To determine their contributions to GABAA receptor (GABAR) channel properties, rat γ2L and δ subunits were acutely co‐expressed with α1 and β3 subtypes in mouse L929 fibroblasts to produce α1β3, α1β3δ or α1β3γ2L GABAR isoforms. With whole‐cell recording, the α1β3 isoform had relatively high sensitivity to GABA (EC50 2.1 μm) and low maximum current amplitude. The α1β1δ isoform had similar sensitivity to GABA (EC50 2.8 μm) and low current amplitude. The α1β3γ2L isoform had lower sensitivity to GABA (EC50 11.6 μm) and higher maximum current amplitude. The single channel conductance of α1β3 channels was low (13 pS) compared with that of α1α3δ and α1β3γ2L channels (27 pS). The single channel kinetic properties of the channels also differed. The α1β3γ2L channel exhibited three open states, while the α1β3 and α1β3δ channels exhibited only two open states with mean dwell times similar to those of the two shorter open states of the α1β3γ2L channel. All three channels exhibited at least five closed states. Bursts of α1β3δ channels consisted primarily of only one or two openings, while those of α1β3 channels contained multiple openings. α1β3γ2L channels exhibited burst kinetics typical for native GABARs with several long openings per burst. These results show that the α1β3 heterodimer formed a GABA‐sensitive channel with complex gating kinetics. Addition of a δ subunit to form the α1β3δ heterotrimer altered the single channel conductance and the kinetic properties of the closed components, but did not affect the GABA sensitivity of the receptor nor the open state kinetics. In contrast, addition of a γ subunit (γ2L subtype) to produce the α1β3γ2L heterotrimer affected the GABA sensitivity, channel conductance and kinetic properties of both open and closed states.

Altered gamma-aminobutyric acid (GABA) function is consistently reported in psychiatric disorders, normal aging, and neurodegenerative disorders and reduced function of GABA interneurons is associated with both mood and cognitive symptoms. Benzodiazepines (BZ) have broad anxiolytic, but also sedative, anticonvulsant and amnesic effects, due to nonspecific GABA-A receptor (GABAA-R) targeting. Varying the profile of activity of BZs at GABAA-Rs is predicted to uncover additional therapeutic potential. We synthesized four novel imidazobenzodiazepine (IBZD) amide ligands and tested them for positive allosteric modulation at multiple α-GABAA-R (α-positive allosteric modulators), pharmacokinetic properties, as well as anxiolytic and antidepressant activities in adult mice. Efficacy at reversing stress-induced or age-related working memory deficits was assessed using a spontaneous alternation task. Diazepam (DZP) was used as a control. Three ligands (GL-II-73, GL-II-74, and GL-II-75) demonstrated adequate brain penetration and showed predictive anxiolytic and antidepressant efficacies. GL-II-73 and GL-II-75 significantly reversed stress-induced and age-related working memory deficits. In contrast, DZP displayed anxiolytic but no antidepressant effects or effects on working memory. We demonstrate distinct profiles of anxiolytic, antidepressant, and/or pro-cognitive activities of newly designed IBZD amide ligands, suggesting novel therapeutic potential for IBZD derivatives in depression and aging.

Although the hippocampus expresses nicotinic acetylcholine receptors (nAChRs) and receives cholinergic innervation, the functional roles of these receptors are not completely understood. Our results indicated that presynaptic nAChRs mediated a calcium influx that enhanced the release of both glutamate and GABA. Fura-2 detection of calcium in single mossy fiber presynaptic terminals indicated that nAChRs directly mediated a calcium influx. In hippocampal neurons in primary culture, both spontaneous vesicular release and evoked release of glutamate and GABA were enhanced by nicotine. The nicotinic current displayed rapid desensitization kinetics, and the response to nicotine was inhibited by alpha-bungarotoxin and methyllcaconitine, suggesting that nAChRs containing the alpha 7 subunit mediated the effect. Modulation of synaptic activity by presynaptic calcium influx may represent a physiological role of acetylcholine in the brain, as well as a mechanism of action of nicotine.

Lithium responsivity in patients with bipolar disorder has been genetically associated with Phosphodiesterase 11A (PDE11A), and lithium decreases PDE11A mRNA in IPSC-derived hippocampal neurons originating from lithium responsive patients. PDE11 is an enzyme uniquely enriched in the hippocampus that breaks down cAMP and cGMP. Here, we determined if decreasing PDE11A expression is sufficient to increase lithium responsivity in mice. In dorsal hippocampus (DHIPP) and ventral hippocampus (VHIPP), lithium-responsive C57BL/6J and 129S6/SvEvTac mice show decreased PDE11A4 protein expression relative to lithium-unresponsive BALB/cJ mice. In VHIPP, C57BL/6J mice also show differences in PDE11A4 compartmentalization relative to BALB/cJ mice. In contrast, neither PDE2A nor PDE10A expression differ among the strains. The compartment-specific differences in PDE11A4 protein expression are explained by a coding SNP at amino acid 499, which falls within the GAF-B homodimerization domain. Relative to the BALB/cJ 499T, the C57BL/6J 499A decreases PDE11A4 homodimerization, which removes PDE11A4 from the membrane. Consistent with the observation that lower PDE11A4 expression correlates with better lithium responsiveness, we found that Pde11a KO mice given 0.4% lithium chow for 3+ weeks exhibit greater lithium responsivity relative to WT littermates in tail suspension, an antidepressant predictive assay, and amphetamine hyperlocomotion, an anti-manic predictive assay. Reduced PDE11A4 expression may represent a lithium-sensitive pathophysiology, because both C57BL/6J and Pde11a KO mice show increased expression of the pro-inflammatory cytokine IL-6 relative to BALB/cJ and PDE11A WT mice, respectively. Our finding that PDE11A4 negatively regulates lithium responsivity in mice suggests that the PDE11A SNPs identified in patients may be functionally relevant.

Stiripentol (STP) has been used as co-therapy for treatment of epilepsy for many years. Its mechanism of action has long been considered to be indirect, as it inhibits the enzymes responsible for metabolism of other anti-convulsant agents. However, a recent report suggested that STP might also act at the neuronal level, increasing inhibitory GABAergic neurotransmission. We examined the effect of STP on the functional properties of recombinant GABA(A) receptors (GABARs) and found that it was a positive allosteric modulator of these ion channels. Its activity showed some dependence on subunit composition, with greater potentiation of alpha3-containing receptors and reduced potentiation when the beta1 or epsilon subunits were present. STP caused a leftward shift in the GABA concentration-response relationship, but did not increase the peak response of the receptors to a maximal GABA concentration. Although STP shares some functional characteristics with the neurosteroids, its activity was not inhibited by a neurosteroid site antagonist and was unaffected by a mutation in the alpha3 subunit that reduced positive modulation by neurosteroids. The differential effect of STP on beta1- and beta2/beta3-containing receptors was not altered by mutations within the second transmembrane domain that affect modulation by loreclezole. These findings suggest that STP acts as a direct allosteric modulator of the GABAR at a site distinct from many commonly used anti-convulsant, sedative and anxiolytic drugs. Its higher activity at alpha3-containing receptors as well as its activity at delta-containing receptors may provide a unique opportunity to target selected populations of GABARs.