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Research Report

Role of adenosine A1 receptor in the perifornical–lateralhypothalamic area in sleep–wake regulation in rats

Md. Noor Alama,b,⁎, Sunil Kumara,c, Seema Raia, Melvi Methipparaa,b,Ronald Szymusiaka,c,d, Dennis McGintya,b

aResearch Service, Veterans Affairs Greater Los Angeles Healthcare System, Sepulveda, CA, USAbDepartment of Psychology, University of California, Los Angeles, CA, USAcDepartment of Medicine, School of Medicine, University of California, Los Angeles, CA, USAdDepartment of Neurobiology, School of Medicine, University of California, Los Angeles, CA, USA

A R T I C L E I N F O

⁎ Corresponding author. Research Service (1Sepulveda, CA 91343, USA. Fax: +1 818 895 95

E-mail address: noor@ucla.edu (M.N. AlamAbbreviations: aCSF, artificial cerebrospi

phenylxanthine, an A1 receptor antagonist; Fmelanin-concentrating hormone; Non-REM,buffered saline

0006-8993/$ – see front matter. Published bydoi:10.1016/j.brainres.2009.09.066

A B S T R A C T

Article history:Accepted 16 September 2009Available online 23 September 2009

The perifornical–lateral hypothalamic area (PF-LHA) has been implicated in the regulationof arousal. The PF-LHA contains wake-active neurons that are quiescent during non-REMsleep and in the case of neurons expressing the peptide hypocretin (HCRT), quiescent duringboth non-REM and REM sleep. Adenosine is an endogenous sleep factor and recent evidencesuggests that adenosine via A1 receptors may act on PF-LHA neurons to promote sleep. Weexamined the effects of bilateral activation as well as blockade of A1 receptors in the PF-LHAon sleep–wakefulness in freely behaving rats. The sleep–wake profiles of male Wistar ratswere recorded during reverse microdialysis perfusion of artificial cerebrospinal fluid (aCSF)and two doses of adenosine A1 receptor antagonist, 1,3-dipropyl-8-phenylxanthine (CPDX;5 μM and 50 μM) or A1 receptor agonist, N6-cyclopentyladenosine (CPA; 5 μM and 50 μM) intothe PF-LHA for 2 h followed by 4 h of aCSF perfusion. CPDX perfused into the PF-LHA duringlights-on phase produced arousal (F=7.035, p<0.001) and concomitantly decreased bothnon-REM (F=7.295, p<0.001) and REM sleep (F=3.456, p<0.004). In contrast, CPA perfusedinto the PF-LHA during lights-off phase significantly suppressed arousal (F=7.891, p<0.001)and increased non-REM (F=8.18, p <0.001) and REM sleep (F=30.036, p<0.001). These resultssuggest that PF-LHA is one of the sites where adenosine, acting via A1 receptors, inhibits PF-LHA neurons to promote sleep.

Published by Elsevier B.V.

Keywords:OrexinHypocretinAdenosinePosterior–lateral hypothalamusSleep

1. Introduction

The perifornical–lateral hypothalamic area (PF-LHA) has beenimplicated in the regulation of behavioral arousal (Gerash-

51A3), Veterans Affairs75.).

nal fluid; CPA, N6-cyclopos-IR, c-Fos protein immunon-rapid eye movemen

Elsevier B.V.

chenko and Shiromani, 2004; Jones, 2005; McGinty andSzymusiak, 2003). The PF-LHA contains a heterogeneouspopulation of neuronal groups as reflected by their state-dependent discharge properties as well as neurotransmitter

Greater Los Angeles Healthcare System, 16111 Plummer Street,

entyladenosine, an A1 receptor agonist; CPDX, 1,3-dipropyl-8-noreactivity; GABA, γ-aminobutyric acid; HCRT, hypocretin; MCH,t sleep; PF-LHA, perifornical–lateral hypothalamic area; TBS, Tris-

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phenotypes. These include cells expressing hypocretin ororexin (HCRT), melanin-concentrating hormone (MCH), γ-aminobutyric acid (GABA) and glutamate. Amongst variousneuronal groups in the PF-LHA, HCRT neurons in particularhave been extensively studied and have been implicated inthe 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-associateddischarge and c-Fos protein immunoreactivity (Fos-IR) and arequiescent during non-REM 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 targetsincluding the basal forebrain, preoptic area, and locuscoeruleus promote waking (Bourgin et al., 2000; Methipparaet al., 2000; Thakkar et al., 2001). Human narcoleptics exhibitHCRT cell loss (Peyron et al., 2000; Thannickal et al., 2000).Symptoms of narcolepsy including excessive sleepiness andcataplexy are also exhibited by dogs with HCRT-2 receptormutation (Lin et al., 1999), HCRT peptide knockout mice(Chemelli et al., 1999), rats with a destruction of HCRT receptorexpressing neurons in the PF-LHA (Gerashchenko et al., 2001)and HCRT/ataxin-3 transgenic mice with destruction of HCRTneurons (Hara et al., 2001). In contrast to HCRT neurons,GABAergic and MCH neurons in the PF-LHA have beenimplicated in sleep regulation. GABAergic and MCH neuronsexhibit sleep-associated Fos-IR (Kumar et al., 2005; Modir-rousta et al., 2005). MCH neurons discharge selectively duringsleep, especially REM sleep, and intracerebroventricularadministration of MCH increases both non-REM and REMsleep (Hassani et al., 2009; Verret et al., 2003).

Adenosine is a ubiquitous neuromodulator that has beenimplicated in the regulation of sleep (Basheer et al., 2004;Benington and Heller, 1995; Datta and Maclean, 2007; Dun-widdie and Masino, 2001; McCarley, 2007). The neuronalproduction of adenosine is coupled with metabolic activityand is higher during waking as compared with sleep (Maquet,1995). Adenosine or its agonists promote sleep and increaseEEG slow wave activity, whereas its antagonists, e.g., caffeineand theophylline, are potent behavioral stimulants andsuppress sleep (Benington et al., 1995; Bennett and Semba,1998; Landolt et al., 1995; Methippara et al., 2005; Radulovackiet 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; Dunwid-die 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 andproduces excitatory effects in central nervous system. Recent-ly, we found that in the lateral preoptic area, A1 receptoractivation produced arousal, whereas A2A receptor activationproduced sleep enhancement, suggesting that adenosine-induced sleep is site- and receptor-dependent (Methippara etal., 2005).

Althoughmany studies examining the role of adenosine asa homeostatic sleep factor have focused on the cholinergicbasal forebrain (Alam et al., 1999; Basheer et al., 2004; Blanco-Centurion et al., 2006; Thakkar et al., 2003a,b), evidencesuggests that adenosine in the PF-LHA may also play a rolein the homeostatic regulation of sleep. For example, immu-nohistochemical evidence suggests that A1 receptors are

localized on HCRT neurons (Thakkar et al., 2002). An in vitropharmacological study suggests that adenosine inhibits HCRTneurons and that this effect is mediated via A1 receptors (Liuand Gao, 2007). A recent in vivo study found that adenosine A1

receptor antagonist, when microinjected into the PF-LHAproduced arousal and suppressed non-REM and REM sleep(Thakkar et al., 2008).

In this study, we examined the effects of bilateralperfusion, using reverse microdialysis, of an adenosine A1

receptor agonist, N6-cyclopentyladenosine, and an A1 receptorantagonist, 1,3-dipropyl-8-phenylxanthine, into the PF-LHAon the sleep–wake profiles of rats. Unlike a microinjectionstudy (Thakkar et al., 2008), drug delivery via reverse micro-dialysis allowed us to examine the long-term effects of thecontinuous perfusion of pharmacological agents that weredelivered without disturbing the animals. Furthermore, it alsoreduced the likelihood that treatment effects could be due tomechanical or inflammatory responses (Quan and Blatteis,1989).

2. Results

2.1. Site of drug delivery

Locations of the microdialysis probes along with the outlinesof membrane that were used for perfusing CPDX and CPA areshown in Fig. 1. Themicrodialysis probes were localized in thePF-LHA and adjoining areas betweenAP −2.8 and −3.3 (Paxinosand Watson, 1998). In earlier studies, we found that perfusionof bicuculline or serotonin into the PF-LHA using the samedrug delivery protocol (Alam et al., 2005; Kumar et al., 2007)affected c-Fos-IR in 500–750 μm radius around the probe.Therefore, based on the probe locations, it is likely that theperfused CPDX and CPA affected areas including perifornicalarea, portions of dorsomedial and ventromedial hypothalamicarea, lateral hypothalamic area, and ventral zona incerta.

2.2. Effects of CPDX on sleep–wakefulness

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±SEM) spent in waking, non-REM, andREM sleep during the 6-h recording period including 2 h oftreatment and 4 h of post-treatment conditions for aCSF-(n=7) and CPDX- (5 μM, n=5 and 50 μM, n=6) treated rats areshown in Fig. 2. aCSF-treated rats spent a significant portion ofthe recording time in sleep including non-REM (56.8±1.6%)and REM sleep (8.9±1.2%) during the 6-h recording periodwithout significant differences between treatment and post-treatment periods. CPDX-treated rats spent significantly moretime in waking (F=7.035, p<0.001) and less time in non-REM(F=7.295, p<0.001) and REM sleep (F=3.456, p=0.004). Of thetwo doses used, 50 μMCPDX-induced behavioral changesweresignificantly greater compared to both aCSF and 5 μM CPDXtreatments during the 2 h of its perfusion. The sleep–wakeprofiles during the post-treatment period in the three groupswere comparable.

The frequencies of waking, non-REM, and REM sleepepisodes of various durations during aCSF vs. 50 μM CPDX

Fig. 1 – Sites of CPDX and CPA perfusions. (A) Photomicrograph of a horizontal section (40× magnification) showing tracts of themicrodialysis probes (large arrows) from an animal that was perfused with aCSF and CPDX. These probes were localized inthe HCRT neuronal field (small arrows), which has beenmagnified (400×) in panel B. (C and D) Reconstruction diagrams (coronalsections) through the PF-LHA showing the outlines and locations of the microdialysis probes used for the delivery of aCSF/CPDX (C) and aCSF/CPA (D). Although, the rostrocaudal locations of the microdialysis probes varied ±0.2 mm, they arerepresented on one plane, which was most commonly encountered. In all these studies, aCSF and drugs were perfusedbilaterally; however, the probes are shown on one side for a better anatomical comparison. Arc, arcuate hypothalamic nucleus;DMH, dorsomedial hypothalamic nucleus; f, fornix; mt, mammillothalamic tract; MTu, medial tuberal nucleus; PeF,perifornical nucleus; VMH, ventromedial hypothalamic nucleus; ZI, zona incerta.

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Fig. 2 – Effects of CPDX on sleep–wakefulness. Percent time(mean±SEM) spent in waking (A), non-REM (B), and REMsleep (C) at 2-h intervals during the 6-h recording periodincluding the first 2 h of treatment (aCSF, 5 μM or 50 μMCPDX perfusion into the PF-LHA) and 4 h of post-treatmentrecordings (aCSF perfusion). In the presence of CPDX in thePF-LHA, spontaneously sleeping rats spent significantlymore time in waking and less time in non-REM and REMsleep. *, as compared to aCSF control; $, as compared to 5 μMCPDX. *, $, p<0.05; **, $$, p<0.01.

Fig. 3 – Effects of CPDX on the frequency of sleep–wakeepisodes. The number (mean±SEM) of waking (A), non-REM(B), and REM sleep (C) episodes during the 2-h recordingperiodwith aCSF and 50μMCPDX perfusion into the PF-LHA.CPDX at 50μMproduced a significant increase inwaking andconcomitant decreases in non-REM and REM sleep episodesduring the treatment period. *, p<0.05.

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perfusion during the 2-h treatment period are shown in Fig. 3.During CPDX perfusion, rats exhibited a significant increase inthe frequency of waking episodes including medium (>30–120 s) and long episodes (>120 s). Animals exhibited signifi-cant reductions in number of medium and long episodes ofnon-REM sleep in response to CPDX. CPDX significantlyreduced the frequency of REM sleep episodes. Fewer medium

and long REM episodes were encountered during aCSFperfusion. During CPDX perfusion, their frequencies alsodecreased but not significantly. The numbers of waking, non-REM and REM sleep episodes during post-treatment conditionsafter aCSF and 50 μM CPDX perfusions were comparable.

2.3. Effects of CPA on sleep–wakefulness

The effects of two doses of CPA vs. aCSF perfused into the PF-LHAonsleep–wakefulnesswere studied ina groupof 9 rats. Thesleep–wake profiles of rats microdialysed with aCSF (n=9) andCPA (5 μM, n=5 and 50 μM, n=8) on percent time (mean±SEM)spent in waking, non-REM, and REM sleep are shown in Fig. 4.

Fig. 4 – Effects of CPA on sleep–wakefulness. Percent time(mean±SEM) spent in waking (A), non-REM (B), and REMsleep (C) at 2-h intervals during the 6-h recording periodincluding the first 2 h of treatment (aCSF, 5 μM and 50 μMCPA perfusion into the PF-LHA) and 4 h of post-treatmentrecordings (aCSF perfusion). CPA at 50 μM suppressedwaking and induced non-REM and REM sleep that lasted upto 2 h post-treatment period. *, as compared to aCSF control;$, as compared to 5 μM CPA. *, $, p<0.05; **, $$, p<0.01.

Fig. 5 – Effects of CPA on the frequency of sleep–wakeepisodes. The frequency (mean±SEM) of various durations ofwaking (A), non-REM (B), and REM sleep (C) episodesduring aCSF and 50 μM CPA perfusion into the PF-LHA. CPAat 50 μM produced a significant decrease in waking andconcomitant increase in non-REM sleep episodes. *, p<0.05.

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aCSF-treated rats were predominantly awake (66.6±1.7%) andspent less time in non-REM (29.7±1.4%) and REM sleep(3.7±0.4%) during the 6-h recording period. There were nosignificant differences between 2-h treatment and 4-h post-treatment conditions. CPA perfusion significantly decreasedthe mean time spent in waking (F=7.891, p<0.001) andincreased the meantime spent in non-REM (F=8.18, p<0.001)and REM sleep (F=30.036, p<0.001). Of the two doses used, 50 μM

CPA induced significantly greater suppression in waking andenhancement of non-REM and REM sleep, compared to bothaCSF and 5 μM CPA treatments during the 2 h of its perfusionand the effects lasted up to the first 2 h post-treatment. Thesleep–wake profiles during the 3–4 h post-treatment period inthe three groups were not significantly different.

The frequencies of waking, non-REM, and REM sleepepisodes during aCSF and 50 μM CPA perfusion during the 2-h treatment period are shown in Fig. 5. The frequency ofwakingepisodes decreased significantly in response to CPA due to asignificantdecrease in thenumberof longepisodes (>120 s). Thenumber of non-REM sleep episodes, in particular long episodesincreased significantly. CPA perfusion also increased the

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frequency of REM sleep episodes, although not significantly(p=0.07). The frequency and duration of waking, non-REM,and REM sleep episodes during post-treatment conditions afteraCSF and 50 μM CPA perfusion were comparable.

3. Discussion

This study demonstrates that blockade of A1-receptor-medi-ated adenosinergic transmission in the PF-LHA by localperfusion of an adenosine A1 receptor antagonist producedarousal with concomitant reductions in non-REM and REMsleep in sleeping rats. Conversely, adenosine A1 receptoractivation by its agonist suppressed waking and increasednon-REM and REM sleep in awake animals. We note (a) that A1

receptor agonist and antagonist produced opposite andreversible behavioral effects; (b) that the microdialysis drugadministration method we used, unlike an earlier microinjec-tion study, provides a better control on drug concentrationand its continuous delivery in undisturbed animals and alsoreduces the likelihood that treatment effects could be due tomechanical or inflammatory responses (Quan and Blatteis,1989; Thakkar et al., 2008); and (c) that in this study themicrodialysis probes used for delivering A1 receptor agonistand antagonist were localized in the PF-LHA (see Fig. 1), andbased on our previous studies of Fos-IR in response tobicuculline and serotonin perfusions into the PF-LHA, it islikely that the perfused drugs affected neuronal population in∼500–750 μm 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 endog-enous adenosine acting via A1 receptor plays a role in theregulation of sleep by inhibiting PF-LHA neurons.

Studies regarding adenosinergic control of sleep havelargely been focused on the basal forebrain neurons (seeIntroduction). The findings of the present study that applica-tion of an A1 receptor agonist and an antagonist in the PF-LHAinduced and suppressed sleep, respectively, indicate that A1

receptor-mediated hypnogenic responses to adenosine arenot confined to the basal forebrain but can be observed inother brain regions including the PF-LHA (Oishi et al., 2008).These findings are consistent and complementary to theprevious report that microinjection of A1 receptor antagonistinto the PF-LHA produced arousal and suppressed spontane-ous non-REM/REM sleep as well as significantly increased thelatency of non-REM sleep during recovery after sleep depriva-tion (Thakkar et al., 2008). We found that A1 receptorantagonist and agonist altered the number of medium andlong episodes. The number of short waking episodes, whichpotentially reflects state initiations, was unaffected. Thissuggests that the majority of neurons in the PF-LHA affectedby adenosine are involved in arousal maintenance.

The PF-LHA contains a predominant population of neuronsthat are involved in cortical activation and behavioral arousal.These includewake/REM-active andwake-active neurons thatare quiescent during non-REM sleep and during both non-REMand REM sleep, respectively (see Introduction). The extracel-lular release of adenosine has been linked with the metabolicstate of the cell. Although the extracellular levels of adenosinein thePF-LHAduring spontaneousor prolongedwaking remain

unknown, it is likely that the activation of PF-LHA neurons,including HCRT neurons, during arousal contributes to aden-osine release locally. In addition, a recent study in transgenicmice in which purinergic gliotransmission was inhibited byselective expression of a dominant-negative SNARE domainunder doxycycline control supports glia as a potent source ofadenosine since these mice exhibited significantly reducedaccumulation of sleep pressure and attenuated responses toadenosinergic A1 receptor antagonist (Halassa et al., 2009). Theaccumulated adenosine, irrespective of its source, wouldeventually inhibit wake-promoting neurons including HCRTneurons via A1 receptor to regulate sleep.

In a complementary study, we found that A1 receptoractivation significantly suppressed the extracellular dischargeactivity of the recorded PF-LHA neurons (unpublished data).This finding along with the behavioral changes as observed inthis study suggests that adenosine exerts its sleep-promotingeffect in the PF-LHA via A1 receptor-mediated inhibition of itsneurons. That PF-LHA neurons are under adenosinergicinfluences is supported by an earlier in vitro study showingthat adenosine via A1 receptor exerts inhibitory influences onHCRT neurons, most potently via presynaptic inhibition of theglutamatergic input or excitatory postsynaptic potential (Liuand Gao, 2007).

Anatomically, HCRT neurons project extensively to majorarousal 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 with6 h of sleep deprivation in rats in which HCRT receptor bear-ing PF-LHA neurons were lesioned with HCRT-2–saporin con-jugate. This finding suggests that the activation of neuronalpopulation in the PF-LHA could be driving basal forebrainrelease of adenosine during arousal (Murillo-Rodriguez et al.,2008).

It is pertinent to note that in the PF-LHA, HCRT neuronsconstitute only a subset of wake-active neurons and thatHCRT neurons are intermingled with various other neuronalgroups including MCH and GABAergic neurons that have beenimplicated 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 thenet physiological output of the PF-LHA is wake-promoting. Forexample, glutamic acid microinjections into the PF-LHAproduce arousal and suppress both non-REM and REM sleep,whereas rats with neurotoxic lesion of this area exhibitincreased non-REM and REM sleep (Alam and Mallick, 2008;Gerashchenko et al., 2001). Although the relative distributionof A1 receptors on various neuronal groups, in particular onGABAergic and MCH neurons, remains unknown, it is likelythat behavioral changes observed in this study were predom-inantly mediated via blockade or activation of wake-promot-ing HCRT and other neurons of unknown neurotransmitterphenotypes. Alternatively, it is possible that adenosinepromotes sleep in part: (a) through the indirect disinhibitionof sleep-active neurons within the PF-LHA; and/or (b) byactivating sleep-promoting neurons, e.g., MCH and GABAergicneurons via A2A receptors. Although the distribution of A2A

receptors in the PF-LHA is not well characterized, the perfusedconcentrations of agonist and antagonist used in this studymay affect A2A receptors as well (Fredholm et al., 2001).

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In conclusion, the findings of this study support that PF-LHA is one of the siteswhere adenosine acting viaA1 receptors,in part, inhibits wake-promoting neurons to promote sleep.

4. Experimental procedures

Experiments were conducted on 16 freely behaving Sprague-Dawley male rats. All the experiments were conducted inaccordance with the National Research Council Guide for theCare and Use of Laboratory Animals and were approved byVeterans Administration Greater Los Angeles HealthcareSystem's Institutional Animal Care and Use Committee. Therats were housed individually and maintained on 12:12 hlight–dark cycle (lights on at 6:00 A.M.) with food and wateravailable ad libitum.

4.1. Surgical implantation

The details of the surgical procedure and experimentalprotocol are described in earlier studies (Alam et al., 2005). Inbrief, rats were stereotaxically implanted with (a) EEG(electroencephalogram) and EMG (electromyogram) electrodesfor the monitoring of the sleep–wake cycles and (b) a bilateralmicrodialysis guide cannulae (23G stainless steel tube) usingthe stereotaxic coordinates, A, −2.9 to −3.1; L, 1.4 to 1.6; H, 4.5to 5.5 (Paxinos and Watson, 1998) such that their tips rested3 mm above the dorsal aspect of the PF-LHA and were blockedwith stylets. Experiments were started at least 10 days aftersurgery and after acclimatization of the animals with therecording environment during the recovery period.

4.2. Experimental protocol

At least 24 h before the experiment, the stylets of themicrodialysis guide cannulae were replaced by microdialysisprobes (semipermeable membrane tip length, 1 mm; outerdiameter, 0.22mm;molecular cutoff size, 50 kDa; Eicom, Japan),fixed with dental acrylic and flushed with artificial cerebrospi-nal fluid (aCSF; composition inmM, 145NaCl, 2.7KCl, 1.3MgSO4,1.2 CaCl2, and 2 Na2HPO4; pH, 7.2) at a flow rate of 2 μl/min for4 h. The time taken by the aCSF solution to travel from thereservoir to the tips of the probes was precisely calculated.

4.2.1. Effects of adenosine A1 receptor agonist/antagonist onsleep–wake cycleStudies suggest that maximum number of PF-LHA neuronsexhibit Fos-IR during lights-off period when rats spentsignificantly more time in waking (Espana et al., 2003;Estabrooke et al., 2001; Kumar et al., 2007). Since in vitrostudies suggest that adenosine exerts inhibitory influences onHCRT neurons via A1 receptor (Liu and Gao, 2007), the effectsof adenosine A1 receptor agonist, N6-cyclopentyladenosine(CPA), on sleep–wakefulness were examined during lights-offperiod between 8.00 P.M. and 3.00 A.M. In contrast, the effects ofA1 receptor antagonist, 1,3-dipropyl-8-phenylxanthine (CPDX)on sleep–wakefulness were examined during lights-on periodbetween 8.00 A.M and 3.00 P.M., when rats are predominantlyasleep. Both CPA and CPDX were dissolved in distilled waterand 0.1N NaOH, respectively, at millimolar concentrations,

and then the stock solutions were diluted to micromolarconcentrations for perfusion. The pH of the perfusing solutionwas adjusted to 7.2. The EEG and EMG of each animal wererecorded during perfusion of aCSF and two doses of CPA (5 μMand 50 μM) or CPDX (5 μM or 50 μM) for 2 h followed by 4 h ofaCSF perfusion. The order of aCSF or drug delivery wasrandomized, and the treatments were spaced by at least24 h. In few cases, the probes either got blocked or leaked, andtherefore, animals did not receive all three treatments. In ourearlier studies, we found that microdialysis probes remainedfunctional 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 wereexamined in different group of animals. At the end of theexperiments, rats were sacrificed and the locations of themicrodialysis probes were histologically confirmed.

After a lethal dose of pentobarbital (100 mg/kg, i.p.), ratswere injected with heparin (500 U, i.p.), and perfusedtranscardially with 30–50 ml of 0.1 M phosphate buffer (pH7.2) followed by 500 ml of 4% paraformaldehyde in phosphatebuffer containing 15% saturated picric acid solution. Thebrains were removed and equilibrated in 10%, 20%, and finally30% sucrose. Horizontal sections were freeze-cut at 30 μmthickness. Alternate sections from the series of sectionsspanning the probe tract were immunostained for HCRT-1protein (Alam et al., 2005; Kumar et al., 2007).

4.3. Data analyses

A single person who was unaware of the experimental condi-tions scored sleep–waking states in 10 s epochs aswaking, non-REM sleep, and REM sleep, according to the method describedearlier (Alam and Mallick, 1990). The sleep–wake data werecomparedat 2h intervals; 2 hof aCSF/drug treatments and4hofpost-treatment with aCSF perfusion. The level of significanceamongst different treatment conditions was determined byusing one-way repeated-measures ANOVAs followed by pair-wise multiple comparisons using Holm–Sidak method. Wefound the strongest effects of CPDX and CPA during the 2 htreatment period, and therefore, the frequencies of sleep–wakeepisodes during aCSF vs. effective doses of CPDX or CPA duringtreatment period were further compared using paired t-test.

Acknowledgments

This work was supported by the US Department of VeteransAffairs Medical Research Service and US National Institutes ofHealth grants NS-050939 (Alam), MH47480 and HL60296(McGinty), and MH63323 (Szymusiak).

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