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. 2020 Dec 29;9:e63493. doi: 10.7554/eLife.63493

Figure 6. POA neurons provide direct inhibition onto VGAT+ PAG neurons, which provide direct inhibition onto PAG-USV neurons.

(A) Viral strategy (left) and schematic (right) for whole-cell patch clamp recordings from fluorescently identified VGAT+ PAG cells while optogenetically activating POAPAG axons. (B) Example image of mCherry-labeled VGAT+ neurons with ChR2-labeled POAPAG axon terminals in the PAG. (C, D) Light-evoked inhibitory postsynaptic currents (IPSCs; observed in n = 26 of 36 VGAT+ neurons recorded from 11 mice) persisted in TTX/4AP and were abolished by bath application of gabazine (n = 10 cells recorded at baseline, n = 22 cells recorded in TTX/4AP, and n = 13 cells also recorded in gabazine including the following pairs: 6 cells recorded in both baseline and TTX/4AP, 3 cells recorded in both baseline and gabazine, and 10 cells recorded in both TTX/4AP and gabazine, p=0.03, one-way ANOVA comparing baseline vs. TTX+4-AP vs. SR-95531, followed by a post-hoc t-test revealing a significant difference between TTX+4-AP vs. SR-95531, p<0.018). IPSC amplitude refers to the peak of the light-evoked current at 0 mV holding potential. Error bars represent S.E.M. (E) Viral strategy (left) and schematic (right) for whole-cell recordings from fluorescently identified CANE-tagged PAG-USV neurons while optogenetically activating local VGAT+ PAG neurons. (F) Example image of mCherry-labeled CANE-tagged PAG-USV neurons and ChR2-labeled VGAT+ PAG neurons. (G,H) Light-evoked IPSCs recorded in TTX/4AP (observed in n = 13 of 16 CANE-tagged cells from four mice) were abolished by gabazine application (N = 10 cells also recorded in gabazine, p<0.001, paired t-test). IPSC amplitude refers to the peak of the light-evoked current at 0 mV holding potential. Error bars represent S.E.M. See also Figure 6—figure supplement 1 and Figure 6—source data 1.

Figure 6—source data 1. Source data for Figure 6D and H.

Figure 6.

Figure 6—figure supplement 1. POA neurons provide direct inhibition onto few PAG-USV neurons.

Figure 6—figure supplement 1.

(A) Viral strategy (left) and schematic (right) for whole-cell patch clamp recordings from fluorescently identified PAG-USV cells while optogenetically activating POAPAG axons. (B) Light-evoked inhibitory postsynaptic currents (IPSCs) were observed in 1 of 23 mCherry-labeled PAG-USV neurons recorded from seven mice. See also Figure 6—figure supplement 1—source data 1.
Figure 6—figure supplement 1—source data 1. Source data for panel B of Figure 6—figure supplement 1.