Increased excitatory synaptic input to granule cells from hilar and CA3 regions in a rat model of temporal lobe epilepsy

One potential mechanism of temporal lobe epilepsy is recurrent excitation of dentate granule cells through aberrant sprouting of their axons (mossy fibers), which is found in many patients and animal models. However, correlations between the extent of mossy fiber sprouting and seizure frequency are weak. Additional potential sources of granule cell recurrent excitation that would not have been detected by markers of mossy fiber sprouting in previous studies include surviving mossy cells and proximal CA3 pyramidal cells. To test those possibilities in hippocampal slices from epileptic pilocarpine-treated rats, laser-scanning glutamate uncaging was used to randomly and focally activate neurons in the granule cell layer, hilus, and proximal CA3 pyramidal cell layer while measuring evoked EPSCs in normotopic granule cells. Consistent with mossy fiber sprouting, a higher proportion of glutamate-uncaging spots in the granule cell layer evoked EPSCs in epileptic rats compared with controls. In addition, stimulation spots in the hilus and proximal CA3 pyramidal cell layer were more likely to evoke EPSCs in epileptic rats, despite significant neuron loss in those regions. Furthermore, synaptic strength of recurrent excitatory inputs to granule cells from CA3 pyramidal cells and other granule cells was increased in epileptic rats. These findings reveal substantial levels of excessive, recurrent, excitatory synaptic input to granule cells from neurons in the hilus and proximal CA3 field. The aberrant development of these additional positive-feedback circuits might contribute to epileptogenesis in temporal lobe epilepsy.     

Galanin expressed in the excitatory fibers attenuates synaptic strength and generalized seizures in the piriform cortex of mice

The neuropeptide galanin is considered to be an endogenous antiepileptic agent, presumably acting via inhibition of glutamate release. Previously, we have demonstrated that in mice ectopically overexpressing galanin in cortical and hippocampal neurons, particularly in granule cells and their axons, the mossy fibers, hippocampal kindling epileptogenesis is suppressed and is associated with attenuated frequency facilitation in mossy fiber-CA3 cell synapses. We hypothesized that changes in synaptic transmission might occur also in other excitatory synapses of the galanin overexpressing (GalOE) mouse, contributing to seizure suppression. Lateral olfactory tract (LOT) synapses, formed by axons of olfactory bulb (OB) mitral cells and targeting piriform cortex (PC) pyramidal cells, ectopically express galanin in GalOE mice. Using whole-cell patch-clamp recordings, we found that excitatory synaptic responses recorded in PC pyramidal cells during high frequency stimulation of the LOT were attenuated in GalOE mice as compared to wild-type controls. This effect was mimicked by bath application of galanin or its agonist galnon to wild-type slices, supporting the notion of ectopic galanin action. Since the high frequency activation induced in vitro resembles epileptic seizures in vivo, we asked whether the observed synaptic inhibition would result in altered epileptogenesis when animals were kindled via the same synapses. In male GalOE mice, we found that the latency to convulsions was prolonged, and once animals had experienced the first stage 5 seizure, generalized seizures were less sustainable. These data indicate that the PC is a possible target for epilepsy treatment by ectopically overexpressing galanin to modulate seizure activity.     

Mossy fiber sprouting and pyramidal cell dispersion in the hippocampal CA2 region in a mouse model of temporal lobe epilepsy

Dentate granule cells and the hippocampal CA2 region are resistant to cell loss associated with mesial temporal lobe epilepsy (MTLE). It is known that granule cells undergo mossy fiber sprouting in the dentate gyrus which contributes to a recurrent, proepileptogenic circuitry in the hippocampus. Here it is shown that mossy fiber sprouting also targets CA2 pyramidal cell somata and that the CA2 region undergoes prominent structural reorganization under epileptic conditions. Using the intrahippocampal kainate mouse model for MTLE and the CA2‐specific markers Purkinje cell protein 4 (PCP4) and regulator of G‐Protein signaling 14 (RGS14), it was found that during epileptogenesis CA2 neurons survive and disperse in direction of CA3 and CA1 resulting in a significantly elongated CA2 region. Using transgenic mice that express enhanced green fluorescent protein (eGFP) in granule cells and mossy fibers, we show that the recently described mossy fiber projection to CA2 undergoes sprouting resulting in aberrant large, synaptoporin‐expressing mossy fiber boutons which surround the CA2 pyramidal cell somata. This opens up the potential for altered synaptic transmission that might contribute to epileptic activity in CA2. Indeed, intrahippocampal recordings in freely moving mice revealed that epileptic activity occurs concomitantly in the dentate gyrus and in CA2. Altogether, the results call attention to CA2 as a region affected by MTLE‐associated pathological restructuring. © 2015 Wiley Periodicals, Inc.     

Structural plasticity of dentate granule cell mossy fibers during the development of limbic epilepsy

Altered granule cell≫CA3 pyramidal cell synaptic connectivity may contribute to the development of limbic epilepsy. To explore this possibility, granule cell giant mossy fiber bouton plasticity was examined in the kindling and pilocarpine models of epilepsy using green fluorescent protein‐expressing transgenic mice. These studies revealed significant increases in the frequency of giant boutons with satellite boutons 2 days and 1 month after pilocarpine status epilepticus, and increases in giant bouton area at 1 month. Similar increases in giant bouton area were observed shortly after kindling. Finally, both models exhibited plasticity of mossy fiber giant bouton filopodia, which contact GABAergic interneurons mediating feedforward inhibition of CA3 pyramids. In the kindling model, however, all changes were fleeting, having resolved by 1 month after the last evoked seizure. Together, these findings demonstrate striking structural plasticity of granule cell mossy fiber synaptic terminal structure in two distinct models of adult limbic epileptogenesis. We suggest that these plasticities modify local connectivities between individual mossy fiber terminals and their targets, inhibitory interneurons, and CA3 pyramidal cells potentially altering the balance of excitation and inhibition during the development of epilepsy. © 2009 Wiley‐Liss, Inc.     

Increased densities of AMPA GluR1 subunit proteins and presynaptic mossy fiber sprouting in the fascia dentata of human hippocampal epilepsy

In human hippocampal epilepsy, there is a consistent pathology of cell loss and reactive synaptic reorganization of ‘excitatory' mossy fibers (MF) into the inner molecular layer (IML) of the fascia dentata (FD). In this study, neo-Timm's histochemistry of MFs and immunocytochemistry of GluR1 were used to determine, in patients with or without hippocampal sclerosis (HS), if there was a correlation between aberrant supragranular (IML) mossy fiber sprouting and increased densities of AMPA GluR1 subunit proteins in the IML of the FD. Computerized quantified densitometric grey values of Timm and GluR1 densities were corrected for the densities of granule cell losses using cell counts. In the IML of the HS group, despite the losses of granule cells, mossy fiber sprouting was significantly greater (P<0.000001) and GluR1 protein densities were significantly higher (P<0.0005) than those of the non-HS group. Unlike supragranular mossy fiber sprouting, which was limited to the IML, the increased GluR1 stainings were distributed throughout the whole molecular layer. For all cases, MF synaptic reorganization in the supragranular ML was correlated with GluR1 subunit protein densities in the IML (R=0.784, P<0.0093). These data demonstrate that in the human epileptic fascia dentata, there are significantly increased AMPA GluR1 subunit proteins associated with aberrant MF synaptic reorganizations. This suggests that the hyperexcitability of sclerotic hippocampus occurs, at least in part, from the associated changes of both presynaptic mossy fiber glutamatergic neoinnervation and increased GluR1 subunit proteins in the dendritic domains of the FD.     

Mossy fibers are the primary source of afferent input to ectopic granule cells that are born after pilocarpine-induced seizures

Granule cell (GC) neurogenesis increases following seizures, and some newborn GCs develop in abnormal locations within the hilus. These ectopic GCs (EGCs) display robust spontaneous and evoked excitatory activity. However, the pattern of afferent input they receive has not been fully defined. This study used electron microscopic immunolabeling to quantitatively evaluate mossy fiber (MF) input to EGCs since MFs densely innervate the hilus normally and undergo sprouting in many animal models of epilepsy. EGC dendrites were examined in tissue from epileptic rats that had initially been treated with pilocarpine to induce status epilepticus and subsequently had spontaneous seizures. MF terminals were labeled with a zinc transporter-3 antibody, and calbindin immunoreactivity was used to label hilar EGCs and GC layer GCs. The pattern of input provided by sprouted MF terminals to EGC dendrites was then compared to the pattern of MF input to GC dendrites in the inner molecular layer (IML), where most sprouted fibers are thought to project. Analysis of EGC dendrites demonstrated that MF terminals represented their predominant source of afferent input: they comprised 63% of all terminals and, on average, occupied 40% and 29% of the dendritic surface in the dorsal and ventral dentate gyrus, respectively, forming frequent synapses. These measures of connectivity were significantly greater than comparable values for MF innervation of GC dendrites located in the IML of the same tissue sections. Thus, EGCs develop a pattern of synaptic connections that could help explain their previously identified predisposition to discharge in epileptiform bursts and suggest that they play an important role in the generation of seizure activity in the dentate gyrus.     

Initial loss but later excess of GABAergic synapses with dentate granule cells in a rat model of temporal lobe epilepsy

Many patients with temporal lobe epilepsy display neuron loss in the dentate gyrus. One potential epileptogenic mechanism is loss of GABAergic interneurons and inhibitory synapses with granule cells. Stereological techniques were used to estimate numbers of gephyrin‐positive punctae in the dentate gyrus, which were reduced short‐term (5 days after pilocarpine‐induced status epilepticus) but later rebounded beyond controls in epileptic rats. Stereological techniques were used to estimate numbers of synapses in electron micrographs of serial sections processed for postembedding GABA‐immunoreactivity. Adjacent sections were used to estimate numbers of granule cells and glutamic acid decarboxylase‐positive neurons per dentate gyrus. GABAergic neurons were reduced to 70% of control levels short‐term, where they remained in epileptic rats. Integrating synapse and cell counts yielded average numbers of GABAergic synapses per granule cell, which decreased short‐term and rebounded in epileptic animals beyond control levels. Axo‐shaft and axo‐spinous GABAergic synapse numbers in the outer molecular layer changed most. These findings suggest interneuron loss initially reduces numbers of GABAergic synapses with granule cells, but later, synaptogenesis by surviving interneurons overshoots control levels. In contrast, the average number of excitatory synapses per granule cell decreased short‐term but recovered only toward control levels, although in epileptic rats excitatory synapses in the inner molecular layer were larger than in controls. These findings reveal a relative excess of GABAergic synapses and suggest that reports of reduced functional inhibitory synaptic input to granule cells in epilepsy might be attributable not to fewer but instead to abundant but dysfunctional GABAergic synapses. J. Comp. Neurol. 518:647–667, 2010. © 2009 Wiley‐Liss, Inc.     

Seizure frequency correlates with loss of dentate gyrus GABAergic neurons in a mouse model of temporal lobe epilepsy

Epilepsy occurs in one of 26 people. Temporal lobe epilepsy is common and can be difficult to treat effectively. It can develop after brain injuries that damage the hippocampus. Multiple pathophysiological mechanisms involving the hippocampal dentate gyrus have been proposed. This study evaluated a mouse model of temporal lobe epilepsy to test which pathological changes in the dentate gyrus correlate with seizure frequency and help prioritize potential mechanisms for further study. FVB mice (n = 127) that had experienced status epilepticus after systemic treatment with pilocarpine 31–61 days earlier were video‐monitored for spontaneous, convulsive seizures 9 hr/day every day for 24–36 days. Over 4,060 seizures were observed. Seizure frequency ranged from an average of one every 3.6 days to one every 2.1 hr. Hippocampal sections were processed for Nissl stain, Prox1‐immunocytochemistry, GluR2‐immunocytochemistry, Timm stain, glial fibrillary acidic protein‐immunocytochemistry, glutamic acid decarboxylase in situ hybridization, and parvalbumin‐immunocytochemistry. Stereological methods were used to measure hilar ectopic granule cells, mossy cells, mossy fiber sprouting, astrogliosis, and GABAergic interneurons. Seizure frequency was not significantly correlated with the generation of hilar ectopic granule cells, the number of mossy cells, the extent of mossy fiber sprouting, the extent of astrogliosis, or the number of GABAergic interneurons in the molecular layer or hilus. Seizure frequency significantly correlated with the loss of GABAergic interneurons in or adjacent to the granule cell layer, but not with the loss of parvalbumin‐positive interneurons. These findings prioritize the loss of granule cell layer interneurons for further testing as a potential cause of temporal lobe epilepsy.     

Axon arbors and synaptic connections of hippocampal mossy cells in the rat in vivo

The axon collateralization patterns and synaptic connections of intracellularly labeled and electrophysiologically identified mossy cells were studied in rat hippocampus. Light microscopic analysis of 11 biocytin-filled cells showed that mossy cell axon arbors extended through an average of 57% of the total septotemporal length of the hippocampus (summated two-dimensional length, not adjusted for tissue shrinkage). Axon collaterals were densest in distant lamellae rather than in lamellae near the soma. Most of the axon was concentrated in the inner one-third of the molecular layer, with the hilus containing an average of only 26% of total axon length and the granule cell layer containing an average of only 7%. Ultrastructural analysis was carried out on three additional intracellularly stained mossy cells, in which axon collaterals and synaptic targets were examined in serial sections of chosen axon segments. In the central and subgranular regions of the hilus, mossy cell axons established a low density of synaptic contacts onto dendritic shafts, neuronal somata, and occasional dendritic spines. Most hilar synapses were made relatively close to the mossy cell somata. At greater distances from the labeled mossy cell (1–2 mm along the septotemporal axis), the axon collaterals ramified predominantly within the inner molecular layer and made a high density of asymmetric synaptic contacts almost exclusively onto dendritic spines. Quantitative measurements indicated that more than 90% of mossy cell synaptic contacts in the ipsilateral hippocampus are onto spines of proximal dendrites of presumed granule cells. These results are consistent with a primary mossy cell role in an excitatory associational network with granule cells of the dentate gyrus. © 1996 Wiley-Liss, Inc.     

Stereologic estimation of hippocampal GluR2/3- and calretinin-immunoreactive hilar neurons (presumptive mossy cells) in two mouse models of temporal lobe epilepsy

Purpose: Hippocampal mossy cells receive dense innervation from dentate granule cells and, in turn, mossy cells innervate both granule cells and interneurons. Mossy cell loss is thought to trigger granule cell mossy fiber sprouting, which may affect granule cell excitability. The aim of this study was to quantify mossy cell loss in two animal models of temporal lobe epilepsy, and determine whether there exists a relationship between mossy cell loss, mossy fiber sprouting, and granule cell dispersion. Methods: Representative hippocampal sections from p35 knockout mice and mice with unilateral intrahippocampal kainate injection were immunolabeled for GluR2/3, two subunits of the amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor and calretinin to identify mossy cells. Mossy fibers were immunostained against synaptoporin. Key Findings: p35 Knockout mice showed no hilar cell death, but moderate mossy fiber sprouting and granule cell dispersion. In the kainate‐injected hippocampus, there was an 80% and 85% reduction of GluR2/3‐ and GluR2/3/calretinin‐positive hilar neurons, respectively, and dense mossy fiber sprouting and significant granule cell dispersion. In the contralateral hippocampus there was a 52% loss of GluR2/3‐, but only a 20% loss of GluR2/3‐calretinin‐immunoreactive presumptive mossy cells, and granule cell dispersion; no mossy fiber sprouting was observed. Significance: These results indicate a probable lack of causality between mossy cell death and mossy fiber sprouting.     

癫痫大鼠海马出芽苔藓纤维突触的超微结构特征

目的:探讨匹罗卡品颞叶癫痫大鼠海马出芽苔藓纤维突触的超微结构特征及其在颞叶癫痫发病机制中的作用。方法:采用Timm组化染色标记出芽苔藓纤维突触末端,在电镜下观察新生突触的类型、比例、定位、以及突触后靶成分。结果:颞叶癫痫大鼠齿状回内分子层可见到银标记的突触末端,出芽苔藓纤维突触主要是轴棘型非对称性突触,其次是轴树型非对称性突触,偶可看到出芽轴突和颗粒细胞体形成突触联系。结论:轴棘型非对称性突触是颞叶癫痫大鼠海马出芽苔藓纤维突触的主要类型,出芽苔藓纤维突触的超微结构特性支持重组突触形成重复的兴奋性环路,而且形成的新的兴奋性环路可能在颞叶癫痫的发生与发展中起重要作用。     

Functional role of mGluR1 and mGluR4 in pilocarpine-induced temporal lobe epilepsy

Altered expression and distribution of neurotransmitter receptors, including metabotropic glutamate receptors (mGluRs), constitute key aspects in epileptogenesis, impaired hippocampal excitability and neuronal degeneration. mGluR1 mediates predominantly excitatory effects, whereas mGluR4 acts as inhibitory presynaptic receptor. Increased hippocampal expression of mGluR1 and mGluR4 has been observed in human temporal lobe epilepsy (TLE). In this study, we address whether genetic mGluR1 upregulation and mGluR4 knock-down influence seizure susceptibility and/or vulnerability of hippocampal neurons by analyzing transgenic animals in the pilocarpine TLE model. Therefore, we generated transgenic mice expressing mGluR1-enhanced green fluorescent protein (EGFP) fusion protein under control of the human cytomegalovirus (CMV) immediate early promoter. Status epilepticus (SE) was induced in (a) mice overexpressing mGluR1-EGFP and (b) mice deficient for mGluR4 (mGluR4 KO) as well as littermate controls. In the acute epileptic stage after pilocarpine application, mGluR4 KO mice showed a significant increase of severe seizure activity, in contrast to mGluR1 transgenics. Analysis of both transgenic mouse lines in the chronic epileptic phase, using a telemetric EEG-/video-monitoring system, revealed a significant increase in seizure frequency only in mGluR1-EGFP mice. In contrast, enhanced neuronal cell loss was only present in the hippocampus of epileptic mGluR4 KO mice. Our results suggest a role for mGluR1 in promoting seizure susceptibility as well as for mGluR4 to counteract excitatory activity and seizure-associated vulnerability of hippocampal neurons. Therefore, our data strongly recommend both mGluRs as potential drug targets to interfere with the development of hippocampal damage and seizure activity in TLE.     

Glutamate transporters alterations in the reorganizing dentate gyrus are associated with progressive seizure activity in chronic epileptic rats.

The expression of glial and neuronal glutamate transporter proteins was investigated in the hippocampal region at different time points after electrically induced status epilepticus (SE) in the rat. This experimental rat model for mesial temporal lobe epilepsy is characterized by cell loss, gliosis, synaptic reorganization, and chronic seizures after a latent period. Despite extensive gliosis, immunocytochemistry revealed only an up-regulation of both glial transporters localized at the outer aspect of the inner molecular layer (iml) in chronic epileptic rats. The neuronal EAAC1 transporter was increased in many somata of individual CA1-3 neurons and granule cells that had survived after SE; this up-regulation was still present in the chronic epileptic phase. In contrast, a permanent decrease of EAAC1 immunoreactivity was observed in the iml of the dentate gyrus. This permanent decrease in EAAC1 expression, which was only observed in rats that experienced progressive spontaneous seizure activity, could lead to abnormal glutamate levels in the iml once new abnormal glutamatergic synaptic contacts are formed by means of sprouted mossy fibers. Considering the steady growth of reorganizing mossy fibers in the iml, the absence of a glutamate reuptake mechanism in this region could contribute to progression of spontaneous seizure activity, which occurs with a similar time course.     

Mossy cells in the mouse dentate gyrus: identification in the dorsal hilus and their distribution along the dorsoventral axis

Previously we showed that large multipolar cells immunoreactive for calretinin and subunits 2 and 3 of amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptors (GluR2/3) clustered in the ventral hilus of the mouse dentate gyrus and revealed that they were mossy cells. Although such large calretinin immunoreactive cells were not seen in the dorsal hilus, our Golgi study revealed the presence of mossy cells in the dorsal hilus. As we observed large intensely GluR2/3 immunoreactive cells in the dorsal hilus, we suggested that these calretinin negative but intensely GluR2/3 positive large cells in the dorsal hilus were also mossy cells. In the present study we confirmed this identification with several methods. The extracellular tracer labeling studies revealed that all of 47 mossy cells identified morphologically were intensely GluR2/3 positive but calretinin negative, whereas none of 22 non-mossy hilar neurons were intensely GluR2/3 positive. Electron microscopically most of intensely GluR2/3 positive somata and dendritic processes showed the characteristic ultrastructural features of mossy cells. Furthermore, the fimbria–fornix–hippocampal commissure transection procedures induced the calretinin expression in some of these dorsal GluR2/3 immunoreactive cells. On the basis of these observations, we concluded that the vast majority of intensely GluR2/3 immunoreactive large cells in the mouse dorsal hilus were mossy cells. Then we evaluated the presumed difference in the distribution of mossy cells along the dorsoventral axis by the disector. The numerical density of mossy cells was about 1.4 times larger at the ventral level than at the dorsal level, indicating that the dorsoventral difference in the distribution of mossy cells in the mouse hilus was far smaller than that previously speculated.     

Hippocampal injury,atrophy, synaptic reorganization,and epileptogenesis after perforant pathway stimulation‐induced status epilepticus in the mouse

Prolonged dentate granule cell discharges produce hippocampal injury and chronic epilepsy in rats. In preparing to study this epileptogenic process in genetically altered mice, we determined whether the background strain used to generate most genetically altered mice, the C57BL/6 mouse, is vulnerable to stimulation‐induced seizure‐induced injury. This was necessary because C57BL/6 mice are reportedly resistant to the neurotoxic effects of kainate‐induced seizures, which we hypothesized to be related to strain differences in kainate's effects, rather than genetic differences in intrinsic neuronal vulnerability. Bilateral perforant pathway stimulation‐induced granule cell discharge for 4 hours under urethane anesthesia produced degeneration of glutamate receptor subunit 2 (GluR2)‐positive hilar mossy cells and peptide‐containing interneurons in both FVB/N (kainate‐vulnerable) and C57BL/6 (kainate‐resistant) mice, indicating no strain differences in neuronal vulnerability to seizure activity. Granule cell discharge for 2 hours in C57BL/6 mice destroyed most GluR2‐positive dentate hilar mossy cells, but not peptide‐containing hilar interneurons, indicating that mossy cells are the neurons most vulnerable to this insult. Stimulation for 24 hours caused extensive hippocampal neuron loss and injury to the septum and entorhinal cortex, but no other detectable damage. Mice stimulated for 24 hours developed hippocampal sclerosis, granule cell mossy fiber sprouting, and chronic epilepsy, but not the granule cell layer hypertrophy (granule cell dispersion) produced by intrahippocampal kainate. These results demonstrate that perforant pathway stimulation in mice reliably reproduces the defining features of human mesial temporal lobe epilepsy with hippocampal sclerosis. Experimental studies in transgenic or knockout mice are feasible if electrical stimulation is used to produce controlled epileptogenic insults. J. Comp. Neurol. 515:181–196, 2009. © 2009 Wiley‐Liss, Inc.     

Hippocampal AMPA and NMDA mRNA levels correlate with aberrant fascia dentata mossy fiber sprouting in the pilocarpine model of spontaneous limbic epilepsy

There is considerable controversy whether aberrant fascia dentata (FD) mossy fiber sprouting is an epiphenomena related to neuronal loss or a pathologic abnormality responsible for spontaneous limbic seizures. If mossy fiber sprouting contributes to seizures, then reorganized axon circuits should alter postsynaptic glutamate receptor properties. In the pilocarpine-status rat model, this study determined if changes in alpha amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and n-methyl-D-aspartic acid (NMDA) receptor subunit mRNA levels correlated with mossy fiber sprouting. Sprague-Dawley rats were injected with pilocarpine (320 mg/kg; i.p.) and maintained in status epilepticus for 6 to 8 hours (pilocarpine-status). Rats were killed during the: (1) latent phase after neuronal loss but before spontaneous limbic seizures (day 11 poststatus; n = 7); (2) early seizure phase after their first seizures (day 25; n = 7); and (3) chronic seizure phase after many seizures (day 85; n = 9). Hippocampi were studied for neuron counts, inner molecular layer (IML) neo-Timm's staining, and GluR1–3 and NMDAR1–2b mRNA levels. Compared with controls, pilocarpine-status rats in the: (1) latent phase showed increased FD GluR3, NMDAR1, and NMDAR2b; greater CA4 and CA1 NMDAR1; and decreased subiculum GluR1 hybridization densities; (2) early seizure phase showed increased FD GluR3, increased CA1 NMDAR1, and decreased subiculum NMDAR2b densities; and (3) chronic seizure phase showed increased FD GluR2; increased FD and CA4 GluR3; decreased CA1 GluR2; and decreased subiculum GluR1, GluR2, NMDAR1, and NMDAR2b levels. In multivariate analyses, greater IML neo-Timm's staining: (1) positively correlated with FD GluR3 and NMDAR1 and (2) negatively correlated with CA1 and subiculum GluR1 and GluR2 mRNA levels. These results indicate that: (1) hippocampal AMPA and NMDA receptor subunit mRNA levels changed as rats progressed from the latent to chronic seizure phase and (2) certain subunit alterations correlated with mossy fiber sprouting. Our findings support the hypothesis that aberrant axon circuitry alters postsynaptic hippocampal glutamate receptor subunit stoichiometry; this may contribute to limbic epileptogenesis. J. Neurosci. Res. 54:734–753, 1998. © 1998 Wiley-Liss, Inc.     

Alteration of NMDA receptor–mediated synaptic interactions in the lateral amygdala associated with seizure activity in a mouse model of chronic temporal lobe epilepsy

Purpose: Because results from both animal models and human temporal lobe epilepsy (TLE) have pointed to synaptic network alterations in the amygdala, we have tested the hypothesis that glutamatergic transmission in the lateral amygdala (LA) is critically involved. Methods: Using the pilocarpine mouse model, LA slices were prepared ex vivo in the recurrent phase of TLE (Pilo group), and LA projection neurons (PNs) were recorded using patch‐clamp techniques. Intrinsic and synaptic properties of LA PNs were analyzed and compared with those in age‐matched saline‐injected controls. Results: Only mild changes were observed in intrinsic properties of LA PNs, whereas both spontaneous excitatory postsynaptic currents (sEPSCs) and miniature EPSCs (mEPSCs) were significantly increased in Pilo as compared to saline controls. This difference was sensitive to AP5, but persisted during action of NBQX, indicating mediation by N‐methyl‐d ‐aspartate (NMDA) receptors. Moreover, these changes were associated with an increase in frequency but not amplitude of mEPSCs, indicative of a contribution of presynaptic mechanisms. Discussion: In conclusion, dynamic changes seem to occur in glutamatergic transmission within the amygdala during TLE, to which a functional upregulation of presynaptic NMDA receptors in LA PNs makes a significant contribution.     

Rapamycin suppresses axon sprouting by somatostatin interneurons in a mouse model of temporal lobe epilepsy

Purpose: In temporal lobe epilepsy many somatostatin interneurons in the dentate gyrus die. However, some survive and sprout axon collaterals that form new synapses with granule cells. The functional consequences of γ‐aminobutyric acid (GABA)ergic synaptic reorganization are unclear. Development of new methods to suppress epilepsy‐related interneuron axon sprouting might be useful experimentally. Methods: Status epilepticus was induced by systemic pilocarpine treatment in green fluorescent protein (GFP)‐expressing inhibitory nerurons (GIN) mice in which a subset of somatostatin interneurons expresses GFP. Beginning 24 h later, mice were treated with vehicle or rapamycin (3 mg/kg intraperitoneally) every day for 2 months. Stereologic methods were then used to estimate numbers of GFP‐positive hilar neurons per dentate gyrus and total length of GFP‐positive axon in the molecular layer plus granule cell layer. GFP‐positive axon density was calculated. The number of GFP‐positive axon crossings of the granule cell layer was measured. Regression analyses were performed to test for correlations between GFP‐positive axon length versus number of granule cells and dentate gyrus volume. Key Findings: After pilocarpine‐induced status epilepticus, rapamycin‐ and vehicle‐treated mice had approximately half as many GFP‐positive hilar neurons as did control animals. Despite neuron loss, vehicle‐treated mice had over twice the GFP‐positive axon length per dentate gyrus as controls, consistent with GABAergic axon sprouting. In contrast, total GFP‐positive axon length was similar in rapamycin‐treated mice and controls. GFP‐positive axon length correlated most closely with dentate gyrus volume. Significance: These findings suggest that rapamycin suppressed axon sprouting by surviving somatostatin/GFP‐positive interneurons after pilocarpine‐induced status epilepticus in GIN mice. It is unclear whether the effect of rapamycin on axon length was on interneurons directly or secondary, for example, by suppressing growth of granule cell dendrites, which are synaptic targets of interneuron axons. The mammalian target of rapamycin (mTOR) signaling pathway might be a useful drug target for influencing GABAergic synaptic reorganization after epileptogenic treatments, but additional side effects of rapamycin treatment must be considered carefully.     

Loss of input from the mossy cells blocks maturation of newly generated granule cells

The objective of this work is to check whether the input from the mossy cells to the inner molecular layer is necessary for the integration and maturation of the newly generated granule cells of the dentate gyrus (DG) in mice, and if after status epilepticus the sprouting of the mossy fibers can substitute for this projection. Newly generated cells were labeled by administration of 5-bromo-deoxyuridine either before or after pilocarpine administration. The neuronal loss in the hippocampus after administration of pilocarpine combined with scopolamine and diazepam seemed restricted to the hilar mossy cells. The maturation of the granule cells was studied using immunohistochemistry for calretinin and NeuN in combination with detection of 5-bromo-deoxyuridine. The sprouting of the mossy fibers was detected using Timm staining for zinc-rich boutons. In normal conditions, granule cells took about 2 weeks to lose the immature marker calretinin. After the loss of the mossy cells, newly generated granule cells remained expressing calretinin for more than a month, until the sprouting of the mossy fibers substituted for the projection of the mossy cells in the inner molecular layer of the DG. Therefore, a proper pattern of connectivity is necessary for the normal development and integration of newly generated granule cells in the adult brain. In a changed environment they cannot adapt transforming in other cell types; simply they are unable to mature. The sprouting of the mossy fibers, although aberrant and a probable source of epileptic activity, may be important for the correct physiology of the granule cells, restoring a likeness of normality in their connective environment. The survival of granule cells incorporated as mature neurons was increased after pilocarpine when compared with normal conditions. Thus, it is likely that the reorganization of the circuitry after the loss of the mossy cells facilitates the survival and incorporation of the newly generated granule cells.