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Progress in Neurobiology 73 (2004) 379–396

Adenosine and sleep–wake regulation

Radhika Basheer1, Robert E. Strecker2, Mahesh M. Thakkar3, Robert W. McCarley*

Neuroscience Laboratory, Department of Psychiatry, Harvard Medical School and Boston VA Healthcare System, Brockton, MA 02301, USA

Received 18 September 2003; accepted 28 June 2004

Abstract

This review addresses three principal questions about adenosine and sleep–wake regulation: (1) Is adenosine an endogenous sleep factor?

(2) Are there specific brain regions/neuroanatomical targets and receptor subtypes through which adenosine mediates sleepiness? (3) What are

the molecular mechanisms by which adenosine may mediate the long-term effects of sleep loss? Data suggest that adenosine is indeed an

important endogenous, homeostatic sleep factor, likely mediating the sleepiness that follows prolonged wakefulness. The cholinergic basal

forebrain is reviewed in detail as an essential area for mediating the sleep-inducing effects of adenosine by inhibition of wake-promoting

neurons via the A1 receptor. The A2A receptor in the subarachnoid space below the rostral forebrain may play a role in the prostaglandin D2-

mediated somnogenic effects of adenosine. Recent evidence indicates that a cascade of signal transduction induced by basal forebrain

adenosine A1 receptor activation in cholinergic neurons leads to increased transcription of the A1 receptor; this may play a role in mediating

the longer-term effects of sleep deprivation, often called sleep debt.

Published by Elsevier Ltd.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380

2. Adenosine in the central nervous system: its neuromodulatory and neuroprotective roles . . . . . . 380

3. Adenosine and sleep . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

3.1. The somnogenic effects of adenosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

3.2. The effects of localized adenosine administration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382

3.3. Adenosine concentration changes during wakefulness: comparison of different brain

regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382

4. Regulation of adenosine levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384

4.1. Effect of prolonged waking on adenosine metabolizing enzymes and transporter . . . . . . . 384

5. Adenosine receptors mediating the somnogenic effects of adenosine . . . . . . . . . . . . . . . . . . . . 385

Abbreviations: A1R, adenosine type A1 receptor; AB-MECA, 4-aminobenzyl-methylcarbonyl-b-D-ribofuranosyl-adenine; ACSF, artificial cerebrospinal

uid; AMP, adenosine monophosphate; ATP, adenosine triphosphate; 2APB, 2-aminoethoxydiphenylborane; BF, basal forebrain; CHA, N6-cyclohexylade-

osine; ChAT, cholineacetyltransferase; CPT, cyclopentyl-1,3-dimethylxanthine; DHBP, 1,10-diheptyl-4,40-bipyridinium; DNA, deoxyribonucleic acid; DNP,

,4-dinitrophenol; DPMA, N6-[2-(3,5-dimethoxyphenyl)-2-(methylphelyl)-ethyl] adenosine; EEG, electroencephalogram; HDB, horizontal band of Broca; Ih,

yperpolarization-activated current; I-kB, inhibitor kappa B; IP3, inositol trisphosphate; LDT, laterodorsal tegmental nucleus; mRNA, messenger ribonucleic

cid; NF-kB, nuclear factor kappa B; NO, nitric oxide; NT1, neurotensin receptor type 1; PKC, protein kinase C; PLC, phospholipase C; PPT, pedenculopontine

egmental nucleus; REM sleep, rapid eye movement sleep; RT-PCR, reverse transcription-polymerase chain reaction; SI, substantia innominata; SST2A,

omatostatin receptor type 2A; XeC, xestospongin C

* Corresponding author. Tel.: +1 508 583 4500x3723; fax: +1 508 586 0894.

E-mail address: [email protected] (R. Basheer), [email protected] (R.E. Strecker), [email protected]

M.M. Thakkar), [email protected] (R.W. McCarley).1 Tel.: +1 617 323 7700x6181; fax: +1 617 363 5592.2 Tel.: +1 508 583 4500x1879; fax: +1 508 586 0894.3 Tel.: +1 508 583 4500x1881; fax: +1 508 586 0894.

301-0082/$ – see front matter. Published by Elsevier Ltd.

oi:10.1016/j.pneurobio.2004.06.004

R. Basheer et al. / Progress in Neurobiology 73 (2004) 379–396380

1. Introduction

Adenosine, a ubiquitous nucleoside, serves as a building

block of nucleic acids and energy storage molecules, as a

substrate for multiple enzymes, and, most importantly for

this review, as an extracellular modulator of cellular activity

(Illes et al., 2000). Since its first description in 1929 by

Drury and Szent-Gyorgyi, adenosine has been widely inves-

tigated in different tissues. The endogenous release of

adenosine exerts powerful effects in a wide range of organ

systems (Olah and Stiles, 1992). For example, adenosine has

a predominantly hyperpolarizing effect on the membrane

potential of excitable cells, producing inhibition in vascular

smooth muscle cells of coronary arteries and neurons in

brain.

Four distinct adenosine receptors, A1, A2A, A2B and A3,

have been identified and their relative distributions exam-

ined (see reviews by Fredholm, 1995; Olah and Stiles, 1995;

Klotz, 2000). The functional significance of these receptors

is of considerable importance for pharmacologic interven-

tion (Ralevic and Burnstock, 1998; Fredholm et al., 2000).

In this paper, the role of adenosine in the central nervous

system is briefly reviewed and is followed by a more

extensive description of its role in the regulation of

sleep–wakefulness. In this description, we survey data on

its selective effects on the basal forebrain (BF) cholinergic

zone, consisting of the horizontal band of Broca (HDB), the

substantia innominata (SI) and the magnocellular preoptic

area (MCPO). We present evidence that the BF effects are

mediated via A1 adenosine receptor activation, and a sub-

sequent signal transduction pathway leading to transcription

factor activation. A possible functional significance for the

selective effects of adenosine on cholinergic neurons is

discussed.

2. Adenosine in the central nervous system: its

neuromodulatory and neuroprotective roles

Adenosine in the central nervous system functions both

as a neuromodulator and as a neuroprotector. Adenosine can

be both a homeostatic modulator and a modulator at the

synapse (Phillis and Wu, 1981; Newby, 1984; Williams,

1989; Cunha, 2001). The most profound effect of adenosine

is inhibitory modulation of cellular activity and neurotrans-

mitter release, and it consequently has been described as a

‘retaliatory modulator’ (Newby, 1984; Dunwiddie, 1985;

Williams, 1989). Its neuromodulatory effects are elicited at

normal physiological levels. The extracellular concentration

of adenosine in brain was initially described to be 30–

50 nM, based on an in vivo cortical cup technique (Phillis

et al., 1989). In vivo microdialysis techniques later led to an

estimate of 180–270 nM in basal forebrain and thalamus

(Porkka-Heiskanen et al., 2000), 40–210 nM in striatum

(Ballarin et al., 1991; Pazzagli et al., 1995) and 109 nM

in cortex (Pazzagli et al., 1994).

In terms of a neuroprotective response, extracellular

adenosine levels have been shown to increase under abnor-

mal cell-threatening conditions such as cell injury, trauma,

ischemia or hypoxia, and adenosine is widely studied as an

endogenous neuroprotective agent in the central nervous

system (Rudolphi et al., 1992; Fredholm, 1997; Ongini and

Schubert, 1998; Von Lubitz, 1999; for review see Latini and

Pedata, 2001). It is believed that the effects on adenosine in

these cell-threatening conditions might be site and event-

specific. It is suggested that increased levels of extracellular

adenosine might exert neuroprotective effects by reducing

excitatory amino acid release and/or Ca2+ influx, as well as

by reducing cellular activity and hence metabolism (Schu-

bert et al., 1997).

Pharmacological agents which enhance extracellular ade-

nosine levels have been shown to reduce neuronal damage in

animal models of cerebral ischemia (Rudolphi et al., 1992;

Park and Rudolphi, 1994; Fredholm, 1997). An increase in

adenosine levels and adenosine A1 receptor activation have

been described as essential to development of ischemic

tolerance (Heurteaux et al., 1995).

In addition, adenosine is also implicated in locomotion,

analgesia, chronic drug use, mediation of the effects of

ethanol, as well as sleep–wake activity (for general review,

see Dunwiddie and Masino, 2001). Throughout the world,

the most widely used pharmacological agent is caffeine, a

6. Adenosine A1 receptor-coupled intracellular signal transduction pathway . . . . . . . . . . . . . . . . . 387

6.1. In basal forebrain adenosine mobilizes intracellular calcium predominantly via the

A1 adenosine receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387

6.2. Adenosine mediates mobilization of calcium from intracellular stores via the IP3 receptor 388

6.3. Adenosine-mediated mobilization of intracellular calcium occurs almost exclusively

in cholinergic neurons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388

7. Effects of sleep deprivation-induced increased levels of adenosine in basal forebrain . . . . . . . . . 388

7.1. Sleep deprivation-induced increase in A1 receptor mRNA in basal forebrain . . . . . . . . . . 389

7.2. Sleep deprivation-induced nuclear translocation and DNA binding activity of NF-kB . . . . 390

8. Functional significance of adenosine-mediated biochemical changes in basal forebrain

cholinergic system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391

9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392

R. Basheer et al. / Progress in Neurobiology 73 (2004) 379–396 381

trimethylxanthine, and related compounds such as theophyl-

lin and theobromine that are present in beverages such as

coffee, tea and cocoa. These competitively antagonize ade-

nosine effects at both the A1 and A2A receptors and are

commonly used as stimulants promoting wakefulness. Both

in humans and rodents, caffeine is shown to reduce sleep

and increase sleep fragmentation (Yanik et al., 1987; Virus et

al., 1990; Landolt et al., 1995; Schwierin et al., 1996;

Fredholm et al., 1999). The Fredholm et al. review (1999)

provides an extensive discussion of the methyxanthines and

their usage.

In the context of this review on behavioral state reg-

ulation the three important questions are: (1) Is adenosine

an endogenous sleep factor? (2) Considering the omni-

presence of adenosine in brain, are there specific brain

regions/neuroanatomical targets and receptors through

which adenosine mediates sleepiness? (3) What are

the molecular mechanisms by which adenosine might

mediate the long-term effects of sleep loss? The following

sections review the literature that has addressed these

questions.

3. Adenosine and sleep

3.1. The somnogenic effects of adenosine

The hypnogenic effects of adenosine were first described

in cats by Feldberg and Sherwood in 1954 and later in dogs

by Haulilca et al., 1973. Since then the sedative, sleep-

inducing effects of systemic and central administrations of

adenosine have been repeatedly demonstrated (e.g., Dun-

widdie and Worth, 1982; Virus et al., 1983; Radulovacki et

al., 1984, 1985; Ticho and Radulovacki, 1991). Well-known

stimulants, caffeine and theophylline, counteract the effects

of adenosine by serving as antagonists at adenosine recep-

tors (Fredholm et al., 1999). One of the best functional

theories for adenosine’s role in sleep–wake behavior

derives from the fact that adenosine, a byproduct of energy

metabolism, may serve as a homeostatic regulator of energy

in brain during sleep, since energy restoration has been

proposed as one of the functions of sleep (Chagoya de

Sanchez et al., 1993; Benington and Heller, 1995). In the

central nervous system, energy metabolism is tightly regu-

lated and the production of adenosine is presumably also

under similar stringent regulatory control (Raichle and

Gusnard, 2002).

Extracellular adenosine concentrations have been shown

to increase with increased metabolism and increased neural

activity (Pull and McIlwain, 1972; McIlwain and Pull, 1979;

Van Wylen et al., 1986; Tobler and Scherschlicht, 1990;

Meghji, 1991; Minor et al., 2001). Wakefulness has approxi-

mately a 30% greater metabolic rate than non-REM sleep

(Maquet et al., 1992, 1997; Madsen et al., 1991; Madsen,

1993). The presence of metabolic activity-dependent release

of adenosine is further supported by the observation that

extracellular adenosine levels in neostriatum and hippocam-

pus were higher during the circadian active period and lower

during the circadian inactive period in rats (Huston et al.,

1996). Differential pulse voltammetry using a glucose sen-

sor in cortex reveal that extracellular glucose levels are

higher during slow wave sleep when compared to waking,

an observation consistent with the idea that energy meta-

bolism, thus glucose utilization/breakdown decreases during

slow wave sleep when compared to waking (Netchiporouk et

al., 2001). The first direct evidence of spontaneous adeno-

sine fluctuations with behavioral state change and prolonged

wakefulness came from our laboratory’s in vivo microdia-

lysis measurements of adenosine in freely behaving animals.

Extracellular adenosine concentrations in consecutive sam-

ples collected every 10 min during a complete sleep cycle,

consisting of waking, slow wave sleep, REMs followed by

waking showed that samples collected during sleep cluster

had significantly lower levels of adenosine (�21%) both for

basal forebrain and thalamus when compared to samples

collected waking (Fig. 1A shows the basal forebrain results)

(Porkka-Heiskanen et al., 1997). (We note the analysis for

the samples collected during REM was inexact due to the

short duration of REM periods.) These observations were in

agreement with the idea that waking-related increased neu-

ronal activity also results in higher levels of extracellular

adenosine. Moreover, prolonged waking induced by gentle

handling of the cats resulted in progressive increase in

adenosine with each hour of waking. Three 10 min samples

for each hour were analyzed. The level of adenosine in

the second hour of waking (30 nM) showed a significant

two-fold increase in the sixth hour of waking and decreased

with subsequent 3 h of recovery sleep (Fig. 1B) (Porkka-

Heiskanen et al., 1997). The monotonic rise in adenosine

concentrations with each hour of prolonged wakefulness

and the slow decline with recovery sleep led to the hypoth-

esis that adenosine was a key mediator of the sleepiness

following prolonged wakefulness, i.e., the homeostatic sleep

drive.

A possible mechanism for the sleep-inducing effects of

adenosine was suggested based on the results from in vitro

electrophysiological studies. In vitro data on adenosine

demonstrated that it had a post-synaptic inhibitory effect

on basal forebrain neurons, as well as neurons in the

cholinergic laterodorsal tegmental nuclei (LDT) (Rainnie

et al., 1994; Arrigoni et al., 2003). Both cholinergic and non-

cholinergic neurons were hyperpolarized by adenosine, an

effect that was mediated by an inwardly rectifying K+

conductance and, in the LDT, also by blockade of the

hyperpolarization-activated current (Ih). In addition, tonic

inhibition of LDT cholinergic neurons via pre-synaptic A1

receptors also has been demonstrated (Arrigoni et al., 2001).

These observations support the notion that adenosine might

promote sleepiness by inhibiting activity and neurotrans-

mitter release of wakefulness-promoting neurons. If an

increase in extracellular adenosine is required to induce

sleep (by inhibiting wakefulness-promoting neurons) then


Fig. 1. Extracellular adenosine during spontaneous waking and sleep and prolonged waking and recovery sleep. (A) Extracellular adenosine during

spontaneous waking and sleep. Adenosine concentrations in consecutive 10 min samples collected from an individual microdialysis probe in BF of cat. W,

wakefulness; S, slow wave sleep; R, rapid eye movement sleep. (B) Mean extracellular adenosine values increased in the basal forebrain during 6 h of prolonged

wakefulness and decreased during the subsequent 3 h of spontaneous recovery sleep. The values are normalized relative to the second hour of deprivation

(Porkka-Heiskanen et al., 1997).

a waking-related increase in adenosine might be seen in all

the wake-active areas.

3.2. The effects of localized adenosine administration

Earlier studies with systemic and ventricular administra-

tions of adenosine had inherent limitations in determining

the site of drug action in brain. Hence, a search for the exact

site of somnogenic action of adenosine was initiated using

the techniques of targeted microinjections and in vivo

microdialysis perfusion. In forebrain, microinjection of

adenosine in the medial preoptic area in rat induced sleep

(Ticho and Radulovacki, 1991). Later studies showed that

the sleep-inducing effect of adenosine in this area was

blocked by the GABAA-benzodiazepine receptor antagonist

flumazenil, suggesting that the effect was mediated via the

GABAA receptor (Mendelson, 2000). In cats, in vivo micro-

dialysis perfusion of adenosine in magnocellular cholinergic

basal forebrain, well known for its wakefulness-promoting

role, as well as brain stem cholinergic areas, the laterodorsal

and pedenculopontine tegmental nuclei (LDT/PPT), pro-

duced a significant reduction in wakefulness (Fig. 2A, BF

and Fig. 2B, LDT) (Portas et al., 1997). Similar effects of

adenosine perfusion on wakefulness were observed in rat

basal forebrain (Fig. 2C) (Basheer et al., 1999). Another

brain stem area, the pontine reticular formation, when

injected with A1 receptor agonist cyclohexyladenosine pro-

duced decreased waking and increased rapid eye movement

sleep (Marks and Birabil, 1998). Thus, administration of

adenosine and adenosine agonists in specific areas of brain

known to be important in behavioral state control reduced

waking and increased sleep. The reader will have noted,

however, that application of pharmacological agents that

produce a behavioral effect does not necessarily imply that

this is the mechanism used in natural brain control of sleep

wakefulness. For this, it is necessary to look at sponta-

neously occurring (i.e., not pharmacologically induced)

alterations in adenosine, discussed in Section 3.3.

3.3. Adenosine concentration changes during wakefulness:

comparison of different brain regions

Based on the pharmacological experiments and the

ubiquitous nature of adenosine, the authors’ initial predic-

tion was that adenosine would increase in all brain regions

with higher neuronal activity in wakefulness and playing a

role in the maintenance of this state. A more global action of

adenosine would also have been predicted from the theory

of Bennington et al. (1995). These predictions were tested in

a recent study using in vivo microdiaysis in conjuction with

EEG recordings of behavioral states; extracellular adeno-

sine levels were measured in six sleep–wake-related brain

regions of the cat: basal forebrain, cerebral cortex, thala-

mus, preoptic area of hypothalamus, dorsal raphe nucleus

and pedunculopontine tegmental nucleus. In all these brain

regions, extracellular adenosine levels showed a similar

decline of 15–20% during (brief) episodes of spontaneous

sleep relative to wakefulness in the cat. However, in the

course of 6 h of imposed sleep deprivation, adenosine levels

increased significantly only in the magnocellular choliner-

gic region of the basal forebrain (to 140% of baseline) and,

to a less-sustained extent, in cortex, but not in the other

regions (Fig. 3). Following sleep deprivation, basal fore-

brain adenosine levels declined very slowly, remaining

significantly elevated throughout a 3 h period of recovery

sleep; but elsewhere, however, levels were either similar to,

or lower than baseline (Porkka-Heiskanen et al., 2000). A

similar increase in basal forebrain adenosine was also

observed in the rat following sleep deprivation (Basheer

et al., 1999). These observations were important in suggest-

ing that the pattern of spontaneous sleep–wake-related

changes in extracellular adenosine differed from the sleep

deprivation-induced changes in extracellular adenosine, the

latter being more specific to the cholinergic basal forebrain.

Thus, the selectivity of adenosinergic increase in basal

forebrain led to the experiments for the better understanding

of the role of sleep deprivation-induced adenosine in basal

forebrain.


Fig. 2. Effect of adenosine (300 mM) perfusion. In cat, perfusion of adenosine in basal forebrain (A) or in LDT (B) resulted in reduced waking and increased

sleep. The behavioral states for ACSF perfusion (open bars) was compared with adenosine perfusion (solid bars). In basal forebrain, waking showed 62%

decrease (P < 0.01), REM sleep increased by 135% (P < 0.01, whereas slow wave sleep did not show significance. For LDT, a 52% (P < 0.01) of decrease in

waking, and significant increases (P< 0.05) in slow wave and REM sleep was observed. (C) In rat, adenosine perfusion in basal forebrain resulted in 25.5% (P<

0.05) waking, while both slow wave and REM sleep showed significant (P < 0.05) increases. A power spectral analysis showed the power in delta band was

significantly increased (P < 0.05) during the adenosine perfusion when compared with ACSF-perfusion baseline period. Panels A and B are adapted from Portas

et al. (1997) and panel C is adapted from Basheer et al. (1999).

The primary effect of the increase in extracellular

adenosine in cholinergic basal forebrain, either due to

sleep deprivation or microdialysis perfusion was on the

ensuing sleep as indicated by an increase in delta power,

i.e., increase in the proportion of the 1–4 Hz frequency

Fig. 3. Adenosine concentrations in six different brain regions during sleep

deprivation and recovery period in cat. In basal forebrain (BF) and cortex,

adenosine levels rise during sleep deprivation although the levels in cortex

decline quicker. In other four areas, thalamus (thal), preoptic anterior

hypothalamus (POAH), pontine pedunculotegmentum (PPT) and dorsal

raphe (DRN) adenosine levels decline slowly during the 6 h of sleep

deprivation. Figure adapted from Porkka-Heiskanen et al. (2000).

range during slow wave sleep (Porkka-Heiskanen et al.,

1997; Basheer et al., 1999, 2000). The increased levels of

delta frequency EEG is suggested to reliably predict the

intensity of sleepiness based on the duration of preceding

wakefulness (Tobler and Borbely, 1990; Franken et al.,

1991). Further support for site specificity of the somno-

genic effects of adenosine came from studies using an

adenosine transporter blocker, nitrobenzylthioinosine

(NBTI) which blocks one of the two equilibrative nucleo-

side transporters (Yao et al., 1997). In vivo microdialysis

perfusion of NBTI increased the levels of extracellular

adenosine by two-fold in basal forebrain as well as in the

control region of thalamus. However, significant decrease

in wakefulness and increase in slow wave as well as REM

sleep was observed only with perfusions in basal forebrain

and not in thalamus (Fig. 4) (Porkka-Heiskanen et al.,

2000). Yet another approach for a selective increase in

adenosine was by localized energy depletion by infusion of

2,4-dinitrophenol (DNP), known to block the mitochon-

drial electron transport chain and ATP production, result-

ing in similar increases in extracellular adenosine in

cholinergic basal forebrain and non-cholinergic neighbor-

ing regions. The increase in the sleep intensity was specific

to DNP-induced accumulation of adenosine in cholinergic

basal forebrain (Kalinchuk et al., 2003b). Together, these


Fig. 4. Effects of the adenosine transporter inhibitor NBTI. (A) In basal

forebrain, NBTI (1 mM) infusion produced significant decrease in waking

and increase in both slow wave and REM sleep. (B) In thalamus, there was

no effect on behavioral states (Porkka-Heiskanen et al., 1997).

results led to investigations exploring the adenosinergic

mechanisms operating due to the sleep deprivation-induced

increase in adenosine in cholinergic basal forebrain.

The site-specific adenosinergic effects on sleep were

shown to require the presence of the A1 adenosine receptor

by another set of experiments. Thakkar et al. (2003) per-

formed bilateral microdialysis perfusion of antisense oligo-

nucleotide to the adenosine A1 receptor in the basal

forebrain in the rat. Knocking down the A1 adenosine

receptor with antisense oligonucleotide perfusion confined

to BF yielded a 60–75% reduction of the delta activity in the

5 h of post-deprivation recovery sleep compared with con-

trol animals perfused with non-sense oligonucleotide or

ACSF. The BF antisense treatment also produced a 50–

60% reduction in non-REM sleep time in post-deprivation

hours 2–5 (Fig. 5). Of particular note, there was no differ-

ence in post-deprivation hour 1, suggesting that other

regions in addition to basal forebrain, perhaps cortex, might

mediate the immediate sleep response following depriva-

tion. The neocortex is suggested because of the initial

deprivation-induced rise in adenosine in the neocortex,

but not in other brain regions outside of basal forebrain

(see Fig. 3).

Studies performed in mice suggested a similar role of

adenosine in basal forebrain acting via the A1 adenosine

receptor. Rebound sleep was inhibited by administration of

A1 receptor antagonist, CPT, after 6 h of sleep deprivation

when the pressure to sleep is enhanced (Stenberg et al.,

2003). However, it should be noted that mice with homo-

zygous constitutive knock-out mutation for A1 adenosine

receptor failed to show any alterations in the sleep–wake

pattern or decrease in the rebound sleep intensity following

6 h of sleep deprivation when compared to their wild-type

counterparts (Stenberg et al., 2003). These data suggest that

in constitutive knock-outs, the absence of A1 receptor from

birth resulted in compensatory mechanisms that maintain

the homeostatic regulation of sleep.

The importance of the role played by adenosine via the A1

receptor in basal forebrain is well supported by the observa-

tion that administration of the A1 receptor specific antagonist,

CPT, in wild type mice inhibits rebound sleep (Stenberg et

al., 2003). Moreover, in the absence of any developmental

compensatory mechanisms, a knock-down of the basal fore-

brain A1 receptor by means of application of antisense to the

A1 receptor, showed a markedly decreased homeostatic

response after prolonged wakefulness (Thakkar et al.,

2003). Together, these results confirm the role of adenosine

in producing a homeostatic response via the A1 receptor

following sleep deprivation (Thakkar et al., 2003). In terms of

site(s) of action of sleep loss increases in adenosine acting via

the A1 receptor, these several studies suggest a relatively site-

specific somnogenic effect of adenosine in the basal fore-

brain, with a lesser effect in neocortex.

4. Regulation of adenosine levels

Adenosine is present both intra- and extracellularly and

the balance is maintained by membrane transporters (Latini

and Pedata, 2001). However, when the energy expenditure

exceeds energy production during metabolic demands of

neuronal activation, adenosine levels increase in the extra-

cellular space. Thus, the higher the activity of the neurons,

the greater is the levels of adenosine and its modulatory

effects (Mitchell et al., 1993; Lloyd et al., 1993; Brundege

and Dunwiddie, 1998). The biochemistry of enzymes

responsible for adenosine production as well as its conver-

sion to inosine or phosphorylation to adenosine monopho-

sphate (AMP) have been well characterized (Fig. 6) (see

reviews by Fredholm et al., 2000; Dunwiddie and Masino,

2001).

4.1. Effect of prolonged waking on adenosine

metabolizing enzymes and transporter

In view of the observed selective increase in the levels of

extracellular adenosine in cholinergic basal forebrain with

prolonged waking, changes in the activity of regulatory

enzymes have been examined following 3 and 6 h of sleep

deprivation in rat. None of the enzymes in BF including

adenosine kinase, adenosine deaminase and both ecto- and

endo-50-nucleotidases showed any change in activity fol-

lowing sleep deprivation (Alanko et al., 2003a; Mackie-

wicz et al., 2003). Adenosine concentrations are is also

regulated by transporters. Two equilibrative and five con-

centrative adenosine transporters are known so far (Bald-

win et al., 1999; Thorn and Jarvis, 1996). The concentrative

transporters are resistant to pharmacological inhibition but


Fig. 5. Effects of basal forebrain perfusion of antisense oligonucleotides against the mRNA of the adenosine A1 receptor compared with controls (ACSF and

non-sense pooled) on recovery sleep following 6 h of sleep deprivation in rats. Note increased wakefulness (A) and decreased non-REM sleep (B) during the first

5 h of the recovery sleep period in the antisense group as compared with controls. There was a significant increase in wakefulness and a decrease in non-REM

sleep during the second, third, fourth, and the fifth hours. REM sleep (C) did not show significant differences. The right part of the graphs (within box) shows

that, for the subsequent 7 h, there was no compensation for the antisense-induced changes in wakefulness and non-REM. Ordinate is mean percentage time spent

in each behavioral state (�S.E.M.) and abscissa is time of day, with lights off occurring at 19:00 h and lights on occurring at 07:00 h. Panel D describes

differences in delta power (1–4 Hz, mean � S.E.M.) for the antisense and the control group for the first 5 h of recovery sleep. Note the significant decrease in the

delta activity in antisense treated animals during each of the 5 h of recovery sleep as compared to the pooled controls (**P < 0.01). Adapted from Thakkar et al.

(2003).

the equilibrative transporters have been characterized, one

being sensitive and another resistant to pharmacological

inhibition by NBTI (Yao et al., 1997). The ‘sensitive’

transporters are important in regulating the levels of ade-

nosine since blocking the ‘sensitive’ transporter with NBTI

resulted in increased levels of extracellular adenosine in

basal forebrain resulting in increased sleepiness (Porkka-

Heiskanen et al., 1997). Six hours of sleep deprivation has

been shown to decrease the [3H] nitrobenzylthioinosine

binding in basal forebrain tissue extracts suggesting a

decline in adenosine transport as a possible mechanism

for the increase in extracellular adenosine during sleep

deprivation (Alanko et al., 2003b).

It is important to note yet another mechanism might play

a role in adenosine increase. Another candidate contributing

to the increased adenosine concentration is the release of

nitric oxide (NO) as demonstrated in several in vitro systems

such as hippocampal slices (Fallahi et al., 1996) and fore-

brain neuronal cultures (Rosenberg et al., 2000). NO has

been implicated in sleep mechanisms (Kapas et al., 1994a,

1994b). The effect of NO can be sleep- or wake-promoting,

based on the site of its release in the brain. For example, NO

promotes waking and REM sleep when released in thalamus

or medial pontine reticular formation (Pape and Mager,

1992; Leonard and Lydic, 1997; Burlet and Cespuglio,

1997; Williams et al., 1997). On the other hand, infusion

of the nitric oxide donor diethylamine-NONOate into cho-

linergic basal forebrain resulting in increased NO release

has been shown to mimic the effects of sleep deprivation by

increasing non-REM sleep (Kalinchuk et al., 2003a). In the

absence of any change in enzyme activity in vivo, these

results suggest that the mechanism for sleep deprivation-

induced increase in extracellular adenosine could be due to

either decline in transporter activity and/or increase in NO

release in basal forebrain during sleep deprivation.

5. Adenosine receptors mediating the somnogenic

effects of adenosine

Evidence is available for both A1 and A2A adenosine

receptor subtypes in mediating the sleep-inducing effects of


Fig. 6. The biochemical pathway detailing the enzymes responsible for

intracellular and extracellular adenosine production as well as its conversion

to inositol or phosphorylation to adenosine monophosphate.

Fig. 7. Effects of adenosine A1 receptor antagonists and agonist. Panel A

shows that microdialysis perfusion of A1 antagonist CPT in basal forebrain

of cats produced a concentration-dependent increase in wakefulness (P <

0.05). Panel B shows that the electrical discharge activity of wake-active

neurons in the basal forebrain was inhibited in a concentration-dependent

manner by local perfusion of an A1 agonist CHA.

adenosine. We first review the A1 receptor studies. Earlier

reports described that i.p. or i.c.v. administration of the

highly selective A1 receptor agonist, N6-cyclopentyladeno-

sine resulted in an increased propensity to sleep and delta

waves during sleep, suggesting a role of the A1 adenosine

receptor (Bennington et al., 1995; Schwierin et al., 1996).

Studies in cat and in rat revealed that the somnogenic

effects of adenosine in the cholinergic region of the basal

forebrain appear to be mediated by the A1 adenosine re-

ceptor, since the unilateral infusion of the A1 receptor

selective antagonist, cyclopentyl-1,3-dimethylxanthine

(CPT) increased waking and decreased sleep (Fig. 7A)

(Strecker et al., 1999, 2000). Moreover, single unit recording

of wake-active neurons in conjunction with in vivo micro-

dialysis of A1 selective agonist, N6-cyclohexyladenosine

(CHA) decreased (Fig. 7B), and A1 selective antagonist,

CPT, increased the discharge activity of the wake-active

neurons (Alam et al., 1999; Thakkar et al., 1999). Recently,

blocking the expression of A1 receptors with microdialysis

perfusion of antisense oligonucleotides designed to hybri-

dize with A1 receptor mRNA and thereby preventing its

translation, resulted in significant reduction in non-REM

sleep and increase in wakefulness. Moreover, as illustrated

in Fig. 5, following microdialysis perfusion of A1 receptor

antisense and 6 h of sleep deprivation, the animals spent a

significantly reduced (50–60%) amount of time in non-REM

sleep with a decrease in delta activity during hours 2–5 in the

post-deprivation period (Thakkar et al., 2003). Thus, ade-

nosine in basal forebrain, acting via the A1 adenosine

receptor is involved in the homeostatic regulation of sleep

both in rats and cats.

In a different brain region, the subarachnoid space below

the rostral basal forebrain, data suggest that prostaglandin

D2 receptor activation-induced release of adenosine exerts

its somnogenic effects via the A2A adenosine receptor

(Matsumura et al., 1994; Urade and Hayaishi, 1999; Mizo-

guchi et al., 2001; Hayaishi, 2002). Infusion of prostaglandin

D2 into the subarachnoid space increased the local extra-

cellular adenosine concentration (Mochizuki et al., 2000).

Data supporting the somnogenic effects of both prostaglan-

din D2 and A2A agonists are induction of c-fos immunor-

eactivity in the ventrolateral preoptic area, this region has

been suggested to be involved in promoting sleep by inhibit-

ing the ascending histaminergic arousal system of the tuber-

omammillary nucleus (Sherin et al., 1998; Scammell et al.,

1998, 2001). Infusion of the A2A agonist CGS 21680 in the

subarachnoid space increased slow-wave sleep, and both

A2A agonist- and prostaglandin D2-induced sleep were

blocked by the A2A antagonist, KF17837. These data pro-

vide the pharmacological evidence for the role of the

A2A receptor in mediating the somnogenic effects of pros-

taglandin D2 (Satoh et al., 1996, 1998, 1999). These data

suggested that prostaglandin D2-induced release of adeno-

sine in a specific area of subarachnoid space below the


Fig. 8. Potential mechanisms through which PGD2 and adenosine A2A

receptor agonists may promote sleep. PGD2 may bind to PGD2 (DP)

receptors in leptomeningeal cells that increase the concentration of ade-

nosine in the subarachnoid space. This adenosine then binds to A2A

receptors in the leptomeninges or in the shell of the accumbens nucleus.

Through synaptic or paracrine signals, these regions then activate sleep-

active neurons in the VLPO that inhibit the TMN and other arousal regions

via GABAergic projections. Adapted from Scammell et al. (2001).

Fig. 9. Dual signaling by A1 adenosine receptor (A1R). Pathway A shows

that adenosine A1 receptor coupled to Gi3 inhibits adenylate cyclase (AD

cyclase), whereas pathway B shows that A1 receptor can activate phospho-

lypase C (PLC) mediated inositol trisphosphate (IP3) production that in turn

can mobilize calcium (Ca2+) from endoplasmic reticulum (ER) and activate

protein kinases.

rostral forebrain, leads to A2A receptor-mediated effects (see

Fig. 8).

However, in the HDB/SI/MCPO area of cholinergic basal

forebrain, only A1 but not A2A receptor mRNA (in situ

hybridization and RT-PCR studies) and protein (receptor

autoradiography) have been detected (Basheer et al., 2001a).

Together, these data provide strong evidence that in HDB/SI/

MCPO area of cholinergic basal forebrain the effects of

adenosine on sleep–wake behavior are mediated through the

A1 adenosine receptor.

6. Adenosine A1 receptor-coupled intracellular signal

transduction pathway

Prolonged waking or sleep restriction produces progres-

sive, additive effects such as decreased neurobehavioral

alertness, decreased verbal learning, and increased mood

disturbances, often referred to as ‘sleep debt’ (Dinges et al.,

1997; Drummond et al., 2000; Van Dongen et al., 2003).

These effects are cumulative over many days and thus,

unlike the shorter-term effects described in previous sec-

tions, are likely to have sleep deprivation- or restriction-

induced alterations in transcription as a basis for these long-

term effects. The next sections describe the authors’ and

others’ investigations of the adenosine signal transduction

pathways that may be responsible for the relevant transcrip-

tional alterations.

The sleep deprivation-induced presence, over several

hours, of increased extracellular adenosine in the cholinergic

basal forebrain suggested the utility of investigating the

intracellular effects of A1 receptor activation that involved

second messenger actions impacting activation of protein

kinases and transcription factors that alter gene expression.

A series of reports from the authors’ laboratory have demon-

strated that A1 adenosine receptors on cholinergic neurons

activate a signal transduction pathway mobilizing intracel-

lular stores of calcium that impacts intracellular changes in

enzyme activities, transcription factor activation, and gene

expression. These data are consistent with reports that the A1

adenosine receptor, coupled to the inhibitory Gi3 subtype of

G-protein, is capable of ‘dual signaling’, i.e., inhibition of

adenylate cyclase and stimulation of phosphlipase C (PLC)

(Gerwins and Fredholm, 1992; Freund et al., 1994; Biber et

al., 1997). Also, Biber et al. (1997) have shown that

increased expression and/or stimulation of A1 receptor

results in PLC activation that, in turn, activates protein

kinase C (PKC) via the production of the second messenger

inositol trisphosphate (IP3) (Berridge, 1993; Fisher, 1995)

(see Fig. 9). The following sub-sections present evidence

that adenosine, in a concentration-dependent manner, mobi-

lizes intracellular calcium via the IP3 receptor located on the

endoplasmic reticulum.

6.1. In basal forebrain adenosine mobilizes intracellular

calcium predominantly via the A1 adenosine receptor

In recent years, the ability to measure changes in intra-

cellular calcium has shown significant technological pro-

gress. Taking advantage of these developments, real time

changes in intracellular calcium in individual neurons have

been measured in 300 mm thick acute brain slices of basal

forebrain using multi-photon microscopy. Adenosine treat-

ment (100 mM) of basal forebrain slices induced an increase

in cytoplasmic calcium reaching a maximum of four- to six-

fold increase in 45 s (Fig. 10A) (Basheer et al., 2002). This

increase was closely matched with an A1 selective agonist,


Fig. 10. Adenosine-mediated mobilization of cytoplasmic calcium. Panel A

shows a typical time course of calcium increase, measured as increase in

calcium orange fluorescence in a live neuron in an acute slice after treatment

with 100 mM adenosine (two photon microscope measurements every

1.37 s). Panel B shows that the significant five-fold increase in intracellular

calcium obtained by adenosine was closely matched by A1 agonist CHA

treatment. Whereas A2 agonist DPMA had no significant effect, there was a

two-fold increase in calcium fluorescence with A3 agonist AB-MECA

treatement (*P < 0.05). Figure adapted from Basheer et al. (2002).

Fig. 11. The adenosine-induced calcium release is from IP3 receptor

regulated internal stores. Panel A shows that increase in intracellular

calcium fluorescence is independent of the presence of calcium in external

medium. The filled bars denote the values observed in the presence of

calcium and open bars represent the values obtained in calcium-free

medium. Panel B shows that pretreatment of slices with 50 mM thapsigargin

to deplete internal stores of calcium abolished the response to adenosine.

Panel C shows that a significant increase in intracellular calcium by

adenosine was unaffected when ryanodine receptor was blocked using

1,10-diheptyl-4,40-bipyridinium (DHBP). Conversely blocking the IP3

receptor using xestospongin C (XeC) or 2-aminoethoxydiphenylborane

(2APB) led to no response to adenosine treatment. In all the panels, *P

< 0.05. Adapted from Basheer et al. (2002).

cyclohexyladenosine (CHA, 100 nM) whereas the A2A

selective agonist, N6-[2-(3,5-dimethoxyphenyl)-2-(methyl-

phelyl)-ethyl] adenosine (DPMA, 100 nM) did not produce

significant change. The A3 selective agonist, 4-aminoben-

zyl-methylcarbonyl-b-D-ribofuranosyl-adenine (AB-MECA,

1 mM), produced a smaller but significant increase (Fig. 10B).

Thus, the adenosine-induced calcium increase appears to be

primarily mediated by the A1 receptor.

6.2. Adenosine mediates mobilization of calcium from

intracellular stores via the IP3 receptor

The increase in cytoplasmic calcium in response to

adenosine was observed in the absence of calcium in the

external medium suggesting its intracellular origin (Fig. 11A).

Furthermore, pretreatment of slices with thapsigargin to

deplete the cells of internal stores failed to show calcium

increase in response to adenosine (Fig. 11B). A major source

is the calcium stores present in the elaborately distributed

network of the endoplasmic reticulum. Both the IP3 and

ryanodine receptor are distributed throughout the endoplas-

mic reticulum and are responsible for releasing calcium

from this internal source (Kostyuk and Verkhratsky, 1994;

Simpson et al., 1995). Blocking IP3 receptor with xes-

tospongin C, a potent cell permeable blocker of IP3 receptor

(Gafni et al., 1997) or with 2-aminoethoxydiphenylborane

(2APB), a functional and membrane permeable IP3 re-

ceptor antagonist (Hamada et al., 1999) prevented calcium

increase, but blocking the ryanodine receptor with

1,10-diheptyl-4,40-bipyridinium did not have any effect


(Fig. 11C). These observations suggest that mobilization of

intracellular calcium is mediated via IP3 receptors and

not ryanodine receptors.

6.3. Adenosine-mediated mobilization of intracellular

calcium occurs almost exclusively in cholinergic

neurons

The basal forebrain contains cells with several neuro-

transmitter phenotypes, including cholinergic, GABAergic,

glutamatergic and peptidergic (Gritti et al., 1993, 1997,

2003; Manns et al., 2001; Semba, 2000; Zabrosky et al.,

1999). A long-standing conundrum is the relative role of

cholinergic and non-cholinergic neurons in mediation of

basal forebrain control of wakefulness. Interestingly, immu-

nohistochemical labeling of basal forebrain sections for

the cholinergic marker, cholineacetyltransferase (ChAT)

showed that all the cells responding to adenosine by mobi-

lizing intracellular calcium were cholinergic in nature

(Fig. 12) and 65% of all the cholinergic cells examined

showed an increase in intracellular calcium in response to

Fig. 12. Adenosine-induced cytosolic calcium increase was seen only in

cholinergic neurons of basal forebrain. The neurors that showed an increase

in calcium (orange fluorescence) after 60 s treatment with adenosine were

also positive for cholinergic marker ChAT (green fluorescence) (yellow

arrowheads). One cholinergic neuron (white arrowhead) does not show

calcium orange flourescence. Adapted from Basheer et al. (2002).

adenosine. There is preliminary evidence that sleep depriva-

tion-induced nuclear translocation of NF-kB is also limited

to cholinergic neurons (Ramesh et al., 2002). These obser-

vations thus present evidence for a selective activation of an

adenosinergic pathway in a subset of BF cholinergic neurons

in response to increased levels of extracellular adenosine.

This suggests the possibility of a functional role of choli-

nergic cells in the response to sleep deprivation.

7. Effects of sleep deprivation-induced increased

levels of adenosine in basal forebrain

One of the notable effects of sleep deprivation-induced

increased levels of extracellular adenosine is the up-regula-

tion of the A1 adenosine receptor mRNA. A common

functional feature of inhibitory receptors (such as the

A1R) is their rapid change in response to agonists, the most

common response being receptor down-regulation, i.e., loss

of receptors from the cell surface following prolonged

exposure to their agonists (Bohm et al., 1997; Grady

et al., 1997). Recent evidence also indicates the presence

of up-regulation of receptors following exposure to agonists

(Souaze, 2001). The receptor-coupled effector pathways

regulate the synthesis and stability of receptor mRNA as

clearly demonstrated for receptors of substance P (Hershey

et al., 1991), b-adrenergic (Collins et al., 1992), serotonin

(5HT2) (Rydelek-Fitzgerald et al., 1993), somatostatin,

(SST2A) (Boudin et al., 2000) and neurotensin receptor

type 1 (NT1) (Souaze, 2001). Thus, prolonged presence of

agonists results either in the down-regulation of its receptor

in order to reduce the response to the over abundant agonist

or up-regulation for continued maintenance of cell sensitivity

to the increasing levels of endogenous agonists. Moreover,

during sleep deprivation when the levels of extracellular

adenosine are increased, an up-regulation of A1 adenosine

receptor is observed at mRNA level.

7.1. Sleep deprivation-induced increase in A1 receptor

mRNA in basal forebrain

To examine A1 receptor regulation in response to sleep

deprivation-induced elevated levels of adenosine, the

authors investigated the ligand-binding efficiency and

mRNA of adenosine A1 and A2A receptors. In situ hybridi-

zation and reverse transcription coupled polymerase chain

reaction (RT-PCR) of total RNA from basal forebrain and

cingulate cortex showed that 6 h of sleep deprivation

resulted in significant increases in A1 receptor mRNA in

basal forebrain but not in cortex (Fig. 13A). This up-regula-

tion of mRNA in basal forebrain was accompanied by

unchanged levels of A1 receptor ligand-binding efficiency

and overall receptor density after 6 h of sleep deprivation.

A2A mRNA and ligand binding was undetectable in this

region (Basheer et al., 2001a). Thus, increased mRNA and

absence of any decrease in ligand binding for A1 receptor


Fig. 13. Effects of sleep deprivation on A1 receptor mRNA and NF-kB DNA binding activity in basal forebrain of rat. Panel A shows the autoradiograph of RT-

PCR product for the A1 receptor and the housekeeping gene cyclophyllin mRNA from basal forebrain and cortex of sleep deprived and control rats. In basal

forebrain, A1 receptor mRNA levels are higher than the sleeping control. No significant change was observed in cortex. In panel B, gel shift assays of the crude

nuclear extracts of basal forebrain shows that NF-kB DNA binding is higher after 3 h of sleep deprivation compared to controls. SD, sleep deprived; C,

undisturbed circadian control; P, probe only loaded, Mut, mutant oligonucleotide.

suggested that sleep deprivation-induced increase in extra-

cellular adenosine might be up-regulating the levels of A1

receptor in order to maintain steady levels of receptor

density and continued response to the agonist, adenosine.

In the absence of any change in the membrane receptor

density after 6 h of sleep deprivation the physiological

significance of the changes observed at the mRNA levels

is not yet clear and well likely require receptor binding assay

after longer period of sleep deprivation. The neuroprotective

effects of adenosine have been suggested to involve up-

regulation of A1 receptor in cortical astrocytes (Biber et al.,

2001). It is possible that the membrane receptor protein

levels also increase at a later time point such as 9 or 12 h of

sleep deprivation. The time course of A1 receptor interna-

lization is debatable, from a t1/2 being 5–15 min in smooth

muscle cell line, DDT1-MF2 cells (Ciruela et al., 1997), or

90 min to 5 h in Chinese hamster ovary cells (Ferguson et

al., 2000; Gao et al., 1999). It is not clear if the absence of

any increase observed after 6 h of sleep deprivation is due to

slow rate of internalization or due to rapid turn over followed

by immediate replacement by the newly synthesized recep-

tor. Based on the mRNA up-regulation observed with sleep

deprivation, there seem to be a positive feed back regulation

of A1 receptor gene transcription.

7.2. Sleep deprivation-induced nuclear translocation

and DNA binding activity of NF-kB

There are many reports of sleep deprivation-induced

increase in the activity of transcription factors (O’Hara et

al., 1993; Cirelli et al., 1995; Basheer et al., 1997; Chen et

al., 1999). One of the documented transcription factors that

binds to the A1R promoter region and enhances transcription

of the A1 receptor (among many other proteins) is NF-kB

(Nie et al., 1998; Hammond et al., 2004). Adenosine, acting

via the A1 receptor, has been shown to activate a signal


transduction pathway leading to protein kinase C (PKC)

activation. Activation of PKC is known to impact many

downstream events including phosphorylation of inhibitory

protein I-kB and the release of NF-kB allowing its trans-

location to the nucleus (Siebenlist et al., 1994; McKinsey

et al., 1997). Indeed there is a sleep deprivation-induced

activation of NF-kB, evinced by an increase in DNA binding

of NF-kB, in the basal forebrain but not in the control region

of cingulate cortex (Fig. 13B). In in vitro slices the adeno-

sine-induced DNA binding of NF-kB was significantly

blocked by pretreatment with the A1 receptor antagonist

CPT, suggesting that A1 receptor activation might be respon-

sible for the NF-kB activation (Basheer et al., 2001b). In

summary, sleep deprivation resulted in the up-regulation of

A1 receptor mRNA and increased NF-kB DNA binding in

basal forebrain. Moreover, pharmacological evidence indi-

cated that the activation of NF-kB was mediated via A1

receptor activation.

As outlined in Fig. 14, the data delineated an adenosi-

nergic pathway, starting from its binding to the A1 subtype

adenosine receptor, proceeding through a second messenger

pathway producing IP3 receptor-mediated intracellular cal-

cium increase and leading to an activation of the transcrip-

tion factor NF-kB. These studies are of interest since NF-kB

has been reported to have a role in regulating the expression

of several sleep regulatory substances, such as interleukin-

1b, TNF-a, nitric oxide synthase, cyclooxygenase-2

Fig. 14. Intracellular signal transduction pathway as deduced from the biochemica

phospholipase C (PLC) mediated production of inositol trisphosphate (IP3) whi

potentially activates protein kinase and subsequent phosphorylation of I-kB a

transcription of genes in cholinergic neurons.

(COX-2) (Borbely and Tobler, 1989; Opp and Krueger,

1991, 1994; Krueger and Majde, 1994; Xie et al., 1994;

Yamamoto et al., 1997). The role of NF-kB in the positive

feedback regulation of the adenosine A1 receptor is currently

being investigated.

8. Functional significance of adenosine-mediatedbiochemical changes in basal forebrain cholinergic

system

In the basal forebrain, both cholinergic and non-choli-

nergic neuronal activity is associated with promoting wake-

fulness (Lo Conte et al., 1982; Szymusiak, 1995; Jones,

1993, 1998; Jones and Muhlethaler, 1999; Semba, 2000).

During spontaneous sleep cycle, the somnogenic effects of

adenosine may be due to the inhibition of neuronal activity

in both cholinergic and non-cholinergic neurons of the basal

forebrain as well as other wake-related areas such as LDT. In

addition, the modulatory effects of sleep deprivation on the

A1 adenosine receptor mRNA and transcription factor NF-

kB activation in the cholinergic basal forebrain, suggest the

significance of an adenosinergic pathway in the long-term

effects of sleep deprivation on the quality of ensuing sleep

and/or the neurobehavioral alertness, cognitive functions

and mood. The intracellular effects of adenosine on calcium

and the activation of NF-kB was observed in cholinergic

l evidences detailed in the review. Adenosine acting on A1 receptor activates

ch in turn releases calcium (Ca2+) from endoplasmic reticulum (ER) that

nd releases the NF-kB dimer to translocate into the nucleus facilitating


neurons. The cholinergic neurons in HDB/SI/MCPO target

the entorhinal cortex, neocortex and amygdala and regulate

aspects of cognition and attention, sensory information

processing and arousal (Nagai et al., 1982; Pearson et al.,

1983; Gallagher and Holland, 1994; Sarter and Bruno, 1997,

2000; Everitt and Robbins, 1997). Cognitive functions such

as learning and memory show a correlated decline with

degenerating cholinergic neurons, as reported in Alzhei-

mer’s patients (see reviews by Everitt and Robbins, 1997;

Wenk, 1997; Baxter and Gallagher, 1997; Baxter and Chiba,

1999; Perry et al., 1978). Wiley et al., 1991 developed a

technique involving 192IgG-saporin-induced lesioning of

p75 nerve growth factor (NGF) receptor containing choli-

nergic cells in rats. The cholinergic lesions using this

technique resulted in severe attentional deficit in a serial

reaction-time task (Muir et al., 1996; McGaughy and Sarter,

1998). The cholinergic basal forebrain is important in

cortical arousal. Animals with lesioned basal forebrain show

decreased arousal and increased slow waves in cortex (Buz-

saki and Gage, 1989; Berntson et al., 2002). The effects of

adenosine on cholinergic basal forebrain are thus potentially

important as the related sleep deprivation-induced ‘cogni-

tive’ effects may be mediated though adenosine.

9. Conclusions

In summary, the data reviewed confirm the somnogenic

role of adenosine in central nervous system. The sleep-

inducing effects are attributed to the inhibition of wakeful-

ness-promoting neurons. The inhibitory effects of adenosine

might be exerted on both cholinergic and non-cholinergic

neurons. The cholinergic basal forebrain is an important area

of brain for mediating the somnogenic effects of adenosine

after prolonged sleep deprivation. The effects of adenosine

in this area are mediated via A1 adenosine receptors.

Furthermore, evidence is provided for the emerging role

of adenosine in mediating the longer-term effects of sleep

deprivation. Sleep deprivation-induced accumulation of

extracellular adenosine is localized to the cholinergic

HDB/SI/MCPO area of basal forebrain. Data from in vitro

studies demonstrate that adenosine, acting via A1 adenosine

receptor, activates inositol trisphosphate receptor-mediated

release of calcium from intracellular stores leading to the

activation of transcription factor NF-kB. Sleep deprivation-

induced adenosine increased the DNA binding activity of

NF-kB and the levels of A1 receptor mRNA, thus providing

evidence for an adenosinergic pathway leading to the acti-

vation of transcriptional process and A1 receptor being one

of the examples of the transcribed genes. The significant

observation was the occurrence of the two events at the

intracellular level, such as adenosine A1 receptor-mediated

mobilization of intracellular calcium and, sleep deprivation-

induced nuclear translocation of NF-kB selectively in a large

population of cholinergic neurons. At this time, the exact

mechanisms that determine the selectivity of cholinergic

population for eliciting the intracellular effects of adenosine

are not clear. In light of the identified role of these choli-

nergic neurons in attention, memory and arousal it is sug-

gested that long-term effects of sleep deprivation on these

aspects of behavior might be partly mediated through ade-

nosine.

Future studies will be directed towards identifying the

array of genes that are activated by adenosinergic activation

of transcription factor NF-kB in cholinergic basal forebrain.

The identification and characterization of genes expressed in

cholinergic neurons will provide new insights into the

mechanisms leading to the cumulative effects of sleep debt.

Acknowledgments

This work was supported by Sleep Medicine Education

and Research Foundation (RB), Department of Veterans

Affairs Medical Research Service Awards (RB, RES),

KO1 award, MH01798 (MMT) and National Institute of

Mental Health, NIMH39683 (RWM).

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Adenosine and sleep–wake regulation

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