Role of the GABA(A) receptor gamma2 subunit in the development of gonadotropin-releasing hormone neurons in vivo.

We have employed transgenic mouse models to examine the functional significance of the gamma2 subunit of the GABA(A) (gamma-aminobutyric acid) receptor to the correct development of gonadotropin-releasing hormone (GnRH) neurons in vivo. In the first experiment, the expression of gamma2 subunit protein by the GnRH phenotype was determined using transgenic mice in which GnRH gene sequences direct the expression of the LacZ reporter to the nucleus of the GnRH neurons. This greatly facilitates the immunocytochemical identification of non-nuclear-located antigens within GnRH neurons and revealed that approximately 25% of juvenile GnRH neurons were immunoreactive for the gamma2 subunit and that this increased to 40% in pubertal mice. In the second experiment, GnRH mRNA expression was examined in the brains of gamma2 subunit knockout mice (gamma2(0/0)) and their wild-type (gamma2+/+) littermates at embryonic day 15 and postnatal days (P) 0 and 11-16 using in situ hybridization. The distribution and numbers of cells expressing GnRH mRNA in gamma2+/+ and gamma2(0/0) mice were not found to differ at any age. However, the GnRH mRNA content of medial septal cells was significantly lower in gamma2(0/0) compared with gamma2+/+ mice at P11-16 (P<0.05) and the same trend was observed for preoptic area neurons. These results demonstrate that while the gamma2 subunit of the GABA(A) receptor is expressed by postnatal GnRH neurons, their embryonic development does not require a functional gamma2 subunit. In contrast, postnatal GnRH mRNA expression was found to be dependent upon signalling through the GABA(A) receptor.

Rescue of gamma2 subunit-deficient mice by transgenic overexpression of the GABAA receptor gamma2S or gamma2L subunit isoforms.

The gamma2 subunit is an important functional determinant of GABAA receptors and is essential for formation of high-affinity benzodiazepine binding sites and for synaptic clustering of major GABAA receptor subtypes along with gephyrin. There are two splice variants of the gamma2 subunit, gamma2 short (gamma2S) and gamma2 long (gamma2L), the latter carrying in the cytoplasmic domain an additional eight amino acids with a putative phosphorylation site. Here, we show that transgenic mice expressing either the gamma2S or gamma2L subunit on a gamma2 subunit-deficient background are phenotypically indistinguishable from wild-type. They express nearly normal levels of gamma2 subunit protein and [3H]flumazenil binding sites. Likewise, the distribution, number and size of GABAA receptor clusters colocalized with gephyrin are similar to wild-type in both juvenile and adult mice. Our results indicate that the two gamma2 subunit splice variants can substitute for each other and fulfil the basic functions of GABAA receptors, allowing in vivo studies that address isoform-specific roles in phosphorylation-dependent regulatory mechanisms.

Postsynaptic clustering of gamma-aminobutyric acid type A receptors by the gamma3 subunit in vivo.

Synaptic localization of gamma-aminobutyric acid type A (GABA(A)) receptors is a prerequisite for synaptic inhibitory function, but the mechanism by which different receptor subtypes are localized to postsynaptic sites is poorly understood. The gamma2 subunit and the postsynaptic clustering protein gephyrin are required for synaptic localization and function of major GABA(A) receptor subtypes. We now show that transgenic overexpression of the gamma3 subunit in gamma2 subunit-deficient mice restores benzodiazepine binding sites, benzodiazepine-modulated whole cell currents, and postsynaptic miniature currents, suggesting the formation of functional, postsynaptic receptors. Moreover, the gamma3 subunit can substitute for gamma2 in the formation of GABA(A) receptors that are synaptically clustered and colocalized with gephyrin in vivo. These clusters were formed even in brain regions devoid of endogenous gamma3 subunit, indicating that the factors present for clustering of gamma2 subunit-containing receptors are sufficient to cluster gamma3 subunit-containing receptors. The GABA(A) receptor and gephyrin-clustering properties of the ectopic gamma3 subunit were also observed for the endogenous gamma3 subunit, but only in the absence of the gamma2 subunit, suggesting that the gamma3 subunit is at a competitive disadvantage with the gamma2 subunit for clustering of postsynaptic GABA(A) receptors in wild-type mice.

Decreased GABAA-receptor clustering results in enhanced anxiety and a bias for threat cues.

Patients with panic disorders show a deficit of GABAA receptors in the hippocampus, parahippocampus and orbitofrontal cortex. Synaptic clustering of GABAA receptors in mice heterozygous for the gamma2 subunit was reduced, mainly in hippocampus and cerebral cortex. The gamma2 +/- mice showed enhanced behavioral inhibition toward natural aversive stimuli and heightened responsiveness in trace fear conditioning and ambiguous cue discrimination learning. Implicit and spatial memory as well as long-term potentiation in hippocampus were unchanged. Thus gamma2 +/- mice represent a model of anxiety characterized by harm avoidance behavior and an explicit memory bias for threat cues, resulting in heightened sensitivity to negative associations. This model implicates GABAA-receptor dysfunction in patients as a causal predisposition to anxiety disorders.

Postsynaptic clustering of major GABAA receptor subtypes requires the gamma 2 subunit and gephyrin.

Most fast inhibitory neurotransmission in the brain is mediated by GABAA receptors, which are mainly postsynaptic and consist of diverse alpha and beta subunits together with the gamma 2 subunit. Although the gamma 2 subunit is not necessary for receptor assembly and translocation to the cell surface, we show here that it is required for clustering of major postsynaptic GABAA receptor subtypes. Loss of GABAA receptor clusters in mice deficient in the gamma 2 subunit, and in cultured cortical neurons from these mice, is paralleled by loss of the synaptic clustering molecule gephyrin and synaptic GABAergic function. Conversely, inhibiting gephyrin expression causes loss of GABAA receptor clusters. The gamma 2 subunit and gephyrin are thus interdependent components of the same synaptic complex that is critical for postsynaptic clustering of abundant subtypes of GABAA receptors in vivo.