A paraventricular hypothalamic nucleus input to ventral of lateral septal nucleus controls chronic visceral pain

Figure 1.. The expression levels of c-fos in the LSV region are significantly increased after CRD stimulation. (A) Schematic of EMG recording for evaluating visceral pain in mice. (B) Representative traces of EMG records. (C) The area under the curve of EMG at 20, 40, 60, and 80 mm Hg in CON and NMD mice (F (3, 15) = 83.68, **P < 0.01, ***P < 0.001, two-way repeated-measure ANOVA followed by Bonferroni post hoc test, n = 6 mice per group). (D) Schematic of c-fos expression evoked by visceral stimulation. (E) Representative images of c-fos–expressed neurons in the LSV region after CRD stimulation. Scale bar, 50 μm. (F) Total number of c-fos–expressed neurons of LSV after CRD stimulation (F (3, 15) = 250.6, ***P < 0.001, one-way ANOVA followed by Tukey post hoc test, n = 4 brain sections from 3 mice for CON, n = 5 brain sections from 3 mice for CON + CRD, NMD and NMD + CRD). (G) Schematic of c-fos expression evoked by mechanical stimulation. (H) Representative images of c-fos–expressed neurons in LSV after von Frey fiber stimulation. Scale bar, 50 μm. (I) Total number of c-fos–expressed neurons of the LSV region after von Frey fiber stimulation (F (3, 14) =8.093, *P < 0.05, one-way ANOVA…

Figure 2.. CRD stimulation induces a sharp increase in calcium signals in LSV neurons. (A) Schematic of the recording system for evaluating calcium signals with fiber photometry and visceral pain behavior evoked by CRD stimuli (top) and representative image of GCaMP6f expression in LSV (bottom). Scale bar, 50 μm. (B and C) Heatmap and average Ca2+ transients of LSV neurons in CON and NMD mice receiving CRD stimulation at 20, 40, 60, and 80 mm Hg, respectively. (D) The area under the curve of calcium activity of LSV neurons in CON and NMD mice receiving CRD stimulation at 20, 40, 60, and 80 mm Hg, respectively (F (3, 15) = 82.26, *P < 0.05, ***P < 0.001, two-way repeated-measure ANOVA followed by Bonferroni post hoc test, n = 6 mice per group). € Averaged peak ΔF/F of calcium activity of LSV neurons in CON and NMD mice receiving CRD stimulation at 20, 40, 60, and 80 mm Hg, respectively (F (3, 15) = 113.8, ***P < 0.001, two-way repeated-measure ANOVA followed by Bonferroni post hoc test, n = 6 mice per group). (F) Schematic of the recording system for evaluating calcium signals with fiber photometry and somatic stimulation evoked by von Frey fiber, tail pinch, and tail brush. (G and H)…

Figure 3.. LSV CaMKIIα-positive neurons are activated in visceral pain behaviors. (A) Representative images of c-fos (green) and CaMKIIα (red) coexpression in LSV (left) and coexpression ratios (right). Scale bar, 50 μm. (B) Representative images of CaMKIIα (red) and NeuN (green) coexpression in LSV (left) and coexpression ratio (right). Scale bar, 50 μm. (C) Schematic of a recording system for evaluating calcium signals with fiber photometry and visceral pain behavior caused by CRD stimuli. (D) Representative image of GCaMP6f expression in LSV CaMKIIα-positive neurons. Scale bar, 50 μm. (E and F) Heatmap and average Ca2+ transients of LSV CaMKIIα-positive neurons in CON and NMD mice receiving CRD stimulation at 20, 40, 60, and 80 mm Hg, respectively. (G) The area under the curve of calcium activity of LSV CaMKIIα-positive neurons in CON and NMD mice receiving CRD stimulation at 20, 40, 60, and 80 mm Hg, respectively (F (3, 15) = 40.44, **P < 0.01, ***P < 0.001, two-way repeated-measure ANOVA followed by Bonferroni post hoc test, n = 6 mice per group). (H) Averaged peak ΔF/F of calcium activity of LSV CaMKIIα-positive neurons in CON and NMD mice receiving CRD stimulation at 20, 40, 60, and…

Figure 4.. Optosilencing of LSV CaMKIIα-positive neurons attenuates visceral hyperalgesia, whereas optoactivation of LSV CaMKIIα-positive neurons promotes adaptive visceral pain behavior. (A) Experimental procedure of optogenetic manipulation of LSV CaMKIIα-positive neurons (left) and schematic of EMG recording for evaluating visceral pain (right). (B) Representative image of viral expression in LSV CaMKIIα-positive neurons (left) and summary view of fiber tip and injection site (right). Black dots and green dots indicate the fiber tip and injection site, respectively. Scale bar, 200 μm. (C) The representative EMG traces of NMD mice were recorded at 20, 40, 60, and 80 mm Hg, respectively. (D) The area under the curve of the EMG at 20, 40, 60, and 80 mm Hg in NMD mice (F (3, 15) = 121.3, ***P < 0.001, two-way repeated-measure ANOVA followed by Bonferroni post hoc test, n = 6 mice per group). (E) Representative EMG traces of CON mice were recorded at 20, 40, 60, and 80 mm Hg, respectively. (F) The area under the curve of EMG at 20, 40, 60, and 80 mm Hg in CON mice (F (3, 15) = 117.0, ***P < 0.001, two-way repeated-measure ANOVA followed by Bonferroni post hoc test, n = 6 mice per group).…

Figure 5.. Dissection of the PVH-LSV circuit. (A) Schematic of the retrograde virus tracing strategy. (B) Typical images of viral expression within the LSV and PVH. Scale bars, 50 µm. (C) Representative images of GFP (green, viral expression) and CaMKIIα (red) coexpression (left) and coexpression ratio in PVH (right). Scale bars, 50 µm. (D) Schematic of the anterograde viral tracing strategy. (E) Typical images of viral expression within the PVH and LSV. Scale bars, 50 µm (left) and 20 µm (right). (F) Schematic of monosynaptic anterograde tracing (left) and representative images of viral expression in LSV. Scale bars, 50 µm. (G) Representative images of c-fos–expressed neurons of PVH after CRD stimulation in CON and NMD mice (left) and the total number of c-fos–expressed neurons (right) (***P < 0.001, unpaired t test, n = 5 brain sections from 3 mice per group). (H) Representative images of c-fos (green) and CaMKIIα (red) coexpression in PVH (left) and coexpression ratios (right, n = 4 brain sections from 3 mice). Scale bars, 50 µm. ANOVA, analysis of variance; CRD, colorectal distention; LSV, lateral septal ventral; NMD, neonatal maternal deprivation; PVH, paraventricular hypothalamic.

Figure 6.. PVH CaMKIIα-positive neurons are involved in regulating visceral hyperalgesia. (A) Schematic of the recording system for evaluating calcium signals with fiber photometry and visceral pain behavior caused by CRD stimuli (top) and representative image of GCaMP6f expression in PVH CaMKIIα-positive neurons (bottom). Scale bar, 50 μm. (B and C) Heatmap and average Ca2+ transients of PVH CaMKIIα-positive neurons in CON and NMD mice receiving CRD stimulation at 20, 40, 60, and 80 mm Hg, respectively. (D) The area under the curve of calcium activity of PVH CaMKIIα-positive neurons in CON and NMD mice receiving CRD stimulation at 20, 40, 60, and 80 mm Hg, respectively (F (3, 15) = 62.90, *P < 0.05, **P < 0.01, two-way repeated-measure ANOVA followed by Bonferroni post hoc test, n = 6 mice per group). (E) Averaged peak ΔF/F of calcium activity of PVH CaMKIIα-positive neurons in CON and NMD mice receiving CRD stimulation at 20, 40, 60, and 80 mm Hg, respectively (F (3, 15) = 72.94, *P < 0.05, **P < 0.01, ***P < 0.001, two-way repeated-measure ANOVA followed by Bonferroni post hoc test, n = 6 mice per group). (F) Experimental procedure of optogenetic manipulation of PVH CaMKIIα-positive…

Figure 7.. Inhibition of PVH–LSV pathway reduces visceral hyperalgesia, whereas activation of PVH–LSV pathway contributes to adaptive visceral pain behavior. (A) Experimental procedure of a dual-virus strategy that specifically regulates the activity of CaMKIIα-positive neurons of the PVH–LSV pathway in NMD mice by combining optogenetic with chemogenetic (left) and schematic of experimental timelines for optogenetic and chemogenetic manipulation (right). (B) Schematic EMG recording for evaluating visceral pain under optogenetic and chemogenetic manipulation. (C) Representative EMG traces of NMD mice were recorded at 20, 40, 60, and 80 mm Hg, respectively. (D) The area under the curve of EMG at 20, 40, 60, and 80 mm Hg in NMD mice (F (3, 18) = 86.90, ***P < 0.001, two-way repeated-measure ANOVA followed by Bonferroni post hoc test, n = 7 mice per group). (E) Experimental procedure of a dual-virus strategy that specifically regulates the activity of CaMKIIα-positive neurons of the PVH–LSV pathway in CON mice by combining optogenetic with chemogenetic (left) and schematic of experimental timelines for optogenetic and chemogenetic manipulation (right). (F) Schematic EMG recording for evaluating…