Introduction

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Nicotine is known to exert important effects on many physiological systems, including the cardiovascular (Hill and Wynder, 1974) and gastrointestinal (GI; Barnett, 1927) systems. Some of these system effects of nicotine may be due to interaction of the drug with receptors in the medulla oblongata (Nagata et al., 1986; Tseng et al., 1993). Hence, the study of nicotinic receptors in the medulla oblongata represents an opportunity to begin to understand the structural diversity of native neuronal nicotinic receptors and how that diversity affects their function.

What was known about nicotinic receptors in the medulla oblongata at the time of the initial submission of this report can be summarized under three headings: 1) physiological responses that occur when nicotinic receptors in the medulla oblongata are activated, 2) morphological studies on the nature of the nicotinic receptor, and 3) a combination of the first two.

With a focus on physiological responses, nicotine microinjected into the dorsal motor nucleus of the vagus (DMV) has been reported to decrease gastric acid secretion (Nagata et al., 1986) and to produce a biphasic effect on gastric motility, namely, an initial decrease followed by an increase in motility (Nagata and Osumi, 1991). Acetylcholine microinjected into the nucleus ambiguous (NA) produces esophageal contractions, which are mediated by a nicotinic receptor (Wang et al., 1991). Nicotine microinjected into the region of the area postrema evokes a biphasic effect on blood pressure; initially, there is an increase followed by a decrease, and the fall in pressure is associated with bradycardia (Kubo and Misu, 1981). Nicotine microinjected into the nucleus tractus solitarius (NTS) causes decreases in blood pressure and heart rate (Robertson et al., 1988). In addition, nicotine microinjected into the rostroventrolateral medulla (RVLM) increases arterial blood pressure (BP) and heart rate (Sapru, 1987; Sundaram and Sapru, 1988; Tseng et al., 1993, 1994). Finally, nicotine applied topically on a site caudal to the RVLM produces a decrease in blood pressure (Feldberg and Guertzenstein, 1976). In the above studies, no information was provided as to the subtype of nicotinic receptor involved in the responses.

With a focus on morphological data, most of the data have been obtained for the DMV and indicate the presence of the 7 subunit (Hunt and Schmidt, 1978; Dominguez Del Toro et al., 1994; Breese et al., 1997) and the 3, 4, and 5 subunits (Wada et al., 1989, 1990; Winzer-Serhan and Leslie, 1997; Zoli et al., 1998). According to Zoli et al. (1998), the 5 subunit signal is weaker than the 3 subunit signal, and they have no data on the 4 subunit. Zoli et al. (1998) provide strong evidence for the presence of the 4 subunit, and the signal is about equal to that for the 3 subunit. Furthermore, they also provide evidence that the 2 subunit is lacking from the DMV. Data obtained for the NTS, specifically the medial NTS, indicate the presence of the 7 subunit (Dominguez Del Toro et al., 1994) and very weak signals for the 3, 4, 5, and 2 subunits (Wada et al., 1989, 1990). The 2 subunit was undetectable (Wada et al., 1989).

With a focus on studies performed that combine physiological responses with morphological studies, to our knowledge only one group of investigators has reported data. Zoli et al. (1998) used receptor autoradiography, in situ hybridization, and patch-clamp recording techniques to identify the nicotinic receptor or receptors in the DMV. Their physiological end point was current changes in response to the application of three different nicotinic receptor agonists to DMV neurons in the brain slices of mice lacking the 2 subunit. Based on their data, they concluded that the major nicotinic receptor subtype in the DMV is the 34 subtype.

Our goal was to use changes in GI and cardiovascular function as end points and characterize the nicotinic receptor or receptors in the medulla. Our long-term goal is to reveal the native neuronal nicotinic receptors in the medulla oblongata responsible for the effects of nicotine on physiological function.

	Experimental Procedures

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Materials. ()-Nicotine tartrate, urethane, -chloralose, L-glutamic acid, and fast green dye were purchased from Sigma Chemical Co. (St. Louis, MO). Hexamethonium dichloride and -bungarotoxin were purchased from Research Biochemicals Inc. (Natick, MA). Dexamethasone was purchased from Elkins-Sinn (Cherry Hill, NJ).

Animal Preparation. Experiments were performed on male Sprague-Dawley rats (n = 207) weighing 250 to 380 g (Taconic, Germantown, NY). Before each experiment, food was withheld overnight but water was provided ad libitum. Animals were anesthetized with an i.p. injection of a cocktail containing 800 mg/kg urethane and 60 mg/kg -chloralose dissolved in 3 ml/kg 0.9% saline. Body temperature was monitored by a rectal thermometer and maintained at 37 ± 1°C with an infrared heating lamp. Before surgery, all animals were pretreated with 0.8 mg of dexamethasone s.c. to minimize brain swelling. Animal care and experimental procedures were performed in accordance with the National Institutes of Health guidelines and with the approval of the Animal Care and Utilization Committee of Georgetown University, Washington, DC.

Surgery. Rats were intubated to maintain an open airway and for instituting artificial respiration when necessary. The carotid artery was cannulated with polyethylene tubing (PE 50) to monitor blood pressure. Blood pressure was recorded using a bridge amplifier connected to a MacLab (ADI Instruments, Milford, MA) data acquisition system. Data were stored on computer (Apple Macintosh G3 connected to MacLab) for analysis at a later time. Special attention was given to avoid damage to the vagus nerves. In some experiments, ligatures were tied around the vagus nerves, and the area was moistened with mineral oil. An intragastric balloon, made from the little finger of a small latex glove, was tied around polyethylene tubing (PE 160) and inserted into the stomach via the fundus. The balloon was positioned in the antrum and secured with a running suture to avoid movement. The tubing was connected to a pressure transducer, which was connected to a bridge amplifier (MacLab; Analog Digital Instruments). The bridge amplifier was fed into the MacLab motherboard, and the signal was recorded by a Macintosh computer. Data were saved for analysis at a later time. The stomach was inflated by introducing warm saline (2-3 ml) into the balloon to achieve baseline pressure of 6 to 15 mm Hg. The animals were then positioned in a stereotaxic apparatus (David Kopf, Tujunga, CA). Muscles covering the occipital part of the skull were carefully removed using a small cautery and spatula until the atlanto-occipital membrane was seen. The membrane and dura were cut using a 16-gauge needle while the area was viewed through a dissection microscope (Bausch & Lomb). The occipital plate was removed by clipping small pieces of bone with small rongeurs. The cerebellum was retracted slightly while using a 26-gauge needle to cut the subarachnoid covering. Calamus scriptorius (CS) was viewed from the dorsal aspect and used as a point of reference (see later).

Microinjection Technique. Nicotine tartrate and L-glutamate were dissolved in 0.9% saline. A histological marker (fast green dye) for studying injection sites was added to drug solutions in a 1 to 2 mg/ml concentration. The pH of all drug solutions was brought to 7.0 to 7.2. Double-barreled pipettes with a tip diameter of between 30 and 60 µm were used. All microinjections were given unilaterally. Injections were given in volumes of 60 nl and administered by hand-controlled pressure. Microinjections were given within 5 s. CS was used as a zero reference point. Stereotaxic coordinates were originally chosen based on histology in Paxinos and Watson (1986). Final DMV coordinates were chosen based on preliminary studies wherein nicotine was found to evoke consistent increases in intragastric pressure (IGP) with minimal effect on BP. Final mNTS coordinates were chosen based on preliminary studies wherein nicotine was found to evoke decreases in both IGP and blood pressure. Coordinates for the DMV ranged from 0.3 to 0.5 mm rostral to CS, medial-lateral 0.3 to 0.5 mm lateral from the midline, and dorsal-ventral 0.5 to 0.7 mm from the dorsal surface of the medulla. Coordinates for the mNTS ranged from 0.3 to 0.5 mm rostral to CS, medial-lateral 0.5 to 0.7 mm lateral from the midline, and from 0.4 to 0.6 mm from the dorsal surface of the medulla.

Chronic Dosing Studies. To study the effects of chronic nicotine treatment on IGP and blood pressure responses, two groups of animals, one control group and one treated with nicotine (0.8 mg/kg base s.c.), were injected twice daily for 10 days. The control group was given vehicle (0.9% saline) twice daily in the same volume as the nicotine-treated group. All solutions were made fresh daily, and the pH was adjusted to 7.3 to 7.4. This dose and treatment of nicotine were chosen based on its ability to up-regulate some nicotinic receptors (Flores et al., 1992) and chronically inactivate a physiologic response to nicotine (Hulihan-Giblin et al., 1990), without causing detrimental behavioral responses.

Protocols Used for Testing Antagonists to Nicotine. To study the ability of hexamethonium (10, 100, and 1000 pmol) and -bungarotoxin (100 pmol) to block nicotine-induced responses, the following protocol was used. Nicotine was microinjected into either the DMV or mNTS. After 15 min, the antagonist was microinjected. At 5 to 10 min after antagonist pretreatment, nicotine was microinjected into the same site. In studying the selectivity of the highest dose of hexamethonium (i.e., 1000 pmol), we also tested against responses evoked by L-glutamate. This is an approach previously used by Wang et al. (1991) to test receptor selectivity of the nicotinic acetylcholine receptor (nAChR) antagonist dihydro--erythroidine.

Histologic Verification. At the end of the experiment, all rats were sacrificed with an overdose of pentobarbital. Brains were removed and fixed in a mixture of 4% paraformaldehyde and 20% sucrose for at least 24 h. The brain was cut into 50-µm-thick coronal sections and stained with neutral red. The location of nuclear groups was studied in relation to microinjection sites using the atlas of Paxinos and Watson (1986).

Data Analysis. Data were analyzed using the Chart Software for data analysis made for MacLab (ADI Instruments). Before microinjections were performed, the lowest points of the IGP trace were obtained over a 3-min control period, and a single value was calculated as the average of all of these points and used as an index of gastric tone. Phasic contractions occurred in some animals but were not always present during the control periods and/or were lacking in a significant number of animals. Hence, phasic contractions were ignored in our study and gastric tone was used as the end point of a gastric response. After microinjections into the mNTS, the minimum value in the IGP trace was taken as the largest drop in gastric tone. For DMV responses to microinjections, the maximum value in the trace was taken as the largest increase in gastric tone. The percentage of change from baseline in IGP was then calculated. Data for IGP are reported as percentage of change from baseline because it was necessary to generate data points that would provide reasonable dose-response curves, because baseline IGP varied between animals. Therefore, to be consistent in reporting data, IGP is reported in percentage of change from baseline. It should be noted that all data that are shown to be statistically significant are significant when analyzed as both raw data and percentage of change from baseline. For blood pressure calculations, the change in mean blood pressure was taken (millimeters of mercury). Data appear as mean (percentage of change from baseline for IGP and change in millimeters of mercury for blood pressure changes) ± S.E.

	Results

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Unilateral Microinjection of Nicotine into DMV: Effects on IGP and BP. Data were obtained from 19 rats and a total of 25 microinjection sites (in some animals, data were obtained from both the right and left DMVs). Nicotine in a dose range of 10 to 1000 pmol was microinjected into the DMV while IGP and systemic BP were monitored. Nicotine in a dose range of 10 to 300 pmol produced dose-related increases in IGP with an ED₅₀ value of 89 pmol (Fig. 1). The highest dose of nicotine tested, 1000 pmol, also produced a significant increase (P < .05) in IGP, but the magnitude of the increase was significantly less (P < .05) than the response obtained with the 300-pmol dose (Fig. 1). The location of the microinjection sites where nicotine elicited these increases in IGP is depicted on coronal brain sections shown in Fig. 2. The dose range of nicotine studied in the DMV had no significant effect on mean BP.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Nicotine dose-response curves for three physiological responses. Nicotine was microinjected (in 60 nl) into two brainstem nuclei, the DMV and the mNTS, while IGP and systemic mean BP were recorded. When nicotine in various doses was microinjected into the DMV, an increase in IGP () was recorded. When nicotine in various doses was microinjected into the mNTS, a decrease in mean arterial pressure () and a decrease in IGP () were elicited. IGP responses are expressed as percentage of change from baseline, whereas mean BP is expressed as change in millimeters of mercury. Each point corresponds to the mean ± S.E. of the responses of four to seven microinjections into separate nuclei (at least three different animals, with the exception of the 10 pmol/60 nl point in the DMV curve, which represents two microinjections in two different animals). Baseline values were IGP, 12.5 ± 2 mm Hg; and BP, 93 ± 3 mm Hg.

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Camera lucida drawings of coronal sections of the medulla showing the microinjection sites of nicotine (10-1000 pmol/60 nl) into the DMV (A). The values to the right of the sections refer to the rostrocaudal distance (mm) from obex. B, actual photograph of the injection site that represents the injection of nicotine in Fig. 3. AP, area postrema; TS, solitary tract; CC, central canal; 12, hypoglossal nucleus. , microinjections of nicotine that elicited increases in IGP (n = 25). , nicotine (100 pmol/60 nl) microinjections that did not elicit a response on IGP (n = 6). ×, vehicle microinjections that did not elicit a response on IGP (n = 3).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Representative chart recordings from one experiment in which nicotine (100 pmol/60 nl) was microinjected into the left DMV before (A) and after (B) ipsilateral (IPSI) cervical vagotomy (Vx). A, nicotine evoked an increase in IGP. B, unilateral ipsilateral (left) cervical vagotomy abolished the IGP response to nicotine. Note: nicotine microinjected into the DMV had no effect on blood pressure.

Unilateral Microinjection of Nicotine into mNTS: Effects on IGP and BP. Data were obtained from 22 rats and a total of 33 microinjection sites (again in some animals, data were obtained from both the right and left mNTSs). Nicotine in a dose range of 0.1 to 1000 pmol was microinjected into the mNTS while IGP, systemic BP, and heart rate were monitored. Data obtained for IGP are summarized in Fig. 1 and indicate that nicotine in a dose range of 0.1 to 300 pmol produced dose-related decreases in IGP. The ED₅₀ value for nicotine-induced decrease in IGP was 0.6 pmol. The highest dose of nicotine tested, 1000 pmol, also produced a significant decrease (P < .05) in IGP, but the magnitude of the decrease was significantly less (P < .05) than the response obtained with the 300-pmol dose (Fig. 1). The location of the microinjection sites where nicotine elicited these decreases in IGP is depicted on coronal brain sections shown in Fig. 4.

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Camera lucida drawings of coronal sections of the medulla showing the microinjection sites of nicotine (0.1-1000 pmol/60 nl) into the mNTS (A). The values to the right of the sections refer to the rostrocaudal distance (mm) from obex. B, actual photograph of the injection site that represents the injection of nicotine in Fig. 5. AP, area postrema; TS, solitary tract; CC, central canal; 12, hypoglossal nucleus. , microinjections of nicotine that elicited decreases in IGP and BP (n = 33). , nicotine (100 pmol/60 nl) microinjections that did not elicit a response on the end points measured (n = 6). ×, vehicle microinjections that did not elicit a response on IGP or BP (n = 3).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Representative chart recordings from one experiment in which nicotine (100 pmol/60 nl) was microinjected into the right mNTS before (A) and after (B) ipsilateral cervical vagotomy and finally after bilateral (BILAT) cervical vagotomy (C). A, nicotine evoked a decrease in both blood pressure and IGP. B, unilateral ipsilateral cervical vagotomy had no effect on nicotine-induced responses evoked from the mNTS. C, bilateral cervical vagotomy abolished nicotine-induced decrease in IGP, whereas the nicotine-induced decrease in blood pressure was unaffected. The drop in IGP in A was from a baseline mean of 12 to 10 mm Hg. Note: the baseline IGP in C was also 12 mm Hg and therefore could drop further on nicotine microinjection into the mNTS.

Effects of Unilateral Microinjection of L-Glutamate into DMV and into mNTS on IGP and BP. L-Glutamate was microinjected into the mNTS and DMV in 5-min intervals to study the ability to elicit responses on IGP and BP. This was performed to show that the desensitization that occurred in response to nicotine microinjections was unique to that agonist. These experiments would show that the effects of stimulating the mNTS or DMV could be elicited after 5 min in response to an all-purpose postsynaptic excitatory agent. In terms of response to second injection of L-glutamate, the data obtained are summarized in Table 1 and indicate that full responses could be obtained for L-glutamate on either IGP or mean BP at the 5-min time point (for DMV and mNTS microinjections).

Effects of Chronic Exposure to Nicotine on IGP and Mean BP Responses Elicited by Acute Microinjection of Nicotine into DMV and mNTS. Rats were treated twice daily for 10 days with either a s.c. injection of nicotine bitartrate (0.8 mg/kg as the base, dissolved in 1 ml of vehicle) or vehicle (0.9% saline). They were then anesthetized 24 h after the last nicotine or vehicle injection and given nicotine by microinjection into either the DMV (100 pmol) or the mNTS (10 pmol). The effects of nicotine on IGP and mean BP were then observed. As shown in Fig. 6, the data obtained in these studies indicate that animals receiving vehicle and then challenged with local microinjections of nicotine exhibited the typical responses. That is, nicotine decreased IGP and blood pressure after microinjection of nicotine into the mNTS and increased IGP after microinjection of nicotine into the DMV. Animals receiving daily injections of nicotine, however, did not exhibit a decrease in gastric pressure when nicotine was microinjected into the mNTS (Fig. 6). Decreases in blood pressure were observed when nicotine was microinjected into this site, and the usual increases in IGP were obtained when nicotine was microinjected into the DMV.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effect of chronic exposure to nicotine on IGP and BP responses elicited by acute administration of nicotine into the DMV and mNTS. Two groups of animals were treated with either saline (open columns) or nicotine (filled columns) for 10 days. Animals were then challenged with acute microinjections of nicotine (100 pmol/60 nl for DMV and 10 pmol/60 nl for mNTS microinjections) while mean BP and IGP were recorded. Baseline values for saline-treated animals: IGP, 13 ± 0.5 mm Hg; and BP, 92 ± 3 mm Hg. Baseline values for nicotine-treated animals: IGP, 12 ± 1 mm Hg; and BP, 95 ± 2 mm Hg. Each bar corresponds to the mean ± S.E. of the responses of 8 to 11 microinjections into separate nuclei (four to six animals) (*P < .05).

Effects of Pharmacological Antagonists Microinjected into DMV and mNTS on Nicotine-Induced Changes in IGP and Mean BP Elicited from DMV and mNTS. The pharmacological antagonists -bungarotoxin and hexamethonium were studied regarding nicotine-induced changes in IGP and mean BP evoked from the DMV and mNTS. For studies of -bungarotoxin, 100 pmol was microinjected 15 min after an initial microinjection of nicotine (100 pmol). Next, a repeat microinjection of nicotine at 100 pmol was made 5 to 10 min after -bungarotoxin. Data obtained from six experiments are summarized in Fig. 7 and indicate that -bungarotoxin pretreatment almost completely blocked nicotine-induced increases in IGP evoked from the DMV. On the other hand, when vehicle for -bungarotoxin (0.9% saline) was tested instead of -bungarotoxin, the repeat administration of nicotine evoked the identical increase in IGP as observed with the initial microinjection of the alkaloid (data not shown). Using the same experimental protocol, we examined the effect of -bungarotoxin at the mNTS. Studies were conducted in six experiments and indicate that -bungarotoxin pretreatment has no effect on nicotine-induced IGP and mean BP changes evoked from the mNTS (Fig. 7).

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Effect of -bungarotoxin microinjection into the DMV and the mNTS on nicotine-induced IGP and BP responses. Nicotine (100 pmol/60 nl) was first microinjected into the mNTS and DMV (open columns) while mean BP and IGP were recorded. After a 10- to 15-min period, -bungarotoxin (100 pmol/60 nl) was microinjected into the same site. Nicotine was then microinjected for a second time (filled columns), and the response was recorded. Baseline values: IGP, 11 ± 1.2 mm Hg; and BP, 91 ± 3 mm Hg. Each bar corresponds to the mean ± S.E. of the responses of six microinjections into separate nuclei (six animals) (*P < .05).

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Effect of hexamethonium microinjection into the DMV and the mNTS on nicotine-induced IGP and BP responses. Nicotine (100 pmol/60 nl) was first microinjected into the mNTS and DMV while mean BP () and IGP ( and ) were recorded. The BP and IGP responses to microinjection of 100 pmol of nicotine were taken as 100% of the response. After a 10- to 15-min period, hexamethonium (10, 100, and 1000 pmol/60 nl) was microinjected into the same site. Nicotine was then microinjected for a second time, 5 min after hexamethonium, and the response was recorded. Baseline values: IGP, 11.3 ± 0.8 mm Hg; and BP, 92 ± 1 mm Hg. Each point corresponds to the mean ± S.E. of the responses of four to six microinjections into separate nuclei (*P < .05, significantly different from the first microinjection of nicotine).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The major findings of this study were that 1) unilateral microinjection of nicotine into the DMV produces a dose-related increase in IGP, due largely to activation of 7 nAChRs. This increase is mediated via excitation of the ipsilateral vagus nerve. 2) Unilateral microinjection of nicotine into the mNTS produces a dose-related decrease in IGP that is mediated via inhibition of both vagus nerves. 3) Unilateral microinjection of nicotine into the mNTS produces a dose-related decrease in BP that is unaffected by vagotomy and is presumably due to inhibition of sympathetic nervous system activity to the vasculature. 4) Chronic exposure of rats to nicotine for 10 days results in a loss of nicotine-evoked decrease in IGP from the mNTS but not a loss of the ability of nicotine to increase IGP from the DMV. Nicotine-induced decreases in BP were also unaffected by chronic exposure of rats to nicotine. 5) Unilateral microinjection of hexamethonium into the mNTS prevents nicotine-induced decreases in IGP and BP, evoked from the mNTS. Unilateral hexamethonium (10 and 100 pmol) microinjection into the DMV does not block the nicotine-induced increase in IGP evoked from this site.

Our results showing that nicotine microinjected into the DMV evokes an increase in gastric function confirms the finding of Nagata and Osumi (1991), wherein a dose of 100 pmol of nicotine microinjected into the DMV caused increases in IGP and gastric motility. The ED₅₀ dose of nicotine for this effect in our study, 89 pmol, is very close to the dose of 100 pmol reported by Nagata and Osumi (1991). We have extended their finding by demonstrating that ipsilateral cervical vagotomy blocks the response, thus proving that nicotine-evoked excitation of gastric function elicited from the DMV is mediated by efferent parasympathetic nerves. The significant antagonism of responses from the DMV by -bungarotoxin suggests that a receptor containing 7 subunits is largely responsible for the nicotine-induced increase in IGP.

Evidence that gastric inhibition evoked by nicotine is due to nicotine acting at the mNTS can be summarized as follows: 1) In our study, nicotine-evoked gastric inhibition from the mNTS occurred with a much lower dose than nicotine-evoked gastric excitation from the DMV, and this fits well with data of Nagata and Osumi (1991) wherein 10 pmol of nicotine produced inhibition and 100 pmol of nicotine was required for excitation. 2) Hexamethonium (10 and 100 pmol) microinjected into the DMV had no effect on nicotine-induced increases in IGP. On the other hand, hexamethonium (10 and 100 pmol) microinjected into the mNTS prevented nicotine-induced changes in IGP. This fits well with the finding of Nagata and Osumi that hexamethonium microinjected into what they assumed was the DMV blocked nicotine-evoked gastric inhibition produced from the same microinjection site. 3) Bilateral vagotomy was required to abolish the nicotine-induced inhibition from the mNTS, whereas only ipsilateral vagotomy was needed to abolish the nicotine-induced excitation from the DMV. This fits with anatomic tracing studies indicating that each DMV provides an ipsilateral projection to the stomach (Blessing et al., 1991), whereas neurons of the mNTS connect to both the right and left DMVs (Blessing et al., 1991). This is also consistent with our earlier physiological data indicating that gastric motility changes produced by unilateral electrical stimulation of the DMV are blocked by ipsilateral vagotomy (Pagani et al., 1985).

Our data suggest that there are at least three different nicotinic receptor subtypes in the DMV and the mNTS that affect gastric function and blood pressure. This is based on the finding that the dose-response curves for increases in IGP (DMV), decreases in IGP, and decreases in BP (mNTS) were all different. Chronic 10-day exposure to nicotine resulted in complete loss of the nicotine-induced decrease in IGP evoked from the mNTS, whereas the other two nicotine-evoked responses were not altered. Finally, -bungarotoxin significantly antagonized the nicotine-evoked response from the DMV but had no effect on either of the nicotine-evoked responses from the mNTS. Conversely, nicotine-evoked responses from the mNTS were more sensitive to hexamethonium (10 and 100 pmol) blockade, whereas these doses had no effect on the nicotine-evoked response from the DMV, suggesting different nAChR subtypes.

Our data indicate that the primary nAChR subtype at the DMV (which modulates IGP) is the 7 subtype. Our best evidence for this is the selective block of nicotine-evoked increase in IGP with -bungarotoxin. We refer to the -bungarotoxin block as selective because the dose of -bungarotoxin used at the DMV had no effect on nicotine-induced responses elicited from the mNTS. We also have preliminary autoradiographic data using ¹²⁵I--bungarotoxin binding demonstrating high-density ligand binding in the DMV of the rat (Ebert et al., 1999). In addition, our most recent findings using immunohistochemistry indicate immunofluorescent staining of DMV neurons with an anti-7-nAChR antibody (Ebert et al., 1999).

Although specific molecular identification of subunits involved in the effects of nicotine from the mNTS was beyond the scope of this study, we propose, based on pharmacological characteristics, that the subtype of nAChR at the mNTS responsible for mediating the nicotine-induced decrease in IGP is the 42 subtype. Our strongest evidence for this is as follows: first, this nAChR was activated by the lowest concentrations of nicotine of the three subtypes investigated, and this fits with data of others demonstrating the high sensitivity of this nAChR subtype to nicotine (Alkondon and Albuquerque, 1993; Chavez-Noriega et al., 1997; Olale et al., 1997). Second, in our study of chronic 10-day exposure to nicotine, desensitization of this mNTS receptor occurred. Data of others demonstrate that an nAChR subtype that does desensitize after chronic nicotine treatment is most likely the 42 subtype (Hulihan-Giblin et al., 1990; Hsu et al., 1996; Olale et al., 1997). In addition, evidence for the presence of the 42 subtype of nAChR in the nucleus tractus solitarius can be found in the published study of Zoli et al. (1998). In this receptor autoradiography study of mice lacking the gene for the 2 nAChR subunit, [³H]cytisine binding in the NTS (which was striking in wild-type 2 mice) was lost.

The subtype of nAChR at the mNTS responsible for mediating nicotine-induced decrease in BP appears to be distinctly different from the nAChR at the mNTS that is responsible for the decrease in IGP and from the nAChR at the DMV that is responsible for the increase in IGP. Studies are under way to elucidate the nature of this nAChR.

It should be noted from our studies that although -bungarotoxin blocked the majority of the nicotine-induced response (shown in Fig. 7) elicited from the DMV, a significant degree of the response remained. This suggests that either our dose of -bungarotoxin was too low to produce a full block of the nicotine-induced response or a portion of the nicotine-induced response was mediated by an nAChR subtype other than the 7 subtype. In yet-to-be-published studies of ours on -bungarotoxin at the DMV, we have found that the dose of -bungarotoxin used in this study was a full blocking dose for the 7 nAChR. Data from the present study using a high dose of hexamethonium (1000 pmol) indicate that the -bungarotoxin-insensitive component of the response is a nicotinic receptor because hexamethonium completely blocks the effect of nicotine at the DMV. To show that 1000 pmol of hexamethonium was not acting in a nonselective manner (i.e., to nonspecifically block all excitatory stimuli); this dose of hexamethonium was shown to have no effect on L-glutamate-evoked increases in IGP from the DMV.

In summary, three important findings were made in our study. The first is that nicotine-induced changes in IGP can result from nicotine acting at least two sites in the dorsal medulla. Nicotine-induced decreases in IGP are due to nicotine acting at the mNTS, whereas nicotine-induced increases in IGP are due to nicotine acting at the DMV. The second is that there are at least three different nAChR subtypes in the dorsal medial medulla oblongata influencing GI and cardiovascular function. One is in the DMV and affects the upper GI tract, and two others are in the mNTS and affect the upper GI tract and the cardiovascular system. The third finding is that the major nAChR in the DMV that affects the upper GI tract is the 7 subtype. In addition, our data plus the data of others suggest that the nAChR subtype in the mNTS responsible for the decrease in IGP may be the 42 subtype.