Adenosinergic modulation of basal forebrain and preoptic/anterior hypothalamic neuronal activity in the control of behavioral state

Abstract

This review describes a series of animal experiments that investigate the role of endogenous adenosine (AD) in sleep. We propose that AD is a modulator of the sleepiness associated with prolonged wakefulness. More specifically, we suggest that, during prolonged wakefulness, extracellular AD accumulates selectively in the basal forebrain (BF) and cortex and promotes the transition from wakefulness to slow wave sleep (SWS) by inhibiting cholinergic and non-cholinergic wakefulness-promoting BF neurons at the AD A1 receptor. New in vitro data are also compatible with the hypothesis that, via presynaptic inhibition of GABAergic inhibitory input, AD may disinhibit neurons in the preoptic/anterior hypothalamus (POAH) that have SWS-selective activity and Fos expression. Our in vitro recordings initially showed that endogenous AD suppressed the discharge activity of neurons in the BF cholinergic zone via the AD A1 receptor. Moreover, in identified mesopontine cholinergic neurons, AD was shown to act post-synaptically by hyperpolarizng the membrane via an inwardly rectifying potassium current and inhibition of the hyperpolarization-activated current, I_h. In vivo microdialysis in the cat has shown that AD in the BF cholinergic zone accumulates during prolonged wakefulness, and declines slowly during subsequent sleep, findings confirmed in the rat. Moreover, increasing BF AD concentrations to approximately the level as during sleep deprivation by a nucleoside transport blocker mimicked the effect of sleep deprivation on both the EEG power spectrum and behavioral state distribution: wakefulness was decreased, and there were increases in SWS and REM sleep. As predicted, microdialyis application of the specific A1 receptor antagonist cyclopentyltheophylline (CPT) in the BF produced the opposite effects on behavioral state, increasing wakefulness and decreasing SWS and REM. Combined unit recording and microdialysis studies have shown neurons selectively active in wakefulness, compared with SWS, have discharge activity suppressed by both AD and the A1-specific agonist cyclohexyladenosine (CHA), while discharge activity is increased by the A1 receptor antagonist, CPT. We next addressed the question of whether AD exerts its effects locally or globally. Adenosine accumulation during prolonged wakefulness occurred in the BF and neocortex, although, unlike in the BF, cortical AD levels declined in the 6th h of sleep deprivation and declined further during subsequent recovery sleep. Somewhat to our surprise, AD concentrations did not increase during prolonged wakefulness (6 h) even in regions important in behavioral state control, such as the POAH, dorsal raphe nucleus, and pedunculopontine tegmental nucleus, nor did it increase in the ventrolateral/ventroanterior thalamic nucleii. These data suggest the presence of brain region-specific differences in AD transporters and/or degradation that become evident with prolonged wakefulness, even though AD concentrations are higher in all brain sites sampled during the naturally occurring (and shorter duration) episodes of wakefulness as compared to sleep episodes in the freely moving and behaving cat. Might AD also produce modulation of activity of neurons that have sleep selective transcriptional (Fos) and discharge activity in the preoptic/anterior hypothalamus zone? Whole cell patch clamp recordings in the in vitro horizontal slice showed fast and likely GABAergic inhibitory post-synaptic potentials and currents that were greatly decreased by bath application of AD. Adenosine may thus disinhibit and promote expression of sleep-related neuronal activity in the POAH. In summary, a growing body of evidence supports the role of AD as a mediator of the sleepiness following prolonged wakefulness, a role in which its inhibitory actions on the BF wakefulness-promoting neurons may be especially important.

Introduction

Initial evidence that adenosine (AD), a purine nucleoside, was an endogenous sleep factor came from pharmacological studies describing the sleep-inducing effects of systemic or intracerebral injections of AD and AD agonist drugs [77], reviewed in [53]. Furthermore, the well known stimulants caffeine and theophylline have since been determined to be antagonists of AD receptors [21]. More recent work has focused on identifying the specific brain structures and receptor subtypes on which AD acts to induce drowsiness and sleep. Acting on the widely distributed A1 receptor, AD is generally an inhibitory neuromodulator reducing the activity of neurons in a number of brain regions, either by directly hyperpolarizing the post-synaptic membrane, or by acting presynaptically to decrease the release of excitatory neurotransmitters, for review see [11], [22], [20]. Presynaptically, A1 mediated reductions in N-type Ca²⁺ currents have been reported in striatal cholinergic interneurons, thereby establishing a cellular mechanism by which AD may serve to reduce acetylcholine release [68]. AD may also have an indirect excitatory effect on post-synaptic neurons, via A1-mediated presynaptic inhibition of inhibitory neurotransmitter release. Although most of the physiological effects of AD in the brain are mediated by the A1 receptor, AD action at the A2 receptor can have neuromodulatory effects that oppose action at A1 receptors, reviewed in [11], [20]. For example, at A2a receptors, found predominantly in the basal ganglia, AD stimulates adenylyl cyclase activity, whereas at A1 receptors AD inhibits the activity of adenylyl cyclase, and thus the formation of cAMP. Although AD has diverse and widespread effects, our recent work, reviewed herein, indicates that the sleep-inducing effects of AD may be largely mediated by AD actions in two adjacent areas in the ventral forebrain.

One hallmark criterion for the identification of a sleep factor is the accumulation of the factor during prolonged wakefulness [32], a property we have described for AD in the basal forebrain (BF) [49]. Interestingly, more recent microdialysis experiments described herein reveal that this sleep deprivation-induced rise in extracellular AD is found only in the BF and, to a somewhat lesser extent, in neocortex, and not in four other subcortical sites examined [48], [69]. Furthermore, it has been recently shown in vivo that local AD infusion decreases the unit activity of those BF neurons with a wakefulness-related discharge pattern [1], [74]. These findings provide strong support for the BF and cortex as critical sites linking sleep deprivation with AD-induced somnolence. In addition to the BF, our recent studies at the cellular level, suggest a mechanism for AD action in the preoptic/anterior hypothalamic area (POAH) that could also promote sleep [45]. Thus, our data support the hypothesis that AD can promote the induction of sleep by two complementary cellular mechanisms, first, by the direct inhibition of the wakefulness-promoting neurons in the cholinergic zone of the BF, and, secondly, a recent observation suggests the possibility of indirect activation of sleep promoting neurons in the POAH via the presynaptic reduction of GABAergic inhibition.

The cholinergic neurons scattered throughout the BF region are thought to play an important role in the regulation of cortical arousal (EEG activation) and the maintenance of wakefulness (W) [30], although the forebrain histaminergic system and brainstem cholinergic, noradrenergic and serotonergic systems are also involved (see Section 3 and [23], [64], [75]). The BF region, referred to herein as the BF cholinergic zone, contains both cholinergic and non-cholinergic (including GABAergic) cortically projecting neurons, of which the majority exhibit their highest discharge activity during W (wake-active neurons) [73], [71]. Prior to the anatomical identification of the central cholinergic pathways, acetylcholine was implicated in the regulation of W based on pharmacological and neurochemical evidence, for review see [62]. For example, cortical acetylcholine release is higher during W and REM sleep than during non-REM sleep (i.e. slow wave sleep, SWS), e.g. [29]. Furthermore, pharmacological increases in acetylcholine concentration induce cortical EEG desynchronization, an effect that is antagonized by atropine, an acetylcholine antagonist, e.g. [33].

In addition to neurons with a wake-active discharge pattern, neurons that discharge preferentially during SWS are found in those ventral forebrain sites in which electrical stimulation produces sleep and lesions produce insomnia, for review see [71]. These sleep-active neurons are most numerous in the POAH (located medial to the cholinergic zone of BF) where they comprise about 25% of the cells recorded in vivo [71]. Neurons that discharge prior to the onset of SWS, a putative characteristic of neurons responsible for the induction of non-REM, are found throughout the BF but are most numerous in the POAH. In the magnocellular BF region these sleep-active cells are predominantly found ventral to the region of dense cholinergic neurons [73]. Lesion and microinjection studies also indicate that the POAH is an important somnogenic brain area (for review see [66], [45]) including the finding that the POAH is a potent site for the sleep-inducing effects of AD [77].

A series of experiments is described below that endeavor to understand the role of endogenous AD in the sleepiness associated with prolonged wakefulness by identifying the brain structures and sequence of cellular events that may mediate the somnogenic properties of AD. Multiple approaches were used including the in vivo neurochemical assessment of extracellular AD levels, local brain microdialysis perfusion of adenosinergic drugs, and both in vivo and in vitro electrophysiological recording. A detailed description of the methodologies is provided for novel procedures, and for those experiments that have not been published elsewhere (see Appendix A).