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Sleep homeostasis is the process by which recovery sleep is generated by prolonged wakefulness. The molecular mechanisms underlying this important phenomenon are poorly understood. Here, we assessed the role of the intercellular gaseous signaling agent NO in sleep homeostasis. We measured the concentration of nitrite and nitrate, indicative of NO production, in the basal forebrain (BF) of rats during sleep deprivation (SD), and found the level increased by 100 ± 51%. To test whether an increase in NO production might play a causal role in recovery sleep, we administered compounds into the BF that increase or decrease concentrations of NO. Infusion of either a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, or a NO synthase inhibitor, Nω-nitro-l-arginine methyl ester (L-NAME), completely abolished non-rapid eye movement (NREM) recovery sleep. Infusion of a NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2diolate (DETA/NO), produced an increase in NREM that closely resembled NREM recovery after prolonged wakefulness. The effects of inhibition of NO synthesis and the pharmacological induction of sleep were effective only in the BF area. Indicators of energy metabolism, adenosine, lactate and pyruvate increased during prolonged wakefulness and DETA/NO infusion, whereas L-NAME infusion during SD prevented the increases. We conclude that an increase in NO production in the BF is a causal event in the induction of recovery sleep.
Sleep loss, induced by prolonged wakefulness, produces a decline in cognitive and motor performance (Dinges et al. 1997), mood disturbances, memory deficits (Chee and Choo 2004) and affects immune function (Bryant et al. 2004). These effects are restored by recovery sleep, which is characterized by prolongation and intensification of both the non-rapid eye movement (NREM) and rapid eye movement (REM) components of sleep. Although the two-process model of sleep regulation (Borbely 1982) accurately describes the expected duration of recovery sleep, the molecular mechanisms that underlie this regulation remain less clear. Endogenous sleep factors – substances that accumulate in the brain during prolonged wakefulness – have been suggested to be mediators of homeostatic sleep regulation (Borbely and Tobler 1989). One potential sleep factor is the inhibitory neuromodulator adenosine (Benington and Heller 1995); during prolonged wakefulness extracellular adenosine concentration increases in the basal forebrain (BF) and induces sleep (Porkka-Heiskanen et al. 1997). As adenosine is an indicator of disturbed energy balance (Dunwiddie and Masino 2001), we hypothesized that during sleep deprivation (SD) continuous activity of the waking-promoting cells in the cholinergic region of the BF (Detari et al. 1984; Szymusiak and McGinty 1986; Szymusiak and McGinty 1989) leads to unfavourable changes in energy demand/supply ratio and consequent adenosine release in this area. Supporting our hypothesis, experimentally induced local energy depletion in the BF increased extracellular adenosine concentration and concurrently induced an increase in sleep (Kalinchuk et al. 2003). Recently, it has been shown that NO can inhibit neuronal energy production (Brorson and Zhang 1999; Maletic et al. 2004; Rosenberg et al. 2000) and stimulate adenosine release from forebrain neurones (Rosenberg 2000 et al.), leading us to consider the possible role of NO in the regulation of behavioural state, and specifically in the induction of recovery sleep.
NO is an intercellular signalling molecule that regulates both physiological and pathophysiological processes in the CNS (Garthwaite and Boulton 1995; Gross and Wolin 1995; Keynes and Garthwaite 2004). NO concentrations undergo state-dependent modulation during the sleep–wake cycle both in the thalamus and the cortex (Burlet and Cespuglio 1997; Williams et al. 1997), but there are no measurements of NO concentrations during prolonged wakefulness in any brain area. Several previous studies have shown that intraperitoneal, subcutaneous or intracerebroventricular administration of inhibitors of the NO-synthesizing enzyme NO synthase (NOS) decreases spontaneous sleep (Kapas et al. 1994; Dzoljic et al. 1996; Monti et al. 1999, 2001; Ribeiro et al. 2000; Monti and Jantos 2005; Ribeiro and Kapas 2005; Cavas and Navarro 2006) whereas NO donors increase it (Kapas and Krueger 1996; Monti and Jantos 2004a), suggesting that NO may have a role as a sleep-facilitating agent. Local injections of NOS inhibitors into the pons, including the cholinergic laterodorsal/pedunculopontine tegmental nuclei (LDT/PPT) as well as the dorsal raphe nucleus, have generally also decreased either REM sleep or both NREM and REM sleep (Datta et al. 1997; Leonard and Lydic 1997; Hars 1999; Monti et al. 1999, 2001). Studies employing local manipulations of NO level in the BF have provided controversial results: injections of NOS inhibitors into the BF have been reported to decrease NREM sleep and increase wakefulness (Monti and Jantos 2004b) or have no effect on sleep (Vazquez et al. 2002), and injection of the NO precursor l-arginine or a NO donor have been reported to be ineffective (Monti and Jantos 2004b). Some studies have also suggested that NO has a pro-arousal (Pape and Mager 1992; Marino and Cudeiro 2003) effect. However, the role of NO in the induction of recovery sleep after SD has been addressed in only one study, in which the NOS inhibitor Nω-nitro-l-arginine methyl ester (L-NAME) administered intraperitoneally decreased NREM sleep recovery (Ribeiro et al. 2000).
We hypothesized that release of NO locally in the BF during prolonged wakefulness, either from intrinsic cells or from terminals of projecting neurones such as those from the LDT/PPT, may be critical for the subsequent increase in sleep, and that the NO release is associated with changes in energy metabolism. To test this hypothesis we either decreased the amount of NO produced during prolonged wakefulness or pharmacologically increased it, and measured the effect of these manipulations on metabolites of energy metabolism and sleep. We also measured the concentrations of NO2– and NO3– (collectively termed NOx–) in the BF during SD, thereby assessing directly the question of whether NO levels change during prolonged wakefulness.
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- Materials and methods
The main finding of the present study is that the production of NO in the basal forebrain during SD is necessary and sufficient to explain the production of NREM recovery sleep. NOx– concentration in the BF rose during SD and fell during recovery sleep; infusion of a NO donor into the BF produced an increase in NREM sleep comparable to that produced by SD, and infusion of a NOS inhibitor or a NO scavenger into the BF prevented the generation of NREM recovery sleep. Remarkably, these effects were restricted to the BF; outside this structure the treatments failed to have effects on sleep. Moreover, increases in adenosine, lactate and pyruvate concentrations in the BF preceded the increase in sleep, whereas inhibition of recovery sleep was not associated with an increase in metabolite concentrations, suggesting a connection between energy metabolism and production of recovery sleep in the basal forebrain area.
We have previously established that SD is accompanied by an increase in extracellular adenosine concentration in the BF (Porkka-Heiskanen et al. 1997; Kalinchuk et al. 2003). In the present study, an NO-associated increase in NREM sleep was accompanied by increased concentrations of adenosine, whereas inhibition of NO synthesis or use of an NO-scavenging compound was associated with unchanged levels of adenosine and no NREM sleep recovery, suggesting that the effect of NO on NREM sleep recovery is mediated through an increase in extracellular adenosine in the BF. This view was further confirmed by the observation that blocking of adenosine receptors with caffeine prevented the DETA/NO-induced increase in sleep. An increase in extracellular adenosine level is a signal of discrepancy between energy demand and availability (Dunwiddie and Masino 2001). We have previously shown that experimentally induced energy depletion in the BF increases local adenosine, lactate and pyruvate levels and induces NREM sleep (Kalinchuk et al. 2003). Inhibition of energy production as a consequence of local NO production may be a specific pathway for generating adenosine release in the vicinity of structures capable of producing sleep. In vitro, NO donors stimulate glycolysis, increasing adenosine, lactate and pyruvate levels, and inhibit oxidative phosphorylation, resulting in depletion of total energy production and a decrease in the ATP/ADP ratio (Rosenberg et al. 2000; Maletic et al. 2004). Neurones and astrocytes respond differently to the increase in NO: astrocytes are able to maintain their energy production by increasing glycolysis by activating 6-phosphofructo-1-kinase, whereas neurones appear to be unable to do this (Almeida et al. 2004). Active inhibition by NO of energy production in a specific brain region – the BF – may be considered as a specific mechanism for induction of sleep in response to prolonged wakefulness. We propose that the concentration of NO increases during prolonged wakefulness, resulting in inhibition of local energy metabolism and production of adenosine, which is the signal for NREM sleep induction.
NO production undergoes state-dependent modulations during the sleep–wake cycle both in the thalamus and the cortex, probably due to state-dependent changes in the activity of neuronal NOS-containing projection neurones (Burlet and Cespuglio 1997; Williams et al. 1997); the NO values in both structures were lower during sleep than during waking, suggesting an activity-related NO increase in the brain. NO concentrations during SD have not been assessed previously. Our results, showing an increased level of NO in the BF during SD, support the hypothesis that NO production is critical for homeostatic sleep regulation. One previous study has shown a decrease in recovery sleep after L-NAME administration during SD (Ribeiro et al. 2000). In that study the recovery sleep was attenuated but not completely abolished as in the present study. Systemic administration of the drug in the previous study, as opposed to local administration in the present study, may explain the partially different outcomes of the two studies.
Our results are in agreement with those of many previous studies, in which either intracerebroventricular or systemic routes of administration of NO donors or NOS inhibitors were used, most often during the spontaneous sleep–wake cycle (reviewed in Gautier-Sauvigne et al. 2005). In the present study, administration of both the NOS inhibitor and the NO scavenger during spontaneous sleep decreased NREM sleep, indicating that a certain NO level is required for the appearance of a normal amount of NREM sleep, and further that the effects of the drugs during SD may have two components: effects on the mechanisms of recovery sleep production (sleep homeostasis) and effects on spontaneous sleep. Remarkably, L-NAME decreased spontaneous sleep only when administered into the BF area, indicating that the effect of NO on spontaneous sleep is also at least partially localized. Two previous studies, employing local microinjections of NOS inhibitors into the BF during spontaneous sleep, reported conflicting data (Vazquez et al. 2002; Monti and Jantos 2004b); one reported changes in sleep and the other did not. The discrepancies can most probably be explained by the sharp localization of the effect, as shown in the present study. Previous studies have also shown that local administration of L-NAME into the pons modulates sleep (Datta et al. 1997; Leonard and Lydic 1997; Hars 1999). NOS is co-localized with acetylcholine in most of the BF nuclei, as well as in the LDT/PPT nuclei that project to the BF (Vincent and Kimura 1992). Both the BF and the LDT/PPT area of the pons contain cholinergic neurones, which regulate the vigilance state by release of acetylcholine (Jones 1991; Leonard and Lydic 1997; Hars 1999). As it has been shown that NO modulates acetylcholine release (Vazquez et al. 2002; Leonard and Lydic 2005), it is possible that the effects of NO on sleep are mediated through cholinergic neurones (Leonard and Lydic 2005).
Adenosine, through A1 receptors, is an inhibitory neuromodulator (Fredholm 1995). Clinically, adenosine is used for neuroprotection in connection with seizures (Boison 2005) and as anti-arrhythmic treatment of supraventricular tachycardia (Hutchinson and Scammells 2004). Adenosine is also a powerful vasodilator (Biaggioni 2004). It stimulates neuronal activity through A2A receptors (Fredholm 1995). Adenosine appears to have a specific function in the regulation of recovery sleep (Porkka-Heiskanen et al. 1997). The site-specificity of the effects in the present study is in agreement with earlier studies, in which the increase in adenosine levels during SD was found to be restricted to the BF (Porkka-Heiskanen et al. 2000; Kalinchuk et al. 2003). The sites at which adenosine, lactate, pyruvate and NO increases were found during SD closely correspond to the area where the cholinergic cells of the BF are situated – the HDB, substantia innominata and MCPO. The present results suggest that recovery sleep may be regulated through waking-active cholinergic cells, whereby adenosine acting on A1 receptors would decrease their firing rate (Rainnie et al. 1994). However, sleep homeostasis was not affected in mice with A1 receptor knockout (Stenberg et al. 2003), and a specific lesion of cholinergic cells did not affect sleep (Kapas et al. 1996; Gerashcenkko et al. 2001). Another possibility is that adenosine could disinhibit the inhibitory GABAergic neurones of the nearby ventrolateral preoptic nucleus (Sherin et al. 1996; Chamberlin et al. 2003) thus promoting sleep. A2A receptor agonist injection into the subarachnoid space promoted sleep (Scammell et al. 2001), suggesting that adenosine could also modulate sleep through activation of A2A receptors. In addition, the effects of caffeine were recently shown to be mediated through A2A receptors (Huang et al. 2005). It is probable that during SD these mechanisms work in synchrony: the increased adenosine level inhibits the waking-active neurones and disinhibits sleep-active neurones through A1 receptors, while activating sleep-active neurones through A2A receptors. The sleep response to increased adenosine levels continues for several hours (Porkka-Heiskanen et al. 1997). We have previously shown that increased adenosine levels in the BF increase the expression of A1 receptors, providing one possible mechanism for the prolonged sleep-promoting effect (Basheer et al. 2001).
An alternative explanation that we considered is that the drugs used in this study might have non-specific effects on sleep that subtract from recovery sleep without actually affecting the fundamental process. For example, infusion of glutamate might block recovery sleep but this does not necessarily mean that glutamate plays a specific role in homeostatic sleep regulation. We do not think that the effects of the drugs used in this study are non-specific because: (i) drugs of varied chemical structure were found to act in a predictable way based on their effect on NO levels: a NOS inhibitor and a NO scavenger inhibited recovery sleep, whereas a NO donor mimicked recovery sleep; (ii) NO2–/NO3– levels increased with SD, indicative of an association between NO production and recovery sleep; and (iii) the effects of drugs blocking recovery sleep were long-lasting. The effects were typically at a maximum not during or immediately after administration, which was during the light/rest period, when a stimulant would be expected to have maximal effect, but during the subsequent dark/active period (Figs 7 and 8). Interestingly, reduction of NO levels in the BF also had long-lasting effects on spontaneous sleep (Figs 9 and 10), suggesting that NO production is a consequence of waking per se and is central in the generation of sleep drive or Process S.
The present results, in addition to confirming NO as a powerful sleep-facilitating agent, provide strong evidence that NO is a critical part of the homeostatic sleep control mechanism regulating effects of prolonged wakefulness, and is, in fact, necessary and sufficient for the generation of recovery sleep. The results further support the hypothesis that local energy depletion in the BF, as reflected in increases in adenosine, lactate and pyruvate levels specifically in this area during SD, may be the initiator of the chain of events that culminate in the induction of recovery sleep. Adenosine, one of the metabolites indicative of energy depletion, and which during SD accumulates in the extracellular space where it can activate adenosine receptors, appears to be the key molecule in the final induction of recovery sleep.