Noradrenergic neurons expressing Fos during waking and paradoxical sleep deprivation in the rat

Abstract

Noradrenaline is known to induce waking (W) and to inhibit paradoxical sleep (PS or REM). Both roles have been exclusively attributed to the noradrenergic neurons of the locus coeruleus (LC, A6), shown to be active during W and inactive during PS. However, the A1, A2, A5 and A7 noradrenergic neurons could also be responsible. Therefore, to determine the contribution of each of the noradrenergic groups in W and in PS inhibition, rats were maintained in continuous W for 3 h in a novel environment or specifically deprived of PS for 3 days, with some of them allowed to recover from this deprivation. A double immunohistochemical labeling with Fos and tyrosine hydroxylase was then performed. Thirty percent of the LC noradrenergic cells were found to be Fos-positive after exposure to the novel environment and less than 2% after PS deprivation. In contrast, a significant number of double-labeled neurons (up to 40% of the noradrenergic neurons) were observed in the A1/C1, A2 and A5 groups, after both novel environment and PS deprivation. After PS recovery and in control condition, less than 1% of the noradrenergic neurons were Fos-immunoreactive, regardless of the noradrenergic group. These results indicate that the brainstem noradrenergic cell groups are activated during W and silent during PS. They further suggest that the inhibitory effect of noradrenaline on PS may be due to the A1/C1, A2 and to a lesser degree to A5 neurons but not from those of the LC as previously hypothesized.

Introduction

Central noradrenergic (NA)-synthetizing neurons are classically recognized as integral parts of the systems that maintain wakefulness (W) and contribute to the inhibition of paradoxical sleep (PS) (Jones, 2005, Jouvet, 1969, Luppi et al., 2006, Sakai et al., 1981). Indeed, enhancing NA transmission with monoamine oxidase inhibitors (such as clorgyline and pargyline) or reuptake blockers (such as imipramine and desimipramine) promotes W and suppresses PS in humans as well as in animals (Cohen et al., 1982, Gervasoni et al., 2002, Jones, 1991, Jouvet, 1969, Mendelson et al., 1982, Nishino and Mignot, 1997, Ross et al., 1995).

Due to their compact organization, at least in the rat, the NA locus coeruleus (LC) cells were extensively recorded across the sleep-wake cycle. It was found that their unit activity is tightly linked to W, since they discharge tonically during this state, decrease their firing rate during slow-wave sleep and become silent during PS (Aston-Jones and Bloom, 1981b, Gervasoni et al., 1998). In contrast, the activity in relation to the sleep-waking cycle of the other brainstem NA cell groups is not known. The present work was undertaken in order to fill this gap by comprehensively examining the distribution of the NA neurons activated when enhancing W or suppressing PS in the rat.

To achieve this goal, we took advantage of the elevated expression of the immediate-early gene c-fos in neurons after a stimulation (Dragunow and Faull, 1989, Kovacs, 1998, Morgan and Curran, 1991), a method widely used to map neurons activated in different experimental situations. The immunohistochemical detection of the protein Fos has been successfully used to localize the brainstem neurons active during PS and/or W (Maloney et al., 1999, Maloney et al., 2000, Shiromani et al., 1995, Verret et al., 2003, Verret et al., 2005, Verret et al., 2006, Yamuy et al., 1993). In the present work, rats either remained awake by placing them in a novel environment (NEv) or were specifically deprived of PS, with some of them allowed to recover from this deprivation. The expression of the protein Fos in NA neurons was visualized by dual immunostaining combining Fos and tyrosine hydroxylase (TH) immunohistochemistry. The NA A1–A7 groups were examined. This work has already appeared in abstract form (Léger et al., 2006, Léger et al., 2007).