THE TERMINALS OF MYENTERIC INTRINSIC PRIMARY AFFERENT NEURONS OF THE GUINEA-PIG ILEUM ARE EXCITED BY 5-HYDROXYTRYPTAMINE ACTING AT 5-HYDROXYTRYPTAMINE-3 RECEPTORS P. P. BERTRAND,*² W. A. A. KUNZE,³ J. B. FURNESS³ and J. C. BORNSTEIN* Departments of *Physiology and ³Anatomy & Cell Biology, University of Melbourne, Parkville, Victoria 3010, Australia Abstract ÐThe aim of this study was to identify the receptor type(s) by which 5-hydroxytryptamine applied to the intestinal mucosa excites the terminals of myenteric AH neurons. The AH neurons have been identi®ed as the intrinsic primary afferent (sensory) neurons in guinea-pig small intestine and 5-hydroxytryptamine has been identi®ed as a possible intermediate in the sensory transduction process. Intracellular recordings were taken from AH neurons located within 1 mm of intact mucosa to which 5-hydroxytryptamine was applied. Trains of action potentials and/or slow depolarizing responses were recorded in AH neurons in response to mucosal application of 5-hydroxytryptamine (10 or 20 mM) or the 5-hydroxytryptamine-3 receptor agonist, 2-methyl-5- hydroxytryptamine (1 or 3 mM), and to electrical stimulation of the mucosa. The 5-hydroxytryptamine-2 receptor agonist, a-methyl-5-hydroxytryptamine (100 mM), and the 5-hydroxytryptamine-1,2,4 receptor agonist, 5-methoxytryptamine (10 mM), did not elicit such responses. The 5-hydroxytryptamine-3 receptor-selective antagonist, granisetron (typically 1 mM), and the 5-hydroxytryptamine-3,4 receptor antagonist, tropisetron (typically 1 mM), each reduced or abolished the responses to 5-hydro- xytryptamine, while the selective 5-hydroxytryptamine-4 receptor antagonist, SB 204070 (1 mM), did not. It is concluded that application of 5-hydroxytryptamine to the mucosa activates a 5-hydroxytryptamine-3 receptor that triggers action potential generation in the mucosal nerve terminals of myenteric AH neurons. q 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: serotonin, intestine, electrophysiology, sensory transduction, sensory neuron. Serotonin (5-hydroxytryptamine, 5-HT) can exert a powerful in¯uence upon the motility and secretory activity of the gastrointestinal tract. The exact nature of these changes depends upon the region of gut studied and the route by which 5-HT is administered. 6±8,36 The large number of 5-HT receptor subtypes present on gut tissues and their wide distri- bution across different functional parts of the enteric circuity are likely to contribute to the observed diversity in responses elicited by 5-HT. 12 For example, the myenteric AH neuron, which functions as an intrinsic primary afferent neuron (IPAN; also referred to as a sensory neuron) in the enteric circuitry, 11 has 5-HT 1A , 5-HT 1P and 5-HT 3 receptors on its cell body, 14,27 and 5-HT 1A and 5-HT 4 receptors on its terminals. 12 The submucosal AH neuron (submucosal IPAN 21 ) has 5-HT 1P and 5-HT 3 receptors on its cell body, but only 5-HT 1P recep- tors on its terminals. 30,35 Extrinsic primary afferent neurons from the vagus have 5-HT 1A and 5-HT 3 receptors on their terminals, 15,16 and the 5-HT-containing enterochromaf®n (EC) cells have 5-HT 3 and 5-HT 4 receptors. 13,32 5-HT, when applied to the mucosa, generates action poten- tials (APs) in the mucosal processes of myenteric AH neurons. 1,4,34 Electrical stimulation of the mucosa elicits a similar response, suggesting that, in addition to a direct effect on the nerve terminal, release of endogenous 5-HT contrib- utes to this response. 3,4 The 5-HT-induced changes in motility and secretion described above may be due to the actions of 5-HT on the terminals of myenteric AH neurons. The purpose of this study is to characterize the receptor type(s) through which mucosal application of 5-HT activates the terminals of the IPANs with cell bodies in the myenteric plexus. EXPERIMENTAL PROCEDURES Tissue preparation All experiments were performed using guinea-pigs (either gender, 160±280 g, Hartley strain, from the University of Melbourne), which were fed a standard laboratory diet until the day of the experiment. Animals were stunned by a blow to the head and killed by severing the carotid arteries and spinal cord in accordance with the guidelines of the University of Melbourne Animal Ethics Committee. A 2- to 3-cm-long segment of ileum, approximately 10±20 cm from the ileocaecal junc- tion, was removed and placed in oxygenated (95% O 2 /5% CO 2 ) physio- logical saline of the following composition (in mM): NaCl, 117; NaH 2 PO 4 , 1.2; MgSO 4 , 1.2; CaCl 2 , 2.5; KCl, 4.7; NaHCO 3 , 25; glu- cose, 11. The physiological saline also contained nicardipine (3 mM) and scopolamine (hyoscine, 1 mM) to relax the smooth muscle and to minimize its movements. The segment was cut open along the line of the mesenteric attachment and pinned mucosal side up in a Petri dish lined with a silastic elastomer. The mucosa, submucosa and circular muscle were removed over half the circumference, leaving the myen- teric plexus with attached longitudinal muscle exposed. The prepara- tion was then transferred to the base of a small recording chamber (volume approximately 2 ml), stretched and pinned ¯at with 80-mm pins. The preparation was superfused with warmed (35±368C) physio- logical saline at a ¯ow rate of 4±6 ml/min. Electrophysiology Myenteric ganglia were visualized at £ 200±300 magni®cation with differential interference contrast optics. Neurons were impaled with glass microelectrodes (120±200 MV tip resistance) containing 2% biocytin in 1 M KCl. Voltage recordings were made in bridge mode 5-HT activates sensory nerve terminals 459 459 Neuroscience Vol. 101, No. 2, pp. 459±469, 2000 q 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00 PII: S0306-4522(00)00363-8 Pergamon www.elsevier.com/locate/neuroscience ²To whom correspondence should be addressed. Tel.: 161-3-8344-4466; fax: 161-3-8344-5818. E-mail address: P.Bertrand@physiology.unimelb.edu.au (P. P. Bertrand). Abbreviations: AP, action potential; EC cells, enterochromaf®n cells; EPSP, excitatory postsynaptic potential; HEPES, N-2-hydroxyethyl- piperazine-N 0 -2-ethanesulphonic acid; 5-HT, 5-hydroxytryptamine (serotonin); IPAN, intrinsic primary afferent neuron; PPP, proximal process potential; RMP, resting membrane potential.