Vagally mediated effects of brain stem dopamine on gastric tone and phasic contractions of the rat

Dopamine administration in the brain stem decreases gastric tone and phasic contractions. The gastric effects of dopamine are mediated via dopamine 2 receptors on neurons of the dorsal motor nucleus of the vagus. The inhibitory effects of dopamine are mediated via inhibition of the postganglionic cholinergic pathway.

Abstract

Dopamine (DA)-containing fibers and neurons are embedded within the brain stem dorsal vagal complex (DVC); we have shown previously that DA modulates the membrane properties of neurons of the dorsal motor nucleus of the vagus (DMV) via DA1 and DA2 receptors. The vagally dependent modulation of gastric tone and phasic contractions, i.e., motility, by DA, however, has not been characterized. With the use of microinjections of DA in the DVC while recording gastric tone and motility, the aims of the present study were 1) assess the gastric effects of brain stem DA application, 2) identify the DA receptor subtype, and, 3) identify the postganglionic pathway(s) activated. Dopamine microinjection in the DVC decreased gastric tone and motility in both corpus and antrum in 29 of 34 rats, and the effects were abolished by ipsilateral vagotomy and fourth ventricular treatment with the selective DA2 receptor antagonist L741,626 but not by application of the selective DA1 receptor antagonist SCH 23390. Systemic administration of the cholinergic antagonist atropine attenuated the inhibition of corpus and antrum tone in response to DA microinjection in the DVC. Conversely, systemic administration of the nitric oxide synthase inhibitor nitro-l-arginine methyl ester did not alter the DA-induced decrease in gastric tone and motility. Our data provide evidence of a dopaminergic modulation of a brain stem vagal neurocircuit that controls gastric tone and motility.

NEW & NOTEWORTHY Dopamine administration in the brain stem decreases gastric tone and phasic contractions. The gastric effects of dopamine are mediated via dopamine 2 receptors on neurons of the dorsal motor nucleus of the vagus. The inhibitory effects of dopamine are mediated via inhibition of the postganglionic cholinergic pathway.

functions of the upper gastrointestinal (GI) tract are influenced and modulated by vagovagal neurocircuits originating within brain stem nuclei located in the dorsal vagal complex [DVC; i.e., nucleus tractus solitarius (NTS), dorsal motor nucleus of the vagus (DMV), and area postrema); vagal motor fibers projecting to the GI tract from the lower third of the esophagus to the splenic flexure derive from the preganglionic neurons of the DMV (36). A vast array of synaptic inputs impinge upon DVC neurons and contribute to the fine tuning of selected circuits devoted to specific functions (8, 36), including inputs originating in the adjacent catecholaminergic A2 area (3, 15, 19, 26). The majority of neurons in the A2 area are dopamine-β-hydroxylase (DβH) but not phenylethanolamine N-methyltransferase immunoreactive (-IR), thus making them noradrenergic rather than dopaminergic or adrenergic (16, 19, 20, 39, 42, 46). A small subgroup of NTS neurons in the A2 area, however, are exclusively tyrosine hydroxylase (TH)-IR (19, 46); hence, they are dopaminergic. These A2 area neurons are the likely source of dopaminergic inputs to DMV neurons (19, 46); however, other areas such as the locus coeruleus, the pontine tegmentum, the ventrolateral medulla, the substantia nigra, and/or the hypothalamus (1, 2, 19, 20, 27, 33, 41) may also provide catecholaminergic, possibly dopaminergic, inputs to DMV neurons.

Using immunohistochemical techniques, Kitahama et al. (23) described a dense network of varicose dopaminergic fibers in the dorsal medullary area, including the DMV, although the source of such inputs were not defined. In association with this robust dopaminergic fiber network, several reports have described a wide distribution of DA2 receptors in the DMV (9, 31, 44); conversely, DA1 receptor immunoreactivity in the DMV is less prominent (17, 31). These observations were supported by electrophysiological data that reported a larger percentage (i.e., >40%) of DMV neurons responding to DA with a DA2-mediated inhibition and a smaller subgroup (i.e., ~25%) that were excited by DA via a DA1-mediated action (46). Thus the overwhelming majority of DMV neurons respond to exogenous application of DA; however these neurons appear to express either DA1 or DA2 receptors but not both (46). Furthermore, despite the likely overlapping sources of dopaminergic and noradrenergic fibers, the response of vagal motoneurons to DA or to norepinephrine was not always similar. Indeed, DMV neurons depolarized by DA were more likely to be inhibited by norepinephrine, whereas neurons unresponsive to DA were more likely hyperpolarized by norepinphrine (46), suggesting different physiological roles for these catecholamines.

Overall there is robust evidence that suggests DA plays a role in the regulation of GI-related brain stem vagal circuits; however, little evidence is available about the effects of brain stem DA to modulate gastric tone and phasic contractions, henceforth referred to as motility.

The aims of the present study were, therefore, to 1) assess the effects of DA application on gastric tone and motility, 2) identify the DA receptor subtype in the DMV responsible for gastric modulation, and 3) identify the postganglionic myenteric pathway(s) activated by brain stem administration of DA.

Preliminary accounts of the this study were presented at the 2015 Digestive Disease Week (35).

MATERIALS AND METHODS

Male Sprague-Dawley rats were housed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited Animal Care Facility maintained at 24°C on a 12-h:12-h light-dark cycle with food and water provided ad libitum.

All procedures were conducted in accordance with the National Institutes for Health guidelines, with the approval of the Penn State University College of Medicine Institutional Animal Care and Use Committee and according to journal policies and regulations on animal experimentation.

In vivo recordings of corpus and antrum tone and motility were conducted in 34 anaesthetized rats; neither microinjections of PBS in the DVC (60 nl) nor administration of PBS on the surface of the fourth ventricle (2 µl) had any significant effect (data not shown).

Gastric studies.

Gastric tone and motility recordings were performed as described previously (6, 34). Briefly, male Sprague-Dawley rats weighing 200–350 g were fasted overnight (water ad libitum) before being anaesthetized deeply with thiobutabarbital (Inactin; Sigma, St Louis, MO; 100–150 mg/kg ip). Rats were intubated with a tracheal catheter and, following a midline laparotomy, two custom-made 6 × 8 mm strain gauges (AT Engineering, Hershey, PA) were sutured to the serosal surface of the anterior gastric corpus and antrum in alignment with the circular smooth muscle; the abdominal incision and the wound margins were sutured with the leads exteriorized. The signals were acquired with a Wheatstone bridge, filtered (low-pass filter cutoff = 0.5 Hz; AT Engineering), amplified (EXP CLSG-2; QuantaMetrics, Newton, PA), and recorded on a computer using Axotape 10 software (Molecular Devices, Sunnydale, CA). Animals were then placed in a stereotaxic frame, and rectal temperature was monitored and maintained at 37 ± 1°C with a heating pad (TCAT 2LV; Physitemp Instruments, Clifton, NJ).

The fourth ventricle was exposed via midline incision and the meningeal membranes were dissected before the exposed brain stem was covered with prewarmed saline during a 60- to 90-min period of stabilization.

The gastric tone and motility traces were monitored throughout the duration of the experiment, and baseline measures were extracted from 5 min before and 15–20 min after drug application; the drug-induced effects on tone were calculated through average value of the calibration measures. Although the basal gastric tone was not preset to a fixed value, the strain gauge transducer was sewn to provide a baseline tension of ~0.5 g; the data reported are thus absolute values of tone displacement over baseline of the 30- to 60-s period centered around the peak effect. Note that the recorded magnitude of the drug-induced effects on tone and motility can be influenced by such factors as the size of the animal and variations in the strain gauge placement. Because these variations may lead to slight differences in responses between individual animals, each animal served as its own control, and motility data were measured as percent changes over baseline. The drug-induced effects on gastric motility were measured in percentage against the normalized value of gastric motility before microinjection (baseline = 100%).

Gastric motility was calculated using the following formula, as described previously (6): motility index percentage = [(n1 × 1) + (n2 × 2) + (n3 × 4) + (n4 × 8)/t] × 100, where n = number of peaks in a particular force range and t = interval time in which the gastric motility is measured (n1 = 20–59 mg, n2 = 60–100 mg, n3 = 101–200 mg, and n4 ≥ 201 mg).

When the response to DA was measured after either atropine or nitro-l-arginine methyl ester (l-NAME) pretreatment, the effect of DA on gastric motility was calculated as the variation in arbitrary units of the motility index from either baseline or from atropine/l-NAME alone.

Experiment design/drugs administration.

A micropipette (25- to 30-μm-tip diameter) was lowered into the left DVC (in mm: +0.4–0.6 rostrocaudal from calamus scriptorius, 0.2–0.4 mediolateral from midline, and −0.5–0.65 dorsoventral from the brain stem surface). Drugs were microinjected in 60-nl volumes using a picospritzer (Toohey, Fairfield, NJ) or applied to the surface of the fourth ventricle (2 µl). All drugs were dissolved in isotonic PBS (in mM: 115 NaCl, 75 Na₂HPO₄, and 7.5 KH₂PO₄, pH 7.4).

A first group of rats was microinjected with DA into the DVC in a dose-dependent manner (0.3–3 nmol/60 nl). A second group of rats underwent vagotomy as described previously (6). Briefly, after the subdiaphragmatic posterior vagus was sectioned, DA (1 nmol/60 nl) was microinjected in the DVC and, 30 min later, the left cervical vagus was severed, disconnecting the remaining vagal outflow to the stomach. DA was microinjected again ~45 min after the cervical vagotomy.

In the third group of rats, 30–45 min after DA microinjection (1 nmol/60 nl) into the DMV, 2 μl of a PBS solution containing either the DA1 or the DA2 dopamine receptor antagonists SCH 23390 and L-741,626 (both at 45 nmol) were applied to the surface of the fourth ventricle, at the level of obex, followed 2–5 min later by a second DA microinjection (1 nmol/60 nl).

The last group of rats, 30–45 min after the first DA microinjection (1 nmol/60 nl), received intravenous drug treatments: 1) atropine methyl nitrate (100 μg/kg iv; nonselective muscarinic antagonist), or 2) l-NAME (10 mg/kg iv, nitric oxide synthase inhibitor) followed 2–5 min later by a second DA microinjection (1 nmol/60 nl) in the DVC.

At the conclusion of the experiment, rats were euthanized with a bilateral pneumothorax and perfused transcardially with 200 ml of saline followed by 200 ml of 4% paraformaldehyde in PBS. The brain stem was removed and postfixed in 4% paraformaldehyde and 20% sucrose for 24–48 h at 4°C and then transferred in a solution containing PBS and 20% sucrose for at least 1 day. The brain stem was then frozen, sliced in 50-μm-thick coronal sections throughout the rostrocaudal extent of the DVC, and counterstained with cresyl violet. Injection sites were identified on a Nikon E400 microscope.

Statistical analysis.

Data were evaluated by comparing the change in response between pre- and posttreatment values within each group using one-way ANOVA followed by post hoc Tukey’s multiple comparison test or paired t-test (Graph Pad Prism; Graph Pad Software, La Jolla, CA) and are reported as means ± SE. In all instances, significance was set at P < 0.05.

RESULTS

Microinjection of dopamine in the DVC decreases gastric motility in a dose-dependent manner.

Microinjections of DA (0.3–3 nmol) in the left DVC decreased gastric tone and motility in 29 of 34 rats (i.e., 85.3%) and increased gastric tone and motility in 5 of 34 rats (i.e., 14.7%). Since it appeared that there were no significant topographic differences in the microinjection site location in rats that responded with inhibition or excitation of gastric tone and motility (Fig. 1), we focused our study on the investigation of the inhibitory effects.

Fig. 1.

Microinjection of dopamine in dorsal vagal complex (DVC) decreases gastric tone and motility. A, left: representative micrograph showing the site of dopamine (DA) microinjection (arrow) in the intermediate DVC. A, right: schematic map of the DMV showing microinjection localization; note that for clarity not all the injection sites have been included. B: representative traces from gastric corpus showing the decrease in tone and motility following DA microinjection (arrows). Oblique bars indicate a 5- to 10-min break in the recording. C: graphic summary showing the decrease in gastric tone and motility in corpus (white bars) and antrum (black bars). Note that the decrease in gastric tone was already maximal at the lower DA dose. N = 8 for each dose. *P < 0.05 vs DA at 0.3 nmol. AP, area postrema; DMV, dorsal motor nucleus of the vagus; CC, central canal; XII, hypoglossal nucleus. D, left: representative traces from gastric corpus (top) and antrum (bottom) showing the duration of the DA-mediated inhibition. Arrows indicate the time of DA (3 nmol/60 nl) microinjection. D, right: graphic summary showing the duration of the decrease in gastric tone following DA microinjection in the DMV.

Microinjections of DA in the left DVC decreased antrum and corpus motility in a dose-dependent manner; both the frequency as well as the amplitude of the phasic contractions were decreased (0.3–3 nmol; n = 8 for each dose); however, the DA-induced inhibition of gastric tone was not dose dependent and appeared maximal even at the lowest dose tested (Fig. 1).

The inhibitory effects of dopamine microinjection lasted up to 25 min; however, there were no significant differences in the duration of the inhibition among the different DA doses or between corpus and antrum responses (Fig. 1).

Dopamine modulation of DMV motoneurons is vagally dependent.

Five rats received a posterior subdiaphragmatic vagotomy, thus severing the vagal motor pathway originating in the right DMV (36). In these animals, microinjection of 1 nmol/60 nl of DA in the left DVC decreased gastric tone (−186 ± 36 and −146 ± 11.1 mg in corpus and antrum, respectively) and motility (−32 ± 15.1 and −40 ± 2.5% in corpus and antrum, respectively) in a manner that is comparable to the decrease observed in vagally-intact rats (tone: −158 ± 13.5 and −199 ± 17.2 mg, in corpus and antrum, respectively; motility: −40 ± 8.4 and −48 ± 6.1% in corpus and antrum, respectively; P > 0.05 for all). The effects of DA microinjection in the left DVC of these rats were abolished completely after the left cervical trunk of the vagus was severed, thus achieving a complete vagotomy (tone: −20 ± 20 and 0 ± 0 mg in corpus and antrum, respectively; motility: −5 ± 31 and 9 ± 5.8% in corpus and antrum, respectively; P < 0.05 for all vs DA in rats with posterior subdiaphragmatic vagotomy; Fig. 2).

Fig. 2.

Dopaminergic inhibition of gastric tone and motility is vagally dependent. A: representative recording from the anterior antrum showing that microinjection of DA (arrow, 1 nmol/60 nl) in the left DVC decreased tone and motility (top trace). In the same animal, following complete vagotomy, a second DA microinjection (arrow) did not affect gastric tone or motility (bottom trace). Oblique bars indicate a 5- to 10-min break in the recording. B: graphic summary showing the decrease in corpus and antrum tone (top) and motility (bottom) pre- and postvagotomy (white and gray bars, respectively; n = 5 for all; *P < 0.05 vs. first DA microinjection); vgtx = after vagotomy.

These data indicate that the effects of DA are mediated by an effect on DMV neurons. Henceforth, the site of microinjection will be referred to as DMV rather than DVC.

The dopamine-induced inhibition of gastric tone and motility is mediated by DA2 receptor activation in the DMV.

To investigate the dopamine receptor responsible for the decreased gastric tone and motility, we conducted a series of experiments in which DA microinjections (1 nmol/60 nl) were performed before and after application of the DA1 or DA2 receptor antagonists, SCH 23390 and L741,626 (45 nmol/2 µl), respectively, to the floor of the fourth ventricle (n = 5–11). Application of either L741,626 or SCH 23390 alone did not alter baseline gastric tone or motility (data not shown, but see traces in Fig. 3).

Fig. 3.

The DA-induced decrease in gastric tone and motility is mediated via DA2-like receptors. A: representative recording showing the decrease in tone and motility (left traces) in the anterior antrum of a rat following DA microinjection in the left DVC. Upon recovery, the DA1 antagonist SCH 23390 (n = 7–11) or the DA2 antagonist L741,626 (n = 5–11) were applied to the floor of the 4th ventricle (both at 45 nmol/2 μl). Application of L741,626 attenuated significantly the inhibitory effects of subsequent microinjections of DA (top trace), whereas SCH 23390 was without effect (bottom trace). Oblique bars indicate a ~45 (1st set) and a 5–10 (2nd set) minutes break in the recording. B: graphic summary showing that pretreatment with the DA2 antagonist L741,626, but not the DA1 antagonist SCH 23390, decreased the inhibitory effects of DA microinjection on corpus and antrum tone and motility. White bars: DA 1 nmol/60 nl; light gray bars: DA + SCH 23390; n = 7–11; black bars: DA + L741,626; n = 5–11. *P < 0.05 vs. DA alone.

DMV microinjection of DA decreased gastric tone and motility in the corpus and antrum. Following return to baseline values and a minimum recovery period of 30 min, the DA2 antagonist L741,626 was applied to the floor of the fourth ventricle 2–5 min before a second DMV microinjection of DA; the DA2 antagonist attenuated the DA-induced decrease in corpus and antrum tone (from −181 ± 24 to −109 ± 16 and from 156 ± 22.2 to −102 ± 35.3 mg in corpus, n = 7, and antrum, n = 6, respectively; P < 0.05 vs DA alone) and motility (from −59 ± 8 to −36 ± 10.6 and from −47 ± 12 to −22 ± 9.5% of baseline in corpus, n = 11, and antrum, n = 5, respectively; P < 0.05 vs DA alone). Conversely, administration of the DA1 antagonist SCH 23390 did not antagonize the DA-induced inhibition of gastric tone and motility (n = 7–11; P > 0.05 for all). Representative traces and summary data are shown in Fig. 3.

These data indicate that the DA-induced inhibition of gastric tone and motility are mediated by activation of DA2-like receptors in the DMV.

The dopamine-induced inhibition of gastric tone and motility is mediated by withdrawal of cholinergic tone.

To investigate the vagal postganglionic pathway affected by DMV microinjection of DA, we conducted a series of experiments in which DA microinjections (1 nmol/60 nl) were performed before and after application of either the muscarinic receptor antagonist atropine (100 μg/kg iv) or the nitric oxide synthase inhibitor l-NAME (10 mg/kg iv).

DMV microinjection of DA decreased gastric tone (−173 ± 16.3 and −245 ± 44 mg in corpus, n = 4, and antrum, n = 4, respectively) and motility [from 100 ± 38.5 to 24 ± 6.5 arbitrary units (AU) and from 167 ± 28.5 to 42 ± 7.3 AU in corpus, n = 4, and antrum, n = 8, respectively]. Following return to baseline values and a minimum recovery period of 30 min, the muscarinic antagonist atropine was administered and, 2–5 min later, a second DVC microinjection of DA induced a significantly attenuated inhibition of corpus and antrum tone (−66 ± 32.8 and −60 ± 20 mg in corpus and antrum, respectively; P < 0.05 vs DA alone; n = 4) and motility (from 50 ± 6.3 to 28 ± 5.1 AU and from 54 ± 9.3 to 33 ± 6.8 AU in corpus and antrum, respectively; P < 0.05 vs DA alone; n = 4–8). Representative traces and summary data are shown in Fig. 4.

Fig. 4.

DA microinjections reduce gastric tone and motility through inhibition of the vagal cholinergic pathway. A: representative traces from the anterior antrum showing that DA (1 nmol/60 nl) microinjection decreases tone and motility (top trace). Following a ~45-min recovery period, application of atropine (100 µg/kg iv) reduced significantly the inhibitory effects of a 2nd DA microinjection (bottom trace). Oblique bars indicate a 5- to 10-min break in the recording. B: graphic summary showing the effect of administration of atropine (left) and nitro-l-arginine methyl ester (l-NAME; right; 10 mg/kg iv) on gastric tone following DA microinjection in the DVC. The DA-induced decrease in gastric tone was reduced significantly by atropine, whereas intravenous administration of l-NAME did not prevent the inhibitory effects of DA. White bars: DA; black: DA + atropine n = 4; gray bars: DA + l-NAME; n = 3. *P < 0.05 vs. baseline. C: scatterplot showing the effects of atropine (left) and l-NAME (right) on gastric motility following DA microinjection in the DVC. The reduction in motility observed after microinjection of DA is prevented by intravenous administration of atropine (n = 4–8), whereas l-NAME (n = 3–4) had no effect. *P < 0.05 vs baseline.

Conversely, administration of the nitric oxide synthase inhibitor l-NAME did not antagonize the DA-induced inhibition of gastric tone and motility (n = 3–4; P > 0.05 for all). Summary data are shown in Fig. 4.

These data indicate that the DA-mediated gastric inhibition is the result of tonic cholinergic withdrawal.

DISCUSSION

In the present study we report that application of exogenous DA on the DVC decreases gastric tone and motility via inhibition of vagal pathways. The inhibitory effects of DA microinjection in the DVC are mediated via activation of DA2 receptors in the DMV, which results in the inhibition of the cholinergic postganglionic pathway.

Our evidence is the following: 1) microinjection of DA in the DVC decreased gastric motility in the vast majority (i.e., 85%) of the rats; 2) the DA-induced decrease in gastric motility was dose dependent; however the decrease in gastric tone was not and appeared to be maximal at the tested doses (0.3–3 nmol); furthermore, the DA-induced decrease in both gastric tone and motility was 3) vagally mediated, since it was prevented by complete vagotomy, and 4) mediated by activation of DA2 receptors in the DMV, since it was attenuated significantly by pretreatment with the DA2 selective antagonist L741,676, but not by the DA1 antagonist SCH 23390; and 5) DA2 receptor-mediated inhibition of DMV neurons resulted in the withdrawal of the tonically active cholinergic postganglionic pathway, since the gastroinhibition was attenuated significantly by pretreatment with a submaximal dose of the muscarinic selective antagonist atropine but not the nitric oxide synthase inhibitor l-NAME.

Our data thus support a role for dopaminergic neurotransmission in the brain stem vagal circuits that control gastric tone and motility.

As mentioned previously, the dopaminergic input to DMV neurons that modulate gastric tone and motility most likely originates from TH-positive neurons of the adjacent A2 area (19, 46), although fibers originating from areas such as A6, A5, A2, A1/C1, A9, and/or the A13 (1, 2, 19, 20, 27, 33, 41) cannot be excluded. While the large majority of the catecholaminergic neurons of the A2 area are also DβH-IR, suggesting that these neurons are noradrenergic or adrenergic, ~10% of the A2 neurons contain TH-IR only, indicating that this subpopulation is exclusively dopaminergic (46). Furthermore, a dense network of dopaminergic-IR fibers innervates both the NTS as well as the DMV (23), suggesting that dopamine may play a prominent role in the modulation of vagal activity.

Investigation of the role played by dopaminergic innervation of vagal neurocircuits regulating gastrointestinal functions, however, is limited.

Brain stem dopamine has been hypothesized to play a role in the control of ingestive as well as reflexive behavior. Given the close proximity of brain stem dopamine receptors with N-methyl-d-aspartate and cholecystokinin receptors (31), Södersten and colleagues (4) put forward the hypothesis that cholecystokinin interacts with DA and glutamate to decrease food intake. This inhibitory action of DA was proposed to occur via activation of both DA1 and DA2 receptors (4). A further role of DA in ingestive behavior was proposed by the same group, which showed that the ingestive responses of sucrose by decerebrate rats were reduced by administration of the nonselective dopaminergic agonist apomorphine (21), thus suggesting that dopaminergic transmission in the brain stem is sufficient for this particular ingestive reflex.

A dopamine-mediated modulation of brain stem neurocircuits was also proposed in relation to nausea and vomiting, esophageal motility, and small intestine secretions. In fact, dopamine was shown to induce the gastric relaxation and decrease in gastric pressure that precedes nausea and vomiting in dogs, possibly via an effect on the chemoreceptive trigger zone (40). Likewise, several groups have reported that mechanical distention of the esophagus or the stomach activates c-FOS in TH-IR, including dopaminergic, brain stem neurons of the A2 area (15, 32, 42). Furthermore, application of the centrally acting DA1/DA2 antagonist haloperidol increased duodenal secretion of bicarbonate, possibly via brain stem vagal pathways (13).

Brain stem dopaminergic neurocircuits may also play a role in pathological conditions. For example, in the 6-OHDA animal model of Parkinson’s disease, concomitant with the decreased number of dopaminergic neurons in the substantia nigra pars compacta, there is an increase in number of catecholaminergic neurons in the A2 area (34, 45), suggesting a potential upregulation of brain stem catecholaminergic neurotransmission from A2 to vagal motoneurons. Interestingly, neurons of the A2 area are involved in swallowing reflexes (5, 11, 18), these observations may provide a mechanistic explanation to the sialorrhea and dysphagia that affect Parkinsonian patients (10, 12, 14, 29). Indeed, microinjections of either DA or the nonselective DA1-DA2 receptor agonist apomorphine in the NTS inhibits the swallowing reflex induced by stimulation of the superior laryngeal nerves (22).

Vagal efferent fibers originating in the preganglionic motoneurons of the DMV modulate gastric tone and motility via tonic projections to excitatory cholinergic, or projections to inhibitory nonadrenergic-noncholinergic (NANC) postganglionic myenteric neurons; hence, gastric relaxation results from vagal modulatory inputs that either inhibit the cholinergic or excite the NANC pathway (36). The loss of effects of DA on gastric tone and motility following complete (i.e., bilateral) vagotomy suggest strongly that the effects of DA in the DVC are vagally-mediated. Furthermore, since the inhibition of gastric tone and motility observed upon DA microinjection in vagally intact animals is similar to that obtained in hemivagotomized animals, these data suggest that the DA effects are mediated by direct activation of ipsilateral-projecting DMV neurons, rather than a combined effect of DA on both DMV neurons and contralateral-projecting NTS neurons.

In the present study, we focused on the DA2-mediated decrease in gastric tone and motility, since the gastric effects were attenuated by pretreatment with the DA2 receptor antagonist L-741,626. Our results confirm previous studies of the inhibitory actions of DA being mediated by DA2 receptor activation (28), and our previous data showing a DA2 receptor-mediated hyperpolarization of DMV neurons suggest that inhibition of these neurons is the likely brain stem target that decreases gastric tone and motility. Indeed, a dense presence of DA2 receptors in the DVC has been reported (31); furthermore, electrophysiological studies have shown that the glutamatergic inputs onto cardiorespiratory vagal neurons are inhibited significantly by DA2 receptor activation (24) and microinjections of DA2 agonists in the nucleus tractus solitarius induced a pressure response (43).

Given the fact that DA2-mediated effects are inhibitory to DMV neurons (46), the only logical vagal mechanism that would ultimately induce gastric relaxation is via inhibition of the cholinergic postganglionic pathway. In fact, DMV neurons are pacemakers (37) and provide a tonic vagal input to excitatory cholinergic and inhibitory NANC postganglionic myenteric neurons that ultimately shapes gastric tone and motility (36). DA2-mediated inhibition of DMV reduces vagal output to myenteric neurons; hence, a DA2-mediated inhibition of vagal projections impinging upon cholinergic myenteric neurons would result in a decrease in gastric tone and motility. Conversely, a DA2-mediated inhibition of vagal projections impinging upon NANC myenteric neurons would result in a limited increase in gastric tone and motility, given the weak influence that tonic NANC pathways have on gastric tone and motility (38). Since pretreatment with the selective acetylcholine muscarinic receptor antagonist atropine, but not the nitric oxide synthase inhibitor l-NAME, attenuated the DA2-mediated gastric inhibition, our data indicate that the gastroinhibitory effects of DA microinjection in the DVC are mediated via inhibition of the excitatory cholinergic pathway in a manner that involves activation of DA2 receptors located on DMV neurons.

Using an electrophysiological approach, we also showed that perfusion with DA1 or DA2 antagonists did not alter the membrane potential of DMV neurons (46). Our present results confirmed that application of either DA1 or DA2 antagonists on the floor of the fourth ventricle per se did not modulate gastric tone or motility significantly, indicating either that the DA released under our experimental conditions is not sufficient to induce measurable alterations of gastric tone or motility or that there are no tonic dopaminergic inputs impinging on gastric-projecting DMV neurons. The first explanation, although, appears to be more likely since preliminary data indicate the presence of a tonic dopaminergic input to DMV neurons arising from neurons of the A9 area (1).

Finally, although the gastric excitatory response to dopamine was limited to ~15% of the animals tested may argue against a prominent role of the excitatory inputs, one has to keep in mind that dopamine has an affinity toward DA2 receptors that is one order of magnitude higher than the affinity toward DA1 receptors (25). Furthermore, one has to consider that brain stem vagal neurocircuits are likely organized along lines of specificity (7, 30), so that sets of neurons, although adjacent, may control different and diverse physiological responses.

In conclusion, our study reports a potentially important role of brain stem vagal DA2 receptors in the modulation of gastric tone and motility; these dopaminergic inputs are likely relevant in the fine tuning of gastrointestinal vagovagal reflexes.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-55530 and DK-99350 and Michael J. Fox Foundation Grant for Parkinson’s Disease (to R. A. Travagli).

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

R.A.T. conceived and designed research; L.A., L.T., C.B., and R.A.T. performed experiments; L.A., L.T., C.B., and R.A.T. analyzed data; L.A., L.T., and R.A.T. interpreted results of experiments; L.A., C.B., and R.A.T. prepared figures; L.A., C.B., and R.A.T. drafted manuscript; L.A., L.T., and R.A.T. edited and revised manuscript; L.A., L.T., C.B., and R.A.T. approved final version of manuscript.