The purine nucleoside adenosine (AD) may play animportant role in the CNS as a neuromodulator of behavioralstate, particularly the sleep/wake cycle (for review,Radulovacki, 1985; Strecker et al., 2000). For example,systemic and intracerebral administration of AD or ADagonists has been shown to decrease wakefulness andincrease sleep (Dunwiddie and Worth, 1982; Ticho andRadulovacki, 1991; Benington et al., 1995; Portas et al., 1997;Fulga and Stone, 1998; Mendelson, 2000).

Recent work supports the hypothesis that a rise ofextracellular AD levels in the arousal-related region of thebasal forebrain (BF) reflects the homeostatic sleep drive. Incats, 6 hrs of prolonged wakefulness produced anapproximately two-fold increase in extracellular BF ADlevels (Porkka-Heiskanen et al., 1997, 2000), and the BF wasa potent site for the sleep-altering effects of AD and ADreceptor antagonists (Strecker et al., 2000; Portas et al.,1997). Three hours of sleep deprivation has also been shownto increase rat extracellular BF AD levels (Basheer et al.,1999). As predicted, single-unit studies revealed that ADinhibits BF wake-active neurons by means of activation of theA1 receptor (Alam et al., 1999; Thakkar et al., 2003a).Furthermore, antisense to the A1 receptor injected locally intoBF increased spontaneous wakefulness and reduced theamount of recovery sleep that followed 6 hrs of total sleepdeprivation (SD) (Thakkar et al., 2003b).

The cumulative findings support the hypothesis that longperiods of spontaneous wakefulness, typical in humans, will beaccompanied by an elevation of both BF AD levels and the

homeostatic sleep drive. Unlike cats, rats are a species with adiurnal pattern of sleep-wakefulness that more closelyresembles that of man, although rats are nocturnal. The presentstudy tested the prediction that rat BF AD levels will elevateduring the dark period when rats are the most spontaneouslyactive and predominantly awake. As a further evaluation ofadenosine as a possible homeostatic sleep regulator, the effectof 6 hrs of SD on rat BF AD levels was examined.

METHODS

Seven adult male Sprague-Dawley rats (Charles RiverLaboratories), weighing between 280 and 350 g, were housedunder constant temperature (23±1°C) and 12:12 light:darkcycle (lights-on 07:00 to 19:00) with food and water availablead libitum. All animals were treated in accordance withAAALAC’s policy on care and use of laboratory animals.Under general anesthesia (i.p. sodium pentobarbital and ani.m.-injected combination of ketamine, xylazine andacepromazine), intracerebral guide cannulas were implanted,targeting the BF, particularly the horizontal diagonal band andsubstantia innominata. At least 16 hrs prior to the beginningof sample collection, a microdialysis probe (CMA11, 2 mmmembrane length, 0.24 mm diameter; CMA/Microdialysis,Stockholm) was inserted through the implanted guide cannulainto the BF (AP -0.3, L 1.8, H -9.0). Probe inlet and outlettubing (FEP tubing; CMA/Microdialysis) transportedartificial cerebrospinal fluid (aCSF = NaCl 147 mM, KCl 3mM, CaCl2 1.2 mM, MgCl2 1.0 mM, pH 7.2) at a flow rate of

The purine nucleoside adenosine may facilitate sleepiness by inhibiting neurons of the magnocellular basal forebrain that areknown to enhance cortical activation during wakefulness. In vivo microdialysis sample collection in rats coupled to microborehigh performance liquid chromatography analysis determined extracellular basal forebrain adenosine levels across the 24 hoursof the day (lights on/off 07:00/19:00). During the dark period, when rats are predominantly active and awake, adenosine levelswere elevated two-fold. Also, basal forebrain adenosine increased during 6 hrs of sleep deprivation and declined slowlythereafter. The data support the hypothesis that basal forebrain adenosine levels may reflect the homeostatic sleep drive thatoccurs daily in animals exhibiting a diurnal pattern of sleep and wakefulness.

CURRENT CLAIM: Extracellular adenosine levels in the basal forebrain exhibit a diurnal fluctuation being almost two-foldhigher in the dark period relative to the light period.

Sleep Research Online 5(4): 155-160, 2003http://www.sro.org/2003/McKenna/155/ Printed in the USA. All rights reserved.

Correspondence: Dr. Robert W. McCarley, Harvard Medical School/Boston VA Healthcare System, Brockton VAMC, Psychiatry 116A,940 Belmont St., Brockton, MA 02301-5596, USA, Tel: 508-583-4500 ext. 2479, Fax: 508-586-0894, E-mail:robert_mccarley@hms.harvard.edu.

1096-214X© 2003 WebSciences

Nocturnal Elevation of Extracellular Adenosine in the Rat Basal Forebrain

James T. McKenna, Lynda J. Dauphin, Kara J. Mulkern,Aaron M. Stronge, Robert W. McCarley and Robert E. Strecker

Department of Psychiatry and Boston VA Healthcare System,Harvard Medical School, Brockton VAMC, Brockton, MA, USA

1.5 µl/min. Samples were collected from the outlet tubingafter exiting the test cage. For all experiments the time delaydue to the dead volume of the system (fluid contained in theprobe and outlet tubing) was taken into account in correlatingneurochemical readings with lights on or lights off. Aspreviously described (Porkka-Heiskanen et al., 1997, 2000;Basheer et al., 1999), 10 µl microdialysis samples wereanalyzed with a microbore high performance liquidchromatography (HPLC) system coupled to a Waters 2487UV detector (detection wavelength = 258 nm).

The diurnal fluctuation of AD in BF was examined in fiverats using 3 hr sampling periods collected for 24 hrs (7:00 AMday 1 to 7:00 AM day 2). The effect of SD on BF AD levelswas investigated in six rats in which hourly samples werecollected, beginning at 12:00 PM. At 1 PM (13:00), the ratswere kept awake by means of gentle handling, including lightolfactory, auditory, and tactile stimulation, similar to variousprevious studies (i.e., Franken et al., 1991, 1995; Porkka-Heiskanen et al., 1997, 2000; Alanko et al., 2003; Mackiewiczet al., 2003; Thakkar et al., 2003a,b). SD ended at 19:00 andthree more 1 hr samples were collected. 10 µl of samplevolume from each of the 3 hr (diurnal experiment) or 1 hrtime periods (sleep deprivation experiment) were analyzed.Four rats contributed data to both experiments. For missingsamples, values were assigned using an arithmetic means ofsurrounding data points (three samples in the diurnal studyand one sample in the SD study) and for two outliers in theSD experiment (values that varied by > 2 S.D. compared toother samples from the same probe). After completion of themicrodialysis experiments, rats were euthanized with CO2.The brain was placed in 4% formaldehyde in 0.1 M phosphatebuffer overnight, followed by transfer to 30% sucrose-0.1 Mphosphate buffer until the tissue block sank. 40 µm coronalsections were cut on a freezing microtome and a series of one-in-six sections was processed with 0.5% cresyl violet to verifythe perfusion site.

RESULTS

Histological analysis confirmed that all seven probes were inthe targeted BF region (see Figure 1).

Diurnal fluctuation. AD levels reached a nadir in the 10:00-13:00 sample of the

light period, and this sample was used as the basal level tocalculate the percent change at other time points. Figure 2shows significant fluctuation in BF AD levels across 24 hrs(N=5 animals; repeated measures ANOVA, F(7,4)=3.638,p<0.01). Absolute levels of BF AD varied from a mean ±SEMof 6.3±1.5 nM (10:00-13:00 time point) to 17.1±4.3 nM(22:00-1:00 time point).

Relative to the light period (open bar on x-axis of Figure2), BF AD levels were higher during the dark period (darkbar). For example, a factorial ANOVA with post hoccomparisons using a conservative test (Bonferroni/Dunn withp-value of 0.0018) revealed significant differences betweenthe 10:00-13:00 time point verses the 19:00-22:00, 22:00-01:00, and 04:00-07:00 time points; and also between the

MCKENNA ET AL.156

Figure 2. Extracellular basal forebrain AD levels are elevated duringthe dark period when rats are known to be most active andpredominantly awake. Three hour samples were collected over a 24hr time span (N=5). The data plotted are means ±SEM expressed asthe percent of baseline (the 10:00-13:00 time point was used asbaseline). Mean AD levels were significantly lower during the lightperiod (white bar under x-axis) compared to the dark period (darkbar under x-axis; see text for details).

Figure 1. Coronal schematic representations of the BF region (-0.26and -0.40 mm caudal of bregma), adapted from Paxinos and Watson(1998), showing the histologically identified location of the ventraltip of each probe (black dots). The microdialysis probe membranewas 250 µm wide x 2 mm long; hence, the brain area sampled by theprobe membrane extends 2 mm above each dot. Calibration: dots are250 µm wide. ac, anterior commissure; acp, anterior commissure,posterior part; HDB, nucleus of the horizontal limb of the diagonalband; LPO, lateral preoptic area; MCPO, magnocellular preopticnucleus; SI, substantia innominata; VP, ventral pallidum.

13:00-16:00 verses 22:00-01:00 hour time points. Asignificant spontaneous increase in extracellular AD BF levelswas therefore observed between the light and the dark period.The variations in adenosine level observed within either thelight period or within the dark period (see Figure 2) were notsignificant (p>0.05).

Six hours total sleep deprivation.As illustrated in Figure 3 (N=6), 60 min samples were

collected from BF beginning at 12:00 to obtain basal ADlevels (12:00-13:00) followed by 6 hrs of total SD (13:00 -19:00), and a 3 hr undisturbed recovery period (19:00-22:00).Absolute levels of BF AD varied from the group mean basallevel of 6.3±1.6 nM (N=6 animals; mean ±SEM; 12:00-13:00time point) to 10.5±1.9 nM at the h4 SD time point.

Extracellular BF AD levels rose significantly abovebaseline levels during the SD period (repeated measuresANOVA, F(6,5)=5.620, p<0.001), consistent with ourlaboratory’s previous findings (Porkka-Heiskanen et al.,1997, 2000; Basheer et al., 1999). Subsequent factorialANOVA and post hoc analysis (Fisher’s PLSD, p-value forsignificance of <0.05) revealed significant differencesbetween basal AD levels and levels after 3, 4, 5, or 6 hrs ofSD.

AD levels initially remained elevated during the recoveryperiod; AD levels in recovery h2 were significantly higherthan baseline, whereas the differences between h1 of therecovery period and baseline approached significance(p=0.0594). AD levels in h3 of the recovery period were notsignificantly different than baseline suggesting a slow returnto basal levels (Fisher’s PLSD post hoc analysis, p-value forsignificance of <0.05, p=0.2909). There were no significantdifferences between AD levels of h3-6 of SD and the first 2hrs of recovery period (Fisher’s PLSD post hoc analysis, allp-values >0.4).

DISCUSSION

This is the first study to demonstrate that extracellular ADlevels in the rat BF are higher during the active period of rats(dark) than during their inactive period (light). A previousstudy has described similar diurnal fluctuations of extracellularAD in the hippocampus and neostriatum of rat (Huston et al.,1996). In contrast, tissue concentrations of AD in the rat frontalcortex have been reported to be lowest during the dark period,suggesting that tissue and extracellular AD levels may beinversely related (Chagoya de Sanchez et al., 1993). Thepresent finding supports the hypothesis that elevated levels ofAD in the BF may reflect the sleepiness associated with periodsof prolonged wakefulness. Based on this hypothesis andprevious data, we had predicted that BF AD levels wouldsteadily rise with the increase in sleep drive associated with anincreasing duration of spontaneous wakefulness as the darkperiod progressed. Indeed, Huston et al. (1996) observed alinear increase in extracellular AD levels in the hippocampusduring the dark period, whereas the pattern they observed in theneostriatum resembled the pattern of BF AD elevationdescribed here, i.e., AD levels were uniformly elevatedthroughout the dark period.

The present finding is also supported by studiesinvestigating the intensity of slow wave activity (SWA) in non-REM sleep (NREM) as a possible correlate of homeostaticsleep drive (Franken et al., 1991, 1995). During the darkperiod, NREM SWA remains uniform and does not show acontinuous elevation across the dark period. This pattern ofSWA resembles that of the dark period BF AD levels describedherein. One possible explanation for why putative measures ofhomeostatic sleep drive are maintained at a steady level duringthe dark period is the fact that rats do not stay awake during theentire dark period. Unlike humans, who are typically awake for14 to 16 hrs without episodes of sleeping, rats sleep up to 35%of the time during the dark (active) period. Conversely, rats areawake for approximately 30-45% of the light period, and 65-75% of the dark period (e.g. Franken et al., 1991; Thakkar etal., 2003b). Typically, when the dark period begins, rats have aninitial period of wakefulness that is followed by brief periods ofsleep (naps) beginning about 90 to 120 minutes later. Sincenaps are known to reduce sleepiness and the homeostatic sleepdrive (Dijk et al., 1987; Cujochem and Dijk, 2003), the rats’brief and periodic episodes of sleep during the dark period maycontribute to maintaining BF AD levels at a uniformly elevatedlevel that corresponds to a fixed level of sleepiness.

Although the possible evaluation of adenosine fluctuationcoinciding with behavioral state changes was not addressed inthe current study, the use of short sample times (e.g., 30 mininstead of 3 hr samples) combined with concomitant electroen-cephalographic assessment of sleep and wakefulness couldaddress this issue. In addition, the effect of locomotor activityon extracellular BF AD is unknown. Huston et al. (1996) foundthat maximal levels of hippocampal AD during the dark periodwere followed by reduced behavioral activity and increased“sleep-like” behavior. Furthermore, the nocturnal elevation inBF AD levels is unlikely to be due solely to circadian influencesince AD levels also increased following 6 hrs of afternoon SD.

157NOCTURNAL RISE IN BASAL FOREBRAIN ADENOSINE

Figure 3. Six hours of total sleep deprivation during the light phase(13:00 – 19:00) produced a rise in extracellular BF AD levels (N=6).AD levels declined slowly at the end of the 6 hrs of SD; by the thirdhour post-SD AD levels were statistically equivalent to baseline. Thebaseline data plotted were collected immediately prior to the SDperiod (12:00-13:00) and the other data plotted are means ±SEMexpressed as the percent of baseline.

The present study also demonstrated that 6 hrs SD (13:00-19:00) produced an increase in extracellular BF ADconcentrations (levels peaked at 181% of basal levels). Themagnitude of this SD-induced increase is comparable to thatpreviously described in the BF for cats (~200% and 140% inPorkka-Heiskanen et al., 1997, 2000) and rats (~225% inBasheer et al., 1999). During a 3 hr recovery period followingSD, AD levels in BF slowly declined. Interestingly, theelevation in BF AD levels during the lights out period in thediurnal experiment (Figure 2) does not seem to occur in the 3hr period beginning at lights out after the 6 hrs of SD (Figure3). This could be explained by the fact that rats sleep ~60% ofthe first 5 hrs after being sleep deprived from 13:00-19:00(lights out at 19:00; Thakkar et al. 2003b), whereas undercontrol conditions rats typically sleep less than 30% of thetime during the 5 hr period after lights out (Borbely andNeuhaus, 1979; Franken et al., 1991).

It is unlikely that the stress of our gentle handlingprocedure is responsible for the present findings since sleepdeprivation by gentle handling is not thought to bepsychologically stressful (Coenen and Van Luijtelaar, 1985;Rechtschaffen et al., 1989). Elevations in BF AD levelsproduced by treadmill-induced sleep loss (unpublished data)appear the same as when gentle handling is used. Indeed, theliterature indicates that sleep deprivation by means of forcedlocomotion does not elevate corticosterone (Tobler et al.,1983; Guzman-Marin et al., 2003) or produces only a smallelevation (Meerlo et al., 2002).

Six hours of SD has been shown to produce site specificincreases of extracellular AD levels in the feline BF andcortex, but not in other subcortical structures, including thehypothalamic preoptic area, pedunculopontine nucleus, dorsalraphe nucleus, and ventroanterior/ventrolateral nuclei of thethalamus (Porkka-Heiskanen et al., 2000). Following the 6 hrsSD, feline BF AD levels slowly declined, whereas levelsdeclined more rapidly in the cortex (Porkka-Heiskanen et al.,1997, 2000). Finally, in all six brain areas examined,extracellular adenosine levels during short periods of sleep incats were ~20% lower than during adjacent periods ofwakefulness (Porkka-Heiskanen et al., 2000). However, thisobservation alone does not fully account for the greaterelevations in AD seen with SD, and in particular, for the factthat the SD-induced rise in AD is site specific (Porkka-Heiskanen et al., 2000).

Ideally, a proposed mechanism should explain the brainsite-specific changes in extracellular AD levels described inthe literature (Huston et al., 1996; Porkka-Heiskanen et al.,2000). Recent reports have described diurnal fluctuation ofmany of the major AD brain enzymes, although thisfluctuation was not identified in all regions studied (Alanko etal., 2003; Mackiewicz et al., 2003). However, the mechanismof the elevation of AD levels during SD appears to beindependent of AD-related enzymatic activity, for SD did notlead to significant fluctuations of AD enzymatic levels in suchsleep-related brain areas as BF (Alanko et al., 2003;Mackiewicz et al., 2003). Alterations in the activity ofnucleoside transporters have also been proposed to beresponsible for this increase of AD levels (Strecker et al.,

2000; McCarley, 2002). Rosenberg et al. (2000) haveproposed that increasing extracellular AD levels in BF may bedue to the local influence of afferents to the BF region,namely from the laterodorsal tegmental/pedunculopontinenuclei, which express nitric oxide synthase. Evidencesuggests that the release of nitric oxide in sleep-relatedregions, including the BF, leads to a rise in extracellularadenosine. A recent study found that preventing mitochondrialsynthesis of ATP in the BF by perfusion of 2,4-dinitrophenolincreased non-REM sleep and induced elevations inextracellular AD, lactate and pyruvate (Kalinchuk et al.,2003). Although the local metabolic effects of this drug weresimilar when perfused in adjacent brain areas, changes insleep were only observed with the BF perfusion. Thus, itremains possible that site specific changes in energymetabolism could mediate the effects of various behavioral,environmental and biochemical factors known to alterextracellular AD levels.

As previously suggested, one of the functions of sleep, and,in particular, slow wave sleep, may be to replenish glycogen,known to be a key cerebral energy reserve, which is depletedduring wakefulness (Benington and Heller, 1995). Such adecrease in energy reserves may result in the production ofadenosine. It has been recently reported that levels of brainglycogen decreased (Kong et al., 2002), and associatedenzymes involved in glycogen synthesis increased (Petit et al.,2002), following periods of extended wakefulness. Glycogenlevels returned to baseline following recovery sleep (Kong etal., 2002), implicating energy metabolic demands as a basis ofthe homeostatic sleep drive. The present data on SD-inducedchanges in BF AD levels are compatible with the glycogenhypotheses and related findings.

In conclusion, during the 24 hr day of the rat, extracellularAD BF levels fluctuated, where highest levels were observedduring the dark period, when the rat is most active.Furthermore, during 6 hrs of SD, AD levels significantlyincreased, and slowly declined in the recovery period after SDwas discontinued. Clearly, further work is needed todistinguish which environmental factors are responsible forthe dark period elevation of extracellular AD, in order todetermine the role of behavioral/locomotor activity, energymetabolism, circadian factors, illumination, and thehomeostatic sleep drive in the regulation of BF AD levels.Particularly, analysis of the spontaneous diurnal fluctuation ina free running animal (complete darkness for 24 hrs per day)could further elucidate if AD fluctuation is due to externalpace maker cues (i.e., light) or due to internal circadianmechanisms.

ACKNOWLEDGMENTS

This work was supported by National Institute of MentalHealth (NIMH 39683), the Department of Veteran AffairsMedical Research Service Awards to RES and RWM, andNational Institutes of Health Fellowship Training Program inSleep, Circadian and Respiratory Neurobiology (1 T32HL07901) to JTM.

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