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The perifornical-lateral hypothalamic area (PF-LHA) has been implicated in the regulation of arousal. The PF-LHA contains wake-active neurons that are quiescent during nonREM sleep and in the case of neurons expressing the peptide hypocretin (HCRT), quiescent during both nonREM and REM sleep. Adenosine is an endogenous sleep factor and recent evidence suggests that adenosine via A1 receptors may act on PF-LHA neurons to promote sleep. We examined the effects of bilateral activation as well as blockade of A1 receptors in the PF-LHA on sleep-wakefulness in freely behaving rats.
The sleep-wake profiles of male Wistar rats were recorded during reverse microdialysis perfusion of artificial cerebrospinal fluid (aCSF) and two doses of adenosine A1 receptor antagonist, 1,3-dipropyl-8-phenylxanthine (CPDX; 5μM and 50μM) or A1 receptor agonist, N6-cyclopentyladenosine (CPA; 5μM and 50μM) into the PF-LHA for 2h followed by 4h of aCSF perfusion. CPDX perfused into the PF-LHA during lights-on phase produced arousal (F=7.035, p <0.001) and concomitantly decreased both nonREM (F=7.295, p<0.001) and REM sleep (F=3.456, p<0.004). In contrast, CPA perfused into the PF-LHA during lights-off phase significantly suppressed arousal (F = 7.891; p <0.001) and increased nonREM (F = 8.18; p <0.001) and REM sleep (F = 30.036; p = <0.001). These results suggest that PF-LHA is one of the sites where adenosine, acting via A1 receptors, inhibits PF-LHA neurons to promote sleep.
The perifornical-lateral hypothalamic area (PF-LHA) has been implicated in the regulation of behavioral arousal (Gerashchenko and Shiromani, 2004; Jones, 2005; McGinty and Szymusiak, 2003). The PF-LHA contains a heterogeneous population of neuronal groups as reflected by their state-dependent discharge properties as well as neurotransmitter phenotypes. These include cells expressing hypocretin or orexin (HCRT), melanin-concentrating hormone (MCH), Gamma-aminobutyric acid (GABA) and glutamate. Amongst various neuronal groups in the PF-LHA, HCRT neurons in particular have been extensively studied and have been implicated in the facilitation and/or maintenance of arousal (Alam et al., 2002; Koyama et al., 2003; Sakurai, 2007; Siegel, 2004; Takahashi et al., 2008). HCRT neurons exhibit wake-associated discharge and c-Fos protein immunoreactivity (Fos-IR) and are quiescent during nonREM and REM sleep (Espana et al., 2003; Estabrooke et al., 2001; Lee et al., 2005; Mileykovskiy et al., 2005). Local applications of HCRT at various projection targets including the basal forebrain, preoptic area and locus coeruleus promote waking (Bourgin et al., 2000; Methippara et al., 2000; Thakkar et al., 2001). Human narcoleptics exhibit HCRT cell loss (Peyron et al., 2000; Thannickal et al., 2000). Symptoms of narcolepsy including excessive sleepiness and cataplexy, are also exhibited by dogs with HCRT-2 receptor mutation (Lin et al., 1999), HCRT peptide knockout mice (Chemelli et al., 1999), rats with a destruction of HCRT-receptor expressing neurons in the PF-LHA (Gerashchenko et al., 2001) and HCRT/ataxin-3 transgenic mice with destruction of HCRT neurons (Hara et al., 2001). In contrast to HCRT neurons, GABAergic and MCH neurons in the PF-LHA have been implicated in sleep regulation. GABAergic and MCH neurons exhibit sleep-associated Fos-IR (Kumar et al., 2005; Modirrousta et al., 2005). MCH neurons discharge selectively during sleep, especially REM sleep and intracerebroventricular administration of MCH increases both nonREM and REM sleep (Hassani et al., 2009; Verret et al., 2003).
Adenosine is a ubiquitous neuromodulator that has been implicated in the regulation of sleep (Basheer et al., 2004; Benington and Heller, 1995; Datta and Maclean, 2007; Dunwiddie and Masino, 2001; McCarley, 2007). The neuronal production of adenosine is coupled with metabolic activity and is higher during waking as compared with sleep (Maquet, 1995). Adenosine or its agonists promote sleep and increase EEG slow wave activity, whereas its antagonists, e.g., caffeine and theophylline, are potent behavioral stimulants and suppress sleep (Benington et al., 1995; Bennett and Semba, 1998; Landolt et al., 1995; Methippara et al., 2005; Radulovacki et al., 1984). Adenosine acts via A1, A2A, A2B and A3 receptors; A1 and A2A receptors are known to mediate the sleep-promoting effects of adenosine (Basheer et al., 2007; Dunwiddie and Masino, 2001; Ribeiro et al., 2002; Scharf et al., 2008). The A1 subtype inhibits adenylate cyclase and is inhibitory, whereas the A2A subtype stimulates adenylate cyclase and produces excitatory effects in central nervous system. Recently we found that in the lateral preoptic area, A1 receptor activation produced arousal whereas A2A receptor activation produced sleep enhancement, suggesting that adenosine-induced sleep is site and receptor dependent (Methippara et al., 2005).
Although many studies examining the role of adenosine as a homeostatic sleep factor have focused on the cholinergic basal forebrain (Alam et al., 1999; Basheer et al., 2004; Blanco-Centurion et al., 2006; Thakkar et al., 2003a; Thakkar et al., 2003b), evidence suggests that adenosine in the PF-LHA may also play a role in the homeostatic regulation of sleep. For example, immunohistochemical evidence suggests that A1 receptors are localized on HCRT neurons (Thakkar et al., 2002). An in vitro pharmacological study suggests that adenosine inhibits HCRT neurons and that this effects is mediated via A1 receptors (Liu and Gao, 2007). A recent in vivo study found that adenosine A1 receptor antagonist, when microinjected into the PF-LHA produced arousal and suppressed nonREM and REM sleep (Thakkar et al., 2008).
In this study we examined the effects of bilateral perfusion, using reverse microdialysis, of an adenosine A1 receptor agonist, N6-cyclopentyladenosine, and an A1 receptor antagonist, 1,3-dipropyl-8-phenylxanthine, into the PF-LHA on the sleep-wake profiles of rats. Unlike microinjection study (Thakkar et al., 2008), drug delivery via reverse microdialysis allowed us to examine the long-term effects of the continuous perfusion of pharmacological agents that were delivered without disturbing the animals. Furthermore, it also reduced the likelihood that treatment effects could be due to mechanical or inflammatory responses (Quan and Blatteis, 1989).
Locations of the microdialysis probes along with the outlines of membrane that were used for perfusing CPDX and CPA are shown in figure-1. The microdialysis probes were localized in the PF-LHA and adjoining areas between AP —2.8 to −3.3 (Paxinos and Watson, 1998). In earlier studies we found that perfusion of bicuculline or serotonin into the PF-LHA using the same drug delivery protocol (Alam et al., 2005; Kumar et al., 2007) affected c-Fos-IR in 500-750μm radius around the probe. Therefore, based on the probe locations, it is likely that the perfused CPDX and CPA affected areas including perifornical area, portions of dorsomedial and ventomedial hypothalamic area, lateral hypothalamic area, and ventral zona incerta.
The effects of two doses of CPDX vs. aCSF perfused into the PF-LHA on sleep-wakefulness were studied in a group of 7 rats. The percent time (mean ± S.E.M.) spent in waking, nonREM, and REM sleep during the 6h recording period including 2h of treatment and 4h of post-treatment conditions for aCSF (n=7) and CPDX (5μM, n=5 and 50μM, n=6) treated rats are shown in figure-2. aCSF treated rats spent a significant portion of the recording time in sleep including nonREM (56.8 ± 1.6%) and REM sleep (8.9 ± 1.2%) during the 6h-recording period without significant differences between treatment and post-treatment periods. CPDX treated rats spent significantly more time in waking (F = 7.035; p <0.001) and less time in nonREM (F = 7.295; p <0.001) and REM sleep (F = 3.456; p = 0.004). Of the two doses used, 50μM CPDX-induced behavioral changes were significantly greater compared to both aCSF and 5μM CPDX treatments during the 2h of its perfusion. The sleep-wake profiles during the post-treatment period in the three groups were comparable.
The frequencies of waking, nonREM and REM sleep episodes of various durations during aCSF vs. 50μM CPDX perfusion during the 2h treatment period are shown in figure-3. During CPDX perfusion, rats exhibited a significant increase in the frequency of waking episodes including medium (>30-120s) and long episodes (>120s). Animals exhibited significant reductions in number of medium and long episodes of nonREM sleep in response to CPDX. CPDX significantly reduced the frequency of REM sleep episodes. Fewer medium and long REM episodes were encountered during aCSF perfusion. During CPDX perfusion their frequencies also decreased but not significantly. The number of waking, nonREM and REM sleep episodes during post-treatment conditions after aCSF and 50μM CPDX perfusion were comparable.
The effects of two doses of CPA vs. aCSF perfused into the PF-LHA on sleep-wakefulness were studied in a group of 9 rats. The sleep-wake profiles of rats microdialysed with aCSF (n=9) and CPA (5μM, n=5 and 50μM, n=8) on percent time (mean ± S.E.M.) spent in waking, nonREM, and REM sleep are shown in figure-4. aCSF treated rats were predominantly awake (66.6 ± 1.7%) and spent less time in nonREM (29.7 ± 1.4%) and REM sleep (3.7 ± 0.4%) during the 6h-recording period. There were no significant differences between 2hr treatment and 4hr post-treatment conditions. CPA perfusion significantly decreased the mean time spent in waking (F = 7.891; p <0.001) and increased the meantime spent in nonREM (F = 8.18; p <0.001) and REM sleep (F = 30.036; p = <0.001). Of the two doses used, 50μM CPA induced significantly greater suppression in waking and enhancement of nonREM and REM sleep, compared to both aCSF and 5μM CPA treatments during the 2h of its perfusion and the effects lasted up to the first 2h post-treatment. The sleep-wake profiles during the 3-4h post-treatment period in the three groups were not significantly different.
The frequencies of waking, nonREM and REM sleep episodes during aCSF and 50μM CPA perfusion during the 2h treatment period are shown in figure-5. The frequency of waking episodes decreased significantly in response to CPA due to a significant decrease in the number of long episodes (>120s). The number of nonREM sleep episodes in particular long episodes of nonREM sleep increased significantly. CPA perfusion also increased the frequency of REM sleep episodes, although not significantly (p=0.07). The frequency and duration of waking, nonREM and REM sleep episodes during post-treatment conditions after aCSF and 50μM CPA perfusion were comparable.
This study demonstrates that blockade of A1-receptor-mediated adenosinergic transmission in the PF-LHA by local perfusion of an adenosine A1 receptor antagonist produced arousal with concomitant reductions in nonREM and REM sleep in sleeping rats. Conversely, adenosine A1 receptor activation by its agonist suppressed waking and increased nonREM and REM sleep in awake animals. We note: a) that A1 receptor agonist and antagonist produced opposite and reversible behavioral effects; b) that the microdialysis drug administration method we used, unlike an earlier microinjection study, provides a better control on drug concentration and its continuous delivery in undisturbed animals and also reduces the likelihood that treatment effects could be due to mechanical or inflammatory responses (Quan and Blatteis, 1989; Thakkar et al., 2008); and c) that in this study the microdialysis probes used for delivering A1 receptor agonist and antagonist were localized in the PF-LHA (see figure-1) and based on our previous studies of Fos-IR in response to bicuculline and serotonin perfusions into the PF-LHA, it is likely that the perfused drugs affected neuronal population in ~500-750μm diameter field around the microdialysis probe (Alam et al., 2005; Kumar et al., 2007). We conclude, therefore, that the observed effects were physiological and that endogenous adenosine acting via A1 receptor plays a role in the regulation of sleep by inhibiting PF-LHA neurons.
Studies regarding adenosinergic control of sleep have largely been focused on the basal forebrain neurons (see introduction). The findings of the present study that application of an A1 receptor agonist and an antagonist in the PF-LHA induced and suppressed sleep, respectively, indicate that A1 receptor-mediated hypnogenic responses to adenosine are not confined to the basal forebrain, but can be observed in other brain regions including the PF-LHA (Oishi et al., 2008). These findings are consistent and complimentary to the previous report that microinjection of A1 receptor antagonist into the PF-LHA produced arousal and suppressed spontaneous nonREM/REM sleep as well as significantly increased the latency of nonREM sleep during recovery after sleep deprivation (Thakkar et al., 2008). We found that A1 receptor antagonist and agonist altered the number of medium and long episodes. The number of short waking episodes, which potentially reflects state initiations, was unaffected. This suggests that the majority of neurons in the PF-LHA affected by adenosine are involved in arousal maintenance.
The PF-LHA contains a predominant population of neurons that are involved in cortical activation and behavioral arousal. These include wake/REM-active and wake-active neurons that are quiescent during nonREM sleep and during both nonREM and REM sleep, respectively (see introduction). The extracellular release of adenosine has been linked with the metabolic state of the cell. Although the extracellular levels of adenosine in the PF-LHA during spontaneous or prolonged waking remain unknown, it is likely that the activation of PF-LHA neurons, including HCRT neurons, during arousal contributes to adenosine release locally. In addition, a recent study in transgenic mice in which purinergic gliotransmission was inhibited by selective expression of a dominant negative SNARE domain under doxycycline control, supports glia as a potent source of adenosine since these mice exhibited significantly reduced accumulation of sleep pressure and attenuated responses to adenosinergic A1 receptor antagonist (Halassa et al., 2009). The accumulated adenosine, irrespective of its source, would eventually inhibit wake-promoting neurons including HCRT neurons via A1 receptor to regulate sleep.
In a complimentary study we found that A1 receptor activation significantly suppressed the extracellular discharge activity of the recorded PF-LHA neurons (unpublished data). This finding along with the behavioral changes as observed in this study suggests that adenosine exerts its sleep-promoting effect in the PF-LHA via A1 receptor mediated inhibition of its neurons. That PF-LHA neurons are under adenosinergic influences is supported by an earlier in vitro study showing that adenosine via A1 receptor exerts inhibitory influences on HCRT neurons, most potently via presynaptic inhibition of the glutamatergic input or excitatory postsynaptic potential (Liu and Gao, 2007).
Anatomically, HCRT neurons project extensively to major arousal systems including the basal forebrain. Interestingly, a recent study found that as compared to the control group, adenosine levels in the basal forebrain did not increase with 6h of sleep deprivation in rats in which HCRT receptor bearing PF-LHA neurons were lesioned with HCRT-2-saporin conjugate. This finding suggests that the activation of neuronal population in the PF-LHA could be driving basal forebrain release of adenosine during arousal (Murillo-Rodriguez et al., 2008).
It is pertinent to note that in the PF-LHA, HCRT neurons constitute only a subset of wake-active neurons and that HCRT neurons are intermingled with various other neuronal groups including MCH and GABAergic neurons that have been implicated in the regulation of sleep (Alam et al., 2005; Estabrooke et al., 2001; Hassani et al., 2009; Kumar et al., 2005; Verret et al., 2003). However, evidence suggests that the net physiological output of the PF-LHA is wake promoting. For example, glutamic acid microinjections into the PF-LHA produce arousal and suppress both nonREM and REM sleep, whereas, rats with neurotoxic lesion of this area exhibit increased nonREM and REM sleep (Alam and Mallick, 2008; Gerashchenko et al., 2001). Although the relative distribution of A1 receptors on various neuronal groups, in particular on GABAergic and MCH neurons, remains unknown it is likely that behavioral changes observed in this study were predominantly mediated via blockade or activation of wake-promoting HCRT and other neurons of unknown neurotransmitter phenotypes. Alternatively, it is possible that adenosine promotes sleep in part: a) through the indirect disinhibition of sleep-active neurons within the PF-LHA; and /or b) by activating sleep-promoting neurons, e.g., MCH and GABAergic neurons via A2A receptors. Although the distribution of A2A receptors in the PF-LHA is not well characterized, the perfused concentrations of agonist and antagonist used in this study may affect A2A receptors as well (Fredholm et al., 2001).
In conclusion, the findings of this study support that PF-LHA is one of the sites where adenosine acting via A1 receptors, in part, inhibits wake-promoting neurons to promote sleep.
Experiments were conducted on 16 freely behaving Sprague-Dawley male rats. All the experiments were conducted in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals and were approved by Veterans Administration Greater Los Angeles Healthcare System’s Institutional Animal Care and Use Committee. The rats were housed individually and maintained on 12:12 h light:dark cycle (lights on at 6:00AM) with food and water available ad libitum.
The details of the surgical procedure and experimental protocol are described in earlier studies (Alam et al., 2005). In brief, rats (n=16) were stereotaxically implanted with a) EEG (electroencephalogram) and EMG (electromyogram) electrodes for the monitoring of the sleep-wake cycles, and b) a bilateral microdialysis guide cannulae (23G stainless steel tube) using the stereotaxic coordinates, A, −2.9 to −3.1; L, 1.4 to 1.6; H, 4.5 to 5.5 (Paxinos and Watson, 1998) such that their tips rested 3 mm above the dorsal aspect of the PF-LHA and were blocked with stylets. Experiments were started at least 10 days after surgery and after acclimatization of the animals with the recording environment during the recovery period.
At least 24h before the experiment, the stylets of the microdialysis guide cannulae were replaced by microdialysis probes (semi-permeable membrane tip length, 1mm; outer diameter, 0.22 mm; molecular cut off size, 50 kDa; Eicom, Japan), fixed with dental acrylic and flushed with artificial cerebrospinal fluid (aCSF; composition in mM, 145 NaCl, 2.7 KCl, 1.3 MgSO4, 1.2 CaCl2, and 2 Na2HPO4; pH, 7.2) at a flow rate of 2 μl/min for 4h. The time taken by the aCSF solution to travel from the reservoir to the tips of the probes were precisely calculated.
Studies suggest that maximum number of PF-LHA neurons exhibit Fos-IR during lights-off period when rats spent significantly more time in waking (Espana et al., 2003; Estabrooke et al., 2001; Kumar et al., 2007). Since in vitro studies suggest that adenosine exerts inhibitory influences on HCRT neurons via A1 receptor (Liu and Gao, 2007), the effects of adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA), on sleep-wakefulness were examined during lights-off period between 8.00PM to 3.00AM. In contrast, the effects of A1 receptor antagonist, 1,3-dipropyl-8-phenylxanthine (CPDX) on sleep-wakefulness were examined during lights-on period, when rats are predominantly asleep. Both CPA and CPDX were dissolved in distilled water and 0.1N NaOH, respectively, at mM concentrations and then the stock solutions were diluted to μM concentrations for perfusion. The pH of the perfusing solution was adjusted to 7.2. The EEG and EMG of each animal were recorded during perfusion of aCSF and 2 doses of CPA (5μM and 50μM) or CPDX (5μM or 50μM) for 2h followed by 4h of aCSF perfusion. The order of aCSF or drug delivery was randomized and the treatments were spaced by at least 24h. In few cases, the probes either got blocked or leaked and therefore, animals did not receive all three treatments. In our earlier studies we found that microdialysis probes remained functional for 5-6 days after implantation(Alam et al., 1999; Kumar et al., 2007), therefore, each animal was used for 5-6 days in this study. The effects of CPA and CPDX were examined in different group of animals. At the end of the experiments, rats were sacrificed and the locations of the microdialysis probes were histologically confirmed.
After a lethal dose of pentobarbital (100 mg/kg, i.p.) rats were injected with heparin (500U, i.p.), and perfused transcardially with 30-50 ml of 0.1 M phosphate buffer (pH 7.2) followed by 500 ml of 4% paraformaldehyde in phosphate buffer containing 15% saturated picric acid solution. The brains were removed and equilibrated in 10%, 20% and finally 30% sucrose. Horizontal sections were freeze-cut at 30μm thickness. Alternate sections from the series of sections spanning the probe tract were immunostained for HCRT-1 protein (Alam et al., 2005; Kumar et al., 2007).
A single person who was unaware of the experimental conditions scored sleep-waking states in 10 sec epochs as waking, non-rapid eye movement (nonREM) sleep and REM sleep, according to the method described earlier (Alam and Mallick, 1990). The sleep-wake data were compared at 2h intervals; 2 hr of aCSF/drug treatments and 4h of post-treatment with aCSF perfusion. The level of significance amongst different treatment conditions was determined by using One Way Repeated Measures ANOVAs followed by pair-wise multiple comparisons using Holm-Sidak method. We found strongest effects of CPDX and CPA during the 2h treatment period and therefore, the frequencies of sleep-wake episodes during aCSF vs. effective doses of CPDX or CPA during treatment period were further compared using paired t-test.
This work was supported by the US Department of Veterans Affairs Medical Research Service and US National Institutes of Health grants, NS-050939 (Alam); MH47480, HL60296 (McGinty); and MH63323 (Szymusiak).
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