RESPONSES OF MYENTERIC S NEURONES TO LOW FREQUENCY STIMULATION OF THEIR SYNAPTIC INPUTS G. ALEX, a N. CLERC, b W. A. A. KUNZE a and J. B. FURNESS a * a Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Vic. 3010, Australia b Laboratoire Inte ¨gration des Informations Sensorielles, Centre National de la Recherche Scienti¢que (CNRS), Marseille, France AbstractöPrevious experiments have shown that prolonged low frequency stimulation of presynaptic inputs causes excitation of AH neurones that considerably outlasts the period of stimulation in the guinea-pig small intestine. The present experiments compare the responses of S neurones (which are motor neurones and interneurones) with responses of AH neurones (intrinsic primary a¡erent neurones) to low frequency stimulation of synaptic inputs. Neurones in the myenteric plexus of isolated segments of guinea-pig small intestine were recorded from with intracellular microelectrodes. During their impalement, the neurones were ¢lled with a marker dye and they were later processed to reveal their shapes and immunohistochemical properties. One group of neurones, inhibitory motor neurones to the circular muscle, was depolarised by stimulation of synaptic inputs at 1 Hz for 100 s to 4 min. With 4-min trains of stimuli, peak depolarisation was 21 þ 2 mV (mean þ S.E.M.), which was reached at about 110 s. Depolarisation was accompanied by increased excitability; before stimulation, a test intracellular pulse (500 ms) triggered 3 action potentials, at the peak of excitability this reached 16 action potentials. Depolarisation began to decline immediately at the end of stimulation. This contrasts with responses of AH neurones, in which depolarisation persisted after the end of the stimulus (peak depolarisation at 300 s). The excitation and depolarisation of inhibitory motor neurones was blocked by the neurokinin 1 tachykinin receptor antagonist, SR140333 (100 nM), but excitation of AH neurones was not a¡ected. Small or no responses to 1 Hz stimulation were recorded from descending ¢lamentous interneurones, longitudinal muscle motor neurones and excitatory circular muscle motor neurones. In conclusion, this study indicates that sustained slow postsynaptic excitation only occurs in AH neurones, and that one type of S neurones, inhibitory motor neurones to the circular muscle, responds substantially, but not beyond the period of stimulation, to activation of synaptic inputs at 1 Hz. This slow excitatory postsynaptic potential evoked by low frequency stimulation is mediated by tachykinins. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: enteric nervous system, synaptic transmission, tachykinins, inhibitory neurones. Several types of events have been recorded at synapses in the myenteric plexus of the small intestine: fast excita- tory postsynaptic potentials (fast EPSPs), slow EPSPs, inhibitory postsynaptic potentials and sustained slow postsynaptic excitation (SSPE) (Wood, 1994; Clerc et al., 1999; Galligan et al., 2000). The enteric nervous system produces 25 or more potential transmitter sub- stances, including peptides, amines, amino acids and the monoxides, NO and CO (Costa et al., 1996; Furness et al., 1999), any of which might be involved in trans- mission at enteric synapses. Despite the large number of potential transmitters, it has taken many years to de¢ne the roles of only a small number. For example, fast EPSPs, which occur in response to single shocks applied to presynaptic ¢bres, were ¢rst reported in the 1970s (Nishi and North, 1973; Hirst et al., 1974), and were generally assumed to be caused by the release of acetyl- choline. It is only in the last 4 years that pharmacological dissection of these events has revealed that there are components of fast EPSPs that are mediated by purines (at P 2X receptors), 5-hydroxytryptamine (5-HT; at 5-HT 3 receptors) and glutamate (at K-amino-3- hydroxy-5-methyl-4-isoxazide propionate receptors) (Lepard et al., 1997; Liu et al., 1997; Lepard and Galligan, 1999; Galligan et al., 2000). Similarly, slow EPSPs, which can be evoked by bursts of high frequency presynaptic stimulation, were described in the 1970s (Wood and Mayer, 1978), but a clear demonstration that tachykinins, acting through neurokinin-3 (NK 3 ) receptors, are transmitters of slow EPSPs has been recent 361 *Corresponding author. Tel. : +61-3-83448859; fax: +61-3- 93475219. E-mail address : j.furness@unimelb.edu.au (J. B. Furness). Abbreviations : AH, designation of neurones with broad action potentials and long-lasting afterhyperpolarisations ; AHP, after- hyperpolarising potential (following an action potential) ; AP, action potentials ; DAB, 3,3P-diaminobenzidine ; EPSP, excitatory postsynaptic potential ; 5-HT, 5-hydroxytryptamine ; MP, mem- brane potential ; NK 1=3 , neurokinin-1/3 (receptor) ; NOS, nitric oxide synthase; P 2X , purine 2X (receptor); R in , input resistance ; RMP, resting membrane potential ; S, designation for enteric neu- rones with tetrodotoxin blocked soma action potentials and prominent fast EPSPs; SR 140333, (S)1-{2-[3-(3,4-dichloro- phenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl}-4-phe- nyl-1-azoniabicyclo[2.2.2]octane chloride ; SR 142801, (S)-(N)-(1- (3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)-4-phe- nylpiperidin-4-yl)-N-methylacetamide ; SSPE, sustained slow postsynaptic excitation ; TTX, tetrodotoxin. NSC 5418 26-2-02 www.neuroscience-ibro.com Neuroscience Vol. 110, No. 2, pp. 361^373, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain PII:S0306-4522(01)00583-8 0306-4522 / 02 $22.00+0.00