匹罗卡品致痫诱发大鼠齿状回颗粒细胞GAP-43 mRNA表达

目的 研究边缘系统癫痫发作后海马颗粒细胞生长相关蛋白（GAP-43）基因表达变化。方法 建立匹罗卡品急、慢性癫痫模型,用原位杂交方法定量检测不同时间点海马颗粒细胞GAP-43mRNA表达。结果 对照组颗粒细胞几乎不表达GAP-43mRNA,匹罗卡品致病后6～12h颗粒细胞表达GAP-43mRNA增高,15～30d呈现第2次高峰。结论 成年大脑海马颗粒细胞在致痫后发生可塑性变化,GAP-43mRNA表达是癫痫大鼠大脑结构性重组（颗粒细胞苔藓纤维出芽）的重要分子机制。     

Matrix metalloproteinase 9 regulates cell death following pilocarpine-induced seizures in the developing brain

Matrix metalloproteinases (MMPs) are involved in tissue repair, cell death and morphogenesis. We investigated the role of the gelatinases MMP-2 and MMP-9 in the pathogenesis of neuronal death induced by prolonged seizures in the developing brain. Seven-day-old rats, MMP-9 knockout mice and transgenic rats overexpressing MMP-9 received intraperitoneal injections of pilocarpine, 250 mg/kg, to induce seizures. After 6-72 h pups were sacrificed, tissue from different brain regions was isolated and expression of MMP-9 mRNA and protein was analyzed by real-time PCR or Western blot. Additionally, brains were fixed and processed for TUNEL-staining, immunohistochemistry and in situ zymography. We found increased numbers of TUNEL-positive cells 24 h after pilocarpine-induced seizures, most pronounced in cortical areas and the dentate gyrus, and less pronounced in thalamus. At 6-24 h, MMP-9 mRNA levels showed significant elevation compared to sham-treated controls; this effect resolved by 48 h, whereas MMP-2 mRNA levels remained stable. Cortical gelatinolytic activity, monitored by in situ zymography, was enhanced following pilocarpine-induced seizures. The MMP inhibitor GM 6001 ameliorated cell death following pilocarpine-induced seizures in infant rats. MMP-9 knockout mice were less susceptible to seizure-induced brain injury. Transgenic rats overexpressing MMP-9 were equally susceptible to seizure-induced brain injury as wild type rats. Our results suggest a significant contribution of MMP-9 to cell death after pilocarpine-induced seizures in the developing brain. As indicated by Western blot analysis, MMP-9 activation may be linked to activation of the Erk/CREB-pathway. The findings implicate involvement of MMP-9 in the pathophysiology of brain injury following seizures in the developing brain.     

Newly generated granule cells show rapid neuroplastic changes in the adult rat dentate gyrus during the first five days following pilocarpine-induced seizures

Long-term neuroplastic changes to dentate granule cells have been reported after seizures and were shown to contribute to recurrent excitatory circuitry. These changes include increased numbers of newborn granule cells, sprouted mossy fibers, granule cell layer dispersion, increased hilar ectopic granule cells and formation of hilar basal dendrites on granule cells. The goal of the current study was to determine the acute progression of neuroplastic changes involving newly generated granule cells after pilocarpine-induced seizures. Doublecortin (DCX) immunocytochemical preparations were used to examine the newly generated granule cells 1-5 days after seizures were induced. The results showed that there are rapid neuroplastic changes to the DCX-labeled cells. At 1 day after seizures were induced, there were significant increases in the percentage of DCX-labeled cells with hilar basal dendrites and in the progenitor cell population. At 2 days after seizures were induced, an increase in the thickness of the layer of DCX-labeled cells occurred. At 3 days after seizures were induced, the number of DCX-labeled cells was significantly increased. At 4 days after seizures were induced, developing synapses were observed on DCX-labeled hilar basal dendrites. Thus, newly generated granule cells in the adult dentate gyrus display neuroplastic changes by 1 day after pilocarpine-induced seizures and further changes occur to this population of cells in the subsequent 4 days. The presence of synapses, albeit developing ones, on hilar basal dendrites during this period indicates that newly generated granule cells become rapidly incorporated into dentate gyrus circuitry following seizures.     

Ultrastructural plasticity of the dentate gyrus granule cells following recurrent limbic seizures: I. Increase in somatic spines

Various paradigms have been used to assess the capacity of the adult brain to undergo activity-dependent morphological plasticity. In this report we have employed recurrent limbic seizures as a means of studying the effects of this form of enhanced neuronal activity on cellular morphology and, in particular, on the incidence of somatic spines on the dentate gyrus granule cells. Seizure activity was induced by the placement of focal, unilateral electrolytic lesions in the dentate gyrus hilus of adult rats. At various intervals postlesion, rats with behaviorally verified seizures were sacrificed, and the hippocampi contralateral, to the lesions were removed and prepared for electron microscopy. Quantitative analysis showed that as early as 5 hours postlesion there was a dramatic increase in the density and morphological complexity of spines on the perikarya of the granule cells in rats that received seizure-producing hilus lesions when compared to granule cells from control rats. Many of the somatic spines received asymmetric synapses. The increase in somatic spines was dependent on seizure activity and persisted for at least 1 month following a single recurrent seizure episode. CA1 pyramidal neurons, which exhibit changes in gene expression in response to hilus lesion-induced seizures but do not normally possess somatic spines, did not exhibit an activity-dependent elaboration of somatic spines. Thus, the seizure-induced elaboration of somatic spines represents an amplification of an existing feature of the granule cells and not an effect occurring throughout hippocampus. These data provide evidence for very rapid and long-lasting structural plasticity in response to brief episodes of seizure activity in the adult brain. © 1994 Wiley-Liss, Inc.     

Ultrastructural plasticity of the dentate gyrus granule cells following recurrent limbic seizures: II. Alterations in somatic synapses

Hilus lesion-induced recurrent limbic seizures cause a dramatic increase in the numbers of somatic spines on dentate gyrus granule cells in the adult rat. Somatic spines are maximally increased 3 h after the initiation of seizures at which time many of these spines form synapses. The present quantitative electron microscopic study assessed the numbers and types of synapses present on the granule cell perikarya and somatic spines of control and experimental seizure rats with the goal of determining if newly elaborated somatic spines arise at the site of pre-existing synapses or are associated with new innervation. Experimental rats were sacrificed 5 h after hilar lesion placement (or 3 h after seizure onset). In both control and hilus-lesioned (HL) rats, 15–20% of the somatic spines could be seen to form synaptic contacts within a single plane of section; these synapses were almost exclusively of the asymmetric type. With the increased incidence of spines in experimental-seizure rats, there was a 6.25-fold greater number of spine synapses in HL versus control rats. There was, in addition, a 60% decrease in the number of asymmetric synapses occurring directly on the granule cell perikarya but no change in the total (spine plus somatic) number of asymmeteric synapses. Although few asymmetric synapses were associated with spines in control tissue, 60–70% of asymmetric synapses were associated with spines in experimental-seizure tissue. In addition, in hilus lesion rats symmetric somatic synapses were increased by 20% on cells in deep stratum granulosum resulting in a dissolution of the superficial-to-deep innervation gradient present in the untreated rat. These findiings support the conclusion that spines induced by seizure activity form at the site of pre-existing asymmetric synapses on the granule cells and demonstrate that brief seizure episodes can rapidly induce marked changes in innervation patterns in the adult brain. © 1994 Wiley-Liss, Inc.     

Ultrastructural features and synaptic connections of hilar ectopic granule cells in the rat dentate gyrus are different from those of granule cells in the granule cell layer

Several investigators have shown the existence of dentate granule cells in ectopic locations within the hilus and molecular layer using both Golgi and retrograde tracing studies but the ultrastructural features and synaptic connections of ectopic granule cells were not previously examined. In the present study, the biocytin retrograde tracing technique was used to label ectopic granule cells following injections into stratum lucidum of CA3b of hippocampal slices obtained from epileptic rats. Electron microscopy was used to study hilar ectopic granule cells that were located 20–40 μm from the granule cell layer (GCL). They had ultrastructural features similar to those of granule cells in the GCL but showed differences, including nuclei that often displayed infoldings and thicker apical dendrites. At their origin, these dendrites were 6 μm in diameter and they tapered down to 2 μm at the border with the GCL. Both biocytin-labeled and unlabeled axon terminals formed exclusively asymmetric synapses with the somata and proximal dendrites of hilar ectopic granule cells. The mean number of axosomatic synapses for these cells was three times that for granule cells in the GCL. Together, these data indicate that hilar ectopic granule cells are postsynaptic to mossy fibers and have less inhibitory input on their somata and proximal dendrites than granule cells in the GCL. This finding is consistent with recent physiological results showing that hilar ectopic granule cells from epileptic rats are more hyperexcitable than granule cells in the GCL.     

Ethanol effects on dentate granule cell LTP

In previous studies we identified a lateral hypothalamic area (LHA) sensitive to ethanol, <5.0 mM, when the perifornical region of the area is perfused with different concentrations of ethanol. Some of these perifornical neurons contain angiotensin (Ang) and project directly to the dentate gyrus where angiotensin is released and inhibits LTP in medial perforant path-dentate granule cell synapses. The AT₁ subtype receptor is involved because pretreatment with losartan, an AT₁ antagonist, prevents Ang II, diazepam, and ethanol impairment of LTP as well as their effects on behavior. There is a possibility that these effects were not specific to the LHA; but might be attributable to direct effects of ethanol on postsynaptic granule cells due to diffusion of the ethanol in the extracellular space or by the circulatory system. The purpose of the present study was to determine a dose effect of ethanol on LTP in these same synapses when the dentate gyrus was perfused with several different concentrations of ethanol under the same conditions in urethane anesthetized rats. Ethanol was administered directly into the dentate gyrus by means of a fine stainless steel cannula attached approximately 1.0 mm from the tip of the glass capillary recording electrode. Results show that the threshold for ethanol in the dentate is higher by a factor of ten, >30 mM and <50mM; and that at higher doses ethanol can have a direct effect on the LHA; and possibly toxic due to increasing ethanol in the blood circulatory system.     

Ethanol effects on dentate granule cell LTP

In previous studies we identified a lateral hypothalamic area (LHA) sensitive to ethanol, < 5.0 mM, when the perifornical region of the area is perfused with different concentrations of ethanol. Some of these perifornical neurons contain angiotensin (Ang) and project directly to the dentate gyrus where angiotensin is released and inhibits LTP in medial perforant path-dentate granule cell synapses. The AT1 subtype receptor is involved because pretreatment with losartan, an AT1 antagonist, prevents Ang II, diazepam, and ethanol impairment of LTP as well as their effects on behavior. There is a possibility that these effects were not specific to the LHA; but might be attributable to direct effects of ethanol on postsynaptic granule cells due to diffusion of the ethanol in the extracellular space or by the circulatory system. The purpose of the present study was to determine a dose effect of ethanol on LTP in these same synapses when the dentate gyrus was perfused with several different concentrations of ethanol under the same conditions in urethane anesthetized rats. Ethanol was administered directly into the dentate gyrus by means of a fine stainless steel cannula attached approximately 1.0 mm from the tip of the glass capillary recording electrode. Results show that the threshold for ethanol in the dentate is higher by a factor of ten, > 30 mM and < 50 mM; and that at higher doses ethanol can have a direct effect on the LHA; and possibly toxic due to increasing ethanol in the blood circulatory system.     

Newly born dentate granule neurons after pilocarpine-induced epilepsy have hilar basal dendrites with immature synapses

Neurogenesis in the subgranular zone of the dentate gyrus persists throughout the lifespan of mammals, and the resulting newly born neurons are incorporated into existing hippocampal circuitry. Seizures increase the rate of neurogenesis in the adult rodent brain and result in granule cells in the dentate gyrus with basal dendrites. Using doublecortin (DCX) immunocytochemistry to label newly generated neurons the current study focuses on the electron microscopic features of DCX-labeled cell bodies and dendritic processes in the dentate gyrus of rats with pilocarpine-induced epilepsy. At the base of the granule cell layer clusters of cells that include up to six DCX-labeled cell bodies were observed. The cell bodies in these clusters lacked a one-to-one association with an astrocyte cell body and its processes, a relationship that is typical for newly born granule cells in control rats. Also, DCX-labeled basal dendrites in the hilus had immature synapses while those in control rats lacked synapses. These results indicate that increased neurogenesis after seizures alters the one-to-one relationship between astrocytes and DCX-labeled newly generated neurons at the base of the granule cell layer. The data also suggest that the synapses on DCX-labeled hilar basal dendrites contribute to the persistence of hilar basal dendrites on neurons born after pilocarpine-induced seizures.     

Mechanisms of colchicine neurotoxicity in the dentate gyrus: Dissociation of seizures and cell death

Extracellular field potentials and the EEG were studied in the dentate gyrus of the rat after intrahippocampal injections of colchicine, which is a relatively selective neurotoxin for dentate granule cells. Injection of colchicine (0.5 μl of a 5-mg/ml solution of colchicine in deionized water) resulted in granule cell hyperexcitability manifested by multispike field potentials in response to stimulation of the excitatory projections from the entorhinal cortex. In anesthetized rats, this state of granule cell hyperexcitability was occasionally accompanied by interictal epileptic spiking or brief electrographic seizures, but granule cell death was observed even in the absence of epileptic activity. Injection of colchicine into the CA1 area of the hippocampus also resulted in multispike field potentials in response to stimulation of the CA3 commissural pathway, but CA1 pyramidal cells were not destroyed by colchicine. Colchicine has been reported to act as a convulsant agent in the dentate gyrus, but it is a relatively selective neurotoxin for dentate granule cells even in the absence of epileptic activity.     

Inhibition of neuronal nitric oxide synthase increases adrenalectomy-induced granule cell death in the rat dentate gyrus

Recent studies have shown that the expression of neuronal nitric oxide synthase (NOS) mRNA is increased after adrenalectomy (ADX). However, the role of increased NO production after ADX in the dentate gyrus is unknown. In this study, the relationship between NO inhibition and apoptosis in the dentate gyrus after ADX was examined. 7-Nitroindazole (7-NI; 30 mg/kg, i.p.), a selective inhibitor of neuronal NOS, was injected 1 day before ADX and subsequently once every 24 h. Then 4 days after ADX, dentate granule cell death was evaluated using silver impregnation and Nissl staining methods. Inhibition of neuronal NOS by 7-NI increased the number of dying granule cells approximately 4-fold in the dentate gyrus of the ADX rats, compared to vehicle-injected ADX controls. These results suggest that increased NO production after ADX may play an endogenous neuroprotective role in the dentate gyrus.     

Lysophosphatidylcholine protects cerebellar granule neurons from apoptotic cell death

Cultured cerebellar granule neurons (CGNs) undergo apoptosis when deprived of depolarizing stimulation and provide an in vitro model system with which to study the effects of neurotrophic substances. Our previous results showed that secretory phospholipases A(2) (sPLA(2)s) protect CGNs from apoptotic cell death under the nondepolarizing condition. In this study, we further analyzed the mechanism whereby sPLA(2) exhibits this effect. Among the primary metabolites of sPLA(2) tested, lysophosphatidylcholine (LPC), but not other lysophospholipids, remarkably rescued CGNs from apoptosis. In contrast, neither arachidonic nor oleic acids displayed neurotrophic effect. Release of LPC into the culture media occurred in response to sPLA(2) treatment, and degradation or sequestration of LPC attenuated the survival-promoting effects of sPLA(2) and LPC. The neurotrophic effect of LPC required the presence of extracellular Ca(2+) and L-type Ca(2+) channel activity, suggesting that Ca(2+) influx across the plasma membrane is evoked by LPC. sPLA(2)- or LPC-induced promotion of CGN survival was suppressed by inhibitors of protein kinase A and phospholipase C, suggesting that they play a role in mediating survival-promoting signal of sPLA(2). The results presented here demonstrate a novel, unexpected neurotrophin-like effect of LPC in the central nervous system.     

Strain differences in seizure-induced cell death following pilocarpine-induced status epilepticus

Mouse strains differ from one another in their susceptibility to seizure-induced excitotoxic cell death. Previously, we have demonstrated that mature inbred strains of mice show remarkable genetic differences in susceptibility to the neuropathological consequences of seizures in the kainate model of status epilepticus. At present, while the cellular mechanisms underlying strain-dependent differences in susceptibility remain unclear, some of this variation is assumed to have a genetic basis. However, it remains unclear whether strain differences in susceptibility to seizure-induced cell death observed following kainate administration are observed following systemic administration of other chemoconvulsants. In rodents, the cholinomimetic convulsant pilocarpine is widely used to induce status epilepticus (SE), followed by hippocampal damage and spontaneous recurrent seizures, resembling temporal lobe epilepsy. This model has initially been described in rats, but is increasingly used in mice. We characterized neuronal pathologies after pilocarpine-induced status epilepticus (SE) in eight inbred strains of mice focusing on the hippocampus. A ramping-up dose protocol for pilocarpine was used and behavior was monitored for 4-5 h. While we did not observe any significant differences in seizure latency or duration to pilocarpine among the inbred strains, we did observe a significant difference in susceptibility to the neuropathological consequences of pilocarpine-induced SE. Of the eight genetically diverse mouse strains screened for pilocarpine-induced status, BALB/cJ and BALB/cByJ were the only two strains that were resistant to the neuropathological consequences of seizure-induced cell death. Additional studies of these murine strains may be useful for investigating genetic influences on pilocarpine-induced status epilepticus.     

Patterns of dentate granule cell responses to perforant path stimulation in epileptic mice with granule cell dispersion

In adult mice, intrahippocampal administration of kainic acid induces a structural modification of the granule cell layer reminiscent of granule cell dispersion (GCD) seen in humans with temporal lobe epilepsy. We tested that GCD might be involved in the patterns of granule cell responses to perforant path stimulation by recording field potentials in vivo after kainic acid-induced status epilepticus until the phase of chronic seizure activity in presence of GCD or after its alteration by K252a co-treatment, an inhibitor of tyrosine kinase activities. Stimulation triggered bursts of multiple population spikes, the number of which progressively increased with time whereas their amplitude decreased in parallel with the progressive decrease in granule cell density. The population spike threshold was reached for a lower excitatory synaptic drive than in controls, as assessed by the initial slope of the field excitatory post-synaptic potential. This indicates that, for identical synaptic responses, granule cells were closer to the firing threshold. Fast inhibition, assessed by paired pulse stimulation, was compromised immediately after the initial status epilepticus, consistent with the rapid loss of most hilar cells. Neither the epileptic course nor the epileptiform responses of the granule cells were modified and manipulation by alteration following GCD manipulation while granule cell neuropeptide-Y immunostaining was substantially decreased. In this mouse model of TLE, granule cells display a progressive increase in epileptiform responses to afferent input until the occurrence of spontaneous seizures. The population spike amplitude decreases in parallel with GCD while the granule cell excitability is enhanced. Consequently, data from field potentials in epilepsy experiments should be interpreted with care, taking into account the possible variations in the neuronal density in the recorded area.     

Long-term increases in dentate granule cell responsivity accompany operant conditioning

The efficacy of synaptic transmission from the perforant path (PP) to the granule cells in the dentate gyrus (DG) of freely moving rats was monitored electrophysiologically over the course of training in an appetitively motivated, discriminated operant paradigm. Every day, 22 hr after behavioral sessions, evoked potentials were recorded from the DG following stimulation of the PP over range of current intensities and the amplitudes of the population spikes were measured. Behavioral conditions involved training in an operant conditioning paradigm or a session of free-feeding. Significant increases in population spike amplitudes were observed over the 8 d of training, but not over the 8 d of free-feeding. This training-induced increase in granule cell responsivity persisted for at least 10 d following the cessation of behavioral trials and was in many ways comparable to long-term potentiation (LTP), subsequently observed in these same rats 24 hr after tetanic stimulation. These data confirm and extend previous reports of synaptic enhancements following conditioning and suggest that such increases in synaptic efficacy may encode some aspect of learning.     

Stimulation of substantia nigra pars reticulata enhances dentate granule cell excitability

We studied the effects of electrical and chemical stimulation of the substantia nigra (SN) on the dentate granule cell (DGC) response to perforant path stimulation. Studies were carried out in both pentobarbital anesthetized and awake freely moving rats. Chemical stimulation was achieved by microinjection of N-methyl-D,L-aspartate (NMDA), an excitatory amino acid. Electrical preconditioning stimulation applied to the area of the SN, but not dorsal to SN, increased the excitability of DGCs. Intracerebral microinjection of NMDA into the contralateral SN pars reticulata (SNR), but not into SN pars compacta or cerebral peduncle, also produced a marked and reversible enhancement of DGC excitability. In both instances, the increased DGC excitability consisted of increased amplitude of the perforant path evoked population spike without change in the slope of the population excitatory postsynaptic potential (pEPSP). We interpret the data to indicate that increasing the output of the SNR increases the excitability of the DGCs, a limbic neuronal population regulating information transfer through hippocampal pathways. Together with our previous finding that decreasing the output of SNR suppressed limbic seizures, these data demonstrate that the SNR exerts a powerful influence on limbic system excitability. Delineating the anatomic pathway mediating this influence could provide valuable insight into the mechanisms underlying basal ganglia-limbic interactions in both physiologic and pathologic conditions.     

Induction of immediate-early gene proteins in dentate granule cells and somatostatin interneurons after hippocampal seizures.

The expression of the protein products of the immediate-early genes c-fos, Fos B, Fos-related proteins (FRAs), c-jun, jun B, jun D and krox-24 was investigated in the rat hippocampus at various times after electrically-induced hippocampal seizures. Hippocampal seizures induced all the immediate-early gene proteins in dentate granule cells with differing time-courses. In addition, Krox-24, Fos and Jun D were also induced in somatostatin-containing interneurons throughout the hippocampus and also in a small percentage of parvalbumin-containing interneurons. Thus, hippocampal seizures induce waves of immediate-early gene protein expression in dentate granule cells and a selective expression of krox-24, Fos and Jun D in hippocampal somatostatin interneurons. These results suggest that biochemical and/or morphological changes occurring in dentate granule cells and somatostatin interneurons after seizures may be regulated by immediate-early gene expression, and that these immediate-early gene proteins may be involved in seizure development in the nervous system.     

Effectiveness of muscimol-containing microparticles against pilocarpine-induced focal seizures

PURPOSE: To investigate the efficacy of in situ lipid-protein-sugar particles (LPSPs) in mitigating the epileptogenic and histologic effects of intrahippocampal pilocarpine in rats. METHODS: LPSPs with and without muscimol were produced by spray-drying, sized by Coulter counter, and muscimol content determined by high-pressure liquid chromatography (HLPC). Particles, free muscimol or saline, were injected into the hippocampi of Sprague-Dawley rats before 40 mM pilocarpine, and seizure activity was scored. The trajectories of behavioral scores between groups were compared with two-way repeated measures analysis of variance. Animals were killed after 2 weeks. Brain sections were stained (Timm and thionin) and scored. RESULTS: LPSPs were 4 to 5 microm in diameter, and contained 0 or 2% (wt/wt) muscimol. In vitro, muscimol was released over a 5-day period. Intrahippocampal injections of normal saline and blank LPSPs did not deter seizure activity from pilocarpine. The rise of the trajectory in behavior scores in animals given LPSPs containing 5 microg muscimol was significantly slower than in those receiving saline, blank particles, or 5 microg of unencapsulated muscimol. There was less apparent neuronal injury and CA3 and supragranular mossy fiber sprouting in hippocampi of animals receiving muscimol-containing particles than in animals that did not receive muscimol. Hippocampi of animals that received 5 microg of encapsulated muscimol showed less supragranular sprouting than did those receiving 5 microg of unencapsulated muscimol, but showed no difference in cell loss or CA3 sprouting. CONCLUSIONS: Focally delivered biodegradable microparticles loaded with muscimol are effective in reducing seizure activity from pilocarpine in animals and mitigate the histologic effects.