e l l type, i t i s not c l e a r whether the response he recorded could have come from the supra or subgranular l a y e r s . - 17 -III The Septo-dentate Pathway. A. The Septum. The septum of the rat l i e s along the midline medial to the l a t e r a l v e n t r i c l e s and just r o s t r a l to the hippocampal region. It i s an elongated structure, widening in a posterior d i r e c t i o n . Several groups of nuclei make up the septum (Young, 1936; Andy & Stephan, 1964; Raisman, 1966). Fibers from the medial sep-t a l and adjacent diagonal band nuclei have been i d e n t i f i e d as projecting to the hippocampus (Daitz & Powell, 1954; Raisman, 1966 ). These nuclei are bordered l a t e r a l l y by the l a t e r a l septal nucleus, caudally by the septo-fimbrial nucleus, and the ventral hippocampal commissure, dorsally by the dorsal septal nucleus, and an t e r i o r - v e n t r a l l y by the nucleus accumbens. Raisman (1966) has described the efferent systems of the septum using degeneration techniques. These are r o s t r a l l y directed f i b e r s to the olfactory tubercule, the medial fore-brain bundle, and over the rostrum of the corpus callosum. The caudally directed f i b e r s include fimbria 1 afferents to the hippocampus and dentate gyrus, a b i l a t e r a l projection into the dorsal fornix, f i b e r s passing to the entorhinal area through the cingulum, and f i b e r s to the habenula, the medial fore-brain bundle, the l a t e r a l preoptic area and the l a t e r a l hypothalamus. There are projections to the septum from the hippocampus, the pyriform cortex, the amygdala, the olfactory tubercle, the hypothalamus, and the midbrain. Thus, the septum makes extensive connections between the limbic system - 18 -of the c e r e b r a l hemispheres and the diencephaIon. A key path-way i n these connections i s the s e p t a l p r o j e c t i o n to the den-t a t e gyrus, which makes e x t e n s i v e e x c i t a t o r y synapses onto key re g i o n s of the d e n d r i t e s of the CA3 and CA4 pyramidal c e l l s (Raisman, 1966 ; Shepherd, 1974 ). These, i n t u r n , p r o j e c t v i a connections with CAl neurons back t o the septum. T h i s septo-dentate-hippocampal p r o j e c t i o n i s i n a d d i t i o n t o a d i r e c t septo-hippocampal pathway. B. C h o l i n e r g i c nature of the Septo-dentate Pathway. Using a combined s u r g i c a l and histochemica1 procedure, Shute & Lewis (1961, 1963) showed that a c e t y l c h o l i n e s t e r a s e (AchE)-containing f i b e r s reach t h e hippocaropel f o r m a t i o n v i a the d o r s a l f o r n i x , f i m b r i a and a l v e u s . They a r i s e from c e l l s b odies which are s i t u a t e d i n the medial s e p t a l nucleus and i n the nucleus of the d i a g o n a l band. Layers of a c e t y l c h o l i n e s t -erase p o s i t i v e s t a i n were found w e l l marked i n the supra-g r a n u l a r l a y e r and l e s s dense i n the i n f r a g r a n u l a r l a y e r of the dentate gyrus. These AchE p o s i t i v e l a y e r s disappear when those pathways are i n t e r r u p t e d (Lewis, et a l . , 1967; McGeer, et a l . , 1969: Storm^rMathisen, 1970, 1972). Recovery of AchE l e v e l s i n the hippocampus a f t e r i n a c t i v a t i o n of AchE by d i -i s o p r o p y l f l u r o p h o s p h a t e (DFP) i n the septum i s dependent upon an i n t a c t f i m b r i a (Chippendale, et a l . , 1974). Furthermore, Ach has been c o l l e c t e d at the d o r s a l s u r f a c e of the hippo-campus and the r e l e a s e can be in c r e a s e d by s t i m u l a t i n g the - 19 -septum (Smith, 1972). The i n c r e a s e i n Ach r e l e a s e a f t e r medial septum s t i m u l a t i o n was a b o l i s h e d by s e c t i o n i n g the f i m -b r i a but not by s e c t i o n i n g the a l v e u s or d o r s a l f o r n i x (Dudar, 1975), even though the a l v e u s and d o r s a l f o r n i x s t a i n f o r c h o l i n e s t e r a s e . M i c r o i o n t o p h o r e t i c a p p l i c a t i o n of a c e t y l -c h o l i n e t o dentate granule c e l l s e x c i t e d n i n e t y percent of them ( S t e i n e r , 1968; Bland, et a l . , 1974). D i r e c t evidence that Ach i s the n e u r o t r a n s m i t t e r at the septb-dentate synapse i s not a v a i l a b l e , although the above evidence s t r o n g l y suggests that t h i s i s the case. D i r e c t evidence would be s u p p l i e d i f Ach was shown t o be r e l e a s e d i n the dentate gyrus a f t e r a s i n g l e a c t i o n p o t e n t i a l i n the med-i a l s e p t a l n u c l e i and i f the e l e c t r i c a l e f f e c t produced c o u l d be mimiced by a p p l i c a t i o n of Ach onto the neuron. C. I n v e s t i g a t i o n of t h e S e p t a l T e r m i n a l s i n the Dentate Gyrus. A pathway from the septum to the dentate gyrus e x i s t s . There i s c o n s i d e r a b l e evidence that t h i s pathway i s c h o l i n e r g i c and passes through the f i m b r i a t o the h i l u s of the dentate gyrus, however the d i s t r i b u t i o n of AchE i n the dentate gyrus does not correspond completely with the p a t t e r n of degener-a t i o n seen a f t e r medial s e p t a l l e s i o n s . Two p a t t e r n s of de-g e n e r a t i o n have been observed. One r e s t r i c t e d t o t h e sub-g r a n u l a r zone of the dentate gyrus, the other i n c l u d i n g a supragranular band. T h i s t h e s i s i s d i r e c t e d towards a r e s -o l u t i o n of t h i s d i s p u t e . - 20 -In order to i d e n t i f y the s e p t a l t e r m i n a l s i n the dentate gyrus, a t r i t i a t e d amino a c i d was i n j e c t e d i n t o the medial septum where i t was i n c o r p o r a t e d i n t o p r o t e i n i n the s e p t a l c e l l bodies and t r a n s p o r t e d t o the nerve t e r m i n a l s i n the dentate gyrus. These t e r m i n a l s can then be i d e n t i f i e d by e i t h e r l i g h t or e l e c t r o n microscopy using a u t o r a d i o g r a p h i c t e c h n i q u e s . T h i s technique has been used t o map pathways i n the b r a i n (Cowan, et a l . , 1972). It has s e v e r a l advantages over degeneration methods? axons passing through the i n -j e c t e d area do not take up the l a b e l (Cowan,et a l . , 1972) whereas axons en passage are i n e v i t a b l y damaged by l e s i o n s . The f i n e s t r u c t u r e of the neurons under study i s not a l t e r e d by e i t h e r d e g e n erative changes or s t a i n i n g d e p o s i t s , and thus, t h e i r d e t a i l e d morphology can be ana l y s e d . With t h i s method i t i s p o s s i b l e to p r e f e r e n t i a l l y l a b e l nerve t e r m i n a l s or axons by v a r y i n g the time from i n j e c t i o n to s a c r i f i c e (Hendrickson, 1972). T h i s i s because of two r a t e s of axoplasmic flow? a f a s t r a t e which t r a n s p o r t s m a t e r i a l s t o the nerve t e r m i n a l s (20 - 1000 mm/day) and a slow r a t e (.5 - 2 mm/day) which d i s t r i b u t e s m a t e r i a l s along the length of the axon (Hendrickson, 1972). Three days a f t e r i n j e c t i o n i n t o the eye of the monkey Hendrickson (1972) r e p o r t e d 53% of the developed s i l v e r g r a i n s were i n nerve t e r m i n a l s i n the l a t e r a l g e n i c u l a t e ? t h i r t y days a f t e r i n j e c t i o n 66% were i n axons. For the reasons mentioned above, i n the present study the a u t o r a d i o g r a p h i c t r a c i n g technique of axoplasmic flow should p r o v i d e a powerful t o o l not only f o r t r a c i n g the whole septo-dentate projection.:, but f o r l o c a l i z i n g t he s p e c i f i c s e p t a l nerve t e r m i n a l s i n the dentate gyrus. - 21 -METHODS - 22 -The amount .of l a b e l l e d p r o t e i n s i n the dentate gyrus a f t e r i n j e c t i o n of t r i t i a t e d l e u c i n e i n t o the medial s e p t a l n u c l e i was measured by t h r e e independent methods. The f i r s t method was t o d i s s e c t t i s s u e from the hippocampal r e g i o n i n c l u d i n g the dentate gyrus, prepare the t i s s u e i n s c i n t i l l a t i o n f l u i d and count the r a d i o a c t i v i t y present i n the t i s s u e by l i q u i d s c i n t i l l a t i o n methods. T h i s procedure was employed to e s t a b l i s h t h a t t r i a t e d p r o t e i n s could be detected i n the dentate gyrus a f t e r a time i n t e r v a l corresponding to the r a t e of f a s t ax-oplasmic t r a n s p o r t . T h i s was necessary i f l a b e l l e d nerve t e r m i n a l s were to be i d e n t i f i e d . Secondly, the d i s t r i b u t i o n of l a b e l l e d p r o t e i n s i n the dentate gyrus a f t e r i n j e c t i o n of t r i t i a t e d l e u c i n e i n t o the medial s e p t a l n u c l e i was observed by p r e p a r i n g b r a i n s e c t i o n s f o r l i g h t m i c r o s c o p i c autoradiography. The r e l a t i v e d i s -t r i b u t i o n of l a b e l i n each l a y e r of the dentate gyrus could be determined by t h i s method. F i n a l l y , the type of nerve t e r m i n a l which was p r e f e r -e n t i a l l y l a b e l l e d by r a d i o a c t i v e p r o t e i n was observed by e l e c t r o n m i c r o s c o p i c autoradiography. T h i s provided a mor-p h o l o g i c a l d e s c r i p t i o n of the s e p t a l nerve t e r m i n a l s i n t h e dentate gyrus. - 23 .-I . Axonal Transport to the Hippocampal Region. A l l the r a t s used i n t h i s study were male r a t s from Woodlyn Farms, O n t a r i o . Twenty-one r a t s weighing 290-320 grams were used t o i n v e s t i g a t e the time course f o r the t r a n -sport of l a b e l l e d p r o t e i n along the axons' of t h e septohippo-3 campal pathway. .35 uC of H- l e u c i n e (New England Nuclear, s p e c i f i c a c t i v i t y 38 Ci/m mole) i n .5 u l i t e r of a r t i f i c i a l c e r e b r o s p i n a l f l u i d (pH 7.4) was i n j e c t e d over 20 minutes. S t e r e o t a x i c i n j e c t i o n s were made using a 10 u l Hamilton s y r i n g e f i t t e d with a 34-guage needle i n t o the medial s e p t a l nucleus at the f o l l o w i n g c o o r d i n a t e s taken from the e a r b a r s : a n t e r i o r 8.9 mm? l a t e r a l 0.0 mm? and d o r s a l 3.5mm.The animals were s a c r i f i c e d by c e r v i c a l f r a c t u r e at 6 and 24 hours, and 4 days. Rates f o r f a s t a x onal t r a n s p o r t have been estimated of 20 - 1000 mm/day and f o r slow axonal t r a n s p o r t of .5 - 2 mm/day. Since the septo-dentate pathway i n r a t i s 4 - 5 mm i n lengt h , f a s t axonal t r a n s p o r t could be expected w i t h i n s i x hours, and slow t r a n s p o r t i n four days. The optimal time from i n j e c t i o n to s a c r i f i c e i n order t o l a b e l nerve t e r m i n a l s was i n v e s t i g a t e d by the use of the above times p l u s a t h i r d i n t e r m e d i a t e time of 24 hours. The b r a i n s were kept on i c e while the septum, caudate nucleus, and hippocampal r e g i o n were d i s s e c t e d out f r e e hand. In order to i n v e s t i g a t e i f the septum p r o j e c t e d d i f f e r e n t i a l l y along the length of the l o n g i t u d i n a l a x i s of the dentate gyrus, the hippocampal r e g i o n was d i v i d e d i n t o two equal a n t e r i o r - 24 -and p o s t e r i o r p o r t i o n s , these p o r t i o n s corresponded to the d o r s a l ( s e p t a l ) p o l e and the v e n t r a l (temporal) pole of the hippocampal r e g i o n . The d i s s e c t e d t i s s u e was weighed and homogenized i n 2 ml. of c o l d 10% TCA, c e n t r i f u g e d at 600 g. for- 5 minutes and the p r e c i p i t a t e was resuspended i n 5% TCA and c e n t r i f u g e d f o r 5 minutes. In the case of the s e p t a l t i s s u e , .5 ml of the supernatant f l u i d was added to 10 ml of a toluene-based s c i n -t i l l a t i o n mixture and counted by l i q u i d s c i n t i l l a t i o n c o u n t i n g . The r e s t of the supernatant was d i s c a r d e d and each p r o t e i n sample was resuspended i n .5 ml soluene and l e f t u n t i l d i s -s o l v e d . .25 ml of the s o l u e n e - p r o t e i n s o l u t i o n were then added t o 10 ml of the toluene-based s c i n t i l l a t i o n mix? 2 drops of a c e t i c a c i d were added to decrease chemilluminescence and samples cooled f o r 60 minutes before counting by l i q u i d s c i n -t i l l a t i o n counting f o r 4 minutes. A l e v e l of background counts per minute was obtained by pr e p a r i n g two v i a l s with .25 ml of soluene, 10 ml of s c i n -t i l l a t i o n mix, 2 drops of HAc and c o o l i n g them f o r 1 hour. T h i s background l e v e l of 23 cpm was s u b t r a c t e d from the cpm obtained f o r each sample and the r e s u l t i n g v a l u e converted t o d i s i n t e g -r a t i o n s per minute by the f o l l o w i n g method: dpm = (cpm - B) • DF . E where: dpm = d i s i n t e g r a t i o n s per minute B = background DF = d i l u t i o n f a c t o r ( i . e . .25 ml of .5 ml s o l u t i o n counted) E = e f f i c i e n c y of counting = 20% f o r 3 H - l e u c i n e . - 25 The r e s u l t i n g dpm were d i v i d e d by t i s s u e weight (mg.) and a n a l y s e d f o r evidence of t r a n s p o r t . - 26 -I I L i g h t Microscope Autoradiography of the Dentate Gyrus. i S i x t e e n r a t s were prepared f o r l i g h t m i c r o s c o p i c auto-3 radiography by i n j e c t i n g 2 uC of H - l e u c i n e (sp. a. = 38 C i / ^mole) i n .5 jul of a phosphate-buffered s o l u t i o n (pH 6.5) over 40 minutes s t e r e o t a x i c a l l y i n t o the medial septum, 9.1 mm a n t e r i o r , o.oo mm l a t e r a l ; 4.0 mm d o r s a l from the earbar center. One animal was not i n j e c t e d with l a b e l and t i s s u e from t h i s b r a i n used as c o n t r o l . At 1 and 10 days the animals were a n a e s t h e s i z e d with Nembutal (50 mg/kg), and k i l l e d by per-f u s i o n with 4% formal s a l i n e , preceded by a .9% s a l i n e r i n s e of the v a s c u l a r system. The whole b r a i n was d i s s e c t e d out and l e f t i n p e r f u s a t e f o r a minimum of one week bef o r e being t r a n s f e r r e d t o sucrose f o r m a l i n . 25um s e c t i o n s were cut i n e i t h e r a c o r o n a l or hor-i z o n t a l plane on a f r e e z i n g microtome and kept i n 2% formald-ehyde u n t i l mounted on c l e a n g l a s s s l i d e s . Both c o n t r o l and t e s t t i s s u e was coated under red safe l i g h t with Kodak NTB 3 emulsion, mixed 1:1 w i t h D r e f t s o l u t i o n , d r a i n e d i n the dark f o r 5 hours, and packaged f o r s t o r a g e . Exposure times of 3-4 weeks at 4° C were used b e f o r e the g r a i n s were developed with Kodak D-19 f o r 3 minutes, f i x e d i n Kodak Rapid F i x , washed i n g e n t l y running water o v e r n i g h t , and s t a i n e d with c r e s y l v i o l e t . G r a i n counts of the whole hippocampus were made under dark f i e l d i l l u m i n a t i o n on a Z e i s s L i g h t microscope. Each of the t h r e e l a y e r s of t h e dentate gyrus were analysed f o r a r e a s 2 of 2600 u which i n c l u d e s the molecular l a y e r , the g r a n u l a r - 27 -l a y e r and the polymorphic r e g i o n of the dentate h i l u s . Both wings of the dentate f o r both hemispheres were counted on 3-4 s l i d e s from t h r e e r a t s f o r the a n a l y s i s of septo-dentate f i b e r s . C o n t r o l a r e a s were taken from l a y e r 3 of the e n t o r h i n a l c o r t e x of the same s l i d e s . - 28 -I I I E l e c t r o n Microscope Autoradiography of the S e p t a l Nerve T e r m i n a l s . Eleven r a t s were i n j e c t e d with e i t h e r 2uC or 4.5 uC of 3 H - l e u c i n e i n the medial septum over 40 mins. Two u n i n j e c t e d r a t s were processed as c o n t r o l animals. Twenty-four hours a f t e r i n j e c t i o n the animals were perfused with 4% paraform-aldehyde, o.5% g l u t a r a l d e h y d e , and 0.54% g l u c o s e i n 0.1 M 3 phosphate b u f f e r (pH 7.4) under Nembutal a n a e s t h e s i a . 1mm cubes were d i s s e c t e d out from the dentate gyrus. The t i s s u e was then l e f t i n aldehyde f i x a t i v e f o r 2 hours, r i n s e d i n b u f f e r , p o s t - f i x e d with b u f f e r e d osmium t e t r o x i d e (1% f o r 2 hours)? dehydrated, and embedded i n an e p o n - a r a l d i t e mixture. Gold s e c t i o n s f o r autoradiography or s i l v e r - g r a y f o r s y n a p t i c c l a s s i f i c a t i o n were cut on e i t h e r a LKB or R e i c h e r t microtome and mounted on uncoated g r i d s f o r c l a s s i f i c a t i o n or on formvar coated g r i d s f o r autoradiography. The s e c t i o n s were not carbon coated. I l f o r d L-4 emulsion was a p p l i e d to the g r i d s prepared f o r autoradiography by the standard loop technique under red s a f e l i g h t , t h e g r i d s were l e f t f o r t h r e e hours t o d r a i n and packaged f o r storage i n black l i g h t - t i g h t boxes. A f t e r 6 to 12 weeks, the s e c t i o n s were developed i n e i t h e r Microdol-x (Rogers, 1967) or D-19 f i x e d and s t a i n e d f o r 10 minutes with lead c i t r a t e . U n i n j e c t e d m a t e r i a l was a l s o processed at the same time. S e c t i o n s not intended f o r auto-radiography were s t a i n e d with both u r a n y l a c e t a t e and lead c i t r a t e , l u t h i c k s e c t i o n s were cut from b l o c k s taken from the dentate gyrus - 29 -and s t a i n e d with t o l u i d i n e b l u e . Only these b l o c k s i n which the g r a n u l a r c e l l l a y e r and polymorphic area of the dentate gyrus could be i d e n t i f i e d were used f o r autoradiography. S e c t i o n s were observed on a P h i l i p s 201 or 300 e l e c t r o n microscope. 500 g r a i n s were c l a s s i f i e d a c c o r d i n g to the s t r u c t u r e u n d e r l y i n g the g r a i n . In the case where the g r a i n o v e r l a y two or more s t r u c t u r e s a c i r c l e with a r a d i u s of 2400 & (Bachmann & S a l p e t e r , 1965) was drawn and the area occupied by each s t r u c t u r e determined. If one s t r u c t u r e occupied more than h a l f of the c i r c l e the g r a i n was a t t r i b -uted t o that s t r u c t u r e , otherwise p r o p o r t i o n a l p r o b a b i l i t i e s 2 were assigned t o each s t r u c t u r e . An area of 4400 u c o v e r i n g the three l a y e r s of the dentate gyrus from a s e c t i o n on which g r a i n counts were performed was photographed at x 8300, p r i n -ted x 2h, and area measurements done on each t i s s u e component. These area measurements were used to c a l c u l a t e the r e l a t i v e g r a i n d e n s i t y f o r each s t r u c t u r e . F i f t y l a b e l l e d synapses were used to c l a s s i f y symmetrical and asymmetrical synapses. T h i s c l a s s i f i c a t i o n of synapses was done on the b a s i s of v e s i c l e s i z e , v e s i c l e shape, d i s t r i b u t i o n p a t t e r n of v e s i c l e s , and t h i c k e n i n g of post s y n a p t i c membrane. - 30 -- 31 -I. Axonal T r a n s p o r t to the Hippocampal Region. In t h i s part of the study d i r e c t h i s t o l o g i c a l obser-v a t i o n of the i n j e c t i o n s i t e was not p o s s i b l e s i n c e the septum was d i s s e c t e d out and prepared f o r s c i n t i l l a t i o n counting of the r a d i o a c t i v i t y i n c o r p o r a t e d i n t o s e p t a l p r o t e i n s . A l l animals s t u d i e d had high a c t i v i t y i n s e p t a l n u c l e i , and ex-tremely low a c t i v i t y i n the caudate nucleus. T h i s was a necessary c o n t r o l f o r seepage of t r i t i a t e d m a t e r i a l from the i n j e c t i o n s i t e i n t o the l a t e r a l v e n t r i c l e s because the caud-a t e nucleus borders on the l a t e r a l v e n t r i c l e s . Low g r a i n counts i n t h i s s t r u c t u r e i n d i c a t e d no d i f f u s i o n i n t o the caudate. It i s u n l i k e l y that the a c t i v i t y measured i n the hippocampal r e g i o n was the r e s u l t of d i f f u s i o n s i n c e an i n -i t i a l a n a l y s i s of t h e counts i n the a n t e r i o r and p o s t e r i o r p o l e s of the hippocampal r e g i o n showed no d i f f e r e n c e s . If a c t i v i t y i n the hippocampal r e g i o n was due to d i f f u s i o n r a t h e r than axonal t r a n s p o r t , higher counts i n the a n t e r i o r ( s e p t a l ) pole would be expected. S i n c e t h e r e was no d i f f e r e n c e , and s i n c e the a c t i v i t y i n the hippocampus was higher than i n the nearby caudate nucleus, counts i n the hippocampal r e g i o n were a t t r i b u t e d to a xonal t r a n s p o r t of p r o t e i n s from the septum to the hippocampal r e g i o n . Data from the a n t e r i o r and p o s t e r i o r p o l e s was pooled i n T a b l e I. At the end of s i x hours th e a c t i v i t y a t t r i b u t e d t o f r e e l e u c i n e was c o n s i d e r a b l y lower than that i n the p r o t e i n i n -d i c a t i n g t h a t most of the t r i t i a t e d l e u c i n e i n j e c t e d i n t o the septum was taken up by the c e l l b odies and i n c o r p o r a t e d i n t o - 32 -TABLE 1 TIME COURSE OF AXONALLY TRANSPORTED H-IEUCINE LABELLED PROTEINS AFTER INJECTION INTO MEDIAL SEPTUM.  Number Mean (dmp/mg) S.D. 6 Hours Hippocampus 21 11.35 6.33 Septum 4 620.75 313.88 Unbound l e u c i n e 5 75.73 Caudate Nucleus 2 <1 24 Hours Hippocampus 21 25.54 18.37 Septum 6 954.24 256.66 Caudate Nucleus 5 <1 4 Days Hippocampus 22 . 69.48 25.98 Septum 6 666.54 184.18 Caudate Nucleus 13 <1 - 33 -p r o t e i n . In order to determine the time course of the t r a n s p o r t of t r i t i a t e d l e u c i n e from the septum to the hippocampal r e g i o n , r a d i o a c t i v e m a t e r i a l appearing i n the hippocampus a f t e r v a r y -ing s u r v i v a l times was an a l y z e d . Using e s t i m a t e s of 20 - 1000 mm/day f o r r a p i d axonal t r a n s p o r t , the appearance of t r i t i a t e d p r o t e i n i n the hippocampal r e g i o n could have been expected at any time up to s i x hours. T a b l e I shows t h a t t r a n s p o r t had occurred at s i x hours but the amount t r a n s p o r t e d was lower than a t twenty-four hours. It would appear that at s i x hours the a c t i v i t y seen was e i t h e r the t a i l i n g edge of a very r a p i d peak of t r a n s p o r t , or the le a d i n g edge of a peak which was t r a n s p o r t e d at a r a t e of approximately 5 mm/day. Since t h i s was twice as f a s t as the f a s t e s t e s t imate f o r slow t r a n s p o r t , the counts seen at twenty-four hours could be a t t r i b u t e d t o f a s t axonal t r a n s p o r t , and the counts a t fou r days t o slow t r a n s p o r t (1 mm/day). In other pathways slow axonal t r a n s p o r t has been estimated t o be from . 5 - 2 mm/day (Hendrickson, 1972). These r e s u l t s i n d i c a t e that nerve t e r m i n a l s i n the dentate gyrus may be expected to be l a b e l l e d twentyrfour hours a f t e r i n j e c t i o n of t r i t i a t e d l e u c i n e i n t o the septum of the r a t . - 34 -II . Distribution of Label in the Dentate Gyrus. In order to analyse which layer of the dentate gyrus receives the septal input, as indicated by the presence of s i lver grains after autoradiographic processing of the tissue, horizontal sections of the hippocampal region were taken. The three layers could be seen dist inct ly in horizontal section (Figure l a , l b ) . In a photograph of the injection si te of one of the animals used in the analysis, heavy radioactive labelling could be seen in the medial septal nuclei (Figure 2a). The density of label in this region was considerably higher than in the neighbouring lateral septal nuclei although diffusion to these areas could not be discounted. At the limits of the septal nuclei bordering the lateral ventricle , diffusion was minimal. This can be seen in Figure 2a and was supported by the low grain counts observed in an area of the caudate nucleus (CN) adjacent to the ventricle (Figure 3 ) . Heavy labelling was seen in the myelinated axons of the fimbria 1 pathway (Figure 2b). It could be seen that the por-tion of the fimbria which borders on the la teral ventricle (lower left ) is much more heavily labelled than the medial portion. Thus, there is a regionalization of the septo-dentate fibers in the fimbria. In the hippocampal region grain counts were done on the three layers of the dentate gyrus and the supropyramida1 (stra-tum radiatum, St . R.) and the subpyramidal (stratum oriens, St . 0.) of the hippocampus. Labelling was seen in both these - 3 5 -l a y e r s of CA3, but not i n CAl, i n agreement with degeneration s t u d i e s by Raisman, et a l . , 1965. The h i g h e s t l a b e l l i n g was observed i n the h i l u s of the dentate gyrus, t h i s i n c l u d e d the polymorphic l a y e r of the dentate gyrus and CA4 of the hippo-campus. Both these r e g i o n s were l a b e l l e d . In the dentate gyrus a l a y e r of heavy l a b e l l i n g was observed i n the sub-g r a n u l a r zone ( F i g u r e 2 c ) . The three l a y e r s of the dentate gyrus, molecular l a y e r (ML), g r a n u l a r l a y e r (GL), and p o l y -morphic l a y e r (PL) a r e i n d i c a t e d i n F i g u r e 2c. I t could c l e a r l y be seen by comparing F i g u r e 2c with F i g u r e 4b, an area i n the e n t o r h i n a l c o r t e x (A.E.) of the same r a t , that no s i g n i f i c a n t l a b e l l i n g occurs i n the molecular l a y e r . Background l a b e l l i n g i s shown i n F i g u r e 4b. F i g u r e 4a em-p h a s i z e d t h i s f i n d i n g . Here the t i p of the medial wing of the dentate gyrus i s shown. The same p a t t e r n of l a b e l l i n g c o uld be seen, the subgranular zone ( l e f t s i d e ) i s l a b e l l e d , w h i l e the supragranular zone ( r i g h t s i d e ) i s not. Furthermore, g r a i n counts on these zones confirmed the observed r e s u l t . T h i s r e s u l t agrees with Raisman's (1965) f i n d i n g s and Cowan's (1975) unpublished r e s u l t s using a u t o r a d i o g r a p h i c a n a l y s i s of a x o n a l l y t r a n s p o r t e d m a t e r i a l , but not with Mosko's (1973). - 36 -I I I . U l t r a s t r u c t u r e of S e p t a l T e r m i n a l s . In order to i d e n t i f y which s t r u c t u r e s i n the subgranular zone of the dentate gyrus were p r e f e r e n t i a l l y l a b e l l e d by s i l v e r g r a i n s , b l o c k s of t i s s u e from t h i s zone were processed f o r the e l e c t r o n m i c r o s c o p i c autoradiography. T a b l e I I g i v e s the r e l a t i v e g r a i n d e n s i t y (RGD), f o r s e v e r a l s t r u c t u r e s i n t h i s zone. A RGD of g r e a t e r than one i n d i c a t e s s i g n i f i c a n t l a b e l l i n g of that s t r u c t u r e . S i l v e r g r a i n s were l o c a l i z e d i n s y n a p t i c s t r u c t u r e s (RGD = 1.72). More synapses than axons were l a b e l l e d i n t h i s zone. T h i s i n d i c a t e s t hat the s e p t a l axons do not simply pass through t h i s r e g i o n , but i n f a c t , do make s y n a p t i c connections with the h i l a r neurons. D e n d r i t e s a r e l a b e l l e d but l e s s so than synapses. C e l l b o d ies and g l i a are not l a b e l l e d . In T a b l e I I I , the type of synapse which i s l a b e l l e d i s pre s e n t e d . Terminal s y n a p t i c boutons onto d e n d r i t i c elements showed h i g h l y p r e f e r e n t i a l l a b e l l i n g . Other types of synapses were e i t h e r not l a b e l l e d (e.g., the mossy f i b e r boutons formed by the axons of g r a n u l e c e l l s ) , or were present i n such s m a l l q u a n t i t i e s (e.g. synapse "en passage") that they could not account f o r the l a b e l l i n g seen with the l i g h t microscope. One of the l a t t e r cases i s shown i n F i g u r e 5. T h i s i s a l a b e l l e d synapse "en passage" passing through the n e u r o p i l of the dentate gyrus. Asymmetrical synapses onto d e n d r i t e s or s p i n e s were the most h i g h l y l a b e l l e d . Of these asymmetrical synapses, f i f t y - 3 7 -percent were onto s p i n e s and h a l f t h a t number onto d e n d r i t e s . T h i s type was the most common i n the h i l a r r e g i o n , occupying 37.4% of the t o t a l s y n a p t i c a r e a . In F i g u r e s 6a and 6b, i t can be seen that the bouton forms a s y n a p t i c c l e f t of 200 -300 A with a pronounced p o s t s y n a p t i c t h i c k e n i n g (300 - 600 & t h i c k n e s s ) and a f i n e r but c l e a r l y d e f i n e d p r e s y n a p t i c t h i c k -ening (50 - 100 $ ) . T h e , s y n a p t i c j u n c t i o n i s s l i g h t l y convex p r e s y n a p t i c a l l y without i n t r u s i o n s or e x t r u s i o n s . The pre-s y n a p t i c s t r u c t u r e c o n t a i n s round e l e c t r o n l u c e n t v e s i c l e s of 400 - 500 A" diameter which a r e moderately packed throughout th e boutons ( F i g u r e s 6a and 6b). A second type of synapse occurred h a l f as f r e q u e n t l y as the former type and was not as h e a v i l y - l a b e l l e d as the p r e -v i o u s l y d e s c r i b e d nerve t e r m i n a l . T h i s synapse made symmet-r i c a l c o n t a c t s onto major d e n d r i t i c t r u n k s . As could be seen i n F i g u r e 6c, the s y n a p t i c c l e f t i s narrower (50 - 100 A) with broken patches of dense m a t e r i a l s y m m e t r i c a l l y arranged on both s i d e s of the c l e f t . T h i s d e n s i t y was marked by an arrow i n F i g u r e 6c. S e v e r a l s m a l l v e s i c l e s (300 A* i n diameter) with some l a r g e v e s i c l e s (700 - 1000 & diameter) were present i n the nerve t e r m i n a l . The symmetrical j u n c t i o n s are as a r u l e more d i f f i c u l t t o f i n d than the asymmetrical j u n c t i o n s and f o r t h i s reason, some p r e t e r m i n a l boutons might possibly* be c l a s s i f i e d as the symmetrical synapses i n the present an-a l y s i s . These r e s u l t s represent the p a t t e r n of l a b e l l i n g i n t h e s t r u c t u r e s of the n e u r o p i l of the subgranular zone of the - 38 -dentate gyrus a f t e r i n j e c t i o n of t r i t i a t e d m a t e r i a l i n t o the s e p t a l n u c l e i . - 39 -TABLE II DISTRIBUTION OF SILVER GRAINS IN DENTATE GYRUS AFTER 3H LEUCINE INJECTION INTO MEDIAL SEPTUM Structure No. O f gra ins % O f gra ins Area % O f area RGD % grains/ % area synapse 135 26.9 661.5 15.6 1.72 dendrite 104 20.7 777.0 17.7 1.17 axon 194 38.6 1777.1 40.6 0.95 soma 55 11.1 752.3 17.1 0.64 g l i a 12 2.39 402.0 9.2 0.26 T o t a l 500 99.7 4369.6 100.8 -- 40 -TABLE III DISTRIBUTION OF SILVER GRAINS IN THE SYNAPSES OF THE DENTATE GYRUS  Structure No. of gra ins % of gra ins Area M % of area RGD % gra i n s / % area ,axo-dendritic or axo-spinous synapse 1) asymmetrical 75 55.6 247.7 37.4 1.49 2) symmetrical 29 21.5 107.6 16.3 1.32 3) unc l a s s i f i e d 5 3.7 1.5 0.2 axo-somatic synapse 6 4.4 29.0 4.4 1.00 synapse en passage 12 8.9 13.5 2.0 4.45 mossy fi b e r bouton 8 5.9 262.2 39.7 0.15 T o t a l 135 100.0 661.5 100.0 - 41 -DISCUSSION - 42 -I n t e r p r e t a t i o n of a u t o r a d i o g r a p h i c l a b e l l i n g must be done with c a u t i o n . D i f f u s i o n from the i n j e c t i o n s i t e cannot e n t i r e l y be e l i m i n a t e d . In t h i s study two s t r u c t u r e s which border on the medial s e p t a l n u c l e i have been r e p o r t e d t o p r o j e c t t o the hippocampal r e g i o n . The f i r s t , the v e n t r a l hippocampal com-missure d i d not i n c o r p o r a t e l a b e l l e d p r o t e i n s i n c e no evidence of l a b e l l i n g was seen i n the molecular l a y e r where these f i b e r s are known to p r o j e c t . The second, the l a t e r a l s e p t a l nucleus was repor t e d by Raisman, 1966 not to p r o j e c t t o the hippo-campus. However, S i e g e l and T a s s o n i (1971) i n d i c a t e d such a p r o j e c t i o n t o the v e n t r a l hippocampus (temporal p o l e ) . The medial s e p t a l n u c l e i p r o j e c t e d t o the d o r s a l hippocampus ( s e p t a l pole) i n t h e i r study. No d i f f e r e n c e s i n l a b e l l i n g between the d o r s a l and v e n t r a l hippocampal r e g i o n were found i n t h i s study e i t h e r by s c i n t i l l a t i o n counting of r a d i o a c t i v i t y i n the hippo-campal r e g i o n or by g r a i n counts done using a l i g h t m i c r o s c o p i c autoradiography. T h i s lack of d i f f e r e n c e could be a t t r i b u t e d t o seepage of t r i t i a t e d l e u c i n e i n t o t h e l a t e r a l septum and subsequent t r a n s p o r t to the v e n t r a l hippocampus. However, ob s e r v a t i o n of F i g u r e 2a re v e a l e d that the d e n s i t y of l a b e l l i n g i n the l a t e r a l septum was s e v e r a l times l e s s than i n the medial septum, and s t i l l no d i f f e r e n c e i n the amounts t r a n s p o r t e d to the d o r s a l and v e n t r a l hippocampus co u l d be d e t e c t e d . The decreased r a d i o a c t i v i t y of the supernatant from the s e p t a l n u c l e i supports other evidence t h a t t r i t i a t e d l e u c i n e , a common amino a c i d i n p r o t e i n , i s i n c o r p o r a t e d i n t o p r o t e i n and t r a n s p o r t e d along the axon (Droz, 1969). There appears - 43 -t o be two r a t e s of t r a n s p o r t along t h e axon. Chippendale, et a l . , (1974) observed that AchE appeared i n the hippocampus 16 -48 hours a f t e r i t s disappearance from t h e septum. T h i s agrees with the f i n d i n g s of the present study t h a t l a b e l l e d p r o t e i n a r r i v e d i n nerve t e r m i n a l s of the dentate gyrus twenty-four hours a f t e r i n j e c t i o n . The f i n d i n g s a l s o c o n f i r m Reports ( G r a f s t e i n , 1967; Hendrickson, 1972) that r a p i d l y t r a n s p o r t e d m a t e r i a l s p r e f e r e n t i a l l y l a b e l nerve t e r m i n a l s . The h e a v i e s t l a b e l l i n g was seen i n the h i l u s of the d e n t a t e . T h i s i s i n agreement with Raisman et a l v ( 1 9 6 5 ) and Mosko et a l v ( 1 9 7 3 ) who saw degenerating t e r m i n a l s i n t h i s r e g i o n , with Shute and Lewis (1961) who observed c h o l i n e s t -e rase p o s i t i v e f i b e r s from the septum ending here, and with Andersen,et al.,(1961) who recorded responses of h i l a r neurons t o s e p t a l s t i m u l a t i o n . A moderately dense l a y e r of s i l v e r g r a i n s was observed i n the subgranular zone of the dentate gyrus. T h i s i s i n agreement wi t h Raisman, et a l . , (1965), Cowan, (1975) j but not with Mosko, et a l . , (1973). An obvious source of e r r o r i n Mosko's study i s the c u t t i n g of f i b e r s i n the v e n t r a l hippocampal commissure which l i e s caudal to the medial septum. The commissural f i b e r s from CAl pass through t h i s commissure and terminate i n the supragranular l a y e r where Mosko et a l . , (1973) found d e g e n e r a t i o n . If even a few of these f i b e r s were damaged by the l e s i o n , t h i s could r e s u l t i n t h e type of degen-e r a t i o n observed by these a u t h o r s . The a u t o r a d i o g r a p h i c t r a c i n g t e c hnique i s s u p e r i o r to degeneration s t u d i e s i n t h i s regard as f i b e r s of passage a r e not a f f e c t e d by t h e i n j e c t i o n of l a b e l , (cowan, et a l . , 1972). - 44 -A c e t y l c h o l i n e s t e r a s e - p o s i t i v e nerve f i b e r s i n the supra-g r a n u l a r zone observed by Shute & Lev;is (1963) are not e x p l a i n -ed by the present f i n d i n g s . But as they observed p r e t e r m i n a l axons w h i l e t h i s study looked at nerve t e r m i n a l s , the r e s u l t s a re not i n c o n f l i c t . The source of the A c h E - p o s i t i v e p r e -t e r m i n a l axons i n the supragranular l a y e r a r e not known. The ma j o r i t y of s i l v e r g r a i n s observed i n : t h i s study were i n nerve t e r m i n a l s . S i l v e r g r a i n s found i n d e n d r i t e s may be a t t r i b u t e d t o some of the l a b e l l e d p r o t e i n c r o s s i n g the s y n a p t i c c l e f t . Evidence that t h i s can occur has been r e p o r -ted by G r a f s t e i n (1967) and Hendrickson (1972). F u r t h e r work i s needed to understand the s i g n i f i c a n c e of t h i s . Two types of l a b e l l e d synapses were observed i n the subgranular zone, an asymmetrical type o c c u r i n g about twice as f r e q u e n t l y as the symmetrical type. One of these nerve t e r m i n a l s may represent the t e r m i n a l of the c h o l i n e r g i c s e p t a l a f f e r e n t s . In t h i s regard, i t i s noteworthy that the asym-m e t r i c a l t h i c k e n i n g of the p o s t s y n a p t i c membrane and round e l e c t r o n - l u c e n t type of v e s i c l e s i n the l a b e l l e d boutons i n t h e dentate gyrus corresponded w e l l with the morphological c h a r a c t e r i s t i c s p r e v i o u s l y a s c r i b e d to c h o l i n e r g i c nerve t e r m i n a l s i n the r a t neostriatum ( H a t t o r i & McGeer 1973, 1974). The other t e r m i n a l could be the r e s u l t of l a b e l d i f f u s i n g i n t o the l a t e r a l septum and l a b e l l i n g t h e d e n t a t e . One argu-ment a g a i n s t t h i s i s that the frequency with which t h i s t e r -minal i s seen i s h a l f that of the other l a b e l l e d t e r m i n a l . Yet the d e n s i t y of l a b e l i n the l a t e r a l septum i s not h a l f t h at i n - 45 -the medial septum; t h e r e f o r e the l a t e r a l s e p t a l p r o j e c t i o n to t h i s area would have to be more than t w i c e as p r o f u s e as the medial s e p t a l p r o j e c t i o n to account f o r the observed l a b e l l i n g . T h i s seems unreasonable s i n c e Raisman observed no p r o j e c t i o n from the l a t e r a l septum and S i e g e l & T a s s o n i observed t h a t the medial and l a t e r a l septum p r o j e c t e d t o d i f f e r e n t segments of the hippocampal r e g i o n and hence wouldn't be observed i n the same e l e c t r o n m i c r o s c o p i c s e c t i o n s . A more l i k e l y ex-p l a n a t i o n i s that one t e r m i n a l a r i s e s from the medial s e p t a l n u c l e i and another from the d i a g o n a l band n u c l e i . Both of these m e d i a l l y s i t u a t e d s e p t a l n u c l e i a r e i n v o l v e d i n the p r o j e c t i o n to the septum (Raisman, et a l . , 1965: Shute & Lewis, 1963). The p o s s i b i l i t y that two pathways a r i s e from t h i s area cannot be d i s c o u n t e d ; p a r t i c u l a r l y , i n view of the f a c t that Andersen s t i l l recorded a dentate response to s e p t a l s t i m u l -a t i o n a f t e r s e c t i o n i n g the f i m b r i a . He concluded that t h e r e i s a s e p t a l pathway running deep to the v e n t r i c u l a r s u r f a c e between the most medial ( d o r s a l f o r n i x ) and most l a t e r a l ( f i m -b r i a ) elements of the f o r n i x system. T h i s does not agree however with the p a t t e r n of l a b e l l i n g observed i n t h i s study, nor with Raisman's degeneration s t u d i e s . - 46 -F i f t y percent of the asymmetrical synapses made c o n t a c t s onto d e n d r i t i c s p i n e s i n the subgranular zone of the dentate gyrus. I d e n t i f i c a t i o n of the p o s t s y n a p t i c element i s not pos-s i b l e i n the present study. Since the g r a n u l e c e l l s do not have b a s i l a r d e n d r i t e s ( C a j a l , 1911), s e p t a l synapses onto gr a n u l e c e l l s must be onto some b a s i l a r p o r t i o n (some or axons) or onto a p i c a l d e n d r i t e s invading the b a s i l a r zone. Yet G o l g i s t a i n s do not show d e n d r i t e s i n t h i s zone, and the m a j o r i t y of s y n a p t i c c o n t a c t s observed were not onto c e l l b o dies. A more l i k e l y p o s s i b i l i t y i s t h a t the s e p t a l t e r m i n a l s contact the d e n d r i t e s of other c e l l types i n t h i s zone. Immediately below the gr a n u l a r l a y e r , two c e l l types a r e found; one type with axons t e r m i n a t i n g i n the g r a n u l a r l a y e r ( p o s s i b l y i n h i b -i t o r y basket c e l l s ) , and another which sends i t s axons to t h e a l v e u s . Below t h i s i s a l a y e r of c e l l s with axons ascending i n t o the molecular l a y e r ( C a j a l , 1911). These c e l l s p r o v i d e a means by which the s e p t a l input to the dentate gyrus could e x c i t e granule c e l l s through e x c i t a t o r y i n t e r n e u r o n s . T h i s would mean that the s e p t a l input to the g r a n u l e c e l l s i s not monosynaptic. The d e n d r i t e s of these c e l l s have been des-c r i b e d by C a j a l (1911). Although they possess fewer spines than the d e n d r i t e s of granule c e l l s , t h i s does not n e c e s s a r i l y e l i m i n a t e them as a p o s s i b l e p o s t s y n a p t i c s t r u c t u r e . F u r t h e r r e s e a r c h to i d e n t i f y the p o s t s y n a p t i c element of t h i s pathway i s i n d i c a t e d . The r o l e of the s e p t a l input to the dentate gyrus can only be a p p r e c i a t e d when a c l e a r understanding of the s y n a p t i c - 47 -o r g a n i z a t i o n of t h i s system i s obtained. The present study c l a r i f i e s some of the pre v i o u s a m b i g u i t i e s about to which l a y e r of the dentate gyrus the s e p t a l a f f e r e n t s p r o j e c t , but r a i s e s q u e s t i o n s concerning the p o s t s y n a p t i c element i n v o l v e d . The v i s u a l i z a t i o n of the s e p t a l t e r m i n a l by e l e c t r o n micro-scopy i n d i c a t e s i t may have s t r u c t u r a l s i m i l a r i t i e s t o known c h o l i n e r g i c nerve t e r m i n a l s but r a i s e s the p o s s i b i l i t y of two s e p t a l t e r m i n a l s , p o s s i b l y one from the medial s e p t a l nucleus and another from the d i a g o n a l band nucleus. Further r e s e a r c h i s needed before the f u l l i m p l i c a t i o n s of these f i n d i n g s to l i m b i c system f u n c t i o n a r e understood. - 48 -BIBLIOGRAPHY - 49 -Andersen, P., Bland, B.H., & Dudar, J.D. O r g a n i z a t i o n of the hippocampal output. Exp. Bra i n Research 17; 152-168, 1973. Andersen, P., Bruland, N., Kaada, B. A c t i v a t i o n of the dentate area by s e p t a l s t i m u l a t i o n , Acta P h y s i o l o g i c a  Scanda navia, 51: 17-28, 1961. Andersen, P., Holmquist, B., and, Voorhoerve, P.E. 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Quantitative histochemistry of a c e t y l -cholinesterase in the rat hippocampal region correlated to histochemica1 staining. Journa1 of Neurochemistry, 17: 739-750, 1970. Storm-Mathison, J . Glutamate decarboxylase in the rat hippo-campal region af t e r lesions of the afferent f i b e r systems. Evidence that the enzyme i s located in i n t r i n s i c neurons. Brain Research 40: 215-235, 1972. Tisdale, A.D. & Naka jima, Y. Fine structure of synaptic v e s i c l e s in two types of nerve terminals in cr a y f i s h stretch receptor organs: Influence of f i x a t i o n methods. Journa1  Comparative Neurology 165: 369-386, 1976. Young, M.W. The nuclear pattern and f i b e r connections of the non-cortical centers of the telencephalon of the rabbit (Lepus cuniculus). Journa1 Compa rat ive Neurology 65: 295-401, 1936. Zimmer, J. I p s i l a t e r a l afferents to the commissural zone of the fascia dentata, demonstrated in decommisurated rats by s i l v e r impregnation. Journa 1 Comparative Neurology 142: 393-416, 1971. - 56 - 57 -F i g u r e 2a H o r i z o n t a l s e c t i o n showing i n j e c t i o n s i t e of one animal used f o r g r a i n counts of the hippocampal r e g i o n . F i g u r e 2b S i l v e r g r a i n s i n the f i m b r i a shown by l i g h t m i c r o s c o p i c autoradiography. The l a t e r a l v e n t r i c a l i s to the lower r i g h t . F i g u r e 2c D i s t r i b u t i o n of s i l v e r g r a i n s i n the polymorphic l a y e r (PL), g r a n u l a r l a y e r (GL) and molecular l a y e r (ML) of the dentate gyrus. - 57a -- 58 -Figure 3 Grain counts in the hippocampal regions CAl, CA3, CA4 and the dentate gyrus. Counts were done in the supra pyramidal layer, stratum radiatum (St. R.) and subpyramidal layer, stratum oriens (St. O.), the three layers of the dentate gyrus: the molecular layer (ML), granular layer (GL), and polymorphic layer (PL). The polymorphic area of the dentate and area CA4 of the hippocampus are counted as one area. Background l a b e l l -ing was seen in the caudate nucleus (CN), and the entorhinal cortex (A.E.). - 58a -st. 0 st.R st. 0 st. R G L M L A E C A l C A 3 C A 4 D G - 59 -F i g u r e 4a T i p of medial wing of dentate gyrus showing d i s t r i b u t i o n of s i l v e r g r a i n s . Subgranular zone i s on the l e f t and mol-e c u l a r l a y e r on the r i g h t . F i g u r e 4b S i l v e r g r a i n s i n E n t o r h i n a l cortex (A.E.) t o show back-ground l a b e l l i n g . F i g u r e 5 U l t r a s t r u c t u r e of subgranular zone of the dentate gyrus. Insert shows a synapse en passage. - 5 9 a -- 60 -F i g u r e 6a Asymmetrical synapse onto a d e n d r i t e i n the dentate gy r u s . F i g u r e 6b Asymmetrical synapse i n the dentate gyrus showing a nerve t e r m i n a l wrapped around a s p i n e . F i g u r e 6c Symmetrical synapse onto d e n d r i t e i n the dentate gyrus. Arrow i n d i c a t e s symmetrical t h i c k e n i n g . - 60a -

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