Seizure-induced molecular changes, sprouting and synaptogenesis of hippocampal mossy fibers.
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Animals↗
Biomedical subjects
A Represa
Publications and source records attributed to A Represa.
Expression of an acidic isoform of calponin in rat brain: western blots on one- or two-dimensional gels and immunolocalization in cultured cells.
Calponin, an actin- and Ca(2+)-calmodulin-binding protein characterized as an inhibitory factor of the smooth-muscle actomyosin activity, has also been shown to be present in some non-muscle cells. However, there is a controversy as to whether calponin is present or not in brain. Several laboratories indicate that this protein is absent in chicken or bovine brains, while Applegate et al. [Applegate, Feng, Green and Taubman (1994) J. Biol. Chem. 269, 10683-10690] have recently reported the identification of an mRNA specific for a 36 kDa non-muscle calponin analogue in homogenates of rat brains. For the first time we demonstrate, by Western blots and in situ immunofluorescence localization using monoclonal as well as affinity-purified polyclonal antibody to gizzard calponin, that a 36-37 kDa and a 35-36 kDa calponin-like proteins are expressed respectively in pig and rat brains and in rat cerebellar cultured cells. The acidic pI (5.2-5.4) of the rat brain protein revealed by isoelectric focusing is in good agreement with that of the protein coded for by the calponin isoform mRNA described by Applegate et al. and is different from that of the protein from chicken gizzard (pI 9.9). Brain calponin-like protein is different from two other Ca(2+)-calmodulin-binding proteins previously identified in brain, namely caldesmon and adducin, and from tropomyosin.
Animals↗
Cell death, gliosis, and synaptic remodeling in the hippocampus of epileptic rats.
Seizures set in motion complex molecular and morphological changes in vulnerable structures, such as the hippocampal complex. A number of these changes are responsible for neuronal death of CA3 and hilar cells, which involves necrotic and apoptotic mechanisms. In surviving dentate granule cells seizures induce an increased expression of tubulin subunits and microtubule-associated proteins, suggesting that an overproduction of tubulin polymers would lead to a remodeling of mossy fibers (the axons of granule cells). In fact, these fibers sprout in the dentate gyrus to innervate granule cell dendrites, creating recurrent excitatory circuits. In contrast, terminal mossy fibers do not sprout in the CA3 field. Navigation of mossy fiber's growth cones may be facilitated by astrocytes, which would exert differential effects by producing and excreting cell adhesion and substrate molecules. In the light of the results discussed here, we suggest that in adult brain activated-resident astrocytes (nonproliferating, tenascin-negative, neuronal cell-adhesion molecule-positive astrocytes) could contribute to the process of axonal outgrowth and synaptogenesis in the dentate gyrus, while proliferating astrocytes, tenascin-positive, could impede any axonal rearrangement in CA3.
Animals↗
Molecular correlates between reactive and developmental plasticity in the rat hippocampus.
Area CA3 of the hippocampus is the most epileptogenic structure of the brain. Various studies have shown that kainate-induced experimental epilepsy in rats and human cases of epilepsy are associated with sprouting of the mossy fibers of the dentate granule neurons and selective loss of pyramidal neurons, notably in the CA3-CA4 areas of Ammon's horn. In experimental models of epilepsy, brief seizure activity initiates a cascade of molecular alterations that will contribute to changes in the expression of numerous genes, which can last several weeks. The products of some of these genes will contribute to the permanent state of enhanced synaptic efficiency, to the sprouting and formation of novel excitatory synapses, and possibly to neuronal cell loss. The expression of genes encoding transcription factors and numerous growth factors is rapidly altered following seizure episodes. Based on observations in vivo and in vitro in cultured hippocampal neurons, it is hypothesized that an interplay between transcription and growth factors, because of their pleiotropic effects on the regulation of effector genes, may be instrumental in coupling transient extracellular stimuli to irreversible cellular alterations.
Animals↗
Distribution of caldesmon and of the acidic isoform of calponin in cultured cerebellar neurons and in different regions of the rat brain: an immunofluorescence and confocal microscopy study.
Caldesmon and calponin are two F-actin-binding and calcium-calmodulin-dependent proteins. In smooth muscle and nonmuscle cells both proteins are localized on actin filaments. Using one- or two-dimensional gel electrophoresis followed by the Western blot technique, and by immunofluorescence studies, we have given evidence that calponin is also present in rat and pig brain. In the present study, for the first time, we demonstrate caldesmon- and calponin-specific immunoreactivities in cerebellar cultured neurons. In the rat central nervous system these antibodies mainly stain neuronal cell bodies and dendrites. By confocal analysis we observed that calponin and caldesmon are located in the actomyosin domain although the total actin and myosin were not saturated. In many cases it is clear that these two proteins are adjacent rather than superimposed in the same domain of the cell. These results are compatible with the functional role of caldesmon and calponin in the regulation of the actomyosin activity as described by others and suggest that they are part of the contractile apparatus of neural cells.
Actins↗
Gliosis and axonal sprouting in the hippocampus of epileptic rats are associated with an increase of tenascin-C immunoreactivity.
Temporal lobe epilepsy is associated with neuronal death, gliosis and sprouting of mossy fibres in the hippocampus of human and rats. In the present study we show that immunoreactivity for tenascin-C (an extracellular matrix glycoprotein) increase in the hippocampus of epileptic rats. However, this increase was only observed in the cases displaying neuronal cell loss and glial reaction (i.e. after kainate treatment but not after kindling). Tenascin-C increase was particularly striking at Ammon's horn, where the antibody labelled both reactive astrocytes (confirmed by double-labelling experiments) and axonal plasma membranes. In the molecular layer tenascin-C immunoreactivity remained unchanged in both kindled or kainate treated rats. It is interesting that increased tenascin-C immunoreactivity was observed within zones in which axonal regeneration did not occur (the CA3 area in kainate-treated animals) whereas zones in which reactive synaptogenesis occurred (such as the CA3 area of kindled rats or the molecular layer of both kindled and kainate-treated rats) were devoid of tenascin-C immunoreactivity. We infer from these results that tenascin-C impedes the terminal sprouting of mossy fibres in CA3 of kainate-treated rats.
Animals↗
Proliferative astrocytes may express fibronectin-like protein in the hippocampus of epileptic rats.
Kainic acid treatment, a model of temporal lobe epilepsy, induces in CA3-CA4 fields of hippocampal complex a neuronal degeneration associated with glial hypertrophy and proliferation. After treatment with kainate, fibronectin (an extracellular matrix protein) immunoreactivity increases in CA3-CA4. Fibronectin antibodies stain proliferative cells (simultaneously labelled by [3H]thymidin) of astrocytic type (double-immunostained by GFAP antibodies). This result constitutes the first direct demonstration of astroglial fibronectin expression in vivo. In the molecular layer of kainate-treated rats there is an axon-terminal degeneration of association-fibers. This is associated with a transient hypertrophy of resident astrocytes but not with any glial proliferation. Reactive astrocytes do not express (or faintly) fibronectin immunoreactivity in this layer. Since fibronectin is involved in astroglial proliferation in vitro, the present observations suggest that astrocytes contribute in vivo to the astroglial proliferation by an autocrin mechanism.
Animals↗
From seizures to neo-synaptogenesis: intrinsic and extrinsic determinants of mossy fiber sprouting in the adult hippocampus.
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Adult↗
Glial reaction after seizure induced hippocampal lesion: immunohistochemical characterization of proliferating glial cells.
Kainic acid treatment (a model of temporal lobe epilepsy) induces Ammon's horn sclerosis, which is characterized by degeneration of CA3 pyramidal neurons and reactive gliosis. In the present study we have combined autoradiographic analysis of 3H-thymidine incorporation and immunocytochemistry to investigate this glial scarring phenomenon. The present results demonstrate that in the fields showing neuronal degeneration (i.e. CA3-CA4 fields of Ammon's horn and dentate hilus) the glial reaction consists of a proliferation and hypertrophy of astrocytes and microglia-macrophages. In the regions showing exclusively terminal axonal degeneration (i.e. the molecular layer of kainate-treated rats), glial cells do not proliferate but astrocytes show a transient hypertrophy. These results also demonstrate that oligodendrocytes do not proliferate in the hippocampus of kainate-treated rats. In agreement with our previous report we find that hippocampal astrocytes from kainate-treated rats express A2B5 immunoreactivity, a marker of type-2 astrocytes. A2B5 immunoreactivity was expressed by astrocytes not only in areas showing glial proliferation such as CA3-CA4 fields, but also in the molecular layer, where astrocytes do not proliferate. This suggests that in the CNS, normal resident astrocytes acquire the phenotypic properties of type-2 astrocytes.
Animals↗
Kainate-induced apoptotic cell death in hippocampal neurons.
We have examined the role apoptosis plays in epileptic brain damage using intra-amygdaloid injection of kainate. With the silver staining technique of Gallyas, argyrophylic (dying) neurons were observed, a few hours after the injection, in the amygdala and in the vulnerable pyramidal neurons of the hippocampal CA3 region. In both areas, cell death has apoptotic features, including: (i) nuclear chromatin condensation and marginalization with light and electron microscopy; (ii) DNA fragmentation with a typical ladder pattern on agarose gel electrophoresis; (iii) positive nuclear labelling with a selective in situ DNA fragmentation staining method. Combined in situ DNA labelling and silver staining showed that the DNA fragmentation occurred in dying neurons. CA1 or granule cells which do not degenerate following intra-amygdaloid injection of kainate were not stained with the in situ DNA labelling or the argyrophylic technique. Administration of diazepam blocked the kainate-induced seizures and prevented DNA fragmentation in CA3 but not in the amygdala. Therefore, apoptosis contributes to the local and distant damage induced by kainate.
Animals↗
Correlation between reactive sprouting and microtubule protein expression in epileptic hippocampus.
Temporal lobe epilepsy in both human and rats is associated with a collateral sprouting of hippocampal mossy fibers (i.e. the axons of granule cells). This sprouting generates abnormal recurrent synaptic connections. We previously showed that in the experimental model of temporal lobe epilepsy induced by an intra-amygdaloid injection of kainate, the synaptic remodeling of mossy fibers was preceded by a transient increased expression of alpha-tubulin in granule cells. This suggests that an overproduction of tubulin polymers may be responsible, at least in part, for the elongation and side-branching of mossy fibers, which occurs 12-30 days after seizures. In the present study we show that this increased expression of alpha-tubulin is accompanied by an increased expression of the microtubule-associated proteins MAP2 and TAU. Thus, using in situ hybridization, we observe that MAP2 messenger RNA levels increased in granule cell bodies and dendrites from day 3 to two weeks after kainate treatment. This rise is associated with a concomitant transient increase of MAP2 immunoreactivity in the granule cell dendrites. TAU messenger RNA also increases in granule cell bodies, while TAU immunoreactivity increases in their axons, the mossy fibers. The time course of these changes parallels that of alpha-tubulin, and develops before and during the axonal mossy fiber sprouting. Since MAP2 and TAU are important for the initiation, elongation and stabilization of neurites, we suggest that the overexpression of these proteins via the formation of microtubules may play an important role in the sprouting of mossy fibers in epileptic rats.
Amygdala↗
NCAM immunoreactivity on mossy fibers and reactive astrocytes in the hippocampus of epileptic rats.
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.
Afferent Pathways↗
Mossy fiber sprouting in epileptic rats is associated with a transient increased expression of alpha-tubulin.
Kainate-induced seizures lead to marked increases of alpha-tubulin mRNA and protein immunoreactivity in the rat dentate gyrus. The increase in alpha-tubulin mRNA was restricted to the granule cell bodies. alpha-Tubulin immunoreactivity was enhanced in granule cell dendrites and axons (the mossy fibers), in the molecular layer. These changes peaked 6-12 days after kainate treatment and preceded the collateral sprouting of mossy fibers which occur 12 to 30 days after seizures. The present results suggest that microtubule formation contributes to the synaptic rearrangements which take place in the hippocampus after seizures.
Amygdala↗
Epilepsy induced collateral sprouting of hippocampal mossy fibers: does it induce the development of ectopic synapses with granule cell dendrites?
In the present study, using Golgi and electron microscopy techniques, experimentally induced epilepsy (kindling and kainate treatment) elicited collateral sprouting of mossy fibers in rat hippocampus. Collateral branches invade the hilus, cross the granule cell layer, and distribute throughout the inner third of the molecular layer. These newly developed collaterals may acquire the typical features of mossy fibers including giant fiber varicosities (mousses), although the mean surface of these mousses was thinner in these collaterals than in terminal branches. Granule cell dendrites may develop giant thorny excrescences, suggesting that the targets of these collaterals are granule cells. Giant synaptic boutons appear in the inner third of molecular layer of epileptic rats. These boutons acquire the morphological features of mossy fiber boutons and made multiple synaptic contacts with dendritic spines. The analysis of the profile types suggests that some of the newly developed collateral mossy fibers made hypotrophic synaptic contacts.
Animals↗
Reactive astrocytes in the kainic acid-damage hippocampus have the phenotypic features of type-2 astrocytes.
Kainic acid treatment, a model of temporal lobe epilepsy, induces Ammon's horn sclerosis characterized by degeneration of CA3 pyramidal neurons and reactive gliosis. We now report that in kainic acid treated rats, reactive astrocytes in the hippocampus are A2B5 immunopositive and express GAP-43 immunoreactivity. A2B5 is a cell surface ganglioside selectively expressed in the glial O-2A lineage (oligodendrocytes and type-2 astrocytes in vitro). Since A2B5-positive cells were also GFAP immunoreactive, our observation suggest that hippocampal-reactive astrocytes in the epileptic process are type-2 astrocytes. GAP-43 is a membrane-associated phosphoprotein involved in neurite outgrowth. In vitro analysis showed that the glial O-2A lineage may express this phosphoprotein. In this study, we found that GAP-43 was coexpressed in astrocytes with A2B5 suggesting that in vivo as in vitro type-2 astrocytes express GAP-43.
Animals↗
Kindling is associated with the formation of novel mossy fibre synapses in the CA3 region.
A Golgi and electron microscopy study of the hippocampal CA3 region was performed on control and kindled Wistar rats. The observations provide evidence that, in epileptic rats, mossy fibres sprout and establish novel synapses with the basilar dendrites of CA3 pyramidal neurons. These newly-developed synapses showed the typical features of mossy synapses observed in the stratum lucidum, including the appearance of complex giant spines. The morphological changes reported here may represent a histopathological substrate for the epilepsy in the absence of overt signs of a hippocampal lesion.
Animals↗
Epileptogenesis and neuronal plasticity: studies on kainate receptor in the human and rat hippocampus.
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Animals↗
Long-term potentiation and sprouting of mossy fibers produced by brief episodes of hyperactivity.
A brief application of high K+ or excitatory amino acids (i.e. kainic acid) generated repetitive synchronized burst that persisted for the duration of the application, in the CA3 field. Once excitability has been enhanced, further stimulation of various inputs evoked burst instead the typical excitatory postsynaptic potential--inhibitory postsynaptic potential sequence evoked in control conditions. These long-lasting changes in synaptic efficacy involved the activation of glutamate receptors of N-methyl-D-aspartate (NMDA) subtype. A brief period of hyperactivity (i.e. kindling of limbic pathways or administration of kainic acid) also resulted in a more delayed synaptic remodeling, notably of hippocampal mossy fibers (i.e. the axons of granule cells that mostly contact the apical dendrites of CA3 pyramidal neurons). Thus mossy fibers sprouted and made multiple ectopic asymmetrical synapses with spines of both granule cells dendrites and basilar dendrites of CA3 pyramidal cells. Finally, sprouting of mossy fibers was associated with a significant rise in the density of kainic acid binding sites (fmol/mg tissue) in the aberrantly innervated zones: the inner third of molecular layer and the stratum oriens of CA3. Saturation studies revealed that this rise did not significantly affect the affinity (Kd values) but the Bmax. In conclusion, brief seizure episodes produced in the hippocampus remarkably long-lasting changes in synaptic efficacy; synaptic density and the mean density of excitatory amino acid receptors of non-NMDA subtype. The role that such plastic changes may play in the permanence of the epilepsy is finally discussed.
Afferent Pathways↗