INTRODUCTION

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IT IS WELL ESTABLISHED that in the rat, as well as in other experimental animals, the nucleus of the solitary tract (NTS) represents the first site where afferents mediating different cardiovascular reflexes make their primary synapse (1). Within the NTS there are discrete zones where different types of afferents terminate. For example, the chemoreceptor projection site is located in a midline region just caudal to the calamus scriptorius, and the baroreceptor and cardiopulmonary afferents are believed to terminate in a region rostral and lateral to it (8, 14, 20, 30). There is a general consensus that a glutamate-like substance is released in the NTS as a neurotransmitter from the terminals of afferents mediating different cardiovascular reflexes (20, 25, 26, 30).

Our knowledge regarding the molecular subcomponents of nicotinic receptors in the central nervous system (CNS) is rapidly expanding. Eleven distinct genes encoding neuronal nicotinic receptor subunits (2-9 and 2-4) have been cloned in the rat (5-7, 11). Multiple combinations of  and  subunits result in the formation of a wide variety of functional neuronal nicotinic receptors with two (e.g., 22, 32, 34, 42) or more (e.g., 345, 324) subunit types. Each combination of subunits confers distinct pharmacological properties to the nicotinic receptors. For example, rat nicotinic receptors composed of five 7 subunits exhibit the following order of affinities: nicotine > cytisine > dimethyl-phenyl-piperizinium (DMPP) > ACh, whereas the order of affinities for 42 is cytisine > nicotine > ACh > DMPP (5, 7). Recently, toxins binding specifically to nicotinic receptors having different subunit compositions have been developed. For example: 1) -bungarotoxin (-BT) isolated from the venom of the snake Bungarus multicintus (21, 36) and -conotoxin ImI isolated from the venom of cone snail Conus imperialis (17) bind specifically to nicotinic receptors containing 7 subunits (10, 18) and 2) -conotoxin AuIB isolated from the venom of the cone snail Conus aulicus binds specifically to nicotinic receptors containing 34 subunits (12). Before the availability of these toxins, several chemicals have been used as blockers of nicotinic receptors. For example, mecamylamine has been reported to be a relatively specific antagonist for nicotinic receptors containing 34 subunits (4, 31).

Nicotinic receptors have been identified by extracellular and intracellular studies on subpopulations of NTS neurons in rat brain stem slice preparations (22) and in dissociated NTS neurons (29). Activation of nicotinic receptors has been reported to release an excitatory amino acid in the NTS (2) or modulate the function of excitatory amino acid receptors in the nucleus ambiguus (NA; 15, 16). However, there are very few detailed studies on the role of nicotinic receptors in the NTS in mediating or modulating cardiovascular function (9, 28). There are no studies in which the role of the subtypes of nicotinic receptors in the NTS mediating cardiovascular responses has been studied. The purpose of this investigation was 1) to carry out a detailed study on the cardiovascular responses elicited by the stimulation of nicotinic receptors in the NTS, 2) to identify the nicotinic receptor subtypes in the NTS region mediating cardiovascular responses, and 3) to test the hypothesis that cardiovascular responses elicited by activation of nicotinic receptors in the NTS are mediated via the excitatory amino acid receptors.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

General procedures. Male Wistar rats (Charles River Laboratories), weighing 350-400 g, were used. Anesthesia was induced by the administration of isoflurane (3% in 100% oxygen) via a nose mask, and the trachea was cannulated with polyethylene tubing. Anesthesia was maintained with tracheal administration of isoflurane (2% in 100% oxygen). One of the femoral veins was cannulated, and urethan (1.2 g/kg) was injected intravenously in five aliquots at 3-min intervals; the tracheal inhalation of isoflurane was discontinued after the administration of first two aliquots of urethan dose. The depth of anesthesia was tested by responses to the pinching of one of the hindpaws. Usually, administration of supplemental doses of urethan was not necessary; however, if the animals showed signs of light anesthesia, additional doses (usually 0.2-0.5 g/kg) were administered intravenously in divided doses. Rectal temperature was maintained at 37 ± 0.5°C. One of the femoral arteries was cannulated, and blood pressure (BP) was monitored via a pressure transducer (Statham P23 Db). The heart rate (HR) was monitored by a tachograph (Grass 7P4) that was triggered by BP waves. The rats were ventilated artificially; the end-tidal CO₂ was estimated from a continuous measurement of expired gas with an infrared CO₂ analyzer modified for use in small animals (Micro-Capnometer, Columbus Instruments) and maintained between 3.5 and 4.5%. All of the tracings were recorded on a polygraph (Grass model 7D).

Microinjection technique. The rats were placed in a prone position in a stereotaxic instrument (David Kopf Instruments) with the bite bar 18 mm below the interaural line. The medulla was exposed by removing the dorsal neck muscles, incising the atlantooccipital membrane, and removing part of the occipital bone and dura. Multibarreled glass micropipettes (tip size 20-40 µm) were used. The micropipette was mounted on a micromanipulator (David Kopf Instruments), and each barrel was connected via polyethylene tubing to one of the channels on a picospritzer (General Valve, Fairfield, NJ). One of the barrels was filled with a 5 mM solution of L-glutamate monosodium (L-Glu, pH 7.4), and other barrels, depending on the experiment, contained nicotine, nicotine receptor antagonists, and normal saline. The micropipette was inserted into the NTS perpendicularly; coordinates for the regions of NTS mediating cardiovascular responses were 0.5 mm rostral to the calamus scriptorius, 0.5 mm lateral to the midline, and 0.5 mm deep from the dorsal surface of the medulla. The micropipette remained at the microinjection site until the observations at that site were completed. The volume of microinjection (50 nl), determined by the displacement of fluid meniscus in the micropipette barrel, was confirmed visually under a modified binocular horizontal microscope (World Precision Instruments) with a graduated reticule in one of the eyepieces. The duration of the injection was 5-10 s.

Statistical analysis. The paired t-test was used when the animals served as their own controls. Differences of more than two means between different groups of rats were determined by analysis of variance followed by Duncan's multiple range test. Differences were considered significant at P < 0.05. All values are expressed as means ± SE.

Drugs and chemicals used. The following drugs or toxins were used: -conotoxin AuIB, -BT, -conotoxin ImI, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD) D()-2-amino-7-phosphonoheptanoic acid (DAP-7), decamethonium, hexamethonium dichloride, L-Glu, -methyl-4- carboxyphenylglycine (MCPG), mecamylamine hydrochloride, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxalline (NBQX), N-methyl-D-aspartic acid (NMDA), nicotine bitartrate, and urethan. All of the solutions for the microinjections were freshly prepared in normal saline containing HEPES, and the pH was adjusted to 7-7.4. The control solutions for microinjections (50 nl) consisted of normal saline containing HEPES with pH adjusted to 7.4. The concentration of drugs injected into the NTS refers to their salts. All of the drugs were procured from Sigma-RBI Chemicals (St. Louis, MO). -Conotoxin AuIB and ImI were synthesized by the previously described methods (12).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The values for mean arterial pressure (MAP) and HR in urethan-anesthetized rats were 123 ± 11 mmHg (n = 16) and 364 ± 41 beats/min (n = 9), respectively.

Concentration response of nicotine. Microinjections (50 nl) of nicotine into the medial NTS (0.5 mm rostral to the calamus scriptorius, 0.5 mm lateral to the midline, and 0.5 mm deep from the dorsal medullary surface) elicited a decrease in MAP in a concentration-dependent manner (Fig. 1A) (n = 15). The responses plateaued at 0.5 µM concentration. The depressor responses to 0.5, 1, and 10 µM concentrations of nicotine were significantly (P < 0.05) greater than those elicited by 0.01 µM concentration. The bradycardic responses to the microinjections of nicotine into the NTS were inconsistent and relatively small. The decrease in HR in response to a 10 µM concentration of nicotine was significantly greater (P < 0.05) than that elicited by other concentrations of nicotine (Fig. 1B) (n = 15). A 10 µM concentration of nicotine was used for further studies, because both the maximum bradycardic and depressor responses were obtained at this concentration.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Concentration response of nicotine. A: mean arterial pressure (MAP) responses. Abscissa, concentrations (Conc; µM); ordinate, decrease in MAP (mmHg). The decreases in MAP, in response to microinjections (50 nl) of nicotine into the medial nucleus of the solitary tract (NTS), ranged from 22 ± 4.5 to 45 ± 2.9 mmHg; the responses plateaued at 0.5 µM concentration. There was no significant difference between the responses elicited by 0.5, 1, and 10 µM concentrations. B: heart rate (HR) responses. Abscissa, concentrations (µM); ordinate, decrease in HR (beats/min). The decreases in HR, in response to microinjections (50 nl) of nicotine into the medial NTS, ranged from 15 ± 1.9 to 34.5 ± 7.7 beats/min (bpm). Maximum responses (both BP and HR) were elicited by a 10 µM concentration; in general HR responses were inconsistent.

Effect of -BT. In this (n = 6) and other series of experiments, using multibarrel micropipettes, microinjections of L-Glu (5 mM, 50 nl) were used to identify the NTS-region from which depressor and bradycardic responses were elicited. After an interval of 5 min, nicotine (10 µM, 50 nl) was microinjected at the same site, and a decrease in MAP was observed. -BT (1 µM, 100 nl) was microinjected at the same site through another barrel of the micropipette. -BT and other toxins or blockers used in this and other series of experiments did not produce any changes in basal MAP or HR. In this and other series of experiments, a larger volume of the nicotinic receptor toxin or blocker (100 nl instead of 50 nl used for the agonist) was used to ensure that most of the nicotinic receptors at the desired site were blocked. After an interval of 3 min, nicotine (10 µM, 50 nl) was again microinjected at the same site and the decrease in MAP was significantly smaller (20% smaller; P < 0.01) compared with the first nicotine-induced response (Fig. 2A). In other experiments (n = 2), an identical protocol was used, except that a higher concentration of -BT (250 µM) was microinjected; the reduction in nicotine-induced decreases in MAP remained at 20%. Similarly, repetition of the same protocol using a 500 µM concentration of -BT did not elicit any further decrease in nicotine-induced depressor responses; the reduction of the depressor response remained relatively small (20%). On the basis of these results, a 1 µM concentration of -BT was selected for further studies. Nicotine-induced decreases in HR before and after the microinjection of -BT (1 µM, 100 nl) were not statistically different (Fig. 3A). To test if microinjections of -BT into the medial NTS exerted any deleterious neuronal effects at the site of injection, L-Glu (5 mM, 50 nl) was microinjected at the same site (n = 5). The L-Glu-induced decreases in MAP (Fig. 2A) and HR (Fig. 3A) before and after the microinjection of -BT (1 µM, 100 nl) were not statistically different.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Effect of nicotinic receptor blockers/toxins on the nicotine-induced MAP responses. In each panel, depressor responses to microinjections (50 nl) of nicotine (10 µM; solid bars) and L-glutamate (L-Glu; 5 mM; hatched bars) into the medial NTS are shown before and after the microinjections of different toxins or blockers. A: nicotine-induced decreases in MAP before and after -bungarotoxin (-BT; 1 µM) were 26.2 ± 2.6 and 21.0 ± 3.0 mmHg, respectively (P < 0.01; paired t-test). Note that in this and other panels, microinjections of respective toxins or blockers did not alter depressor responses to microinjections of an unrelated agonist, L-Glu, at the same site (hatched bars). For example, L-Glu-induced decreases in MAP before and after -BT (1 µM) were 21.6 ± 1.8 and 20.6 ± 1.6 mmHg, respectively (P > 0.05; paired t-test). B: nicotine-induced decreases in MAP before and after microinjections of -conotoxin ImI (1 µM) were 28.1 ± 7 and 17.5 ± 4.4 mmHg (P < 0.05). C: the decreases in MAP before and after microinjections of -conotoxin AuIB (1 µM) were 29.7 ± 5.3 and 6.3 ± 1.7 mmHg, respectively (P < 0.01). D: the decreases in MAP before and after microinjections of mecamylamine (Mec; 1 mM) were 32.4 ± 6.3 and 10 ± 1.8 mmHg, respectively (P < 0.05). E: the decreases in MAP before and after microinjections of hexamethonium (C6; 1 mM) were 28.8 ± 4.3 and 15.3 ± 3.2 mmHg, respectively (P < 0.05). F: the decreases in MAP before and after microinjections of -BT (Bung; 1 µM) and mecamylamine (1 mM) were 34.2 ± 2.4 and 0.6 ± 0.4 mmHg, respectively (P < 0.01).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Effect of nicotinic receptor blockers/toxins on the nicotine-induced HR responses. In each panel, bradycardic responses to microinjections (50 nl) of nicotine (10 µM; solid bars) and L-Glu (5 mM; hatched bars) into the medial NTS are shown before after the microinjections of different toxins or blockers. A: nicotine-induced decreases in HR before and after -BT (1 µM) were 32.5 ± 7 and 21.3 ± 3.9 beats/min, respectively (P > 0.05; paired t-test). Note that in all panels, microinjections of respective toxins or blockers did not alter bradycardic responses to microinjections of an unrelated agonist, L-Glu, at the same site; for example, L-Glu-induced decreases in HR before and after -BT (1 µM) were 59.2 ± 11.1 and 60.4 ± 13.5 beats/min, respectively (P > 0.05). B: nicotine-induced decreases in HR before and after microinjections of -conotoxin ImI (1 µM) were 9 ± 1 and 6.5 ± 1.5 beats/min (P > 0.05). C: the decreases in HR before and after microinjections of -conotoxin AuIB (1 µM) were 14.5 ± 4.7 and 9 ± 6.4 beats/min, respectively (P > 0.05). D: the decreases in HR before and after microinjections of mecamylamine (1 mM) were 5 ± 2.2 and 2 ± 2 beats/min, respectively (P > 0.05). E: the decreases in HR before and after microinjections of hexamethonium (1 mM) were 16.7 ± 5.3 and 14.7 ± 5.1 beats/min, respectively (P > 0.05). F: the decreases in HR before and after microinjections of -BT (1 µM) and mecamylamine (1 mM) were 9 ± 3.3 and 1 ± 1 beats/min, respectively (P < 0.05).

Effect of ImI. The protocol in this (n = 4) and other series of experiments using different toxins or blockers was similar to that described for -BT. The nicotine-induced decrease in MAP after the microinjection of ImI (1 µM, 100 nl) into the NTS was significantly smaller (38% smaller; P < 0.05) compared with the first nicotine response (Fig. 2B). The differences in nicotine-induced decreases in HR before and after the microinjection of ImI were not statistically significant (Fig. 3B). The decreases in MAP (Fig. 2B) and HR (Fig. 3B) induced by microinjections of L-Glu (5 mM, 50 nl) into the NTS before and after the microinjection of ImI at the same site were not statistically different.

Effect of AuIB. The decrease in MAP induced by a microinjection of nicotine into the NTS after the microinjection of AuIB (1 µM, 100 nl) at the same site was significantly smaller (79%; P < 0.01) compared with the first nicotine response (Fig. 2C) (n = 5). In these experiments, nicotine-induced decreases in HR before and after the microinjection of AuIB (100 µM) into the NTS were not statistically different (Fig. 3C). In other experiments (n = 10), an identical protocol was used except that different concentrations (2, 5, 50, 75, and 100 µM) of AuIB were microinjected into the NTS. The reduction in nicotine-induced depressor responses by microinjections of these higher concentrations of AuIB into the NTS remained comparable to that induced by a 1 µM concentration of AuIB. For example, after the microinjection of a 100 µM concentration of AuIB into the NTS, the nicotine-induced decrease in MAP was significantly smaller (P < 0.05) compared with the first nicotine response; the reduction in nicotine-induced depressor response was 74%. The decreases in MAP (Fig. 2C) and HR (Fig. 3C) induced by microinjections of L-Glu (5 mM, 50 nl) into the NTS before and after the microinjection of AuIB (1 µM, 100 nl) were not statistically different. Even at higher concentrations (100 µM, 100 nl), microinjections of AuIB into the NTS did not exert any deleterious neuronal effects at the site of injection; the L-Glu-induced decreases in MAP before and after the microinjection of AuIB were not statistically different.

Effect of mecamylamine. In this group of rats (n = 5), the nicotine-induced decrease in MAP after the microinjection of mecamylamine (1 mM, 100 nl) was significantly smaller (69%; P < 0.05) compared with the first nicotine-induced response (Fig. 2D). Nicotine-induced decreases in HR before and after the microinjection of mecamylamine were not statistically different (Fig. 3D). Mecamylamine did not exert any deleterious neuronal effects at the site of injection. The L-Glu (5 mM, 50 nl)-induced decreases in MAP (Fig. 2D) and HR (Fig. 3D) before and after the microinjection of mecamylamine were not statistically different.

Effect of hexamethonium. In this group of rats (n = 5), the nicotine-induced decrease in MAP after the microinjection of hexamethonium (1 mM, 100 nl) into the NTS was significantly smaller (47%; P < 0.05) compared with the first nicotine response (Fig. 2E). Nicotine-induced decreases in HR before and after the microinjection of hexamethonium were not statistically different (Fig. 3E). Hexamethonium did not exert any deleterious neuronal effects at the site of injection. The L-Glu (5 mM, 50 nl)-induced decreases in MAP (Fig. 2E) and HR (Fig. 3E) before and after the microinjection of hexamethonium were not statistically different.

Effect of sequential microinjections of -BT and mecamylamine. In this group of animals (n = 5), the medial NTS was identified by microinjections of L-Glu (5 mM, 50 nl) that induced the usual decreases in MAP and HR (Figs. 4A and 2F). Microinjection of nicotine (10 µM, 50 nl) at the same site elicited a decrease in MAP (Figs. 4B and 2F), with little or no change in HR (Figs. 4B and 3F). After an interval of 7 min, -BT (1 µM, 100 nl) was microinjected at the same site. As stated earlier, the toxin alone produced no changes in MAP or HR. Three minutes after the injection of the toxin, nicotine (10 µM, 50 nl) was again microinjected at the same site; the decrease in MAP was reduced (20%) (Fig. 4C) compared with the first nicotine-induced response. Seven minutes after that, mecamylamine (1 mM, 100 nl) was microinjected at the same site and, within 3 min of mecamylamine injection, nicotine (10 µM, 50 nl) was injected at the same site. The depressor and bradycardic responses to nicotine were almost completely (98.2%; P < 0.01) blocked (Figs. 4D, 2F, and 3F). After this almost complete blockade of nicotinic receptors in the NTS, by sequential injections of -BT and mecamylamine, microinjections of L-Glu into the NTS elicited a small but significant (P < 0.05) increase in the depressor responses (Fig. 2F), whereas the HR responses remained unaltered (Fig. 3F).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Tracings showing complete blockade of nicotine (Nico)-induced cardiovascular responses by combined microinjections of mecamylamine and -BT (Bungarotx) into the medial NTS (multibarreled micropipettes were used so that various agents could be microinjected at the same site). A: medial NTS was identified by microinjections of L-Glu (5 mM, 50 nl; small arrow); a decrease in MAP (50 mmHg) and HR (85.7 beats/min) was elicited. B: after 5 min, nicotine (10 µM) was microinjected (50 nl) at the same site; a decrease in MAP (50 mmHg) was observed but no bradycardia was elicited. After 7 min, -BT (1 µM) was microinjected at the same site (large arrow between B and C); no changes in BP or HR were induced by this toxin. C: 3 min later, nicotine (10 µM) was again microinjected at the same site; the depressor response (35.7 mmHg) was smaller (20% reduction) compared with the response in A. After 7 min, mecamylamine (1 mM) was microinjected at the same site (large arrow between C and D); it did not alter BP or HR. D: 3 min later, nicotine injection (10 µM) into the NTS was repeated; the response to nicotine was completely blocked. E: within 3 min, L-Glu was again microinjected at the same site; the depressor response (60.7 mmHg) was greater (21.4%) compared with the first response to L-Glu.

Effect of blockade of excitatory amino acid receptors. With the use of multibarreled micropipettes, the depressor region of NTS was identified by unilateral microinjections of L-Glu in another group of rats (n = 7). Within 5 min, nicotine (10 µM, 50 nl) was microinjected at the same site and a decrease in MAP was observed (Fig. 5). After an interval of 7 min, DAP-7 (10 mM, 100 nl), NBQX (2 mM, 100 nl), and MCPG (40 mM, 100 nl) were microinjected sequentially at the same site within 2-min intervals. Two minutes after the last microinjection (i.e., MCPG), nicotine (10 µM, 100 nl) was again microinjected at the same site; the nicotine-induced decrease in MAP was not significantly different from initial nicotine responses (Fig. 5).

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Effect of excitatory amino acid receptor (EAAR) blockade on nicotine-induced responses. Nicotine (10 µM) was microinjected (50 nl) into the medial NTS before and after the blockade of EAA receptors. EAA receptor blockade was accomplished by microinjecting (50 nl each) sequentially (within intervals of 2 min), D()-2-amino-7-phosphonoheptanoic acid (10 mM), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxalline (2 mM), and -methyl-4-carboxyphenylglycine (40 mM) into the same site; these doses of the EAAR blockers have been previously shown to be specific for their respective agonists (7). Nicotine-induced decreases in MAP before and after EAAR blockade were 46.4 ± 5.9 and 47.9 ± 8.4 mmHg, respectively (P > 0.05). These results indicate that the depressor responses induced by microinjections of nicotine into the medial NTS are not mediated via EAARs.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

There are very few reports in which nicotine has been directly microinjected into the NTS to activate nicotinic receptors (9, 28). In one of these reports (9), microinjections of nicotine into the medial NTS (1 mm caudal to 0.5 mm rostral to the calamus scriptorius, 0-0.5 mm lateral to the midline, and 0.2-0.5 mm deep) elicited a biphasic response; an initial increase was followed by a decrease (20-48 mmHg) in BP. In another study (28), microinjections (60 nl) of nicotine (0.06-6.15 mM) into the NTS (0-0.5 mm rostral to the calamus scriptorius, 0.35-0.5 mm lateral to midline, and 0.4-0.55 mm deep from the dorsal surface of the medulla) elicited depressor (10-42 mmHg) and bradycardic (10-50 beats/min) responses. Our results are in general agreement with aforementioned reports (9, 28) in that depressor and bradycardic responses were elicited by microinjection of nicotine into the medial NTS. Our bradycardic responses were not robust, and we did not observe the initial increase in BP that was reported by others (9). Because of the relatively large volumes (200 nl) and concentrations of nicotine (0.3-3 mM) used by others in their studies (9), the initial increase in BP is likely due to the spread of nicotine to the chemoreceptor projection zone in the midline region of the commissural subnucleus of NTS. Activation of this chemoreceptor projection site in the NTS has been reported to elicit pressor responses (8, 14, 20).

On the basis of the current knowledge regarding the organization of medullospinal cardiovascular centers, the mechanism by which nicotine exerts its depressor and bradycardic action can be postulated as follows. Activation of nicotinic receptors in the medial NTS results in excitation of secondary NTS neurons that, in turn, excite neurons located in the caudal ventrolateral medullary depressor area (CVLM) (32). Excitation of the CVLM neurons results in the inhibition of neurons in the ventrolateral medullary pressor area (RVLM) via a GABAergic mechanism (33-35). Inhibition of the RVLM neurons decreases the excitatory input to sympathetic preganglionic neurons located in the intermediolateral cell column (23, 24) and results in a depressor response and a slight bradycardic response. The bradycardic response may involve a direct projection from the NTS to the NA, where the preganglionic parasympathetic neurons that provide vagal innervation to the heart are located (15, 16). Excitation of the NTS neurons may activate the aforementioned NA neurons and elicit bradycardia; however, microinjections of nicotine into the NTS elicited relatively small and inconsistent HR responses, suggesting that the distribution of nicotinic receptors on the secondary NTS neurons involved in bradycardic responses is not dense. Alternatively, the neurons mediating HR responses may be more sensitive to anesthesia causing the attenuation of HR responses to microinjections of L-Glu into the NTS.

Microinjections of -BT and ImI into the medial NTS at concentrations known to be specific for nicotinic receptors containing 7 but not 34 subunits (10) elicited only 20-38% attenuation of depressor responses induced by microinjections of nicotine (10 µM) at the same site. These observations suggest that a relatively small proportion (20-38%) of nicotinic receptors containing 7 subunits is involved in mediating the depressor responses induced by microinjections of nicotine into the medial NTS.

On the other hand, microinjections of AuIB at concentrations (1 µM) known to be specific for nicotinic receptors containing 34 subunits (12) elicited an ~79% attenuation of depressor responses induced by microinjections of nicotine (10 µM) into the NTS. Mecamylamine, an agent believed to be a nicotinic receptor channel blocker as well as a competitive antagonist, also elicited a 69% block of nicotine-induced responses. Recently, mecamylamine has been shown to be a potent noncompetitive inhibitor of neuronal 34 subtype nicotinic receptor (4, 31). The inhibition of 34 subtype of nicotinic receptor by mecamylamine has the characteristics of an open channel block and exhibits voltage dependence (31). Hexamethonium elicited an ~47% block of nicotine-induced responses. This agent, although not very specific, has also been reported to be a potent antagonist at nicotinic receptors containing 34 subunits (4, 27). Collectively, the results obtained by using AuIB, mecamylamine, and hexamethonium suggest that a relatively greater proportion (~47-80%) of nicotinic receptors containing 34 subunits is involved in mediating the depressor responses induced by microinjections of nicotine into the medial NTS.

The aforementioned results suggest that a heterogenous population of nicotinic receptors is present in the medial NTS, making it necessary to use a combination of antagonists to completely block the responses to microinjections of nicotine into the NTS. When -BT (1 µM) and mecamylamine (1 mM) were microinjected sequentially into the NTS (using multibarreled micropipettes) before the microinjection of nicotine, the depressor responses were 98% blocked. Combined microinjections of -BT and mecamylamine into the NTS did not attenuate the responses to L-Glu, indicating that no deleterious effects on the NTS neurons occurred and the attenuation of the nicotine response was in fact due to the blockade of the nicotinic receptors.

In the CNS, nicotinic receptors are located on the presynaptic terminals as well as on the neuronal soma. In other areas of the brain, e.g., in the NA, nicotine has been reported to excite parasympathetic cardiac neurons by enhancing glutamatergic neurotransmission via both presynaptic and postsynaptic mechanisms (15, 16). In these studies (15, 16), the presynaptic facilitation of glutamatergic neurotransmission was blocked by -BT, whereas the postsynaptic augmentation of non-NMDA currents was mediated via -BT-insensitive nicotinic receptors. We hypothesized that a similar situation may exist in the NTS, i.e., activation of nicotinic receptors in the NTS may activate both presynaptic and postsynaptic excitatory amino acid receptors, depolarize NTS neurons and elicit a depressor response. However, complete blockade of NMDA, non-NMDA, and metabotropic excitatory amino acid receptors in the NTS did not attenuate or abolish the depressor responses to microinjections of nicotine at the same site. In these experiments, the concentrations of DAP-7, NBQX, and MCPG were selected from our previous studies (8). At these concentrations, these antagonists blocked completely and specifically the responses to their respective agonists (i.e., NMDA, AMPA, and ACPD, respectively). The observation that the complete blockade of excitatory amino acid receptors in the NTS had no effect on the nicotine responses suggested that presynaptic nicotinic receptors in this region did not significantly contribute to the nicotine-induced cardiovascular responses. Complete blockade of nicotinic receptors in the NTS on one side did not alter basal BP or HR. This observation does not necessarily indicate that the cholinergic mechanisms involving nicotinic receptors in the NTS are not tonically active. Perhaps a bilateral blockade of nicotinic receptors in the NTS may be necessary to reveal if these receptors are tonically active. Similar observations have been reported for excitatory amino acid receptors in the NTS, which are known to be tonically active, and bilateral blockade of these receptors in the NTS is in fact necessary to elevate basal levels of BP and HR (26).

We conclude that the predominant nicotinic receptors in the NTS involved in mediating depressor responses may contain 34 subunits, because AuIB elicited 60-70% attenuation of nicotine-induced depressor responses. A smaller component (20-30%) of nicotine-induced depressor response may involve nicotinic receptors containing 7 subunits, because ImI and -BT produced a 20-30% attenuation of nicotine-induced responses. For complete blockade of nicotine-induced cardiovascular responses, it was necessary to block nicotinic receptors containing both types (i.e., 34 and 7) of subunits. It is not clear, however, whether the nicotinic receptors containing 7 and 34 subunits are located on the same or different NTS neurons.

Perspectives