8 Werle, hA. J. and Herrera, A. A. (1988) J. Neurobiol. 19, 465-481 9 Lullman-Rauch, R. hA. (1971) Z. Zellforsch. 121,593-603 10 Letinsky, M. S., Fischbeck, K. H. and McMahan, U. J. (1976)J. Neurocytol. 5, 691-718 11 Van Essen, D. C., Gordon, H., Soha, J. hA. and Fraser, S. E. (1990) J. Neurobiol. 21,223-249 12 Frank, E., Gautvik, K. and Sommerschild, H. (1975) Acta Physiol. Scand. 95, 66-76 13 Balice-Gordon, R. J. and Lichtman, J. W. (1989) Soc. Neurosci. Abstr. 15, 165 14 Wigston, D. J. (1989) J. NeuroscL 9, 639-647 15 Nabekura, J. and Lichtman, J. W. (1989) Soc. Neurosci. Abstr. 15, 165 16 O'Brien, R. A. D., Ostberg, A. J. and Vrbov~, G. (1978)J. Physiol. (London) 282, 571-582 17 hAorrison-Graham, K. (1983) Dev. Biol. 99, 298-311 18 Van Essen, D. C. (1982) in Neuronal Development (Spitzer, N. C., ed.), pp.

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333-376, Plenum Press 19 Changeux, J. P. and Danchin, A. (1976) Nature 264, 705-712 20 Purves, D. and Lichtman, J. (1985) Principles of Neural Development pp. 290-295, Sinauer 21 Bennett, hA. R. and Robinson, J. (1989) Proc. R. Soc. London Ser. B 235, 299-320 22 Connold, A. C., Evers, J. V. and Vrbov~., G. (1986) Dev. Brain Res. 28, 99-107 23 Kuromi, H. and Kidokoro, Y. (1984) Dev. Biol. 103, 53-61 24 Ziskind-Conhaim, L., Geffen, I. and Hall, Z. W. (1984) J. Neurosci. 4, 2346-2349 25 Liu, D. W. and Westerfield, hA. (1989) Soc. Neurosci. Abstr. 15, 1262 26 Hirokawa, N. and Heuser, J. E. (1982) J. Neurocytol. 11,487-510 27 Bloch, R. J. and Pumplin, D. W. (1988) Am. J. PhysioL 254, C345-C364 28 Balice-Gordon, R. J. and Lichtman, J. W. (1990)J. NeuroscL 10, 894-908 29 Fraser, S. E. and Poo, M-hA. (1982) Curr. Top. Dev. Biol. 17, 77-100

30 Nitkin, R. hA. etal. (1987) J. Cell BioL 105, 2471-2478 31 Hunter, D. D., Shah, V., hAerlie, J. P. and Sanes, J. R. (1989) Nature 338, 229-234 32 Anglister, L. and hAcMahan, U. J. (1985) J. Cell Biol. 101,735-743 33 Salpeter, hA. hA., Cooper, D. L. and Levitt-Gilmour, T. (1986) J. Cell Biol. 103, 1399-1403 34 Burden, S. J., Sargent, P. B. and MchAahan, U. J. (1979)J. Cell Biol. 82, 412-425 35 hAcMahan, U. J. and Slater, C. R. (1984) J. Cell Biol. 98, 1453-1473 36 Reist, N. E., hAagill, C. and hAchAahan, U. J. (1987) J. Cell Biol. 105, 2457-2469 37 Sanes, J. R., Marshall, L. hA. and hAchAahan,U. J. (1978)J. Cell BioL 78, 176-198 38 Glicksman, M. A. and Sanes, J. R. (1983) J. Neurocytol. 12,661-671 39 Lichtman, J. W. and Balice-Gordon, R. J. (1990) J. Neurobiol. 21, 99-106 40 van Mier, P. and Lichtman, J. W. (1989) Soc. Neurosci. Abstr. 15, 334

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Brief seizure episodesinduce long-term potentiation and mossy fibre sprouting in the hippocampus Yehezkel Ben-Ari and Alfonso Represa YehezkelBen-Ariand Alfonso Represaare at the Unit#de Neurobio/ogieet Physiopathologiedu Developpement, INSERMU29, 123 Bd de Port-Royal,75674 ParisCedex14, France.

Much of our present understanding of the cellular mechanisms of /earning and memory derives from studies on the hippocampus in which long-term potentiation (LTP) of synaptic transmission is produced by a train of high-frequency electrical stimulation 1 or by potassium channelblockers2. The hlppocampusis also a se12ure-prone region and recent studies have revealed that brief seizure episodes produce remarkably long-lasting changes which are reminiscent of 'classicar LTP3.A brief seizure episodealso sets in motion a cascadeof events that includes changesin gene expression, sprouting of fibres and the establishment of new synaptic contacts. Thispaper reviews this use-dependentstructural rearrangement of the neuronal network and discussesits possible role in epilepsyand asa modelof plasticity in the adult nervoussystem.

Granule cells and the CA3 region: organization and unique features Since the general organization of the hippocampal formation has been reviewed elsewhere 4 we shall only refer to the principal neuronal elements of the granule cell-CA3 region which are of direct relevance to the topic of this review. The granule cells (see Fig. 1 ) constitute a gateway to the hippocampus since they are the target of the perforant path, a major afferent bundle that originates in the entorhinal cortex. Granule cells play a pivotal role in hippocampal function since their elimination by early postnatal X-ray irradiation induces behavioural deficits in the rat, similar to those produced by bilateral hippocampal lesion s. The axons o f the granular cells, the mossy fibres (see Fig. 1), establish synaptic contacts with inter° neurones in the inferior region and with the proximal part of the apical dendrites of giant CA3 312

pyramidal neurones. Mossy fibre terminals contain and release glutamate 6,7, which generates an EPSP acting primarily on quisqualate and kainate subtypes of glutamate receptors 8. At the electron microscopic level, the mossy fibre synapses appear as multiinvaginated boutons (up to 101~m in diameter) establishing multiple contacts with giant dendritic spines 9. Because of the proximity to the cell body of the pyramidal neurone (less than 200 I~m), the synaptic input will efficiently control the excitability of its target 1°. The giant pyramidal neurones o f CA3 receive, in addition to the mossy fibre input, extrinsic projections originating from the contralateral hippocampus (commissural fibres) and other cortical or subcortical afferents. CA3 pyramidal neurones are also directly interconnected by a powerful excitatory recurrent collateral system which impinges on the upper dendritic region. This recurrent collateral system, which is glutamatergic and involves N M D A (N-methyI-D-aspartate) receptors (see below) plays a major role in the sensitivity of CA3 neurones to epileptogenic conditions 11,12. Characteristic features o f mossy fibres. The mossy fibres have other distinct features that permit their visualization and underlie their important role in the control of the excitability of the CA3 region. (1) Mossy fibres contain and release opioid peptides, such as dynorphin and enkephalins, which generate epileptiform activity in the CA3 region 13,~4. (2) The granule cells of fascia dentata and CA1 pyramidal cells can be heavily immunostained with calbindin28K (CaBP-D-28K)~5,~6; mossy fibres also become

© 199o,ElsevierSciencePublishersLtd,(UK) 0166-2236/90/$0200

TINS, VOI. 13, NO. 8, 1990

immunostained, whereas CA3 pyramidal neurones are devoid of staining in control conditions. Like other calcium (Ca2+)-binding proteins, CaBP-D-28K probably participates in the regulation of intracellular Ca 2~ levels 17. (3) In humans and in various animal species, mossy fibre terminals contain the highest concentrations of zinc found in the brain (i.e. 150-250 pM zinc18'19); zinc is found in large, dense core vesicles 2°,2~ and is taken up and released from mossy fibres 21,22. The Timm stain, which reveals zinc, can be used to study changes in mossy fibre innervation 2°. The function of zinc in mossy fibres has not been elucidated; however, it blocks NMDA channels 23,24, reduces GABAergic responses24 and reduces Ca 2+ currents (probably due to an intracellular release of Ca2+) 25. (4) Kainic acid (KA) is a structural analogue of glutamate with potent excitotoxic and convulsant properties 26,27. KA binds to a high-affinity site (Kd = 12-20 nM) with a slow dissociation rate and to a rapidly dissociating low-affinity site (Kd = 5 0 - 1 0 0 nM) 28 The high-affinity site has a non-uniform distribution, with the highest density in the terminal region of the mossy fibre in human and rat hippocampus, permitting the use of quantitative autoradiographic techniques to label the mossy fibres 29,3°. Lesion experiments suggest that a substantial proportion of the high-affinity KA binding sites are located presynaptically on the mossy fibre terminals 3~. KA has particularly powerful excitatory effects on CA3 pyramidal neurones 3,32,33. It also reduces GABAAand GABAB-mediated inhibition 33,34 and attenuates several repolarizing K + conductances as well as Ca2+ currents 34,35. KA produces seizures and brain damage, which have been used as an experimental model of temporal lobe epilepsy 26 ,27 . CA3 neurones are the most vulnerable in the brain to the neurotoxic effects of KA, and mossy fibres have been implicated in this preferential action, presumably due to the excitotoxic release of glutamate, zinc and other factors 27.

Convergence of NMDA-dependent and NMDAindependent LTP on CA3 pyramidal neurones A high-frequency train of electrical stimuli to the mossy fibres in the hilar region produces a longlasting potentiation of mossy fibre EPSPs in CA3 neurones. In contrast to the LTP produced in CA1 by stimulation of the Schaffer collateral or in the granule cells by stimulation of the perforant path 36, this LTP is not blocked by selective NMDA antagonists and is due to a rise in the intracellular concentration of calcium ([Ca2+]~), probably mediated by the activation of voltage-dependent Ca2+ channels 37. However, a high-frequency stimulation train given to the commissural-associational projections produces, in the apical dendrite of CA3 pyramidal cells, an LTP that is blocked by NMDA receptor antagonists; the terminal field of the commissuralassociational projection, unlike the mossy fibre region, is enriched in NMDA receptors 3a. The convergence of NMDA-dependent and NMDAindependent forms of Ca2+-mediated LTP on the dendrites of the same neurone is, to the best of our knowledge, unique and may underlie the prominent TINS, VoL 13, No. 8, 1990

effects of hyperactivity on CA3 neurones (see below).

Seizure-induced LTP in the CA3 region Bath application of convulsant drugs or procedures, such as bicuculline, pentylenetetrazol, high K + medium or K + channel blockers, generates epileptiform activity in the CA3 region, which propagates subsequently to other hippocampal regions (see, for example, references in Ref. 3). This epileptiform activity is characterized by the frequency of recurrent synchronized bursts (interictal bursts). However, Ben-Ari and Gho 3 have found that a brief epileptiform episode also produces a long-lasting potentiation of synaptic transmission which is reminiscent of LTP; thus, as shown in Fig. 2, a brief bath application of KA to hippocampal slices evoked interictal bursts that persisted for 10-15 min. After washing out KA, electrical stimulation of the mossy fibres or other inputs to CA3 neurones, which previously elicited an EPSP-IPSP (inhibitory postsynaptic potential) sequence now evoked an allor-none interictal burst. This effect persisted for the time the preparation lasted (for up to 4 h) 3. The

A

c,

Fig. 1. (A) Diagram of the hippocampal network. (B) Coronal section of rat hippocampus to demonstrate the distribution of Timm-stained mossy fibres in a control rat. Abbreviations: Ent, entorhinal cortex; FD, fascia dentata; F, fimbria; MF, mossy fibre; pp, perforant pathway; rc, recurrent collateral; Sc, Scha ffer collateral. 313

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Fig. 2. A brief bath application of kainic acid (KA) induced long-lasting changes which involve NMDA (N-methyI-D-aspartate) receptors. (A,B) Records from the same CA3 pyramidal neurone. (A) D-APV [D-(-)-2-amino-5-phosphonovaleric acid] was continuously superfused; mossy fibre stimulation is first shown to evoke a typical EPSP-IPSP sequence (left), addition of KA (dark bar) induced a spontaneous burst which disappeared a few minutes after washing out of KA. Mossy fibre stimulation 20 min later evoked an EPSP-IPSP sequence. (B) In control medium (without DAPV) 45 min after washing out of KA, stimulation of the mossy fibres evoked a burst instead of the EPSP-IPSP response. This new response persisted for the duration of the preparation. (Seealso Ref. 3.)

314

generates a persistent evoked synchronized burst in CA3 (Ref. 41). Parallel observations using chronic models of epilepsy have extended these findings. Kindling is an animal model of temporal lobe epilepsy in which repetitive daily stimulation of limbic structures generates a persistent modification of synaptic responsiveness42. Daily application of NMDA receptor antagonists prior to the electrical stimulation delays the development of the kindling state, but fails to suppress established kindled seizures43,44. These observations reflect the major difference between the process of epileptogenesis and that of seizure manifestation: NMDA receptor antagonists are more likely to prevent the former than to block the latter 3,45. They also suggest that processes involved in LTP play a role in epileptogenesis. Seizure-induced mossy fibre sprouting in experimental epilepsy

In KA-treated rats there is a collateral sprouting of mossy fibres, which cross the granule cell layer to innervate the inner third of the molecular layer31,46,47. This sprouting is associated with the development of new aberrant synaptic contacts 47. Mossy fibres long-lasting effect of a brief seizure episode has also form a new aberrant infrapyramidal band in the several features in common with LTP. (1) It is due CA3 field of the contralateral hippocampus 31 neither to a persistent reduction in GABAergic Furthermore, in the reinnervated zones there is a inhibition nor to a sustained increase in cell excit- significant increase in the density of KA binding sites ability. (2) Addition of NMDA receptor antagonists, (124_+5 fmol/mg protein and 165+6 fmol/mg such as D-(-)-2-amino-5-phosphonovaleric acid protein in control and epileptic rats, respectively), (APV), before and during KA application prevented but no change in affinity (Kd values were 12_+2 nM these long-lasting effects without blocking the and 14+3 riM, respectively)31. seizure episode 3. In contrast, NMDA antagonists In kindled rats, mossy fibres form an aberrant failed to block the giant EPSPs once these were band in both the supragranular layer of the fascia induced. Therefore, as in LTP, activation of the dentata 48,49 and the CA3 infrapyramidal layer NMDA receptor channel complex is necessary for (stratum oriens) 49. These aberrant bundles are the induction but not for the maintenance of this enriched in zinc and immunoreactive to CaBP-Duse-dependent model of persistent changes in 28K antibody. There is also a significant increase in synaptic properties. It is likely that the brief recurrent KA binding in the supragranular layer of the fascia burst increased [Ca2+] i by the activation of NMDA dentata and in the CA3 infrapyramidal layer receptors; this could generate, as for LTP, a chain of (205-+18% and 392-+40% of control, respectively). events leading to the persistent potentiation of This increase is specifically limited to the regions synaptic transmission 39. A similar long-lasting innervated by the mossy fibres (CA3 and fascia change has been observed in CA3 neurones follow- dentata, but not CA1) 49. A qualitative electron ing other treatments that induce a transient epilepti- microscopic study suggested that mossy fibres form activity, including high K+ (Ref. 3) or repetitive establish aberrant synaptic contacts with granular trains of high-frequency electrical stimulation of the dendrites 48. This sprouting is conspicuous even mossy fibres (in vitro kindling4°). Furthermore, bath before the first generalized epileptic events, i.e. application of the K+ channel blocker, mast cell- localized afterdischarges are sufficient to induce degranulating peptide, which in CA1 induces LTP2, sprouting that persists for several months 48. ~NS, VoL 13, No.& 1990

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Mossy fibre sprouting in human patients with epilepsy Childhood epilepsy ~°. The group of children studied suffered from a severe epilepsy that lasted for several weeks. The underlying disorders had different aetiologies. The only common feature was the occurrence of frequent severe generalized seizures. Hippocampal tissue samples were dissected within a postmortem delay of 24 h; with a short delay like this, the density of KA binding sites is not altered in control conditions 3°. Neuropathological examinations did not reveal histological lesions in the hippocampus. Compared with age-matched controls, the epilepsy cases showed a clear increase in Timm deposits in the pyramidal-oriens layers of CA3. Furthermore, there was a marked rise of KA binding sites (Fig. 3) in CA3 (from 49.6+14 to 125+24 fmol/mg tissue) and in the fascia dentata (from 24+5 to 51_+7 fmol/mg tissue), but not in CA1 (22+7 versus 32+5fmol/mg tissue). This increase probably represents a change in the density of sites without a change in binding affinity since the Kd in epileptic hippocampi (19.6 nM) is quite similar to that previously reported in human hippocampal membrane preparations 51. Adult temporal lobe epilepsy. Seizure-induced sprouting of mossy fibres has also been reported in adult human patients with temporal lobe epilepsy 52-s4 (see Table I). The hippocampal specimens, which presented a typical sclerosis of Ammon's horn characterized by a lack of CA4 neurones, were obtained surgically and the mossy fibres stained either by the Timm procedure or immunohistochemically using dynorphin-A antibodies. Mossy fibre sprouting was conspicuous in the molecular layer of the fascia dentata but not in the CA3 field. Ultrastructural analysis of the hippocampus from one epileptic patient suggested that the supragranular Timm deposits correspond to mossy fibre terminals 53. Are the new mossy fibre synapses functional? The histochemical and autoradiographic data reported above clearly suggest that mossy fibres sprout during epileptic processes. Electron microscopic studies indicate that this sprouting is associated with the development of new synapses, although quantitative studies will be necessary to establish the presence of the characteristic giant dendritic spines in the postsynaptic membranes facing the mossy boutons. It is at present unclear whether mossy fibre collaterals form a functional recurrent excitatory circuit. Tauck and Nadler 55 reported that 12-21 days after intraventricular KA administration, antidromic stimulation of the mossy fibres elicited multiple population spikes in granule cells in contrast to the single population spikes obtained in control slices. However, intracellular studies are required to provide more compelling evidence on the synaptic nature of this recurrent loop and on the pharmacology of these aberrant synapses. It will also be of interest to examine the effects of the 'double' innervation of mossy fibres (supra- and infrapyramidal) on the excitability of CA3 neurones. TINS, VoL 13, No. 8, 1990

point:

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A recent study suggested a significant correlation between the density of an aberrant supragranular mossy fibre band and the frequency of spontaneous epileptic seizures in KA-treated rats56. Although this correlation concerned only a proportion of the rats, the animals that exhibited chronic spontaneous seizures showed significantly more sprouting than animals that did not have spontaneous seizures. Therefore, in human and experimental epilepsy there appears to be a collateral sprouting of mossy fibres and the establishment of aberrant connections. This is likely to enhance the excitability of granule cells and CA3 pyramidal cells, notably because of the prominent excitatory effects of KA, dynorphin and zinc. What are the mechanisms of seizure-induced collateral mossy fibre sprouting? The cascade of causally related events that leads from seizures to mossy fibre sprouting is not at present known; there are clearly a number of gaps in the information that we have. However, it is possible to propose the following mechanisms. Rise in [ C a 2 + ] i . As for LTP39, a brief seizure episode triggers a large Ca2+ influx in hippocampal neurones 57-59. Extensive evidence suggests that Control

Control

Kindled

Kindled

Fig. 3. Photomicrographs depictin& the sprouting of hippocampal mossy fibres in the CA3 field of kindled rats. (A,C) Timm staining. Note the increased density of silver deposits in the stratum oriens of CA3 (arrows) in kindled rats. (B,D) [3H]KA autoradiography. Note the increased density of [3H]KA binding sites, notably in the stratum oriens of CA3 (arrows). Abbreviations: lu, stratum lucidum of CA3; o, stratum oriens of CA3; p, pyramidal cell layer. (See also Ref. 49.) 315

TABLE I. Sprouting of mossy fibres in human tissue

increase the expression of the proto-oncogenes c-fos, c-jun, Subjects No. of cases Cases with mossy Markers Refs jun-b and zif/268; this is first seen fibre sprouting in the granule cells of the fascia Childhood Seven postmortem 100% Timm staining. 50 dentata (after 10-30 min) and epilepsy hippocampi Quantitative subsequently in CA3 and other generalized autoradiography limbic regions 6°-64, with a patseizures tern that is reminiscent of the (4.3 years)a progressive spread of paroxysmal activity revealed in these models Temporal lobe 35 surgically 83% Dynorphin-A 52 by depth electrical recordings and epilepsy (age not removed immunoreactivity 2-deoxyglucose autoradiography reported) a hippocampi (Refs 27, 63). The induction of Partial complex or Ten surgically 100% Timm staining 53 c-fos expression is among the partial complex removed and, from one most rapid genomic responses prosecondary hippocampi patient, electron duced by neuronal activity and generalized microscopy studies in vitro have shown that seizures c-fos expression is directly stimu(32 years)a lated by voltage-dependent Ca2+ channels 65, nerve growth factor Temporal lobe Ten surgically 70% Timm staining 54 and second messenger systems, epilepsy removed and dynorphin-A without the necessity of inter(29.2 years)a hippocampi immunostaining vening protein synthesis 63,66. AlaAverage age at the time of surgery or necropsy. though the exact role of these genes is not identified, it has been the rise in Ca2+ concentration activates Ca 2+- suggested that these proto-oncogenes are nuclear dependent proteases, protein kinase C and Ca2+/ 'third messengers '63 with an important role in calmodulin kinase systems, which may be instru- coupling short-term events, elicited by extracellular mental in mediating long-term changes in synaptic stimuli, to long-term alterations in target gene transmission 39. expression 63'64'66. A possible candidate target gene Activation of immediate-early genes. Brief limbic for regulation by Fos and Jun is preproenkephalin seizures in whole-animal models of epilepsy rapidly (see Ref. 63). Interestingly, recent studies have shown that the high-frequency stimulation that produces LTP, also induces activation of immediateearly genes, particularly in the dentate granule cells67,68; as in LTP, this increased expression is prevented by NMDA antagonists 67,68. Changes in gene expression. Limbic seizures also induce a delayed increase in the enkephalin and dynorphin content of mossy fibre terminal boutons and in mRNAs that encode for preproenkephalins and neuropeptide y69,7o. These changes, which are particularly prominent in the granule cell-CA3 system, are specific, since other mRNAs within the granule cells are either unaffected or, in the case of preprodynorphin, reduced in the same seizure |.. episode 7°. Gall and Isackson 71 have also found that epileptic seizures dramatically increase (20-28-fold) Z mRNA for nerve growth factor (NGF). This increase is first seen in granule cells of the fascia dentata (within 1 h of seizure onset) and like induction of c-fos expression, it is followed by a rise in NGF mRNA in other limbic structures, including CA3 and the amygdala. The granule cell-CA3 region has the highest NGF and I3-NGF mRNA content in the brain72,73; NGF is released in this region, taken up and retrogradely transported to the cell bodies of septal cholinergic neurones 74 where it plays an important role in the survival and expression of cholinergic functions 7s. NGF increases neurite Fig. 4. Schematic representation of proposed neuronal network changes extension in several preparations 75-78, and intrainduced by seizures in the CA3 region. Dotted lines represent aberrant fibres hippocampal injections of antiserum to NGF inhibit and synapses. Abbreviations: MF, mossy fibre; lu, stratum lucidum; py, the sympathohippocampal sprouting induced by stratum pyramidum; or, stratum oriens; rc, recurrent collateral fimbria/fornix transection 79. NGF antiserum also 316

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Dolns collateral sprouting and synaptic contacts may be continuously formed in adult hippocampus 81. It is not unreasonable to suggest that mechanisms and factors (such as NGF), which modulate developmental plasticity and induce mitogenesis, have remained active in this region of the adult nervous Concluding remarks and future prospects Taken together, these findings emphasize the system and may mediate use-dependent plasticity. Whatever the exact mechanisms, these studies need for a more dynamic view of the circuit and mechanisms involved in epilepsy. Clearly, brief represent another example of the important impact seizure episodes induce persistent changes in the that the study of epilepsy has on our understanding neuronal network, epilepsy resulting not only from of how the brain works. We suggest that usedeficits associated with neuronal loss but also from dependent sprouting and neosynaptogenesis in the the formation of aberrant excitatory connections. It adult brain play a role in epilepsy and may share will be interesting to determine the causal relation- common mechanisms with other physiological or ship between the sequence of events leading from morphological plasticities. hyperexcitability to sprouting. There are several areas of research that will be particularly exciting. To determine the pattern of enhanced physiologi- Selected references 1 Bliss, T. V. P. and Lemo, T. (1973) J. Physiol. (London) 232, cal activity required to produce the long-lasting 331-356 changes in excitability. Seizure-induced LTP is pro2 Cherubini, E., Ben-Ari, Y., Gho, M., Bidart, J. N. and duced in CA3 neurones by 50-60 paroxysmal Lazdunski, M. (1987) Nature 328, 70-73 bursts3; the type of stimulation more effective in 3 Ben-Ari, Y. and Gho, M. (1988) J. PhysioL (London) 404, inducing classical LTP mimics the pattern of action 365-384 4 Cotman, C. W., Monaghan, D. T., Ottersen, O. P. and potentials observed during the burst 3. Whether this Storm-Mathisen, J. (1987) Trends Neurosci. 10, 273-280 pattern is sufficient to induce changes in gene 5 Bayer, S. A., Brunner, R. L., Hine, R. and Altman, J. (1973) expression and morphology is at present unclear. If Nature (London) 242,222 more persistent seizures are required to produce the 60ttersen, O. P. and Storm-Mathisen, J. (1984) in Handbook of Chemical Neuroanatomy (Vol. 3) (BjSrklund, A., Hbkfelt, latter it will be important to determine the type of T. and Kuhar, M. J., eds), pp. 141-226, Elsevier intracellular messenger involved. 7 Crawford, I. L. and Connor, J. D. (1973) Nature 244, To determine how the rise in [Ca2+]i produced by 442-443 seizure in the proximal part of the apical dendrite of 8 Neuman, R. S., Ben-Ari, Y., Gho, M. and Cherubini, E. (1988) CA3 neurones (mossy fibre zone) interacts with that Neurosci. Lett. 92, 64-68 9 Amaral, D. G. and Dent, J. A. (1981) J. Comp. Neurol. 195, produced in the distal part of the same dendrite 51-86 (commissural-associational synapse). Will Ca2+ 10 Brown, T. H. and Johnston, D. (1983) J. Neurophysiol. 50, induce changes in gene expression in these cells? 487-507 This situation offers a unique example of con- 11 Miles, R. and Wong, R. K. S. (1986) J. Physiol. (London) 373, 371-373 vergence of NMDA-clependent and NMDAindependent forms of LTP in different locations of 12 Traub, R. D. and Wong, R, K. S. (1982) Science 216, the same neurone, and enables a direct comparison 13 745-747 Stengaard-Pedersen, K., Fredens, K. and Larsson, L. I. (1983) between the effects of a rise in [Ca2+]i produced by Brain Res. 273, 81-96 the activation of voltage-dependent Ca2+ channels 14 Bloom, F. E. (1983) Annu. Rev. Pharmacol. Toxicol. 23, 151-170 and one produced by the activation of NMDA 15 Sloviter, R. S. (1989)J. Comp. Neurol. 280, 183-196 receptors. 16 Bousez-Dumesnil, N., Thomasset, M. and Ben-Ari, Y. (1989) To examine whether other fibre connections in Brain Res. 486, 165-169 CA3 sprout following brief seizures. Obvious candi- 17 Baimbridge, K. G. and Miller, J. J. (1982) Brain Res. 245, 223-229 dates are the recurrent excitatory collaterals (Fig. 4). Extensive theoretical and experimental evidence 18 Hu, K. H. and Friede, R. L. (1986) J. Neurol. 15, 677-685 Frederickson, C. J., Klitenick, M. A., Manton, W. I. and indicates that these play an essential role in trigger- 19 Kirpatrick, J. B. (1983) Brain Res. 273, 335-339 ing the synchronized burst in CA3 neurones and are 20 Haug, F. M. S. (1973) Adv. Anat. Embryol. Cell Biol. 47, responsible for the seizure-prone properties of this 1-71 21 Ibata, Y. and Otsuka, N. (1969)J. Histochem. Cytochem. 17, circuit11,12. 171-175 To determine neosynaptogenesis quantitatively, 22 Aniksztejn, L., Charton, G. and Ben-Ari, Y. (1987) Brain Res. in particular to examine whether the aberrant 404, 58-64 synapses induce the development of the characteris- 23 Peters, S., Koh, J. and Choi, D. W. (1987) Science 236, 589-593 tic giant postsynaptic spines in both CA3 pyramidal neurones and granule cells. This phenomenon rep- 24 Westbrook, G. L. and Mayer, M. L. (1987) Nature 328, resents a model of induction, by an afferent system, 25 640-643 Sire, J. A. and Cherubini, E. Neuroscience (in press) of major postsynaptic changes that can be readily 26 Nadler, J. V. (1981) Life Sci. 29, 2031-2042 visualized. 27 Ben-Ari, Y. (1985) Neuroscience 14, 375-403 To determine why the seizure preferentially 28 London, E. D. and Coyle, J. T. (1978) Mol. Pharmacol. 15, 492-505 switches on a cascade of changes in gene expression 29 Unnerstall, J. R. and Wamsley, J. K. (1983) Eur. J. Pharmacol. in the granule cells. In common with cerebellar and 86, 361-371 olfactory bulb granule cells, hippocampal granule 30 Tremblay, E., Represa, A. and Ben-Ari, Y. (1985) Brain Res. 343, 378-382 cells have a postnatal neurogenesis in rats; new

delays the development of kindling 8°. Therefore, although speculative, it is likely that seizure-induced release of NGF contributes to seizure-induced mossy fibre sprouting.

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Acknowledgements The authors are grateful to O. 8arbin and A. Nistri for critically reading the manuscript.

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