The frontiers of sleep

MEETING

REPORT

The frontiers of sleep In early June, in the house of the European Parliament in Strasbourg, the Human Frontier Science Program celebrated its tenth anniversary with a note of pride for its many accomplishments and optimism for the future. One of two scientific meetings held to mark the occasion was on the regulation and functions of sleep*, an appropriate subject for a Human Frontier initiative. Given that the function of sleep is still unknown, the great accumulation of experimental results available is still in need of a rational synthesis. If sleep research is a frontier, it is not because of its age. Indeed, the Strasbourg meeting could well have been a 50th anniversary celebration. In 1949, Moruzzi and Magoun published their seminal discovery of the ascending reticular activating system1, a region of the brainstem reticular formation that is responsible for determining whether we are awake or asleep, conscious or unconscious. Through such discoveries in the 1940s and 1950s, sleep research was at the forefront of what came to be called neuroscience. In 1953, the discovery of rapid eye movement (REM) sleep2, a hitherto unsuspected state in which we spend almost five years of our life, came to mark the heydays of sleep research. Indeed, within neuroscience, sleep research is now an old discipline, so old that it can be subdivided into an AM and a PM phase (ante- and post-MEDLINE) of approximately equal length.

Success stories: the cellular electrophysiology of sleep This meeting provided an excellent assessment of remarkable progress and of equally remarkable ignorance. The characterization of the electrophysiological correlates of sleep and waking at the single-cell level is certainly on the success side. Over the past decade, this area of research has reached an enviable degree of scientific maturity, fulfilling the reductionist’s dream of explaining the sleep EEG in terms of ion channels and elementary currents. [This is not to say that the EEG is useless: new techniques, such as sophisticated coherence analysis (Peter Achermann, Zürich, Switzerland) and independent component analysis (Terrence Sejnowski, La Jolla, CA, USA) can reveal interesting differences between brain regions.] The work of Mircea Steriade (Quebec, Canada) has shown, for example, that slow oscillations (⬍1 Hz) coordinate various kinds of sleep rhythms, such as slow waves (1–4 Hz), spindles (8–13 Hz) and even short high-frequency bursts (40 Hz)3. The work of David McCormick *The Regulation of Sleep. Held in Strasbourg, France; 31 May – 2 June 1999.

(New Haven, CT, USA) has dissected the ionic conductances underlying several of these oscillations and their modulation by the ascending reticular activating system. This acts through noradrenaline, ACh, histamine and metabotropic glutamate receptors, largely through the production of Ins(1,4,5)P3. It now appears that metabotropic glutamate receptors are also responsible for the thalamic activation produced by cortical neurons located in layer VI, which configure a veritable ‘descending activating system’4. Another success story has been the dissection of the brainstem neural populations that generate REM sleep. This success story can now be repeated in the hypothalamus and basal forebrain, where neuronal populations are located that are responsible for coordinating slow-wave sleep (SWS) and harmonizing it with REM sleep. Robert McCarley (Brockton, MA, USA) presented compelling evidence that adenosine accumulates in the basal forebrain during prolonged waking and inhibits neurons through A1 receptors5. Osamu Hayaishi’s studies (Osaka, Japan) also point to a key role for adenosine in the regulation of sleep and waking, although such a role would be downstream of prostaglandin D2. While some inconsistencies need to be resolved, much is expected from the characterization of the cell types involved, of their afferents and efferents, and of the chemicals that regulate their activity.

REM sleep, depression and narcolepsy Other areas of intensive research relate to the mapping of brain areas that are particularly activated during different sleep states, such as REM sleep. At the meeting, remarkable agreement emerged between data obtained in humans using PET (Pierre Maquet, Liege, Belgium)6, and data obtained using FOS mapping in cat (Michel Jouvet, Lyon, France) and rat. Briefly, REM sleep, which is generated by brainstem centers discovered by Jouvet 40 years ago7, is associated with a marked activation of limbic areas, such as the amygdala and the anterior cingulate. Maquet presented data suggesting that brain areas activated during practice tasks were reactivated during REM sleep (when subjects might have been dreaming vividly of those tasks). This observation bears some resemblance to the reactivation demonstrated by Wilson and McNaughton during SWS in the rat. However, the specificity of the reactivation during sleep needs to be demonstrated, as it can also occur during quiet waking after training in the rat8. Sleep researchers are also interested in clinical aspects of their discipline. After many

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years of investigations in a dog model, Emmanuel Mignot (Palo Alto, CA, USA) is focusing on the isolation of the gene for narcolepsy, a disorder characterized by the intrusion of REM-sleep components during waking. Joëlle Adrien (Paris, France) and J. Christian Gillin (San Diego, CA, USA) reported on the intriguing connections between sleep and depression9. This is a complicated subject, but it seems clear that total sleep deprivation or selective REM deprivation relieve depression, if only until one goes to sleep again. Conversely, treatment with some antidepressants, especially monamine oxidase inhibitors, eliminates REM sleep in humans for several months, and yet no obvious adverse consequences seem to emerge. Is REM sleep our daily endogenous depressant, is it harmless, but also useless, or are we missing something?

Circadian and homeostatic aspects of sleep At a different level, a key advance has been the development of a theoretical and experimental framework for distinguishing between two fundamental components of sleep: the homeostatic and circadian (Alexander Borbély, Zürich, Switzerland)10. The former refers to the fact that the longer one stays awake, the more one needs to sleep, the latter to the change in sleep propensity with the time of day. These two components can be dissociated in humans using forced de-synchrony protocols, in which sleep is scheduled to occur at all circadian phases (Derk-Jan Dijk, Boston, MA, USA). Paradoxically, the circadian drive for waking peaks immediately before habitual sleep-time, and that for sleep immediately before wake-time. This balances the homeostatic drive to sleep most effectively and helps to consolidate long periods of waking and sleep11. The circadian component of sleep is comparatively well understood. Its function is clearly to adapt the succession of behavioral states to the alternation of day and night, and its mechanisms are becoming known to the last detail through genetic and molecular studies. For example, using two-photon microscopy, Martha Gillette (Urbana, IL, USA) has shown that glutamate and ACh induce distinct phase changes of the suprachiasmatic nucleus circadian clock via distinct temporal and spatial patterns of Ca2⫹ release from intracellular stores. The homeostatic component – the true essence of sleep – remains instead a veritable mystery. Many studies have shown that this component is reflected in the amount of slow waves in the EEG (Borbély). For example, slow-wave activity is greatly increased after sleep deprivation. The discovery that

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TINS Vol. 22, No. 10, 1999

Giulio Tononi and Chiara Cirelli The Neurosciences Institute, San Diego, CA 92121, USA.

417

MEETING

REPORT

G. Tononi and C. Cirelli – The frontiers of sleep

states of hibernation, or even of daily torpor, are also followed by an increase of slowwave activity is of great interest and suggests that sleep might permit an active recovery process that is not possible during lowtemperature hypometabolic states (Irene Tobler, Zürich, Switzerland). However, while the growth factors, cytokines and other molecules that influence the local regulation of sleep are being elucidated (James Krueger, Pullman, WA, USA; Ferenc Obál, Szeged, Hungary12), the nature of the recovery process is completely unknown. Yet the homeostatic component of sleep appears to be a rather universal phenomenon, being present in all mammals and birds studied so far. At this meeting, some initial evidence was presented indicating that Drosophila might also have sleep-like states. Not only do flies have an increased sensory threshold during prolonged periods of rest, but they show a rest rebound if they are deprived of rest for 12 h, suggesting a homeostatic control. Moreover, the analysis of changes in gene expression across the rest– activity cycle in both rats and flies is beginning to reveal a number of molecular correlates of behavioral states (Giulio Tononi, San Diego, CA, USA).

Why sleep? As promising as the new studies reported at this meeting might be, their importance must be measured against the fundamental, embarrassing and persisting shame of sleep research: our ignorance about the functions of sleep. Why do we sleep at all? Why do we need to spend a third of our life in a state of ‘abject mental annihilation’, as Sherrington once called it? The quest for discovering the functions of sleep is both a nursery and a graveyard for countless new theories and hypotheses. Among such hypotheses, some of the most popular see sleep as somehow involved in learning and memory. Albeit with due circumspection, a few hypotheses of this nature were voiced at this meeting. For example, during the spindles and slow waves of sleep, which are associated with bursting discharges, it is plausible, although as yet not proven, that a massive amount of Ca2⫹ might enter neurons. In this context, Michael Berridge (Cambridge, UK) presented his concept of the endoplasmic reticulum as a ‘neuron within a neuron’: a continuous network that extends throughout the cell, and up into the dendrites and spines. This network serves both as a sink for Ca2⫹ entering through voltage- and receptor-gated channels, and as a source for Ca2⫹ transients that can give rise to regenerative phenomena, including Ca2⫹ waves. The release of Ca2⫹ is a signaling mechanism that can powerfully regulate both neural excitability and plasticity, in part mediated by changes in gene expression. The idea has, thus, occurred to many that SWS could be a time when 418

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Ca2⫹ influx can lead to gene expression and thereby to long-term plastic changes. However, while Ca2⫹ homeostasis might be involved in sleep, the evidence indicates that, Ca2⫹ entry or not, little or no new information can be acquired during sleep. Nor perhaps should it be, as we are not exposed to the outside world but only to the intrinsic activity of our brain (although Jouvet intriguingly suggests that REM sleep might serve to re-program circuits that preserve biological individuality in the face of a changing environment). Moreover, it is becoming increasingly clear that the expression of genes normally associated with plasticity is induced during waking, not during sleep. For example, the phosphorylation of CREB and other transcription factors and the expression of several immediate–early genes are high during waking and low during sleep. It was shown at this meeting that Arc (activity-regulated cytoskeletal protein), an immediate–early gene that is currently the focus of great interest because of its unique property of selectively marking activated synapses, is also induced during waking and not during sleep (Tononi). Indeed, regardless of whether sleep is important for plasticity, it is becoming apparent that a comparison of sleep and waking might represent an ideal tool for examining cellular and molecular correlates associated with the induction of plastic changes. Such a comparison has already revealed that, under physiological conditions and without the need for any experimental manipulation, substantial differences exist in protein phosphorylation and gene expression between when the brain is plastic (awake) and when it is not (asleep). It has also been determined that a key reason for the low levels of various markers of plasticity during sleep is the inactivity of specific neuromodulatory systems, such as the noradrenergic system (but not the serotoninergic system)13. Moreover, new data indicate that after long-term sleep deprivation, levels of such markers of plasticity remain low. Studies that are now under way in a few laboratories (Borbély, Krueger, Tononi) will exploit sleep, waking and sleep deprivation in order to investigate the consequences of sleep on plastic changes triggered during waking. The meeting ended with heated questioning and soul searching. Is sleep a local or a global phenomenon? Can single neurons sleep? Can one investigate this in the slice or even in cell cultures? Do neurons tire of the waking activity? And what would be the cause? What special benefit does sleep provide that the brain needs it so desperately? Does sleep promote synaptic growth or remodeling? And is sleep really for the brain? Do SWS and REM sleep serve the same function, complementary functions or no functions at all? Craig Heller (Stanford, CA, USA) made the important point that active

sleep, a peculiar state of sleep found early in development, might be a precursor of both SWS and REM sleep, and not just of REM sleep, as was generally thought. This would correspond well with the recent demonstration by Siegel that the echidna, a primitive monotreme, also shows a mixed precursor sleep state14. Perhaps the most-obvious impression produced by this excellent meeting was the contrast between our growing experimental refinement and our ignorance about fundamental issues. There is no other area of neuroscience, except perhaps consciousness, that is so remarkably familiar and yet so difficult to define scientifically. Whoever enters sleep research with high expectations would be well served by a generous endowment of what John Keats called negative capability: ‘that is when man is capable of being in uncertainties, mysteries, doubts, without any irritable reaching after fact and reason’. This virtue, which Keats thought associated with poetic prowess, might also profit a scientist adventuring in a field littered with corpses of beautiful theories. Indeed, the fact that we still do not know what sleep is for must be counted as both an insult and a challenge to a scientist’s intelligence. It also provides one of the last opportunities to face a true mystery. Indeed, there is perhaps no better time to lift nature’s veil than when she is asleep. If you are up to such a challenge, try embarking on an investigation of the functions of sleep.

Note added in proof The gene responsible for canine narcolepsy was identified recently as the hypocretin receptor 2 gene15. References 1 Moruzzi, G. and Magoun, H.W. (1949) Electroencephalog. Clin. Neurophysiol. 1, 455–473 2 Aserinsky, E. and Kleitman, N. (1953) Science 118, 273–274 3 Destexhe, A., Contreras, D. and Steriade, M. (1999) J. Neurosci. 19, 4595–4608 4 McCormick, D.A. and Bal, T. (1997) Annu. Rev. Neurosci. 20, 185–215 5 Porkka-Heiskanen, T. et al. (1997) Science 276, 1265–1268 6 Maquet, P. et al. (1996) Nature 383, 163–166 7 Jouvet, M., Michel, F. and Courjon, J. (1959) C. R. Soc. Biol. 153, 1024–1028 8 Kudrimoti, H.S., Barnes, C.A. and McNaughton, B.L. (1999) J. Neurosci. 19, 4090–4101 9 Gillin, J.C. et al. (1984) in Neurobiology of Mood Disorders (Post, R.M. and Ballenger, J.C., eds), pp. 157–189, Williams and Wilkins 10 Borbély A.A. (1982) Hum. Neurobiol. 1, 195–204 11 Czeisler, C.A. et al. (1999) Science 284, 2177–2181 12 Krueger, J.M., Obal, F., Jr and Fang, J. (1999) J. Sleep Res. 8 (Suppl. 1), 53–59 13 Cirelli, C., Pompeiano, M. and Tononi, G. (1996) Science 274, 1211–1215 14 Siegel, J.M. et al. (1998) Philos. Trans. R. Soc. London Ser. B 353, 1147–1157 15 Lin, L. et al. (1999) Cell 98, 365–376