Volume 157, Issue 2, Pages 522-536.e2 (August 2019) Extrinsic Primary Afferent Neurons Link Visceral Pain to Colon Motility Through a Spinal Reflex in Mice  Kristen M. Smith-Edwards, Sarah A. Najjar, Brian S. Edwards, Marthe J. Howard, Kathryn M. Albers, Brian M. Davis  Gastroenterology  Volume 157, Issue 2, Pages 522-536.e2 (August 2019) DOI: 10.1053/j.gastro.2019.04.034 Copyright © 2019 AGA Institute Terms and Conditions

Gastroenterology 2019 157, 522-536. e2DOI: (10. 1053/j. gastro. 2019 Copyright © 2019 AGA Institute Terms and Conditions

Figure 1 Characterization of spontaneous and evoked GCaMP activity in myenteric neurons. (A, B) Time-lapse color-coded image (left) and ΔF/F0 traces (right) of myenteric neurons without stimulation in (A) ex vivo and (B) in vivo preparations. Each pixel in a collection of GCaMP images is assigned a color corresponding to when that particular pixel reached peak fluorescence. (C) There were no significant differences in percentage of spontaneously active cells between ex vivo and in vivo preparations. (D, E) HEX significantly decreased the proportion of myenteric neurons that displayed spontaneous GCaMP activity (D) ex vivo and (E) in vivo. (F) Amplitudes of responses were not significantly different across frequencies. (G) Percentage of responsive neurons per myenteric ganglion increased with increasing frequency. (H) Time-lapse color-coded image (Hi), ΔF/F0 traces of responsive myenteric neurons (Hii), and resulting tissue movement (Hiii: black, circular muscle axis; gray, longitudinal muscle axis) to 20-Hz electrical stimulation of the colon (red arrow). (I) TTX significantly decreased the percentage of responsive neurons per ganglion. (J) TTX significantly decreased evoked movement. (K–M) No significant differences in the amplitude of (K) evoked responses or (L) response latency were measured in ex vivo and in vivo experiments, but (M) movement latency was significantly increased in vivo. ∗P < .05, ∗∗P < .01, (C, K–M) unpaired and (D, E, I–J) paired Student t test, 1-way analysis of variance (ANOVA), Tukey post hoc test (F, G). Gastroenterology 2019 157, 522-536.e2DOI: (10.1053/j.gastro.2019.04.034) Copyright © 2019 AGA Institute Terms and Conditions

Figure 2 Stimulation oral and anal to the imaging field activates distinct neural circuits and leads to different patterns of smooth muscle contractility. (A, B) Time-lapse color-coded images (i) and ΔF/F0 traces (ii) of responses to stimulation oral (left) and anal (right) stimulation in (A) ex vivo and (B) in vivo preparations. (C, E) Percentage of neurons per ganglion that exhibit early (i) and late (ii) responses due to oral and anal stimulation in (C) ex vivo and (E) in vivo preparations. (D, F) Movement traces in the circular (i) and longitudinal (ii) muscle axes evoked by oral (black) and anal (gray) stimulation in (D) ex vivo and (F) in vivo preparations. Positive values represent lengthening (circular) or movement (longitudinal) in the oral direction; negative values represent shortening (circular) or movement (longitudinal) in the anal direction.(G, H) Scatterplot of movement coordinates from (G) ex vivo and (H) in vivo preparations. (I–L) HEX significantly decreased the percentage of responsive neurons to (I, K) oral and (J, L) anal stimulation in vivo and ex vivo. (C, E, I–L) ∗P < .05, ∗∗P < .01, paired Student t tests. Gastroenterology 2019 157, 522-536.e2DOI: (10.1053/j.gastro.2019.04.034) Copyright © 2019 AGA Institute Terms and Conditions

Figure 3 Specific activation of ExPANs with capsaicin (CAP) does not reproduce myenteric neuron responses and smooth muscle contractions induced by capsaicin applied to the colon. (A) Schematic of ex vivo preparation in which 2 chambers keep the L6 DRG and dorsal and ventral roots separate from the colon. (B) Time-lapse color-coded images of DRG responses to electrical stimulation of the dorsal root (left) and CAP (10 μmol/L, right); electrical stimulation produced responses significantly greater in amplitude compared with CAP (Bii). (C) GCaMP traces of spontaneous activity in myenteric neurons (top) in response to CAP applied to the DRG (middle) or applied to the colon (bottom); arrow indicates the addition of CAP to bath solution. (D, E) CAP applied to the DRG had no effect on myenteric neuron activity, whereas CAP applied to the colon significantly increased myenteric neuron activity: (D) percentage of responding neurons and (E) response amplitude compared with levels of spontaneous activity. (F) CAP applied to the DRG had no effect on tissue movement, whereas CAP applied to the colon significantly increased tissue movement. ∗P < .05, ∗∗P < .01, (B) paired Student t test or (D–F) repeated-measures 1-way ANOVA. M, mol/L; Max, maximum. Gastroenterology 2019 157, 522-536.e2DOI: (10.1053/j.gastro.2019.04.034) Copyright © 2019 AGA Institute Terms and Conditions

Figure 4 Extrinsic parasympathetic pathways, but not ExPANs, directly influence myenteric neuron activity and produce smooth muscle contractions in the colon. (A) Image of colon nerve ex vivo preparation with dorsal and ventral roots cut and spinal cord removed from spinal column. Black dots indicate imaging fields. (B) GCaMP signal in L6 DRG neurons (Bi), time-lapse color-coded image of myenteric neuron activity (Bii, Biii), and evoked tissue movement (Biv) due to dorsal root stimulation (red arrow). (C) GCaMP signal in L6 DRG neurons (note the presence of a few ventral root afferents) (Ci), time-lapse color-coded image of myenteric neuron activity (Cii and Ciii), and tissue movement (Civ) due to ventral root stimulation (red arrow). (D) Percentage of neurons that respond to dorsal and ventral root stimulation when spinal cord is removed. (E, F) HEX significantly decreased the (E) percentage of responses and (F) tissue movement due to ventral root stimulation. (G) Scatterplot of movement coordinates due to ventral root stimulation (red) overlaid on the oral/anal movement scatterplot from Figure 2 for comparison. ∗P < .05, ∗∗P < .01, using paired Student t test (E, F). Gastroenterology 2019 157, 522-536.e2DOI: (10.1053/j.gastro.2019.04.034) Copyright © 2019 AGA Institute Terms and Conditions

Figure 5 ExPANs indirectly influence myenteric neuron activity and produce smooth muscle contractions in the colon. (A) Image of colon nerve ex vivo preparation with the spinal cord intact. White dots indicate fields. (B) Time-lapse color-coded image (Bi), ΔF/F0 traces of responsive myenteric neurons (Bii), and tissue movement (Biii) due to dorsal root stimulation with spinal cord intact (red arrow). (C) Time-lapse color-coded image (Ci), ΔF/F0 traces of responsive myenteric neurons (Cii), and tissue movement (Ciii) due to dorsal root stimulation (red arrow). (D) Amplitude (ΔF/F0) of GCaMP responses to ventral and dorsal root stimulation. (E) Latency of responses to ventral and dorsal root stimulation. (F) Percentage of active myenteric neurons during recordings (per 1-second bin) in different experimental conditions. (G) Scatterplot of movement coordinates due to dorsal root stimulation (blue) overlaid on oral/anal/ventral root scatterplot from Figures 2 and 5 for comparison. ∗∗P < .01 paired Student t test (D). Gastroenterology 2019 157, 522-536.e2DOI: (10.1053/j.gastro.2019.04.034) Copyright © 2019 AGA Institute Terms and Conditions

Figure 6 Optogenetic stimulation of ExPANs activates neurons in the SPN and DCM. (A) Lumbosacral spinal cord section from littermate control after blue-light stimulation with no c-fos expression in the SPN (Aii and Aiv) or DCM (Aiii). (B) Lumbosacral spinal cord section from TRPV1-ChR2YFP mouse shows yellow fluorescent protein expression in sensory afferents and c-fos expression in activated cells in response to optogenetic stimulation. Note the close apposition of afferent fibers to c-fos–positive cells in the SPN (Bii, Biv) and DCM (Biii). (C) Electromyography recordings of the visceromotor response evoked by blue-light stimulation in TRPV1-ChR2 (Ci) and control mice (Cii). (D, E) Numbers of c-fos–positive cells in the (D) SPN and (E) DCM were significantly higher in TRPV1-ChR2 mice compared with controls. ∗P < .05, unpaired Student t test. Gastroenterology 2019 157, 522-536.e2DOI: (10.1053/j.gastro.2019.04.034) Copyright © 2019 AGA Institute Terms and Conditions

Figure 7 Diagram of hypothesized and newly proposed pathways of lumbosacral ExPANs to the colon. (A) ExPAN terminals localized to the myenteric plexus have led to the hypothesis that ExPANs can directly influence myenteric neuron activity via release of neuropeptides at their peripheral terminals. (B) Our findings indicate that rather than local modulation, ExPANs indirectly influence myenteric neuron activity and colon motility via central modulation through a sensory-parasympathetic spinal reflex. M, mucosa; MP, myenteric plexus; SP, submucosal plexus. Gastroenterology 2019 157, 522-536.e2DOI: (10.1053/j.gastro.2019.04.034) Copyright © 2019 AGA Institute Terms and Conditions