5'-Nucleotidase activity of mossy fibers in the dentate gyrus of normal and epileptic rats.

Sprouting of mossy fibers in the hippocampus of rats that underwent limbic epileptogenesis by amygdala kindling or kainate injection was studied at the light microscopic and ultrastructural levels by cytochemical demonstration of the enzyme 5'-nucleotidase. This adenosine-producing ectoenzyme has previously been shown to characterize malleable terminals during brain development and lesion-induced synaptogenesis, but to be otherwise associated with glial membranes. At the light microscopic level, kainate-treated but not control or kindled rats showed 5'-nucleotidase activity in the CA3 region and in the inner molecular layer of the dentate gyrus. At the ultrastructural level, in control animals, the synapses of the molecular and granular layers were enzyme negative. Only some mossy fiber boutons of the dentate hilus exhibited 5'-nucleotidase activity. In epileptic rats, synaptic labeling within the hilus appeared more intense. Moreover, 5'-nucleotidase-containing terminals within the inner molecular layer, presumably ectopic mossy fiber boutons, were found in both kindled and kainate-treated rats. It is concluded that, in both the normal and epileptic hippocampus, 5'-nucleotidase is associated with axons capable of a plastic sprouting response. The synaptic enzyme may attenuate the glutamatergic transmission of mossy fibers, in particular of the aberrant mossy fibers in epileptic rats, by producing the inhibitory neuromodulator adenosine. Alternatively, 5'-nucleotidase may influence synapse formation by its putative non-enzymatic, adhesive functions.     

Glial reaction after seizure induced hippocampal lesion: immunohistochemical characterization of proliferating glial cells

Summary 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 of³H-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 rype-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.     

Limbic seizures cause pronounced changes in the expression of neurokinin B in the hippocampus of the rat.

Immunohistological and in situ hybridization techniques were used to study the influence of kainic acid-induced seizures and of pentylenetetrazol kindling on neurokinin B immunoreactivity and neurokinin B mRNA in the rat hippocampus. Pronounced increases in neurokinin B immunoreactivity were observed in the terminal field of mossy fibres 10-60 days after intraperitoneal injection of kainic acid. These slow but persistent increases in immunoreactivity were accompanied by markedly enhanced expression of neurokinin B mRNA in the granule cells and in hilar interneurons adjacent to the granule cell layer. These changes were preceded by transient increases in neurokinin B mRNA and immunoreactivity in CA1 pyramidal cell layer two and 10 days after kainic acid, which, however, subsided later on. Pentylenetetrazol kindling caused similar increases in neurokinin B mRNA expression in granule cells and in CA1 pyramidal cells, but not in hilar interneurons. In CA1, increased neurokinin B message was present two days after termination of the kindling procedure but not after 10 days. Sixty days after kainic acid injection, neurokinin B immunoreactivity extended to the inner-third of the molecular layer of the dentate gyrus. After pentylenetetrazol kindling, a neurokinin B-immunoreactive band was observed in the infrapyramidal region of CA3. Lesions of the dentate granule cells by local injection of colchicine in kainic acid-treated rats abolished the supragranular neurokinin B-positive staining, whereas it was almost unchanged after transection of the ventral hippocampal commissure. These observations suggest that neurokinin B immunoreactivity may be located in ipsilateral mossy fibres undergoing collateral sprouting to the inner molecular layer or to the infrapyramidal region in CA3, respectively. Preprotachykinin A mRNA, which encodes for neurokinin A and substance P, and substance P immunoreactivity were not changed in the hippocampus of epileptic rats compared with untreated animals. The observed changes in neurokinin B immunoreactivity and mRNA indicate that specific functional and morphological changes may be induced in hippocampal neurons by recurrent limbic seizures.     

Activity-dependent changes in synaptophysin immunoreactivity in hippocampus,piriform cortex,and entorhinal cortex of the rat

Synaptophysin, an integral membrane glycoprotein of synaptic vesicles, has been widely used to investigate synaptogenesis in both animal models and human patients. Kindling is an experimental model of complex partial seizures with secondary generalization, and a useful model for studying activation-induced neural growth in adult systems. Many studies using Timm staining have shown that kindling promotes sprouting in the mossy fiber pathway of the dentate gyrus. In the present study, we used synaptophysin immunohistochemistry to demonstrate activation-induced neural sprouting in non-mossy fiber cortical pathways in the adult rat. We found a significant kindling-induced increase in synaptophysin immunoreactivity in the stratum radiatum of CA1 and stratum lucidum/radiatum of CA3, the hilus, the inner molecular layer of the dentate gyrus, and layer II/III of the piriform cortex, but no significant change in layer II/III of the entorhinal cortex, 4 weeks after the last kindling stimulation. We also found that synaptophysin immunoreactivity was lowest in CA3 near the hilus and increased with increasing distance from the hilus, a reverse pattern to that seen with Timm stains in stratum oriens following kindling. Furthermore, synaptophysin immunoreactivity was lowest in dorsal and greatest in ventral sections of both CA3 and dentate gyrus in both kindled and non-kindled animals. This demonstrates that different populations of sprouting axons are labeled by these two techniques, and suggests that activation-induced sprouting extends well beyond the hippocampal mossy fiber system.     

Neuropeptide Y biosynthesis is markedly induced in mossy fibers during temporal lobe epilepsy of the rat

Neuropeptide Y (NPY) immunoreactivity and gene expression was investigated in the hippocampus after kainic acid-induced seizures and pentylenetetrazol kindling in the rat. Pronounced increases of NPY immunoreactivity were found in the terminal field of mossy fibers in both animal models. In kainic acid-treated rats the peptide progressively accumulated in the hilus and the stratum lucidum of CA3, 5-60 days after injection of the toxin and, at the later intervals, extended to the supragranular molecular layer of the dentate gyrus indicating sprouting of these neurons. Unilateral injection of colchicine into the hilus abolished NPY staining of the mossy fibers. Using in situ hybridization, in both animal models markedly enhanced expression of prepro-NPY mRNA was observed in the granular layer, containing the perikarya of the mossy fibers. It is suggested that sustained expression of the neuromodulatory neuropeptide NPY, in addition to the observed plastic changes, may contribute to altered excitability of hippocampal mossy fibers in epilepsy. Neither somatostatin immunoreactivity nor gene expression were enhanced in granule cells/mossy fibers.     

Expression of synaptopodin, an actin-associated protein, in the rat hippocampus after limbic epilepsy

Synaptopodin, a 100 kD protein, associated with the actin cytoskeleton of the postsynaptic density and dendritic spines, is thought to play a role in modulating actin-based shape and motility of dendritic spines during formation or elimination of synaptic contacts. Temporal lobe epilepsy in humans and in rats shows neuronal damage, aberrant sprouting of hippocampal mossy fibers and subsequent synaptic remodeling processes. Using kainic acid (KA) induced epilepsy in rats, the postictal hippocampal expression of synaptopodin was analyzed by in situ hybridization (ISH) and immunohistochemistry. Sprouting of mossy fibers was visualized by a modified Timm's staining. ISH showed elevated levels of Synaptopodin mRNA in perikarya of CA3 principal neurons, dentate granule cells and in surviving hilar neurons these levels persisted up to 8 weeks after seizure induction. Synaptopodin immunoreactivity in the dendritic layers of CA3, in the hilus and in the inner molecular layer of the dentate gyrus (DG) was initially reduced. Eight weeks after KA treatment Synaptopodin protein expression returned to control levels in dendritic layers of CA3 and in the entire molecular layer of the DG. The recovery of protein expression was accompanied by simultaneous supra- and infragranular mossy fiber sprouting. Postictal upregulation of Synaptopodin mRNA levels in target cell populations of limbic epilepsy-elicited damage and subsequent Synaptopodin protein expression largely co-localized with remodeling processes as demonstrated by mossy fiber sprouting. It may thus represent a novel postsynaptic molecular correlate of hippocampal neuroplasticity.     

Optical recording study of granule cell activities in the hippocampal dentate gyrus of kainate-treated rats

In the epileptic hippocampus, newly sprouted mossy fibers are considered to form recurrent excitatory connections to granule cells in the dentate gyrus and thereby increase seizure susceptibility. To study the effects of mossy fiber sprouting on neural activity in individual lamellae of the dentate gyrus, we used high-speed optical recording to record signals from voltage-sensitive dye in hippocampal slices prepared from kainate-treated epileptic rats (KA rats). In 14 of 24 slices from KA rats, hilar stimulation evoked a large depolarization in almost the entire molecular layer in which granule cell apical dendrites are located. The signals were identified as postsynaptic responses because of their dependence on extracellular Ca(2+). The depolarization amplitude was largest in the inner molecular layer (the target area of sprouted mossy fibers) and declined with increasing distance from the granule cell layer. In the inner molecular layer, a good correlation was obtained between depolarization size and the density of mossy fiber terminals detected by Timm staining methods. Blockade of GABAergic inhibition by bicuculline enlarged the depolarization in granule cell dendrites. Our data indicate that mossy fiber sprouting results in a large and prolonged synaptic depolarization in an extensive dendritic area and that the enhanced GABAergic inhibition partly masks the synaptic depolarization. However, despite the large dendritic excitation induced by the sprouted mossy fibers, seizure-like activity of granule cells was never observed, even when GABAergic inhibition was blocked. Therefore, mossy fiber sprouting may not play a critical role in epileptogenesis.     

大鼠脑缺血长期存活后海马可塑性的研究

本实验试图进一步探讨经长期存活后，脑缺血及不全脑缺血大鼠海马的可塑性变化，并验证它们之间的关系。脑缺血模型采用pulsinelli四血管结扎及其改变法，苔藓纤维显示采用Timm染色法．结果显示，大鼠脑缺血20muin、再存活90d后，海马CAI区细胞几乎全部丧失，CA2／CA3也出现严重的细胞消亡，CAI区明显萎缩，多数伴有苔藓纤维的侧枝抽芽，同期不全脑缺血大鼠海马未见或仅见局限于CAI的部分细胞丢失，齿状回或CA3区也伴有苔藓纤维抽芽．提示脑缺血经长期存活后引起的苔藓纤维的侧枝抽芽并不依赖于CAI区的细胞死亡．     

Involvement of highly polysialylated neural cell adhesion molecule (PSA-NCAM)-positive granule cells in the amygdaloid-kindling-induced sprouting of a hippocampal mossy fiber trajectory

The mossy fiber system in the hippocampus of amygdaloid-kindled rats was examined by using highly polysialylated neural cell adhesion molecule (PSA-NCAM) as a marker for immunohistochemical detection of immature dentate granule cells and mossy fibers in combination with bromodeoxyuridine (BrdU) labeling of newly generated granule cells. Statistically significant increases in BrdU-labeled cells and PSA-NCAM-positive cells occurred in the dentate gyrus following kindling. The increase in PSA-NCAM-immunoreactive neurites was confined to the entire stratum lucidum of CA3. Immunoelectron-microscopic examination also revealed that PSA-NCAM-positive immature synaptic terminals of the sprouting mossy fibers increased in the stratum lucidum of CA3 in the kindled rats. The increase in the numbers of PSA-NCAM-positive granule cells correlated well with the increase in the immunopositive neurites and synaptic terminals on the mossy fiber trajectory. The increase in these PSA-NCAM-immunopositive structures is thought to reflect the enhancement of sprouting and synaptogenesis of mossy fibers by a subset of granule cells newly generated during amygdaloid-kindling and suggests that the reorganization of the mossy fiber system on the normal trajectory at least in part contributes to the acquisition and maintenance of an epileptogenic state.     

Increased dendritic excitability in hippocampal ca1 in vivo in the kainic acid model of temporal lobe epilepsy: a study using current source density analysis

We used kainic acid in rats as an animal model of temporal lobe epilepsy, and studied the synaptic transmission in hippocampal subfield CA1 of urethane-anesthetized rats in vivo. Dendritic currents were revealed by field potential mapping, using a single micropipette or a 16-channel silicon probe, followed by current source density analysis. We found that the population excitatory postsynaptic potentials in the basal dendrites and distal apical dendrites of CA1 were increased in kainate-treated as compared with control rats following paired-pulse, but not single-pulse, stimulation of CA3b or medial perforant path. In contrast, the trisynaptic midapical dendritic response in CA1 following medial perforant path stimulation was decreased in kainate-treated as compared with control rats. Increased coupling between excitatory postsynaptic potential and the population spike in CA1 was found after kainate seizures. Short-latency, presumably monosynaptic CA1 population spikes following medial perforant path stimulation was found in kainate-treated but not control rats. An enhancement of dendritic excitability was evidenced by population spikes that invaded into or originated from the distal apical dendrites of CA1 in kainate-treated but not control rats. Reverberation of hippocampo-entorhinal activity was evidenced by recurrent excitation of CA1 following CA3b stimulation in kainate-treated but not control rats. Blockade of inhibition by intraventricularly administered bicuculline induced excitatory potentials in CA1 that were stronger and more prolonged in kainate-treated than control rats. The bicuculline-induced excitation was mainly blocked by non-N-methyl-D-aspartate receptor antagonists. We conclude that kainate seizures induced disinhibition in CA1 that unveiled excitation at the basal and distal apical dendrites, resulting in enhancement of the direct entorhinal cortex to CA1 input and reverberations via the hippocampo-entorhinal loop. These changes in the output of the hippocampus from CA1 are likely detrimental to the behavioral functions of the hippocampus and they may contribute to increased seizure susceptibility after kainate seizures.     

Comparative immunohistochemistry of synaptic markers in the rodent hippocampus in pilocarpine epilepsy

Pilocarpine-induced epileptic state (Status epilepticus) generates an aberrant sprouting of hippocampal mossy fibers, which alter the intrahippocampal circuits. The mechanisms of the synaptic plasticity remain to be determined. In our studies in mice and rats, pilocarpine-induced seizures were done in order to gain information on the process of synaptogenesis. After a 2-month survival period, changes in the levels of synaptic markers (GAP-43 and Syn-I) were examined in the hippocampus by means of semi-quantitative immunohistochemistry. Mossy fiber sprouting (MFS) was examined in each brain using Timm's sulphide-silver method. Despite the marked behavioral manifestations caused by pilocarpine treatment, only 40% of the rats and 56% of the mice showed MFS. Pilocarpine treatment significantly reduced the GAP-43 immunoreactivity in the inner molecular layer in both species, with some minor differences in the staining pattern. Syn-I immunohistochemistry revealed species differences in the sprouting process. The strong immunoreactive band of the inner molecular layer in rats corresponded to the Timm-positive ectopic mossy fibers. The staining intensity in this layer, representing the ectopic mossy fibers, was weak in the mouse. The Syn-I immunoreactivity decreased significantly in the hilum, where Timm's method also demonstrated enhanced sprouting. This proved that, while sprouted axons displayed strong Syn-I staining in rats, ectopic mossy fibers in mice did not express this synaptic marker. The species variability in the expression of synaptic markers in sprouted axons following pilocarpine treatment indicated different synaptic mechanisms of epileptogenesis.     

Hippocampal cell proliferation and epileptogenesis after audiogenic kindling are not accompanied by mossy fiber sprouting or Fluoro-Jade staining

Repetitive sound-induced seizures, known as audiogenic kindling (AK), gradually induce the transference of epileptic activity from brainstem to forebrain structures along with behavioral changes. The aim of our work was to correlate the behavioral changes observed during the AK with possible alterations in neuronal proliferation, cell death, hippocampal mossy fiber sprouting and in the EEG pattern of Wistar audiogenic rats, a genetically susceptible strain from our laboratory.Susceptible and non-susceptible animals were submitted to repeated sound stimulations for 14-16 days and hippocampal mitotic activity was studied through the incorporation of bromodeoxyuridine (BrdU). Cell death and mossy fiber sprouting were assessed, respectively, by using Fluoro-Jade and Timm staining, 2 and 32 days after the last kindling stimulation. In addition, we used immunofluorescent double labeling for a glial and a mitotic marker to evaluate newly born cell identity. Some animals had hippocampus and amygdala electrodes for EEG recordings.Our results show that kindled animals with 6-11 generalized limbic seizures (class IV-V) had increased cell proliferation in the dentate gyrus when compared with animals with zero or one to three seizures. BrdU-positive cells labeled on day 2 and on day 32 were both GFAP negative. In the later group, rounded and well-defined BrdU-positive/GFAP-negative nuclei were seen in different portions of the granule cell layer. We did not observe any Fluoro-Jade or differential Timm staining in kindled animals at both killing times. However, EEG recordings showed intense epileptic activity in the hippocampus and amygdala of all animals with limbic seizures.Therefore, our data indicate that AK-induced limbic epileptogenicity is able to increase the hippocampal mitotic rate, even though it does not seem to promote neuronal death or mossy fiber sprouting in the supragranular layer of the dentate gyrus.     

听源性惊厥易感大鼠点燃后海马结构内的突触素表达

为探讨听源性惊厥点燃对突触可塑性的影响，采用免疫细胞化学方法结合体视学分析，研究了Ｗｉｓｔａｒ种系的听源性惊厥易感大鼠（Ｐ７７ＰＭＣ）惊厥和点燃后海马结构内突触素ｐ３８表达的差异。结果显示：（１）Ｐ７７ＰＭＣ大鼠１次惊厥后，海马结构内ｐ３８免疫反应产物呈明显的板层样分布；（２）Ｐ７７ＰＭＣ大鼠点燃后，ｐ３８免疫反应产物的定位分布与１次惊厥后相比较，没有明显改变，但是，ｐ３８免疫反应产物的密度普遍增     

Continuous infusion of neurotrophin-3 triggers sprouting,decreases the levels of TrkA and TrkC,and inhibits epileptogenesis and activity-dependent axonal growth in adult rats

Neurotrophin-3 (NT-3), a member of the neurotrophin family of neurotrophic factors, is important for cell survival, axonal growth and neuronal plasticity. Epileptiform activation can regulate the expression of neurotrophins, and increases or decreases in neurotrophins can affect both epileptogenesis and seizure-related axonal growth. Interestingly, the expression of nerve growth factor and brain-derived neurotrophic factor is rapidly up-regulated following seizures, while NT-3 mRNA remains unchanged or undergoes a delayed down-regulation, suggesting that NT-3 might have a different function in epileptogenesis. In the present study, we demonstrate that continuous intraventricular infusion of NT-3 in the absence of kindling triggers mossy fiber sprouting in the inner molecular layer of the dentate gyrus and the stratum oriens of the CA3 region. Furthermore, despite this NT-3-related sprouting effect, continuous infusion of NT-3 retards the development of behavioral seizures and inhibits kindling-induced mossy fiber sprouting in the inner molecular layer of the dentate gyrus. We also show that prolonged infusion of NT-3 leads to a decrease in kindling-induced Trk phosphorylation and a down-regulation of the high-affinity Trk receptors, TrkA and TrkC, suggesting an involvement of both cholinergic nerve growth factor receptors and hippocampal NT-3 receptors in these effects. Our results demonstrate an important inhibitory role for NT-3 in seizure development and seizure-related synaptic reorganization.     

Perforant path lesioning induces sprouting of CA3-associated fibre systems in mouse hippocampal formation

In comparison to the rat, the anatomy of the mouse hippocampus, and in particular the response to entorhinal cortex lesioning, is less well characterised. Here we studied the axonal sprouting response after lesioning of the entorhinodentate perforant path projection in young adult SJL/J and C57BL/6 mice. We found that lesioning led to translaminar sprouting of Timm stained regio inferior hippocampus (CA3)-associated fibre systems into the denervated termination zones of the CA3 and dentate gyrus, from the adjacent non-denervated stratum radiatum of CA3. Differences were seen in the Timm staining pattern of the two strains of mice, while the response to lesioning appeared similar albeit less pronounced than that observed in the rat. We also observed an intensified acetylcholine esterase staining reflective of cholinergic sprouting in the denervated perforant path termination zones, which was particularly prominent in areas with sprouting of Timm stained CA3-associated fibres. Finally, we showed that some of the sprouting fibres within the CA3 were myelinated, due to an increased density of silver impregnated myelinated fibres in this region after lesioning. These results show that the basic characteristics of the response to perforant path lesioning in mice are similar to those in the rat, but suggest that the magnitude of the response in the two species is different.     

Seizures induce simultaneous GABAergic and glutamatergic transmission in the dentate gyrus-CA3 system

Monosynaptic and polysynaptic responses of CA3 pyramidal cells (PC) to stimulation of the dentate gyrus (DG) are normally blocked by glutamate receptor antagonists (GluRAs). However, after kindled seizures, GluRAs block the monosynaptic excitatory postsynaptic potential (EPSP) and isolate a monosynaptic inhibitory postsynaptic potential (IPSP), suggesting that mossy fibers release GABA. However, kindling epilepsy induces neuronal sprouting, which can underlie this fast inhibitory response. To explore this possibility, the synaptic responses of PC to DG stimulation were analyzed in kindled epileptic rats, with and without seizures, and in nonepileptic rats, immediately after a single pentylenetetrazol (PTZ)-induced seizure, in which sprouting is unlikely to have occurred. Excitatory and inhibitory synaptic responses of PC to DG stimulation were blocked by GluRAs in control cells and in cells from kindled nonseizing rats, confirming that inhibitory potentials are disynaptically mediated. However, a fast IPSP could be evoked in kindled epileptic rats and in nonepileptic rats after a single PTZ-induced seizure. The same response was induced after rekindling the epileptic nonseizing rats. This IPSP has an onset latency that parallels that of the control EPSP and is not altered under low Ca(2+) medium or halothane perfusion. In addition, it was reversibly depressed by L(+)-2-amino-4-phosphonobutyric acid (L-AP4), which is known to inhibit transmitter release from mossy fibers. These results demonstrate that seizures, and not the synaptic rearrangement due to an underlying epileptic state, induce the emergence of fast inhibition in the DG-CA3 system, and suggest that the mossy fibers underlie this plastic change.     

Immunoreactive corticotropin releasing factor in adult and developing rat cerebellum: its presence in climbing and mossy fibres

Corticotropin Releasing Factor (CRF) immunoreactivity was localized within the rat central nervous system with immunocytochemical methods, using a highly specific anti-serum to rat-CRF. In adult rats abundant CRF immunoreactivity (CRF-ir) was found in perikarya of the inferior olivary nucleus, in climbing fibres in the molecular layer, and mossy fibres in the granular layer of the cerebellum and in the cerebellar nuclei. CRF-ir climbing fibres were found in a longitudinal zonal pattern throughout the molecular layer. In developing cerebellum only a few CRF-positive fibres were seen on postnatal day 8. From day 8 onwards a gradual increase in CRF-ir was found in both the molecular and granular layer with formation of CRF-positive nests around Purkinje cellbodies starting at day 12. Subsequent outgrowth into the molecular layer could be followed, leading to an adult level of CRF-ir in the cerebellum at approximately day 22 postnatally. CRF-ir in mossy fibres has as adult appearance from approximately day 18. The temporal and morphological changes of CRF-ir in fibres in the cerebellum of immature rats followed closely the morphological development of climbing and mossy fibres. The present findings indicate that CRF (or related substances) are already present in both climbing and mossy fibres during their outgrowth into the cerebellum.     

Short- and long-term changes in CA1 network excitability after kainate treatment in rats

Neuron loss, axon sprouting, and the formation of new synaptic circuits have been hypothesized to contribute to seizures in temporal lobe epilepsy (TLE). Using the kainate-treated rat, we examined how alterations in the density of CA1 pyramidal cells and interneurons, and subsequent sprouting of CA1 pyramidal cell axons, were temporally associated with functional changes in the network properties of the CA1 area. Control rats were compared with animals during the first week after kainate treatment versus several weeks after treatment. The density of CA1 pyramidal cells and putative inhibitory neurons in stratum oriens was reduced within 8 days after kainate treatment. Axon branching of CA1 pyramidal cells was similar between controls and animals examined in the first week after kainate treatment but was increased several weeks after kainate treatment. Stimulation of afferent fibers in brain slices containing the isolated CA1 region produced graded responses in slices from controls and kainate-treated rats tested <8 days after treatment. In contrast, synchronous all-or-none bursts of spikes at low stimulus intensity (i.e., "network bursts") were only observed in the CA1 several weeks after kainate treatment. In the presence of bicuculline, the duration of evoked bursts was significantly longer in CA1 pyramidal cells weeks after kainate treatment than from controls or those examined in the first week posttreatment. Spontaneous network bursts were also observed in the isolated CA1 several weeks after kainate treatment in bicuculline-treated slices. The data suggest that the early loss of neurons directly associated with kainate-induced status epilepticus is followed by increased axon sprouting and new recurrent excitatory circuits in CA1 pyramidal cells. These changes characterize the transition from the initial acute effects of the kainate-induced insult to the eventual development of all-or-none epileptiform discharges in the CA1 area.     

Differential upregulation of extracellular matrix molecules associated with the appearance of granule cell dispersion and mossy fiber sprouting during epileptogenesis in a murine model of temporal lobe epilepsy

We have investigated changes in the extracellular matrix of the hippocampus associated with the early progression of epileptogenesis in a murine model of temporal lobe epilepsy using immunohistochemistry. In the first week following intrahippocampal injection of the glutamate agonist, domoate, there is a latent period at the end of which begins a sequential upregulation of extracellular matrix (ECM) molecules in the granule cell layer of the dentate gyrus, beginning with neurocan and tenascin-C. This expression precedes the characteristic dispersion of the granule cell layer which is evident at 14 days post-injection when the first recurrent seizures can be recorded. At this stage, an upregulation of the chondroitin sulfate proteoglycan, phosphacan, the DSD-1 chondroitin sulfate motif, and the HNK-1 oligosaccharide are also observed. The expression of these molecules is localized differentially in the epileptogenic dentate gyrus, especially in the sprouting molecular layer, where a strong upregulation of phosphacan, tenascin-C, and HNK-1 is observed but there is no expression of the proteoglycan, neurocan, nor of the DSD-1 chondroitin sulfate motif. Hence, it appears that granule cell layer dispersion is accompanied by a general increase in the ECM, while mossy fiber sprouting in the molecular layer is associated with a more restricted repertoire. In contrast to these changes, the expression of the ECM glycoproteins, laminin and fibronectin, both of which are frequently implicated in tissue remodelling events, showed no changes associated with either granule cell dispersion or mossy fiber sprouting, indicating that the epileptogenic plasticity of the hippocampus is accompanied by ECM interactions that are characteristic of the CNS.     

Phosphorylated and non-phosphorylated neurofilament proteins: distribution in the rat hippocampus and early changes after kainic acid induced seizures

The regional distribution of neurofilament proteins in the rat hippocampus and their early changes after kainic acid induced seizures were investigated immunocytochemically with antibodies against light weight neurofilament, phosphorylated and non-phosphorylated heavy weight neurofilament. The light weight and non-phosphorylated heavy weight neurofilaments were distributed more unevenly than the phosphorylated neurofilament. The perikarya and processes of pyramidal cells in the CA3 field contained the highest light weight and non-phosphorylated heavy weight neurofilaments, while the perikarya of granule cells contained only few light weight neurofilament and the perikarya of CAI pyramidal cells were even devoid of immunoreactivity of both light and heavy weight neurofilaments. The fiber staining of the light weight and non-phosphorylated heavy weight neurofilaments, especially the former, was less in the CAI field and molecular layer of dentate gyrus. The phosphorylated neurofilament immunoreactivity was identified only in axons. Mossy fibers, the axons of granule cells, contained the light weight and phosphorylated heavy weight neurofilaments, but not the non-phosphorylated neurofilament. Seven days after the kainic acid induced seizures, the phosphorylated neurofilament staining was greatly reduced in the CAI and inner molecular layer of the dentate gyrus, probably resulting from the axonal degeneration of the Schaffer collaterals and the commissural/associational fibers. Furthermore, the non-phosphorylated neurofilament appeared in the mossy fibers of the CA3 stratum lucidum, which normally do not express such immunoreactivity. The results indicate that the neurofilaments are altered following the neuronal degeneration and post lesional plasticity caused by the kainic acid administration. Therefore, the examination of various phosphorylated neurofilaments may offer a comprehensive understanding of major hippocampul pathways, axonal plasticity and the possible roles of neurofilaments in the hippocampus following excitotoxic insults.