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Journal of Physiology (2002), 545.3, pp. 1007-1016
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.021337

Nicotinic ACh receptor subtypes on gastrointestinally projecting neurones in the dorsal motor vagal nucleus of the rat

Niaz Sahibzada *†, Manuel Ferreira Jr *, Bernice Williams †, Adam Wasserman *, Stefano Vicini ‡ and Richard A. Gillis *

	ABSTRACT

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To determine the predominant nicotinic ACh receptor (nAChR) located on neurones in the dorsal motor nucleus of the vagus (DMV) that project to the gastrointestinal tract, we used the rat brainstem slice preparation and whole-cell recordings of DMV neurones identified by retrograde DiI tracing to pharmacologically characterize nAChRs. Pressure ejection of acetylcholine (ACh, 250 µM for 200 ms) from a patch pipette placed ~10-20 µm from the surface of the recorded cell produced an inward current in most DMV neurones sampled. The average currents for neurones projecting to the fundus, antrum and caecum were 149 ± 38 (n = 25), 115 ± 18 (n = 29) and 117 ± 23 pA (n = 6), respectively. Blockade of the 7 subtype of nAChR with either -bungarotoxin (-BGT) or methyllycaconitine (MLA) counteracted 60-75 % of the ACh-evoked current in DMV neurones projecting to the fundus, antrum and caecum. In neurones projecting to the fundus and the antrum, currents resistant to -BGT were significantly blocked by dihydro--erythroidine (10-20 nM), an antagonist of the 42 subtype of nAChR. In neurones projecting to the caecum, currents resistant to -BGT were significantly depressed by a low concentration of mecamylamine (1 µM). Cytisine (100 µM), an agonist of nAChRs that contain the 7 or the 4 subunit, evoked significant currents in caecum-projecting neurones that were previously exposed to -BGT. In contrast, cytisine had no effect on DMV neurones previously exposed to -BGT that project to the fundus or antrum. Our data indicate that the prevailing nAChR subtype in DMV neurones projecting to the GI tract is the 7 subtype. In addition, we obtained evidence for the co-expression of the 42 nAChR subtype on DMV neurones projecting to the fundus and antrum, and the 34 nAChR subtype on DMV neurones projecting to the caecum.

(Received 26 March 2002; accepted after revision 26 September 2002; first published online 25 October 2002)
Corresponding author N. Sahibzada: Department of Pharmacology, Georgetown University Medical Center, Washington, DC 20007, USA. Email: sahibzan{at}georgetown.edu

	INTRODUCTION

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Recently Zoli et al. (1998) concluded that there are four classes of brain nicotinic receptors based, in part, on experimental data obtained from 2 mutant mice, and, in part on in situ hybridization data obtained in the rat. These four classes were the 7 (type 1); receptors containing the 2 subunit, most probably the 42 subtype (type 2); receptors containing the 4 subunit (34, possibly the 345 (type 3); and a fourth type (type 4) which also involves the 4 subunit, but which appears to also contain the 4 and/or the 2 subunit. These investigators have also determined the predominant localizations in the brain for each of the four receptor classes. In the case of the type 1 or 7 nAChR, they concluded that this receptor was located primarily in the cortex and limbic areas (Zoli et al. 1998). Type 2, which primarily consists of the 42 nAChR, was described as located throughout the brain. In contrast, the type 3 or 34 nAChR was stated as being located in the dorsocaudal medulla oblongata, specifically in the nucleus tractus solitarius (NTS), dorsal motor nucleus of the vagus (DMV) and area postrema. It was also listed as being located in the medial habenula and interpeduncular nucleus. Type 4 or those nAChRs that co-assemble with the 4 subunit were reported to be predominantly located in the lateral medial habenula and dorsal interpeduncular nucleus.

Since the classification of CNS nAChRs by Zoli et al. (1998), data have been reported for the dorsocaudal medulla, specifically on nAChRs located in the DMV, that are at odds with the conclusion that the 34 nAChR is the predominant nAChR subtype at this brainstem site. For example, we have used an in vivo anaesthetized rat preparation to characterize the important functional nAChRs in this nucleus of the dorsocaudal medulla oblongata. Our results indicated that the 7 nAChR subtype is the major nAChR in this nucleus (Ferreira et al. 2000, 2001), and were based on microinjection studies of nicotine (and antagonists of nAChRs) into the DMV while monitoring gastric function, and in vitro autoradiography and immunohistochemistry of the DMV. We also demonstrated that this 7 nAChR was located at a postsynaptic site (Ferreira et al. 2001). Zaninetti et al. (1999) reported that the 7 and the 42 nAChRs were completely responsible for mediating the excitatory effects of acetylcholine elicited from DMV neurones. No evidence for the existence of an 34 nAChR subtype in the DMV was obtained. Their data was based on using patch clamp recording techniques and pharmacological agents in brain slice preparations containing the DMV.

In situ hybridization data (Fig. 7b and c, Zoli et al. 1998) indicate that the 3 subunits are present in the DMV, but are located laterally within this nucleus. If so, our previous study (Ferreira et al. 2001) would not have detected the 34 subtype of nAChR in the DMV, because changes in gastric function were used to monitor receptor subtype and the lateral DMV projects to the caecum (Altschuler et al. 1991). Furthermore, in Zaninetti et al. (1999), no information was provided as to whether the DMV neurones studied electrophysiologically exited the brain and reached the periphery, let alone innervated the caecum.

In view of the contradictory findings as to which is the predominant nAChR in the DMV, we employed the in vitro brain slice preparation and characterized the nAChRs in DMV neurones that project to two locations in the stomach and to one location in the caecum. Our results indicate that the major functional nAChR in the DMV is the 7 nAChR. This is true regardless of whether we are studying DMV neurones projecting to the antrum, fundus or caecum. Interestingly, the 42 and the 34 nAChR subtypes play a significant but minor role in the DMV control of the stomach and caecum, respectively.

	METHODS

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All procedures were performed in accordance with NIH guidelines and with the approval of the Animal Care and Utilization Committee of Georgetown University, Washington, DC, USA.

Retrograde tracer application

To identify DMV neurones that project to the gastrointestinal region of interest, the fluorescent retrograde tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes) was used (Mendelowitz & Kunze, 1991; Browning et al. 1999). Sprague-Dawley rats (postnatal days 17-25) of either sex were anaesthetized with isoflurane (4 % induction; 2 % maintenance with room air), and an abdominal laparotomy was performed. Crystals of DiI were applied to the serosal surface of the gastric fundus, antrum and caecum. The application site was embedded in a fast-hardening epoxy resin (Bipax, Tra-Con, Inc.) that was allowed to dry for several minutes before the entire surgical area was washed with warm saline. The wound was closed with 4-0 silk sutures and the animal allowed to recover for 5-15 days. Peanut oil was applied around the incision area to guard against animals interfering with the wound healing process. In a few experiments, adult animals were treated in similar fashion, and gastric- and caecum-projecting neurones were identified with retrograde DiI tracing.

Slice preparation

Brainstems were removed subsequent to decapitation, which was undertaken during isoflurane anaesthesia. They were placed in ice-cold (4 °C) artificial cerebrospinal fluid (ACSF) solution (pH 7.4) containing (mM): NaCl 120; KCl 3.1; NaHCO₃ 26; K₂HPO₄ 1; CaCl₂ 2; MgCl₂ 1; glucose 5; sucrose 10; ascorbic acid 0.4; myo-inositol 3 and sodium pyruvate 2. The solution was oxygenated with a carbogen mixture of 95 % O₂ and 5 % CO₂. Coronal brainstem sections (250 µm) containing the DMV (1 mm rostral and 1 mm caudal to the calamus scriptorus) were obtained using a vibratome. Prior to recording, the slices were incubated in oxygenated ACSF solution at 37 °C for 1 h.

Electrophysiological recording

Brain slices were transferred to a recording chamber (volume 700 µl) attached to the stage of a Nikon E600-FN microscope. They were continuously perfused with ACSF solution at room temperature (21-22 °C) (composition (mM): NaCl 120; KCl 3.1; NaHCO₃ 26; K₂HPO₄ 1; CaCl₂ 2; MgCl₂ 1; glucose 5 and sucrose 10 (pH 7.4)). The solution was continuously exposed to a mixture of 95 % O₂ and 5 % CO₂ to enrich it with oxygen and to maintain the pH (7.4). Picrotoxin (50 µM) and atropine (10 µM) were added to this solution to block GABA receptor chloride channels and muscarinic cholinergic receptors, respectively.

Neurones in the DMV area were identified visually by infrared-differential interference contrast (IR-DIC) and fluorescence optics via a CCD camera (Dage S-75) (Fig. 1A, C and D). A 60 water immersion objective lens (Nikon) was used for identifying and approaching neurones. To avoid photolytic damage, initial exposure to episcopic fluorescence illumination was brief (< 2 s). Once the experiment was terminated, the neurone underwent a longer fluorescence exposure to ensure that it was labelled. Only one neurone per slice was recorded.

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Figure 1. Technique of recording from a retrogradely labelled DMV neurone projecting to the fundus A, photomicrograph showing the position of the patch electrode (PE) in relation to the ACh 'puffer' pipette (PS). B, inward current in response to a 5 s application of ACh (250 µM) to a DMV neurone voltage clamped at -60 mV. C and D, infrared and fluorescence photomicrographs showing the fundus cell from which the recording in B was made. Abbreviations: AP, area postrema; CC, central canal; DMV, dorsal motor nucleus of the vagus; TS, tractus solitarius; XII, hypoglossal nucleus. Scale bars: A = 134 µm; C = 20 µm.

Whole-cell voltage clamp (holding potential -60 mV) recordings of fluorescent DMV neurones were made with patch electrodes (5-6 M) containing a solution (pH 7.2) composed of (mM): potassium gluconate 145; EGTA 5; MgCl 2.5; Hepes 10; Na-ATP 5; and Na-GTP 0.2. The patch pipette was coupled to an amplifier (Axopatch 1-D; Axon Instruments Inc.) and its signal filtered (5 kHz), digitized (Digidata 1200C; Axon Instruments Inc.) and stored on a PC running pCLAMP 8 software (Axon Instruments Inc.) for later analysis. Series resistance was < 10 M and was continuously monitored.

Drug application

To study drugs with agonist properties, rapid focal application was used to minimize loss of effect due to receptor desensitization (Cuevas & Berg, 1998). Focal application was performed using a pressure ejection technique. Pulses of receptor agonist drugs (ACh, 250 µM; cytosine, 100 µM; nicotine, 100 µM; choline, 10 mM) were delivered by pressure from a second patch pipette (10 M; 5-15 p.s.i.) via a Picospritzer (World Precision Instruments Inc.). The pipette was positioned under visual guidance ~10-20 µm away from the surface of the soma of the recorded neurone (Fig. 1A). The interval between drug applications was > 1 min.

Rationale for drug concentrations selected was as follows: ACh was the primary drug studied and was used at a concentration of 250 µM in the spritz pipette. This concentration is in the range used by others who have applied ACh in this way to neurones (Frazier et al. 1998; Zaninetti et al. 1999). The duration of ACh application as well as other receptor agonist drugs employed (i.e. nicotine, cytisine and choline) was 200 ms. Preliminary results indicated that this duration allowed the peak response to be detected without any desensitization and occurred within minutes (see Fig. 1B and Fig. 2A). Nicotine and cytisine were used with pipette concentrations similar to that of ACh since our earlier studies indicated that these three agonists produce similar responses at concentrations of 3-100 µM in DMV slices (Bertolino et al. 1997). Furthermore, these agonists are roughly equipotent and equi-effective at the 7 nAChR subtype (Chavez-Noriega et al. 1997). The dose for choline (10 mM), a relatively selective 7 nAChR agonist, was derived from the studies of Alkondon et al. (1997).

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Figure 2. Pressure ejection of ACh (250 µM for 200 ms) on a DMV neurone projecting to the fundus evokes an inward current A, recording showing that ACh application (interstimulus interval > 1 min) produces inward currents that are not attenuated by repeat application of the agonist. B, enlarged representation of the inward current portion indicated between the dashed lines in A.

Antagonists of nAChRs were delivered to the DMV neurone via bath application. The protocol followed was to first obtain stable current responses from pressure ejection of one of the aforementioned nAChR agonist drugs. Next, the slice was exposed either to -bungarotoxin (-BGT), methyllycaconitine (MLA), dihydro--erythroidine (DHE) or mecamylamine. After 5 min of exposure to a nAChR antagonist, the agonist drug was retested. To guard against possible drug interactions, the order in which a drug was applied varied from neurone to neurone such that a similar number of neurones was exposed first to either one drug or another.

The selected concentrations of -BGT (100 nM) and MLA (10 nM) were based on data from others which show them to be selective for blocking the 7 nAChR (Gopalakrishnan et al. 1995; Wong et al. 1995; Palma et al. 1996; Cuevas & Berg, 1998; Frazier et al. 1998; Genzen et al. 2001). The selected concentrations of DHE (10 and 20 nM) were based on findings of others that concentrations in this range are relatively selective for blocking the 42 nAChR subtype (Alkondon & Albuquerque, 1993; Marks et al. 1999; Genzen et al. 2001). The selected concentration of mecamylamine (1 µM) was based on the data of Frazier et al. (1998), who reported that it had no effect on the 7 nAChR subtype. Finally, the concentrations of atropine and picrotoxin employed in our study were based on data from our previous published studies (Travagli et al. 1991; Bertolino et al. 1997).

Data analysis

In general, data reported are presented as means ± S.E.M. In the case of rise time and half-width current measurements, data are reported as means ± S.D. Responses to nAChR agonist drugs are given as the peak amplitude of whole-cell currents expressed in picoamps. The effects of antagonists on responses to specific agonists are expressed as the percentage reductions of the peak amplitude. The full response of an agonist with no antagonist present was designated as 100 %. Statistical significance was determined using Student's paired t test (P < 0.05).

Source of drugs

Except for DiI (Molecular Probes, Eugene, OR, USA) and isoflurane (Abbot Laboratories), all of the drugs used in our study were purchased from Sigma Chemical Co. (St Louis, MO, USA).

	RESULTS

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Data obtained using antagonists of the 7 nAChR subtype on ACh-induced responses of DMV neurones projecting to the stomach (fundus and antrum) and to the caecum

7 nAChR on neurones projecting to the fundus. Pressure ejection of ACh (250 µM for 200 ms) produced an inward current in 25 of the 33 DiI-labelled neurones. The peak amplitude of the current was 149 ± 38 pA (range: 21-383 pA; n = 25), which was reached in 28 ± 49 ms (Fig. 2). The half-width value of the current averaged 454 ± 201 ms. The response was reproducible if the interval between ACh applications was > 1 min (Fig. 2A). The ACh-evoked current from fundus-projecting DMV neurones could be recorded in the presence of tetrodotoxin (TTX) at a 1 µM concentration. Prior to perfusion of the brain slice preparation with TTX, ACh application evoked a current of 175 ± 46 pA (n = 5). In the presence of TTX, ACh still evoked a robust current from these neurones (151 ± 45 pA, P > 0.05 comparing ACh-evoked current responses before and after TTX, n = 5). These results indicate that ACh was exerting its effect by acting directly on the recorded neurone rather than activating it through a presynaptic site and subsequent release of an excitatory neurotransmitter.

Bath application of -BGT, 100 nM, for 5 min produced a significant antagonism (P < 0.05) of ACh-evoked responses in 25 neurones. The average response of the 25 ACh-responsive neurones was 42 ± 9.5 pA after 5 min exposure to -BGT. This amounted to a 72 ± 5.1 % (range: 19-100 %) reduction in the peak amplitude of ACh-evoked current by -BGT. An experiment depicting the antagonistic interaction between ACh and -BGT is shown in Fig. 3A.

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Figure 3. Blockade of ACh-elicited inward current by 7 nAChR antagonists in retrogradely labelled neurones projecting to the fundus A, blockade of ACh-induced current by -BGT (100 nM). B, similar inhibition of inward current by MLA at a dose (10 nM) specific for the 7 subtype. Note: assessment of recovery from nAChR antagonists blockade was based upon slice perfusion in ACSF for at least 15 min.

In nine of the 25 neurones studied, 5 min exposure to -BGT resulted in nearly complete blockade of the ACh-evoked response. The block in these nine cells averaged 92 ± 5.5 % (range: 88-100 %). The distribution of the percentage reductions plotted as a histogram (not shown), did not show them to be a separate population with exclusively 7 nAChRs but merely one end of a continuum.

To determine if the -BGT block was reversible, recovery was followed in nine neurones that included those completely (n = 4) or partially (n = 5) blocked by -BGT exposure. A partial recovery of 53 ± 10.1 % of the ACh-evoked current (87 ± 22 pA compared with 156 ± 29 pA) was observed after an interval of at least 15 min following removal of -BGT from the superfusate (e.g. Fig. 3A). There was no significant difference in recovery from -BGT inhibition between neurones that were completely blocked or partially blocked.

For studies with MLA, recordings were made from a total of 18 DMV neurones that had been labelled via application of DiI to the fundus. Pressure ejection of ACh (250 µM for 200 ms) produced an inward current in 10 of the 18 neurones studied. The peak amplitude was 105 ± 28 pA (range: 31-332 pA; n = 10). Bath application of MLA (10 nM) for 5 min produced a significant antagonism (P < 0.05) of ACh-evoked responses in the 10 neurones. The average response of the 10 neurones following exposure to MLA was 26 ± 8 pA, which amounted to a 75 ± 7.0 % (range: 19-92 %) reduction in the peak amplitude of the ACh-evoked current. An experiment depicting the antagonistic interaction between ACh and MLA appears as Fig. 3B.

Because our previous in vivo DMV studies in adult animals did not detect a blocking effect of MLA against nicotine (Ferreira et al. 2001), we determined whether this lack of effect of MLA was related to the age of the animal or to the agonist employed (i.e. nicotine vs. ACh). Thus, studies were carried out with MLA and nicotine in brainstem slices of neonate and adult animals (270-300 g). In neonate animals, nicotine (100 µM) was tested on six neurones. Five responded, and the peak amplitude of the current was 84 ± 22 pA. Repeat application of nicotine given at 5 min intervals produced reproducible responses. Perfusion of the preparation with MLA (10 nM) reduced the nicotine-induced response to 29 ± 10 pA, or by 62 ± 10 %. Corresponding data obtained for nicotine in adult DMV neurones projecting to the fundus were 76 ± 23 pA (control), 33 ± 8 pA after 5 min exposure to MLA, and the resulting block was 53 ± 6.0 % (n = 5, i.e. 5 of 5 neurones responded to nicotine).

7 nAChR on neurones projecting to the antrum. Pressure ejection of ACh (250 µM for 200 ms) produced an inward current in 29 of 37 DiI-labelled DMV neurones. ACh produced a rapidly rising inward current with a peak amplitude of 115 ± 18 pA (range: 25-445 pA; n = 29). It attained its peak amplitude in 52 ± 19 ms and a half-width value of 259 ± 120 ms. The ACh response was reproducible, providing the interval between ACh application to the neurone was > 1 min. A typical example is illustrated in Fig. 4A.

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Figure 4 Blockade of ACh-induced whole-cell current by bath application of -BGT (100 nM) in an antrum-projecintg (A) and in a caecum-projecting (B) DMV neurone.

Bath application of -BGT (100 nM) for 5 min produced a significant (P < 0.05) antagonism of ACh-evoked responses in the 29 neurones studied. Repeat application of ACh after -BGT exposure resulted in a 65 ± 5.0 % (40 ± 8 pA; range: 16-100 pA) reduction in the peak amplitude of ACh-evoked current. An experiment depicting the antagonist interaction between ACh and -BGT appears as Fig. 4A.

In six of the 29 neurones studied, 5 min exposure to -BGT resulted in nearly complete inhibition (98 ± 2 %; range: 88-100 %) of the ACh-evoked response.

Next, MLA was tested for its ability to antagonize the effect of ACh on DMV neurones projecting to the antrum. In these neurones (n = 6) pressure ejection of ACh (250 µM for 200 ms) produced an inward current whose peak amplitude was 70 ± 19 pA (range: 30-147 pA). Following bath application of MLA (10 nM), this response was reduced to 19 ± 1.0 pA, which amounts to a 61 ± 10 % (range: 34-89 %) reduction in the peak amplitude of ACh-evoked current.

7 nAChR on neurones projecting to the caecum. Six cells were studied and four responded to pressure ejection of ACh (250 µM for 200 ms). ACh produced a rapidly rising inward current that attained its peak amplitude in 76 ± 31 ms and had a half-width value of 358 ± 201 ms. The average peak amplitude of the ACh-evoked current was 117 ± 23 pA (range: 61-162 pA). A typical example is illustrated in Fig. 4B. As long as the interval between ACh application to the neurone was > 1 min, the ACh-evoked response was reproducible.

Bath application of -BGT (100 nM) for 5 min produced a significant (P < 0.05) antagonism of ACh-evoked responses. In the four neurones studied, repeat application of ACh after -BGT resulted in a 75 ± 14 % (range: 36-100 %) reduction in the peak amplitude (21 ± 8 pA) of the ACh-elicited current. An experiment depicting the antagonistic interaction between ACh and -BGT appears as Fig. 4B.

Studies of choline applied to DMV neurones projecting to the stomach. To further confirm the presence of the 7 nAChR subtype on DMV neurones, the selective 7 nAChR agonist choline (Alkondon et al. 1997) was applied to both fundus (n = 5) and antrum (n = 3) labelled cells. Choline (10 mM) induced a fast-decaying current in both cell types that was sensitive to -BGT (Fig. 5). The mean peak amplitude for both fundus and antrum cells was 100 ± 26 pA (range: 71-190 pA) and 82 ± 36 pA (range: 42-154 pA), respectively. Exposure to -BGT for 5 min resulted in > 80 % attenuation of the choline-induced current (fundus, 16 ± 1.2 pA; antrum, 12 ± 2.6 pA).

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Figure 5 Inhibition of the choline (10 mM)-evoked response by bath application of -BGT (100 nM) in a fundus-projecting DMV neurone. Note the fast decaying profile of the whole-cell current that is characacteristic of the 7 nAChR response.

Data obtained using antagonists of the 42 nAChR subtype on ACh-induced responses of DMV neurones projecting to the stomach (fundus and antrum) and to the caecum

Focusing on the ACh-evoked current responses that were resistant to -BGT in neurones projecting to the fundus, we tested a second antagonist, dihydro--erythroidine (DHE). We chose this agent because Zaninetti et al. (1999) recently reported that the combination of MLA and DHE blocked nearly all of the current responses produced by locally applied ACh to DMV neurones. However, in their study, no attempt was made to identify where the DMV neurone under study was projecting within the GI tract. Concentrations of DHE ranging from 10-20 nM were used, and were tested either after -BGT was tested and present in the perfusing solution (n = 5) or before -BGT was tested (n = 6) (total neurones evaluated with the combination of the two blockers was 11). Data are summarized in Table 1, and a representative experiment is shown in Fig. 6. As can be noted, combining DHE with -BGT produced an additional significant block (i.e. 14 ± 3.5 %, P < 0.05). Blockade with the combination amounted to 83 %.

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Figure 6 Attenuation of ACh (250 µM) response by a combination of antagonists that have been shown to be specific for the 7 and 42 nAChR subtypes. Note: assessment of recovery from nAChR antagonists blockade was based upon slice perfusion in ACSF for at least 15 min.

Focusing on the ACh-evoked current responses that were resistant to -BGT in neurones projecting to the antrum, we tested DHE either after -BGT was tested and present in the perfusing solution (n = 5), or before -BGT was present (n = 8) (total neurones evaluated with the combination of the two antagonists was 13). Data are summarized in Table 1. As can be noted, combining DHE with -BGT produced an additional significant block (23 ± 4.8 %, P < 0.05). Blockade with the combination amounted to 81 %. As in the case of the fundus, the above data indicate that DMV neurones projecting to the antrum are associated with the 7 and the 42 nAChR sbtypes.

Focusing on the ACh-evoked current responses that were resistant to -BGT in neurones projecting to the caecum (Table 1), it should be noted that in three of the four neurones blockade by -BGT was 78, 86 and 100 %. Hence, there was little current left to evaluate the antagonistic effect of DHE.

Additional pharmacological studies of DMV neurones projecting to the stomach (fundus and antrum) and to the caecum

The above data suggest that at least two subtypes of nAChRs are present on the DMV neurones projecting to the fundus, namely 7 and 42. To assess whether a 4 subtype is also present, experiments were performed using cytisine. This is an agonist at nAChRs containing the 4 subunit (Papke & Heinemann, 1994) and the 7 subtype (Chavez-Noriega et al. 1997). Provided that the interstimulus interval was > 5 min (Fig. 7), pressure ejection of cytisine (100 µM for 200 ms) evoked a fast reproducible inward current. To discern the presence or absence of the 4 subunit in fundus-projecting neurones, two types of experiments were performed. The first was to perfuse the brain slice with -BGT (100 nM) for 5 min and then pressure eject cytisine onto the neurone. In the four neurones studied using this protocol, no effect of cytisine was observed. The second experiment type was to pressure eject cytisine first, and then retest the agonist in the presence of a 5 min bath application of 100 nM -BGT. Three neurones were evaluated and two of the three responded to cytisine. The average current response was 147 pA. However, during perfusion of the slice with -BGT, the current response was reduced to 5 pA for a 95 % block of the response.

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Figure 7. Pressure ejection of cytisine (100 µM for 200 ms) on a DMV neurone projecting to the fundus evokes an inward current A, repeat cytisine application elicits reproducible inward currents that are resistant to attenuation, provided the interstimulus interval > 5 min. B, enlarged time scale representation of the inward current portion indicated between the dashed lines in A.

Cytisine was also pressure ejected onto antrum-projecting neurones (n = 5) in order to determine the presence of a 4 subunit. Cytisine elicited a current response whose mean amplitude was 47 ± 7.0 pA. Perfusion of the slice with -BGT resulted in a 100 % block of the cytisine-evoked response.

To address the question of whether a 4 subtype of nAChR is present on DMV neurones projecting to the caecum, experiments identical to those described for evaluating the presence of a 4 subtype on fundus- and antrum-projecting neurones were performed. Following bath perfusion of the brain slice preparation with -BGT (100 nM) for 5 min, cytisine was pressure ejected onto the neurone. Of the four neurones tested, three responded to cytisine with current responses of 18, 20 and 20 pA (mean = 19 ± 0.7 pA; P < 0.05). Adding DHE (10 or 20 nM) to the perfusing solution had little effect on these cytisine-evoked currents. In the next series of experiments, we pressure ejected cytisine first, and then retested the agonist in the presence of a 5 min bath perfusion of 100 nM -BGT. In the eight neurones that were evaluated, cytisine evoked current responses that averaged 54 ± 9.0 pA. Perfusion of the slice preparation with -BGT (100 nM) reduced this current to 25 ± 4 pA for a 50 ± 7.0 % block of the response. In four of the eight neurones, mecamylamine (1 µM) was added to the perfusing solution. In these neurones, following -BGT block of 52 ± 11 %, mecamylamine further blocked 41 ± 10 % of the remaining cytisine-induced response.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Our results indicate that the 7 subtype is by far the most prevalent nAChR subtype in DMV neurones projecting to the GI tract. Based on whole-cell patch clamp pharmacological studies of retrogradely labelled DMV neurones projecting to the fundus, antrum and caecum, greater than 60 % of the ACh-induced excitatory response of these neurones is mediated by 7 nAChRs. We demonstrate this by using two specific blockers of the 7 nAChR subtype, namely -BGT and MLA. The data we have obtained for DMV fundus-projecting neurones are closely matched by our in vivo rat studies where we have observed that approximately 60 % of the gastric response produced by nicotine microinjected into the DMV is due to nicotine exciting neurones that are of the 7 subtype (Ferreira et al. 2000, 2001). Our present data are also consistent with previous autoradiographic and immunohistochemical studies of 7 nAChRs on DMV neurones. Using [¹²⁵I]-BGT and a specific antibody to the 7 subunit, we demonstrated a high concentration of these nAChRs in the DMV of the rat, and these nAChRs largely disappeared in rats with chronic vagal nerve axotomy (Ferreira et al. 2001).

Other investigators have obtained data supportive of our conclusion that the 7 nAChR is the predominant nAChR in the DMV. Most impressive to us are the early findings of Hunt & Schmidt (1978). These investigators demonstrated that [¹²⁵I]-BGT, a radioligand that selectively binds to 7 nicotinic receptors, exhibits an intense degree of binding in the DMV. Equally impressive are the functional data of Zaninetti et al. (1999), where whole-cell recordings were performed on various motor nuclei in brainstem slices of rats. ACh was delivered by pressure microinjection to DMV neurones visualized using infrared videomicroscopy (but not identified by retrograde tracer placed in the GI tract). Thirteen vagal motoneurones were studied and 62 ± 4 % of the ACh-evoked current was blocked by an 7 nAChR subtype antagonist, namely MLA (10 nM). Additionally, immunocytochemical localization studies of the 7 subunit in the rat CNS by Dominguez del Toro et al. (1994), revealed a very strong signal in the DMV. Even studies of expression of nAChR 7 mRNA and [¹²⁵I]-BGT binding in human postmortem brain revealed considerable labelling in the DMV (Breese et al. 1997). It is perplexing to us that Zoli and colleagues (1998) were not informed by their studies that the 7 nAChR predominates in the DMV. We speculate that these investigators focused on forebrain areas for documenting the presence of the 7 nAChR, and used the hindbrain as CNS tissue for searching for other nAChR subtypes.

As noted in the Introduction, Zoli and colleagues (1998), in contrast to us, concluded that the predominant nicotine receptor in the DMV was the 34 subtype. Inspection of some of their data as well as earlier data of Wada et al. (1989), suggested that the 34 nAChR subtype might be located in the lateral portion of the nucleus. This is, in fact, what we have found. Our studies of lateral DMV projections to the caecum indicated that although the majority of the nAChRs present were the 7 subtype (~75 %), a small but significant number of the functional nAChRs in the lateral area were probably the 34 subtype. Evidence for this is based on the finding that -BGT-resistant ACh-induced responses were significantly antagonized by 1 µM mecamylamine (a concentration too low to affect the 7 nAChR subtype, Frazier et al. 1998). In addition, DHE in a concentration that blocked some of the ACh-evoked excitation in the fundus- and antrum-projecting neurones was ineffective in blocking any of the agonist-induced changes in the caecum-projecting neurones. In terms of fundus- and antrum-projecting neurones, we found no evidence for functional expression of the 34 nAChR subtype.

We also obtained pharmacological evidence for the expression of the 42 subtype of nAChR in fundus- and antrum-projecting neurones based on utilizing the drug DHE. Again, the percentage of the ACh-evoked response of DMV neurones mediated by this nAChR subtype was relatively small (14 % in fundus-projecting neurones and 23 % in antrum-projecting neurones). Likewise, Zaninetti (1999) reported that the DHE-sensitive component of the ACh-induced current was relatively less than the MLA-sensitive component, i.e. 36 % as compared to 62 % for the 7-mediated component.

Relating the present findings to our published results from in vivo studies in the anaesthetized rat has been both fruitful and frustrating. As indicated above, -BGT was able to counteract 60 % of the nicotine-evoked increase in intragastric pressure produced by microinjecting these agents into the DMV (Ferreira et al. 2001). However, at that time we were unclear as to which nAChR subtype was responsible for mediating the remaining 40 % of the response. Based on the present data obtained with DHE on ACh-evoked responses in fundus-projecting neurones, we have tested DHE in vivo. Our results indicate that DHE given after -BGT blocks the remaining portion of the -BGT-resistant effect of nicotine on intragastric pressure (M. Ferreira, N. Sahibzada & R. A. Gillis, unpublished data). On the other hand, in our in vivo studies we were unable to show any blocking effect of selective 7 nAChR subtype doses of MLA on nicotine-evoked increases in intragastric pressure (Ferreira et al. 2001). In our brain slice studies reported here, we did see blockade of ACh- and nicotine-evoked currents in fundus-projecting neurones by MLA. Similarly, Zaninetti et al. (1999) originally reported that MLA effectively counteracted ACh-evoked currents in the DMV neurones studied in a brain slice preparation. As indicated in Results, our inability to see a blocking effect of MLA in vivo is not due to older animals being used or to the use of nicotine as the nAChR agonist. At this point, we have no explanation for the lack of blocking effect of MLA on the 7 nAChR subtype in our in vivo rat preparation.

In our previous in vivo rat study (Ferreira et al. 2001), data obtained raised two questions that we will attempt to address. First, are the 7 subunits at the DMV assembled as homomers or as heteromers? Second, are the 7 nAChRs in the DMV synaptically activated? Our current findings suggest that the 7 subunits are assembled as homomers. Evidence for this is that both -BGT and MLA, which in low concentrations exhibit selectivity for blocking the 7 homomeric subtype of receptor, produced equivalent robust antagonistic effects against ACh. Failure of 'low' concentrations of MLA to block the ACh-evoked response while -BGT is effective has been used as evidence of an 7 heteromeric subtype of receptor (Yu & Role, 1998). In addition, rapid reversibility of -BGT-induced block of ACh-evoked currents (within 5 min) upon washout of the antagonist is evidence for an 7 heteromeric subtype of receptor (Cuevas et al. 2000). We did not detect the 'rapid reversibility' of -BGT-induced block in our DMV neurones. Finally, the presence of a choline-evoked current is characteristic of the 7 homomeric nAChR subtype (Alkondon et al. 1997; Klink et al. 2001) and this current was detected in our DMV neurones.

Regarding the question of whether the 7 nAChRs in the DMV are synaptically activated, we have recently found that microinjection of physostigmine into the DMV of anaesthetized adult rats while monitoring intragastric pressure (IGP; technique described in Ferreira et al. 2001), results in an increase in IGP (M. Ferreira and R. A. Gillis, unpublished data). Presumably, the increase in IGP is due to synaptically released ACh that has been protected from enzymatic inactivation by the anticholinesterase agent, physostigmine. When physostigmine is followed by microinjection of -BGT into the same DMV site in a dose that has no effect per se in non-physostigmine-treated animals, there is an additional robust increase in IGP (M. Ferreira and R. A. Gillis, unpublished data). The ACh, protected by inhibition of cholinesterase, we speculate, acts on both glutamatergic and GABAergic nerve terminals to release glutamate and GABA onto DMV neurones. Indeed, in our earlier published study, we demonstrated that acetylcholine at the DMV acts presynaptically to release GABA (Bertolini et al. 1997). Assuming an excess of glutamate, the net effect of physostigmine is an increase in IGP. We suggest that the nAChR on GABAergic terminals (but not on glutamatergic terminals) is of 7 nAChR subtype. Hence, addition of -BGT (after physostigmine) will block ACh-induced release of GABA and result in a further increase in IGP. An effect of -BGT in the DMV after inhibition of cholinesterase is consistent with the idea that 7 nAChRs in the DMV are tonically activated by synaptically released ACh. This speculation is also consistent with choline acetyltransferase-like immunoreactivite processes in the DMV (Ruggiero et al. 1994). The source of these cholinergic afferents could be the medial subnucleus of the tractus solitarius (Ruggiero et al. 1990), and/or the pedunculopontine tegmental nucleus (Rinaman et al. 1999).

In conclusion, taking together the findings of the present study, our earlier findings (Ferreira et al. 2000, 2001), and data from other investigators (Hunt & Schmidt, 1978; Breese et al. 1997; Zaninetti et al. 1999) the predominant nAChR in the DMV is, without question, the 7 nAChR subtype.

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Top Abstract Introduction Methods Results Discussion References

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Acknowledgements

This work was supported by National Institutes of Health grants NS-36035 (N.S.), RO1 DK29975, RO1 DK56920 (R.A.G.) and a supplement to grant RO1 DK29975 (M.F.).

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