Fig. 8: Respiratory and behavioral changes after activation of the CeA-PVT circuit.

a–g Neural tracing analysis of downstream targets of CeA^GABA neurons by delivering a virus encoding mWGA into the CeA from Vgat-Cre mice. Postsynaptic neurons of CeA^GABA neurons were detected in several brain regions involved in emotional control. Scale bars, 20 μm. h Quantification of the number of mWGA⁺ neurons in different regions (n = 4 mice). i Schematic of the chemogenetic strategy to specifically activate CeA^GABA neurons projecting to the PVT. j Immunohistochemical detection of hM3Dq-mCherry expression in the CeA. Scale bar, 100 μm. k RNAscope-FISH images showing colocalization of hM3Dq (red) and Slc32a1 (green) mRNA. Scale bars, 50 μm. l Distribution of RF (bin size 0.5 Hz) in a 40-min assay following saline and CNO injections (n = 8 mice per group). Activation of LPBN-projecting CeA^GABA neurons produced a shift to rapid breaths. p < 0.0001 for 2.5 and 3 Hz. m The injection of CNO (i.p.) significantly increased RF at 10-, 20- and 30-min mark compared to saline treatment. n = 8 mice per group. 10 min, p = 0.0159; 20 min, p = 0.0024; 30 min, p = 0.0022. n Accumulative duration spent in active and quiescent states during 40-min plethysmography recordings. Activation of CeA^GABA neurons produced prolonged active time and reduced quiescent periods. o Accumulative duration of each behavior. p Schematic of the neural tracing strategy to identify CeA^GABA neurons that concurrently target both the PVT and LPBN. q Representative images showing identification of CeA^GABA neurons projecting to the LPBN (red) and PVT (green), and their overlap (composite color). The view indicated by yellow dashed rectangle (top) was enlarged in bottom images. Scale bars, 50 μm. r Quantification of the number of mCherry⁺, eYFP⁺ and overlap neurons (n = 3 mice). s Quantitative analysis of overlap neurons accounted for 26.3% ± 5.1% of the total mCherry⁺ neurons and 20.0% ± 3.1% of the total eYFP⁺ neurons (n = 3 mice). t Schematic of architecture of CeA^GABA neurons that form the CeA-PVT and CeA-LPBN circuits. ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001 by two-way ANOVA followed by Šídák’s multiple comparisons test (l, m), two-tailed unpaired t test (n, o). All data are presented as the mean ± SEM. Source data are provided as a Source Data file. The materials depicted in (g, i, p) are created in BioRender. Xiaoyi, W. (2025) https://BioRender.com/i81e326. Abbreviations: aca anterior commissure, anterior part Aq aqueduct, D3V dorsal 3rd ventricle, dlPAG dorsolateral periaqueductal gray, DRD dorsal raphe nucleus, dorsal part, DRI dorsal raphe nucleus, interfascicular part, DRL dorsal raphe nucleus, lateral part, DRN dorsal raphe nucleus, DRV dorsal raphe nucleus, ventral part, lBNST nucleus of stria terminalis, lateral division, LHb lateral habenular nucleus, lPAG lateral periaqueductal gray, lvBNST nucleus of stria terminalis, lateralventral division, mBNST nucleus of stria terminalis, medial division, MD mediodorsal thalamic nucleus, mlf medial longitudinal fasciculus, mvBNST nucleus of stria terminalis, medialventral division, PBP parabrachial pigmented nucleus of the ventral tegmental area, PN paranigral nucleus, PVT paraventricular thalamic nucleus, SNR substantia nigra, reticular part, vlPAG ventrolateral periaqueductal gray, VTA ventral tegmental area.