Fig. 7: Photostimulation of CeA^GABA neurons produces diverse effects on electrophysiological activity of LPBN^Glu neurons projecting to the preBötC.

a, b Schematic of the virus injection strategy and experimental setup for cell-attached and whole-cell patch-clamp slice recordings in LPBN^Glu neurons projecting to the preBötC, while illuminating (frequency: 10 Hz; width, 20 ms; power: 20 mW, duration: 10 s) axon terminals of CeA^GABA neurons projecting to the LPBN. c Action potentials were evoked by photostimulation of ChR2-expressing CeA^GABA neurons, as measured by whole-cell patch clamp slice recordings. d Immunohistochemical detection of ChR2 expression in the CeA (left, red), eYFP expression in the preBötC (middle, green), and axon terminals of CeA^GABA neurons and LPBN neurons projecting to the preBötC (right). Scale bars, 100 μm. e RNAscope-FISH images of colocalization of ChR2 (red), Slc32a1 (pink) and Gad2 (green) mRNAs in CeA^GABA neurons. Scale bar, 50 μm. f, g Quantitative analysis of the efficiency and specificity of ChR2-mCherry in CeA^GABA neurons. n = 4 mice. h–k Example traces showing that photostimulation produced different responsive patterns of firing in two subsets of LPBN^Glu neurons projecting to the preBötC (h, j). Firing rate histograms (10 s bin size; top traces) derived from cell-attached recordings (bottom traces) demonstrate increased firing rates in Type 1 neurons (i, p = 0.0073, n = 6 cells from 5 mice) and decreased firing rates in Type 2 neurons (k, p = 0.0009, n = 12 cells from 5 mice). l Pie chart showing the proportion of three subgroups of LPBN^Glu neurons projecting to the preBötC based on firing response patterns to photostimulation: Type 1 (n = 6, 14.6 %), responsive neurons with increased firing rate; Type 2 (n = 12, 29.3%), responsive neurons with decreased firing rate; Type 3 (n = 23, 56.1%), unresponsive neurons with unaltered firing rate. n = 41 neurons with spontaneous firing from 5 mice. m–p Typical traces showing changes in action potentials in the presence of illumination (m, o). Firing rate histograms (10 s bin size; top traces) derived from whole-cell recordings (bottom traces) demonstrate increased firing rates in Type 1 neurons (n, p = 0.0372, n = 3 neurons from 5 mice) and decreased firing rates in Type 2 neurons (p, p = 0.0031, n = 12 neurons from 5 mice). q Pie chart showing the proportion of three types of LPBN^Glu neurons projecting to the preBötC based on whole-cell recordings (n = 3 for Type 1, n = 12 for Type 2, n = 9 for Type 3, n = 26 neurons from 5 mice). r Typical traces showing that light-evoked (width: 5 ms, power: 20 mW) IPSCs recorded in LPBN^Glu neurons were inhibited by TTX (1 μM). Such an inhibition was reversed by 4-AP (100 μM). The evoked IPSCs were completely eliminated by PTX (50 μM). s Pie chart showing the percentage of preBötC-projecting LPBN^Glu neurons with light-evoked IPSCs: responsive (n = 13 neurons from 5 mice) and unresponsive (n = 28 neurons from 5 mice). t, u Quantification of the amplitude of light-evoked IPSCs of responsive neurons. t, p = 0.0012, aCSF vs. TTX; p = 0.0098, TTX vs. TTX + 4-AP (n = 13 neurons from 5 mice); u, p = 0.0002 (n = 13 neurons from 5 mice). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 by two-tailed paired t test (i, k, n, p), two-tailed Wilcoxon matched-pairs signed rank test (u), and two-tailed Friedman test with Dunn’s multiple comparisons test (t). All data are presented as the mean ± SEM. Source data is provided as a Source Data file. The materials depicted in (a, b) are created in BioRender. Xiaoyi, W. (2025) https://BioRender.com/i81e326.