In cerebellar granule cells, delta subunit-containing GABA(A) receptors are found exclusively at extrasynaptic sites, but their subcellular distribution in other brain areas is poorly understood. We examined the anatomical localization and physiological activation of these receptors in adult mouse dentate gyrus granule cells. Immunocytochemistry revealed a high density of delta subunits in the molecular layer and a much lower density in the cell body layer. At the ultrastructural level, immunogold-labeled delta subunits were found at the edge of symmetric synapses on granule cell dendrites. Functional correlates of this perisynaptic localization were obtained by comparing inhibitory responses in delta subunit-deficient (delta-/-) and wild-type (wt) mice. In whole-cell recordings at 22 degrees C, the weighted decay time constants (tau(w)) of spontaneous IPSCs (sIPSCs) were significantly longer in wt mice but were similar at 34 degrees C, reflecting the role of temperature-dependent GABA uptake in shaping sIPSC decay. IPSCs evoked by minimal stimulation (eIPSCs) near the somata had similar tau(w) in delta-/- and wt mice, but eIPSCs elicited from dendritic sites decayed significantly more slowly in wt mice, consistent with a higher density of delta subunit-containing receptors in the molecular layer. The tau(w) of dendritic eIPSCs of wt mice were shortened by ZnCl2 (10 microm), reflecting the high Zn2+ sensitivity of delta subunit-containing GABA(A) receptors, and were prolonged by the GAT-1 GABA transporter inhibitor NO711 (10 microm). Our results demonstrate a perisynaptic localization of delta subunit-containing GABA(A) receptors and indicate that these receptors can be activated by GABA overspill in the molecular layer.

Complex changes in GABA A receptors (GABA A Rs) in animal models of temporal lobe epilepsy during the chronic period include a decrease in the ␦ subunit and increases in the ␣4 and ␥2 subunits in the dentate gyrus. We used postembedding immunogold labeling to determine whether the subcellular locations of these subunits were also altered in pilocarpine-treated epileptic mice, and related functional changes were identified electrophysiologically. The ultrastructural studies confirmed a decrease in ␦ subunit labeling at perisynaptic locations in the molecular layer of the dentate gyrus where these subunits are critical for tonic inhibition. Unexpectedly, tonic inhibition in dentate granule cells was maintained in the epileptic mice, suggesting compensation by other GABA A Rs. An insensitivity of the tonic current to the neurosteroid tetrahydrodeoxy-corticosterone was consistent with decreased expression of the ␦ subunit. In the pilocarpine-treated mice, ␣4 subunit labeling remained at perisynaptic locations, but increased ␥2 subunit labeling was also found at many perisynaptic locations on granule cell dendrites, consistent with a shift of the ␥2 subunit from synaptic to perisynaptic locations and potential partnership of the ␣4 and ␥2 subunits in the epileptic animals. The decreased ␥2 labeling near the center of synaptic contacts was paralleled by a corresponding decrease in the dendritic phasic inhibition of granule cells in the pilocarpine-treated mice. These GABA A R subunit changes appear to impair both tonic and phasic inhibition, particularly at granule cell dendrites, and could reduce the adaptive responses of the GABA system in temporal lobe epilepsy.

In central neurons, a tonic conductance is activated by ambient levels of the inhibitory transmitter GABA. Here, we show that in dentate gyrus granule cells, where tonic inhibition is mediated by ␦ subunit-containing GABA A receptors, this conductance is augmented by low concentrations (30 mM) of ethanol. In contrast, the tonic inhibition mediated by ␣5 subunit-containing receptors of CA1 pyramidal cells is not affected. The effect of ethanol on tonic inhibition specifically reduces the excitability of the dentate gyrus and identifies the ␦ subunit-dependent tonic inhibition as a likely site of ethanol action in the brain.

Expanded polyglutamine (polyQ) proteins in Huntington's disease (HD) as well as other polyQ disorders are known to elicit a variety of intracellular toxicities, but it remains unclear whether polyQ proteins can elicit pathological cell-cell interactions which are critical to disease pathogenesis. To test this possibility, we have created conditional HD mice expressing a neuropathogenic form of mutant huntingtin (mhtt-exon1) in discrete neuronal populations. We show that mhtt aggregation is a cell-autonomous process. However, progressive motor deficits and cortical neuropathology are only observed when mhtt expression is in multiple neuronal types, including cortical interneurons, but not when mhtt expression is restricted to cortical pyramidal neurons. We further demonstrate an early deficit in cortical inhibition, suggesting that pathological interactions between interneurons and pyramidal neurons may contribute to the cortical manifestation of HD. Our study provides genetic evidence that pathological cell-cell interactions elicited by neuropathogenic forms of mhtt can critically contribute to cortical pathogenesis in a HD mouse model.

No abstract

The endoribonuclease, Dicer, is indispensible for generating the majority of mature microRNAs (miRNAs), which are posttranscriptional regulators of gene expression involved in a wide range of developmental and pathological processes in mammalian central nervous system. While functions of Dicer-dependent miRNA pathways in neurons and oligodendrocytes have been extensively investigated, little is known about the role of Dicer in astrocytes. Here we report the effect of Cre-loxP mediated conditional deletion of Dicer selectively from postnatal astroglia on brain development. Dicer-deficient mice exhibited normal motor development and neurological morphology prior to postnatal week 5. Thereafter mutant mice invariably developed a rapidly fulminant neurological decline characterized by ataxia, severe progressive cerebellar degeneration, seizures, uncontrollable movements and premature death by postnatal week 9–10. Integrated transcription profiling, histological and functional analyses of cerebella showed that deletion of Dicer in cerebellar astrocytes altered the transcriptome of astrocytes to be more similar to an immature or reactive-like state prior to the onset of neurological symptoms or morphological changes. As a result, critical and mature astrocytic functions including glutamate uptake and antioxidant pathways were substantially impaired, leading to massive apoptosis of cerebellar granule cells and degeneration of Purkinje cells. Collectively, our study demonstrates the critical involvement of Dicer in normal astrocyte maturation and maintenance. Our findings also reveal non-cell autonomous roles of astrocytic Dicer-dependent pathways in regulating proper neuronal functions and implicate that loss of or dysregulation of astrocytic Dicer-dependent pathways may be involved in neurodegeneration and other neurological disorders.

Axonal sprouting of excitatory neurons is frequently observed in temporal lobe epilepsy, but the extent to which inhibitory interneurons undergo similar axonal reorganization remains unclear. The goal of this study was to determine whether somatostatin (SOM)-expressing neurons in stratum (s.) oriens of the hippocampus exhibit axonal sprouting beyond their normal territory and innervate granule cells of the dentate gyrus in a pilocarpine model of epilepsy. To obtain selective labeling of SOM-expressing neurons in s. oriens, a Cre recombinase-dependent construct for channelrhodopsin2 fused to enhanced yellow fluorescent protein (ChR2-eYFP) was virally delivered to this region in SOM-Cre mice. In control mice, labeled axons were restricted primarily to s. lacunosum-moleculare. However, in pilocarpine-treated animals, a rich plexus of ChR2-eYFP-labeled fibers and boutons extended into the dentate molecular layer. Electron microscopy with immunogold labeling demonstrated labeled axon terminals that formed symmetric synapses on dendritic profiles in this region, consistent with innervation of granule cells. Patterned illumination of ChR2-labeled fibers in s. lacunosum-moleculare of CA1 and the dentate molecular layer elicited GABAergic inhibitory responses in dentate granule cells in pilocarpine-treated mice but not in controls. Similar optical stimulation in the dentate hilus evoked no significant responses in granule cells of either group of mice. These findings indicate that under pathological conditions, SOM/GABAergic neurons can undergo substantial axonal reorganization beyond their normal territory and establish aberrant synaptic connections. Such reorganized circuitry could contribute to functional deficits in inhibition in epilepsy, despite the presence of numerous GABAergic terminals in the region.

Ethanol alters the distribution and abundance of PKC␦ in neural cell lines. Here we investigated whether PKC␦ also regulates behavioral responses to ethanol. PKC␦ Ϫ/Ϫ mice showed reduced intoxication when administered ethanol and reduced ataxia when administered the nonselective GABA A receptor agonists pentobarbital and pregnanolone. However, their response to flunitrazepam was not altered, suggesting that PKC␦ regulates benzodiazepine-insensitive GABA A receptors, most of which contain ␦ subunits and mediate tonic inhibitory currents in neurons. Indeed, the distribution of PKC␦ overlapped with GABA A ␦ subunits in thalamus and hippocampus, and ethanol failed to enhance tonic GABA currents in PKC␦ Ϫ/Ϫ thalamic and hippocampal neurons. Moreover, using an ATP analog-sensitive PKC␦ mutant in mouse L(tk Ϫ ) fibroblasts that express ␣4␤3␦ GABA A receptors, we found that ethanol enhancement of GABA currents was PKC␦-dependent. Thus, PKC␦ enhances ethanol intoxication partly through regulation of GABA A receptors that contain ␦ subunits and mediate tonic inhibitory currents. These findings indicate that PKC␦ contributes to a high level of behavioral response to ethanol, which is negatively associated with risk of developing an alcohol use disorder in humans.