Characterization of the in vitro effects of 5-hydroxytryptamine (5-HT) on identified neurones of the rat dorsal motor nucleus of the vagus (DMV)

Abstract

1 Whole cell patch clamp techniques were used on thin brainstem slices to investigate the effects of 5-hydroxytryptamine (5-HT) on gastrointestinal-projecting dorsal motor nucleus of the vagus (DMV) neurones. Neurones were identified as projecting to the stomach (n=122) or intestine (n=84) if they contained the fluorescent tracer Dil after it had been applied to the gastric fundus, corpus or antrum/pylorus or to the duodenum or caecum. 2 A higher proportion of intestinal neurones (69%) than gastric neurones (47%) responded to 5-HT with a concentration-dependent inward current which was antagonized fully by the 5-HT2A receptor antagonist ketanserin (1 microM). 3 Stimulation of the nucleus tractus solitarius (NTS) induced inhibitory synaptic currents that were reduced in amplitude by application of the 5-HT1A receptor agonist 8-OHDPAT (1 microM) or the 5-HT1A/1B receptor agonist TFMPP (1 microM) in 61% and 52% of gastric- and intestinal-projecting neurones, respectively. 5-HT also significantly reduced the frequency but not the amplitude of spontaneous inhibitory currents. 4 These data show that 5-HT excites directly a larger proportion of intestinal projecting neurones than gastric-projecting neurones, as well as inhibiting synaptic transmission from the NTS to the DMV. These data imply that the response to DMV neurones to 5-HT may be determined and classified by their specific projections.

Figure 1

Exogenously applied 5-HT induces a concentration-dependent inward current in gastrointestinal-projecting DMV neurones. (A) Representative traces from one gastric-projecting DMV neurone illustrating the concentration-dependent nature of the inward current induced by superfusion of 5-HT. Neurones were voltage-clamped at −50 mV before application of 5-HT for a period of time sufficient for the induced current to reach a stable plateau (60–150 s). The inward current did not desensitize, even at high concentrations. The vertical lines indicate an interruption in recording of 120, 110 and 135 s for the top, middle and bottom trace, respectively. A recovery period of at least 10 min was allowed between successive applications. (B) Concentration-response curve for gastric- and intestinal-projecting DMV neurones (voltage clamped at −50 mV) in response to exogenously applied 5-HT. The current induced by 5-HT is expressed as a percentage of the maximal response. Note that the EC₅₀ value for the inward current in intestinal neurones (10 μM) is significantly larger than that of gastric neurones (4 μM) ^*P<0.05.

Figure 2

5-HT decreases the duration of the action potential afterhyperpolarization and increases the action potential duration. To evoke a single action potential, neurones were current clamped at −55 mV before passing a short duration (16 ms) depolarizing current pulse of intensity sufficient to evoke the firing of one action potential at its offset. (A) Illustrate the response of a corpus-projecting neurone; (B) Illustrate the response of a caecal-projecting neurone to 5-HT (30 μM). In both gastric-projecting neurones (e.g. A) and intestinal-projecting (e.g. B) DMV neurones 5-HT decreased the amplitude of the afterhyperpolarization (left traces) as well as increasing the duration of the action potential (right traces).

Figure 3

The postsynaptic responses to 5-HT are mediated via activation of 5-HT₂ receptors only. All neurones were voltage-clamped at −50 mV before application of 5-HT (30 μM) for periods sufficient to allow the induced inward current to reach a stable plateau. Following complete recovery and a wash-out period of at least 10 min, the response of the same neurone to 5-HT receptor selective agonists or the response to 5-HT in the presence of 5-HT receptor selective antagonists were assessed. Receptor antagonists were superfused for a minimum of 10 min before re-application of receptor agonists. (A) The inward current induced by superfusion with 5-HT (30 μM; left panel) was prevented by prior application of the 5-HT_2A receptor antagonist, ketanserin (1 μM). Similarly, the 5-HT₂ receptor agonist α-methyl 5-HT (3 μM; black trace, right panel), mimicked the inward current induced by 5-HT, a response that was also prevented entirely by the 5-HT_2A receptor antagonist, ketanserin (1 μM). (B) Unlike 5-HT (30 μM; left panel), the 5-HT_1A receptor agonist, 8-OHDPAT (1 μM) had no postsynaptic actions. Similarly, preincubation with the 5-HT_1A receptor antagonist, NAN-190 (1 μM, right panel) did not attenuate the 5-HT-induced inward current. (C) Preincubation with the 5-HT_3/4 receptor antagonist ICS 205-930 (tropisetron, 3 μM) did not attenuate the inward current induced by 5-HT (30 μM).

Figure 4

Activation of presynaptic 5-HT_1A receptors, decreases the amplitude of evoked IPSC. (A,B) Neurones were voltage-clamped at −50 mV before evoked IPSC were induced in gastrointestinal-projecting DMV neurones by stimulation of the NTS. Pairs of IPSC were evoked 250–750 ms apart (left panel). Following superfusion with 5-HT (30 μM), the amplitude of the evoked IPSC were reduced. This reduction in amplitude of IPSC is highlighted in the bar chart (right panel) which compares the amplitude of the first and second pulse (C1 and C2, respectively) and their ratio (C2/C1) before and after superfusion with 5-HT (30 μM). Calibration bar=400 ms, 200 pA. (C) The 5-HT-induced reduction in IPSC amplitude was attenuated by prior exposure to 5-HT_1A receptor antagonist, NAN-190 (1 μM; left traces) but not by the 5-HT_2A receptor antagonist, ketanserin (1 μM; middle traces) or the 5-HT_3/4 receptor antagonist, ICS 205-930 (tropisetron, 3 μM; right traces). Calibration bar=400 ms, 200 pA.