Stress-induced neuroplasticity in the gastric response to brainstem oxytocin in male rats

Abstract

Previous studies have shown that pharmacological manipulations with stress-related hormones such as corticotropin-releasing factor and thyrotropin-releasing hormone induce neuroplasticity in brainstem vagal neurocircuits, which modulate gastric tone and motility. The prototypical antistress hormone oxytocin (OXT) has been shown to modulate gastric tone and motility via vagal pathways, and descending hypothalamic oxytocinergic inputs play a major role in the vagally dependent gastric-related adaptations to stress. The aim of this study was to investigate the possible cellular mechanisms through which OXT modulates central vagal brainstem and peripheral enteric neurocircuits of male Sprague-Dawley rats in response to chronic repetitive stress. After chronic (5 consecutive days) of homotypic or heterotypic stress load, the response to exogenous brainstem administration of OXT was examined using whole cell patch-clamp recordings from gastric-projecting vagal motoneurons and in vivo recordings of gastric tone and motility. GABAergic currents onto vagal motoneurons were decreased by OXT in stressed, but not in naïve rats. In naïve rats, microinjections of OXT in vagal brainstem nuclei-induced gastroinhibition via peripheral release of nitric oxide (NO). In stressed rats, however, the OXT-induced gastroinhibition was determined by the release of both NO and vasoactive intestinal peptide (VIP). Taken together, our data indicate that stress induces neuroplasticity in the response to OXT in the neurocircuits, which modulate gastric tone and motility. In particular, stress uncovers the OXT-mediated modulation of brainstem GABAergic currents and alters the peripheral gastric response to vagal stimulation.

NEW & NOTEWORTHY The prototypical antistress hormone, oxytocin (OXT), modulates gastric tone and motility via vagal pathways, and descending hypothalamic-brainstem OXT neurocircuits play a major role in the vagally dependent adaptation of gastric motility and tone to stress. The current study suggests that in the neurocircuits, which modulate gastric tone and motility, stress induces neuroplasticity in the response to OXT and may reflect the dysregulation observed in stress-exacerbated functional motility disorders.

INTRODUCTION

The pathophysiology of functional gastrointestinal disorders (FGIDs), including functional dyspepsia and irritable bowel syndrome, remains to be elucidated fully, although impairment or disruption of the brain-gut axis is suggested to contribute to its development (1–3). FGIDs are highly correlated with stress (1, 4–7), and in both humans and animal models, stressful situations alter gastrointestinal (GI) motor functions, including delayed gastric emptying and accelerated colonic transit (8–13).

Chronic exposure to variable, unpredictable stress (i.e., chronic heterotypic stress, CHe) induces both structural and functional plasticity in brain regions involved in controlling hypothalamic-pituitary-adrenocortical (HPA) activity and autonomic responses to stress (14). Such neuroplasticity associated with stress maladaptation results in behavioral and physiological adjustments, including delayed gastric emptying and enhanced colonic contractile activity (11, 15, 16). Many of the effects of chronic stress on gastric functions are related to an upregulation of corticotropin-releasing factor (CRF) release following stress maladaptation. Indeed, CRF itself delays gastric emptying and decreases gastric tone and motility, but facilitates colonic motility via a central mechanism that involves brainstem vagal neurocircuits (11, 12, 17–19). Furthermore, the central application of oxytocin (OXT) attenuates the increased CRF mRNA expression and antagonizes the delayed gastric emptying in response to CHe (11). Conversely, many rodent species, including Sprague-Dawley rats used in the present study, show an adaptive response to a repeated stressor (i.e., chronic homotypic stress, CHo). Such adaptation is evident by attenuation in the activity in the hypothalamic-pituitary-adrenocortical (HPA) axis, restored gastric emptying and colonic motility compared with either acute stress or CHe. This adaptive response of GI motility in stress adaptation is related to an upregulation of OXT expression in the paraventricular nucleus of the hypothalamus (PVN) and in the hypothalamic-vagal neurocircuits that control GI functions (11, 20).

Among other actions, the brain-gut axis modulates gastric tone and motility (21). Visceral sensory signals from the upper GI tract are conveyed to neurons of the nucleus tractus solitarius (NTS), which integrates the information and transfers it to the adjacent vagal preganglionic neurons of the dorsal motor nucleus of the vagus (DMV), mainly via GABAergic neurotransmission (21–23). The efferent vagal fibers from the DMV modulate gastric tone and motility via activation of postganglionic cholinergic excitatory and/or nonadrenergic noncholinergic (NANC) inhibitory pathways that involve nitric oxide (NO) or vasoactive intestinal peptide (VIP; 21, 24).

The vagal neurocircuits that regulate gastric functions undergo an extraordinary degree of plasticity in response to a variety of intrinsic and extrinsic factors, such as stress. Recently, our group has shown that following acute stress plastic rearrangements occur in brainstem vagal GABAergic neurocircuits in response to stress-related hormones, such as CRF, OXT, or norepinephrine (NE) (21, 25, 26).

In naïve rats, oxytocinergic projections from the parvocellular subdivisions of the PVN mediate many well-documented physiological functions in the gut through vagal pathways, including gastric relaxation via activation of a nitrergic pathway (27–32). The anxiolytic and antistress effects of central OXT release are also well documented. Within the PVN, OXT inhibits the expression and release of CRF as well as inhibits the activity of the HPA axis (33, 34). Furthermore, parvocellular OXT neurons innervate brainstem autonomic neurons to modulate autonomic and sensory systems associated with stress responses. In particular, hypothalamic OXT reverses the delayed gastric emptying and impaired gastric motility following acute or CHe, whereas antagonism or knockout of OXT receptors prevents the adaptive GI responses, following CHo (9, 11, 13, 35, 36). We have also shown that CHo increases the density of PVN-DMV oxytocinergic projections (26), suggesting that this neurocircuit may play a relevant role in the restoration of appropriate GI functions following adaptation to stress. The precise mechanisms by which OXT neuroplasticity occurs within GI-related brainstem neurocircuits to facilitate stress adaptation, however, remains obscure. The aim of this study was to investigate the possible cellular mechanisms through which OXT modulates central vagal brainstem and peripheral enteric neurocircuits in response to chronic stress in male rats.

MATERIALS AND METHODS

Ethical Approval

Male Sprague-Dawley rats (7–10 wk of age at the start of experiments) were paired housed in an American Association for the Accreditation of Laboratory Animal Care accredited Animal Care Facility maintained at 24°C on a 12:12 h light/dark cycle with food and water provided ad libitum. All procedures were conducted in accordance with the National Institutes of Health guidelines, following ARRIVE guidelines, and with the approval of the Penn State University College of Medicine Institutional Animal Care and Use Committee (No. 0420), and conform to the principles and regulations as described in the editorial by Grundy (37).

Stress Paradigms

The stress procedures were described previously (13, 20, 26). Briefly, rats were divided randomly into three groups: 1) control, 2) chronic homotypic stress (CHo; in this model rats adapt to stress, i.e., rats become stress resilient), and 3) chronic heterotypic (CHe; in this model rats do not adapt to stress, i.e., rats remain stress-sensitive). Rats in the control group were handled daily for ∼10 min without further stress induction; the CHo group underwent 2 h of restraint stress daily, for five consecutive days, in a cylinder that did not allow movement; and the CHe group underwent different stressors each day for five consecutive days: 1) 2 h restraint, 2) forced swim, i.e., 20 min in a container (36L × 26W × 24H) filled with water at room temperature, such that their hind paws could not touch the bottom of the container; 3) water avoidance, i.e., 90 min on a round platform (6 cm diameter) in a container with room temperature water at a level just below the top of the platform; 4) cold, i.e., 2 h in a cage maintained at 4°C; and 5) 2 h restraint. The stress procedures were conducted between 9:00 AM and 11:00 AM. Although both day 1 and 5 consisted of restraint stress, the other stressors were applied in random order. The experimental procedures (see In Vitro Electrophysiological Recordings and In Vivo Gastric Recordings) were conducted immediately after the final stress load, i.e., on day 5. The number of fecal pellets excreted during each stress paradigm were noted as a surrogate marker of stress response.

In Vitro Electrophysiological Recordings

Patch-clamp recordings were made from 11 rats (n = 3 control, n = 4 CHo, and n = 4 CHe). Rats were anesthetized deeply with isoflurane (5% with air) before administration of a bilateral pneumothorax and the brainstem was removed rapidly and immersed in ice-cold Krebs solution. Coronal brain slices containing the dorsal vagal complex (DVC; NTS, DMV, and area postrema) were cut at 300 µm thickness using a vibratome and incubated in oxygenated Krebs solution at 30°C for at least 90 min before use. A single brainstem slice was transferred in a perfusion chamber, held in place with a nylon mesh on the stage of a microscope (Nikon E600FN), and maintained at 32 ± 1°C with continuous perfusion with Krebs solution.

Whole cell patch-clamp recordings were conducted in medial, i.e., presumed gastric-projecting DMV neurons, using a glass pipette with 2–5 MΩ tip resistance, when filled with potassium chloride intracellular solution. Recordings were conducted using a single-electrode voltage-clamp amplifier (Axopatch 200 A; Molecular Devices, Union City, CA). Data were filtered at 2 kHz, digitized via a Digidata 1440 A interface, and analyzed using pClamp10 software (Molecular Devices). Only recordings with a series resistance <20 MΩ were used.

Miniature inhibitory postsynaptic currents (mIPSCs; i.e., action potential independent events) were recorded from DMV neurons at a holding potential of −50 mV in slices perfused with 1 mM kynurenic acid and 0.3 µM tetrodotoxin.

OXT (150 nM) was dissolved in the perfusing Krebs solution (29, 31) and applied for a period of time sufficient for the response to reach a plateau or for a maximum of 5 min. Neurons were allowed to recover for at least 15 min before perfusion with 30 nM CRF (5 min), followed by washout (10 min) and reapplication of OXT. mIPSCs were analyzed offline with Clampfit (Molecular Devices) or MiniAnalysis software (Synaptosoft, Leonia, NJ). A minimum variation of 20% in mIPSC frequency or amplitude was taken as an indication of a response.

Krebs solution was composed of the following (in mM): 126 NaCl, 25 NaHCO₃, 2.5 KCl, 1.2 MgCl₂, 2.4 CaCl₂, 1.2 NaH₂PO₄, and 11 dextrose, maintained at pH 7.4 by bubbling with 95% O₂-5% CO₂. Potassium chloride intracellular solution was composed of the following (in mM): 140 KCl, 1 CaCl₂, 10 HEPES, 10 EGTA, 2 Na₂ATP, and 0.25 NaGTP, adjusted to pH = 7.36 with KOH.

Phosphate-buffered saline (PBS) was composed of the following (in mM): 124 NaCl, 26 NaHCO₃, and 2 KH₂PO₄, pH = 7.4.

In Vivo Gastric Recordings

Gastric tone and motility recordings were made from 37 rats (n = 10 control, 14 CHo, and 13 CHe). Rats were fasted overnight (water ad libitum) and anesthetized with thiobutabarbital sodium (Inactin; 100–150 mg/kg ip). The anesthesia level was monitored continuously throughout the experiment. The core temperature was kept at 37°C with a heating pad. Once a deep plan of anesthesia was achieved (absence of palpebral reflex), rats were intubated with a tracheal catheter, the jugular vein was isolated and a catheter was inserted to allow intravenous injections. A midline laparotomy was performed to expose the anterior gastric wall. Encapsulated miniature strain gauges (6 × 8 mm; AT Engineering, Hershey, PA) were sutured to the serosal surface of the anterior gastric corpus and antrum in alignment with the gastric circular smooth muscle, and the laparotomy was closed with a 5-0 suture with the strain gauge leads exteriorized. The signals of the strain gauges were amplified (EXP CLSG-2; QuantaMetrics, Newton, PA), filtered (low pass cut off = 0.1 Hz; AT Engineering), digitized via a Digidata 1320 interface, and recorded using AxoScope software (Molecular Devices). Rats were then placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA), and the lower medulla was exposed via blunt dissection. The meningeal membranes above the vagal trigone were dissected, and the exposed lower medulla was covered with a gauze soaked in prewarmed saline for at least 1 h to stabilize.

A glass micropipette (20–30 µm tip diameter) was directed into the DVC (in mm: 0.4–0.5 rostrocaudal from calamus scriptorius, 0.2–0.3 mediolateral from midline, 0.6–0.65 dorsoventral from the brainstem dorsal surface). Drugs were dissolved in phosphate-buffered saline (PBS) and microinjected in 60-nL volumes over a period of 2 min via a picospritzer (Parker Hannifin, Hollis, NH). Fluorescent microspheres (Fluoresbrite, Polysciences Inc, Warrington, PA) were included in the injectate for post hoc verification of the injection site. Gastric tone and motility were monitored for 5 min before drug application and for at least 15 min after the microinjection. Gastric tone and motility were allowed to recover for a minimum of 1 h between injections.

Gastric tone was measured as absolute tone variation (in milligram) from the baseline. Gastric motility was calculated using the following formula, as described previously (26, 38):

$$\text{motility index }=(N_{\text{1}}·\text{1}+N_{\text{2}}·\text{2}+N_{\text{3}}·\text{4}+N_{\text{4}}·\text{8})/t\times \text{1}00,$$

where N equals the number of peaks in a particular force range (N₁ = 25–50 mg, N₂ = 51–100 mg, N₃= 101–200 mg, N₄ > 200 mg), and t equals the time interval over which the gastric motility was measured. The effect of drugs on gastric motility was measured relative to the averaged value of gastric motility before microinjection (baseline = 100%). The NO synthase inhibitor nitro-l-arginine methyl ester (l-NAME, 10 mg/kg), the VIP antagonist (50 µg/kg), or the muscarinic agonist bethanechol (50 µg/kg) were administered intravenously before the repeated OXT microinjection.

At the conclusion of the experiment, rats were fixed via transcardial perfusion with 0.1 M PBS followed by paraformaldehyde (PFA, 4%) in 0.1 M PBS. Brainstems were removed and postfixed in 4% PFA for 48 h and then transferred to 0.1 M PBS with 20% sucrose for 2 days. The brainstem was frozen, and coronal sections (50 µm thickness) throughout the rostrocaudal extent of the DVC were cut using a microtome. Every fourth slice was mounted to identify the injection site using a Nikon E400 microscope.

Data Analysis

Data were analyzed by investigators blinded to the treatment. Data were tested, and normal distribution and equal variances in the sampled distribution were found. Student’s t tests were used for parametric data; one-way ANOVA followed by a post hoc Tukey’s comparison was used for intergroup comparisons using GraphPad Prism 5 (GraphPad Software, San Diego, CA). Data are reported as means ± SE with significance defined as P < 0.05.

Drugs

Oxytocin, l-NAME, bethanechol, and atosiban were purchased from Sigma-Aldrich (St. Louis, MO), and VIP antagonist was purchased from Bachem (Torrance, CA).

RESULTS

Stress Adaptation Decreased Fecal Pellet Output

There was no significant difference in number of fecal pellets excreted between the homotypic (CHo, 8.1 ± 1.06, n = 19) and the heterotypic (CHe, 7.3 ± 0.93, n = 14) groups on the first day of stress (unpaired t test: t₃₁ = 0.6402, P = 0.5268). At the end of the fifth day of CHo stress, however, the number of fecal pellets expelled were significantly fewer than those expelled on day 1 (2.9 ± 0.66, paired t test, t₁₈ = 4.082, P = 0.0007), suggesting that CHo rats showed adaptation to stress. Data are summarized in Fig. 1. Conversely, at the end of the fifth day of CHe stress, the fecal pellets expelled were not different than the numbers of pellets expelled on day 1 (5.3 ± 1.04, paired t test, t₁₃ = 2.002, P = 0.0666), suggesting that CHe rats did not show adaptation to stress but, rather, remained stress sensitive. Data are summarized in Fig. 1.

Figure 1.

Summary graphic showing the number of fecal pellets excreted during the restraint stress load at day 1 (open bar) and at day 5 (filled bar). Rats that underwent five consecutive days of chronic, repetitive stress (chronic homotypic stress, Cho, n = 19 rats) excreted a significantly lower number of fecal pellets at day 5 than at day 1 (paired t test t₁₈ = 4.082, P = 0.0007). Conversely, rats that underwent five consecutive days of chronic, nonrepetitive stress (chronic heterotypic stress, CHe, n = 14 rats) excreted a similar number of fecal pellets at day 1 and day 5 (paired t test t₁₃ = 2.002, P = 0.0666). *P < 0.05 vs. day 1.

Oxytocin Modulates Inhibitory Neurotransmission to Gastric-Projecting DMV Neurons from Stressed but Not from Naive Male Rats

We conducted a series of experiments in which we analyzed the effects of OXT on mIPSCs, since GABAergic transmission onto DMV neurons is the main regulator of vagal output (21). There was no difference in the baseline frequency (pulse per second; pps) or amplitude (pA) of miniature inhibitory postsynaptic currents (mIPSCs) in DMV neurons from naïve (0.395 ± 0.028 pps and 114 ± 13.5 pA; n = 9), CHo (0.584 ± 0.058 pps and 97 ± 10.6 pA, n = 10), and CHe rats (0.604 ± 0.137 pA and 103 ± 5.7 pA, n = 7; one way ANOVA and Tukey’s multicomparison test, F_2,23 = 3.179, P = 0.0604 for frequency; F_2,22 = 0.6367, P = 0.5385 for amplitude; Fig. 2C).

Figure 2.

Representative traces showing the effects of oxytocin perfusion on miniature inhibitory postsynaptic currents (mIPSCs) in DMV neurons. Representative traces showing that oxytocin did not have any effect on mIPSCs in neurons from a naïve rat (A); however, perfusion with oxytocin reduced the frequency of mIPSCs in neurons from a CHe rat (B). C: summary graphics showing the baseline frequency (top) and amplitude (bottom) of mIPSCs of DMV neurons from naïve (n = 9 rats), CHo (n = 10 rats), and CHe (n = 7 rats) groups. There were no differences among these groups (one way ANOVA and Tukey’s multi comparison test, F_2,23 = 3.179, P = 0.0604 for frequency; F_2,22 = 0.6367, P = 0.5385 for amplitude). Bars represent the mean responses. D: summary graphic showing the decreased frequency of mIPSC induced by oxytocin. Percentage of frequency decrease induced by OXT alone (left; naïve, n = 9; Cho, n = 10; Che, n = 7) and by repeated application of OXT in slices pretreated with CRF (right; naïve, n = 5; Cho, n = 7; Che, n = 4). Neurons in which oxytocin was assessed only once appear as solid symbols, whereas open symbols of similar shape represent data from the same neuron before and after CRF application. (Paired t test; *P < 0.05 vs. baseline. Bar represents the mean response). E: summary graphic showing the percentage of DMV neurons in which oxytocin perfusion decreased the frequency of mIPSCs. Numbers embedded in the columns represent the number of responsive vs. nonresponsive neurons to OXT alone and after repeated application of OXT in slices pretreated with CRF. Note that CHo and CHe uncovered an oxytocin-induced reduction of mIPSCs frequency. χ² test, P < 0.05. CHe, chronic heterotypic stress; CHo, chronic homotypic stress; CRF, corticotropin-releasing factor; DMV, dorsal motor nucleus of the vagus; OXT, oxytocin.

In nine neurons from three naïve male rats, perfusion of brainstem slices with OXT-reduced mIPSC frequency in two neurons (both from the same rat) but not in the remaining seven neurons (t₈ = 0.7962, P = 0.4489) and had no effect on mIPSC amplitude (t₈ = 1.179, P = 0.2770) in any of the neurons tested (Fig. 2). After perfusion with CRF, a second perfusion with OXT decreased the frequency but not the amplitude of mIPSC in three of five neurons (t₄ = 3.373, P = 0.0140 for frequency and t₄ = 0.5292, P = 0.3123 for amplitude; Fig. 2, A and C).

In contrast, in 10 of 10 neurons from four CHo rats, perfusion with OXT decreased mIPSC frequency significantly (t₉ = 4.320, P = 0.0010) without altering the amplitude (t₉ = 0.9820, P = 0.3518). After perfusion with CRF, a second perfusion with OXT decreased the frequency but not the amplitude of mIPSC in seven of seven neurons (t₆ = 4.000, P = 0.0036 for frequency and t₆ = 0.1741, P = 0.4337 for amplitude).

The OXT-induced inhibition of mIPSCs frequency was similar between the first and second application (t₆ = 2.222, P = 0.0680; Fig. 2D).

In six of seven neurons from four CHe rats, perfusion with OXT decreased mIPSC frequency significantly (t₆ = 4.946, P = 0.0013) without affecting the amplitude (t₆ = 0.2599, P = 0.8036). After perfusion with CRF, a second perfusion with OXT decreased the frequency but not the amplitude of mIPSC in four of four neurons (t₃ = 3.266, P = 0.0235 for frequency and t₃ = 1.924, P = 0.0750 for amplitude). The OXT-induced inhibition of mIPSCs was similar between the first and second application (t₃ = 1.403, P = 0.2551; Fig. 2, B and D).

The percentage of neurons in which OXT decreased mIPSCs frequency was significantly different between naïve, CHe, or CHo groups (χ² test, P = 0.0060; Fig. 2E).

These data suggested that, similar to that observed in DMV neurons from naive rats treated with CRF, stress in male rats uncovers the modulation of GABAergic synapses by OXT.

We then conducted a series of in vivo experiments to investigate whether the postganglionic effectors and/or the response of gastric tone and motility to exogenous OXT (150 pmoles/60 nL) application was altered in chronically stressed animals.

In 37 rats (10 naïve, 14 CHo, and 13 CHe) microinjection of OXT in the DVC decreased corpus tone by −109 ± 9.40 mg and motility to 63 ± 4.9% of baseline. There was no significant difference in the OXT-induced decrease in corpus tone or motility among the groups (one-way ANOVA: F_2,34 = 0.4403, P = 0.6474 for tone, and F_2,34 = 0.0364, P = 0.9643 for motility; Fig. 3). Similarly, in 33 rats (10 naive, 10 CHo, and 13 CHe) microinjection of OXT in the DVC decreased antrum tone by −101 ± 6.92 mg and motility to 52 ± 3.6% of baseline. There was no significant difference in the OXT-induced decrease in antrum tone or motility among the groups (one-way ANOVA: F_2,30 = 1.087, P = 0.3500 for tone, and F_2,34 = 0.3262, P = 0.7239 for motility; Fig. 4).

Figure 3.

Oxytocin-induced effects of corpus tone and motility are mediated by different postganglionic pathways following stress in male rats. A: representative traces from a naive male rat showing that microinjection of OXT (150 pmoles) in the dorsal vagal complex (DVC) decreases corpus tone and motility (left). After intravenous administration of nitro-l-arginine methyl ester (l-NAME), the inhibitory effect of oxytocin was prevented (middle). After intravenous administration of VIP antagonist, a second microinjection of OXT induced a similar inhibition as in control (right). Oblique bars represent a 1- to 2-min break in the recording. B: data points showing the corpus tone response of individual male rats to microinjection of OXT alone (left column), in the presence of l-NAME (left middle column), in the presence of VIP antagonist (right middle column), and in the presence of bethanechol (Beth, right column) for naïve males (left, black, n = 8 rats), CHo (middle, red, n = 11 rats), and CHe (right, blue, n = 11 rats). Bars represent the mean responses. *P < 0.05 vs. oxytocin alone. C: data points showing the corpus motility response of individual male rats to microinjection of OXT alone (left column), in the presence of l-NAME (left-middle column), in the presence of VIP antagonist (right middle column), and in the presence of bethanechol (Beth, right column) for naïve males (left, black), CHo (middle, red), and CHe (right, blue). Bar represents the mean responses. *P < 0.05 vs. oxytocin alone. CHe, chronic heterotypic stress; CHo, chronic homotypic stress; OXT, oxytocin; VIP, vasoactive intestinal peptide.

Figure 4.

Oxytocin-induced effects of antrum tone and motility are mediated by different postganglionic pathways following stress in male rats. A: representative traces from a CHo male rat showing that microinjection of OXT (150 pmoles) in the dorsal vagal complex (DVC) decreases antrum tone and motility (left). After an increase in tone and motility induced by an intravenous administration of VIP antagonist, a second microinjection of OXT induced a similar inhibition as in control (right). B: data points showing the antrum tone response of individual male rats to microinjection of OXT alone (left column), in the presence of l-NAME (left middle column), in the presence of VIP antagonist (right middle column), and in the presence of bethanechol (Beth, right column) for naïve males (left, black, n = 8 rats), CHo (middle, red, n = 11 rats), and CHe (right, blue, n = 11 rats). Some data point are greater than−300 mg and have been collected in the greater than −300. Bar represents the mean responses. C: data points showing the antrum motility response of individual male rats to microinjection of OXT alone (left column), in the presence of l-NAME (left middle column), in the presence of VIP antagonist (right middle column), and in the presence of bethanechol (Beth, right column) for naïve males (left, black), CHo (middle, red), and CHe (right, blue). Bar represents the mean responses. *P < 0.05 vs. oxytocin alone. CHe, chronic heterotypic stress; CHo, chronic homotypic stress; l-NAME, nitro-l-arginine methyl ester; OXT, oxytocin; VIP, vasoactive intestinal peptide.

In all groups, pretreatment with the OXT receptor antagonist atosiban (30 nmoles/2 µL on the floor of the iv ventricle) increased corpus tone by 47 ± 15 mg without changing corpus motility (100 ± 5.8% of baseline). In the presence of atosiban, the response to a repeated microinjection of OXT on corpus tone and motility (−51 ± 8.3 mg and 87 ± 6.7% of baseline, paired t test, t₃₂ = 6.438, P < 0.0001 for tone, t₂₈ = 3.231, P = 0.0016 for motility vs. OXT alone) was attenuated significantly. In all groups, pretreatment with atosiban increased antrum tone by 73 ± 23.8 mg without changing antrum motility (105 ± 10.3% of baseline). In the presence of atosiban, the response to a repeated microinjection of OXT on antrum tone and motility (−41 ± 8.5 mg and 108 ± 12.1% of baseline, paired t test, t₃₂ = 6.145, P < 0.0001 for tone, t₃₂ = 4.824, P < 0.0001 for motility vs. OXT alone) was attenuated significantly (not shown).

These data indicate the specificity of the OXT receptor activation and suggest that there is a tonic release of OXT that modulates corpus and antrum tone but not motility.

We then conducted a series of experiments to investigate the mechanism of the OXT-mediated relaxation.

In all groups administration of the NO synthase inhibitor, l-NAME, VIP antagonist, or bethanechol increased both corpus and antrum tone and motility, similarly with the only exception of antrum tone, which was increased by l-NAME administration significantly more in CHe versus naïve or CHo rats (one-way ANOVA: F_2,29 = 5.910, P = 0.0074). Data are summarized in Table 1.

Table 1.

Effects of peripheral antagonists

	Corpus		Antrum
	Tone, mg	Motility, % of baseline	Tone, mg	Motility, % of baseline
Naïve rats
l-NAME	69 ± 19.4	114 ± 9.6	85 ± 25	175 ± 42
	n = 8	n = 8	n = 8	n = 8
VIP antagonist	57 ± 21.7	143 ± 11.6	57 ± 25	124 ± 24
	n = 8	n = 8	n = 8	n = 8
Bethanechol	438 ± 145	242 ± 48	748 ± 385	273 ± 74
	n = 7	n = 7	n = 7	n = 7
CHo rats
l-NAME	94 ± 32	105 ± 8.9	71 ± 23	119 ± 11
	n = 11	n = 11	n = 11	n = 11
VIP antagonist	80 ± 21.5	121 ± 9.8	95 ± 59	131 ± 21
	n = 11	n = 11	n = 11	n = 11
Bethanechol	784 ± 131	313 ± 36	374 ± 112	273 ± 23
	n = 10	n = 10	n = 10	n = 10
CHe rats
l-NAME	87 ± 21.7	121 ± 22.7	228 ± 50a	199 ± 33
	n = 11	n = 11	n = 11	n = 11
VIP antagonist	57 ± 16.6	134 ± 26.9	81 ± 35	122 ± 11
	n = 11	n = 11	n = 11	n = 11
Bethanechol	890 ± 269.1	375 ± 101.8	340 ± 74	246 ± 29
	n = 10	n = 10	n = 10	n = 10

Values are means ± SE; n, number of rats. CHo, chronic homotypic stress; l-NAME, nitro-l-arginine methyl ester. ^aP < 0.05 vs. antrum tone, l-NAME-Naïve, and CHo (t₃ = 5.910, P = 0.0074).

In Male Rats Oxytocin Engages Different Vagal Efferent Pathways following Stress Adaptation

We have reported recently that in naïve male rats the corpus response to OXT microinjection in the DVC is antagonized by systemic administration of l-NAME (29, 31). We confirmed these data by showing that in 8 naïve male rats microinjection of OXT in the DVC decreased corpus tone and motility (Fig. 3). In the presence of l-NAME, repeated application of OXT induced a significantly lower inhibition of corpus tone and motility (t₇ = 6.722, P = 0.0003 and t₇ = 2.642, P = 0.0333 vs. OXT alone, respectively; Fig. 3).

Since vagal inhibitory actions are also mediated by the peripheral release of vasoactive intestinal peptide (VIP), we tested the effects of OXT microinjections in the DVC after pretreatment with VIP antagonist. In eight naïve male rats in which microinjection of OXT decreased corpus tone and motility (Fig. 3) in the presence of the VIP antagonist, the second OXT injection induced a similar decrease in corpus tone and motility as during perfusion without the VIP antagonist (t₇ = 1.811, P = 0.1131 and t₇ = 0.1118, P = 0.9141 vs. OXT alone, respectively; Fig. 3).

To confirm that the gastroinhibitory actions of OXT did not involve inhibition or withdrawal of the tonically active cholinergic pathway, in seven naïve male rats in which microinjection of OXT in the DVC decreased corpus tone and motility, a repeat microinjection of OXT in the presence of bethanechol induced a similar decrease in corpus tone and motility (t₆ = 1.041, P = 0.3381 and t₆ = 1.033, P = 0.3414 vs. oxytocin alone, respectively; Fig. 3).

These data indicate that, in naïve male rats, the inhibitory effect of OXT on corpus tone and motility is mediated solely via a NO-dependent postganglionic pathway, and does not involve either activation of the inhibitory VIP pathway or withdrawal of the excitatory cholinergic pathway.

In 11 CHo male rats in which the first OXT microinjection induced a decrease in corpus tone and motility, in the presence of l-NAME, the second microinjection of OXT reduced corpus tone and motility to a significantly lesser extent (t₁₀ = 2.713, P = 0.0218 and t₁₀ = 2.246, P = 0.0485 vs. OXT alone, respectively; Fig. 3). In contrast to naïve rats, however, the inhibition of corpus tone but not motility in response to repeated OXT injection was reduced significantly in the presence of the VIP antagonist (t₉ = 3.492, P = 0.0058 and t₉ = 1.028, P = 0.3282 vs. OXT alone, respectively). Finally, as in naïve rats, bethanechol did not affect the gastroinhibitory effects of OXT (t₉ = 1.272, P = 0.2353 and t₉ = 1.751, P = 0.1139 vs. OXT alone, respectively; Fig. 3).

These data indicate that, in contrast to naïve male rats, the OXT-induced relaxation of corpus tone in CHo male rats involves the peripheral release of both NO and VIP, whereas the OXT-induced decrease in corpus motility involves the peripheral release of NO only.

In contrast to naïve rats and CHo rats, however, in 11 CHe rats, the OXT-induced decrease in corpus tone and motility was unaffected by l-NAME (t₁₀ = 0.9054, P = 0.3865 and t₁₀ = 0.8427, P = 0.4191 vs. OXT alone, respectively; Fig. 3). Conversely, in the presence of the VIP antagonist, the OXT-induced decrease in corpus tone and motility was reduced significantly (t₁₀ = 2.608, P = 0.0261 and t₁₀ = 2.419, P = 0.0361 vs. OXT alone, respectively; Fig. 3). In the presence of bethanechol, the second microinjection of OXT induced a similar decrease in corpus tone and motility (t₉ = 1.541, P = 0.1577 and t₉ = 1.136, P = 0.2852 vs. oxytocin alone, respectively; Fig. 3).

These data indicate that, unlike naïve or CHo rats, the effects of OXT microinjection in the DVC on corpus tone in male CHe rats occur via a mechanism that involves the peripheral release of VIP only.

In naïve male rats, the effect of OXT-induced inhibition of antrum tone was significantly reduced by pretreatment with l-NAME (t₇ = 3.245, P = 0.0071) but not the VIP antagonist (t₇ = 0.3723, P = 0.7207) or bethanechol (t₆ = 2.230, P = 0.0673; Fig. 4). Conversely, the OXT-induced decrease of antrum motility was significantly reduced by pretreatment with the VIP antagonist (t₇ = 2.884, P = 0.0118) and l-NAME (t₆ = 2.307, P = 0.0303) but not bethanechol (t₆ = 2.031, P = 0.0885; Fig. 4).

In CHo male rats, the effect of OXT-induced inhibition of antrum tone was significantly reduced by pretreatment with l-NAME (t₁₀ = 2.558, P = 0.0142) but not by the VIP antagonist (t₁₀ = 0.3105, P = 0.3813) or bethanechol (t₉ = 0.8846, P = 0.3994; Fig. 4). Conversely, the OXT-induced decrease of antrum motility was significantly reduced by pretreatment with both l-NAME (t₈ = 2.532, P = 0.0176) and the VIP antagonist (t₁₀ = 2.113, P = 0.0304) but not bethanechol (t₉ = 1.032, P = 0.3292; Fig. 4).

In CHe male rats, the effect of OXT-induced inhibition of antrum tone was significantly reduced by pretreatment with the VIP antagonist (t₁₀ = 2.663, P = 0.0119) but not by pretreatment with l-NAME (t₁₀ = 0.8002, P = 0.2211) or bethanechol, which instead increased the OXT response (t₉ = 2.950, P = 0.0162; Fig. 4). Conversely, the OXT-induced decrease of antrum motility was significantly reduced by pretreatment with the VIP antagonist (t₁₀ = 5.022, P = 0.0003) but not l-NAME (t₁₀ = 0.4857, P = 0.3188) or bethanechol (t₉ = 1.467, P = 0.0882; Fig. 4).

These data suggest that the OXT-induced relaxation of the antrum is mediated by the peripheral release of NO in naïve and CHo male rats. As in the corpus, however, the OXT-induced antrum relaxation in CHe male rats is due to the release of VIP. In contrast, the OXT-induced reduction of antral motility is determined by the peripheral release of VIP in all groups (naïve, Cho, and CHe) with NO release additionally participating in the reduction of antral motility in the CHo group only.

DISCUSSION

In the present study, we report that, in male rats, the vagal neurocircuits engaged by exogenous administration of OXT vary according to the type of chronic stress load. In particular, we have shown that 1) OXT modulates GABAergic currents impinging onto DMV neurons in stressed but not in naïve, rats; 2) pretreatment with CRF uncovers the in vitro OXT response in naïve rats but does not alter the OXT response in stressed rats; 3) OXT microinjection in the DVC induces corpus relaxation and decreased motility via peripheral release of NO in naïve rats, peripheral release of VIP in CHe rats, and peripheral release of both NO and VIP in CHo rats; and 4) OXT microinjection in the DVC induces antrum relaxation via peripheral NO release and reduced motility via both NO and VIP release in naïve rats, via VIP pathways only in CHe rats, and via both NO and VIP release in CHo rats.

Taken together, our data suggest that chronic stress induces plasticity in the brainstem vagal neurocircuits that modulate gastric tone and motility.

The peripheral responses to microinjection of OXT observed in CHo rats suggest a response intermediate between that observed in naïve versus CHe rats, perhaps due to an incomplete recovery from the stress load. Indeed, Although the OXT-induced gastroinhibition is similar across all groups, the difference appears only in the peripheral pathways engaged. In fact, adaptation to stress involves the activation of an otherwise masked VIP pathway in addition to the already engaged nitrergic pathway, yet the final gastroinhibitory response is similar. This observation suggests that stress susceptibility may involve a reduction in either the contribution or efficiency of the nitrergic pathway, and the activation of a VIPergic pathway. Conversely, stress, or the lack of resilience to stress, results in a switch to a VIPergic only pathway, whereas adaptation to stress involves recovery of the original nitrergic pathway. These observations appear to imply that vagal postganglionic NO and VIP pathways are not only engaged in different pathophysiological conditions, but may also have different contributions to gastric motility and emptying.

The functions of the upper GI tract, including gastric tone and motility, are modulated by the pacemaker activity of vagal preganglionic DMV neurons that regulate the release of inhibitory nonadrenergic noncholinergic neurotransmitters, mainly NO and VIP, or the release of excitatory cholinergic neurotransmission by myenteric neurons (21). The spontaneous activity of DMV neurons is shaped by tonic GABAergic inputs originating largely from NTS neurons (21). The GABAergic inputs to DMV neurons undergo a significant degree of plasticity, and many neuroactive substances, pharmacological mediators, as well as pathophysiological conditions such as acute stress or surgical vagal deafferentation can modulate this synapse (21). Our previous studies have shown that OXT does not appear to modulate the GABAergic inputs between NTS and DMV in naïve, nonstressed rats. Pretreatment with the prototypical stress hormone CRF or blockade of vagal afferent fiber activity uncovers the ability of OXT to modulate GABAergic currents (29, 31). We have shown previously that such uncovering of the OXT response is associated with an elevation in the levels of cAMP within the GABAergic synapse (29, 31), which induces the trafficking of OXT, as well as other, internalized receptors (31, 39–41). Here we confirm that perfusion with OXT does not modulate mIPSCs frequency in DMV neurons from naïve rats unless pretreated with CRF (31) and expand our previous observation by reporting that OXT decreases mIPSCs frequency in the significant majority of DMV neurons from chronically stressed rats. In these stressed rats, OXT decreases GABAergic currents per se, and pretreatment with CRF does not increase this inhibition, suggesting that the stress response may have induced long-lasting changes in the vagal GABAergic synapse.

In the present manuscript, we also confirmed that, in naïve male rats, DVC application of OXT induces gastric relaxation via peripheral release of NO (29). In naïve rats, following pretreatment with CRF (as a means to pharmacologically mimic acute stress) OXT induces corpus relaxation via activation of different vagal efferent pathways, specifically those that involve the postganglionic release of VIP (31). This observation is mimicked in the response observed in CHe rats, suggesting that stress switches the inhibitory pathway from NO only to VIP only. In CHo rats, however, our observations suggest a re-emergence of this NO pathway. Although the time frame of the shift of the activation of the different neurocircuits is, at the moment, unclear, our previous studies showed that trafficking of internalized receptors occurs within a few minutes of acute stress (or perfusion with neuroactive substances that increase cAMP levels within the GABAergic synapse) and unless there is a constant stimulus that keeps these cAMP levels elevated, receptors are reinternalized after ∼1 h (reviewed in Ref. 21).

Previous studies by Takahashi’s group reported that CHe increased the hypothalamic levels of CRF significantly (11). This observation may explain the data reported herein, in which we show that the 1) in vitro OXT effects in CHe male rats were not exacerbated by exogenous perfusion with CRF, and 2) the in vivo OXT effects were no longer antagonized by pretreatment of l-NAME but rather were attenuated by pretreatment with the VIP antagonist, indicating a rearrangement of the brainstem vagal neurocircuits, which then results in the engagement of different vagal pathways impinging on myenteric neurons. Further studies are necessary to confirm whether this effect is associated with CHe-dependent elevation in brainstem CRF levels, although, based on the data presented herein, we propose the following interpretation: in naïve conditions, OXT excites DMV neurons directly (42) but does not inhibit GABAergic inputs to DMV neurons. The DMV neurons excited by OXT directly are involved in the peripheral release of NO, which induces a gastroinhibitory response. Meanwhile, in these naïve conditions, GABAergic synaptic inputs provide a robust and selective inhibition of the DMV neurons that modulate the peripheral release of VIP from myenteric neurons, hence administration of the VIP antagonist does not alter the OXT-mediated gastroinhibition. Conversely, in CHe rats, the lack of adaptation to stress increases the hypothalamic CRF inputs to the brainstem which, in turn, allows OXT-mediated presynaptic actions to inhibit GABA release. This decrease in GABA release is likely to relieve the inhibition that the DMV exerts on the efferent VIPergic pathway.

The in vivo responses of CHo rats to OXT microinjection, in contrast, could be interpreted as a response intermediate between that of naïve and CHe rats. CHo is known to increase the density of OXT positive fibers in the brainstem vagal complex and increase OXT expression in the PVN (20), which ultimately decreases CRF expression and release. Thus, the stress-induced gastric dysregulation would be attenuated both via direct actions of OXT, via actions to reduce the effects of CRF while, at the same time, CRF also uncovered further presynaptic actions of OXT, which drive efferent nitrergic pathways. It is also possible that, because CHo is associated with upregulation of OXT inputs to the DVC that the direct excitation of DMV neurons is more prominent, decreasing the importance of inhibition of GABAergic inputs.

In fact, the percentage of DMV neurons, in which OXT inhibits mIPSC frequency, is similar in CHo and CHe rats, and the inhibition in corpus tone in response to OXT microinjection is attenuated significantly by pretreatment with both l-NAME and the VIP antagonist. The combined involvement of both nitrergic and VIPergic pathways suggests a partial recovery from neuroplasticity that occurs in response to CHe and may be a strategy by which male rats restore appropriate gastric motor functions following stress adaptation. Alternatively, the continued involvement of the VIPergic pathway may represent the remnants of neurocircuitry alterations in response to prolonged periods of stress that have not yet recovered to baseline conditions.

Given that the incidence of FGIDs is more prominent in females, a shortcoming of the present study is the use of only male rats. Our previous work (38) has shown that gastric tone and motility of female rats is influenced heavily by the circulating levels of endogenous estrogen (43), and stress is known to interact with estrogen signaling and function to exacerbate FGID symptoms (8). Understanding the mechanistic basis of these sex and stress-related interactions at the level of vagal neurocircuits controlling gastric functions will be important subjects for future studies.

In summary, we have shown that in male rats, chronic stress uncovered the ability of OXT to modulate GABAergic neurotransmission and shift the postganglionic pathways in response to brainstem OXT application. Although both CHo and CHe seem to have the same ability to uncover the OXT responsiveness of GABAergic currents, it appears to occur via engaging different vagal neurocircuits. A corollary of the main discoveries reported herein is that the responses to OXT appear to provide further evidence that distinct brainstem vagal neurocircuits control the diverse portions of the GI tract, in this particular case the gastric corpus and antrum, as put forward in Ref. 44 and reviewed in Ref. 21. Despite the overall response being gastroinhibition, clearly, the pathways engaged have some differential effect at the level of the stomach, and that is why engagement of the VIP pathway (in the absence of the NO pathway, i.e., CHe rats) still causes a delay in gastric emptying. Reintroduction of the NO pathway (i.e., in CHo rats) appears to be important in restoring appropriate gastric emptying, despite the continued presence of the VIP output.

DATA AVAILABILITY

The data that support the findings of this study are available on request from the corresponding author.

GRANTS

This study was supported by National Institute of Health Grant DK 120170 (to K. N. Browining) and, in part, by a grant with the Pennsylvania Department of Health (to R. A. Travagli) using Tobacco CURE Funds (The Department specifically disclaims responsibility for any analyses, interpretations, or conclusions).

DISCLOSURES

No competing interests by any of the authors. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; all persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

AUTHOR CONTRIBUTIONS

R.A.T. conceived and designed research; Y.J. and J.E.Z. performed experiments; Y.J., K.N.B., and R.A.T. analyzed data; Y.J., K.N.B., and R.A.T. interpreted results of experiments; Y.J., K.N.B., and R.A.T. prepared figures; Y.J., K.N.B., and R.A.T. drafted manuscript; Y.J., J.E.Z., K.N.B., and R.A.T. edited and revised manuscript; Y.J., J.E.Z., K.N.B., and R.A.T. approved final version of manuscript.