Electrophysiological evidence of monosynaptic excitatory transmission between granule cells after seizure-induced mossy fiber sprouting

Mossy fiber sprouting is a form of synaptic reorganization in the dentate gyrus that occurs in human temporal lobe epilepsy and animal models of epilepsy. The axons of dentate gyrus granule cells, called mossy fibers, develop collaterals that grow into an abnormal location, the inner third of the dentate gyrus molecular layer. Electron microscopy has shown that sprouted fibers from synapses on both spines and dendritic shafts in the inner molecular layer, which are likely to represent the dendrites of granule cells and inhibitory neurons. One of the controversies about this phenomenon is whether mossy fiber sprouting contributes to seizures by forming novel recurrent excitatory circuits among granule cells. To date, there is a great deal of indirect evidence that suggests this is the case, but there are also counterarguments. The purpose of this study was to determine whether functional monosynaptic connections exist between granule cells after mossy fiber sprouting. Using simultaneous recordings from granule cells, we obtained direct evidence that granule cells in epileptic rats have monosynaptic excitatory connections with other granule cells. Such connections were not obtained when age-matched, saline control rats were examined. The results suggest that indeed mossy fiber sprouting provides a substrate for monosynaptic recurrent excitation among granule cells in the dentate gyrus. Interestingly, the characteristics of the excitatory connections that were found indicate that the pathway is only weakly excitatory. These characteristics may contribute to the empirical observation that the sprouted dentate gyrus does not normally generate epileptiform discharges.     

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.     

Contributions of mossy fiber and CA1 pyramidal cell sprouting to dentate granule cell hyperexcitability in kainic acid-treated hippocampal slice cultures

Axonal sprouting like that of the mossy fibers is commonly associated with temporal lobe epilepsy, but its significance remains uncertain. To investigate the functional consequences of sprouting of mossy fibers and alternative pathways, kainic acid (KA) was used to induce robust mossy fiber sprouting in hippocampal slice cultures. Physiological comparisons documented many similarities in granule cell responses between KA- and vehicle-treated cultures, including: seizures, epileptiform bursts, and spontaneous excitatory postsynaptic currents (sEPSCs) >600 pA. GABAergic control and contribution of glutamatergic synaptic transmission were similar. Analyses of neurobiotin-filled CA1 pyramidal cells revealed robust axonal sprouting in both vehicle- and KA-treated cultures, which was significantly greater in KA-treated cultures. Hilar stimulation evoked an antidromic population spike followed by variable numbers of postsynaptic potentials (PSPs) and population spikes in both vehicle- and KA-treated cultures. Despite robust mossy fiber sprouting, knife cuts separating CA1 from dentate gyrus virtually abolished EPSPs evoked by hilar stimulation in KA-treated but not vehicle-treated cultures, suggesting a pivotal role of functional afferents from CA1 to dentate gyrus in KA-treated cultures. Together, these findings demonstrate striking hyperexcitability of dentate granule cells in long-term hippocampal slice cultures after treatment with either vehicle or KA. The contribution to hilar-evoked hyperexcitability of granule cells by the unexpected axonal projection from CA1 to dentate in KA-treated cultures reinforces the idea that axonal sprouting may contribute to pathologic hyperexcitability of granule cells.     

Enhanced but fragile inhibition in the dentate gyrus in vivo in the kainic acid model of temporal lobe epilepsy: a study using current source density analysis

Temporal lobe epilepsy is related to many structural and physiological changes in the brain. We used kainic acid in rats as an animal model of temporal lobe epilepsy, and studied the neural interactions of the dentate gyrus in urethane-anesthetized rats in vivo. Our initial hypothesis was that sprouting of mossy fibers, the axons of the granule cells, increases proximal dendritic excitatory currents in the inner molecular layer of the dentate gyrus. Extracellular currents were detected in vivo using current source density analysis. Backfiring the mossy fibers in CA3 or orthodromic excitation of the granule cells through the medial perforant path induced a current sink at the inner molecular layer. However, the sink or inferred excitation at the inner molecular layer was not increased in kainic acid-treated rats and the sink actually correlated negatively with the degree of mossy fiber sprouting. It is inferred that the latter sink was mediated mainly by association fibers and not by recurrent mossy fibers. After kainic acid treatment, paired-pulse inhibition of the population spikes in the dentate gyrus was increased. In contrast, reverberant activity that involved looping around an entorhinal-hippocampal circuit was increased in kainic acid-treated rats, compared to control rats. The increase of inhibition in kainic acid-treated rats was readily blocked by a small dose of GABA(A) receptor antagonist bicuculline. The latter dose of bicuculline induced paroxsymal spike bursts in kainic acid-treated but not control rats, demonstrating that the increased inhibition in dentate gyrus was fragile.In conclusion, after kainic acid induced seizures, the dentate gyrus in vivo showed an increase in inhibition that appeared to be fragile. The hypothesized increase in proximal dendritic excitation due to mossy fiber sprouting was not detected. However, the fragile inhibition could explain the seizure susceptibility in patients with temporal lobe epilepsy.     

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.     

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.     

NMDA receptor-dependent plasticity of granule cell spiking in the dentate gyrus of normal and epileptic rats

Because granule cells in the dentate gyrus provide a major synaptic input to pyramidal neurons in the CA3 region of the hippocampus, spike generation by granule cells is likely to have a significant role in hippocampal information processing. Granule cells normally fire in a single-spike mode even when inhibition is blocked and provide single-spike output to CA3 when afferent activity converging into the entorhinal cortex from neocortex, brainstem, and other limbic regions increases. The effects of enhancement of N-methyl-D-aspartate (NMDA) receptor-dependent excitatory synaptic transmission and reduction in gamma-aminobutyric acid-A (GABA(A)) receptor-dependent inhibition on spike generation were examined in granule cells of the dentate gyrus. In contrast to the single-spike mode observed in normal bathing conditions, perforant path stimulation in Mg(2+)-free bathing conditions evoked graded burst discharges in granule cells which increased in duration, amplitude, and number of spikes as a function of stimulus intensity. After burst discharges were evoked during transient exposure to bathing conditions that relieve the Mg(2+) block of the NMDA receptor, there was a marked increase in the NMDA receptor-dependent component of the EPSP, but no significant increase in the non-NMDA receptor-dependent component of the EPSP in normal bathing medium. Supramaximal perforant path stimulation still evoked only a single spike, but granule cell spike generation was immediately converted from a single-spike firing mode to a graded burst discharge mode when inhibition was then reduced. The induction of graded burst discharges in Mg(2+)-free conditions and the expression of burst discharges evoked in normal bathing medium with subsequent disinhibition were both blocked by DL-2-amino-4-phosphonovaleric acid (APV) and were therefore NMDA receptor dependent, in contrast to long-term potentiation (LTP) in the perforant path, which is induced by NMDA receptors and is also expressed by alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptors. The graded burst discharge mode was also observed in granule cells when inhibition was reduced after a single epileptic afterdischarge, which enhances the NMDA receptor-dependent component of evoked synaptic response, and in the dentate gyrus reorganized by mossy fiber sprouting in kindled and kainic acid-treated rats. NMDA receptor-dependent plasticity of granule cell spike generation, which can be distinguished from LTP and induces long-term susceptibility to epileptic burst discharge under conditions of reduced inhibition, could modify information processing in the hippocampus and promote epileptic synchronization by increasing excitatory input into CA3.     

AMPA receptor alterations precede mossy fiber sprouting in young children with temporal lobe epilepsy

Following neurological injury early in life numerous events, including excitotoxicity, neural degeneration, gliosis, neosynaptogenesis, and circuitry reorganization, may alone or in concert contribute to hyperexcitability and recurrent seizures in temporal lobe epilepsy. Our studies provide new evidence regarding the temporal sequence of key elements of hippocampal reorganization, mossy fiber sprouting and glutamate receptor subunit up-regulation, in a subset of young temporal lobe epileptic patients. Without evidence of mossy fiber sprouting, the youngest age group (3-10 years old) of mesial temporal lobe epileptic patients demonstrated enhanced glutamate receptor subunit profiles, suggesting that the dendritic change precedes axonal sprouting. However, sclerotic hippocampal specimens from epileptic patients ages 12-15 years old had the characteristic features of glutamate receptor up-regulation and mossy fiber sprouting first identified in the adult, indicating that reconstructed circuits appear early in the course of the disease. Non-sclerotic hippocampal specimens from lesion associated temporal lobe epileptic patients of all age groups showed minimal cell loss, sparse staining of glutamate receptor subunits in the dentate gyrus, and little or no mossy fiber sprouting. These compelling findings suggest a progressive sequence of events in the reorganization of the dentate gyrus of sclerotic hippocampal specimens. We suggest that cell loss and up-regulation of glutamate receptor subunits appear early in temporal lobe epilepsy and contribute to the synaptic plasticity that may facilitate the subsequent sprouting of mossy fiber collaterals which compound an already precipitous state of decline. The combination of pre-synaptic and post-synaptic changes serves as a potential substrate for hyperexcitability.     

癫痫发作敏感大鼠齿状回苔状纤维侧枝发芽

目的　探讨海马齿状回苔状纤维侧枝发芽与癫痫发作敏感性形成之间的关系。方法　在颈部皮下注射惊厥剂量的海人酸 (KA ,10mg/kg)诱发大鼠出现癫痫发作后 ,采用Timm’s染色法 ,分别在注射KA后3d、7d和 1个月 3个时间点观察致痫大鼠海马齿状回内苔状纤维发芽的情况。结果　Timm’s染色发现 ,注射KA后 7d ,海马齿状回分子层内带和颗粒细胞上层出现苔状纤维的异常发芽 ,注射KA后 1个月海马齿状回内Timm’s染色颗粒颜色加深 ,范围增大。提示海马苔状纤维发芽形成的时间过程与癫痫发作敏感性形成的时间过程一致。结论　海马齿状回分子层内带和颗粒细胞上层出现异常的苔状纤维发芽可能与癫痫发作敏感性形成有关。     

海人酸诱导癫痫大鼠齿状回神经肽Y能苔状纤维侧枝发芽

目的：探讨海马苔状纤维侧枝发芽与癫痫发作敏感性形成的关系。材料与方法：在海人酸诱发大鼠出现癫痫发作后,采用免疫组织化学染色法观察大鼠海马齿状回内神经肽Ｙ能纤维的异常发芽。结果：首次证实在癫痫发作后７天时,在海马齿状回内分子层就已出现神经肽Ｙ能纤维的异常增生。结论：这可能是对癫痫发作后齿状回门区神经肽Ｙ能神经元缺失的一种代偿性变化。     

Lack of effect of mossy fiber-released zinc on granule cell GABA(A) receptors in the pilocarpine model of epilepsy

The recurrent mossy fiber pathway of the dentate gyrus expands dramatically in the epileptic brain and serves as a mechanism for synchronization of granule cell epileptiform activity. It has been suggested that this pathway also promotes epileptiform activity by inhibiting GABA(A) receptor function through release of zinc. Hippocampal slices from pilocarpine-treated rats were used to evaluate this hypothesis. The rats had developed status epilepticus after pilocarpine administration, followed by robust recurrent mossy fiber growth. The ability of exogenously applied zinc to depress GABA(A) receptor function in dentate granule cells depended on removal of polyvalent anions from the superfusion medium. Under these conditions, 200 microM zinc reduced the amplitude of the current evoked by applying muscimol to the proximal portion of the granule cell dendrite (23%). It also reduced the mean amplitude (31%) and frequency (36%) of miniature inhibitory postsynaptic currents. Nevertheless, repetitive mossy fiber stimulation (10 Hz for 1 s, 100 Hz for 1 s, or 10 Hz for 5 min) at maximal intensity did not affect GABA(A) receptor-mediated currents evoked by photorelease of GABA onto the proximal portion of the dendrite, where recurrent mossy fiber synapses were located. These results could not be explained by stimulation-induced depletion of zinc from the recurrent mossy fiber boutons. Negative results were obtained even during exposure to conditions that promoted transmitter release and synchronized granule cell activity (6 mM [K(+)](o), nominally Mg(2+)-free medium, 33 degrees C). These results suggest that zinc released from the recurrent mossy fiber pathway did not reach a concentration at postsynaptic GABA(A) receptors sufficient to inhibit agonist-evoked activation.     

Seizures, cell death, and mossy fiber sprouting in kainic acid-treated organotypic hippocampal cultures

Sprouting of the mossy fiber axons of the dentate granule cells is a structural neuronal plasticity found in the mature brain of epileptic humans and experimental animals. Mossy fiber sprouting typically arises in experimental animals after repeated seizures and may contribute to the hyperexcitability of the epileptic brain. Investigation of the molecular triggers and spatial cues involved in mossy fiber sprouting has been hampered by the lack of an optimal in vitro model for studying this rearrangement. For an in vitro model to be feasible, the circuitry and receptors involved in convulsant-induced mossy fiber sprouting would have to be localized near the granule cells, rather than being dependent on long-range brain interconnections. However, it is not known whether this is the case. We report here that that application of the convulsant, kainic acid, to organotypic hippocampal explant cultures induces seizures, neuronal cell death, and subsequent dramatic mossy fiber sprouting with a similar laminar preference and time-course to that seen in intact animals. Prolonged (48 h) but not transient (4 h) kainic acid treatment caused regionally selective neuronal cell death. Cultures treated with kainic acid for a prolonged period displayed a time- and dose-dependent increase in supragranular Timm staining reflective of increased mossy fiber innervation to this area. Direct visualization of mossy fiber axons with neurobiotin-labeling revealed that mossy fibers in kainic acid-treated cultures exhibited a dramatic increase in supragranular axonal branch points and synaptic boutons. The cellular and molecular determinants required for kainic acid-induced cell death and subsequent mossy fiber reorganization thus appear to be intrinsic to the hippocampal slice preparation, and are preserved in culture. Given the ease with which functional inhibitors or pharmacological agents may be utilized in this system, slice cultures may provide a powerful model in which to study the molecular components involved in triggering mossy fiber outgrowth and underlying its laminar specificity. Elucidation of these molecular pathways will likely have both specific utility in clarifying the functional consequences of mossy fiber sprouting, as well as general utility in understanding of synaptic reorganization in the mature central nervous system.     

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.     

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.     

Role of mossy fiber sprouting and mossy cell loss in hyperexcitability: a network model of the dentate gyrus incorporating cell types and axonal topography

Mossy cell loss and mossy fiber sprouting are two characteristic consequences of repeated seizures and head trauma. However, their precise contributions to the hyperexcitable state are not well understood. Because it is difficult, and frequently impossible, to independently examine using experimental techniques whether it is the loss of mossy cells or the sprouting of mossy fibers that leads to dentate hyperexcitability, we built a biophysically realistic and anatomically representative computational model of the dentate gyrus to examine this question. The 527-cell model, containing granule, mossy, basket, and hilar cells with axonal projections to the perforant-path termination zone, showed that even weak mossy fiber sprouting (10-15% of the strong sprouting observed in the pilocarpine model of epilepsy) resulted in the spread of seizure-like activity to the adjacent model hippocampal laminae after focal stimulation of the perforant path. The simulations also indicated that the spatially restricted, lamellar distribution of the sprouted mossy fiber contacts reported in in vivo studies was an important factor in sustaining seizure-like activity in the network. In contrast to the robust hyperexcitability-inducing effects of mossy fiber sprouting, removal of mossy cells resulted in decreased granule cell responses to perforant-path activation in agreement with recent experimental data. These results indicate the crucial role of mossy fiber sprouting even in situations where there is only relatively weak mossy fiber sprouting as is the case after moderate concussive experimental head injury.     

Reassessment of the effects of cycloheximide on mossy fiber sprouting and epileptogenesis in the pilocarpine model of temporal lobe epilepsy

A feature of animal models of temporal lobe epilepsy and the human disorder is hippocampal sclerosis and Timm stain in the inner molecular layer (IML) of the dentate gyrus, which represents synaptic reorganization and may be important in epileptogenesis. We reassessed the hypothesis that pre-treatment with cycloheximide (CHX) prevents Timm staining in the IML following pilocarpine (PILO)-induced status epilepticus (a multifocal model of temporal lobe epilepsy), but allows epileptogenesis (i.e., chronic spontaneous seizures) after a latent period. Hippocampal slices from PILO-treated rats without Timm stain in the IML after CHX treatment were hypothesized to lack the electrophysiological abnormalities suggestive of recurrent excitation. The primary experimental groups were as follows: 1) CHX (1 mg/kg) 30-45 min prior to administration of PILO (320 mg/kg ip, 2) only PILO, and 3) only saline (0.5 ml, IP). The CHX pre-treatment significantly decreased the number of rats that responded to PILO with status epilepticus compared to rats that received only PILO. Pre-treatment with CHX did not significantly alter the spontaneous motor seizure rate post-treatment compared to treatment with PILO alone in those animals from each group that developed status epilepticus during PILO treatment. Timm stain in the IML was not significantly different between the PILO- and PILO+CHX-treated rats. Using quantitative methods, CHX did not prevent hilar, CA1, or CA3 neuronal loss compared to the PILO-treated rats. Extracellular responses to hilar stimulation in 30 microM bicuculline and 6 mM [K(+)](o) demonstrated all-or-none bursting in both the CHX+PILO- and PILO-treated rats but not in control rats. Whole cell recordings from granule cells, using glutamate flash photolysis to activate other granule cells, showed that both the CHX+PILO- and PILO-treated rats had excitatory synaptic interactions in the granule cell layer, which were not found after saline treatment. Some rats responded to PILO (with or without CHX pre-treatment) with only one or a few seizures at treatment, and some of these animals (n = 4) demonstrated spontaneous motor seizures within 2 mo after treatment. Timm staining and neuron loss in this group were not clearly different from saline-treated rats. These results suggest that in the PILO model, pre-treatment with CHX does not affect mossy fiber sprouting in the IML of epileptic rats and does not prevent the formation of recurrent excitatory circuits. However, the develoment of spontaneous motor seizures, in a small number of rats, could occur without detectable hippocampal neuron loss or mossy fiber sprouting, as assessed by the Timm stain method.     

Modest increase in extracellular potassium unmasks effect of recurrent mossy fiber growth

The recurrent mossy fiber pathway of the dentate gyrus expands dramatically in many persons with temporal lobe epilepsy. The new connections among granule cells provide a novel mechanism of synchronization that could enhance the participation of these cells in seizures. Despite the presence of robust recurrent mossy fiber growth, orthodromic or antidromic activation of granule cells usually does not evoke repetitive discharge. This study tested the ability of modestly elevated [K(+)](o), reduced GABA(A) receptor-mediated inhibition and frequency facilitation to unmask the effect of recurrent excitation. Transverse slices of the caudal hippocampal formation were prepared from pilocarpine-treated rats that either had or had not developed status epilepticus with subsequent recurrent mossy fiber growth. During superfusion with standard medium (3.5 mM K(+)), antidromic stimulation of the mossy fibers evoked epileptiform activity in 14% of slices with recurrent mossy fiber growth. This value increased to approximately 50% when [K(+)](o) was raised to either 4.75 or 6 mM. Addition of bicuculline (3 or 30 microM) to the superfusion medium did not enhance the probability of evoking epileptiform activity but did increase the magnitude of epileptiform discharge if such activity was already present. (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (1 microM), which selectively activates type II metabotropic glutamate receptors present on mossy fiber terminals, strongly depressed epileptiform responses. This result implies a critical role for the recurrent mossy fiber pathway. No enhancement of the epileptiform discharge occurred during repetitive antidromic stimulation at frequencies of 0.2, 1, or 10 Hz. In fact, antidromically evoked epileptiform activity became progressively attenuated during a 10-Hz train. Antidromic stimulation of the mossy fibers never evoked epileptiform activity in slices from control rats under any condition tested. These results indicate that even modest changes in [K(+)](o) dramatically affect granule cell epileptiform activity supported by the recurrent mossy fiber pathway. A small increase in [K(+)](o) reduces the amount of recurrent mossy fiber growth required to synchronize granule cell discharge. Block of GABA(A) receptor-mediated inhibition is less efficacious and frequency facilitation may not be a significant factor.     

Characteristics of local excitatory circuits studied with glutamate microapplication in the CA3 area of rat hippocampal slices

1. Local neuronal circuits in CA3 of hippocampal slices were studied by recording excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) intracellularly during glutamate microapplication in CA3. Control experiments validated this approach by providing evidence that glutamate microdrops stimulated neurons but not axons-of-passage or axon terminals in CA3. 2. Glutamate microdrops (10-20 mM, 10-20 microns diam) increased the firing frequency of extracellularly recorded dentate granule cells for 5-10 s when applied to their somata but not when applied to their mossy fiber axons and terminals in the hilus and in CA3. 3. Glutamate microapplications to granule cell somata, but not to mossy fiber axons, also increased the frequency of intracellularly recorded EPSPs in CA3 pyramidal cells for 5-10 s. This provided a second line of evidence that glutamate did not cause firing in mossy fiber axons synapsing in CA3. 4. In slices where the CA3 region was surgically separated from the dentate gyrus and CA2, glutamate microdrops placed in the CA3 stratum pyramidale within 400 microns of intracellularly recorded pyramidal cells increased the frequency of EPSPs and IPSPs. Tetrodotoxin (1 microgram/ml) blocked these increases in PSP frequency, indicating that they did not result from glutamate-induced depolarization and associated transmitter release from presynaptic terminals. Increases in PSP frequency were interpreted to reflect glutamate activations of CA3 neurons with local synaptic connections to recorded cells. 5. Low concentrations of picrotoxin (PTX, 5-10 microM) blocked glutamate-induced increases in IPSP frequency and often revealed increases in EPSP frequency where they were not previously observed. This suggests that recurrent inhibitory circuits normally mask or block transmission through recurrent excitatory pathways in CA3. 6. In five experiments following PTX treatment (7.5-10 microM), large and prolonged (up to 2 min) increases in EPSP frequency were observed in CA3 pyramidal cells to glutamate microapplications in CA3. Rhythmic epileptiform bursts eventually occurred in two of these cases, suggesting that the protracted increases in EPSP frequency represent a form of reverberating excitation during a transition from normal to epileptic states. 7. Sixteen CA3 pyramidal cells were recorded in PTX (5-10 microM) during glutamate microapplications at 200 and 400 microns on each side of the recording site. The most consistent glutamate-induced increases in EPSP frequency occurred to microapplications 200 microns from recording sites on the hilar side.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Functional changes in neuropeptide Y- and somatostatin-containing neurons induced by limbic seizures in the rat

The influence of sustained epileptic seizures evoked by intraperitoneal injection of kainic acid on the gene expression of the neuropeptides somatostatin and neuropeptide Y and on the damage of neurons containing these peptides was studied in the rat brain. Injection of kainic acid induced an extensive loss of somatostatin and, though less pronounced, of neuropeptide Y neurons in the inner part of the hilus of the dentate gyrus. Neuropeptide Y-immunoreactive neurons located in the subgranular layer of the hilus, presumably pyramidal-shaped basket cells, were spared by the treatment. Although neuropeptide Y messenger RNA was not detected in granule cells of control rats, it was found there after kainic acid seizures at all time intervals investigated (12 h to 90 days after injection of kainic acid). High concentrations of neuropeptide Y messenger RNA were especially observed 24 h after injection of kainic acid. At this time neuropeptide Y messenger RNA was also transiently observed in CA1 pyramidal cells. Neuropeptide Y synthesis in granule cells in turn gave rise to an intense immunoreactivity of the peptide in the terminal field of mossy fibers which persisted for the entire time period (90 days) investigated. In addition, neuropeptide Y messenger RNA concentrations were also drastically elevated in presumptive basket cells located at the inner surface of the granule cell layer, especially at the “late” time intervals investigated (30–90 days after kainic acid).

These data support the concept that extensive activation of granule cells by limbic seizures contributes to the observed neuronal cell death in CA3 pyramidal neurons and interneurons of the hilus. Consecutively, basket cells containing neuropeptide Y and presumably GABA might be activated and participate in recurrent inhibition of granule cells. Neuropeptide Y-immunoreactive fibers observed in the inner molecular layer at “late” time intervals after kainic acid may result either from collateral sprouting of mossy fibers or from basket cells extensively expressing the peptide.

It is speculated that neuropeptide Y synthesized and released at a high rate from granule cells and basket cells may exert a protective action against seizures.