Fig. 4
The activity of CaMKIIα neurons in the LPBN is increased after formalin injection. A-C Immunofluorescence staining of c-Fos and CaMKIIα in the LPBN after formalin injection. A Representative image of co-labeling of CaMKIIα-positive neurons (red) and c-Fos immunoreactivity (green) in the LPBN 1.5 h after saline or formalin injection. B Density of c-Fos-positive cells in the LPBN. C Proportions of CaMKIIα-positive cells co-expressing c-Fos in the LPBN (n = 6 sections from three mice per group). D–H In vivo calcium imaging of CaMKIIα neurons in the LPBN after formalin injection. D Schematic of unilateral LPBN stereotaxic injection of AAV-CaMKIIα-EGFP or AAV-CaMKIIα-GCaMP6s in naive mice and representative image of expression of the GCaMP6s in the CaMKIIα neurons of LPBN. The arrow indicates the implant location. Scale bar, 100 μm. E Schematic of the recording system for the Ca2+ signal in the CaMKIIα neurons of LPBN with fiber photometry in naive mice. F Average fluorescence signals (ΔF/F) of GCaMP6s (red) and EGFP (grey) recorded from CaMKIIα neurons in the LPBN after formalin injection; baseline was a 5 min period before formalin injection. G Mean ΔF/F of GCaMP6s and EGFP fluorescence signals from CaMKIIα neurons in the LPBN in phase 1 (0–5 min), interphase (5–15 min) and phase 2 (15–45 min). H Heatmap of ΔF/F for all individual mice with GCaMP6s (n = 5). Data are mean ± SEM. *p < 0.05. Two-tailed unpaired t test in B, C, and two-way ANOVA followed by the Tukey’s multiple comparisons test in E