Expression of mRNA and functional alpha1‐adrenoceptors that suppress the GIRK conductance in adult rat locus coeruleus neurons

Locus coeruleus neurons in adult rats express binding sites and mRNA for α1-adrenoceptors even though the depolarizing effect of α1-adrenoceptor agonists on neonatal neurons disappears during development. In this study intracellular microelectrodes were used to record from locus coeruleus neurons in brain slices of adult rats and reverse transcription-polymerase chain reaction (RT – PCR) was used to investigate the mRNA expression of α1- and α2-adrenoceptors in juvenile and adult rats. The α1-adrenoceptor agonist phenylephrine had no effect on the membrane conductance of locus coeruleus neurons (Vhold −60 mV) but decreased the G protein coupled, inward rectifier potassium (GIRK) conductance induced by α2-adrenoceptor or μ-opioid agonists. The GIRK conductance induced by noradrenaline was increased in amplitude when α1-adrenoceptors were blocked with prazosin. RT – PCR of total cellular RNA isolated from microdissected locus coeruleus tissue demonstrated strong mRNA expression of α1a-, α1b- and α1d-adrenoceptors in both juvenile and adult rats. However, only mRNA transcripts for the α1b-adrenoceptors were consistently detected in cytoplasmic samples taken from single locus coeruleus neurons of juvenile rats, suggesting that this subtype may be responsible for the physiological effects seen in juvenile rats. Juvenile and adult locus coeruleus tissue expressed mRNA for the α2a- and α2c-adrenoceptors while the α2b-adrenoceptor was only weakly expressed in juveniles and was not detected in adults. The results of this study show that α1-adrenoceptors expressed in adult locus coeruleus neurons function to suppress the GIRK conductance that is activated by μ-opioid and α2-adrenoceptors. Keywords: α1-adrenoceptors, α2-adrenoceptors, μ-opioid receptors, GIRK conductance, locus coeruleus, gene expression Introduction The function of α1-adrenoceptors expressed by noradrenergic locus coeruleus neurons changes during development. In developing rats, locus coeruleus neurons are excited by local (iontophoretic) applications of noradrenaline that are too low to activate inhibitory α2-adrenoceptors (Nakamura et al., 1988). This effect of noradrenaline is mimicked by the α1-adrenoceptor agonist phenylephrine, and the actions of both agonists are blocked by the selective α1-adrenoceptor antagonist HEAT (2-beta[4-hydroxyphenyethlaminomethy]tetralone). Both excitatory and inhibitory effects on cell firing also occur when endogenous noradrenaline is released following electrical stimulation of the dorsal noradrenergic bundle. The α1-adrenoceptors are located directly on noradrenergic locus coeruleus neurons, as both noradrenaline and phenylephrine depolarize these cells in brain slices prepared from immature mice or rats (Finlayson & Marshall 1984; 1986; Williams & Marshall, 1987). During maturation the excitatory effects of α1-adrenoceptors become progressively more difficult to detect and effectively disappear in adult rats (Williams & Marshall, 1987; Nakamura et al., 1988). Despite the apparent loss of function, α1-adrenoceptors continue to be expressed in the adult locus coeruleus with autoradiographic binding detected using the α1-adrenoceptor antagonists, [3H]prazosin (Chamba et al., 1991) and [125I]HEAT (Jones et al., 1985). While in situ hybridization has localized mRNA for the α1a- and α1b-adrenoceptors in adult locus coeruleus neurons (Day et al., 1997), little is known of the expression of particular α1-adrenoceptor subtypes locus coeruleus neurons of juvenile rats. Notwithstanding the loss of excitatory effects of α1-adrenoceptor agonists in adults (Williams & Marshall, 1987; Nakamura et al., 1988), there is limited evidence that these receptors continue to be functional in adults. Small increases in the frequency of spontaneous action potential firing have been produced with praxosin in brain slices. These were attributed to block of α1-adrenoceptors activated by endogenous noradrenaline release but no mechanism was identified that could explain such an effect (Ivanov & Aston-Jones, 1995). It has also been reported that α1-adrenoceptors can increase noradrenaline release from synaptosomes prepared from locus coeruleus terminals in cortex and hippocampus (Pastor et al., 1996). In the report by Williams & Marshall (1987) it is noted as an unpublished observation that in adult locus coeruleus neurons, prazosin can increase the outward current produced by noradrenaline. This current is produced by GIRK channels that are opened by α2-adrenoceptor stimulation (Egan et al., 1983; Williams et al., 1985). In this study we investigated whether α1-adrenoceptors in locus coeruleus neurons can function to modulate signalling pathways that open GIRK channels. We also used reverse transcription-polymerase chain reaction (RT – PCR) to determine which of the three cloned α1-adrenoceptor subtypes is present in locus coeruleus tissue of juvenile and adult rats, and in cytoplasmic samples from single juvenile locus coeruleus neurons.