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Long-term potentiation and discrimination learning Skelton, Ronald William 1982

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LONG-TERM POTENTIATION AND DISCRIMINATION LEARNING by RONALD WILLIAM SKELTON B.Sc, Bishop's University, 1973 M.A., Concordia University, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PSYCHOLOGY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November 1982 (c) Ronald William Skelton, 1982 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 ^Sp) t v A s * / y  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date /?-e^~-.<4-t~^ "2- , / 9 * DE-6 (3/81) i i ABSTRACT Recent electrophysiological studies have shown that e l e c t r i c a l brain stimulation (EBS) can produce l a s t i n g increases in synaptic e f f i c a c y , defined as the quantitative relationship between pre- and postsynaptic a c t i v i t y . However, the behavioural significance of t h i s long-term potentiation (LTP) has yet to be demonstrated c l e a r l y . The p r i n c i p a l objective of t h i s thesis was to determine whether increased synaptic e f f i c a c y , produced by high-frequency EBS, enhances behavioural responses to a fixed amount of presynaptic a c t i v i t y . Following i d e n t i f i c a t i o n of EBS parameters capable of monitoring synaptic e f f i c a c y for long periods without producing LTP, a paradigm was developed in which a single-pulse of EBS in the perforant path (PP) acquired stimulus control over the temporal pattern of operant responses. In this paradigm, postsynaptic evoked potentials in the dentate gyrus (DG) produced by the PP EBS were used to monitor synaptic e f f i c a c y on every t r i a l of conditioning and also to measure one component of the neural a c t i v i t y generated by the EBS stimulus c o n t r o l l i n g the operant responses. In Experiment 3, high-frequency stimulation of the PP produced- LTP at the PP-DG synaptic interface and f a c i l i t a t e d subsequent acqui s i t i o n of stimulus control by the single pulse PP EBS. This effect could not have been due to s e n s i t i z a t i o n or to the stimulus properties of the high-frequency t r a i n s . The f i n a l experiment confirmed the importance of the EBS-evoked a c t i v i t y in the DG to the stimulus control by the PP EBS. The rate of. acq u i s i t i o n was d i r e c t l y related to the magnitude of the evoked potentials and two-stage b i l a t e r a l lesions of the PP in a s p e c i f i c sequence reduced the p r o b a b i l i t y of behavioural responses to the EBS. Taken together, these results indicate that the behavioural consequence of excitatory neural a c t i v i t y can be enhanced by an increase in synaptic e f f i c a c y . As such, they provide considerable support for the v a l i d i t y of LTP as a model of neural changes subserving learning and . for physiological theories of memory based on modifications in the- strength of synaptic connections. i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES vi LIST OF FIGURES v i i ACKNOWLEDGEMENTS v i i i CHAPTER 1: INTRODUCTION 1 Hippocampal Anatomy 6 Hippocampal Electrophysiology 10' Long Term Potentiation 16 Synaptic E f f i c a c y And Learning 25 Stimulus Properties Of Brain Stimulation 30 Experiments 35 CHAPTER 2: Long-Term Potentiation And Low-Frequency Stimulation . ... 37 METHODS 40 Implantation And Histology 40 Stimulation 42 Data Acquisition And Analysis 42 RESULTS 43 DISCUSSION 50 CHAPTER 3: Stimulus Properties Of Angular Bundle Stimulation 55 METHODS . 59 Implantation And Histology 59 Electrophysiological Procedures 60 Behavioural Apparatus 61 Behavioural Procedures 62 RESULTS 66 DISCUSSION 73 CHAPTER 4:Long-term Potentiation And Stimulus Control 77 METHODS 81 Implantation And Histology 81 Electrophysiological Procedures 82 Behavioural Apparatus 83 Behavioural And Experimental Procedures 84 RESULTS 86 DISCUSSION 97 CHAPTER 5: The Effect Of Perforant Path Lesions On Stimulus Control 103 METHODS 110 Implantation And Histology 110 Electrophysiology 113 Acquisition Of Discrimination • 114 Perforant Path Lesions 116 RESULTS 119 Acquisition Of Discrimination ,120 Perforant Path Lesions 132 DISCUSSION 144 Acquisition Of Discrimination 144 Perforant Path Lesions 153 CHAPTER 6: GENERAL DISCUSSION 160 BIBLIOGRAPHY 181 v i LIST OF TABLES Table I: P o p u l a t i o n Spike Amplitudes at S t a r t and End of Experiment 2 69 Table I I : Evoked P o t e n t i a l s and EBS-DS D i s c r i m i n a t i o n 90 v i i LIST OF FIGURES Figure 1.1: Hippocampal anatomy 8 Figure 1.2: Evoked potentials and long-term potentiation.... 13 Figure 2.1: Tetanic stimulation and synaptic e f f i c a c y 44 Figure 2.2: Stimulation at 0.2 Hz and synaptic e f f i c a c y 45 Figure 2.3: Histology 46 Figure 2.4: Low-frequency stimulation and synaptic e f f i c a c y . 49 Figure 3.1: Response contigencies and measurements 63 Figure 3.2: Electrode placements 67 Figure 3.3: Sample Evoked potentials 70 Figure 3.4: Discrimination ratios 71 Figure 4.1: Electrode Placements 87 Figure 4.2: Population Spike Amplitudes 91 Figure 4.3: Sample Evoked Potentials 93 Figure 4.4: Discrimination Ratios 94 Figure 5.1: Relative Positions of Stimulating, Recording and Lesion Electrodes 111 Figure 5.2: Lesion Sequences 119 Figure 5.3: Acquisition of Discrimination 121 Figure 5.4: Sample Evoked Potentials and Electrode Locations 125 Figure 5.5: Electrode Placements, FAST group 128 Figure 5.6: Electrode Placements, SLOW group 129 Figure 5.7: D i s t r i b u t i o n of EBS-DS Electrode Locations 131 Figure 5.8: Centres and Maximal Excursions of PP Lesions.... 133 Figure 5.9: Lesions and Daily Discrimination Ratios 135 Figure 5.10: Lesions and Averaged Discrimination Ratios 137 Figure 5.11: Behavioural and Neural Effects of PP lesions... 139 v i i i ACKNOWLEDGEMENTS I would l i k e to ex press - my deepest gratitude' to my advisor", Dr. A.G. P h i l l i p s , for his encouragement and d i r e c t i o n during every stage of t h i s thesis. I would l i k e to thank Dr. J.J. M i l l e r for his invaluable guidance and support on a l l of the electrophysiological aspects of t h i s work, and Dr. D.M. Wilkie for his important contributions in the areas of animal learning and memory, as well as for his assistance with the data analysis. I would also l i k e to thank the members of my examination committee for their helpful comments on various drafts of t h i s thesis. To a large degree, my perspectives have been shaped by interactions ' with many graduate students and technical assistants. In p a r t i c u l a r , I would l i k e to thank S. Assaf, T. Richardson, M. Oliver, S. Kehl, F. LePiane, D. Paul, M. Spetch and D. T r e i t for the time, energy and insights they provided. The friendships of Shaun and Lori Gray, and Chip Gerfen were mainstays of my graduate career. I owe my greatest debt of gratitude to my wife, Chris, whose u n f a i l i n g support and encouragement made the completion of t h i s thesis possible. This research was funded by N.S.E.R.C. grant #7808 to Dr. A.G. P h i l l i p s . I recieved personal support from N.S.E.R.C. postgraduate fellowships (1978-79, 1980-81) and from a K i l l i a m pre-doctoral fellowship (1981-82). 1 CHAPTER 1: INTRODUCTION Tr a d i t i o n a l l y , the analysis of learning has been approached from both behavioural and physiological perspectives. The behavioural approach is concerned with defining the environmental circumstances necessary for learning to occur, the features of an experience which are remembered and the effects of time and subsequent events upon the memory of e a r l i e r experiences. The goal of the physiological approach i s to account for a l l of the behavioural aspects of learning in terms of changes in the central nervous system, or in other words, to reveal the neural bases of learning and memory. There are many ways in which the neural bases of learning and memory can be studied. Several excellent reviews of much of the research in thi s area have been written, describing a variety of applicable techniques including various combinations of neuroanatomical, neurochemical or neurophysiological approaches (Kandel & Spencer, 1968; McGeer, Eccles & McGeer, 1978, chap. 15; Thompson, Patterson & Teyler, 1972; Rosenzweig & Bennett, 1976; Sokolov, 1977). Each of these has incorporated many level s of analysis, from the macroscopic study of *whole brain regions (e.g., Greenough, 1976, pp. 255-278), to the sub-microscopic investigation of events at the molecular level in a single synapse (e.g., Lee, Schottler, Oliver & Lynch, 1980). In th i s thesis, an attempt w i l l be made to relate one kind of electrophysiological change in a r e l a t i v e l y small population of c e l l s to one type of neuronal change which might occur during 2 learning and which might be a component of the neural bases of learning. The c r u c i a l component of any neural model of learning and memory is the process used to explain how information transmission within the nervous system is modified by experience. A number of models agree that the most l i k e l y location of neuronal changes during learning i s the synapse and at least f i v e d i f f e r e n t mechanisms for information storage through synaptic modifications have been proposed (Brindley, 1967, 1969; Burke, T9'66; Eccles, 1'953; Hebb, 1949;- Kandei Spencer, 1968; Shimbel, 1950). One of these, the "Use Pr i n c i p l e " , proposes that the strength of synaptic connections is increased by successful ( i . e . , spike-generating) use (Hebb, 1949). In other words, behavioural experiences which activate p a r t i c u l a r synapses are thought to cause a "trace" or "track" to be l a i d into the nervous system through a system of interconnected f a c i l i t a t e d synapses (Hebb, 1949). Although the "Use P r i n c i p l e " has often been considered to be plausible (e.g. Brindley, 1967; Goddard, 1980, pp. 231-247), u n t i l recently, there was very l i t t l e evidence that the synapses of central neurons can undergo long-lasting a l t e r a t i o n s . Within the past ten years, neurophysiological research has demonstrated a process in the hippocampus whereby brief periods of neuronal a c t i v i t y can produce an enduring increase in postsynaptic responsiveness to presynaptic a c t i v i t y ( B l i s s & Gardner-Medwin, 1973; B l i s s & Lomo, 1973). This phenomenon, c a l l e d long-term potentiation (LTP), has profound implications 3 for investigations of the neural bases of learning and memory because i t shares with learning two fundamentally important c h a r a c t e r i s t i c s : immediacy and permanency. In a recent summary, Bullock (1977, pp. 234-235) l i s t s 48 variables governing the integration and transformation of neural input by individual neurons and neural networks. Presumably, variations in almost any of these influences could a l t e r the flow of neural a c t i v i t y through the nervous system and thereby encode memories. The present choice of focus upon LTP, which is a modification of only two or' three of these variables, i's based on the s u i t a b i l i t y of LTP as a model of the neuronal changes which could and might subserve learning. This s u i t a b i l i t y was suggested in the o r i g i n a l reports (Bliss & Gardner-Medwin, 1973; B l i s s & Lomo, 1973) and has subsequently been reiterated in a number of reviews of both LTP and mechanisms of learning ( B l i s s , 1979; Doty, 1979, pp. 53-63; Eccles, 1979; Kandel, 1976, pp. 476-489; Lynch, G a l l & Dunwiddie, 1977, pp. 113-126; Tsukahara, 1981). The following sections w i l l examine some of evidence for changes in the synaptic e f f i c a c y in mammalian central nervous systems and discuss the properties of LTP which make i t such an a t t r a c t i v e model of the neuronal changes subserving learning and memory. One of the primary reasons that LTP holds such promise as a model of the neuronal changes subserving learning is because there have been few other instances where non-pathogenic manipulations of the nervous system have been found to produce changes in synaptic transmission which l a s t for more than a few 4 seconds. Lasting changes in synaptic transmission were reported in 1947 (Larrabee & Bronk, 1947) and electrophysiological research over the next twenty-five years culminated in the demonstration of LTP. Progress was marked by ever-increasing durations of synaptic f a c i l i t a t i o n and by the demonstration of the e f f e c t in central rather than peripheral tissue. The general p r i n c i p l e used in studying changes in synaptic transmission was the measurement of the postsynaptic responses to a fixed presynaptic input, generated by e l e c t r i c a l stimulation. Lasting increases in the amplitude of theu postsynaptic responses-following high frequency e l e c t r i c a l stimulation revealed a l t e r a t i o n s in the input/output relations of the synaptic system. These alt e r a t i o n s are c a l l e d potentiation (Eccles, 1953; Katz, 1966, p. 157). The f i r s t demonstration of post-tetanic potentiation (PTP) was a 2-3 min augmentation of autonomic ganglion responses produced by 10 sec of 15 Hz "tetanic" ( i . e . , high-frequency) stimulation (Larrabee & Bronk, 1947). A similar change was soon observed in a monosynaptic spinal reflex by Lloyd (1949) who showed a postsynaptic response increase l a s t i n g 6 minutes beyond the termination of 36 sec of 500 Hz stimulation. The maximum l i m i t of 2 hrs appeared to be reached by Spencer and Wigdor (1965) who administered 90 min of stimulation at 500 Hz. At about the same time, the f i r s t comprehensive studies on the electrophysiology of the hippocampus were being completed (Gloor, Sperti & Vera, 1963; Gloor, Vera & Spe r t i , 1963; Gloor, Vera & Sperti, 1964). In the l a s t of these papers, evidence was 5 presented showing an 8 sec PTP of granule c e l l s responses in the dentate gyrus (DG) following 7 sec of 20 Hz stimulation of the entorhinal cortex (Gloor, et a l . 1964). The f i r s t indication of t r u l y long-lasting potentiation appeared in abstract form in 1971 ( B l i s s & Gardner-Medwin, 1971) and two years later the complete documentation of t h i s remarkable phenomenon appeared (B l i s s & Gardner-Medwin, 1973; B l i s s & Lomo, 1973). The four most important features of LTP shown by these o r i g i n a l demonstrations were: 1) the brevity of the stimulation (3-20' sec), 2*)- the immediacy of the- effect (15 sec), 3) the magnitude of the potentiation (300%), and 4) the duration of the potentiation (8 hrs - 16 weeks). In order to gain a f u l l appreciation of the phenomenon of LTP and i t s implications for theories of learning and memory, i t is necessary to have some grasp of the anatomy and electrophysiology of the the hippocampus, the structure in which LTP was discovered and has been studied most extensively. The next two sections w i l l describe the most relevant features of hippocampal anatomy and electrophysiology and subsequent sections w i l l describe the measurement of synaptic e f f i c a c y and the c h a r a c t e r i s t i c s of LTP which are most relevant to i t s v a l i d i t y as a model of the neural changes subserving learning. 6 Hippocampal Anatomy The major structures and connections of the hippocampal and parahippocampal formations are well documented and have been known for a long time. The early anatomists studied Golgi-stained material from a variety of sub-primate mammals and described the i n t r i c a t e structures in great d e t a i l (Lorente de No, 1934; Ramon y Cajal, 1911). Later studies replicated and extended the o r i g i n a l observations (Blackstad, 1956; Raisman, Cowan & Powell, 1965). Although subsequent studies have used better lesions- and stains or anterograde' and' retrograde' tracers to refine the descriptions and reveal some new connections, there have been few major contradictions of the early work. The hippocampus is an a r c h i p a l l i a l structure, the oldest portion of the cerebral cortex. In the rat brain, i t i s situated just anterior to the cerebellum, transversely oriented, folded up under and completely covered by the cerebral cortex and corpus callosum (Zeman & Innes, 1963, pp. 144-146). The hippocampal formation is subdivided on the basis of the major c e l l types into the subiculum, four "CA" (Cornu Ammonis) subfields and the DG (Lorente de No, 1934; Ramon y Cajal, 1911). In the hippocampus proper, the large pyramidal c e l l s of areas CA1, CA2, CA3, and CA4 are arranged in a single folded sheet of c e l l s and are more densely packed than in any region of the cortex. The s l i g h t l y smaller granule c e l l s of the DG are even more t i g h t l y packed into a separate sheet of c e l l s that is folded around one edge of the pyramidal c e l l sheet. Although there are a considerable number of interneurons in the 7 hippocampal formation (Amaral, 1978), pyramidal and granule c e l l s outnumber a l l other c e l l types (Ramon y Cajal, 1911; Shepherd, 1974, pp. 266-267). The outer extremity of the hippocampus, the subiculum, i s contiguous with the cingulate cortex (Lorente de No, 1934). The major c o r t i c a l afferent to the hippocampus and DG i s from the entorhinal cortex via the perforant path (Ramon y Cajal, 1911; Shepherd, 1974, p. 262). Subcortical structures are extensively interconnected with the hippocampal formation through the fimbria—fornix (Andersen, Bland & Dud'ar, 1 973;' Raisman et a l . , 1965). Figure 1.1 i l l u s t r a t e s the most important functional interconnections and the sp a t i a l relationships between the major neuronal elements. As a consequence of the arrangement of the major groups of c e l l s in sheets, neuronal elements in the hippocampus are arranged in layers. The di f f e r e n t components of the pyramidal and granule c e l l s , proximal and d i s t a l apical dendrites, c e l l bodies, axons and basal dendrites, are a l l r e s t r i c t e d to individual layers (Lorente de No, 1934; Ramon y Cajal, 1911). This laminar segregation of neuronal elements is maintained even to the scale of diff e r e n t segments of the dendritic trees. In the DG in p a r t i c u l a r , afferent projections from d i f f e r e n t structures form "en passage" synapses on par t i c u l a r portions of the dendritic tree and there is very l i t t l e s p a t i a l overlap between the afferent fibres from d i f f e r e n t areas. Fibres from the l a t e r a l and medial entorhinal c o r t i c i e s are topographically arranged on the outer two thirds of the Figure 1.1: Hippocampal anatomy. Schematic representation of the major c e l l groups . and interconnections of the l e f t hippocampal formation, as viewed in .horizontal section. The projection from the entorhinal cortex (EC) to the dentate gyrus (DG) i s the perforant path (PP). The discontinuity of the PP after i t leaves the angular bundle (AB) s i g n i f i e s the upwards projection of the PP f i b r e s . In contrst, the connections of the DG-CA3 and CA3-CA1 are s o l i d , i n dicating that these f i b r e s are confined in each hippocampal l e v e l (lamella). The locations of the stimulating (STIM) and recording (REC) electrodes used throughout t h i s thesis are also shown. Cali b r a t i o n s : anterior (ANT) and l a t e r a l (LAT) d i r e c t i o n s ; 0.5 mm. 9 dendritic tree (Hjorth-Simonsen, 1972; Hjorth-Simonsen & Jeune, 1972; McNaughton & Barnes, 1977; Steward, 1976). Commissural fibres from the con t r a l a t e r a l CA3 and CA4 regions share the inner t h i r d of the granule c e l l dendritic tree with the associational fibres from a n t e r i o r - i p s i l a t e r a l CA3 (Gottlieb & Cowan, 1973; Hjorth-Simonsen, 1977; Lauberg, 1979). The route taken by the PP fib r e s from the entorhinal cortex to the DG and hippocampus is well known and has been described comprehensively (Hjorth-Simonsen & Jeune, 1972). The dorsal-ventral axis of the entorhinal cortex is mapped onto the rostro-caudal axis of the DG (Hjorth-Simonsen, 1972). The fibres of the PP enter the angular bundle from the entorhinal cortex, travel r o s t r a l l y , medially and dorsally along i t and then leave at various points along i t s length, usually posterior and ventral to the target regions of the DG. The fibres then ascend dorsally and r o s t r a l l y through the subiculum to perforate the hippocampal fi s s u r e , primarily at the crest (medial point) of the DG but to a certain extent, along the entire length of the fissure (Hjorth-Simonsen & Jeune, 1972). The PP is a group of fib r e s defined by their origins and projections. In contrast, the angular bundle i s a large, v i s i b l e structure, defined by i t s position and shape (Lorente de No, 1934; Ramon y Cajal, 1911). It i s the caudal extension of the dorsal hippocampal commissure lying subjacent to the posterior portion of the corpus callosum, the splenium. The angular bundle contains a number of efferent fibres from entorhinal cortex, primarily those of the PP but also including those of the 10 crossed temporo-amnionic pathway (Blackstad, 1956; Raisman et a l . , 1965; Steward, 1976; Swanson & Cowan, 1977; Wyss, 1981). Afferent f i b r e s from dentate, CA3, CA1 and subicular regions descend through the angular bundle to the entorhinal and p e r i r h i n a l c ortices (Blackstad, 1956; Hjorth-Simonsen, 1971; Steward & S c o v i l l e , 1976; Swanson & Cowan, 1977; Swanson, Sawchencko & Cowan, 1981). Clea r l y , the angular bundle i s not the same structure as the PP and yet i t is d i f f i c u l t to estimate the r e l a t i v e numbers of PP and non-PP fibres contained- in i t . One- study which used electron-micrographs to assess terminal density following entorhinal lesions reported that the majority of entorhinal projections terminated in the DG (Nafstad, 1967). The angular bundle has been described as a "bottleneck" through which a l l PP fibres flow (Lomo, 1971a) and the largest potentials evoked by stimulation of the angular bundle have been those recorded from the DG (Andersen, 1975, pp. 155-176; Lomo, 1971a). Thus, the PP may, at some levels of the angular bundle, comprise the majority of the angular bundle and yet at some points along their courses, these two structures are quite d i s t i n c t from one another. Hippocampal Electrophysiology The dense aggregation of c e l l types and s p a t i a l segregation of neuronal elements have important consequences for neurophysiological research. In the hippocampus i t i s possible to record spike a c t i v i t y of single c e l l s (units) (e.g., Ranck, 11 1973), the slowly fluctuating a c t i v i t y in the dendritic regions (EEG) (e.g., Vanderwolf, Kramis, G i l l e s p i e & Bland, 1975, pp. 101-128) and the potentials evoked by stimulation of afferent f i b r e bundles (e.g., Gloor et a l . , 1963). As w i l l be shown, i t is this l a t t e r type of recording which i s most important to the investigation of changes in synaptic transmission. In order to measure changes in synaptic e f f i c a c y , both pre-and postsynaptic a c t i v i t y levels must be known or measured. In the hippocampus, the dense aggregation, consistent orientation and laminar arrangement of the major c e l l types^ make i t possible to record the summed postsynaptic a c t i v i t i e s from a large number of c e l l s . This i s because e l e c t r i c a l stimulation produces a fixed amount of a c t i v i t y in a presynaptic f i b r e bundle that in turn leads to large f i e l d potentials (Gloor et a l . , 1963; Lomo, 1971a). The stimulation of the presynaptic elements can be very selective because the afferent f i b r e bundles are s p a t i a l l y separate from each other and from the postsynaptic c e l l s . This process has been studied and described comprehensively in the PP-DG system (Andersen, B l i s s & Skrede, 1971a; Gloor et a l . , 1963; Lomo, 1971a). E l e c t r i c a l stimulation of the PP produces synchronous action potentials in these f i b r e s , which in turn produce synchronous excitatory postsynaptic potentials (EPSP's) in the dendrites of the dentate granule c e l l s . The s p a t i a l arrangement of the postsynaptic c e l l s allows the e x t r a c e l l u l a r ionic currents to summate and produce large e x t r a c e l l u l a r f i e l d responses (Gloor et a l . , 1963). The large, negative f i e l d s in 1 2 the dendritic layer r e f l e c t the average EPSP levels in the granule c e l l population and are c a l l e d population EPSP's (Fig 1.2A). Strong activation of the PP w i l l lead to action potentials in the granule c e l l bodies, which also produce large e x t r a c e l l u l a r f i e l d s . The summed, ex t r a c e l l u l a r f i e l d s of these spikes appear on evoked potentials recorded from the c e l l layer as a sharp negative-going population spike, superimposed on a slower positive wave (Gloor et a l . , 1963; Lomo, 1971a). The amplitude of the population spike is determined by the number and synchrony of the contributing- granule c e l l discharges' (Andersen et a l . , . 1971a). The slow positive wave observed in the c e l l u l a r region is in part an imperfect r e f l e c t i o n of the population EPSP in the dendritic layer, and in part a r e f l e c t i o n of a number of h i l a r events (Gloor et a l . , 1963; Lomo, 1971a). The granule c e l l s of the dentate region l i e in a single sheet, wrapped around the end of the CA3-CA4 layer, forming an "upper" and a "lower" blade. The area between the blades is the hi l u s of the DG (Lorente de No, 1934; Ramon y Cajal, 1911). An electrode driven v e r t i c a l l y through the anterio-dorsal DG w i l l pass sequentially though the dendritic layer of the upper blade, the c e l l layer, the hi l u s and then the c e l l and dendritic layers of the lower blade. Sequential records of evoked potentials following PP stimulation taken at selected v e r t i c a l intervals reveal large changes in the waveforms between locations along the electrode t r a c t . Taken together, these records comprise a depth p r o f i l e , c h a r a c t e r i s t i c of thi s region (Gloor et a l . , 1963; Lomo, 1971a). For example, the EPSP is negative in the 13 Figure 1 . 2 : Evoked potentials and long-term potentiation. A. Population EPSP: A ty p i c a l evoked potential recorded from the dendritic region of the DG following stimulation of the PP.The portion of the wave representing the population EPSP i s delimited by the v e r t i c a l dashed l i n e s . B. Population spike: An evoked potential recorded from the hil u s of the DG following PP stimulation. The amplitude (amp.) of the population spike is measured from the peak p o s i t i v i t y to the peak negativity. C. Long-term potentiation: An evoked potential from the same electrode s i t e as in B., following stimulation at the same current intensity, 2 hrs after tetanic stimulation. msec 15 dendrites of the upper and lower blades, but posit i v e in the c e l l layers and h i l u s . Large changes in the amplitude of the EPSP are observed between r e l a t i v e l y close dendritic locations and therefore, the evoked potential waveform provides a valuable index of electrode depth which pre c i s e l y i d e n t i f i e s the location of the recording electrode. In contrast, the amplitude of the population spikes within the h i l u s remains r e l a t i v e l y constant through a considerable variation in depth (Lomo, 1971a) and so h i l a r placements of recording electrodes are considered superior to dendritic 1 , placements i f stable evoked potentials are to be recorded over long periods ( B l i s s & Gardner-Medwin, 1973; Douglas & Goddard, 1975). The amplitude of the population spike can be used to indicate changes in synaptic e f f i c a c y . While the presynaptic input to the neurons is determined only by the stimulation int e n s i t y , the postsynaptic response i s free to vary under the influence of changes in transmitter release, receptor s e n s i t i v i t y or the resting p o t e n t i a l , resistance or capacitance of the postsynaptic membrane (Bullock, 1977, p 234; Katz, 1966, pp. 157-158; Shepherd, 1974, pp. 58-76). Fluctuations in population EPSP's r e f l e c t the sum of any or a l l of these influences. The amplitude of the population spike r e f l e c t s not only the changes which a f f e c t the EPSP, but also the factors which a f f e c t the translation of EPSP's into action potentials, i . e . the output of the granule c e l l s (Andersen et a l . , 1971a, Lomo, 1971a). In other words, the amplitude of the population spikes r e f l e c t s everything a f f e c t i n g synaptic e f f i c a c y , the 16 input/output relationship of a synaptic system (Alger & Teyler, 1976; B l i s s & Lomo, 1973; Wilson, 1981). Thus, population spikes are not only the most stable measurement of synaptic e f f i c a c y in chronic animals but also the most comprehensive. In thi s thesis, a l l e l ectrophysiological measurements were confined to the amplitudes of population spikes recorded from freely-moving animals. Figure 1.2B shows how population spike amplitudes were measured. Long Term- Potentiation Reviews of both LTP ( B l i s s , 1979; Eccles, 1 9 7 9 ; Lynch, et a l . , 1977, pp. 1 1 3-128) and mechanisms of learning (Doty, 1979, pp. 53-63; Kandel, 1976, pp. .476-489; Tsukahara, 1981) make i t very clear that many authors consider LTP to be a suitable model of the neuronal changes subserving learning because of the par t i c u l a r combination of properties possessed by LTP. Four of these properties have previously been mentioned in connection with the o r i g i n a l demonstrations of LTP, namely: 1) brevity of causal event, 2) immediacy of onset, 3) magnitude of change and 4) persistence. A t y p i c a l change in population spike amplitude following tetanic stimulation (LTP) is shown in Figure 1.2B,C. The following paragraphs w i l l examine these c h a r a c t e r i s t i c s in more d e t a i l , describing studies which have confirmed and extended the o r i g i n a l reports, and showing why pa r t i c u l a r combinations of these c h a r a c t e r i s t i c s of LTP are similar to the ch a r a c t e r i s t i c s of learning. The most important combination of properties exhibited by 17 LTP must c e r t a i n l y be immediacy, d u r a b i l i t y and induction by non-pathological causes. Although there have been a number of examples of long-lasting changes in the nervous system, most notably sprouting (e.g., Lynch & Cotman, 1975, pp. 123-154), p r o l i f e r a t i o n of c o r t i c a l dendrites (e.g., Greenough, 1976, pp. 255-278), and macromolecular changes (e.g., Matthies, 1982, pp. 1-15), none of these have shown functional changes which transpire rapidly. Although the exact latency of LTP has not been c l e a r l y defined, increases* in synaptic e f f i c a c y are me a su rabble- within a minute or two of the offset of the tetanic stimulation. In their o r i g i n a l report, B l i s s and Lomo (1973) considered the 15 sec delay between stimulation offset and the appearance of LTP to be an important d i s t i n c t i o n between the new phenomenon, LTP, and the well known PTP. In another report however, a large increase in synaptic e f f i c a c y was observed within a second of stimulation o f f s e t , but t h i s increase was followed by a gradual decline in synaptic e f f i c a c y over a ten minute period (McNaughton, Douglas & Goddard, 1978). Because the i n i t i a l , transient increase was attributed to PTP, these data too were used as evidence for a difference between PTP and LTP. Regardless of whether one or two processes are used to account for the immediate onset of LTP, there is no doubt that synaptic eff i c a c y i s increased very quickly. It i s not clear that the absolute maximum duration of LTP has ever been tested. An endurance test beyond the 16 weeks o r i g i n a l l y reported (Bliss & Gardner-Medwin, 1973) or even the 18 seven weeks reported in a subsequent paper (Douglas & Goddard, 1975) would probably test electrode design, more than LTP duration. Both extra-long demonstrations of LTP employed trains of tetanic stimulation repeated at i n t e r v a l s . The separation of these t r a i n s by minutes ( B l i s s & Gardner-Medwin, 1973) or days (Douglas & Goddard, 1975) revealed a c r u c i a l property of LTP; the a b i l i t y to integrate successive inputs over very long i n t e r v a l s . This long integration period between successive events becomes even' more important when related' to the brevity pf the stimulation required for each individual event. The LTP that lasted for 16 weeks was produced by 9 trains each l a s t i n g 15 sec, presented at 30 min intervals (Bliss & Gardner-Medwin, 1973) and the 7-week LTP was produced by 11 d a i l y tetanic trains l a s t i n g only 2.0 sec each (Douglas & Goddard, 1975). Other t r a i n durations which have been reported to produce LTP include 4 sec ( B l i s s & Lomo, 1973), 1 sec (Lee et a l . , 1980) and 20 msec (Douglas, 1977). The s i m i l a r i t i e s between these temporal c h a r a c t e r i s t i c s and those of o n e - t r i a l , m u l t i - t r i a l and multi-learning paradigms are obvious. The v a l i d i t y of LTP as a model rests p a r t l y upon the parameters of the e l e c t r i c a l stimulation required to produce LTP. For example, i t i s quite clear that the stimulation does not need to produce pathological epileptiform a c t i v i t y in order to cause LTP (Douglas, 1977; Douglas & Goddard, 1975). In fact, much less stimulation is required for LTP than PTP (Bliss & Lomo, 1973). Studies on the effects of stimulation frequency and 19 intensity have revealed a s i m i l a r i t y between neuronal conditions required for LTP and the patterns of spontaneous neuronal a c t i v i t y observed in the hippocampus. The following section w i l l describe these studies and show how the parameters of the e l e c t r i c a l stimulation required to produce LTP support the use of LTP as a model of a neural basis of learning. It has been f a i r l y d i f f i c u l t to estimate the absolute lower l i m i t s of frequency and intensity required to produce LTP. From the outset, i t was clear that these two parameters interact ( B l i s s & Lomo, 1973): high- intensity (60 V), high frequency (15 Hz) stimulation led to LTP while stimulation at lower i n t e n s i t i e s (10, 30 V) or at a lower frequency (0.5 Hz) did not. A l l subsequent analyses of the intensity requirements of LTP have used rather high stimulation frequencies (100-400 Hz) and have found the current threshold of LTP to be quite close to the threshold of the population spike (McNaughton et a l . , 1978; Wilson , 1981; Yamamoto & Sawada, 1981). Goddard (1980, pp. 231-247) has used t h i s relationship to argue that the magnitude of the input required to produce LTP is not unlike the inputs which occur naturally. The frequencies of stimulation which are necessary for the production of LTP appear to l i e well within the f i r i n g repertoires of hippocampal units. Almost a l l of the previously c i t e d studies have used frequencies between 10 and 100 Hz, and c e l l s in the hippocampal and dentate regions of freely moving animals have been shown to f i r e spontaneously 2 to 150 times per second (Ranck, 1973). Even though there i s l i t t l e question that 20 higher stimulation frequencies lead to greater magnitude LTP (B l i s s & Gardner-Medwin, 1973; Douglas & Goddard, 1975; Dunwiddie & Lynch, 1978; Yamamoto & Sawada, 1981), the minimum frequency of stimulation capable of producing LTP remains controversial. The importance of stimulation frequency to the production of LTP has been used to infer properties of the mechanisms of LTP (Lynch, et a l . , 1978, pp. 113-128) and the neural conditions responsible for changes in synaptic e f f i c a c y during learning (Goddard, 1980, pp. 231-2 47). One- attempt to determine' the ef f e c t s of stimulation frequency used stimulation currents just above the population spike threshold, and purportedly showed that only 100 Hz stimulation produced "pure" LTP, whereas lower frequencies produced a graded combination of f a c i l i t a t i o n and depression (Dunwiddie & Lynch, 1978). Since then, a l l studies on the mechanisms of LTP conducted by t h i s group of researchers have employed 100 Hz stimulation exclusively (Browning, Dunwiddie, Bennett, Gispen, & Lynch, 1979; Lee, Oliver, Schottler and Lynch, 1981, pp. 189-212; Lee et a l . , 1980; Lynch, Browning and Bennett, 1979). Replication of a preliminary report of LTP produced by 0.2 Hz (Douglas & Goddard, 1975) could provide an estimate of the v a l i d i t y of these positions and might esta b l i s h the frequency l i m i t below which i t is "safe" to use stimulation to monitor synaptic e f f i c a c y without changing i t . The f i r s t experiment in this thesis w i l l be a careful examination of the e f f e c t s of low frequency stimulation on synaptic e f f i c a c y in the PP-DG system. 21 So far, two lines of evidence supporting the v a l i d i t y of LTP as a model have been presented. F i r s t , the temporal c h a r a c t e r i s t i c s of LTP were shown to be similar to certain fundamental c h a r a c t e r i s t i c s of learning. In t h i s context, the most important features of LTP were the brevity of the required stimulation, the immediacy and permanency of the increased synaptic e f f i c a c y and the length of the intervals over which successive trains of stimulation have cumulative e f f e c t s . Second, the frequencies and i n t e n s i t i e s of the e l e c t r i c a l stimulation were shown to induce neuronal a c t i v i t y similar to spontaneous hippocampal a c t i v i t y . A t h i r d l i n e of evidence, the generality and s p e c i f i c i t y of LTP, are other features which endorse the use of LTP as a model of learning. Generality refers to the- number of systems in the brain capable of undergoing LTP while s p e c i f i c i t y refers to the l i m i t i n g of LTP to the fibres activated by the high-frequency stimulation. To date, LTP has been found to occur in most hippocampal systems. In addition to the previously described PP-DG system, LTP has also been produced in the following systems; the mossy fibre-CA3 projection (Alger & Teyler, 1976; Eccles, 1979), fimbria-CA3 (Yamamoto & Swada, 1981), the Schaffer c o l l a t e r a l -CA1 fibres (Alger & Teyler, 1976; Andersen, Sundberg, Sveen & Wigstrom, 1977; Dunwiddie & Lynch, 1978; Lynch, Dunwiddie & Gribkoff, 1977; Teyler, Alger, Bergman & Livingston, 1977), commissural-CA1 (Buzsaki, 1981) and septum to dentate projection (McNaughton & M i l l e r , Note 1). To date, there have been only two reports of LTP in non-hippocampal systems, both in the s t r i a t e 22 (visual) cortex. Lee (1981) showed LTP in a c o r t i c a l s l i c e preparation from adult rats whereas Komatsu, Toyama, Maeda & Sakaguchi (1981) showed that LTP could be produced in the cortex of cats only between the ages of 2 and 6 weeks. It i s d i f f i c u l t to know whether the abundance of evidence for LTP in the hippocampus, compared to the r e l a t i v e paucity in other regions of the brain, is due to a functional difference between the hippocampus and other structures or more simply, to the r e l a t i v e ease with which synaptic e f f i c a c y can be monitored in hippocampal systems. Although- i t is' clear that the spinal cord does not undergo LTP (Lloyd, 1949), a great deal more research with refined techniques i s needed to determine whether LTP is a general property of central neurons. S p e c i f i c i t y of neuronal changes i s an important c r i t e r i o n by which to evaluate any putative model of learning, because there i s a great deal of evidence indicating that learning can be very s p e c i f i c to p a r t i c u l a r stimuli and features of the environment (e.g., Mackintosh, 1974, chap. 7, 9, 10). In most, systems studied to date, LTP has been found to be homosynaptic, i. e . r e s t r i c t e d to the fibres activated by the tetanic stimulation ( B l i s s , 1979). The alternative to homosynaptic potentiation i s heterosynaptic potentiation, the production of an increased postsynaptic responsiveness to inputs other than the afferents d i r e c t l y stimulated ( B l i s s , 1979). Homosynaptic potentiation has been reported in the granule c e l l s of the DG (McNaughton & Barnes, 1977; McNaughton et a l . , 1978; Wilson, Levy & Steward, 1981), the CA1 pyramidal c e l l s (Andersen et a l . , 23 1977; Dunwiddie & Lynch, 1978; Lynch et a l . , 1977), and the CA3 pyramidal c e l l s (Yamamoto & Sawada, 1981). However, there is at least one report of heterosynaptic potentiation in CA3 c e l l s (Eccles, 1979). A l l of these studies have had to contend with comparisons of rather large f i b r e bundles. At least one author in the area has expressed the hope that future studies w i l l be able to show that LTP can be r e s t r i c t e d to a single fib r e within a bundle ( B l i s s , 1979). Perhaps the most compelling c r i t i c i s m of the v a l i d i t y of LTP as a model of the neural changes- in learning is one which has been directed at the "Use P r i n c i p l e " in general (Doty, 1959;. Thompson et a l . , 1972). These authors point out that only one input is required for the "Use P r i n c i p l e " to operate. In contrast, learning i s c l e a r l y superior under conditions involving at least two s t i m u l i , when at least one of these i s a "motivating" stimulus and when the motivating stimulus i s r e l i a b l y preceded by a neutral stimulus. Further, the repeated presentations of a single stimulus w i l l often lead to a decrement in the o r i g i n a l response, i . e . , habituation. Although thi s c r i t i c i s m speaks only to those who would propose LTP as a model of a l l of the changes in learning, i t highlights the dangers of s i m p l i s t i c inferences and the need to expand the analysis of LTP to the question of whether LTP i s affected by concurrent a c t i v i t y in two or more neural systems. There have been two studies which have indicated an interaction between the a c t i v i t y in two d i f f e r e n t systems and the production of LTP. In the f i r s t , LTP was produced by the 24 simultaneous ac t i v a t i o n of the l a t e r a l and medial perforant paths (McNaughton et a l . , 1978). The importance of co-operativity among these afferent fibres (spatial summation) was shown by the absence of LTP when i d e n t i c a l stimulation parameters were delivered to medial and l a t e r a l paths independently. In the second, more relevant study, the magnitude and duration of LTP in the PP-DG system were increased by high-frequency stimulation of the medial r e t i c u l a r formation for a few seconds immediately after tetanic stimulation of the PP (Laroche & Bl'och, 1982, pp. 575-588). Although no attempt was made to discover the effects of high frequency r e t i c u l a r stimulation upon synaptic e f f i c a c y in the untetanized PP-DG system, this study c l e a r l y showed that LTP in one system could be modulated by later a c t i v i t y in another. The electrophysiological c h a r a c t e r i s t i c s of LTP have provided one kind of support for the v a l i d i t y of LTP as a model of neuronal changes which might subserve learning. However, another kind of support is absolutely essential for LTP to be considered to be an accurate model. It must be shown that increases in synaptic e f f i c a c y occur during learning. In addition, a causal relationship must be established between these neuronal changes and the behavioural changes c a l l e d learning. To date, most analyses of neuronal a c t i v i t y in conditioning paradigms have provided only indir e c t evidence for the occurrence of LTP-like changes during learning. 25 S y n a p t i c E f f i c a c y a n d L e a r n i n g I n o r d e r t o e s t a b l i s h a l i n k b e t w e e n s y n a p t i c e f f i c a c y a n d l e a r n i n g , i t i s n e c e s s a r y t o h a v e a n i n d e x o f b o t h p r e - a n d p o s t s y n a p t i c a c t i v i t y l e v e l s , a n d t o p r o v i d e e v i d e n c e f o r a r e l a t i o n s h i p b e t w e e n e l e c t r o p h y s i o l o g i c a l a n d b e h a v i o u r a l c h a n g e s . E v e n t h o u g h t h e r e h a v e b e e n few s t u d i e s a b l e - t o mee t t h e s e c r i t e r i a , t h e r e h a v e b e e n a number o f i n d i r e c t i n d i c a t i o n s t h a t c h a n g e s i n s y n a p t i c e f f i c a c y o c c u r d u r i n g l e a r n i n g . One p r o m i s i n g a p p r o a c h f o l l o w e d by D e a d w y l e r a n d h i s c o l l e a g u e s f o r a number o f y e a r s h a s b e e n 1 the- a n a l y s i s o f c o n d i t i o n i n g - i n d u c e d c h a n g e s i n s e n s o r y e v o k e d p o t e n t i a l s . The f i r s t o f t h r e e r e p o r t s r e v e a l e d s l o w d e p o l a r i z a t i o n s i n t h e DG w h i c h i n c r e a s e d o v e r t h e c o u r s e o f c o n d i t i o n i n g . T h e s e s l o w w a v e s a p p e a r e d d u r i n g a f i x e d i n t e r v a l a f t e r t o n e s w h i c h p r e d i c t e d w a t e r . The a m p l i t u d e s o f t h e s e s l o w w a v e s w e r e c o r r e l a t e d w i t h t h e l a t e n c y o f t h e b e h a v i o u r a l r e s p o n s e s t o t h e t o n e s d u r i n g a c q u i s i t i o n , c r i t e r i o n p e r f o r m a n c e a n d e x t i n c t i o n p h a s e s o f t h e e x p e r i m e n t ( D e a d w y l e r , Wes t & L y n c h , 1 9 7 9 ) . I n c r e a s e d s y n a p t i c e f f i c a c y was c o n s i d e r e d t o be one p o s s i b l e m e c h a n i s m f o r t h e d e v e l o p m e n t o f t h e s e s l o w w a v e s . I n s u b s e q u e n t , more r e f i n e d a n a l y s e s o f t h i s p h e n o m e n o n , some o f t h e o r i g i n a l a u t h o r s h a v e p r o v i d e d u n e q u i v o c a l e v i d e n c e a g a i n s t a r o l e f o r i n c r e a s e d s y n a p t i c e f f i c a c y i n t h e d e v e l o p m e n t o f t h e s e n s o r y e v o k e d p o t e n t i a l s o b s e r v e d i n t h e i r p a r a d i g m ( D e a d w y l e r , Wes t & R o b i n s o n , I 9 8 l a , b ) . Upon c l o s e r a n a l y s i s , t h e s i n g l e d e p o l a r i z i n g wave o b s e r v e d i n t h e o r i g i n a l s t u d y ( D e a d w y l e r e t a l . , 1979) was f o u n d t o be a c o m p o s i t e o f 26 two s y n a p t i c w a ves, one f o l l o w i n g t h e o t h e r . The f i r s t , s h o r t l a t e n c y wave was shown t o be a c o n s e q u e n c e o f a c t i v i t y i n t h e PP, b u t t h i s wave d i m i n i s h e d o v e r t h e c o u r s e o f c o n d i t i o n i n g ( D e a d w y l e r e t a l . , 1 9 8 1 a ) . The s e c o n d wave i n c r e a s e d d u r i n g t h e c o u r s e o f c o n d i t i o n i n g , b u t t h i s wave a p p e a r e d t o r e s u l t f r o m a c o m m i s s u r a l i n p u t , m o d u l a t e d by i n h i b i t i o n a r i s i n g f r o m t h e s e p t u m ( D e a d w y l e r e t a l . , 1 9 8 1 a ) . T h e s e s t u d i e s i l l u s t r a t e t h e d a n g e r s o f i n f e r r i n g c h a n g e s i n s y n a p t i c e f f i c a c y f r o m d a t a b a s e d o n l y on p o s t s y n a p t i c r e s p o n s e s t o u n m e a s u r e d i n p u t s . The a n a l y s i s o f h i p p o c a m p a l u n i t a c t i v i t y d u r i n g l e a r n i n g h a s p r o v i d e d i n d i r e c t e v i d e n c e f o r i n c r e a s e s i n s y n a p t i c e f f i c a c y d u r i n g l e a r n i n g . A c a r e f u l m a p p i n g s t u d y o f u n i t a c t i v i t y i n 18 f o r e b r a i n s t r u c t u r e s d u r i n g P a v l o v i a n c o n d i t i o n i n g r e v e a l e d d r a m a t i c c h a n g e s i n t h e p y r a m i d a l c e l l s a c t i v i t y i n CA3 ( O l d s , D i s t e r h o f t , S e g a l , K o r n b l u t h & H i r s h , 1 9 7 3 ) . S u b s e q u e n t s t u d i e s have shown t h a t t h e f i r i n g p a t t e r n o f h i p p o c a m p a l p y r a m i d a l c e l l s i n t h e p r e s e n c e o f c o n d i t i o n e d s t i m u l i ( C S ) , c h a n g e s o v e r t h e c o u r s e o f c o n d i t i o n i n g a n d e v e n t u a l l y comes t o p r e d i c t t h e f o r c e - d y n a m i c s o f t h e c o n d i t i o n e d , n i c t i t a t i n g membrane r e s p o n s e ( B e r g e r , Laham & Thompson, 1980; B e r g e r & Thompson, I 9 7 8 a , b ; H o e h l e r & Thompson, 1980; P a t t e r s o n , B e r g e r & Thompson, 1 9 7 9 ) . The r e l a t i o n s h i p b e t w e e n t h e c h a n g e s i n h i p p o c a m p a l u n i t a c t i v i t y a n d t h e d e v e l o p m e n t o f t h e c o n d i t i o n e d r e s p o n s e h a s been d e m o n s t r a t e d t h r o u g h t h e use o f s e v e r a l b e h a v i o u r a l p r o c e d u r e s i n c l u d i n g p s e u d o - and b a c k w a r d s - c o n d i t i o n i n g ( B e r g e r & Thompson, 1 9 7 8 b ) , e x t i n c t i o n ( B e r g e r & Thompson, 1982) and t h r e s h o l d t e s t s 27 (Kettner & Thompson, 1982). In the present context, the most interesting evidence has derived from a comparison of unit a c t i v i t i e s in the entorhinal cortex and CA3 (Clark, Berger & Thompson, 1978). Over the course of conditioning, a l t e r a t i o n s in the a c t i v i t y of units in the entorhinal cortex were very small, but appeared e a r l i e r in conditioning than the topographically similar changes in hippocampal pyramidal c e l l a c t i v i t y (Clark et a l . , 1978). According to an interpretation published elsewhere, a c t i v i t y in the entorhinal units formed- a "template" of the conditioned-response which was then amplified and mapped onto the c e l l s of the hippocampus through a process resembling LTP (Berger et a l . , 1980). Although t h i s view i s supported by measures of pyramidal c e l l a c t i v i t y changes produced by LTP (Deadwyler, Gribkoff, Cotman & Lynch, 1976), the acceptance of t h i s conclusion must await evidence that the entorhinal c e l l s which change e x c i t a b i l i t y during learning were connected d i r e c t l y to the corresponding hippocampal units. A somewhat stronger though s t i l l indirect l i n k between hippocampal unit a c t i v i t y , learning and increases in synaptic e f f i c a c y has been provided by a study which examined the effects of p o s t - t r i a l stimulation on unit a c t i v i t y and LTP. The a b i l i t y of p o s t - t r i a l stimulation of the medial r e t i c u l a r formation to f a c i l i t a t e learning is well known (Bloch, 1976, pp. 583-590; McGaugh, Gold, Handwerker, Jensen, Martinez, Meligeni & Vasquez, 1979, pp. 151-164). Laroche and Bloch (1982, pp. 575-588) were able to link r e t i c u l a r stimulation to hippocampal unit changes 28 and LTP by showing that p o s t - t r i a l r e t i c u l a r stimulation not only accelerated the learning-induced changes in hippocampal units of one group of animals, but also increased the magnitude and duration of LTP in another group of animals given tetanic stimulation of the PP. Even though a l l three effects were not shown in a single group of animals, the authors concluded that the effects of r e t i c u l a r stimulation on learning could be explained by the changes in hippocampal unit a c t i v i t y and synaptic e f f i c a c y (Laroche & Bloch, 1982, pp. 583-590). The question of the relationship between' the hippocampus, learning and LTP has also been approached using h i s t o l o g i c a l techniques. Dendritic spines from the DG of mice exposed to a single 2-hr conditioning session are reportedly larger than corresponding spines in mice fed non-contingently (Fifkova & Van Harreveld, 1978). The s i m i l a r i t y between these results and previously reported effects of high-frequency stimulation of the PP (Van Harreveld & Fifkova, 1975) led the authors to c i t e LTP as the mechanism responsible for both changes and their data as proof that LTP subserves learning (Fifkova & Van Harreveld, 1978). However, these data and their interpretation have been questioned on several grounds. F i r s t , no evidence of successful conditioning was provided (Fifkova & Van Harreveld, 1978). Second, no evidence of LTP was presented in either of the e a r l i e r papers (Fifkova & Van Harreveld, 1977; Van Harreveld & Fifkova, 1975). Third, the changes in the spines lasted less than four hours (Fifkova & Van Harreveld, 1977). Fourth, 29 subsequent studies in which proper electrophysiological procedures were employed found very d i f f e r e n t patterns of morphological changes and provided strong evidence that the areas of dendritic spines were not changed by LTP (Lee et a l . , 1981, pp. 189-212; Lee et a l . , 1980). In summary, none of these studies of unit a c t i v i t y , sensory-evoked potentials or dendritic spine areas were able to provide dir e c t evidence of increases in synaptic e f f i c a c y during learning. Clearly, there are many problems inherent in trying to observe neuronal changes' during learning- and infer a role for' such a l t e r a t i o n s in synaptic e f f i c a c y in memory. What i s needed is a more direct approach that could overcome the three main problems of 1) l o c a l i z i n g the stimulus-related neural a c t i v i t y in the brain, 2) assessing changes in synaptic e f f i c a c y and 3) providing evidence for a causal relationship between the neuronal and behavioural changes. One direct approach would be to use* e l e c t r i c a l brain stimulation (EBS) of a well-defined pathway as a conditioned or discriminative stimulus (CS or DS) in a learning paradigm. Stimulus control by EBS would demonstrate the existence of a r e l a t i o n s h i p between behaviour and a fixed amount of neural a c t i v i t y in a l o c a l i z e d brain region. Measurements of EBS-evoked postsynaptic a c t i v i t y would provide an index of synaptic e f f i c a c y . If the EBS were to activate a system known to exhibit LTP, the effects of tetanic stimulation and consequent LTP upon the rate of conditioning could be studied. Through the use of tetanic stimulation as an experimental manipulation i t should be 30 possible to infer a causal r e l a t i o n s h i p between changes in synaptic e f f i c a c y and changes in behaviour. The following sections w i l l describe previous uses of EBS as a stimulus in a behavioural context and establish the f e a s i b i l i t y of this approach. Stimulus Properties of Brain Stimulation It has been established that EBS of many d i f f e r e n t regions of the brain can serve as CS or DS in a variety of learning paradigms (Doty, 1969') . This a b i l i t y to- acquire stimulus' control over responding demonstrates that EBS can have "stimulus properties". In other words, the EBS has some properties in common with exteroceptive s t i m u l i , even though the neural a c t i v i t y generated by the EBS i s not necessarily the sameas that generated by any other conditioned or unconditioned s t i m u l i . The exact nature of the perceptual properties of an EBS stimulus i s irr e l e v a n t . The stimulus properties of EBS are operationally defined by the a b i l i t y to acquire control over responding in any conditioning paradigm, in the same way that a CS is defined by i t s use in a Pavlovian conditioning paradigm and a DS is defined by i t s use in an operant conditioning paradigm. There is a great deal of evidence for the s i m i l a r i t y between conditioning with exteroceptive and EBS s t i m u l i . The rate of conditioning and transfer of stimulus control between EBS and exteroceptive stimuli indicate that an animal learns about both in very similar ways, in either Pavlovian of operant 31 paradigms. This appears to be true regardless of whether the EBS is used as the CS (Doty, 1969; Mis, Norman, Hurley, Lohr & Moore, 1974; Moore, Marchant, Norman, & Kwaterski, 1973; Woody & Yarowski, 1973), the unconditioned stimulus (Doty, 1969; Kandel & Benevento, 1973; Mis, Gormezano & Harvey, 1979; Pirch, Corbus & Napier, 1981; Powell & Moore, 1980; Shinkman & Bruce, 1979), or both (Heinricher, Rosenfeld & Dowman, 1981). Although there are instances where careful behavioural analysis has revealed differences between EBS and exteroceptive stimuli (e.g., Ramer & Wilkie, 1977), on the whole, the use" of EBS stimuli to reveal neuronal changes in learning would appear to be j u s t i f i e d . Considerable success has been achieved in the use of conditioned EBS to trace the flow of information through the nervous system. For example, one early study compared the effects of two methods of surgical i s o l a t i o n of homotopic c o r t i c a l gyri to show that the- fibres mediating the stimulus properties of c o r t i c a l EBS were corti c o f u g a l rather than i n t r a c o r t i c a l (Rutledge & Doty, 1962). A related study has revealed a fundamental difference between the functions of the anterior commissure and the splenium of the corpus callosum in the interhemispheric communication of neural a c t i v i t y during and after learning (Doty & Overman, 1977, pp. 75-88). Tests of discrimination and generalization between EBS of d i f f e r e n t areas have two very straightforward hypotheses. S p e c i f i c a l l y , there should be generalization and rapid transfer of stimulus control between anatomically connected s i t e s when both l i e along the neural path mediating the stimulus 32 properties. In addition, there should be r e l i a b l e discrimination between s i t e s which are anatomically "disconnected" or which subserve d i f f e r e n t behavioural functions (Doty, 1969). In general, experimental findings have been consistent with these hypotheses. Doty (1969) c i t e s examples of complete stimulus generalization between two electrode sites confined to the auditory cortex, s t r i a t e cortex, r e t i c u l a r formation or medial lemniscus. Generalization or rapid transfer between electrode s i t e s have- also' been- reported' for' homo t o p i c locations- in l e f t and right s t r i a t e cortex (Doty, 1965), auditory cortex (Swadlow & Schneiderman, 1970), and caudate nuclei (Manning & Schneiderman, 1970). Rapid transfer has been shown to occur between d i r e c t l y connected s i t e s l i k e the l a t e r a l geniculate nuclei and the visu a l cortex (Doty, 1965) or the l a t e r a l hypothalamus and the septum (Stutz, Rossi, Hastings & Brunner, 1974). Good discrimination has generally occurred between sites not connected d i r e c t l y (Stutz et a l . , 1974), or between electrode placements where higher intensity stimulation has d i f f e r e n t motivational properties (Doty, 1969; Hupka, 1970; Stutz, 1968). These examples show one way in which the the functional importance of anatomical connections has been established. In the analysis of the neuronal changes subserving learning, the main advantage offered by the use of EBS as a stimulus i s the degree to which the neural a c t i v i t y known to control behaviour can be kept constant and l o c a l i z e d to a 33 s p e c i f i c region of the brain. Exteroceptive stimuli usually produce neural a c t i v i t y in a number of regions of the brain, the le v e l of a c t i v i t y may vary greatly between t r i a l s and much of this a c t i v i t y could be completely unrelated to the behavioural reaction (Olds et a l . , 1973). A paradigm in which EBS i s a stimulus permits the accurate l o c a l i z a t i o n and qua n t i f i c a t i o n of neural a c t i v i t y in the spec i f i e d system, and thereby makes i t possible to evaluate changes in synaptic e f f i c a c y during learning. A recent, very elegant' study employing these principles' of l o c a l i z a t i o n of neural a c t i v i t y has provided the strongest evidence to date for the occurrence of increases in synaptic e f f i c a c y during learning (Tsukahara, Oda & Notsu, 1981). In thi s study, the CS was e l e c t r i c a l stimulation of fib r e s known to project from the cerebral peduncle to the red nucleus and thence through two other synaptic connections to the biceps muscle. Repeated pairings of the CS and footshock led to the development of consistent responses to previously subthreshold stimulation and to a systematic decrease in the threshold of the withdrawal response. The data from CS-only, US-only, backwards- and randomly-paired groups c l e a r l y demonstrated that the effect was due to conditioning and not to s e n s i t i z a t i o n or to the effects of the EBS per se. In this study, there was evidence for changes in the input/output relationship of the cor t i c o f u g a l f i b r e s and c e l l s of the red nucleus. The latency of the electrographic response in the biceps muscle confirmed the conduction of the EBS-evoked 3 4 a c t i v i t y by the t r i s y n a p t i c pathway. Additionally, analysis of a second input to the c e l l s of the red nucleus showed that neither the overal l e x c i t a b i l i t y of the c e l l s of the red nucleus nor the e f f i c a c y of the other synapses mediating the response were affected by the conditioning. Although the authors favoured the formation of new synapses as the explanation of the increase in synaptic e f f i c a c y , the data were consistent with the changes observed with LTP. The only important evidence not provided was that derived from direct recording of stimulation-evoked a c t i v i t y in the c e l l s of the red nucleus'. Although t h i s study employed the p r i n c i p l e of l o c a l i z i n g neural a c t i v i t y through the use of an EBS-CS, the analysis of the relationship between synaptic changes and learning could be considered to -be passive. Behavioural changes were induced through conditioning and were then shown to be correlated with a l t e r a t i o n s in neuronal a c t i v i t y or e x c i t a b i l i t y . A more di r e c t approach to the analysis of the relationship between behavioural and neuronal alt e r a t i o n s would be to induce a neuronal change and test for alt e r a t i o n s in behaviour. The neuronal change of primary interest here is LTP. The major d i f f i c u l t y with t h i s approach is ensuring that the neuronal change is produced in a synaptic system relevant to the behaviour being observed. This problem can be overcome through the use of EBS as a conditioned or discriminative stimulus. There are three stages in the analysis of the effects of LTP in a system subserving the stimulus properties of EBS. F i r s t , the stimulus properties of EBS in a hippocampal system 3 5 must be demonstrated. Then, the effects of LTP produced by tetanic stimulation upon the stimulus properties must be determined. F i n a l l y , the relationship between the neuronal a c t i v i t y monitored in the hippocampal system and the neural a c t i v i t y subserving the stimulus properties of the EBS must be c l e a r l y shown. The three behavioural experiments in t h i s thesis are each addressed to one of these three stages. Experiments The- primary objective of this thesis rs to provide evidence for a r e l a t i o n s h i p between LTP and learning by showing that increases in synaptic e f f i c a c y produced by tetanic stimulation a f f e c t the translation of a fixed amount of neuronal a c t i v i t y into a behavioural reaction. Single-pulse EBS of the angular bundle w i l l be used to produce a fixed amount of neuronal a c t i v i t y in the PP. Evoked potentials recorded from the DG w i l l confirm that each stimulation of the angular bundle produces direc t a ctivation of the PP and synaptic activation of dentate granule c e l l s . This same single-pulse stimulation w i l l be examined for stimulus properties in a behavioural context. The tetanic stimulation w i l l be applied to the f i b r e s of the PP through the same stimulating electrodes as the single-pulse stimulation and the effect of the resulting LTP on the stimulus properties of angular bundle stimulation w i l l be assessed. This thesis w i l l be comprised of four experiments. The f i r s t w i l l be an electrophysiological investigation of LTP produced by low frequency stimulation. The other three w i l l 36 examine the stimulus properties of angular bundle stimulation in an operant paradigm. The f i r s t behavioural experiment w i l l e s tablish whether single-pulse stimulation of the angular bundle that activates the PP can acquire stimulus control over responding for food. The second behavioural experiment w i l l determine whether an increase in the synaptic e f f i c a c y of the PP-DG system ( i . e . , LTP) w i l l a f f e c t the rate of acqui s i t i o n of stimulus control by the EBS. The t h i r d experiment w i l l examine the relationship between the stimulus properties of the angular bundle' stimulation and' the activation of the PP. S p e c i f i c a l l y , whether the behavioural significance of angular bundle stimulation depends to some degree upon the i n t e g r i t y of the PP between the stimulating and recording s i t e s . This w i l l be accomplished through the use of lesions anterior to the stimulation s i t e that w i l l s e l e c t i v e l y damage the fibres of the PP. 37 CHAPTER 2: Long-Term Potentiation and Low-Frequency Stimulation. Despite the p r o l i f e r a t i o n of studies on the phenomenon of LTP, important questions remain as to the c r i t i c a l parameters of the e l e c t r i c a l stimulus that are necessary for the production of LTP. Several studies have shown that the magnitude of LTP is related to the stimulation frequency (Alger & Teyler, 1976; Barrionuevo, Schottler & Lynch, 1980; B l i s s & Lomo, 1973; Douglas & Goddard, 1975; McNaughton et a l . , 1978) but the minimum frequency capable of producing LTP has not yet been c l e a r l y defined. It is absolutely essential to i d e n t i f y stimulation parameters that do not produce LTP, in order to monitor synaptic e f f i c a c y over long periods. This is especially true in the context of t h i s thesis, which examines the relationship between changes in synaptic e f f i c a c y and changes in behaviour. Early studies of LTP in the PP-DG systems of both anesthetized and freely-moving rabbits showed that stimulation at frequencies of 10, 15, 20 and 100 Hz produced LTP but 0.5 Hz stimulation did not ( B l i s s & Gardner-Medwin, 1973? B l i s s & Lomo, 1973). In a later study using freely-moving rats, LTP was found to be produced r e l i a b l y by stimulation of the PP at a number of frequencies greater than 10 Hz (Douglas & Goddard, 1975). In t h i s report, i t was mentioned that stimulation at lower rates (3 or 0.2 Hz) produced LTP only in a minority of cases (Douglas & Goddard, 1975). 38 Low frequency stimulation has also been found to produce a depression of postsynaptic responses. In one study, the magnitude of increases in population spike amplitudes was found to be proportional to the frequency of stimulation (Dunwiddie & Lynch, 1978). Stimulation of the Schaffer c o l l a t e r a l (Sch) - CA1 system in the in v i t r o hippocampus at 100 Hz and at a voltage just above the threshold of the population spike led to large increases in population spike amplitudes but stimulation at 33 and 15 Hz produced much smaller changes. Stimulation at 5 Hz produced only marginal potentiation while 1 Hz stimulation actually depressed the evoked responses (Dunwiddie & Lynch, 1978) . The analysis of the depression produced by 1 Hz stimulation was extended in a recent study of the Sch-CA1 system in anesthetized rats (Barrionuevo et a l . , 1980). Subthreshold stimulation at 1 Hz for 100 sec was found to depress population EPSP's for 2-15 min. Subsequent stimulation with 100 pulses at 100 Hz led to stable increases, but a second 100 pulse t r a i n of stimulation at 1 Hz reversed t h i s potentiation, reducing the EPSP's to the o r i g i n a l control l e v e l s . The authors suggested that low-frequency stimulation activated a hippocampal mechanism which reversed the otherwise durable LTP produced by high-frequency stimulation. In summary, there have been c o n f l i c t i n g reports as to the existence and direction of long-term changes produced by stimulation at frequencies of 1 Hz or less. The present study re-examined the long-term changes in synaptic e f f i c a c y produced 3 9 by low frequency stimulation of the PP-DG system of freely moving rats. The measurement of synaptic e f f i c a c y was made as comprehensive as possible through analysis of input/output (I/O) curves. A number of fixed inputs were produced by stimulation over a large range of i n t e n s i t i e s and measurements of population spike amplitudes provided a measure of the output of the PP-DG system. Several laboratories have used I/O curves routinely to provide a description of the changes in postsynaptic responsiveness (Alger & Teyler, 197'&; B l i s s & Gardner-Medwin, 1973; B l i s s & Lomo, 1973; Teyler et a l . , 1977; Wilson, 1 9 8 1 ; Wilson et a l . , 1981 ). These studies have revealed .the importance of evaluating responses at more than one stimulation intensity. In most of these reports, high-frequency stimulation was shown to produce leftward s h i f t s in the I/O curves. These s h i f t s usually resulted from three changes in the population responses; 1) decreases in threshold, 2) large r e l a t i v e increases at low i n t e n s i t i e s and 3) large absolute increases at high i n t e n s i t i e s . To date, there have been no systematic attempts to compare entire I/O curves in order to evaluate changes in synaptic e f f i c a c y . In the present experiment, I/O curves were co l l e c t e d d a i l y and successive curves were compared by c a l c u l a t i n g the geometric area under each curve. Because th i s measure was sensitive to decreases in threshold and absolute increases in population spike amplitudes at a l l i n t e n s i t i e s tested, i t provided a comprehensive and sensitive evaluation of changes in synaptic e f f i c a c y . 40 The present experiment examined the long-term effects of PP stimulation at three low frequencies (0.2, 0.1 and 0.04 Hz) on responses in the DG. The areas of I/O curves c o l l e c t e d at 24 hr intervals were compared to the areas of curves c o l l e c t e d on the f i r s t (baseline) day of testing and percentage scores were calculated for each animal on each day. The only stimulation pulses used in t h i s study were those pulses necessary for the generation of I/O curves. The primary objective of this study was to estimate the minimum frequency of e l e c t r i c a l stimulation capable of producing LTP. METHODS Implantation and Histology Male albino Wistar rats weighing 300-350 gms, were implanted under sodium pentobarbital (Nembutal) anesthesia with recording and stimulating electrodes in the h i l u s of the DG and in the PP respectively. The recording electrodes were constructed from single strands of teflon-coated wire and the monopolar stimulating electrodes were 00 stainless steel insect pins insulated to within 0.4 mm of their t i p s . The in d i f f e r e n t and current electrodes were stainl e s s steel s k u l l screws joined together with 36 ga s t a i n l e s s steel wire. A l l electrodes and wires were soldered to gold-plated pins (Amphenol 220-P02) prior to implantation. The electrodes were positioned s t e r e o t a x i c a l l y in the DG (AP: 3.3 mm posterior to bregma, L: 1.8 mm l a t e r a l to the 41 midline, and V: 3.5 mm ventral to the surface of the cortex) and PP (AP: 8.1 mm posterior to bregma, L: 4.3 mm l a t e r a l to the midline and V: 2.5 mm ventral to the surface of the cortex). The s k u l l was l e v e l between bregma and lambda and both electrodes were on the l e f t side of the brain. Single, constant current, cathodal square wave pulses of 0.1 msec duration were delivered at 0.2 Hz and evoked potentials were monitored as the electrodes were lowered into the target areas. The t o t a l number of pulses delivered during surgery was usually less than 40. Recording electrodes were positioned for maximal h i l a r responses and the stimulating electrodes were positioned so that the thresholds of the population spikes were less than 100 pA. The electrodes were then fixed in place with dental a c r y l i c and the animals were given two weeks to recover from the ef f e c t s of surgery. At the end of the experiment, the animals were anesthetized with urethane (1.5 mg/kg) and the locations of the electrode t i p s were marked by passing a 25 pA DC anodal current for 30 sec. The animals were k i l l e d with an overdose of anesthetic and perfused with 0.15M saline followed by 2% potassium ferrocyanide in 10% formalin. The locations of the stimulating and recording electrode s i t e s were determined from 50 um, thionin-stained, frozen sections. 42 S t i m u l a t i o n N i n e a n i m a l s w e r e u s e d t o a s s e s s t h e e f f e c t s o f s t i m u l a t i o n a t t h r e e l o w f r e q u e n c i e s : 0 . 2 , 0 .1 a n d 0 . 0 4 Hz ( 1 / 5 , 1 /10 a n d 1 / 2 5 s e c r e s p e c t i v e l y ) . S i x o f t h e s e a n i m a l s , t w o a t e a c h f r e q u e n c y , w e r e g i v e n 30 p u l s e s e a c h d a y , i n an a s c e n d i n g s e r i e s o f 10 c u r r e n t i n t e n s i t i e s : 2 0 , 4 0 , 6 0 , 8 0 , 1 0 0 , 1 5 0 , 2 0 0 , 2 5 0 , 300 a n d 400 p A . T h r e e o t h e r a n i m a l s w e r e u s e d t o a s s e s s t h e e f f e c t s o f an i n c r e a s e d number o f p u l s e s a t 0 .1 H z . T h e s e a n i m a l s r e c e i v e d 98 p u l s e s e a c h d a y i n a s e r i e s o f 14 i n t e n s i t i e s ( 1 0 , 3 0 , 50 a n d 120 p A w e r e i n s e r t e d ' i n t o t h e p r e v i o u s s e r i e s o f c u r r e n t i n t e n s i t i e s ) . I n t h e s e a n i m a l s , s e v e n p u l s e s w e r e d e l i v e r e d a t e a c h i n t e n s i t y . One a d d i t i o n a l a n i m a l was u s e d t o a i d t h e c o m p a r i s o n o f t h e e f f e c t s o f l o w - a n d h i g h -f r e q u e n c y s t i m u l a t i o n . T h i s a n i m a l r e c e i v e d t e t a n i c s t i m u l a t i o n i m m e d i a t e l y a f t e r t h e f i r s t d a y ' s t e s t i n g w i t h 98 p u l s e s a t 0 .1 H z . T h i s t e t a n i c s t i m u l a t i o n (200 H z ) c o n s i s t e d o f 20 t r a i n s o f 10 p u l s e s a t 250 pA w i t h 10 s e c i n t e r v a l s b e t w e e n t h e t j r a i n s . D a t a A c q u i s i t i o n a n d A n a l y s i s T e s t i n g was c o n d u c t e d a t a b o u t t h e same t i m e e a c h d a y w h i l e t h e a n i m a l s moved f r e e l y a b o u t a wooden box 30 cm s q u a r e . T h r o u g h o u t t h e s t u d y , t h e s t i m u l a t i o n p u l s e s w e r e c o n t i n u o u s l y m o n i t o r e d on a s t o r a g e o s c i l l o s c o p e . The e v o k e d p o t e n t i a l s a t e a c h c u r r e n t i n t e n s i t y w e r e a m p l i f i e d , f i l t e r e d ( 0 . 1 - 3 k H z ) , d i s p l a y e d on a s t o r a g e o s c i l l o s c o p e a n d f e d i n t o a PDP 1 1 / 1 0 c o m p u t e r f o r a v e r a g i n g a n d h a r d c o p y p r o d u c t i o n . A s h o p - b u i l t p r e - a m p l i f i e r w i t h a h i g h i n p u t i m p e d a n c e (>10 M Ohm) a n d a g a i n 43 of 10 was located on the lead close to the animals head and connected to the recording electrode. Daily I/O curves were generated by p l o t t i n g population spike amplitudes (output) against current i n t e n s i t i e s (input). The geometric area under each I/O curve was calculated by treating the I/O curve and the X-axis as a polygon and using the general formula for the areas of r e c t i l i n e a r figures (Durrant, Kingston, Sharp & Kerr, 1961, p. 30). In order to assess changes in synaptic e f f i c a c y , the area of the I/O curves obtained from each rat on each day were compared' to control areas- measured on the f i r s t test day and a percentage score was calculated. The steps in the conversion of the data from evoked potentials to percentage area are summarized in Figures 2.1 and 2.2. RESULTS A l l stimulating electrodes were located in the PP and a l l recording electrodes were located in the h i l u s of the DG (Fig 2.3). At higher current i n t e n s i t i e s (200-400 p.A) the evoked potentials of a l l animals contained unambiguous population spikes. The observed differences between animals in the thresholds, latencies and maximal amplitudes of the population spikes could a l l be attributed to minor differences in electrode placements. In the one animal given tetanic stimulation, the observed changes in the I/O curves were similar to those previously reported to follow tetanic stimulation (Alger & Teyler, 1976; F i g u r e 2.1: T e t a n i c s t i m u l a t i o n and s y n a p t i c e f f i c a c y . A. Evoked p o t e n t i a l s at three c u r r e n t i n t e n s i t i e s sampled j u s t b efore and 24 hrs a f t e r t e t a n i c s t i m u l a t i o n (200 Hz) . C a l i b r a t i o n s : 2 mV, 5msec. B. Input/output (I/O) curves b e f o r e and -24 hrs a f t e r 200 Hz s t i m u l a t i o n . The area bounded by the post t e t a n i c I/O curve was c a l c u l a t e d and compared to the area under the p r e - t e t a n i c curve (shaded). C. The percentage i n c r e a s e s of I/O curve area on the 4 t e s t days f o l l o w i n g t e t a n i c s t i m u l a t i o n . MS Test Day Figure 2.2: Stimulation at 0.2 Hz and synaptic efficacy A. Evoked potentials at three current intensities on Days 1 and 7 of testing. Calibrations: 2 mV, 5 msec. B. Complete I/O curves are shown for one animal on Days 1 (closed circles) and 7 (open c i r c l e s ) . The area under the I/O curve from Day 1 (shaded) served as the control value against which other I/O curve areas were compared. C. Percentage increases in the area under the I/O curve of one animal over 7 days of testing. 46 Figure 2.3: Histology. The locations of stimulating ( T ) and recording (•) electrodes are shown on p a r t i a l coronal sections redrawn from Konig and Rlip p e l (1963)'. Left posterior cortex and' l e f t anterio-dOrsal hippocampus are depicted. The approximate distance (mm) of each section from Bregma i s indicated. 4? 48 B l i s s & Gardner-Medwin, 1973; B l i s s & Lomo, 1973; Teyler et a l . , 1977; Wilson, 1981; Wilson et a l . , 1981). The amplitudes of the population spikes were larger at a l l current i n t e n s i t i e s but the percentage increase varied (Fig 2.1A,B). The net result was a 56% increase in the area of the I/O curve as measured the following day (Fig 2.1B,C). The magnitude of the LTP produced by the tetanic stimulation was greatest on Day 1 and declined over the next 3 days (Fig 2.1C). Low-frequency stimulation of the PP produced alt e r a t i o n s in responses in the DG' similar to those observed following' high frequency stimulation (Figs 2.1C, 2.2C). Gradual, cumulative increases in synaptic effi c a c y were observed in both animals tested with 30 pulses per day at 0.2 Hz (Figs 2.2C, 2.4A). After seven days of testing, the areas under the I/O curves exceeded 200% of control values (Fig 2.4A). This change in area was due to increases in population spike amplitudes at a l l i n t e n s i t i e s tested and to a decrease in the threshold of the population spikes (e.g., Fig 2.2B). Stimulation at 0.1 Hz produced mixed e f f e c t s , ranging from a depression (Fig 2.4B), to a potentiation of population spike amplitudes (Fig 2.4D). Increases in synaptic e f f i c a c y were evident following stimulation with 98 pulses per day but not following 30 pulses per day. Although the rate of increase in I/O curve area over the f i r s t test days appeared to be greater at 0.1 Hz than at 0.2 Hz, i t should be noted that asymptotic l e v e l s were reached after approximately the same number of pulses, regardless of frequency; 210 pulses at 0.2 Hz, 1 9 8 41 F i g u r e 2.4: Low-frequency s t i m u l a t i o n and s y n a p t i c e f f i c a c y A., B. & C. The e f f e c t s of d a i l y t e s t i n g with 0.2, 0.1 or 0.04 Hz r e s p e c t i v e l y . Each curve shows the changes i n the area under the I/O curves of i n d i v i d u a l animals, t e s t e d d a i l y with an s e r i e s of 30 p u l s e s at ascending i n t e n s i t i e s . D. The changes i n the areas under the I/O curves of three animals t e s t e d d a i l y with a s e r i e s of 98 at ascending i n t e n s i t i e s . 50 pulses at 0.1 Hz. Stimulation with 30 pulses at 0.04 Hz produced no appreciable, systematic changes in synaptic e f f i c a c y . DISCUSSION The results of the present experiment indicated that LTP was produced by e l e c t r i c a l stimulation at frequencies lower than 1 Hz. Stimulation at 0.2 Hz, and in two cases, at 0.1 Hz led to changes in population spike amplitudes very similar to al t e r a t i o n s previously reported to follow tetanic stimulation (Alger & Teyler, 1976; B l i s s & Gardner-Medwin, 1973; B l i s s & Lomo, 1973; Teyler et a l . , 1977; Wilson, 1981; Wilson et a l . , 1981). In some animals, the percentage increases in the areas of the I/O curves produced by stimulation at low frequencies were as great as those produced by stimulation at 200 Hz in one animal. Stimulation at a lower frequency (0.04 Hz) appeared to have no e f f e c t s upon synaptic e f f i c a c y . There was some indication of an interaction between the frequency of stimulation and the number of pulses per day. In two of three cases, 98 pulses per day led to potentiation whereas 30 pulses per day at the same frequency produced either no effect or a depression of responses in the DG. These results confirm a brief report describing LTP following low-frequency stimulation in the PP-DG system of freely-moving rats (Douglas & Goddard, 1975). In that study, 136 pulses per day of stimulation at 3 or 0.2 Hz and 300 uA led to the development of LTP in five of 11 cases. However, the results 51 were not presented in d e t a i l and no d i s t i n c t i o n was made between animals given 3 Hz and 0.2 Hz stimulation. In addition, recording s i t e s in the h i l u s of the DG were not considered separately from those in the dendritic layers of the DG or CA1. The present study extends these findings in a number of important respects. F i r s t , a l l responses were recorded from the same area of the brain, the hilus of the DG. Second, the e f f e c t s of low-frequency stimulation were examined across a broad range of stimulation i n t e n s i t i e s . Third, the lower l i m i t of frequencies capable of" producing LTP was extended to 0.1 Hz, and' the minimum number of pulses per day required to produce LTP at low frequencies was reduced to 30. The present study therefore provides a more refined and detailed analysis of the effects of low-frequency stimulation. The demonstration of LTP following stimulation at 0.1 and 0.2 Hz stands in sharp contrast to previous reports of a depression of population responses produced by stimulation at 1 Hz (Barrionuevo et a l . , 1980; Dunwiddie & Lynch, 1978). There are three major methodological differences which could account for the discrepancy; f i r s t , the type of preparation used (freely-moving vs. unanesthetized rat or the in v i t r o hippocampal s l i c e ) ; second, the hippocampal systems studied (PP-DG vs. Sch-CA1); or t h i r d , the intensity of the stimulation used to measure and change synaptic e f f i c a c y (low to high vs. subthreshold or threshold). Although the e f f e c t s of very low frequency stimulation ( i . e . , < 1 Hz) have never been systematically compared in 52 d i f f e r e n t hippocampal preparations or systems, i t is unlikely that either of these two factors could account for the discrepancy between the present and previous findings. Stimulation at a moderate frequency (15 Hz) produces LTP of comparable magnitude in the PP-DG of unanesthetized and anesthetized rabbits (Bliss & Gardner-Medwin, 1973; B l i s s & Lomo, 1973), in the Sch-CA1 system of in vivo (anesthetized) and in v i t r o preparations (Teyler et a l . , 1977), and in the PP-DG, mossy fibre-CA3 and Sch-CA1 systems in v i t r o (Alger & Teyler, 1976) . The most l i k e l y explanation for the contrasting e f f e c t s of low-frequency stimulation i s the difference in the current i n t e n s i t i e s used in the present and previous studies. The interaction of stimulation frequency and intensity was noted in the preceding chapter. Stimulation at frequencies greater than 100 Hz leads to LTP even at current i n t e n s i t i e s very near the population spike threshold ( B l i s s & Lomo, 1973; McNaughton et a l . , 1978; Wilson, 1981; Yamamoto & Sawada, 1981). In contrast, stimulation at frequencies below 20 Hz produces LTP only at current i n t e n s i t i e s well above th i s threshold (Alger & Teyler, 1976; B l i s s & Lomo, 1973; Douglas & Goddard, 1975; Dunwiddie & Lynch, 1978; Yamamoto & Sawada, 1981). Both studies which reported a depression of responses following 1 Hz stimulation used current i n t e n s i t i e s at or below population spike threshold (Barrionuevo et a l . , 1980; Dunwiddie & Lynch, 1978). In the present study, much of the stimulation used was well above threshold and LTP was observed following 0.2 Hz stimulation. 53 Six years ago, i t was suggested that low-frequency, high-intensity stimulation might potentiate responses while low-frequency, low-intensity stimulation might depress them (Alger & Teyler, 1976). The difference between the present and previous findings supports t h i s conclusion. Furthermore, the present findings indicate that a s i g n i f i c a n t reduction in any of a number of stimulation parameters may reverse the e f f e c t of stimulation from potentiation to depression. S p e c i f i c a l l y , the results showed that a reduction of frequency from 0.2 to 0.1 Hz or a reduction of the number of pulses from 98 to 30 per day could reverse the d i r e c t i o n of the change in synaptic e f f i c a c y from potentiation to depression (Figs 2.4A,D vs. 2.4B). These results also indicated that the parameters of stimulation used to monitor synaptic e f f i c a c y can be reduced to the point where no a l t e r a t i o n s in synaptic e f f i c a c y are produced (Fig 2.4C). The present study supports the conclusion of Barrionuevo et a l . (1980), but with an important q u a l i f i c a t i o n . The hippocampus may indeed possess a mechanism for reversing otherwise durable increases in synaptic e f f i c a c y , but t h i s mechanism would appear to be influenced by more than just the frequency of neuronal a c t i v i t y . The well-established interaction between stimulation frequency and intensity provides additional evidence that the mechanism responsible for LTP is sensitive to more than just stimulation frequency. In the f i n a l analysis, the most important feature of the present study is the evidence that stimulation at 0.04 Hz does not a l t e r synaptic e f f i c a c y but that stimulation at 0.1 Hz does. 54 These results have important p r a c t i c a l applications. Many studies of p l a s t i c i t y in the hippocampus require a stable baseline of evoked a c t i v i t y against which to measure changes in synaptic e f f i c a c y . In a l l of these studies, i t i s absolutely es s e n t i a l to ensure that the stimulation used to monitor synaptic e f f i c a c y does not a l t e r the preparation under study (Alger & Teyler, 1976). The present findings indicate that stimulation used to monitor synaptic e f f i c a c y should be presented at a frequency less than 0.1 Hz or fewer than 30 times each day. This consideration is p a r t i c u l a r l y important in attempts to establish a relationship between changes in synaptic e f f i c a c y and changes in behaviour. 55 CHAPTER 3; Stimulus Properties of Angular Bundle Stimulation The focus of this thesis is on the use of an EBS stimulus to i d e n t i f y the relationship between changes in synaptic e f f i c a c y and changes in behaviour. The f i r s t major step in this endeavour i s taken by the study described in t h i s Chapter, namely, the demonstration that single-pulse stimulation of the angular bundle can acquire stimulus control over responding. The analysis of the stimulus properties of single-pulses and angular bundle stimulation both represent advances in the use of EBS stimuli in a behavioural context. There were a number of reasons for selecting the angular bundle as the s i t e of the EBS. Angular bundle stimulation activates the fibres of the PP and produces postsynaptic c e l l f i r i n g s in the DG (Andersen, et a l . , 1971a; Lomo, 1971a). Therefore, the implantation of both stimulating and recording electrodes in the PP-DG system can be accurately guided by the waveforms of the evoked potentials obtained during surgery (Chap. 2). In addition, evoked potentials obtained throughout the course of a study provide confirmation that each stimulation pulse activates the PP. F i n a l l y , the PP-DG i s the only hippocampal system in which LTP has been studied using f r e e l y -moving animals ( B l i s s & Gardner-Medwin, 1973; Deadwyler et a l . , 1976; Douglas, 1977; Douglas & Goddard, 1975). Consequently, the PP-DG is the only system in which stable evoked potentials and LTP have been demonstrated over periods greater than one day. 56 Single-pulses were used as the EBS to measure synaptic e f f i c a c y throughout conditioning-, without inducing changes". The s e n s i t i v i t y of the PP-DG system to e l e c t r i c a l stimulation was shown c l e a r l y in the previous chapter. By using single-pulses and recording evoked potentials, synaptic e f f i c a c y could be monitored on every t r i a l and any changes in the neuronal a c t i v i t y induced by the EBS stimulus could be assessed. In the context of t h i s thesis, i t is very important to be able to evaluate the postsynaptic response to stimulation and to measure how these responses change under the influence of conditioning and tetanic stimulation. Even though evoked a c t i v i t y can be recorded following trains of pulses, the interpretation of these evoked potentials is d i f f i c u l t . Single-pulse stimulation produces a r e l a t i v e l y simple a c t i v a t i o n -i n h i b i t i o n sequence in dentate granule c e l l s (Assaf & M i l l e r , 1978). In contrast, stimulation with two or more pulses produces complicated events, whose nature i s determined largely by the intervals between the pulses (Alger & Teyler, 1976; Assaf & M i l l e r , 1978; Lomo, 1971a,b). Multiple pulse stimulation can even produce epileptiform afterdischarges which have d r a s t i c , long-lasting consequences (Douglas & Goddard, 1975; Goddard, Mclntyre & Leech, 1969). The long-term e f f e c t s of tetanic stimulation on the complex postsynaptic responses to trains of stimulation have not been documented. The major d i f f i c u l t y inherent in any attempt to demonstrate stimulus control by a single pulse of EBS is the p o s s i b i l i t y that each pulse might not produce s u f f i c i e n t neural a c t i v i t y to 57 influence behaviour. Observations of the animals during electrophysiological t e s t i n g (Chap. 2) revealed no orienting responses nor any a l t e r a t i o n in ongoing behaviours following single-pulse stimulation. In addition, there have been no reports of successful conditioning with a single-pulse EBS stimulus used from the outset of tra i n i n g , although in some studies, single pulses have e l i c i t e d a response previously conditioned to trains of pulses (Doty, 1969; Freeman, 1962; Woody and Yarowsky, 1973). If single-pulse, angular bundle stimulation has any stimulus' properties', these'' would be shown best under circumstances conducive to the acquis i t i o n of stimulus control by a weak, brief stimulus. This consideration guided the design of the behavioural paradigm and experimental procedures used in the present study. There were four relevant features of the behavioural paradigm. F i r s t , the task used was appetitive, with food reinforcement, so that many t r i a l s could be presented each day without overly stressing the animals. Second, the measured response was simple for the animals to perform and was unambiguously related to the food: a response was recorded every time the animals examined the inside of their food cup. Third, the DS signalled the start of a 10 sec period in which a response would lead to the immediate delivery of food. Thus, a close response-reinforcer co r r e l a t i o n was present during the post-DS period. Fourth, the food was delivered at the end of the 10 sec i f no response was made, in order to maintain a high DS-reinforcer c o r r e l a t i o n . The presentations of the DS and food 58 were spaced by variable i n t e r v a l s , so that the time since the previous reinforcer had l i t t l e or no predictive value, and the only r e l i a b l e predictor of the response-reinforcement contingency period was the DS. This paradigm was b a s i c a l l y a successive operant discrimination (go/no-go) task because responses were reinforced only during a brief period after the DS and not during the i n t e r t r i a l i n t e r v a l when only background stimuli were present. Two other methodological procedures were incorporated to f a c i l i t a t e the acquisition of stimulus - control by the' EBS-DS'. Prior to the onset of conditioning with the EBS-DS, a l l animals were trained with a brief (0.1 sec) tone as the DS u n t i l stimulus control by the tone was established. It was hoped that this pre-training with an exteroceptive stimulus and consequent exposure to the apparatus and reinforcement contingencies would speed the acqu i s i t i o n of stimulus control by the EBS-DS. Studies of stimulus control transfer between exteroceptive stimuli have shown that acquisition of rates in. succesive operant discrimination (go/no-go) tasks are f a c i l i t a t e d by previous experience with this type of discrimination (Mackintosh, 1974, pp. 600-601). Most important in the present context i s the previously successful use of pre-training with exteroceptive stimuli prior to conditioning with an EBS stimulus (Freeman, 1962). In addition to t h i s pre-training, special care was taken to eliminate or mask exteroceptive stimuli correlated with the delivery of the EBS-DS; i . e . , noises and l i g h t flashes 59 associated with the operation of the electronic equipment. In order to test for stimulus control by such st i m u l i , three test days were added at the end of the experiment. On these days, the current intensity of the EBS was set to zero and no DS was presented. METHODS Implantation and Histology The surgical and' h i s t o l o g i c a l procedures described in the previous chapter were used, with only minor modifications. B r i e f l y , 14 male hooded Sprague-Dawley rats (CBFL, Montreal) weighing 400-450 gms were implanted with monopolar stimulating and recording electrodes in the PP and the h i l u s of the DG respectively. In order to prevent hearing loss, atraumatic earbars were used to hold the animals in the stereotaxic instrument. Electrode construction and placements, current return and i n d i f f e r e n t recording electrodes, anesthesia and electrophysiological procedures were a l l as previously described. In addition, a threaded p l a s t i c connecter of a P l a s t i c Products electrode (MS303) was positioned over the right p a r i e t a l bone and fixed in place with a l l of the other electrodes, using dental cement. This electrode connector was used to anchor the lead wires to a l l of the other electrodes during behavioural t e s t i n g . Tetracycline (Pfizer) was sprinkled into the wound prior to suturing and the animals were given at 60 least seven days to recover from surgery. After the completion of testing, the animals were anesthetized with urethane and the f i n a l locations of the electrode t i p s were marked by passing small anodal currents through the electrodes. The electrode locations were determined from thionin-stained, frozen sections. Electrophysiological procedures Throughout the behavioural testing, the stimulation consisted cf' 0\ 1 msec, cathodal, square- wave constant current pulses, controlled by two Ortec stimulators (Model 4701) and fed through three Grass Stimulus Isolation Units (SIU5A). The current i n t e n s i t i e s of the stimulation pulses were continuously monitored on a storage oscilloscope. The output from the recording electrodes were fed through six-channel commutators d i r e c t l y into a Tektronics (5000 series) storage oscilloscope for amplification (1 M Ohm input impedance), f i l t e r i n g (0.1-3 kHz) and display (negative up). Population spike amplitudes were measured d i r e c t l y from the oscilloscope screen, from the peak p o s i t i v i t y to the peak negativity. After recovery from surgery, the animals were screened to ensure that the electrodes were s t i l l functioning normally and positioned properly. Evoked potentials were recorded following one (250-300 pA) pulse. Animals were considered suitable for further study i f the evoked potential showed a population spike amplitude greater than 1.0 mV, at a peak latency less than 5.0 msec. 61 During behavioural testing, the current i n t e n s i t i e s were set to 500 uA and the amplitude of each population spike was measured d i r e c t l y from the oscilloscope screen. Mean and standard error of the mean (SEM) values were calculated d a i l y for each animal. Behavioural Apparatus A l l testing was conducted in a darkened room containing the electronic equipment and three black sound-attenuating chambers (60 x 60 x 60 cm) with one-way mirrors on the' doors' of the chambers. A NOVA 3/12 computer controlled a l l stimulus presentations, response contingencies and response measurements. The equipment was designed to produce stimulation pulses s i l e n t l y ( i . e . , no relays) and a radio masked the sounds of the exper imenter. Each chamber contained a Plexiglass test box (30 x 30 x 45 cm high), a six-channel commutator ( A i r f l y t e CAY-675-6), electrode leads, a p e l l e t dispenser (Colburn Instruments; E14-02), and a tone generator ("Sonalert": Mallory SC628, 2.8 kHz). A brass food cup (2.5 x 3.0 x 1.0 cm deep; S c i e n t i f i c Prototype) protruded from a side wall, 4.0 cm above the grid f l o o r . It was pivoted and attached to a microswitch mounted outside the box such that a downwards force >0.25 N on the l i p would depress the food cup and operate the microswitch. Operations of the microswitches defined the onsets and offsets of the animals' responses. Early in behavioural testing, these responses usually resulted from e f f o r t s to search the food cups for p e l l e t s . 62 Hinged tubes constructed from p l a s t i c window-screen connected the food-cups to the p e l l e t dispensers. Behavioural Procedures Animals who s a t i s f i e d the electrophysiological c r i t e r i a (N=8) were food deprived and s t a b i l i z e d to 85% body weight over an 18 day period. Water was available ad l i b in the home cages, which were maintained on a 12 hr light-dark cycle (light= 8:00 am to 8:00 pm). Each animal was tested about the same time each day, between- 9-:00: am and 7:00 pm. Daily test sessions consisted of 40 t r i a l s , presented on a V-I 2 min schedule. I n t e r t r i a l intervals were selected randomly, without replacement, from a set of 9 values with a mean of 120 sec and a range of 30 to 120 sec. Response onsets and offsets were measured only during the t r i a l s . T r i a l s consisted of a 10 sec baseline period, a brief DS, a 10 sec (max) post-DS period and the delivery of a food p e l l e t . The onset of the f i r s t response in the post-DS period terminated the t r i a l and led to the immediate delivery of the food p e l l e t . In e f f e c t , the DS defined two time periods: one (A) extending forwards to the onset of the f i r s t response (10 sec max), the other (B) extending backwards to the offset of the last response preceding the DS (again, 10 sec max). A discrimination r a t i o was calculated according to the r a t i o : 1 - (A/(A+B)). Figure 3.1 i l l u s t r a t e s the relationship between reinforcement, the DS, responses and the components (A & B) of the discrimination r a t i o . 63 Figure 3.1: Response contigencies and measurements. The relationship between the discriminative stimulus (DS), food p e l l e t s (FOOD), responses (RESP), and components of the discrimination r a t i o are shown. A discrimination r a t i o of 1 - (a / (a+b)) was calculated on each t r i a l . The events in three t y p i c a l t r i a l s are depicted in A, B and C. A. Indiscriminate responding: RESP's are dist r i b u t e d randomly with respect to the DS. The intervals "a" & "b" are approximately equal and the discrimination r a t i o is close to 0.50. B. No RESP to DS: t r i a l i s terminated after 10 sec by delivery of FOOD. Interval "a" (10 sec) i s greater than i n t e r v a l "b" so the discrimination r a t i o on this t r i a l i s less than 0.50. C. Discriminate responding: No RESP's are made in the Pre-DS period, the last 10 sec of the i n t e r - t r i a l i n t e r v a l . A RESP is made immediately after the DS. Interval "a" is much less than "b" and the discrimination r a t i o is between 0.50 and 1.00. This pattern of responding and high discrimination ratios indicate stimulus control by the DS. D S F O O D R E S P V l - 2 m i n P r e - D S P e r i o d P o s t - D S P e r i o d (10 s e c ) II < 1 0 s e c ) n  II H n n n D S F O O D R E S P J l JL D S F O O D R E S P Jl_ II UL a H 65 As control of responding by the DS increases, the value of this r a t i o approaches 1. Discrimination ratios were calculated on a l l t r i a l s and averaged over blocks of 10 and sessions of 40 t r i a l s . A mean r a t i o >0.70 for the session was selected as the c r i t e r i o n l e v e l of performance on the basis of observations of behavioural patterns. At c r i t e r i o n performance, an animal's behaviour on the majority of t r i a l s t y p i c a l l y consisted of s i t t i n g q uietly prior to the DS and an immediate orientation and approach the food cup following the DS. Discrimination ratios were not calculated on trial's in which the* DS-was- presented during an on-going response, but the majority of the t r i a l s l o st through these "missed" t r i a l s took place during the early stages of conditioning. There were three phases to the experiment: 1) Tone as DS, 2) EBS as DS and 3) No DS. During the f i r s t phase, the DS was a 0.1 sec tone (2.8 kHz). Testing in thi s phase was continued for at least seven days or u n t i l c r i t e r i o n performance had been achieved for two consecutive days. After reaching the c r i t e r i o n , each animal was transferred i n d i v i d u a l l y to Phase 2, on the following day. During Phase 2, the DS was EBS, a single, 0.1 msec (500 uA) pulse stimulation of the PP, and the tone was no longer presented. Testing was continued as before for nine days. In phase 3, the current intensity of the EBS-DS was set to zero and no DS was e x p l i c i t l y presented. This last phase was designed to show that responding in Phase 2 was controlled by the EBS rather than the environmental events correlated with i t . 66 RESULTS A l l recording and stimulating electrodes were in the DG and angular bundle respectively (Fig. 3.2). The d i s t r i b u t i o n of recording s i t e s was: four in the h i l u s , three in the c e l l body layer and one in the dendritic layer of the granule c e l l s . The waveforms of the evoked potentials observed during the experiment indicated that the stimulating electrodes were in the PP. The amplitudes of the population spikes ranged from 3.2 to 10.2 mV at the start of Phase 2 (EBS as DS) and 1.9 to 7.6 mV at the end (Table 1, Fig 3.3) Over the seven days of Phase 1, the DS (tone) gained control over the temporal dimension of the animals' responding. The mean discrimination r a t i o increased from 0.50 on Day 1 to 0.77 on Day 7. A repeated measures analysis of variance (ANOVA) showed these changes to be s i g n i f i c a n t (F (6,42)=31.81, p< 0.0001 ) . On the f i r s t day of phase 2, the EBS had no v i s i b l e e f f e c ts upon behaviour. The mean discrimination r a t i o was equal to 0.50, and the stimulation e l i c i t e d no v i s i b l e orienting responses, motor twitches, or alterations in on-going behaviours such as grooming, s n i f f i n g , rearing, chewing, freezing or s i t t i n g s t i l l . Over the nine days of Phase 2, the mean discrimination r a t i o rose from 0.50 to 0.76 (Fig 3.4) as responding came under the control of the stimulation pulses (repeated measures ANOVA, F(8,56)=15.79, p< 0.0001). Over the course of conditioning the amplitudes of the population spikes tended to decline (Table I) 67 Figure 3.2: Electrode placements. The locations of the stimulating (T) and recording (•) electrodes are depicted on p a r t i a l coronal sections redrawn from Konig and Kl i p p e l (1963). Left posterior c o r t i c i e s and l e f t anterio-dorsal hippocampi are shown. Each section has a number showing the estimated distance (in mm) of the section posterior to Bregma. 6 8 69 TABLE I RAT DAY 1 DAY 9 2 4.62 1 .86 4 6.10 3.38 5 3.20 2.72 6 2.16 5.36 7 3.04 2.60 8* - -9 6.99 7.56 10 1 0.20 4.56 TABLE I: Mean population spike amplitudes (mV) at start and end of Phase 2 (ESB as DS). * Evoked potentials were not co l l e c t e d from t h i s animal due to a faulty recording electrode. 70 Figure 3.3: Sample Evoked potentials. Pictures of evoked potentials recorded in the dentate gyrus following stimulation of the perforant path. One example from each of 4 animals, c o l l e c t e d post-operatively. Calibrations: 2mV, 5 msec 71 Figure 3.4: Discrimination r a t i o s . The mean discrimination ratios and standard errors (SEM) are plotted against the days of testing in a l l three phases of the exper iment. 73 As expected, responses during the three days of Phase 3 were d i s t r i b u t e d randomly with respect to the' DS; the" mean-discrimination r a t i o s ranged from 0.51 to 0.53. Because the individual ratios on the last day of Phase 2 and the f i r s t day of Phase 3 had non-overlapping ranges, no s t a t i s t i c a l analyses were considered necessary to confirm the difference. DISCUSSION This study provides a clear demonstration of the stimulus properties of single-pulse angular bundle stimulation. At the conclusion of testing in both Tone- and EBS-DS phases, performance was at comparable l e v e l s , even though there was no immediate positive transfer between stimuli and the acquis i t i o n of stimulus control by the EBS-DS required approximately twice as many t r i a l s as acqui s i t i o n by the Tone-DS. The absence of discriminated responding in the f i n a l , No-DS, phase c l e a r l y indicated that exteroceptive stimuli from the electronic equipment had acquired no stimulus control. The use of one pulse per t r i a l stands in contrast to the use of t r a i n s of pulses in most other studies of the stimulus properties of EBS (Doty, 1969). Single pulses produce r e l a t i v e l y simple neural a c t i v i t y which can be measured quite e a s i l y (Gloor, Vera & Sper t i , 1963; Lomo, 1971a; Ranck, 1975). In electrophysiological applications, single pulses have revealed many features of neuronal function (e.g., Ratz, 1966) and have 74 usually provided the standard against which other forms of stimulation have been compared (e.g., Schmidt, 1976). By showing the stimulus properties of single-pulse stimulation, the present study has l a i d a foundation for a number of new and refined approaches to the analysis of the relationship between neuronal a c t i v i t y and behaviour. Stimulation of the angular bundle activated the PP input to the hippocampus, which in turn activated the dentate granule c e l l s • in a discrete, well-defined manner (Lomo, 1971a). The demonstration of" the- stimulus properties of angular bundle stimulation provides a new way of r e l a t i n g hippocampal processing of neuronal a c t i v i t y to behaviour. Prior to the completion of the experiments in this thesis, there were no other reports showing behavioural reactions produced by, or conditioned to angular bundle (or PP) stimulation. One report has since appeared in the l i t e r a t u r e , confirming the major finding of the present study. Through the use of electrophysiological procedures similar to those used here, stimulation of the angular bundle at 15 and 100.Hz was shown to acquire stimulus control over responding in a one-way shock avoidance paradigm (Ott, Ruthrich, Reymann, Lindenau & Matthies, 1982, pp. 441-452). Although electrophysiological data were presented in that report, they were d i f f i c u l t to interpret. Changes in synaptic e f f i c a c y in the PP-DG system were evaluated by recording evoked potentials from single test'pulses delivered before, during and after each da i l y session of conditioning. The slope of the 75 population EPSP's were found to increase over the course of conditioning,, while the amplitudes of the population spikes declined (Ott et a l . , 1982, pp. 441-452). Although the authors attributed these changes to the effects of conditioning, the absence of "EBS-only" controls makes i t d i f f i c u l t to rule out the p o s s i b i l i t y that the 15 or 100 Hz stimulation alone was s u f f i c i e n t to a l t e r synaptic e f f i c a c y . Indeed, given the weight of evidence for the a b i l i t y of 15 Hz stimulation to produce LTP (Alger & Teyler, 1976; B l i s s & Gardner-Medwin; 1973; B l i s s & Lomo, 1973'; Teyler- et a l . , 19*77), i t is surprising- that potentiation of the population spikes was not observed. Perhaps the 400 stimulation t r a i n s , of approximately 50 pulses each, spread over 10 days were not presented at a s u f f i c i e n t l y high current i n t e n s i t y . The authors did not disclose the current i n t e n s i t i e s used. Nevertheless, the demonstration of the stimulus properties of angular bundle stimulation in that report (Ott et a l . , 1982) confirms one major finding of the present study. In the present experiment, a c q u i s i t i o n of stimulus control during the EBS-DS phase did not appear to be accompanied by increased synaptic e f f i c a c y in the PP-DG system. In fact, population spike amplitudes tended to decline. These changes would not be predicted from the results obtained in the red nucleus (Tsukahara et a l . , 1981), where synaptic e f f i c a c y appeared to increase during the conditioning of an EBS-CS. In the neuronal system studied by Tsukahara et a l . (1981), there were only three synapses between the stimulation and the muscle 76 that executed the conditioned response. Because none of the efferents of any hippocampal subfields terminate in either the pyramidal or extrapyramidal motor systems (Raisman et a l . , 1965; Swanson & Cowan, 1977), i t is very l i k e l y that many synapses were involved in the translation of the PP stimulation into an overt response. Presumably, any of a number of these synapses could have been modified during conditioning. The tendency of population spike amplitudes to decrease during the EBS-DS phase could have been a consequence of conditioning, a depression induced- by the EBS or merely the result of gradual tissue degeneration at the tips of the stimulating or recording electrodes. Therefore, the evoked potential data c o l l e c t e d during t h i s study do not provide evidence for or against a relationship- between changes in synaptic e f f i c a c y and changes in behaviour. The evoked potential data show only that increased synaptic e f f i c a c y at the f i r s t synapse orthodromic to the stimulation s i t e i s not a necessary condition for acquis i t i o n of stimulus control by an EBS stimulus. There can now be l i t t l e doubt that EBS in the angular bundle, even when limited to one pulse per t r i a l , has stimulus properties. The paradigm developed and used in the present study provides a means of l o c a l i z i n g and measuring neural a c t i v i t y d i r e c t l y related to the performance of a behavioural response. In the next chapter, t h i s paradigm w i l l be used to evaluate the behavioural effects of LTP on the acquis i t i o n of stimulus control by angular bundle stimulation. 77 CHAPTER 4: Long-term Potentiation and Stimulus Control The experiment described in this chapter examines the functional significance of LTP by determining whether tetanic stimulation a l t e r s the tra n s l a t i o n of neural a c t i v i t y into behaviour. L o g i c a l l y , the hypothesis of th i s experiment is almost a truism. That i s : i f the stimulus properties of the EBS depend upon the a c t i v i t y in di r e c t l y - d r i v e n neurons, then the strength of these properties must be related to the l e v e l of evoked a c t i v i t y in the postsynaptic c e l l s . Therefore, any factor which increases the output of the postsynaptic c e l l s should increase the behavioural significance of the EBS. The present experiment tested this hypothesis by comparing the acqui s i t i o n of stimulus control by EBS-DS in a control group to acqui s i t i o n in an experimental group that received tetanic stimulation of the PP just before the onset of conditioning with the EBS-DS. There have been two previous attempts to use LTP to a l t e r behaviour. Barnes (1979) compared young (10-16 month) and old (28-34 month) rats for spontaneous alternation in a T-maze, before and after high-frequency stimulation (400 Hz) and found paradoxical r e s u l t s . The tetanic stimulation produced s i g n i f i c a n t l y more LTP in the young rats but the effect on spontaneous alternation was more pronounced in the old rats. The performance of young rats was almost unaffected by the stimulation while the performance of the old rats declined to chance l e v e l s . This inverse relationship between 78 electrophysiological and behavioural consequences did not appear to be p a r t i c u l a r l y strong. No s t a t i s t i c a l differences between groups were reported for the behavioural measures and the within-group difference between day and night tests was greater than the e f f e c t s of the stimulation. Therefore, these results f a i l to illuminate the relationship between changes in synaptic e f f i c a c y and behaviour. In the second study (Campbell & Milgram, 1980), high frequency, high-intensity stimulation (60 Hz, 60 pulses per day, 30 days) of CA3 f a c i l i t a t e d subsequent acqu i s i t i o n of s e l f -stimulation through the same electrodes. The rate of acquisition in animals given low-frequency, high-intensity stimulation (1 Hz, 60 pulses per day, 30 days) was the same as in implanted controls. This effect appeared to be s p e c i f i c to the EBS reinforcer because the acquisition of lever-pressing for food was the same in a l l three groups. In this study, the only evidence of l a s t i n g neuronal changes was the development of motor seizures (kindling) in the high-frequency group and there was nothing to indicate that the low-frequency stimulation did not produce LTP. Even though stimulation which produces kindling may also produce LTP (Racine, Newberry & Burnham, 1977), the reverse is not true. The development of motor seizures requires stimulation that produces epileptiform afterdischarges (Racine, 1972), LTP does not (Douglas & Goddard, 1975). Therefore, the study by Campbell and Milgram (1980) shows only that kindling a l t e r s the motivational significance of hippocampal stimulation and the link between LTP and learning provided by this study is 79 rather tenuous. In the present experiment, the use of an EBS stimulus and the analysis of evoked potentials should overcome the major problems of re l a t i n g changes in synaptic e f f i c a c y to changes in behaviour. The effects of the tetanic stimulation on synaptic e f f i c a c y in the PP-DG w i l l be documented and the behaviourally relevant stimulus (the EBS-DS) w i l l be known to activate fibres of the PP. The major d i f f i c u l t y in the present experiment was the selection of the EBS-DS current i n t e n s i t y . At low current i n t e n s i t i e s , the stimulus properties^ of the EBS-DS might- be-i n s u f f i c i e n t to allow a c q u i s i t i o n of control over responding. At high current i n t e n s i t i e s , the tetanic stimulation might not produce a s u f f i c i e n t elevation of population spike amplitudes to cause a s i g n i f i c a n t difference in acqui s i t i o n rate. The best procedure for selecting the EBS-DS current intensity might have been to choose an intensity value for each animal on the basis of individual I/O curves. However, the stimulation required to produce these curves would have exposed each animal to the EBS-DS prior to the onset of conditioning. Non-reinforced pre-exposures of animals to conditioned stimuli retards subsequent conditioning (Mackintosh, 1974, pp. 36-40). Therefore, the EBS-DS current intensity could not be based on I/O curves and so a midrange intensity value of 250 ph was selected, on the basis of electrophysiological p i l o t studies. At this point on the I/O curves of most p i l o t animals ( i . e . , at 250 JJA) , tetanic stimulation produced the largest r e l a t i v e increases in population spike amplitudes. It was hoped that at th i s 80 intensity, the electrophysiological differences between control and experimental animals would be maximal. In the previous experiment (Chap. 3), a 500 jjA EBS -DS acquired stimulus control in about 9 days. It was hoped that acqui s i t i o n of stimulus control by a 250 pA EBS -DS would take longer and provide a greater number of days over which the performance of control and experimental animals might be d i f f e r e n t . A number of experimental procedures were incorporated to minimize the direct transfer of stimulus control from- the EBS in-the electrophysiological tests to the EBS-DS in the behavioural tests. F i r s t , a l l electrophysiological tests were administered while the animals were in a p l a s t i c "carrying" box, which was very d i f f e r e n t from the behavioural test chambers. Second, a l l electrophysiological and behavioural tests were separated by at least 2 hrs. F i n a l l y , the number of non-reinforced stimulation pulses administered prior to the onset of conditioning was kept to an absolute minimum. As in the previous experiment, a l l animals were tested in three consecutive phases: 1) Tone-DS, 2) EBS-DS, and 3) No DS. The animals in the experimental group were given tetanic stimulation 2 hrs before the f i r s t session of the EBS-DS phase. The trains of high-frequency pulses were preceded and followed by single pulse stimulation at low-frequency, in order to determine baseline lev e l s and the immediate effects of the tetanic stimulation. The control animals were given only low-frequency stimulation which equated the test duration and number 81 of evoked population spikes (see METHODS). As before, population spike amplitudes were measured for each animal on every t r i a l of the EBS-DS phase. METHODS Implantation and Histology The surgical and h i s t o l o g i c a l procedures described in Chapter 2 were used, with only minor modifications. B r i e f l y , 22 male hooded' Sprague-Dawl'ey' rats" (CBFL, Montreal) weighing 300-450 gms were implanted with monopolar stimulating and recording electrodes in the PP and the h i l u s of the DG respectively. Atraumatic earbars were used during surgery to avoid damaging the eardrums. Electrode construction and placements, current return and i n d i f f e r e n t recording electrodes, anesthesia and electrophysiological procedures were a l l as previously described. Threaded p l a s t i c connectors from P l a s t i c Products electrodes were again part of the s k u l l assembly and were used to anchor the lead wires during behavioural testing. Tetracycline (Pfizer) was sprinkled into the wound prior to suturing and the animals were given at least 14 days to recover from surgery. At the conclusion of testing, electrode placements were determined in the usual manner. 82 Electrophysiological Procedures. As before, the temporal parameters of the stimulation pulses were controlled by two Ortec stimulators, but in th i s experiment, the current i n t e n s i t i e s were controlled by two WPI (#305) constant-current stimulus i s o l a t i o n units (output impedance = 200 K Ohms). One storage oscilloscope was used to monitor current i n t e n s i t i e s while another was used for the c o l l e c t i o n , amplification, f i l t e r i n g and display of the evoked potentials. The amplitudes of a l l evoked population spikes were measured d i r e c t l y from' the' screen and- in addition, the amplified, f i l t e r e d signals from the oscilloscopes were fed into a D i g i t a l PDP 11/03 (MINC) microcomputer for storage, analysis and display. Each evoked potential was sampled at 50 psec/pt for a 25 msec period after the EBS-DS. The animals were screened for electrode function and placements two weeks after surgery. Potentials evoked by single, 0.1 msec, 250 pA pulses to the PP were measured, photographed, d i g i t i z e d , stored and plotted. These evoked potentials were used to select the 12 best animals and to ensure that there were no gross differences between the population spike amplitudes of the control and experimental groups. The population spike amplitude and latency c r i t e r i a were as previously described. The animals were tested behaviourally in two di f f e r e n t sets, one two weeks after the other, with each set having three experimental and three control animals. The only difference between the sets was in the storage of evoked potentials. During the testing of the f i r s t set of animals, the only available data 83 storage device was a floppy-disk drive ( D i g i t a l , RX02, 1/4 Mbyte). Because the operation of t h i s device produced a loud noise correlated with each EBS-DS, the evoked potential data could not be stored during the EBS-DS phase. Consequently, a l l population spike amplitudes were measured d i r e c t l y from the oscilloscope screens (cf. Chap. 3). During behavioural testing of the second set of animals, a s i l e n t cartridge-disk drive ( D i g i t a l , RL02 drive; 20 Mbytes) was available and used to store the evoked potentials. The population spike amplitudes of this second' set of animals were- measured from these d i g i t i z e d waveforms as well as from the the oscilloscope screens. The estimates of population spike amplitudes derived from these two sources were always within 5% of each other and so the measurements from the oscilloscope screens were used for a l l animals. Behavioural Apparatus The sound-attenuating chambers, electrode leads, commutators, p e l l e t dispensers, tone generators, Plexiglas test boxes and pivoted food cups with response-measuring microswitches were a l l the same as in the previous experiment (Chap. 3). Stimuli, response contingencies and measurements were controlled by the NOVA 3/12 minicomputer located in a separate room. The only equipment change was the addition of three warning l i g h t s , located outside the sound-attenuating chambers and v i s i b l e to the experimenter but not the animals. These l i g h t s were illuminated during each pre-DS period to aid the 84 measurement of the population spikes from the oscilloscope screens and to prepare the MINC computer for evoked potential data c o l l e c t i o n . Behavioural and Experimental Procedures. The animals were food deprived to 85% body weight over an 18 day period and then tested in three consecutive phases: 1) Tone-DS, 2) ESB-DS and 3) No DS. As before, d a i l y testing consisted of 40 t r i a l s , presented on a VI-2 min schedule, each t r i a l having a 10 sec pre-DS period, a brief DS, a' 10 sec (max) post-DS period and a food p e l l e t d elivery. The f i r s t response in the post-DS period terminated the t r i a l and led to the immediate delivery of the food p e l l e t . Discrimination r a t i o s , based on the timing of responses in the pre- and post-DS periods, were calculated for every t r i a l and averaged for each d a i l y session as in the previous experiment (cf., Chap.3). Two hours before the f i r s t session with the EBS-DS, the control and experimental animals were each given a series of stimulation pulses in a carrying box placed outside the sound attenuating chambers. The control animals received three sets of 10 (250 pA) single pulses at low frequency. The experimental animals were given tetanic stimulation consisting of 100 pulses at high frequency, preceded and followed by 10 single pulses. The tetanic stimulation was delivered in 10 trains of 10 pulses at 200 Hz, with the trains presented at the same low frequency as the pre- and post-tetanic single pulses. Single pulses and trains were presented at 0.1 Hz (1/10 sec) for the f i r s t set of 85 animals, and 0.04 Hz (1/25 sec) for the second set of animals. In t h i s way, the control animals were given one pulse of stimulation for every 10 pulse t r a i n given to the experimental animals. It was expected that only one population spike would be produced by each t r a i n of PP stimulation at 200 Hz (Lomo, 1971a; McNaughton et a l . , 1978). Because the stimulation frequencies of the single pulses and trains were also matched (e.g., 1 pulse or 1 t r a i n every 10 sec), the stimulation series delivered to the control and experimental animals balanced both the duration of the series- and' the number of evoked population spikes. The population spike amplitudes produced by the 10 pre-tetanic pulses of the experimental animals and the 30 single pulses of the control animals provided the baselines against which a l l subsequent averaged population spike amplitudes were compared. A l l animals remained in the Tone-DS phase u n t i l the ac q u i s i t i o n of stimulus control was complete. Acquisition usually required 7 days. The EBS-DS phase lasted for 17 days and was followed by the 2 day, No DS phase. The EBS-DS current i n t e n s i t i e s in the two l a t t e r phases were 250 and 0 pA respectively. A l l animals were given one f i n a l e lectrophysiological test, on a day after the completion of behavioural testing. Evoked potentials were c o l l e c t e d following 30 single pulses at 0.04 Hz and 250 J JA, while the animals moved freely about the p l a s t i c carrying box used for the other electrophysiological test procedures. The electrophysiological c r i t e r i o n for LTP was a 20% increase in population spike amplitudes during the f i n a l test and also for at least three 86 days of the EBS-DS phase. RESULTS The electrode placements of the 10 animals that s a t i s f i e d the electrophysiological and behavioural c r i t e r i a are shown in Figure 4.1. A l l stimulating and a l l but one of the recording electrodes were in the angular bundle and DG respectively. The d i s t r i b u t i o n of electrode placements was: experimental animals -three h i l a r , one dendritic; control animals - four h i l a r , one dendritic and one on the outer border of the DG. The evoked potentials recorded from this l a t t e r s i t e were i d e n t i c a l to those c o l l e c t e d from h i l a r placements. A l l evoked potentials were consistent with the various h i l a r and dendritic recording elctrode placements and showed that the EBS-DS activated the fi b r e s of the PP. The locations of the stimulating electrodes were within the range of placements observed in the la s t experiment and there were no consistent differences in the placements of the electrodes in control and experimental animals. Two animals were dropped from the experiment, both from the experimental group. One did not show discriminated responding to the Tone-DS, even after 400 t r i a l s . The other animal f a i l e d to s a t i s f y the electrophysiological c r i t e r i a for LTP; there was no elevation in population spike amplitudes following the tetanic stimulation. During the tetanic stimulation, the behaviour of the 87 Figure 4.1: Electrode Placements. The locations of the stimulating electrodes of the experimental (T) and control (V) animals are displayed on posterior c o r t i c a l coronal sections. The locations of the recording electrodes of the experimental (•) and control (o) animals are depicted on p a r t i a l coronal ' sections showing only the anterio-dorsal hippocampus. A l l electrodes were on the l e f t side of the brain and a l l sections were redrawn from Konig and Klippel (1963). S8 8.9 8 9 a n i m a l s a n d t h e s l o w - w a v e a c t i v i t y i n t h e DG were w a t c h e d c o n t i n u o u s l y . E x p l o r a t o r y r e s p o n s e s s u c h a s s n i f f i n g , r e a r i n g and l o c o m o t i o n u s u a l l y f o l l o w e d e a c h t e t a n i c t r a i n , e v e n when t h e a n i m a l s were q u i e s c e n t p r i o r t o t h e s t i m u l a t i o n . A l t h o u g h t h e s l o w - w a v e a c t i v i t y i n t h e DG was d e p r e s s e d f o r s e v e r a l s e c o n d s f o l l o w i n g e a c h t r a i n , no e p i l e p t i f o r m a f t e r d i s c h a r g e s p i k e s were e v e r o b s e r v e d . The f o u r a n i m a l s i n t h e e x p e r i m e n t a l g r o u p p r o v i d e d c l e a r e v i d e n c e o f LTP. P r i o r t o t e t a n i c s t i m u l a t i o n , t h e a m p l i t u d e s o f t h e p o p u l a t i o n - s p i k e s - i n t h e e x p e r i m e n t a l g r o u p were s m a l l e r t h a n t h o s e o f t h e c o n t r o l g r o u p , b u t n o t s i g n i f i c a n t l y (t_(8) = 0.35, NS; s e e T a b l e I I ) . A f t e r t h e t e t a n i c s t i m u l a t i o n a n d t h r o u g h o u t t h e EBS-DS p h a s e , t h e mean p o p u l a t i o n s p i k e a m p l i t u d e s o f t h e e x p e r i m e n t a l g r o u p w e re a b o v e 120% o f t h e b a s e l i n e v a l u e s ( F i g . 4 . 2 ) . I n c o n t r a s t , t h e p o p u l a t i o n s p i k e a m p l i t u d e s o f t h e c o n t r o l g r o u p r e m a i n e d a t o r b e l o w b a s e l i n e l e v e l s o v e r t h i s same p e r i o d . A l t h o u g h t h e b e t w e e n - g r o u p d i f f e r e n c e d u r i n g t h e EBS-DS p h a s e f a i l e d t o a c h i e v e t h e t r a d i t i o n a l c r i t e r i o n f o r s t a t i s t i c a l s i g n i f i c a n c e ( r e p e a t e d m e a s u r e s ANOVA, F ( 1 , 8 ) = 3 . 7 1 , p < 0 . 0 9 ) , when t h e e n v i r o n m e n t a l c o n d i t i o n s o f t h e b a s e l i n e t e s t s were d u p l i c a t e d a t t h e end o f t h e e x p e r i m e n t , t h e b e t w e e n - g r o u p d i f f e r e n c e was s i g n i f i c a n t i n t h e e x p e c t e d d i r e c t i o n ( t ( 8 ) = 2 . 3 8 , p<0.05, o n e - t a i l e d t e s t ) . F i g u r e 4.3 shows r e p r e s e n t a t i v e e v o k e d p o t e n t i a l s f r o m a n i m a l s i n t h e c o n t r o l a n d e x p e r i m e n t a l g r o u p s . The mean d i s c r i m i n a t i o n r a t i o s f o r b o t h g r o u p s i n t h e t h r e e p h a s e s o f t h e e x p e r i m e n t a r e shown i n F i g u r e 4.4. I n t h e Tone-DS 90 TABLE 11 Evoked Potentials and EBS-DS Discrimination EXPER mean CONTROL mean EVOKED POTENTIALS BEHAV RAT BASELINE FINAL FINAL FINAL (mV) (mV) (%) RATIO 5 4.09 5.18 127 0.73 9 5.3-9- 6!.2 3 1 1 6 0.71 1 0 2.50 5.53 221 0.77 1 7 1 .56 3.08 1 97 0.74 3.39 5.00 165 0.74 6 3.72 5.82 1 56 0.77 8 2.76 3.67 133 0. 57 1 2 6.38 3.82 60 0.52 1 4 1 .79 1 .53 85 0.50 18 1 .63 1 .36 83 0.57 21 12.55 4.89 39 0.75 4.81 3.52 93 0.61 Mean population spike amplitudes of the experimental (EXPER) and control animals at the beginning and end of the experiment. Percentage scores are FINAL/BASELINE x 100. The measurement of behaviour (BEHAV) is the mean discrimination r a t i o on the l a s t three days of the EBS-DS phase. 91 Figure 4.2: Population Spike Amplitudes. The mean and SEM values of the population spike amplitudes, expressed as a percentage of single pulse baseline l e v e l s , are shown for the experimental (•) and control (o) groups on the f i r s t and la s t electrophysiological tests and a l l 17 days of the EBS-DS phase. <?3 P P — D G E V O K E D POPULATION SP IKES O o P R E - T E T A N U S DAY 1 DAY 17 • • J J J Figure 4.3: Sample Evoked Potentials. Averaged evoked potentials from one animal in the experimental group and one in the control group, c o l l e c t e d during the baseline test (PRE-TETANUS) and on Days 1 and 17 of the EBS-DS phase. Triangles (T) mark the population spikes. C a l i b r a t i o n s : 2 mV, 5 msec. 94 Figure 4.4: Discrimination Ratios. The mean and SEM discrimination ratios for the experimental (•) and control (o) groups are shown for a l l three phases of the experiment. The discrimination r a t i o of 0.70 was the c r i t e r i o n performance l e v e l . 96 phase, discrimination ratios rose to c r i t e r i o n levels ( i . e . , > 0.70) within four or five days. A repeated measures ANOVA confirmed the significance of the change over days (F_(6,48) = 57.0, p < 0.01), and did not reveal any s i g n i f i c a n t difference between the groups (F(1,8) = 0.07, NS) or the learning rates (group by day interaction, J?(6,48) = 0.51, NS). At the beginning of Phase 2, and for several days thereafter, the mean discrimination ratios of both groups were very close to 0.50, indicating that the ESB-DS had l i t t l e stimulus control (Fig. 4.4). The mean discrimination ratios of both groups remained below 0.60 u n t i l the tenth day of the EBS-DS phase. At th i s point, they diverged sharply. By Day 15, the EBS-DS had acquired control over the responses of a l l of the animals in the experimental group, while the mean of the control group remained low. The significance of th i s difference was shown by the group by day interaction term of a repeated measures ANOVA (F(16,128) = 1.90, p < 0.03). As in the previous experiment, the low discrimination ratios in the No-DS phase showed that responding to the EBS-DS was due to the stimulation and not to exteroceptive stimuli correlated with EBS-DS. Even though the acquis i t i o n of stimulus control by the EBS-DS was s i g n i f i c a n t l y better in the experimental group, the mean discrimination r a t i o of the control group also rose (Fig. 4.4). This increase was almost e n t i r e l y due to the responses of the two control animals with the largest evoked potentials (Table I I ) . The population spike of one of these animals (#21) was extremely large before the EBS-DS phase (baseline) and though i t 97 decreased, i t was s t i l l the second largest evoked potential in the control group on the f i n a l t e s t . The evoked potentials of the other animal (#6) s a t i s f i e d a l l the c r i t e r i a of LTP. The average response to the la s t 10 pulses of the baseline stimulation was 40% larger than the average response to the f i r s t 10 pulses. In addition, evoked potentials of comparable magnitude were observed on 9 days of the EBS-DS phase and the f i n a l evoked potentials were 56% greater than the baseline responses. On the last 3 days of the EBS-DS phase, the discrimination ratios' of both animals were above the behavioural c r i t e r i o n for stimulus control (Table I I ) . The data from these two control animals were consistent with the experimental e f f e c t . The best stimulus control by the EBS-DS was observed in the animals with the largest evoked potentials, regardless of the o r i g i n of the difference in population spike amplitude - tetanic stimulation, low-frequency stimulation or electrode placement. DISCUSSION The present results show a relationship between changes in synaptic e f f i c a c y and behaviour. The acquis i t i o n of stimulus control by the EBS-DS was faster in the animals given tetanic stimulation than in those given only low-frequency stimulation prior to conditioning. Both rates were slower than the acq u i s i t i o n rate in the previous experiment (Chap. 3), where the current intensity was 500 juA. Studies of c l a s s i c a l conditioning 98 with exteroceptive stimuli have shown that stronger stimuli acquire stimulus control faster than weaker ones (Mackintosh, 1974, pp. 41-42). The acquisition rates in the present and previous experiments indicate that the e f f e c t i v e stimulus strength of the EBS-DS was greatest with a 500 ph stimulus and least with a 250 ph stimulus in animals not given tetanic stimulation. The intermediate rate of acq u i s i t i o n in the experimental group indicates that the tetanic stimulation increased the stimulus strength of the 250 ph EBS-DS. The EBS-DS activated the fibres' of the PP and' the granule c e l l s of the DG. As a consequence of the tetanic stimulation, the granule c e l l responses to the EBS-DS in the animals of the experimental group were enhanced for the duration of the EBS-DS phase. This change in synaptic e f f i c a c y in the PP-DG system can account for the difference in the stimulus strengths of the EBS-DS. The results indicated that through a change in synaptic e f f i c a c y , a fixed amount of neural a c t i v i t y in the PP was better able to acquire stimulus control over responding. In other words, the behavioural consequence of neural a c t i v i t y in a given system can be influenced by synaptic e f f i c a c y in postsynaptic systems. Although the results confirm the experimental hypothesis and appear to ident i f y a relationship between the electrophysiological and behavioural consequences of tetanic stimulation, there are two other interpretations which must be considered. The differences in acq u i s i t i o n rates could have been due to 1) electrode placements or 2) positive transfer of 99 stimulus properties from the tetanic stimulation to the single-pulse EBS-DS. There was no evidence that the group difference was due to the placements of the stimulating electrodes or the number of relevant fibres activated by the 250 pA stimulation. A l l stimulation s i t e s were clustered in a limited region of the angular bundle and there were no consistent differences in the d i s t r i b u t i o n s of s i t e s in the two groups. In addition, the population spike amplitudes of the experimental group before the tetanic stimulation- were s l i g h t l y smaller than those of the control group. Observations of the animals during the tetanic stimulation c e r t a i n l y indicated the presence of stimulus properties; orienting and exploratory responses frequently followed each tetanic stimulation. However, there are four l i n e s of evidence indicating that transfer of stimulus control cannot account for the experimental e f f e c t . F i r s t , there was acquisition of stimulus control by the EBS-DS in two animals not given tetanic stimulation. Second, posi t i v e transfer would have been apparent at the start of the EBS-DS phase, but the discrimination ratios of the two groups were v i r t u a l l y i d e n t i c a l for 10 days. Third, only very few trains were given and yet transfer between trains and single-pulse EBS has been demonstrated only after a great number of EBS-reinforcer pairings (Freeman, 1962; Woody & Yarowsky, 1973). Fourth, the tetanic trains were presented in the absence of a reinforcer, a procedure which usually does not lead to the a c q u i s i t i o n of stimulus control (Tsukahara et a l . , 100 1981). Non-reinforced pre-exposures of conditioned and discriminative stimuli tend to retard, not f a c i l i t a t e subsequent conditioning (Mackintosh, 1974, pp. 36-40). Therefore, any explanation of the present results based on the stimulus properties of the tetanic stimulation i s untenable. The evoked potential data of the present experiment showed that the postsynaptic responses to the EBS-DS in the DG were increased by the tetanic stimulation. Even though the significance l e v e l of the population spike increase could be questioned, i t must be noted that the- evoked potentials col l e c t e d during the EBS-DS phase were recorded under less than ideal circumstances. The animals were freely-moving and engaged in a variety of d i f f e r e n t behaviours at the time of each EBS-DS. Consequently, the amplitudes of the population spikes could have been influenced by any number of non-PP inputs to the DG. The depressed evoked responses of the control group indicates the o v e r a l l i n h i b i t o r y nature of these non-PP influences. It should be emphasized that when the evoked responses of the two groups were tested in a controlled environment at the end of the experiment, the population spike amplitudes of the experimental group were s i g n i f i c a n t l y larger. It cannot be assumed that only synapses near the recording electrode were activated by the EBS-DS and affected by the tetanic stimulation. Changes in the postsynaptic responses to the EBS-DS in other terminal areas of fibres projecting from the stimulation s i t e s could also have subserved the experimental e f f e c t . The next chapter examines the known anatomy of the 1 0 1 angular bundle and through the use of lesions, compares the contributions of PP and non-PP fibres to the stimulus properties of the EBS-DS. Although the relationship between the postsynaptic a c t i v i t y in the DG and the neural a c t i v i t y mediating the stimulus properties of the EBS-DS has yet to be defined empirically, the present results s t i l l provide evidence that behaviour i s influenced by synaptic e f f i c a c y . Any explanation of the present results based on a physiological process other than LTP would have to rely on a functional change capable' of l a s t i n g at least 10 days. In the present experiment, the increased population spike amplitudes showed that the tetanic stimulation produced a long-lasting functional change, LTP. There is no evidence for any other type of functional change, in present experiment or in the l i t e r a t u r e . In summary, the presence of LTP in the PP-DG was accompanied by an increased behavioural reaction to stimulation of the angular bundle. The a c t i v i t y recorded from the DG might have been correlated with, or responsible for the stimulus properties of the EBS-DS. In the absence of any other behavioural or electrophysiological processes to account for the enduring e f f e c t s of the tetanic stimulation, these results provide dir e c t evidence for the influence of synaptic e f f i c a c y on behaviour. They show that neural a c t i v i t y which might otherwise have no effect on behaviour can acquire control i f the synaptic e f f i c a c y in postsynaptic systems is increased. Accordingly, learning can be viewed as alte r a t i o n s in the 1 02 translations of neural a c t i v i t y into overt, observable responses. In th i s scheme, changes in synaptic e f f i c a c y would be important components in one of the neural bases of learning and memory. 1 03 CHAPTER 5: The Effect of Perforant Path Lesions on Stimulus Control The major advantage of using e l e c t r i c a l stimulation of the PP as a discriminative stimulus i s the degree to which the a c t i v i t y generated by the stimulation can be l o c a l i z e d to a s p e c i f i c structure in the brain. This l o c a l i z a t i o n of a c t i v i t y should allow a more direct analysis of the neural changes involved in learning than i s possible in paradigms using environmental s t i m u l i , which produce neural a c t i v i t y in unspecified regions of the brain. Despite this advantage, i t must also be recognized that EBS produces a c t i v i t y in not just one, but many di f f e r e n t structures in the region of the electrode t i p . Furthermore, the stimulus properties of the EBS are probably due to only a portion of the t o t a l evoked a c t i v i t y . Any advantage of using EBS as a DS or CS could be e a s i l y lost by a t t r i b u t i n g the stimulus properties to a system not actually subserving them. This i s p a r t i c u l a r l y important in the present context where the stimulus properties of the angular bundle stimulation are attributed p r i n c i p a l l y to activation of the PP. Up to th i s point in the thesis, two indirect l i n e s of evidence have been used to est a b l i s h the role of PP activation in the stimulus properties of the EBS-DS. F i r s t , the evoked a c t i v i t y recorded from the DG during implantation and testing showed that the stimulation activated the PP d i r e c t l y and the granule c e l l s of the DG synaptically (Chap. 3 & 4). Second, tetanic stimulation of the PP which increased the evoked 1 04 a c t i v i t y in the DG, f a c i l i t a t e d the ac q u i s i t i o n of stimulus control by the EBS-DS. In the present experiment, an attempt was made to provide more direct evidence for the importance of PP act i v a t i o n by tryi n g to separate the contributions of PP and non-PP systems to the stimulus properties of the EBS-DS. The major neuronal elements near the placements of the EBS-DS electrodes can be divided into three categories: 1) c e l l bodies dorsal and ventral to the corpus callosum and angular bundle respectively, 2) uncrossed ascending and descending u n i l a t e r a l f i b r e s and 3) ascending 1 and' descending commissural f i b r e s . The clustering of the stimulating ( i . e . , EBS-DS) electrodes in the angular bundle observed in the previous experiments (Chap. 3 & 4) and the r e l a t i v e neuronal densities of gray and white matter indicate that d i r e c t excitation of the c e l l bodies probably contributed l i t t l e to the stimulus properties of the EBS-DS. The other two alternatives were much more l i k e l y to be involved. The u n i l a t e r a l , ascending projections of the fibres in the region of the electrode t i p s include PP fi b r e s originating in the entorhinal cortex and terminating in the DG (Hjorth-Simonsen & Jeune, 1972), subicular cortex iWyss, 1981), CA1 (Steward, 1976) and CA3 (Hjorth-Simonsen & Jeune, 1972). The u n i l a t e r a l , descending projections include fibres which project to the entorhinal cortex and p e r i r h i n a l cortex from CA1 (Swanson, Sawchenko & Cowan, 1981), CA3 (Hjorth-Simonsen, 1971), the subiculum (Beckstead, 1978), presubiculum (Beckstead, 1978), and parasubiculum (Segal, 1977) (see also Swanson & Cowan, 1977 or 105 Swanson, Wyss & Cowan, 1978 for extensive d e s c r i p t i o n s ) . There are many commissural systems in the v i c i n i t y of the EBS-DS electrodes. The dorsal surface of the angular bundle is covered by the corpus callosum and the angular bundle i s i t s e l f the caudal portion of the dorsal hippocampal commissure. Commissural f i b r e s could therefore include those of a) the splenium of the corpus callosum; homotopic interconnections of the temporal, o c c i p i t a l and r e t r o s p l e n i a l c o r t i c i e s (Zeman & Innes, 1963, pp. 160-163), b) dorsal hippocampal commissure; homotopic interconnections' - of the subicu'lum, presubTcul'um and parasubiculum (Swanson & Cowan, 1977) and c) crossed temporo-ammonic and temporo-dentate paths; connections of the entorhinal cortex to the contralateral subiculum (Swanson & Cowan, 1977), CA1 (Steward, 1976), CA3 (Wyss, 1981), entorhinal cortex (Wyss, 1981) and r o s t r a l portions of the DG (Wyss, 1981). The innervation of the DG by projections of the c o n t r a l a t e r a l PP was not found in the rat u n t i l quite recently (Goldowitz, White, Steward, Lynch & Cotman, 1975; Zimmer & Hjorth-Simonsen, 1975). Subsequent studies showed that the topographical organization of the crossed temporo-dentate projection was very similar to the i p s i l a t e r a l projections of the PP (Steward, 1976). Although these fibres project into most of the DG r o s t r a l to the point where the dorsal hippocampal commissure crosses the midline (Wyss, 1981), within this r e l a t i v e l y large area there are only very few f i b r e s (Goldowitz et a l . , 1975; Steward & S c o v i l l e , 1976). The sparsity of the innervation i s probably the main reason why t h i s projection 106 escaped detection for so many years (Goldowitz et a l . , 1975). The route taken by the PP fib r e s from the entorhinal cortex to the DG and hippocampus is well known (Hjorth-Simonsen & Jeune, 1972). The dorsal-ventral axis of the entorhinal cortex is mapped onto the rostro-caudal axis of the DG and hippocampus. Many of the fibres of the PP travel horizontally from their points of or i g i n in the entorhinal cortex and converge in the angular bundle as i t arches up and forward. Perforant path fibres leave the angular bundle/dorsal hippocampal commissure along i t s length. Fibres originating 1 in- the ventral entorhinal cortex remain in the angular bundle for only a short distance before they perforate through the subicular regions horizontally, to innervate the posterior-ventral DG and hippocampus. Fibres from the most dorsal sections of the entorhinal cortex remain in the dorsal hippocampal commissure u n t i l just before t h i s bundle decussates (crosses the midline). At this point the PP fibres are immediately dorsal to the most r o s t r a l extreme of the DG and descend almost v e r t i c a l l y into i t . Most of the PP fibres leave the angular bundle posterior and ventral to their target regions of the DG and ascend dorsally and r o s t r a l l y through the subiculum. Many perforate the hippocampal fissure at the crest (medial point) of the DG but some perforate along the entire length of the fissu r e (Hjorth-Simonsen & Jeune, 1972). Although the exact route taken by the crossed temporo-dentate projection has not been described, i t can be assumed that the fibres project d i r e c t l y r o s t r a l from the point where they cross the midline in the dorsal hippocampal 1 07 commissure. In summary, these anatomical studies indicated that PP fibr e s activated by the EBS-DS probably included projections to the i p s i l a t e r a l DG, the i p s i l a t e r a l CA1, CA3 and subiculum and, to a lesser extent, the r o s t r a l homotopic areas in the contralateral hemisphere. The non-PP fibres activated by the EBS-DS probably included the interconnections of the homotopic posterior c o r t i c a l and entorhinal regions. A l l of the non-PP fibres and commissural PP fibres remain in the corpus callosum or the- angular bundle/dorsal hippocampal commissure to cross the midline 3-4 mm anterior to the EBS-DS electrode placements. In contrast, almost a l l the PP fibres leave the angular bundle posterior to t h i s crossing and perforate through the subiculum. On the basis of this anatomy, i t appeared to be possible to make a lesion in the subiculum, anterior to the stimulating electrode, and produce considerable damage to the PP. The target position of the lesion in thi s study was ventral to the corpus callosum and ventro-lateral to the dorsal hippocampal commissure. The lesion had to be s u f f i c i e n t l y r o s t r a l to avoid the spread of damage back to the region of the EBS-DS electrode. At the same time, the lesion had to be caudal enough to transect most of the PP fibres activated by the EBS-DS. These lesions were not expected to produce substantial damage of c e l l bodies in the region of the EBS-DS electrode, f i b r e s of the corpus callosum, or fibres interconnecting the l e f t and right entorhinal c o r t i c i e s . Although these lesions would not discriminate between the 108 PP projections to the subiculum,.CA1, CA3 and DG, the r e l a t i v e densities of PP innervation to these four areas suggested that the projection from the entorhinal cortex to the DG predominated. Most anatomical papers r e f r a i n from stating quantitative comparisons of innervation densities, but even cursory examination of almost any of the published photographs (e.g., Swanson et a l . , 1978) reveals large discrepancies in innervation d e n s i t i e s . Electron microscopic analysis of degenerating terminals in DG, CA3 and CA1 following a lesion to medial entorhinal cortex revealed a density r a t i o of 48:16:1 for these three areas (Nafstad, 1967). On the basis of the assumption that the stimulus properties of the EBS-DS depended primarily on activation of the u n i l a t e r a l projections of the PP, transection of these f i b r e s was expected to a l t e r the stimulus control of the EBS-DS. Small e l e c t r o l y t i c lesions were used to make these transections for a number of .reasons. The lesion electrodes could be and were implanted at the same time as the EBS-DS and recording electrodes. Evoked potentials from the DG ensured that a l l lesion- electrodes were accurately positioned in the PP. Because the lesions were made by passing anodal current through the electrodes, i t was possible to delay the lesions u n t i l each animal reached a comparable le v e l of EBS-DS discrimination. In addition, the actual lesion procedure was so simple that only l i g h t anesthesia was necessary at the time of the lesion and the stress to the animals was minimized. As with any lesion, i t was necessary to control for non-109 s p e c i f i c e f f e c t s . S p e c i f i c a l l y , a l t e r a t i o n s in behaviour unrelated to the conduction, transmission or immediate i p s i l a t e r a l processing of the EBS evoked a c t i v i t y . Therefore, an additional group of control animals was given lesions in the homotopic subiculum, contralateral to the EBS-DS electrode. Due to the uncertainties of electrode implantation and a c q u i s i t i o n of stimulus control by the EBS-DS, recording and lesion electrodes were implanted b i l a t e r a l l y in every animal. This permitted the assignment of equal numbers of animals to control and experimental groups after the a c q u i s i t i o n of stimulus control by the EBS-DS was complete. The b i l a t e r a l recording electrodes served two purposes. They were used to guide the implantation of the b i l a t e r a l lesion electrodes and to monitor the a c t i v i t y in both l e f t and right DG during the period immediately following the EBS-DS. This study was an attempt to identi f y the r e l a t i o n s h i p between a c t i v i t y in the i p s i l a t e r a l PP projection to the anterior DG and the stimulus control by the EBS-DS. The main purpose was to show that the evoked a c t i v i t y in the PP-DG was not just correlated with the neural events mediating the stimulus properties but was actually an important component of these events. Such a demonstration would provide ample j u s t i f i c a t i o n for the use of the present discrimination paradigm in the analysis of the roles of synaptic e f f i c a c y and hippocampal processing in the control of behaviour. 110 METHODS Implantation and Histology Twenty-five male hooded Sprague-Dawley rats (500-600 gm) were operated under Nembutal anesthesia using the procedures described previously. Two monopolar recording electrodes and a monopolar stimulating electrode were positioned s t e r e o t a x i c a l l y into the l e f t and right DG and the l e f t posterior PP respectively, using the same co-ordinates as in the previous experiments. Two additional electrodes were implanted b i l a t e r a l l y into the PP, using co-ordinates which were 2 mm more anterior and 1 mm more medial than the co-ordinates of f i r s t PP electrode. For the sake of c l a r i t y , these electrode placements are c a l l e d anterior or posterior PP. These names are based only on the r e l a t i v e positions of the electrodes and do not imply any other anatomical or functional d i s t i n c t i o n s between two portions of the PP. S i m i l a r l y , the terms con t r a l a t e r a l and i p s i l a t e r a l refer to the r e l a t i o n between a lesion or a recording electrode and the posterior PP electrode unless s p e c i f i c a l l y stated otherwise. Thus a "cont r a l a t e r a l , anterior PP" electrode i s r o s t r a l to the posterior PP electrode and on the other side of the brain, but i s not necessarily in a co n t r a l a t e r a l projection of the PP. The r e l a t i v e positions of the PP and the stimulating, recording and lesioning electrodes are shown in F i g . 5.1. The stereotaxic co-ordinates of the five electrodes to be implanted were too close together to permit the use of five I K L R F i g u r e 5 . 1 : R e l a t i v e P o s i t i o n s of S t i m u l a t i n g , Recording and Le s i o n E l e c t r o d e s . S i m p l i s t i c r e p r e s e n t a t i o n of the l o c a t i o n s of the a n t e r i o r (ANT) and p o s t e r i o r (POST) s t i m u l a t i n g (STIM) e l e c t r o d e s with r e s p e c t to the e n t o r h i n a l c o r t e x (EC), dentate gyrus (DG) and f i b r e s of the p e r f o r a n t path (PP) i n both the l e f t (L) and r i g h t (R) hemispheres. The POST STIM e l e c t r o d e was i n the same p o s i t i o n as the EBS-DS e l e c t r o d e s i n p r e v i o u s experiments (Chap. 3 & 4 ) . 1 1 2 independent electrode c a r r i e r s . Therefore, the b i l a t e r a l recording electrodes and the two l e f t PP stimulating electrodes were both implanted as pairs in two separate c a r r i e r s . In order to compensate for the curvature of the cortex, the l e f t PP stimulating electrode was mounted in the c a r r i e r such that the t i p of the anterior PP electrode was 0.5 - 0.7 mm dorsal to the t i p of the posterior electrode. During implantation, single-pulse stimulation (500 _uA, 0.2 Hz) was delivered through a l l three stimulating electrodes and evoked potentials were recorded from the DG' electrode i p s i l a t e r a l to each stimulation s i t e . As far as possible, the depths of a l l electrodes were adjusted to maximize the amplitudes and thresholds of the population spikes. The pair of recording electrodes was implanted to a depth which positioned the l e f t and i f possible, both electrodes in the h i l u s , as shown by the waveform of the potentials evoked by stimulation through the electrodes i p s i l a t e r a l to each. The depths of the two l e f t PP electrodes were adjusted to optimize the position of only the posterior (EBS-DS) electrode. The right anterior PP stimulating electrode was implanted to a depth which optimized the evoked potential in the right DG. No consideration was given to the waveforms of evoked potentials c o n t r a l a t e r a l to any of the stimulating electrodes. In five of the animals, the positions of the electrodes were not considered acceptable and the operations on these were terminated before completion. The anchor for the leads, the current return and ind i f f e r e n t electrodes were a l l as described previously. The 1 13 electrodes were fixed in place with dental cement, Tetracycline was applied to the wound and the animals were allowed at least two weeks to recover from the effects of surgery. After a l l testing was concluded, the animals were anesthetized with urethane and a 25 uA DC anodal current was passed through each electrode to produce iron deposits at the t i p s . The animals were perfused with saline followed by a 2% solution of potassium ferrocyanide in 10% formalin to stain the iron deposits and f i x the tissue. The tips of the b i l a t e r a l recording and PP stimulating' electrodes were located on thionin stained, coronal sections. In addition, the positions of the lesion electrode t i p s and the lesion damage were reproduced from sections taken at the point of maximal coronal spread of damage. Electrophysiology Throughout the study, the EBS consisted of 0.1 msec constant current, cathodal square wave pulses which were continuously monitored on a storage oscilloscope. The evoked potentials from both recording electrodes were passed through a commutator and a dual-channel, shop-built preamplifier (AC, gain=1.0, input impedance > 10 M Ohm) to storage oscilloscopes for amplification, f i l t e r i n g (0.1-3 kHz) and display. The amplified, f i l t e r e d signals were fed to a D i g i t a l (MINC) microcomputer which multiplexed and d i g i t i z e d (at 65 usec/point) each evoked potential for 33 msec following the stimulation pulse. The evoked potentials were stored on a s i l e n t cartridge disk ( D i g i t a l ; RL02, 10 Mbyte) for analysis l a t e r . At the end of 1 14 the experiment, the evoked potentials c o l l e c t e d from each.animal on each day were averaged together and the amplitudes of the averaged population spikes i p s i l a t e r a l and contr a l a t e r a l to the stimulation were calculated. Two weeks after surgery, the animals were screened for electrode location and function. Single (300 uA) pulses were passed through each of the three stimulating electrodes and b i l a t e r a l evoked potentials were c o l l e c t e d . Animals were considered suitable for further study i f the sum of the population spike- amplitudes of four evoked potentials exceeded 4.0 mV. Three of these evoked potentials were those i p s i l a t e r a l to each of the three stimulating electrodes. The fourth was the evoked potential in the right DG produced by stimulation through the EBS-DS electrode, located in the l e f t angular bundle. In a l l , 14 animals s a t i s f i e d this c r i t e r i o n and were food deprived to 85% body weight over an 18 day period. » Acquisition of Discrimination. The behavioural apparatus and procedures were i d e n t i c a l to those described in the previous chapter. B r i e f l y , stimulus control by a Tone-DS was established f i r s t and then testing with the single pulse EBS-DS was begun. Daily sessions consisted of 40 t r i a l s , presented on a VI-2 min schedule. The f i r s t response to the food-cup after the DS was rewarded by the immediate delivery of a food p e l l e t . In the absence of a response within 10 sec of the DS, a food p e l l e t was delivered non-contingently. As before, discrimination ratios based on the temporal pattern 1 15 of responding in the 10 sec periods before and after the DS were calculated on every t r i a l and averaged over the entire session. Stimulus control by the Tone-DS was established within seven days. On the f i r s t and last days of testing with the Tone-DS, I/O curves were derived for the EBS-DS and i p s i l a t e r a l DG electrode combination (cf. Chap. 2). On 10 of the 40 t r i a l s on these two days, a single pulse of EBS was presented at the onset of the Tone-DS. As in Chapter 2, 10 current i n t e n s i t i e s were presented in an ascending series ranging from 20-400- uA, but this time, each intensity was presented only once. Analysis of the evoked potentials and the I/O curves derived from them revealed the current i n t e n s i t i e s required to produce a threshold (1.0 mV) and a maximal population spike in each animal. The current intensity later used for the EBS-DS was 250 uA or the intensity required to produce a submaximal population spike, whichever current was greater. After 15 days of testing with the single pulse EBS-DS, the animals who had not acquired the discrimination (n=7) were tested with a multi-pulse EBS-DS, which consisted of 10, 200 Hz pulses. This stimulation was comparable to the tetanic stimulation used in Chapters 2 and 4. The tetanic stimulation was expected to produce LTP and thereby increase the strength of the stimulus properties of the EBS without increasing the population of directly-stimulated neural elements. Multi-pulse EBS-DS's at the same current i n t e n s i t i e s used in the acqui s i t i o n phase were delivered on the f i r s t 30 t r i a l s 1 16 of the 16th day, and for the f i r s t 10 t r i a l s on subsequent days u n t i l c r i t e r i o n discrimination had been achieved. On a l l other t r i a l s , the EBS-DS was a single pulse. Testing with the single-pulse EBS-DS was continued for three additional days to confirm the permanency of the e f f e c t . While this t r a i n i n g was being completed, those animals who had learned to discriminate the single-pulse EBS-DS were being tested in the next phases of the experiment. For convenience, these two set of animals were c a l l e d FAST and SLOW on the basis of the r e l a t i v e rates of acquisi t i o n of stimulus control by the single-pulse- EBS-DS. Perforant Path Lesions. A l l lesions were made under ether anesthesia by passing a 1.0 mA DC anodal current through one of the anterior PP electrodes. Lesions were made on the evening of the last EBS-DS acqu i s i t i o n day and post-lesion testing was begun the following day. Only the animals in the FAST group were considered suitable for the analysis of the effects of PP lesions for two reasons. F i r s t , the reasons for the differences in the acquis i t i o n rates of the FAST and SLOW groups were unknown, although differences in electrode placements and evoked a c t i v i t y were suspected. It was decided that the FAST and SLOW groups might not constitute a single homogeneous population, at least with respect to the neural systems involved in the stimulus properties of the EBS. Because the animals in the FAST group acquired the discrimination of the single pulse EBS-DS without any additional 1 1 7 trai n i n g , these animals were considered to be a better preparation in which to study the effects of PP lesions. The second reason for focusing the analysis of PP lesion effects on the animals of the FAST group was the uncertainty about specifying a l l of the neurophysiological and behavioural consequences of the multi-pulse EBS-DS's. Even though the synaptic e f f i c a c y of the PP-DG system was monitored, possible changes in other PP terminal areas or non-PP systems activated by the EBS were not. Furthermore, high-frequency stimulation of the PP produces ac t i v i t y ' in- the DG' that i s very d i f f e r e n t from the a c t i v i t y evoked by single pulses (Lomo, 1971a). It seemed possible that even after subsequent retraining with the single-pulse EBS-DS, the animals in the SLOW group might continue to use a d i f f e r e n t component of the EBS-evoked a c t i v i t y than that used by the animals in the FAST group. Therefore, lesions through the anterior PP electrodes were given only to the animals of the FAST group, and the animals in the SLOW group were used for a d i f f e r e n t , preliminary study. The animals in the FAST group were divided into two subgroups and given a u n i l a t e r a l lesion i p s i l a t e r a l (n=4) or contral a t e r a l (n=3) to the EBS-DS electrode, through the appropriate anterior PP electrode. Discrimination performance was evaluated for three days before and after this l e s i o n , with the EBS-DS current i n t e n s i t i e s used in the acquisition phase of the experiment. By th i s point in the experiment, each animal in the FAST group represented a considerable investment of time and e f f o r t . 118 In order to derive the maximum usefulness from these animals, i t was decided to perform a second lesion in each animal, through the anterior PP lesioning electrode not used for the f i r s t l e s i o n . These lesions would complete a two-stage, b i l a t e r a l lesion of the PP, anterior to the EBS-DS electrode. Figure 5.2 shows the progression of these lesions in the two subgroups of animals. The names "Ipsi-contra" and "Contra-ipsi" designate the two subgroups and indicate the sequence in which the i p s i l a t e r a l ( i p s i ) and contra l a t e r a l (contra) PP lesions were made. Prior to the second lesions, an e f f o r t was made to increase the s e n s i t i v i t y of the animals to decrements in the stimulus strength of the EBS-DS. The current intensity of the EBS-DS was reduced to the minimum value capable of maintaining c r i t e r i o n discrimination performance. After two days of tests to determine the current thresholds of EBS-DS stimulus control, each animal was given a day of testing with the new, low-intensity EBS-DS and then lesions were made according to the procedures described previously. It was believed that these second lesions might extend the analysis of the role of structures contralateral to the EBS-DS, or replicate the effects of the f i r s t , u n i l a t e r a l lesions. I l l F i g u r e 5.2: L e s i o n Sequences Smaller v e r s i o n s of the diagram in previous f i g u r e i l l u s t r a t e the p r o g r e s s i o n of experimental events and the f u n c t i o n s of the e l e c t r o d e s at each stage. Evoked p o t e n t i a l s (EP) were c o l l e c t e d from both r e c o r d i n g e l e c t r o d e s d u r i n g the a c q u i s i t i o n (ACQ) phase. The subgroup names IP-CON and CON-IP s p e c i f y whether the l e s i o n i p s i l a t e r a l (IP) to the EBS-DS preceded or fo l l o w e d the c o n t r a l a t e r a l (CON) l e s i o n . Black c i r c l e s i n d i c a t e the presence of a l e s i o n and "LESION" i n d i c a t e s the e l e c t r o d e through which the most recent l e s i o n was made. 1 20 RESULTS Acquisition of Discrimination The Tone-DS acquired stimulus control over the temporal pattern of the operant responses of a l l animals in 7 days. After 15 days of testing with single pulses, the EBS-DS had acquired control over the responses of the animals in the FAST but not the SLOW group. Figure 5.3 c l e a r l y shows that the two groups d i f f e r e d in the number of days of testing before the onset of discrimination learning' ( f i r s t r a t i o < 0.60). There were three stages in the a c q u i s i t i o n of stimulus control by the EBS-DS observed in each animal. F i r s t , the latency to the onset of a c q u i s i t i o n . This stage was characterized by random fluctuations of the daily discrimination ratios between the l i m i t s of 0.45 and 0.55. Second, acquisition time. In t h i s stage, discrimination r a t i o s rose progressively from 0.60 to 0.70. Third, asymptotic performance. The discrimination ratios in this stage varied only s l i g h t l y from day to day and always remained above 0.70. Learning rate, i . e . , the number of days for the EBS-DS to acquire c r i t e r i o n stimulus control, was the sum of the f i r s t two stages. The latency to the onset of a c q u i s i t i o n was generally longer than the acquisition time. In the FAST group, there were 5-13 days between the start of the EBS-DS phase and the f i r s t r a t i o above 0.60 but only 2-7 days from that day to the f i r s t day of c r i t e r i o n performance. For each animal, these two measures were highly correlated (r(5)=0.86, p < 0.05). 121 Figure 5.3: Acquisition of Discrimination. The discrimination ratios (mean and SEM, n's=7,7) are shown for the Tone-DS, the f i r s t 15 days of the single-pulse EBS-DS phase and the 6 days of testing with multi-pulse EBS-DS. The animals were divided into FAST (•) and SLOW (o) groups on the basis of acquisition rate; multi-pulse EBS-DS's were given only to the animals in the SLOW group, after 15 days of single-pulse EBS-DS. A l l mean values were calculated from t r i a l s using the single-pulse EBS-DS. C r i t e r i o n discrimination r a t i o = 0.70. 123 The multi-pulse EBS-DS's delivered to the animals in the SLOW group after day 15 had both immediate and enduring, behavioural and electrophysiological e f f e c t s . The multi-pulse EBS-DS always e l i c i t e d an unconditioned orienting response, unlike the single pulse EBS-DS which never did. Furthermore, acqui s i t i o n of stimulus control was e s s e n t i a l l y complete within 3 days of the f i r s t multi-pulse EBS-DS presentations even though the responses of the SLOW group were v i r t u a l l y random on the preceding 15 days. In fact, the ac q u i s i t i o n time of stimulus control by the- multi-pulse EBS-DS in the SLOW' group was s i g n i f i c a n t l y less than the acquis i t i o n time in the FAST group (FAST, SLOW means= 5.3, 3.3 days, t(l2)=2.26, _p<0.05). It must be emphasized that after the f i r s t day of multi-pulse EBS-DS's, 75% of a l l the t r i a l s had single-pulse EBS-DS's. Nevertheless, the discrimination ratios of the SLOW animals rose to le v e l s (nonsignificantly) greater than those of the FAST animals (FAST, SLOW means = 0.75, 0.79, t(12) = 1.90 r NS). The electrophysiological consequences of the multi-pulse stimulations were also very clear. The amplitudes of the evoked population spikes increased during the multi-pulse EBS-DS sessions and remained larger following the return to single-pulse EBS-DS. In six of the seven animals, the increase was greater than 40% and the maximum increase was 177%. This contrasts sharply with the s t a b i l i t y of the evoked potentials over the 15 days of testing with single-pulse EBS-DS: only one of the 14 animals had a larger (15%) population spike on Day 15 when compared to Day 1. The increases in population spike 1 24 amplitudes caused by the multi-pulse EBS-DS's were s u f f i c i e n t to bring the evoked potentials in the SLOW group up to the same le v e l as those previously observed in the FAST animals (SLOW = 5 .35 mV, FAST = 5 .08 mV). In order to determine which features of the EBS were responsible for the differences in the acquisition rates of the FAST and SLOW groups, each animal was scored on four measures: 1) the amplitudes of the i p s i l a t e r a l and contra l a t e r a l population spikes, 2) population spike thresholds, 3) the locations of the EBS-DS electrodes, and 4) the EBS-DS current intensity r e l a t i v e to population spike threshold. Examples of evoked potentials and electrode locations are presented in Figure 5 . 4 . This figure i l l u s t r a t e s some of the major differences between the animals in the FAST and SLOW groups. The clearest was the difference in the t o t a l amount of evoked a c t i v i t y , as measured by the sum of the population spike amplitudes i p s i l a t e r a l and contra l a t e r a l to the EBS-DS (FAST, SLOW means = 7 . 5 , 4 . 3 mV, t ( 1 2 ) = 2 . 4 0 , p < 0 . 0 4 ) . This difference was due in part to a s i g n i f i c a n t difference in contr a l a t e r a l a c t i v i t y (FAST, SLOW means = 1 .6 , 0.1 mV, £ ( 1 2 ) = 3 . 6 2 , p < 0 .01) and in part to a non-significant difference in i p s i l a t e r a l a c t i v i t y (FAST, SLOW means = 5 . 1 , 4 .2 mV, t_(l2) = 1 .56 , NS). The t o t a l amount of evoked a c t i v i t y was also the factor which showed the best c o r r e l a t i o n with the learning rate for a l l animals ( r ( l 2 ) = 0 . 7 3 , p < 0 . 0 l ) . Within the animals in the FAST group, th i s c o r r e l a t i o n was very high (_r(5)=0.83, p<0 .0 l ) and appeared to be due to the latency of acquis i t i o n (_r(5)=0.83, 1 2 5 Figure 5 . 4 : Sample Evoked Potentials and Electrode Locations. The locations of sample stimulating ( T ) and b i l a t e r a l recording (•)•electrodes are depicted on p a r t i a l coronal sections of the l e f t posterior cortex and the l e f t and right anterio-dorsal hippocampi respectively. The approximate distance from Bregma of each section i s shown in mm. Evoked potentials recorded from the l e f t DG ( i p s i l a t e r a l to the stimulation) and from the right DG, (contralateral to the stimulation) are shown to the l e f t and right of the coronal sections. Note the short latency (3 msec) latency of the population spike (arrow) in the i p s i l a t e r a l evoked potential as compared to the much longer latency ( 8 msec) of the same component in the evoked potential co n t r a l a t e r a l to the EBS-DS. The information in these figures derives from two representative animals: A and B are from the animals who acquired the discrimination of the EBS-DS the fastest and the slowest respectively. Stimulation intensity = 300 uA. Cal i b r a t i o n s : 2 mV, 5 msec. 1 27 p<0.02), rather than the acquis i t i o n time (r(5)= -0.52, NS). The correlations within the FAST group alone are quite important because the difference between groups in the amount of con t r a l a t e r a l evoked a c t i v i t y might have been partly due to a difference in the locations of the contra l a t e r a l recording electrodes (Fig 5.5, 5.6). Even though 24 of the 28 recording •electrodes were properly positioned in the hilus of the l e f t or right DG, and a l l four of the inaccurately placed electrodes were in dendritic regions of the right (contralateral) DG, three of these dendritic placements" were in animals of the SLOW group. Because evoked potentials obtained from dendritic electrode locations may not have provided an accurate estimate of the magnitude of the co n t r a l a t e r a l evoked a c t i v i t y , the differences in the evoked potentials of the two groups may have been exaggerated. Consequently, the between-group difference in cont r a l a t e r a l recording electrode placements may have been partly responsible for the correlation between evoked potentials and learning rate. However, the previously mentioned correlation between learning rate and t o t a l evoked a c t i v i t y in the animals of FAST group indicates that the rate of learning was indeed related to the magnitude of the a c t i v i t y evoked by the EBS-DS. Additional evidence for the importance of PP fi b r e a c t i v a t i o n to the stimulus properties of the EBS came from a s i g n i f i c a n t difference in the mean population spike thresholds of the two groups (FAST, SLOW means= 50, 140 uA, t(12)=3.5, P<0.01) and a high c o r r e l a t i o n between learning rate and threshold (_r( 1 2) =0. 64, p<0.02). Although the cor r e l a t i o n had to F i g u r e 5.5: E l e c t r o d e Placements, FAST group. The l o c a t i o n s of the r e c o r d i n g e l e c t r o d e s (•) and the p o s t e r i o r s t i m u l a t i n g e l e c t r o d e s (T) are shown on p a r t i a l c o r o n a l s e c t i o n s of the a n t e r i o - d o r s a l hippocampus and p o s t e r i o r c o r t e x , redrawn from Konig and K l i p p e l (1973). The approximate d i s t a n c e (mm) of each s e c t i o n from Bregma are shown. Figure 5.6: Electrode Placements, SLOW group Positions of electrodes of animals in SLOW group as in previous Figure. 130 be interpreted with caution because i t was derived mostly from the between-group differences, i t was consistent with a relat i o n s h i p between the proximity of the EBS-DS electrode to the PP and the strength of the EBS-DS stimulus properties. H i s t o l o g i c a l analysis of the locations of the stimulating electrodes provided further evidence of this relationship. A l l of the stimulating electrodes were in the angular bundle (Fig 5.5, 5.6), but the locations of the electrodes in the animals in the FAST group were more t i g h t l y clustered in a central area of this pathway. This difference"can be c l e a r l y seen in Figure 5.7, a plot of the anterior-posterior versus the medial-lateral co-ordinate of each EBS-DS electrode. In order to determine whether the rate of learning was s i g n i f i c a n t l y related to the dispersion of the electrodes, the mean anterior-posterior and l a t e r a l co-ordinates were calculated and then each electrode location was ranked on the basis of distance to this central point. A comparison of the rank scores showed that the difference between the groups was s i g n i f i c a n t (FAST, SLOW median ranks = 4, 11, Mann-Whitney U(7,7) = 4, P<0.01) and the c o r r e l a t i o n between rank and the rate of learning across a l l animals was good (rho(12)=0.69, p<0.0l). The f i n a l indication of the dependence of acquisition rate upon the degree of PP act i v a t i o n came from the analysis of the EBS-DS current i n t e n s i t i e s . In the previous experiments, a high current EBS-DS (500 pA; Chap. 3) acquired stimulus control faster than a moderate (250 JJA; Chap. 4) EBS-DS. However, in the present experiment, faster rates of acquisition could not be 111 1 0 11 • • / 5 / o 1 2 7 3 2 1 3 / , • o o o • -I 14 1 / • O / / • o / J L / i 4 . 6 4 . 4 4 . 2 4 . 0 L A T E R A L C 0 - 0 R D I N A T E (m m) 8 . 2 m 3D o 3D I TJ o 8.4 H m 3D O 3D O 8 . 6 O i O 3D O Z 8 . 8 > m ' / CO 3 Figure 5.7: D i s t r i b u t i o n of EBS-DS Electrode Locations. The anterior-posterior co-ordinates of the stimulating electrodes as revealed by h i s t o l o g i c a l analysis are plotted against the medial-lateral co-ordinates. C i r c l e s and squares d i f f e r e n t i a t e the electrode locations of the animals in the FAST and SLOW groups and the numbers above each symbol show the learning-rate rank of each animal. The curved dashed l i n e s show the approximate boundaries of the angular bundle. 1 32 attributed to absolute current intensity, because the average current intensity of the animals in the SLOW group was in fact higher than that of the FAST group (FAST, SLOW means = 264, 330 p.A t(l2)=2.3, p=0.055, NS). A s i g n i f i c a n t and consistent r e l a t i o n between the amount of stimulation and acquis i t i o n rate appeared only when the EBS-DS current i n t e n s i t i e s were expressed as a percentage of the population spike threshold (FAST, SLOW means= 610, 330%, U12)= 1.96, p<0.05, one-tailed t e s t ) . This difference, due largely to the group difference in population spike thresholds"' described' above', indicated once more that stimulation of the PP and not non-PP elements in the angular bundle or the surrounding tissue was responsible for a major component of the stimulus properties of the EBS-DS. Perforant Path Lesions The behavioural effects of b i l a t e r a l lesions through the anterior PP electrodes were studied only in the animals of the FAST group. The centres and maximum excursions ( i . e . , spread) of these lesions are shown in Figure 5.8. In general, the tips of the anterior electrodes (the lesion centres) were in the subiculum, very close to the hippocampal fi s s u r e , and none were in the dorsal hippocampal commissure. The damage to the hippocampal formation was limited to the dorsal t i p of the posterior DG and the overlying subiculum, caudal to the decussation of the dorsal hippocampal commissure. The damage to structures underneath the DG was minimal, but the lesions did tend to spread up the electrode shafts and produce some damage 733 IP-CON CON-IP F i g u r e 5.8: Centres and Maximal E x c u r s i o n s of PP L e s i o n s . The l o c a t i o n s of the l e s i o n e l e c t r o d e t i p s (T) appear i n s i d e the b o l d l i n e s which d e l i n e a t e the maximum spread of damage produced by the two-stage, b i l a t e r a l , a n t e r i o r PP l e s i o n s . The f i r s t , u n i l a t e r a l , l e s i o n s (1) were e i t h e r i p s i l a t e r a l (IP) or c o n t r a l a t e r a l (CON) to the EBS-DS e l e c t r o d e s i n the IP-CON and CON-IP subgroups r e s p e c t i v e l y . The second l e s i o n s (2) were made through the remaining a n t e r i o r PP e l e c t r o d e , l o c a t e d i n e i t h e r the l e f t (L) or r i g h t (R) hemisphere. The EBS-DS was always on the l e f t s i d e of the b r a i n . These s e c t i o n s , redrawn from Konig and K l i p p e l (1973) corresponded to c o r o n a l s e c t i o n s 6.5 mm p o s t e r i o r t o Bregma. 134 to the overlying regions of the cortex. One feature of the lesions not shown c l e a r l y in Figure 5.8 was a tendency for the lesions to be constricted at the l e v e l of the corpus callosum. Generally, the lesions were barely wider than the width of the electrode shafts and damage to the f i b r e bundles in the region was minimal. The consequences of the u n i l a t e r a l ( f i r s t ) lesions were very surprising. The lesions i p s i l a t e r a l to the EBS-DS actually appeared to improve discrimination performance, rather than impair i t , whereas the c o n t r a l a t e r a l lesions', instead of leaving the performance unchanged, appeared to produce a s l i g h t d e f i c i t (Figs. 5.9, 5.10). The consequences of the second lesions were more consistent with the experimental expectations. The lesions i p s i l a t e r a l to the EBS-DS (Contra-ipsi subgroup) produced a r e l a t i v e l y severe d e f i c i t in discrimination performance and the contralateral lesions (Ipsi-contra subgroup) had no effect upon the discrimination (Figs. 5.9, 5.10). A repeated measures ANOVA confirmed two important features of the data: the second lesions produced greater d e f i c i t s than the f i r s t (F(1,5)=13.94, p<0.02) and the d e f i c i t s of the Contra-i p s i subgroup were s i g n i f i c a n t l y greater than those of the I p s i -contra group (F ( 1 , 5) = 1 9 . 40 , p<0.0l). Planned tests (Dunn's Jt) showed that after each lesion, the subgroup means were s i g n i f i c a n t l y d i f f e r e n t (p<0.05) but the only s i g n i f i c a n t within-subgroup difference between pre- and post-lesion discrimination r a t i o s was in the subgroup Contra-ipsi, following the second lesion (p<0.05). Because the second lesions completed 135 Figure 5.9: Lesions and Daily Discrimination Ratios. The mean (and SEM) discrimination ratios for the days preceding and following lesions of the anterior PP. Lesion 1 was i p s i l a t e r a l (IP) or con t r a l a t e r a l (CON) to the EBS-DS. Lesion 2 was the converse. Closed c i r c l e s (•) designate the ratios of IP-CON group; open c i r c l e s (o), the CON-IP group. D A Y S ( Pre / P o s t - L e s i on ) 137 L E S I O N 1 L E S I O N 2 B e f o r e A f t e r B e f o r e A f t e r F i g u r e 5.10: Lesions and Averaged D i s c r i m i n a t i o n R a t i o s . The d i s c r i m i n a t i o n r a t i o s of each animal were averaged over the days preceding and f o l l o w i n g each l e s i o n . The subgroups means (and SEM's) shown here provide a c l e a r e r p i c t u r e of the l e s i o n e f f e c t s . A b b r e v i a t i o n s , symbols and subgroups as i n pr e v i o u s f i g u r e . 138 a two-stage, b i l a t e r a l PP lesion in both subgroups, and yet only one subgroup showed s i g n i f i c a n t d e f i c i t s in discrimination, i t would appear that "lesion-sequence" was a c r i t i c a l factor. The lesions were examined for gross, consistent differences between the damage produced in the l e f t and right hemispheres, or between the Ipsi-contra and Contra-ipsi subgroups. Few were found. The damage to the overlying cortex, corpus callosum and DG regions was equal for both sides and subgroups (Fig. 5.8). There was s l i g h t l y more clu s t e r i n g of lesion centres in the subiculum i p s i l a t e r a l to the EBS-DS but there were no consistent differences in the A-P l e v e l s . In order to refine the analysis, the neural effects of each lesion were quantified by four measures: 1) the amplitudes of i p s i l a t e r a l population spikes evoked by stimulation through the lesion electrodes, at the time of screening, 2) lesion-induced change (%) in the population spikes i p s i l a t e r a l to the EBS-DS, 3) v e r t i c a l distance between the t i p of the lesion electrode and the DG, and 4) the maximum cross-sectional area (size) of the lesion, including only the portion of the lesion underneath the corpus callosum and dorsal hippocampal commissure. The subgroup means on a l l fiv e variables are shown in Figure 5.11. The data were subjected to a c o r r e l a t i o n a l analysis (multiple regression) that determined whether any weighted combination of the four lesion-related variables could predict the behavioural changes. In other words, whether the behavioural e f f e c t s were determined by the sizes, accuracies or evoked potential effects of the lesions. It should be noted that this 139 Figure 5.11: Behavioural and Neural E f f e c t s of PP lesions. Subgroup means (and SEM) of individual scores for 5 variables used in multiple regression analysis. Every animal eventually received two lesions, one i p s i l a t e r a l (IP) and the other contralateral (CON) to the EBS-DS. The labels below the horizontal axes specify which lesion was made and also the designations of the subgroups. The numbers within the bargraphs indicate how many lesions had been made when the effects were tested, and the l e t t e r s (L or R) indicate whether the measure was taken from the l e f t or right hemisphere. Note: the EBS-DS was always in the l e f t , angular bundle. A. Behaviour: Lesion-induced changes in discrimination ratios expressed as residual change scores in order to remove any correlations between the pre- and post-lesion measures. Negative numbers show discrimination d e f i c i t s . B. EBS-DS Evoked A c t i v i t y : Lesion-induced percent changes in the amplitudes of the population spikes e l i c i t e d by the EBS-DS and recorded from the i p s i l a t e r a l DG. (Post-lesion / Pre-lesion x 100). C. PP Fibre Density at Lesion S i t e s : Population spike amplitudes (mV) e l i c i t e d by 300 uA stimulation through l e f t or right lesion electrode and recorded from the DG i p s i l a t e r a l to the stimulation. Amplitude considered to be proportional to the number of PP fibres activated by stimulation and subsequently damaged by the l e s i o n . D. Lesion Size: Cross-sectional area of lesions taken at the point of maximal diameter in coronal plane. E. V e r t i c a l distance from DG: Measured from the centre of the lesions to the hippocampal f i s s u r e . A N T E R I O R P P P O P U L A T I O N S P I K E S ( m v ) E B S - D S P O P U L A T I O N S P I K E C % C H A N G E ) O o z o b o o o D I S T A N C E F R O M DGf jnm) ro ui m o o ro o o o^o o o ro I L E S I O N A R E A C m m 2 j o Ul o o CD R E S I D U A L C H A N G E IN R A T I O I + o o z o o z o —J— o o ro o o| o z o z H o o I z o 141 multiple regression analysis compared the individual scores and did not s p l i t the animals into their respective subgroups. The behavioural changes produced by the f i r s t lesion were compared to the accuracy and size of only the f i r s t lesions. This analysis f a i l e d to find any s i g n i f i c a n t r e l a t i o n between variations in the lesion measures and variations in the behavioural e f f e c t s . The behavioural changes which followed the second lesion could have been due to either the second lesions alone or the cumulative effects of both lesions. Consequently, t'he behavioral effects of the second lesions were compared to the second-lesion and sum-of-lesion values of the four neural parameters. This analysis showed that the changes in discrimination performance following the second lesion could be predicted by a combination of the t o t a l effects of both lesions (R-squared=99%, F(4,2)= 1,018, p<0.0l), and that a l l four lesion-related variables provided s i g n i f i c a n t contributions (p<0.05). The two most heavily weighted ( i . e . , important) factors were measures of the relati o n s h i p between the electrodes and the PP. The amplitudes of the population spikes observed following stimulation through the lesioning electrodes at the time of screening was the most important factor, followed by percent changes in EBS-DS evoked population spike amplitudes, lesion size and lesion-centre to DG distances in a r a t i o of 3 : 2.1 : 1.2 : 1 (Beta values; -0.85, +0.60, -0.33 & +0.28, respectively). This multiple regression analysis indicated that the magnitude of the d e f i c i t in discrimination was related primarily 1 42 to the accuracy of the lesion placements in the PP and the effe c t of the lesions on the EBS-DS evoked a c t i v i t y in the i p s i l a t e r a l DG. The di r e c t i o n of the relation between lesion size and change in discrimination r a t i o revealed that the larger d e f i c i t s in discrimination were produced by the smaller lesions. In short, the behavioural e f f e c t s of the b i l a t e r a l lesions seemed to depend upon a s p e c i f i c disruption of the PP-DG system. I n i t i a l l y , there appeared to be a discrepancy between the interpretations of the ANOVA of the behavioural data and the multiple- regression analysis of" the- lesion variables. The main conclusion from the ANOVA was that lesion-sequence (ipsi-contra vs contra-ipsi) was a very important factor. In contrast, the multiple regression analysis, which did not include l e s i o n -sequence as a factor, showed that the behavioural changes produced by the lesions were due to damage of the PP and changes in the evoked a c t i v i t y to the EBS-DS. This discrepancy disappeared when the evoked pote n t i a l data were considered in d e t a i l because lesion-sequence was seen to be a c r u c i a l factor determining the magnitude and di r e c t i o n of the lesion effect on the i p s i l a t e r a l evoked potentials. Comparisons of i p s i l a t e r a l population spike amplitudes showed that i p s i l a t e r a l lesions, whether given before or after the c o n t r a l a t e r a l lesions, reduced the i p s i l a t e r a l evoked a c t i v i t y (35 and 45% respectively). In contrast, c o n t r a l a t e r a l lesions f a c i l i t a t e d the EBS-DS evoked a c t i v i t y , and the magnitude of th i s f a c i l i t a t i o n was highly dependent upon le s i o n -sequence. The f i r s t c o n t r a l a t e r a l lesion produced only a 17% 143 increase in the mean amplitude of the population spikes, but the second c o n t r a l a t e r a l lesion increased the mean amplitude by 309%. The population spike amplitudes (in mV) of the animals in the Ipsi-contra subgroup were actually larger af t e r the second lesion than they were before the f i r s t lesion, even though an i p s i l a t e r a l lesion of the PP and a reduction in current intensity had taken place during the intervening days. A repeated measures ANOVA of the percent changes in population spike amplitudes revealed that lesion-sequence was indeed a s i g n i f i c a n t factor ( l e s i o n number by group interaction: F(1,5)=13.41, p<0.02). Pairwise post hoc tests between subgroup means showed that the threefold increase in the evoked potentials in the Ipsi-contra subgroup was s i g n i f i c a n t l y d i f f e r e n t from a l l other lesion-induced changes in evoked a c t i v i t y (Newman-Keuls' tests: p<0.05). The large increases in evoked a c t i v i t y in the damaged PP following the con t r a l a t e r a l lesions were both unexpected and revealing. No similar interactions between the PP's of di f f e r e n t hemispheres have been reported previously. Nor has there been any previous indication of such a rapid compensatory mechanism for e l e c t r o l y t i c l esions. The changes in the evoked a c t i v i t y i p s i l a t e r a l to the EBS-DS following the lesion to the cont r a l a t e r a l side provide an explanation for the d i f f e r e n t i a l e f f e c t s of the two b i l a t e r a l lesions. Namely, . only one lesion sequence produced a behavioural d e f i c i t because only one lesion sequence produced a s i g n i f i c a n t disruption of the combined EBS-DS evoked a c t i v i t y in the PP-DG systems on both sides of the 144 brain. DISCUSSION The data from both acqu i s i t i o n and lesion phases of th i s present experiment indicated the importance of the PP and b i l a t e r a l hippocampal structures to the stimulus properties of the EBS-DS. The rate of acquisition of stimulus control was related to the clu s t e r i n g of electrode locations in proximity to the f i b r e s of the PP and the resultant differences in evoked a c t i v i t y . The changes in stimulus control produced by the lesions were related to the proximity of b i l a t e r a l lesion electrodes to the PP fibres on both sides of the brain and to changes in the i p s i l a t e r a l evoked a c t i v i t y . These data constitute r e l a t i v e l y strong evidence that the stimulus properties of the EBS-DS were due in large part to the evoked a c t i v i t y in the i p s i l a t e r a l and contr a l a t e r a l DG, produced by activation of the PP. Acquisition of Discrimination The o r i g i n a l purpose of this phase of the experiment was to establish stimulus control by the EBS-DS so that the effect of lesioning the PP on the stimulus properties could be tested. However, during the early EBS-DS phase, i t became clear that a c q u i s i t i o n rates were bimodally d i s t r i b u t e d . After 15 days, half of the animals had reached c r i t e r i o n performance while the other animals s t i l l showed no behavioural responses to the EBS-1 45 DS. This unexpected difference in acquisition rates made i t possible to id e n t i f y several features of the EBS-DS that were systematically related stimulus cont r o l . Namely, the a c t i v i t y evoked by the EBS-DS, the ra t i o of the EBS-DS current intensity to population spike threshold and the placements of the stimulating electrodes in the angular bundle. Each of these features are discussed in d e t a i l below. In seeking an explanation for the differences in the acqu i s i t i o n rates of the FAST and SLOW groups, general procedural variables can- be' discounted immediately. For example, there were no consistent differences between the groups with respect to absolute body weight, food deprivation, test session time of day or test chambers. Furthermore, responding in the Tone-DS phase was approximately the same, in terms of both acqui s i t i o n rate and f i n a l performance l e v e l s . In a l l respects, the animals of both groups were treated in an i d e n t i c a l manner and d i f f e r e d only in responsiveness to the EBS-DS. Before the start of the EBS-DS phase, a l l animals were assigned to one of two groups on the basis of I/O curves. One of these groups was to receive i p s i l a t e r a l lesions; the other, contr a l a t e r a l lesions. This procedure must not be confused with the segregation of animals into the FAST and SLOW groups. A l l animals were expected to learn the discrimination. As i t turned out, half of the animals assigned to each of the i p s i l a t e r a l and contr a l a t e r a l lesion groups were unable to detect the single-pulse EBS-DS, and were consequently c l a s s i f i e d as SLOW. The equal number of animals in FAST and SLOW groups occurred only by 1 46 chance. As in the previous experiment (Chap. 4), the differences between the acq u i s i t i o n rates of the two groups appeared to be due to quantitative differences in the stimulus strength of the EBS-DS, rather than to q u a l i t a t i v e differences per se. The animals of the FAST group had latencies to the onset of acquisi t i o n that were approximately twice as long as the acquisi t i o n times. Once acqui s i t i o n began, i t proceeded at a f a i r l y constant rate. It seemed that the major impediment to acquisi t i o n was a d i f f i c u l t y i n detecting the stimulus and not in forming an association once the stimulus was detected. This conclusion received further support from the rapid ac q u i s i t i o n of stimulus control in the SLOW group after the f i r s t multi-pulse EBS-DS day. Even though the responses of the animals in the SLOW group were v i r t u a l l y random on the 15 preceding days, once multi-pulse EBS-DS presentations were begun, acqui s i t i o n of stimulus control was es s e n t i a l l y complete after only 3 days. It must be emphasized that during the acqu i s i t i o n time of the SLOW group ( i . e . , the second and t h i r d days of multi-pulse t e s t i n g ) , 75% of a l l t r i a l s were with the single-pulse EBS-DS. Therefore, the majority of discriminated responses during this time were controlled by the previously i n e f f e c t i v e stimulus, the single-pulses. Clearly, the SLOW animals were not inherently poorer learners or performers. The previous absence of discrimination could only have been due to stimulus properties whose strength was below the detection threshold. 147 The animals c l e a r l y had no d i f f i c u l t y detecting the multi-pulse EBS-DS) as almost every t r a i n e l i c i t e d an orienting response. Learning to attend to the relevant stimulus dimension or modality i s a well-known early component of discrimination learning (Mackintosh, 1974, pp. 589-607). For example, when animals receive pre-training on a simple discrimination, subsequent acquisition of a d i f f i c u l t discrimination i s f a c i l i t a t e d i f the relevant stimulus dimension is common to both tasks (Mackintosh, 1974, pp. 593-601). The multi-pulse EBS-DS was c l e a r l y a stronger stimulus' and may have attuned the SLOW animals to the stimulus properties of the weaker, single-pulse stimulus. To summarize, the behavioural difference between the FAST and SLOW groups appeared to be a matter of the stimulus strength of the EBS-DS; how well i t could be detected. The most reasonable explanation for the SLOW animals' i n a b i l i t y to respond to the single-pulses prior to multi-pulse testing was the weakness of the EBS-DS stimulus properties. From this conclusion, i t followed that the electrophysiological differences between the FAST and SLOW animals could provide important insights into the factors c o n t r o l l i n g the stimulus strength of angular bundle stimulation. Analysis of stimulating electrode placements, population spike thresholds, EBS-DS current i n t e n s i t i e s and evoked a c t i v i t y showed that a l l four of these factors were related to the rate of a c q u i s i t i o n , but not independently. The ultimate factor was evoked a c t i v i t y in the DG of both hemispheres. Total population 1 48 spike amplitudes were not only s i g n i f i c a n t l y larger in the FAST animals, they were also correlated with the rate of ac q u i s i t i o n . The electrophysiological effects of the tetanic stimulation in the SLOW group provided additional evidence that stimulus control depended upon the magnitude of the EBS-DS evoked a c t i v i t y . Although "cueing" by the multi-pulses provided a sati s f a c t o r y explanation for the immediate onset of acquis i t i o n in the SLOW animals, i t did not explain why acquis i t i o n rates and f i n a l performance levels were so high. Changes in the EBS-DS evoked a c t i v i t y did provide an explanation. The tetanic stimulation increased i p s i l a t e r a l evoked a c t i v i t y by an average of 102%, thereby r a i s i n g the population spike amplitudes into the same range as those in the FAST animals. Unfortunately, the acquisi t i o n rates and f i n a l performance levels of the animals in the SLOW group were too consistent to permit meaningful c o r r e l a t i o n a l analysis between the behavioural and evoked potential data. Nevertheless, i t was clear that the evoked a c t i v i t y was at least partly responsible for the maintenance of stimulus c o n t r o l . The differences in the EBS-DS stimulus strengths between the FAST and SLOW groups prior to multi-pulse stimulation were traced back to differences in the placements of the stimulating electrodes. The electrodes of the animals in the FAST group were clustered around a point in the angular bundle where the concentration of PP fibres was probably highest. Caudal to the point of cl u s t e r i n g , the angular bundle fans out and becomes much more dispersed (Fig. 5.5, 5.6). Lomo (1971 a) showed that PP 1 49 fib r e s also fan out in the r o s t r a l d i r e c t i o n ; more a c t i v i t y was e l i c i t e d by stimulation through electrodes in caudal placements than in r o s t r a l placements (Lomo, 1971a). The threefold difference in population spike thresholds showed that the electrodes of the animals in the FAST group were indeed closer to the fibres of the PP than the electrodes of the SLOW animals. The I/O curves of the animals who eventually comprised the SLOW group showed that high currents were required to produce threshold and asymptotic population spikes. Consequently, these animals were assigned r e l a t i v e l y high current i n t e n s i t i e s for the EBS-DS. The thresholds and hence the EBS-DS current i n t e n s i t i e s were lower in the FAST group. However, i t was clear that the variable c o n t r o l l i n g stimulus strength and acquisition rate was not the absolute value of the EBS-DS current intensity, but the ra t i o of this value to the population spike threshold. This r a t i o had a mean value of about six in the FAST group and three in the SLOW group. In other words, acqui s i t i o n was faster in some animals not because current i n t e n s i t i e s were higher, but because the current i n t e n s i t i e s used were a larger multiple of the population spike threshold. This finding completed the link between the placements of the stimulating electrodes and stimulus strength. In the FAST group, electrodes were clustered in the angular bundle near the point of the highest concentration of PP f i b r e s . Consequently, less current was required to produce a threshold population spike and an equal amount of current activated more PP f i b r e s . Current i n t e n s i t i e s of both groups were r e l a t i v e l y equal because 150 a l l were within the l i m i t s of 250 and 400 uA. Because more PP fi b r e s were activated in the animals of the FAST group, the evoked a c t i v i t y was greater, and hence, was more e a s i l y detected. The v a l i d i t y of t h i s l i n e of reasoning was supported by the s t a t i s t i c a l relationships between each one of these factors and acquisition rates. Although the multi-pulse EBS-DS was unquestionably a stronger stimulus than the single-pulses, the electrophysiological correlates of t h i s difference could not be spe c i f i e d . Each train evoked only a single population spike, because the i n t e r - p u l s e - i n t e r v a l was 5 msec and a l l responses after the f i r s t were inhi b i t e d (Gloor et a l . , 1964; Lomo, 1971a,b). Responses evoked by trains of stimulation at frequencies greater than 100 Hz have not been studied in great d e t a i l . Hence, the excitatory effect of these trains on c e l l s in CA3 i s unknown and i t could have been larger or smaller than the effects of single-pulses. S i m i l a r l y , the inhibitory periods that follow PP stimulation have been studied only with respect to single-pulse stimulation. Personal observations of the slow wave a c t i v i t y in the DG indicated that a c t i v i t y in the DG was suppressed for several seconds after each tetanic t r a i n . This stands in marked contrast to the usual 100 to 200 msec period of i n h i b i t i o n that follows a single pulse (Assaf & M i l l e r , 1978). Clearly, a number of electrophysiological effects of the tetanic stimulation could have contributed to the stimulus strength of the multi-pulse EBS-DS and much more research i s required for these to be properly understood. 151 The present study provided the f i r s t demonstration that stimulation in the angular bundle can f i r e the c e l l s in the cont r a l a t e r a l DG. Previously, stimulation of the entorhinal cortex, even at high intensity, produced only a small wave in the c o n t r a l a t e r a l DG (Steward, Cotman & Lynch, 1973). Although thi s wave was shown to arise from a c t i v i t y in the CA3 i p s i l a t e r a l to the stimulation, a subsequent study (Deadwyler, West, Cotman & Lynch, 1975) showed that even high intensity stimulation of this commissural pathway would not e l i c i t population spikes. The evoked a c t i v i t y recorded from the c o n t r a l a t e r a l DG in the present study could have been the consequence of a c t i v i t y in one of two alternative routes. A c t i v i t y could have been conducted either by the d i r e c t , crossed PP projection (Goldowitz et a l . , 1975), or by the temporo-temporal. projections of the entorhinal cortex (Blackstad, 1956; Wyss, 1981) and then the i p s i l a t e r a l PP projections in the c o n t r a l a t e r a l hemisphere. The fibres that cross from one entorhinal cortex have not been studied in s u f f i c i e n t d e t a i l to establish whether they are c o l l a t e r a l s of PP fibres or separate projections. Therefore, i t is not known how many synapses would be involved in conduction along t h i s l a t t e r route and the latency of a response propagated along these fibres cannot be predicted. There is evidence both for and against mediation of the cont r a l a t e r a l evoked a c t i v i t y by the crossed temporo-dentate component of the PP. Previous studies have shown that after a substantial lesion of the c o n t r a l a t e r a l entorhinal cortex and a 152 long period of recovery (e.g., 30 days), stimulation in the intact entorhinal cortex e l i c i t s a population spike from the cont r a l a t e r a l DG (Steward et a l . , 1973; Wilson, 1981; Wilson et a l . , 1979, 1981). The latency of t h i s response was 6 msec, in contrast to the 8 msec response latency observed in the present study. Although th i s 2 msec difference is not s u f f i c i e n t grounds for rejecting the crossed PP as a candidate pathway, i t suggests that the responses may arise via d i f f e r e n t routes. Remember, the contr a l a t e r a l population spikes recorded in the present study have not been- observed" previously • in intact animals. It seems foolhardy to accept one possible route in favour of the other based on so l i t t l e data. Even though the o r i g i n of this c o n t r a l a t e r a l evoked a c t i v i t y remains uncertain, i t must be emphasized that this a c t i v i t y was related to the stimulus strength of the EBS-DS. Taken together, the results from the a c q u i s i t i o n phase confirmed the importance of a c t i v i t y in the PP-DG to the stimulus properties of angular bundle stimulation. The rate of acqu i s i t i o n and hence the stimulus strength of the EBS-DS, was dependent upon the magnitude of the evoked a c t i v i t y in the DG of both hemispheres. Variations in evoked a c t i v i t y were traced back to the relationship between population spike thresholds and EBS-DS current i n t e n s i t y . The EBS-DS current i n t e n s i t i e s were r e l a t i v e l y constant but there were large differences in the thresholds. The ultimate factor c o n t r o l l i n g both the magnitude of the evoked a c t i v i t y and the EBS-DS stimulus strength was the proximity of the stimulating electrodes to the PP fibres in the 153 angular bundle. In conclusion, t h i s phase of the study provided much new evidence lin k i n g a c t i v i t y in the PP-DG to the stimulus properties of the EBS-DS. Perforant Path Lesions The effects of PP lesions on stimulus control by the EBS-DS showed that the i p s i l a t e r a l projection of the PP and the evoked a c t i v i t y in the anterior DG was partly, but not exclusively, responsible for the stimulus properties of the EBS-DS. Lesions anterior and i p s i l a t e r a l to the stimulation s i t e s , when preceded by a homotopic lesion in the contra l a t e r a l hemisphere, produced s i g n i f i c a n t reductions in stimulus control by a low current EBS-DS. U n i l a t e r a l lesions in either hemisphere, and b i l a t e r a l lesions in the reverse sequence had no s i g n i f i c a n t e f f e c t s on the discrimination r a t i o s . Because the observed d e f i c i t s followed only one of three types of lesions, they could not have been due to non-specific d e b i l i t a t i o n s of motor responses. The anatomy of the PP and the structures subserving the stimulus properties of the EBS-DS proved to be much more complicated than anticipated. The PP fibres projecting to the anterior DG were c l e a r l y not a l l collected into a single tight bundle at the le v e l of the lesions, nor were the stimulus properties solely dependent upon evoked a c t i v i t y near the recording electrodes. It is important to note that the lesions i p s i l a t e r a l to the EBS-DS did not eliminate the evoked a c t i v i t y in the DG and even b i l a t e r a l lesions produced only p a r t i a l disruptions of stimulus control. The identity of the structures 1 54 responsible for the evoked a c t i v i t y and the stimulus control p e r s i s t i n g after the lesions are the f i r s t issues that must be addressed. Clearly, the i p s i l a t e r a l lesions did not transect a l l the fibres projecting from the EBS-DS s i t e to the anterior DG. The f i r s t and second i p s i l a t e r a l lesions reduced the population spikes by only 35 and 45% respectively. It should be emphasized that mapping studies prior to the experiment and the electrophysiological procedures used during surgery ensured that the- lesion electrodes' were placed in the most dense aggregation of PP fibres at the A-P l e v e l of the lesions. The persistence of the evoked a c t i v i t y after the lesion showed that the fibres of the PP must be dispersed through a f a i r l y extensive region of tissue. It was not clear whether the undamaged PP projections were contained in the dorsal hippocampal commissure or a l t e r n a t i v e l y , had projected into the DG posterior and ventral to the lesions. In view of the amount of evoked a c t i v i t y in the anterior DG after the lesions, there was l i t t l e need to seek further explanation of the continued stimulus control by the EBS-DS. The undamaged fibres projecting to the anterior DG may well have sustained stimulus control a l l by themselves. Nevertheless, i t should be recognized that many fib r e s activated by the EBS-DS could have projected to regions of the DG posterior to the recording s i t e . As mentioned in the previous section, stimulation through caudal electrodes produces a c t i v i t y in a much larger portion of the DG than stimulation through r o s t r a l 155 electrodes (Lomo, 1971a). This i s a consequence of PP fibres leaving the angular bundle at various points along i t s length (Hjorth-Simonsen & Jeune, 1972). Therefore, many of the PP fib r e s activated by the EBS-DS may have projected to more caudal regions of the DG and would not have been damaged by the anterior lesions. These f i b r e s are excellent candidates for the mediation of stimulus control after the lesions. Having dealt with the issue of the structures that could have mediated stimulus control after the lesions, the next issue to be addressed concerns the structures that were responsible for the lesion-induced d e f i c i t s in discriminated responding. Through the careful consideration of the neural effects of the lesions, and the circumstances in which d e f i c i t s were observed, i t was possible to identif y the factors that were primarily responsible for the decrements in discriminated responding. It w i l l be recalled that u n i l a t e r a l lesions produced no s i g n i f i c a n t changes in stimulus control. After these lesions, the current intensity of the EBS-DS given to each animal was lowered to the minimum value capable of sustaining c r i t e r i o n responding. Subsequently, the PP lesion i p s i l a t e r a l to the EBS-DS produced a s i g n i f i c a n t decrease in the discrimination ratios but the con t r a l a t e r a l lesion did not. When the procedure and results are described in th i s manner, the sequence of the lesions and the EBS-DS current intensity would both appear to have been important determinants of the lesion e f f e c t . Closer examination of the factors considered by the multiple regression showed that the i n i t i a l transection of 1 56 c o n t r a l a t e r a l PP fibres was partly responsible for the behavioural effects of the subsequent i p s i l a t e r a l l e s i o n . The lesion-induced changes in the discrimination ratios were d i r e c t l y related to the proximity of the lesion electrodes to the i p s i l a t e r a l projections of the PP in both hemispheres. It w i l l be recalled that proximity was estimated by passing a 300 uA pulse through the lesion electrodes in the l e f t and right hemispheres and recording evoked potentials from the DG i p s i l a t e r a l to the stimulation. Presumably, the amplitudes of these population spikes, c o l l e c t e d before the' onset of conditioning, were determined primarily by the numbers of PP fibres activated by the fixed current pulses. This in turn provided an estimate of the density of PP fibres in the v i c i n i t y of each lesion electrode and served as one index of the PP damage subsequently produced by the lesions. The relationship between the measures of the proximity of the lesion s i t e s in both hemispheres to the PP and the d e f i c i t s in discrimination produced by the second lesions indicated that the behavioural change resulted from the combined ef f e c t s of the c o n t r a l a t e r a l -i p s i l a t e r a l sequence of the lesions. The explanation of why only t h i s p a r t i c u l a r sequence was e f f e c t i v e depends primarily on an analysis of the changes in evoked potentials after each l e s i o n . The f i r s t c o n t r a l a t e r a l lesion had no effect on the evoked a c t i v i t y produced by the EBS-DS in the i p s i l a t e r a l DG. In contrast, when animals received the i p s i l a t e r a l - c o n t r a l a t e r a l sequence, the second (contralateral) lesion induced a dramatic increase in the population spike 1 57 amplitudes. This increase was greater than 300% and was observed less than 24 hrs after the lesions. In addition, the population spike amplitudes e l i c i t e d by the low-current EBS-DS after the second (contralateral) lesion were larger than those e l i c i t e d by the higher current EBS-DS before the f i r s t ( i p s i l a t e r a l ) lesion in the same animals. Clearly, the augmentation was more than s u f f i c i e n t to compensate for the neural effects of the f i r s t l e s i o n . Therefore, i t i s hardly surprising that the contralateral lesions in these animals produced no d e f i c i t s in stimulus control. The neural changes underlying t h i s augmentation of the population spike could not be spec i f i e d but may have been due in whole or in part to the previous damage in the i p s i l a t e r a l pathway. Interestingly, the f i r s t c o n t r a l a t e r a l lesion had no effect on evoked responses mediated by an intact i p s i l a t e r a l PP projection. Previous investigations of functional p l a s t i c i t y in the PP-DG have focussed on the reinnervation of deafferented regions by fibres from the con t r a l a t e r a l hemisphere (e.g., Steward et a l . , 1973; Wilson et a l . , 1981). L i t t l e or no attention has been paid to transmission along partly damaged pathways or to interactions between lesions in homotopic portions of the PP. Only further investigation could determine whether the increases resulted from changes in synaptic e f f i c a c y in the DG, s p e c i f i c to the PP f i b r e s , or from a generalized increase in granule c e l l e x c i t a b i l i t y , produced by a release from tonic i n h i b i t i o n . Despite the lack of a concise physiological explanation, these lesion-induced increases in 158 evoked a c t i v i t y remain important because they provide a very plausible explanation for the difference in behavioural e f f e c t s of the two b i l a t e r a l lesion sequences. One structure that was probably uninvolved in the lesion effect was the crossed temporo-dentate projection of the PP. These fibres are very few in number (Goldowitz et a l . , 1975) and project only to the most anterior portions of the co n t r a l a t e r a l DG, r o s t r a l to the decussation of the dorsal hippocampal commissure (Wyss, 1981). Although the route of these fibres has not been described', i t would be reasonable to expect the fib r e s to remain in the commissure at least u n t i l after crossing the midline. Because the lesions were caudal to thi s decussation and ventro-lateral to the commissural bundle, there was probably very l i t t l e damage to the crossed temporo-dentate f i b r e s . The fibres may have been partly responsible for the stimulus properties of the EBS-DS but not the behavioural d e f i c i t s induced by the lesions. Direct damage to the DG below the lesion electrodes or to the subiculum adjacent to the electrodes did not provide a satis f a c t o r y explanation of the lesion e f f e c t s . The l e s i o n -induced changes in discrimination ratios were inversely related to the v e r t i c a l distance from the lesion electrodes to DG and to the size of the lesions. In other words, the largest d e f i c i t s in discrimination were induced by lesions that produced the least amount of damage to the DG and subiculum. Clearly, the lesion effect resulted from damage to some other structure. As mentioned e a r l i e r , the direct relationship between the 159 behavioural effects of the lesions and the proximity of the lesion electrodes to fibres of the PP showed that the lesion effect was most probably due to damage to the PP both i p s i l a t e r a l and contralateral to the EBS-DS. In summary, the results from t h i s phase of the experiment reaffirmed the role of the i p s i l a t e r a l projections of the PP in mediation of the stimulus properties of angular bundle stimulation. In addition, they also suggested a role for the c o n t r a l a t e r a l PP, though the nature of t h i s role was not c l e a r . Throughout the entire experiment, population spike- amplitudes proved to be a useful and r e l i a b l e measure of the neural a c t i v t y generated by the EBS-DS. Taken together, the results from both phases of the present experiment confirmed that the a c t i v i t y recorded from the DG was indeed relevant to stimulus control by single-pulse angular bundle stimulation. 160 CHAPTER 6: GENERAL DISCUSSION Long before the discovery of LTP, changes in the flow of neural a c t i v i t y through synapses had been proposed as a neural substrate of learning and memory (Brindley, 1967, 1969; Burke, 1966; Eccles, 1953; Hebb, 1949; Kandel & Spencer, 1968; Shimbel, 1950). The phenomenon of LTP shows that neurons in the mammalian hippocampus and perhaps many brain regions have the a b i l i t y to undergo enduring a l t e r a t i o n s in synaptic e f f i c a c y following r e l a t i v e l y brief periods of act i v a t i o n but this c a p a b i l i t y hardly proves that changes in synaptic e f f i c a c y subserve learning. In order to establish whether LTP i s a suitable model of the neuronal changes subserving learning, i t is necessary to show that a) the conditions necessary for LTP could occur without e l e c t r i c a l stimulation, b) increases in synaptic e f f i c a c y influence behavioural responses and c) synaptic e f f i c a c y in systems mediating conditioned responses increases during learning. The experiments in this thesis addressed the f i r s t two issues d i r e c t l y , and the t h i r d i n d i r e c t l y . The electrophysiological experiment (Chap. 2) examined the minimum frequency of EBS capable of producing LTP and showed that EBS pulses as infrequent as once every 10 seconds can induce LTP. Two experiments (Chaps. 3 & 5) were devoted to developing a behavioural paradigm in which a discrete stimulus generated a constant amount of a c t i v i t y in an i d e n t i f i e d neural system. In these studies, postsynaptic a c t i v i t y generated by an EBS-DS was 161 monitored continuously in order to estimate the magnitude of the neural a c t i v i t y e l i c i t e d by each DS and to observe whether synaptic e f f i c a c y was increased by conditioning. In addition, the evoked a c t i v i t y in the DG was shown to be a fundamental component of the EBS-DS stimulus properties. The most important experiment (Chap. 4) provided a demonstration of the behavioural consequences of increased synaptic e f f i c a c y . S p e c i f i c a l l y , this study showed that potentiation of synaptic e f f i c a c y in a system carrying stimulus information' could increase the probability of a behavioural response to a fixed amount of presynaptic a c t i v i t y . Even though the DS was e l e c t r i c a l stimulation rather than an exteroceptive stimulus, and the potentiation was produced by tetanic stimulation rather than conditioning, the f i n d i n g s o f this study have important implications for many forms of learning. The results from a l l of the experiments in t h i s thesis w i l l be reviewed below and the implications of each for the neural bases of learning and memory w i l l be discussed. In the f i r s t (electrophysiological) experiment, very low frequency stimulation (0.2, 0.1 Hz) increased synaptic e f f i c a c y in a manner that was indistinquishable from the potentiation produced by tetanic stimulation (Chap. 2). S p e c i f i c a l l y , population spike amplitudes rose and thresholds f e l l gradually over the 7 days of testing. The data l e f t l i t t l e doubt that high frequency a c t i v i t y was not essential for the production of LTP and provided further evidence that the neural conditions required to change synaptic e f f i c a c y for long periods l i e well 162 within the l i m i t s of normal neural a c t i v i t y . Comparisons between thi s study and e a r l i e r investigations of the effects of low frequency stimulation (e.g., Dunwiddie & Lynch, 1978), suggested that both synchrony and frequency of afferent a c t i v i t y are important determinants of LTP. At the conclusion of the behavioural experiments, the stimulus properties of single-pulse, angular bundle stimulation had been demonstrated in three separate r e p l i c a t i o n s , using 26 d i f f e r e n t animals. Although the latency to the onset of acqu i s i t i o n of' stimulus' control was longer for the- EBS-DS than for the Tone-DS, the learning curves and f i n a l performance levels were about the same for both s t i m u l i . The absence of discriminated responses to low or zero current EBS stimuli showed that stimulus control was due to the neural a c t i v i t y produced by the stimulation and not to any exteroceptive stimuli generated by the electronic equipment. Because the single-pulse EBS-DS e l i c i t e d no motor twitches or unconditioned orienting responses, i t was possible to infer that the stimulus properties were due to a c t i v i t y in central, rather than peripheral neurons. The importance of the p a r t i c u l a r stimulus used in the present experiment cannot be overemphasized. One advantage common to a l l EBS stimuli in comparison to extreoceptive stimuli i s the a b i l i t y to generate a fixed amount of a c t i v i t y in a pre-determined structure within the brain. The single-pulse, angular bundle EBS used in this thesis was a refined example of this general p r i n c i p l e . Most importantly, the use of very brief pulses at a constant current assured that each pulse produced a 163 single action potential in each of a fixed number of fibres near the electrode t i p s (Ranck, 1975). In addition, each pulse e l i c i t e d a large, e a s i l y interpreted, postsynaptic evoked potential in the DG, making i t possible to monitor the electrode placement and synaptic eff i c a c y on every t r i a l during conditioning. F i n a l l y , the long intervals between the single-pulses ensured that the EBS-DS did not produce LTP. These evoked potentials in the DG were -clearly related to the stimulus properties of the EBS-DS; one experiment was devoted to" i d e n t i f y i n g t h i s relationship (Chap. 5')'". In this experiment, the rate of acquisition of stimulus control by the EBS-DS was governed largely by the magnitude of the evoked a c t i v i t y in the DG, which in turn depended upon the number of PP f i b r e s activated by the EBS pulses. Supporting t h i s conclusion was evidence that the current intensity of the EBS-DS and the placements of the stimulating electrodes were important factors in determining the a c q u i s i t i o n rate, only with respect to their influence on the magnitude of the b i l a t e r a l evoked a c t i v i t y in the DG. S p e c i f i c a l l y , a c q u i s i t i o n was fastest in those animals who had stimulating electrodes positioned closest to the fibres of the PP, EBS-DS current i n t e n s i t i e s well above the i p s i l a t e r a l population spike threshold, and clear population spikes in the c o n t r a l a t e r a l DG. The rate of a c q u i s i t i o n provided a valuable index of the stimulus strength of the EBS-DS. The r e l a t i v e l y constant ac q u i s i t i o n times and the highly variable latencies to the onset of ac q u i s i t i o n showed that the major impediment to ac q u i s i t i o n 1 6 4 lay in the i n i t i a l detection of the EBS-DS (Chap. 5). From th i s i t was inferred that the stimulus strength was very close to the threshold for detection, and that d i f f e r e n t i a l a c q u i s i t i o n rates were due to r e l a t i v e l y minor differences in stimulus strength. The e f f e c t s of b i l a t e r a l PP lesions in thi s same experiment (Chap. 5), provided additional confirmation of the role played by the PP in the stimulus properties of angular bundle stimulation. Despite the fact that one set of b i l a t e r a l lesions reduced but did not eliminate the evoked responses in the DG, these lesions" s t'i 11 produced s i g n i f i c a n t d e f i c i t s in discrimination performance. These d e f i c i t s in stimulus control were attributed to b i l a t e r a l destruction of the PP, in a sequence which did not permit the appearance of a compensatory neural change. This experiment showed that a c t i v i t y in the i p s i l a t e r a l projection of the PP to the anterior DG played an important, but not exclusive role in mediating the stimulus properties of the EBS-DS. Analysis of the evoked potentials recorded from the DG following each EBS-DS throughout conditioning showed that increased synaptic e f f i c a c y in the PP-DG was not essential for the ac q u i s i t i o n of stimulus control. Population spike amplitudes did not increase systematically, unless tetanic stimulation was delivered as part of the experimental procedure (Chap. 3, 4 & 5). Of course, t h i s evidence does not exclude the p o s s i b i l i t y that changes in synaptic e f f i c a c y occurred postsynaptic to the DG and were c r i t i c a l l y involved in the learning. It should be noted that there i s a s l i g h t discrepancy 1 65 between the present observations and those of a recent study where' acq u i s i t i o n of stimulus control by a • PP EBS-CS was accompanied by alt e r a t i o n s in the DG responses to single pulse stimulation (Ott et a l . , 1982, pp. 441-452). In that study, multi-pulse stimulation at high frequency (15 or 100 Hz) acquired control over shock avoidance responses. Concurrently, the amplitudes of population EPSP's but not population spikes in the DG increased. The authors of that study (Ott et a l . , 1982, pp. 441-452) concluded that the observed changes in synaptic e f f i c a c y were caused by the conditioning experience and subserved the acq u i s i t i o n of stimulus control by the multi-pulse EBS-CS. Unfortunately, these conclusions were confounded by several problems with the methods and data. Even though the stimulation consisted of approximately 240 pulses each day at high frequency, there was no attempt to control for the physiological effects of the EBS. Therefore, the changes in the evoked potentials may have reflected LTP and could have been completely unrelated to the learning. More importantly, the absence of changes in population spike amplitudes showed that the changes in neural responses to the EBS were limited to the dendritic region of the granule c e l l s . From th i s i t can be concluded that the transynaptic evoked a c t i v i t y in CA3 was unchanged by the learning experience. It is d i f f i c u l t to see how such a limited a l t e r a t i o n in neural responses could have affected behaviour. The fundamental differences .between that study (Ott et a l . , 1982, pp. 441-452) and the experiments in this thesis must be 166 c l e a r l y understood. The single-pulses used here ensured that the stimulation used to monitor synaptic e f f i c a c y and provide a behaviourally relevant stimulus (Chaps. 3, 4 & 5) was incapable of producing LTP (Chap. 2). Furthermore, the basic measure of synaptic e f f i c a c y used here was the population spike; a measure which indicated the magnitude of evoked a c t i v i t y passed on to the next synaptic system, the CA3. For these reasons, the present demonstration that changes in synaptic e f f i c a c y are unnecessary for acq u i s i t i o n of stimulus control provided a strong challenge to the findings' and conclusions of Ott et a l . (1982, pp. 141-152). Actually, the presence or absence of an effect of conditioning on synaptic e f f i c a c y in the PP-DG was only a tangential issue in this thesis. The central issue was whether increased synaptic eff i c a c y in the PP-DG can influence the behavioural consequences of presynaptic a c t i v i t y generated by single-pulse stimulation of the angular bundle. In this context, the most important observation in this thesis was that tetanic stimulation f a c i l i t a t e d the subsequent ac q u i s i t i o n of stimulus control by a EBS-DS (Chap. 4). For several reasons, i t was possible to attribute this f a c i l i t a t i o n to an increase in the the stimulus strength of the EBS-DS, caused by the potentiation of synaptic e f f i c a c y in the PP-DG. The a b i l i t y to interpret the effects of tetanic stimulation in terms of changes in stimulus strength was partly due to the finding of the previously discussed experiment that showed the EBS-DS stimulus strength to be d i r e c t l y related to the magnitude 167 of the evoked a c t i v i t y in the DG (Chap. 5). A great deal of evidence also came from within the LTP .experiment i t s e l f (Chap. 4). S p e c i f i c a l l y , the evoked potentials c o l l e c t e d prior to the tetanic stimulation were the same for both groups before, but not after the tetanic stimulation. These evoked potentials indicated the production of LTP and showed that the EBS-DS evoked a c t i v i t y was greater in the experimental animals than in the control animals, throughout the course of conditioning. Furthermore, the 10 day delay between the tetanic stimulation and the appearance of a difference in the- discrimination performance of the control and experimental groups showed that the f a c i l i t a t i o n of acqui s i t i o n was not due to a transfer of stimulus control from the tetanic stimulation to the sin g l e -pulse EBS-DS. For a l l of these reasons, the eff e c t of tetanic stimulation on acqui s i t i o n of stimulus control by the EBS-DS provided a convincing demonstration that enhanced synaptic e f f i c a c y , when present in a neural system related to a conditioned response, can increase the pro b a b i l i t y of a behavioural response to a fixed amount of presynaptic a c t i v i t y . Further evidence for the influence of synaptic e f f i c a c y on behaviour came from e f f e c t s of the multi-pulse EBS-DS on stimulus control by single pulses (Chap. 5). It w i l l be r e c a l l e d that half of the animals tested showed no evidence of stimulus control by the single-pulse EBS-DS even after 600' t r i a l s (15 days). However, after only one day of testing with a multi-pulse EBS-DS, the discrimination ratios of a l l animals exceeded previous l e v e l s . Over the next two days of conditioning, when 168 75% of a l l t r i a l s used a single-pulse EBS-DS, discrimination ratios increased rapidly up to and beyond c r i t e r i o n l e v e l s . On the following days when every t r i a l used a single-pulse EBS-DS, discrimination performance continued to improve and soon reached levels higher than any previously observed with an EBS-DS in this thesis. Even though the rapid onset of a c q u i s i t i o n was probably due to the obvious stimulus properties of the EBS t r a i n s , the rapid a c q u i s i t i o n and continued discrimination performance to the single-pulse EBS-DS was probably due to the increased synaptic e f f i c a c y ( i . e . , LTP1) produced by the multi-pulse stimulation. Thus, the e f f e c t of increased synaptic e f f i c a c y on behavioural responses to a constant amount of neural a c t i v i t y was demonstrated twice; once with tetanic stimulation delivered in a separate environment p r i o r to conditioning, and once within the conditioning paradigm i t s e l f . These results show that the effects of LTP on the processing of neural a c t i v i t y extend well beyond the f i r s t synapse after the stimulated f i b r e s . The translation of evoked a c t i v i t y in the DG into the complex behavioural responses measured in the present paradigm must have involved many synapses and probably, many changes in neuronal interactions. After a l l , the unconditioned responses to high-intensity, high frequency stimulation was a complex exploratory response, not a simple motor twitch or a f u l l y developed approach and investigation of a food-hopper (Chap. 2, 4 & 5). This demonstration of the behavioural effects of LTP provided convincing evidence that there were no compensatory changes in 169 synapses postsynaptic to the DG. In other words, the present findings discount the previously overlooked p o s s i b i l i t y that the e f f e c t s of LTP in one system could be n u l l i f i e d by a depression in a s e r i a l synapse. The primary goal of t h i s thesis was to demonstrate a behavioural consequence of increased synaptic eff i c a c y and thereby support the v a l i d i t y of LTP as a model of neuronal changes subserving learning. It should be clear that the present studies examined the behavioural e f f e c t s of a neural modi f ication^ thought to be present at the completion- of a certain phase(s) of learning. No attempt was made to mimic any gradual neuronal changes during conditioning, or the way in which reinforcers might interact with a neural "trace" of the stimulus. The e f f e c t s of LTP on acq u i s i t i o n of stimulus control by the EBS-DS are best described as evidence indicating that an amplification of synaptic transmission is capable of increasing the p r o b a b i l i t y of a behavioural response to a fixed amount of presynaptic a c t i v i t y . A very general, l o g i c a l r elationship could be inferred from the present findings. Namely, i f presynaptic a c t i v i t y in a given neural system is related to behaviour, and the synaptic e f f i c a c y of that system is increased by tetanic stimulation or by conditioning, then the relationship between the presynaptic a c t i v i t y and the behavioural response w i l l be strengthened. However, before t h i s inference could be considered v a l i d , in whole or in part, careful consideration must be given to the question of whether the present results generalize to 1 70 exteroceptive stimuli or must be limited to EBS s t i m u l i . It could be argued that the a b i l i t y to generalize from the ef f e c t of LTP on an EBS-DS to conditioning with exteroceptive stimuli depends largely on the degree of s i m i l a r i t y between the neural a c t i v i t y generated by EBS and exteroceptive s t i m u l i . Clearly, there were several features of the a c t i v i t y generated by the EBS-DS which were quite d i f f e r e n t from neural a c t i v i t y caused by exteroceptive s t i m u l i . The activation of the PP by the EBS-DS was extremely synchronous, independent of a c t i v i t y in other brain regions, primarily u n i l a t e r a l and' coactive with many other fibres which were s p a t i a l l y but not functionally related. Of these differences, only the synchrony of the a c t i v i t y would tend to make a given amount of PP a c t i v i t y more rather than less l i k e l y to influence behaviour. Therefore, the most important point to consider is whether increased synaptic e f f i c a c y only a f f e c t s the postsynaptic responses to synchronous afferent a c t i v i t y and not to the more asynchronous a c t i v i t y generated by natural s t i m u l i . This seemingly complex issue can be d i s t i l l e d into the much simpler question of whether LTP influences the normal synaptic transmission from individual f i b r e s to individual c e l l s or whether the changes in synaptic e f f i c a c y affect only synchronous transmission between populations of c e l l s . Unfortunately, i t is almost impossible to to address th i s question d i r e c t l y by f i r i n g a single afferent fib r e and recording from a single neuron postsynaptic to i t . However, there is e l e c t r o p h y s i o l o g i c a l , anatomical and neurochemical evidence that LTP a f f e c t s small 171 populations of, and perhaps i n d i v i d u a l , c e l l s . The electrophysiological evidence derives from analysis of the changes in I/O curves produced by tetanic stimulation. S p e c i f i c a l l y , the r e l a t i v e increases in population spike amplitudes and population EPSP's are often largest at the lowest i n t e n s i t i e s , when fewest fibres are activated by the EBS (Chap. 2; Alger & Teyler, 1976; B l i s s & Lomo, 1973; Wilson, 1981). The anatomical evidence comes from electron microscopic analysis of LTP-induced changes in synaptic boutons and dendritic spines (Lee, et a l , 1980; Lee et a l , 1 9 8 1 , pp. 189-212). These studies demonstrated u l t r a s t r u c t u r a l changes in individual terminals. F i n a l l y , investigations of neurotransmitter changes following LTP have shown that tetanic stimulation that produces LTP also increases glutamate receptors on hippocampal neurons (Baudry, Oliver, Creager, Wieraszko & Lynch, 1980) and glutamate release from PP fibres (Dolphin, Errington & B l i s s , 1982). In view of a l l of this converging evidence, i t would appear to be very l i k e l y that LTP enhances synaptic transmission between in d i v i d u a l c e l l s and a f f e c t s the transmission of asynchronous and synchronous a c t i v i t y to an equal degree. Therefore, the fact that the neural a c t i v i t y evoked by the EBS-DS i s more synchronous than the a c t i v i t y generated by exteroceptive stimuli cannot by i t s e l f be used to d i s c r e d i t any generalization from the present findings to conditioning with exteroceptive s t i m u l i . To reiterate t h i s generalization, the effect of tetanic stimulation on ac q u i s i t i o n of stimulus control by the EBS-DS suggests that changes in synaptic e f f i c a c y 1 7 2 influence the p r o b a b i l i t y of a behavioural response to a fixed amount of presynaptic neural a c t i v i t y . In the present paradigm, the PP served as an example of an afferent fib r e system carrying stimulus information. Even though the animals learned to respond behaviourally to PP stimulation, t h i s finding does not necessarily indicate that the PP is in any way related to behavioural responses to exteroceptive s t i m u l i . The stimulus properties of EBS have been observed in numerous brain regions, many of which are c l e a r l y not involved in the processing of exteroceptive stimuli (Doty, 1969'). For example, Tsukahara et a l . (1981) showed that EBS in a t r i s y n a p t i c pathway from the red nucleus to the biceps muscle can acquire stimulus control over a withdrawal reflex. Few would use these data to argue for a resemblance between the perceptual properties of the EBS and those of a tone. Nevertheless, i t may prove worthwhile to consider whether the EBS-DS in the present paradigm produced a c t i v i t y in structures normally involved with conditioning of exteroceptive s t i m u l i . The presynaptic a c t i v i t y produced in the PP by the EBS-DS may have had d i f f e r e n t stimulus properties than a c t i v i t y in t h i s pathway during conditioning with exteroceptive s t i m u l i . In the present paradigm, the PP a c t i v i t y predicted food and the animals learned to respond appropriately. In contrast, the PP a c t i v i t y evoked by sensory events seems to encode stimulus "novelty" or "unexpectedness" (Deadwyler et a l . , 1979; Deadwyler et a l . , 1981a) . As mentioned e a r l i e r (see Chap. 1), analysis of slow-wave 1 73 potentials in the DG has shown that sensory-evoked a c t i v i t y in the PP decreases during conditioning and during habituation (Deadwyler et a l . , 1981a). After conditioning or habituation, these evoked potentials reappear i f the frequency of the tones, or the intervals between tones is altered. These data suggest that a c t i v i t y in the PP does not encode either the positive or the negative predictive value of exteroceptive s t i m u l i , but rather the occurrence of a "mismatch" between a sensory event and an expected event. This being the case, then the EBS-DS did not duplicate exactly the neural a c t i v i t y in the PP normally present during conditioning with exteroceptive s t i m u l i . Nevertheless, there are reasons to believe that the postsynaptic a c t i v i t y in the DG generated by the EBS-DS may have been similar to DG a c t i v i t y present during normal learning. There is a considerable body of evidence indicating that sensory-evoked unit a c t i v i t y in the DG, CA1 and CA3 is systematically related to stimulus control (see Chap. 1 or Berger & Thompson, 1978; Disterhoft & Segal, 1978; Olds et a l . , 1973; West, Christian, Robinson, & Deadwyler, 1981). S p e c i f i c a l l y , through the pairing of sensory events with reinforcers, these events acquire the a b i l i t y to e l i c i t behavioural responses that are correlated with changes in hippocampal unit a c t i v i t y . Some of the authors in t h i s f i e l d have suggested that increased synaptic e f f i c a c y may be responsible for the increased responsiveness of units in the hippocampus (e.g., Berger & Thompson, 1978; Clark et a l . , 1978; Deadwyler et a l . , 1976, 1979). In view of these changes in unit 174 a c t i v i t y in the DG during conditioning with sensory events, i t would appear that the effect of tetanic stimulation on the stimulus strength of the EBS-DS may have been a reasonable approximation of changes in the DG a c t i v i t y during normal learning. The question remains as to the type or process of learning that is most closely modeled by the present demonstration of LTP's effect on a c q u i s i t i o n . One behavioural phenomenon that bears a s u p e r f i c i a l resemblance to the increase in behavioural responses to the EBS-DS i s s e n s i t i z a t i o n . S e n s i t i z a t i o n is demonstrated whenever the presentation of a strong stimulus enhances the unconditioned response to a weaker stimulus (Grether, 1938; Sharpless & Jasper, 1956). Because the two stimuli need not be temporally contiguous, t h i s phenomenon i s considered to be a non-associative form of learning. It should be noted that in purely behavioural studies, the two^stimuli are not usually d i f f e r e n t i n t e n s i t i e s of the same sensory modality but are usually two quite d i f f e r e n t events, such as a buzzer and footshock (e.g., Harris, 1943). Usually, the effect of intense stimuli on subsequent responses to weaker versions of the same stimulus is not tested, presumably because the s e n s i t i z i n g stimulus (e.g., footshock) usually e l i c i t s a very strong behavioural reaction and the expected e f f e c t on weaker versions of the same stimulus i s so obvious". However, in the present paradigm, the strong and weak stimuli d i f f e r e d only in the number of EBS pulses. Therefore, i t is essential to consider whether the effect of tetanic 175 stimulation on a c q u i s i t i o n was due to behavioural s e n s i t i z a t i o n , or a l t e r n a t i v e l y , may have modeled a process subserving s e n s i t i z a t i o n . There are a number of reasons why s e n s i t i z a t i o n does not provide a s a t i s f a c t o r y explanation for the effect of tetanic stimulation on a c q u i s i t i o n of stimulus control by the EBS-DS. For example, s e n s i t i z a t i o n i s usually a transient e f f e c t , l a s t i n g minutes or, at the most, hours (Grether, 1938; Harris, 1943). In addition, s e n s i t i z a t i o n i s a general increase in arousal (Sharpless & Jasper, 1956) and' as- such would not be expected to f a c i l i t a t e acquisition of discrimination ratios in the paradigm used here. Support for this conclusion derives primarily from p i l o t studies conducted during the development of the present paradigm. In these studies using only a Tone-DS, animals that were tested at 80% of their free-feeding weight instead of 85%, responded continuously throughout the i n t e r -t r i a l intervals and never achieved discrimination ratios above 0.55. S i m i l a r l y , animals tested during the evening when behavioural a c t i v i t y levels were highest were also unable to learn the predictive significance of the Tone-DS. In sum, increased arousal was highly detrimental to a c q u i s i t i o n . Two additional points suggest that behavioural s e n s i t i z a t i o n did not contribute to the the effect of tetanic stimulation on acquisition of stimulus control by the EBS-DS. F i r s t , tetanic stimulation appeared to have no effect on discriminated responding to a Tone-DS. Although the data were not presented in this thesis, four d i f f e r e n t p i l o t studies were 176 conducted, using a t o t a l of 16 experimental and 10 control animals. None of these studies provided the s l i g h t e s t indication of a f a c i l i t a t o r y effect of tetanic stimulation on acquisition rates. Second, electrophysiological studies have c l e a r l y demonstrated that LTP i s limited to the afferent fibres activated by the tetanic trains (Chap. 1; Andersen et a l . , 1977; Dunwiddie & Lynch, 1978; Eccles, 1979; Lynch et a l . , 1977; McNaughton et a l . , 1978; Wilson et a l . , 1981). In t h i s regard, LTP i s very unlike behavioural s e n s i t i z a t i o n . Taken together the electrophysiological and the' behavioural data make i t seem very unlikely that the effects of tetanic stimulation on acquisition were due to behavioural s e n s i t i z a t i o n . There may be some role for synaptic potentiation in s e n s i t i z a t i o n , but the time course of this phenomenon indicates that PTP rather than LTP may provide the closest model. Post-tetanic potentiation, l i k e s e n s i t i z a t i o n , i s an increase in responsiveness that l a s t s for only a few minutes (e.g., LLoyd, 1947; McNaughton et a l . , 1978). The s p e c i f i c i t y of PTP has not been examined in d e t a i l so i t i s not known whether PTP is a result of a generalized increase in postsynaptic e x c i t a b i l i t y to many d i f f e r e n t inputs. However, i t is possible to speculate that behavioural s e n s i t i z a t i o n could be a consequence of transient PTP in afferent fibres carrying stimulus information from many di f f e r e n t modalities. A behavioural phenomenon that i s far more analagous to the f a c i l i t a t i o n of a c q u i s i t i o n produced by tetanic stimulation is the "acquired distinctiveness of cues" (Mackintosh, 1974, pp. 1 77 589-603). This phenomenon i s a well-known early component of discrimination learning and consists of the focussing of attention on p a r t i c u l a r stimuli or features of stimuli that distinguish a DS from the background, or two stimuli from one another. It w i l l be recalled that in the present studies (Chap. 4 & 5), the increased synaptic e f f i c a c y in the PP-DG appeared to have i t s effect by s e l e c t i v e l y amplifying the neural a c t i v i t y produced by the EBS-DS. This in turn appeared to increase the stimulus strength of the PP a c t i v i t y (Chap. 4). Conceivably, increased synaptic e f f i c a c y during' discrimination learning with exteroceptive stimuli could operate by s e l e c t i v e l y increasing the neural a c t i v i t y generated by a p a r t i c u l a r DS or one feature of two DSs in neuronal systems mediating perception. This process would act to increase the prominence or salience of the relevant stimulus and direct attention towards i t . Up to t h i s point in the discussion, the effect of LTP on ac q u i s i t i o n has been viewed as an a l t e r a t i o n in the processing of stimulus information. The relationship of a c t i v i t y in the PP with behaviour was considered to be similar to that of any afferent pathway that might encode the occurrence of an exteroceptive stimulus. However, perhaps the a c t i v i t y generated by PP stimulation could also be considered as being representative of a much broader class of events. Namely, afferent a c t i v i t y that bears any r e l a t i o n to behaviour. According to t h i s perspective, changes in synaptic e f f i c a c y would a f f e c t the processing of not only information about exteroceptive events, but also information from internal 178 receptors (e.g., from the gut or muscles). In addition, the behavioural effects of neural a c t i v i t y from brain areas involved in complex processing could also be enhanced by increased synaptic e f f i c a c y . The implication of this position is that changes in synaptic e f f i c a c y would have the power to increase the probability of a response to any external or internal stimulus or change the nature of the behavioural response e n t i r e l y . These broad implications from the present findings are the same as the' implications of LTP- i t s e l f . As such-, they have' been widely recognized and voiced by a number of authors ( B l i s s , 1979; Doty, 1979, pp. 53-63; Eccles, 1979; Kandel, 1976, pp. 476-489; Lynch et a l . , 1977, pp. 113-126; Tsukahara, 1981). Perhaps the most comprehensive treatment of t h i s issue has been provided by Goddard (1981, pp. 231-247), in a very interesting update of Hebb's (1949) synaptic model of learning. Goddard (1981, pp. 231-247), l i k e almost a l l theorists since Hebb (1949), began with Hebb's basic assumption that the strength of synaptic connections can be modified. A second important assumption was that every modification in behaviour i s a consequence of an a l t e r a t i o n in the flow of a c t i v i t y through the central nervous system. The inference usually made from these two assumptions is that changes in the strength of synaptic connections ( i . e . , synaptic efficacy) are largely responsible for the behavioural a l t e r a t i o n s c a l l e d learning. A l l synaptic models of learning recognize that neural processing consists of more than just the synaptic transmission 179 of action potentials from one neuron to another in a long chain of s e r i a l synapses. Every postsynaptic response i s determined by a large number of factors, including: the sum of tonic and phasic excitatory and inhibitory inputs, dendritic and somatic geometry, membrane c h a r a c t e r i s t i c s and e x c i t a b i l i t y cycles. Synapses attenuate inputs and each c e l l performs some transformation on a l l afferent a c t i v i t y . Furthermore, any given behaviour i s probably a consequence of concurrent a c t i v i t y in many converging neural systems. Despite the complexity of the determinants of information flow and behavioural responses-, the increased synaptic e f f i c a c y that characterizes LTP could exert a very powerful influence by reducing the attenuation of s p e c i f i c excitatory inputs and thereby make the difference between the success and f a i l u r e of synaptic transmission. Potentiation of one or many sets of synapses in a s e r i a l chain could allow a previously i n e f f e c t i v e amount of neural a c t i v i t y to reach the motor neurons. A l t e r n a t i v e l y , potentiation in the synapses along one multi-synaptic pathway and not another could guide the flow of information and result the appearance of a new response to an old stimulus. Almost a l l synaptic models of learning since Hebb's (1949) concur in thi s view of the neural events subserving learning (Brindley, 1967, 1969; Burke, 1966; Eccles, 1953; Kandel & Spencer, 1968; Shimbel, 1950). The models d i f f e r only with respect to the mechanisms subserving the proposed changes, the neural events required to produce the changes or the regions of the brain where these changes occur. The common basis of the 180 theories in changes in synaptic e f f i c a c y shows the inherent appeal of t h i s concept. However, i t must be emphasized that u n t i l the discovery of LTP, there was l i t t l e " proof that central neurons possessed the a b i l i t y to undergo changes necessary for l a s t i n g a l t e r a t i o n s in synaptic transmission. The studies in th i s thesis are important because they demonstrate that increased synaptic e f f i c a c y can indeed influence the behavioural response to an otherwise fixed amount of neural a c t i v i t y . This thesis provides one of the f i r s t pieces of evidence to validates a basic assumption common to a l l synaptic models of' learning. Namely, an increase in synaptic e f f i c a c y can enhance the behavioural consequences of neural a c t i v i t y . What remains to be shown is that t h i s kind of neural modification occurs as a consequence of experience and encodes the resulting behavioural change. 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