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The commissural projection of the hilus of the area dentata in the rat Willcox, John Edward Francis 1983

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THE COMMISSURAL PROJECTION OF THE HILUS OF THE AREA DENTATA IN THE RAT by JOHN EDWARD FRANCIS WILLCOX B.A., The University of Manitoba, 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Physiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1983 c) John Edward Francis Willcox, 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 The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (.3/81) ABSTRACT Stimulation of the commissural afferents to the hippocampal formation evoked responses in the regio superior, regio i n f e r i o r , and area dentata. Two waves were found in the stratum moleculare of the fasci a dentata: the presynaptic volley (MN1), and the population EPSP (MN2). MN2 was caused by simultaneous a c t i v i t y in both the commissural and association f i b r e s . Granule c e l l s were r e l i a b l y evoked by a commissural stimulus. The excit a t i o n of a granule c e l l was followed by a long period of i n h i b i t i o n . The long period of i n h i b i t i o n and the absence of a granule c e l l population spike were attributed to feed-forward i n h i b i t i o n . The commissural current source in the area dentata was hidden by f i e l d potentials generated in adjacent structures that were activated simultaneously by the commissural stimulus. In the h i l a r region a compound pyramidal c e l l action potential that had both orthodromic and antidromic components was found (HN2). A presynaptic volley in the commissural afferents was located in the commissural terminal zone of the regio i n f e r i o r (HN1). The P wave was f i r s t misinterpreted as the commissural current source in the granule c e l l s , but further investigation l o c a l i z e d i t to the stratum pyramidale of the regio i n f e r i o r . It was not d e f i n i t e l y characterized, but i t appeared to be due to passive recovery processes that occurred afte r the propagation of action potentials in the pyramidal i i i cells of the regio inferior. Hilar neurons project to the contralateral and ipsilateral area dentata, and to the ipsilateral and/or the contralateral regio superior. i v TABLE OF CONTENTS Abstract i i List of Figures v i i i List of Tables x Abreviations xi 1 INTRODUCTION 1 1.1 ANATOMY OF THE HIPPOCAMPAL REGION 1 1.2 ANATOMY OF THE HIPPOCAMPAL FORMATION 5 1.2.1 Hippocampus 5 1.2.2 Fascia Dentata 11 1.2.3 Hilar Region 11 1.3 IPSILATERAL CONNECTIONS 14 1.3.1 Retrohippocampal Formation 14 a. Entorhinal Cortex 14 b. Parasubiculum 16 c. Area Retrosplenialis 16 d. Presubiculum 16 1.3.2 Hippocampal Formation 17 a. Subiculum 17 b. Hippocampus 18 c. Area Dentata ...20 d. Lamellar Organization 25 ' V e. Basket Cells . 25 1.3.3 Summary Of The Ipsilateral Connections 26 1.4 THE COMMISSURAL CONNECTIONS 27 1.4.1 Commissures 27 1.4.2 Retrohippocampal Formation ' 28 a. Entorhinal Cortex 28 b. Parasubiculum 29 c. Area Retrosplenialis 30 d. Presubiculum 30 1.4.3 Hippocampal Formation 30 a. Subiculum 30 b. Hippocampus ....31 c. Area Dentata 36 1.4.4 The Course Of The Interhippocampal Fibres ....40 1.5 PHYSIOLOGY OF THE PERFORANT PATH INPUT TO THE AREA DENTATA 41 1.5.1 Single Unit Responses 41 1.5..2 Population Field Potentials 44 a. Introduction 44 b. Perforant Path Responses 47 1.6 PHYSIOLOGY OF THE COMMISSURAL INPUT TO THE AREA DENTATA 49 1.6.1 Single Unit Responses 49 1.6.2 Population Field Potentials 49 1.7 THE PRESENT STUDY 50 2 METHODS 57 v i 2.1 SURGICAL PROCEDURES 57 2.2 STIMULATION AND RECORDING 58 2.3 DATA ANALYSIS 60 2.4 HISTOLOGY 63 2.5 MECHANICAL LESIONS 63 2.6 CHEMICAL LESIONS 65 3 RESULTS 66 3.1 POPULATION FIELD POTENTIALS 66 3.1.1 Introduction 66 3.1.2 The Molecular Layer 66 3.1.3 Hilar Region 69 3.2 UNIT RESPONSES 7 5 3.2.1 Stratum Granulosum 75 3.2.2 Hilar Region 81 3.3 VENTRAL PSALTERIUM LESIONS 88 3.4 KAINIC ACID LESIONS 91 4 DISCUSSION 95 4.1 FIELD POTENTIALS 95 4.1.1 MN 1 95 4.1.2 MN2 96 4.1.3 HN1 96 4.1.4 HN2 97 4.1.5 HN3 99 v i i 4.1.6 P 99 4.2 COMMISSURAL SINK-SOURCE IN THE FASCIA DENTATA 100 4.3 COMMISSURAL INHIBITION OF GRANULE CELLS 105 4.4 ABSENCE OF A COMMISSURAL GRANULE CELL POPULATION SPIKE 106 4.5 TERMINAL FIELDS OF THE HILAR NEURONS 107 5 SUMMARY 111 APPENDIX 116 BIBLIOGRAPHY 119 v i i i LIST OF FIGURES 1 Gross Anatomy Of The Hippocampal Region 4 2 Horizontal Section Of The Hippocampal Region ...7 3 Coronal Section Of The Dorsal Hippocampus 10 4 Coronal Section Of The Hilar Region 13 5 Commissural And Association Cells Of The Hilus 23 6 Distribution Of Hilar Commissural Terminals 33 7 Schematic Diagram Of The Projections Of The Commissural System Innervating The Area Dentata 56 8 Histological Localization Of A Stimulating Electrode Placement 62 9 Identified Waveforms In The Area Dentata 68 10 Characteristics Of The Commissural Evoked Response In The Area Dentata 71 11 Localization Of The Commissural P Wave 74 12 Latency Histograms Of Granule Cells Evoked By Perforant Path And Commissural Stimuli 78 13 The Effect Of Perforant Path And Commissural Stimuli On The Spontaneous Activity Of Granule Cells ...80 14 Commissural Stimulus Intensity And The P r o b a b i l i t y Of Granule Cell Discharge 83 15 Latency Histograms Of Hilar Neurons Evoked By A Commissural Stimulus 85 16 Identification Of Hilar Neurons Evoked Antidromically By A Commissural Stimulus 87 i x 17 Effect Of A Ventral Psalterium Lesion On Commissural Evoked Responses 90 18 Effect Of A Kainic Acid Lesion On Commissural Evoked Responses 93 19 Hilar Input To The Contralateral Regio Superior 109 20 Connections Of Hilar Neurons 114 21 Extra-hilar Responses 118 X LIST OF TABLES I Major Divisions Of The Hippocampal Region 2 II Stereotaxic Co-ordinates 59 III Latencies Of Unit Responses 76 ABREVIATIONS C Centigrade CSD current source density EPSP excitatory post-synaptic potential GABA gamma amino butyric acid HRP horse radish peroxidase Hz Herz IP intraperitoneal IPSP inhibitory post-synaptic potential IS-SD i n i t i a l segment-somato dendritic kg kilogram M molar mg milligram mm millimeter msec millisecond mvolt milli v o l t 1 1 INTRODUCTION 1.1 ANATOMY OF THE HIPPOCAMPAL REGION The structures of the hippocampal region are subdivided as illustrated in Table 1. They begin at the rostral pole near the septum, curve along the lateral ventricles in a caudal and ventral direction, and terminate in the temporal pole (figure 1). The curved shape of the hippocampal structures resembles a ram's horn, hence the name cornu ammonis, or Ammon's horn. This-means that unqualified use of the terms rostral, caudal, dorsal,, and ventral can be misleading because the major axis of the hippocampal region is rotated through an angle of 180°. The longest axis of the hippocampal region, running from the septal pole to the temporal pole, is known as the septo-temporal axis. The septal one-third of the septo-temporal axis of the hippocampal formation is known as the dorsal hippocampal formation, and the temporal one-third is known as the ventral hippocampal formation. The middle one-third is called the posterior hippocampal formation. The hippocampal formation is distinctive due to the close-packing of the major types of neurons: the pyramidal and the granule cells, into two thin sheets. The sheets are folded over one-another, looking like two interlocked horseshoes when viewed 2 . TABLE I. MAJOR DIVISIONS OF THE HIPPOCAMPAL REGION Hippocampal Region Hippocampal Formation Retrohippocampal Formation j—Subiculum Hippocampus-Area Dentata i — Presubiculum Area Retr o S p l e n i a l i s • Parasubiculum Entorhinal Cortex Regio Superior Regio I n f e r i o r Fascia Dentata -Hilus FIGURE 1 GROSS ANATOMY OF THE HIPPOCAMPAL REGION EC entorhinal cortex F fimbria HCA hippocampus VP ventral psalterium a plane of the coronal section in figure 3 b plane of the horizontal section in figure 2 5 in cross-section (figure 2). This arrangement is seen in most of the septo-temporal extent of the hippocampal formation. The fascia dentata seen in a coronal section is a u-shaped structure that curves around the medial edge of the lower layer of pyramidal cells (figures 2 & 3). It consists of a tightly packed layer of granule c e l l s . The curvature of the longtitudinal axis causes the area dentata to rotate through an arc of 180°. Consequently, the layer of granule cells that was dorsal near the septal pole is ventral in the temporal pole. The two blades have been distinguished by their proximity to the hippocampal fissure which follows the curvature of the hippocampal formation. The upper blade is proximal to the hippocampal fissure and the lower blade is d i s t a l . The topographical relationships between the structures of the hippocampal region can be seen most clearly in a horizontal section through the posterior hippocampal formation (figure 2). 1.1 ANATOMY OF THE HIPPOCAMPAL FORMATION 1.2.1 Hippocampus Lorente de No (1934), using a Golgi stain, divided the hippocampus into four fields: CA1, CA2, CA3, and CA4 (figure 3). CA1 is a zone of tightly packed pyramidal c e l l s . CA3 is a zone 6 FIGURE 2 H o r i z o n t a l S e c t i o n Of The Hippocampal Region AD a r e a d e n t a t a AR a r e a r e t r o s p l e n i a l i s F f i m b r i a LEC l a t e r a l e n t o r h i n a l c o r t e x MEC m e d i a l e n t o r h i n a l c o r t e x P a r a p a r a s u b i c u l u m PC p e r i r h i n a l c o r t e x Pre p r e s u b i c u l u m RI r e g i o i n f e r i o r RS r e g i o s u p e r i o r S s u b i c u l u m T h i s f i g u r e was adapted from: Swanson and Cowan, (1977); Habets, (1980); and Hjorth-Simonsen and Jeune, (1972). 7 8 o f p y r a m i d a l c e l l s d i s t i n g u i s h e d f r o m t h o s e o f CA1 by t h e i r l a r g e r s i z e , d i f f e r e n t d e n d r i t i c a r b o r i z a t i o n , S c h a f f e r c o l l a t e r a l e f f e r e n t s , and mossy f i b r e a f f e r e n t s . CA3 i s d i v i d e d i n t o t h r e e s u b - f i e l d s : t h e l a t e r a l CA3a; t h e i n t e r m e d i a t e CA3b; a n d t h e m e d i a l CA3c. F i e l d CA2 i s a s m a l l t r a n s i t i o n z o n e o f p y r a m i d a l c e l l s t h a t s e p a r a t e s CA1 and CA3. I t i s i d e n t i f i e d i n z i n c s t a i n s by an e x p a n s i o n o f t h e mossy f i b r e t e r m i n a l zone known a s t h e 'end b u l b ' ( M c L a r d y 1 9 6 3 ) . CA2 h a s n o t been r e c o g n i z e d a s a d i s t i n c t e n t i t y i n r e c e n t c l a s s i f i c a t i o n s b e c a u s e i t s s u b - c o r t i c a l p r o j e c t i o n f i e l d s a r e t h e same a s t h o s e o f CA3 (Swanson a n d Cowan, 1 9 7 7 ) , a n d i t i s p h y s i o l o g i c a l l y i d e n t i c a l t o CA3 ( A n d e r s e n , 1 9 5 9 ) . Ramon y C a j a l ( 1 9 6 8 ) a n d B l a c k s t a d ( 1 9 5 6 ) h a v e d i v i d e d t h e h i p p o c a m p u s i n t o two f i e l d s : r e g i o s u p e r i o r , w h i c h i s i d e n t i c a l t o CA1; a n d r e g i o i n f e r i o r , w h i c h i s CA2 and CA3 lumped t o g e t h e r . The r e g i o s u p e r i o r i s d i v i d e d i n t o s e v e r a l l a y e r s ( f i g u r e 3 ) . The a l v e u s c o n t a i n s t h e a x o n s o f t h e p y r a m i d a l c e l l s . The s t r a t u m o r i e n s i n c l u d e s t h e b a s a l d e n d r i t e s and t h e e m e r g i n g a x o n s o f t h e p y r a m i d a l c e l l s . The p y r a m i d a l c e l l s o m a t a f o r m a c o m p a c t l a y e r i n t h e s t r a t u m p y r a m i d a l e . The t h i c k p r o x i m a l a p i c a l d e n d r i t e s make up t h e s t r a t u m r a d i a t u m , and t h e t h i n n e r d i s t a l a p i c a l d e n d r i t e s c o n s t i t u t e t h e s t r a t u m l a c u n o s u m -m o l e c u l a r e . The r e g i o i n f e r i o r h a s t h e same l a y e r s a s t h e r e g i o s u p e r i o r w i t h t h e a d d i t i o n o f t h e s t r a t u m l u c i d u m on t h e p r o x i m a l a p i c a l d e n d r i t e s , a d j a c e n t t o t h e s t r a t u m p y r a m i d a l e ( f i g u r e 3 ) . The s t r a t u m l u c i d u m c o n t a i n s t h e mossy f i b r e t e r m i n a l s . 9 FIGURE 3 (A) Coronal Section Of The Dorsal Hippocampus (B) The Strata Of CA1 And The Area Dentata (C) The Strata Of CA3 alv alveus s. or stratum oriens s. pyr stratum pyramidale s. rad stratum radiatum s. l . - m stratum lacunosum-moleculare s. luc stratum lucidum h. f h i l a r f i s sure s. mol stratum moleculare s. gran stratum granulosum h i l h i l u s 10 11 1.2.2 Fascia Dentata The fascia dentata has two layers (figure 3). The stratum granulosum is the layer of tightly packed granule c e l l somata. The c e l l bodies are packed so closely together that there is very l i t t l e extracellular space (Blackstad and Dahl, 1962). The stratum moleculare is the region of granule c e l l dendrites. The distal dendrites are marked by a predominance of long, thin postsynaptic spines; whereas, the proximal dendrites are characterized by stubby spines and shaft synapses (Nafstad, 1967). 1.2.3 Hilar Region The hilar region contains the pyramidal c e l l s of CA3c and the hilus of the fascia dentata. It is bounded by the concave face of the stratum granulosum, and a line joining the lateral edges of the fascia dentata (figure 4). Amaral (1978) has identified twenty-one different types of neuron in the hilar region. The hilus of the fascia dentata is bounded by the concave face of the stratum granulosum, and by a line joining the lateral edges of the upper and lower blades of the fascia dentata to the medial edge of the regio inferior (figure 3). Lorente de No (1934) considered the hilus to be a part of the hippocampus, and called i t zone CA4. However, more recent research includes the hilus in the area dentata (Blackstad, Figure 4 Coronal Section Of The Hilar Region h hilus h.r. hilar region Fascia dentata 14 1956; Amaral, 1978). The most prevalent neurons of the hilus are the mossy ce l l s , which, in contrast to the other major neurons of the hippocampal formation, are randomly scattered (Amaral, 1 978) . 1 .3 I PSILATERAL CONNECTIONS 1.3.1 Retrohippocampal Formation a. Entorhinal Cortex The entorhinal cortex receives input from several sensory modalities. The rat, cat, and monkey a l l have an olfactory input to the entorhinal cortex from the prepyriform cortex (Cragg,l960; Hjorth-Simonsen, 1972; van Hoesen and Pandya, 1975a,'1975b; Beckstead, 1978; Habets, et al 1980a, 1980b). In the monkey the somatic, visual, and auditory cortices converge on the prorhinal and perirhinal cortices, which in turn project to the entorhinal cortex (Jones and Powell, 1970; van Hoesen and Pandya, 1975a; van Hoesen et a l , 1975). Entorhinal afferents from the sensory cortex have not been demonstrated in the rat, but there are inputs from adjacent prorhinal and cingulate cortices (Srebro et a l , 1979). The entorhinal cortex of the rat receives subcortical afferents from the medial septum, amygdala, thalamus, locus coeruleus, ventral tegmentum, and dorsal raphe 15 (Domesick, 1976; Beckstead, 1978). The hippocampal formation sends efferents from the regio inferior, regio superior, and subiculum to the entorhinal cortex (Hjorth-Simonsen, 1971; Krettek and Price, 1977; Swanson and Cowan, 1977; Beckstead, 1978; Kohler et a_l, 1978). Retrohippocampal inputs from the presubiculum and the prosubiculum terminate in the entorhinal cortex (Swanson and Cowan, 1977; Beckstead, 1978; Kohler et a l , 1978). The entorhinal cortex is the source of the largest input to the hippocampal formation through the fibre tract known as the perforant path (Ramon y Cajal, 1968; Lorente de No, 1934). The perforant path fibres are small unmyelinated axons that form en  passage synapses with the granule c e l l dendrites (Laatsch and Cowan, 1966; Nafstad, 1967). The medial and lateral entorhinal cortices can be distinguished on the basis of their different projection fields. The medial entorhinal cortex sends fibres along the medial perforant path to the middle one-third of the stratum moleculare layer of the fascia dentata, and the lateral entorhinal cortex projects to the outer one-third of the molecular layer through the lateral perforant path (Hjorth-Simonsen, 1972; Hjorth-Simonsen and Jeune, 1972; Steward, 1976; Steward and Scoville, 1976; McNaughton and Barnes, 1977; McNaughton, 1980). The medial and lateral entorhinal cortices also project topographically onto the stratum lacunosum-moleculare of the regio superior and the regio inferior (Steward, 1976; Steward and Scoville, 1976). 16 b. Parasubiculum The parasubiculum receives afferents from the nucleus reuniens of the thalamus, the amygdala, and the endopyriform cortex (Krettek and Price, 1977; Herkenham, 1978). Hippocampal afferents come from the regio inferior (Swanson and Cowan, 1977). The parasubiculum sends efferent fibres to the medial entorhinal cortex (Swanson and Cowan, 1977). c. Area Retrospenialis There is no data available on the ipsilateral connections of the area retrosplenialis. d. Presubiculum The presubiculum receives afferents from the thalamus, cingulate cortex, regio inferior, and the hilus (Domesick, 1969; Swanson et al., 1977; Herkenham, 1978). The presubiculum of the guinea pig receives a subicular projection (Sorensen and Shipley, 1979). The presubiculum projects to the thalamus, the cingulate cortex, and the mammilary nucleus (Meibach and Siegel, 1977; Sikes et^  a_l, 1977; Swanson and Cowan, 1977). In the monkey and the guinea pig there are efferents to the entorhinal cortex 17 (Shipley, 1975; Rosene, 1976). 1.3.2 Hippocampal Formation a. Subiculum The subiculum is innervated by fibres from the medial septum, thalamus, hypothalamus, locus coeruleus, amygdala, endopyriform cortex, raphe nucleus, regio inferior, regio superior, and hilus (Raisman et §_1, 1 965; Conrad et a l , 1974; Pickel et a l , 1974; Krettek and Price, 1977; Meibach and Siegel, 1977; Beckstead, 1978; Herkenham, 1978; Swanson et a l , 1978; Berger et a l , 1980). The prorhinal and perirhinal cortices innervate the subiculum in the monkey (van Hoesen and Pandya, 1975b). The subiculum is the major source of extrinsic fibres from the hippocampal region. It projects to the hypothalamus, lateral septum, mammillary nucleus, thalamus, perirhinal area, and the hippocampus (Lavail et a l , 1973; Meibach and Siegel, 1977; Swanson and Cowan, 1977; Beckstead, 1978; Berger, 1980b). The subiculum of the monkey innervates widespread areas of the cerebral cortex (Rosene and van Hoesen, 1977). 18 b. Hippocampus (i) Regio Superior The regio superior receives extrinsic afferents from the entorhinal cortex (above). The stratum radiatum of the regio superior is innervated by fibres called Schaffer collaterals which originate in the regio inferior pyramidal cells and perhaps the hilus. The Schaffer collaterals are unmyelinated, and they make en passage synapses in the stratum radiatum of the regio superior. There is some dispute over the precise source of the Schaffer collaterals. Lorente de No (1934) showed diagrams of hilar neurons that had Schaffer collaterals. This was indirectly confirmed by Zimmer (1971). It was more directly corroborated by Hjorth-Simonsen (1972), and by Swanson et a l , (1978) who injected t r i t i a t e d leucine into the hilus and found that i t was transported to the Schaffer collateral terminal zone in the regio superior. Amaral (1978), however, could not find any hilar neurons with Schaffer collaterals in his Golgi preparations. Amaral's data was supported by Laurberg (1979), and Laurberg and Sorensen (1981). The projection fields of the regio superior are entirely ipsilateral, and terminate in the perirhinal and entorhinal cortices, the lateral septum, and the subiculum (Andersen et a l , 1973; Hjorth-Simonsen, 1973; Swanson and Cowan, 1977; Swanson et a l , 1978; Finch and Babb, 1980b). Collateral fibres of the regio superior pyramidal c e l l axons synapse on the dendrites of 19 basket c e l l s in the s t r a t u m o r i e n s . The basket c e l l s have axons t h a t rami fy w i t h i n the s t ra tum p y r a m i d a l e and synapse on the p y r a m i d a l c e l l somata ( L o r e n t e de No, 1934; B l a c k s t a d and F l o o d , 1963). ( i i ) Regio I n f e r i o r The r e g i o i n f e r i o r r e c e i v e s a f f e r e n t s from the med ia l septum, l o c u s c o e r u l e u s , and raphe n u c l e u s (Raisman et a_l, 1965; P i c k e l et a l , 1974; P a s q u i e r and R e i n a s o - S u a r e z , 1976; Meibach and S i e g e l , 1977). I n t r i n s i c a f f e r e n t s are d e r i v e d from the f a s c i a d e n t a t a (Raisman et a l , 1965; B l a c k s t a d et a l , 1970; Swanson et a l , 1978), sub icu lum and e n t o r h i n a l c o r t e x (above) . A l o n g t i t u d i n a l a s s o c i a t i o n system c o n n e c t i n g CA3a a l o n g the s e p t o - t e m p o r a l a x i s has been r e p o r t e d (Lorente de No, 1934; Swanson et a l , 1978; L a u r b e r g , 1979). The r e g i o i n f e r i o r p r o j e c t s b i l a t e r a l l y to the l a t e r a l septum, and i p s i l a t e r a l l y to the c i n g u l a t e a r e a , p r e s u b i c u l u m , e n t o r h i n a l c o r t e x , s u b i c u l u m , and the h i l u s ( L o r e n t e de No, 1934; H j o r t h - S i m o n s e n , 1971; Deadwyler e_t a l , 1975b; Swanson and Cowan, 1977; Swanson e_t a_l, 1978; Berger et al, 1980; L a u r b e r g and Swanson, 1981). I t has been shown, u s i n g s imul taneous r e t r o g r a d e l a b e l l i n g from d i f f e r e n t a r e a s , tha t the p y r a m i d a l c e l l s of the r e g i o i n f e r i o r have m u l t i p l e c o l l a t e r a l s which i n n e r v a t e the c o n t r a l a t e r a l and i p s i l a t e r a l hippocampus and the septum (Swanson et a_l, 1980). C o l l a t e r a l branches of r e g i o i n f e r i o r p y r a m i d a l c e l l s synapse on a d j a c e n t basket c e l l 20 d e n d r i t e s . The basket c e l l s form r e c i p r o c a l c onnections with the pyramidal c e l l s (Lorente de No, 1934). c. Area Dentata ( i ) H i l u s The h i l u s r e c e i v e s a f f e r e n t s from the medial septum, hypothalamus, median raphe nucleus, l o c u s c o e r u l e u s (Raisman et a l , 1965; P i c k e l , 1974; Moore and H a l a r i s , 1975; Pasquier and Reinoso-Suarez, 1976), e n t o r h i n a l c ortex (above), and the granule c e l l s of the f a s c i a dentata (Laatsch and Cowan, 1966; Amaral, 1978). The i p s i l a t e r a l e f f e r e n t s of the h i l a r c e l l s remain e n t i r e l y w i t h i n the hippocampal formation (Swanson et a_l, 1978). There are p r o j e c t i o n s to the r e g i o i n f e r i o r , and r e g i o s u p e r i o r (Swanson et a_l, 1978). The neuronal i n t e r a c t i o n s w i t h i n the h i l u s are complex (Amaral, 1978). The h i l u s i s the source of an i p s i l a t e r a l a s s o c i a t i o n path to the proximal one t h i r d of the d e n d r i t e s of the granule c e l l s (Zimmer, 1971; G o t t l i e b and Cowan, 1972 and 1973; Lynch et a l , 1976; Swanson et a l , 1978; F r i c k e and Cowan, 1978; Laurberg, 1979; Laurberg and Sorensen, 1981). T h i s a s s o c i a t i o n system o r i g i n a t e s i n the h i l u s (Amaral, 1978; Swanson et a l , 1978; Laurberg, 1979; Laurberg and Sorensen, 1981). The a s s o c i a t i o n and commissural p r o j e c t i o n s of the h i l u s terminate i n the same 21 zone of the stratum moleculare of the fascia dentata, and there is speculation that both systems compete for the same terminals (Gottlieb and Cowan, 1972; Fricke and Cowan, 1978; O'Leary et a l , 1979a, 1979b). Mossy c e l l s , unaligned pyramidal c e l l s , and fusiform cells in the hilus have been implicated as probable sources of the association fibres in the mouse and the rat (Amaral, 1978; West et a l , 1979, Berger et a l , 1981). Axons of the association system reach their terminal zone by either penetrating the granule c e l l layer, or passing through the stratum radiatum of the medial regio inferior and curving around the lateral edge of the stratum granulosum to reach the proximal one-third of the stratum moleculare (figure 5). The association fibres extend through the stratum moleculare to the junction of the upper and lower blades, and reach the lower blade from the medial direction (Zimmer, 1971; Lynch e_t a l , 1 976; Swanson et a l , 1978). There is a septo-temporal difference in the distribution of the association terminals. The association fibres originating in the septal one-half of the hilus are distributed widely over the septo-temporal extent of the fascia dentata, while those originating in the temporal one-half of the hilus show less septo-temporal spread (Zimmer, 1971; Swanson e_t a l , 1978; Fricke and Cowan, 1978). Association cells in the septal two-thirds of the hilus project mainly to the upper blade of the fascia dentata, whereas, those from the temporal one-third project primarily to the lower blade (Fricke and Cowan, 1978). FIGURE 5 The Commissural And Association Cells Of The Hilus. (i) Association c e l l . ( i i ) Commissural c e l l with the paths of the efferent axon according to: (a) Gottlieb and Cowan, (1973); 4b) Swanson et a l , (1978); (c) Laurberg and Sorensen, (1981). 23 24 ( i i ) F a s c i a D e ntata The f a s c i a d e n t a t a r e c e i v e s a f f e r e n t s from the e n t o r h i n a l c o r t e x , and t h e h i l u s (above). The e x i s t e n c e of a s e p t a l i n n e r v a t i o n of the f a s c i a d e n t a t a i s the s o u r c e of some disagreement (see A s s a f , 1978 f o r a r e v i e w ) . The g r a n u l e c e l l e f f e r e n t f i b r e s a r e c a l l e d mossy f i b r e s . These a r e s m a l l u n m y e l i n a t e d axons t h a t a r e c h a r a c t e r i z e d by l a r g e p r e s y n a p t i c v a r i c o s i t i e s ( L a a t s c h and Cowan, 1966; Yamamoto, 1972; Andersen, 1975). They emerge from t h e h i l a r s i d e of the s t r a t u m granulosum and d i s t r i b u t e numerous c o l l a t e r a l s j u s t beneath the s t r a t u m granulosum. These c o l l a t e r a l s form a dense f i b r e p l e x u s t h a t i s i n c o n t a c t w i t h the d e n t a t e basket c e l l s (Amaral, 1978; Swanson et a_l, 1978). The b a s k e t c e l l axons make r e c i p r o c a l s y n a p t i c c o n t a c t w i t h the g r a n u l e c e l l s i n the s t r a t u m granulosum. The basket c e l l s a r e p r o b a b l y r e s p o n s i b l e f o r the r e c u r r e n t i n h i b i t i o n found i n the f a s c i a d e n t a t a (Ramon y C a j a l , 1968; L o r e n t e de No, 1934; Andersen e_t a l , 1966; Lomo, 1971; Amaral, 1978; S t r u b l e et a l , 1978). The main e f f e r e n t branches of the mossy f i b r e s form b u n d l e s i n the h i l u s which converge on the r e g i o i n f e r i o r making en passage synapses on the a p i c a l d e n d r i t e s of the h i l a r neurons t h a t they e n c o u n t e r (Ramon y C a j a l , 1968; L o r e n t e de No, 1934; McLardy, 1960: L a a t s c h and Cowan, 1966; B l a c k s t a d and K j a e r h e i m , 1978; G a a r k s j a e r , 1978). The mossy f i b r e t e r m i n a l zone i n the r e g i o i n f e r i o r forms the s t r a t u m l u c i d u m , as d e s c r i b e d e a r l i e r . 25 d. Lamellar Organization of the Hippocampal Formation The serial connection between: the entorhinal cortex, fascia dentata, regio inferior, regio superior, and subiculum has been studied extensively. A l l of the fibres in this pathway have been described as having a parallel orientation in a tansverse plane (Andersen et §_1, 1971; Rawlins and Green, 1977). The entorhinal cortex projection to the fascia dentata is topical (Hjorth-Simonsen and Jeune, 1972), and the stimulation of a small number of perforant path fibres activated a narrow band of granule cells (Lomo, 1971; Rawlins and Green, 1977). The mossy fibres have a parallel orientation (Garksjaer, 1978), but the projection of the regio inferior to the regio superior showed a substantial divergence (Hjorth-Simonsen, 1973; Swanson et al 1978; Laurberg, 1979; Leung, 1979a). The regio superior fibres in the alveus that innervate the subiculum also have a parallel orientation (Rawlins and Green,1977; Swanson and Cowan, 1977; Swanson et a l , 1978; Leung, 1979b). With the exception of the diverging fibres from the regio inferior to the regio superior, the stimulation of any of these neurons will activate a narrow, tranverse band of postsynaptic c e l l s . e. Basket"Cells of the Hippocampal Formation Basket c e l l s are found mainly on the output side of pyramidal and granule c e l l s : the stratum oriens and the hilus. Seress and Pokorny (1981) located 65% of the dentate basket 26 cells in the hilus, adjacent to the stratum granulosum; 30% were in the stratum moleculare, and the remainder were in the stratum granulosum. The axon of a single basket c e l l diverges extensively, forming axosomatic synapses over a large area of the stratum pyramidale or the stratum granulosum (Ramon y Cajal, 1968; Lorente de No, 1934; Amaral, 1978; Struble et a l , 1978; Seress and Pokorny, 1981). In the fascia dentata the basket c e l l dendrites are aligned in a plane that is perpendicular to the predominantly transverse orientation of the perforant path axons (Struble et a l , 1978; Seress and Pokorny, 1981). The basket cells and granule cells are reciprocally connected (above). This arrangement would cause a narrow band of perforant path fibres to activate a small transverse segment of the fascia dentata, and inhibit the surrounding granule c e l l s . The dendrites of basket cells ramify within the hilus, and extend through the stratum granulosum to spread through the stratum moleculare (Laatsch and Cowan, 1966; Amaral, 1978; Seress and Pokorny, 1981). Lorente de No (1934) found no basket c e l l synapses in the hilus. 1.3.3 Summary Of The Ipsilateral Connections The majority of the cortical and subcortical afferents of the hippocampal region terminate in the medial and lateral entorhinal cortices. The subiculum is the source of most of the hippocampal region efferent fibres. The intrinsic circuits of the hippocampal region form a predominantly unidirectional path 27 from the entorhinal cortex to the subiculum. The serial pathway involving the entorhinal cortex, fascia dentata, regio inferior, regio superior, and subiculum is reinforced by connections from the entorhinal cortex to the regio inferior, regio superior, and the subiculum. There may be a circuit from the fascia dentata to the hilus, and from the hilus to the regio inferior, regio superior, and subiculum; but this is currently under dispute. Small reciprocal cicuits have been demonstrated between the hilus and the fascia dentata; the pyramidal cells and the pyramidal basket c e l l s ; and between the granule cells and the dentate basket c e l l s . 1.4 THE COMMISSURAL CONNECTIONS 1.4.1 Commissures The hippocampal formations of each hemisphere communicate with one-another through fibres that are found in the dorsal psalterium and ventral psalterium. The dorsal psalterium contains axons that project to the entorhinal cortex, parasubiculum, subiculum, the stratum lacunosum-moleculare of the regio superior and the regio inferior, and the fascia dentata (Blackstad, 1956; Sperti et a l , 1970; Gol.dowitz et a l , 1975; Swanson and Cowan, 1977). The ventral psalterium contains the interhemispheric projections from the hippocampal region to 28 the hippocampus, fascia dentata, presubiculum, and possibly also the parasubiculum, and the entorhinal cortex (Lorente de No, 1934; Blackstad, 1956; Andersen, 1957; Cragg and Hamlyn, 1957; Gottlieb and Cowan, 1973; Swanson and Cowan, 1977; Swanson et a l , 1978). Axons are arranged in the ventral psalterium in an organized fashion according to their origin. Fibres from the septal hippocampus occupy the ventral posterior part of the ventral psalterium, and fibres from the temporal hippocampal region occupy the more rostral part of the ventral psalterium (Gottlieb and Cowan, 1973; Swanson et a l , 1978). Fibres from the lateral regio inferior (CA3a) are dorsal to those from the medial regio inferior (CA3c) and the hilus (Swanson et a l , 1978). 1.4.2 Retrohippocampal Formation a. Entorhinal Cortex The entorhinal cortex innervates the contralateral entorhinal cortex, subiculum, hippocampus, and fascia dentata (Lorente de No, 1934; Blackstad, 1956; Steward et a l , 1974; Steward and Scoville, 1976; Zimmer and Hjorth-Simonsen, 1976; Swanson and Cowan, 1977; Steward, 1980). The innervation of the contralateral subiculum is sparse (Blackstad, 1956). The projection to the contralateral stratum lacunosum-moleculare of the regio superior and the regio inferior is minor in the rat 29 (Blackstad, 1956), and possibly nonexistent in the rabbit (Cragg and Hamlyn (1957). The commissural projection from the entorhinal cortex to the fascia dentata is considered to be significant by one laboratory (Swanson and Cowan, 1977); however, other groups have found that this projection was sparse in intact animals, and proliferated only after the ipsilateral entorhinal-cortex was destroyed (Steward et a l , 1974; Zimmer and Hjorth-Simonsen, 1975; Steward and Scoville, 1976; Steward, 1980) . The entorhinal cortex receives contralateral afferents from the entorhinal cortex (above), parasubiculum, and presubiculum (Swanson and Cowan, 1977; Beckstead, 1978). A l l of the interhemispheric fibres to and from the entorhinal cortex, with the exception of those afferents from the parasubiculum, cross in the dorsal psalterium (Blackstad, 1956; Swanson and Cowan, 1977). b. Parasubiculum The parasubiculum sends efferents to the contralateral entorhinal cortex and parasubiculum through the ventral psalterium (Blackstad, 1956; Swanson and Cowan, 1977). Afferents to the parasubiculum come from the contralateral parasubiculum and entorhinal cortex (Blackstad, 1956; Steward and Scoville, 1976). 30 c. Area Retrosplenialis The area retrosplenialis receives interhemispheric afferents from the subiculum (Blackstad, 1956). It has no known interhemispheric efferents (Blackstad, 1956). d. Presubiculum Presubicular efferents innervate the contralateral presubiculum, parasubiculum, and entorhinal cortex. Commissural afferents come from the presubiculum (Blackstad, 1956; Steward and Scoville, 1976). 1.4.3 Hippocampal Formation a. Subiculum The subiculum has no interhemispheric efferents (Swanson and Cowan, 1977). It receives sparse contralateral afferents from the entorhinal cortex, regio inferior, and hilus (Blackstad, 1956; Steward and Scoville, 1976). 31 b. Hippocampus (i) Regio Superior The regio inferior has no interhemispheric efferents (Raisman et a_l, 1 965; Andersen et a l , 1973; Segal and Landis, 1974; Hjorth-Simonsen and Laurberg, 1977). The regio inferior receives contralateral afferents from the entorhinal cortex (above), regio inferior, and the hilus (Gottlieb and Cowan, 1973; Swanson et a l , 1978). Some data (figure 6) suggested that the regio inferior was the only source of afferents to the contralateral regio inferior, and the hilus projected only to the contralateral fascia dentata (Hjorth-Simonsen and Laurberg, 1977; Laurberg, 1979; Berger et a l , 1981; Laurberg and Sorensen, 1981). However, small injections of tri t i a t e d amino acids that were restricted to the hilus (Swanson et a l , 1978) labelled terminals in the contralateral regio superior, regio inferior, and area-dentata (figure 6). The interhippocampal fibres to the contralateral regio superior separate into two groups in the contralateral hemisphere. One group leaves the fimbria and passes through the alveus over the regio superior and terminates in the stratum oriens. The second group of fibres passes through the stratum pyramidale of the regio superior and follows the same course as the Schaffer collaterals; terminating in the stratum radiatum of FIGURE 6 T e r m i n a l D i s t r i b u t i o n Of The H i l a r C o m m i s s u r a l  P r o j e c t i o n . ^ H j o r t h - S i m o n s e n and L a u r b e r g , ( 1 9 7 7 ) ; L a u r b e r g , ( 1 9 7 9 ) ; L a u r b e r g a n d S o r e n s e n , ( 1 9 8 1 ) . Swanson e t a l ( 1 9 7 8 ) . 33 34 the regio superior (Gottlieb and Cowan, 1973; Swanson et a l , 1978). The distribution of commissural terminals is not uniform throughout the regio superior. The septal regions of the stratum radiatum and stratum oriens of the regio superior are more densely innervated than the temporal regions (Gottlieb and Cowan, 1973; Laurberg, 1979). There is disagreement over the relative densities of the terminals in the stratum oriens and-the stratum radiatum. Andersen (1959; 1960a) found that the apical dendrites of the regio superior had a lower response threshold than the basal dendrites to a stimulus applied to the contralateral hippocampus. Lesions of the ventral psalterium resulted in heavy degeneration of both the stratum oriens and the stratum radiatum (Blackstad, 1956). Gottlieb and Cowan (1973) and Laurberg (1979), however, found a gradient of terminal density along the septo-temporal axis of the regio superior following lesions of the contralateral regio inferior: the density of terminals in the stratum oriens and the stratum radiatum was equal in the septal regions, but the number of terminals in the stratum radiatum declined, relative to the number of terminals in the stratum oriens, in the temporal regions. In another experiment using small injections of tr i t i a t e d amino acids, Swanson e_t al (1978) found that the labelling was more intense in the contralateral stratum radiatum when the injections were localized the hilus, but injections restricted to the regio inferior caused more intense labelling in the stratum oriens. The confusion over the distribution of the commissural 35 terminals in the regio superior arose because a lesion of the ventral psalterium (Blackstad, 1956) would not distinguish between hilar and regio inferior efferents; and a lesion of the regio inferior would sever nearby hilar efferent axons also. Small injections of labelled amino acids would isolate the hilar and regio inferior efferent sytems if the marker did not diffuse too far. The figure presented by Swanson et a_l (1978) shows a hilar injection with no evidence of diffusion into the regio inferior. ( i i ) Regio Inferior The regio inferior, as described above, sends commissural fibres to the subiculum, regio superior, and regio inferior. It does not innervate either the contralateral hilus, or fascia dentata (Swanson and Cowan, 1977; Swanson et a_l, 1978). The innervation of the regio inferior is more pronounced in the stratum oriens than in the stratum radiatum (Swanson e_t a l , 1978). The commissural projection of the regio inferior is homotopic insofar as i t is most dense at the homotopic region, but there is extensive divergence along the medio-lateral axis of the regio superior and the regio inferior. There is also substantial divergence along the septo-temporal axis of the hippocampal formation. The septo-temporal spread of the hippocampal commissural efferents is more pronounced from fibres originating in the septal half of the hippocampus than i t is from those fibres from the temporal half (Gottlieb and 36 Cowan,1973; Fricke and Cowan, 1978; Laurberg, 1979). The regio inferior receives input from the contralateral entorhinal cortex (above), regio inferior (above), and hilus (Gottlieb and Cowan, 1973; Swanson et a l , 1978). c. Area Dentata (i) Hilus The hilus is the only source of fibres to the contralateral hilus and area dentata (Hjorth-Simonsen and Laurberg, 1977; Swanson et a l , 1978; Laurberg, 1979; West et aj., 1979, Laurberg and Sorensen, 1981). The cel l s of origin of the commissural projection are the modified pyramidal c e l l s , mossy ce l l s , and fusiform cells of the hilus (Amaral, 1978; Swanson et a l , 1978; West et a l , 1979; Berger et a l , 1981). There is some dispute over the extent of the projection f i e l d of the hilar neurons (figure 5). Swanson et al (1978) showed that small injections of tri t i a t e d amino acids restricted to the hilus caused labelled material to be distributed widely bilaterally throughout the hippocampal formation. Hjorth-Simonsen and Laurberg (1977) and Laurberg (1979), however, found no terminal degeneration outside of the ipsilateral and contralateral area dentata when hippocampal lesions were restricted to the hilus. Terminal degeneration could only be provoked in the hippocampus when lesions involved the regio 37 inferior. Injections of HRP (Laurberg, 1979; Berger et a l , 1981), or true blue (Laurberg and Sorensen, 1981) into the regio superior failed to show any retrograde labelling of hilar c e l l s in either hemisphere. The hilus was labelled only when injections were made into the fascia dentata (Berger e_t a l , 1981). Lorente de No (1934) showed examples of hilar neurons that had collaterals projecting to both the regio superior and the fimbria (his figures 31 and 32). Amaral (1978), however, was unable to find any hilar neurons that had Schaffer collaterals, although he found numerous cel l s that had axons entering the fimbria. The innervation of the contralateral area dentata is not s t r i c t l y homotopic. Efferents from each region of the hilus have terminals along a large septo-temporal extent of the area dentata, and those fibres originating in the septal two-thirds of the hilus diverge mmore widely than those originating in the temporal one-third (Fricke and Cowan, 1978; Swanson e_t a_l, 1978; Laurberg, 1979; Laurberg and Sorensen, 1981). The terminals are more dense in the homotopic region and become relatively sparse in the anterior and posterior directions (Swanson et a_l, 1978). A septo-temporal difference is also found in the hilar efferents to the two blades of the fascia dentata. Neurons in the septal one-third of the hilus innervate the upper blade more intensively than the lower blade, but those in the temporal two-thirds of the hilus innervate the lower blade more intensively than the upper blade (Fricke and Cowan, 1978). Hilar fibres 38 project primarily to the stratum radiatum of the contralateral regio superior and regio inferior (above). The hilus receives interhippocampal afferents through the ventral psalterium (Lorente de No, 1934; Cragg and Hamlyn, 1957; Raisman et a l , 1965; Gottlieb and Cowan, 1973). The terminals are more dense in the temporal hilus than in the septal hilus (Hjorth-Simonsen and Laurberg, 1977). Commissural terminals are found on the dendrites of hilar neurons, not on the somata (Gottlieb and Cowan, 1973). Blackstad (1956) was unable to find evidence of terminal degeneration in the hilus following lesions of the ventral psalterium, but this may have been due to the rapid disappearance of degeneration products in the hilus (Hjorth-Simonsen and Laurberg, 1977). (i i) Fascia Dentata The fascia dentata has no contralateral efferents (Raisman et a l , 1965; Blackstad et a l , 1970; Gottlieb and Cowan, 1973; Swanson and Cowan, 1977). The interhemispheric afferents of the fascia dentata originate in the entorhinal cortex and the hilus (above). The crossed entorhinal efferents to the fascia dentata terminate in the outer two-thirds of the stratum moleculare in a laminar fashion, corresponding to the lamination of the ipsilateral entorhinal efferents: the medial entorhinal cortex projects to the middle one-third of the stratum moleculare, and 39 the lateral entorhinal cortex projects to the outer one-third of the stratum moleculare (Steward, 1976). The entorhinal projection to the contralateral fascia dentata innervates the lower blade more intensively than i t does the upper blade (Goldowitz et a l , 1975; Steward, 1976). The commissural fibres from the hilus terminate in the lower one-third of the stratum moleculare, proximal to the terminal f i e l d of the lateral entorhinal cortex (Blackstad, 1956; Raisman et a l , 1965; Laatsch and Cowan, 1967; Gottlieb and Cowan, 1973; Lynch et a_l, 1976; Hjorth-Simonsen and Laurberg, 1977; Fricke and Cowan, 1978). The terminals are found on dendritic spines and shafts of what are presumed to be granule cells (Blackstad et a l , 1965, 1967, 1970; Alksne et a l , 1966; Laatsch and Cowan, 1966,1967). These decussating fibres are not distributed uniformly in the fascia dentata: the terminal density in the septal region is greater than the terminal density in the temporal region (Raisman e_t a_l, 1965; Gottlieb and Cowan, 1973; Mosko et a_l, 1973; Hjorth-Simonsen and Laurberg, 1977; Swanson et a l , 1978; Laurberg, 1979). The comparative densities of the commissural terminals in the upper and lower blades of the fascia dentata were described earlier. 1.4.4 The Course Of Interhippocampal Fibres 40 The ipsilateral path that the interhippocampal fibres follow before they enter the fimbria has not been established definitively (figure 5). Gottlieb and Cowan (1973), using tr i t i a t e d leucine as a marker, reported that commissural fibres moved rostrally and laterally through the stratum radiatum of the regio inferior before they entered the alveus. Swanson and Cowan (1977) found that the commissural fibres passed in the rostral direction through the stratum oriens of the regio inferior, and did not enter the alveus until they were near the septal pole of the hippocampus. Laurberg and Sorensen (1981) claimed that the commissural fibres entered the alveus immediately and then moved towards the septal pole of the hippocampal. formation before entering the fimbria. The commissural fibres enter the contralateral fimbria adjacent to the stratum oriens of the regio inferior (Gottlieb and Cowan, 1973; Laurberg, 1979). These fibres break up into several groups in the contralateral hemisphere. Two of them innervate the stratum oriens and the stratum radiatum of the regio superior (above). A third group of fibres enters the septal portion of the alveus between the fimbria and the lateral edge of the lower blade of the fascia dentata, and then travels in the temporal direction. Fibres leave this bundle and enter the stratum oriens of the regio inferior before going into the hilus. Some of these fibres penetrate the layer of granule cells and synapse in the stratum moleculare (Gottlieb and Cowan, 1973; 41 Swanson et a l , 1978). The fourth group of fibres also moves in the temporal direction through the stratum oriens of the regio inferior, but innervates the fascia dentata by passing around the lateral edge of the upper blade and extending around the stratum moleculare to the medial extreme of the fascia dentata, and then bending around the lower blade (Gottlieb and Cowan, 1973; Swanson et a l , 1978). 1.5 THE PHYSIOLOGY OF THE PERFORANT PATH INPUT TO THE FASCIA DENTATA 1.5.1 Single Unit Responses A principal neuron of the hippocampal formation typically responds to the stimulation of an intrinsic input with a single impulse followed by a relatively long period of inhibition of spontaneous activity. This sequence of excitation-inhibition has been documented in the entorhinal cortex and subiculum (Finch and Babb, 1980a and 1980b), hippocampus (Spencer and Kandel, 1961; Andersen e_t a_l, 1964a and 1964b; MacVicar and Dudek, 1980), and the fascia dentata (Andersen e_t §_1, 1966; Lomo, 1968, 1971; Dudek et a l , 1975; Assaf, 1978). Excitation of a c e l l corresponded with an EPSP recorded with an intracellular electrode (Andersen et a l , • 1964a; Andersen et a_l, 1966; Dudek et a l , 1975), and the period of inhibition corresponded with an 42 IPSP recorded with an intracellular electrode (Andersen et a l , 1966; Lomo, 1968). Dudek et a l (1975) failed to see IPSPs in evoked granule c e l l s , but that may have been due to their use of KCl-fi l l e d electrodes for their intracellular recording (Dichter, 1973). The activation-inhibition sequence is probably an example of recurrent inhibition caused by the pyramidal cell-basket c e l l , and granule cell-basket c e l l reciprocal interactions (Spencer and Kandel, 1961; Andersen e_t a l , 1964a; Andersen et a l , 1966; Lomo, 1971). It occurred following the antidromic stimulation of pyramidal cells and granule cells (Kandel and Spencer, 1961; Lomo, 1968). The basket cells probably use the putative inhibitory transmitter gamma-amino butyric acid - GABA (Storm-Mathieson, 1977). Interneurons have, been located which fir e repetetively for the duration of the recorded IPSP in the hippocampus (Andersen et a l , 1967; Finch and Babb,l977). The divergence of the basket c e l l axons in the stratum pyramidale and the stratum granulosum would cause inhibition in the neurons bordering the evoked c e l l s . Neurons recorded in the areas of 'surround inhibition' would show an IPSP without a preceding EPSP (Spencer and Kandel, 1961). Basket cells in the fascia dentata have axons that spread 1mm in the longtitudinal direction on each side of the soma (Struble et a_l, 1978) Lomo (1968) found that when a narrow transverse strip of granule cells was activated by a perforant path stimulus, the inhibition was maximum at a distance of 0.5mm on either side of the evoked c e l l s . 43 Neural circuits causing recurrent excitation have been proposed for the hippocampus. They may involve collateral fibres of pyramidal cells that contact pyramidal cells directly (Lorente de No, 1934; Lebovitz, 1971; Livingstone, 1980; MacVicar and Dudek, 1980); or indirectly through excitatory interneurons (MacVicar and Dudek, 1980). Recurrent excitation has been reported in the fascia dentata of cats following activation of the perforant path fibres (Habets et a l , 1980a). Andersen et al (1967) postulated an inhibitory feedforward circuit in the hippocampus but the evidence was not compelling. Feedforward inhibition may exist in the area dentata where the basket c e l l dendrites extend into the stratum moleculare. Entorhinal, septal, commissural, and associational efferents could make direct contact with inhibitory interneurons. Lomo (1968) found no evidence of feedforward inhibition in the fascia dentata following a perforant path stimulus. Habets et al (1980a) did not find any evidence of feedforward inhibition in the fascia dentata of the cat following stimulation of the lateral entorhinal cortex. 44 1.5.2 Population Field Potentials a. Introduction Neural activity presupposes the movement of electrical charges across the neural membrane. The movement of a charge through a resistance such as a membrane or through the extracellular medium involves a loss of electrical potential. This voltage drop can be measured by a sensitive voltmeter. When a neural membrane is depolarized, positive charges carried by ions such as sodium pass through the depolarized membrane into the intracellular compartment, creating a net negative charge in the extracellular space. The positive charge moves through the intracellular compartment to a part of the membrane that has not been depolarized, and i t passes into the extracellular space in the form of positive ions such as sodium (Llinas and Nicholson, 1974). This creates a net positive charge in the extracellular space outside of the non-depolarized membrane that surrounds the depolarized areas. When a patch of membrane is hyperpolarized a positive charge is created in the extracellular space adjacent to the affected membrane by the release of positive potassium ions from the c e l l , or by the movement of negative chloride ions into the c e l l . Peripheral to this a negative potential is created in the extracellular space where positive charges move into the c e l l . A region of membrane that is the site of the movement of 45 positive charges into the neuron is known as a current sink; and a region of membrane that is the site of the movement of positive charges out of the neuron is known as a current source. An EPSP creates an active sink in the postsynaptic membrane and a passive source around i t ; whereas, an IPSP creates an active current source in the hyperpolarized membrane and a passive sink around i t (Llinas and Nicholson, 1974). Field potentials are additive: potentials of equal but opposite sign will cancel, and those of the same sign will sum algebraically. A number of synchronously active current generators in close proximity with their sinks and sources uniformly aligned will create large f i e l d potentials. The f i e l d potentials of randomly scattered current generators will cancel one-another. Field potential analysis is done with a recording electrode that is lowered through the presumed current generators while the evoked potentials are measured at regular spatial intervals. If the current generators are arranged in a straight row or a flat sheet with the sinks and sources aligned, a sink is presumed to . exist where the highest amplitude negative wave is recorded; and a source is presumed to exist where the highest amplitude of a positive wave is recorded. These assumptions do not hold for a curvilinear array of generators (Lorente de No, 1953; Nicholson, and Llinas, 1974; Klee and Rail, 1977). The onset of the. sink and source generated by the same input should be simultaneous, but the shape of the f i e l d -46 potentials created w i l l not necessarily be mirror-images because the configuration of the potential f i e l d is affected by the geometry of the active neurons (Gloor, 1963; Nicholson and Llinas, 1974). Field potentials can be measured at a distance from their sinks and sources, and, because f i e l d potentials sum algebraically, large distant generators can mask small local ones. Consequently, the identification of a region of maximum negative potential or positive potential on a single recording track does not necessarilly mean that a local sink or source has been found. Several parallel penetrations may be required to localize a current generator (Llinas and Nicholson, 1974; Klee and Rail, 1977; Leung, 1979c). When a sink or source has been localized, i t must s t i l l be determined whether i t is active or passive in order to identify i t as as EPSP or an IPSP. Field potential analysis must be combined with a detailed knowledge of the anatomy and the physiology of the system under investigation before any definitive conclusions can be drawn about the nature of sinks and sources (Lorente de No, 1953; Llinas and Nicholson, 1974). b. Perforant Path Responses The hippocampal formation is p a r t i c u l a r l y amenable to fi e l d potential analysis because its principal neurons are organized into compact sheets with their axons on one side of 47 the somata, and their dendrites uniformly oriented on the other side. The afferent terminals from different sources are usually found in seperate layers on the dendrites. Laminar f i e l d -potential analyses of the hippocampal formation following the stimulation of various afferents have been made by several laboratories (reviewed in Andersen, 1975; Lopes da Silva, 1978). The field-potentials evoked in the fascia dentata following stimulation of the perforant path will be presented as an example. This data was f i r s t produced in the rabbit (Lomo, 1971), and has been verified in the rat (Assaf, 1978). When a recording electrode was lowered through the hippocampal fissure as the medial perforant path fibres were stimulated, a short-latency negative wave was recorded (figure 9a). This wave had its maximum amplitude in the perforant path terminal zone in the middle one-third of the stratum moleculare. It corresponded with an EPSP recorded in a granule c e l l with an intracellular electrode. This negative wave represented synchronously-evoked EPSPs in the stratum moleculare. It decreased in amplitude as the recording electrode was moved ventrally through the stratum moleculare, and i t reversed polarity just above the stratum granulosum. When the cerebral cortex and the over-lying regio superior were removed, the negativity also reversed polarity in the outer one-third of the stratum moleculare. A small, triphasic (positive-negative-positive) wave was sometimes found in the perforant path terminal zone in the 48 stratum moleculare. It could follow a short stimulus train of 320Hz, and i t was resistant to anoxia (Lomo, 1971). This wave represented the presynaptic volley in the perforant path fibres. At high stimulus intensities, a negative wave appeared on the positivity in the stratum granulosum and the hilus. This wave had its maximum amplitude in the stratum granulosum, and i t corresponded with the discharges of single units in the stratum granulosum. It was caused by the synchronous discharge of a large number of granule cel l s , and is referred to as the granule c e l l population spike. A long-latency positive wave was recorded in the stratum granulosum. This corresponded in latency with an intracellular IPSP found in granule cells (Lomo, 1968, 1971). 1.6 PHYSIOLOGY OF THE COMMISSURAL INPUT TO THE FASCIA DENTATA 1.6.1 Single Unit Responses The most thorough analysis of the responses in the fascia dentata evoked by commissural stimuli was made by Deadwyler et al (1975). Spontaneously active granule cells that were evoked by a commissural stimulus were subsequently inhibited for up to one second. This was ascribed to recurrent inhibition due to basket c e l l activity. Evoked granule cells did not become 49 synchronized in a population spike even at high stimulus intensities (Deadwyler et a_l, 1975; Buzsaki and Czeh, 1981). This was thought to be a result of either the relatively sparse innervation of the fascia dentata by the commissural system (Deadwyler et a l , 1975), or the operation of feed-forward inhibition in the commissural input to the fascia dentata (Buzsaki and Czeh, 1981). 1.6.2 Population Field Potentials Stimulation of the contralateral hippocampal input evoked a negative wave in the stratum moleculare. The maximum amplitude of this wave occurred in the commissural terminal zone in the proximal one-third of the granule c e l l dendrites. Single units in the stratum granulosum were evoked after the onset of the negative wave, suggesting that i t was an active sink evoked in the stratum moleculare by the commissural stimulus (Deadwyler et a l , 1975). At the granule c e l l layer, this negativity was succeeded by a positive wave with a similar onset latency. The i n i t i a l part of this wave was thought to be the passive sink of the active source in the stratum moleculare. The later part of this wave was thought to be the active source evoked in the stratum granulosum by inhibitory basket c e l l activity. 50 1.7 THE PRESENT STUDY Electrophysiological investigations of the commissural input to the fascia dentata have raised several questions which were the incentive for the present study. Granule c e l l single unit responses to commissural stimuli have not been definitively characterized. Cragg and Hamlyn (1957), and von Euler et al (1958) were able to evoke granule cells by stimulating the fimbria, but Gloor e_t al (1963) were unable to evoke a response in the fascia dentata with a fimbrial stimulus. Deadwyler e_t al (1975) found that a commissural stimulus caused a pattern of excitation-inhibition in granule c e l l s . McNaughton e_t §_1 (1978) found that the response to a commissural stimulus in the fascia dentata was purely inhibitory, and they proposed that commissural afferents terminated on dentate basket cells rather than on granule c e l l s . Assaf (1978), Buzsaki and Czeh (1981), and Buzsaki and Eidelberg (1981) have reported that the predominant response of granule cells to commissural stimuli was inhibition without prior excitation, although a minority of cells responded with excitation followed by inhibition. The relatively low density of commissural terminals on granule cells has been given as the explanation of the absence of a commissural-granule c e l l population spike; whereas, the higher density perforant path innervation of the fascia dentata 51 enables a perforant path stimulus to evoke a granule c e l l population spike (Deadwyler et aJ., 1975). The commissural fibres terminate on efficient shaft and spine synapses, but perforant path fibres terminate on less effective long, thin spine synapses (Nafstad,1967; Diamond et a l , 1970; Rail, 1970). In addition, the more proximal location of the commissural synapses should result in less attenuation of the EPSP (Rail, 1970). These two factors might compensate for the lower commissural terminal density. The inability of Deadwyler et a_l ( 1975) to evoke a commissural-granule c e l l population spike might have been due to the inappropriate location of their stimulating electrode. Their stimulus co-ordinates and their figure (Deadwyler e_t a_l, 1975, figure 1) locate their stimulating electrode in the lateral part of the regio inferior, but anatomical investigation has demonstrated that the commissural system projecting to the fascia dentata originates entirely within the hilus (above). A stimulating electrode located in the lateral regio inferior might excite hilar fibres that pass near this location on the way to the fimbria (figure 5), but that may not be the optimal site for evoking a response in the fascia dentata. An electrode in or near the hilar region might provide more effective stimulus for evoking a granule c e l l population spike. Deadwyler et al (1975) described the i n i t i a l part of the commissurally-ev.oked positive wave in the stratum granulosum as the passive source of the active sink that was evoked in the stratum moleculare. However, they said that the positivity began 52 between 0.5msec and 1.0msec after the onset of the negativity. If the positive wave were generated by the active sink, i t s onset latency should have been the same as that of the negative wave (Gloor et a l , 1963; Llinas and Nicholson, 1974). If Deadwyler et al (1975) were stimulating the lateral regio inferior (above), then they would probably evoke a significant response in the contralateral regio inferior because this is one of the projection fields of CA3. Responses evoked in the regio inferior might be recorded in the fascia dentata, and they would obscure the locally generated waves. Afferent and efferent commissural fibres are found in the hilus, alveus, fimbria and ventral psalterium (figure 6). Stimulation of any of these structures would evoke an orthodromic response in the fascia dentata, and both orthodromic and antidromic responses in the hilar region. Deadwyler e_t a_l (1975) claimed that they found no antidromic responses in the hilus at the stimulus intensities used to evoke responses in the fascia dentata. They did find two classes of unit responses distinguished by their latencies which they localized in the stratum granulosum. One group of responses had an average latency of 2.0msec, and the other group had latencies that ranged from 3.0 to 5.0msec. The earlier responses occurred o.5msec prior to the onset of the negativity of the population EPSP. These unit responses followed the commissural presynaptic volley in the stratum moleculare by 0.2msec, so they would not have been axonal spikes in the commissural fibres that pass through the stratum granulosum on the way to the stratum 53 moleculare. These short latency spikes may have been hilar unit responses that were volume-conducted to the stratum granulosum, or they may have been responses in the hilus adjacent to the stratum granulosum that were mistakenly identified as occuring in the stratum granulosum. Population f i e l d potentials and unit responses in the hilar region have not yet been examined very thoroughly. Buzsaki and Czeh (1981) found short latency antidromic responses in the hilus just beneath the stratum granulosum when they stimulated the commissural input. Cragg and Hamlyn (1957) and Andersen (1959, 1960) recorded a short-latency, high amplitude negative wave in the hilar region when the commissural afferents were stimulated. It was preceded by a. low amplitude negativity, and followed by a high amplitude, long duration positive wave. The characteristics of these waves have not been elaborated.. The hilus is the source of projections to both the ipsilateral and contralateral fascia dentatae (above). The suggestion has been made that both systems might originate in the same cel l s (Gottlieb and Cowan, 1973; Swanson et a_l, 1978; West et a l , 1979). Lorente de No (1934) illustrated a hilar neuron that had an axon projecting to the fimbria, and a collateral branch pentrating the ipsilateral stratum granulosum (his figure 9, c e l l #22). Swanson et al (1980) , using double retrograde labelling of single neurons, showed that single hippocampal neurons in the regio inferior had associational, commissural, and septal collaterals. Laurberg and Sorensen (1981) used double retrograde labelling of single neurons to 54 show that single hilar neurons innervated both the ipsilateral and contralateral fascia dentatae. A stimulating electrode in the hilus would orthodromically activate efferent fibres to the contralateral fascia dentata, and i t would antidromically activate commissural fibres afferent to the stimulating electrode (figure 7 ) . The antidromic stimulus would invade the somata of the cells of origin in the contralateral hilus, and i t would also travel up collateral axons that innervate the ipsilateral stratum moleculare. The negativity found in the stratum moleculare would be a composite of the EPSPs generated by the direct orthodromic projection, and those generated by the antidromic stimulation of the association collateral. The present study was undertaken to c l a r i f y and resolve the aforementioned details. The following points were addressed: 1. How does a granule c e l l respond to a commissural stimulus? 2. Is there a commissurally-evoked granule c e l l population spike? 3. Demonstrate the commissural sink and source in the fascia dentata. 4. Characterize the commissural responses in the hilar region. 5. Distinguish the commissural and associational responses in the stratum moleculare. FIGURE 7 Schematic Diagram Of The Projections Of The  Commissural System That Innervate The Area Dentata. FD fascia dentata R recording electrode S stimulating electrode VP ventral psalterium orthodromic component antidromic component 57 2 METHODS 2.1 SURGICAL PROCEDURES Acute recording experiments were performed on male Wistar rats weighing 150 to 350 grams. They were anaesthetized with urethane (1.2 to 1.5 mg/kg i.p.). Body temperature was maintained at a temperature of 37° C. using a heating pad connected to a temperature regulator (EKEG Electronics) and a rectal probe. The animals were fixed in a stereotaxic frame with the incisor bar set at 4.2mm below zero. Skull flaps were removed in order to expose the underlying cortex for ease of manipulating the stimulating and recording electrodes. The flaps extended from bregma to 9.0mm posterior to bregma, and from the midline to 5.0mm lateral to the midline, exposing the medial entorhinal cortex, dorsal hippocampal formation, and ventral psalterium of one side of the brain. A flap was also removed on the contralateral side, extending from 2.0mm posterior to bregma to 4.0mm posterior to bregma, and from the midline to 4.0 mm lateral to the midline; exposing the contralateral dorsal hippocampal formation. The dura was then removed and the cortex was covered with 0.9%M saline that was warmed to 37°C. 58 2.2 STIMULATION AND RECORDING Concentric bipolar stainless steel electrodes with a tip separation of 0.3mm or 0.5mm were used for electrical stimulation. Square-wave pulses of 0.1msec duration were delivered through a stimulus isolation unit at a frequency of 0.5Hz. The stimuli were triggered by a Digitimer D4030 stimulus programmer. Extracellular recording was carried out with glass micropipettes that had been pulled to a -fine tip and then broken-back to an outside diameter of 2.0 microns. They were f i l l e d with a 4.0M solution of NaCl, and pipettes having a resistance of 2.0 to 4.0 megohms were selected for use. Some electrodes were f i l l e d with the dye Pontamine sky blue for the marking of recording sites. The stimulating and recording electrodes were positioned according to the co-ordinates given in Table II. The best position for a stimulating electrode to evoke a response in the contralateral area dentata was found by using the procedure described below. A stimulating electrode was lowered into the medial entorhinal cortex. Then a recording electrode was placed in the perforant path terminal zone of the fascia dentata using the ipsilateral perforant path evoked responses as a guide. The recording electrode was then lowered 100 microns in order to reach the probable location of the commissural terminal zone 59. TABLE I I . STEREOTAXIC COORDINATES OF STIMULATING AND RECORDING PLACEMENTS. PLANE STIMULATING ANTERIOR MEDIO- DORSO-SITE POSTERIOR LATERAL VENTRAL Entorhinal 8.1 mm 4.3mm 2.5mm cortex Commissural 3.5 2.5 3.5 Ventral 1.5 1.0 3.0 psalterium RECORDING SITE Area Dentata 3.5 1.5-2.5 3.0-4.0 60 (Deadwyler et a l , 1975). A stimulating electrode was then lowered into the contralateral area dentata and adjusted until the evoked response showed a maximum negativity (the MN2 wave of figure 9). An example of a good stimulating site is shown in figure 8. When the stimulating site was found, an evoked response profile was constructed by stimulating the contralateral hilar region and recording the commissural responses at different depths in the area dentata. 2.3 DATA ANALYSIS The electrical responses were fed to a custom-built preamplifier and then to an amplifier before being displayed on an oscilloscope (Tektronix D-44). Population f i e l d potentials were averaged on a PDP 11/10 computer. Single c e l l responses were sent to a custom-built amplitude discriminator, and then relayed to the PDP 11/10 for rasters and post-stimulus histograms. The PDP 11/10 displayed the data on a visual terminal (Tektronix 4010) and permanent records were transcribed on an interactive digital plotter (Tektronix 4662). FIGURE 8 Histological Localization Of The Stimulating Electrode. The dark spot on the slice is an iron deposit made at the tip of the stimulating electrode. 62 63 2.4 HISTOLOGY .Stimulating sites were marked by passing an anodal current (15 to 20 microamperes for 20 seconds) through the central tip of the stimulating electrode and then perfusing the animals intracardially with a solution of potassium ferrocyanide-formalin. This left a blue deposit on the tissue surrounding the electrode (figure 8). Recording sites were identified by ejecting Pontamine sky blue from the recording electrode with a 10 microampere current for 10 to 15 minutes. At the end of each experiment, the rats were deeply anaesthetized with an additional injection of urethane, and they were then perfused intracardially with normal saline and then with a potassium ferrocyanide-formalin solution. The brain was then removed and le f t overnight in a formalin solution for fixation. The brain was cut into 50 micron thick coronal sections, and stained with either Thionine or neutral red. 2.5 MECHANICAL LESIONS The rats were anaesthetized with sodium pentobarbital (50 mg/kg i.p.) and positioned in a stereotaxic frame with the 64 incisor bar set 4.2mm below zero. A hole was dr i l l e d in the cranium from bregma to a point 3.0mm posterior to bregma. The hole was 1.0mm wide and centred 1.0mm lateral to the midline. An incision was then made in the dura. Lesions were made with a knife blade that was designed to f i t into a Kopf stereotaxic blade holder. To make lesions of the commissural fibres in the ventral psalterium, the blade was stereotaxically positioned 0.5mm posterior to bregma and 0.5mm to 1.0mm lateral to the midline. The blade was lowered 4.0mm below the cortical surface, and then moved 2.5mm caudally, severing the fibre tract. The septo-hippocampal fibres were severed with a knife with a hinged blade that was attached to a Kopf stereotaxic electrode holder. The hinge allowed the knife to rotate in the coronal plane. The blade was inserted into the hole d r i l l e d for the ventral psalterium lesion at a point 0.5mm posterior to bregma, and 1.0mm lateral to the midline. The blade was set at a 60° angle to the horizontal plane, and lowered below the cortex for 6.0mm along this angle. The knife was then rotated just past a line perpendicular to the surface of the cortex. Acute recordings were made 7-14 days after the lesion was made. 65 2.6 CHEMICAL LESIONS Unilateral lesions of the regio inferior were made with kainic acid. The rats were anaesthetized with sodium pentobarbital (50mg/kg i.p.), and a burr-hole was dr i l l e d in the cranium at the following stereotaxic co-ordinates (incisor bar set at 4.2mm below zero): 1.5mm posterior to bregma and 1.2mm lateral to the midline. A Hamilton syringe was inserted into the hole to a depth of 4.5mm below the surface of the cortex, and 0.4 microlitres of kainic acid were injected into the lateral ventricle over a period of 30 minutes. Acute experiments were performed • 7-14 days later. The extent of the kainic acid lesion was assessed histologically following the acute experiments. 66 3 RESULTS 3.1 POPULATION FIELD POTENTIALS 3.1.1 Introduction Stimulation of the commissural input evoked six identifiable population responses in the area dentata. These were: the MN1 and MN2 waves of the stratum moleculare of the area dentata (figure 9); and the HN1, HN2, HN3, and P waves of the hilar region (figure 9). 3.1.2 The Molecular Layer The MN1 wave was a small amplitude, short-latency waveform that was capable of following a short stimulus train of 200Hz. This wave probably represented the presynaptic fibre volley in the commissural afferents to the fascia dentata (Deadwyler et a l , 1975) . The MN2 wave reached i t s maximum amplitude in the stratum moleculare, between 50 and 100 microns above the stratum granulosum, and approximately 100 microns below the perforant path population EPSP maximum response. It had an average onset latency of 3.1msec, and i t would not follow a short 200Hz FIGURE 9 Identified Waveforms Of The Area Dentata. (a) Laminar field-potential analysis of the perforant path evoked responses. (b) Schematic diagram of the area dentata showing the track of the recording electrode in (a) and (c). The depth in microns below the hippocampal fissure is indicated. R recording electrode track r . i . regio inferior s.g. stratum granulosum (c) Commissural responses. Elements of the complex waveform are identified by the symbols listed below. + MN1 MN2 HN1 HN2 HN3 68 69 stimulus train. It was probably the response identified by Deadwyler et al (1975), and Assaf and Miller (1981) as the commissurally-evoked population EPSP in the fascia dentata. When the recording electrode was moved closer to the stratum granulosum, the MN2 wave diminished in amplitude (figure 9). A negative wave could be evoked in the commissural zone of the stratum moleculare by a stimulating electrode that was located in the stratum radiatum of the contralateral regio superior. As the stimulating electrode was lowered towards the fascia dentata, this negativity gradually decreased in amplitude, and the MN2 began to appear. The latency of the MN2 wave was shorter than the stratum radiatum evoked negativity. 3.1.3 Hilar Region The HN1 wave had a short latency, small amplitude, short duration, and was capable of following a short stimulus train of 200Hz. It was found in the apical dendrites of the pyramidal cells of the medial regio inferior (figure 9). HN2 was visible in the apical dendrites of the pyramidal cells of the medial regio inferior (figure 9). It had a slightly longer latency than the HN1 wave, and it had a small amplitude. Closer to the stratum pyramidale the latency of HN2 was shorter than i t was in the stratum radiatum and its amplitude was greater (figure 10). It followed a short, 200 Hz stimulus train, FIGURE 10 Characteristics Of The Commissurally Evoked Responses  In The Area Dentata. (a) Depth profiles of the MN2 • • and P * * waves. The amplitude of the responses was measured with the prestimulus baseline set at zero. MN2 was measured at the latency of the peak negativity (•), and P was measured at the latency of the peak positivity (*). (b) Laminar profile of the commissural responses. The depth below the hippocampal fissure in microns is indicated adjacent to the response. (c) Schematic illustration of a granule c e l l (above) and a pyramidal c e l l (below) at the indicated depth below the hippocampal fissure. (d) Latency-amplitude characteristics of HN2. The solid line shows the amplitude of HN2 at the depth indicated on the vertical axis. The dotted line shows the latency of the peak amplitude of HN2 at the appropriate depth. 71 3.5 msec 72 b u t w i t h a s u b s t a n t i a l d e c r e m e n t i n a m p l i t u d e . I n t h e s t r a t u m o r i e n s t h e a m p l i t u d e o f HN2 was s m a l l e r a n d i t s l a t e n c y was s h o r t e r ( f i g u r e 1 0 ) . I t f o l l o w e d a s h o r t , 200Hz s t i m u l u s t r a i n w i t h no l o s s o f a m p l i t u d e . The HN3 wave was r e c o r d e d i n t h e h i l a r r e g i o n ( f i g u r e 9 ) . I t s l a t e n c y was a p p r o x i m a t e l y 3.0 t o 3.5msec. I t d i d n o t show any a m p l i t u d e f a c i l l i t a t i o n when t h e c o m m i s s u r a l e l e c t r o d e was s t i m u l a t e d s e q u e n t i a l l y , a n d i t was n o t a s s o c i a t e d w i t h any u n i t a r y d i s c h a r g e s . The P wave began n e a r t h e s t r a t u m g r a n u l o s u m a n d had i t s maximum a m p l i t u d e i n t h e h i l u s n e a r t h e s t r a t u m p y r a m i d a l e o f t h e r e g i o i n f e r i o r ( f i g u r e s 9 a n d 1 0 ) . The e a r l y p a r t o f t h e P wave was o b s c u r e d by t h e HN2 wave, b u t i t s o n s e t l a t e n c y a p p e a r e d t o be s i m i l a r t o t h a t o f t h e MN2 wave. When t h e a m p l i t u d e o f t h e p o s i t i v i t y was c o m p a r e d a t d i f f e r e n t l o c a t i o n s a l o n g t h e m e d i o - l a t e r a l a x i s o f t h e h i p p o c a m p a l f o r m a t i o n , i t i n c r e a s e d a s t h e r e c o r d i n g e l e c t r o d e was moved c l o s e r t o t h e s t r a t u m p y r a m i d a l e o f t h e r e g i o i n f e r i o r , b u t i t was n o t e v i d e n t a t a l l i n t h e most m e d i a l r e g i o n s o f t h e h i l u s ( f i g u r e 1 1 ) . A p o s i t i v e wave c o u l d be r e c o r d e d t h r o u g h o u t t h e a r e a d e n t a t a when a s t i m u l a t i n g e l e c t r o d e was l o c a t e d i n t h e s t r a t u m o r i e n s o f t h e t h e c o n t r a l a t e r a l r e g i o s u p e r i o r . T h i s wave h a d i t s peak a m p l i t u d e i n t h e h i p p o c a m p a l r e g i o n . T h e r e was no c l e a r e v i d e n c e i n d i c a t i n g t h a t t h e r e was a p a s s i v e c u r r e n t s o u r c e i n t h e s t r a t u m g r a n u l o s u m when t h e c o m m i s s u r a l i n p u t was s t i m u l a t e d . FIGURE 11 Localization Of The Commissurally-evoked P-wave. (a) Schematic illustration of the area dentata showing the placement of the recording electrode in 11b. r . i . regio inferior s.g. stratum granulosum (b) Commissurally-evoked responses recorded at the locations shown in 11a. The HN2 wave diminishes and the P wave disappears as the recording electrode is moved medial to the regio inferior. 74 75 3.2 UNIT RESPONSES 3.2.1 Stratum Granulosum The latencies of granule c e l l responses to commissural and perforant path stimuli are shown in Table III and in figure 12. Neurons evoked in the stratum granulosum by a commissural stimulus were the same neurons that were evoked by a perforant path stimulus (figure 12). These granule cells had an average response latency of 4.6msec to a commissural stimulus, and 4.5msec to a perforant path stimulus. The activation of a spontaneously firi n g granule c e l l by a commissural stimulus was invariably followed by a long period of quiescence. When the commissural and perforant path stimuli were compared at stimulus intensities that were just adequate to evoke a granule c e l l with 75% of the stimuli, the perforant path stimulus inhibited the spontaneous activity of the granule c e l l for 50 to 150msec; but the commissural stimulus inhibited the granule c e l l for 150 to 250msec (figure 13). Spontaneously active granule cells were never inhibited by a commissural stimulus without prior activation. Increasing the intensity of a perforant path stimulus can increase the synchrony of the discharges of a population of granule c e l l s , and create a population spike (Lomo, 1971). A granule c e l l population spike was never evoked by a commissural TABLE I I I . LATENCIES OF UNIT RESPONSES R e c o r d i n g S i t e St i m u l a t i o n S i t e Number o f C e l l s T e s t e d Mean L a t e n c y (msec) Commissural a n t i d r o m i c 22 2 .7 H i l a r R e g i o n Commissural o r t h o d r o m i c 28 3 .7 P e r f o r a n t P a t h 35 5.8 F a s c i a D e n t a t a Commissural P e r f o r a n t P a t h 64 63 4 . 6 4 . 5 FIGURE 12 Latency Histograms Of Granule Cells Evoked By  Perforant Path And Commissural Stimuli. (a) Latency distribution of perforant path evoked granule c e l l s . (b) Latency distribution of commissurally evoked granule c e l l s . The insets show oscilloscope traces of a granule c e l l evoked by both perforantpath and commissural stimuli. FIGURE 13 The Effect Of Perforant Path And Commissural Stimuli  On The Spontaneous Discharge Of Granule Cells. (a) Post-stimulus histogram showing the characteristic activation-inhibition sequence of a granule c e l l following a perforant path stimulus .The insert shows an oscilloscope record of several super-imposed sweeps. (b) Illustration of a similar response pattern in the neuron following a commissural stimulus. Note the longer duration of the inhibition. CD > CO o 81 stimulus. A commissural stimulus could evoke a population spike in the stratum pyramidale of the regio superior and the regio inferior. As the intensity of a commissural stimulus was increased over the threshold intensity for evoking a granule c e l l , the latency of the dicharge decreased, and the neuron responded to a larger percentage of the stimuli. Above an optimal intensity, the effectiveness of a stimulus decreased, and the neuron responded to a smaller percentage of the stimuli (figure 14). 3.2.2 Hilar Region Single unit responses in the hilar region had an average latency of 3.3msec to a commissural stimulus, and 5.8msec to a perforant path stimulus (Table III and Figure 15). The hilar c e l l s evoked by a perforant path stimulus had a high threshold, and they usually occurred after the perforant path evoked granule c e l l population spike (figure 16c). Unitary responses recorded in the hilar region had the characteristics of antidromically activated ce l l s in 22 of 50 cases. These antidromic cells had a constant latency, followed a short, 200Hz stimulus train, and, when spontaneously active, demonstrated collision-extinction (figure 16a, 16b). They occasionally showed a break on the i n i t i a l positivity (figure 16a and 16d) that was similar to inflections on extracellular responses that have been identified as manifestations of an FIGURE 14 Relationship Between Commissural Stimulus Intensity  And The Probability Of Firing Of A Granule C e l l . The threshold stimulus intensity for evoking a granule c e l l has been assigned a value of 1. The x-axis has been measured in multiples of the threshold intensity. After the stimulus intensity exceeded a strength of three times the threshold, the probability of evoking a granule c e l l was reduced. FIGURE 15 Latency Histograms Of Hilar Neurons Evoked By  Commissural Stimuli. (a) Latency distribution of hilar neurons evoked by antidromic stimulation. The insert shows an oscilloscope trace of an antidromically evoked hilar neuron. (b) Orthodromic c e l l s . The insert shows an ortho-dromically evoked hilar neuron.The insert shows an oscilloscope trace of an orthodromically evoked hilar neuron. 85 A. B. Latency (msec) FIGURE 16 Identification Of An Antidromically Evoked Hilar  Neuron. (a) Commissurally-evoked hilar neuron. (b) Collision: the evoked response is blocked by the spontaneous action potential. (c) The same hilar neuron evoked by a perforant path stimulus. (d) The same neuron responding to a 200Hz stimulus. 5 msec 88 i n i t i a l segment-somato dendritic (IS-SD) break (Kandel et a l , 1961; Eccles et a l , 1966; Llinas et a l , 1969). This IS-SD break was not evident in the synaptically-evoked response (figure 16c), or in the spontaneous spike (figure 16b). The antidromic units had an average latency of 2.7msec to a commissural st imulus. Latency variability to commissural stimuli was demonstrated in 28 of 50 hilar unit responses. These ce l l s had an average latency of 3.7msec. The response latencies of these hilar orthodromic and antidromic ce l l s to commissural stimuli corresponded with the duration of the HN2 wave (2-4msec). The HN2 wave probably represents a commissurally-evoked pyramidal c e l l population spike in the regio superior that has both orthodromic and antidromic components. 3.3 LESIONS OF THE VENTRAL PSALTERIUM Lesions of the ventral psalterium were performed on several animals (n=14). These lesions caused the degeneration of the direct commissural afferents to the fascia dentata, but spared the commissural efferents (figure 17). If the ventral psalterium or the fimbria were stimulated, a recording electrode in the stratum moleculare of the fascia dentata would record an MN2 FIGURE 17 The Effect Of A Ventral Psalterium Lesion On The  Commissurally Evoked Population Field Potential. (a) Schematic illustration of the commissural system following a lesion of the ventral psalterium. The fibres distal to the lesion have degenerated, and a stimulating electrode in the ventral psalterium antidromically activates the surviving fibres that are proximal to the lesion. R recording electrode S stimulating electrode VP ventral psalterium orthodromic fibres antidromic fibres (b) The commissurally evoked MN2-wave recorded in the stratum moleculare (above), and the hilar respon-ses (below) following the lesion of the ventral psalterium. VP 91 wave only i f the antidromically stimulated neurons had efferent branches that innervated the ipsilateral fascia dentata (figure 17a). HN1 was missing in the lesioned animals, but the HN2 and P waves were both prominent (figure 17b). The MN2 wave could be generated in the commissural-association terminal zone of the stratum moleculare when the lesioned ventral psalterium was stimulated (figure 17b). Granule cells could be evoked in the stratum granulosum during the i n i t i a l phase of the MN2 wave indicating that this was probably an active sink evoked in the stratum moleculare. 3.4 KAINIC ACID LESIONS Kainic acid is a neurotoxin that is an analogue of the putative neurotransmitter glutamate. When i t is injected into the lateral ventricle i t destroys the pyramidal cells of the regio inferior ipsilateral to the injection, without causing notable damage to the other structures of the hippocampal formation (figure 18a). The removal of hilar and regio inferior cells would result in the loss of the commissural projection and the ipsilateral association system of that hilus (figure 18b). The only access to the contralateral area dentata would be through the direct commissural projection. When the intact hilus was stimulated an MN2 wave was recorded in the commissural-association terminal FIGURE 18 The Effect Of A Kainic Acid Lesion On The Commissural  Response. (C) Histological section of kainic acid-treated hippocampus showing the degeneration of the hilar and regio inferior neurons ipsilateral to the rec-ording electrode. (B) Schematic illustration of the stimulating and rec-ording electrodes in the kainic acid-treated hipp-ocampus. Note the absence of the antidromic input. (C) Responses in the stratum moleculare (above) and the hilus (below). The HN1, HN2, and P waves are missing. 93 93 (^ ) 94 zone of the contralateral fascia dentata, but no HN1, HN2, HN3, or P waves were found in the hilar region (figure 18c). 95 4 DISCUSSION 4.1 FIELD POTENTIALS The f i e l d potentials evoked in the area dentata by a commissural stimulus have six distinct waveforms: the MN1, and MN2 waves of the stratum moleculare; and the HN1, HN2, HN3, and P waves of the hilar region. 4.1.1 MN1 The MN1 wave was caused by synchronous discharges in the presynaptic fibres of the commissural projection to the stratum moleculare. The ab i l i t y of this response to follow a high frequency stimulus train, i t s short latency, and its similarity to the response identified by Deadwyler et al (1975) as a presynaptic volley, confirm this interpretation. 96 4.1.2 MN2 The MN2 wave had its maximum amplitude in the commissural-associational terminal zone of the stratum moleculare. It failed to follow a high frequency stimulus train, and the amplitude of the response was increased by a preceding stimulus applied to the commissural stimulating electrode, so i t was probably a synaptic response. The short latency of the response indicated that i t was monosynaptic. The onset of MN2 preceded the action potentials of evoked granule c e l l s , so i t was an active sink in the stratum moleculare, and indicated that the commissural fibre volley had caused a population EPSP in the granule c e l l s . This corresponds with the interpretation of Deadwyler et a_l (1975), and Assaf and Miller (1981). The fact that the MN2 wave could be evoked in rats that had kainic acid lesions, or lesions of the ventral psalterium, shows that MN2 is caused by the summation of two separate inputs: the direct commissural projection, and the indirect associational system. 4.1.3 HN1 The HN1 wave was found in the apical dendrites of the regio inferior pyramidal c e l l s . Its location in the commissural terminal zone, i t s a b i l i t y to follow a high-frequency stimulus train, and i t s absence following the removal of commissural afferents by a lesion of the ventral psalterium, indicate that 97 this response was caused by synchronous discharges in the presynaptic fibes that terminate in the stratum radiatum of the regio inferior. 4.1.4 HN2 HN2 was found in the lateral part of the hilar region. The amplitude and latency of HN2 varied as the depth of the recording electrode was changed. The latency of the wave was shortest in the stratum oriens of the regio inferior. At this depth the response was capable of following a high-frequency stimulus with no change in amplitude or latency. The latency and the amplitude of HN2 increased at the stratum pyramidale. The amplitude of the response was maximum at this depth. The latencies of single unit discharges in the stratum pyramidale coincided with the latency of HN2, so i t was the pyramidal c e l l population spike. When recorded in the stratum pyramidale, HN2 followed a brief, high frequency stimulus train with a constant latency, but with a subsantial loss of amplitude. These characteristics of HN2 were caused by antidromic action potentials that arrived in the pyramidal c e l l axons in the stratum oriens and then invaded the pyramidal c e l l somata in the stratum pyramidale. An action potential arriving at a c e l l body is delayed by the increased load presented by the larger volume of the soma (Rail and Shepherd, 1970; Llinas and Nicholson, 1974). This caused the IS-SD break seen in single units (Brock et a l , 1953; Coombs et a l , 1957), and the increase 98 in the latency of the population response when i t arrived at the stratum pyramidale. The surface area of the c e l l bodies is larger than the surface area of the axons, so a larger current can flow into the extra-cellular space, thereby, increasing the amplitude of the response (Lorente de No, 1953). The increased volume of the somatic compartment decreases the safety-factor for conduction of the response (Brock et a_l, 1 953; Coombs e_t a l , 1957). This would be the cause of the decreased amplitude of HN2 at the stratum pyramidale when a high frequency stimulus was used. Some of the loss of amplitude at high stimulus frequencies might have been due to the loss of an orthodromic component of HN2 that was unable to follow the stimulus. Single unit orhodromic and antidromic responses corresponded in latency with HN2. However, separate orthodromic and antidromic components could not be distinguished in the population response. The amplitude of HN2 decreased and the latency increased as the recording electrode was raised into the apical dendrites of the pyramidal c e l l s . This decremental response is typical of an impulse that is being conducted into thin distal dendrites (Lorente de No, 1953). Although dendritic spikes have been found in the pyramidal cells of cats, (Spencer and Kandel, 1961), and guinea pigs (Wong e_t a l , 1979), i t cannot be ascertained from this data whether or not these impulses were being conducted actively or passively into the dendrites. 99 4.1.5 HN3 The HN3 wave did not show any potentiation or inhibition by a preceding stimulus, and i t was not associated with the discharge of single c e l l s . The identity of this wave remains uncertain. 4.1.6 P The P wave was visible near the stratum granulosum. The early part of the positivity was hidden by HN2, but i t s onset latency seemed to be similar to that of MN2. Consequently, i t has been identified- as the passive source of the active sink, MN2 (Deadwyler et a l , 1975). As a f i r s t approximation, on a single recording track, the highest amplitude of a fie l d potential should be found close to the generator of that potential. The maximum amplitude of the P wave was found in the hilar region, in the stratum pyramidale of the regio inferior, not in the stratum granulosum. The identification of the P wave as a pyramidal c e l l wave is supported by its absence in the kainic acid preparations in which there were no pyramidal c e l l s . The P wave was absent in the medial hilus where there are no pyramidal c e l l s . The P wave is not the passive source of an active sink created in the'pyramidal c e l l dendrites by commissural EPSPs in the stratum radiatum because i t occurred in the rats with 100 lesions of the ventral psalterium, and they had no commissural afferents. It was also present when the contralateral regio superior was stimulated, and there is no orthodromic projection from the regio superior to the contralateral regio inferior. The P wave may have been caused by passive recovery processes in the pyramidal cells following the transmission of an antidromic action potential into the pyramidal c e l l dendrites. It seems unlikely that the P wave is caused entirely by an active source generated in the stratum pyramidale by inhibitory basket cells that synapse there, because the onset of the P wave, and the HN2 overlapped. That would not be sufficient time for activity to complete the pyramidal c e l l to basket c e l l to pyramidal c e l l c i r c u i t . 4.2 COMMISSURAL SINK-SOURCE IN THE FASCIA DENTATA The absence of a current source for a commissurally-evoked sink in the stratum moleculare (MN2) is puzzling. The non-reversing negativity occurred in a region of active granule c e l l s , because single units were evoked by a commissural stimulus in the stratum granulosum where no positivity was evident. The lack of a commissural reversal of the molecular layer positivity was illustrated in an earlier publication (Steward et a l , 1978), and predicted in a computer simulation of population f i e l d potentials of the fascia dentata (Habets et a l , 101 1978). The computer model showed that the source should appear in the distal dendrites of the granule c e l l s . However, i t also predicted that medial entorhinal cortex stimulation would not produce a source in the stratum granulosum, yet this source has been prominently and consistently evoked in the stratum granulosum with perforant path stimulation (Lomo,1971; McNaughton, 1980; Assaf and Miller, 1981). A non-reversing negativity was found in the caudate nucleus when the motor cortex was stimulated, but no explanation was offered by the authors (Blake et a l , 1976). The perforant path evoked sink in the stratum moleculare was associated with a prominent source that could be recorded in the stratum granulosum, and the hilar region. The explanation for the difference in the sink-source relationship might be found in the structural differences between the perforant path and the commissural innervations of the fascia dentata. The commisssural fibres terminate on the proximal dendrites of the granule ce l l s , but the perforant path fibres terminate on the more distal dendrites. It has been demonstrated that the perforant path sink in the middle one-third of the granule c e l l dendrites creates a large current source in the stratum granulosum, and a smaller one in the distal dendrites (Lomo, 1971). The proximal location of the commissural terminals might allow a larger proportion of the current to leave the intracellular compartment through the distal dendrites, and leave a smaller source in the stratum granulosum. Negative 1 02 currents generated in the apical dendrites of the regio superior by commissural stimulation might be large enough to be recorded in the distal dendrites of the fascia dentata. These would sum with the positive currents and cancel them (Habets et a l , 1980a). Lomo (1971) had to remove the regio superior in order to record the distal dendritic source in the upper blade of the fascia dentata when the perforant path was stimulated. The lower blade of the fascia dentata is close to the lateral ventricle. Cerebro-spinal f l u i d has a low impedance, and i t can conduct current from distant sources (Nicholson, 1981). The basal dendrites of the regio inferior are close to the lateral ventricle, and they might generate current flow that reaches the lower blade of the fascia dentata. This might mask a current source in the distal dendrites of the granule ce l l s of the lower blade. Another anatomical difference between the commissural and perforant path inputs to the area dentata that might explain the difference in the sink-source relationships, is the fact that the commissural fibres terminate in the hilus, but the perforant path fibres do not. EPSPs evoked in hilar cells by commissural stimuli would cause negative fields that might cancel positive fields generated in the stratum granulosum. Among the target the target cells that might create these fields are the basket c e l l s . Basket cells are large neurons that are found mostly in the hilus near the stratum granulosum (Amaral, 1978; Seress and Pokorny, 1981). The basket cells have large dendrites that ramify within the hilus, and project through the stratum 103 granulosum into the stratum moleculare. They would be capable of receiving a large synaptic input and might be able to generate large extracellular fields. Basket ce l l s may have survived the kainic acid treatment. They would not have been noticed because they l i e close to the stratum granulosum, and the histological stains that were used did not clearly discriminate between different types of neurons. Two experiments showed what might have been a small current source evoked in the stratum granulosum by a commissural stimulus. The positive wave had a small amplitude, and i t could not be determined from the data whether or not i t was a small locally generated f i e l d , or a large distant f i e l d . The data from these experiments show that laminar f i e l d potential analysis is a weak analytical tool. It should not be used unless its limitations are understood. The greatest weakness is the inability to distinguish between local fields and distant fields. Field potential analysis can be improved by making several parallel electrode penetrations through a structure in order to create isopotential maps to identify local fields (Llinas et a l , 1969; Llinas and Nicholson, 1974). Field potential analysis cannot be used accurately unless the anatomy of the structure under investigation is clearly understood (Llinas et a_l, 1969). Field potentials are easiest to interpret when the soma of the active cells form a flat sheet with their dendrites on one side of the soma, and their axons emerging from the opposite side. The synaptic input should be 104 restricted to one side of the soma, and no other nearby structures should be affected simultaneously by the input. This type of cellular array is what Lorente de No (1953) called an 'open f i e l d ' , and i t is ideal for the generation of a large extracellular dipole. The identification of sinks and sources becomes more d i f f i c u l t when the c e l l layer forms a sphere or a curved surface; when the dendrites are radially oriented, irregular, or multipolar; or when the synaptic activity occurs simultaneously on more than one side of the layer of somata (Lorente de No,1953; Biedenbach and Freeman, 1964; Llinas and Nicholson, 1974; Klee and Rail, 1971). Although the area dentata appears to be structurally simple, closer inspection shows that there are deviations from the ideal open f i e l d . The fascia dentata is curved; the commissural system innervates the apical and basal side of the stratum pyramidale and the stratum granulosum; and the dendritic arborization of the hilar cells and the basket cells is irregular. The best methods available for the analysis of sinks and sources is either the technique of Rail and Shepherd (1968) which incorporates the curvature of an array of generators; or current source density (CSD) analysis (Nicholson and Freeman, 1975) which eliminates the effects of distant current sources. CSD analysis has been used in the regio superior (Leung, 1979c), the perforant path input to the fascia dentata (Jeffreys, 1979; Habets et a l , 1980a), and the mossy-fibre activation of the regio inferior (Yamamoto 1972). 105 4.3 COMMISSURAL INHIBITION OF GRANULE CELLS Spontaneously active granule cells that were evoked by a commissural stimulus were inhibited for a longer period of time than granule cells that were evoked by a perforant path stimulus. If the same population of granule cells were evoked by_ both stimuli, and if the inhibition of granule cells were due entirely to recurrent inhibition, then the duration of the inhibition from both inputs should be similar. The basket cells which are thought to be responsible for the inhibition are found in the inner stratum moleculare and just below the stratum granulosum. The dendrites of these basket cells spread through the stratum moleculare and the hilus (Amaral, 1978; Seress and Pokorny, 1981); the perforant path and commissural terminal zones. This makes feedforward inhibition possible. Habets et a_l 1980a) could not find any evidence of feedforward inhibition in the perforant path input to the fascia dentata of the cat. Commissural feedforward inhibition combined with recurrent inhibition might be the cause of the prolonged commissural inhibition of the granule c e l l s . Buzsaki and Eidelberg (1981) found cells in the area dentata that fired repetetively at short latency to a commissural stimulus. They called these cells inhibitory interneurons, but there was nothing in their data that would lead one to believe that they were inhibitory rather than excitatory. 106 The commissural stimulus also causes orthodromic and antidromic activation of hilar neurons. If these hilar neurons make synaptic contact with the dentate basket c e l l s , then they would add to the commissural stimulation of the inhibitory c e l l s . 4.4 ABSENCE OF A COMMISSURAL GRANULE CELL POPULATION SPIKE The inability of a commissural stimulus to evoke a granule c e l l population spike cannot be readily explained. The presence of a population spike indicates that a large number of neurons have been activated, and that they have fired synchronously (Andersen et a l , 1971a). The commissural and perforant path systems seem to evoke a similar population of granule c e l l s , so if there are a sufficient number of cells to evoke a perforant path granule c e l l population spike, then there should also be enough cells to evoke a commissural granule c e l l population spike. The range of latencies of the evoked granule cells to commissural and perforant path stimuli, below the stimulus threshold intensity of the perforant path population spike, was similar; so the granule cells seem to have a similar degree of synchrony of discharge to both inputs. Increasing the intensity of the perforant path stimulus evoked a population spike. When the intensity of the commissural stimulus was raised beyond an optimal level, a granule c e l l was evoked less frequently. 107 Increasing the intensity of the commissural stimulus inhibited granule cells and prevented a population spike. Buzsacki and Eidelberg (1981) suggested that feedforward inhibition might be the reason why there is no commissural granule c e l l population spike. A commissural stimulus did evoke population spikes in the regio superior and the regio inferior. 4.5 TERMINAL FIELDS OF THE HILAR NEURONS Anatomical evidence has shown that the same hilar neurons innervate the ipsilateral and contralateral fascia dentata. This was verified electrophysiological^ with the ventral psalterium lesions. There is a dispute over whether or not the same hilar neurons also project to the ipsilateral • and contralateral hippocampus. The negative wave that was evoked in the fascia dentata by a stimulating electrode in the contralateral regio superior shows that the commissural neurons do innervate the ipsilateral or the contralateral hippocampus, or both. The regio superior has no projection to the area dentata, and the only input to the fascia dentata is from hilar neurons. Consequently, that negativity must be caused either by the antidromic stimulation of a hilar efferent to the regio superior, orthodromic invasion of efferent collaterals to the contralateral fascia dentata (figure 19a); or by the antidromic FIGURE 19 Hilar Input To The Contralateral Regio Superior. A stimulating electrode in the regio superior can evoke poplation f i e l d responses and single granule cells in the fascia dentata. This can only occur through either (or both) of the pathways illustrated here. R S recording electrode stimulating electrode 110 stimulation of a commissural projection to the regio superior, followed by the orthodromic invasion of a collateral efferent to the ipsilateral fascia dentata (figure 19b). This supports the anatomical data of Swanson and Cowan (1977). 111 5 SUMMARY Stimulation of the hilar region evoked six different waveforms in the contralateral area dentata. Two of these, the MN1 and MN2 waves, were associated with the commissural activation of granule c e l l s . Three waves were caused by activity in the regio inferior: HN1, HN2, and P. HN3 was found in the hilar region, but i t was not associated with the discharge of any neurons and its identity remains unknown. MN2 was the extracellular sign of a granule c e l l population EPSP, caused by the simultaneous activation of both commissural and association inputs to the area dentata. MN1 was the presynaptic discharge in the commissural fibres afferent to the fascia dentata. The subsequent discharge of single cells in the granule c e l l layer at short latency means that the commissural projection to the fascia dentata was excitatory rather than inhibitory. However, the long duration of the inhibition that followed the excitation of granule c e l l s , and the absence of a commissural granule c e l l population spike, may have been due to the concurrent activation of granule cells and inhibitory basket c e l l s . HN2 was a pyramidal c e l l population spike in the regio inferior caused by the orthodromic and antidromic activation of pyramidal cells by a commissural stimulus. The changes in the latency and amplitude of HN2 at different depths of the hilar 1 12 region were characteristic of the antidromic propagation of an impulse through a population of c e l l s . HN1 was the presynaptic volley in the commissural fibres afferent to the regio inferior. The P wave was caused by activity in the regio inferior. Its identity is uncertain, but i t may have been due to passive recovery processes that followed the antidromic propagation of the impulses that constituted HN2. This work has emphasized the importance of a careful interpretation of f i e l d potential data. The P wave can easily be mistaken for a commissurally-evoked current source in a population of granule cells because its latency is similar to what one would expect for -the reversal of MN2. The close proximity of the fascia dentata and the regio inferior make i t easy to misinterpret the source of f i e l d potentials. It was probably the concurrent generation of f i e l d potentials by the commissural stimulus in other areas that masked the current source in the fascia dentata. The HN4 wave (see Appendix) could be misinterpreted as a granule c e l l population spike because it could sometimes be recorded near the stratum granulosum, and its latency was similar to what would be anticipated for a granule c e l l population spike. Hilar neurons innervate the ipsilateral and contralateral granule cells (association and commissural projections), and the ipsilateral and/or contralateral regio superior. Single cells may project to a l l fields of the hippocampal formation. The connections of the hilar neurons are summarized in figure 20. FIGURE 20 C o n n e c t i o n s Of The H i l a r Commissural Neurons. B b a s k e t c e l l G g r a n u l e c e l l H h i l a r c o m m i s s u r a l neuron to regio superior to regio inferior contralateral hippocampus from contralateral hilus 1 15 Data described in this paper confirmed that single hilar projection cells innervate both the ipsilateral and contralateral area dentata. There was additional evidence which demonstrated that the neurons innervating the fascia dentata also project to the ipsilateral and/or contralateral regio superior. This electrophysiological data, combined with the anatomical evidence that the hilus is the source of fibres that innervate most of the ipsilateral and contralateral hippocampus (Swanson et al 1978), indicates that there may be single neurons in the hilus that have widely branching collaterals that innervate both the ipsilateral and contralateral hippocampi. There is also indirect evidence that these same hilar neurons also make synaptic contact with the dentate basket cel l s , and form feedforward inhibition. 116 APPENDIX A spike-like wave could be recorded in the lateral part of the hilar region (figure 21). This response showed amplitude f a c i l i t a t i o n when the commissural electrode was stimulated sequentially. It was associated with the discharge of single units in the stratum pyramidale of the regio inferior (figure 21). The amplitude of this wave was maximum outside the hilar region. 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