Molecular, Circuit, and Stress Response Characterization of Ventral Pallidum Npas1-Neurons

Representative images of DIO-eYFP and tdTomato expression in multiple brain regions of the Npas1-cre mouse. The first and fourth columns (from the left) show the expression of the eYFP (in green) within the brain regions, while the second and fifth columns show the expression of the tdTomato (in red). The third and sixth columns show the merged images. Scale bar, 100 µm. ac, Anterior commissure; LS, lateral septum; MS, medial septum; Hb, habenula; MHb, medial habenula; PVT, paraventricular nucleus of the thalamus; LH, lateral hypothalamus; opt, optical nerve; Thal, thalamus; LV, lateral ventricle; 3V, third ventricle; PAG, periaqueductal gray; Aq, cerebral aqueduct.

Cell type-specific profiling of mRNA from VP Npas1⁺ neurons reveal potential functions and regulators these neurons. A, Validation of Npas1 enrichment in ribosome-associated mRNA from VP Npas1⁺ neurons used in genetic profiling. B, Volcano plot of DEGs within Npas⁺ VP cells versus non-cell type-specific VP cells. Supporting data listing the significantly enriched genes from VP Npas1⁺ neurons compared with nonspecific VP neurons are available in Extended Data Figure 3-1. C, Top hits from Biological Process and Molecular Function GO (Gene Ontology) term analysis and an example from one of the top hits, regulation of neurogenesis, with the degree of fold change of genes within the GO term. Supporting data listing top hits from Biological Process and Molecular Function GO term analysis in VP Npas1⁺ neurons are available in Extended Data Figure 3-2. D, Two top hits from upstream regulator prediction analysis using the top two GO terms identified, neurogenesis and development of nervous system. Predicted DEG targets of each regulator are identified with arrows along with their fold change analyzed in VP Npas1⁺ neurons.

Validation of DREADDs activation on Npas1⁺ neurons. A, Representative current-clamp recordings from VP Npas1⁺ neurons in vitro expressing the hM4Di (left) or hM3Dq (right) receptor. Following CNO wash-on, hM4Di cells (n = 4) showed a decrease in spiking, and hM3Dq cells (n = 3) showed an increase in spiking in response to depolarizing current steps. B, A subset of hM3Dq cells (n = 8) that did not have an increase in spiking displayed enhanced depolarization but also a reduction in membrane resistance. *p < 0.05.

Chemogenetic activation of hM3Dq receptors in VP Npas1⁺ neurons increases susceptibility to the SSDS in male mice. A, Representative timeline of the experiments involving the manipulation of VP Npas1⁺ neurons during the SSDS. B–D, Effects of the chemogenetic activation of hM3Dq receptors in VP Npas1⁺ neurons during the SSDS in the SI test and FST. E–G, Effects of chemogenetic activation of hM4Di receptors in VP Npas1⁺ neurons during the SSDS in the SI and FST. Data are presented as the mean plus the individual values obtained for each animal (n = 5–9 animals/group). *p < 0.05, **p < 0.01, ***p < 0.001.

Chemogenetic activation of hM4Di receptors in VP Npas1⁺ neurons decreases susceptibility to the CSDS in male mice. A, Representative timeline of the experiments involving the activation of hM4Di receptors in VP Npas1⁺ neurons during the CSDS. B–D, Effects of the chemogenetic activation of hM4Di receptors in VP Npas1⁺ neurons during the CSDS in the SI and FST. Data are presented as the mean plus the individual values obtained for each animal (n = 6–8 animals/group). *p < 0.05, **p < 0.01, ***p < 0.001.

Chemogenetic activation of hM3Dq receptors in VP Npas1⁺ neurons is anxiogenic in male mice. A, Representative timeline of the experiments involving the activation of hM3Dq receptors in VP Npas1⁺ neurons during behavioral tests that evaluate anxiety-like and depressive-like behaviors. B–E, effects of chemogenetic activation of hM3Dq receptors in VP Npas1⁺ neurons during the EPM test. F, G, effects of the chemogenetic activation of hM3Dq receptors in VP Npas1⁺ neurons during the OFT. H, I, Effects of the chemogenetic activation of hM3Dq receptors in VP Npas1⁺ neurons during the SI. J, Effects of chemogenetic activation of hM3Dq receptors in VP Npas1⁺ neurons during the ST. K, L, Effects of chemogenetic activation of hM3Dq receptors in VP Npas1⁺ neurons on the sucrose preference and intake on the SPT. Data are presented as the mean plus the individual values obtained for each animal (n = 8–9 animals/group). *p < 0.05.