Jaideep Kapur

A gain-of-function mutation in the sodium channel gene Scn2a results in seizures and behavioral abnormalities

The GAL879-881QQQ mutation in the cytoplasmic S4-S5 linker of domain 2 of the rat brain IIA sodium channel (Na(v)1.2) results in slowed inactivation and increased persistent current when expressed in Xenopus oocytes. The neuron-specific enolase promoter was used to direct in vivo expression of the mutated channel in transgenic mice. Three transgenic lines exhibited seizures, and line Q54 was characterized in detail. The seizures in these mice began at two months of age and were accompanied by behavioral arrest and stereotyped repetitive behaviors. Continuous electroencephalogram monitoring detected focal seizure activity in the hippocampus, which in some instances generalized to involve the cortex. Hippocampal CA1 neurons isolated from presymptomatic Q54 mice exhibited increased persistent sodium current which may underlie hyperexcitability in the hippocampus. During the progression of the disorder there was extensive cell loss and gliosis within the hippocampus in areas CA1, CA2, CA3 and the hilus. The lifespan of Q54 mice was shortened and only 25% of the mice survived beyond six months of age. Four independent transgenic lines expressing the wild-type sodium channel were examined and did not exhibit any abnormalities. The transgenic Q54 mice provide a genetic model that will be useful for testing the effect of pharmacological intervention on progression of seizures caused by sodium channel dysfunction. The human ortholog, SCN2A, is a candidate gene for seizure disorders mapped to chromosome 2q22-24.

Status Epilepticus Increases the Intracellular Accumulation of GABAA Receptors

Status epilepticus is a neurological emergency that results in mortality and neurological morbidity. It has been postulated that the reduction of inhibitory transmission during status epilepticus results from a rapid modification of GABAA receptors. However, the mechanism(s) that contributes to this modification has not been elucidated. We report, using an in vitro model of status epilepticus combined with electrophysiological and cellular imaging techniques, that prolonged epileptiform bursting results in a reduction of GABA-mediated synaptic inhibition. Furthermore, we found that constitutive internalization of GABAA receptors is rapid and accelerated by the increased neuronal activity associated with seizures. Inhibition of neuronal activity reduced the rate of internalization. These findings suggest that the rate of GABAA receptor internalization is regulated by neuronal activity and its acceleration contributes to the reduction of inhibitory transmission observed during prolonged seizures.

Subunit-Specific Trafficking of GABAA Receptors during Status Epilepticus

It is proposed that a reduced surface expression of GABAA receptors (GABARs) contributes to the pathogenesis of status epilepticus (SE), a condition characterized by prolonged seizures. This hypothesis was based on the finding that prolonged epileptiform bursting (repetitive bursts of prolonged depolarizations with superimposed action potentials) in cultures of dissociated hippocampal pyramidal neurons (dissociated cultures) results in the increased intracellular accumulation of GABARs. However, it is not known whether this rapid modification in the surface-expressed GABAR pool results from selective, subunit-dependent or nonselective, subunit-independent internalization of GABARs. In hippocampal slices obtained from animals undergoing prolonged SE (SE-treated slices), we found that the surface expression of the GABAR β2/3 and γ2 subunits was reduced, whereas that of the δ subunit was not. Complementary electrophysiological recordings from dentate granule cells in SE-treated slices demonstrated a reduction in GABAR-mediated synaptic inhibition, but not tonic inhibition. A reduction in the surface expression of the γ2 subunit, but not the δ subunit was also observed in dissociated cultures and organotypic hippocampal slice cultures when incubated in an elevated KCl external medium or an elevated KCl external medium supplemented with NMDA, respectively. Additional studies demonstrated that the reduction in the surface expression of the γ2 subunit was independent of direct ligand binding of the GABAR. These findings demonstrate that the regulation of surface-expressed GABAR pool during SE is subunit-specific and occurs independent of ligand binding. The differential modulation of the surface expression of GABARs during SE has potential implications for the treatment of this neurological emergency.

Ketamine controls prolonged status epilepticus

New treatments are needed to control prolonged status epilepticus given the high failure rate of current therapies. In an animal model of status epilepticus based on electrical stimulation of the hippocampus, rats demonstrate at least 5 five-hours of seizure activity following stimulation. Phenobarbital (70 mg/kg) administered 15 min after stimulation effectively controlled seizures in 66% of animals (n=6). When phenobarbital (70 mg/kg) was administered 60 min after stimulation, seizures were controlled in 25% of animals (n=4). Ketamine (100 mg/kg) administered 15 min after stimulation did not control seizures in any animal (n=4). But when ketamine was administered one hour after stimulation it effectively controlled seizures in all animals (n=4). Increasing doses of ketamine were administered 60 min after stimulation to generate a dose-response curve. The ketamine dose response (fraction of seizure free rats) data were fit to a sigmoid curve to derive an ED(50) of 58 mg/kg. These findings suggest that prolonged status epilepticus becomes refractory to phenobarbital but can be effectively controlled by ketamine. For patients experiencing prolonged status epilepticus that is refractory to phenobarbital, ketamine may be an alternative to general anesthesia.

Distribution of α1, α4, γ2, and δ subunits of GABAA receptors in hippocampal granule cells

GABAA receptors are pentamers composed of subunits derived from the alpha, beta, gamma, delta, theta, epsilon, and pi gene families. alpha1, alpha4, gamma2, and delta subunits are expressed in the dentate gyrus of the hippocampus, but their subcellular distribution has not been described. Hippocampal sections were double-labeled for the alpha1, alpha4, gamma2, and delta subunits and GAD65 or gephyrin, and their subcellular distribution on dentate granule cells was studied by means of confocal laser scanning microscopy (CLSM). The synaptic versus extrasynaptic localization of these subunits was inferred by quantitative analysis of the frequency of colocalization of various subunits with synaptic markers in high-resolution images. GAD65 immunoreactive clusters colocalized with 26.24+/-0.86% of the alpha1 subunit immunoreactive clusters and 32.35+/-1.49% of the gamma2 subunit clusters. In contrast, only 1.58+/-0.13% of the alpha4 subunit immunoreactive clusters and 1.92+/-0.15% of the delta subunit clusters colocalized with the presynaptic marker GAD65. These findings were confirmed by studying colocalization with immunoreactivity of a postsynaptic marker, gephyrin, which colocalized with 27.61+/-0.16% of the alpha1 subunit immunoreactive clusters and 23.45+/-0.32% of the gamma2 subunit immunoreactive clusters. In contrast, only 1.90+/-0.13% of the alpha4 subunit immunoreactive clusters and 1.76+/-0.10% of the delta subunit clusters colocalized with gephyrin. These studies demonstrate that a subset of alpha1 and gamma2 subunit clusters colocalize with synaptic markers in hippocampal dentate granule cells. Furthermore, all four subunits, alpha1, alpha4, gamma2, and delta, are present in the extrasynaptic locations.

A combination of ketamine and diazepam synergistically controls refractory status epilepticus induced by cholinergic stimulation

Purpose: New treatments are needed for status epilepticus (SE) that is refractory to drugs modulating GABAA receptors, and NMDA receptor antagonists are candidate drugs.

Selective Loss of Dentate Hilar Interneurons Contributes to Reduced Synaptic Inhibition of Granule Cells in an Electrical Stimulation-Based Animal Model of Temporal Lobe Epilepsy

Neuropeptide‐containing hippocampal interneurons and dentate granule cell inhibition were investigated at different periods following electrical stimulation‐induced, self‐sustaining status epilepticus (SE) in rats. Immunohistochemistry for somatostatin (SOM), neuropeptide Y (NPY), parvalbumin (PV), cholecystokinin (CCK), and Fluoro‐Jade B was performed on sections from hippocampus contralateral to the stimulated side and studied by confocal laser scanning microscopy. Compared to paired age‐matched control animals, there were fewer SOM and NPY‐immunoreactive (IR) interneurons in the hilus of the dentate gyrus in animals with epilepsy (40–60 days after SE), and 1, 3, and 7 days following SE. In the hilus of animals that had recently undergone SE, some SOM‐IR and NPY‐IR interneurons also stained for Fluoro‐Jade B. Furthermore, there was electron microscopic evidence of the degeneration of SOM‐IR interneurons following SE. In contrast, the number of CCK and PV‐IR basket cells in epileptic animals was similar to that in controls, although it was transiently diminished following SE; there was no evidence of degeneration of CCK or PV‐IR interneurons. Patch‐clamp recordings revealed a diminished frequency of inhibitory postsynaptic currents in dentate granule cells (DGCs) recorded from epileptic animals and animals that had recently undergone SE compared with controls. These results confirm the selective vulnerability of a particular subset of dentate hilar interneurons after prolonged SE. This loss may contribute to the reduced GABAergic synaptic inhibition of granule cells in epileptic animals. J. Comp. Neurol. 500:876–893, 2007.

Activity-dependent scaling of GABAergic synapse strength is regulated by brain-derived neurotrophic factor.

The homeostatic plasticity hypothesis suggests that neuronal activity scales synaptic strength. This study analyzed effects of activity deprivation on GABAergic synapses in cultured hippocampal neurons using patch clamp electrophysiology to record mIPSCs and immunocytochemistry to visualize presynaptic GAD-65 and the gamma2 subunit of the GABA(A) receptor. When neural activity was blocked for 48 h with tetrodotoxin (TTX, 1 microM), the amplitude of mIPSCs was reduced, corresponding with diminished sizes of GAD-65 puncta and gamma2 clusters. Treatment with the NMDA receptor antagonist APV (50 microM) or the AMPA receptor antagonist DNQX (20 microM) mimicked these effects, and co-application of brain-derived neurotrophic factor (BDNF, 100 ng/mL) overcame them. Moreover, when neurons were treated with BDNF alone for 48 h, these effects were reversed via the TrkB receptor. Overall, these results suggest that activity-dependent scaling of inhibitory synaptic strength can be modulated by BDNF/TrkB-mediated signaling.

Photothrombotic brain infarction results in seizure activity in aging Fischer 344 and Sprague Dawley rats

This study was designed to determine whether photothrombotic brain infarction could result in epileptic seizures in adult animals. Male Fischer 344 (F344) rats at 2, 6, 12, 24, and 30 months of age and male Sprague Dawley (SD) rats at 2 and 6 months of age underwent photothrombotic brain infarction with the photosensitive dye rose bengal by focusing a wide (6 mm) or narrow (3 mm) diameter white light beam on the skull overlying left hemisphere anterior frontal, midfrontal, frontoparietal, or parietal areas. Animals were monitored with video and EEG recordings. Morphological analysis of infarct size was performed with a computer-assisted image analysis system. The primary finding of this study was that epileptic seizures were recorded in post-mature rats 2 months after lesioning the frontoparietal cortex with large photothrombotic infarcts that extended to the cortical-subcortical interface. These seizures were characterized behaviorally by motor arrest, appeared to originate in the periinfarct area, and could be distinguished from inherited spontaneous bilateral cortical discharges by the morphology, frequency, duration, and laterality of the ictal discharges. Small cortical lesions were ineffective in producing seizures except for one animal that demonstrated recurrent prolonged focal discharges unaccompanied by behavioral change. Stage 3 seizures were observed in a small number of mid-aged and aged animals lesioned with large infarcts in anterior frontal and frontoparietal areas. These results suggest that the technique of photothrombosis can be used to produce neocortical infarction as a means to study mechanisms of secondary epileptogenesis.

Evidence for a chronic loss of inhibition in the hippocampus after kindling: electrophysiological studies

Rats were kindled with either of 2 protocols: (1) a rapidly recurring hippocampal seizure (RRHS) paradigm in which 10 sec stimulus trains were delivered every 5 min through hippocampal electrodes; and (2) a traditional approach in which 1 sec stimulus trains were given to the amygdala once daily. Three groups of kindled rats were prepared: (1) one of amygdala-kindled rats that had experienced 9-15 seizures; (2) one of RRHS-kindled rats that had experienced 96 seizures; and (3) one of RRHS-overkindled rats that had experienced 144-336 seizures. After a 1 month seizure-free period, the animals were anesthetized with urethane and measurements were made on the potency of paired pulse inhibition in the CA1 region of the hippocampus. All groups of kindled animals were found to have significantly less paired pulse inhibition than control rats. This decrement was confined to interpulse intervals less than or equal to 70 msec. The amount of inhibition lost correlated with the number of seizure that had occurred. The GABAergic agonist muscimol restored paired pulse inhibition in kindled animals for interpulse intervals less than or equal to 70 msec towards normal values. These results indicate that not only RRHS, but also other modes of kindling, reduced GABAergic inhibition in the CA1 region of the hippocampus and that this diminution was long-lasting, if not permanent.