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. Indexing termssomatostatin; neuropeptide Y; colocalization; status epilepticus Temporal lobe epilepsy (TLE) is a common form of acquired medically refractory partial epilepsy. The pathogenesis of TLE has been investigated intensively by human studies and animal models, which have provided several explanations for the increased excitability of neurons in the epileptic hippocampus. These potential mechanisms include sprouting of new axon collaterals resulting in the formation of recurrent excitatory connections on granule cells, changes in the intrinsic excitability of neurons (McNamara, 1999), and the altered expression of neurotransmitter receptors, such as NMDA and GABA A receptors (Coulter, 2001 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptOne controversial and intensely investigated pathogenic mechanism is altered GABAmediated inhibition. Neuroanatomical studies in human surgical specimens and experimental animal models of TLE have demonstrated a loss of inhibitory interneurons in the hilus of the dentate gyrus of the hippocampus (de Lanerolle et al., 1989;Sloviter, 1991;Obenaus et al., 1993;Mathern et al., 1995;Houser and Esclapez, 1996;Kobayashi and Buckmaster, 2003). These neuroanatomical studies are supported by electrophysiological studies demonstrating diminished paired pulse inhibition of CA1 pyramidal neurons and dentate granule cells (DGCs) (Kapur et al., 1989;Lothman et al., 19...

GABA A receptors are pentamers composed of subunits derived from the α, β, γ, δ, θ, ε, and π gene families. α1, α4, γ2, and δ 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 α1, α4, γ2, and δ 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 α1 subunit immunoreactive clusters and 32.35±1.49% of the γ2 subunit clusters. In contrast, only 1.58±0.13% of the α4 subunit immunoreactive clusters and 1.92±0.15% of the δ 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 α1 subunit immunoreactive clusters and 23.45±0.32% of the γ2 subunit immunoreactive clusters. In contrast, only 1.90±0.13% of the α4 subunit immunoreactive clusters and 1.76±0.10% of the δ subunit clusters colocalized with gephyrin. These studies demonstrate that a subset of α1 and γ2 subunit clusters colocalize with synaptic markers in hippocampal dentate granule cells. Furthermore, all four subunits, α1, α4, γ2, and δ, are present in the extrasynaptic locations.

Neurosteroid sensitivity of GABAA receptor mediated inhibition of the hippocampal dentate granule cells (DGCs) is reduced in animal models of temporal lobe epilepsy. However, the properties and subunit composition of GABAA receptors mediating tonic inhibition in DGCS of epileptic animals have not been described. In the DGCs of epileptic animals, allopregnanolone and L-65708 sensitivity of holding current was diminished and δ subunit was retained in the endoplasmic reticulum and its surface expression was decreased the in the hippocampus. Ro15–4513 and lanthanum had distinct effects on holding current recorded from DGCs of control and epileptic animals. The pharmacological properties of GABAA receptors maintaining tonic inhibition in DGCs of epileptic animals were similar to those containing the α4βxγ2 subunits. Furthermore, surface expression of the α4 subunit increased and a larger fraction of the subunit was co-immunoprecipitated with the γ2 subunit in hippocampi of epileptic animals. Together these studies revealed that functional α4βxδ and α5βxγ2 receptors were reduced in the hippocampi of epileptic animals, and that novel α4bxγ2 receptors contributed to the maintenance of tonic inhibition. The presence of α4βxγ2 receptors resulted in low GABA affinity and neurosteroid sensitivity of tonic currents in the DGCs of epileptic animals that could potentially increase seizure vulnerability. These receptors may represent a novel therapeutic target for anticonvulsant drugs without sedative actions.

In animal models of temporal lobe epilepsy (TLE), neurosteroid sensitivity of GABA A 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 GABA A 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[1,5␣][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 ␣4 subunit. Expression of the ␣4 subunit and its colocalization with the synaptic marker GAD65 was increased in epileptic DGCs. Location of the ␣4 subunit in relation to symmetric (inhibitory) synapses on soma and dendrites of control and epileptic DGCs was examined with postembedding immunogold electron microscopy. The ␣4 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 ␣4 subunit-containing receptors are present at synapses and participate in synaptic transmission. These changes may facilitate seizures in epileptic animals.

Organophosphates (OP) inhibit the enzyme cholinesterase and cause accumulation of acetylcholine, and are known to cause seizures and status epilepticus (SE) in humans. The animal models of SE caused by organophosphate analogs of insecticides are not well characterized. SE caused by OPs paraoxon and diisopropyl fluorophosphate (DFP) in rats was characterized by electroencephalogram (EEG), behavioral observations and response to treatment with the benzodiazepine diazepam administered at various stages of SE. A method for SE induction using intrahippocampal infusion of paraoxon was also tested. Infusion of 200 nmol paraoxon into the hippocampus caused electrographic seizures in 43/52 (82.7%) animals tested; and of these animals, 14/43 (30%) had self-sustaining seizures that lasted 4–18 hours after the end of paraoxon infusion. SE was also induced by peripheral subcutaneous injection of diisopropyl fluorophosphate (DFP, 1.25 mg/kg) or paraoxon (1.00 mg/kg) to rats pretreated with atropine (2 mg/kg) and 2-pralidoxime (2-PAM,50 mg/kg) 30 minutes prior to OP injection. SE occurred in 78% paraoxon–treated animals and in 79% of DFP-treated animals. Diazepam (10 mg/kg) was administered 10 min and 30 min after the onset of continuous EEG seizures induced by paraoxon and it terminated SE in a majority of animals at both time points. DFP-induced SE was terminated in 60% animals when diazepam was administered 10 minutes after the onset of continuous EEG seizure activity but diazepam did not terminate SE in any animal when it was administered 30 minutes after the onset of continuous seizures. These studies demonstrate that both paraoxon and DFP can induce SE in rats but refractoriness to diazepam is a feature of DFP induced SE.

We combined a study of the subcellular distribution of the ␣1, ␣2, ␣4, ␤1, ␤2/3, ␥2, and ␦ subunits of the GABA A receptor with an electrophysiological analysis of GABA A receptor currents to determine the types of receptors expressed on cultured hippocampal pyramidal neurons. The immunocytochemistry study demonstrated that ␣1, ␣2, ␤2/3, and ␥2 subunits formed distinct clusters of various sizes, which were colocalized with clusters of glutamate decarboxylase (GAD) immunoreactivity at rates ranging from 22 to 58%. In contrast, ␣4, ␤1, and ␦ subunits were distributed diffusely over the cell soma and neuronal processes of cultured neurons and did not colocalize with the synaptic marker GAD. Whole-cell GABA A receptor currents were moderately sensitive to GABA and were modulated by diazepam. The whole-cell currents were also enhanced by the neurosteroid allopregnanolone (10 nM). Tonic currents, measured as changes in baseline current and noise, were sensitive to Zn 2ϩ , furosemide, and loreclezole; they were insensitive to diazepam. These studies suggest that two kinds of GABA A receptors are expressed on cultured hippocampal neurons. One kind of receptor formed clusters, which were present at GABAergic synapses and in the extrasynaptic membrane. The ␣1, ␣2, ␤2/3, and ␥2 subunits were contained in clustered receptors. The second kind was distributed diffusely in the extrasynaptic membrane. The ␣4, ␤1, and ␦ subunits were contained in these diffusely distributed receptors. The properties of tonic currents recorded from these neurons were similar to those from recombinant receptors containing ␣4, ␤1, and ␦ subunits.

Inhibitory neurosteroids, molecules generated in glia from circulating steroid hormones and de novo from cholesterol, keep seizures in check in epileptic animals. They can enhance inhibitory transmission mediated by gamma‐aminobutyric acid receptors and have anticonvulsant action. ANN NEUROL 2010;67:689–693

Homeostatic control of synaptic efficacy is often mediated by dynamic regulation of excitatory synaptic receptors. Here, we report a novel form of homeostatic synaptic plasticity based on regulation of shunt currents that control dendritosomatic information transfer. In cortical pyramidal neurons from wild-type mice, HCN1 channels underlie a dendritic hyperpolarization-activated cationic current (I h ) that serves to limit temporal summation of synaptic inputs. In HCN1 knock-out mice, as expected, I h is reduced in pyramidal neurons and its effects on synaptic summation are strongly diminished. Unexpectedly, we found a markedly enhanced bicuculline-and L-655,708-sensitive background GABA A current in these cells that could be attributed to selective upregulation of GABA A ␣5 subunit expression in the cortex of HCN1 knock-out mice. Strikingly, despite diminished I h , baseline sublinear summation of evoked EPSPs was unchanged in pyramidal neurons from HCN1 knock-out mice; however, blocking tonic GABA A currents with bicuculline enhanced synaptic summation more strongly in pyramidal cells from HCN1 knock-out mice than in those cells from wild-type mice. Increasing tonic GABA A receptor conductance in the context of reduced I h , using computational or pharmacological approaches, restored normal baseline synaptic summation, as observed in neurons from HCN1 knock-out mice. These data indicate that upregulation of ␣5 subunit-mediated GABA A receptor tonic current compensates quantitatively for loss of dendritic I h in cortical pyramidal neurons from HCN1 knock-out mice to maintain normal synaptic summation; they further imply that dendritosomatic synaptic efficacy is a controlled variable for homeostatic regulation of cortical neuron excitability in vivo.