Article Figures & Data
Figures
- Figure 1.
NMD enhances neuronal excitability of the CL and EMG activity. A, The experimental protocol of CRD stimulation. B, c-Fos expression increased after CRD stimulation at the CL region. Scale bars: 20 µm. The white boxes depict the area shown in the box of the CL region (n = 3 slices, **p < 0.01, two-sample t test). Scale bars: 50 µm. C, Representative traces illustrating sEPSCs of a CL neuron from CON and NMD mice. Bar plot showing the significant increase sEPSC frequency (CON, n = 6 cells; NMD, n = 7 cells, **p < 0.01, two-sample t test). D, Representative action potential traces from the CON and NMD groups evoked by 80-, 120-, and 160-pA current stimulation (left). Bar plot illustrating the higher firing frequency of neurons in NMD mice than in CON mice in response to different injected current intensity (right; CON, n = 6 cells; NMD, n = 5 cells, *p < 0.05, **p < 0.01, two-way RM ANOVA). E, Changes in the action potential threshold in CON and NMD mice (CON, n = 6 cells; NMD, n = 6 cells, **p < 0.01, two-sample t test). F, The RP was not altered after NMD (n = 6 cells, p > 0.05, two-sample t test). G, The experimental protocol of extracellular and EMG recording. H, Representative traces of extracellular and EMG recording of the CON and NMD groups. Bar plot showing the action potentials per second of the extracellular recordings and AUC of the EMG recordings at the same distention pressure (CON, n = 8 mice; NMD, n = 8 mice, **p < 0.01, two-way RM ANOVA). I, Correlation analysis showing a positive correlation between EMG and extracellular recordings at 20 and 60 mmHg CRD stimulation (r = 0.5489, p = 0.0341). See also Extended Data Figure 1-1.
- Figure 2.
Excitatory glutamatergic neurons in the CL participate in the visceral pain experience of NMD mice. A, Representative images of c-Fos neurons (green) and CaMKII neurons (red) co-expression in the CL (left) and the percentage co-expressed in the CL (right). Scale bars: 50 µm. White arrows point to representative neurons. B, Schematic of optogenetic experiments in CON and NMD mice (left); timeline of optogenetic experiments (middle); representative images of viral expression in the CL (right). Scale bars: 20 µm. The white boxes depict the area shown in the box of the CL. Scale bars: 50 µm. C, Sample traces and summarized data showing the effects of photostimulation (eNpHR, n = 8 mice, ***p < 0.001, two-way RM ANOVA). D, Summarized data showing the effects of photostimulation (EYFP, n = 9 mice, p > 0.05, two-way RM ANOVA). E, Sample traces and summarized data showing the effects of photostimulation (hChR2, n = 8 mice, **p < 0.01, two-way RM ANOVA). F, Summarized data showing the effects of photostimulation (EYFP, n = 8 mice, p > 0.05, two-way RM ANOVA). G, Representative images of c-Fos neurons (red) and glutamatergic neurons (green) co-expression in the CL (left) and the percentage of co-expression in the CL (right). Scale bars: 50 µm. White arrows point to representative neurons. H, Schematic of optogenetic (left) and representative images of viral expression in the CL (right). Scale bars: 20 µm. The white boxes depict the area shown in the box of the CL. Scale bars: 50 µm. I, Sample traces and summarized data showing the effects of photostimulation (eNpHR, n = 6 mice, ***p < 0.001, two-way RM ANOVA). J, Summarized data showing the effects of photostimulation (EYFP, n = 5 mice, p > 0.05, two-way RM ANOVA). K, Sample traces and summarized data showing the effects of photostimulation (hChR2, n = 8 mice, ***p < 0.0001, two-way RM ANOVA). L, Summarized data showing the effects of photostimulation (EYFP, n = 6 mice, p > 0.05, two-way RM ANOVA).
- Figure 3.
Stimulation of the CL terminal alters ACC activity and the visceral pain experienced by NMD mice. A, Schematic of the anterograde virus tracing strategy. Typical images of AAV9-hSyn-EGFP injection sites within the CL (left) and viral expression in the ACC (right). Scale bars: 20 µm. The white boxes depict the area shown in the box of the CL. Scale bars: 50 µm. B, Schematic of the virus tracing strategy and representative images of EYFP neurons (green) and CaMKII neurons (red) co-expression in the ACC. Scale bars: 20 µm. The white boxes depict the area shown in the box of the CL. Scale bars: 50 µm. C, Schematic of the retrograde virus tracing strategy. Typical images of AAV2/R-hSyn-EGFP injection sites within the ACC (left) and viral expression in the CL (right). Scale bars: 20 µm. The white boxes depict the area shown in the box of the CL. Scale bars: 50 µm. D, Representative images of GFP neurons (green) and CaMKII neurons (red) co-expression in the CL. Scale bars: 50 µm. The percentage of GFP and CaMKII-positive neurons in the CL. White arrows point to representative neurons. E, Schematic of optogenetic and in vivo recording of NMD mice. F, Sample traces (left) and summarized data (right) showed the firing rate of ACC neurons in the NMD mice before, during, and after light photostimulation with multiple channel recordings (n = 8 mice, *p < 0.05, **p < 0.01, one-way ANOVA). G, Summarized data showing the effects of photostimulation (eNpHR, n = 11 mice, ***p < 0.001, two-way RM ANOVA). H, Summarized data showing the effects of photostimulation (EYFP, n = 10 mice, p > 0.05, two-way RM ANOVA). I, Schematic of optogenetic and in vivo recording of CON mice. J, Sample traces (left) and summarized data (right) showing the firing rate of ACC neurons in the CON mice before, during, and after light photostimulation with multiple channel recordings (n = 8 mice, *p < 0.05, **p < 0.01, one-way ANOVA). K, Sample traces and summarized data showing the effects of photostimulation (hChR2, n = 11 mice, *p < 0.05, two-way RM ANOVA). L, Summarized data showing the effects of photostimulation (EYFP, n = 8 mice, p > 0.05, two-way RM ANOVA).
- Figure 4.
NMD enhances neuronal excitability of the ACC and EMG activity. A, The expression of c-Fos at the ACC increased after CRD stimulation (n = 4 slices, ***p < 0.001, two-sample t test). Scale bars: 20 µm. The white boxes depict the area shown in the box of the CL. Scale bars: 50 µm. B, Representative traces of action potential frequency recorded in the ACC of CON and NMD mice (left). Changes in action potential frequency in CON and NMD mice (right; CON, n = 6 cells, NMD, n = 8 cells, *p < 0.05, two-way RM ANOVA). C, Changes in action potential threshold in CON and NMD mice (n = 8 cells, *p < 0.05, two-sample t test). D, The RP threshold was not significantly altered between CON and NMD mice (n = 8 cells, p > 0.05, two-sample t test). E, Representative traces of sEPSCs recorded in the ACC of CON and NMD mice. Bar plots of the amplitude and frequency of sEPSCs recorded in the ACC of CON and NMD mice (n = 8 cells, *p < 0.05, two-sample t test). F, Representative traces of extracellular recording in the ACC of CON and NMD mice. Bar plots describing the action potentials per second of extracellular recording at the same distention pressure (n = 8 mice, **p < 0.01, two-way RM ANOVA). G, Representative traces of EMG recording of CON and NMD mice. Bar plot describing the AUC of EMG recording at the same distention pressure (n = 8 mice, **p < 0.01, two-way RM ANOVA). H, Correlation analysis showing a positive correlation between the EMG and extracellular recordings in NMD mice at 20 and 60 mmHg CRD stimulation (r = 0.6434, p = 0.0177).
- Figure 5.
ACC glutamatergic excitatory neurons participate in visceral pain of NMD mice. A, Representative images of c-Fos neurons (green) and CaMKII neurons (red) co-expression in the ACC of NMD mice (left) and the percentage of co-expression in the ACC (right). Scale bars: 50 µm. White arrows point to representative neurons. B, Schematic of optogenetic (left) and representative images of viral expression in the ACC (right). Scale bars: 20 µm. The white boxes depict the area shown in the box of the ACC. Scale bars: 50 µm. C, Sample traces and summarized data showing the effects of photostimulation (eNpHR, n = 8 mice, *p < 0.05, two-way RM ANOVA). D, Summarized data showing the effects of photostimulation (EYFP, p > 0.05, n = 7 mice, two-way RM ANOVA). E, Sample traces and summarized data showing the effects of photostimulation (hChR2, n = 7 mice, **p < 0.01, two-way RM ANOVA). F, Summarized data showing the effects of photostimulation (EYFP, n = 10 mice, p > 0.05, two-way RM ANOVA). G, Representative images of c-Fos neurons (red) and glutamatergic neurons (green) co-expression in the ACC of VGlu2-Cre mice (left), and the percentage of co-expression in the ACC (right). Scale bars: 50 µm. White arrows point to representative neurons. H, Schematic of optogenetic (left) and representative images of viral expression in the ACC (right). Scale bars: 20 µm. The white boxes depict the area shown in the box of the CL. Scale bars: 50 µm. I, Sample traces and summarized data showing the effects of photostimulation (eNpHR, n = 6 mice, *p < 0.05, two-way RM ANOVA). J, Summarized data showing the effects of photostimulation (EYFP, n = 5 mice, p > 0.05, two-way RM ANOVA). K, Sample traces and summarized data showing the effects of photostimulation (hChR2, n = 6 mice, **p < 0.01, two-way RM ANOVA). L, Summarized data showing the effects of photostimulation (EYFP, n = 9 mice, p > 0.05, two-way RM ANOVA).
- Figure 6.
ACC regulation affects CL-mediated chronic visceral pain in mice. A, Schematic of optogenetic and chemogenetic protocol. B, Representative images of viral expression in the CL (top) and ACC (bottom). Scale bars: 20 µm. The white boxes depict the area shown in the box of the ACC. Scale bars: 50 µm. C, The sample traces of EMG. D, Summarized data showing the effects of photostimulation. Blue-light stimulation of the ACC can increase chronic visceral pain, and chemogenetic stimulation can reverse chronic visceral pain, in CON mice by inhibition the excitability of neurons in the ACC (n = 8 mice, *p < 0.05, two-way RM ANOVA). E, Schematic of optogenetic and chemogenetic protocol. F, Representative images of viral expression in the CL (top) and ACC (bottom). Scale bars: 20 µm. The white boxes depict the area shown in the box of the ACC. Scale bars: 50 µm. G, The sample traces of EMG. H, Summarized data showing the effects of photostimulation. Yellow-light stimulation of the ACC can significantly decrease chronic visceral pain, and chemogenetic stimulation can reverse chronic visceral pain, in NMD mice by activation the excitability of neurons in the ACC (n = 6 mice, *p < 0.05, **p < 0.01, two-way RM ANOVA).
- Figure 7.
Alteration of NMDAR subunit expression and function in the PSD of ACC of NMD mice. A, Western blottings showing increased NR2A and NR2B subunits of NMDAR and enhanced CaMKIIα activation in the PSD of the NMD mice ACC, compared with CON mice ACC. B, Quantification of NR2A/PSD95 as in panel A (n = 3 mice, **p < 0.01, two-sample t test). Quantification of NR2B/PSD95 as in panel A (n = 3 mice, **p < 0.01, two-sample t test). Quantification of GluR1/PSD95 as in panel A (n = 3 mice, p > 0.05, two-sample t test). Quantification of GluR2/PSD95 as in panel A (n = 3 mice, p > 0.05, two-sample t test). Quantification of p-CaMKIIα/CaMKIIα normalized to CON mice ACC as in panel A (n = 3 mice, ***p < 0.001, two-sample t test). Quantification of CaMKIIα/PSD95 as in panel A (n = 3 mice, p > 0.05, two-sample t test). C, D-AP5 or KN-93 increased visceral pain threshold in NMD mice (n = 8 mice, **p < 0.01, ***p < 0.001, two-sample t test). D, Schematic of chemogenetic and D-AP5 injected protocol in CON mice. Sample traces and summarized data showing the effects of chemogenetic activation the CL neuron terminal in the ACC and D-AP5 inhibition the NMDAR in the ACC (n = 8 mice, **p < 0.01, two-way RM ANOVA).
- Figure 8.
A working model showing that a direct CLGlu-ACCGlu neural circuit mediates chronic visceral pain. A, CRD induces firings of both CL and ACC neurons and visceral pain sensation of CON mice. Blue-light activation of CL glutamatergic neuron terminals in ACC region evoked a lot of more firings of ACC neurons accompanied by an enhanced visceral pain response, which was reversed by chemogenetic inhibition of ACC glutamatergic neurons of CON mice. B, CRD induces a lot of more firings of both CL and ACC neurons and an enhanced visceral pain response of NMD mice when compared with CON mice. Yellow-light inhibition of CL glutamatergic neuron terminals in ACC region suppressed both the ACC neural activity and the visceral hypersensitivity of NMD mice, which was reversed by chemogenetic activation of ACC glutamatergic neurons.
: indicating a neuron under physiologic condition; : indicating a neuron under hyperactive condition.
Extended Data
Extended Data Figure 1-1
CFA does not increase c-Fos expression, neural excitability, and synaptic transmission of the CL, and the effects of optogenetically modulating of CL on visceral pain responses. Connect to Figure 1. A, The left hindpaw of a mouse at 3 d after CFA injection and NS. CFA induced changes in the paw withdrawal frequency (n = 6 mice, *p < 0.05, two-sample t test). B, The expression of c-Fos in the contralateral CL did not increase after CFA injection into the left paw (NS, n = 6 slices; CFA, n = 7 slices, p > 0.05, two-sample t test). Scale bars: 20 µm. The white boxes depict the area shown in the box of the CL. Scale bars: 50 µm. C, The expression of c-Fos in the ipsilateral CL did not increase after CFA injection into the left paw (NS, n = 5 slices; CFA, n = 5 slices, p > 0.05, two-sample t test). Scale bars: 20 µm. The white boxes depict the area shown in the box of the CL. Scale bars: 50 µm. D, Representative traces of sEPSCs recorded in the CL of NS and CFA mice. Bar plots of the frequency and amplitude of sEPSCs recorded in the CL of NS and CFA mice (n = 6 cells, p > 0.05, two-sample t test). E, Representative traces of action potential frequency recorded in the ACC of NS and CFA mice. Bar plots of the action potential frequency in NS and CFA mice (n = 6 cells, p > 0.05, two-way RM ANOVA). F, Changes in action potential threshold and RP threshold were not significantly difference in NS and CFA mice (n = 6 cells, p > 0.05, two-sample t test). G, Schematic of optogenetic experiments in CFA and NS mice and timeline of optogenetic experiments. Representative images of viral expression in the CL. Scale bars: 20 µm. The white boxes depict the area shown in the box of the CL. Scale bars: 50 µm. H, Summarized data showing the effects of photostimulation in CFA mice (eNpHR, n = 8 mice, p > 0.05; hChR2, n = 8 mice, p > 0.05, two-sample t test). I, Summarized data showing the effects of photostimulation in NS mice (eNpHR, n = 8 mice, p > 0.05; hChR2, n = 8 mice, p > 0.05, two-sample t test). J, Schematic of optogenetic experiments in CFA and NS mice and timeline of optogenetic experiments. Representative images of viral expression in the CL. Scale bars: 20 µm. The white boxes depict the area shown in the box of the CL. Scale bars: 50 µm. K, Summarized data showing the effects of photostimulation in CFA mice (eNpHR, n = 8 mice, p > 0.05, two-sample t test). L, Summarized data showing the effects of photostimulation in NS mice (hChR2, n = 8 mice, p > 0.05, two-sample t test). Download Figure 1-1, TIF file.
