Fig. 4: Photostimulation of CeAGABA neurons activates breathing under quiescence and anesthesia conditions. | Nature Communications

Fig. 4: Photostimulation of CeAGABA neurons activates breathing under quiescence and anesthesia conditions.

From: GABAergic neurons in central amygdala contribute to orchestrating anxiety-like behaviors and breathing patterns

Fig. 4

a Schematic of the optogenetic strategy involving the delivery of a virus encoding ChR2 or a control virus lacking ChR2 in Vgat-Cre mice. b Immunohistochemical validation of ChR2 expression. Left image: eYFP+ (left, green) and ChR2-mCherry+ (right, red) neurons in the CeA and tracks of fibers implantation. The middle and right confocal images are enlarged views of the regions denoted by the squares in the left image. Scale bars, 200 µm. c Whole-cell patch clamp slice recordings of light-evoked action potentials in a CeAGABA neuron confirming the excitatory effect of ChR2 activation. d RNAscope-FISH images showing colocalization of ChR2 (red) and Slc32a1 (green) mRNA. Scale bar, 50 μm. e Quantitative analysis of the efficiency and specificity of ChR2-mCherry expression. n = 4 mice. f Schematic diagram of optogenetics and WBP. g Typical respiratory waveforms showing the photostimulation effects on ventilation. h Distribution of RF (bin size 0.5 Hz) over a 1-min assay. n = 10 per group. p < 0.0001 for 2 and 2.5 Hz, p = 0.0008 for 3.5 Hz, p = 0.0025 for 4 Hz. i Quantitative analyses of the effects of illumination at different frequencies on breathing parameters. n = 7 for eYFP, n = 10 for ChR2. 5 Hz: p = 0.0004 for RF, p = 0.0142 for MV; 10 Hz: p = 0.0001 for RF, p < 0.0001 for MV; 20 Hz: p < 0.0001 for RF, p < 0.0001 for MV; ChR2-laser off vs. ChR2-laser on. j Schematic of PND recordings in bilaterally vagotomized, mechanically ventilated, anesthetized mice. k Representative traces showing that photostimulation of CeAGABA neurons significantly increased PND activity. l, m Quantification of group data (n = 11 per group). l, p < 0.0001. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA followed by Šídák’s multiple comparisons test (h), two-tailed paired t-test (i, l) and two-tailed Wilcoxon matched-pairs signed rank test (m). All data are presented as the mean ± SEM. Source data is provided as a Source Data file. The materials depicted in (a, f, j) are created in BioRender. Xiaoyi, W. (2025) https://BioRender.com/i81e326. PND phrenic nerve discharge.

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