Specificity of Synaptic Growth in Brain: Remodeling Induced by Kainic Acid Lesions

Specificity of Synaptic Growth in Brain: Remodeling Induced by Kainic Acid Lesions CARL W. COTMAN Department of Psychobiology, University of California, Irvine, Calif. 9271 7 (U.S.A)

INTRODUCTION The basic circuitry of the brain is formed during development. However, brain circuitry is not fixed once it is formed. Even in the adult it is plastic, mutable, so that many perturbations, from environmental to experimental, can initiate new functional connections. Data providing the strongest support for this concept arise from studies of the responses elicited by partial denervation. Destruction of a portion of the input to a target often allows the remaining undamaged inputs to grow and create new functional synapses. This phenomenon is commonly referred to as axon sprouting or reactive synaptogenesis. The initial suggestion that partial denervation can induce new synapse formation originated from studies of the neuromuscular junction. In 1885, Exner suggested that the rapid recovery of neuromuscular contraction following partial transection of a motor nerve, but prior to regeneration, might be the result of fiber sprouting and the growth of new connections by the residual fibers. In the late forties and early fifties Edds (1950), Hoffmann (1950) and others showed by anatomical and histochemical methods that this was in fact the case. Studies of reactive fiber growth in response t o denervation were extended t o nerve-nerve connections in the sympathetic ganglion by Murray and Thompson in 1957, t o the spinal cord in 1958 by Liu and Chambers and to the brain in 1969 by Raisman. The basic paradigm in all such studies involves removing a portion of the input by various experimental means, and examining, over time, the response of the remaining afferents using anatomical, chemical or electrophysiological methods. It is now clear that new connections will form in response to denervation in a number of areas throughout the brain (see Cotman, 1978). At present, studies in the hippocampus provide the most thorough documentation of the nature of these events in the brain and their underlying mechanisms. GENERAL PRINCIPLES As a result of studies in the hippocampus and a variety of other brain systems, it is possible to derive the following general conclusions (Cotman and Lynch, 1976; Cotman and Nadler, 1978).

204 (1) Partial denervation stimulates some fibers to grow and form new synapses. These synapses usually are similar in overall appearance t o the original ones. In the cases where the synapses have been tested they are functional. (2) In mature animals synapses first form at approximately 5-9 days after a lesion; synaptogenesis may continue for 1-2 months. (3) The rate of reactive fiber growth can be changed. It can be accelerated if a small lesion precedes, by a few days, a larger one; it can be slowed down by the administration of glucocorticoids. (4) The capacity to form new synapses in response to a lesion persists throughout the life of an animal. It occurs in aged animals as well as mature adults although in aged animals the rate is slower and the extent of growth is reduced. (5) Synapse formation is selective in that only particular afferents respond in particular places to particular lesions. An afferent reactive t o one type of lesion may not respond to another. (6) In the adult nervous system, an efferent cannot reinnervate a new cell type. Existing inputs to a cell are increased and/or reorganized. Synapses may grow into areas of a dendritic field not previously innervated, i.e. grow outside their normal lamina. In all cases studied so far reactive afferents are adjacent to a denervated zone.

CURRENT MAJOR ISSUES The most critical issue at present is to understand the basis of selectivity. Why do only particular afferents grow t o particular places? The answer to this question would give considerable insight into reactive synaptogenesis. It has often been suggested that reactive synaptogenesis may not simply be a repair process, but that it may reflect an underlying mechanism the brain has at its disposal to remodel its circuitry throughout life. Studies on the specificity of synapse formation induced by lesions may provide clues on the hierarchy of changes normally permissible and their underlying mechanisms. More directly, knowledge of which fibers will reconnect should provide a basis to predict the usefulness of circuitry rearrangements for the recovery of function after brain damage. Will the new connections aid or hinder recovery? It might be possible to encourage beneficial changes and discourage nonbeneficial ones. In this paper I shall describe the basic changes which take place in the hippocampus following select lesions and I will examine some of the basic mechanisms insofar as they are understood. Through the use of kainic acid we have recently been able t o gain new insights into the nature of reactive synaptogenesis. Kainic acid was isolated in 1953 by Murakami and coworkers as the active ingredient in a crude extract of the red marine alga Digenea simplex used in the treatment of ascariasis. Subsequently it has been found that kainic acid is a cytotoxic agent structurally related to glutamate which destroys cell bodies while sparing those afferents impinging on these cells (see McCeer et al., 1978; Nadler, 1978). The use of kainic acid in the hippocampus has enabled us to selectively destroy cell groups previously inaccessible t o experimental manipulation. This has made it possible to examine a number of questions on selectivity which previously could not be studied in detail. Is selectivity in reactive synaptogenesis related at all t o the nature of the transmitter? To the nearest fibers? To the most abundant afferent? To the extent of denervation? To selective initiation of growth only in particular fibers? Besides serving as an experimental approach for understanding selectivity, the use of

205 kainic acid provides, for the first time, a means to experimentally imitate the cell loss that occurs in the hippocampus as a result of stroke, epilepsy and senile dementia. The hippocampus is well known to be a primary target of these dibilitating diseases (Minckler, 1971; Blackwood and Corsellius, 1976). What,if any, is the nature of synaptic growth and recovery following the loss of intrinsic cell populations? BASIC ORGANIZATION OF THE HIPPOCAMPUS The hippocampal formation consists of the dentate gyrus and the hippocampus proper which is divided into subfields CA1 , CA2, CA3 and CA4 (see Fig. 1). The hippocampal formation offers a number of important advantages for studies on reactive growth. It contains predominately two cell types, pyramidal cells in the hippocampal fields and granule cells in the dentate gyrus. These neurons and their axonal and dendritic processes are well separated by a large scale regional architecture. The circuitry within the hippocampus is relatively simple and well defined. Extrinsic inputs arise primarily from the septum, entorhinal cortex and various brain stem nuclei. Intrinsic inputs include several intrahippocampal projections as well as reciprocal connections between the two hippocampi. These, plus a few interneurons, appear to account for the vast majority of all connections. The connections are organized in discrete lamina, as we shall discuss (see Cotman and Nadler (1978) for a recent comprehensive review of hippocampal circuitry).

Fig. 1. Schematic of the hippocampus showing the major subfields and connections.

INTRAVENTRICULAR KAINIC ACID We have studied the effects of intraventricularly administered kainic acid as a means of selectively eliminating hippocampal neurons. We have found that the type of response obtained depends markedly on the dose administered (Nadler et al., 1978a, c). Low doses (0.1-0.3 pg) act quite selectively on CA3 pyramidal neurons. By 3 days after kainic acid administration, sections stained with cresyl violet show an extensive reduction in CA3 neurons (see Fig. 2). Most pyramidal cells are lost from areas CA3a and CA3b and a few are absent

206

Fig. 2. Effect of intraventricular kainic acid on hippocampal neurons. a: vehicle injected; b: 0.3 c: 0.8 pg; d: 1.1 pg. All pictures are from animals 3 days after the injection.

pg;

from area CA3c. Cell loss is greatest in the septal portion of the hippocampus. Granule cells, CA2, and CA1 neurons are not killed at this dose. At higher concentrations (approximately 1.1 pg), kainic acid has a remarkable effect. In addition to the loss of the CA3 neurons, subfields CA4 and CA1 are destroyed. Dentate granule cells are resistant to kainic acid as are CA2 cells. Intermediate doses (0.8 pg) produce a relatively selective lesion of CA3 and CA4 pyramidal cells. More than 90% of CA3 cells are lost as well as most CA4 cells except those at the apex of the dentate. CA1 is unaffected as are CA2 pyramidal cells and dentate granule cells. Some subicular neurons are lost, Doses above about 3 pg are usually fatal, and in the few animals which survive, hippocampal destruction is massive. Thus, kainic acid appears to destroy specific intrinsic hippocampal neurons as a function of the applied dose. From our studies, we have identified the order of sensitivity to kainic acid as CA3a pyramids > CA3b pyramids, CA3c pyramids > CA4 pyramids = subicular pyramids > CA1 pyramids > CA2 pyramids 9 dentate granule cells. Kainic acid does not appear to damage extrinsic afferents or intrinsic connections other than those emanating from the cells which are destroyed. The septal projection, for example, remains intact. The presence of acetylcholinesterase (AChE) in the cholinergic septohippocampal fibers provides a simple, convenient means of studying this projection. Following kainic acid there is no demonstrable loss in AChE staining in the hippocampus indicating that the septal afferents remain intact, despite the loss of intrinsic hippocampal neurons.

207 Before turning t o a discussion of reactive growth, it is appropriate t o comment on the selectivity of kainic acid. It is clear that kainic acid at low concentrations acts selectively on the hippocampus. Intraventricular kainic acid also affects structures outside the hippocampus when used at high concentrations. For example, at low doses (approximately 0.5 pg) there is occasional damage in the lateral septum adjacent to the site of injection but little damage elsewhere. Thus, at these or lower doses, kainic acid produces a fairly pure hippocampal lesion. With increasing doses, degenerating neurons appear in some of the thalamic nuclei, amygdala and deep layers of the cerebral cortex. No degenerating cells are seen in areas known to project to the hippocampal formation, except in nucleus reuniens of the thalamus and layer 111 of the entorhinal cortex. The striatum and hypothalamus are invariably spared, except for the lateral nucleus and premammillary area at high doses. The basis of selectivity in the hippocampus is not known, but it is not due to selective diffusion since, within the hippocampus, the CA2 field and granule cells are spared even when CA1 or CA4 neurons are destroyed. Also, areas directly adjacent to the injection site, such as the medial septum, are unaffected. Kainic acid appears to exert its effect directly on the hippocampus and not through an action elsewhere in the brain since low doses injected directly into the hippocampus produce a similar hierarchy of destruction. In order to obtain selective destruction of hippocampal neurons by intraventricular administration, we have found it necessary to carefully regulate the dose and the time over which the drug is administered. We inject kainic acid over a 30 min period at a fairly constant rate in order to minimize local concentration gradients and non-selective destruction. Rapid administration of high concentrations directly into brain tissue destroys most cells, illustrating that kainic acid can be non-selective when not precisely controlled. We have found that the effects of kainic acid are not confined t o adults but can be used as an effective cytotoxic agent in developing animals. Intraventricular kainic acid, as in adults, generally spares granule cells and acts preferentially on the hippocampal subfields. The main difference appears to be that the effective doses required are approximately 10-fold higher. REORGANIZATION WITHIN THE HIPPOCAMPUS AFTER KAINIC ACID LESIONS As mentioned above, one of our goals in pursuing studies with kainic acid is to examine the response to a clinically relevant form of hippocampal damage. Kainic acid lesions in rats closely mimic the selective destruction of hippocampal neurons seen in man following various neural pathologies. The CA3 field in the rat is homologous t o the end folium in man, a subfield of hippocampus particularly susceptible t o disease. The CA1 area plus subiculum of the rat is homologous t o the Somner sector in man, an area also susceptible t o disease. CA2 pyramidal cells, homologous to h2 cells in man, and dentate granule cells are nearly always spared. Thus, the lesion produced by 1.1 pg of kainic acid appears very similar to that which results from various human pathologies. In view of this finding, and the potential for mechanistic analyses not possible with other lesions, we performed a variety of histological and ultrastructural studies to determine whether the connections destroyed by kainic acid are replaced. Rats were injected bilaterally with an intermediate dose of kainic acid and the extent of reinnervation was studied. As noted above, intermediate doses of kainic acid (0.8 pg) destroy nearly every CA3 and CA4 pyramidal cell. In normal or vehicle-injected rats, the synaptic density in stratum radiatum of CA1 is about 35 synapses/100-pm2. In animals sacrificed

208 1-7 days after kainic acid injection, there is extensive presynaptic degeneration in this zone and the synaptic density falls to about 6 synapses/100 p m 2 . This reflects the loss of the dense innervation by the Schaffer collateral and commissural fibers. Many of the residual synapses feature boutons with flattened vesicles in symmetric contact, characteristics thought to signify inhibitory connections. A few asymmetric excitatory contacts are also preserved. Do the synapses return over time and, if so, what are their sources? When stratum radiatum is examined at 41 or 55 days after kainic acid administration, the synaptic density is normal (35 synapses/100 pm’), and the newly formed synapses resemble those normally present. In fact, in the electron microscope there is little, if any, difference between the recovered neuropil and that seen in normal animals. The dimensions of stratum radiatum are not affected by the drug treatment. Thus, it must be that extensive new growth has occurred. The total number of intact synapses increases at least 6-f0ld, illustrating one of the most extensive examples of reactive synaptogenesis yet reported. Which afferents contributed to this recovery? Few of the afferents adjacent to stratum radiatum appear t o grow. It might be expected that the mossy fibers which are left without their target cells might extend into CA1. Normally, the mossy fibers terminate sharply at the CA3-CA2 border. After kainic acid lesions, we never saw a case where the mossy fibers invaded the CA2 or the CA1 subfields. Thus, afferents without their target do not always mobilize and take the nearest target. Similarly, septal fibers retain their usual boundaries. AChE histochemistry shows that the septo-hippocampal fibers do not change their innervation in stratum radiatum. Finally, autoradiographic studies of the temporo-ammonic tract show that this afferent does not grow into the denervated zone from its normal termination in an adjacent lamina, stratum lacunosum moleculare. Thus, translaminar growth by adjacent fibers innervating the CA1 field does not appear responsible for reconstituting the population of synapses. At present, we have not identified the source of the new synapses. It appears likely that they arise from interneuronal proliferation, CA1 collaterals or extensive growth of fibers from the few residual CA3 pyramidal cells. These data indicate a strong selectivity in the systems responsible for reinnervation. It is interesting to note that kainic acid, administered t o developing animals in doses which destroy the CA3 field, promotes the growth of mossy fibers into the CA1 field (Fig. 3). Thus, it is clear that some of the constraints present in mature animals are not active in developing animals.

Fig. 3. Growth of the mossy fiber projection into the CA1 field when the CA3 field is destroyed in immature rats by kainic acid. No such changes occur following kainic acid destruction in mature animals. The figure o n the left shows the pattern in normal animals, that on the right from kainic acid treated animals. The arrow shows part of the new innervation by the mossy fibers.

209

Kainic acid at intermediate doses selectively denervates the dentate gyrus. The normal organization of inputs on the granule cell dendrites is illustrated in Fig. 4. The outer threefourths of the dendritic field consist primarily of a dense innervation from the ipsilateral entorhinal cortex and a sparse innervation from the septum and contralateral entorhinal cortex. The inner one-fourth of the dendritic field contains primarily the commissural and associational fibers originating from the contralateral and ipsilateral pyramidal cells of CA4, respectively. It contains, in addition, a very few septal fibers and mossy fibers (collaterals of the granule cells). In previous work, it has been shown that residual afferents extensively rewire their circuitry following an entorhinal lesion (Cotman and Lynch, 1976; Cotman and Nadler, 1978). Septal fibers and temporo-ammonic fibers proliferate within the denervated zone; PP/CPP

Normal

septol

C/A

C /A

I

I

Fig. 4. Organization of inputs on dendrites of granule cells in the dentate gyrus. Upper: normal organization of inputs. Middle: reorganization of inputs following unilateral removal of the entorhinal cortex in adult rats. Lower: organization of inputs in adult animals following unilateral removal of entorhinal cortex in the neonate.

210

commissural and associational fibers grow and form a new boundary pathway into the denervated zone. Bilateral injections destroy both the commissural and associational projections which terminate in the inner one-fourth of the granule cell dendritic field. This type of lesion provides a particularly unique opportunity to examine selectivity of growth within a defined and highly plastic population of neurons. Kainic acid lesions provide a way of examining the nature of the reinnervation response on a single cell type t o different forms o f denervation and allow the extent of denervation t o be varied. What is the nature of the reinnervation process? Destruction of the contralateral hippocampal CA4 neurons results in the loss of approximately 20% of the synaptic input, which is restored over time (McWilliams et al., 1975; McWilliams and Lynch, 1978). The new synapses appear t o originate from the associational system. Septal fibers and mossy fibers d o not appear to change. What happens when approximately 50% of the input to the commissural-associational zone is lost? Kainic acid injected bilaterally at intermediate doses (about 0.8 pg) increases the extent of denervation because it partially destroys the associational system. Again, electron microscopic analysis illustrates that the synaptic population returns t o nomial within 50 days (Nadler and Cotman, in preparation). Septal fibers d o not appear t o change as indicated by a normal AChE staining pattern. However, mossy fibers grow extensive collaterals and proliferate specifically within the denervated zone (see Fig. 5A). It is surprising that the septal system does not react; the

Fig. 5. Rearrangement of affercnt input in the commissural-associational zone as a result of progressive denervation. A: removal of approximately 50% of the synapses releases the growth of mossy fibers but not septal fibers, 105 days after injection; left normal, right denervated. B: removal of approximately 80% of the synapses releases the growth of septal fibers; left normal, right denervated. g = granule cells, m = molecular layer of dentate gyrus.

21 1 response of this system appears directly related to the extent of denervation. If higher doses of kainic acid are used so that nearly all the synapses in the commissural-associational zone are removed, the entire dendritic layer above the supragranular band stains uniformly for AChE (Fig. 5B). Entorhinal projections remain unchanged after all 3 lesions. These results are summarized in Fig. 6. Clearly, the nature of the response in the hippocampus depends markedly o n the nature of the lesion. There is a progressive hierarchy of reorganization depending precisely on the extent of denervation. In addition, the response depends on the nature of the particular afferent removed. Growth may be specific with no translaminar rearrangements as described for the CA1 field or dentate commissural-associational zone. Alternately, extensive translaminar growth may be displayed as shown by the expansion of commissural-associational fibers after an entorhinal lesion. What mechanisms appear to account for this remarkably specific growth?

E A

S

C

v 0

MF

E -C/A

S B

M'F

S 0

MF

Fig. 6. Diagram illustrating the changes in synaptic input caused by progressive removal of synapses from the commissural-associational zone. Upper: minimal denervation. Middle: intermediate denervation. Lower: maximal denervation.

212 MECHANISMS OF REINNERVATION Transmitter identity clearly does not account for the specificity. The septal system, for example, is cholinergic (see Storm-Mathisen, 1977) and grows in place of systems which clearly do not use acetylcholine as their transmitter. The entorhinal projections and the commissural and associational projections appear to use glutamate and/or aspartate as their neurotransmitters (Nadler et al., 1977, 1978b;Sandoval et al., 1978;White et al., 1978,1979). Similarly, the distance between the cells of origin of the reactive fibers and the denervated terminal field does not account for selectivity. For example, septal neurons are further from the zone of denervation than entorhinal neurons. Yet septal fibers react to extensive commissural-associational denervation whereas entorhinal fibers never do. Competition based on the abundance of particular afferent systems also does not appear to account for selectivity. The mossy fibers and the septal input are present, it appears, in similar abundance and, as we have discussed, mossy fibers grow under conditions in which the septal fibers do not. It should be noted that it may be that the mossy fiber and supragranular septal inputs are innervating interneurons rather than granule cells. Nonetheless, one system reacts before the other so that the differential response cannot be related to selective denervation in either case. A differential innervation pattern would need t o exist and there is no evidence at present for this. Other examples also tend to rule out competition based strictly on afferent abundance (see Cotman and Nadler, 1978). Selectivity might lie, at least in part, in the initiation process so that only certain fibers will grow under certain circumstances. Or, it might lie in some mechanism beyond initiation such as a specific affinity between afferents and target cells. Finally, there may be a selective suppression of growth. A simple experiment can be performed which provides a way to determine if selective initiation accounts for the differential responses. As noted above, septal fibers will proliferate in response t o a complete or partial entorhinal lesion or an, e.g., extensive commissuralassociational lesion. If initiation underlies the selective response in the commissural-associational zone, it should be possible t o initiate growth with minor commissural-associational denervation by simultaneously performing an entorhinal and a partial commissural-associational lesion. That is, once the septal system is reactive, it should react generally. Alternatively, if processes other than initiation are involved, there should be no change. We found that the AChE pattern is the same in response to an entorhinal lesion or a combination entorhinal and partial commissural-associational lesion (Cotman and Nadler, 1978, and in preparation). It does appear, however, that with complete commissural-associational lesions the characteristic expansion of the clear zone in the inner molecular layer does not occur. This zone expands in response t o an entorhinal lesion alone. Geometry is clearly an essential factor in establishing the pattern of reinnervation and, at one level of analysis, it accounts for some of the selectivity. Afferents must be adjacent t o a denervated zone in order to react, indicating that the presence of a normal field is prohibitive to growth. Proximity, however, while necessary is an insufficient growth condition in itself. As we have discussed, fibers adjacent to a denervated zone may not react, e.g., entorhinal fibers after a complete commissural-associational lesion. It appears that specific membrane properties and factors that inhibit growth are the most acceptable explanation for the selectivity at present. The presence of growth suppression factors was postulated by Cajal in order t o account for the termination of growth during development. Evidence favoring such factors in axon sprouting has been rallied recently by Diamond et al. (1976) based on studies on the sala-

213 mander hind limb. Reducing axoplasmic flow (via colchicine treatment) without actually destroying fibers triggers sprouting by adjacent untreated fibers. We have performed similar experiments in the CNS (Goldowitz and Cotman, 1978). We found that colchicine treatment which does not cause a net loss of synapses can promote synapse formation. These data are consistent with a regulation of synaptic growth by so-called negative trophic factors. Thus, as in the peripheral nervous system, it may be that growth suppression factors supplied t o the terminal by axoplasmic flow are responsible for maintaining the brain in a suppressed state of growth. There may be an interactive effect between the presence of existing fibers and the growth of new fibers. Growth, it appears, may need to be constantly held back. In the course of development and reinnervation, neurons accept a finite and fixed number of synapses. Denervation, whether through the actual loss of synapses or a disturbance of their metabolic impact on their target, releases synaptic growth. Progressive denervation may allow a progressive release of growth in residual afferents. CRITICAL AFFERENT MODEL OF LAMINATION The notion that certain afferents can suppress the growth of others appears t o explain a number of our observations. Specifically it is proposed that the commissural-associational system can suppress or repel the growth of the septal system. The septal fibers do not react in the commissural-associational zone until all, or nearly all, of these fibers are depleted. In contrast, septal fibers respond t o a partial, as well as complete, entorhinal lesion. Moreover, septal fibers in the inner molecular layer appear t o be displaced from part of their field by reactive commissural-associational fibers as evidenced by an expanded clear zone. This clear zone exactly corresponds to that of expanded commissural-associational fibers which sprout partway into the denervated entorhinal area. It may be that these expanded commissuralassociational fibers have forced the septal fibers out of their normal zone. If this is the case, removal of commissural-assocational fibers would prevent the clearing of septal fibers. Rats were treated with kainic acid in order to destroy commissural-associational fibers, given a unilateral entorhinal lesion 30 days later and stained for AChE 15 days post-lesion. The pattern of AChEstainingshowed that the AChE clear zone failed to develop. Thus, it appears that commissural-associational fibers can repel reactive septal fibers. Commissural-associational fibers appear t o serve as a critical afferent which can establish the pattern of septal reactivity and reorganization. SUMMARY The data discussed in this chapter illustrate that highly selective synaptic growth can occur in the adult brain. In order to trigger synaptic growth we have used kainic acid. Kainic acid kills certain neurons in the hippocampus when injected at low concentrations into the lateral ventricle. The CA3 subfield is most sensitive t o the cytotoxic action followed by CA4, CA1 and subiculum. The CA2 field and dentate gyrus are particularly resistant to kainic acid. The lesions produced by kainic acid in rats resemble, in many respects, the types of lesions produced in man as a result of epilepsy, senile dementia and stroke. In man, the Somner sector (homologous to CA1 and subiculum in rat) and the end folium (homologous

214 t o CA3 field in rat) are particularly vulnerable to cell loss. Over time, we find that the residual connections reorganize and a new circuitry emerges. Denervated cells become repopulated with new synaptic connections. The circuits which recover after intrinsic hippocampal cell loss depend not only on the cell group lost but on the extent of their loss. Particular fibers grow only in particular areas. In the commissuralassociational zone of the dentate gyrus fibers react sequentially as a function of the extent of denervation. It appears as if, in some cases, those fibers most related to the lost input react. When a particular extent of denervation is achieved, the reaction becomes less specific. Growth is always selective and hierarchical. The mechanisms which underlie selectivity are complex indeed. At present, it does not seem that transmitter identity, afferent competition, or proximity t o the denervated zone can account for the findings. Moreover, selective initiation of growth t o particular fibers can be ruled out in some cases. It is argued that the reactivity of septal fibers is selectively restrained by the commissuralassociational fibers and further, that reactive commissural-associational fibers can actually displace reactive septal fibers. Thus, growth suppression may play a critical role in reactive synaptogenesis. It may be that growth is normally retarded but can be released when such factors are reduced either by destruction of inputs or by reducing the axoplasmic flow of substances along afferent fibers. Thus, it may be that synapses are always trying to form in the mature brain and that there is a constant struggle between maintaining and changing synaptic connections.

REFERENCES Blackwood, W. andcorsellius, J.A.N. (Eds.) (1976) Greenfield’s Neuropathology. Edward Arnold, London. Cotman, C.W. (Ed.) (1978) Neuronal Plasticity. Raven Press, New York. Cotman, C.W. and Lynch, G.S. (1976) Reactive synaptogenesis in the adult nervous system. In Neuronal Recognition, S. Barondes (Ed.), Raven Press, New York, pp. 69-108. Cotman, C.W. and Nadler, J.V. (1978) Reactive synaptogenesis in the hippocampus. In Neuronal Plasticity, C.W. Cotman (Ed.), Raven Press, New York, pp. 227-271. Diamond, J., Cooper, E., Turner, C. and MacIntyre, L. (1976) Trophic regulation of nerve sprouting. Science, 193: 311-377. Edds, Jr., M.V. (1950) Collateral regeneration of residual motor axons in partially denervated muscles. J. exp. Zool., 113: 517. Exner, S. (1885) Notiz zur der Frage von der Fuservertheilung mehrerer Nerven in einem Musket. Arch. gen. Physiol., 36: 572. Goldowitz, D. and Cotman, C.W. (1978) Evidence that neurotrophic interactions control synapse formation in the adult rat brain. Brain Res., Submitted. Hoffmann, H. (1950) Local reinnervation in partially denervated muscle: a histophysiological study. Aust. . I exp. . Biol med. Sci., 28: 383-397. Liu, C.N. and Chambers, W.W. (1958) Intraspinal sprouting of dorsal root axons: development of new collaterals and preterminals following partial denervation of the spinal cord in the cat. Arch. Neurol. Psychiat. (Chic.), 79: 46. McGeer, E.G., Olney, J.W. and McCeer, P.L. (Eds.) (1978) Kainic Acid a s a Tool in Neurobiology. Raven Press, New York. McWilliams, J.R. and Lynch, G.S. (1978) Terminal proliferation and synaptogenesis following partial deafferentation. J. comp. Neurol., 180: 581-615. McWilliams, J.R., Lynch, G.S. and Cotman, C.W. (1975) Synaptic reinnervation in the inner molecular layer of the dentate gyrus following partial deafferentation: an electron microscopic study in the rat. Neurosci. Abstr., 1: 517. Minckler, J. (Ed.) (1971) Pathology o f t h e Nervous System, Vo12. McGraw-Hill, New York.

215 Murakami, S., Takernoto, T., Shimizu, 2. and Daigo, K. (1953) Effective principle of Digenea. Jap. J. Pharm. Chem., 25: 571-574. Murray, J.G. and Thompson, J.W. (1957) The occurrence and function of collateral sprouting in the sympathctic nervous system of the cat. J. Physiol. (Lond.), 135: 133. Nadler, J.V. (1978) Kainic acid: neurophysiological and neurotoxic action. Life Sci., in press. Nadler, J.V., White, W.F., Vaca, K.W., Redburn, D.A. and Cotman, C.W. (1977) Characterization of putative amino acid transmitter release from slices of rat dentate gyrus. J. Neurochem., 29: 279290. Nadler, J.V., Perry, B.W. and Cotman, C.W. (1978a) Preferential vulnerability to intraventricular kainic acid. In Kainic Acid as a Tool in Neurobiology, E.G. McGeer, J.W. Olney and P.L. McGeer (Eds.), Raven Press, New York, pp. 591-596. Nadler, J.V., White, W.F., Vaca, K.W., Perry, P.W. and Cotman, C.W. (1978b) Biochemical correlates of transmission mediated by glutamate and aspartate. J. Neurochem., 31: 147-155. Nadler, J.V., Perry, B. and Cotman, C.W. ( 1 9 7 8 ~ )Intraventricular kainic acid in hippocampal pyramidal cells. Nature (Lond.), 271: 676-617. Raisman, G. (1969) Neuronal plasticity in the septal nuclei of the adult rat. Brain Res., 14: 25. Sandoval, M.E., Horch, P. and Cotman, C.W. (1978) Evaluation of glutamate as a hippocampal ncurotransmitter: glutamate uptake and release from synaptosomes. Brain Res., 142: 285-299. Storm-Mathisen, J. (1977) Localization of transmitter candidates in the brain: the hippocampal formation a s a mode1.h-ogr. Neurobiol., 8: 119-181. White, W.F., Nadler, J.V., Hamberger, A., Cotman, C.W. and Cummins, J.T. (1978) Glutamate as transmitter of hippocampal perforant path. Nature (Lond.), 270: 356-351. White, W.F., Nadler, J.V. and Cotman, C.W. (1979) The effect of acidic amino acid antagonists o n synaptic transmission in the hippocampal formation in vitro. Brain Res., 164: 177-194.