Neurotensin excitation of serotonergic neurons in the rat nucleus raphe magnus: ionic and molecular mechanisms

Abstract

To understand the cellular and molecular mechanisms by which neurotensin (NT) induces an analgesic effect in the nucleus raphe magnus (NRM), whole-cell patch-clamp recordings were performed to investigate the electrophysiological effects of NT on acutely dissociated NRM neurons. Two subtypes of neurons, primary serotonergic and secondary non-serotonergic cells, were identified from acutely isolated NRM neurons. During current-clamp recordings, NT depolarized NRM serotonergic neurons and evoked action potentials. Voltage-clamp recordings showed that NT excited serotonergic neurons by enhancing a voltage-insensitive and non-selective cationic conductance. Both SR48692, a selective antagonist of subtype 1 neurotensin receptor (NTR-1), and SR 142948A, a non-selective antagonist of NTR-1 and subtype 2 neurotensin receptor (NTR-2), failed to prevent neurotensin from exciting NRM serotonergic neurons. NT-evoked cationic current was inhibited by the intracellular administration of GDP-β-S. NT failed to induce cationic currents after dialyzing serotonergic neurons with the anti-G_αq/11 antibody. Cellular Ca²⁺ imaging study using fura-2 showed that NT induced the calcium release from the intracellular store. NT-evoked current was blocked after the internal perfusion of heparin, an IP₃ receptor antagonist, or BAPTA, a fast Ca²⁺ chelator. It is concluded that neurotensin enhancement of the cationic conductance of NRM serotonergic neurons is mediated by a novel subtype of neurotensin receptors. The coupling mechanism via G_αq/11 proteins is likely to involve the generation of IP₃, and subsequent IP₃-evoked Ca²⁺ release from intracellular stores results in activating the non-selective cationic conductance.

Introduction

The midbrain periaqueductal gray (PAG) and the rostral ventromedial medulla (RVM), which includes nucleus raphe magnus (NRM), nucleus reticularis gigantocellularis pars alpha (NRGα), and nucleus reticularis paragigantocellularis (NRPG), are major components of the brainstem descending antinociceptive pathway that modulates the nociceptive transmission in the dorsal horn of spinal cord and trigeminal sensory complex (Fields et al., 1991). The PAG sends an excitatory innervation to the rostral ventromedial medulla, which in turn projects to the spinal dorsal horn and inhibits pain transmission (Basbaum and Fields, 1984, Fields et al., 1991). Within the rostral ventromedial medulla, NRM, a serotonergic nucleus, is an essential component of the brainstem antinociceptive neuronal circuitry. Multiple lines of evidence indicate that serotonergic cells play a major role in NRM-mediated antinociceptive effects. NRM serotonergic neurons send a dense projection to the spinal dorsal horn and trigeminal sensory complex (Skagerberg and Bjorklund, 1985). Electrical stimulation of the NRM inhibits the nociceptive activity of spinal and trigeminal dorsal horn neurons (Basbaum and Fields, 1984). It has been reported that intrathecal application of serotonin inhibits the evoked activity of nociceptive neurons in the spinal dorsal horn and produces an analgesic effect (Yaksh and Wilson, 1979). Furthermore, analgesia induced by the stimulation of PAG or RVM has been shown to be partially reduced by the intrathecal administration of serotonin antagonist (Hammond and Yaksh, 1984).

Central administration of neurotensin, a tridecapeptide, produces an antinociceptive effect (Behbehani, 1992). An extensive body of evidence suggests that neurotensin produces an analgesic effect by modulating the neuronal activity of NRM. Immunohistochemical studies indicated that PAG neurons containing neurotensin project to the rostral ventromedial medulla and form the synapses with NRM serotonergic neurons (Lakos and Basbaum, 1988). Autoradiographic studies showed that a high density of neurotensin receptors is expressed in the nucleus raphe magnus (Kessler et al., 1987). When microinjected into the NRM, neurotensin produces a potent antinociceptive effect by inhibiting the noxious stimulus-evoked activity of nociception-specific neurons of spinal dorsal horn (Fang et al., 1987, Urban and Gebhart, 1997). Neurotensin has been shown to excite basal forebrain, dorsal raphe, substantia nigra, supraoptic and ventral tegmental neurons (Farkas et al., 1994, Jiang et al., 1994, Kirkpatrick and Bourque, 1995, Wu et al., 1995, Jolas and Aghajanian, 1996). Therefore, it is very likely that neurotensin induces an analgesic effect by directly exciting serotonergic neurons and activating the NRM-spinal cord antinociceptive pathway. In the present study, this hypothesis was tested by investigating the ionic and molecular mechanisms by which neurotensin modulates the excitability of acutely dissociated rat NRM neurons with the aid of whole-cell patch-clamp recordings and intracellular Ca²⁺ fluorescence measurement.