Introduction

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The DMV of the medulla oblongata is a major site of origin of vagal preganglionic fibers to the gastrointestinal tract. This nucleus, together with the nTS, is often considered as the DVC. Numerous studies have shown that various neurotransmitters microinjected into the DVC alter gastrointestinal (Krowicki and Hornby, 1995) and cardiovascular function (van Giersbergen et al., 1992). Other brainstem nuclei, such as the nROb (Hornby et al., 1990; Rogers et al., 1980), maintain direct anatomical connections with the DVC, and microinjection of agents into the nROb can either increase or decrease gastric motor function (Krowicki and Hornby, 1995; Tache et al., 1995). In addition, there is good physiological and anatomical evidence supporting the involvement of these nuclei in cardiovascular regulation. The caudal nROb controls sympathetic outflow to the cardiovascular system (Jansen et al., 1995; King and McCall, 1992; Coleman and Dampney, 1995) and the preganglionic cardiomotor neurons innervating the heart reside in the nAmb (Dampney, 1994), and to a lesser extent, in the DMV (Standish et al., 1995).

In the mammalian brain, VIP is a major regulatory peptide that fulfills many of the classical criteria for neurotransmission (Fahrenkrug, 1993). In the DVC, VIP-positive cell bodies and fibers (Palkovits et al., 1982; Roberts et al., 1980; Sims et al., 1980) and VIP binding sites are noted (Martin et al., 1987; Shaffer and Moody, 1986). In the raphe nuclei, VIP-immunoreactive fibers are present (Roberts et al., 1980; Batten, 1995). However, at the present time, no information on the role of VIP in the lower brainstem nuclei to control gastric motor or cardiovascular function is available, although intracerebroventricular administration of VIP in the rat was reported to increase blood pressure via increased sympathetic outflow (Endo et al., 1991).

We recently reported that microinjection of VIP into the nROb at a single dose of 10 pmol decreases gastric motor function in the rat (Krowicki et al.,1996d). In the present study we describe the gastric motor and MAP changes in response to microinjection of VIP at a complete range of doses into the DVC, nROb and nAmb.

	Materials and Methods

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Animals

Twenty-three male Sprague-Dawley rats (235-420 g), purchased from Charles River Laboratories (Wilmington, MA), were used in all experiments. They were maintained in a temperature-controlled environment and on a 12-h light/dark cycle with free access to food and water except the night preceding the experiment. At this time, the animals were deprived of food (18-20 h) but allowed free access to water.

General Methods

All procedures were performed on the animals with the approval of the LSUMC Institutional Animal Care and Use Committee. Experiments were done in rats initially anesthetized with ketamine and xylazine mixture (50 and 5 mg/kg i.m., respectively) and then (25 min later) with -chloralose (80 mg/kg) through an indwelling catheter placed in the left femoral vein. A separate catheter was placed in the left femoral artery and connected to a pressure transducer (Viggo-Spectramed, model P23XL, Oxnard, CA) and polygraph (model 7E, Grass Instrument Co., Quincy, MA) for direct measurement of blood pressure. Heart rate was monitored by a tachograph triggered by the arterial pressure pulse (model 7P4H, Grass Instrument Co., Quincy, MA). A tracheotomy was performed in all animals to ease respiration with the respiratory assist mode of a small animal respirator (Kent Scientific Corp., Litchfield, CT). An intraluminal latex balloon was used to record intragastric pressure and small strain gauges mounted on the surface of the stomach were used for continuous recording of pyloric circular smooth muscle and greater curvature longitudinal smooth muscle contractile activity (Krowicki and Hornby, 1993a). The animals were then placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) for controlled administration of drugs into the different areas of the lower brainstem. In some animals, bilateral vagotomy was performed at the midcervical level in the presence of full surgical anesthesia. The vagi were carefully separated from the left and right common carotid arteries and silk snares were loosely placed around them, then vagotomy was achieved by avulsion. Rectal temperature was maintained between 37.0 and 37.5°C.

Microinjection Technique

The dorsal surface of a medulla was exposed by a limited craniotomy for VIP microinjections into the DVC, nROb and nAmb. Seven-barreled glass micropipettes (20-30 µm total external tip diameter; Dagan Corp., Minneapolis, MN) were connected to a nitrogen-pressured, pneumatic pico-pump (model PV 830, World Precision Instruments, New Haven, CT). For experiments in which range of doses were used, one barrel was prefilled with vehicle (see below); four others with VIP (0.1, 1, 10 and 100 pmol/30 nl); and the sixth one with 1% pontamine sky blue. The micropipette was then lowered into the right DVC (0.5 mm rostral to the obex, 0.5 mm lateral from the midline and 0.4-0.5 mm down from the surface), the nROb (0.7 mm rostral to the obex, in the midline and 1.2-1.3 mm below the surface at the level of the obex) and the right nAmb (0.7 mm rostral to the obex, 1.5 mm lateral from the midline and 1.3 mm below the surface at the level of the obex), according to the atlas of Paxinos and Watson (1986). The micropipette was left in place until all microinjections into each medullary site were performed.

Histological Localization

After completion of the microinjections into the specific medullary site, 30 nl 1% pontamine sky blue was injected through the cannula, and the rats were perfused with saline followed by a solution of 4% paraformaldehyde. Sections of brainstem (40-50 µm) were stained with neutral red, and placement of the pipette tip in the DVC, nROb and nAmb was verified by examination of the histological sections. Only results from those rats in which histology documented adequate placement within the appropriate brainstem nucleus were included for final evaluation. Specifically, in one experiment the tip of the micropipette was localized outside the DVC, and in two other experiments it was located outside the nAmb.

Drugs

Vasoactive intestinal polypeptide (American Peptide Co., Sunnyvale, CA) was dissolved in 0.9% saline containing 0.1% bovine serum albumin radioimmunoassay grade (vehicle; Sigma Chemical Co., St. Louis, MO).

Data Analysis

Peak changes in intragastric pressure were expressed as differences between peak or nadir values of intragastric pressure after injections and the base line (mean intragastric pressure) calculated for a period of 2 min before injection. Additionally, the area of the response in intragastric pressure for each treatment was calculated with a microcomputer-based imaging system (Imaging Research Inc., Ontario, Canada). For this purpose the base lines were extended across the period of the response to the point at which intragastric pressure had returned to base line, and the area of the response was calculated as the area enclosed between the base line and the curve of the response. Areas of the response were considered as positive and negative values to give an estimate of the overall increase or decrease in intragastric pressure, respectively. Gastric smooth muscle contractility was quantified by MMI calculated within 2 min before and 10 min after administration of the solutions and expressed as a difference between post- and preinjection values of MMI, based on Ormsbee and Bass (1976), as reported previously (Krowicki and Hornby, 1993b). MAP was calculated as diastolic pressure + 1/3 of the pulse pressure.

Specific Experimental Design

Dose-response study. In 18 rats, micropipettes were lowered in random order into the right DVC, nROb or nAmb, and vehicle and VIP (0.1, 1, 10, 50 and 100 pmol/site) were microinjected into the medullary regions with 15- to 45-min intervals between injections. In our experience these intervals are sufficient to avoid tachyphylaxis to VIP after repeated administration of the peptide at the same dose. Six of these rats received microinjections of vehicle and VIP into all the brainstem nuclei under investigation. In five of the same animals, at the end of the experiments, to ascertain the anatomic specificity of the gastric responses to microinjections of VIP into the DVC and the nROb, VIP was microinjected at a dose of 100 pmol in a similar volume into the brainstem medulla 0.7 mm rostral to the obex, 1.6 mm lateral from the midline and 0.4 mm down from the surface (Krowicki and Hornby, 1993b).

Bilateral vagotomy. Two of the rats used for a dose-response study and five additional animals were used for investigating the vagus nerve involvement in the gastric motor effects of VIP in the DVC (10 pmol) and nROb (50 pmol). Bilateral vagotomy at the midcervical level was performed after typical responses to the peptide in the DVC and/or nROb were obtained, followed by repeat microinjection of VIP (the micropipette was left in the site) 30 to 60 min after vagotomy.

Statistical Methods

The differences between groups were assessed by paired t-test or by one-way or one-way repeated measures analysis of variance followed by Student-Newman-Keuls multiple comparisons test. Values of P < .05 were considered to be statistically significant.

	Results

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Gastric motor effects of VIP in the DVC. Figure 1 shows the effects of vehicle and VIP (0.1, 1, 10, 50 and 100 pmol), microinjected into the DVC on intragastric pressure, expressed as peak change from base line and the total area of the response, and pyloric and greater curvature region smooth muscle contractile activity. VIP significantly increased intragastric pressure (both peak responses and areas of the response) at doses from 1 to 100 pmol when compared with the effect of vehicle. Increases in pyloric smooth muscle contractile activity achieved statistical significance only after microinjection of VIP at the highest dose of 100 pmol (fig. 1). This is because of a transient decrease in pyloric contractile activity which, in some animals, preceded much longer lasting excitation in response to the peptide. MAP increased after VIP at doses of 1, 10 and 100 pmol (fig. 2). The changes in greater curvature contractile activity, caused by interanimal variation, did not attain statistical significance. A composite drawing of microinjection sites in the DVC is shown in figure 3. A simple spatial dissection of the magnitude of the responses related to the site of microinjection illustrates that maximal increases in peak intragastric pressure after microinjection of VIP, at a dose of 10 pmol, are noted in animals in which the injection is located in the intermediate medial DVC at, or close to, the level of the area postrema (peak changes from 3.0 to 12.0 cm H₂O). Small increases in peak intragastric pressure occurred after microinjection in the lateral DVC (peak change from base line of 2.5 cm H₂O) and minimal changes after microinjections in the adjacent neuropil (peak changes from base line from 0 to 0.5 cm H₂O).

View larger version (K): [in this window] [in a new window]   Fig. 1.   Effects of vehicle and VIP (0.1, 1, 10, 50 and 100 pmol) microinjected into the DVC on intragastric pressure (PRIGP, peak response; ARIGP, area of the response), pyloric circular muscle (PCA) and greater curvature longitudinal muscle (GCCA) contractile activity. Data are mean (bar = S.E.) changes from base line for number of animals indicated next to the x-axis. *Statistically significant when compared with the effect of vehicle.

View larger version (K): [in this window] [in a new window]   Fig. 2.   Effects of vehicle and VIP microinjected into the DVC (0.1, 1, 10 and 100 pmol) or nROb (1, 10 and 100 pmol) on MAP. Data are mean (bar = S.E.) changes from base line for six animals. *Statistically significant when compared with the effect of vehicle.

View larger version (K): [in this window] [in a new window]   Fig. 3.   Spatial dissection of the magnitude of the response in relation to the location of the tip of the micropipette for all microinjections (10 pmol of VIP) in the region of the right DVC, nROb, nAmb and neuropil adjacent to DVC (controls). The original sections were drawn by use of a drawing tube attached to a Nikon Labophot microscope and transposed onto representative sections arranged from most caudal (top left) to most rostral (bottom right). Squares indicate increases in peak intragastric pressure of 0.6 to 1.5 cm H2O (light stipple), 1.6 to 3.5 cm H2O (moderate stipple) and >3.5 cm H2O (heavy stipple). Triangles indicate decreases in peak intragastric pressure of 0.6 to 2.4 cm H2O (moderate stipple) and >2.5 cm H2O (heavy stipple). Double circles indicate minimal changes in peak intragastric pressure of 0.5 to 0.5 cm H2O. Numbers refer to approximate distance from the obex (mm). Abbreviations: AP, area postrema; DMV, dorsal motor nucleus of the vagus; IO, inferior olive; mlf, medial longitudinal fasciculus; nAmb, nucleus ambiguus; nROb, nucleus raphe obscurus; nRPa, nucleus raphe pallidus; nTS, nucleus of the solitary tract; P, pyramid; RVL, rostroventrolateral medulla; XII, hypoglossal nucleus.

View larger version (K): [in this window] [in a new window]   Fig. 4.   Representative chart recording of one experiment in which VIP (1 and 100 pmol) was microinjected into the DVC. (A) VIP at a dose of 1 pmol evoked increases in intragastric pressure, pyloric circular and greater curvature longitudinal muscle contractile activity. A small increase in arterial blood pressure was also observed in this animal. (B) Similar but more evident increases in intragastric pressure and pyloric contractile activity are seen after VIP at a dose of 100 pmol in the same animal.

Gastric motor effects of VIP in the nROb. Figure 5 shows the effects of vehicle and VIP (1, 10, 50 and 100 pmol) microinjected into the nROb on gastric motor function. In contrast to the effect of VIP in the DVC, VIP in the nROb evoked decreases in intragastric pressure responses (peak and total areas of the response) at doses of 10, 50 and 100 pmol. The changes in the area of the response, but not peak response, were dose-related. Similarly, there was an inhibition of pyloric smooth muscle contractility after VIP microinjection into the nROb at doses of 10, 50 and 100 pmol. The changes in greater curvature contractile activity did not achieve statistical significance. MAP was elevated after VIP was microinjected into the nROb at a dose of 100 pmol (fig. 2). A composite drawing of microinjection sites in the nROb is shown in figure 3. All microinjections into the nROb decreased intragastric pressure; however, the greatest responses were noted in the more caudally located microinjections.

View larger version (K): [in this window] [in a new window]   Fig. 5.   Effects of vehicle and VIP (1, 10, 50 and 100 pmol), microinjected into the nROb, on intragastric pressure (PRIGP, peak response; ARIGP, area of the response), pyloric circular muscle (PCA) and greater curvature longitudinal muscle (GCCA) contractile activity. Data are mean (bar = S.E.) changes from base line for the number of animals indicated next to the x-axis. *Statistically significant when compared with the effect of vehicle.

View larger version (K): [in this window] [in a new window]   Fig. 6.   Representative chart recording of one experiment in which VIP (1 and 100 pmol) was microinjected into the nROb. (A) VIP at a dose of 1 pmol evoked small decreases in intragastric pressure, pyloric circular and greater curvature longitudinal muscle contractile activity. A small increase in arterial blood pressure was also observed. (B) Marked inhibition of gastric motor function and an increase in blood pressure are seen after VIP at a dose of 100 pmol in the same animal.

VIP in the nAmb. In five animals VIP (1, 10 and 100 pmol) was microinjected in the vicinity of the nAmb. No discernible changes in the gastric motor and cardiovascular function were observed (table 1). A composite drawing of microinjection sites in the nAmb is shown in figure 3 and shows that the microinjections extend into the nAmb and adjacent medullary reticular areas in most cases. Microinjections more rostrally located could have affected the subjacent rostroventrolateral medulla.

Vagotomy. The effect of bilateral vagotomy on gastric motor and cardiovascular responses to VIP microinjections into the DVC (10 pmol) and the nROb (50 pmol) was investigated in seven animals, and the results are shown in table 2. In the DVC, VIP-induced increases in intragastric pressure and pyloric smooth muscle contractile activity were abolished. Vagotomy also abolished the decrease in intragastric pressure and pyloric contractile activity observed in response to VIP in the nROb. Vagotomy by itself evoked no changes in gastric motor and cardiovascular function (table 3).

Control microinjections. These microinjections resulted in no discernible change in gastric motor function, and the location of these sites is illustrated in the composite drawing (fig. 3). In these animals positive gastric responses to the same dose of VIP microinjected into the DVC and nROb were obtained either before or after these control microinjections.

	Discussion

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The major finding of the present study is that VIP, microinjected into the DVC, increases intragastric pressure and gastric contractile activity, whereas its microinjection into the nROb inhibits gastric motor function. All gastric motor effects of VIP in these nuclei are mediated through vagal pathways.

In the DVC, microinjection of VIP increases gastric motor function. The greatest responses are noted in the medial DVC; therefore, the most likely mechanism of VIP to increase gastric motor function in the DVC is via a direct excitation of preganglionic motor neurons, either in the DMV, or possibly in the subnucleus gelatinosus of the nTS, where the dendritic arbor of these motor neurons receive extensive synaptic input (Rinaman et al., 1989) as part of the vago-vagal reflex pathways. The greatest concentration of preganglionic neurons innervating the stomach occurs at the level of the medial intermediate DMV (Fox and Powley, 1985; Shapiro and Miselis, 1985) where VIP microinjections evoked the greatest gastric responses. At the present time, we are not able to perform a more sophisticated spatial dissection of the effective sites of VIP within the DVC to determine which of these sites may be responsible for the observed gastric effects. Microinjections of VIP into the lateral DVC or into the adjacent neuropil are largely ineffective. To our knowledge, the effect of VIP on neuronal excitability in the DVC has not been investigated. However, VIP has been shown to enhance neuronal excitability in hippocampal slices of the rat (Haas and Gahwiler, 1992) and to excite dorsal horn neurons in the cat (Jeftinija et al., 1982). In the DVC, small increases in MAP were consistently noted after injection of 1 to 100 pmol of VIP. Because this site is known to relay baroreceptor information to the rostroventrolateral medulla and control sympathetic tone (Spyer, 1990), it is possible that VIP modulates the baroreceptor reflex.

VIP containing afferents to the DVC originate from both descending pathways and primary sensory neurons in autonomic ganglia. VIP immunoreactivity has been demonstrated in cells in the nodose ganglion that provide VIP-immunoreactive input to the DVC (Helke and Hill, 1988). However, Palkovits et al. (1982) have demonstrated that the majority of VIP innervation of the nTS is derived not from nodose ganglion cells but from neurons intrinsic to the nucleus. Another likely source for neuronal VIP in the DVC may be also the central nucleus of the amygdala, which is a brain area that maintains direct connections with the DVC (Schwaber et al., 1982), although it is controversial as to whether the central subnucleus contains VIP-immunoreactive neurons (Roberts et al., 1980; Sims et al., 1980).

It is also possible that VIP of a peripheral origin may affect gastric motor function in the DVC. The proximity of the DVC to the cerebrospinal fluid bathing the fourth ventricle and its close anatomical association to the area postrema provides likely routes through which circulating agents may reach specific receptors in the DVC. The highest density of VIP receptors in the brain is found in the area postrema (Martin et al., 1987; Shaffer and Moody, 1986). In addition, VIP binding sites occur in the nTS (Martin et al., 1987; Shaffer and Moody, 1986), and this nucleus can also be exposed to circulating agents directly via its own specialized and permeable microcirculation (Gross et al., 1990; Broadwell and Sofroniew, 1993). It has been demonstrated that intravenous infusion of VIP has a delayed inhibitory effect on gastric acid secretion in anesthetized rats, which became more evident after subdiaphragmatic vagotomy (Nassar et al., 1995). This implicates a central excitatory effect of peripheral VIP administration, with the DVC as a likely target site of action. Normal plasma levels of VIP are extremely low, ranging from 0 to 190 pg/ml (O'Dorisio et al., 1989); however, plasma VIP levels are markedly elevated in patients with VIP-secreting tumors, with the mean concentration of 702 pg/ml (Long et al., 1981) or 956 pg/ml (O'Dorisio et al., 1989). The syndrome produced by these tumors is often referred to as the Verner-Morrison or WDHA syndrome and includes changes in gastrointestinal transit. Since increased gastric motor activity may be accompanied by an increase in gastric emptying and gastrointestinal transit, it is possible that some of the gastrointestinal effects of VIP-secreting tumors are caused by an action of VIP in the DVC.

Vasoactive intestinal polypeptide shows high sequence homology with PACAP and PACAP is usually considered to be a potential ligand of VIP receptors (Sreedharan et al., 1995), although the structural similarity between VIP and PACAP receptors does not exceed 52% (Lutz et al., 1995; Adamou et al., 1995). In the nROb, VIP microinjection decreases gastric motor function and this effect is unlikely to be mediated through PACAP receptors, because microinjection of PACAP38 into the same site evokes dose-dependent increases in gastric tone (Krowicki et al., 1996d). Similarly, VIP evokes no changes in gastric motor function after microinjection into the nAmb, whereas PACAP has an excitatory effect when administered into the same nucleus (Krowicki et al., in press). Pharmacological evidence also suggests the existence of multiple VIP receptors (Usdin et al., 1994) with functional support for the existence of at least two forms of VIP receptors in the brain based on their sensitivity to GTP (Hill et al., 1992). The distinct regional distribution of GTP-sensitive and GTP-insensitive VIP binding sites in brain areas may reflect differential coupling to adenylate cyclase in lower brainstem nuclei (Palkovits et al., 1993) and may be reflected in differences in the effects of VIP in the DVC and nROb.

Several hypotheses could account for the inhibitory effects of VIP on gastric motor function in the nROb. The most straightforward explanation is that VIP selectively activates pathways to the nTS that decrease gastric tone and contractility through visceral afferent-efferent connections (Rinaman et al., 1989). This may include several neurotransmitters in the nTS, including catecholamines (Siaud et al., 1989), substance P (Spencer and Talman, 1986) or nitric oxide (Krowicki et al., 1997). All of these substances have been shown to decrease gastric motor function in the DVC. Specifically, we have recently demonstrated that the gastric inhibitory effect of substance P, microinjected into the nROb, is mediated via nitric oxide in the DVC (Krowicki and Hornby, 1996a), and it is likely that VIP works the same way, although this remains to be tested.

The peripheral pathways and mechanisms of VIP in the nROb-induced gastric relaxation are not elucidated by the present study. Relaxation of the stomach is mediated by vagal nonadrenergic, noncholinergic pathways (de Ponti et al., 1987), which utilize nitric oxide (Krowicki and Hornby, 1996b; Meulemans et al., 1995), VIP (Li and Rand, 1990), ATP and GABA (Krantis et al., 1995). So far, we have shown the gastric relaxation, evoked by VIP microinjected into the nROb, is not abolished by peripheral GABA_A antagonism by bicuculline methiodide (Krowicki and Hornby, 1996c); however, the involvement of other neurotransmitters has not yet been investigated. Gastric relaxation could also be a result of sympathetic activation. It is unlikely that the gastric relaxation evoked by VIP, microinjected into the nROb, involves sympathetic (splanchnic) pathways because vagotomy abolishes the effect. Although there is evidence that splanchnic nerve activation mediates gastric relaxation via inhibition of cholinergic nerves, Andrews and Lawes (1984) have shown that splanchnic stimulation directly relaxes the stomach. Thus, the absence of gastric relaxation by VIP in the nROb in vagotomized animals is consistent with this response being vagally, rather than sympathetically, mediated.

We were initially concerned that increases in intragastric pressure evoked by VIP in the DVC may be secondary to the evoked increases in blood pressure. However, the fact that increases in MAP were evoked by VIP (10 and 100 pmol) microinjected into the nROb, similar to those evoked after microinjection into the DVC, means that this is unlikely to be the case, because, in the nROb, VIP-induced decreases in intragastric pressure and gastric contractility were associated with increases in MAP.

In the present study, gastric and cardiovascular effects were not observed upon microinjection of VIP into the nAmb. In other studies, putative neurotransmitters in this nucleus control heart rate and, in some instances, gastric motor function (Williford et al., 1981; Ishikawa et al., 1988). In regard to the absence of effects on the stomach motor function after microinjection of VIP into the region of the nAmb it is unclear, from anatomical studies, the extent to which neurons in the nAmb innervate the stomach (reviewed in Krowicki and Hornby, 1995). At best, only a few cells are labeled in the nAmb after retrograde tracer is applied to the stomach (Shapiro and Miselis, 1985). The existence of direct projections from nAmb to DVC was once reported (Portillo and Pasaro, 1987) but has not been confirmed by other investigators. Therefore, it is perhaps not surprising that minimal effects on gastric function are noted after microinjection of VIP. Some of the observed gastric effects after microinjection of other agents into the region of the nAmb, such as thyrotropin-releasing hormone (Ishikawa et al., 1988), may be caused by activation of reticular inputs to DVC which are scattered throughout the medullary tegmentum in this region (Rogers et al., 1980). In some cases, after the highest (100 pmol) dose of VIP in the nAmb, there was a small (not statistically significant) and delayed (about 10 min) increase in intragastric pressure. However, we cannot exclude the possibility that this is caused by diffusion from the site of microinjection either to the reticular areas or the DVC itself. It is may not be surprising that cardiovascular effects were not noted because Batten (1995) reports that VIP fibers are not in close anatomical association with cardiac vagal neurons in the nAmb.

In conclusion, the major finding of the present study is that microinjection of VIP in hindbrain sites evokes opposing, but vagally mediated, effects on gastric motor function. Microinjection of VIP into the DVC results in gastric motor excitation, whereas microinjection of the peptide into the nROb evokes gastric inhibition in anesthetized rats. The precise pathways and mechanisms by which these effects are mediated await further investigation.