Figure 1
From: Neuropsychiatric Phenotypes Produced by GABA Reduction in Mouse Cortex and Hippocampus
Outline of transgenic approach and validation of Gad1 genetic ablation. (a) Schematic diagram showing generation of the conditional knockout of Gad1, Ppp1r2-Cre+/-:Gad1loxP/loxP, referred to as mutant and of the triple transgenic mouse that allows visualization of Cre-targeted cells by enhanced YFP (EYFP), Ppp1r2-Cre+/-:Gad1loxP/loxP: Rosa26 loxP-Stop-LoxP – EYFP+/-, referred to as YFP-mutant. (b) Double immunolabeling in the mPFC of GAD67 (red) and YFP (green) in mPFC of 10-week-old mice showing the YFP cells positive for GAD67. The table shows %YFP cells positive for GAD67 (out of 135 cells in the controls; 132 cells in the mutants in mPFC) in both genotypes demonstrating GAD67 deletion virtually all YFP-positive cells in mutant mice. (c) Representative western blot bands and their quantification (in graph) showing that GAD67 protein is reduced in somatosensory cortical tissue homogenates of 10-week-old mutants (t(10)=3.09, p<0.01) while GAD65 is increased (t(11)=3.04, *p<0.01). (d) Tissue GABA content in the control floxed mice (white, n=14) and mutant mice (black, n=13). GABA level is lower in mPFC (t(25)=2.69, *p=0.0013), frontal cortex (FC, t(25)=4.07, *p=0.0004) and hippocampus (Hipp, t(25)=2.09, *p=0.046), but not in striatum (t(25)=0.12, p=0.91) or cerebellum (Cere, t(25)=1.03, p=0.31). (e) Gad1 ablation in GABAergic interneurons reduces the amplitude of evoked IPSCs in the anterior cingulate cortex (ACC). Plot of evoked IPSC amplitudes in slices from control and mutant mice (two-way ANOVA, F(1,88)=64.5 for genotype main effect, *p<0.001). Inset: Representative traces from control and mutant groups showing the difference in amplitudes at the same stimulation intensity. (f) Pyramidal neurons in mutant mPFC are more excitable compared to controls. Summary graph of the firing frequency of pyramidal cells with the mutant group exhibiting significantly higher number of action potentials in response to somatic current injection (two-way ANOVA, F(1,187)=27.1 for genotype main effect, *p<0.001). Inset: Representative recordings showing the firing patterns of pyramidal neurons, for a current injection of 100 pA. All Data are mean±SEM; animal number or cell number n is indicated in parentheses or plot bars.
