Alterations in c-fos expression after different experimental procedures of sleep deprivation in the cat

BRAIN RESEARCH

E L S E V I E R Brain Research 735 (1996) 108-118

Research report

Alterations in c-fos expression after different experimental procedures of sleep deprivation in the cat

Laurence Ledoux, Jean-Pierre Sastre *, Colette Buda, Pierre-Herv~ Luppi, Michel Jouvet D@artement de Mddecine Expdrimentale, INSERM U52 CNRS ERS 5645, Universit~ Claude Bernard, 8 avenue Rockefeller, 69373 Lyon Cedex 08,

France

Accepted 21 May 1996

Abstract

In the present study, we sought to examine the expression of the c-fos proto-oncogene in the cat brain after two different procedures of 24 h sleep deprivation. A first group of cats was gently sleep-deprived; they were awoken by a gentle touch of the hand (n = 5). A second group was sleep-deprived by the water tank technique which is a stressful deprivation (n = 4). A third group was placed 2 h on the water tank and was therefore stressed but not sleep-deprived (n = 2). A fourth group (control group of basal and unspecific Fos expression) was not sleep-deprived (n = 5). These four groups allowed us to separate Fos expression due to stress from Fos expression due to sleep deprivation. On the one hand, compared with controls cats, an important increase in Fos expression, detected by immunohistochemistry, was observed in the preoptic area of sleep-deprived cats by both gentle and stressful methods. On the other hand, there was a significant increase in Fos expression in the lateral hypothalamus of gently deprived cats as compared with control cats. These data indicate that c-fos expression can be employed as a marker of some putative homeostatic mechanism regulating sleep. The only sites in which there was a significant increased number of c-fos expressing neurons were located in the preoptic area which is known to be involved in sleep and in the lateral hypothalamic area.

Keywords: Sleep deprivation; Cat; c-fos; Preoptic area; Stress; Sleep

1. Introduct ion

Several hypothalamic and pontobulbar structures involved in the sleep-waking cycle have been demarcated by lesion experiments, neuron recording, anatomical, neurochemical and pharmacological studies. Some structures, however, may be unknown. Moreover, in a structure taking part in the sleep regulation, only a specific population of neurons might be involved. Neuron recording allows to determine the participation of specific neurons but can be applied simultaneously to only a few cells, the neurochemical identity of which cannot be completely demonstrated.

Recently, some authors have proposed that the expression of c-fos can serve as a marker of neuronal activity [51 ] and also of genomic activation. C-los is an immediate early gene (IEG) because its transcription is rapidly induced by extracellular stimulation [36-38] to regulate tar- get gene transcription [25]. C-fos shows increased mRNA

* Corresponding author. Fax: + 33 7877-7172; E-mail: jouvet @cism- sun.univ-lyon l.fr

levels 5 rain after the stimulation and protein peaks in the nucleus 1 to 2 h after [59]. Fos immunochemistry can provide a functional map of the brain for a particular stimulus with single-cell resolution. It also gives the oppor- tunity of demonstrating the neurochemical identity of neurons expressing Fos by double-immunostaining in combination with a cytoplasmatic marker. C-fos can be induced by many types of stimuli such as nociceptive [20], auditory [15], olfactory stimulation [56], drug administration [18], cortical lesions [22], stress [21], anesthesia [32] and behav- ioral studies [64]. Consequently, experimental conditions have to be chosen with care to obtain a Fos expression specific to a single stimulus.

The study of c-fos in relation to the sleep-waking cycle is recent, and different methods have been used. In rats, c-fos expression was studied either after sleep deprivation and spontaneous wakefulness [45,46] or auditory stimulation which increases paradoxical sleep [34]. In cats, c-fos expression has been observed only after carbachol injection in the pons [61,62,68,69].

In order to study neurons involved in the triggering of sleep, Fos expression was studied after a 24 h sleep

0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PI1 S0006- 8993(96)00599-9

L. Ledoux et al./Brain Research 735 (1996) 108-118 109

deprivation in cats. Sleep deprivation was chosen to acti- vate neurons involved in the sleep-onset because sleep deprivation is known to increase the sleep pressure according to homeostatic process [5]. The first study of Fos expression in relation to sleep deprivation was carried out by Pompeiano et al. [45] in rats. They have studied Fos expression after gentle sleep deprivation or spontaneous wakefulness. Since cats do not have a robust circadian rhythm which would permit to study spontaneous waking as in rat, our study compares Fos expression after gentle sleep deprivation with Fos expression after stressful sleep deprivation or controls.

2. Materials and methods

A total of 16 cats (2.5-4 kg, male) were used in this study. They were divided into four groups: gently sleep- deprived cats (GD) n = 5 (U129, A130, G130, J130 and V130), controls of gently deprived cats (CGD) n = 5 (X129, W129, El30, K130 and O130), stressed and deprived cats (SD) n = 4 (V129, Z130, T131 and G132) and stressed cats (SC) n = 2 (S131 and D132).

2.1. Electrode implantation

Under pentobarbital anesthesia (Nembutal, 25 mg /kg , i.v.), GD and CGD cats were chronically implanted with extradural electrodes for the polygraphic monitoring of sleep-wakefulness states. Stainless steel screws were placed bilaterally over the orbits to record the electro-oculogram (EOG) and over the frontal and occipital cortices to record the electroencephalogram (EEG). Nuchal muscle activity was detected by bared loops of stranded stainless steel wire inserted deep in the neck muscles. All the electrodes were connected to a plug (Connectral) which was ce- mented to the skull with dental acrylic resin. No electrode was implanted in the lateral geniculate nucleus because cortical lesions induce Fos expression [10,70].

2.2. Polygraphic data recording and analysis

The cats were then placed in a sound-attenuated ther- mostated cage with 24 h continuous dim light and fed at 8.30 a.m. every day. Polygraphic recording (24 h /day) began about 3 - 4 days after the surgical operation, it was handscored minute by minute according to previously described criteria for wakefulness (W), light slow-wave sleep (SWS1), deep slow-wave sleep (SWS2) and paradoxical sleep (PS) [44].

2.3. Sleep deprivation

After two weeks spent in the cage (habituation period), GD cats were sleep-deprived for 24 h (n = 2) or for 15 h (n = 3): they were awoken by a gentle touch of the hand

when the polygraphic record showed the beginning of SWS2 or PS. Each CGD cat was polygraphically recorded in parallel of one GD cat during the habituation period and the sleep deprivation. CGD cats were placed in the same environmental conditions (light, temperature, sound, feed time) as GD cats but were allowed to sleep during the sleep deprivation of the GD cats. They were sacrificed less than 1 h after them. CGD cats were necessary to control basal and unspecific Fos expression.

2.4. Stress and deprivation

SD cats were sleep-deprived by the flower pot method for 24 h whereas SC cats were placed on the flower pot for only 2 h. The water tank technique is known to induce stress [7,27,39,63,67]. The group of animals placed 24 h on the tank (SD cats), was sleep-deprived and stressed. In similar conditions, Sallanon et al. [55] in our laboratory have found that the amounts of sleep were: SWS1; 31.3 _ 2.3%, SWS2; 1.6 _+ 1% and PS; 0 versus SWS1; 37.1 _+ 2,2% SWS2; 9.6 _+ 1% and PS; 13.7 _+ 0.5% in control. The group placed only 2 h on the tank (SC cats), was stressed but not sleep-deprived since cats can be spontaneously awake during 2 h. SD and SC cats were sacrificed between 3 and 5 p.m. at the end of the period spent on the flower pot. These two groups form stress controls.

2.5. Perfusion and fixation

At the end of the sleep deprivation, animals were sacrificed under deep pentobarbital anesthesia (30 mg/kg , i.v.) by transcardial perfusion of Ringer Lactate with hep- arine (5 UI /ml) , followed by a perfusion of an ice-cold fixative containing 4% paraformaldehyde, 0.1% picric acid in 0.1 M phosphate buffer (PB, pH 7.4). The brains were cut into several blocks and post-fixed for 48 h at 4°C in 0.1 M PB containing 2% paraformaldehyde and 0.1% picric acid. These blocks were transferred to a 30% sucrose in 0.1 M PB for 48 h at 4°C, and then 25 I~m frozen frontal sections were cut on a cryostat. They were collected in 0.1 M phosphate buffer saline (PBS) containing 0.3% Triton X-100 (PBST) and 0.1% sodium azid (PBST-Az).

2.6. Immunocytochemistry

Sections were incubated for 96 h at 4°C in 0.1 M PBST-Az containing a rabbit polyclonal antibody to fos (1:1000, Oncogene Science, Uniondale, NY). After two rinses in PBST, sections were transferred for 1.5 h to a biotinylated goat antibody to rabbit immunoglobulin solu- tion (Vector Laboratories, Burlingame, CA) at 1:2000 at room temperature. After two more rinses, sections were incubated for 1.5 h in an Avidin Biotin HRP Complex (Elite Kit from Vector Laboratories, Burlingame, CA) diluted at 1:400 in PBST at room temperature. After rinses, sections were developed for 15 min in 0.05 M

110 L. Ledoux et al. / Brain Research 735 (1996) 108-118

T r i s - H C 1 b u f f e r ( p H 7 .6 ) c o n t a i n i n g 0 , 0 2 % 3 - 3 ' - d i a m i n o -

b e n z i d i n e s o l u t i o n , 0 . 0 0 3 % h y d r o g e n p e r o x i d e a n d 0 . 6 %

n i c k e l a m m o n i u m s u l f a t e . T h e r e a c t i o n w a s s t o p p e d b y

w a s h e s in a P B S T - A z s o l u t i o n . S e c t i o n s w e r e c o u n t e r -

s t a i n e d b y n e u t r a l red . T h e i m m u n o c y t o c h e m i s t r y o f s ec -

t i o n s f r o m t h e b r a i n o f o n e g e n t l y s l e e p - d e p r i v e d ca t w a s

c o n d u c t e d s i m u l t a n e o u s l y w i t h t h e i m m u n o c y t o c h e m i s t r y

o f s e c t i o n s f r o m t h e b r a i n o f o n e c o n t r o l cat .

T h e p o l y c l o n a l a n t i b o d y u s e d is s p e c i f i c to F o s p r o t e i n

( r e s i d u e s 4 to 17): it r e c o g n i z e s n e i t h e r t he f o s - r e l a t e d

a n t i g e n s n o r t he f o s - a s s o c i a t e d p r o t e i n [19] in t h e rat . S i n c e

t h i s s p e c i f i c i t y h a s n o t b e e n p r o v e d in t h e ca t w e wi l l u s e

F L I ( f o s - l i k e i m m u n o r e a c t i v i t y ) i n s t e a d o f c - f o s i m m u n o -

r e a c t i v i t y .

T h e n u m b e r o f p o s i t i v e ce l l s w a s q u a n t i f i e d w i t h a n

i m a g e a n a l y z e r ( B i o c o m ) in b r a i n s t r u c t u r e s o f p a r t i c u l a r

i n t e r e s t f o r t h e s l e e p - w a k e c y c l e a n d b r a i n s t r u c t u r e s w h i c h

p r e s e n t i m p o r t a n t F o s i m m u n o s t a i n i n g ( s e e T a b l e 1). F o r

e a c h cat , t h e q u a n t i f i c a t i o n w a s d o n e fo r e a c h p l a n e f r o m

A 8 to A 1 6 a n d fo r A 0 . 6 , P2 , P3 , P 1 2 . 7 a n d P 1 3 . 5 o f

Table I Quantitative evaluation of Fos expression levels in different brain regions of controls of gently sleep-deprived cats (A = (B = GD) and stressed and sleep-deprived cats (C = SD)

CGD), gently sleep-deprived cats

Regions A: CGD = 5 B: GD = 5 C: SD = 4 n Multiple-range test

moy S.E.M. moy S.E.M. moy S.E.M.

Cingulate cortex 0.5 0.3 ll) 5 47 8.6 25 AB; C Sulcus ectosylvius anterior: ECSA 0 0 2.3 1.5 3.2 1.7 18 Caudate nucleus: CA 0.7 0.3 9.1 2.6 5.5 2.0 70 Septofimbrial nucleus of the septum: SFN 2 1.2 6.2 1.5 11 2.5 37 AB; BC Nucleus accumbens: ACC 1.2 0.6 6.9 2.4 7.6 3.9 23 Nucleus of the anterior commissure: ACN 0.5 (1.2 3.3 1 4.4 1.3 35 Nucleus of the stria terminalis: ST 2.5 0.9 15 3.9 26 5.7 39 AB; BC Nucleus of the diagonal band of Broca: DB B 5 1.5 5.3 2.1 35 12.1 22 Substancia innominata: SI 0.4 0.2 1.1 0.6 12 7.4 20 Preoptic area lateral division: LPO 7.5 1.6 26 4. l 56 8.4 55 AB; C Preoptic area median division: MPO 25 5.7 65 8.6 93 12.9 56 A; BC Supraoptic nucleus: SON 1 0.5 0.6 0.2 3.9 1.8 52 Suprachiasmatic nucleus: SCH 51 15 63 10 85 22.4 38 Anterior hypothalamic nucleus: AH 0.3 0.2 11 5.9 19 11.5 18 Paraventricular nucleus: PAH 5.2 1.7 3.3 0.8 1 I 4.3 59 Periventricular complex: PEH 5.2 1.3 6.7 1.3 10 2.2 63 Dorsomedial hypothalamic nucleus: DMH 5 1 9.8 3 15 4.9 37 Ventromedial hypothalamic nucleus: VMH 1.9 0.5 3.8 0.9 14 5.7 26 AB; C lnfundibular nucleus: IN 2.7 0.7 3.3 0.6 20 3.4 73 AB; C Dorsal hypothalamic nucleus: DH 2.7 0.8 15 3.6 4.5 2.0 17 AC; BC Lateral hypothalamic area: HLA 2.9 0.5 9.4 1.2 23 3.8 134 A; B; C Dorsal hypothalamic area: HDA 11 2.5 24 3.5 32 7.3 109 AB; BC Posterior hypothalamic area: HPA 5.4 1.1 15 2.9 61 11.8 6/) AB; C Tuberomamillary nucleus: TM 1 0.3 2.8 1.5 14 4.4 15 AB; C Anterior mamillary nucleus: MA 3.2 1.9 6.6 2.2 76 31.1 19 AB; C Premamillary nucleus: SMX 2 0.8 5.2 2 4.8 2.3 21 Supramamillary nucleus: MS 0.3 0.2 2.3 1.1 4 1.4 21 Medial mamillary nucleus: MM 0.8 0.4 5.6 2.4 7.3 2.4 21 Lateral mamillary nucleus: ML 0.3 0.2 0 0 0.3 0.3 18 Nucleus of the field of Forel: FF 2.4 0.6 6.8 2.1 17 4.8 23 AB; BC Subthalamic nucleus: SUB /).6 0.4 l) 0 0.3 0.3 24 Periacqueductal gray: PAG 3.6 0.8 13 3 20 8.2 24 Pontine gray: PGM 10 2.7 37 5.7 44 17.7 24 Colliculi 5.6 2.2 8.6 3.2 21 11.2 24 Locus coeruleus proper: LC 1.1 0.4 1.4 0.5 2.9 1.3 25 Locus coeruleus alpha: LCa 0.1 0.1 0.1 0.1 2.4 0.8 26 AB; C Peri-locus coeruleus alpha: LCp&ia 0 0 0.2 0.1 0.4 0.3 26 Locus subcoeruleus: LSC 0.1 0.1 0.6 0.2 1.8 0.7 48 AB; BC Nucleus laterodorsalis tegmenti: Ldt 0.5 0.3 0.7 /).4 5 1.5 44 AB; C Raphe dorsalis: DRM 0 0 3 1.3 8.4 7.4 14 Cuneiformis nucleus: CNF 3.9 0.8 4.5 1 4.5 1.9 28 Parabrachialis lateralis: PbL 1.2 0.6 2.1 0.6 1.5 0.5 48 Parabrachialis medialis: PbM 0.2 0.1 0.2 0.1 1.1 0.5 46 Kolliker Fuse: KF 0.6 0.3 1.3 0.6 1.4 1.0 23

The relationship among experimental groups (A, B and C) was examined by multiple range analysis with the level of significance set at P < 0.05. The experimental groups are separated by a semi-colon when they are statistically different (n: total number of half-sections examined by structure in the three different groups). Nomenclature according to Bleier [4], Reinoso-Suarez [48], Berman et al. [2,3] and Sakai [52].

L. Ledoux et al. / Brain Research 735 (1996) 108-118 111

Berman's atlas [2,3]. Finally, the variations of the number of FLI in the different groups was statistically analyzed by using one-way analysis of variance. The relationships among experimental groups was further examined by multiple-range test with the level of significance set at P < 0.05.

3. Results

3.1. Polygraphic recording

intensity of nuclear immunostaining of different neurons within a section even in the case of neighboring ceils.

The difference in the number of Fos-like immunoreac- tive cells between the different groups is described below. Only in the suprachiasmatic nucleus (SCH), do the variations in the number of cells not seem to depend on the experimental paradigm (stress, sleep deprivation) as shown in Table 1.

3.3. Comparison between gently sleep-deprived cats (GD) and controls of gently sleep-deprived cats (CGD)

During sleep deprivation, only SWS 1 was allowed. The experimenter gently touched the cat when the polygraphic recording showed the first signs of SWS2 or PS. Each cat presents a specific sleep deprivation pattern, the number of interventions varying from 0 to 30 per h. Some cats were almost spontaneously sleep-deprived while others had to be frequently awoken.

3.2. General presentation of c-fos immunoreactivity

Fos-like immunoreactivity was restricted to the cell nucleus as shown in Fig. 1. There were variations in the

Fos-like positive cells were scattered throughout all the lateral preoptic area (LPO) and medial preoptic area (MPO) (Figs. 1 and 2). As shown in Table 1 and Fig. 2, a significant difference between GD and CGD cats was observed in the medial division of the preoptic area. There was a significantly larger number of FLI neurons in the MPO of the GD cats (65 _+ 8.6) than in CGD (25 _+ 5.7). This number is the basal expression of Fos which is therefore low. There was no significant difference in Fos expression in the lateral preoptic area, the stria terminalis, the nucleus of the diagonal band of Broca, the cingulate cortex, the supraoptic nucleus and the brainstem. However,

0

100 ~m

t

d

l i b

Fig. 1. A: a microphotograph of a frontal section of the brain (A14 of Berman's atlas), illustrating the distribution of Fos immunohistochemistry in the preoptic area and the suprachiasmatic nucleus of a sleep-deprived cat. The section is counterstained with neutral red. LPO,MPO: lateral, medial preoptic area; SCH: suprachiasmatic nucleus; V3: third ventricule; OC: optic chiasma. B: microphotograph illustrating the nuclear Fos immunohistochemistry (the section is counterstained with neutral red).


A

SI

G E N T L Y D E P R I V E D C O N T R O L S T R E S S E D A N D D E P R I V E D

SI

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: ' ~ " " S I " : ° . .

i" • -.. ~ : " DBH-. ~" " DBH "I

• : . : ' : ~ . - . . . . ' " ..

• S T I C

• :- , ,~6 . • ~ ' ~ " . L ~ ~. . . . - ,

"i" " ' ~ " •

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Fig. 2. The distribution of Fos immunoreactivity in three cats, a gently deprived cat (U129), a control cat (X129) and a stressed and deprived cat (G132). A series of Biocom drawings of 25 Ixm frontal sections from rostral (A) to caudal (I) illustrates the distribution of Fos immunoreactivity after gentle sleep deprivation, no treatment or stressful sleep deprivation. Each dot represents a Fos positive neuron. ACC: nucleus accumbens; ACN: nucleus of the anterior commissure; CA: caudate nucleus; DBB: nucleus of the diagonal band of Broca; DH: dorsal hypothalamic nucleus; DMH: dorsomedial hypothalamic nucleus; EN: entopeduncular nucleus; FF: nucleus of the field of Forel; HAA: anterior hypothalamic nucleus; HDA: dorsal hypothalamic area; HL: lateral habenular nucleus; HLA: lateral hypothalamic area; HM: medial habenular nucleus; HPA: posterior hypothalamic area; IC: internal capsule; INF: infundibular nucleus; LGL: lateral geniculate nucleus; LPO: lateral preoptic area division; MA: anterior mamillary nucleus; ML: lateral mamillary nucleus; MM: medial mamillary nucleus; MPO: median preoptic area; MS: supramamillary nucleus; OT: optic tract; PAH: paraventricular nucleus; PARA: anterior paraventricular nucleus of the thalamus; PEH: periventricular complex; PVH: parvocellular hypothalamic nucleus; PP: pes pedunculi; RE: reticular complex of the thalamus; RF: retroflex bundle; SCH: suprachiasmatic nucleus; SFN: septofimbrial nucleus of the septum; SI: substantia innominata; SMT: stria medullaris thalami; SMX: premamillary nucleus; SUB: subthalamic nucleus; SON: supraoptic nucleus; SPF: subparafascicular nucleus; ST: nucleus of the stria terminalis; TM: tuberomamillary nucleus; VMH: ventromedial hypothalamic nucleus: ZI: nucleus of the zona incerta (Bleier [4], Reinoso-Suarez, [48]; Berman et al. [3]).

L. Ledoun et al. /Brain Research 735 (1996) 108-118 113

GENTLY DEPRIVED STRESSED AND

DEPRIVED

Fig. 2 (continued).

there were significantly more FL1 neurons in the lateral hypothalamic area of the GD groups than in the control. The distribution of Fos-like immunoreactivity in one GD

cat (U129) and one CGD cat (X129) is illustrated in Fig. 2.

3.4. Comparison between stressed and depriued cats (SD) and stressed cats (SC)

In SD and SC groups Fos expression was found in a large number of neurons in the cingulate cortex (47 Ifr 9 vs. 95.7 k 91, the colliculi (21 f 11 vs. 46.7 f 20) and the pontine gray (44 f 18 vs. 110 k 4). An important differ-

ence in the number of Fos-like positive cells was found in both the medial and the lateral division of the preoptic area. In SD cats a large or very large number of Fos-like positive cells (93 k 13 in the MPO and 56 + 8 in the LPO) was observed whereas in SC cats, Fos was expressed in a few or a moderate number of neurons (23.3 f 4 in the MPO and 13 f 4.2 in the LPO). A lower difference has been found in the stria terminalis, the dorsal hypothalamic area, the anterior mamillary nucleus and the dorsal raphe nucleus (Table 1). In other structures no important varia- tion was observed.

The distribution of Fos-like immunoreactivity in one


GENTLY DEPRIVED

G

IC

O

I

C O N T R O L

k/

ic

STRESSED AND DEPRIVED

H

T P S

PA

[

"

Fig. 2 (continued).

SD cat (G132) is illustrated in Fig. 2 together with the Fos immunoreactivi ty distribution of one GD cat (U129) and one CGD cat (X129). These three cats are examples and should not be considered as representative of all cats.

3.5. Comparison between gently deprived cats (GD) and stressed and deprived cats (SD) and stressed cats (SC)

The number of Fos-l ike positive cells was higher in the cingulate cortex, the periaqueductal gray, the pontine gray and the colliculi in the stressed and deprived cats and in the stressed cats with respect to both gently deprived cats and controls of gently deprived cats (Table 1). A lower

difference was observed in the supraoptic nucleus and the diagonal band of Broca between stressed cats (SD and SC) and GD and CGD cats. In the preoptic area there were more FLI neurons in the medial and lateral preoptic area of the SD cats than in the GD and the SC cats (MPO: 23.3 _+ 4; LPO: 13 + 4 for the SC cats, see Table 1 for SD and GD groups).

3.6. C-fos expression in other areas involved in sleep regulation

In the reticular magnocellular nucleus and in all the pontine regions known to be active either during paradoxi-

L. Ledoux et al. / Brain Research 735 (1996) 108-118 115

cal sleep, (laterodorsalis tegmenti, locus coeruleus peri alpha, locus subcoernleus, parabrachialis lateralis nuclei) or during waking (locus coernleus proper, raphe dorsalis) [23,52,53] no important difference in the levels of Fos-like positive cells was observed between the CGD and GD groups. However the number of FLI neurons was significantly higher in SD group than in both CGD and GD groups for the nuclei laterodorsalis tegmenti, locus subcoeruleus and locus coeruleus alpha (Table 1). In the posterior hypothalamus, neurons of the tuberomamillary area involved in waking (principally histaminergic neurons) [30] did not express more Fos protein in GD cats than in CGD cats (Table 1).

4. Discussion

The main result may be summarized as follows: a sleep deprivation, whether it be gentle or stressful, induces a significant increase in Fos protein in the medial preoptic area and the lateral hypothalamic area and a slighter, non-significant, increase in the stria terminalis of gently deprived cats (GD) and of stressed and deprived cats (SD) with respect to control (CGD) and stressed cats (SC). Besides, in the cingulate cortex and some pontine regions, there are more Fos-like positive cells in stressed and deprived cats (SD) and stressed cats (SC) than in gently deprived cats (GD).

Fos immunoreactivity has been useful to map the cell activation in studies of nociception [28] and audition [15]. But some studies have shown that Fos expression differs from that of 2-deoxyglucose [ 12] suggesting that Fos is not expressed in all activated cells. One hypothesis is that multiple but not all second messengers induce c-fos expression [36]. This restriction has to be kept in mind. Moreover, c-fos being induced by different kinds of stimuli [38], experimental conditions must be chosen with care to obtain a Fos expression specific of the sleep deprivation induction. It is important to place cats in stable conditions of temperature, sound and light [50] because important hyperthermia [57] and a high-sound level [29] induce Fos expression in the preoptic area (POA). The light influence on c-fos expression depends on circadian rhythms [1,13,14,47,58] but cats do not have a well-known circadian rhythm. Finally, the perfusion was done less than 5 rain after anesthesia to eliminate the synthesis of Fos protein known to occur after anesthetic injection [32].

Therefore the most important problem is stress. As extensively reported, stress induces Fos in many areas [8] that are not all directly related to stress. Thus the sleep deprivation has to be conducted with as little stress as possible. In order to achieve this goal, cats were accustomed to the experimenter who took care of them every day. In addition, during the sleep deprivation, SWS1 was allowed in order to avoid too much stress. Furthermore, to test the influence of stress, cats were placed on the water

tank for 24 h (SD) or 2 h (SC) (see Section 2, Materials and methods). Both groups were stressed but only SD cats were sleep-deprived since even in our CGD it was not unfrequent to observe spontaneous arousal lasting 2 h or more. After such a procedure, a larger number of positive cells was observed in the cingulate cortex of SD and SC cats - a region where neurons express Fos in case of stress [21] - with respect to GD and CGD cats. Besides, approxi- mately the same difference was found in the number of Fos positive cells in the medial preoptic area between SD and SC cats and between GD and CGD cats. For this reason, it is likely that a large number of Fos positive neurons in the medial preoptic area after sleep deprivation is related to the sleep deprivation per se and not only to stress. Moreover, since the number of Fos positive cells is higher in the medial preoptic area of stressed and deprived cats than in the medial preoptic area of gently deprived cats, Fos expression in the stressed and deprived cats might represent Fos expression due to deprivation plus Fos expression due to stress.

We have observed differences in Fos expression between cats of the same group. These differences illustrate the difficulty in studying sleep regulation in cats. Indeed, rats always belong to the same genetic strain, which is not the case with cats. Some cats sleep much more than others and there is no satisfactory 'sleep pressure' index enabling us to measure homeostatic regulation. We could have measured the sleep pressure by depriving the cats and registering their rebound before the experiment. But in preliminary experiments, we have shown that some cats became accustomed to the procedure. Moreover, an habituation prevents Fos expression [33,43].

In rats, several groups have studied the expression of the c-fos proto-oncogene (mRNA or protein or both) during the sleep-waking cycle. Using Northern blot analysis, O'Hara et al. [40] have shown that in the hypothalamus the c-fos mRNA level was very low, whereas the highest level of c-fos mRNA appears, after a sleep deprivation, in the cerebellum, an area in which we did not observe any Fos positive cells. Grassi-Zucconi et al. [17] also using North- ern blot analysis have observed that the c-fos mRNA expression in the pons is generally high during the active period but low during the rest period. On the one hand, this study is in agreement with the data of Pompeiano et al. [45,46] who have studied both c-fos mRNA (by in situ hybridization) and protein expression after sleep deprivation or spontaneous wakefulness. They have observed high levels of Fos positive cells in the POA, especially the medial preoptic area. However, contrary to the results of these authors [66], in our cats the increase in FLI neurons in the pons, was not significant. On the other hand, the increase in paradoxical sleep (PS) duration induced either by auditory stimulation or by sleep deprivation is accom- panied by an increase in Fos expression in various structures of the brainstem known to have PS-on cells, in the suprachiasmatic nucleus and in the lateral and dorsal hy-


pothalamic areas but not in the preoptic area [34,35]. This would suggest that after a certain amount of sleep recovery, the c-fos transcriptional cascade is turned off.

In cats, Fos expression in the pons and medulla has been studied after carbachol injections in the medial pontine reticular formation [61,62] or the rostral pontine tegmentum [68,69]. Such injections are followed by PS-like hypersomnia. Shiromani et al. [61,62] have observed higher levels of Fos positive cells in many pontine structures known to be implicated in PS generation, in carbachol- treated animals with respect to controls. Moreover, they found more Fos positive cells with longer PS bouts than with shorter PS bouts. Yamuy et al. [68,69] examined a more caudal part of the brainstem and their data are in agreement with those of Shiromani et al. [61,62]. But among regions which express high levels of Fos positive cells in carbachol-treated animals, some are known to have a specific discharge rate during PS (medial pontine reticular formation, laterodorsalis tegmenti and pedunculopon- tine tegmental nuclei) while others (locus coeruleus and dorsal raphe nuclei) have a discharge rate which stops during PS. The same observation could be made concerning Fos expression in the preoptic area: many electrophysi- ological data show that a majority of neurons in the preoptic area have a higher discharge rate during sleep than during waking [24,41], whereas Szymusiak and McGinty [65] demonstrated that there were more wake active cells in the adjacent basal forebrain area than sleep active cells. In our sleeping control cats we have not found any correlation between the number of Fos-like positive cells in the medial and lateral preoptic area and the amount of SWS2 and PS during the 60 and 180 min preceding the sacrifice. As shown in Table 1 there is a significant increase in c-fos like activity in the medial preoptic area of the gently deprived cats. Moreover this increase is even more important in stressed deprived cats. For this reason it is logical to assume that the expression of the fos like protein during the preceding 60 to 120 rain is mostly related with waking. However there are numerous physiological data which suggest that the preoptic area is the only region for which the lesion with ibotenic acid [54] record- ings [65] and pharmacological experiments [31] are in favor of a important hypnogenic function. Moreover, in the rat, local injection of c-los antisense in the medial preoptic area is followed by a secondary insomnia [6]. Accordingly, it is not unlikely that the c-fos like protein level might also represent a molecular correlate of the homeostatic control of sleep since it has been shown that stressed deprivation is followed by a larger rebound of sleep than gentle deprivation [42]. Thus it may be suggested that Fos could be expressed in wake active neurons and represents an index of previous wakefulness if not of a sleep need or a sleep pressure. It has been suggested earlier that the release of 5HT during wake would be involved in the preparation of sleep by triggering the synthesis of hypnogenic sub- stances within the preoptic area [9]. Further studies are

needed in order to determine whether fos-like immunore- active cells in the preoptic area receive serotoninergic innervation and whether Fos is associated with the increased expression of neuropeptides known to be implicated in the control of sleep (as VIP) [ 11,16,49]. Moreover, our data do not disclose any significant difference between c-los like expression in both the diagonal band of Broca and the substantia innominata. This may indicate that those two structures may not regulate sleep in the same way as the preoptic area. Finally, we do not have any explanation concerning the increase in FLI neurons in the lateral hypothalamic area since this structure is not yet known to participate in the regulation of sleep.

Since the acceptance of the present paper, an important article appeared concerning c-fos expression in the hypothalamus of the rat after sleep deprivation. Sherin et al. [60] by using a retrograde tracer, the cholera toxin B, in combination with c-fos immunocytochemistry have found a small group of c-fos positive cells in the ventrolateral preoptic area, projecting to the posterior hypothalamus, which number correlates positively with the amount of recovery sleep after sleep deprivation. It is difficult to compare our data since they have been obtained in different species. Moreover we have not sacrificed our cats during the rebound of sleep which would have followed both gentle and stressed deprivation. However, as dis- cussed above, we have not found any correlation between the number of Fos-like positive neurons in the medial and lateral preoptic area or any other hypothalamic area and the amount of SWS2 and PS during either the 60 or 180 min which preceded the sacrifice in our sleeping control group (CGD). This is different from the findings of Sherin et al. [60].

Further experiments are certainly needed in order to understand our results. It has been shown that there is a direct projection from the medial preoptic area to the ventrolateral periaqueductal gray: a key structure control- ling PS [26].

Acknowledgements

The investigation in this report was supported by IN- SERM U52, CNRS ERS 5645 and DRET 91-130. The authors want to thank Dr. Sakai for his helpful advice concerning the hypothalamus anatomy, Catherine Limoge for correction of the English and Gabriel Debilly for his assistance in statistical data analysis.

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Alterations in c-fos expression after different experimental procedures of sleep deprivation in the cat