Cholinergic and noncholinergic brainstem neuronsexpressing Fos after paradoxical (REM) sleep deprivationand recovery

Laure Verret, Lucienne Leger, Patrice Fort and Pierre-Herve LuppiCNRS UMR 5167, Institut Federatif des Neurosciences de Lyon (IFR 19), Faculte de medecine RTH Laennec, 7, rue GuillaumeParadin, 69372 Lyon Cedex 08, France

Keywords: acetylcholine, muscle atonia, rat, REM sleep, reticular

Abstract

It is well accepted that populations of neurons responsible for the onset and maintenance of paradoxical sleep (PS) are restricted tothe brainstem. To localize the structures involved and to reexamine the role of mesopontine cholinergic neurons, we compared thedistribution of Fos- and choline acetyltransferase-labelled neurons in the brainstem of control rats, rats selectively deprived of PS for! 72 h and rats allowed to recover from such deprivation. Only a few cholinergic neurons from the laterodorsal (LDTg) andpedunculopontine tegmental nuclei were Fos-labelled after PS recovery. In contrast, a large number of noncholinergic Fos-labelledcells positively correlated with the percentage of time spent in PS was observed in the LDTg, sublaterodorsal, alpha and ventralgigantocellular reticular nuclei, structures known to contain neurons specifically active during PS. In addition, a large number of Fos-labelled cells were seen after PS rebound in the lateral, ventrolateral and dorsal periaqueductal grey, dorsal and lateralparagigantocellular reticular nuclei and the nucleus raphe obscurus. Interestingly, half of the cells in the latter nucleus wereimmunoreactive to choline acetyltransferase. In contrast to the well-accepted hypothesis, our results strongly suggest that neuronsactive during PS, recorded in the mesopontine cholinergic nuclei, are in the great majority noncholinergic. Our findings furtherdemonstrate that many brainstem structures not previously identified as containing neurons active during PS contain cholinergic ornoncholinergic neurons active during PS, and these structures may therefore play a key role during this state. Altogether, our resultsopen a new avenue of research to identify the specific role of the populations of neurons revealed, their interrelations and theirneurochemical identity.

Introduction

It is now well accepted that populations of neurons localized in thedorsal part of the pontine reticular formation, the mesopontinecholinergic nuclei and the ventromedial medullary reticular formationplay a crucial role in the genesis of paradoxical sleep (PS), also termedrapid eye movement (REM) sleep (Jouvet, 1962; Jones, 1991). Indeed,long periods of PS have been elicited in cats and rats by pharmaco-logical stimulation of the dorsal part of the pontine reticular formationnamed the sublaterodorsal nucleus (SLD) in the latter species(Baghdoyan et al., 1987; Vanni-Mercier et al., 1989; Yamamotoet al., 1990; Boissard et al., 2002). Further, it contains neuronsspecifically active during PS (review in Sakai et al., 2001). Such typeof neurons has been also recorded in the laterodorsal (LDTg) andpedunculopontine (PPTg) tegmental cholinergic nuclei (El Mansariet al., 1989; Steriade et al., 1990; Kayama et al., 1992; Sakai &Koyama, 1996) and the alpha and ventral gigantocellular reticularnuclei (GiA and GiV), located in the ventromedial medullary reticularformation (Kanamori et al., 1980). It has been hypothesized that LDTgand PPTg neurons are cholinergic and responsible for EEG activationduring PS (review in Jones, 1991) while those of the GiA and GiV are

glycinergic and responsible for the muscle atonia of PS (Sakai, 1988;Fort et al., 1993; Rampon et al., 1996; Boissard et al., 2002).The lack of information concerning the activity of neurons in other

brainstem structures with regards to PS has been partially overcomeby the use of the immunohistochemical detection of Fos, the proteinproduct of the immediate–early gene c-fos (Dragunow & Faull, 1989;Morgan & Curran, 1991). Activated neurons can be mapped at alarger scale than with electrophysiology and their neurochemicalnature determined. Using this method, authors have shown that thenumbers of Fos-positive cells counted in some discrete nuclei of thebrainstem are positively or negatively correlated with the amount ofPS induced either by carbachol microinjection (Shiromani et al.,1992; Yamuy et al., 1993; Shiromani et al., 1996; Yamuy et al.,1998) or by PS deprivation (Merchant-Nancy et al., 1995; Maloneyet al., 1999, 2000), in both cat and rat. However, there are significantdiscrepancies in these studies. In particular, two of them reported thatin the LDTg and PPTg the number of Fos-expressing cells identifiedas cholinergic is positively correlated with the amount of PS(Shiromani et al., 1996; Maloney et al., 1999) while another studyfailed to confirm these findings (Yamuy et al., 1998). In addition, theSLD and the GiV were reported to contain a number of Fos-labelledcells positively correlated with PS only in two of these studies(Yamuy et al., 1993; Merchant-Nancy et al., 1995).Therefore, the aim of the present study was to resolve previous

discrepancies by mean of a careful analysis in rats of the distribution

Correspondence: Dr P. H. Luppi, as above.E-mail: luppi@sommeil.univ-lyon1.fr

Received 13 August 2004, revised 25 January 2005, accepted 2 February 2005

European Journal of Neuroscience, Vol. 21, pp. 2488–2504, 2005 ª Federation of European Neuroscience Societies

doi:10.1111/j.1460-9568.2005.04060.x

of the neurons expressing Fos in the brainstem following PSdeprivation, PS recovery after deprivation and control conditions. Inaddition, it was aimed to determine whether the cholinergic neuronsfrom the LDTg and PPTg are Fos-immunoreactive after PS recoveryand therefore indeed correspond to the PS-active neurons recorded inthese nuclei.

Materials and methods

Animals and surgery

Male Sprague-Dawley rats (280–320 g, IFFA Credo, L’Arbresle,France) were anaesthetized with chloral hydrate (1 g ⁄ kg, i.p.) andmounted conventionally in a stereotaxic frame (David Kopf,Epinay-sur-Seine, France) with ear bars and a head holder. Thebone was exposed and cleaned. Three stainless steel screws werefixed in the parietal and frontal parts of the skull and three wireelectrodes inserted into the neck muscles to monitor the electro-encephalogram (EEG) and the electromyogram (EMG), respect-ively.

The bone was then covered with a thin layer of acrylic cement(Superbond; Sun Medical Co, Moriyama, Shiga, Japan). A six-pinconnector was embedded in a mount of dental cement with the EEGscrews and wires. Animals were allowed 2 days recovery fromsurgery in individual Plexiglas containers before starting the habitu-ation to the recording cable. Animals were cared for according to theGuide for the Care and Use of Laboratory Animals (NIH Publication80–23; Authorization n! 03–505 of the French Ministry of Agricul-ture).

Recording and PS deprivation

Each rat was kept in its container and connected to a cable attached toa commutator and suspended with a balanced boom to allow freemovement of the animal within the container. During the baseline dayand in the control condition, the floor of the container was coveredwith woodchips. The animal had ad libitum access to food and waterin dispensers that hung within easy reach on the side of the container.A 12-h light–dark cycle was maintained in the recording room (withlights on 06.00–18.00 h). The rats were placed in the recordingcontainer (30 cm diameter, 40 cm high) and connected to the cable8 days before baseline recording to allow for habituation to therecording environment. EEG and EMG recordings were collected on acomputer via a CED interface using Spike 2 software (CambridgeElectronic Design, UK).

PS deprivation was performed for ! 72 h using the flowerpottechnique, which has previously been shown to cause a selectivedeprivation of PS in rats (Mendelson et al., 1974; Maloney et al.,1999). Each rat was placed on a platform which was surrounded bywater and large enough (6.5 cm in diameter) to hold the animal duringwaking (W) and slow wave sleep (SWS), but preventing it fromengaging in PS bouts because of the loss of muscle tone accompany-ing this state and the correlated risk of falling into the water. Food andwater containers were positioned so as to be easily accessible to theanimal on the platform.

The experimental protocol was performed over a 5-day period inthree groups of four rats: control (PSC), PS deprivation (PSD) andPS rebound (PSR) groups. On the first day, a baseline recording wasperformed for all animals. During the last 4 days of the experiment,the animals from the PSC group remained on a bed of woodchips intheir container before being anaesthetized for histological perfusion(at 17.00 h). In the PSD condition, the animals were placed on the

platform on the second day of the experiment (at 13.00 h) until theywere killed, at 16.30 h. On the two first days of deprivation, theanimals were removed from the platform for 30 min to permitcleaning of their containers. In the PSR condition the animals were,like the PSD animals, placed on the platform. After 72 h of PSdeprivation they were put back at 13.00 h into control conditions, i.ein a container with a dry bed of woodchips to allow for recovery ofPS. After ! 30 min of exploration and grooming, PSR animals fellasleep. They were anaesthetized for perfusion 150 min after thebeginning of the first PS phase, which appeared with a latency of37.5 ± 5.2 min.

Polygraphic recordings

Vigilance states were discriminated with the cortical EEG and neckEMG. During W, desynchronized (or activated) low-amplitude EEGwas accompanied by a sustained EMG activity with phasic bursts.SWS was clearly distinguished by high-voltage slow waves (1.5–4.0 Hz) and spindles (10–14 Hz) and the disappearance of phasicmuscular activity in an immobile animal with closed eyes. A decreasein the EEG amplitude associated with a flat EMG (i.e. muscle atonia)signalled the onset of PS episodes further characterized by apronounced theta rhythm (5–9 Hz). For each vigilance state, aspectral analysis of the EEG was performed on-line using the fast-Fourier transform.

Histological and immunohistochemical procedures

The animals were perfused with a Ringer’s lactate solution containing0.1% heparin, followed by 500 mL of a fixative composed of 4%paraformaldehyde and 0.2% picric acid in 0.1 m phosphate buffer(PB; pH 7.4). The brains were postfixed for 2 h in the same fixative at4 !C and then stored at 4 !C for at least 2 days in 0.1 m PB with 30%sucrose. They were rapidly frozen with CO2 gas. Coronal sections25-lm-thick were obtained with a cryostat and stored in 0.1 m PB,pH 7.4, containing 0.9% NaCl, 0.3% Triton X-100 (PBST) and 0.1%sodium azide (PBST-Az).For double immunostaining experiments, free-floating sections were

successively incubated in: (i) a rabbit antiserum to Fos (1 : 5000;Ab-5; Oncogene, CA, USA) in PBST-Az for 3 days at 4 !C; (ii) abiotinylated goat antirabbit IgG (1 : 2000; Vector Laboratories,Burlingame, CA, USA) for 90 min at room temperature; and (iii) anABC-HRP solution (1 : 1000; Elite kit, Vector Laboratories) for90 min at room temperature. The sections were immersed in a 0.05-mTris-HCl buffer (pH 7.6) containing 0.025% 3,3¢-diaminobenzidine-4HCl (DAB; Sigma, Saint Quentin Fallavier, France), 0.003% H2O2

and 0.6% nickel ammonium sulphate for 10 min at room temperature.The reaction was stopped by two rinses in PBST-Az. The free-floatingsections were then incubated in: (i) a goat antiserum to cholineacetyltransferase (ChAT; 1 : 2000; Chemicon, CA, USA) in PBST-Azfor 3 days at 4 !C; (ii) a biotinylated donkey antigoat IgG (1 : 2000;Vector Laboratories, CA, USA) for 90 min at room temperature; and(iii) an ABC-HRP solution (1 : 1000; Elite kit, Vector Laboratories)for 90 min at room temperature. Finally, the sections were immersedfor 15 min at room temperature in the same DAB solution as abovebut without nickel. The sections were then mounted on gelatin-coatedslides, dried, dehydrated and coverslipped with DePex.Controls in the absence of primary antibodies were routinely run to

ensure the absence of nonspecific single or dual immunostaining in thematerial. As specified by the supplier, the Fos antiserum was madeagainst a synthetic peptide corresponding to the N-terminal part

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(residues 4–17) of human Fos. This part of the protein displays 100%homology between human and rat (van Straaten et al., 1983; Curranet al., 1987) and no homology with Fos-related antigens such as Fos B,Jun B, Fra-1 and Fra-2 (Blast 2 sequences, NCBI). In agreement withprevious studies, very few immunoreactive (IR) nuclei were observed inthe rat brain with this Fos antiserum following perfusion during daylight(Semba et al., 2001; Ro et al., 2003; Gong et al., 2004).

Cell counts and analysis of immunohistochemical data

Because the distribution of the Fos-IR and Fos–ChAT double-labelledneuronswas identical on both sides of the brain, only hemi-sectionswereanalysed. Drawings of double-immunostained sections were made witha Axioscope microscope (Zeiss, Germany) equipped with a motorizedX–Y-sensitive stage and a video camera connected to a computerized

Fig. 1. Schematic distribution of ChAT-IR (small grey dots), Fos-IR (small black dots) and Fos–ChAT (large black dots) double-labelled neurons in 15 coronalsections taken at 400-lm intervals through the full rostrocaudal extent of the pons and medulla in a representative animal for control (left hand side), PS-deprivation(middle) and PS-rebound (right hand side) conditions. (A) Sections from )7.40 and )7.80 from Bregma. (B) Sections from )8.20 to )9.00 from Bregma.(C) Sections from )9.40 to )10.20 from Bregma. (D) Sections from )10.60 to )11.80 from Bregma. (E) Sections from )12.20 to )13.00 from Bregma.Abbreviations for the names of the structures are in the main list.

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image analysis system (Mercator; ExploraNova, La Rochelle, France).Single- and double-labelled neurons were plotted on sections taken at400-lm intervals through the full rostrocaudal extent of the pons and

medulla (15 sections between AP)7.40 and)13.00 from Bregma). Thethree categories of neurons were counted per structure automaticallyusingMercator. When a structure was present on more than one section,

Fig. 1. Continued

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the sum of all the neurons counted on all the sections was made. Thecountswere not performed in nuclei unlikely to play any role in the onsetand maintenance of PS, in particular the motor and sensory nuclei such

as the superior and inferior colliculus, the pontine nuclei, nuclei of thelateral lemniscus, cochlear nuclei, cranial motor nuclei, sensorytrigeminal nuclei, cerebellum, superior and inferior olivary complexes

Fig. 1. Continued

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and vestibular nuclei. However, to provide qualitative information wedecided to display Fos-IR neurons localized in these structures in thedrawings of Fig. 1.

The atlas of Paxinos & Watson (1997) was used as a reference forall structures except the medial PPTg (PPTgM), which was delineatedaccording to Maloney et al. (1999), the SLD which was named and

Fig. 1. Continued

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delineated according to Swanson (1998) and the lateral paragiganto-cellular (LPGi) and rostroventrolateral reticular nuclei, which weregrouped under the name of LPGi.

Statistical analysis

anova tests were performed on the different vigilance states acrossexperimental conditions. A post hoc PLSD Fisher test was used toidentify significant pairwise differences. The same tests wereperformed to compare cell counts among the animals groups in thedifferent brainstem structures. Cell counts were correlated withphysiological variables using a simple linear regression analysis. Allstatistics were performed using Statview 5.0.

Results

Quantification of the sleep and waking phases

The PS deprivation procedure was effective in producing a nearcomplete elimination of PS for ! 75 h in the PSD and 72 h in the PSRgroups. During the 150 min prior to killing, the quantities of PSdiffered significantly between the group of control animals (PSC) andthe PSD and PSR groups (all P < 0.05). The state of PS constituted16% of the last 150 min prior to killing in the PSC group, 0% in thePSD group and 49% in the PSR group (Table 1). The PSD animalsspent 30% and the PSR 31% of their time in SWS, significantly lessthan those from the PSC group (48%; P < 0.05). The PSD grouppresented a large quantity of W (70%) compared to the PSC (36%)and PSR (20%) groups. The increase in PS quantities during the last

Fig. 1. Continued

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150 min prior to killing in the PSR group compared to the PSC groupwas due to a significant increase in the duration of the PS bouts(Table 1; P < 0.01). The number of bouts did not vary significantlybetween the two groups (Table 1).

Distribution of the Fos-immunoreactive neurons

As expected, the Fos immunolabelling was restricted to the nucleus ofthe labelled cells. The diameter of the labelled nuclei was of 8–15 lm.The distribution of Fos-IR neurons in the brainstem greatly differedbetween the three experimental groups of rats (Table 2 and Fig. 1). Inthe PSC group, only a few to a moderate number of cells was found inall brainstem structures while in the PSD and PSR groups largenumbers of neurons were seen with a globally different distribution(Table 2 and Fig. 1). In the PSD group, no significant Fos labelling wasseen in brain areas activated in response to acute stress (Cullinan et al.,1995), areas such as the dorsomedial nucleus of the hypothalamus, thelateral portion of the retrochiasmatic area (not illustrated) and the locuscoeruleus (LC). Altogether, these results confirm previous onesshowing that, in the brainstem, very few neurons express Fos duringthe rest phase when the animals are maintained in their familiarenvironment (Herdegen et al., 1995; Semba et al., 2001; Ro et al.,2003; Gong et al., 2004). They further indicate that the Fosimmunostaining in the PSD group is not due to stress.

The number of Fos-IR neurons was quantified in 27 brainstemstructures including all structures from the reticular formation, theperiaqueductal grey and the cholinergic and monoaminergic nuclei(Tables 2 and 3 and Fig. 1). Correlations between the number ofFos-IR and Fos–ChAT double-labelled neurons and the amount of thethree vigilance states were calculated for all structures (Table 4). Tosimplify the text, only the significant correlations are mentioned.

Three structures displayed a significantly higher number of Fos-IRneurons in the PSD condition than in the PSR and PSC conditions.These were the dorsal raphe (DRN), lateral parabrachial (PBL) andraphe pallidus (RPa) nuclei (Table 2 and Figs 2C and D, and 4Gand H). In the DRN, the number of Fos-IR cells was significantly andpositively correlated with the percentage of time spent in W duringthe final 150-min recording period (R " 0.65, P < 0.05). In the PBL,the Fos-IR neurons were spread over all subdivisions in the PSDcondition whereas many of them were concentrated in the centralportion of the central lateral subnucleus (Bester et al., 1997) in thePSR condition (Figs 1C, and 2C and D). The number of Fos-labelledcells was significantly higher in the PSD condition than the PSCcondition in the deep mesencephalic reticular nucleus (DPMe). The

nucleus of the solitary tract (Sol) displayed a higher number of Fos-IRneurons in the PSD condition, but this did not reach significancebecause of large variability among the animals (Fig. 1E).In 12 brainstem structures, the number of Fos-IR neurons was

significantly higher in the PSR condition than in the PSD conditionand moreover the PSC condition (Table 2). A very high level ofsignificance (P < 0.001) between PSR and PSD conditions and apositive correlation with the percentage of time spent in PS during thefinal 150-min recording period was reached in three nuclei, namely theSLD (R " 0.83, P < 0.001), dorsal paragigantocellular nucleus(DPGi; R " 0.84, P < 0.001) and GiA (R " 0.65, P < 0.05; Table 4).This is due to a substantial number of Fos-IR neurons labelledspecifically in these three nuclei after PS rebound (Figs 1C–E, 2E andF, and 4A and B) and to moderate interindividual variability (Table 2).Interestingly, the number of Fos-IR cells in the DPGi was alsonegatively correlated with the percentage of time spent in W

Table 1. Time spent in waking, slow-wave sleep and paradoxical sleep

PSC PSD PSR

Vigilance statesW (%) 35.6 ± 2.4 69.6 ± 6.8** 20.4 ± 3.9*,###

SWS (%) 48.0 ± 2.6 30.4 ± 6.8* 30.6 ± 4.8*PS (%) 16.4 ± 0.3 0 ± 0* 49.1 ± 6.4**,###

PS boutsNumber (n) 21.3 ± 2.3 – 23.8 ± 5.3Duration (min) 1.2 ± 0.1 – 2.3 ± 0.3**

Percentage of time spent in waking (W), slow wave sleep (SWS) and para-doxical sleep (PS) scored per 10-s epoch over the last 150 min before killing,and number and duration of PS bouts during the same period for Control (PSC,n " 4), PS-deprived (PSD, n " 4) and PS-recovery (PSR, n " 4) rats. Signi-ficance values indicated for individual points are *P < 0.05 and**P < 0.01 vs. PSC, ###P < 0.001 between PSR and PSD.

Table 2. Numbers of Fos-IR neurones in the brainstem

n PSC PSD PSR

Periaqueductal greydPAG 4 8.0 ± 5.4 29.8 ± 5.6 144.5 ± 56.2*,#

lPAG 4 9.3 ± 4.8 50.0 ± 9.6* 47.8 ± 11.5*vlPAG 5 15.5 ± 5.7 68.5 ± 3.9** 85.3 ± 16.0***CGPn 3 4.3 ± 2.3 21.8 ± 14.7 35.5 ± 13.3

Rostral rapheDRN 4 4.0 ± 2.1 31.0 ± 7.9** 7.5 ± 4.7#

MnR 4 0.3 ± 0.3 3.3 ± 1.3 15.3 ± 4.2**,##

Pontomesencephalic tegmentiPPTg 5 3.0 ± 2.4 20.0 ± 3.9 24.3 ± 9.1*PPTgM 3 0 ± 0 0 ± 0 11.8 ± 3.4**,#

PPTg + PPTgM 3.3 ± 2.3 23.3 ± 4.1 37.0 ± 12.3*,#

LDTg 5 13.5 ± 7.0 22.5 ± 2.6 63.0 ± 15.6**,#

LDTgV 3 1.8 ± 0.5 11.0 ± 5.4 23.3 ± 6.0**LDTg + LDTgV 15.3 ± 7.1 33.5 ± 6.0 86.3 ± 21.0**,#

Mesopontine reticular formationDPMe 4 3.8 ± 1.0 28.0 ± 7.2* 59.5 ± 17.8**SLD 3 0.5 ± 0.5 6.0 ± 1.4 46.8 ± 9.9***,###

PnO 6 6.8 ± 1.4 34.3 ± 16.7 66.3 ± 26.7*PnC 3 6.5 ± 1.9 18.0 ± 4.1 64.3 ± 18.3**,#

Dorsolateral ponsPBL 1 6.8 ± 4.2 73.8 ± 1.7*** 42.5 ± 8.3**,##

LC 3 0.3 ± 0.3 1.3 ± 0.8 0.8 ± 0.5KF 1 2.0 ± 0.9 6.5 ± 2.0 4.0 ± 2.4PCRt 6 7.8 ± 3.6 51.0 ± 19.8 63.8 ± 29.4Sol 5 8.3 ± 4.3 51.5 ± 28.7 13.0 ± 6.8

Medullary reticular formationGi 6 3.3 ± 1.3 14.5 ± 9.9 33.8 ± 10.7*GiA 4 0.5 ± 0.3 10.3 ± 3.7 31.8 ± 4.1***,###

GiV 3 0.3 ± 0.3 4.5 ± 1.9 35.3 ± 9.9**,##

DPGi 6 3.3 ± 1.1 1.8 ± 0.6 49.0 ± 10.9***,###

LPGi 6 6.0 ± 1.9 52.3 ± 8.1* 99.5 ± 23.3**,#

Caudal rapheRMg 5 0.5 ± 0.5 1.5 ± 0.5 6.5 ± 2.3*,#

ROb 5 0.3 ± 0.3 3.8 ± 1.1 27.3 ± 6.0***,##

RPa 5 0.3 ± 0.3 27.0 ± 7.0** 7.8 ± 2.8#

Number (mean ± SEM) of Fos-IR neurons calculated in the brainstem forcontrol (PSC, n " 4), PS-deprived (PSD, n " 4) and PS-recovery (PSR,n " 4) rats. Cells were counted from a total of 15 sections at 400-lm intervalsthrough the full rostrocaudal extent of the pons and medulla. The values dis-played are the average across four animals in each group of the sum of Fos-labelled neurons counted on one or several sections (column n) depending onthe rostrocaudal extent of the structures. Significance values indicated forindividual points are: *P < 0.05, **P < 0.01 and ***P < 0.001 vs. PSC;#P < 0.05, ##P < 0.01 and ###P < 0.001 between PSR and PSD.

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(R " –0.61, P < 0.05). A high level of significance (P < 0.01) and astrong positive correlation with the percentage of time spent in PSduring the final 150-min recording period was seen in three othernuclei, namely the median raphe (MnR; R " 0.75, P < 0.01), ventralgigantocellular (GiV; R " 0.71, P < 0.01) and raphe obscurus (ROb;R " 0.76, P < 0.01) nuclei (Table 4). The MnR contained a moderatenumber of Fos-IR neurons in the PSR condition (Fig. 1B), whereas theGiV and ROb showed a substantial number of Fos-positive neurons(Fig. 1E). In the GiV, a conspicuous group of neurons showing anintensely labelled nucleus was systematically seen among the exitingfibres of the hypoglossal nerve (Fig. 4C and D).A low level of significance (P < 0.05) was obtained in six

additional areas, which are the dorsal periaqueductal grey (dPAG),pedunculopontine tegmental (PPTg together with PPTgM), laterodor-sal tegmental (LDTg and LDTgV, the ventral part of the LDTg),caudal pontine reticular (PnC), raphe magnus (RMg) and lateralparagigantocellular nuclei (LPGi) (Table 2). The dPAG and LPGidisplayed the highest number of Fos-IR neurons among all thestructures quantified while the RMg contained the lowest number. Inbetween were the LDTg (Figs 1B and C, and 3) and the PnC (Fig. 1Cand D), which contained a substantial number of Fos-IR neurons(Table 2). Across conditions, the numbers of Fos-IR cells weresignificantly positively correlated with the percentage of time spent inPS during the final 150-min recording period in the PPTgM

(R " 0.71, P < 0.01), PnC (R " 0.65, P < 0.05) and LDTg(R " 0.63, P < 0.05; Table 4).It is worth noting that in four additional regions the number of Fos-

IR neurons was significantly higher in the PSR condition than in thePSC condition but only tended to be higher than the PSD condition.These were the DPMe, oral pontine (PnO) and gigantocellular reticularnuclei and the LDTgV, which all contained substantial numbers ofFos-IR neurons in the PSR condition (Table 2, Fig. 1).In two structures, the number of Fos-IR neurons did not differ

between the PSD and PSR conditions but was much higher than in thePSC condition. These structures are the lateral and ventrolateralperiaqueductal grey (lPAG and vlPAG; Fig. 2A and B). In two areas,more Fos-IR cells were seen in the PSR and PSD conditions than inthe PSC condition but this did not reach statistical significance. Thesewere the central grey of the pons and the parvicellular reticular nucleus

Table 3. Double-labelled Fos–ChAT neurones in the brainstem: numbers andas percentages of singly ChAT- and Fos-labelled neurones

PSC PSD PSR

PPTg + PPTgMFos–ChAT 0 ± 0 0 ± 0 2.3 ± 1.7ChAT (%) 0 ± 0 0 ± 0 0.9 ± 0.6Fos (%) 0 ± 0 0 ± 0 3.8 ± 2.7

LDTg + LDTgVFos–ChAT 0 ± 0 0 ± 0 3.8 ± 1.8*,#

ChAT (%) 0 ± 0 0 ± 0 2.3 ± 1.3Fos (%) 0 ± 0 0 ± 0 3.5 ± 1.4

PCRtFos–ChAT 0 ± 0 1.5 ± 0.9 0.5 ± 0.3ChAT (%) 0 ± 0 6.3 ± 3.7 1.0 ± 0.6Fos (%) 0 ± 0 6.3 ± 4.3 2.8 ± 1.7

GiFos–ChAT 0 ± 0 0 ± 0 0.8 ± 0.3**,##

ChAT (%) 0 ± 0 0 ± 0 5.5 ± 2.1Fos (%) 0 ± 0 0 ± 0 3.7 ± 2.2

DPGiFos–ChAT 0 ± 0 0 ± 0 3.8 ± 1.4**,##

ChAT (%) 0 ± 0 0 ± 0 55.1 ± 5.8Fos (%) 0 ± 0 0 ± 0 12.5 ± 8.2

LPGiFos–ChAT 0 ± 0 0 ± 0 8.0 ± 1.7***,###

ChAT (%) 0 ± 0 0 ± 0 23.8 ± 13.6Fos (%) 0 ± 0 0 ± 0 8.3 ± 1.7

RObFos–ChAT 0 ± 0 0 ± 0 11.5 ± 3.1**,##

ChAT (%) 0 ± 0 0 ± 0 66.8 ± 11.6Fos (%) 0 ± 0 0 ± 0 42.7 ± 7.8

Number (mean ± SEM) of double-labelled neurons (Fos–ChAT) and meanpercentage (± SEM) of Fos–ChAT neurons vs. singly ChAT-IR (%ChAT)and Fos-IR (%Fos) cells for control (PSC, n " 4), PS-deprivation(PSD, n " 4) and PS-rebound (PSR, n " 4) rats. For cell count-ing, see Table 2. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. PSC;#P < 0.05, ##P < 0.01 and ###P < 0.001 between PSR and PSD.

Table 4. Relationship between the number of Fos-IR and Fos–ChAT cellcounts and the states of sleep

Regression coefficient (R)

Fos-IR Fos–ChAT

W SWS PS W SWS PS

Periaqueductal greydPAG –0.02 –0.04 0.07 – – –lPAG 0.20 –0.50 0.05 – – –vlPAG –0.04 –0.38 0.23 – – –CGPn –0.15 –0.20 0.27 – – –

Rostral rapheDRN 0.64* –0.23 –0.54 – – –MnR –0.49 –0.45 0.75** – – –

Pontomesencephalic tegmentiPPTg 0.09 –0.56 0.22 –0.37 –0.12 0.44PPTgM –0.38 –0.57 0.71** –0.14 –0.22 0.27PPTg + PPTgM –0.06 –0.59 0.40 –0.38 –0.15 0.47LDTg –0.43 –0.34 0.63* –0.41 –0.31 0.59LDTgV –0.34 –0.36 0.55 –0.37 –0.38 0.59LDTg + LDTgV –0.42 –0.36 0.63* –0.40 –0.34 0.61*

Mesopontine reticular formationDPMe –0.15 –0.59 0.51 – – –SLD –0.54 –0.48 0.83*** – – –PnO –0.17 –0.35 0.37 – – –PnC –0.38 –0.46 0.65* – – –

Dorsolateral ponsPBL 0.56 –0.59 –0.25 – – –LC 0.21 –0.34 –0.02 – – –KF 0.48 –0.51 –0.22 – – –

Medullary reticular formationPCRt 0.02 –0.17 –0.11 0.49 –0.50 –0.23Sol 0.30 0.12 –0.38 – – –Gi –0.32 –0.24 0.47 –0.50 –0.10 0.58GiA –0.44 –0.35 0.65! – – –GiV –0.45 –0.45 0.71** – – –DPGi –0.84* –0.37 0.84*** –0.55 –0.03 0.60*LPGi –0.22 –0.47 0.49 –0.56 –0.04 0.88****

Caudal rapheRMg –0.34 –0.42 0.58 – – –ROb –0.57 –0.30 0.76** –0.59 –0.26 0.83***RPa 0.50 –0.17 –0.42 – – –

Relationship between the number of Fos-IR and Fos–ChAT cell countsand states of sleep as assessed by a simple linear regression (testingfor W, SWS and PS amounts). The sign + or – of the regressionoefficient (R) indicates the direction of the covariation. *P < 0.05; !P < 0.02;**P < 0.01; ***P < 0.001; ****P < 0.0001.

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of the medulla oblongata (Table 2 and Fig. 1). Finally, two nuclei, theKolliker–Fuse and the LC, showed a small or very small number ofFos-IR neurons in all conditions (Table 2).

Distribution of the ChAT-immunoreactive neuronsexpressing Fos

The distribution of ChAT-IR neurons, as observed in our experiment,was similar to that described previously (Tatehata et al., 1987; Jones,

1990; Ruggiero et al., 1990). Apart from the well-known mesopontinegroup and cranial motor nuclei, ChAT-IR cells were visible in severalmedullary nuclei, namely the periolivary nuclei, medial vestibularnucleus, reticular formation close to the ventromedial solitary tractnucleus, DPGi, LPGi and ROb, and in a band of neurons whichstretched dorsoventrally across both the parvicellular reticular nucleus(PCRt) and the lateral part of the gigantocellular nucleus (Fig. 1C–E).In the LPGi, a medial group of relatively small and moderately ChAT-IR neurons was clearly differentiated from the larger and more IR cellsbelonging to the external nucleus ambiguus (Fig. 4E and F). Neurons

Fig. 2. Photomicrographs of Fos (nuclear staining) and ChAT (diffuse cytoplasmic staining) double-immunostained sections at the pontine level, from PSD (lefthand side) and PSR (right hand side) rats. (A and B) Photomicrographs showing the vlPAG in (A) PSD and (B) PSR animals. Numerous Fos-labelled neurons areobserved in this structure in both conditions. Aq, Sylvius aqueduct. (C and D) Photomicrographs showing the PBL in (C) PSD and (D) PSR animals. In the PSDcondition, Fos-IR cells are spread over all the PBL while they form a compact group in the central part of the nucleus in the PSR condition. (E and F)Photomicrographs showing the SLD in (E) PSD and (F) PSR rats. Note the presence of a large number of Fos-labelled cells specifically in the PSR rat. These cellsare not immunoreactive to ChAT and are therefore not cholinergic. Scale bars, 100 lm.

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Fig.3.

Fig.4.

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stretching vertically along the midline and approaching the fourthventricle (Fig. 4A and B) were considered as belonging to ROb.

Double-labelled Fos–ChAT neurons were almost exclusively ob-served in the PSR group (Table 3 and Fig. 1). The only region showingdouble-labelled neurons in both PSD and PSR conditions was the PCRtin which very few Fos–ChAT cells were observed (Table 3). In the PSRgroup, occasional cholinergic motoneurons were double-labelled in theoculomotor nuclei. The periolivary nuclei and the medial vestibularnucleus also contained a few double-labelled cells (Fig. 1C–E).

Surprisingly, the mesopontine group (PPTg, PPTgM, LDTg andLDTgV) contained a very small number of double-labelled cells(Fig. 3). These neurons represented a small percentage (3–5%) of thesingly Fos-IR cells and an even lower percentage (0.8–4%) of thesingly ChAT-IR cells (Table 3). In contrast, medullary structures suchas the ROb, the LPGi and to a lesser extent the DPGi contained asubstantial number of double-labelled cells (Table 3 and Figs 1 and4B). In the DPGi, double-labelled cells displayed a preferential dorsallocation, near the ependymal layer (Figs 1D and E, and 4B). In theLPGi, their distribution was restricted to the medial half of the nucleus(Figs 1E and 4F). In the ROb, 43% of the singly Fos-IR and 67% ofthe singly ChAT-IR neurons were double-labelled (Table 3). In theDPGi and LPGi, the double-labelled neurons corresponded to 12 and8% of the singly Fos-IR neurons and to 55 and 24% of the singlyChAT-IR neurons, respectively.

Across experimental conditions, the number of double-labelled cellswas significantly positively correlated with the percentage of timespent in PS during the final 150-min recording period in the LPGi(R " 0.88, P < 0.001), ROb (R " 0.83, P < 0.001), LDTg + LDTgVas a whole (R " 0.61, P < 0.05) and DPGi (R " 0.59, P < 0.05;Table 4).

Discussion

In the present study, by comparing Fos staining in control rats, ratssubjected to a selective PS deprivation and rats allowed to recover fromsuch deprivation, we identified at the cellular level populations ofbrainstem neurons with an activity positively or negatively correlatedwith PS. We first confirmed that the SLD, LDTg and GiV, threestructures previously identified by lesion, pharmacological and elec-trophysiological studies as being essential components of the networkresponsible for PS genesis, contained a large number of Fos-labelledneurons following PS hypersomnia. In addition, we further reveal thatthe dPAG, the DPGi and the LPGi, structures unknown to be activeduring PS, contained a large number of Fos-labelled cells following PShypersomnia. In contrast, structures such as the DRN, the PBL and theRPa contained Fos-labelled neurons specifically after PS deprivation,indicating that these neurons might be very active in the absence of PS.Finally, we found that cholinergic mesopontine neurons were rarelyimmunoreactive to Fos following PS hypersomnia, suggesting that the

vast majority of these neurons are not activated during PS. In contrast,numerous cholinergic neurons immunoreactive to Fos were seen inseveral medullary structures such as the LPGi and ROb following PShypersomnia, indicating that these medullary cholinergic neuronsmight play an important role during PS. In the following, we firstdiscuss the methodological issues of our work before confronting ourresults with the literature and discussing their functional significance.

Methodological considerations

Our study is based on the assumption that activated cells display Fosimmunoreactivity after a delay of ! 1 h with a peak 1–3 h after thestimulation followed by a progressive decrease and a disappearance ofstaining after 4 h (Dragunow & Faull, 1989). We choose the Fosmethod to identify the cells activated during PS because, despite somedrawbacks, it still constitutes the best method available to identify at acellular level activated neurons. Indeed, although neurons with low orphasic discharge rates do not always express Fos or, conversely, Foslabelling reflects in some neurons changes in calcium-mediatedcellular processes other than increases in neuronal discharge (Morgan& Curran, 1986; Dragunow & Faull, 1989), it is still accepted thatmost of the activated neurons express Fos.To maximize our chance of obtaining Fos immunostaining reflecting

activity positively or negatively correlated with PS, we submitted therats for a long period of time (72 h) to a very effective and selectivePS-deprivation method (the flowerpot method). During the deprivation,the animals presented 69% of W, 30% of SWS and 0% of PS. We thenallowed a group of these deprived animals to recover for 3 h, a periodduring which they presented 49% of PS, 30% of SWS and 20% of W.By comparison, control rats exhibited 16% of PS, 48% of SWS and35% of W. Because none of these three groups of animals presentedonly one vigilance state during the 3 h preceding perfusion, one mightconclude that the Fos labelling observed is not specific to any of thestates of vigilance. However, several points strongly suggest that theFos-labelled cells after PS recovery correspond to neurons activeduring PS. First, we have demonstrated that the Fos labelling obtainedin the PSR group is positively correlated with the amount of PS fornumerous structures but not with that of SWS and W. Second,numerous Fos-labelled cells were observed in three structures alreadyknown to contain PS-active cells, namely the LDTg, SLD and GiV,while a few or no cells were observed in W- or SWS-active structuressuch as the LC (present results), the hypocretin-containing cells (Verretet al., 2003b) or the ventrolateral preoptic area (Verret et al., 2003a).Third, the distribution of labelled cells was significantly differentbetween the PSD and PSR groups in the large majority of brainstemstructures. Altogether, these points strongly suggest that the Fos-labelled cells in the PSR group correspond to neurons active during PS.It is important to stress, however, that it is not possible with the Fosmethod to determine whether or not the labelled neurons are active

Fig. 4. Photomicrographs of medullary sections double-immunostained with Fos (black nuclear staining) and ChAT (brown cytoplasmic staining) of a PSD (lefthand side) and a PSR (right hand side) rat. (A and B) Photomicrographs showing the DPGi and ROb in (A) a PSD and (B) a PSR rat. Note the presence of Fos-IRand Fos–ChAT double-labelled cells (arrows) in these two nuclei specifically in the PSR rat. 4V, fourth ventricle; mlf, medial longitudinal fasciculus. (C and D)Photomicrographs showing the GiV in (C) a PSD and (D) a PSR rat. A cluster of Fos-IR neurons is localized in the GiV region just dorsal to the inferior olivarycomplex in between the fibres of the hypoglossal nerve (12n) specifically in the PSR condition. (E and F) Photomicrographs showing the LPGi in (E) a PSD and(F) a PSR rat. Note that more Fos-IR neurons are visible in the LPGi in the PSR rat than in the PSD rat. Two double-immunostained neurons (designated by arrowsand enlarged) are visible in the medial part of the LPGi specifically in the PSR condition. (G and H) Photomicrographs showing the RPa in (G) a PSD and(H) a PSR rat. Note the presence of numerous Fos-IR cells in the RPa specifically in the PSD rat. Scale bars, 100 lm.

Fig. 3. Photomicrographs showing the LDTg on three sections double-immunostained with Fos (black nuclear staining) and ChAT (brown cytoplasmic staining) in(A) PSC, (B) PSD and (C) PSR rats. A large number of singly Fos-IR neurons intermingled with cholinergic neurons are observed in the PSR condition, whereas afew to occasional Fos-labelled cells are present in PSD and PSC conditions, respectively. Only one double-immunostained neuron is visible in the PSR rat (arrow).Aq, Sylvius aqueduct. Scale bars, 100 lm.

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selectively during PS. Further, they could be responsible for PS onsetand maintenance or could be involved in physiological changes takingplace during this state.By contrast, most of the Fos labelling obtained in the PSD group

does not seem to be correlated with one of the vigilance states or withthe chronic stress induced by the procedure. Indeed, excepting for theDRN, neurons previously shown to be Fos-immunoreactive after W orstress (Pompeiano et al., 1992; Tononi et al., 1994; Cullinan et al.,1995) were not labelled in our PSD group. These results suggest thatW-active and stress-related neurons are not active during PSdeprivation although we cannot completely exclude the possibilitythat such neurons are active but not labelled with Fos due toautoregulation of Fos expression known to occur in the case ofexposure to chronic stimuli (Sassone-Corsi et al., 1988; Schonthalet al., 1989; Herdegen et al., 1995; Herdegen & Leah, 1998). Themost likely explanation is therefore that the Fos-positive neurons inthe PSD group correspond to neurons increasing their activity duringthe deprivation to avoid the onset of PS. These neurons can be eitherdirectly responsible for the inhibition of the onset of PS via inhibitionof PS-executive neurons or indirectly involved in such inhibition, forexample by increasing the postural tonus of the animals to preventthem falling into the water.In summary, it seems likely that the Fos-labelled neurons in our PSR

group correspond to neurons active during PS while those labelled inour PSD group correspond to neurons inhibiting the onset of PS.

Functional significance of the Fos and Fos–ChAT labellingsvisualized

Structures known to be implicated in PS onset and maintenancecontaining Fos-labelled cells specifically after PS recovery

Six pontine and two medullary structures previously hypothesized tobe implicated in PS onset and maintenance, namely the LDTg,LDTgV, PPTg, PPTgM, SLD, PnC, GiA and GiV contained asubstantial to large number of Fos-labelled neurons in the PSR group.The number of neurons was highly significantly increased in the PSRgroup compared to the two other conditions in the SLD, GiA and GiV.In the cholinergic nuclei, only a few of the Fos-labelled neurons wereimmunoreactive to ChAT. In addition, although the number of Fos-labelled neurons was high in the PnC it must be stressed that thelabelled neurons were in fact dispersed in this large structure,suggesting that only a small percentage of the neurons from thisnucleus are active during PS.Our results are only in partial agreement with the two previous

similar studies. Fos-labelled neurons were indeed also observed in thePPTg, LDTg and SLD but not in the PnC, GiA and GiV by Merchant-Nancy et al. (1995) after 60–90 min recovery from a 48-h PSdeprivation performed with the flowerpot technique. At variance withthese and our results, Maloney et al. (1999) observed with the sameprotocol as ours excepting a shorter PS deprivation (48 h in place of72 h) significantly more Fos-labelled neurons only in the LDTg andthe PnO in the PSR group compared to the PSC and PSD groups. Theyfurther showed significantly more ChAT–Fos double-labelled cells inthe LDTg and PPTg in the PSR group than the PSC and PSD groups.The differences between these and our study could be due to theshorter duration of the PS deprivation used and therefore the smalleramount of PS in their recovery group compared to our study(Merchant-Nancy et al., 19% of PS; Maloney et al., 28%; our study,49%). It could also be due to a lower specificity and sensitivity of theFos antibodies previously used. As stated by Merchant-Nancy et al.

(1995), their Fos antibody indeed also recognizes Fos-related antigenswell known to be strongly expressed in basal daylight condition incontrast to Fos which is not expressed in significant amounts in thatcondition. Such cross-reactivity could also explain why Maloney et al.(1999) did not observe an increase in the number of Fos-labelled cellsin their PSR groups in the SLD, GiV and GiA. Indeed, they reported alarge number of Fos-labelled cells in their control group in all countedbrainstem structures while we found only a few or a small number ofsuch cells in those structures in our control group (for example theycounted 66 Fos-labelled cells in the GiV per section in their PSCgroup whereas we found less than one cell per section in the samecondition). Such high numbers in their PSC group probably preventedthem from reaching statistically significant differences betweenexperimental groups for several structures.In addition, our findings that the SLD, GiA and GiV contained Fos-

positive cells specifically after PS recovery are strongly supported byour recent results in rats and previous ones in cats. We indeed recentlyshowed that desinhibition or excitation of the SLD by iontophoreticadministration of GABAA antagonists (bicuculline or gabazine) orkainic acid, a glutamate agonist, induces a state resembling PS(Boissard et al., 2002). In contrast, administration of the samecompounds in the adjacent PnO and PnC produced active waking(Boissard et al., 2002). These and our present results altogether highlysuggest that the SLD contains a large number of neurons specificallyactive during PS, playing a key role in its onset and maintenance,whereas the PnO and PnC are structures primarily involved in otherfunctions with a small subset of neurons playing a role in the state ofPS.In agreement with previous studies in cats (Sakai, 1988; Yamuy

et al., 1993; Morales et al., 1999), we also recently demonstrated, witha combination of Fos staining after 90 min of a state of PS induced bybicuculline injection in the SLD and anterograde PHA-L tracing, thatthe SLD provides an excitatory projection to the GiA and GiV(Boissard et al., 2002). Indeed, PHA-L anterogradely labelled fibrescoming from the SLD were seen around numerous Fos-labelled cellbodies in the GiA and GiV. The location of the Fos-labelled cells inthat previous study was similar to that found here in the PSR group,confirming that these two structures contain numerous neurons activeduring PS. Our and other previous studies (Holstege & Bongers, 1991;Fort et al., 1993; Rampon et al., 1996; Boissard et al., 2002) furthersuggest that these neurons are glycinergic and are responsible for thehyperpolarization of motoneurons during PS, leading to the muscleatonia occurring during this state.We observed only a small number of Fos–ChAT double-labelled

neurons in the mesopontine cholinergic nuclei (LDTg, LDTgV, PPTgand PPTgM) in the PSR group and none in the PSC and PSD groups.Of great interest, the LDTg contained a large number of ChAT-negative Fos-labelled neurons and the other nuclei a substantialnumber of such neurons specifically in the PSR group. Maloney et al.(1999) found a large number of Fos and a substantial number of Fos–ChAT double-labelled cells in these structures in all experimentalgroups, with significantly more Fos-labelled neurons in the PSR groupthan the PSD group only in the LDTg. They also found slightly butsignificantly more Fos–ChAT neurons in the LDTg and the PPTgM inthe PSR group than the PSD group. In two additional studies in catsexamining immunostaining for Fos and ChAT following a strongincrease in PS quantities induced by carbachol injection into the dorsalpontine tegmentum, either a small proportion of the cholinergic cellswere Fos-positive (Shiromani et al., 1996) or no ChAT cells weredouble-labelled (Yamuy et al., 1998). In the latter study, a largenumber of ChAT-negative Fos-labelled cells were, however, observedin the LDTg and PPTg. The authors further demonstrated that a minor

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proportion of these neurons were immunoreactive to GAD andtherefore GABAergic. These results are in agreement with those ofMaloney et al. (1999) showing a significantly higher number of GAD–Fos-labelled neurons within the LDTg, LDTgV, PPTgM and PPTg inthe PSR group than in the PSD and PSC groups. These and our resultsindicate that the LDTg and to a minor extent the other cholinergicnuclei contain a substantial to large number of noncholinergic Fos-labelled cells specifically after PS recovery, a minor proportion ofwhich are GABAergic and the majority of which use anotherneurotransmitter. These results further importantly suggest that theneurons active during PS recorded previously in these nuclei (ElMansari et al., 1989; Kayama et al., 1992) are in the vast majoritynoncholinergic. The present results are at variance with the hypothesisthat cholinergic mesopontine neurons play a crucial role in PS onsetand maintenance. This hypothesis was based on results obtained incats showing that direct administration of cholinomimetics into thepontine reticular formation causes a dose-dependent enhancement ofPS (review in Baghdoyan, 1997). Further, it has been shown in thesame species that neurotoxic lesion of the LDTg–PPTg decreases theamount of PS (Jones & Webster, 1988) and that acetylcholine releaseis significantly increased in the pontine reticular formation over W andSWS (Kodama et al., 1990; Leonard & Lydic, 1995). However, directadministration of cholinomimetics in the rat pontine reticularformation was recently shown to have either no effect on PS or toinduce only a small increase in PS quantities (review in Boissard et al.,2002). These and our present results suggest that mesopontinecholinergic neurons might not play a crucial role in PS onset andmaintenance in rats, in contrast to cats.

Structures unknown to be implicated in PS containingFos-labelled cells specifically after PS recovery

Several structures previously not identified as containing neuronsactive during PS contained a substantial to large number of Fos-labelled neurons specifically in the PSR group. These structures werethe dPAG, the LPGi, the DPGi, the MnR and the ROb. A small to alarge proportion of the Fos-labelled neurons from some of themedullary structures were immunoreactive to ChAT.

To our knowledge, only the DPGi has been implicated in the controlof PS. Indeed, we and others recently hypothesized that it contains theGABAergic neurons responsible for the cessation of activity of thenoradrenergic LC neurons during PS (Luppi et al., 1999; Kaur et al.,2001). Supporting this claim, the DPGi constitutes a major GABA-ergic afferent to the LC (Aston-Jones et al., 1986; Ennis & Aston-Jones, 1989; Luppi et al., 1995; Peyron et al., 1995), and applicationof bicuculline to noradrenergic LC neurons during PS restores theiractivity, indicating that GABA is responsible for their inactivationduring this state (Gervasoni et al., 1998). In addition, it has beenshown that bilateral stimulation of the DPGi significantly increased PSquantities, an effect blocked by picrotoxin application into the LC(Kaur et al., 2001). Further, we found that the DPGi contained a largenumber of cells labelled both with Fos and retrogradely in animalsthat received an injection of cholera toxin b subunit (CTb) in the LCand then were subjected to 72 h of PS deprivation and 3 h of PSrecovery (Verret et al., 2003a). In these experiments, a smaller butsignificant number of CTb–Fos double-labelled cells was alsoobserved in the LPGi, indicating that it could also contain a subsetof the GABAergic neurons responsible for the inactivation ofnoradrenergic LC neurons during PS. Supporting this claim, neuronswith an activity specific to PS have been recorded in the cat LPGi(Sakai, 1988). It is nevertheless unlikely that all Fos-labelled neurons

found specifically in the LPGi in the PSR group play such role. Inaddition, because the LPGi also contained significantly more Fos-labelled cells in the PSD than in the PSC group, some of the Fos-labelled neurons in the PSR group could be active during both PSdeprivation and PS recovery. Studies of the afferents and efferents ofthe LPGi Fos-labelled neurons after PS deprivation and recovery arenecessary to determine their role in particular with regards to thesympathetic nervous system because the LPGi is known to play acrucial role in its control (review in Sun, 1995).The ROb contained a substantial number of Fos-labelled neurons

specifically in the PSR group. This nucleus is known to containserotonergic neurons innervating the spinal cord (Arvidsson et al.,1992). However, it has been shown that these neurons are not activeduring PS (Sakai et al., 1983; Jacobs et al., 2002). Nevertheless,neurons specifically active during PS have also been recorded in thecat ROb (Sakai, 1988). In the present study, we found that half of theFos-labelled neurons in the PSR group were immunoreactive to ChATand therefore cholinergic. We also found that a smaller subset(! 10%) of the Fos-labelled neurons from the LPGi and DPGi wereimmunoreactive to ChAT in the PSR group. Although these choliner-gic neurons have already been described, their physiological role isstill unknown. Of relevance to this matter, electrical stimulation of thenucleus ROb has been shown to induce a pattern of sympatheticactivity similar to that occurring naturally during PS (Futuro-Neto &Coote, 1982). Additional neuroanatomical and physiological studiesare needed to determine whether cholinergic neurons of the ROb areindeed involved in such phenomena during PS.Finally, the dPAG was the brainstem structure containing the largest

number of Fos-labelled neurons following PS rebound. This is quitesurprising because this structure has never been implicated in PSregulation and is classically involved in fear, anxiety and vocalization(review in Behbehani, 1995). Additional studies are again needed todetermine the role of these neurons during PS.

Structures containing Fos-positive neurons after both PSdeprivation and PS recovery

Four structures contained more Fos-labelled cells in the PSR and PSDgroups than the PSC group, namely the lPAG and vlPAG, the DPMeand the PBL. The location of the neurons within the PBL differedbetween the two conditions, indicating that two different populationsof neurons are active during PS deprivation and PS recovery. Themajority of labelled neurons were indeed clustered in the central lateralsubnucleus in the PSR group while they were more diffusely localizedin the PBL in the PSD group. Because these structures were notpreviously implicated in PS control, additional studies are necessary todetermine their role.Numerous Fos-labelled neurons were observed with a similar

distribution in the lPAG and vlPAG and the DPMe in both PSR andPRD groups. They might therefore correspond to neurons activeduring both PS deprivation and PS recovery or to two different typesof neurons active specifically during one of these conditions. Anumber of previous studies suggest that the lPAG and vlPAG containneurons specifically active during PS while the most ventral part of theperiaqueductal grey and the adjacent DPMe contain GABAergicneurons ceasing firing during PS. Indeed, we have shown that thelPAG and vlPAG contain GABAergic neurons projecting to the DRNand LC nuclei (Peyron et al., 1995; Luppi et al., 1999; Gervasoniet al., 2000). Further, we showed that the lPAG and vlPAG containCTb–Fos double-labelled cells in animals that received an injection ofCTb in the LC and then were subjected to 72 h of PS deprivation and

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3 h of PS recovery (Verret et al., 2003a). Besides, it has been shown incats and rats that ejection of muscimol, a GABA agonist, in the mostventrolateral part of the periaqueductal grey induces a strong increasein PS quantities (Sastre et al., 1996; Boissard et al., 2000). Further, weshowed that this area and the adjacent DPMe contain GABAergicneurons projecting to the SLD and we hypothesized that these neuronsare responsible for the inactivation of the PS-active neurons of theSLD during W and SWS (Boissard et al., 2003). Altogether these andour present results suggest that the lPAG and vlPAG and the DPMecontain a mixture of neurons specifically active or inactive during PS.Additional electrophysiological, neuroanatomical and pharmacologi-cal studies are necessary to confirm the existence of these two types ofneurons.

Structures containing Fos-labelled neurons specifically after PSdeprivation

The DRN and RPa contained a substantial number of Fos-labelledcells specifically in the PSD group. These results suggest that theseneurons might be strongly active in the absence of PS. They couldbe serotonergic because these nuclei are known to contain seroton-ergic neurons which show tonic activity during W, which isdecreased during SWS, and no activity during PS (Trulson &Jacobs, 1979; Heym et al., 1982). Double-labelling experiments withserotonin and Fos are necessary to test this hypothesis. However, notall brainstem serotonergic neurons seem to be active in the PSDgroup because only a small proportion of DRN neurons wereimmunoreactive to Fos and only a few Fos-labelled neurons wereseen in other raphe nuclei such as the MnR and the ROb in thisexperimental group. Studies of the Fos-labelled cells found in theDRN and RPa might be important because they could play a crucialrole in the inhibition of PS-executive neurons during W and SWS,although recent pharmacological results suggest that serotonergicneurons from the DRN play no role in PS generation (Sakai &Crochet, 2001). In that context, neurons from the LC were not Fos-labelled in any of our experimental groups. This is not surprising forthe PSR group because noradrenergic LC neurons are known to beinactive during PS (Aston-Jones & Bloom, 1981; Gervasoni et al.,1998). However, it has been hypothesized that these neurons play akey role in the inhibition of PS during W and SWS. Moreover,Maloney et al. (1999) reported, using nearly the same protocol thanours, a decrease in the number of Fos–tyrosine hydroxylase-positiveneurons in the PSR group compared to the PSC and PSD groups.However, the difference found was only weakly significant andconcerned a very small number of LC noradrenergic neurons. Inaddition, Merchant-Nancy et al. (1995) found no significant changein the number of Fos-labelled neurons in the LC between theircontrol and PS recovery groups. Similarly, Yamuy et al. (1998)found no significant change in the number of Fos–tyrosinehydroxylase double-labelled cells between control and animals withlarge amounts of pharmacologically induced PS. From these and ourresults, it might therefore be concluded that noradrenergic LCneurons are not strongly active during PS deprivation and thereforemight play a rather minor role in the inhibition of PS onset.In conclusion, our results confirm that the SLD, LDTg, GiA and

GiV play a crucial role in PS onset and maintenance. In contrast,they suggest that the cholinergic mesopontine neurons might notplay an important role. In addition, our results reveal that manyadditional brainstem structures contain a large number of neuronsactive during PS. We in particular found that cholinergic neuronsfrom the ROb, neurons from the dLPG, lPAG, vlPAG, LPGi and

PBL were immunoreactive to Fos after PS recovery. All thesenuclei might therefore also play a crucial role in PS onset andmaintenance.These results strongly suggest that the neuronal network implicated

in PS genesis involves many more populations of neurons thanpreviously thought. They open the way to neuroanatomical andphysiological studies to identify the specific role of each populationrevealed. Studies combining Fos staining with that of neurotransmit-ters, enzymes, receptors, anterograde and retrograde tracers are alreadyunder way in our laboratory to more fully identify the populationsrevealed. In vivo and in vitro electrophysiological recordings are alsonow necessary to characterize the firing activity of these populationsof neurons across the sleep–waking cycle.

AcknowledgementsThis work was supported by CNRS (CNRS UMR5167) and Universite C.Bernard Lyon I. L.V. received a PhD grant from the French Ministere de laRecherche and Lilly Institute. We also thank Anne Berod for her photographicassistance.

Abbreviations3, oculomotor nucleus; 4, trochlear nucleus; 5, trigeminal nucleus; 6, abducensnucleus; 7, facial nucleus; 7n, facial nerve; 10, vagus nucleus; 12, hypoglossalnucleus; Amb, ambiguus nucleus; CGPn, central grey of the pons; ChAT,choline acetyltransferase; CnF, cuneiform nucleus; CTb, cholera toxin bsubunit; dPAG, dorsal periaqueductal grey; DPGi, dorsal paragigantocellularnucleus; DPMe, deep mesencephalic nucleus; DRN, dorsal raphe nucleus;DTg, dorsal tegmental nucleus; EEG, electroencephalogram; EMG, electromy-ogram; g7, genu of the facial nerve; Gi, gigantocellular reticular nucleus; GiA,gigantocellular reticular nucleus, alpha part; GiV, gigantocellular reticularnucleus, ventral part; IC, inferior colliculus; IO, inferior olivary nucleus; IP,interpeduncular nucleus; IR, immunoreactive; KF, Kolliker–Fuse nucleus; LC,locus coeruleus; LDTg, laterodorsal tegmental nucleus; LDTgV, laterodorsaltegmental nucleus, ventral part; lfp, longitudinal fasciculus of the pons; LL,lateral leminiscus; lPAG, lateral periaqueductal grey; LPGi, lateral paragigan-tocellular and rostroventrolateral reticular nuclei; mlf, medial longitudinalfasciculus; MnR, median raphe nucleus; MVe, medial vestibular nucleus; PB,phosphate buffer; PBL, lateral parabrachial nucleus; PBST, PB containing 0.9%NaCl and 0.3% Triton X-100; PBST-Az, PBSTwith 0.1% sodium azide; PCRt,parvicellular reticular nucleus; Pn, pontine nuclei; PnC, pontine reticularnucleus, caudal part; PnO, pontine reticular nucleus, oral part; PPTg,pedunculopontine tegmental nucleus; PPTgM, pedunculopontine tegmentalnucleus, medial part; PS, paradoxical sleep; PSC, PS control; PSD, PSdeprivation; PSR, PS rebound; py, pyramidal tract; RMg, raphe magnusnucleus; ROb, raphe obscurus nucleus; RPa, raphe pallidus nucleus; rs,rubrospinal tract; scp, superior cerebellar peduncle; SLD, sublaterodorsalnucleus; SO, superior olivary nucleus; Sol, nucleus of the solitary tract; Sp5,spinal trigeminal nucleus; sp5, spinal trigeminal tract; SWS, slow-wave sleep;Tz, nucleus of the trapezoid body; vlPAG, ventrolateral periaqueductal grey;VTg, ventral tegmental nucleus; W, waking.; xscp, decussation of the superiorcerebellar peduncle.

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