Figure 4. γ2 is essential for GABA_AR synaptic localization in the brain.

(A, B) Localization of GABA_AR subunits in the cerebellar granule cell (GC)-γ2 knockout (KO) mice and age matched controls without Cre expression (Control). Inhibitory presynaptic VGAT and excitatory postsynaptic GluN1 were co-stained. (A) Loss of γ2 was observed specifically in the granular layer in γ2 GC-KO mice, whereas α1 and β2 remained. The images are representative of four independent experiments. (B and C) High-magnification representative images showed protein distribution on each glomerulus. Inhibitory inputs project to outer edges of the glomerulus, whereas excitatory inputs project to inner edges of the glomerulus. In the γ2 GC-KO, the fraction of α1 colocalized with VGAT was reduced, whereas the fraction of GluN1 colocalized with α1 was increased (n = 30 areas/2 animal each). (D–F) Spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded from granule cells in acute cerebellar slices, and representative traces are shown (D). In γ2 GC-KO mice, sIPSC frequency (E) and amplitude (F) were dramatically reduced, but not completely eliminated (n = 4 bins (E), n = 69–1740 events (F), see Materials and methods). The asterisk indicates a sIPSC recorded from a γ2 GC-KO mouse. Picrotoxin (100 µM) blocked all sIPSCs. (G and H) Representative images show localization of gephyrin in γ2 GC-KO and control mice. Gephyrin colocalized with VGAT at the glomerular periphery in controls. In the γ2 GC-KO, the fraction of gephyrin colocalized with VGAT was reduced, and at the same time, the fraction of GluN1 colocalized with gephyrin was reduced (n = 30 areas/2 animal each). Scale bars: 60 μm (A), 5 μm (B, G). Data are given as mean ± s.e.m.; p values were determined using student’s t test.