5-HT1A receptor-mediated activation of G-protein-gated inwardly rectifying K+ current in rat periaqueductal gray neurons
Introduction
5-Hydroxytryptamine (5-HT, serotonin) as a neurotransmitter and a neuromodulator has a widespread distribution and multiple functions including sleep, cognition, sensory perception, motor activity, temperature regulation, appetite, sexual behavior, hormone secretion, and nociception in the mammalian central nervous system. Cellular actions of 5-HT are mediated by up to 14 distinct, cell-membrane-located, receptor subtypes (Hoyer and Martin, 1997).
The midbrain periaqueductal gray (PAG) is believed to be an important component in the endogenous pain control system (Basbaum and Fields, 1984). This pain-suppression system is activated by electrical stimulation of the PAG (Millan et al., 1987) and by local injection of morphine-like narcotics or opioid peptides at the PAG (Fields et al., 1991). The PAG region contains an abundance of 5-HT-immunoreactive processes (Clements et al., 1985) and several types of 5-HT receptors (Pompeiano et al., 1992).
There are some controversies about the roles of 5-HT in the analgesia elicited from the PAG. Some in vivo studies have suggested that 5-HT antagonists microinjected into the PAG inhibit the antinociception produced by electrical stimulation of the PAG (Coimbra and Brandão, 1997) and by microinjection of opioid peptides into the PAG (Schul and Frenk, 1991). In contrast, antinociception produced by electrical stimulation of the PAG was attenuated following microinjection of 5-HT at the site of stimulation in the PAG (Hämäläinen and Lovick, 1997).
The direct effects of 5-HT on the PAG neuronal activities performed in slice preparations have revealed that the firing activities of PAG neurons were inhibited by 5-HT (Behbehani et al., 1993, Lovick, 1994, Stezhka and Lovick, 1994). Especially, Behbehani et al. (1993) have reported that 5-HT exerts 5-HT1A receptor-mediated actions on neurons in all regions of the PAG, and that activation of 5-HT1A receptors primarily leads to an inhibition of PAG cells that was associated with a hyperpolarization of the membrane and an increase in K+ conductance (Behbehani et al., 1993).
In many central neurons, 5-HT, acting through 5-HT1A receptors, activates an inwardly rectifying K+ current leading to hyperpolarization of the hippocampal neurons (Andrade et al., 1986), of the rat dorsal raphe neurons (Penington et al., 1993, Katayama et al., 1997), of the rat hippocampal neurons (Oh et al., 1995), of the caudal raphe neurons (Bayliss et al., 1997), of the rat facial motoneuron (Larkman and Kelly, 1998), of the dissociated hippocampal CA3 neurons (Sodickson and Bean, 1998), and of the rat neonatal pyramidal cells (Takigawa and Alzheimer, 1999). Furthermore, it has been reported that 5-HT-activated inwardly rectifying K+ channels are coupled to pertussis-toxin-sensitive G-proteins (Andrade et al., 1986, Penington et al., 1993, Oh et al., 1995, Katayama et al., 1997, Bayliss et al., 1997).
However, the physiological and pharmacological characteristics of the K+ channels involved in the 5-HT-induced hyperpolarization of the PAG neuron and the intracellular action mechanisms of 5-HT on the PAG neuron have not yet been elucidated. In the present study, we have examined the 5-HT receptor subtypes, the pharmacological profile of the K+ channel, the involvement of G-proteins, and the effects of intracellular second messenger modulators on the 5-HT effect in acutely dissociated PAG neurons under voltage clamp conditions.
Section snippets
Isolation of PAG neurons
According to previous reports from our laboratory (Min et al., 1996, Han et al., 1999), acutely isolated single PAG neurons were prepared. In brief, 10- to 16-day-old Sprague-Dawley rats of both sexes were decapitated under pentobarbital sodium anesthesia (50 mg/kg, i.p.). The brain was removed and the transverse slices (400 μm thickness) were made with a microslicer (DTK-1000, DSK, Japan). Slices were preincubated in the incubation solution that had been saturated with 95% O2 and 5% CO2 at
5-HT-activated inward currents (I5-HT) in the PAG neurons
At a holding potential (VH) of −60 mV in the external solution containing 30 mM K+, 5-HT activated inward currents in 30–40% of the PAG neurons tested, and the other 60–70% of the neurons did not respond to 5-HT (Fig. 1A). Most of 5-HT-responsive cells had oval-shaped somas with two to three dendrites, while 5-HT-nonresponsive cells had round-, triangular-, and rectangular-shaped somas with multiple dendrites. In general, 5-HT-responsive cells had medium-sized (10–20 μM) somas, while
Discussion
The purpose of this study was to characterize the electrophysiological properties and action mechanisms of 5-HT-induced activation of the K+ channel in acutely dissociated rat PAG neurons using the nystatin-perforated and conventional whole-cell patch-clamp recording modes under voltage-clamp conditions. There was no significant difference in the distribution of the 5-HT responsive cells in all regions (dorsal, dorsolateral, ventral, and ventrolateral) of the PAG. Among the PAG neurons tested,
Acknowledgements
The study was supported by a research grant (the program year of 2000) from Kyung Hee University to Young-Wuk Cho.
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