Developmental changes in miniature IPSC (mIPSC) kinetics have been demonstrated previously in cerebellar neurons in rodents. We report that these kinetic changes in mice are determined primarily by developmental changes in GABA A receptor subunit expression. mIPSCs were studied by whole-cell recordings in cerebellar slices, prepared from postnatal day 11 (P11) and P35 mice. Similar to reports in granule neurons, wild-type cerebellar stellate neuron mIPSCs at P11 had slow decay kinetics, whereas P35 mIPSCs decayed five times faster. When mIPSCs in cerebellar stellate neurons were compared between wild-type (ϩ/ϩ) and GABA A receptor ␣1 subunitdeficient (Ϫ/Ϫ) littermates at P35, we observed dramatically slower mIPSC decay rates in Ϫ/Ϫ animals. We took advantage of the greater potency of imidazopyridines for GABA current potentiation with ␣1 subunit-containing receptors to characterize the relative contribution of ␣1 subunits in native receptors on inhibitory synapses of cerebellar granule neurons. Zolpideminduced prolongation of mIPSC decay was variable among distinct cells, but it increased during development in wild-type mice. Similarly, Zolpidem prolongation of mIPSC decay rate was significantly greater in adult ϩ/ϩ mice than in knock-outs. We propose that an increased ␣1 subunit assembly in postsynaptic receptors of cerebellar inhibitory synapses is responsible for the fast inhibitory synaptic currents that are normally observed during postnatal development. Key words: GABA receptor; gene knock-out; patch-clamp; inhibitory synapses; development; GABASpontaneous IPSCs (sIPSCs) and miniature IPSCs (mIPSCs) [recorded in the presence of tetrodotoxin (TTX)] in mammalian neurons have been shown to become faster with development (Brickley et al., 1996;Draguhn and Heinemann, 1996;Tia et al., 1996;Brussard et al., 1997;Hollrigel and Soltesz, 1997;Pouzat and Hestrin, 1997;Dunning et al., 1999;Okada et al., 2000). Several mechanisms have been proposed to explain these changes, including changes in expression of specific GABA A receptor (GABA A -R) subunits (Tia et al., 1996; Dunning et al., 2000;Okada et al., 2000) and changes in reuptake mechanisms (Draguhn and Heinemann, 1996). Recent studies with overexpression of the ␣1 subunit of the GABA A receptor in thalamic neurons have indicated that the enhanced expression of the ␣1 subunit could be responsible for faster mIPSCs (Okada et al., 2000). This hypothesis is supported by work in recombinant systems that demonstrate that rapid application of brief pulses of GABA to outside-out excised patches generates faster currents with ␣1␤2␥2 than with other subunit combinations (Verdoorn, 1994;Gingrich et al., 1995;Lavoie et al., 1997;McClellan and Twyman, 1999).sIPSCs have been characterized extensively in granule and stellate neurons in rat cerebellar slices (Llano and Gerschenfeld, 1993;Brickley et al., 1996Brickley et al., , 1999Tia et al., 1996;Nusser et al., 1997;Kondo and Marty, 1998). The decay time of sIPSCs in granule neurons has been shown to undergo a developmental...

Angelman syndrome (AS) is a severe neurodevelopmental disorder resulting from a deletion/mutation in maternal chromosome 15q11–13. The genes in 15q11–13 contributing to the full array of the clinical phenotype are not fully identified. This study examines whether a loss or reduction in the GABAAreceptor β3subunit (GABRB3) gene, contained within the AS deletion region, may contribute to the overall severity of AS. Disrupting the gabrb3 gene in mice produces electroencephalographic abnormalities, seizures, and behavior that parallel those seen in AS. The seizures that are observed in these mice showed a pharmacological response profile to antiepileptic medications similar to that observed in AS. Additionally, these mice exhibited learning and memory deficits, poor motor skills on a repetitive task, hyperactivity, and a disturbed rest–activity cycle, features all common to AS. The loss of the single gene, gabrb3, in these mice is sufficient to cause phenotypic traits that have marked similarities to the clinical features of AS, indicating that impaired expression of the GABRB3 gene in humans probably contributes to the overall phenotype of Angelman syndrome. At least one other gene, the E6-associated protein ubiquitin-protein ligase (UBE3A) gene, has been implicated in AS, so the relative contribution of the GABRB3 gene alone or in combination with other genes remains to be established.

Studies using molecular modeling, genetic engineering, neurophysiology/pharmacology, and whole animals have advanced our understanding of where and how inhaled anesthetics act to produce immobility (minimum alveolar anesthetic concentration; MAC) by actions on the spinal cord. Numerous ligand- and voltage-gated channels might plausibly mediate MAC, and specific amino acid sites in certain receptors present likely candidates for mediation. However, in vivo studies to date suggest that several channels or receptors may not be mediators (e.g., gamma-aminobutyric acid A, acetylcholine, potassium, 5-hydroxytryptamine-3, opioids, and alpha(2)-adrenergic), whereas other receptors/channels (e.g., glycine, N-methyl-D-aspartate, and sodium) remain credible candidates.

Synchronized neural activity is believed to be essential for many CNS functions, including neuronal development, sensory perception, and memory formation. In several brain areas GABA(A) receptor-mediated synaptic inhibition is thought to be important for the generation of synchronous network activity. We have used GABA(A) receptor beta3 subunit deficient mice (beta3-/-) to study the role of GABAergic inhibition in the generation of network oscillations in the olfactory bulb (OB) and to reveal the role of such oscillations in olfaction. The expression of functional GABA(A) receptors was drastically reduced (>93%) in beta3-/- granule cells, the local inhibitory interneurons of the OB. This was revealed by a large reduction of muscimol-evoked whole-cell current and the total current mediated by spontaneous, miniature inhibitory postsynaptic currents (mIPSCs). In beta3-/- mitral/tufted cells (principal cells), there was a two-fold increase in mIPSC amplitudes without any significant change in their kinetics or frequency. In parallel with the altered inhibition, there was a significant increase in the amplitude of theta (80% increase) and gamma (178% increase) frequency oscillations in beta3-/- OBs recorded in vivo from freely moving mice. In odor discrimination tests, we found beta3-/- mice to be initially the same as, but better with experience than beta3+/+ mice in distinguishing closely related monomolecular alcohols. However, beta3-/- mice were initially better and then worse with practice than control mice in distinguishing closely related mixtures of alcohols. Our results indicate that the disruption of GABA(A) receptor-mediated synaptic inhibition of GABAergic interneurons and the augmentation of IPSCs in principal cells result in increased network oscillations in the OB with complex effects on olfactory discrimination, which can be explained by an increase in the size or effective power of oscillating neural cell assemblies among the mitral cells of beta3-/- mice.

Alcohol use disorder (AUD) is heritable, but the genetic basis for this disease remains poorly understood. Although numerous gene variants have been associated with AUD, these variants account for only a small fraction of the total risk. The idea of inheritance of acquired characteristics, i.e. “epigenetic inheritance,” is re-emerging as a proven adjunct to traditional modes of genetic inheritance. We hypothesized that alcohol drinking and neurobiological sensitivity to alcohol are influenced by ancestral alcohol exposure. To test this hypothesis, we exposed male mice to chronic vapor ethanol or control conditions, mated them to ethanol-naïve females, and tested adult offspring for ethanol drinking, ethanol-induced behaviors, gene expression, and DNA methylation. We found that ethanol-sired male offspring had reduced ethanol preference and consumption, enhanced sensitivity to the anxiolytic and motor-enhancing effects of ethanol, and increased Bdnf expression in the ventral tegmental area (VTA) compared to control-sired male offspring. There were no differences among ethanol- and control-sired female offspring on these assays. Ethanol exposure also decreased DNA methylation at the BdnfÆpromoter of sire's germ cells and hypomethylation was maintained in the VTA of both male and female ethanol-sired offspring. Our findings show that paternal alcohol exposure is a previously unrecognized regulator of alcohol drinking and behavioral sensitivity to alcohol in male, but not female, offspring. Paternal alcohol exposure also induces epigenetic alterations (DNA hypomethylation) and gene expression changes that persist in the VTA of offspring. These results provide new insight into the inheritance and development of alcohol drinking behaviors.

The alpha6 subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) has been implicated in mediating the intoxicating effects of ethanol and the motor ataxic effects of general anesthetics. To test this hypothesis, we used gene targeting in embryonic stem cells to create mice lacking a functional alpha6 gene. Homozygous mice are viable and fertile and have grossly normal cerebellar cytoarchitecture. Northern blot and reverse transcriptase-polymerase chain reaction analyses demonstrated that the targeting event disrupted production of functional alpha6 mRNA. Autoradiography of histological sections of adult brains demonstrated that diazepam-insensitive binding of [3H]Ro15-4513 to the cerebellar granule cell layer of wild-type mice was completely absent in homozygous mice. Cerebellar GABA(A)-R density was unchanged in the mutant mice; however, the apparent affinity for muscimol was markedly reduced. Sleep time response to injection of ethanol after pretreatment with vehicle or Ro15-4513 did not differ between genotypes. Sleep time response to injection of pentobarbital and loss of righting reflex and response to tail clamp stimulus in mice anesthetized with volatile anesthetics also did not differ between genotypes. Thus, the alpha6 subunit of the GABA(A)-R is not required for normal development, viability, and fertility and does not seem to be a critical or unique component of the neuronal pathway mediating the hypnotic effect of ethanol and its antagonism by Ro15-4513 in mice. Similarly, the alpha6 subunit does not seem to be involved in the behavioral responses to general anesthetics or pentobarbital.

GABA type-A (GABA-A) receptors containing the α2 subunit (GABRA2) are expressed in most brain regions and are critical in modulating inhibitory synaptic function. Genetic variation at the GABRA2 locus has been implicated in epilepsy, affective and psychiatric disorders, alcoholism and drug abuse. Gabra2 expression varies as a function of genotype and is modulated by sequence variants in several brain structures and populations, including F2 crosses originating from C57BL/6J (B6J) and the BXD recombinant inbred family derived from B6J and DBA/2J. Here we demonstrate a global reduction of GABRA2 brain protein and mRNA in the B6J strain relative to other inbred strains, and identify and validate the causal mutation in B6J. The mutation is a single base pair deletion located in an intron adjacent to a splice acceptor site that only occurs in the B6J reference genome. The deletion became fixed in B6J between 1976 and 1991 and is now pervasive in many engineered lines, BXD strains generated after 1991, the Collaborative Cross, and the majority of consomic lines. Repair of the deletion using CRISPR- Cas9 -mediated gene editing on a B6J genetic background completely restored brain levels of GABRA2 protein and mRNA. Comparison of transcript expression in hippocampus, cortex, and striatum between B6J and repaired genotypes revealed alterations in GABA-A receptor subunit expression, especially in striatum. These results suggest that naturally occurring variation in GABRA2 levels between B6J and other substrains or inbred strains may also explain strain differences in anxiety-like or alcohol and drug response traits related to striatal function. Characterization of the B6J private mutation in the Gabra2 gene is of critical importance to molecular genetic studies in neurobiological research because this strain is widely used to generate genetically engineered mice and murine genetic populations, and is the most widely utilized strain for evaluation of anxiety-like, depression-like, pain, epilepsy, and drug response traits that may be partly modulated by GABRA2 function.

One of the great strengths of the mouse model is the wide array of genetic tools that have been developed. Striking examples include methods for directed modification of the genome, and for regulated expression or inactivation of genes. Within neuroscience, it is now routine to express reporter genes, neuronal activity indicators and opsins in specific neuronal types in the mouse. However, there are considerable anatomical, physiological, cognitive and behavioral differences between the mouse and the human that, in some areas of inquiry, limit the degree to which insights derived from the mouse can be applied to understanding human neurobiology. Several recent advances have now brought into reach the goal of applying these tools to understanding the primate brain. Here we describe these advances, consider their potential to advance our understanding of the human brain and brain disorders, discuss bioethical considerations, and describe what will be needed to move forward.