ATP is a neurotransmitter (NT) of fast excitatory postsynaptic potentials (fEPSP) in myenteric neurons. These studies will focus on pharmacologic, elecrophysiologic and immunohistochemical characterization of purinergic receptors and neurons expressing these receptors in the intact myenteric plexus of t he guinea pig ileum. Conventional electrophysiological and immunohistochemical methods will be used to identify neurons expressing purinergic receptors. Using single electrode voltage clamp, current evoked by 'fast flow' application of agonists will determine rank-order potency and purinoceptor subtype. The purinergic component of the fEPSP will be isolated by superfusion of hexamethonium to block t he cholinergic component. To determine if ATP and acetylcholine (ACH) are cotransmitted from the same fiber: 1) the stimulus strength necessary to evoked a purinergic component to the fEPSP will be determined., 2) the preparation will be incubated with guanethidine which blocks NT release from sympathetic neurons and 3) the preparation will be incubated with botulinum neurotoxin which prevents ACH release from nerve terminals. Because release of ACH is presynaynayptically regulated (17) and ATP may be cotransmitted with ACH [42], the presynaptic regulation of the purinergic component of the fEPSP by serotonin, norepinephrine and opiate will be investigated. Specificity of t he drugs used in these studies will be tested against purely cholinergic fEPSPs and depolarizations by puffs of ACH onto the neuron. Throughout these experiments, recording electrodes will be filled with NeurobiotinR to correlate the electrophysiology and pharmacology of the neuron with its cell morphology. To investigate the role of P2x-expressing neurons in the neurocircuitry of the gut, markers for motor neurons and interneurons will be localized. Nitric oxide (NO) is a NT released from inhibitory motor neurons in the myenteric plexus [19]. Localization of NO synthase immunoreactivity (NOS-ir) will identify inhibitory motorneurons [19]. Immunoreactivity for ACH will be used as a marker for excitatory motorneurons and interneurons [14]. These studies will promote further understanding of noncholinergic/nonadrenergic control of gastrointestinal motility.
