Abolition of zolpidem sensitivity in mice with a point mutation in the GABAA receptor gamma2 subunit.

Agonists of the allosteric benzodiazepine site of GABAA receptors bind at the interface of the alpha and gamma subunits. Here, we tested the in vivo contribution of the gamma2 subunit to the actions of zolpidem, an alpha1 subunit selective benzodiazepine agonist, by generating mice with a phenylalanine (F) to isoleucine (I) substitution at position 77 in the gamma2 subunit. The gamma2F77I mutation has no major effect on the expression of GABAA receptor subunits in the cerebellum. The potency of zolpidem, but not that of flurazepam, for the inhibition of [3H]flunitrazepam binding to cerebellar membranes is greatly reduced in gamma2I77/I77 mice. Zolpidem (1 microM) increased both the amplitude and decay of miniature inhibitory postsynaptic currents (mIPSCs) in Purkinje cells of control C57BL/6 (34% and 92%, respectively) and gamma2F77/F77 (20% and 84%) mice, but not in those of gamma2F77I mice. Zolpidem tartrate had no effect on exploratory activity (staircase test) or motor performance (rotarod test) in gamma2I77/I77 mice at doses up to 30 mg/kg (i.p.) that strongly sedated or impaired the control mice. Flurazepam was equally effective in enhancing mIPSCs and disrupting performance in the rotarod test in control and gamma2I77/I77 mice. These results show that the effect of zolpidem, but not flurazepam, is selectively eliminated in the brain by the gamma2F77I point mutation

Protein Quality and the Protein to Carbohydrate Ratio within a High Fat Diet Influences Energy Balance and the Gut Microbiota In C57BL/6J Mice

peer-reviewedMacronutrient quality and composition are important determinants of energy balance and the gut microbiota. Here, we investigated how changes to protein quality (casein versus whey protein isolate; WPI) and the protein to carbohydrate (P/C) ratio within a high fat diet (HFD) impacts on these parameters. Mice were fed a low fat diet (10% kJ) or a high fat diet (HFD; 45% kJ) for 21 weeks with either casein (20% kJ, HFD) or WPI at 20%, 30% or 40% kJ. In comparison to casein, WPI at a similar energy content normalised energy intake, increased lean mass and caused a trend towards a reduction in fat mass (P = 0.08), but the protein challenge did not alter oxygen consumption or locomotor activity. WPI reduced HFD-induced plasma leptin and liver triacylglycerol, and partially attenuated the reduction in adipose FASN mRNA in HFD-fed mice. High throughput sequence-based analysis of faecal microbial populations revealed microbiota in the HFD-20% WPI group clustering closely with HFD controls, although WPI specifically increased Lactobacillaceae/Lactobacillus and decreased Clostridiaceae/Clostridium in HFD-fed mice. There was no effect of increasing the P/C ratio on energy intake, but the highest ratio reduced HFD-induced weight gain, fat mass and plasma triacylglycerol, non-esterified fatty acids, glucose and leptin levels, while it increased lean mass and oxygen consumption. Similar effects were observed on adipose mRNA expression, where the highest ratio reduced HFD-associated expression of UCP-2, TNFa and CD68 and increased the diet-associated expression of b3-AR, LPL, IR, IRS-1 and GLUT4. The P/C ratio also impacted on gut microbiota, with populations in the 30/ 40% WPI groups clustering together and away from the 20% WPI group. Taken together, our data show that increasing the P/C ratio has a dramatic effect on energy balance and the composition of gut microbiota, which is distinct from that caused by changes to protein quality.KN is supported by the Teagasc Vision Programme on Obesity, which also funded the work detailed in this manuscript. LM is supported by a Teagasc PhD Walsh Fellowship. HMR is supported by SFI PI (11/PI/1119)

Alcohol-induced motor impairment caused by increased extrasynaptic GABAA receptor activity

Neuronal mechanisms underlying alcohol intoxication are unclear. We find that alcohol impairs motor coordination by enhancing tonic inhibition mediated by a specific subtype of extrasynaptic GABA(A) receptor (GABAR), α6β3δ, expressed exclusively in cerebellar granule cells. In recombinant studies, we characterize a naturally occurring single-nucleotide polymorphism that causes a single amino acid change (R100Q) in α6 (encoded in rats by the Gabra6 gene). We show that this change selectively increases alcohol sensitivity of α6β3δ GABARs. Behavioral and electrophysiological comparisons of Gabra6(100R/100R) and Gabra6(100Q/100Q) rats strongly suggest that alcohol impairs motor coordination by enhancing granule cell tonic inhibition. These findings identify extrasynaptic GABARs as critical targets underlying low-dose alcohol intoxication and demonstrate that subtle changes in tonic inhibition in one class of neurons can alter behavior