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NCAM immunoreactivity on mossy fibers and reactive astrocytes in the hippocampus of epileptic rats
106
Brailt Research. ~2~ (1993)IIIt~ 111~ ,~, 1993 Elsevier Science Publishers B.V. All rights reserved 1~01~6-89t~3/93/$1)6.1}~1
BRES 19333
NCAM immunoreactivity on mossy fibers and reactive astrocytes in the hippocampus of epileptic rats Jgr6me Niquet, Isabel Jorquera, Yehezkel Ben-Ari and Alfonso Represa INSERM U29, Paris (France)
(Accepted 1 June 1993)
Key words: Cell adhesion molecule; Kainate treatment; Astrocyte; Gliosis; Mossy fiber sprouting
Sprouting and synaptogenesis of mossy fibers develop in adult hippocampus after epilepsy. In control conditions, mossy fibers constitute the main afferent pathway to the Ammon's horn, where they mainly innervate CA3 pyramidal cells, but after treatment with the convulsant agent, kainate, mossy fibers also innervate granule cell dendrites generating recurrent excitatory circuits which may contribute to the maintenance of the epileptic condition. In the present study we show an enhanced immunoreactivity to neural cell adhesion molecules (NCAMs), a family of membrane glycoproteins involved in axonal growth. NCAM immunoreactivity is enriched on cytoplasmic membranes of axon shafts that are likely to be mossy fiber collaterals. NCAM immunoreactivity was also observed on the cytoplasmic membranes of reactive astrocytes, at the axon-glial contacts. Our results therefore suggest that there is an interaction of newly developed mossy fibers with other fibers and glial cells. This interaction may be mediated by NCAMs. Taking into account the trophic properties of NCAMs we suggest that they regulate the sprouting, growing and synaptogenesis of mossy fibers in epileptic conditions.
INTRODUCTION Reactive sprouting and synaptogenesis of hippocampal mossy fibers (MFs), the axons of dentate granule cells, may constitute the morphological substrate of temporal lobe epilepsy (reviewed in ref. 5). This has been well analyzed in the experimental model of temporal lobe epilepsy induced by the administration of the neurotoxin, kainate (KA) 28. Thus, collaterals of MFs m a d e heterotopic contacts with granular cell dendrites 28, generating recurrent excitatory circuits. These newly developed contacts would increase the hipp o c a m p a l excitability and facilitate the synchronization of a greater n u m b e r of cells, contributing to the maintenance of the epileptic condition 5'36. Neural cell adhesion molecules ( N C A M ) are a complex family of m e m b r a n e glycoproteins (see refs. 13,21,32 for reviews). In vitro studies suggested that N C A M s mediate adhesion between neuronal elements and are powerful inducers of neurite outgrowth 1°-12. This trophic ability is m e d i a t e d by the homophilic interaction between N C A M in the n e u r o n and N C A M in the substratum m'12. N C A M binding was m o d u l a t e d
by the amounts of polysialic acid (PSA) linked on the extracellular domain of the molecule m'2°'33. Thus, the removal of P S A from N C A M increases its adhesive properties 2°'33 and reduces its trophic effects 1°. N C A M s are present in adult rodent brains as three major glycosylated polypeptides poorly enriched in PSA, with sizes about 180, 140 and 120 kDa. In contrast, immature brain showed highly polysialilated N C A M s (embryonic NCAM) 23. K A treatment induces a reactive gliosis in the hippocampus, which is mainly characterized by an astroglial hypertrophy in both CA3 (stratum oriens and radiatum) and dentate gyrus (hilus and molecular layer) 29. These reactive astrocytes have been found to express in the C A 3 - C A 4 field the PSA-rich N C A M 23. If the glial expression of N C A M s contributes to the collateral sprouting of MFs it should interact with axonal N C A M s . Nevertheless M F sprouts do not express embryonic N C A M in KA-treated rats 23. In the present report we study the light and electron microscope (EM) patterns of N C A M immunoreactivity in the fascia dentata of K A - t r e a t e d rats, using an antibody recognizing the whole family of N C A M s ~5.
Correspondence: A. Represa, INSERM U29, 123 Bd. Port Royal, H6pital de Port-Royal, 75674 Paris Cedex 14, France. Fax: (33) (1) 46 34 16 56.
107
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Fig. 1. MF sprouting after KA treatment. A: control dentate gyrus stained by the Timm procedure depicting the distribution of MF terminals. Note that Timm deposits are restricted to the plexiform layer and the stratum lucidum. B: camera lucida reconstruction of a Golgi-impregnated granule cell from a control rat. The MF gives a few collaterals. Dotted lines represent the granule cell layer and the pyramidal cell layer, respectively. C: 30 days after KA treatment Timm-stained MF terminals were observed throughout the hilus; they also form an aberrant band in the inner third of the molecular layer (arrows). D: camera lucida reconstruction of Golgi-impregnated granule cells from a rat treated with KA 12 days before. Note that a few collaterals attempt the molecular layer (arrows). Bar = 200/~m in A,C and 100 ~m in B,D.
MATERIALS
AND METHODS
Animals Male Wistar rats 1180-200 g) were used throughout these experiments. They had access to food and water ad libitum and were housed in individual cages under diurnal lighting conditions, with lights on from 08.00 to 20.00 h. For lesion experiments, animals were anaesthetized with chloralhydrate and submitted to unilateral injections of KA (1.2 /xg of KA was dissolved in 0.3 /~1 of phosphate buffer (PB), pH 7.4) into the amygdala under stereotaxic guidance at the following coordinates according to the Atlas of Albe-Fessard~: A, 6 ram; L, 4 ram; H, 2 mm. Animals showing typical KA-induced limbic motor seizures for at least 2 h upon recovery from anaesthesia were kept for the experiments. These animals were sacrificed 3, 6, 8, 12, 20, 30 and 60 days after treatment. Animals injected with 0.3 ~tt PB at the same stereotaxic coordinates were used as controls. Lesions were analyzed by Cresyl violet and hematein-eosin. Only those animals showing a complete lesion of C A 3 - C A 4 fields were retained for this study.
Biochemical analysis The dentate gyrus was rapidly dissected from control (n = 3) and KA-treated rats (12, 20 and 30 days after KA; n = 3 for each group). The tissues were thawed and homogenized by sonication in 50 m M Tris-HCl (pH 8.0) containing 1 m M MgCI2, l m M phenyl methyl sulfonyl fluoride (PMSF). NP40 was added to a final concentration of 1% and the suspension left for 10 mn on ice. The suspension was clarified by centrifugation (20 mn at 10,000× g) and the supernatant mixed with SDS sample buffer 22. Samples (100 /xg proteins each) were analyzed by one-dimensional SDS-PAGE using 6% polyacrylamide gels. Proteins were transferred during 5 h at 60 V to a nitrocellulose m e m b r a n e 37. After the transfer, the m e m b r a n e was stained with Ponceau red. The blot was saturated for 30 min in 5% skim milk in Tris-HCl, 150 m M NaCI, pH 8.0 (TBS) at room temperature. The nitrocellulose m e m b r a n e was then incubated overnight with anti-NCAM (1:2,000 in TBS containing 0.05% Tween 20 and 0.5% milk; TBS-T). After several washes, the blot was incubated with a alkaline phosphatase-conjugated goat anti-rabbit IgG 11:2,000; Sigma) in TBS-T buffer for 2 h. Immunoreactive proteins were revealed with B C I P / N B T substrate.
Histochemical staining Control (n = 5), KA-treated (3, 8, 12, 20, 30 or 60 days after injection; n = 5 per delay) rats were perfused with 0.4% sodium sulphide followed by 4% paraformaldehyde to stain MFs according to a modified Timm sulphide silver method 19. The Timm-stained sections (20 /zm thick coronal sections) were counter-stained with Cresyl violet, dehydrated, cleared with xylene and m o u n t e d with Permount. Sprouting was also analyzed with a modified rapid Golgi stain e7 in rats 6 (n = 5), 12 (n = 5) and 30 (n = 5) days after KA. In brief 100 /zm thick vibratome slices of hippocampus were placed for 24 h in 2.4% potassium dichromate containing 0.2% osmium tetroxide and then placed between two slides in 0.75% silver nitrate solution for 24 h. The sections were then dehydrated, cleared with xylene and mounted with Permount. Drawings were made with a camera lucida using either a 2 0 × , 4 0 × or 100x objective lens.
Immunocytological procedures U n d e r deep anaesthesia, control rats (n = 8) and KA-treated rats (3 (n = 2), 6 (n = 2), 8 (n = 4), 12 (n = 7), 20 (n = 10), 30 (n = 10) and 60 (n = 5) days after treatment) were perfused through the heart with PB containing 2% paraformaldehyde. After perfusion, brains were removed and kept in the same fixative for 24 h, then in PB containing 15% sucrose at 4°C (24 h). Coronal sections ( 4 0 / x m ) of the dorsal hippocampus were conserved at 4°C in PB containing 0.1% sodium azide. All procedure labelling was made at room temperature. Sections from control rats were processed simultaneously, with the same solutions, with sections from animals sacrificed at different times after KA. In brief, sections were immersed first for
5 rain m methanol containing I ' , ~ I 1 , O , t~ mhihi! cndogcnou~ pcroxidases, then 1 h in PB containing (i.2~; gelalin f'BG and 1).41; triton. Sections were incubated overnight at room tcmpcralurc with anti-NCAM antibodies in PB-G. After washing, sections were incubated with goat anti-rabbit polyelonal antibody ( B o e h r i n g e r - M a n nheim; 1:100) and peroxidase anti-peroxidase complex (Boehringcr Mannheim; 1:200). The labelling was revealed with diaminobenzidine (0.05%) and H 2 0 2 (().01'~.) in Tris-HCl. Imnaunocytochemical controls were performed using a normal rabbit serum or goat Ig(i instead of antibodies.
Electron microscopy Control (n - 11) and KA-treated 112 (n = 3). 20 (n - 6L 30 (n 2) and 60 (n = 1) days after treatment) were intracardially perfused with 1'7( paraformaldehyde l% glutaraldehyde in PB. The brains were removed and immersed for 12 h in 1% paraformaldehyde. Coronal slices of dorsal hippocampus (80/zm thick), were incubated overnight with anti-NCAM antibodies as described below. After developing the immunoperoxidase reaction, the slices were post-fixed with 0.2ci osmium tetroxidc in PB, en bloc stained with uranyl acetate, dehydrated and embedded in araldite. Ultrathin sections of dentate gyrus were cut and examined in a Philips electron microscope. =
RESULTS
Mossy fiber sprouting after KA treatment In control rats, Timm-stained MFs formed a dense pack of fibers both in the hilus, where they were restricted to the plexiform layer (collateral branches of MFs), and in CA3 where they were restricted to the lucidum layer (terminal branches; Fig. 1A); in fact MF terminals innervate both hilar interneurons and CA3 pyramidal cells 3'6"14"1~.In KA-treated animals we found, in agreement with previous observations 2~'3°'36, a collateral sprouting of MFs. Thus, with Timm staining we observed that in KA-treated rats silver deposits were not restricted to the plexiform layer and invaded the deeper layers of hilus (Fig. 1C). In addition Timm deposits formed an ectopic band in the inner third of the molecular layer (Fig. 1C). This ectopic band was first observed by 12 days after KA, but it was much more developed 30 days later. With Golgi staining we confirmed that MF collaterals overlapped the border of the plexiform layer and branched throughout the deeper layers of hilus in rats sacrificed 12 or 30 days after KA (Fig. 1D). These collaterals may also cross through the granule cell layer and reach the molecular layer where they synaptically contact granule cell dendrites 2s. This collateral MF sprouting was already conspicuous by 12 days after KA. At shorter times (6 days) after KA we never observed signs of sprouting, and the pattern of Golgi staining was quite similar to that of controls. The intensity of MF branching was much more intense in rats sacrificed 30 days after KA 28. The changes in the distribution of MFs were permanent since they persisted in animals sacrificed 12 months after KA 2~. The observation of EM preparations reveals that the hilus of KA-treated animals was almost exclusively
109 constituted by axons and glial cells. Very scarce dendrites and neuronal somas of interneurons were observed. Very few synaptic contacts were therefore seen in this field. In the molecular layer of KA-treated rats the unique apparent change was the presence of reactive glial cells (large extensions enriched in gliofilaments) and the appearance of a few MF boutons28; the axonal and dendritic patterns were similar to those in control rats.
Western blot analysis In Western blots from control adult rats, NCAM antibodies labelled three distinct bands, with apparent molecular weights of 180, 140 and 120 kDa, respectively (Fig. 2). These bands most likely correspond to the three major adult isoforms of NCAM (NCAM-180, NCAM-140 and NCAM-120). Blots obtained from control and KA-treated rats did not show qualitative differences, as depicted in Fig. 2.
Immunocytochemistry of NCAM in control hippocampus In agreement with previous data 26, in control rats the hippocampal pattern of NCAM immunoreactivity was heterogeneous. The more intensely immunostained regions were the stratum lucidum of CA3 and the plexiform layer of hilus (Fig. 3A,B). Therefore, N C A M immunoreaetivity was mainly distributed throughout the terminal field of MFs 26. Moderate patterns of immunoreactivity were found in the stratum
180
-
140
-
120
-
C
KA
Fig. 2. Immunoblot analysis of NCAM immunoreactivity in the dentate gyrus of control (lane 1) and KA-treated (30 days after KA; lane 2) rats. Three different bands were obtained with apparent molecular weights of 180, 140 and 120 kDa, in agreement with the molecular weights of the three major adult isoformsof NCAMs.
oriens and radiatum of Ammon's horn, as well as within the inner third of the molecular layer. In agreement with previous studies 26, NCAM immunoreactivity was restricted to the cytoplasmic membranes of pyramidal cells, granule cells, interneurons and MFs (Fig. 4). This immunoreactivity was intense at the zone of cell-cell contacts (Fig. 4), including cell bodies and dendrites (pyramidal and granule cells). Dendritic spines were in some cases imrnunoreactive but we did not observe any immunostaining of postsynaptic densities. The cytoplasmic membrane of MFs (collateral branches in the plexiform layer and terminal ones in the lucidum) was the most densely immunostained structure in the hippocampus (Fig. 4A,B). Mossy fibers may be easily identified because they form bundies of unmyelinated, thin, well-fasciculated axons from which giant characteristic MF boutons originate 6. The MF staining mainly concerns the axon shafts and rarely involves synaptic boutons. Immunoreactivity was mainly enriched at the zones of axon-axonal contact and axon-dendritic or somatic contacts (arrow in Fig. 4). In the present study, glial cells were not immunopositive, as well as other axonal fibers (i.e. the axons of the perforant path which impinge on the more distal part of granule cell dendrites).
Immunocytochemistry pocampus
of NCAM in KA-treated hip-
Ipsilateral to the KA injection, the staining pattern obtained with NCAM antibodies significantly changed in the hippocampal complex. Thus, in rats sacrificed 6, 8, 10 and 12 days after injection, there was a lack of neuronal staining in C A 3 - C A 4 because of the neuronal death in these fields, whereas the background staining significantly increased in these fields 20, 30 or 60 days after treatment. Also in the dentate gyrus, the staining pattern was modified in KA-treated rats: it was no longer limited to the plexiform layer and extended throughout the hilus (Fig. 3C,D). In fact, the laminar pattern of NCAM immunostaining in the hilus was lost, as was the laminar pattern of Timm-stained MFs (compare Figs. 1 and 3). The striking correlation of NCAM immunoreactivity with the pattern of Timm staining suggests that collateral sprouts of MFs were NCAM immunoreactive. EM analysis of KA-treated hippocampus revealed that NCAM antibodies stained in the hilus of KAtreated rats both axonal profiles and astrocytes. Thus, NCAM antibodies stained throughout the hilus fibers with the morphologic features of MFs (Fig. 5): they were unmyelinated, thin, well-fasciculated axons; they gave rise to a few large-complex boutons, although these boutons rarely made synaptic contacts (most tar-
ll{t
Fig. 3. NCAM immunoreactivity. A,B: in control hippocampus N C A M antibodies essentially labelled the plexiform layer (arrow heads) and the stratum lucidum, overlaying the terminal fields of MFs. The inner third of the molecular layer was also immunorcactive. (LD: 30 days after KA treatment N C A M immunoreactivity distributed throughout the hilus like sprouted MFs (compare with Fig. l('). Bar = 200 ~ m in A,C and 80 ~ m in B,D. G, granule cell layer; H, hilus: m, molecular layer.
get cells had been destroyed by the treatment). NCAM immunoreactivity was restricted to the cytoplasmic membrane, at the axon-axonal (Fig. 5) and axon-glial (arrows in Fig. 6) contacts. In the granule cell layer, as in the inner third of the molecular layer, fibers were also immunoreactive at the contact with granule cell bodies and dendrites and glial extensions. In the present study, reactive astrocytes were NCAM immunopositive after KA treatment. Astroglial immunoreactivity was observed on the cytoplasmic membrane of soma and extensions; this immunoreactivity
was particularly intense at the zone of contacts with axons (arrows in Fig. 6). DISCUSSION
The present study showed an increase in area showing NCAM immunoreactivity in the hippocampus of KA-treated rats. This change spatio-temporally correlates with both the sprouting of MFs and the glial reaction observed in the same animals. NCAM immunoreactivity was indeed particularly enriched in the
Fig. 4. N C A M antibodies stain the cytoplasmic m e m b r a n e s of MFs in control hippocampus. A,B: EM photographs depicting the N C A M immunostaining in the pyramidal cell l a y e r - s t r a t u m lucidum of CA4 (A) and in the plexiform layer (B). Only cytoplasmic m e m b r a n e s of pyramidal cell bodies and MFs were immunopositive, notably at the axon-axonal and a x o n - n e u r o n a l cell body (arrows) contacts. Note the characteristic fasciculation of MFs. Bar = 1 /~m.
112
Fig. 5. NCAM immunoreactivity after KA treatment in the hilus of KA-treated rats. A: numerous immunostained axons were observed throughout the hilus; they are likely to be MFs. Thus they are well-fasciculated and a few synaptic boutons of complex shape and great size originate from them (*). These synaptic boutons rarely made synaptic contacts since most of target elements had been destroyed by the treatment, lmmunoreactivity is clearly seen at the axon-axonal contacts. Cytoplasmic membranes of synaptic boutons were not stained. Bar = 1 ;~m.
cytoplasmic membrane of axon shafts and glial cells in the zones of MF sprouting, mainly the hilus. The present results suggest that NCAMs were expressed by
MF sprouts and reactive glial cells. NCAMs may therefore contribute to the synaptic rearrangement of MFs observed in epileptic conditions.
Fig. 6. NCAM immunoreactivity after KA treatment in the hilus of KA-treated rats. A,B: EM photographs depicting astroglial NCAM immunoreactivity at the contact with axons (arrows) which are also immunoreactive at the axonaxonal contacts (arrow heads). Bar = 0.5/~m.
114 The optic and EM changes here reported were observed in every treated animal. This is likely to be due to the rigorous selection of experimental animals. Thus, we only analyzed KA-treated rats showing a complete lesion of C A 3 - C A 4 fields, all of them showing a very similar pattern of MF sprouting.
NCAM immunoreactil~ity in adult control hippocampus Previous reports showed that N C A M immunoreactivity and m R N A expression correlated in CNS with processes of neurite outgrowth and synaptogenesis during postnatal development 2'~6'17'25'26'34.These molecules persisted in adult brain structures, such as the hippocampus ~'w'23'26'35, in which they may play a role in stabilizing cell-cell contacts. In adult hippocampus there is a clear compartmenration on the distribution of the three major N C A M isoforms: NCAM-180 was mainly expressed on dendrites 24, whereas it was absent from MFs. In contrast NCAM was observed both in dendrites (of granule ceils, pyramidal neurons and interneurons) and axons (essentially MFs) 2~. We therefore suggest that MFs mainly express NCAM-140 a n d / o r NCAM-120 and that both isoforms may sustain their characteristic fasciculation. In the present study, in control hippocampus, glial cells were not stained by N C A M antibodies.
N C A M immunoreactirity after KA treatment As previously reported 2s, after KA treatment there is a dramatic growing and sprouting of MFs which invade both the deeper fields of the hilus and, after passing through the granule cell layer, the inner third of the molecular layer. The hilus of KA-treated animals was almost exclusively composed of glial cells and axons. Goigi- and Timm-stained preparations suggest that most of these hilar axons are newly developed MF collaterals. Thus, like normal MFs, they appear as bundles of thin, unmyelinated, well-fasciculated axons which may from originate MF boutons 2s. In the present study we show a striking spatio-temporal correlation among the MF sprouting and the changes on N C A M immunoreactivity, mainly in the hilus. This suggests that MF sprouts express NCAMs. Furthermore, at EM levels, all axons in the hilus of KA-treated rats are N C A M positive, mainly at the zones of axon-axonal and axon-astrocyte contacts. Nevertheless fibers other than MFs may also be labelled by N C A M antibodies. Thus, most of these hilar axons display features of MFs: they form bundles of thin, well-fasciculated, unmyelinated fibers from which may originate complex giant synaptic boutons. These fibers were not immunostained by antibodies against
NCAM-180 (unpublished results), suggesting that axonal sprouts express NCAMs of 14(/or 12(J kDm In agreement with a previous observation ~, reactive astrocytes in the hilus of KA-treated rats were NCAM immunoreactive; the immunorcactivity was restricted to the cytoplasmic membrane at the contacl with axons, suggesting that NCAM mediate axonal-glial interaction.
NCAMs may contribute to M F sprouting Previous in vitro analysis on the role of NCAM showed that these molecules are powerful inducers of neurite outgrowth from central 11 or peripheral I°~-~ neurons. This capacity is directly related to the homophilic binding of NCAM to NCAM molecules, and are therefore modulated by polysialic acid (PSA) w. In fact, N C A M isoforms enriched in PSA, which are mainly present at embryonic stages 7'3~'34, arc less adhesive z°'33 but better inducers of neurite outgrowth ~ than adult isoforms. Reactive astrocytes in the hippocampus of KA-treated rats were immunoreactive to an antibody that recognizes the PSA-rich forms of N C A M 2-~. Glial cells therefore provide a faint adhesive substrate for MFs. In contrast, after KA treatment, MFs and other eventual axonal sprouts were not labelled by these antibodies 23. Axonal sprouts would therefore express NCAM- 140 and - 120 poorly enriched on polysialic residues. This NCAM expression may exert a robust axonaxonal adhesion, stronger than the axon-glial one. In the model proposed by Ruthishauser 3~ at the neuromuscular junction, activity-dependent sprouting was associated with an increase in axonal PSA levels (weaker axon-axonal interaction); this sprouting developed over a substrate which was more adhesive. On the contrary in the hippocampus, the stronger interaction is the axon-axonal, whereas the axon-glial interactions is the weaker. In our study the intense fasciculation of MFs may result from the strong axon-axonal interaction mediated by adult NCAM isoforms, whereas the collateral branching and growth may be caused by the weaker interaction of MFs with the PSA-enriched glial substrate. The antagonism among these 2 different forces may also explain the slow pattern of growing in the hippocampus of epileptic rats. Thus, MF collaterals never go beyond the borders of dentate gyrus despite the restricted number of target cells there proposed (KA treatment destroys most of the interneurons in this field); the first MF collaterals need at least 12 days to attempt the molecular layer and 30 days seem to be required to complete this growing -~°. The morphoregulatory activities of NCAM may be mediated by a specific modulation of intracellular sig-
115 naling systems 9'38, and may also be related to an interaction with cytoskeleton proteins. On this respect it is interesting to note that NCAMs (most likely NCAM180 and NCAM-140) seem to modulate tyrosine phosphorylation of tubulin in growth cone membranes from fetal rat brains 4. This phosphorylation may contribute to the axonal growing by controlling the polymerization of tubulin. It is tempting to suggest that extracellular (NCAMs) and intracellular (cytoskeleton proteins) factors cooperate to facilitate MF sprouting. Other adhesion or substrate proteins may influence the axonal branching and outgrowth (i.e. L1 also expressed by MFs) 26, NCAM being just a link in the complex processes of epilepsy-induced sprouting. The understanding of the complex mechanisms involved on such axonal growing certainly require a systematic analysis of environmental (i.e. cell adhesion molecules), diffusible (i.e. neurotrophines) and neuronal (i.e. cytoskeleton proteins) factors. Acknowledgements. We are very grateful to Dr. C. Goridis for suggestions and comments and kindly providing NCAM antibodies. We are also grateful to S. Guidasci for photographs, and to D. Diabira for technical assistance. REFERENCES 1 Albe-Fessard, D., Stutinski, F. and Libouban-le-Touze, S., Atlas st~r~otaxique du diencephale du rat blanc, CNRS, Paris, 1971. 2 Alcantara, A.A., Pfenninger, K.H. and Greenough, W.T., 5B4CAM expression parallels neurite outgrowth and synaptogenesis in the developing rat brain, J. Comp. NeuroL, 319 (1992) 337-348. 3 Amaral, D.G. and Dent, J.A., Development of the mossy fibers of the dentate gyrus. I. A light and electron microscopic study of the mossy fibers and their expansions, J. Comp. NeuroL, 195 (1981) 51-86. 4 Atashi, J.R., Klinz, S.G., Ingraham, C.A., Matten, W.T., Schachner, M. and Maness, P.F., Neural cell adhesion molecules modulate tyrosine phosphorylation of tubulin in nerve growth cone membranes, Neuron, 8 (1992) 831-842. 5 Ben-Ari, Y. and Represa, A., Brief seizure episodes induce long-term potentiation and mossy fibre sprouting in the hippocampus, Trends Neurosci., 13 (1990) 312-318. 6 Blackstad, T.W. and Kjaerheim, A., Special axo-dendritic synapses in the hippocampal cortex: electron and light microscopic studies on the layer of the mossy fibers, J. Comp. Neurol., 117 (1961) 113-159. 7 Boisseau, S., Nedelec, J., Poirier, V., Rougon, G. and Simonneau, M., Analysis of high PSA N-CAM expression during mammalian spinal cord and peripheral nervous system development, Det~elopment, 112 (1991) 69-82. 8 Bonfanti, L., Olive, S., Poulain, D.A. and Theodosis, D.T., Mapping of the distribution of polysialylated neural cell adhesion molecule throughout the central nervous system of the adult rat: an immunocytochemical study, Neuroscience, 49 (1992) 419-436. 9 Doherty, P., Ashton, S.V., Moore, S.E. and Walsh, F.S., Morphoregulatory activities of NCAM and N-cadherin can be accounted for by G protein-dependent activation of L- and N-type neuronal Ca 2+ channels, Cell, 67 (1991) 21-33. 10 Doherty, P., Cohen, J. and Walsh, F.S., Neurite outgrowth in response to transfected N-CAM changes during development and is modulated by polysialic acid, Neuron, 5 (1990) 209-219. 11 Doherty, P., Fruns, M., Seaton, P., Dickson, G., Barton, C.H.,
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116 molecules have different binding properties, Nature, 304 (1983) 347-349. 34 Seki, T. and Arai, Y., Expression of highly polysialylated NCAM in the neocortex and piriform cortex of the developing and the adult rat, Anat. Embryol., 184 (1991)395-401. 35 Seki, T. and Arai, Y., The persistent expression of highly polysialylated NCAM in the dentate gyrus of the adult rat, Neurosci. Res., 12 (1991) 503-513. 36 Tauck, D.L. and Nadler, J.V., Evidence for functional mossy fiber
sprouting in hippocampat formation of kainic acid treated rats, .t Neurosci., 5 (1985) 1016 1022. 37 Towbin, tt.T., Staehclin. T. and Gordon, S.. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets, Proc. natl. Acad. Sci. USA, 76 (1979) 4350-4354. 38 Von Bohlen und Halbach, F., Taylor, J. and Schachner, M., Cell type-specific effects of the neural adheskm molecules LI anti N-CAM on diverse second messenger systems, Eur. J. Neurosci, 4 (1992) 896-909.
Brailt Research. ~2~ (1993)IIIt~ 111~ ,~, 1993 Elsevier Science Publishers B.V. All rights reserved 1~01~6-89t~3/93/$1)6.1}~1
BRES 19333
NCAM immunoreactivity on mossy fibers and reactive astrocytes in the hippocampus of epileptic rats Jgr6me Niquet, Isabel Jorquera, Yehezkel Ben-Ari and Alfonso Represa INSERM U29, Paris (France)
(Accepted 1 June 1993)
Key words: Cell adhesion molecule; Kainate treatment; Astrocyte; Gliosis; Mossy fiber sprouting
Sprouting and synaptogenesis of mossy fibers develop in adult hippocampus after epilepsy. In control conditions, mossy fibers constitute the main afferent pathway to the Ammon's horn, where they mainly innervate CA3 pyramidal cells, but after treatment with the convulsant agent, kainate, mossy fibers also innervate granule cell dendrites generating recurrent excitatory circuits which may contribute to the maintenance of the epileptic condition. In the present study we show an enhanced immunoreactivity to neural cell adhesion molecules (NCAMs), a family of membrane glycoproteins involved in axonal growth. NCAM immunoreactivity is enriched on cytoplasmic membranes of axon shafts that are likely to be mossy fiber collaterals. NCAM immunoreactivity was also observed on the cytoplasmic membranes of reactive astrocytes, at the axon-glial contacts. Our results therefore suggest that there is an interaction of newly developed mossy fibers with other fibers and glial cells. This interaction may be mediated by NCAMs. Taking into account the trophic properties of NCAMs we suggest that they regulate the sprouting, growing and synaptogenesis of mossy fibers in epileptic conditions.
INTRODUCTION Reactive sprouting and synaptogenesis of hippocampal mossy fibers (MFs), the axons of dentate granule cells, may constitute the morphological substrate of temporal lobe epilepsy (reviewed in ref. 5). This has been well analyzed in the experimental model of temporal lobe epilepsy induced by the administration of the neurotoxin, kainate (KA) 28. Thus, collaterals of MFs m a d e heterotopic contacts with granular cell dendrites 28, generating recurrent excitatory circuits. These newly developed contacts would increase the hipp o c a m p a l excitability and facilitate the synchronization of a greater n u m b e r of cells, contributing to the maintenance of the epileptic condition 5'36. Neural cell adhesion molecules ( N C A M ) are a complex family of m e m b r a n e glycoproteins (see refs. 13,21,32 for reviews). In vitro studies suggested that N C A M s mediate adhesion between neuronal elements and are powerful inducers of neurite outgrowth 1°-12. This trophic ability is m e d i a t e d by the homophilic interaction between N C A M in the n e u r o n and N C A M in the substratum m'12. N C A M binding was m o d u l a t e d
by the amounts of polysialic acid (PSA) linked on the extracellular domain of the molecule m'2°'33. Thus, the removal of P S A from N C A M increases its adhesive properties 2°'33 and reduces its trophic effects 1°. N C A M s are present in adult rodent brains as three major glycosylated polypeptides poorly enriched in PSA, with sizes about 180, 140 and 120 kDa. In contrast, immature brain showed highly polysialilated N C A M s (embryonic NCAM) 23. K A treatment induces a reactive gliosis in the hippocampus, which is mainly characterized by an astroglial hypertrophy in both CA3 (stratum oriens and radiatum) and dentate gyrus (hilus and molecular layer) 29. These reactive astrocytes have been found to express in the C A 3 - C A 4 field the PSA-rich N C A M 23. If the glial expression of N C A M s contributes to the collateral sprouting of MFs it should interact with axonal N C A M s . Nevertheless M F sprouts do not express embryonic N C A M in KA-treated rats 23. In the present report we study the light and electron microscope (EM) patterns of N C A M immunoreactivity in the fascia dentata of K A - t r e a t e d rats, using an antibody recognizing the whole family of N C A M s ~5.
Correspondence: A. Represa, INSERM U29, 123 Bd. Port Royal, H6pital de Port-Royal, 75674 Paris Cedex 14, France. Fax: (33) (1) 46 34 16 56.
107
B
. ..~..-'i';:.'.'.'.'..9:.
~.....::....-
D
Fig. 1. MF sprouting after KA treatment. A: control dentate gyrus stained by the Timm procedure depicting the distribution of MF terminals. Note that Timm deposits are restricted to the plexiform layer and the stratum lucidum. B: camera lucida reconstruction of a Golgi-impregnated granule cell from a control rat. The MF gives a few collaterals. Dotted lines represent the granule cell layer and the pyramidal cell layer, respectively. C: 30 days after KA treatment Timm-stained MF terminals were observed throughout the hilus; they also form an aberrant band in the inner third of the molecular layer (arrows). D: camera lucida reconstruction of Golgi-impregnated granule cells from a rat treated with KA 12 days before. Note that a few collaterals attempt the molecular layer (arrows). Bar = 200/~m in A,C and 100 ~m in B,D.
MATERIALS
AND METHODS
Animals Male Wistar rats 1180-200 g) were used throughout these experiments. They had access to food and water ad libitum and were housed in individual cages under diurnal lighting conditions, with lights on from 08.00 to 20.00 h. For lesion experiments, animals were anaesthetized with chloralhydrate and submitted to unilateral injections of KA (1.2 /xg of KA was dissolved in 0.3 /~1 of phosphate buffer (PB), pH 7.4) into the amygdala under stereotaxic guidance at the following coordinates according to the Atlas of Albe-Fessard~: A, 6 ram; L, 4 ram; H, 2 mm. Animals showing typical KA-induced limbic motor seizures for at least 2 h upon recovery from anaesthesia were kept for the experiments. These animals were sacrificed 3, 6, 8, 12, 20, 30 and 60 days after treatment. Animals injected with 0.3 ~tt PB at the same stereotaxic coordinates were used as controls. Lesions were analyzed by Cresyl violet and hematein-eosin. Only those animals showing a complete lesion of C A 3 - C A 4 fields were retained for this study.
Biochemical analysis The dentate gyrus was rapidly dissected from control (n = 3) and KA-treated rats (12, 20 and 30 days after KA; n = 3 for each group). The tissues were thawed and homogenized by sonication in 50 m M Tris-HCl (pH 8.0) containing 1 m M MgCI2, l m M phenyl methyl sulfonyl fluoride (PMSF). NP40 was added to a final concentration of 1% and the suspension left for 10 mn on ice. The suspension was clarified by centrifugation (20 mn at 10,000× g) and the supernatant mixed with SDS sample buffer 22. Samples (100 /xg proteins each) were analyzed by one-dimensional SDS-PAGE using 6% polyacrylamide gels. Proteins were transferred during 5 h at 60 V to a nitrocellulose m e m b r a n e 37. After the transfer, the m e m b r a n e was stained with Ponceau red. The blot was saturated for 30 min in 5% skim milk in Tris-HCl, 150 m M NaCI, pH 8.0 (TBS) at room temperature. The nitrocellulose m e m b r a n e was then incubated overnight with anti-NCAM (1:2,000 in TBS containing 0.05% Tween 20 and 0.5% milk; TBS-T). After several washes, the blot was incubated with a alkaline phosphatase-conjugated goat anti-rabbit IgG 11:2,000; Sigma) in TBS-T buffer for 2 h. Immunoreactive proteins were revealed with B C I P / N B T substrate.
Histochemical staining Control (n = 5), KA-treated (3, 8, 12, 20, 30 or 60 days after injection; n = 5 per delay) rats were perfused with 0.4% sodium sulphide followed by 4% paraformaldehyde to stain MFs according to a modified Timm sulphide silver method 19. The Timm-stained sections (20 /zm thick coronal sections) were counter-stained with Cresyl violet, dehydrated, cleared with xylene and m o u n t e d with Permount. Sprouting was also analyzed with a modified rapid Golgi stain e7 in rats 6 (n = 5), 12 (n = 5) and 30 (n = 5) days after KA. In brief 100 /zm thick vibratome slices of hippocampus were placed for 24 h in 2.4% potassium dichromate containing 0.2% osmium tetroxide and then placed between two slides in 0.75% silver nitrate solution for 24 h. The sections were then dehydrated, cleared with xylene and mounted with Permount. Drawings were made with a camera lucida using either a 2 0 × , 4 0 × or 100x objective lens.
Immunocytological procedures U n d e r deep anaesthesia, control rats (n = 8) and KA-treated rats (3 (n = 2), 6 (n = 2), 8 (n = 4), 12 (n = 7), 20 (n = 10), 30 (n = 10) and 60 (n = 5) days after treatment) were perfused through the heart with PB containing 2% paraformaldehyde. After perfusion, brains were removed and kept in the same fixative for 24 h, then in PB containing 15% sucrose at 4°C (24 h). Coronal sections ( 4 0 / x m ) of the dorsal hippocampus were conserved at 4°C in PB containing 0.1% sodium azide. All procedure labelling was made at room temperature. Sections from control rats were processed simultaneously, with the same solutions, with sections from animals sacrificed at different times after KA. In brief, sections were immersed first for
5 rain m methanol containing I ' , ~ I 1 , O , t~ mhihi! cndogcnou~ pcroxidases, then 1 h in PB containing (i.2~; gelalin f'BG and 1).41; triton. Sections were incubated overnight at room tcmpcralurc with anti-NCAM antibodies in PB-G. After washing, sections were incubated with goat anti-rabbit polyelonal antibody ( B o e h r i n g e r - M a n nheim; 1:100) and peroxidase anti-peroxidase complex (Boehringcr Mannheim; 1:200). The labelling was revealed with diaminobenzidine (0.05%) and H 2 0 2 (().01'~.) in Tris-HCl. Imnaunocytochemical controls were performed using a normal rabbit serum or goat Ig(i instead of antibodies.
Electron microscopy Control (n - 11) and KA-treated 112 (n = 3). 20 (n - 6L 30 (n 2) and 60 (n = 1) days after treatment) were intracardially perfused with 1'7( paraformaldehyde l% glutaraldehyde in PB. The brains were removed and immersed for 12 h in 1% paraformaldehyde. Coronal slices of dorsal hippocampus (80/zm thick), were incubated overnight with anti-NCAM antibodies as described below. After developing the immunoperoxidase reaction, the slices were post-fixed with 0.2ci osmium tetroxidc in PB, en bloc stained with uranyl acetate, dehydrated and embedded in araldite. Ultrathin sections of dentate gyrus were cut and examined in a Philips electron microscope. =
RESULTS
Mossy fiber sprouting after KA treatment In control rats, Timm-stained MFs formed a dense pack of fibers both in the hilus, where they were restricted to the plexiform layer (collateral branches of MFs), and in CA3 where they were restricted to the lucidum layer (terminal branches; Fig. 1A); in fact MF terminals innervate both hilar interneurons and CA3 pyramidal cells 3'6"14"1~.In KA-treated animals we found, in agreement with previous observations 2~'3°'36, a collateral sprouting of MFs. Thus, with Timm staining we observed that in KA-treated rats silver deposits were not restricted to the plexiform layer and invaded the deeper layers of hilus (Fig. 1C). In addition Timm deposits formed an ectopic band in the inner third of the molecular layer (Fig. 1C). This ectopic band was first observed by 12 days after KA, but it was much more developed 30 days later. With Golgi staining we confirmed that MF collaterals overlapped the border of the plexiform layer and branched throughout the deeper layers of hilus in rats sacrificed 12 or 30 days after KA (Fig. 1D). These collaterals may also cross through the granule cell layer and reach the molecular layer where they synaptically contact granule cell dendrites 2s. This collateral MF sprouting was already conspicuous by 12 days after KA. At shorter times (6 days) after KA we never observed signs of sprouting, and the pattern of Golgi staining was quite similar to that of controls. The intensity of MF branching was much more intense in rats sacrificed 30 days after KA 28. The changes in the distribution of MFs were permanent since they persisted in animals sacrificed 12 months after KA 2~. The observation of EM preparations reveals that the hilus of KA-treated animals was almost exclusively
109 constituted by axons and glial cells. Very scarce dendrites and neuronal somas of interneurons were observed. Very few synaptic contacts were therefore seen in this field. In the molecular layer of KA-treated rats the unique apparent change was the presence of reactive glial cells (large extensions enriched in gliofilaments) and the appearance of a few MF boutons28; the axonal and dendritic patterns were similar to those in control rats.
Western blot analysis In Western blots from control adult rats, NCAM antibodies labelled three distinct bands, with apparent molecular weights of 180, 140 and 120 kDa, respectively (Fig. 2). These bands most likely correspond to the three major adult isoforms of NCAM (NCAM-180, NCAM-140 and NCAM-120). Blots obtained from control and KA-treated rats did not show qualitative differences, as depicted in Fig. 2.
Immunocytochemistry of NCAM in control hippocampus In agreement with previous data 26, in control rats the hippocampal pattern of NCAM immunoreactivity was heterogeneous. The more intensely immunostained regions were the stratum lucidum of CA3 and the plexiform layer of hilus (Fig. 3A,B). Therefore, N C A M immunoreaetivity was mainly distributed throughout the terminal field of MFs 26. Moderate patterns of immunoreactivity were found in the stratum
180
-
140
-
120
-
C
KA
Fig. 2. Immunoblot analysis of NCAM immunoreactivity in the dentate gyrus of control (lane 1) and KA-treated (30 days after KA; lane 2) rats. Three different bands were obtained with apparent molecular weights of 180, 140 and 120 kDa, in agreement with the molecular weights of the three major adult isoformsof NCAMs.
oriens and radiatum of Ammon's horn, as well as within the inner third of the molecular layer. In agreement with previous studies 26, NCAM immunoreactivity was restricted to the cytoplasmic membranes of pyramidal cells, granule cells, interneurons and MFs (Fig. 4). This immunoreactivity was intense at the zone of cell-cell contacts (Fig. 4), including cell bodies and dendrites (pyramidal and granule cells). Dendritic spines were in some cases imrnunoreactive but we did not observe any immunostaining of postsynaptic densities. The cytoplasmic membrane of MFs (collateral branches in the plexiform layer and terminal ones in the lucidum) was the most densely immunostained structure in the hippocampus (Fig. 4A,B). Mossy fibers may be easily identified because they form bundies of unmyelinated, thin, well-fasciculated axons from which giant characteristic MF boutons originate 6. The MF staining mainly concerns the axon shafts and rarely involves synaptic boutons. Immunoreactivity was mainly enriched at the zones of axon-axonal contact and axon-dendritic or somatic contacts (arrow in Fig. 4). In the present study, glial cells were not immunopositive, as well as other axonal fibers (i.e. the axons of the perforant path which impinge on the more distal part of granule cell dendrites).
Immunocytochemistry pocampus
of NCAM in KA-treated hip-
Ipsilateral to the KA injection, the staining pattern obtained with NCAM antibodies significantly changed in the hippocampal complex. Thus, in rats sacrificed 6, 8, 10 and 12 days after injection, there was a lack of neuronal staining in C A 3 - C A 4 because of the neuronal death in these fields, whereas the background staining significantly increased in these fields 20, 30 or 60 days after treatment. Also in the dentate gyrus, the staining pattern was modified in KA-treated rats: it was no longer limited to the plexiform layer and extended throughout the hilus (Fig. 3C,D). In fact, the laminar pattern of NCAM immunostaining in the hilus was lost, as was the laminar pattern of Timm-stained MFs (compare Figs. 1 and 3). The striking correlation of NCAM immunoreactivity with the pattern of Timm staining suggests that collateral sprouts of MFs were NCAM immunoreactive. EM analysis of KA-treated hippocampus revealed that NCAM antibodies stained in the hilus of KAtreated rats both axonal profiles and astrocytes. Thus, NCAM antibodies stained throughout the hilus fibers with the morphologic features of MFs (Fig. 5): they were unmyelinated, thin, well-fasciculated axons; they gave rise to a few large-complex boutons, although these boutons rarely made synaptic contacts (most tar-
ll{t
Fig. 3. NCAM immunoreactivity. A,B: in control hippocampus N C A M antibodies essentially labelled the plexiform layer (arrow heads) and the stratum lucidum, overlaying the terminal fields of MFs. The inner third of the molecular layer was also immunorcactive. (LD: 30 days after KA treatment N C A M immunoreactivity distributed throughout the hilus like sprouted MFs (compare with Fig. l('). Bar = 200 ~ m in A,C and 80 ~ m in B,D. G, granule cell layer; H, hilus: m, molecular layer.
get cells had been destroyed by the treatment). NCAM immunoreactivity was restricted to the cytoplasmic membrane, at the axon-axonal (Fig. 5) and axon-glial (arrows in Fig. 6) contacts. In the granule cell layer, as in the inner third of the molecular layer, fibers were also immunoreactive at the contact with granule cell bodies and dendrites and glial extensions. In the present study, reactive astrocytes were NCAM immunopositive after KA treatment. Astroglial immunoreactivity was observed on the cytoplasmic membrane of soma and extensions; this immunoreactivity
was particularly intense at the zone of contacts with axons (arrows in Fig. 6). DISCUSSION
The present study showed an increase in area showing NCAM immunoreactivity in the hippocampus of KA-treated rats. This change spatio-temporally correlates with both the sprouting of MFs and the glial reaction observed in the same animals. NCAM immunoreactivity was indeed particularly enriched in the
Fig. 4. N C A M antibodies stain the cytoplasmic m e m b r a n e s of MFs in control hippocampus. A,B: EM photographs depicting the N C A M immunostaining in the pyramidal cell l a y e r - s t r a t u m lucidum of CA4 (A) and in the plexiform layer (B). Only cytoplasmic m e m b r a n e s of pyramidal cell bodies and MFs were immunopositive, notably at the axon-axonal and a x o n - n e u r o n a l cell body (arrows) contacts. Note the characteristic fasciculation of MFs. Bar = 1 /~m.
112
Fig. 5. NCAM immunoreactivity after KA treatment in the hilus of KA-treated rats. A: numerous immunostained axons were observed throughout the hilus; they are likely to be MFs. Thus they are well-fasciculated and a few synaptic boutons of complex shape and great size originate from them (*). These synaptic boutons rarely made synaptic contacts since most of target elements had been destroyed by the treatment, lmmunoreactivity is clearly seen at the axon-axonal contacts. Cytoplasmic membranes of synaptic boutons were not stained. Bar = 1 ;~m.
cytoplasmic membrane of axon shafts and glial cells in the zones of MF sprouting, mainly the hilus. The present results suggest that NCAMs were expressed by
MF sprouts and reactive glial cells. NCAMs may therefore contribute to the synaptic rearrangement of MFs observed in epileptic conditions.
Fig. 6. NCAM immunoreactivity after KA treatment in the hilus of KA-treated rats. A,B: EM photographs depicting astroglial NCAM immunoreactivity at the contact with axons (arrows) which are also immunoreactive at the axonaxonal contacts (arrow heads). Bar = 0.5/~m.
114 The optic and EM changes here reported were observed in every treated animal. This is likely to be due to the rigorous selection of experimental animals. Thus, we only analyzed KA-treated rats showing a complete lesion of C A 3 - C A 4 fields, all of them showing a very similar pattern of MF sprouting.
NCAM immunoreactil~ity in adult control hippocampus Previous reports showed that N C A M immunoreactivity and m R N A expression correlated in CNS with processes of neurite outgrowth and synaptogenesis during postnatal development 2'~6'17'25'26'34.These molecules persisted in adult brain structures, such as the hippocampus ~'w'23'26'35, in which they may play a role in stabilizing cell-cell contacts. In adult hippocampus there is a clear compartmenration on the distribution of the three major N C A M isoforms: NCAM-180 was mainly expressed on dendrites 24, whereas it was absent from MFs. In contrast NCAM was observed both in dendrites (of granule ceils, pyramidal neurons and interneurons) and axons (essentially MFs) 2~. We therefore suggest that MFs mainly express NCAM-140 a n d / o r NCAM-120 and that both isoforms may sustain their characteristic fasciculation. In the present study, in control hippocampus, glial cells were not stained by N C A M antibodies.
N C A M immunoreactirity after KA treatment As previously reported 2s, after KA treatment there is a dramatic growing and sprouting of MFs which invade both the deeper fields of the hilus and, after passing through the granule cell layer, the inner third of the molecular layer. The hilus of KA-treated animals was almost exclusively composed of glial cells and axons. Goigi- and Timm-stained preparations suggest that most of these hilar axons are newly developed MF collaterals. Thus, like normal MFs, they appear as bundles of thin, unmyelinated, well-fasciculated axons which may from originate MF boutons 2s. In the present study we show a striking spatio-temporal correlation among the MF sprouting and the changes on N C A M immunoreactivity, mainly in the hilus. This suggests that MF sprouts express NCAMs. Furthermore, at EM levels, all axons in the hilus of KA-treated rats are N C A M positive, mainly at the zones of axon-axonal and axon-astrocyte contacts. Nevertheless fibers other than MFs may also be labelled by N C A M antibodies. Thus, most of these hilar axons display features of MFs: they form bundles of thin, well-fasciculated, unmyelinated fibers from which may originate complex giant synaptic boutons. These fibers were not immunostained by antibodies against
NCAM-180 (unpublished results), suggesting that axonal sprouts express NCAMs of 14(/or 12(J kDm In agreement with a previous observation ~, reactive astrocytes in the hilus of KA-treated rats were NCAM immunoreactive; the immunorcactivity was restricted to the cytoplasmic membrane at the contacl with axons, suggesting that NCAM mediate axonal-glial interaction.
NCAMs may contribute to M F sprouting Previous in vitro analysis on the role of NCAM showed that these molecules are powerful inducers of neurite outgrowth from central 11 or peripheral I°~-~ neurons. This capacity is directly related to the homophilic binding of NCAM to NCAM molecules, and are therefore modulated by polysialic acid (PSA) w. In fact, N C A M isoforms enriched in PSA, which are mainly present at embryonic stages 7'3~'34, arc less adhesive z°'33 but better inducers of neurite outgrowth ~ than adult isoforms. Reactive astrocytes in the hippocampus of KA-treated rats were immunoreactive to an antibody that recognizes the PSA-rich forms of N C A M 2-~. Glial cells therefore provide a faint adhesive substrate for MFs. In contrast, after KA treatment, MFs and other eventual axonal sprouts were not labelled by these antibodies 23. Axonal sprouts would therefore express NCAM- 140 and - 120 poorly enriched on polysialic residues. This NCAM expression may exert a robust axonaxonal adhesion, stronger than the axon-glial one. In the model proposed by Ruthishauser 3~ at the neuromuscular junction, activity-dependent sprouting was associated with an increase in axonal PSA levels (weaker axon-axonal interaction); this sprouting developed over a substrate which was more adhesive. On the contrary in the hippocampus, the stronger interaction is the axon-axonal, whereas the axon-glial interactions is the weaker. In our study the intense fasciculation of MFs may result from the strong axon-axonal interaction mediated by adult NCAM isoforms, whereas the collateral branching and growth may be caused by the weaker interaction of MFs with the PSA-enriched glial substrate. The antagonism among these 2 different forces may also explain the slow pattern of growing in the hippocampus of epileptic rats. Thus, MF collaterals never go beyond the borders of dentate gyrus despite the restricted number of target cells there proposed (KA treatment destroys most of the interneurons in this field); the first MF collaterals need at least 12 days to attempt the molecular layer and 30 days seem to be required to complete this growing -~°. The morphoregulatory activities of NCAM may be mediated by a specific modulation of intracellular sig-
115 naling systems 9'38, and may also be related to an interaction with cytoskeleton proteins. On this respect it is interesting to note that NCAMs (most likely NCAM180 and NCAM-140) seem to modulate tyrosine phosphorylation of tubulin in growth cone membranes from fetal rat brains 4. This phosphorylation may contribute to the axonal growing by controlling the polymerization of tubulin. It is tempting to suggest that extracellular (NCAMs) and intracellular (cytoskeleton proteins) factors cooperate to facilitate MF sprouting. Other adhesion or substrate proteins may influence the axonal branching and outgrowth (i.e. L1 also expressed by MFs) 26, NCAM being just a link in the complex processes of epilepsy-induced sprouting. The understanding of the complex mechanisms involved on such axonal growing certainly require a systematic analysis of environmental (i.e. cell adhesion molecules), diffusible (i.e. neurotrophines) and neuronal (i.e. cytoskeleton proteins) factors. Acknowledgements. We are very grateful to Dr. C. Goridis for suggestions and comments and kindly providing NCAM antibodies. We are also grateful to S. Guidasci for photographs, and to D. Diabira for technical assistance. REFERENCES 1 Albe-Fessard, D., Stutinski, F. and Libouban-le-Touze, S., Atlas st~r~otaxique du diencephale du rat blanc, CNRS, Paris, 1971. 2 Alcantara, A.A., Pfenninger, K.H. and Greenough, W.T., 5B4CAM expression parallels neurite outgrowth and synaptogenesis in the developing rat brain, J. Comp. NeuroL, 319 (1992) 337-348. 3 Amaral, D.G. and Dent, J.A., Development of the mossy fibers of the dentate gyrus. I. A light and electron microscopic study of the mossy fibers and their expansions, J. Comp. NeuroL, 195 (1981) 51-86. 4 Atashi, J.R., Klinz, S.G., Ingraham, C.A., Matten, W.T., Schachner, M. and Maness, P.F., Neural cell adhesion molecules modulate tyrosine phosphorylation of tubulin in nerve growth cone membranes, Neuron, 8 (1992) 831-842. 5 Ben-Ari, Y. and Represa, A., Brief seizure episodes induce long-term potentiation and mossy fibre sprouting in the hippocampus, Trends Neurosci., 13 (1990) 312-318. 6 Blackstad, T.W. and Kjaerheim, A., Special axo-dendritic synapses in the hippocampal cortex: electron and light microscopic studies on the layer of the mossy fibers, J. Comp. Neurol., 117 (1961) 113-159. 7 Boisseau, S., Nedelec, J., Poirier, V., Rougon, G. and Simonneau, M., Analysis of high PSA N-CAM expression during mammalian spinal cord and peripheral nervous system development, Det~elopment, 112 (1991) 69-82. 8 Bonfanti, L., Olive, S., Poulain, D.A. and Theodosis, D.T., Mapping of the distribution of polysialylated neural cell adhesion molecule throughout the central nervous system of the adult rat: an immunocytochemical study, Neuroscience, 49 (1992) 419-436. 9 Doherty, P., Ashton, S.V., Moore, S.E. and Walsh, F.S., Morphoregulatory activities of NCAM and N-cadherin can be accounted for by G protein-dependent activation of L- and N-type neuronal Ca 2+ channels, Cell, 67 (1991) 21-33. 10 Doherty, P., Cohen, J. and Walsh, F.S., Neurite outgrowth in response to transfected N-CAM changes during development and is modulated by polysialic acid, Neuron, 5 (1990) 209-219. 11 Doherty, P., Fruns, M., Seaton, P., Dickson, G., Barton, C.H.,
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