Altered expression and localization of hippocampal A-type potassium channel subunits in the pilocarpine-induced model of temporal lobe epilepsy

Altered ion channel expression and/or function may contribute to the development of certain human epilepsies. In rats, systemic administration of pilocarpine induces a model of human temporal lobe epilepsy, wherein a brief period of status epilepticus (SE) triggers development of spontaneous recurrent seizures that appear after a latency of 2-3 weeks. Here we investigate changes in expression of A-type voltage-gated potassium (Kv) channels, which control neuronal excitability and regulate action potential propagation and neurotransmitter release, in the pilocarpine model of epilepsy. Using immunohistochemistry, we examined the expression of component subunits of somatodendritic (Kv4.2, Kv4.3, KChIPl and KChIP2) and axonal (Kv1.4) A-type Kv channels in hippocampi of pilocarpine-treated rats that entered SE. We found that Kv4.2, Kv4.3 and KChIP2 staining in the molecular layer of the dentate gyrus changes from being uniformly distributed across the molecular layer to concentrated in just the outer two-thirds. We also observed a loss of KChIP1 immunoreactive interneurons, and a reduction of Kv4.2 and KChIP2 staining in stratum radiatum of CA1. These changes begin to appear 1 week after pilocarpine treatment and persist or are enhanced at 4 and 12 weeks. As such, these changes in Kv channel distribution parallel the acquisition of recurrent spontaneous seizures as observed in this model. We also found temporal changes in Kv1.4 immunoreactivity matching those in Timm's stain, being expanded in stratum lucidum of CA3 and in the inner third of the dentate molecular layer. Among pilocarpine-treated rats, changes were only observed in those that entered SE. These changes in A-type Kv channel expression may contribute to hyperexcitability of dendrites in the associated hippocampal circuits as observed in previous studies of the effects of pilocarpine-induced SE.     

Disruption of ClC-2 expression is associated with progressive neurodegeneration in aging mice

Heterozygous mutations in ClC-2 have been associated in rare cases with increased susceptibility to generalized, idiopathic epilepsy. Initially, it was hypothesized that mutations in ClC-2 may be associated with epilepsy due to a direct role for ClC-2 in the modification of hippocampal neuronal excitability. However, the absence of an overt seizure-susceptibility phenotype in young ClC-2 knockout (KO) mice rendered this hypothesis- implausible. A recent study of older ClC-2 KO mice (>6 months) revealed abnormalities in the myelin of central axons and a subtle defect in the neuronal function in the central auditory pathway. These findings prompted us to re-examine hippocampal neuron morphology and excitability in older ClC-2 KO mice. Interestingly, electrocorticographic recordings obtained in older mice revealed spontaneous interictal spikes which are a marker of perturbed hippocampal neurotransmission with a resultant increase in excitation. This electrophysiological defect was associated with astrocyte activation and evidence of neuronal degeneration in the CA3 region of the hippocampus of these older mice. Together, these findings raise the possibility that ClC-2 expression plays a subtle neuroprotective role in the aging hippocampus.     

Reactive oxygen species generated by NADPH oxidase are involved in neurodegeneration in the pilocarpine model of temporal lobe epilepsy

Reactive oxygen species (ROS) appear to be involved in several neurodegenerative disorders. We tested the hypothesis that oxidative stress could have a role in the hippocampal neurodegeneration observed in temporal lobe epilepsy induced by pilocarpine. We first determined the spatio-temporal pattern of ROS generation, by means of detection with dihydroethidium oxidation, in the CA1 and CA3 areas and the dentate gyrus of the dorsal hippocampus during status epilepticus induced by pilocarpine. Fluoro-Jade B assays were also performed to detect degenerating neurons. ROS generation was increased in CA1, CA3 and the dentate gyrus after pilocarpine-induced seizures, which was accompanied by marked cell death. Treatment of rats with a NADPH oxidase inhibitor (apocynin) for 7 days prior to induction of status epilepticus was effective in decreasing both ROS production (by an average of 20%) and neurodegeneration (by an average of 61%). These results suggest an involvement of ROS generated by NADPH oxidase in neuronal death in the pilocarpine model of epilepsy.     

Network and pharmacological mechanisms leading to epileptiform synchronization in the limbic system in vitro

Seizures in patients presenting with mesial temporal lobe epilepsy result from the interaction among neuronal networks in limbic structures such as the hippocampus, amygdala and entorhinal cortex. Mesial temporal lobe epilepsy, one of the most common forms of partial epilepsy in adulthood, is generally accompanied by a pattern of brain damage known as mesial temporal sclerosis. Limbic seizures can be mimicked in vitro using preparations of combined hippocampus-entorhinal cortex slices perfused with artificial cerebrospinal fluid containing convulsants or nominally zero Mg(2+), in order to produce epileptiform synchronization. Here, we summarize experimental evidence obtained in such slices from rodents. These data indicate that in control animals: (i) prolonged, NMDA receptor-dependent epileptiform discharges, resembling electrographic limbic seizures, originate in the entorhinal cortex from where they propagate to the hippocampus via the perforant path-dentate gyrus route; (ii) the initiation and maintenance of these ictal discharges is paradoxically contributed by GABA (mainly type A) receptor-mediated mechanisms; and (iii) CA3 outputs, which relay a continuous pattern of interictal discharge at approximately 1Hz, control rather than sustain ictal discharge generation in entorhinal cortex. Recent work indicates that such a control is weakened in the pilocarpine model of epilepsy (presumably as a result of CA3 cell damage). In addition, in these experiments electrographic seizure activity spreads directly to the CA1-subiculum regions through the temporoammonic pathway. Studies reviewed here indicate that these changes in network interactions, along with other mechanisms of synaptic plasticity (e.g. axonal sprouting, decreased activation of interneurons, upregulation of bursting neurons) can confer to the epileptic, damaged limbic system, the ability to produce recurrent limbic seizures as seen in patients with mesial temporal lobe epilepsy.     

Neuregulin 1 regulates excitability of fast-spiking neurons through Kv1.1 and acts in epilepsy

Dysfunction of fast-spiking, parvalbumin-positive (FS-PV) interneurons is implicated in the pathogenesis of epilepsy. ErbB4, a key Neuregulin 1 (NRG1) receptor, is mainly expressed in this type of interneurons, and recent studies suggest that parvalbumin interneurons are a major target of NRG1-ErbB4 signaling in adult brain. Thus, we hypothesized that downregulation of NRG1-ErbB4 signaling in FS-PV interneurons is involved in epilepsy. We found that NRG1, through its receptor ErbB4, increased the intrinsic excitability of FS-PV interneurons. This effect was mediated by increasing the near-threshold responsiveness and decreasing the voltage threshold for action potentials through Kv1.1, a voltage-gated potassium channel. Furthermore, mice with specific deletion of ErbB4 in parvalbumin interneurons were more susceptible to pentylenetetrazole- and pilocarpine-induced models of epilepsy. Exogenous NRG1 delayed the onset of seizures and decreased their incidence and stage. Moreover, expression of ErbB4, but not ErbB2, was downregulated in human epileptogenic tissue. Together, our findings suggest that NRG1-ErbB4 signaling contributes to human epilepsy through regulating the excitability of FS-PV interneurons. ErbB4 may be a new target for anticonvulsant drugs in epilepsy.     

Synaptic connections from multiple subfields contribute to granule cell hyperexcitability in hippocampal slice cultures

Limbic status epilepticus and preparation of hippocampal slice cultures both produce cell loss and denervation. This commonality led us to hypothesize that morphological and physiological alterations in hippocampal slice cultures may be similar to those observed in human limbic epilepsy and animal models. To test this hypothesis, we performed electrophysiological and morphological analyses in long-term (postnatal day 11; 40-60 days in vitro) organotypic hippocampal slice cultures. Electrophysiological analyses of dentate granule cell excitability revealed that granule cells in slice cultures were hyperexcitable compared with acute slices from normal rats. In physiological buffer, spontaneous electrographic granule cell seizures were seen in 22% of cultures; in the presence of a GABA(A) receptor antagonist, seizures were documented in 75% of cultures. Hilar stimulation evoked postsynaptic potentials (PSPs) and multiple population spikes in the granule cell layer, which were eliminated by glutamate receptor antagonists, demonstrating the requirement for excitatory synaptic transmission. By contrast, under identical recording conditions, acute hippocampal slices isolated from normal rats exhibited a lack of seizures, and hilar stimulation evoked an isolated population spike without PSPs. To examine the possibility that newly formed excitatory synaptic connections to the dentate gyrus contribute to granule cell hyperexcitability in slice cultures, anatomical labeling and electrophysiological recordings following knife cuts were performed. Anatomical labeling of individual dentate granule, CA3 and CA1 pyramidal cells with neurobiotin illustrated the presence of axonal projections that may provide reciprocal excitatory synaptic connections among these regions and contribute to granule cell hyperexcitability. Knife cuts severing connections between CA1 and the dentate gyrus/CA3c region reduced but did not abolish hilar-evoked excitatory PSPs, suggesting the presence of newly formed, functional synaptic connections to the granule cells from CA1 and CA3 as well as from neurons intrinsic to the dentate gyrus. Many of the electrophysiological and morphological abnormalities reported here for long-term hippocampal slice cultures bear striking similarities to both human and in vivo models, making this in vitro model a simple, powerful system to begin to elucidate the molecular and cellular mechanisms underlying synaptic rearrangements and epileptogenesis.     

Conditional transgenic suppression of M channels in mouse brain reveals functions in neuronal excitability, resonance and behavior

In humans, mutations in the KCNQ2 or KCNQ3 potassium-channel genes are associated with an inherited epilepsy syndrome. We have studied the contribution of KCNQ/M-channels to the control of neuronal excitability by using transgenic mice that conditionally express dominant-negative KCNQ2 subunits in brain. We show that suppression of the neuronal M current in mice is associated with spontaneous seizures, behavioral hyperactivity and morphological changes in the hippocampus. Restriction of transgene expression to defined developmental periods revealed that M-channel activity is critical to the development of normal hippocampal morphology during the first postnatal weeks. Suppression of the M current after this critical period resulted in mice with signs of increased neuronal excitability and deficits in hippocampus-dependent spatial memory. M-current-deficient hippocampal CA1 pyramidal neurons showed increased excitability, reduced spike-frequency adaptation, attenuated medium afterhyperpolarization and reduced intrinsic subthreshold theta resonance. M channels are thus critical determinants of cellular and neuronal network excitability, postnatal brain development and cognitive performance.     

Hippocampal pathology in the human neuronal ceroid-lipofuscinoses: distinct patterns of storage deposition, neurodegeneration and glial activation

The neuronal ceroid-lipofuscinoses (NCLs) are recessively inherited lysosomal storage diseases, currently classified into 8 forms (CLN1-CLN8). Collectively, the NCLs constitute the most common group of progressive encephalopathies of childhood, and present with visual impairment, psychomotor deterioration and severe seizures. Despite recent identification of the underlying disease genes, the mechanisms leading to neurodegeneration and epilepsy in the NCLs remain poorly understood. To investigate these events, we examined the patterns of storage deposition, neurodegeneration, and glial activation in the hippocampus of patients with CLN1, CLN2, CLN3, CLN5 and CLN8 using histochemistry and immunohistochemistry. These different forms of NCL shared distinct patterns of neuronal degeneration in the hippocampus, with heavy involvement of sectors CA2-CA4 but relative sparing of CA1. This selective pattern of degeneration was also observed in immunohistochemically identified interneurons, which exhibited a graded severity of loss according to phenotype, with calretinin-positive interneurons relatively spared. Furthermore, glial activation was also regionally specific, with microglial activation most pronounced in areas of greatest neuronal loss, and astrocyte activation prominent in areas where neuronal loss was less evident. In conclusion, the NCLs share a common pattern of selective hippocampal pathology, distinct from that seen in the majority of temporal lobe epilepsies.     

Dynamic seizure-related changes in extracellular signal-regulated kinase activation in a mouse model of temporal lobe epilepsy

Extracellular signal-regulated kinase (ERK) is highly sensitive to regulation by neuronal activity and is critically involved in several forms of synaptic plasticity. These features suggested that alterations in ERK signaling might occur in epilepsy. Previous studies have described increased ERK phosphorylation immediately after the induction of severe seizures, but patterns of ERK activation in epileptic animals during the chronic period have not been determined. Thus, the localization and abundance of phosphorylated extracellular signal-regulated kinase (pERK) were examined in a pilocarpine model of recurrent seizures in C57BL/6 mice during the seizure-free period and at short intervals after spontaneous seizures. Immunolabeling of pERK in control animals revealed an abundance of distinctly-labeled neurons within the hippocampal formation. However, in pilocarpine-treated mice during the seizure-free period, the numbers of pERK-labeled neurons were substantially decreased throughout much of the hippocampal formation. Double labeling with a general neuronal marker suggested that the decrease in pERK-labeled neurons was not due primarily to cell loss. The decreased ERK phosphorylation in seizure-prone animals was interpreted as a compensatory response to increased neuronal excitability within the network. Nevertheless, striking increases in pERK labeling occurred at the time of spontaneous seizures and were evident in large populations of neurons at very short intervals (as early as 2 min) after detection of a behavioral seizure. These findings suggest that increased pERK labeling could be one of the earliest immunohistochemical indicators of neurons that are activated at the time of a spontaneous seizure.     

Slow feedback inhibition in the Ca3 area of the rat hippocampus by synergistic synaptic activation of mGluR1 and mGluR5

Interneurons are critical in regulating the excitability of principal cells in neuronal circuits, thereby modulating the output of neuronal networks. We investigated synaptically evoked inhibitory responses in CA3 pyramidal cells mediated by metabotropic glutamate receptors (mGluRs) expressed somatodendritically by interneurons. Although pharmacological activation of mGluRs in interneurons has been shown to enhance their excitability, the inability to record mGluR-mediated synaptic responses has precluded detailed characterization of mGluR function in hippocampal interneurons. We found that a single extracellular pulse in CA3 stratum pyramidale was sufficient to induce disynaptic inhibitory responses mediated by postsynaptic mGluRs of the interneurons in CA3 pyramidal cells of hippocampal slice cultures. The disynaptic inhibitory response followed a short-latency monosynaptic inhibitory response, and was observed at stimulus intensities evoking half-maximal monosynaptic IPSCs. Synergistic activation of mGluR1 and mGluR5 was required to induce the full inhibitory response. When recordings were obtained from interneurons in CA3 stratum radiatum or stratum oriens, a single extracellular stimulus induced a slow inward cationic current with a time course corresponding to the slow inhibitory response measured in pyramidal cells. DCG IV, a group II mGluR agonist, which specifically blocks synaptic transmission through mossy fibres, had no effect on mGluR-mediated synaptic responses in interneurons, suggesting that feed-forward inhibition via mossy fibres is not involved. Thus, postsynaptic mGluR1 and mGluR5 in hippocampal interneurons cooperatively mediate slow feedback inhibition of CA3 pyramidal cells. This mechanism may allow interneurons to monitor activity levels from populations of neighbouring principal cells to adapt inhibitory tone to the state of the network.     

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

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

Hippocampal neurons and glia in epileptic EL mice

Reactive changes in hippocampal astrocytes are frequently encountered in association with temporal lobe epilepsy in humans and with drug or kindling-induced seizures in animal models. These reactive changes generally involve increases in astrocyte size and number and often occur together with neuronal loss and synaptic rearrangements. In addition to producing astrocytic changes, seizure activity can also produce reactive changes in microglia, the resident macrophages of brain. In this study, we examined the effects of recurrent seizure activity on hippocampal neurons and glia in the epileptic EL mouse, a natural model of human multifactorial idiopathic epilepsy and complex partial seizures. Timm staining was used to evaluate infrapyramidal mossy fiber organization and the optical dissector method was used to count Nissl-stained neurons in hippocampus of adult (about one year of age) EL mice and nonepileptic C57BL/6J (B6) and DDY mice. Immunostaining forglial fibrillary acidic protein (GFAP) and Iba1, an actin cross-linking molecule restricted to macrophages and microglia, was used to evaluate astrocytes and microglia, respectively. The EL mice experienced about 25–30 complex partial seizures with secondary generalization during routine weekly cage changing. No significant differences were found among the mouse strains for Timm staining scores or for neuronal counts in the CA1 and CA3 pyramidal fields or in the hilus. However, the number of GFAP-positive astrocytes was significantly elevated in the stratum radiatum and hilus of EL mice, while microglia appeared hyper-ramified and were more intensely stained in EL mice than in the B6 or DDY mice in the hilus, parietal cortex, and pyriform cortex. The results indicate that recurrent seizure activity in EL mice is associated with abnormalities in hippocampal astrocytes and brain microglia, but is not associated with obvious neuronal loss or mossy fiber synaptic rearrangements. The EL mouse can be a useful model for evaluating neuron-glia interactions related to idiopathic epilepsy.     

Acute and chronic responses to the convulsant pilocarpine in DBA/2J and A/J mice

Characterizing the responses of different mouse strains to experimentally-induced seizures can provide clues to the genes that are responsible for seizure susceptibility, and factors that contribute to epilepsy. This approach is optimal when sequenced mouse strains are available. Therefore, we compared two sequenced strains, DBA/2J (DBA) and A/J. These strains were compared using the chemoconvulsant pilocarpine, because pilocarpine induces status epilepticus, a state of severe, prolonged seizures. In addition, pilocarpine-induced status is followed by changes in the brain that are associated with the pathophysiology of temporal lobe epilepsy (TLE). Therefore, pilocarpine can be used to address susceptibility to severe seizures, as well as genes that could be relevant to TLE. A/J mice had a higher incidence of status, but a longer latency to status than DBA mice. DBA mice exhibited more hippocampal pyramidal cell damage. DBA mice developed more ectopic granule cells in the hilus, a result of aberrant migration of granule cells born after status. DBA mice experienced sudden death in the weeks following status, while A/J mice exhibited the most sudden death in the initial hour after pilocarpine administration. The results support previous studies of strain differences based on responses to convulsants. They suggest caution in studies of seizure susceptibility that are based only on incidence or latency. In addition, the results provide new insight into the strain-specific characteristics of DBA and A/J mice. A/J mice provide a potential resource to examine the progression to status. The DBA mouse may be valuable to clarify genes regulating other seizure-associated phenomena, such as seizure-induced neurogenesis and sudden death.     

Physical training reverts hippocampal electrophysiological changes in rats submitted to the pilocarpine model of epilepsy

Physical exercise and fitness programs in patients with epilepsy are still a matter of controversy. Effects of physical exercise in animals with epilepsy have been demonstrated. To further investigate the possible mechanisms by which physical activity interferes with epileptogenesis, the present work was aimed to study the effect of aerobic exercise on "in vitro" hippocampal electrophysiological parameters observed in rats submitted to the pilocarpine model of epilepsy. Electrophysiological changes were monitored by extracellular field potentials recorded from CA1 area. Control rats and rats with epilepsy were submitted to an aerobic exercise program. The number of population spikes (PS) and slope of field excitatory postsynaptic potentials (fEPSP) were analyzed. Trained rats with epilepsy exhibited a reduction in PS when compared with nontrained rats with epilepsy in different concentrations of extracellular potassium or bicuculline. Physical training also enhanced the late phase of LTP in rats with epilepsy. Our results indicate that physical training reduces CA1 hyperresponsiveness and can modify synaptic plasticity in rats submitted to the pilocarpine model of limbic epilepsy.     

Investigation of the neuronal aggregate generating seizures in the rat tetanus toxin model of epilepsy

A key question in epilepsy is the organization and size of the neuronal networks necessary for generating seizures. Hypotheses include: a single focal neuronal network drives seizure discharges across the brain, which may or may not be identical with the circuits that generate interictal spikes; or multiple neuronal networks link together in re-entrant loops or other long-range networks. It remains unclear whether any of these hypotheses apply to spontaneous seizures in freely moving animals. We used the tetanus toxin chronic model of epilepsy to test the different predictions made by each hypothesis about the propagation and interaction of epileptic discharges during seizures. Seizures could start in either the injected or noninjected dorsal hippocampus, suggesting that seizures have multifocal onsets in the tetanus toxin model. During seizures, individual bursts propagated in either direction, both between the right and left dorsal hippocampi, and between CA3 and the dentate gyrus in the same hippocampus. These findings argue against one site "driving" seizures or seizures propagating around a limbic loop. Specifically, the side leading each burst switched a median of three times during the first 20 s of a seizure. Analysis of bursts during seizures suggested that the network at each recording site acted like a neuronal oscillator. Coupling of population spikes in right and left CA3 increased during the early part of seizures, but the cross-correlation of their whole-discharge waveforms changed little over the same period. Furthermore, the polarity of the phase difference between population spikes did not follow the phase difference for complete discharges. We concluded that the neuronal aggregate necessary for seizures in our animals comprises multiple spatially distributed neuronal networks and that the increased synchrony of the output (population spike firing) of these networks during the early part of seizures may contribute to seizure generation.     

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

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