Neurotensin excitation of serotonergic neurons in the rat nucleus raphe magnus: ionic and molecular mechanisms
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.
Section snippets
Acute isolation of nucleus raphe magnus neurons
Neurons of the rat nucleus raphe magnus (NRM) were acutely dissociated according to the procedures described previously (Wu et al., 1995, Wu and Wang, 1996). Briefly, 14 to 16-days old Sprague–Dawley rats were terminally anesthetized with sodium pentobarbital and decapitated. The whole brain was quickly removed, and 300 μm-thick brainstem slices containing the NRM were prepared by using a Vibratome slicer in ice-cold PIPES-buffered Ringer solution containing (in mM): NaCl 120, KCl 5, NaHCO3 20,
Identification of two subtypes of acutely isolated NRM neurons
Two subtypes of neurons, primary and secondary cells, were observed from acutely dissociated NRM neurons. Primary neurons did not fire action potentials spontaneously and were large oval cells with two to three primary thick processes (diameter across the long axis=30–45 μm, Fig. 1(A)) (Pan et al., 1993, Pan et al., 1997). In agreement with a previous study showing that primary cells are serotonergic neurons (Pan et al., 1993), tryptophan hydroxylase (TH) immunohistochemical staining indicated
Discussion
To gain insight into the cellular and molecular mechanisms by which neurotensin produces an analgesic effect in the NRM, we investigated the electrophysiological effect of neurotensin on acutely dissociated NRM neurons. Consistent with previous studies using brain slices (Pan et al., 1993, Pan et al., 1997), the present study identified two subtypes of acutely isolated NRM neurons, primary and secondary cells. Our results demonstrate that neurotensin selectively depolarizes and excites primary
Acknowledgements
This work was supported by the fund from the National Science Council (NSC89-2320-B-182-057) and the Chang Gung Medical Research Foundation (CMRP 555).
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