Paradoxical (REM) sleep genesis: The switch from anaminergic–cholinergic to a GABAergic–glutamatergic hypothesis

Pierre-Herve Luppi *, Damien Gervasoni, Laure Verret, Romain Goutagny, Christelle Peyron,Denise Salvert, Lucienne Leger, Patrice Fort

UMR5167 CNRS, Faculte de Medecine Laennec, Institut Federatif des Neurosciences de Lyon (IFR 19), Universite Claude Bernard Lyon I, 7,Rue Guillaume Paradin, 69372 Lyon cedex 08, France

Abstract

In the middle of the last century, Michel Jouvet discovered paradoxical sleep (PS), a sleep phase paradoxically characterized by corticalactivation and rapid eye movements and a muscle atonia. Soon after, he showed that it was still present in ‘‘pontine cats’’ in which all struc-tures rostral to the brainstem have been removed. Later on, it was demonstrated that the pontine peri-locus coeruleus a (peri-LCa in cats,corresponding to the sublaterodorsal nucleus, SLD, in rats) is responsible for PS onset. It was then proposed that the onset andmaintenanceof PS is due to a reciprocal inhibitory interaction between neurons presumably cholinergic specifically active during PS localized in thisregion andmonoaminergic neurons. In the last decade, we have tested this hypothesis with ourmodel of head-restrained rats and functionalneuroanatomical studies. Our results confirmed that the SLD in rats contains the neurons responsible for the onset and maintenance of PS.They further indicate that (1) these neurons are non-cholinergic possibly glutamatergic neurons, (2) they directly project to the glycinergicpremotoneurons localized in the medullary ventral gigantocellular reticular nucleus (GiV), (3) the main neurotransmitter responsible fortheir inhibition during waking (W) and slow wave sleep (SWS) is GABA rather than monoamines, (4) they are constantly and tonicallyexcited by glutamate and (5) theGABAergic neurons responsible for their tonic inhibition duringW and SWS are localized in the deepmes-encephalic reticular nucleus (DPMe). We also showed that the tonic inhibition of locus coeruleus (LC) noradrenergic and dorsal raphe(DRN) serotonergic neurons during sleep is due to a tonicGABAergic inhibition byneurons localized in the dorsal paragigantocellular retic-ular nucleus (DPGi) and the ventrolateral periaqueductal gray (vlPAG). We propose that these GABAergic neurons also inhibit the GAB-Aergic neurons of the DPMe at the onset and during PS and are therefore responsible for the onset and maintenance of PS.! 2007 Published by Elsevier Ltd.

Keywords: Brainstem; Hypothalamus; Acetylcholine; GABA; Glutamate

1. The identification of the brainstem executive structures

Back in 1959, Michel Jouvet and Francois Michel dis-covered in cats a phase of sleep characterized by a completedisappearance of the muscle tone, and paradoxically asso-ciated with a cortical activation and rapid eye movements(REM) (Jouvet and Michel, 1959). In view of its singular-ity, they proposed to call this state paradoxical sleep (PS).It corresponded to REM sleep, the state described in 1953by Aserinsky and Kleitman and that correlates with dreamactivity in humans (Aserinsky and Kleitman, 1953; Dementand Kleitman, 1957). The occurrence of muscle atoniabrought Jouvet to propose that PS was a distinct sleep state

0928-4257/$ - see front matter ! 2007 Published by Elsevier Ltd.doi:10.1016/j.jphysparis.2007.05.006

Abbreviations: 5HT, serotonin; CTb, cholera toxin B subunit; DPGi,dorsal paragigantocellular reticular nucleus; DPMe, deep mesencephalicreticular nucleus; DRN, dorsal raphe nucleus; eVLPO, extended ventro-lateral preoptic nucleus; GABA, gamma-aminobutyric acid; GAD, glu-tamate decarboxylase; GiV, ventral gigantocellular reticular nucleus; Hcrt,hypocretin (orexin); LC, locus coeruleus; LDT, laterodorsal tegmentalnucleus; LPGi, lateral paragigantocellular reticular nucleus; MCH, mel-anin concentrating hormone; NA, noradrenaline; Pc, parvocellular retic-ular nucleus; peri-LCa, peri-locus coeruleus alpha nucleus; PH, posteriorhypothalamus; PPT, pedunculopontine nucleus; PnO, pontis oralis nucl-eus; PnC, pontis caudalis nucleus; PS, paradoxical sleep; SLD, sublate-rodorsal nucleus; SWS, slow wave sleep; vlPAG, ventrolateralperiaqueductal gray; W, waking.* Corresponding author. Tel.: +33 4 78 77 10 40; fax: +33 4 78 77 10 22.E-mail address: luppi@sommeil.univ-lyon1.fr (P.-H. Luppi).

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Journal of Physiology - Paris 100 (2007) 271–283

and a true vigilance state independent of slow wave sleep(SWS) and waking (W). Over the next 40 years followingits discovery, Jouvet and co-workers pursued the study ofPS. Supporting the theory of the duality of sleep, theyshowed that PS was present in mammals and birds butabsent in amphibians and reptiles in contrast to SWS. Jou-vet also demonstrated that PS onset and maintenancedepended upon structures di!erent from those regulatingSWS and W. He first showed that PS persists after decor-tication, after cerebellar ablation or transections of thebrainstem rostral to the pons. In contrast, transections atthe posterior limit of the pons suppressed PS (Jouvet,1962a). He also demonstrated that a state resembling PSis still visible in the ‘‘pontine cat’’, a preparation in whichall the structures rostral to the pons are removed (Jouvet,1962a). These results indicated that brainstem structuresare necessary and su"cient to cause and maintain the stateof PS, a concept that is still valid today. Subsequently, Jou-vet and others showed that electrolytic and chemicallesions of the dorsal part of the pontis oralis (PnO) andcaudalis (PnC) nuclei specifically suppress PS (Carli andZanchetti, 1965; Jouvet, 1962b; Jouvet and Delorme,1965; Sastre et al., 1981; Webster and Jones, 1988) indicat-ing that these nuclei contain the neurons responsible for PSonset and maintenance.

Jouvet (1962a) was also the first to demonstrate thatcholinergic mechanisms play a major role in PS generationsince peripheral atropine administration suppressed PS,whereas anticholinesterase compounds increased PS. Then,George et al. (1964) discovered that bilateral injections ofcarbachol, a cholinergic agonist, into the PnO and PnCpromote PS. It was later shown that PS is induced withthe shortest latency when carbachol is unilaterally injectedin a small area of the dorsal PnO and PnC (Baghdoyan,1997; Garzon et al., 1998; Lai and Siegel, 1990; Vanni-Mer-cier et al., 1989; Yamamoto et al., 1990), named peri-locuscoeruleus a (peri-LCa) (Sakai et al., 1981, 1979b) and cor-responding to the sublaterodorsal nucleus (SLD) definedby Swanson (1998) in rats (Fig. 1). It also approximatelycorresponds to the dorsal subcoeruleus nucleus defined inrats by Paxinos and Watson (1997). Sakai and co-workersfrom Jouvet’s laboratory (Sakai, 1985; Sakai et al., 1981,2001; Sakai and Koyama, 1996) found that the greatmajority of the pontine neurons with a tonic activity occur-ring specifically during PS (PS-on neurons) were located inthe peri-LCa. They divided these neurons in two popula-tions (Sakai and Koyama, 1996). A first population con-sisted of neurons (i) located in the dorsal and rostralperi-LCa, (ii) inhibited by carbachol, a cholinergic agonistand (iii) projecting rostrally to the intralaminar thalamic

MCHGABA

GivGlycine

Spinalmotoneurons

Thalamus

LCNA

DPMeGABA

SLDGlutamate

DPGi, LPGiGABA

DRN5HT

Hcrt

Inhibitory pathways

Excitatory pathways

vlPAGGABA

Fig. 1. Model of the network responsible for PS onset and maintenance. The onset and maintenance of PS would result from the activation of PS-onglutamatergic neurons from the SLD. PS-on neurons from the SLD would induce muscle atonia via direct projections to glycinergic premotoneuronslocalised in the GiV. They would also induce EEG activation via direct thalamic projections. The activation of SLD PS-on neurons would be due to theremoval of tonic inhibitions arising mainly from GABAergic PS-o! neurons localized in the DPMe and to a minor extent noradrenergic (LC) andserotonergic (DRN) PS-o! neurons. The cessation of activity of the PS-o! neurons at the onset and during PS would be due to a tonic inhibition issuedfrom GABAergic PS-on neurons localized in the DPGi and the vlPAG. Hypothalamic MCH and Hcrt neurons would participate in the regulation of PSvia direct inhibitory and excitatory projections to the DPMe PS-o! GABAergic neurons and reciprocal inhibitory interactions. Yellow: PS-on structures,Grey: PS-o! structures, Green: target structures.

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nuclei of the thalamus, the posterior hypothalamus and thebasal forebrain (Fig. 1). A second population was formedby PS-on neurons (1) excited by carbachol, (2) distributedin all parts of the peri-LCa and (3) projecting caudally tothe ventral gigantocellular nucleus (GiV) also named mag-nocellular reticular nucleus (Mc) localized in the ventrome-dial medullary reticular formation (Fig. 1) (Sakai et al.,1981, 1979b). Based on these and other results, it was pro-posed that neurons of the first type are responsible for thecortical activation during PS, whereas neurons of the sec-ond type caused the muscle atonia via descending excit-atory projections to glycinergic premotoneurons withinthe GiV (Fort et al., 1990, 1993; Jones, 1991b; Luppiet al., 1988; Sakai et al., 2001; Sakai and Koyama, 1996).In addition, the GiV contains spinal-projecting PS-on neu-rons (Sakai et al., 1979a; Siegel et al., 1979) and its cyto-toxic lesion induces a decrease in PS amounts and anincrease in muscle tone during PS (Holmes and Jones,1994). Further, intracellular recordings of motoneuronscombined with strychnine applications demonstrated thatglycine is responsible for the tonic hyperpolarization ofthe spinal, hypoglossal and trigeminal motoneurons (Chaseet al., 1989; Kohlmeier et al., 1996; Soja et al., 1991;Yamuy et al., 1999) and we have shown that the GiV con-tains a large contingent of glycinergic neurons (Fort et al.,1990, 1993; Rampon et al., 1996a). These glycinergic neu-rons project directly to spinal motoneurons (Holstege andBongers, 1991) while those of the parvocellular and parvo-cellular alpha nuclei directly project to the trigeminalmotor nucleus (Li et al., 1996; Rampon et al., 1996b).The release of both glycine and GABA increases in thespinal cord and the hypoglossal motor nucleus during theatonia produced by a cholinergic stimulation of the peri-LCa (Kodama et al., 2003). In addition, we recentlyshowed that glycinergic neurons from these nuclei expressFos after a pharmacological induction of PS (Boissardet al., 2002; Morales et al., 2006). Moreover, followingthe induction of PS by carbachol injections in the peri-LCa, Fos-labelled cells were found in the GiV, and wereshown to project to the trigeminal motor nucleus (Moraleset al., 1999). At variance with all these data, it was recentlyshown in rats that lesion with the cell-specific toxin orexinB-saporin of the ventromedial medulla does not reduce theatonia of PS. The authors further demonstrated that 10%of the neurons of the SLD projecting to the spinal cordare Fos-positive after PS increases (Lu et al., 2006). Fur-ther, they reported that anterogradely labelled terminalsfrom SLD neurons surround spinal glycinergic premoto-neurons. However, since the authors only recorded theneck muscles, their results cannot be extended to othergroups of muscles in particular the cranial ones since gly-cinergic neurons innervating cranial motoneurons arelocalized in the GiV and the parvocellular reticular nucleus(Pc). Second, they do not exclude the possibility that boththe GiV and the spinal glycinergic premotoneurons are co-responsible of the atonia of the neck muscles during PS.Complete lesions of the glycinergic neurons from the GiV

and those of the spinal cord are necessary to resolve thisissue.

2. The reciprocal aminergic–cholinergic interaction model

Hobson et al. (1975) were the first to propose that theonset of PS was due to a reciprocal inhibitory interactionbetween cholinergic PS-on neurons and monoaminergicPS-o! neurons. They were soon followed by Sakai et al.(1981) who proposed a slightly revised model. This well-accepted hypothesis was formulated following the findingsthat serotonergic neurons from the dorsal raphe nucleus(DRN) and noradrenergic neurons from the locus coeru-leus (LC) cease firing specifically during PS, i.e. have a mir-ror activity to PS-on neurons (Fig. 1) (Aghajanian andVanderMaelen, 1982; Aston-Jones and Bloom, 1981; Hob-son et al., 1975; McGinty and Harper, 1976). Supportingthis theory, drugs enhancing serotonergic and noradrener-gic transmission, in particular monoamine oxidase inhibi-tors and serotonin/norepinephrine reuptake blockers,specifically suppress PS (Gervasoni et al., 2002; Jones,1991b; Jouvet, 1969). However, the sites where themonoamines and particularly serotonin exert their PS-sup-pressant e!ect remain unclear. Indeed, injections of norepi-nephrine, epinephrine or benoxathian (an a2 adrenergicagonist) into the peri-LCa inhibit PS, but injections ofserotonin have no e!ect (Crochet and Sakai, 1999a,b;Tononi et al., 1991). Besides, norepinephrine inhibits thenon-cholinergic PS-on neurons via a2-adrenoceptor, andhas no e!ect on the presumed cholinergic PS-on neuronsfrom the peri-LCa, while serotonin has no e!ect on bothtypes of neurons (Sakai and Koyama, 1996). Monoaminescould also act on PS-on neurons localized in other struc-tures than the peri-LCa, like the GiV (Luppi et al., 1988)or the pedunculopontine tegmental (PPT) and laterodorsaltegmental cholinergic nuclei (LDT) (not shown in Fig. 1)(Horner and Kubin, 1999). Indeed, the PPT and LDT con-tain PS-on neurons although the great majority of the neu-rons in these nuclei are tonically active during both W andPS (Datta and Hobson, 1994; Datta and Siwek, 2002;Datta et al., 2001; Kayama et al., 1992). In support of thishypothesis, infusion of fluoxetine in the LDT or the medialpontine reticular formation decreased PS, an e!ect blockedby pre-treatment with a selective 5-HT1A receptor antago-nist (Monti and Jantos, 2005).

In conclusion, a large number of data supported thehypothesis that the onset and maintenance of PS is dueto reciprocal inhibitory interactions between cholinergicPS-on neurons and PS-o! monoaminergic neurons. How-ever, it is important to stress here that the results stronglysupporting the respective inhibitory and stimulatory rolesof the monoamines and acetylcholine in PS genesis wereobtained using local application of agonists. Indeed, weare still lacking the demonstration that local applicationsof monoamines and acetylcholine antagonists influencePS genesis and this would be the only way to unambigu-ously demonstrate the role of the endogenous monoamines

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and acetylcholine in the genesis of PS. Further, since themonoaminergic–cholinergic hypothesis, a large number ofnew results strongly suggested that the PS-on neurons fromthe SLD are glutamatergic instead of cholinergic. In addi-tion, we have shown in rats that the removal of a tonicGABAergic inhibition was a more crucial event for the acti-vation of PS-on neurons from the SLD that the removal ofthe monoaminergic inhibition. These results were obtainedby combining single unit recordings, local pharmacology bymicroiontophoresis in the unanesthetized head-restrainedrat, and anterograde and retrograde tracing combined withFos and neurochemical identification of labelled-cells (Bois-sard et al., 2002, 2003; Darracq et al., 1996; Gervasoni et al.,1998, 2000; Verret et al., 2005, 2006). In the next chapters,we present these results and propose a new theory on theneuronal network responsible for PS.

3. Evidence that the neurons responsible for PS genesis areglutamatergic instead of cholinergic

We first demonstrated in cats that the great majority ofthe neurons in the peri-LCa projecting to the GiV are notcholinergic (Luppi et al., 1988). We more recently showedthat carbachol iontophoresis into the SLD, the rat equiva-lent of the peri-LCa, induces a W state with increased mus-cle activity and has no e!ect on SLD PS-on neurons(Boissard et al., 2002). These results indicate important dif-ferences between rats and cats in the pharmacological sen-sitivity of the SLD PS-on neurons. In agreement with ourresults, carbachol injections into the di!erent subdivisionsof the rat pontine tegmentum only slightly enhanced PS(Bourgin et al., 1995; Gnadt and Pegram, 1986; Shiromaniand Fishbein, 1986; Velazquez-Moctezuma et al., 1989) orhave no reliable e!ect (Deurveilher et al., 1997). Further,although an increase in the number of PPT and LDT cho-linergic neurons containing Fos has been observed after PSrecovery following PS deprivation (Maloney et al., 1999),we observed only occasional cholinergic neurons contain-ing Fos in these nuclei and the SLD in our recent studyusing the same paradigm (Verret et al., 2005). In addition,neurotoxic lesions of the PPT and LDT in rats induce nochange in PS quantities (Lu et al., 2006). Finally, M2 andM4 muscarinic receptors knockout mice displayed nochange in PS while M3 knockout mice only showed a smalldecrease in PS quantities (Goutagny et al., 2005a). In addi-tion, a number of results suggest that SLD PS-executiveneurones are glutamatergic. Indeed, neurons containingmRNA for vesicular glutamate transporter 2 (VGLUT2)have been described in the SLD (Lu et al., 2006). It wasalso previously shown that glutamate release in the GiVincreases specifically during PS (Kodama et al., 1998) andinjection of non-NMDA glutamate agonists in the GiVsuppresses muscle tone (Lai and Siegel, 1991). Altogether,these results clearly indicate that the SLD PS-executiveneurons are not cholinergic and apparently not cholinocep-tive. They further suggest that they are glutamatergicalthough a direct demonstration is still lacking.

4. Demonstration that GABAergic neurons localized in thedeep mesencephalic reticular nucleus (DPMe) gate the onsetof PS

We reported that a long-lasting PS-like hypersomnia canbe induced with a short latency in the head-restrained ratby microiontophoretic applications of bicuculline or gab-azine, both GABAA antagonists, specifically within theSLD (Boissard et al., 2002). We also recorded in the SLDneurons that are specifically active during PS and excitedby bicuculline or gabazine iontophoresis (Boissard et al.,2000). Studies in freely moving rats have reproduced ourresults (Pollock and Mistlberger, 2003; Sanford et al.,2003). In cats, pressure injection of bicuculline and to a les-ser extent of phaclofen (a GABAB antagonist) in the peri-LCa induces a strong increase in PS quantities with shortlatencies. In contrast, the application of muscimol (aGABAA agonist) or baclofen (a GABAB agonist) inducedW (Xi et al., 1999, 2001). Furthermore, the microinjectionof scopolamine (a muscarinic receptor antagonist) did notblock the induction of PS by bicuculline (Xi et al., 2004),indicating that acetylcholine is not involved in the e!ectof bicuculline. Altogether, these data indicate that theremoval of a tonic GABAergic input present on SLD PS-on neurons during both W and SWS is the key event forthe onset of PS.

By combining retrograde tracing with cholera toxin Bsubunit (CTb) and glutamic acid decarboxylase (GAD)immunostaining, we recently showed that the GABAergicinnervation of SLD neurons arises from both interneuronsand distant neurons located in the pontine and deep mes-encephalic reticular nuclei and to a minor extent fromhypothalamic and medullary structures (Boissard et al.,2003). Besides, Xi et al. (1999) found in cats that admin-istration of antisense oligonucleotides against GADmRNA in the peri-LCa, produces a significant decreasein W and an increase in PS. On the other hand, Maloneyet al. (2000) in rats found that the number of Fos express-ing GABAergic neurons in the PnO decreased followingPS rebound, suggesting that GABAergic neurons fromthis structure are active during W and SWS and inactiveduring PS. Finally, it has been shown in cats (Sastreet al., 1996, 2000), rats (Boissard et al., 2000) and guineapigs (Vanini et al., 2007) that muscimol injections in themost ventrolateral part of the periaqueductal gray(vlPAG) and in the region of the deep mesencephalic retic-ular nucleus just ventral to it (DPMe) induce a strongincrease in PS quantities (Fig. 1). It was also reported incats that muscimol applications limited to the DPMeinduced an increase in PS quantities while those in thevlPAG had no e!ect (Crochet et al., 2006). Finally, elec-trolytic lesions in cats of the DPMe (Petitjean et al.,1975) and chemical lesions in rats of the vlPAG and theDPMe (designed in their paper as lateral pontine tegmen-tum, LPT) were also followed by an increase in PS quan-tities (Lu et al., 2006). In our study, we reported a strongbut non-GABAergic projection to the SLD from the

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vlPAG and a mixed GABAergic and non-GABAergicprojection from the DPMe (Boissard et al., 2003). Fromall these results, we propose that GABAergic neuronslocated in the DPMe project to and directly inhibit thePS-executive neurons from the SLD specifically duringW and SWS. The non-GABAergic projection from thevlPAG could be glutamatergic and responsible for thetonic glutamatergic input present during all vigilancestates on the SLD-executive neurons (see below) (Boissardet al., 2002).

5. Evidence that SLD neurons responsible for PS onset andmaintenance are tonically excited by glutamate

We recently showed that the iontophoretic injection ofkainic acid (a glutamate agonist) in the SLD excites thePS-on neurons and induces a PS-like state (Boissardet al., 2002). Further, the PS-like state induced by bicucul-line iontophoresis in the SLD was reversed by the applica-tion of kynurenate, a wide spectrum antagonist ofexcitatory amino acids (Boissard et al., 2002). In agreementwith these results, the microdialysis injection of kainic acidin the cat peri-LCa induces a PS-like state (Onoe andSakai, 1995). Altogether, these results suggest that PS-onneurons in the SLD receive a tonic glutamatergic inputduring all sleep–waking states. They further suggest thatfollowing the removal of the tonic GABAergic input atthe onset of PS, an unmasked glutamatergic input isresponsible for the tonic activity of the SLD PS-on neuronsthroughout PS. The glutamatergic neurons providing thisconstant excitatory input to SLD PS-on neurons shouldbe located in the brainstem, although forebrain glutamater-gic neurons could also participate. Indeed, as shown by thepersistence of a PS–non-PS cycle in the ‘‘pontine cat’’, thestructures responsible for the genesis of PS are restricted tothe brainstem (Jouvet, 1962a). Accordingly, such glutama-tergic inputs can arise from the numerous non-GABAergicneurons projecting to the SLD and localized in the vlPAG,the mesencephalic, pontine and parvocellular reticularnuclei (not illustrated).

Inputs to the SLD originating in the primary motor areaof the frontal cortex, the bed nucleus of the stria terminalis,and the central nucleus of the amygdala might also partic-ipate in the activation of the SLD PS-on neurons. Indeed,corticofugal pyramidal cells are glutamatergic. UsingfMRI, Maquet et al. (1996) found in humans that theregional blood flow in the amygdaloid complex is positivelycorrelated with PS. Furthermore, the electrical stimulationin rats of the central nucleus of the amygdala increases thefrequency of pontine waves recorded in or just above theSLD during PS (Deboer et al., 1998). From these results,we hypothesized that the frontal cortex and the centralnucleus of the amygdala and functionally-related bednucleus of the stria terminalis provide an excitatory gluta-matergic projections to PS-on neurons from the SLD.Functional neuroanatomical studies are now needed to

directly identify the glutamatergic neurons projecting tothe SLD constantly activating the PS-on neurons.

6. GABAergic neurons responsible for the inactivation ofmonoaminergic neurons during PS

According to the classical ‘‘reciprocal interaction’’model (McCarley and Hobson, 1975; Sakai et al., 1981),the cessation of firing of the noradrenergic and serotoner-gic neurons at the onset of PS is the result of active PS-spe-cific inhibitory processes originating from PS-on cells.These neurons were first hypothesized to be cholinergicand localized in the peri-LCa, LDT and PPT. However,acetylcholine excites LC noradrenergic neurons and is onlyweakly inhibitory on serotonergic DRN neurons (Guyenetand Aghajanian, 1979; Koyama and Kayama, 1993). It wastherefore suggested that they might use GABA or glycineinstead as an inhibitory neurotransmitter (Jones, 1991a;Luppi et al., 1991). To test this hypothesis, we investigatedthe e!ect of bicuculline or gabazine and strychnine (a gly-cine antagonist) on the activity of LC noradrenergic andDRN serotonergic cells. The antagonists were applied bymicroiontophoresis during W, SWS and PS on monoamin-ergic neurons recorded in the head-restrained unanesthe-tized rat (Darracq et al., 1996; Gervasoni et al., 1998,2000).

Iontophoretic applications of bicuculline, gabazine orstrychnine during SWS or PS restored a tonic firing inLC noradrenergic and DRN serotonergic neurons (Dar-racq et al., 1996; Gervasoni et al., 1998, 2000). Applicationof these antagonists during W induced a sustained increasein discharge rate. These experiments revealed the existenceof tonic GABA and glycinergic inputs to the LC and DRNthat are e!ective during all vigilance states. Importantly,we found that when the e!ect of strychnine occurred duringtransitions between PS and W, the discharge rate of the LCor DRN neurons further increased at the onset of W. Incontrast, during the e!ect of bicuculline, the discharge rateof a LC or DRN neuron did not change at the transitionbetween PS and W. These results strongly suggested thatthe release of GABA but not that of glycine is responsiblefor the inactivation of LC noradrenergic neurons andDRN serotonergic during PS. At variance with our results,Levine and Jacobs (1992) have found in cats that the ion-tophoretic application of bicuculline reversed the typicalsuppression of neuronal activity of DRN serotonergic neu-rons during SWS but not during PS. In addition, Sakai andCrochet (2000) found that the microdialysis infusion ofbicuculline in cats had no e!ect on DRN serotonergic neu-rons during PS and suspected a non-specific excitatorye!ect of bicuculline in our experiments. This latter proposalappeared unlikely since we reproduced the e!ect of bicucul-line with gabazine, another specific GABAA antagonist(unpublished results). Our results are further supportedby the microdialysis studies of Nitz and Siegel (1997a,b),showing in cats a significant increase in GABA release inthe DRN and LC during PS as compared to W and SWS

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and, in contrast, no detectable changes in glycine concen-trations. Based on these and our results, we suggest thatduring W, the LC and DRN cells are under a tonic GAB-Aergic inhibition that increases during SWS and even fur-ther, during PS, and that such increase in GABAergicinhibition is responsible for the inactivation of these neu-rons during the sleep states. In contrast, the glycinergictonic inhibition would be constant across the sleep–wakingcycle and thus control the general excitability of LC andDRN neurons.

Double-staining experiments combining GAD and CTbimmunostaining further showed that the LC and DRNreceive GABAergic inputs from neurons located in a largenumber of distant regions from the forebrain to themedulla (Gervasoni et al., 2000; Luppi et al., 1999). Indeed,we observed a substantial number of GAD-immunoreac-tive neurons in the preoptic area, the lateral hypothalamicarea, the vlPAG and the DPGi that project to the LC andDRN (Gervasoni et al., 2000; Luppi et al., 1999). Based onphysiological and electrophysiological data (see above), weexpect that one or several of these GABAergic a!erents are‘‘switched on’’ specifically at the onset of and during PSepisodes and are responsible for the inhibition of brainstemmonoaminergic neurons during PS. It was recently pro-posed that GABAergic neurons located in the extendedventrolateral preoptic nucleus (eVLPO) might be responsi-ble (Lu et al., 2002). Indeed, a small number of the Fos-labelled neurons found in this nucleus after PS increasesproject to the LC and DRN (Lu et al., 2002). However,although we also observed in our recent study Fos-labelledneurons in the eVLPO after PS rebound, these neuronswere only occasionally retrogradely labelled from the LC(Verret et al., 2006). In addition, PS-like episodes still occurin pontine or decerebrate cats (Jouvet, 1972). Moreover, PSepisodes induced by carbachol injections in the pons ofdecerebrate animals are still associated with a cessationof activity of serotonergic neurons of the raphe obscurusand pallidus nuclei (Woch et al., 1996). Therefore, it is verylikely that the vlPAG and/or the DPGi contain the GAB-Aergic neurons responsible for the inhibition of monoam-inergic neurons during PS (Gervasoni et al., 2000; Luppiet al., 1999). Supporting this hypothesis, local applicationsof bicuculline block the DPGi-evoked inhibition of LCneurons (Ennis and Aston-Jones, 1989) and focal ionto-phoretic applications of NMDA in the vlPAG induce bicu-culline sensitive IPSPs in DRN serotonergic neurons (Liuet al., 2000). Further, Yamuy et al. (1995) showed that aftera long period of PS induced by a pontine injection of car-bachol, a large number of Fos-positive cells are visible inthe DRN and the vlPAG. Moreover, Maloney et al.(1999) observed in the periaqueductal gray an increase inFos-positive GAD immunoreactive neurons after a PSrebound induced by deprivation. To directly determineamong the GABAergic a!erents to the LC, those activeduring PS, we recently combined iontophoretic applicationof CTb in the LC with Fos staining in rats deprived of PS,rats with enhanced recovery PS after selective deprivation,

and control rats. Using this method, we observed a largenumber of CTb and Fos double-immunostained neuronsin the DPGi and a substantial number in the vlPAG andthe lateral paragigantocellular reticular nucleus (LPGi)specifically after PS rebound (Fig. 1) (Verret et al., 2006).From these results, we propose that the GABAergic neu-rons responsible for the inhibition of the LC noradrenergicneurons during PS are mainly, but not exclusively, locatedin the DPGi. In order to further test this hypothesis, werecorded the spontaneous activity of neurons from theDPGi across the sleep–waking cycle in head-restrainedrats. Neurons with an activity specific to PS (PS-on neu-rons) were found within this nucleus (Goutagny et al.,2005b), supporting the idea that it contains the GABAergicneurons responsible for the cessation of activity of the nor-adrenergic neurons of the LC during PS. This hypothesis isalso supported by a recent study showing that electricalstimulation of the area of the DPGi induces an increasein PS quantities (Kaur et al., 2001). It is however likely thatthe GABAergic neurons inhibiting the serotonergic neu-rons of the DRN are mainly localized in the vlPAG ratherthan the DPGi since the latter structure provides only aweak GABAergic projection to the DRN (Gervasoniet al., 2000). In conclusion, our results indicate that GAB-Aergic neurons from the vlPAG and the DPGi tonicallyinhibit the monoaminergic neurons during PS. We recentlyproposed that these GABAergic neurons could also inhibitduring PS the PS-o! GABAergic neurons from the DPMe(Luppi et al., 2004). If this hypothesis is correct, the onsetof firing of DPGi and vlPAG PS-on neurons constitute thetriggering event in the induction of PS. The mechanismsresponsible for the activation of these neurons remain tobe identified. It can nevertheless be proposed that it canbe due to their intrinsic properties and/or inputs fromstructures regulating the homeostasis of PS like the poster-ior hypothalamus (see below). At variance with our theory,it was recently shown that a large proportion of the Fos-labelled neurons localized in the SLD after PS increasesare GABAergic neurons projecting to the GABAergicPS-o! neurons from the DPMe. These results indicate thatthe SLD contain both glutamatergic and GABAergic PS-on neurons. From these results, it was proposed that thegenesis of PS is due to a reciprocal inhibitory interactionbetween GABAergic DPMe and SLD neurons (Lu et al.,2006). However, the authors did not report in their studywhether the DPGi, the vlPAG or other brainstem struc-tures than the SLD also contain GABAergic PS-on neu-rons projecting to the DPMe. Experiments are inprogress in the laboratory to determine which one of thetwo hypotheses is correct.

7. Beyond the glutamatergic–GABAergic neuronal network

As described above, great progresses were made in therecent years to identify the neuronal network responsiblefor PS onset and maintenance. There are however a num-ber of questions remaining for the future: (1) by which

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mechanisms and pathways the brain switches from SWS toPS and (2) what are the mechanisms responsible for thehomeostasis of PS, i.e. the occurrence of a recovery of PSquantities after deprivation?

We indeed need to determine the mechanisms responsi-ble for the ‘‘switch on’’ of the PS-on structures in the mid-dle of a SWS phase and their ‘‘switch o!’’ after a periodwith a length quite stable for a given species but highly var-iable across species depending on their metabolism rate.Indeed, bigger the animals are, longer their episodes ofPS are (Siegel, 2005). Another important underestimatedcharacteristic of the episodes of PS is that they start aftera quite long period of an intermediate state during whichthe EEG display a mix of spindles and theta whereas theyterminate abruptly by a short microarousal (Gervasoniet al., 2004). These characteristics suggest that the mecha-nisms allowing to enter or exit PS episodes might be di!er-ent. Additional studies are needed to explore suchhypothesis. It would be also necessary to determine therelationship between the GABAergic neurons responsiblefor the onset of SWS localized in the VLPO and the net-work responsible for PS.

Concerning the homeostasis of PS, it has been shownthat PS recovery following PS deprivation was abolishedin ‘‘pontine cats’’ (Jouvet, 1988). These results suggest thatthe brainstem contains the structures responsible for PSbut not those responsible for its homeostatic regulation.

Our recent studies comparing the distribution of Fos-labelled neurons in the brainstem of control rats, of ratsselectively deprived of PS for approximately 72 h and ofrats allowed to recover from such deprivation gave someclues on the localization and nature of the neurons respon-sible for the homeostasis of PS (Verret et al., 2003, 2005,2006). Further, they revealed that other structures thanthose identified previously contain neurons strongly activeduring PS indicating that the neuronal network responsiblefor its genesis was perhaps not yet completely identified.Indeed, if a large number of Fos-labelled cells, positivelycorrelated with the percentage of time spent in PS, wasobserved in the structures described above as beinginvolved in PS genesis such as the LDT, SLD and GiV, alarge number of Fos-labelled cells was also seen in theeVLPO, lateral and posterior hypothalamus, dorsal peri-aqueductal gray, LPGi and the nucleus raphe obscurus.Interestingly, half of the cells in the latter nucleus wereimmunoreactive to choline acetyltransferase (Verret et al.,2005). These findings demonstrate that many structuresnot previously identified contain neurons active duringPS that might therefore play a key role during this state.A number of results exposed in detail below indicate thatamong them, the posterior hypothalamus could be respon-sible for PS homeostasis.

7.1. The posterior hypothalamus

A large body of data indicates that the posteriorhypothalamus (PH) plays a crucial role in vigilance states

regulation. First, von Economo reported that posteriorhypothalamic and midbrain junction lesions result in sleep-iness based on observations of patients from the encephali-tis lethargica epidemic during World War 1 (VonEconomo,1926). In subsequent years, hypersomnia was reported inmonkeys (Ranson, 1939), rats (Nauta, 1946) and cats (Swettand Hobson, 1968) following electrolytic lesions of the PH.Transient hypersomnia was also observed after lesion withibotenic acid (Sallanon et al., 1988; Swett and Hobson,1968) or inactivation by local application of muscimol, aGABAA agonist (Lin et al., 1989; Sallanon et al., 1989).Further, it was shown that the waking e!ect of modafiniland amphetamine is suppressed by the injection ofmuscimol in the cat PH (Lin et al., 1992, 1996). Besides,Vanni-Mercier et al. (1984) recorded neurons active onlyor mainly during waking in the PH of cat by extracellularrecordings.

Reviewing carefully the literature, we also found evi-dence that the PH plays a role in PS regulation. First,despite the fact that a PS-like state still occurred in ‘‘pon-tine cats’’, indicating that the brainstem as su"cient to pro-duce PS, PS recovery following PS deprivation wasabolished in these animals (Jouvet, 1988). These resultssuggest that the brainstem contain the structures responsi-ble for PS but not those responsible for its homeostatic reg-ulation. Besides, neurons specifically active during PS wererecorded in the PH (Alam et al., 2002; Koyama et al., 2003;Steininger et al., 1999). Further, PS quantities weredecreased after muscimol injection in the PH (Lin et al.,1989; Sallanon et al., 1989). Recent data summarized belowconfirmed that the PH contains populations of neuronsinvolved in PS control.

7.2. The hypocretin (orexin) neuronal population

Narcolepsy, a sleep disorder characterized by excessivedaytime sleepiness and cataplexy, may be caused by thelack of hypocretin (Hcrt) mRNA and peptides in Humans(Peyron et al., 2000) or a disruption of the Hcrt receptor 2or its ligands in dogs and mice (Chemelli et al., 1999; Linet al., 1999). Hcrt-containing neurons are located exclu-sively in the dorsomedial, lateral and perifornical hypo-thalamic areas (Peyron et al., 1998). Hypocretins aretwo peptides, Hcrt-1 (orexin-A) and Hcrt-2 (orexin-B),generated from a single preprohypocretin (de Leceaet al., 1998; Peyron et al., 1998; Sakurai et al., 1998).Axons from these neurons are found in the hypothala-mus, LC, raphe nuclei, tuberomamillary nucleus, midlinethalamus, all levels of spinal cord, sympathetic and para-sympathetic centers, and many other brain regions(Peyron et al., 1998; van den Pol, 1999). The two G pro-tein-coupled receptors of the hypocretins (HcrtR-1 andHcrtR-2) (Sakurai et al., 1998) also show a widespreadand heterogeneous pattern of expression throughout theCNS (Hervieu et al., 2001; Marcus et al., 2001; Trivediet al., 1998). A large body of data indicates that Hcrt cellsare specifically active during W. It has first been shown

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that Hcrt cells are C-Fos-positive after a period of naturalor pharmacologically induced W, such as treatments withstimulants like amphetamine or modafinil. On the con-trary, they are not C-Fos-positive after PS reboundindicating that Hcrt neurons are inactive during PS(Torterolo et al., 2003; Verret et al., 2003). Consistentwith these observations, it has been shown by continuousmicrodialysis or CSF puncture through 24 h that the levelof Hcrt released in situ or in the CSF is higher during theactive period than the quiet period in rat (Yoshida et al.,2001) and in monkey (Zeitzer et al., 2003). Further, it hasbeen shown that Hcrt cells are silent during SWS andtonic periods of PS, with occasional burst discharge inphasic PS. They discharge during active W, they havemoderate and approximately equal levels of activity dur-ing grooming and eating and a maximal activity duringexploratory behaviour (Lee et al., 2005; Mileykovskiyet al., 2005). As for the monoaminergic neurons (seeabove), the cessation of activity of Hcrt cells during sleepis likely caused by a tonic GABAergic inhibition. Indeed,local application of bicuculline in the perifornical regioninduces W and C-Fos labelling of the Hcrt but not ofthe intermingled neurons containing the melanin concen-trating hormone (MCH) (Alam et al., 2005; Goutagnyet al., 2005c). The VLPO neurons could be responsibleof such inhibition during SWS while the eVLPO orMCH neurons could be responsible of such inhibitionduring PS (see below).

A large number of results further indicate that Hcrtneurons participate in the induction and maintenance ofW via direct excitatory projections to the populations ofneurons previously identified as being responsible for W.Indeed, HcrtR-1 and HcrtR-2 are densely packed inmonoaminergic and cholinergic nuclei involved in theinduction of W (Jones, 1993). Moreover, ICV administra-tion and local injections of Hcrt in the LC induce W(Bourgin et al., 2000; Hagan et al., 1999). It has been alsoshown that the waking e!ect of Hcrt-1 when injected inthe lateral ventricle can be blocked by systemic injectionsof an antagonist of the H1 histaminergic receptors(Yamanaka et al., 2002), and is abolished in H1 knockoutmice (Huang et al., 2001). In vitro, Hcrt applicationsstrongly increase the firing rate of LC noradrenergic neu-rons (Hagan et al., 1999; Horvath et al., 1999), dopami-nergic VTA neurons (Nakamura et al., 2000),serotonergic dorsal raphe neurons (Brown et al., 2001;Liu et al., 2002), and histaminergic neurons of the tubero-mammillary nucleus (Bayer et al., 2001; Eriksson et al.,2001). These data are therefore consistent with a globalstimulatory e!ect of Hcrt on monoaminergic and cholin-ergic tone to maintain W. In addition, we previously pro-posed that Hcrt neurons could provide an excitatoryprojection to the DPMe PS-o! GABAergic neurons andby this way inhibit the onset of PS (Fig. 1) (Luppiet al., 2004). The absence of this pathway in narcolepticpatients could be specifically responsible for the cataplec-tic attacks. This hypothesis supported by the presence of

Hcrt-immunoreactive fibers in the DPMe (Lu et al.,2006; Peyron et al., 1998) remain to be tested.

7.3. The MCH/GABAergic neuronal population

We recently demonstrated that the PH contain a verylarge number of Fos-labelled neurons specifically in ratsperfused after a PS rebound compared to those perfusedat the end of the PS deprivation (Verret et al., 2003). Wefurther demonstrated that the majority of these neuronsare immunoreactive to MCH. These results suggest thatMCH neurons are specifically and strongly active duringPS. Our data are in agreement with electrophysiologicalstudies showing the presence in the PH of neurons stronglyactive during PS (Alam et al., 2002; Koyama et al., 2003;Steininger et al., 1999). To determine whether MCHplays a role in PS regulation, we performed ICV adminis-trations of MCH. Injections of 0.2, 1 and 5 lg induced adose-dependant increase in PS quantities compared tosaline due to an increase in the number of PS bouts butnot of their duration. To a minor extent, an increase inthe amount of SWS was also observed after MCHadministration.

Since MCH neurons co-contain GABA, are active dur-ing PS and that MCH is rather an inhibitory peptide (Gaoand van den Pol, 2001), MCH neurons likely promote PSindirectly by inhibiting neurons, themselves inhibiting thePS executive neurons during W and SWS. The monoamin-ergic neurons in the brainstem, the histaminergic neuronsin the tuberomammillary nucleus and the Hcrt neuronsall belong to this category. They are active during W,decrease or nearly cease their activity during SWS andare silent during PS (Gervasoni et al., 1998, 2000; Leeet al., 2005; Mileykovskiy et al., 2005; Steininger et al.,1999). Further, based on electron and light microscopicobservations, it has been shown that MCH and Hcrt neu-rons are interconnected (Bayer et al., 2002; Guan et al.,2002) (Fig. 1). We therefore propose that MCH neuronsprimarily induce SWS and PS via combined MCH/GAB-Aergic inhibitory actions on the intermingled Hcrt neurons(Fig. 1) and to a minor extent on the monoaminergic neu-rons. They could also increase PS quantities via a directinhibitory projection on the DPMe PS-o! GABAergic neu-rons (Boissard et al., 2002, 2003). Since PS still occurs in‘‘pontine cats’’, it is unlikely that MCH and Hrct neuronsare necessary to induce PS as stated above. We thereforeproposed that they could be rather responsible of PShomeostasis since PS rebound is abolished in ‘‘pontinecats’’. Additional studies are however needed to explorethis hypothesis.

8. Conclusion: a new network model for the onset andmaintenance of PS (Fig. 1)

Based on our and others results, we propose the follow-ing model (Fig. 1). The activation of PS-on glutamatergicneurons from the SLD underlies the onset and mainte-

278 P.-H. Luppi et al. / Journal of Physiology - Paris 100 (2007) 271–283

nance of PS. Ascending SLD PS-on glutamatergic neuronsinduce cortical activation via their projections to intralam-inar thalamic relay neurons in conjunction with W/PS-oncholinergic and glutamatergic neurons of the LDT andPPT, mesencephalic and pontine reticular nuclei and thebasal forebrain. Descending PS-on glutamatergic SLD neu-rons induce muscle atonia via their excitatory projectionsto glycinergic premotoneurons localized in the GiV.

During W and SWS, SLD PS-on neurons are inhibitedby a tonic GABAergic input arising from GABAergicPS-o! neurons localized in the DPMe. Noradrenergicand serotonergic PS-o! neurons, although not essential inthis matter, also participate in the inactivation of SLD neu-rons particularly during W. At the onset of, and through-out PS, the monoaminergic neurons cease firing becauseof an active GABAergic inhibition from PS-on neuronslocated in the DPGi and the vlPAG. These GABAergicPS-on neurons at the same time also inhibit the GABAer-gic PS-o! neurons from the DPMe. They are therefore theinitiators of PS. Their activity might be due to intrinsicpacemaker properties and influenced by inputs from auto-nomic regulatory structures.

The activity of the SLDPS-on neurons during PS is due tothe strong glutamatergic excitatory input present during allvigilance states blocked duringWand SWSby the inhibitoryinputs from the GABAergic and monoaminergic PS-o!neurons. The glutamatergic input arise from one or severalnon-GABAergic brainstem a!erents to the SLD (e.g. thevlPAG, the DPMe, pontine reticular nuclei and the Pc).

Forebrain neurons are not necessary for PS onset andmaintenance. However, Hcrt and MCH hypothalamic neu-rons participate in the homeostasis of PS through recipro-cal connections and their respective excitatory andinhibitory projections on the brainstem monoaminergicand GABAergic PS-o! neurons.

Acknowledgments

This work was supported by CNRS (UMR 5167 andFRE 2469) and Universite Claude Bernard Lyon 1.

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