Characterization of neuromuscular transmission and projections of muscle motor neurons in the rat stomach

Abstract

The stomach is the primary reservoir of the gastrointestinal tract, where ingested content is broken down into small particles. Coordinated relaxation and contraction is essential for rhythmic motility and digestion, but how the muscle motor innervation is organized to provide appropriate graded regional control is not established. In this study, we recorded neuromuscular transmission to the circular muscle using intracellular microelectrodes to investigate the spread of the influence of intrinsic motor neurons. In addition, microanatomical investigations of neuronal projections and pharmacological analysis were conducted to investigate neuromuscular relationships. We found that inhibitory neurotransmission to the circular muscle is graded with stimulus strength and circumferential distance from the stimulation site. The influence of inhibitory neurons declined between 1 and 11 mm from the stimulation site. In the antrum, corpus, and fundus, the declines at 11 mm were about 20%, 30%, and 50%, respectively. Stimulation of inhibitory neurons elicited biphasic hyperpolarizing potentials often followed by prolonged depolarizing events in the distal stomach, but only hyperpolarizing events in the proximal stomach. Excitatory neurotransmission influence varied greatly between proximal stomach, where depolarizing events occurred, and distal stomach, where no direct electrical effects in the muscle were observed. Structural studies using microlesion surgeries confirmed a dominant circumferential projection. We conclude that motor neuron influences extend around the gastric circumference, that the effectiveness can be graded by the recruitment of different numbers of motor neuron nerve terminals to finely control gastric motility, and that the ways in which the neurons influence the muscle differ between anatomical regions.

NEW & NOTEWORTHY This study provides a detailed mapping of nerve transmission to the circular muscle of the different anatomical regions of rat stomach. It shows that excitatory and inhibitory influences extend around the gastric circumference and that there is a summation of neural influence that allows for finely graded control of muscle tension and length. Nerve-mediated electrical events are qualitatively and quantitatively different between regions, for example, excitatory neurons have direct effects on fundus but not antral muscle.

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INTRODUCTION

The stomach is an important organ of the gastrointestinal tract that functions as a primary reservoir for ingested materials and as the site of major breakdown of content to digestible fluid and particles that are mixed and propelled through the pyloric sphincter to the duodenum (1, 2). To exert these functions, the stomach has two regions: a proximal region that can change its volume considerably to accommodate different amounts of gastric content and a distal region for mixing and propulsion (1, 3). Imaging of stomach movements indicates that there is a fine control of proximal stomach volume and, in the distal stomach, movement consists of a series of contractile rings that are graded in amplitude, commencing proximally and progressing distally in co-ordination with pyloric sphincter opening (4–7). Gastric volume and the coordinated contractions and relaxations are mainly controlled through the enteric nervous system (ENS), which contains both inhibitory and excitatory gastric motor neurons and its connections with the central nervous system (CNS) (8–10). Large numbers of enteric neurons are intrinsic to the stomach, with over 400,000 in rats and guinea pigs (11, 12). The reasons for such large numbers may include the need to finely control gastric muscle movements (1). Especially in the distal stomach, the arrangement of the motor innervation is expected to align with the observed circumferential orientation of muscle movement and contractile activity (4–7, 13).

Functional gastrointestinal disorders (FGIDs), which generally have no effective treatment, affect more than 40% of the world’s population and can severely impact quality of life (14). In the case of the stomach, gastroparesis is a disorder characterized by significantly delayed gastric emptying and symptoms include abdominal pain, early satiety, nausea, and vomiting (15, 16). Some underlying causes of gastroparesis have been associated with changes in the intrinsic neuronal circuitry, particularly the loss of inhibitory motor neurons (15, 17).

In the current study, we have addressed deficiencies in knowledge of the functional organization of the gastric motor neurons, especially to investigate whether the spread and summation of influences from intrinsic inhibitory and excitatory motor neurons in different gastric regions are consistent with the observations that have been made of gastric motility patterns. We have used intracellular microelectrodes to record from gastric smooth muscle cells (SMCs) to investigate the motor innervation. We have augmented these functional observations with structural investigations of neuronal projections and pharmacological analysis of the way in which they influence the muscle.

MATERIALS AND METHODS

Animals/Tissue Source

The procedures were approved by the Florey Institute of Neuroscience and Mental Health Animal Ethics Committee (approval 21-010) and by the University of Melbourne Animal Ethics Committee (approval 24935). Sprague-Dawley rats were sourced from either Ozgene ARC, Murdoch, Western Australia, or the internal University of Melbourne colony. Rats were housed in a 12/12-h light-dark cycle and supplied with food and water ad libitum before any experiments. Stomach samples were collected from 89 rats, 41 females and 48 males, 6–10 wk old, 132–293 g for females and 175–516 g for males. Rats were euthanized with an intraperitoneal injection of pentobarbital sodium (100 mg/kg) to deeply anesthetize followed by severing the carotid arteries once reflexes were abolished. For electrophysiology experiments, all rats were taken between 9–10 AM.

Immunohistochemistry in Thick Mounts and Cryostat Sections

Fresh stomachs were collected from rats that were euthanized as described in Animals/Tissue Source. The stomach was excised and placed into phosphate-buffered saline (PBS: 0.15 M NaCl in 0.01 M sodium phosphate buffer, pH 7.2) containing nicardipine (1 µM) and opened and pinned to a balsa board before being immersed in fixative (2% paraformaldehyde and 0.2% picric acid in 0.1 M sodium phosphate buffer, pH 7.0) overnight at 4°C. Fixative was washed out with dimethyl sulfoxide (DMSO), 3 × 10 min, followed by PBS, 3 × 10 min, and tissue was stored in PBS-azide (0.1% sodium azide in PBS) at 4°C until being prepared for thick mounts or sections.

Regions of stomach to be sectioned were placed in 30% sucrose in PBS-azide (PBS-sucrose-azide) overnight at 4°C, followed by an overnight incubation in a mixture of optimal cutting temperature (OCT) compound (Trajan Scientific and Medical, Ringwood, Australia) and PBS-sucrose-azide in a 1:1 ratio. Tissue blocks were embedded in 100% OCT medium and frozen in isopentane cooled by liquid nitrogen. Cryostat sections (20 μm) were cut and mounted onto SuperFrostPlus microscope slides (Menzel-Glaser; Thermo Fisher, Scoresby, Australia). Sections were then air-dried for 1 h, blocked with normal horse serum (NHS) and Triton (10% NHS plus 0.1% Triton X-100 in PBS) for 30 min at room temperature (RT) and then incubated in a mixture of primary antibodies: sheep antineuronal nitric oxide synthase (nNOS) and rabbit anti-tachykinin (TK) diluted in antibody diluent (0.29 M NaCl, 0.01 M sodium phosphate buffer, 0.1% sodium azide pH 7.1) (see Supplemental Table S1 for antibody details). Sections were incubated with diluted primary antibodies overnight at 4°C. Sections were then washed with 3 × 10 min PBS followed by a 1.5-h incubation in a mixture of Alexa Fluor secondary antibodies diluted in antibody diluent at RT (see Supplemental Table S2). Sections were coverslipped with Dako fluorescence mounting media (Agilent, Santa Clara). Slides were examined at ×20 magnification using a Zeiss LMS800 confocal microscope (Zeiss, Jena, Germany).

Thick-mount preparations are full-thickness muscularis externa preparations in which the muscle layers are not separated. Thick mounts were processed using the Ce3D clearing and immunohistochemical method that has been optimized for gastrointestinal tissues (18, 19) with the following changes: preparations were blocked with NHS for two nights at RT and then incubated in primary antibodies, human anti-Hu C/D, and mouse antivasoactive intestinal peptide (VIP) (see Supplemental data) for 4 days at 4°C. Preparations were washed 3 × 30 min before being transferred into a mixture of Alexa Fluor secondary antibodies (see Supplemental Table S1) and incubated overnight at 4°C. Samples were washed again before a 5-day incubation in Ce3D clearing solution with changes daily for the first 2 days. Tissue was mounted in the Ce3D clearing solution on a microscope slide and imaged using a ×10 objective with a BioRad MR1C1024 confocal scanning laser system installed on a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany).

Electrophysiology

Neurotransmission to the circular muscle was investigated in strips that were cut parallel to the circular muscle (Supplemental Fig. S1). Rats were anesthetized and culled as described above. The strips were on average 23-mm long and 5-mm wide (average across all regions). They were pinned to a Sylgard elastomer base of an organ bath in Krebs physiological saline [final molarities, with respect to ions (mM): 144 Na⁺, 4.76 K⁺, 1.2 Mg²⁺, 2.5 Ca²⁺, 127.8 Cl⁻, 25 equivalent ${\text{HCO}}_3^{-}$, 1.06 ${\text{H}}_2{\text{PO}}_4^{-}$, 1.2 $\mathrm{S}{\mathrm{O}}_4^{2-}$, and 11.1 glucose], bubbled with 95% O₂-5% CO₂. The physiological saline was maintained at 34–35°C. The strips were stretched to an average of 127 ± 8% (all data shown hereafter as mean ± SD unless stated otherwise) of their lengths in the resting state. The strips parallel to the circular muscle spanned from the ventral to dorsal surface with the greater curvature placed at 6 mm from the stimulating electrodes, the strip was then stretched to span 7 mm past the transmural electrodes on the ventral surface. Intracellular recordings from the muscle were made at distances of 1, 3, 5, 7, 9, and 11 mm lateral to the transmural electrodes, which was circumferentially along the ventral surface, across the greater curvature to the dorsal surface. These strips were on average 63 ± 20% of the total stomach circumference, however just over 50% of the strip was recorded from, therefore, the recordings and data generated come from ∼32% of the total circumference (further details in results). Fundus and corpus strips were taken at least 5 mm proximal or distal, respectively, to the limiting ridge closest to the esophageal groove and at least 10 mm lateral from the esophageal groove. Antrum strips were taken at least 5 mm proximal to the pyloric sphincter.

Intracellular impalements into the circular muscle layer of the stomach were made using sharp glass microelectrodes, prepared from borosilicate glass capillaries (GC100F-15, 1 mm OD, 0.58 mm ID, Harvard Apparatus, Holliston, MA) using a P-87 Flaming/Brown Micropipette Puller (Sutter Instruments, California) and filled with 0.5 M KCl. Electrode impedances ranged from 100 to 190 MΩ. Impalements were made through the serosal surface and longitudinal muscle. The longitudinal muscle of the rat stomach is ∼10- to 20-µm thick (13). The recording electrode was advanced beyond this distance to ensure that the tip was in the circular muscle, and in some experiments, this location was confirmed using microelectrodes containing carboxyfluorescein (5%). At the end of the recording, 0.5 -nA hyperpolarizing current pulses (0.2 s duration at 2.5 Hz for 2 min) were used to inject carboxyfluorescein into the recorded SMC (20). The impalement was then held for an additional 2 min before the electrode was withdrawn. The tissue was then removed from the organ bath, placed on a microscope slide, and imaged using an AxioImager microscope (Zeiss, Germany). This confirmed that recordings were made from the circular muscle layer. The organ bath was irrigated at a flow rate of 3 mL/min with physiological saline containing 400 µM of hexamethonium chloride, a nicotinic receptor antagonist to prevent ganglionic transmission, and 3 µM nicardipine, an L-type calcium channel blocker, to prevent muscle contraction. To record inhibitory junction potentials (IJPs), excitatory junction potentials (EJPs) were blocked pharmacologically using 1 µM hyoscine hydrochloride, a muscarinic receptor antagonist. To record EJPs, IJPs were pharmacologically blocked using N-nitro-l-arginine (l-NNA; 100 µm) plus MRS2500 (1 µm) to block nitrergic and purinergic (P2Y1) transmission, respectively. In addition, Ano1 subtype, calcium-activated chloride channel (CaCC) antagonists, first-generation antagonist niflumic acid (NFA), and second-generation antagonist CaCCinh-A01 and a SK channel blocker, apamin were used to investigate the influence of interstitial cells on neurotransmission (see discussion).

Data were recorded using an AxoClamp2B amplifier (Axon Instruments, California) in bridge mode using a 0.1 × LUT head-stage. Signals were fed into a CED Power 1401 interface before being processed and analyzed in Spike2 software version 7 (Cambridge Electronic Design, Cambridge, UK). Stimulation used silver wire transmural electrodes, one electrode was placed below, and one electrode was placed above the muscle strip, both perpendicular to the circular muscle bundles. The silver wire was insulated, except for the region that was in contact with the muscle strip. Stimulating electrodes were connected to an Iso-flex stimulus isolator (Microprobes, Gaithersburg, MD) linked to a Master-8 stimulator (A.M.P.I., Jerusalem, Israel). An oscilloscope was also used to monitor signals. A Leica micromanipulator was used to position the recording microelectrodes. Cells in the proximal stomach that had a recorded resting membrane potential (RMP) more hyperpolarized than −30 mV were used, whereas in the distal stomach cells with an RMP of ≤40 mV were used. Electrical stimuli (1-ms pulse width, of increasing amplitudes, 5–90 V) were delivered either as a single pulse or as a short, high-frequency train (5 pulses separated by 20 ms). Junction potentials were evoked only when the cell was at RMP, between, not during, slow waves. In the current experiments, these stimulus parameters in the presence of tetrodotoxin (100 nM) elicited no muscle response. At least three responses to each stimulus pattern and voltage were recorded in the same cell and the averaged waveform was used for analysis.

Lesion Microsurgeries (Double Myotomies and Myectomies)

Rats were anesthetized with a mixture of xylazine (9.1 mg/kg, Ellar Laboratories, Tullamarine, Australia) and ketamine (54.5 mg/kg, Troy Laboratories, Glendenning, Australia) given intraperitoneally. The peritoneal cavity of anesthetized rats was opened along the abdominal midline and the ventral surface of the stomach was exposed. Pairs of longitudinal cuts (myotomies), 1, 2, or 4 mm apart, were made through the muscularis externa to the depth of the submucosa, leaving the submucosa and mucosa intact. The ventral surface of the corpus where the cuts were to be made was moistened with PBS containing nicardipine (1 µM) making the lesions. The cuts were 10–12 mm in length across the ventral corpus from proximal to distal. A droplet of colloidal carbon (India ink, Royal Talens Australia) was placed at each end of the myotomy cuts to aid in their location when the stomach was removed for fixation. Rats were taken for investigation of the consequences of the myectomies 10 days later. For each myotomy operation, there were four animals per group, two males and two females. Immunohistochemistry (detailed above) was performed on sections taken between and parallel to the cuts (Supplemental Fig. S7).

Myectomy rats were anesthetized, and the stomach was exposed as described above. To create myectomy site, four incisions forming a rectangle were made across the ventral to dorsal corpus, across the greater curvature. Ligatures were placed at the corners to aid in locating the region of lesion when the stomach was removed for fixation. The longitudinal muscle and myenteric plexus between the incisions were removed, which created a rectangular myectomy region which was ∼1.0 × 0.7 cm. The circular muscle, submucosa, and mucosa layers were left intact. After surgery, the rats were returned to their home cages and provided with free access to food and water. For each myectomy operation, there were four animals per group, two males and two females. They were taken for investigation of the consequences of the myectomies 6 days later. Thick-mount immunohistochemistry was completed as detailed above.

After surgery, the rats were returned to their home cages, monitored daily, and provided with free access to food and water. Cefazolin (50 mg/kg subcutaneous, Clifford Hallam Healthcare, Melbourne, Australia) and Temgesic (0.05 mg/kg, subcutaneous where appropriate, Clifford Hallam Healthcare) were administered postoperatively. No postoperative behavioral changes were observed.

Compounds

The compounds used were the following: apamin [0.1 µM; in ethanol (EtOH), Alomone Labs, Jerusalem, Israel], CaCCinh-A01 (5 µM; in DMSO, Sigma-Aldrich, Sydney, Australia), 5(6)-carboxyfluorescein [5% in 20 mM Tris buffer (pH = 7.0), Sigma-Aldrich], hexamethonium (400 µM, Sigma-Aldrich), hyoscine hydrochloride (scopolamine hydrochloride; 1 µM, Sigma-Aldrich), nicardipine (1 µM or 3 µM; in EtOH, Sigma-Aldrich), niflumic acid (NFA, 100 µM; in EtOH, Sigma-Aldrich), l-NNA (100 µM; stock in 1 M HCl, Sigma-Aldrich) and MRS2500 (1 µM, Tocris, Bristol, UK), and tetrodotoxin (TTX, 0.1 µM, Abcam). Unless stated otherwise, compounds were dissolved in deionized water. These are all final concentrations of these compounds; working solutions were prepared to ensure concentrations of EtOH, DMSO, and HCl were below 0.1% of stock concentration after dilution in circulating physiological saline.

Compounds were perfused in Krebs solution at ∼3 mL/min continuously through the bath. Where drugs were scarce, some were recirculated using 100 mL or 200 mL of Krebs solution.

Data Analysis and Statistics

Experimental data were analyzed in Spike2, ImageJ, and Excel. Statistical analysis and graphs were completed in GraphPad Prism v8.0 (GraphPad software, San Diego, CA). To assess the effects of distance from the stimulating electrode and stimulus strength on the components of junction potentials, the characteristics of components were determined using the waveform average and cursors aligned with transitions between components of the responses. The amplitude of the junction potentials was calculated as the difference in membrane potential at the event peak with the average RMP 2 s before the stimulus. The data obtained in each preparation were normalized to the average response to a single 30-V stimulus obtained in the cells located 1 mm from the stimulating electrode from the same preparation. Average IJP amplitudes, without normalization to the 1-mm 30-V response, across all three anatomical regions at all stimulus voltages are provided in the Supplemental data (Supplemental Fig. S6). This stimulus voltage was chosen as it produced an intermediate response above threshold for all cells but not a supramaximal response. Data for pharmacological investigations were not normalized. Data were analyzed using unpaired t tests or a one-way ANOVA with Tukey’s multiple comparisons, respectively. Significance was set at P ≤ 0.05.

The effects of the lesion microsurgeries (double myotomies) on the sections were quantitatively analyzed using ×20 magnification tile scans from the center of the section (see Supplemental data for image analysis details).

RESULTS

Gastric Regions and Dimensions

We have investigated the regional innervation of the rat stomach in which the regions were defined as explained below (Fig. 1). Regions are referred to in terms of the rat equivalents of the anatomical divisions that are applied to human stomach, fundus, corpus, and antrum (Fig. 1A). They can also be referred to in the rat by mucosal specialization (Fig. 1B) or muscle functions (Fig. 1C). Anatomically, the fundus is proximal to a line from the groove between the stomach and the esophagus (angle of His in human) and the opposite greater curvature (Fig. 1C). Distal to this are the corpus and antrum.

Figure 1.

Regions of the rat stomach, based on images of full stomachs dissected from rats after the feeding phase, and fixed whole. A: regions defined by conventional anatomy defining the fundus, corpus, and antrum. B: regions defined by specialization of the mucosa. A stratified squamous (aglandular) epithelial lining occurs in the fundus, proximal corpus, and the esophageal groove that surrounds the opening of the esophagus and extends along a small region of the lesser curvature. It is continuous with the aglandular lining of the esophagus. The remainder of the corpus and the antrum have a glandular lining. The position of the limiting ridge, a boundary between the glandular and aglandular parts, differed between individual rats as indicated by the proximal shaded region in B. Distal shaded region indicates the corpus/antral boundary where there is transition zone of changing glandular cell types. C: regions defined by functional movements. The proximal stomach is the gastric reservoir, and the distal stomach is the region of mixing and proximo-distal propulsion. The pyloric sphincter regulates the timing and particle sizes of aspirates that are propelled into the duodenum. See text for further details.

The rat stomach has a region in which the lining is aglandular, very similar to and continuous with the esophageal lining (Fig. 1B). The aglandular region includes the fundus, the esophageal groove as far as its junction with the antrum, and variable amounts of the proximal corpus. The linings of the remainder of the corpus and the antrum are glandular (Fig. 1B). The transition between the aglandular and glandular parts of the rodent stomach is marked by a fold of mucosa, the limiting ridge, that varies between individual rats in the position that it meets the greater curvature (13, 21) (Fig. 1B). The glandular mucosa has a large region of oxyntic glands dominated by parietal and chief cells and a region of antral glands where gastrin cells occur (22). Between these is a transition zone (approximate position of corpus/antral boundary shaded in Fig. 1B), where both parietal cells and gastrin cells are found (22). The glands adjacent (distal) to the limiting ridge at the lesser curvature are, histologically, antral glands. In terms of muscle function, the rat stomach, like single-compartment stomachs of other mammals, consists of a proximal region that changes volume to accommodate gastric content and a distal region that mixes and propels the food (Fig. 1C) (3).

In the current study, we used the anatomical regions to define where electrophysiology recordings were made. The circumferential lengths of the muscle strip preparations that were recorded were on average 24 mm from the fundus (∼54% of the total gastric circumference), 23 mm from the corpus (51% of the total circumference), and 26 mm from the antrum (93% of the total circumference). All strips were ∼5 mm wide (proximo-distal). These lengths were measured after strips were removed from the regions and placed in buffered saline containing nicardipine to relax the muscle and thus prevent muscle contraction.

Electrophysiological Properties

Resting membrane potentials (RMPs) of smooth muscle cells (SMCs) in the fundus, corpus, and antrum were −35.6 ± 3.8 mV, −48.8 ± 7.0 mV, and −50.2 ± 5.0 mV, respectively, with the fundus being significantly more depolarized than the other regions (fundus vs. corpus P < 0.0001, fundus vs. antrum P < 0.0001, corpus vs. antrum P = 0.014). Slow waves were observed in muscle strips from the antrum and corpus (distal stomach) but never in the fundus strips (proximal stomach). Slow waves were observed in 94.4% of antral strips and 60.5% of corpus strips. Across the distal stomach, amplitudes of slow waves above RMP differed between strips and between cells, ranging from 4 to 24 mV, at frequencies that ranged from 3 to 6 cycles per minute (cpm). The time to peak amplitude was on average 6.6 ± 2.7 s (Fig. 2B). Within corpus preparations, the slow-wave amplitude and frequency were on average 10.8 ± 4.7 mV and 4.8 ± 0.6 cpm (n = 10 cells, 7 strips). In the antrum, amplitude and frequency were on average 13.7 ± 6.4 mV and 3.7 ± 0.7 cpm (n = 12 cells, 6 strips). Frequency of slow waves was significantly greater in the corpus preparations closer to the predicted pacemaker region compared with the antrum preparations, which were disconnected from this pacemaker region in these experiments.

Figure 2.

Compound inhibitory junction potentials (IJPs) and associated afterdepolarizations (After-Dep). A: compound IJP and afterdepolarization in response to a single stimulus pulse (at the arrow at cursor 1), applied to intramural axons in an antral muscle strip. Dotted lines indicate the placement of cursors for measurements for the fast and slow components of the IJP and the afterdepolarization. B: a series of spontaneous slow waves (SWs) recorded via an intracellular microelectrode in the antrum. One wave is shown on an expanded timebase matching that of A. C: example traces of the IJP responses and afterdepolarization events seen in the three anatomical regions. D: quantification of the durations of the fast and slow IJP components across the fundus, corpus, and antrum. E: durations of the afterdepolarizations and the duration to peak of the response across all regions. F: amplitudes of the afterdepolarization events post IJP. In these experiments, the measured responses shown were evoked by a single 90-V pulse in cells that were located 3 mm circumferential to stimulus site. Data shown in D as means ± SE; data shown in E and F shown as min to max with line as median. ****P < 0.0001.

Inhibitory Transmission

Transmural stimulation activates both the inhibitory and excitatory axons that supply the muscle. To investigate the electrical events in response to stimulation of the inhibitory axons, cholinergic excitatory transmission was blocked by adding hyoscine hydrochloride (100 µM) to the physiological saline perfusing the organ bath.

In gastric SMCs in the presence of hyoscine, a single suprathreshold stimulus pulse evoked a biphasic (compound) IJP, with an initial fast phase followed by a slow phase and often an afterdepolarization. The forms of IJPs and the afterdepolarization differed substantially between the anatomically defined gastric regions and even within regions there were differences between cells (Fig. 2C). In the fundus and corpus, the afterdepolarization duration was ∼1 s, whereas in the antrum, it was significantly (P < 0.0001) longer, ∼8.5 s (Fig. 2E). The afterdepolarization was also significantly (P < 0.0001) larger in amplitude in the antrum compared with the fundus (Fig. 2F). In all regions, the fast phase of the hyperpolarization was prominent and similar in duration. However, the overall duration of the IJP in the fundus and corpus was briefer than in the antrum. In the antrum, the fast phase was followed by a more obvious slow component (Fig. 2, C and D). In most cases, the compound IJP in the antrum was followed by a large depolarizing event (Fig. 2, A and C) that was often similar in shape, amplitude, and duration to spontaneous slow waves (SW) in the same preparations (Fig. 2B). The slow depolarizing event that followed the IJP in the antrum had a median duration of around 8 s and variable amplitude, averaging 6 mV with amplitudes up to 25 mV observed (Fig. 2, C, E, and F). Different pharmacological agents were used to identify the mechanisms involved in the genesis of each IJP component.

Because the afterdepolarization resembled the slow wave (SW), and the generation of SW involves an opening of Ano1 chloride channels in Interstitial cell of Cajal (ICC) in which E_Cl is depolarized relative to RMP (23, 24), we investigated effects of extracellular Cl⁻ substitution and Ano1 chloride channel blockers (Fig. 3). Replacement of 50% of the NaCl in the Krebs solution with Na gluconate, which reduced Cl⁻ to 68.8 mM, had no significant effect on the afterdepolarization. Replacing all the NaCl, thus reducing extracellular Cl⁻ to 9.8 mM, abolished the afterdepolarization (Fig. 3A). The CaCC blockers niflumic acid and CaCCinh-A01 substantially reduced both the afterdepolarization amplitude and area under the curve (AUC; Fig. 3, B and C). However, both the fast component and the slow component of the IJP were also substantially reduced by lowering extracellular Cl⁻ to 9.8 mM or by Ano1 channel blockers. Thus, the reduction of the slow afterdepolarization may be due to the effects of Cl⁻ substitution on the IJP that now fails to trigger the subsequent depolarizing event.

Figure 3.

Investigation of chloride conductance dependence of the compound IJP (inhibitory junction potential) and afterdepolarization (After-dep) in the antrum. A: effect of reducing external chloride from 128.6 mM to 9.8 mM by substitution of sodium chloride for sodium gluconate. Although there was no significant reduction of membrane potential, the afterdepolarization and the slow IJPs were almost abolished and the fast IJPs were substantially reduced (n = 14 control and n = 12 chloride substitute cells, 2 rats, 1 female and 1 male). B: niflumic acid (NFA); there was no significant difference in RMP, the afterdepolarization and the slow IJPs were substantially reduced (n = 12 control and n = 8 NFA cells, 5 rats, 2 females and 3 males). C: CaCCinh-AO1 hyperpolarized the smooth muscle (P < 0.0001) and reduced both components of the IJP and the afterdepolarization (n = 7 control and n = 13 CaCCinhA01 cells, 3 rats, 1 female and 2 males). Inset in each panel shows the membrane potential in cells in control (standard physiological solution) and with the chloride substitution or antagonist. Measured responses were evoked by a single 90-V pulse in cells that were located 3 mm circumferential to stimulus sites. Data shown as means ± SE. ****P < 0.0001, ***P = 0.0005, **P = 0.002, *P = 0.01. RMP, resting membrane potential.

There is evidence that the purinergic component of inhibitory transmission to the gastrointestinal muscle involves the opening of small conductance potassium (SK) channels whose opening is prevented by the SK blocker apamin (25). We therefore investigated the effects of apamin on the IJP in the antrum, which we found reduced the AUC for both the fast and slow components and reduced the peak amplitude of the IJP (Fig. 4A). It caused small reductions in the afterdepolarization and did not change RMP. The NOS inhibitor l-NNA reduced both components of the compound IJP and the afterdepolarization in both the antrum and the fundus but did not change RMP (Fig. 4, B and C).

Figure 4.

Compound inhibitory junction potential (IJP) and afterdepolarization in the antrum and fundus following small conductance potassium channel (SK) block, or block of nitrergic transmission. A: effect of the SK blocker apamin in the antrum (n = 13 control and n = 6 apamin, 2 male rats). B: effect of NOS inhibitor l-NNA in the antrum (n = 10 control and n = 19 l-NNA, 2 rats, 1 female and 1 male). C: effects of NOS inhibitor l-NNA in the fundus (n = 13 control and n = 16 l-NNA, 2 rats, 1 female and 1 male). The measured responses were evoked by a single 90-V pulse in cells that were located 3 mm circumferential to stimulus sites. Data shown as means ± SE. ****P < 0.0001, ***P = 0.0006, **P = 0.005, *P = 0.01. l-NNA, N-nitro-l-arginine; NOS, nitric oxide synthase.

Grading of Inhibitory Responses with Circumferential Distance

The relationship between response amplitude and distance was mapped using the same protocol across all three regions as detailed in Fig. 5A. Strips were purposefully investigated around the greater curvature (Fig. 5A) because circumferential contractions of the intact stomach include the ventral and dorsal surfaces and the greater curvature between them. In the fundus, IJPs declined in amplitude in a linear manner from the point of stimulation, with a decline to half amplitude occurring at ∼6 mm from the site of stimulation, when stimulus strengths above 40 V were used (Fig. 5B and Supplemental Figs. S2, S3, and S4). In the circular muscle of the corpus, the distance for the decline to half amplitude was ∼8 mm (Fig. 5C and Supplemental Fig. S3) and in the antrum, the decline to half amplitude was beyond the point of recording (Fig. 5D and Supplemental Fig. S4).

Figure 5.

Diminution of inhibitory junction potential (IJP) amplitudes in the circular muscle with increased distances from stimulating electrodes. A: diagram showing in yellow the sites from which strips were removed for investigation of transmission in vitro. B, C, and D: relations between IJP amplitudes and stimulus strength at 1, 3, 5, 7, 9, and 11 mm from the stimulating electrodes in circular muscle strips from the fundus (B), corpus (C), and antrum (D). Data are normalized to the 1-mm 30-V pulse shown as means ± SE with a linear regression line. See Supplemental Figs. S2–S4 for subject numbers.

Grading of Inhibitory Responses with Stimulus Strength

The amplitudes of IJPs were graded with both stimulus strength and distance from the site of stimulation. To investigate the effect of change in stimulus strength, IJPs evoked by single stimuli of different strengths delivered at ∼10-s intervals were recorded in cells located 1, 5, and 9 mm from stimulating electrode (Figs. 5 and 6 and Supplemental Figs. S2, S3, and S4). This stimulation interval was adopted to avoid the facilitation of IJP amplitudes that can occur with interstimulus intervals of 2 s or less (26). At each recording site, 11 increments in stimulus strength ranging from 5 to 90 V (5 V increases in stimulus strength to 20 V and then 10 V increases from 20 V to 90 V) were used. When the stimulus was increased from 5 to 90 V for circular muscle strips from the fundus, there was a near linear increase in IJP amplitude with each increase of the stimulus strength (Fig. 6, A and B), suggesting that at least 10 nerve fibers influence each single SMC (see discussion). There was also a graded increase in IJP amplitude with each increase in stimulus strength in both the corpus and antrum, but in both these cases, there was steeper relationship between 5 and 30 V stimulation compared with the 40- to 90-V stimulation (Fig. 6, C and D).

Figure 6.

Increase of inhibitory junction potential (IJP) amplitude in the circular muscle with increased stimulus strength. A: superimposed records of individual IJPs in an individual smooth muscle cell of the fundus in response to single transmural electrical pulses (at the arrow, ranging from 5 to 90 V, different stimulus strengths shown in different colors with the 90-V pulse shown in black). B–D: relations between IJP amplitudes and stimulus strength at 1, 5, and 9 mm from the stimulating electrodes in circular muscle strips from the fundus (B), corpus (C), and antrum (D). Data are normalized to the 1-mm 30-V pulse shown as means ± SE. Linear regression lines (5–30 V regression adjusted to cross XY zero, 40–90 V standard linear regression). See Supplemental Figs. S2–S4 for subject numbers.

Excitatory Transmission

To investigate excitatory EJPs, NOS was inhibited with l-NNA (100 µM) and purinergic transmission was inhibited with the P2Y1 antagonist MRS2500 (1 µM). Under these conditions, fast EJPs were recorded from the circular muscle of the fundus (Fig. 7A) and corpus (Fig. 7B), but not from the antrum (Fig. 7C), with either single pulses or 50-Hz trains. In the fundus, there was a hyperpolarization that followed the nerve-mediated depolarization, this was especially prominent after the train stimulation. This may be because we did not block the pituitary adenyl cyclase activating peptide (PACAP)/VIP receptors and thus this component could be a residual IJP response that is neuropeptide mediated. In the antrum, there was a residual hyperpolarizing event under the same conditions.

Figure 7.

Junction potentials after blocking inhibitory transmission with the NOS inhibitor l-NNA and the P2Y1 purine receptor antagonist MRS2500 elicited by single pulses (dots) or trains of pulses (multiple dots). Junction potentials are shown at either 1 mm (gray shading) or 3 mm away from the stimulating electrodes. The amplitudes and shapes of junction potentials differed between regions. Excitatory junction potentials occurred in the fundus (A) and corpus (B), but not in the antrum (C). l-NNA, N-nitro-l-arginine; NOS, nitric oxide synthase.

In the fundus, where EJPs could be recorded consistently, the relationship between response amplitude and distance was mapped (Fig. 8) using the protocol as described above. These data are similar to those of the IJP mapping, the EJP amplitude in the circular muscle increased with stimulus strength (Fig. 8A) and diminished with distance from stimulating electrodes, with a decline to half amplitude occurring at ∼11 mm from the stimulus site (Fig. 8B).

Figure 8.

Influence of excitatory neurons on the membrane potential of the circular muscle in the fundus. A: relations between EJP amplitudes and stimulus strengths at 1, 5, and 9 mm from the stimulating electrodes in circular muscle strips. Linear regression line (5–30 V regression corrected to cross XY zero, 40–90 V standard linear regression). B: relations between EJP amplitudes at 1, 2, 5, 7, 9, and 11 mm distances from the stimulating electrodes in circular muscle strips from the fundus. Data are normalized to the 1-mm 30-V pulse shown as means ± SE. See Supplemental data for subject numbers. EJP, excitatory junction potential.

Structural Investigation of Circumferential Projections of Neurons

The projections of muscle motor neurons in the circumferential direction, parallel to the circular muscle, were investigated by cutting the nerve fibers innervating the circular muscle bundles and waiting for the axons that were severed from their cell bodies to die (Fig. 9, A–C). Two parallel lesions through the muscularis externa in the corpus, perpendicular to the circular muscle bundles (a double myotomy), were made at either 1–2 mm or 4 mm apart. Ten days after the surgery, the operated region was collected and fixed for immunohistochemistry. Sections were cut between the myotomy lesions (Fig. 9C and Supplemental Fig. S7) to determine if there was a loss of circumferentially innervating nerve fibers, originating from cell bodies outside the myotomy region. Immunohistochemical localization of neuronal nitric oxide synthase (nNOS) was used to locate axons of inhibitory neurons and tachykinins (TK) to locate axons of excitatory neurons (Supplemental Table S1).

Figure 9.

Double myotomy surgery and its consequences for innervation of the circular muscle in the corpus. A: image of the myotomies, in vivo, immediately after completion of the cuts. The myotomy cuts are defined by a small amount of blood that has entered the cuts. B: appearance of the stomach soon after removal from the rat, 10 days later. Colloidal carbon remnants can be seen in the cuts (arrows). C: diagram showing positions of myotomy cuts (purple). Green dotted line indicates the position that sections were taken from. Blue lines indicate the direction of the circular muscle bundles. D–G: representative immunohistochemistry of sections cut perpendicular to the circular muscle, showing cross sections of neural NOS (nNOS; green) and TK (orange) fiber bundles. D: low power image of nNOS and TK fibers in control tissue. E: low-power image of nNOS and TK fibers, at the green dotted line between myotomy lesions (1–2 mm lesion example). F: high-power image of fiber cross sections in control. G: high-power image of fiber cross sections in myotomy. H: quantitative data showing the reduction of fiber bundle area, in the circular muscle from between lesions of 4 mm separation, compared with control, and a reduction between lesions of 1–2 mm separation. ****P < 0.0001, ***P = 0.0005, **P = 0.002, *P = 0.01. CM, circular muscle; LM, longitudinal muscle; MY, myenteric plexus; nNOS, neuronal nitric oxide synthase; TK, tachykinins.

Quantification of cross-sectional areas of fiber bundles in the circular muscle, for both the inhibitory and excitatory neuron markers, was found to be significantly reduced between the myotomy sites when compared with control tissue (Fig. 9, D–H), which shows there was a degeneration of axons entering circumferentially from outside the myotomy region. Lesions with 4 mm separation significantly reduced the average size (µm²) of the fiber bundles compared with the control from 10.5 ± 1.9 to 7.0 ± 1.1 µm² (P = 0.005) for nNOS immunoreactive fiber bundles and 13.7 ± 2.0 to 9.6 ± 1. 1.0 µm² (P = 0.019) for TK immunoreactive bundles. Lesions with 1–2 mm separation significantly reduced the average sizes of the fiber bundles compared with control to 5.8 ± 1.0 µm² (P = 0.0001) for nNOS immunoreactive fiber bundles and 6.7 ± 2.0 µm² (P < 0.0001) for TK immunoreactive bundles (P < 0.0001).

Structural Investigation of Proximo-Distal Projections of Neurons

The electrophysiological and structural investigations above indicate that the axons of muscle motor neurons innervating the circular muscle project in the circumferential direction in all three anatomical regions. It has also been established that the vast majority of inhibitory motor neurons project an initial axon that extends from proximal to distal and excitatory motor neurons project distal to proximal before diverging into a terminal arbor with a dense array of varicosities (points of neurotransmitter release), which circumferentially run close to and innervates the individual SMCs (8, 27). In the following experiments, we investigated the structural proximo-distal spread of VIP immunoreactive inhibitory nerve fibers through the use of gastric myectomy surgery.

The thick mounts showed long varicose nerve fibers extending circumferentially revealed by anti-VIP and myenteric ganglia revealed by anti-Hu C/D (Fig. 10). The lack of Hu C/D immunoreactivity in the myectomy region indicated that the myenteric plexus was successfully removed. The edge of the myectomy lesion is visible in Fig. 10A. There was reduced fiber density ∼1 mm distal to the myectomy region (Fig. 10C). This demonstrates that the axons of inhibitory motor neurons arise for local neurons that project short distances distally in the stomach. The regions ∼1.5 mm distal to the region without myenteric ganglia showed the full dense innervation where the myenteric ganglia and innervation patterns were normal (Fig. 10, A and D).

Figure 10.

Myectomy surgery and its consequences for innervation of the circular muscle in the corpus to investigate proximo-distal fiber distributions. A: representative low-power immunohistochemistry of thick-mount preparation immediately distal to corpus myectomy site (yellow box in B). Immunoreactivity of inhibitory neurons, VIP (green), and Hu C/D (orange) showing neuronal cell bodies. Myectomy example demonstrates a reduction of innervation distal to the lesion site (purple in B) followed by a gradient of increased innervation further from the lesion. B: representation of myectomy lesion (purple) and the location image in A was taken from (yellow box). Further details see Supplemental data. C: high-power image on the edge of the lesion site showing reduced density of nerve fibers. D: high-power image at distance distal from lesion, cell bodies are present, and more fibers are present. VIP, vasoactive intestinal peptide.

DISCUSSION

Dominant Circumferential Influences of Muscle Motor Neurons on the Circular Muscle

The electrophysiological and structural investigations of the current study both indicate that the enteric motor neurons that supply the circular muscle project their axons a short distance proximo-distally and a much longer distance around the circumference of the stomach (Fig. 11). We found that the functional circumferential influence spanned at least 11 mm in all regions, but the decline to half amplitude varied between the fundus, corpus, and antrum. This is consistent with studies in which projections of muscle motor neurons in the guinea pig stomach have been investigated by labeling with retrogradely transported dyes (8, 27) and with descriptions of the patterns of contraction of the distal stomach in circumferential (annular) bands in vivo and in vitro in rat and other species (7, 28–30). The circumferential orientation of influence of the circular muscle motor neurons is augmented by the electrical properties of gastric muscle cells, which are aligned circumferentially in each gastric region and form an electrical syncytium (13, 31). The arrangement of the smooth muscle cells (SMCs) and their electrical interconnections results in an electrically determined space constant of 3.0 ± 0.2 mm in the circumferential direction, but only ∼0.5 mm in the proximo-distal direction, perpendicular to the circular muscle bundles (32). Thus, the influences of the neurons in the circumferential direction extend beyond their physical lengths (diagrammatical representation in Fig. 11B).

Figure 11.

Depiction of the innervation of the circular muscle deduced from the current study and data in the literature. A: diagram of the anatomy of the rat stomach, showing the directions of muscle bundles of the circular muscle coat (blue lines). B: relationship of a single inhibitory motor neuron to the circular muscle, below showing the approximate distance the axon projects physically (solid bar) and its longer region of influence due to electrotonic spread (indicated by dots). The motor neurons give rise to branching terminals that follow the direction of the muscle. C: multiple inhibitory motor neurons innervate bundles of electrically coupled smooth muscle cells. D: representative cell body of the motor neuron. Its axon runs a short distance orthogonal to the circular muscle to a branch point. E: individual SMCs form bundles of cells that are interconnected by bands of smooth muscle. Different responses to nerve stimulation that are recorded by intracellular electrodes within an individual muscle cell could be affected by the cell’s relation to the muscle bundles, bands, and nerve terminals. For example, if the cell is in the center of a bundle (1), at the edge of a bundle (2), or within connecting bands (3). SMCs, smooth muscle cells.

Summation of Motor Neuron Effects

Functionally, in terms of movement, the single-compartment mammalian stomach has two parts, proximal and distal (1, 3, 33) (Fig. 1C), which corresponds neither to the anatomical regions (fundus, corpus, and antrum) (Fig. 1A) nor to the regions marked by mucosal specializations (Fig. 1B). The proximal part changes volume to accommodate different amounts of content, and the distal part mixes and propels the content. In the rat, the annular contractions that propel the gastric content in the distal stomach are ∼6 mm wide on average (7). A region of corpus that is 6 mm wide (proximo-distal) and 10 mm long (a circumferential distance up to which motor neuron influences spread) contains ∼4,420 ± 500 (means ± SE) NOS neurons, which includes inhibitory motor neurons to the circular muscle, longitudinal muscle, muscularis mucosae, and NOS motor neurons to intramural arteries (12). The circular muscle is the largest and most densely innervated of these targets, and if one third of the neurons are assumed to innervate the circular muscle, such a region would contain the cell bodies of ∼1,500 ± 170 inhibitory (NOS) neurons supplying the circular muscle. Retrograde tracing studies of gastric motor neurons have been made in the guinea pig (8, 27). Given the similar sizes of guinea pigs and rats, their similar gastric morphologies, and similarities of their gastric enteric neuron numbers (11), it would suggest that it is legitimate to extrapolate between these species. The retrograde tracing studies indicate that very large numbers of neurons supply small regions of circular muscle (8). These authors used 100- to 200-µm diameter beads coated with a dried lipophilic dye (DiI) pressed onto the circular muscle to label neurons that projected to the region of the bead. Nerve fibers that were labeled with DiI were traced in the direction of the circular muscle for up to 20 mm, and it was found that 144 ± 28 myenteric nerve cells were labeled from single beads, ∼30% of which were NOS neurons (8). Thus, any SMC that is recorded is predicted to be influenced by 10–100 s of neurons, which is consistent with the grading of responses with intensity of stimulation that we recorded in this study. We hypothesize that increasing the stimulus amplitude resulted in greater responses due to the recruitment of more motor axons. Because the axons are found at different points within the muscle layers, they can be predicted to have different thresholds resulting from their different relationships to the stimulating electrodes. The large numbers of motor neurons supplying small regions suggest that there may be redundancy of innervation and that proportionally small losses of neurons in gastric disorders (15) may not, by themselves, have significant consequences for gastric control.

Although the grading of response amplitudes with stimulus strength that we have described provides a functional correlate of the multineuronal inputs predicted by structural studies, it underestimates the number of neurons that converge on muscle cells within the circular muscle. This is because it is not possible to separate the numerous nerve fibers by stimulus thresholds and because the stimulus parameters used did not reach maximal activation of all of the neurons. The maximum amplitudes of the IJPs recorded throughout this study in the stomach were smaller than the amplitudes of IJPs that have been reported from other regions of the gastrointestinal tract. We investigated inhibitory transmission in some of the colons from the same animals under the same conditions, which confirmed that IJPs in the colon had 3–4 times greater amplitude compared with the stomach.

Differences in Neuromuscular Control across Regions

Paralleling the functional differences seen at an organ level, our intracellular electrophysiological data also show differences in the characteristics of neuromuscular transmission between anatomical regions (fundus, corpus, and antrum, Fig. 1A). Muscle cells in the fundus were more depolarized (−37 mV) at rest compared with those of the corpus (−49 mV) or antrum (−50 mV). This is consistent with a previous report that muscle cells in the aglandular region of rat stomach had resting membrane potentials of −40 to −45 mV, whereas those in the glandular stomach of the rat were −50 to −55 mV (34). Furthermore, also similarly to Xue et al. (34), slow waves (SWs) were recorded by intracellular microelectrodes in the distal, not proximal, rat stomach. A more depolarized membrane potential in the fundus has also been reported to occur in the dog, guinea pig, and human stomachs (35–37).

There is convincing evidence that both excitatory and inhibitory motor neurons that supply the muscle of the gastrointestinal tract release cotransmitters. The majority of excitatory neurons contain and release two excitatory transmitters, acetylcholine (ACh) and tachykinins (TK), and close analysis of experimental data indicates that the inhibitory neurons also act through co-transmission, using an ATP-like transmitter, NO, and peptides (closely related PHI, VIP, and PACAP), but to different extents depending on region and species (8, 38–42). An example of species difference is that in the mouse, the antral IJP lacks a slow component and appears to be entirely attributable to purinergic transmission (43), whereas in the current study in rats, we have found a prominent NO-mediated slow component in the antrum. We have not attempted an analysis of the relative roles of the cotransmitters in the different gastric regions, which would require systematic multiple-drug pharmacology experiments, using different combinations of antagonists, different orders of application, and the titration of drug concentrations to ensure effectiveness without nonspecific actions for each of the three stomach regions. Differences in the pharmacology of inhibitory transmission at different frequencies of stimulation have also been reported, with the NO component being more prominent and the purinergic component being attenuated at higher frequencies of stimulation in the colon (40). We have not investigated this difference, having conducted our pharmacological investigation on IJPs in response to single stimuli. An important future study will be to systematically investigate cotransmitter contributions to responses at different frequencies in the different gastric regions.

We found regional differences in the properties of excitatory and inhibitory neurotransmission. In the case of excitatory transmission, single pulses applied to intramural nerve fibers evoked depolarizing events, EJPs, in the fundus, with smaller and more inconsistent EJPs in the corpus, and no depolarizing events in the antrum. This agrees with earlier investigations in which EJPs were not recorded in the guinea pig antrum, even after blocking cholinesterases to enhance acetylcholine availability, but were recorded in the fundus (36, 44). Thus, it was only in the fundus that we were able to use electrophysiological recordings from the muscle to determine the projections of excitatory motor neurons (Fig. 8). Although the excitatory neurons do not evoke depolarizing junction potentials in the distal stomach, spontaneous SWs were recorded in the majority of preparations and excitatory enteric neurons are clearly functionally effective in the antrum based on other data in the literature. Excitatory neuron activation enhances SW amplitudes and the annular contractions associated with antral SWs (44, 45). In the mouse antrum, stimulation of intramural nerve endings enhanced SW frequency, an effect that was prevented by muscarinic receptor block with atropine (46). The enhancement of SWs was not observed in ICC-deficient (W/W^v) mice (46). Thus, in the antrum, the principal site of action of the excitatory neurons appears to be the ICC, not the SMC.

In the case of inhibitory transmission to the muscle, hyperpolarizing events, IJPs, occurred in all regions investigated, but their time courses and associated afterdepolarizations differed. The compound IJP varied in duration across the anatomical regions, with the fast component being prominent across all regions and the slow component being more obvious in the antrum compared with the fundus and corpus (Fig. 2). Investigations of the cellular basis of inhibitory control indicate that the effects on the smooth muscle are mediated both directly via muscle cells and indirectly via other components, including ICC and platelet-derived growth factor receptor-α (PDGFRα) fibroblast-like cells (47) that form part of the electrically coupled syncytium, known as the SIP syncytium, consisting of SMCs, ICCs, and PDGFRα cells (47). Thus, the electrical events recorded from SMCs are only a window on the events that occur in the neighboring SMCs and in the other cell types (31, 47). Our pharmacological studies demonstrated that both the fast and slow IJPs were impacted by blocking both CaCC using niflumic acid and SK channels with apamin, suggesting that both the fast and slow components have a dependence on interstitial cells. It is probable that this impact on both components of the junction potential is influenced by the fast and slow components having some temporal overlap. A detailed analysis of the integration and locations within the SIP syncytium where inhibitory signals act is complex to undertake and is beyond the scope of the present investigations.

It should be noted that EJPs and IJPs are to a significant extent an artifact of synchronous stimulation of the excitatory and inhibitory axons, respectively. Under physiological conditions, axons discharge asynchronously and the membrane is depolarized with oscillations during the period of nerve activity, without discrete junctional events being detected (48, 49).

Afterdepolarizations

Brief depolarizations, lasting ∼1 s following fast IJPs, were observed in the fundus. Afterdepolarizations were also observed in the corpus with longer durations. In the antrum, the IJP repolarized more slowly due to the greater prominence of the slow component of the IJP, and the afterdepolarizations had median durations of 8 s (Fig. 2). Depolarizations that follow hyperpolarizations, observed by Hodgkin and Huxley in squid giant axons and termed by them as anode break excitation, are a common feature of excitable cells that are due to K current inactivation and Na current activation persisting after the release of hyperpolarization (50). These depolarizing events have been recorded from intestinal muscle following hyperpolarization by large external electrodes in the presence of tetrodotoxin (51). We hypothesize that the brief afterdepolarizations observed in the fundus are contributed to by anode break excitation (sometimes also called rebound excitation) as previously described in axons and SMCs (50, 51). In the antrum, we hypothesize that the slower afterdepolarizations (lasting ∼8 s) are SWs because they have a similar time course and amplitude to spontaneously occurring SWs. Spontaneous SWs had an average time to peak of 6.6 ± 2.7 s, and the afterdepolarizations in the antrum had a time to peak of 4.4 ± 2.1 s. Long-duration depolarizations following IJPs were previously reported in the canine colon where they were also concluded to be SWs (52). In a more recent study, postinhibitory excitation in the colon has been shown to be dependent on nitrergic transmission and generation of Ano1-dependent excitation in ICC (53). We hypothesize that the SW-like events are initiated, sometimes prematurely, when the hyperpolarization of the IJP is conducted electrotonically within the SIP syncytium to ICC-MY where SWs are generated (47). This hypothesis requires further investigation.

Local Inhomogeneities

It was notable that postjunctional events recorded from different SMCs in the same region differed considerably from each other (Figs. 2 and 7). For example, in the antrum, some SMC recordings contained a prominent slow component of the IJP, whereas others showed very little of this component. On the other hand, the fast component was consistently observed (Fig. 2). The afterdepolarization amplitude also differed between cells, not in a consistent relationship to the amplitude of the IJP. The tissue itself is not homogeneous, consisting of muscle bundles that branch and are joined by connecting bands (13). Cross-sectional widths of muscle bundles vary from less than 100 µm to ∼1 mm and connecting bands (Fig. 11) are commonly ∼50 µm wide (13). Thus, part of the explanation of the differences may be that recordings were made from cells that had different spatial relationships with neighboring cells (e.g., located at centers or edges of bundles or in connecting bands, Fig. 11F), different electrical connectivity with other SMCs and with ICC and PDGFRα cells, and/or different innervation patterns. These possibilities are a major challenge for investigation, which would ideally require simultaneous recording of activity in each of the cell types to analyze.

Possible Consequences for Gastric Physiology

As we have discussed above, our investigations indicate that large numbers of motor neurons influence all regions of the stomach. From this observation, we hypothesize that the fine grading of gastric volume and gastric emptying that is observed in vivo is contributed to by engagement of different proportions of the motor neuron populations. Grading may also be achieved by changing the frequency of neuron firing. In the proximal stomach, where there are no ICC-MY and therefore no slow waves, excitatory and inhibitory neurons act directly on the muscle, thus directly influencing the volume of this region. In the distal stomach, excitatory and inhibitory neurons both have effects via interstitial cells. This is consistent with the neurons having finely graded effects on the SWs, and hence on the propulsive contractions that empty the distal stomach. The large numbers of motor neurons that influence the circular muscle in all parts of the stomach may provide an innervation reserve that, to some degree, protects gastric function from loss of neurons. Thus, deficits in neuromuscular control may not be simply due to the loss of motor neurons but may involve changes in other properties, for example, neurotransmission efficiency.

Conclusions

This paper provides the first extensive functional mapping of neural transmission to SMCs of the circular muscle across the different anatomical regions of the rat stomach. It reveals that there is a dense functional innervation by multiple motor neurons that affects each individual SMC. Excitatory neurons have a direct influence on the membrane potentials of SMCs in the fundus, but not in the antrum, with their effects in the corpus being variable and intermediate. The inhibitory neurons generate hyperpolarization in all regions. In the antrum, the slow component of the hyperpolarization (the IJP) is prominent, and this is commonly followed by a rebound depolarization. We deduce that the influence of motor neurons involves direct innervation of SMCs and also indirect interactions through ICCs and PDGFRα cells. The motor neurons run circumferentially around the stomach and influence the excitability of the circular muscle in a way that is consistent with the annular patterns of contraction that are observed in vitro and in vivo in the distal stomach.

DATA AVAILABILITY

Data will be made available upon reasonable request.

SUPPLEMENTAL DATA

Supplemental Tables S1 and S2 and Supplemental Figs. S1–S7: 10.5281/zenodo.10086283.

GRANTS

This work was supported by National Institutes of Health Grant, The Virtual Stomach (1OT2OD030538), Principal Investigators Leo Cheng (University of Auckland), and Z. Liu (University of Michigan).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.R.D., M.J.S., X.W., Z.L., and J.B.F. conceived and designed research; M.R.D., B.H., and J.B.F. performed experiments; M.R.D., B.H., and J.B.F. analyzed data; M.R.D., M.J.S., X.W., Z.L., and J.B.F. interpreted results of experiments; M.R.D., B.H., and J.B.F. prepared figures; M.R.D. and J.B.F. drafted manuscript; M.R.D., B.H., X.W., Z.L., and J.B.F. edited and revised manuscript; M.R.D., B.H., M.J.S., X.W., Z.L., and J.B.F. approved final version of manuscript.