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Mossy fiber sprouting in epileptic rats is associated with a transient increased expression of α-tubulin
Neuroscience Letters, 156 (1993) 149-152 © 1993 ElsevierScientificPublishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00
149
NSL 09622
Mossy fiber sprouting in epileptic rats is associated with a transient increased expression of a-tubulin A. Represa, H. Pollard, J. M o r e a u , G. Ghilini, M. K h r e s t c h a t i s k y a n d Y. Ben-Ari 1NSERM U29, Paris (France)
(Received 12 February 1993; Revisedversionreceived 2 April 1993;Accepted 5 April 1993) Key words: Hippocampus;Kainate;Seizure; Mossyfiber sprouting; Tubulin; In situ hybridization;Immunocytochemistry
Kainate-inducedseizures lead to marked increases of a-tubulin mRNA and protein immunoreactivityin the rat dentate gyrus. The increase in a-tubulin mRNA was restrictedto the granulecell bodies, a-Tubulin immunoreactivitywas enhancedin granulecell dendrites and axons (the mossy fibers), in the molecularlayer. These changespeaked 6 12 days after kainate treatment and preceded the collateral sprouting of mossyfibers which occur 12to 30 days after seizures.The presentresults suggestthat microtubuleformationcontributesto the synapticrearrangementswhichtake place in the hippocampusafter seizures.
One of the most striking changes observed in epileptic brains is the abnormal sprouting and synaptogenesis of hippocampal mossy fibers (mf, the axons of dentate granule cells) (reviewed in ref. 2). Thus, mf which mainly innervate CA3 pyramidal cells in control conditions, also innervate hippocampal granule cell dendrites (in the inner third of the molecular layer) in epileptic brains [14, 16, 17], generating recurrent excitatory circuits [17]. It has therefore been postulated that these synaptic remodelings contribute to the maintenance of the epilepsy. This has been well analyzed in the experimental model of temporal lobe epilepsy induced by the convulsive agent kainate (KA [1]). The expression of cytoskeletal proteins increases within neurons during periods of axonal or dendritic growths. This has been well illustrated for fl-tubulin [3, 10], a-tubulin [11] and the microtubule-associated protein MAP-2 [10, 12]. The expression of tubulin also increases in neuronal cells after injury [13, 18], preceding axonal or dendritic regenerations. The production of microtubules which results from the assemby of tubulins and microtubule-associated proteins, may therefore sustain the elongation and side-branching of both axons and dendrites. In the rat, several isotypes ofa-tubulins are generated at the transcriptional level [7-9]. a-Tubulin mRNAs display a variety of temporal and spatial patterns of expresCorrespondence." A. Represa, INSERM U29, 123 Bd. Port Royal, 75014 Paris, France. Fax: (33) (1) 46341656.
sion during development of the CNS. Tal-tubulin m R N A is expressed at high levels in neuronal cell populations at a time when they are extending processes, whereas T26 a-tubulin m R N A is expressed constitutively at low levels in most or all neural cell types throughout development, with some enrichment in the proliferative zones [11]. We have examined the effects of seizures induced by intra-amygdaloid injection of KA [1] upon the expression of a-tubulin in the hippocampus, by combining in situ hybridization and immunocytochemical techniques. Male Wistar rats (180-200 g) were submitted to unilateral injections of KA (1.2/2g) into the amygdala, as previously described [15]. Animals showing typical KA-induced limbic motor seizures for at least 2 h upon recovery from anaesthesia were kept for the experiments. Animals injected with phosphate buffer at the same stereotaxic coordinates were used as controls. The hippocampal complex ipsilateral to the injection site was analyzed. In view of hybridization studies, the rats were sacrificed at various time intervals after the onset of the behavioral seizure activity, (n=3, for each time point analyzed). In situ hybridization was performed on 15-/2mthick frontal brain cryostat sections using an [35S]dATPlabeled oligonucleotide probe [4] complementary to the sequences of both the T26 and T a l mRNAs [6] as previously described [4]. The sections were apposed to Amersham fl-max films for 1 week. Some sections were further processed by dipping into Amersham LM 1 emulsion and counterstained with thionin after processing. Controls
150
for the specificity of the in situ hybridization reaction consisted in the addition of 200-fold excess of non-radioactive probe in the hybridization solution, which resulted in a total absence of signal. In contrast, no change in the hybridization pattern was observed when an unrelated oligonucleotide, such as an excess of unlabeled MAP-2 probe was used for competition. Sections from control and KA-treated rats were incubated with the same hybridization mixtures and apposed to the same film. A quantitative analysis was performed on autoradiograms by densitometry (Samba/2005, TITN, Alcatel, France). Three different sections per rat were analyzed. For immunocytochemistry, control rats (n=5) and KA-treated rats were perfused with 2% paraformaldehyde, 3 (n=4), 4 (n=2), 6 (n=4), 12 (n=6), 20 (n=4), 30
(n=4) days after treatment. The brains were either frozen and cut (40/lm thick) or embedded in paraffin and cut (7 /.zm). The sections were incubated overnight with mouse anti c¢-tubulin antibodies (1:1000; Amersham) and further developed according to the peroxidase anti-peroxidase technique. Immunocytochemical controls were performed using either a normal rabbit serum or mouse immunoglobulins instead of primary antibodies. Alternate sections were stained with Cresyl violet to monitor the extent of lesions. In keeping with an earlier study [11], under control conditions, ~x-tubulin hybridization signal was localized almost exclusively in the bodies of pyramidal cells, granule cells, and interneurons, (Fig. 1A). Within 3 days after injection, a 30--50% increase in ~-tubulin m R N A level was observed in the pyramidal and granule cell layers (Fig. 2). This elevation was transient. Six days after KA, c¢-tubulin m R N A levels returned to control values in the pyramidal cells of CAI subfield, whereas in CA3 the hybridization signal disappeared (Fig. 2), due to the degeneration of the pyramidal cells in this area [1]. In contrast, in dentate granule cells, the increased hybridization signal was maintained for more than 12 days after injection (Figs. I B and 2). In this layer, the hybridization signal returned close to the control value 20 days after seizures.
I
•
~1oo
3
Fig. 1. Up-regulation of ~-tubulin mRNA m the rat hippocampus upon KA administration. Frontal sections were hybridized ,vith a ~sS-labeled oligonucleotide probe. A C: low magnification dark field photographs of autoradiograms from representative sections through the dorsal hippocampal formation from control rats (saline-injected) (A) and rats killed 12/B) and 20 (C) days alter KA. Arrowheads indicate the loss in the hybridization signal, resulting from the neuronal lesions in CA3 CA4 subfields. D,E: bright field emulsions showing ct-tubulin expressing granule cells in a control rat (D) and a rats sacrified 12 days after KA. dg, dentate gyrus.
t 6
12
20
60 days
Fig. 2. Quantitative analysis as a function of time, of ~-tubulin m R N A levels from in situ hybridization experiments. Values are plotted as percent (+S.E.M.) of control densities measured on autoradiograms from rats sacrificed 3 days after saline administration. Data are combined from three experiments, (n=2 3 animals per groups in each experiment). )'-axis is % of control m R N A level. "P<0.005 (Student's test), as compared to controls. O, granular cell layer of the dentate gyrus: , CA1 pyramidal cell layer; [Z], CA3 pyramidal cell layer. 3 days after KA treatment c¢-tubulin expression is increased in all neuronal cells analyzed. Between 3 and 12 days after KA treatment, the sustained tubulin expression is restricted to the dentate gyrus. Levels close to control values are tbund later on.
151 In our control conditions, tubulin immunoreactivity (IR) was diffusely distributed in dendrites of granule cells, pyramidal cells and interneurons (Fig. 3A,C,E). Cell bodies and axons (including mossy fibers) were not stained by this antibody. Treatment with KA induced a transient increase in tubulin IR in the hippocampus (Fig. 3B). This increase was conspicuous in both the inner third of the molecular layer where the antibody likely stains proximal granule cell dendrites (Fig. 3D) and in the stratum lucidum of CA3 field where the antibody stains mossy fibers (Fig. 3E). These changes were first observed 4 days after K A administration; they persisted in rats 12 and 20 days after KA and had returned to control levels 30 days after KA. In CA3, there was a lack of dendritic IR due to the loss of pyramidal cells (Fig. 3F). No significant changes were observed in CA1 (Fig. 3B). The present results demonstrate that 0~-tubulin m R N A and protein signals increase in the dentate gyrus after 3 and 4 days of KA treatment, respectively, and peak on
day 12. This depicts a striking correlation between m R N A and protein expression, although while the m R N A level is significantly increased as early as 3 days post KA, 4 days are necessary to obtain a clear, significant elevation of ~-tubulin IR in the granule cells. Likewise ~-tubulin m R N A signal returns to control values 20 days after KA treatment whereas the protein level returns to control values 30 days after KA. This enhanced expression of ~-tubulin likely reflects an overproduction of ~-tubulin subunits by granule cells, which could be transported as either assembled microtubules or unassembled tubulins (reviewed in ref. 5), from cell bodies to dendrites and axons (mf). Since microtubule-associated proteins such as MAPs or TAU proteins modulate tubulin polymerization, it will be of interest to study whether the expression of these proteins is altered after KA treatment. The present changes develop in the dentate gyrus, before and during axonal m f sprouting. In fact, mf collaterals begin to sprout around 12 days after KA treatment
Fig. 3. ~-'rubulin immunoreactivityincreases in the hippocampus of KA-treated rats. In control hippocampus (A,C,E) ~-tubulin IR is present in the dendrites of granule cells (C) and in the pyramidal neurons of CA1-CA4 fields (E). The IR is absent from axons. After 4 days of KA treatment, Tubulin IR increases in both granule-cell dendrites (mainly the proximal third; D) and mf in the stratum lucidum (arrows in B and F), while it disappears from CA3 pyramidal dendrites due to the neuronal loss. Paraffinsections. Bar--300/lm in A,B and 60/.tm in C-F. dg, dentate gyrus; g, granule cell layer; H, hilus; Lu, stratum lucidum; m, molecularlayer; mf, mossy fibers; O, stratum oriens; P, pyramidal cell layer.
152 [14, 15]. 30 d a y s a f t e r K A t h e r e are n o f u r t h e r c h a n g e s in the p a t t e r n o f m f d i s t r i b u t i o n [14, 15]. T h e p r o d u c t i o n o f t u b u l i n s likely c o n t r i b u t e s to the g r o w i n g a n d t h e sideb r a n c h i n g o f mf. T u b u l i n p r o d u c t i o n a n d e x p r e s s i o n by g r a n u l e cell d e n d r i t e s m a y in a d d i t i o n c o n t r i b u t e to the p o s t s y n a p t i c c h a n g e s i n d u c e d in g r a n u l e cells by the dev e l o p m e n t o f h e t e r o t y p i c m f s y n a p t i c c o n t a c t s [14]. A u t h o r s are g r a t e f u l to D. D i a b i r a a n d I. J o r q u e r a for t e c h n i c a l a s s i s t a n c e a n d to S. G u i d a s c i f o r p h o t o g r a p h s . Financial support from the Association Fran~aise contre les M y o p a t h i e s is a k n o w l e d g e d . 1 Ben-Ark Y., Limbic seizures and brain damage produced by kainic
2
3
4
5
6
7
acid: mechanisms and relevance to human temporal lobe epilepsy, Neuroscience, 14 (1985) 375-403. Ben-Ari, Y. and Represa, A., Brief seizure episodes induce longterm potentiation and mossy fibre sprouting in the hippocampus, Trends Neurosci., 13 (1990) 312 318. Bond, J.F. and Farmer, S.R., Regulation of tubulin and actin mRNA production in rat brain: expression of a new beta-tubulin mRNA with development, Mol. Cell Biol., 3 (1983) 1333 1342. Charriaut-Marlangue, C., Pollard, H., Kadri-Hassani, N., Khrestchatisky, M., Moreau, J., Dessi, F., Kang, K.1. and Ben-Ari. Y., Increase in specific proteins and mRNAs following transient anoxia-aglycemia in rat CA1 slices, Eur. J. Neurosci., 4 (1992) 766776. Cleveland, D.W. and Hoffman, P.N., Slow axonal transport models come full circle: evidence that microtubule sliding mediates axon elongation and tubulin transport, Cell, 67 (1991) 453-456. Ginzburg, J., Behar, L., Givol, D. and Littauer, U.Z., The nucleotide sequence of rat ~-tubulin: 3'-end characteristics and evolutionary conservation, Nucleic Acids Res., 9 (1986) 2691 2697. Ginzburg, J., Teichmann, A., Griffin, W.S.T. and Littauer, U.Z., Differential expression of ~-tubulin in rat cerebellum as revealed by in situ hybridization., FEBS Lett., 194 (1981) 161 164.
8 Lemischka, I.R., Farmer, S.R., Racaniello, V.R. and Sharp, RA._ Nucleotide sequence and evolution of mammalian ~-tubulin mRNA., J. Mol. Biol., 150 (1981) 101 -120. 9 Lewis, S.A., Lee, M.G.-S. ans Cowan, N.J., Five mouse tubulin isotypes and their regulated expression during development, J. Cell Biol., 101 (1985) 852 861. 10 Matus, A., Bernhardt, R., Bodmer, R. and Alaimo, D., Microtubuteassociated protein 2 and tubulin are differently distributed in the dendrites of developing neurons, Neuroscience, 17 (1986) 37 I 389. 11 Miller, F.D., Naus, C.C.G., Durant, M., Bloom, F.E. and Milner. R.J., lsotypes of ~-tubulin are differentially regulated during neuronal maturation, J. Cell Biol., 105 (1987) 3065-3073. 12 Nunez, J., Immature and mature variants of MAP2 and tau proteins and neuronal plasticity, Trends Neurosci., I 1 (1988) 477~79. 13 Phillips, L.L. and Steward, O., Increases in mRNA for cytoskeletal proteins in the denervated neuropil of the dentate gyrus: an in situ hybridization study using riboprobes for fl-actin and fl-tubulin, Mol. Brain Res., 8 (1990) 249 257. 14 Represa, A., Jorquera, I.. Le Gal La Salle, G. and Ben-Ari, Y., Epilepsy induced collateral sprouting of hippocampal mossy fibers: does it induce the development of ectopic synapses with granule cell dendrites, Hippocampus, in press. 15 Represa, A., Tremblay, E. and Ben-Ari, Y., Kainate binding sites in the hippocampal mossy fibers: localization and plasticity, Neuroscience, 20 (1987) 739 748. 16 Sutula, T., Xiao-Xian, H., Cavazos, J. and Scott, G., Synaptic reorganization in the hippocampus induced by abnormal functional activity, Science, 239 (1988) 1147 1150. 17 Tauck, D.L. and Nadler, J.V., Evidence for functional mossy fiber sprouting in hippocampal formation of kainic acid treated rats, J. Neurosci., 5 (1985) 1016-1022. 18 Tetzlaff, W., Alexander, S.W., Miller, F.D. and Bisby, M.A., Response of facial and rubrospinal neurons to axotomy: changes in mRNA expression for cytoskeletal proteins and GAP-43, J. Neurosci., 11 (1991) 2528-2544.
149
NSL 09622
Mossy fiber sprouting in epileptic rats is associated with a transient increased expression of a-tubulin A. Represa, H. Pollard, J. M o r e a u , G. Ghilini, M. K h r e s t c h a t i s k y a n d Y. Ben-Ari 1NSERM U29, Paris (France)
(Received 12 February 1993; Revisedversionreceived 2 April 1993;Accepted 5 April 1993) Key words: Hippocampus;Kainate;Seizure; Mossyfiber sprouting; Tubulin; In situ hybridization;Immunocytochemistry
Kainate-inducedseizures lead to marked increases of a-tubulin mRNA and protein immunoreactivityin the rat dentate gyrus. The increase in a-tubulin mRNA was restrictedto the granulecell bodies, a-Tubulin immunoreactivitywas enhancedin granulecell dendrites and axons (the mossy fibers), in the molecularlayer. These changespeaked 6 12 days after kainate treatment and preceded the collateral sprouting of mossyfibers which occur 12to 30 days after seizures.The presentresults suggestthat microtubuleformationcontributesto the synapticrearrangementswhichtake place in the hippocampusafter seizures.
One of the most striking changes observed in epileptic brains is the abnormal sprouting and synaptogenesis of hippocampal mossy fibers (mf, the axons of dentate granule cells) (reviewed in ref. 2). Thus, mf which mainly innervate CA3 pyramidal cells in control conditions, also innervate hippocampal granule cell dendrites (in the inner third of the molecular layer) in epileptic brains [14, 16, 17], generating recurrent excitatory circuits [17]. It has therefore been postulated that these synaptic remodelings contribute to the maintenance of the epilepsy. This has been well analyzed in the experimental model of temporal lobe epilepsy induced by the convulsive agent kainate (KA [1]). The expression of cytoskeletal proteins increases within neurons during periods of axonal or dendritic growths. This has been well illustrated for fl-tubulin [3, 10], a-tubulin [11] and the microtubule-associated protein MAP-2 [10, 12]. The expression of tubulin also increases in neuronal cells after injury [13, 18], preceding axonal or dendritic regenerations. The production of microtubules which results from the assemby of tubulins and microtubule-associated proteins, may therefore sustain the elongation and side-branching of both axons and dendrites. In the rat, several isotypes ofa-tubulins are generated at the transcriptional level [7-9]. a-Tubulin mRNAs display a variety of temporal and spatial patterns of expresCorrespondence." A. Represa, INSERM U29, 123 Bd. Port Royal, 75014 Paris, France. Fax: (33) (1) 46341656.
sion during development of the CNS. Tal-tubulin m R N A is expressed at high levels in neuronal cell populations at a time when they are extending processes, whereas T26 a-tubulin m R N A is expressed constitutively at low levels in most or all neural cell types throughout development, with some enrichment in the proliferative zones [11]. We have examined the effects of seizures induced by intra-amygdaloid injection of KA [1] upon the expression of a-tubulin in the hippocampus, by combining in situ hybridization and immunocytochemical techniques. Male Wistar rats (180-200 g) were submitted to unilateral injections of KA (1.2/2g) into the amygdala, as previously described [15]. Animals showing typical KA-induced limbic motor seizures for at least 2 h upon recovery from anaesthesia were kept for the experiments. Animals injected with phosphate buffer at the same stereotaxic coordinates were used as controls. The hippocampal complex ipsilateral to the injection site was analyzed. In view of hybridization studies, the rats were sacrificed at various time intervals after the onset of the behavioral seizure activity, (n=3, for each time point analyzed). In situ hybridization was performed on 15-/2mthick frontal brain cryostat sections using an [35S]dATPlabeled oligonucleotide probe [4] complementary to the sequences of both the T26 and T a l mRNAs [6] as previously described [4]. The sections were apposed to Amersham fl-max films for 1 week. Some sections were further processed by dipping into Amersham LM 1 emulsion and counterstained with thionin after processing. Controls
150
for the specificity of the in situ hybridization reaction consisted in the addition of 200-fold excess of non-radioactive probe in the hybridization solution, which resulted in a total absence of signal. In contrast, no change in the hybridization pattern was observed when an unrelated oligonucleotide, such as an excess of unlabeled MAP-2 probe was used for competition. Sections from control and KA-treated rats were incubated with the same hybridization mixtures and apposed to the same film. A quantitative analysis was performed on autoradiograms by densitometry (Samba/2005, TITN, Alcatel, France). Three different sections per rat were analyzed. For immunocytochemistry, control rats (n=5) and KA-treated rats were perfused with 2% paraformaldehyde, 3 (n=4), 4 (n=2), 6 (n=4), 12 (n=6), 20 (n=4), 30
(n=4) days after treatment. The brains were either frozen and cut (40/lm thick) or embedded in paraffin and cut (7 /.zm). The sections were incubated overnight with mouse anti c¢-tubulin antibodies (1:1000; Amersham) and further developed according to the peroxidase anti-peroxidase technique. Immunocytochemical controls were performed using either a normal rabbit serum or mouse immunoglobulins instead of primary antibodies. Alternate sections were stained with Cresyl violet to monitor the extent of lesions. In keeping with an earlier study [11], under control conditions, ~x-tubulin hybridization signal was localized almost exclusively in the bodies of pyramidal cells, granule cells, and interneurons, (Fig. 1A). Within 3 days after injection, a 30--50% increase in ~-tubulin m R N A level was observed in the pyramidal and granule cell layers (Fig. 2). This elevation was transient. Six days after KA, c¢-tubulin m R N A levels returned to control values in the pyramidal cells of CAI subfield, whereas in CA3 the hybridization signal disappeared (Fig. 2), due to the degeneration of the pyramidal cells in this area [1]. In contrast, in dentate granule cells, the increased hybridization signal was maintained for more than 12 days after injection (Figs. I B and 2). In this layer, the hybridization signal returned close to the control value 20 days after seizures.
I
•
~1oo
3
Fig. 1. Up-regulation of ~-tubulin mRNA m the rat hippocampus upon KA administration. Frontal sections were hybridized ,vith a ~sS-labeled oligonucleotide probe. A C: low magnification dark field photographs of autoradiograms from representative sections through the dorsal hippocampal formation from control rats (saline-injected) (A) and rats killed 12/B) and 20 (C) days alter KA. Arrowheads indicate the loss in the hybridization signal, resulting from the neuronal lesions in CA3 CA4 subfields. D,E: bright field emulsions showing ct-tubulin expressing granule cells in a control rat (D) and a rats sacrified 12 days after KA. dg, dentate gyrus.
t 6
12
20
60 days
Fig. 2. Quantitative analysis as a function of time, of ~-tubulin m R N A levels from in situ hybridization experiments. Values are plotted as percent (+S.E.M.) of control densities measured on autoradiograms from rats sacrificed 3 days after saline administration. Data are combined from three experiments, (n=2 3 animals per groups in each experiment). )'-axis is % of control m R N A level. "P<0.005 (Student's test), as compared to controls. O, granular cell layer of the dentate gyrus: , CA1 pyramidal cell layer; [Z], CA3 pyramidal cell layer. 3 days after KA treatment c¢-tubulin expression is increased in all neuronal cells analyzed. Between 3 and 12 days after KA treatment, the sustained tubulin expression is restricted to the dentate gyrus. Levels close to control values are tbund later on.
151 In our control conditions, tubulin immunoreactivity (IR) was diffusely distributed in dendrites of granule cells, pyramidal cells and interneurons (Fig. 3A,C,E). Cell bodies and axons (including mossy fibers) were not stained by this antibody. Treatment with KA induced a transient increase in tubulin IR in the hippocampus (Fig. 3B). This increase was conspicuous in both the inner third of the molecular layer where the antibody likely stains proximal granule cell dendrites (Fig. 3D) and in the stratum lucidum of CA3 field where the antibody stains mossy fibers (Fig. 3E). These changes were first observed 4 days after K A administration; they persisted in rats 12 and 20 days after KA and had returned to control levels 30 days after KA. In CA3, there was a lack of dendritic IR due to the loss of pyramidal cells (Fig. 3F). No significant changes were observed in CA1 (Fig. 3B). The present results demonstrate that 0~-tubulin m R N A and protein signals increase in the dentate gyrus after 3 and 4 days of KA treatment, respectively, and peak on
day 12. This depicts a striking correlation between m R N A and protein expression, although while the m R N A level is significantly increased as early as 3 days post KA, 4 days are necessary to obtain a clear, significant elevation of ~-tubulin IR in the granule cells. Likewise ~-tubulin m R N A signal returns to control values 20 days after KA treatment whereas the protein level returns to control values 30 days after KA. This enhanced expression of ~-tubulin likely reflects an overproduction of ~-tubulin subunits by granule cells, which could be transported as either assembled microtubules or unassembled tubulins (reviewed in ref. 5), from cell bodies to dendrites and axons (mf). Since microtubule-associated proteins such as MAPs or TAU proteins modulate tubulin polymerization, it will be of interest to study whether the expression of these proteins is altered after KA treatment. The present changes develop in the dentate gyrus, before and during axonal m f sprouting. In fact, mf collaterals begin to sprout around 12 days after KA treatment
Fig. 3. ~-'rubulin immunoreactivityincreases in the hippocampus of KA-treated rats. In control hippocampus (A,C,E) ~-tubulin IR is present in the dendrites of granule cells (C) and in the pyramidal neurons of CA1-CA4 fields (E). The IR is absent from axons. After 4 days of KA treatment, Tubulin IR increases in both granule-cell dendrites (mainly the proximal third; D) and mf in the stratum lucidum (arrows in B and F), while it disappears from CA3 pyramidal dendrites due to the neuronal loss. Paraffinsections. Bar--300/lm in A,B and 60/.tm in C-F. dg, dentate gyrus; g, granule cell layer; H, hilus; Lu, stratum lucidum; m, molecularlayer; mf, mossy fibers; O, stratum oriens; P, pyramidal cell layer.
152 [14, 15]. 30 d a y s a f t e r K A t h e r e are n o f u r t h e r c h a n g e s in the p a t t e r n o f m f d i s t r i b u t i o n [14, 15]. T h e p r o d u c t i o n o f t u b u l i n s likely c o n t r i b u t e s to the g r o w i n g a n d t h e sideb r a n c h i n g o f mf. T u b u l i n p r o d u c t i o n a n d e x p r e s s i o n by g r a n u l e cell d e n d r i t e s m a y in a d d i t i o n c o n t r i b u t e to the p o s t s y n a p t i c c h a n g e s i n d u c e d in g r a n u l e cells by the dev e l o p m e n t o f h e t e r o t y p i c m f s y n a p t i c c o n t a c t s [14]. A u t h o r s are g r a t e f u l to D. D i a b i r a a n d I. J o r q u e r a for t e c h n i c a l a s s i s t a n c e a n d to S. G u i d a s c i f o r p h o t o g r a p h s . Financial support from the Association Fran~aise contre les M y o p a t h i e s is a k n o w l e d g e d . 1 Ben-Ark Y., Limbic seizures and brain damage produced by kainic
2
3
4
5
6
7
acid: mechanisms and relevance to human temporal lobe epilepsy, Neuroscience, 14 (1985) 375-403. Ben-Ari, Y. and Represa, A., Brief seizure episodes induce longterm potentiation and mossy fibre sprouting in the hippocampus, Trends Neurosci., 13 (1990) 312 318. Bond, J.F. and Farmer, S.R., Regulation of tubulin and actin mRNA production in rat brain: expression of a new beta-tubulin mRNA with development, Mol. Cell Biol., 3 (1983) 1333 1342. Charriaut-Marlangue, C., Pollard, H., Kadri-Hassani, N., Khrestchatisky, M., Moreau, J., Dessi, F., Kang, K.1. and Ben-Ari. Y., Increase in specific proteins and mRNAs following transient anoxia-aglycemia in rat CA1 slices, Eur. J. Neurosci., 4 (1992) 766776. Cleveland, D.W. and Hoffman, P.N., Slow axonal transport models come full circle: evidence that microtubule sliding mediates axon elongation and tubulin transport, Cell, 67 (1991) 453-456. Ginzburg, J., Behar, L., Givol, D. and Littauer, U.Z., The nucleotide sequence of rat ~-tubulin: 3'-end characteristics and evolutionary conservation, Nucleic Acids Res., 9 (1986) 2691 2697. Ginzburg, J., Teichmann, A., Griffin, W.S.T. and Littauer, U.Z., Differential expression of ~-tubulin in rat cerebellum as revealed by in situ hybridization., FEBS Lett., 194 (1981) 161 164.
8 Lemischka, I.R., Farmer, S.R., Racaniello, V.R. and Sharp, RA._ Nucleotide sequence and evolution of mammalian ~-tubulin mRNA., J. Mol. Biol., 150 (1981) 101 -120. 9 Lewis, S.A., Lee, M.G.-S. ans Cowan, N.J., Five mouse tubulin isotypes and their regulated expression during development, J. Cell Biol., 101 (1985) 852 861. 10 Matus, A., Bernhardt, R., Bodmer, R. and Alaimo, D., Microtubuteassociated protein 2 and tubulin are differently distributed in the dendrites of developing neurons, Neuroscience, 17 (1986) 37 I 389. 11 Miller, F.D., Naus, C.C.G., Durant, M., Bloom, F.E. and Milner. R.J., lsotypes of ~-tubulin are differentially regulated during neuronal maturation, J. Cell Biol., 105 (1987) 3065-3073. 12 Nunez, J., Immature and mature variants of MAP2 and tau proteins and neuronal plasticity, Trends Neurosci., I 1 (1988) 477~79. 13 Phillips, L.L. and Steward, O., Increases in mRNA for cytoskeletal proteins in the denervated neuropil of the dentate gyrus: an in situ hybridization study using riboprobes for fl-actin and fl-tubulin, Mol. Brain Res., 8 (1990) 249 257. 14 Represa, A., Jorquera, I.. Le Gal La Salle, G. and Ben-Ari, Y., Epilepsy induced collateral sprouting of hippocampal mossy fibers: does it induce the development of ectopic synapses with granule cell dendrites, Hippocampus, in press. 15 Represa, A., Tremblay, E. and Ben-Ari, Y., Kainate binding sites in the hippocampal mossy fibers: localization and plasticity, Neuroscience, 20 (1987) 739 748. 16 Sutula, T., Xiao-Xian, H., Cavazos, J. and Scott, G., Synaptic reorganization in the hippocampus induced by abnormal functional activity, Science, 239 (1988) 1147 1150. 17 Tauck, D.L. and Nadler, J.V., Evidence for functional mossy fiber sprouting in hippocampal formation of kainic acid treated rats, J. Neurosci., 5 (1985) 1016-1022. 18 Tetzlaff, W., Alexander, S.W., Miller, F.D. and Bisby, M.A., Response of facial and rubrospinal neurons to axotomy: changes in mRNA expression for cytoskeletal proteins and GAP-43, J. Neurosci., 11 (1991) 2528-2544.