Volume 152, Issue 6, Pages 1407-1418 (May 2017) Optogenetic Demonstration of Functional Innervation of Mouse Colon by Neurons Derived From Transplanted Neural Cells  Lincon A. Stamp, Rachel M. Gwynne, Jaime P.P. Foong, Alan E. Lomax, Marlene M. Hao, David I. Kaplan, Christopher A. Reid, Steven Petrou, Andrew M. Allen, Joel C. Bornstein, Heather M. Young  Gastroenterology  Volume 152, Issue 6, Pages 1407-1418 (May 2017) DOI: 10.1053/j.gastro.2017.01.005 Copyright © 2017 AGA Institute Terms and Conditions

Figure 1 Immunohistochemical characterization of enteric neurospheres generated from fNS and pNS Wnt1::Cre;ChR2-EYFP mouse intestine. (A) Many HuC/D+ cells (red) are present in both fNS and pNS. Quantification of the percentage of neurosphere (4′,6-diamidino-2-phenylindole [DAPI]-stained) cells that show Hu or Sox10 immunostaining (minimum of 100 DAPI nuclei analyzed from each neurosphere). There is no significant difference between fNS and pNS in the percentage of Hu+ or Sox10+ cells (t tests, P > .05). (B; upper panels) High magnification images showing Hu immunostaining (red), DAPI staining (blue) and merged image of Hu (red), DAPI (blue) and enhanced green fluorescent protein (EGFP) (green). White asterisks indicate some Hu+ cells. (B; lower panels) Sox10 immunostaining (red), DAPI staining (blue) and merged image of Sox10 (red), DAPI (blue) and EGFP (green). White asterisks indicate Sox10+ cells. (C) A subpopulation of Hu cells (red) shows nNOS immunostaining (white, white asterisks) in both fNS and pNS. (D) A subpopulation of Hu cells (red) shows ChAT immunostaining (white, white arrows) in both fNS and pNS. (E) Quantification of the percentage of Hu+ cells that show nNOS immunostaining (minimum of 50 Hu+ cells analyzed from each neurosphere). There is no significant difference between fNS and pNS in the percentage of Hu+ cells that is also nNOS+ (t test, P > .05). (F) Low-magnification images of fNS and pNS showing that most EdU+ cells are located near the peripheries of fNS and pNS. Quantitative data are presented as mean ± SD, and were obtained from a minimum of 3 different experiments. Gastroenterology 2017 152, 1407-1418DOI: (10.1053/j.gastro.2017.01.005) Copyright © 2017 AGA Institute Terms and Conditions

Figure 2 Transplantation of ChR2-expressing neurospheres into the colon of postnatal mice. (A) The intestines of E14.5 or P3−5 Wnt1::Cre;ChR2EYFP mice in which all neural crest-derived cells express ChR2 were dissociated, ChR2EYP+ cells isolated by flow cytometry and neurospheres were generated. Recipient 3- to 4-week-old wild-type mice were anesthetized, the distal colon exposed and held in place with black cotton, and then neurospheres were transplanted into pockets made in the external layers. Four to 16 weeks later, the recipient mice were killed, the colon was removed, opened along the mesenteric border for electrophysiological recordings from the circular muscle after electrical or light stimulation. To record IJPs, the preparations were stimulated oral to the recording site (electrode shown in blue) while EJPs were recorded after stimulation anal to the recording site (yellow). (B) External muscle layers of an opened preparation of colon into which a ChR2EYFP-expressing neurosphere had been transplanted 4 weeks previously. The edge of the neurosphere is at the top, out of the field of view. ChR2EYFP+ cells have migrated from the transplant site and formed ganglia (arrows). (C) ChR2EYP+ expressing varicose fibers in the colonic circular muscle of a recipient. The fibers shown are distal to graft-derived cell bodies. (D, E) Some ChR2EYFP+ (graft-derived) nerve fibers show NOS immunoreactivity (D), while others show vesicular acetylcholine transporter (VAChT) immunostaining (E). NOS and VAChT are markers of nitrergic (inhibitory) and cholinergic (excitatory) neurons, respectively. Gastroenterology 2017 152, 1407-1418DOI: (10.1053/j.gastro.2017.01.005) Copyright © 2017 AGA Institute Terms and Conditions

Figure 3 Properties of IJPs evoked by selective stimulation of graft-derived neurons. (A) Intracellular recordings from colonic circular muscle cells in a mouse that did not contain any ChR2-expressing cells. Unlike electrical stimulation, laser stimulation did not result any changes in membrane potential. Note, to mark the time of onset of the light stimulus, a 5-ms hyperpolarizing current pulse (50 pA) was passed through the recording electrode at the time of triggering the laser (arrow). (B) Electrical or light stimulation (arrows) evoked IJPs in the circular muscle of the colon containing transplanted neurospheres derived from the E14.5 intestine. Both electrical- and light-evoked IJPs were reduced by the P2Y1 receptor antagonist, MRS2179 (10 μM), and abolished by a combination of MRS2179 (10 μM) and the NOS inhibitor, NOLA (100 μM). Note, to mark the time of onset of the light stimulus, a 5-ms hyperpolarizing current pulse (50 pA) was passed through the recording electrode at the time of triggering the laser (arrow). (C) Quantification of effects of MRS2179 (MRS, 10 μM) and NOLA (100 μM) on electrical-stimulated (Electrical Stim) and light-stimulated (Light Stim) IJPs in preparations into which neurospheres derived from the E14.5 gut (fetal neurospheres) had been transplanted. (D) Effects of MRS2179 (MRS, 10 μM) and NOLA (100 μM) on electrical-stimulated and light-stimulated IJPs in preparations into which neurospheres derived from the P3−5 gut (postnatal neurospheres) had been transplanted. The amplitude of the light-evoked IJPs is larger than light-evoked IJPs in preparations containing neurospheres derived from the E14.5 gut (C). Data are presented as mean ± SEM. Con, Control. Gastroenterology 2017 152, 1407-1418DOI: (10.1053/j.gastro.2017.01.005) Copyright © 2017 AGA Institute Terms and Conditions

Figure 4 Properties of EJPs evoked by stimulation of graft-derived neurons. (A) Electrical or light stimulation (arrows) evoked EJPs in the circular muscle of the colon containing transplanted neurospheres derived from the E14.5 intestine. Both electrical- and light-evoked EJPs were abolished by the muscarinic receptor antagonist, hyoscine. (B) Quantification of effects of hyoscine (Hyo) on electrical-stimulated (Electrical stim) and light-stimulated (Light Stim) EJPs in preparations into which neurospheres derived from the E14.5 gut had been transplanted. Hyoscine does not wash out. (C) Hyoscine also abolished electrical- and light-evoked EJPs in preparations containing neurospheres derived from the postnatal gut. Data are presented as mean ± SEM. Con, Control. Gastroenterology 2017 152, 1407-1418DOI: (10.1053/j.gastro.2017.01.005) Copyright © 2017 AGA Institute Terms and Conditions

Figure 5 Transplanted progenitors generate interneurons and inhibitory motor neurons. (A) In fNS recipients, hexamethonium significantly reduced the amplitude of light- and electrically evoked IJPs. (B) In pNS recipients, hexamethonium had no significant effect on light-evoked IJPs, but significantly reduced the amplitude of electrically evoked IJPs. (C, D) 5 μM TNP-ATP, which blocks both P2X2 and P2X3 receptors, significantly reduced light- and electrically evoked IJP amplitude in both fNS (C) and pNS (D) recipients. (E, F) Hexamethonium plus TNP-ATP significantly reduced light- and electrically evoked IJP amplitude in both fNS (E) and pNS (F) recipients. Data are presented as mean ± SEM. Con, control; W, wash. Gastroenterology 2017 152, 1407-1418DOI: (10.1053/j.gastro.2017.01.005) Copyright © 2017 AGA Institute Terms and Conditions

Figure 6 Graft-derived neurons receive synaptic inputs from other graft-derived neurons and endogenous neurons. (A) Intracellular electrophysiological recording from a graft-derived neuron. Light evoked a depolarization with possible truncated action potential at the start as well as a large incoming fast excitatory postsynaptic potential (FEPSP). (A’) Electrical stimulation evoked a short burst of FEPSPs in the same neuron. (B, B’) Recording from a different graft-derived neuron. (B)-Light stimulus response at −60 mV (more depolarized than the neuron shown in A), which triggers an action potential (AP). Some smaller FESPSPs are also received during the stimulus. (B’) Light stimulus at −80 mV evokes depolarization and FEPSPs. The intracellular recordings were not held for sufficient time for pharmacologic analyses. The light-evoked depolarizations in both neurons confirm that they expressed ChR2 and hence were graft-derived. Gastroenterology 2017 152, 1407-1418DOI: (10.1053/j.gastro.2017.01.005) Copyright © 2017 AGA Institute Terms and Conditions