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ON THE FUNCTIONAL SIGIFICANCE OF C-FOS INDUCTION DURING THE SLEEP-WAKING CYCLE

On the Functional Significance of c-fos Induction During the Sleep-waking Cycle

Chiara Cirelli MD, PhD and Giulio Tononi MD, PhD The Neurosciences Institute, San Diego, California 92121 Abstract: A striking finding in recent years has been that the transition from sleep to waking is accompanied in many brain regions by a widespread activation of c-fos and other immediate-early genes (IEGs). IEGs are induced by various electrical or chemical signals to which neural cells are exposed and their protein products act as transcription factors to regulate the expression of other genes. After a few hours of sleep, the expression of these transcription factors in the brain is absent or restricted to very few cells. However, after a few hours of spontaneous waking or sleep deprivation, the expression of c-fos and other IEGs is high in cerebral cortex, hypothalamus, septum, and several thalamic and brainstem nuclei. While cells expressing c-fos during waking are widely distributed, they represent only a subset of all neurons in any given area. These observations raise several questions: Why is c-fos expressed during waking and not during sleep? Is waking always accompanied by c-fos induction? Which subset of cells express c-fos during waking and why only a subset? Once c-fos has been induced, what are the functional consequences of its activation? In this review, we summarize our current understanding of the meaning of c-fos activation in the brain in relation to the sleep-waking cycle and suggest that c-fos induction in the cerebral cortex during waking might be related to the occurrence of plastic phenomena. Key words: c-fos; immediate early gene; locus coeruleus; plasticity; rat; sleep deprivation INTRODUCTION THE REMARKABLE CHANGES IN NEURAL FIRING PATTERNS THAT OCCUR BETWEEN SLEEP AND WAKING are one of the best-established aspects of the cellular physiology of behavioral states.1 Recently, a new perspective on the cellular correlates of sleep and waking has been opened by the discovery that sleep and waking are associated not only with changes in neural activity, but also with striking differences in the expression of certain genes. While a systematic investigation of differences in gene expression between sleep, waking, and sleep deprivation is still in progress,2-3 for a few well-studied genes it appears possible to draw some initial conclusions and to attempt some functional interpretations. Preeminent among such genes is c-fos, the best known among the so-called immediate early genes (IEGs), a category of genes that are rapidly induced by many extracellular stimuli. The first experiments demonstrating a modulation in the expression of c-fos during the sleep-waking cycle and after sleep deprivation were performed in the early nineties. 4-7 These studies supported previous findings of changes in brain RNA and protein content across the sleep-waking cycle3 and provided the first clear indication that the

Accepted for publication February 2000 Address correspondence to: Dr. Chiara Cirelli, The Neurosciences Institute, 10640 John J. Hopkins Drive, San Diego, CA 92121, USA., Tel: 858-626 2110, Fax: 858-626 2199, E-mail: [email protected] SLEEP, Vol. 23, No. 4, 2000 9

expression of specific genes may be subject to significant modulations in the course of sleep and waking. Soon thereafter, several laboratories demonstrated that mRNA and protein levels of NGFI-A (nerve growth factor-induced A), Jun B, and other IEGs are also modulated by sleep and wakefulness in various brain areas. Surprisingly, this dramatic modulation of gene expression between spontaneous sleep and waking is frequently overlooked in the vast literature that relies on IEGs mapping for functional studies of brain activity. For example, a recent 120-page review lists hundreds of studies of IEGs induction in the brain due to various experimental manipulations, but fails to mention that spontaneous waking per se is able to induce IEGs expression in many brain regions.8 This paper will focus on state-dependent changes in the expression c-fos, although other IEGs will also be briefly discussed. First, we consider the general cellular and molecular mechanisms involved in c-fos induction in the brain in response to different types of stimulation. We describe in some detail how the pattern of c-fos expression changes in the brain in relation to spontaneous or pharmacologically induced sleep, spontaneous waking, short-term and long-term sleep deprivation, and arousal induced by psychostimulants. We discuss the use of c-fos as a marker of cellular activation and the "late" genes that have been identified as potential c-fos targets. Finally, we examine the functional significance of c-fos induction during waking by considering the evidence relating c-fos expression to celluOn the Functional Significance of c-fos--Cirelli et al

lar stress, learning, and plasticity. C-FOS AS AN IMMEDIATE EARLY GENE Immediate early genes such as c-fos share the property that their transcription is induced via preexisting cell proteins without requiring de novo protein synthesis. This property is analogous to that of the IEGs of some viruses and bacteriophages, which are expressed immediately after the infection of the cell in the absence of cellular protein synthesis. In fact, the gene fos was first identified as a retroviral gene (v-fos) present in the Finkel-Biskis-Jinkins osteosarcoma virus -- an oncogene that has transforming ability when overexpressed. C-fos is the normal cellular gene (or proto-oncogene) from which v-fos evolved. The protein products of many proto-oncogenes are involved in signal transduction cascades, and include receptor ligands, tyrosine kinases, non-tyrosine kinase receptors, nuclear receptors, and serine-threonine kinases. A significant number of proto-oncogenes, such as c-fos and other members of the fos (fra-1, fra-2, fosB) and jun (c-jun, junB, junD) families, encode nuclear proteins. The protein product of c-fos, Fos, is a transcription factor that, by binding to DNA regulatory regions, can control the expression of many other "target" genes. C-fos is expressed in a limited number of tissues, including amniotic and placental tissue, fetal liver, adult bone marrow and growing bone, and developing central nervous system9. Overexpression of c-fos in transgenic mouse lines specifically affects bone, cartilage, and hematopoietic cell development, while homozygous mice lacking c-fos show delay in growth and sexual maturation, osteopetrosis, and altered haematopoiesis secondary to altered bone development9-13. Effective Stimuli Since its discovery,14 it has been clear that c-fos is a general sensor that can be rapidly induced by incoming stimuli at the cell membrane and then convert them into long-term responses that require gene activation, such as cell division, growth, and memory formation. For this central role, c-fos has been described as a "master switch" of cellular regulatory activities.15 In the CNS, c-fos can be induced by many different stimuli, such as noxious, acoustic, thermal, visual, and somatosensory stimuli. With the exception of GABA and glycine, all classical neurotransmitters and neuromodulators have been found to activate c-fos in at least some experimental conditions. These include glutamate (via NMDA, AMPA/kainate, and metabotropic receptors), dopamine, serotonin, norepinephrine and epinephrine (via and receptors), acetylcholine (via muscarinic and nicotinic receptors), and histamine. Trophic factors (EGF, NGF, FGF, PDGF), NO (via cGMP), IL-6, potassium-induced

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membrane depolarization (via activation of voltage-sensitive Ca++ channels) and direct stimulators of second messenger pathways (e.g., phorbol esters) also induce c-fos, together with a long list of drugs, including amphetamines, cocaine, haloperidol, morphine and caffeine (see below). It is probably a simpler task to list those stimuli that do not induce c-fos. To our knowledge, postsynaptic inhibition has never been associated with the induction of c-fos expression in postsynaptic cells. Indeed, c-fos expression is reduced when motoneurons are postsynaptically inhibited.16 Adenosine and its agonists, which generally act as inhibitory neuromodulators, reduce c-fos expression in the spinal cord after pain stimulation17 (A1 receptors), in the suprachiasmatic nucleus after light-induced phase-shift18 (A1 receptors), and in the striatum after amphetamine exposure19 (A2A receptors). However, stimulation with A2A receptor agonists (which results in activation of adenylate cyclase) increases c-fos expression in mesolimbic areas20. It has also been suggested that one of the mechanisms by which somatostatin exerts its inhibitory actions is through blockade of c-fos expression.21 Temporal Pattern of Induction In response to a stimulus, one observes first an increase in c-fos mRNA levels, which is soon followed by the synthesis of Fos protein. C-Fos mRNA can be induced within 20 minutes of stimulus onset, whereas the induction of Fos protein requires a period of up to 90 min.22,23 High levels of Fos are generally observed for several hours and then progressively decline.24 For instance, Fos is induced and disappears within four to eight hours of an osmotic stimulation and within four to five hours following administration of convulsant agents.25 After a light pulse as short as five minutes, Fos peaks in the suprachiasmatic nucleus after one to two hours and disappears within six hours.26 C-Fos induction is more effective when a novel stimulus is applied, or when the animal is stimulated after a period of sensory deprivation. For instance, c-fos mRNA levels in cerebral cortex, septum, and hippocampus are increased after acute restraint stress but are lower than in controls after repeated stress.27 The so-called "refractory period" for c-fos induction lasts for several hours after the primary stimulus. Both Fos and Fos-related proteins (Fras) are probably responsible for the repression of c-fos transcription after the primary stimulus by acting on the regulatory sites of the c-fos promoter.8 The Control of c-fos Transcription The signal transduction pathways by which many different stimuli can induce or silence c-fos expression in the CNS have been well characterized. The c-fos promoter contains at least four specific regulatory elements, a serum response element (SRE), a Ca++ and cAMP response eleOn the Functional Significance of c-fos--Cirelli et al

ment (CaRE-CRE), a sis-inducible element (SIE), and an AP-1 binding site (FAP)28. The CaRE-CRE element mediates c-fos induction by increased intracellular Ca++ (e.g,. through L-type Ca++ channels) and cAMP (e.g., through dopamine, norepinephrine, and adenosine). The CaRECRE element has been shown to be particularly relevant for the transcriptional activation of c-fos. The increases in Ca++ and cAMP activate the Ca++-calmodulin dependent protein kinases (CaMKII and IV, in particular) and protein kinase A, and these kinases phosphorylate the transcription factor CREB on serine 13329 (P-CREB). P-CREB in turn acti-

vates c-fos transcription. Although usually described as functionally distinct, the four transcriptional control elements are strictly dependent upon each other and act as a single interdependent transcription complex to regulate c-fos expression. Experiments in transgenic mice that carry point mutations in each of the four sites show that all are essential for the normal function of the c-fos promoter. For example, a point mutation in SIE or FAP causes a dramatic loss of c-fos induction by depolarizing stimuli, although the CaRE-CRE is intact.28 C-FOS EXPRESSION IN THE BRAIN DURING THE SLEEPWAKING CYCLE Experiments performed in rats,4-7,30-37 mice,38 ground squirrels,39 Syrian hamsters,40 and cats41 have consistently found that the expression of c-fos in the brain is very low or absent after a few hours of sleep/inactivity, while it is high if the animal has been awake for a few hours. The pattern of c-fos expression after a few hours (3 to 24) of total sleep deprivation is similar throughout the entire brain to the pattern observed after spontaneous wakefulness.31 This indicates that c-fos expression is associated with waking per se, rather than with circadian or other factors. In rat and cat, a detailed anatomical mapping of c-fos expression in the entire brain across behavioral states is available 5,31,41,42 and will be summarized below. During physiological sleep, c-fos expression is low throughout the brain. With the exception of the ventrolateral preoptic area, which will be discussed later, no brain region has been found to show a positive correlation between c-fos expression (at either mRNA or protein levels) and the amount of sleep in the previous three to twelve hours in rats 5,31 or in the previous one to three hours in cats.41 A few cells show very low levels of Fos immunostaining in the cerebral cortex, in particular in piriform, cingulate, and perirhinal cortices, as well as in some hypothalamic and thalamic nuclei, superior and inferior colliculi, and central gray. However, this faint immunostaining is found in the same areas in which Fos levels are much higher after waking, suggesting that it may merely represent what is left from the previous waking induction. In agreement with this conclusion, it has been observed that, in both rats32 and mice,38 there is a negative correlation between the percentage of total or slow-wave sleep in the one to two hours preceding sacrifice and the number of Fos positive neurons in the cerebral cortex. During spontaneous or forced waking, the expression of c-fos is high in most regions of the neocortex and allocortex, including frontal, motor, parietal, temporal, occipital, cingulate, insular, piriform, and entorhinal areas (Figure 1). In the hypothalamus, c-fos is induced in the medial and lateral preoptic areas, in the posterior hypothalamic area, and in the supramammillary nuclei. Induction of c-fos is also

11 On the Functional Significance of c-fos--Cirelli et al

Figure 1--c-fos mRNA expression in a rat that spent ~80% of the last 5 h asleep (left column), in a rat that spent ~80% of the last 5 h asleep but was continuosly awake for the last 30 min before sacrifice (middle column), and in a rat that spent ~75% of the last 5 h awake (right column). The first 2 rats were sacrificed during the light period at the same circadian time (~ 15.00), while the last animal was sacrificed during the dark period (~2.00). Sections A-F are in craniocaudal order. Abbreviations: AON, anterior olfactory nucleus; BS, bed nucleus of the stria terminalis; CA, Ammons horn; Cb, cerebellum; CG, central gray; Den, dorsal endopiriform nucleus; Ent, entorhinal cortex; Fr, frontal cortex; LC, locus coeruleus; MG, medial geniculate nucleus; MPA, medial preoptic area; Occ, occipital cortex; Par, parietal cortex; Pn, pontine nuclei; PRh, perirhinal cortex; PrS, presubiculum; RN, red nucleus; RS, retrosplenial cortex; SC, superior colliculus; VPN, ventroposterior complex of thalamic nuclei. Bar is 5mm. Modified from ref 5. SLEEP, Vol. 23, No. 4, 2000

seen in the septum, in the verticle and horizontal limbs of the diagonal band of Broca, in the amygdala, and in the thalamus (Fig. 1). In the verticle and horizontal limbs of the diagonal band of Broca, but not in the medial septal nucleus, several Fos positive cells have been shown to be cholinergic.42a In the thalamus, Fos staining is seen in several midline nuclei (paraventricular, rhomboid, reuniens) and intralaminar nuclei (centromedial and centrolateral) but not in the "specific" thalamic nuclei or in the thalamic reticular nucleus. In the brainstem, c-fos expression is high in the superior and inferior colliculi, central gray, dorsal raphe, locus coeruleus, and parabrachial nuclei (Figure 1). In the cerebral cortex, Fos expression during waking is increased in all layers. However, the cells expressing Fos protein represent a small fraction of all the cells present in a given section (Fig. 2). Only a few studies have attempted neurochemically to identify the cortical cells expressing cfos. According to preliminary double labeling experiments, c-fos is induced in pyramidal projection neurons rather than in cortical interneurons.43 In the visual cortex, the number of cortistatin positive cells increases after sleep deprivation and some of them express c-fos.44 In the basal forebrain, Fos immunostaining does not colocalize with that for the low affinity nerve growth factor receptor, suggesting that in this region neurons expressing c-fos during waking are not cholinergic45. The expression of c-fos is not strictly proportional to the amount of previous waking, as indicated by studies of sleep deprivation ranging from 3 to 24 hours. In most brain regions, and in particular in the cerebral cortex, the overall

levels of c-fos are higher after 3 than after 24 hours of sleep deprivation.31 After long-term sleep deprivation performed with the disk-over-water apparatus for 5­14 days, only a few scattered cells expressing Fos are present in the cerebral cortex, with no specific localization to any cortical area or cortical layer (Cirelli and Tononi, unpublished results). Thus, the pattern of c-fos expression in the cerebral cortex after sustained periods of waking is more similar to the one observed after a few hours of sleep than to the one observed after a few hours of waking. C-FOS EXPRESSION IN SPECIFIC BRAIN AREAS c-fos Expression in the Medial Preoptic Area During Waking The results reviewed above suggest that, in general, while c-fos expression is higher during waking than during sleep, it does not constitute a reliable indicator of the duration of previous waking. There may be, however, some exceptions. In particular, in hypothalamic regions such as the medial preoptic area (MPA), the expression of Fos remains high even after long-term sleep deprivation (Cirelli and Tononi, unpublished results). The MPA of the hypothalamus has been extensively implicated in the regulation of sleep by lesion and stimulation studies. In addition, recording studies have demonstrated a population of waking-on cells, which increase their firing rate just before behavioral arousal.46 Data listed below suggest that the amount of cfos expression in MPA may either reflect previous waking or, alternatively, a homeostatic need for sleep. 1) In rats, c-fos expression in MPA is at least as high after 24 hours of sleep deprivation as after three hours of sleep deprivation31 and it is consistently elevated after long-term sleep deprivation (Cirelli and Tononi, unpublished results). 2) In cats, c-fos expression is significantly increased in MPA after sleep deprivation by gentle handling.41 The increase in the number of Fos positive cells in this area is higher in stressed sleep deprived cats than in gently deprived cats.41 Interestingly, sleep rebound is more significant after stressful sleep deprivation than after gentle sleep deprivation.47 3) c-Fos expression in MPA is consistently increased after treatment with methamphetamine, methylphenidate, modafinil, and caffeine which result in prolonged waking.48-49 4) In old rats, a reduced expression of c-fos with respect to young rats has been described in the hypothalamus, including the MPA, in response to 12 hours of sleep deprivation.50 Old rats also show a reduced amount of slow-wave sleep with respect to young rats during recovery sleep after 12 hours of sleep deprivation.50 5) In rats, injections in MPA of c-fos antisense, but not of sense, oligonucleotides has been show to block the expression of Fos detected immunocytochemically after periods of spontaneous waking.51 While normal rats or rats inject12 On the Functional Significance of c-fos--Cirelli et al

Figure 2--Fos protein expression in the parietal cortex after 3 h of spontaneous sleep (A), 3 h of sleep deprivation by gentle handling (B), and 3 h of spontaneous waking (C). The rats in A and B were sacrificed during the light period at the same circadian time (~ 13.00), while the rat in C was sacrificed during the dark period (~1.00). Many Fos positive cells are present in both B and C but almost none in A. Note, however, that even in B and C only a subset of cortical neurons are expressing Fos protein. Bar is 100µm.

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ed with c-fos sense oligonucleotides tend to stay awake during the dark hours and to sleep during the light hours, rats bilaterally injected in the afternoon with c-fos antisense oligonucleotides slept less than controls on the day after the injection.51 The increased amount of waking appeared to be physiological and it was not followed by a compensatory increase in sleep. The inhibition of the expression of Fos in the preoptic hypothalamus by antisense oligonucleotides may thus interfere with mechanisms that assess the duration and intensity of prior waking and/or with homeostatic executive mechanisms that bring about sleep. c-fos Expression in the Ventrolateral Preoptic Area During Sleep While the expression of c-fos during sleep is very low throughout the brain, in the ventrolateral preoptic nucleus (VLPO) of the anterior hypothalamus, a positive correlation was found between the number of Fos positive cells and the percentage of sleep in the preceding hour.34 After 9­12 hours of sleep deprivation, the number of VLPO Fos positive cells was higher in rats that were allowed to sleep for one hour with respect to those that were sacrificed immediately after sleep deprivation. Therefore, c-fos expression in VLPO was related to the sleeping process per se rather than to the need for sleep.34 More recently, c-fos expression in VLPO was found to be higher in nocturnal rodents (rats) sacrificed during or just after the light (rest) period than during the dark (activity) period.37 By contrast, in the diurnal rodent Arvicanthis niloticus, the number of Fos immunoreactive cells in VLPO was higher during the dark (rest) period than during the light (activity) period.52 These findings are of interest in view of the electrophysiological demonstration of sleep-on neurons around this region.46 Sleep-on neurons have also been reported in neighboring basal forebrain and hypothalamic areas, including the MPA.46 Whether such scattered sleep-on neurons may also express c-fos during sleep remains to be demonstrated. In areas such as the MPA, in which c-fos is known to be strongly induced by waking, it will be essential to demonstrate that sleep-related c-fos positive cells are positively correlated with the amount of sleep and are localized differently with respect to those activated by waking. A few studies have analyzed c-fos expression after pharmacological treatments that induce sleep. After injection in the subarachnoid space, prostaglandin D2 increases slowwave sleep and also induces c-fos expression in the basal meninges, VLPO, bed nucleus of the stria terminalis, central nucleus of the amygdala (lateral subdivision) and parabrachial nucleus (external lateral subnuclei).53 Since this pattern of c-fos expression differs from that seen during physiological sleep, it could reflect, at least in part, prostaglandin D2 stimulation rather than slow-wave sleep

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per se. In agreement with this conclusion, when an increase in sleep is pharmacologically obtained by adenosine injection in the basal forebrain, Fos positive cells are mainly induced in the ipsilateral basal forebrain but not in the other regions where Fos is observed after prostaglandin D2 infusion.54 Moreover, injection of an adenosine A2A agonist in the subarachnoid space underlying the rostral basal forebrain induces sleep and increases c-fos expression mainly in the shell of the accumbens nucleus and in the olfactory tubercle but not in many other areas activated by prostaglandin D2 or adenosine.55 In summary, c-fos expression identifies two closely related areas of the anterior hypothalamus that could be involved in complementary aspects of sleep regulation. Fos positive cells are consistently induced in MPA during spontaneous waking, sleep deprivation, and pharmacologicallyinduced arousal. Moreover, inhibiting Fos buildup through c-fos antisense oligonucleotides prevents the homeostatic increase of sleep after prolonged waking. Fos positive MPA cells may therefore detect the build up of sleep pressure during waking. Preliminary results show that these cells are not GABAergic,56 but no data are currently available regarding the anatomical pathways that could mediate their effects on the sleep-waking cycle. On the other hand, VLPO cells express c-fos during physiological sleep, during slow-wave sleep rebound after sleep deprivation, and after at least some pharmacological treatment that induce sleep. Some of these cells contain the synthetic enzyme for GABA and have been shown to project directly to histaminergic cells of the tuberomammillary nucleus, to the locus coeruleus, and to serotoninergic nuclei.57 They could therefore inhibit several components of the ascending arousal system.57 A role of c-fos in the regulation of sleep has also been inferred by preliminary data showing that c-fos knockout mice have a 33% decrease in total sleep time compared with wild types and heterozygotes.58 However, the results of these knockout experiments are difficult to interpret due to the complexity of the phenotype. Mice with a null mutation in c-fos do not show any obvious alterations in the embryonic and neonatal phenotype of the nervous system.10 However, adult c-fos deficient mice show subtle behavioral abnormalities; they are less responsive to external stimuli,10 fail to respond normally to stress,9 and are impaired in complex learning tasks.59 It is not clear whether abnormalities in such complex functions are due to structural or anatomical brain alterations per se or whether they are secondary to bone structural changes. For instance, learning deficits could simply be due to the bone abnormalities leading to an impairment of hearing.59 c-fos Expression During REM Sleep As mentioned above, the synthesis of c-fos mRNA

On the Functional Significance of c-fos--Cirelli et al

requires ~20 minutes and that of Fos 30­60 minutes. Due to these time constraints, the absence of a positive correlation between physiological REM sleep episodes (lasting ~2 minutes in rats and ~6 minutes in cats) and c-fos induction is not surprising. In the rat, the number of cortical Fos positive cells and the amount of REM sleep in the 2 hours preceding sacrifice are negatively correlated, although the correlation is not as strong as for slow-wave sleep.32 The absence of Fos staining after variable periods of sleep that included multiple REM sleep episodes suggest that physiological REM sleep is not associated with Fos expression. Nonetheless, a number of studies have attempted to identify neuronal populations that are "activated" during REM sleep. To this end, Fos expression has been extensively studied after pharmacological manipulations that substantially increase the duration of REM sleep episodes, in particular carbachol microinjection in the pons.60-65 Microinjection of the mixed cholinergic agonist carbachol in the dorsal pontine tegmentum induces a state comparable to natural REM sleep, but much longer in duration.66 After carbachol injections, Fos expression increases in many portions of the medial and lateral reticular formation of the pons and medulla (in medium or small size neurons but not in giant neurons), in nuclei of the dorsolateral rostral pons (central gray, laterodorsal nucleus, pedunculopontine nucleus, locus coeruleus, parabrachial nuclei), raphe nuclei, vestibular nuclei, and in the abducens nucleus. By contrast, c-fos expression is reduced in trigeminal, facial, and hypoglossal neurons. The majority of Fos positive neurons in the pons are neither noradrenergic, nor cholinergic or serotoninergic, while some may be GABAergic.62,64,65 It appears that, to some extent, the longer the episode of REM sleep induced by carbachol, the higher the levels of c-fos expression. However, if the duration of the REM episode exceeds two hours, c-fos expression does not differ from controls.60,61 Neurons in the septum, hippocampus, lateral geniculate nucleus and occipital cortex, which are known from recording studies to be active during REM sleep, do not express Fos protein after carbachol injections.60,63 It is difficult to determine to what extent Fos expression after pharmacologically induced REM-like states faithfully maps the specific sets of neurons that are responsible for the generation of REM sleep as opposed to those that are directly stimulated by the drug. However, for the same total amount of REM sleep, c-fos expression is higher in animals treated with carbachol than in controls.60 This suggests that Fos expression in at least some brainstem cells is related to the injection of carbachol rather than to the occurrence of REM sleep per se. The injection of the GABAA agonist muscimol in the posterior periaqueductal gray of the cat is also effective in inducing a prolonged REM-like state. Depending on the precise injection site, muscimol can either have no effect, or produce prolonged episodes of waking, slow-wave

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sleep, or REM sleep.67 Following muscimol injection, a positive correlation has been found between the number of Fos positive cells and the amount of REM-like states in the previous three hours in the laterodorsal tegmental nucleus of the pons, in the supramammillary nucleus of the hypothalamus, in the septum, hippocampus, cingulate cortex, amygdala, stria terminalis, and in the accumbens nucleus.68 Non-pharmacological methods have also been used to increase the duration of REM sleep episodes. In a series of studies,69,70 the amount of REM sleep was reported to increase from 8% of the total recording time during the baseline period to 10­13% after auditory stimulation, and to ~ 20% after sleep deprivation for 48 hours on the inverted flower pot technique. After these treatments, a significant induction of c-fos was found in the suprachiasmatic nucleus, amygdala, dorsal hypothalamus, and many brainstem nuclei, including the pedunculopontine and laterodorsal tegmental nuclei, the subcoeruleus, and the solitary tract nucleus. In another study,71 after ~ 50h of sleep deprivation with the inverted flower pot technique, REM sleep percentage doubled with respect to baseline levels (from 15% to 28%). Double labeling studies performed after three hours of recovery showed that, in the pontomesencephalic tegmentum, the number of cholinergic/Fos positive and GABAergic/Fos positive cells was increased, while that of noradrenergic/Fos positive and serotoninergic/Fos positive cells was decreased. These changes were positively correlated with the amount of REM sleep during the recovery period. In a recent study, rats were sleep deprived of total sleep or selectively of REM sleep for four to eight days using the disk-over-water apparatus.72 This method does not involve pharmacological manipulations and induces a rebound of REM sleep that is more robust than that observed with any other method previously used.73 Moreover, the method controls for aspecific effects of the deprivation procedure through the presence of a yoked control that receives the same amount of stimulation. Some totally sleep deprived (TSD) rats and their yoked controls (TSC) were sacrificed immediately after sleep deprivation, while others (TSD-R and TSC-R) were allowed to recover sleep ad libitum for three to five hours before sacrifice. During the last three hours before sacrifice, TSD and TSC rats had 0% of REM sleep while TSD-R and TSC-R rats had 59% and 30%, respectively. In several forebrain regions, including anterior cingulate, retrosplenial, entorhinal cortex, lateral septum, amygdala, supramammillary nucleus, dentate gyrus, CA1 region of the ventral hippocampus, and subiculum, a positive correlation was found between the amount of REM sleep in the last three hours and the number of Fos positive cells. The amount of Fos staining in these regions was high in TSD-R rats, intermediate in TSC-R rats, and low in TSD and TSC rats. A positive correlation between

On the Functional Significance of c-fos--Cirelli et al

REM sleep and Fos expression was also found in the following brainstem regions: pretectal nuclei, deep layers of the superior colliculus, pontine nuclei, lateral parabrachial nucleus, subcoeruleus nucleus alpha, deep mesencephalic nucleus, pontine reticular nuclei oralis and caudalis, and gigantocellular reticular nucleus, including its ventral portion. Many regions showing high levels of Fos expression during REM rebound after long-term sleep deprivation correspond to those expressing Fos during REM sleep after muscimol injection in the periaqueductal gray. Many of these regions show prominent REM-related theta activity in rats and cats.74 They also partially match the brain regions showing an increase in blood flow during REM sleep in humans.75-77 In contrast to the limbic cortical regions listed above, in most other cortical areas, including frontal, insular (Figure 3), piriform, parietal (Figure 4), temporal, perirhinal, and occipital cortex, the expression of Fos protein was minimal in TSD-R rats. The few Fos positive cells that were present in these areas were exclusively located in the deep layers of the cortex. Levels of c-fos expression were low also in the striatum and in most thalamic nuclei. These results differ strikingly from those observed after periods of waking of similar duration, in which many Fos positive cells are found in all cortical regions and in all cortical layers.

MECHANISMS RESPONSIBLE FOR THE EXPRESSION OF CFOS DURING WAKING A recent series of experiments78 examined the mechanisms responsible for the high expression of c-fos and NGFI-A during spontaneous waking but not during sleep. It is well known that the activity of neuromodulatory systems with diffuse projections, such as the noradrenergic locus coeruleus (LC), is modulated by the behavioral state of the animal. During sleep, LC neurons fire tonically at very low levels, whereas during waking, LC neurons fire tonically at higher levels and emit phasic, short bursts of action potentials when triggered by salient events.79,80 LC neurons release norepinephrine, which can modify neural activity and excitability as well as the expression of certain genes, including IEGs.81 To establish whether the activity of the noradrenergic system is involved in the regulation of c-fos expression, unilateral injections of 6-hydroxydopamine were used in rats to selectively destroy the LC of one side. In these animals, the raw EEG and its power density spectrum were not significantly different between the lesioned and the intact side one to two weeks following the lesion. After either spontaneous waking or sleep deprivation for three hours, Fos levels on the intact side were high and comparable to those observed in normal animals after periods of waking. However, Fos expression was almost abolished in cortical areas and hippocampus on the lesioned side (Figure 5). A similar decrease on the lesioned side was also observed for NGFI-A protein. In another series of experiments, rats were treated with DSP-4, a neurotoxin that selectively ablates noradrenergic axon terminals originating from LC but not from noradrenergic non-LC neurons.82 After DSP-4 treatment, noradrenergic terminals are almost entirely and bilaterally destroyed in the cerebral cortex, hippocampus, thalamus, tectum, cerebellum, and spinal cord, but they are only moderately affected in the hypothalamus, including the MPA.82 As after unilateral LC lesions, waking behavior and waking EEG were normal. However, in these animals the expression of Fos and NGFI-A after three hours of sleep deprivation was significantly reduced with respect to control rats in most cortical areas although not in the hypothalamus.78 These data show that, in the absence of an intact noradrenergic system, waking behavior accompanied by lowvoltage fast activity patterns is not sufficient for the normal induction of c-fos and other IEGs in many cortical areas. By the same token, it is likely that the low levels of expression of c-fos and other IEGs during sleep can be accounted for, at least in part, by the low level of LC activity in this behavioral state. The recent data demonstrating low levels of Fos expression in non-limbic areas during massive REM sleep rebounds after long-term sleep deprivation also support this hypothesis. Since both REM sleep and waking are associated with low-voltage fast activity and high metabolic rate in the cerebral cortex, the cessation of noradrenergic

15 On the Functional Significance of c-fos--Cirelli et al

Figure 3--Many Fos positive cells are present in the agranular insular cortex and in the dorsal part of the anterior olfactory nucleus (A) and in the perirhinal cortex (B) of a rat that was sleep deprived for 8 h before sacrifice (left column) but not in the corresponding section of a rat which spent 75% of the previous 8 h in spontaneous sleep (mostly non-REM sleep; right column). No Fos positive cells are present in the same regions in a rat that spent 50% of the previous 2 h in REM sleep (middle column). In this rat REM rebound followed 4 days of total sleep deprivation obtained by the disk-over-water method. Scale bar = 100 µm.

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activity during REM sleep may be a key factor for the reduced expression of c-fos in most cortical areas during this behavioral state. While there are a number of mechanisms by which norepinephrine release during waking might facilitate the induction of IEGs, an important pathway involves the phosphorylation of CREB, which can act in turn on the promoters for c-fos and NGFI-A. Using an antibody specific for the phosphorylated Ser-133 residue of CREB, the expression of P-CREB was examined in brain sections from rats in which the LC had been lesioned on one side. It was found that P-CREB levels were considerably reduced in cortical areas depleted of noradrenergic fibers, in close correspondence with the decrease in Fos and NGFI-A immunostaining. Given the similar distribution of Fos expression during waking and of -adrenergic receptors in the cortex,78 this result suggests that norepinephrine released by LC terminals during waking may induce Fos by acting through a pathway that leads from -receptors to CREB phosphorylation. At present, it is not clear whether the expression of c-fos during waking is enabled by the tonic firing of LC cells during this behavioral state or by the short-lasting, phasic increases in LC discharge which are observed with various salient stimuli.83,84 Some data are consistent with the latter possibility. For example, in freely moving rats exploring a new environment, LC cells show a robust phasic response when the animals encounter a new object.84 The response is short lasting and rapidly habituates on the second or subsequent visits to the same object.84 Similarly, Fos expression in the cerebral cortex is more efficiently induced by novel rather than by familiar stimuli85-88 as well as by short rather than by long periods of sleep deprivation.31 Other studies indicating that the level of activity of noradrenergic LC cells is important for IEGs expression in many brain areas come from different experimental

paradigms. For instance, it has been shown that high basal expression of NGFI-A in the cortex requires not only a degree of sensory stimulation but also the presence of a noradrenergic tone.89 Local unilateral infusion of 6-hydroxydopamine in LC strongly reduces the induction of c-fos by yohimbine or stress in the ipsilateral but not contralateral frontal, cingulate, and piriform cortices.90 The expression of Fos in the olfactory bulb after olfactory stimulation was also shown to require an intact noradrenergic system.91 Furthermore, destruction of descending noradrenergic pathways from LC to the spinal cord reduces the behavioral response to formalin injection in the paw as well as formalin-induced Fos expression in the spinal cord.92 In addition, adrenergic receptor antagonists strongly reduce the induction of c-fos in the brain at birth.93 Finally, a recent study showed that both basal and visually evoked expression of c-fos was significantly reduced in the visual cortex of young rats in which noradrenergic fibers had been destroyed by DSP-4 treatment94. The significant effect of the noradrenergic system on cfos expression during waking appears to be specific. This is demonstrated by studies of the role of the serotoninegic system, which, like the noradrenergic system, has diffuse projections and fires at a high level during waking and at a low level during sleep.95 However, recent experimental results indicate that, in contrast to the effects of noradrenergic lesions, diffuse lesions of cortical serotoninergic fibers do not affect c-fos expression in the cerebral cortex during waking (Shaw PJ and Tononi G, in preparation). It remains to be determined whether other neuromodulatory systems with diffuse projections, such as the cholinergic and the histaminergic systems, are involved in c-fos expression during waking. DISSOCIATIONS BETWEEN WAKING AND C-FOS EXPRESSION As discussed above, in animals with lesions of the noradrenergic system there is a clear-cut dissociation between waking and the expression of c-fos and other IEGs. In these animals, waking episodes appear normal from a behavioral and EEG standpoint. However, spontaneous or forced waking for three hours is associated with c-fos expression levels that are much lower than those normally seen during waking and comparable to those seen during sleep.78 This demonstrates that waking as such is not necessarily associated with the induction of c-fos. Evidently, how and where c-fos expression is increased depends on additional factors, such as the level of noradrenergic activity. Additional evidence of dissociation between waking and c-fos expression comes from the study of the effects of psychostimulants. Amphetamine, methylphenidate, modafinil and caffeine all have arousing activity, but induce very different patterns of c-fos expression in the brain.48,49,96-98 Amphetamine strongly increases c-fos levels in most corti16 On the Functional Significance of c-fos--Cirelli et al

Figure 4--Fos positive cells in the parietal cortex after 8 h of sleep deprivation (A), 2 h of REM rebound (B), and 8 h of spontaneous sleep (C). Same rats as in Fig. 3. Scale bar = 100 µm.

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cal areas, caudate-putamen, accumbens nucleus, septum, and anterior preoptic nucleus. Scattered Fos positive neurons are also present in the superior and inferior colliculi and in the central gray. No Fos protein positive cells are present in the midline and intralaminar thalamic nuclei, nor in the dorsal raphe, parabrachial nucleus, and locus coeruleus. C-fos expression after methylphenidate administration is very similar to that observed after amphetamine treatment. Caffeine in low doses induces c-fos expression in a few cortical areas. After high doses of caffeine, c-fos expression increases in most cortical areas, caudate-putamen, accumbens nucleus, amygdala, MPA, paraventricular thalamic nucleus, substantia nigra pars reticulata, parabrachial nucleus, locus coeruleus, and nucleus of the solitary tract. By contrast, c-fos expression following modafinil administration is mainly localized in the anterior hypothalamic nucleus and adjacent areas, while it is very low in cortical and striatal regions. The marked differences in Fos expression induced by different arousal-promoting drugs may be explained by their different mechanisms of action. Amphetamine acts by releasing catecholamines, and its arousing effects can be almost completely prevented by inhibiting catecholamine synthesis99. Caffeine's arousal effect is mediated in part by an increase in catecholaminergic transmission, in particular noradrenergic and nigrostriatal dopaminergic transmission, as well as by a decrease in adenosinergic transmission.49 By contrast, the arousing effect of Modafinil is not prevented by inhibiting catecholamine synthesis and it may be mediated by indirect mechanisms, perhaps through decreased GABAergic transmission48 or through activation of hypocretin(orexin)-containing neurons in the lateral hypothalamus.100 Thus, the elevation of c-fos expression occurring after psychostimulants that increase catecholamine activity, such as amphetamine and caffeine, but not after modafinil, whose mechanism of action seems to be independent of the availability of endogenous catecholamines101 appears to be consistent with a crucial role for norepinephrine in IEGs induction during waking. INTERPRETING CHANGES IN C-FOS EXPRESSION ACROSS SLEEP AND WAKING IN FUNCTIONAL TERMS The discovery that the expression of IEGs is strongly modulated across behavioral states has added a new dimension to the study of sleep and waking. For example, the suppression of the need for sleep obtained through the injection of c-fos antisense oligonucleotides in the MPA indicates a causal link between the expression of c-fos and the homeostatic regulation of sleep. However, for most other brain areas, and especially for the cerebral cortex, the significance of such strong modulation in gene expression still eludes us. Several possibilities need to be considered.

C-fos as a Marker of Cellular Activation Since c-fos responds to so many different stimuli, and through so many different pathways, it has been widely used as an indirect but convenient marker of cellular activation.102-106 However, as mentioned before, it is still unclear what "cellular activation" means and what kind of physiological activity is signaled when Fos expression is induced. It is by now clear that the "activation" as indicated by c-fos does not always correspond to that shown by imaging techniques such as 2-deoxi-glucose (2-DG) and positron emission tomography.102,107,108 In some cases 2DG signal intensity increases while Fos does not or, conversely, some manipulations lead to increase in Fos expression without changes in glucose metabolism. For instance, Modafinil increases Fos expression but not glucose utilization in the anterior hypothalamus.97-98 Moreover, c-fos expression but not 2-DG signal intensity is increased in the paraventricular nucleus after water deprivation,102 or in the CA1 region of the hippocampus after ischemic damage.107 On the other hand, focal epilepsy induced by epicortical application of penicillin induces Fos expression only within the epileptic focus, while glucose metabolism is increased also in the homotopic contralateral cortex.109 Perhaps more intriguing is the fact that c-fos data often do not match well with single-unit recording data. For instance, many waking-on neurons in the basal forebrain and brainstem reticular formation, which fire at a higher rate in waking than in sleep, do not express Fos in spontaneous waking or after sleep deprivation.5,31,53 Many brain areas known to be important in waking, including thala-

Figure 5--Expression of c-fos after 3h of spontaneous waking following unilateral neurotoxic lesions of the locus coeruleus. A. Tyrosine hydroxylase immunocytochemistry of a rat with a lesion of the left locus coeruleus sacrificed after 3h of spontaneous waking during the dark hours. The panels show left and right parietal cortex, respectively. Scale bar: 200 µm. B. Expression of Fos protein. Photomicrographs from adjacent sections show that Fos staining after spontaneous waking is high on the intact side (right) and almost absent on the lesioned side (left). Modified from ref. 76. 17 On the Functional Significance of c-fos--Cirelli et al

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mus, basal forebrain, posterior hypothalamus and mesopontine tegmentum, do not express Fos during amphetamine-, methylphenidate-, or modafinil-induced wakefulness.48 During REM rebound after sleep deprivation, only a very small number of cholinergic cells in the dorsal pontine tegmentum express Fos.71 Also, Fos is not expressed in dorsal root ganglia or in the gracile nucleus after noxious stimulation,110 or in the lateral vestibular nucleus after labyrinthectomy.111 Electrical or chemical stimulation leads to an increase in the firing of most LC cells, but only a few noradrenergic neurons are Fos positive.112 Similarly, only a few cells in the retina express c-fos in response to flashing light, a stimulus known to produce an electrophysiological response in virtually all retinal neurons.113,114 In the suprachiasmatic nucleus, Fos expression is induced only upon photic stimulation during the dark phase but not during the light phase, even though retinal illumination during either phase results in a similar increase in firing.105 Moreover, Fos is expressed in only a few neurons of a cortical or subcortical tonotopic column after a tone stimulation that, according to electrophysiological criteria, activates the overwhelming majority of units in such a column.108 Furthermore, even after the strongest stimulation of whiskers only subsets of neurons express Fos in the corresponding barrel cortex.115 During kindled seizures there is a qualitative but not a quantitative correlation between the number of seizure-induced population action potentials and the magnitude of c-fos mRNA increase in the granule cells of the hippocampus, and pretreatment with NMDA antagonists reduce c-fos induction despite an increase in the number of granule cell population action potentials.116 Finally, it has been shown in the magnocellular neurons of the supraoptic nucleus that the induction of c-fos requires receptor activation and not simply spike activity, because the same number of action potentials is effective in increasing Fos expression if caused by carbachol injection but not if produced by antidromic activation.117 Altogether, these observations indicate that neuronal depolarization and an increase in firing rate per se are not enough to induce c-fos in many neurons. In some cases, cells expressing Fos may share a particular cellular phenotype. For instance, the majority of striatal neurons that express Fos after cocaine contain substance P.118 In other cases, however, Fos positive cells within a particular brain nucleus do not seem to have any common neuronal or glial phenotype. Light-induced Fos expression in the SCN is localized within the ventral subdivision of the nucleus, in a pattern similar to the site of termination of visual inputs. In this area ~10% of Fos positive cells are peptidergic,119 ~10% are astrocytes,120 and the remaining 80% do not belong to an identified cell type. It appears, therefore, that factors beyond a cell's metabolic rate, its level of depolarization, or its specific phenotype may determine the degree of c-fos expression in

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neural cells. Several recent studies in cell culture suggest that one such factor may be represented by a significant increase in intracellular Ca.++29 The role of Ca++ has been studied in great detail in relation to glutamatergic neurotransmission. As was mentioned above, glutamate can induce c-fos expression in vitro by acting through both NMDA and non-NMDA receptors. In both cases c-fos induction is Ca++ dependent,121 although the route of Ca++ entry and the intracellular signaling pathways engaged may differ significantly depending on the particular receptor subtypes and cell lines.122-125 Furthermore, depending of its route of entry, calcium can influence different transcriptional responses.29 Irrespective of the intracellular pathways involved, however, the general picture that is emerging is that the amount of glutamate receptor stimulation necessary to induce c-fos is substantial. In cultured hippocampal neurons, for instance, the exposure to a high (but not toxic) dose of NMDA for 30­45 seconds significantly increases intracellular Ca++ levels but not c-fos mRNA levels, and a near-maximal increase in c-fos transcription is only obtained after 10 min exposure.121 In cultured cortical neurons, strong synaptic activation through both NMDA and non-NMDA receptors is needed to activate voltagesensitive Ca++ channels and subsequently c-fos expression.126 In addition, it has recently been shown that in cultured striatal neurons the induction of c-fos requires the activation of voltage-sensitive Ca++ channels of L-type.125 L-type Ca++ channels are only activated by strong depolarization and do not inactivate, thus permitting massive Ca++ influx.125 Some recent evidence in vivo also indicates that the induction of c-fos requires a conspicuous and sustained increase in synaptic activity. For instance, in anesthetized rats, the electrical stimulation of the motor cortex induces Fos expression in striatal and nigral cells only if a minimal number of ~80,000 cortical shocks per stimulation session is delivered.127 In vivo, calcium entry through voltage-sensitive Ca++ channels has been shown to mediate the induction of c-fos caused by convulsant agents,e.g.,128 and angiotensin II.129 In view of the demonstration that the noradrenergic system plays a key role in the expression of Fos during normal waking, if would be important to know whether norepinephrine promotes calcium entry either directly or upon glutamatergic depolarization. Unfortunately, the evidence on this point is missing. It is known, however, that other neuromodulators or agents that act by increasing cAMP levels and PKA activity (as norepinephrine does) can influence the activity of Ca++ channels.130 For instance, in striatal neurons, the induction of cfos caused by stimulation of dopamine D1 receptors, which activate the cAMP pathway, is dependent on functional NMDA receptors and Ca.++131 In summary, no definitive conclusion can yet be drawn concerning the signals that are necessary and sufficient to

On the Functional Significance of c-fos--Cirelli et al

induce c-fos. The evidence shows that Fos induction is generally associated with membrane depolarization and increases in firing rate. However, membrane depolarization and increases in firing seem to be effective only when they are associated with a strong and persistent activation of neurotransmitter receptors (glutamatergic and others), leading to substantial changes in intracellular Ca++ levels. Whether and under what conditions a rise of Ca++ is sufficient to induce c-fos, remains to be determined. c-fos as a Marker of Genetic Activation The fact that c-fos is an IEG and encodes a transcription factor explains why, irrespective of the mechanisms of its activation, much interest has focused on the identification of its "target" genes. However, the identification of such genes has turned out to be technically very demanding. An important reason for such difficulties is that Fos does not regulate transcription by itself but must associate in dimers with a member of the jun family (either c-jun, junB, or junD) to form an AP-1 complex. The AP-1 complex can then influence both the basal and the enhanced transcription of the large number of genes that have an AP-1 consensus sequence (TGAC/GTCA) in their promoter. Members of the jun family are not always induced by the same conditions and to same extent as c-fos and therefore the composition of the dimer may vary. For example, jun D is not induced by most of the stimuli effective for c-fos and c-jun is only weakly induced by an intracellular increase in cAMP and does not respond to membrane depolarization.8 jun B, on the other hand, is induced by cAMP and membrane depolarization as strongly as c-fos.8 Accordingly, cjun mRNA levels do not change in relation to the light/dark cycle or in response to sleep deprivation,7,132 while jun B expression shows a spontaneous circadian oscillation in cerebral cortex and striatum (but not in other areas) with higher levels during the subjective night.33,132 jun B expression is also higher in waking than in sleep in the cerebral cortex33,133. Some data suggest that the dimers Fos:junB should be more abundant during waking, while c-Jun:c-Jun should prevail in sleep.33 The functional consequences of these interactions are still unclear, but they are clearly relevant. For instance, when junB extensively dimerizes with Fos, displaced c-jun molecules might form dimers with other transcription factors such as ATF-2 or CBP and consequently bind consensus sequences different from the AP1 site.8 Despite these difficulties, proenkephalin,134 nerve growth factor135 and tyrosine hydroxylase136 have been identified as target genes for c-fos. Other potential targets include the adipocyte P2 gene induced during adipocyte differentiation,137 the mouse a1(III) collagen gene,138 and the human collagenase gene induced during cell transformation.139 C-fos is also involved in the platelet-derived

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growth factor-mediated increase in the expression of transin, a matrix-degrading secreted metalloprotease,140 in prodynorphin expression after inflammation,141 in choline acetyltransferase expression in basal forebrain cholinergic neurons,142 and in the induction of the tissue inhibitor of metalloprotease 1143. C-fos also decreases the expression of cytoskeletal proteins such as ezrin and tropomyosin and promotes morphological transformation.144 Finally, c-fos is known to negatively regulate the human urokinase gene and the human NAD(P)H:quinone oxidoreductase1 gene, which encodes an enzyme that protects cells from redox cycling and oxidative stress. The significance of such negative regulation is unknown.145 Finally, it should be mentioned that systematic investigations of differences in gene expression between sleep and waking have indicated that only a very small fraction of the mRNAs expressed in cerebral cortical tissue is modulated by behavioral state.2 Many of these genes are IEGs (e.g., cfos, NGFI-A, NGFI-B, Arc, IER5) and others are genes encoded by the mitochondrial genome that are rapidly influenced by neural activity. At this time, none of the presumed targets of c-fos has been found to be modulated by sleep and waking. Whether some of the other genes induced by prolonged waking2-3 may be target of c-fos remains to be investigated. C-fos Expression, Plasticity, and Metabolic Stress While the cellular targets of c-fos and other IEGs are poorly understood, considerable interest has focused on the possible relationship between the expression of IEGs and the cellular correlates of learning and memory. Unfortunately, while many studies have found a correlation between the expression of c-fos and the induction of plastic changes necessary for learning and memory, it was often unclear whether the correlation was with learning per se or merely with the motor component of the task.146,147 In a few cases, however, a clear relation between c-fos expression and plasticity has been established. In a rat model in which motor activity and learning were effectively dissociated, Fos expression was shown to increase in the motor cortex in association with motor skill learning and synaptic growth. Fos upregulation was not maintained during practice once the skill was acquired.148 In mice, a strong Fos induction in several forebrain areas paralleled the ability to acquire a freezing response to novel conditioned stimuli. Preexposure to the stimulus resulted in reduced Fos production as well as impaired acquisition of conditioned fear.88 Inhibition of Fos synthesis using a c-fos antisense oligonucleotide blocked the acquisition and extinction of learned taste aversion,149,150 retention of a passive avoidance task151 and of a brightness discrimination task.152 Finally, c-fos expression in the medial prefrontal cortex of the rat has recently been linked to the acquisition of averOn the Functional Significance of c-fos--Cirelli et al

sive learning in a paradigm in which the role of learning could be dissociated from that of novelty and stress.153 The fact that c-fos expression is dependent on the noradrenergic system (see above) is also suggestive of a relation between the expression of this IEG during waking and the induction of plastic changes. The activation of the noradrenergic system is important for the initiation of plastic changes in the brain, including developmental plasticity, learning, and long-term potentiation.78,81 To mediate such changes norepinephrine acts through different mechanisms: 1) it promotes the responsiveness of neurons to extrinsic, salient stimuli by increasing their "signal-tonoise" ratio; 2) it increases the rate of metabolic processes; 3) it controls the biochemical pathways that lead to the phosphorylation of transcription factors such as CREB and to the induction of c-fos and other IEGs. During waking, norepinephrine is released diffusely all over the brain when novel salient events cause a burst of activity in LC. During sleep, when LC cells discharge at a low level or become silent, norepinephrine is not released and all these mechanisms, including the induction of IEGs, become ineffective. How would the expression of c-fos be related to the initiation of plastic phenomena? Learning is associated with neuronal morphological changes, and protein and RNA synthesis inhibitors can cause amnesia as well as prevent such structural changes. In principle, Fos could directly promote the transcription of proteins necessary for plastic changes, for instance, those used in the formation of new synapses. The established role of Fos in the expression of growth factors (NGF) and growth factors-induced proteases such as transin support this hypothesis, as well as its participation in the expression of collagen, collagenase, and tissue inhibitor of metalloprotease 1. Prodynorphin is also a target of Fos and a gene potentially involved in synaptic plasticity.154 A related possibility is to consider c-fos as a marker of "metabolic stress" rather than a direct marker of plasticity,155 although these two possibilities are not mutually exclusive. In particular, c-fos could play a role in cellular processes associated with basic cell functioning that are upregulated during periods of plastic change. As was mentioned above, c-fos has been implicated in the expression of two enzymes involved in the synthesis of neurotransmitters, tyrosine hydroxylase and choline acetyltransferase. Thus, c-fos induction could be instrumental to the restoration of neurotransmitters, enzymes, and synaptic vesicles that are utilized during the increased metabolic activity associated with learning. If the hypothesis of a connection between the induction of c-fos during waking and the initiation of plastic changes in the brain is correct, the low levels of c-fos expression during sleep would imply that such plastic phenomena occur less significantly, if at all, during this behavioral state. Indeed, several other genes, whose induction has

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been linked to the initiation of plastic phenomena, such as P-CREB, NGFI-A, BDNF, BiP, and Arc, are also expressed at higher levels in waking than in sleep.156 These molecular observations fit nicely with the fact that we cannot acquire new information during sleep.157,158 This appears to make functional sense, in that the brain should learn how to successfully adapt to the environment only when it is awake. As stated above, the inactivity during sleep of the noradrenergic system and possibly of other neuromodulatory systems with diffuse projections may be a major factor in preventing the induction of plasticity-related genes during either slow-wave sleep or REM sleep despite the continuing neuronal activity. On the other hand, if waking is not interrupted by periods of sleep, the induction of plastic changes may also be impaired. It was noted above that the levels of c-fos are remarkably low in the cerebral cortex and in many other subcortical areas during long-term sleep deprivation. This suggests that plasticity and learning, inasmuch as they require activation of c-fos and other IEGs, may be impaired after prolonged waking and may need intervening periods of sleep. Preliminary results indicate that several other plasticity-related genes, including NGFI-A, BiP and Arc, which are induced by short periods of waking, return to low levels after a few days of sleep deprivation.156 CONCLUSIONS Studies of c-fos expression in sleep and waking have provided a large amount of new information about the cellular substrates of behavioral state control. These studies have revealed remarkable differences between waking and sleep at the cellular level. Moreover, c-fos is proving extremely useful to map neural populations that control waking, NREM sleep, and REM sleep with cellular resolution and over the entire brain. As important as these new results are, they should be interpreted with some caution. C-fos may act at times as a master switch of cellular regulatory activities that is rapidly induced by stimuli at the cell membrane and converts them into long-term responses requiring gene activation, such as cell division, growth, and memory formation. However, the activation of c-fos cannot be considered as synonimous with the activation shown by imaging and/or by electrophysiological studies. While the induction of c-fos is usually associated with membrane depolarization and an increase in firing rate, and may require a massive entry of calcium, it is unknown whether these factors are by themselves sufficient to trigger it. It is also essential to remember, in interpreting patterns of c-fos expression in relation to behavioral state, that Fos protein, once induced, may persist for up to four to eight hours. Finally, c-fos is a transcription factor that regulates the expression of several late target genes, but only a few of them have been clearly identified so far, making it difficult to determine the functional consequences of c-fos inducOn the Functional Significance of c-fos--Cirelli et al

tion. Experiments performed in several species have consistently found that, during sleep, c-fos expression is very low in most brain regions except for a few scattered cells in the hypothalamus (especially in the VLPO), while during waking it is high in other hypothalamic areas (MPA), cerebral cortex, septum, thalamus, and brainstem. Fos positive cells are induced in the MPA during spontaneous waking, shortand long-term sleep deprivation, and pharmacologicallyinduced arousal. C-fos antisense oligonucleotides that block the accumulation of Fos protein in the MPA during prolonged waking prevent the subsequent homeostatic increase of sleep. Therefore, Fos positive (presumptive non-GABAergic) MPA cells may detect the build-up of sleep pressure during waking. On the other hand, VLPO cells express c-fos during physiological sleep, during slowwave sleep rebounds after sleep deprivation, and after some pharmacological treatments that induce sleep. Some Fos positive VLPO cells contain the synthetic enzyme for GABA and, through direct projections to histaminergic, noradrenergic, and serotoninergic neurons, may inhibit several components of the ascending arousal system. The MPA and the VLPO are thus potentially involved in complementary aspects of sleep regulation. In the cerebral cortex, the expression of c-fos is much higher in waking than in sleep and is largely controlled by the noradrenergic system. However, even during waking, only a subset of cortical neurons express c-fos, and the reason is unclear. One possibility is that cortical cells expressing Fos protein during waking may be those that undergo plastic changes initiated by massive calcium entry. The induction of Fos in these cells could be important for the occurrence of such plastic changes, for example by regulating the synthesis of neurotrophic and morphogenetic proteins such as NGF, collagen, collagenase, prodynorphin, and several others. A related possibility is that the induction of c-fos may be necessary for the restoration of neurotransmitters, enzymes, and synaptic vesicles that are utilized during the increased metabolic activity and cellular stress that are associated with wakefulness and specifically with learning. ACKNOWLEDGMENTS This work was carried out as part of the experimental neurobiology program at The Neurosciences Institute, which is supported by Neurosciences Research Foundation. The Foundation receives major support for this program from Novartis. C.C. is a Joseph Drown Foundation Fellow. REFERENCES

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