Neuronal pathways and transmission to the lower esophageal sphincter of the guinea pig  Shiyong Yuan, Marcello Costa, Simon J.H. Brookes  Gastroenterology  Volume 115, Issue 3, Pages 661-671 (September 1998) DOI: 10.1016/S0016-5085(98)70145-3 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Schematic illustration of arrangements of the preparation. The stomach was opened along the lesser curvature, continuing through the LES and up the right side of the esophagus. The mucosa and submucosa were removed, and focal electrical stimulation was applied via a pair of fixed stimulating electrodes (0.3 mm apart) placed on the surface of the LES. Another pair of electrodes could be repositioned on the esophagus, upper stomach, or vagus nerve. Intracellular recordings were made from exposed circular muscle cells of the LES. Gastroenterology 1998 115, 661-671DOI: (10.1016/S0016-5085(98)70145-3) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Diagram of a preparation without mucosa and submucosa in which focal electrical stimulation was applied to the upper stomach along an arc (○ and ●) about 5 mm from the LES to detect pathways from the upper stomach to the LES. Typically, gastric stimulation evoked IJPs at 8 (●) of 20 (○) stimulating sites, located primarily on the gastric sling muscle and fundal circular muscle (n = 4), indicating the presence of functional pathways from the stomach to the LES. Gastroenterology 1998 115, 661-671DOI: (10.1016/S0016-5085(98)70145-3) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of 100 μmol/L L-NOARG, 0.5 μmol/L apamin, and 1 μmol/L hyoscine on junction potentials recorded from a single smooth muscle cell of the LES, evoked by focal electrical stimulation (50 V; 0.2 millisecond for 0.5 second) on the LES at different frequencies (1 pulse; 5, 10, and 20 Hz). (A–F) Example of the effect of the drugs on the responses. (A) Circular muscle cells of the LES had small irregular spontaneous oscillation of the membrane potential (black bar in A). Electrical stimulation evoked frequency-dependent IJPs in the LES. (A) Control responses to different frequencies of electrical pulses. (B) These inhibitory responses were greatly reduced by the application of L-NOARG. The residual inhibitory responses were blocked by subsequent addition of apamin, showing (C) EJPs that were abolished by (D) further addition of hyoscine. (E) Inhibitory responses recovered partially after washing for 25 minutes. (F) Time courses of electrical pulses. All responses were blocked by 0.6 μmol/L tetrodotoxin (not shown). The effects of the drugs on mean amplitude of the response are summarized on the right side of the figure (n = 6). The amplitudes of the IJP were frequency dependent and reproducible at intervals of 5 minutes (see the first three control responses). Gastroenterology 1998 115, 661-671DOI: (10.1016/S0016-5085(98)70145-3) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Effects of hexamethonium on IJPs recorded from a single cell of the LES evoked by focal electrical stimulation of vagus nerve (50 V; 20 Hz; 1 millisecond for 1 second) or the LES (0.2 millisecond for 1 second). (A) Example of the effects of the drug on the responses (left, vagal stimulation; right, LES stimulation). (a) In the presence of 1 μmol/L hyoscine, control responses to stimulation applied to the different sites. Note that IJPs evoked by stimulation on the surface of the LES are typically larger than those evoked by vagal stimulation. (b) After addition of 100 μmol/L hexamethonium, responses to vagal stimulation were abolished but the responses to focal LES stimulation were only slightly reduced. (c) Time courses of different sites of stimulation. (B) Summary of the effects of the drugs on mean amplitude of the response (n = 4). IJPs elicited by vagal or LES stimulation were reproducible at intervals of 5 minutes. (a) Hexamethonium (100 μmol/L) abolished the IJPs evoked by vagal stimulation within 10 minutes but only reduced the amplitude of IJPs elicited by (b) stimulation on the LES (paired t test between the mean amplitudes before and after the drug: P < 0.05; t = 3.8; df = 3). Gastroenterology 1998 115, 661-671DOI: (10.1016/S0016-5085(98)70145-3) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Effects of hexamethonium on IJPs recorded from a single cell of the LES evoked by electrical stimulation (50 V; 20 Hz; 0.2 millisecond for 1 second) of esophagus 25–30 mm above the LES and the LES. (A) Example of the effects of the drug on the responses (left, esophageal stimulation; right, LES stimulation). (a) Control responses. (b) Hexamethonium (100 μmol/L) abolished the responses to esophageal stimulation but only partially reduced the responses to focal LES stimulation. (c) Time course of stimulation pulses. (B) Summary of the effects of the drugs on mean amplitude of the response (n = 4). Both electrical stimuli evoked inhibitory responses in LES, but the amplitudes of IJPs to the LES stimulation are normally larger than those to esophageal stimulation. These IJPs were reproducible at intervals of 5 minutes (see the first three control responses in a and b in B; n = 4). Hexamethonium (100 μmol/L) abolished the responses to esophageal stimulation within 5 minutes but only reduced the IJPs elicited by LES stimulation (paired t test between the mean amplitudes before and after the drug; P < 0.05; t = 8.7; df = 3). All responses partially recovered after washout for 5 minutes. This suggests that long descending pathways in the esophagus to inhibitory motor neurons of the LES are mediated via nicotinic synapses, similar to vagal efferent pathways. Gastroenterology 1998 115, 661-671DOI: (10.1016/S0016-5085(98)70145-3) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Example of an experiment showing the effects of hexamethonium on IJPs recorded from a single cell of the LES evoked by focal electrical stimulation (50 V; 20 Hz; 0.2 millisecond for 1 second) of upper gastric region (left) and LES (right). (A) Control responses evoked by different sites of stimulation. (B) Hexamethonium (100 μmol/L) only significantly reduced the amplitudes of the IJPs to LES stimulation but not to the upper gastric region stimulation. (C) The time courses of the stimulation. Gastroenterology 1998 115, 661-671DOI: (10.1016/S0016-5085(98)70145-3) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Labeling of nerve fibers by DiI applied to the gastric sling muscle. (A) Very few anterogradely labeled nerve fibers can be seen in the muscle bundles of LES (arrows), but (B) a large number of motor axons in gastric sling muscle was labeled by DiI (arrows), suggesting that the two muscles are innervated by different, nonoverlapping populations of motor neurons. A similar pattern of anterogradely labeled fibers can be seen in four of four preparations tested. Gastroenterology 1998 115, 661-671DOI: (10.1016/S0016-5085(98)70145-3) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Typical map of nerve cell bodies and nerve fibers labeled by DiI applied to six sites in the myenteric plexus of the esophagus approximately 2 mm oral to the LES (open diamonds near origin of axes). Note that in this preparation, the stomach and esophagus were opened along the greater curvature to show the LES away from the sling muscle. After 4 days in organ culture, DiI-labeled nerve cell bodies (○) were visible within the esophagus and under the LES (▨) but not in the stomach. DiI-labeled nerve fibers were present in the upper stomach (▩) extending a few millimeters beyond the boundary of the LES. A number of nerve fibers were mapped individually from the DiI application sites running down into the upper stomach (solid lines). The position of the gastric sling muscle fibers is indicated by the dashed lines. Gastroenterology 1998 115, 661-671DOI: (10.1016/S0016-5085(98)70145-3) Copyright © 1998 American Gastroenterological Association Terms and Conditions