Fig. 6: The CeA-LPBN circuit primarily regulates breathing patterns in behaviorally quiescent state.

a Illustration of the viral injection strategy for chemogenetic stimulation of CeA^GABA neurons projecting to the LPBN. b Immunohistochemical detection of hM3Dq expression in the CeA. Scale bar, 100 μm. c RNAscope-FISH images showing colocalization of hM3Dq (red) and Slc32a1 (green) mRNA in CeA^GABA neurons projecting to the LPBN. Scale bars, 20 μm. d Distribution of RF (bin size 0.5 Hz) over a 40-min assay of saline and CNO injections (n = 8 mice per group). Activation of CeA^GABA neurons projecting to the LPBN produced a shift to rapid breaths. p < 0.0001 for 1.5 and 2 Hz. e Time-course of RF changes over a 60-min recording following saline and CNO injections. RF significantly increased at 30-min mark (p = 0.0425). n = 8 mice per group. f Accumulative duration spent in active and quiescent states during 40-min plethysmography assay (n = 8 mice per group). Activation of CeA^GABA neurons projecting to the LPBN had no effect on both active and quiescent durations. g Accumulative duration of each behavior (n = 8 per group). Compared to the saline group, sniff was reduced in the CNO group (p = 0.0262). h Schematic of the genetic ablation strategy by bilateral injections of a virus encoding Casp3 into the LPBN. i–k Following ablation of LPBN neurons projecting to the preBötC, photostimulation (10 Hz) of axon terminals of CeA^GABA neurons projecting to the LPBN had no significant effect on breathing parameters (n = 6 per group). l Schematic illustrating bilateral ablation of LPBN^Glu neurons projecting to the preBötC. m–o Following ablation of LPBN^Glu neurons projecting to the preBötC, photostimulation (10 Hz) of axon terminals of CeA^GABA neurons projecting to the LPBN produced no significant effect on breathing parameters (n = 9 per group). p Schematic of bilateral ablation of LPBN^GABA neurons projecting to the preBötC. q–s Following ablation of LPBN^GABA neurons projecting to the preBötC, photostimulation (10 Hz) of axon terminals of CeA^GABA neurons projecting to the LPBN significantly increased ventilation (n = 9 for per group). q, p = 0.0045; r, p = 0.0479; s, p = 0.0028. ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001 by two-way ANOVA followed by Šídák’s multiple comparisons test (d, e), two-tailed unpaired t-test (f, g: moving, sniff, eupnea, sleep), two-tailed Mann–Whitney test (g: groom) and two-tailed paired t test (i–k, m–o, q–s). All data are presented as the mean ± SEM. Source data is provided as a Source Data file. The materials depicted in (a, h, l, p) are created in BioRender. Xiaoyi, W. (2025) https://BioRender.com/i81e326.