Zakaria Mtchedlishvili

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.

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.

Diminished allopregnanolone enhancement of GABAA receptor currents in a rat model of chronic temporal lobe epilepsy

1 Neurosteroid modulation of GABAA receptors present on dentate granule cells (DGCs) acutely isolated from epileptic (epileptic DGCs) or control rats (control DGCs) was studied by application of GABA with or without the modulators and by measuring the amplitude of peak whole‐cell currents. 2 In epileptic DGCs, GABA efficacy (1394 ± 277 pA) was greater than in control DGCs (765 ± 38 pA). 3 Allopregnanolone enhanced GABA‐evoked currents less potently in epileptic DGCs (EC50= 92.7 ± 13.4 nm) than in control DGCs (EC50= 12.9 ± 2.3 nm). 4 Pregnenolone sulfate inhibited GABA‐evoked currents with similar potency and efficacy in control and epileptic DGCs. 5 Diazepam enhanced GABA‐evoked currents less potently in epileptic (EC50= 69 ± 14 nm) compared to the control DGCs (EC50= 29.9 ± 5.7 nm). 6 There were two different patterns of zolpidem modulation of GABAA receptor currents in the epileptic DGCs. In one group, zolpidem enhanced GABAA receptor currents but with reduced potency compared to the control DGCs (EC50= 134 ± 20 nmvs. EC50= 52 ± 13 nm). In the second group of epileptic DGCs zolpidem inhibited GABAA receptor currents, an effect not observed in control DGCs. 7 Epileptic DGCs were more sensitive to Zn2+ inhibition of GABAA receptor currents (IC50= 19 ± 6 μm) compared to control (IC50= 94.7 ± 7.9 μm). 8 This study demonstrates significant differences between epileptic and control DGCs. We conclude that (1) diminished sensitivity of GABAA receptors of epileptic DGCs to allopregnanolone can increase susceptibility to seizures; (2) reduced sensitivity to diazepam and zolpidem, and increased sensitivity to Zn2+ indicate that loss of allopregnanolone sensitivity is likely to be due to altered subunit expression of postsynaptic GABAA receptors present on epileptic DGCs; and (3) an inverse effect of zolpidem in some epileptic DGCs demonstrates the heterogeneity of GABAA receptors present on epileptic DGCs.

Diminished Neurosteroid Sensitivity of Synaptic Inhibition and Altered Location of the α4 Subunit of GABAA Receptors in an Animal Model of Epilepsy

In animal models of temporal lobe epilepsy (TLE), neurosteroid sensitivity of GABAA receptors on dentate granule cells (DGCs) is diminished; the molecular mechanism underlying this phenomenon remains unclear. The current study investigated a mechanism for loss of neurosteroid sensitivity of synaptic GABAA receptors in TLE. Synaptic currents recorded from DGCs of epileptic animals (epileptic DGCs) were less frequent, larger in amplitude, and less sensitive to allopregnanolone modulation than those recorded from DGCs of control animals (control DGCs). Synaptic currents recorded from epileptic DGCs were less sensitive to diazepam and had altered sensitivity to benzodiazepine inverse agonist RO 15-4513 (ethyl-8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5α][1,4]benzodiazepine-3-carboxylate) and furosemide than those recorded from control DGCs. Properties of synaptic currents recorded from epileptic DGCs appeared similar to those of recombinant receptors containing the α4 subunit. Expression of the α4 subunit and its colocalization with the synaptic marker GAD65 was increased in epileptic DGCs. Location of the α4 subunit in relation to symmetric (inhibitory) synapses on soma and dendrites of control and epileptic DGCs was examined with postembedding immunogold electron microscopy. The α4 immunogold labeling was present more commonly within the synapse in epileptic DGCs compared with control DGCs, in which the subunit was extrasynaptic. These studies demonstrate that, in epileptic DGCs, the neurosteroid modulation of synaptic currents is diminished and α4 subunit-containing receptors are present at synapses and participate in synaptic transmission. These changes may facilitate seizures in epileptic animals.

Development of γ‐aminobutyric acidergic synapses in cultured hippocampal neurons

The formation and maturation of γ‐aminobutyric acid (GABA)‐ergic synapses was studied in cultured hippocampal pyramidal neurons by both performing immunocytochemistry for GABAergic markers and recording miniature inhibitory postsynaptic currents (mIPSCs). Nascent GABAergic synapses appeared between 3 and 8 days in vitro (DIV), with GABAA receptor subunit clusters appearing first, followed by GAD‐65 puncta, then functional synapses. The number of GABAergic synapses increased from 7 to 14 DIV, with a corresponding increase in frequency of mIPSCs. Moreover, these new GABAergic synapses formed on neuronal processes farther from the soma, contributing to decreased mIPSC amplitude and slowed mIPSC 19–90% rise time. The mIPSC decay quickened from 7 to 14 DIV, with a parallel change in the distribution of the α5 subunit from diffuse expression at 7 DIV to clustered expression at 14 DIV. These α5 clusters were mostly extrasynaptic. The α1 subunit was expressed as clusters in none of the neurons at 7 DIV, in 20% at 14 DIV, and in 80% at 21 DIV. Most of these α1 clusters were expressed at GABAergic synapses. In addition, puncta of GABA transporter 1 (GAT‐1) were localized to GABAergic synapses at 14 DIV but were not expressed at 7 DIV. These studies demonstrate that mIPSCs appear after pre‐ and postsynaptic elements are in place. Furthermore, the process of maturation of GABAergic synapses involves increased synapse formation at distal processes, expression of new GABAA receptor subunits, and GAT‐1 expression at synapses; these changes are reflected in altered frequency, kinetics, and drug sensitivity of mIPSCs. J. Comp. Neurol. 495:497–510, 2006.

Increased neurosteroid sensitivity of hippocampal GABAA receptors during postnatal development.

To investigate developmental changes in neurosteroid modulation of GABA(A) receptors, whole-cell currents were elicited by applying GABA with allopregnanolone or pregnenolone sulfate (PS) to dentate granule cells (DGCs), acutely isolated from 7-14-day-old and adult rats. GABA evoked larger currents from dentate granule cells acutely isolated from adult rats (adult DGCs) than from neonatal DGCs, due to increased efficacy (1662+/-267 pA in adult DGCs versus 1094+/-198 pA in neonatal DGCs, P=0.004), and current density (0.072+/-0.01 pA/microm(2) in neonatal rat DGCs to 0.178+/-0.02 pA/microm(2) in adult DGCs), but unchanged potency (EC(50) was 18.5+/-2 microm in adult DGCs, and 26.6+/-7.9 microm in neonatal DGCs, P=0.21). Allopregnanolone sensitivity of GABA(A) receptor currents increased during development due to an increased potency (21.1+/-4.7 nM in adult DGCs versus 94.6+/-9 nM in neonatal DGCs, P=0.0002). The potency and efficacy of PS inhibition of GABA(A) receptor currents were remained unchanged during development (13+/-6 microm and 13.2+/-5.9 microm, P=0.71 and 85.5%+/-3.5% and 83.6%+/-0.8%, P=0.29, respectively). To investigate possible mechanism of developmental changes in GABA(A) receptor properties, in situ hybridization for alpha1, alpha4 and gamma2 subunit mRNAs was performed in dentate gyrus of the two age groups. Qualitatively, alpha1 subunit mRNA was expressed at low levels in neonatal rats while it was well expressed in adult rats. The alpha4 and gamma2 subunits were well expressed in the dentate gyrus of adult and neonatal rats. Immunohistochemical staining for alpha1 subunit in hippocampal slices from neonatal and adult rats was examined under confocal laser scanning microscope. This demonstrated that cell bodies and dendrites of granule cells are moderately positive for the alpha1 staining in adult rats but weakly so in neonatal rats. Higher-magnification images demonstrate large number of clusters of alpha1-subunit in the cell bodies of dentate granule cells of adult rat but rare clusters in granule cells of neonatal rats. Maturation of GABA(A) receptors in DGCs is characterized by increased number of GABA(A) receptors that are more sensitive to endogenous neurosteroid allopregnanolone, which might be related to increased expression of alpha1 subunit.

Characterization of the convulsant action of pregnenolone sulfate.

Pregnenolone sulfate (PS) is an endogenous neurosteroid synthesized by glial cells, which acts as a potent convulsant when injected intracerebroventricularly and intraperitoneally. PS is found in relatively high concentrations in the hippocampus. But its convulsant action in the hippocampus has not been characterized. A range of PS doses were infused directly into the right hippocampus of 42 rats, which were subsequently monitored for behavioral and electrographic seizures. At the highest dose (4 micromol), PS produced status epilepticus (SE) and severe behavioral convulsions. As the dose of PS was reduced, the fraction of rats having SE diminished (ED50 for SE = 2.7 micromol). At doses lower than 300 nmol, PS infusion produced discrete electrographic seizures (ED50 = 68 nmol) associated with mild behavioral seizures. Both the behavioral seizure score (BSS) and the total number of seizures during the observation period changed in a dose-dependent manner. In separate experiments in cultured hippocampal neurons, PS enhanced NMDA-evoked whole-cell currents (EC50 = 16 microM). The results demonstrate that the hippocampus is highly sensitive to the convulsant effects of PS and that the enhancement of NMDA currents could contribute to the convulsant action of PS.

HORMONES AND GENDER | Neurosteroid Modulation of GABA A Receptor-Mediated Synaptic Transmission in an Animal Model of Temporal Lobe Epilepsy

Temporal lobe epilepsy (TLE) is often accompanied by plasticity of hippocampal neurons and their synaptic connectivity. A potential mechanism that may contribute to increased hyperexcitability of these neurons is altered GABAA receptor-mediated synaptic inhibition. Neurosteroids can modulate the GABAA receptors and seizure activity. This article summarizes our observations on neurosteroid modulation of the GABAA receptors in the hippocampal dentate granule cells obtained from an animal model of temporal lobe epilepsy.