From synapse to behavior: rapid modulation of defined neuronal types with engineered GABAA receptors

Abstract

In mammals, identifying the contribution of specific neurons or networks to behavior is a key challenge. Here we describe an approach that facilitates this process by enabling the rapid modulation of synaptic inhibition in defined cell populations. Binding of zolpidem, a systemically active allosteric modulator that enhances the function of the GABAA receptor, requires a phenylalanine residue (Phe77) in the gamma2 subunit. Mice in which this residue is changed to isoleucine are insensitive to zolpidem. By Cre recombinase-induced swapping of the gamma2 subunit (that is, exchanging Ile77 for Phe77), zolpidem sensitivity can be restored to GABAA receptors in chosen cell types. We demonstrate the power of this method in the cerebellum, where zolpidem rapidly induces significant motor deficits when Purkinje cells are made uniquely sensitive to its action. This combined molecular and pharmacological technique has demonstrable advantages over targeted cell ablation and will be invaluable for investigating many neuronal circuits.

Figure 1

Strategy to restrict zolpidem sensitivity to selected neural types, using Cre recombinase to drive a γ2I77 to γ2F77 subunit swap. The genotype, the pentameric GABA_A receptors formed from α, β and γ2 (γ2F77 or γ2I77) subunits and corresponding brain sections, with zolpidem sensitivity indicated in red, are shown for three groups of mice. Homologous recombination in embryonic stem cells is used to introduce a Phe77 to isoleucine point mutation and to flank exon 4 with loxP sites. The resultant mice are crossed with mice expressing Cre recombinase and the zolpidem-sensitive γ2 subunit under the control of the Purkinje cell-selective L7 promoter to generate PC-γ2-swap mice. In the Purkinje cells of these mice, Cre catalyzes recombination between the loxP sites, resulting in excision and loss of exon 4, and thus deletion of the γ2I77 subunit (gray). In its place, the γ2F77^GFP transgene produces the wild-type zolpidem-sensitive γ2 subunit (red), tagged with GFP at its amino terminus.

Figure 2

Summary of mouse genotypes. In situ hybridization autoradiographs show the expression of various GABA_A receptor γ2 subunit mRNA in brain sections from γ2I77lox, PC-Δγ2 and PC-γ2-swap adult mice. The corresponding transgene content (genotype) of the mice is indicated above the images (arrowheads, loxP sites; Ex 4, exon 4). The γ2I77lox and PC-Δγ2 autoradiographs were obtained with a γ2-specific (exon 4) oligonucleotide. To visualize selectively the γ2F77^GFP mRNA, the PC-γ2-swap image was obtained with a GFP-specific oligonucleotide. Magnifications of the boxed areas are shown at the bottom. In γ2I77lox cerebellum, exon 4-containing γ2I77 mRNA is present in all layers of the cerebellum, but is most evident in the granule cell (Gr) and Purkinje cell (PC) layers. In PC-Δγ2 cerebellum, γ2I77 mRNA is absent from the Purkinje cell layer. In PC-γ2-swap brains, γ2F77^GFP mRNA is present only in Purkinje cells. Scale bars, 2 mm and 300 μm.

Figure 3

Synaptic expression of the γ2F77^GFP subunit in Purkinje cells of adult PC-γ2-swap mice. A confocal microscopic image shows immunoreactivity for GFP (green), the α1 subunit of the GABA_A receptor (blue) and GAD (red) in the cerebellum. Note the short segments of GFP immunoreactivity surrounding the somata and dendrites of Purkinje cells. Glomeruli in the granule cell layer are immunonegative for GFP. Inset shows magnified images of the boxed area, indicating that individual GFP patches also contain α1 subunit immunoreactivity and are apposed to GAD-immunoreactive structures corresponding to GABAergic nerve terminals. Other GAD-positive structures apposed to α1 subunit immunoreactive segments represent GABAergic terminals that innervate interneuron (stellate/basket cell) dendrites. Scale bars, 15 μm and 2 μm (inset).

Figure 4

Potentiation of GABA_A receptor-mediated mIPSCs by zolpidem in Purkinje cells of PC-γ2-swap mice. (a) Continuous 20-s segments of whole-cell recordings (-70 mV) from Purkinje cells of γ2I77lox (postnatal day 140), PC-Δγ2 (P109) and PC-γ2-swap (P134) mice. mIPSCs (downward current deflections) are absent from the PC-Δγ2 recording, but appear normal in the PC-γ2-swap recording. (b) Superimposed averaged mIPSCs in the absence (black) and presence (red) of zolpidem in γ2I77lox (top) and PC-γ2-swap (bottom) mice, before (left) and after (right) peak scaling (same cells as in a). Smooth red or black lines on the decaying phase of the scaled traces (gray) are fits of double-exponential functions. Zolpidem (1 μM) increased the amplitude and decay of the mIPSCs recorded from the PC-γ2-swap Purkinje cell, but had no effect on those recorded from the γ2I77lox cell. (c) Pooled data showing the effect of zolpidem on peak amplitude, τ_w and charge transfer. Columns indicate means and vertical error bars indicate s.e.m. (**P < 0.01, ***P < 0.001, Mann Whitney U-test; n = 8 and 5 for γ2I77lox and PC-γ2-swap mice, respectively).

Figure 5

Motor performance on a rotarod and horizontal beam after systemic administration of zolpidem. (a) The rotarod was accelerated from 5 to 30 r.p.m. over 180 s. Data for γ2I77lox littermate control (n = 20), PC-γ2-swap (n = 20) and PC-Δγ2 (n = 5) mice are presented as the daily means ± s.e.m. of six trials for males and females combined (*P < 0.05 for PC-γ2-swap versus γ2I77lox control mice on training day 1, repeated measures ANOVA and Newman-Keuls post hoc tests). (b) Effects of zolpidem on motor performance. Shown is the effect of cumulative doses (3+3+3 mg/kg i.p., total 9 mg/kg) of zolpidem on the rotarod performance of γ2I77lox control (n = 9) and PC-γ2-swap (n = 5) male mice. The rotarod was accelerated from 5 to 30 r.p.m. over 180 s (***P < 0.001 between mouse lines and P < 0.001 versus saline injection within the PC-γ2-swap line, one-way ANOVA and Newman-Keuls post hoc tests). Data are presented as means ± s.e.m. (c) Effect of 12 mg/kg of i.p. zolpidem on the walking beam performance of γ2I77lox control (n = 9) and PC-γ2-swap (n = 5) male mice. Mice were pre-trained for 4 d to traverse the 100-cm long, 0.8-cm diameter wooden beam. Columns represent means and vertical error bars s.e.m. (***P < 0.001 between mouse lines and P < 0.001 versus saline injection within the PC-γ2-swap line, two-way ANOVA and Newman-Keuls post hoc tests).