Figure 2. Chemogenetic inhibition of pyramidal neurons in the LA, but not BA impairs auditory fear conditioning. A, hM4Di-mCherry fluorescence in the LA of a CaMKIICre(+) mouse, 4 weeks after infusion of AAV8-hSyn-DIO-hM4Di-mCherry. Scale bar, 50 μm. B, Schematic summarizing the distribution of hM4Di-mCherry fluorescence in the LA (spanning –1.22mm to –1.94mm posterior from bregma) of CaMKIICre(+) mice evaluated in E. C, CNO-induced inhibition of an hM4Di-expressing LA pyramidal neuron. Rheobase was measured before (baseline) and after application of CNO (10 μm). The current-step protocol is depicted below the traces. The first step to elicit spiking is highlighted in red; the trace shown is the response to the denoted current step. D, Rheobase summary for hM4Di-expressing LA pyramidal neurons, before and after CNO application (t(3)=4.4, *p = 0.021). Each experiment is shown as connected circles (n = 4). E, Impact of LA pyramidal neuron inhibition on auditory fear conditioning. CaMKIICre(+)/hM4Di (black) and CaMKIICre(−)/hM4Di (white) mice were trained using a 3 CS/3 US paradigm. CNO (2 mg/kg, i.p.) was administered 30 min before training. Freezing behavior during training is plotted on the left. The yellow bars represent CS presentations, and the red bars represent US presentations. There was no main effect of genotype (F(1,54)=2.6, p = 0.14). The plots on the right show freezing during context (t(9)=4.0, **p = 0.003) and cue (t(9)=2.9, *p = 0.017) recall tests. The bars represent the mean ± SEM, with dots next to the bars denoting individual data points (n = 5–6/group). Sex differences were not assessed for this experiment. F, hM4Di-mCherry fluorescence in the BA of a CaMKIICre(+) mouse, 4 weeks after infusion of AAV8-hSyn-DIO-hM4Di-mCherry. Scale bar, 50 μm. G, Schematic summarizing the distribution of hM4Di-mCherry fluorescence in the BA (coronal view, spanning –1.22mm to –1.94mm posterior from bregma) of CaMKIICre(+)/hM4Di mice evaluated in C. H, CNO-induced inhibition of an hM4Di-expressing BA pyramidal neuron. Rheobase was measured before (baseline) and after application of CNO (10 μm), as described for C. I, Rheobase summary for hM4Di-expressing BA pyramidal neurons, before and after CNO application (t(6)=7.6, ***p < 0.001). Each experiment is shown as connected circles (n = 7). J, Impact of BA pyramidal neuron inhibition on fear learning. CaMKIICre(+)/hM4Di (red) and CaMKIICre(−)/hM4Di (white) mice were trained using a 3 CS/3 US paradigm. CNO (2 mg/kg, i.p.) was administered 30 min before training. Freezing behavior during training is plotted on the left. The yellow bars represent CS presentations, and the red bars represent US presentations. There was a significant main effect of genotype (F(1,132)=4.9, p = 0.038; CS3: ***p < 0.001, post-CS3: *p = 0.023). The plots on the right show freezing during context (t(22)=1.9, p = 0.074) and cue (t(29)=0.1, p = 0.905) tests. Error bars represent the mean ± SEM, with dots next to the bars denoting individual data points (n = 5/6–7/5 males/females per group).