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. Author manuscript; available in PMC: 2024 Jul 1.
Published in final edited form as: Nature. 2023 Jun 28;619(7969):332–337. doi: 10.1038/s41586-023-06246-7

Figure 2. LH-Nts evoked calcium signals in the VTA are regulated by GABA release and Ntsr1 signaling.

Figure 2.

(a-b) Schematics for Cre-dependent CRISPR targeting (top) and viral injections (bottom left). (a) Quantification and example traces of Li-IPSCs recorded in VTA-GABA neurons with control CRISPR or sgVgat expression in LH-NTS neurons (bottom right; control, n=11 cells from N=3 mice; sgVgat n=7 cells from N=3 mice; **P<0.01, scale bar: 25 pA, 10 ms). (b) Quantification and example traces of currents evoked by 1 μM bath NTS (bottom right; control, n=10 cells from N=3 mice; sgNtsr1 n=8 cells from N=2 mice; *P<0.05, scale bar: 20 s, 50 pA). (c) Proposed model of calcium signals in VTA-DA neurons. (d) Schematic of viral injection and fiber implantation (left) and expression of Chrimson-tdTomato and GCaMP6m in the LH and VTA with representative optical fiber placements for stimulation and imaging (right). (e) Average GCaMP6m fluorescence (Z-score) following 1 s (top) or 3 s (bottom) stimulation at different frequencies. (f-g) Average area under the curve (AUC) of the Z-scored GCaMP6m fluorescence during (f) and for 10 s following (g) stimulation at different frequencies (3 s stim). (h) Average GCaMP6m fluorescence (Z-score) at the onset of 3 s, 40 Hz optical stimulation. (i) Average slope of the initial rise in GCaMP6m fluorescence at different stimulus frequencies (3 s stim). (j) Average GCaMP6m fluorescence decay following termination of 3 s stimulation, normalized to peak. (k) Average decay time constant (tau) of GCaMP6m fluorescence at different stimulus frequencies for 3 s stimulation. (e-k, control N=8, sgVgat N=7, sgNtsr1 N=6, sgVgat+sgNtsr1 N=5; Post-hoc comparisons: *P<0.05, **P<0.01, ***P<0.001, red asterisk sgVgat vs. control; blue asterisk sgNtsr1 vs. control; purple asterisk sgVgat+sgNtsr1 vs. control.) See Supplementary Table 2 for details on statistical tests. Data are presented as mean ± SEM.