Excitatory synaptic input to granule cells increases with time after kainate treatment

Temporal lobe epilepsy is usually associated with a latent period and an increased seizure frequency following a precipitating insult. After kainate treatment, the mossy fibers of the dentate gyrus are hypothesized to form recurrent excitatory circuits between granule cells, thus leading to a progressive increase in the excitatory input to granule cells. Three groups of animals were studied as a function of time after kainate treatment: 1-2 wk, 2-4 wk, and 10-51 wk. All the animals studied 10-51 wk after kainate treatment were observed to have repetitive spontaneous seizures. Whole cell patch-clamp recordings in hippocampal slices showed that the amplitude and frequency of spontaneous excitatory postsynaptic currents (EPSCs) in granule cells increased with time after kainate treatment. This increased excitatory synaptic input was correlated with the intensity of the Timm stain in the inner molecular layer (IML). Flash photolysis of caged glutamate applied in the granule cell layer evoked repetitive EPSCs in 10, 32, and 66% of the granule cells at the different times after kainate treatment. When inhibition was reduced with bicuculline, photostimulation of the granule cell layer evoked epileptiform bursts of action potentials only in granule cells from rats 10-51 wk after kainate treatment. These data support the hypothesis that kainate-induced mossy fiber sprouting in the IML results in the progressive formation of aberrant excitatory connections between granule cells. They also suggest that the probability of occurrence of electrographic seizures in the dentate gyrus increases with time after kainate treatment.     

Neuropeptide-Y immunoreactivity in the pilocarpine model of temporal lobe epilepsy

 Neuropeptide-Y (NPY) is expressed by granule cells and mossy fibres of the hippocampal dentate gyrus during experimental temporal lobe epilepsy (TLE). This expression may represent an endogenous damping mechanism since NPY has been shown to block seizure-like events following high-frequency stimulation in hippocampal slices. The pilocarpine (PILO) model of epilepsy is characterized by an acute period of status epilepticus followed by spontaneous recurrent seizures and related brain damage. We report peroxidase-antiperoxidase immunostaining for NPY in several brain regions in this model. PILO-injected animals exhibited NPY immunoreactivity in the region of the mossy fibre terminals, in the dentate gyrus inner molecular layer and, in a few cases, within presumed granule cells. NPY immunoreactivity was also dramatically changed in the entorhinal cortex, amygdala and sensorimotor areas. In addition, PILO injected animals exhibited a reduction in the number of NPY-immunoreactive interneurons compared with controls. The results demonstrate that changes in NPY expression, including expression in the granule cells and mossy fibres and the loss of vulnerable NPY neurons, are present in the PILO model of TLE. However, the significance of this changed synthesis of NPY remains to be determined. Received: 19 August 1996 / Accepted: 21 March 1997     

Recurrent mossy fiber pathway in rat dentate gyrus: synaptic currents evoked in presence and absence of seizure-induced growth

A common feature of temporal lobe epilepsy and of animal models of epilepsy is the growth of hippocampal mossy fibers into the dentate molecular layer, where at least some of them innervate granule cells. Because the mossy fibers are axons of granule cells, the recurrent mossy fiber pathway provides monosynaptic excitatory feedback to these neurons that could facilitate seizure discharge. We used the pilocarpine model of temporal lobe epilepsy to study the synaptic responses evoked by activating this pathway. Whole cell patch-clamp recording demonstrated that antidromic stimulation of the mossy fibers evoked an excitatory postsynaptic current (EPSC) in approximately 74% of granule cells from rats that had survived >10 wk after pilocarpine-induced status epilepticus. Recurrent mossy fiber growth was demonstrated with the Timm stain in all instances. In contrast, antidromic stimulation of the mossy fibers evoked an EPSC in only 5% of granule cells studied 4-6 days after status epilepticus, before recurrent mossy fiber growth became detectable. Notably, antidromic mossy fiber stimulation also evoked an EPSC in many granule cells from control rats. Clusters of mossy fiber-like Timm staining normally were present in the inner third of the dentate molecular layer at the level of the hippocampal formation from which slices were prepared, and several considerations suggested that the recorded EPSCs depended mainly on activation of recurrent mossy fibers rather than associational fibers. In both status epilepticus and control groups, the antidromically evoked EPSC was glutamatergic and involved the activation of both AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors. EPSCs recorded in granule cells from rats with recurrent mossy fiber growth differed in three respects from those recorded in control granule cells: they were much more frequently evoked, a number of them were unusually large, and the NMDA component of the response was generally much more prominent. In contrast to the antidromically evoked EPSC, the EPSC evoked by stimulation of the perforant path appeared to be unaffected by a prior episode of status epilepticus. These results support the hypothesis that recurrent mossy fiber growth and synapse formation increases the excitatory drive to dentate granule cells and thus facilitates repetitive synchronous discharge. Activation of NMDA receptors in the recurrent pathway may contribute to seizure propagation under depolarizing conditions. Mossy fiber-granule cell synapses also are present in normal rats, where they may contribute to repetitive granule cell discharge in regions of the dentate gyrus where their numbers are significant.     

Suppression of pilocarpine-induced status epilepticus and the late development of epilepsy in rats

Status epilepticus (SE) has been related to subsequent development of epilepsy. The present work was aimed at elucidating the relationship between the duration of pilocarpine- (PILO)-induced SE and the subsequent development of epilepsy in rats. The latency for the appearance of the first spontaneous seizure, the frequency of spontaneous seizures, the cell density in the hippocampal formation and the density of supragranular neo-Timmstaining were monitored. At 30 min, 1, 2 and 6 h after the beginning of SE, animals were treated with diazepam plus pentobarbital. In non-treated rats, SE remitted spontaneously. Animals exhibiting 30 min of PILO-induced SE did not develop spontaneous seizures. Hippocampal cell counts and the density of neo-Timm staining in these animals were similar to those observed in control rats. In the other groups longer SE durations were related to: shorter latency for the appearance of the first spontaneous seizure, increased number of the spontaneous recurrent seizures, severe cell loss in the hippocampal formation, or increased supragranular neo-Timm staining. These data suggest that more than 30 min of SE is required to produce hippocampal damage with subsequent synaptic reorganization of the mossy fibre pathway that could account for SRSs observed in the PILO model of epilepsy.     

Protective effects of mossy fiber lesions against kainic acid-induced seizures and neuronal degeneration

The effects of a hippocampal mossy fiber lesion have been determined on neuronal degeneration and limbic seizures provoked by the subsequent intracerebroventricular administration of kainic acid to unanesthetized rats. Mossy fiber lesions were made either by transecting this pathway unilaterally or by destroying the dentate granule cells unilaterally or bilaterally with colchicine. All control rats eventually developed status epilepticus and each temporally discrete seizure that preceded status epilepticus was recorded from the hippocampus ipsilateral to the kainic acid infusion before the contralateral hippocampus. A mossy fiber lesion of the ipsilateral hippocampus prevented the development of status epilepticus in 26% of subjects and in 52% of subjects seizures were recorded from the contralateral hippocampus before the ipsilateral hippocampus. Unlike electrographic records from other treatment groups, those from rats which had received a bilateral colchicine lesion exhibited no consistent pattern indicative of seizure propagation from one limbic region to another. A bilateral, but not a unilateral, mossy fiber lesion also dramatically attenuated the behavioral expression of the seizures. Regardless of its effects on kainic acid-induced electrographic and behavioral seizures, a mossy fiber lesion always substantially reduced or completely prevented the degeneration of ipsilateral hippocampal CA3-CA4 neurons. This protective effect was specific for those hippocampal neurons deprived of mossy fiber innervation. Neurons in other regions of the brain were protected from degeneration only when the mossy fiber lesion also prevented the development of electrographic status epilepticus. These results suggest that the hippocampal mossy fibers constitute an important, though probably not an obligatory, link in the circuit responsible for the spread of kainic acid seizures. Degeneration of CA3-CA4 neurons appears to depend upon (1) the duration of hippocampal seizure activity and (2) an as yet undefined influence of or interaction with the mossy fiber projection which enhances the neurodegenerative effect of the seizures.     

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.     

A chronic histopathological and electrophysiological analysis of a rodent hypoxic-ischemic brain injury model and its use as a model of epilepsy

Ischemic brain injury is one of the leading causes of epilepsy in the elderly, and there are currently no adult rodent models of global ischemia, unilateral hemispheric ischemia, or focal ischemia that report the occurrence of spontaneous motor seizures following ischemic brain injury. The rodent hypoxic-ischemic (H-I) model of brain injury in adult rats is a model of unilateral hemispheric ischemic injury. Recent studies have shown that an H-I injury in perinatal rats causes hippocampal mossy fiber sprouting and epilepsy. These experiments aimed to test the hypothesis that a unilateral H-I injury leading to severe neuronal loss in young-adult rats also causes mossy fiber sprouting and spontaneous motor seizures many months after the injury, and that the mossy fiber sprouting induced by the H-I injury forms new functional recurrent excitatory synapses. The right common carotid artery of 30-day old rats was permanently ligated, and the rats were placed into a chamber with 8% oxygen for 30 min. A quantitative stereologic analysis revealed that the ipsilateral hippocampus had significant hilar and CA1 pyramidal neuronal loss compared with the contralateral and sham-control hippocampi. The septal region from the ipsilateral and contralateral hippocampus had small but significantly increased amounts of Timm staining in the inner molecular layer compared with the sham-control hippocampi. Three of 20 lesioned animals (15%) were observed to have at least one spontaneous motor seizure 6-12 months after treatment. Approximately 50% of the ipsilateral and contralateral hippocampal slices displayed abnormal electrophysiological responses in the dentate gyrus, manifest as all-or-none bursts to hilar stimulation. This study suggests that H-I injury is associated with synaptic reorganization in the lesioned region of the hippocampus, and that new recurrent excitatory circuits can predispose the hippocampus to abnormal electrophysiological activity and spontaneous motor seizures.     

Consequences of recurrent seizures during early brain development.

It is well documented that prolonged seizures (status epilepticus) can cause neuronal injury and result in synaptic reorganization in certain brain regions. However, the effect of recurrent, relatively short seizures in young animals on subsequent brain development is not known. To study the consequences of recurrent seizures on the developing brain, we subjected immature rats to a total of 50 flurothyl-induced seizures from postnatal day 11 until day 23. Immunohistochemistry for c-fos was performed to characterize the pattern of neuronal activation following the seizures. Cell counting of dentate granule cells, CA3, CA1, and hilar neurons, using unbiased stereological methods, and the silver impregnation method were used to evaluate neuronal death following the recurrent seizures. Timm and Golgi staining were performed four weeks after the 50th seizure to evaluate the effects of recurrent seizures on synaptic organization. Our results show that recurrent flurothyl-induced seizures progressively increased excitability of the brain, as revealed by a dramatic increase in the extent and intensity of c-fos immunostaining. While no cell loss was detected in the hippocampus with either Cresyl Violet or silver stains, animals experiencing multiple daily seizures developed increased mossy fiber sprouting in both the supragranular layer of the dentate gyrus and the infrapyramidale layer of the CA3 region. Golgi staining confirmed that there was an increase in mossy fibers in the pyramidal cell layer. Our results suggest that serial recurrent seizures in the immature brain can lead to significant changes in mossy fiber distribution even though the seizures do not cause significant hippocampal cell loss.     

Survival of dentate hilar mossy cells after pilocarpine-induced seizures and their synchronized burst discharges with area CA3 pyramidal cells.

The clinical and basic literature suggest that hilar cells of the dentate gyrus are damaged after seizures, particularly prolonged and repetitive seizures. Of the cell types within the hilus, it appears that the mossy cell is one of the most vulnerable. Nevertheless, hilar neurons which resemble mossy cells appear in some published reports of animal models of epilepsy, and in some cases of human temporal lobe epilepsy. Therefore, mossy cells may not always be killed after severe, repeated seizures. However, mossy cell survival in these studies was not completely clear because the methods did allow discrimination between mossy cells and other hilar cell types. Furthermore, whether surviving mossy cells might have altered physiology after seizures was not examined. Therefore, intracellular recording and intracellular dye injection were used to characterize hilar cells in hippocampal slices from pilocarpine-treated rats that had status epilepticus and recurrent seizures ('epileptic' rats). For comparison, mossy cells were also recorded from age-matched, saline-injected controls, and pilocarpine-treated rats that failed to develop status epilepticus.Numerous hilar cells with the morphology, axon projection, and membrane properties of mossy cells were recorded in all three experimental groups. Thus, mossy cells can survive severe seizures, and those that survive retain many of their normal characteristics. However, mossy cells from epileptic tissue were distinct from mossy cells of control rats in that they generated spontaneous and evoked epileptiform burst discharges. Area CA3 pyramidal cells also exhibited spontaneous and evoked bursts. Simultaneous intracellular recordings from mossy cells and pyramidal cells demonstrated that their burst discharges were synchronized, with pyramidal cell discharges typically beginning first.From these data we suggest that hilar mossy cells can survive status epilepticus and chronic seizures. The fact that mossy cells have epileptiform bursts, and that they are synchronized with area CA3, suggest a previously unappreciated substrate for hyperexcitability in this animal model.     

Lithium-pilocarpine-induced status epilepticus in immature rats result in long-term deficits in spatial learning and hippocampal cell loss

Rat pups age of 14 postnatal day (P14) were subjected to lithium-pilocarpine (Li-PC) model of status epilepticus (SE). Control rats (n=6) were given an equivalent volume of saline intraperitoneally. Behavioral testing began on P60 including the Morris water maze, the radial arm maze, and the rotarod test. Brain were then analyzed with cresyl violet stain for histological lesions and evaluated for mossy fiber sprouting with the Timm stain. We observed spatial memory deficits both in the Morris water maze and radial arm maze in Li-PC-treated rat. There was no motor impairment in Li-PC-treated rat by the rotarod test. Two of six Li-PC-treated rats showed cell loss in hippocampal CA1 subfield. The Timm staining pattern was similar in both control and Li-PC-treated rats. Result of this study suggests that Li-PC-induced SE in immature rats cause long-term cognitive deficit and permanent cell loss in hippocampal CA1, but spare motor impairment.     

The lesional and epileptogenic consequences of lithium-pilocarpine-induced status epilepticus are affected by previous exposure to isolated seizures: effects of amygdala kindling and maximal electroshocks

In temporal lobe epilepsy, the occurrence of seizures seems to correlate with the presence of lesions underlying the establishment of a hyperexcitable circuit. However, in the lithium-pilocarpine model of epilepsy, neuronal damage occurs both in the structures belonging to the circuit of initiation and maintenance of the seizures (forebrain limbic system) as in the propagation areas (cortex and thalamus) and in the circuit of remote control of seizures (substantia nigra pars reticulata). To determine whether or not we could protect the brain from lesions and epileptogenesis induced by status epilepticus and identify cerebral structures involved in the genesis of epilepsy, we studied the effects of the chronic exposure to non-deleterious seizures, either focalized with secondary generalization (amygdala kindling, kindled-pilocarpine rats), or primary generalized (ear-clip electroshocks, electroshock-pilocarpine rats) on neuronal damage and epileptogenesis induced by lithium-pilocarpine status epilepticus. These animals were compared to rats subjected to status epilepticus but not pretreated with seizures (sham-kindled-pilocarpine or sham-electroshock-pilocarpine rats). Compared to sham-pilocarpine rats, neuronal damage was prevented in the limbic system of the kindled-pilocarpine rats, except in the hilus of the dentate gyrus and the entorhinal cortex, while it was enhanced in rats pretreated with electroshocks, mainly in the entorhinal and perirhinal cortices. Most sham-kindled- and sham-electroshock-pilocarpine rats (92-100%) developed recurrent seizures after a silent period of 40-54days. Likewise, all kindled-pilocarpine rats developed spontaneous seizures after the same latency as their sham controls, while only two of 10 electroshock-pilocarpine rats became epileptic after a delay of 106-151days.The present data show that the apparent antiepileptic properties of electroshocks correlate with extensive damage in midbrain cortical regions, which may prevent the propagation of seizures from the hippocampus and inhibit their motor expression. Conversely, the extensive neuroprotection of the limbic system but not the hilus and entorhinal cortex provided by amygdala kindling does not prevent epileptogenesis. Thus, the hilus, the entorhinal and/or perirhinal cortex may be key structure(s) for the establishment of epilepsy.     

Post-treatment with rapamycin does not prevent epileptogenesis in the amygdala stimulation model of temporal lobe epilepsy

Approximately 30% of all epilepsy cases are acquired. At present there is no effective strategy to stop epilepsy development after the precipitating insult. Recent data from experimental models pointed to the mTOR pathway, which can be potently inhibited by rapamycin. However, data on the antiepileptic and antiepileptogenic properties of rapamycin are conflicting. Therefore, we tested whether rapamycin post-treatment influences epileptogenesis in the amygdala stimulation model of temporal lobe epilepsy in rats. Animals were treated with rapamycin (6 mg/kg) or vehicle daily for 2 wks, beginning 24 h after stimulation. Sham-operated animals were treated with rapamycin or vehicle but were not stimulated. Animals were video-EEG monitored to detect spontaneous seizures. Animals were sacrificed 4 wks later and brains were collected for Timm staining. There were no significant differences in the number of stimulated rats developing epilepsy; latency to first spontaneous seizure; number of seizures, or seizure frequency in epileptic animals. The area occupied by mossy fibers was significantly increased in stimulated vs. sham-operated animals but was not different in animals treated with rapamycin vs. vehicle. Collectively, our data suggest that the antiepileptic or antiepileptogenic action of rapamycin is not a universal phenomenon and might be limited to certain experimental models or experimental conditions.     

Selective innervation of embryonic hippocampal transplants by adult host dentate granule cell axons

Fragments containing different cytoarchitectonic fields were dissected out of late embryonic rat hippocampal primordia and transplanted into the hippocampus or septum of adult syngeneic hosts. Field CA3 transplants contained clusters of large, angular (pyramidal) cell bodies surrounded by a radiating corona of dendrites. These cells stained selectively with our monoclonal antibody Py, and a proportion were labelled by [3H]thymidine administered on the 15th day of embryonic life. Field CA1 transplants contained smaller, angular, Py-negative cells, which formed elongated laminae rather than globular clusters. The ability of the host dentate granule cells to project to the transplants was examined by (1) the Timm stain for mossy fibres, (2) electron microscopy of Golgi-impregnated CA3 pyramidal neurons in the transplants, and (3) quantitative electron microscopic assessment of the proportions of large mossy fibre terminals in the synaptic population of the transplants. The Timm stain showed that CA3 transplants received a projection from host dentate granule cells when the transplants were placed in direct contact with the axons in the host mossy fibre pathway. As in the normal host field CA3, the ingrowing mossy fibres terminated selectively on the juxtacellular regions of the dendritic tree and ignored the major part of the dendrites in the radiating corona. The electron micrographs showed that within this territory the host mossy fibres formed synaptic terminals with all the complex features typical of normal mossy fibres, and were presynaptic to complex spines arising from the juxtacellular region of Golgi-impregnated donor CA3 pyramidal cells. The quantitative electron microscopic study demonstrated that the mossy fibre-innervated juxtacellular regions of the field CA3 transplants had up to 20% of the normal density of mossy fibre synapses found in the stratum lucidum of field CA3 in situ. CA3 transplants which were placed in the septum, remote from the host mossy fibres, had either trivial numbers of mossy fibre synapses or none. This confirmed that the abundant mossy fibre terminals in the intrahippocampal CA3 transplants were of host origin, and not due to donor dentate granule cells inadvertently included in the grafts. The selectivity of the host dentate projection for field CA3 transplants was demonstrated by the observation that CA1 transplants in the same locations received only slight mossy fibre projections in the Timm stain, and in electron micrographs their synaptic population had only insignificant numbers of large mossy fibre terminals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mossy fiber-granule cell synapses in the normal and epileptic rat dentate gyrus studied with minimal laser photostimulation.

Dentate granule cells become synaptically interconnected in the hippocampus of persons with temporal lobe epilepsy, forming a recurrent mossy fiber pathway. This pathway may contribute to the development and propagation of seizures. The physiology of mossy fiber-granule cell synapses is difficult to characterize unambiguously, because electrical stimulation may activate other pathways and because there is a low probability of granule cell interconnection. These problems were addressed by the use of scanning laser photostimulation in slices of the caudal hippocampal formation. Glutamate was released from a caged precursor with highly focused ultraviolet light to evoke action potentials in a small population of granule cells. Excitatory synaptic currents were recorded in the presence of bicuculline. Minimal laser photostimulation evoked an apparently unitary excitatory postsynaptic current (EPSC) in 61% of granule cells from rats that had experienced pilocarpine-induced status epilepticus followed by recurrent mossy fiber growth. An EPSC was also evoked in 13-16% of granule cells from the control groups. EPSCs from status epilepticus and control groups had similar peak amplitudes ( approximately 30 pA), 20-80% rise times (approximately 1.2 ms), decay time constants ( approximately 10 ms), and half-widths (approximately 8 ms). The mean failure rate was high (approximately 70%) in both groups, and in both groups activation of N-methyl-D-aspartate receptors contributed a small component to the EPSC. The strong similarity between responses from the status epilepticus and control groups suggests that they resulted from activation of a similar synaptic population. No EPSC was recorded when the laser beam was focused in the dentate hilus, suggesting that indirect activation of hilar mossy cells contributed little, if at all, to these results. Recurrent mossy fiber growth increases the density of mossy fiber-granule cell synapses in the caudal dentate gyrus by perhaps sixfold, but the new synapses appear to operate very similarly to preexisting mossy fiber-granule cell synapses.     

Bilateral kainic acid lesions in the rat hilus induce non-linear additive mossy fiber neoinnervation

Kainic acid (KA) lesions of the rat hilus model hippocampal sclerosis and temporal lobe epilepsy. Unilateral hilar cell loss denervates the associational afferents normally projecting to the inner molecular layer (IML) granule cell dendrites, followed by ipsilateral mossy fiber (MF) sprouting. Hilar neurons also project through the hippocampal commissure to the contralateral IML. This study compared densities of IML MF sprouting following unilateral versus bilateral low dose KA lesions, using Neo-Timm stain 30 days later. Unilateral KA (0.4 μg) caused only dense ipsilateral MF sprouting. Bilateral lesions with lower doses of KA (0.1 with 0.2 or 0.3 μg) induced dense bilateral MF sprouting. However, the same low doses of KA injected unilaterally did not induce significant sprouting ipsilaterally or contralaterally. These data show that denervations of both associational and commissural afferents to the same IML dendritic zones of granule cells induce non-linear, additive bilateral MF neoinnervations.     

Recurrent excitatory connectivity in the dentate gyrus of kindled and kainic acid-treated rats

Repeated seizures induce mossy fiber axon sprouting, which reorganizes synaptic connectivity in the dentate gyrus. To examine the possibility that sprouted mossy fiber axons may form recurrent excitatory circuits, connectivity between granule cells in the dentate gyrus was examined in transverse hippocampal slices from normal rats and epileptic rats that experienced seizures induced by kindling and kainic acid. The experiments were designed to functionally assess seizure-induced development of recurrent circuitry by exploiting information available about the time course of seizure-induced synaptic reorganization in the kindling model and detailed anatomic characterization of sprouted fibers in the kainic acid model. When recurrent inhibitory circuits were blocked by the GABA(A) receptor antagonist bicuculline, focal application of glutamate microdrops at locations in the granule cell layer remote from the recorded granule cell evoked trains of excitatory postsynaptic potentials (EPSPs) and population burst discharges in epileptic rats, which were never observed in slices from normal rats. The EPSPs and burst discharges were blocked by bath application of 1 microM tetrodotoxin and were therefore dependent on network-driven synaptic events. Excitatory connections were detected between blades of the dentate gyrus in hippocampal slices from rats that experienced kainic acid-induced status epilepticus. Trains of EPSPs and burst discharges were also evoked in granule cells from kindled rats obtained after > or = 1 wk of kindled seizures, but were not evoked in slices examined 24 h after a single afterdischarge, before the development of sprouting. Excitatory connectivity between blades of the dentate gyrus was also assessed in slices deafferented by transection of the perforant path, and bathed in artificial cerebrospinal fluid (ACSF) containing bicuculline to block GABA(A) receptor-dependent recurrent inhibitory circuits and 10 mM [Ca(2+)](o) to suppress polysynaptic activity. Low-intensity electrical stimulation of the infrapyramidal blade under these conditions failed to evoke a response in suprapyramidal granule cells from normal rats (n = 15), but in slices from epileptic rats evoked an EPSP at a short latency (2.59 +/- 0.36 ms) in 5 of 18 suprapyramidal granule cells. The results are consistent with formation of monosynaptic excitatory connections between blades of the dentate gyrus. Recurrent excitatory circuits developed in the dentate gyrus of epileptic rats in a time course that corresponded to the development of mossy fiber sprouting and demonstrated patterns of functional connectivity corresponding to anatomic features of the sprouted mossy fiber pathway.     

Changes in mIPSCs and sIPSCs after kainate treatment: evidence for loss of inhibitory input to dentate granule cells and possible compensatory responses

How inhibition is altered after status epilepticus and the role of inhibition during epileptogenesis remain unsettled issues. The present study examined acute (4-7 days) and chronic (>3 mo) changes of GABA(A) receptor-mediated inhibitory synaptic input to dentate granule cells after kainate-induced status epilepticus. Whole cell patch-clamp techniques were used to record spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) in the presence of 6,7-dinitroquinoxaline-2,3-dione and dl-2-amino-5-phosphonopentanoic acid to block glutamatergic excitatory synaptic transmission. In both groups, mean sIPSC frequency of dentate granule cells from the saline- and kainate-treated rats was not significantly different. However, mIPSC frequency from the kainate-treated rats of both groups was approximately 30% lower than that of the respective saline controls. The mean amplitude of sIPSCs and mIPSCs from kainate-treated rats was not reduced in either the acute or chronic groups. The mean 10-90% rise time of IPSCs was not altered in kainate-treated rats, but the decay time constant was slightly longer than in controls, and the charge transfer 4-7 days after kainate treatment was significantly larger. The similar reduction of mIPSC frequency (i.e., approximately 30%) in the two groups of kainate-treated rats suggests a decreased inhibitory input to dentate granule cells (presumably due to a partial loss of inhibitory interneurons that innervate them) without recovery during epileptogenesis. The lack of effect on sIPSC frequency and the decreased mIPSC frequency in both groups suggests a possible compensatory increase in firing rate of interneurons, which may involve a hypothetical reduction of inhibitory input to the remaining interneurons.     

High ratio of synaptic excitation to synaptic inhibition in hilar ectopic granule cells of pilocarpine-treated rats

After experimental status epilepticus, many dentate granule cells born into the postseizure environment migrate aberrantly into the dentate hilus. Hilar ectopic granule cells (HEGCs) have also been found in persons with epilepsy. These cells exhibit a high rate of spontaneous activity, which may enhance seizure propagation. Electron microscopic studies indicated that HEGCs receive more recurrent mossy fiber innervation than normotopic granule cells in the same animals but receive much less inhibitory innervation. This study used hippocampal slices prepared from rats that had experienced pilocarpine-induced status epilepticus to test the hypothesis that an imbalance of synaptic excitation and inhibition contributes to the hyperexcitability of HEGCs. Mossy fiber stimulation evoked a much smaller GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSC) in HEGCs than in normotopic granule cells from either control rats or rats that had experienced status epilepticus. However, recurrent mossy fiber-evoked excitatory postsynaptic currents (EPSCs) of similar size were recorded from HEGCs and normotopic granule cells in status epilepticus-experienced rats. HEGCs exhibited the highest frequency of miniature excitatory postsynaptic currents (mEPSCs) and the lowest frequency of miniature inhibitory postsynaptic currents (mIPSCs) of any granule cell group. On average, both mEPSCs and mIPSCs were of higher amplitude, transferred more charge per event, and exhibited slower kinetics in HEGCs than in granule cells from control rats. Charge transfer per unit time in HEGCs was greater for mEPSCs and much less for mIPSCs than in the normotopic granule cell groups. A high ratio of excitatory to inhibitory synaptic function probably accounts, in part, for the hyperexcitability of HEGCs.     

Neuropeptide Y regulates recurrent mossy fiber synaptic transmission less effectively in mice than in rats: Correlation with Y2 receptor plasticity

A unique feature of temporal lobe epilepsy is the formation of recurrent excitatory connections among granule cells of the dentate gyrus as a result of mossy fiber sprouting. This novel circuit contributes to a reduced threshold for granule cell synchronization. In the rat, activity of the recurrent mossy fiber pathway is restrained by the neoexpression and spontaneous release of neuropeptide Y (NPY). NPY inhibits glutamate release tonically through activation of presynaptic Y2 receptors. In the present study, the effects of endogenous and applied NPY were investigated in C57Bl/6 mice that had experienced pilocarpine-induced status epilepticus and subsequently developed a robust recurrent mossy fiber pathway. Whole cell patch clamp recordings made from dentate granule cells in hippocampal slices demonstrated that, as in rats, applied NPY inhibits recurrent mossy fiber synaptic transmission, the Y2 receptor antagonist (S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6H)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide (BIIE0246) blocks its action and BIIE0246 enhances synaptic transmission when applied by itself. Y5 receptor agonists had no significant effect. Thus spontaneous release of NPY tonically inhibits synaptic transmission in mice and its effects are mediated by Y2 receptor activation. However, both NPY and BIIE0246 were much less effective in mice than in rats, despite apparently equivalent expression of NPY in the recurrent mossy fibers. Immunohistochemistry indicated greater expression of Y2 receptors in the mossy fiber pathway of normal mice than of normal rats. Pilocarpine-induced status epilepticus markedly reduced the immunoreactivity of mouse mossy fibers, but increased the immunoreactivity of rat mossy fibers. Mossy fiber growth into the inner portion of the dentate molecular layer was associated with increased Y2 receptor immunoreactivity in rat, but not in mouse. These contrasting receptor changes can explain the quantitatively different effects of endogenously released and applied NPY on recurrent mossy fiber transmission in mice and rats.     

Plasticity of both excitatory and inhibitory synapses is associated with seizures induced by removal of chronic blockade of activity in cultured hippocampus

One factor common to many neurological insults that can lead to acquired epilepsy is a loss of afferent neuronal input. Neuronal activity is one cellular mechanism implicated in transducing deafferentation into epileptogenesis. Therefore the effects of chronic activity blockade on seizure susceptibility and its underlying mechanisms were examined in organotypic hippocampal slice cultures treated chronically with the sodium channel blocker, tetrodotoxin (TTX), or the N-methyl-D-aspartate receptor (NMDAR) antagonist, D-2-amino-5-phosphonovaleric acid (D-APV). Granule cell field potential recordings in physiological buffer revealed spontaneous electrographic seizures in 83% of TTX-, 9% of D-APV-, but 0% of vehicle-treated cultures. TTX-induced seizures were not associated with membrane property alterations that would elicit granule cell hyperexcitability. Seizures were blocked by glutamate receptor antagonists, suggesting that plasticity in excitatory synaptic circuits contributed to seizures. The morphology of granule cells and their mossy fiber axons remained largely unchanged, and the number of synapses onto granule cells measured immunohistochemically was not increased in TTX- or D-APV-treated cultures. However, voltage-clamp recordings revealed that miniature excitatory postsynaptic current frequency and kinetics were increased and miniature inhibitory postsynaptic current kinetics were decreased in D-APV- and TTX-treated cultures compared with vehicle. Changes were more profound and qualitatively different in TTX- compared with D-APV-treated cultures, consistent with the dramatic effects of TTX treatment on seizure expression. We propose that chronic blockade of action potentials by TTX induces homeostatic responses including plasticity of both excitatory and inhibitory synapses. Removal of TTX unmasks the impact of these synaptic plasticities on local circuit excitability, resulting in spontaneous seizures.