α4βδ GABAA receptors (GABARs) have low CNS expression, but their expression is increased by 48 h exposure to the neurosteroid THP (3α-OH-5α[β]-pregnan-20-one). THP also increases the efficacy of δ-containing GABARs acutely, where GABA is a partial agonist. Thus, we examined effects of THP (100 nM) and full GABA agonists at α4β2δ (gaboxadol, 10 μM, and β-alanine, 10 μM – 1 mM), on surface expression of α4β2δ. To this end, we used an α4 construct tagged with a 3XFLAG (F) epitope or measured expression of native α4 and δ. HEK-293 cells or cultured hippocampal neurons were transfected with α4Fβ2δ and treated 24 h later with GABA agonists, THP, GABA plus THP or vehicle (0.01% DMSO) for 0.5 h – 48 h. Immunocytochemistry was performed under both non-permeabilized and permeabilized conditions to detect surface and intracellular labeling, respectively, using confocal microscopy. The high efficacy agonists and GABA (1 or 10μM) plus THP increased α4β2δ surface expression up to 3-fold after 48 h, an effect first seen by 0.5 h. This effect was not dependent upon the polarity of GABAergic current, although expression was increased by KCC2. Intracellular labeling was decreased while functional expression was confirmed by whole cell patch clamp recordings of responses to GABA agonists. GABA plus THP treatment did not alter the rate of receptor removal from the surface membrane, suggesting that THP-induced α4β2δ expression is likely via receptor insertion. Surface expression of α4β2δ was decreased by rottlerin (10 μM), suggesting a role for PKC- δ. These results suggest that trafficking of α4β2δ GABARs is regulated by high efficacy states.

Fluctuations in circulating levels of ovarian hormones have been shown to regulate cognition (Sherwin and Grigorova, 2011. Fertil. Steril. 96, 399–403; Shumaker et al., 2004. JAMA. 291, 2947–2958), but increases in estradiol on the day of proestrus yield diverse outcomes: In vivo induction of long-term potentiation (LTP), a model of learning, is reduced in the morning, but optimal in the afternoon (Warren et al., 1995. Brain Res. 703, 26–30). The mechanism underlying this discrepancy is not known. Here, we show that impairments in both CA1 hippocampal LTP and spatial learning observed on the morning of proestrus are due to increased dendritic expression of α4βδ GABAA receptors (GABARs) on CA1 pyramidal cells, as assessed by electron microscopic (EM) techniques, compared with estrus and diestrus. LTP induction and spatial learning were robust, however, when assessed on the morning of proestrus in α4−/− mice, implicating these receptors in mediating impaired plasticity. Although α4βδ expression remained elevated on the afternoon of proestrus, increases in 3α-OH-THP (3α-OH-5α-pregnan-20-one) decreased inhibition by reducing outward current through α4βδ GABARs (Shen et al., 2007. Nat. Neurosci. 10, 469–477), in contrast to the usual effect of this steroid to enhance inhibition. Proestrous levels of 3α-OH-THP reversed the deficits in LTP and spatial learning, an effect prevented by the inactive metabolite 3β-OH-THP (10 mg/kg, i.p.), which antagonizes actions of 3α-OH-THP. In contrast, administration of 3α-OH-THP (10 mg/kg, i.p.) on the morning of proestrus improved spatial learning scores 150–300%. These findings suggest that cyclic fluctuations in ovarian steroids can induce changes in cognition via α4βδ GABARs that are dependent upon 3α-OH-THP.

Increases in expression of α4βδ GABAA receptors (GABARs), triggered by fluctuations in the neurosteroid THP (3α-OH-5α[β]-pregnan-20-one), are associated with changes in mood and cognition. We tested whether α4βδ trafficking and surface expression would be altered by in vitro exposure to flumazenil, a benzodiazepine ligand which reduces α4βδ expression in vivo. We first determined that flumazenil (100 nM – 100 μM, IC50=~1 μM) acted as a negative modulator, reducing GABA (10 μM)-gated current in the presence of 100 nM THP (to increase receptor efficacy), assessed with whole cell patch clamp recordings of recombinant α4β2δ expressed in HEK-293 cells. Surface expression of recombinant α4β2δ receptors was detected using a 3XFLAG reporter at the C-terminus of α4 (α4F) using confocal immunocytochemical techniques following 48 h exposure of cells to GABA (10 μM) + THP (100 nM). Flumazenil (10 μM) decreased surface expression of α4F by ~60%, while increasing its intracellular accumulation, after 48 h. Reduced surface expression of α4β2δ after flumazenil treatment was confirmed by decreases in the current responses to 100 nM of the GABA agonist gaboxadol. Flumazenil-induced decreases in surface expression of α4β2δ were prevented by the dynamin blocker, dynasore, and by leupeptin, which blocks lysosomal enzymes, suggesting that flumazenil is acting to increase endocytosis and lysosomal degradation of the receptor. Flumazenil increased the rate of receptor removal from the cell surface by 2-fold, assessed using botulinum toxin B to block insertion of new receptors. These findings may suggest new therapeutic strategies for regulation of α4β2δ expression using flumazenil.

Summary Aims To investigate whether there exists a cardio‐protective effect of Fasudil, a selective Rho kinase (ROCK) inhibitor, in an experimental murine model of acute viral myocarditis. Methods Male BALB/c mice were randomly assigned to three groups: control, myocarditis treated with placebo and myocarditis treated with Fasudil (n = 40 animals per group). Myocarditis was established by intraperitoneal injection with coxsackievirus B3 (CVB3). Twenty‐four hours after infection, Fasudil was intraperitoneally administered for 14 consecutive days. Twenty mice were randomly selected from each group to monitor a 14‐day survival rate. On day 7 and day 14, eight surviving mice from each group were sacrificed and their hearts and blood were obtained to perform serological and histological examinations. Expression of ROCKs, IL‐17, IL‐1b, TNFα, RORgt, and Foxp3 were quantified with RT‐PCR. Plasma levels of TNF alpha, IL‐1 beta, and IL‐17 were measured by ELISA. In addition, protein levels of IL‐17 and ROCK2 in cardiac tissues were analyzed with Western blot. Results Fasudil treatment significantly increased survival, attenuated myocardial necrotic lesions, reduced CVB3 replication and expression of ROCK2 and IL‐17 in the infected hearts. This treatment also imposed a T‐cell subpopulation shift, from Th17 to Treg, in cardiac tissues. Conclusions ROCK pathway inhibition was cardio‐protective in viral myocarditis with increased survival, decreased viral replication, and inflammatory response. These findings suggest that Fasudil might be a potential therapeutic agent for patients with viral myocarditis.