Chengsan Sun

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.

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 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.

Homeostatic regulation of synaptic excitability: tonic GABA(A) receptor currents replace I(h) in cortical pyramidal neurons of HCN1 knock-out mice.

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 GABAA current in these cells that could be attributed to selective upregulation of GABAA α5 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 GABAA 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 GABAA 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 α5 subunit-mediated GABAA 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.

GABAA Receptor Internalization during Seizures

Summary:  A rapid modification in the postsynaptic γ‐aminobutyric acid (GABAA) receptor population occurs during the prolonged seizures of status epilepticus (SE). This rapid modification contributes to a reduction in GABA‐mediated inhibition and the development of benzodiazepine pharmacoresistance. Previous hypotheses to explain the modification have included an alteration in the structural composition or posttranslational modification of the receptors. In a cultured hippocampal neuron model, we found that there was differential subcellular distribution of GABAA receptor subunits and that the constitutive internalization of GABAA receptors containing a β2/3 subunit was rapid and activity‐dependent. Based on this finding, we posit that an activity‐dependent increase in the rate of internalization of synaptic GABAA receptors during SE contributes to the reduction in inhibitory transmission and the development of benzodiazepine pharmacoresistance.

Receptors with low affinity for neurosteroids and GABA contribute to tonic inhibition of granule cells in epileptic animals

Neurosteroid sensitivity of GABA(A) 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 GABA(A) receptors mediating tonic inhibition in DGCs of epileptic animals have not been described. In the DGCs of epileptic animals, allopregnanolone and L-655708 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 suggesting that the pharmacological properties of GABA(A) 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 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.

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.

Endogenous neurosteroid synthesis modulates seizure frequency

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

Characterization of status epilepticus induced by two organophosphates in rats

Organophosphates (OPs) 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 200nmol 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 4-18h after the end of paraoxon infusion. SE was also induced by peripheral subcutaneous injection of diisopropyl fluorophosphate (DFP, 1.25mg/kg) or paraoxon (1.00mg/kg) to rats pretreated with atropine (2mg/kg) and 2-pralidoxime (2-PAM, 50mg/kg) 30min prior to OP injection. SE occurred in 78% paraoxon-treated animals and in 79% of DFP-treated animals. Diazepam (10mg/kg) was administered 10min and 30min 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 10min after the onset of continuous EEG seizure activity but diazepam did not terminate SE in any animal when it was administered 30min 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.

Loss of cholecystokinin-containing terminals in temporal lobe epilepsy

Altered GABA-mediated inhibition is proposed to play a role in the pathogenesis of epilepsy. Previous studies have demonstrated a loss of somatostatin-containing GABAergic interneurons innervating granule cells in epileptic animals. However, the reorganization of synapses between interneurons and granule cells has not been investigated. We studied synapse organization in an animal model of temporal lobe epilepsy (TLE) using continuous hippocampal stimulation. The distribution of axon terminals and inhibitory synapses on granule cell dendrites was studied using a combination of immunohistochemistry and pre-embedding electron microscopy techniques. A whole-cell patch-clamp technique was applied to study the functional changes in GABAergic input from different interneurons. In epileptic animals, the density of cholecystokinin (CCK)-immunoreactive (IR) fibers and α2 subunit containing GABAA receptors in the inner molecular layer of the dentate gyrus was reduced. Quantitative immuno-electron microscopy study revealed that the ratio of CCK-containing symmetric synapses to the total symmetric synapses was reduced. The frequency of GABAergic synaptic currents (sIPSC) was decreased and their amplitude was increased. The inhibitory effect of the activation of cannabinoid 1 (CB1) receptors was also reduced in epileptic animals. Isolation of CCK- and parvalbumin (PV)-containing GABAergic inputs by N- and P/Q-type calcium channel blockers respectively suggested that GABA release from CCK-containing interneurons was selectively reduced in epileptic rats. This study found that there was a loss of CCK-containing GABAergic synapses to granule cells both morphologically and functionally. These studies add to our understanding of the mechanisms that contribute to altering GABAergic inhibition of granule cells in TLE.