The PS deprivation procedure was effective in producing a near complete elimination of PS in the group of rats perfused after the 72 h of deprivation (PSD group) and also in that subjected to the same deprivation and then allowed to recover for three hours before perfusion (PSR group). These two groups of rats presented no significant difference in their quantities of PS (PSD: 4.4 ± 2.5 min, PSR, 4.8 ± 3.0 min), SWS (PSD: 1348.5 ± 80.0 min, PSR, 1375.6 ± 136.6 min) and W (PSD: 2965.9 ± 83.2 min, PSR, 2929.3 ± 129.2 min) during the 72 h of deprivation.

During the 150 min prior to sacrifice, the quantities of PS were significantly different in the PSD and PSR groups and in the group of control animals (PSC group) (p < 0.05). The state of PS constituted 16 % of the last 150 minutes prior to sacrifice in the PSC group, 0 % in the PSD group and 44 % in the PSR group (Table 1). There was a significant decrease in the time spent in slow wave sleep (SWS) in PSD (30 %) and PSR (33 %) conditions as compared to the PSC (48 %) condition (p < 0.05). The PSD group presented a large quantity of waking (W) (70%) compared to the PSC (36%) and PSR (23%) groups. The increase in PS quantities during the last 150 minutes prior to sacrifice in the PSR group compared to the PSC group was due to a significant increase in the duration of the PS bouts (PSC, 1.2 ± 0.1 min; PSR, 2.5 ± 0.2 min) (p < 0.001). The number of bouts did not vary significantly between the two groups (PSC, 21.2 ± 2.3; PSR, 27.2 ± 3.9).

The number of Fos+ neurons located in the posterior hypothalamus as a whole was highly significantly increased in the PSR group compared to the PSC and PSD groups (Figs. 1 and 2) (Table 1). The mean number of Fos+ neurons was superior in the PSD group compared to the PSC group but it did not reach statistical significance (Table 1). Across conditions, the number of Fos+ neurons in the posterior hypothalamus was positively correlated with the percent PS (R = 0.648, p < 0.05) and showed no correlation with the percent wake (R = 0.337) or the percent SWS (R = 0.418).

The largest number of Fos+ neurons in the PSR group was localized in the lateral hypothalamic area (151.6 ± 12.2 neurons per hemi-section). A substantial number of labeled cells was also observed in the rostral part of the zona incerta (77.0 ± 17.6 neurons per hemi-section), perifornical nucleus (55.7 ± 4.9 neurons per hemi-section), dorsal hypothalamic area (56.2 ± 9.7 neurons per hemi-section) and ventromedial hypothalamic nucleus (50.0 ± 15.7 neurons per hemi-section). A small number of cells was localized in the dorsomedial hypothalamic nucleus (36.7 ± 6.8 neurons per hemi-section) and the caudal part of the zona incerta (22.5 ± 3.6 neurons per hemi-section). In all these structures, excepting the lateral and dorsal hypothalamic areas, the number of Fos+ cells in the PSC and PSD group was very limited and not statistically different between the two groups and significantly inferior to the PSR group. In the lateral and dorsal hypothalamic areas, the number of Fos+ neurons was significantly superior in the PSD group compared to the PSC group (p < 0.05) and significantly inferior compared to the PSR group (P < 0.05).

In all groups, the arcuate nucleus of the hypothalamus contained only a few Fos+ cells.

In all groups, only a few to occasional hypocretin neurons expressed Fos (PSR, 2%; PSD, 3%; PSC, 0.1% of all the hypocretin neurons counted) (Fig. 1A) (Table 1). The number of Hypocretin+/Fos+ cells was neither correlated with the percent waking (R = 0.396), the percent SWS (R = -0.455) nor the percent PS (R = -0.19).

In contrast to hypocretin, the majority of the MCH+ cells were immunoreactive to Fos in the recovery condition (Fig. 1B, Fig. 2C) whereas only a few were Fos+ in the deprivation and control conditions (PSR, 58%; PSD, 0.2%; PSC, 0.1% of all the MCH neurons counted) (Fig. 2) (Table 1). Across conditions, the number of MCH+/Fos+ cells was significantly and positively correlated with the percent time spent in PS (R = 0.917, p < 0.0001) and negatively correlated with the percent waking (R = -0.65, p < 0.05). It was not significantly correlated with the percent SWS (R = -0.28). In the PSR condition, the MCH+/Fos+ neurons made up for 76 % of the Fos+ neurons localized in the perifornical area, 60% of the Fos+ neurons in the lateral hypothalamic area and 34% of the Fos+ neurons in the rostral zona incerta.

The injection of 0.02, 0.2, 1 and 5 μg of MCH into the left lateral ventricle of rats at dark onset induced a dose-dependant and significant increase in the amount of PS (Fig. 3, Table 2). Injection of 10 μg of MCH had no significant effect. The strongest increases in PS quantities were obtained with 0.2 and 1 μg MCH doses. The maximum increase with these two doses compared to NaCl was seen during the first two hours (0.2 μg, 198%; 1 μg, 174%) (p < 0.01). The increase of PS quantities following 0.2 and 1 μg injections was visible during 8 and 4 h, respectively (Fig. 3, Table 2). The injection of 0.02 μg of MCH induced a smaller increase in PS quantity compared to NaCl with a maximum increase again during the first two hours (137%) (p < 0.05). With this dose, PS quantities remained significantly increased only during the first four hours compared to saline. The 5 μg MCH dose significantly increased PS quantities compared to NaCl but only during the third and fourth hours (119%)(p < 0.01).

For the 0.2, 1 and 5 μg doses, the increase in PS quantities was due to an increase in the number of bouts of PS and not in their duration (Fig. 4). In the case of the 0.02 μg injections, the number of bouts and their duration were not significantly modified. For all doses, the latency of the first PS episode was unchanged compared to NaCl.

In addition to the effect on PS, a significant increase in SWS and a decrease in W quantities were obtained after administration of 0.2 and 1 μg of MCH compared to NaCl (p < 0.05) (Fig. 3, Table 2). Injection of 10 μg of MCH also induced an increase in SWS and a decrease in W quantities compared to NaCl, which did not reach statistical significance. No effect on SWS and W was seen with 0.02 and 5 μg doses compared to NaCl. The increase in SWS quantities following 0.2 and 1 μg MCH injections was maximal during the first two hours (0.2 μg, 69%; 1 μg, 73%) (p < 0.05) (Fig. 3, Table 2). The maximum decrease in W amounts was seen during the first 2 hours for 0.2 μg (-25%) and the third and fourth hours for 1 μg (-35%). The 0.2 and 1 μg MCH injections significantly increased SWS and decrease W quantities during 2 and 6 h compared to NaCl, respectively (Fig. 3, Table 2).

All animals were housed and cared for according to the National Institute of Health "Guide for the care and use of laboratory animals" (NIH publication 80–23). Male Sprague-Dawley rats (280–320 g, n = 25; IFFA Credo) were prepared for electroencephalogram (EEG) and electromyogram (EMG) monitoring under chloral hydrate anaesthesia (400 mg/kg, i.p.) as previously described 23. They were placed under a 12 h/12 h light/dark cycle (lights on from 6:00 AM to 6:00 PM) and constant temperature (23°C) in a Plexiglas jar for at least 8 days of habituation.

EEG and EMG recordings were collected on a computer via a CED interface using the Spike 2 software (Cambridge Electronic Design). Vigilance states were discriminated with the aid of EEG and EMG data as previously described 23.

PS deprivation was performed during 72 h using the classical flowerpot method 181937. Food and water were accessible ad libitum to animals. A 24 h baseline recording was first performed for all animals. Then, the rats were divided in control (PSC, n = 4), PS deprivation (PSD, n = 4) and PS rebound (PSR, n = 4) groups. The animals of the PSC group remained in their recording jar for 5 days before anaesthesia and perfusion at 5:00 PM. The animals of the PSD group were placed on platforms (6.5 cm in diameter) over water at 1:00 PM for three days and then anesthetized and perfused at ≅ 4:30 PM. The animals of the PSR group were placed on platforms for 72 h of PS deprivation and then back in their home cage for ≅ 3 h to allow a recovery of PS. They were anaesthetized for perfusion 150 min exactly after the first PS episode.

The PSC, PSD and PSR animals were perfused with a Ringer's lactate solution containing 0.1% 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). Brains were postfixed for 2 h at 4°C and then stored for 2 days at 4°C in PB with 30% sucrose. The brains were rapidly frozen with CO₂ gas, sliced in 25 μm-thick coronal sections, and stored in PB containing 0.9% NaCl, 0.3% Triton X-100 and 0.1% sodium azide (PBST-Az). They were successively incubated in (i) a rabbit antiserum to Fos (1:5,000; Oncogene) in PBST-Az for 3 days at 4°C; (ii) a biotinylated goat antirabbit IgG solution (1:2,000; Vector Laboratories); and (iii) an ABC-HRP solution (1:1,000; Elite kit, Vector Laboratories) both for 90 min at room temperature. Finally, the sections were immersed in a 0.05 M Tris-HCl buffer (pH 7.6) containing 0.025% 3,3'-diaminobenzidine-4 HCl (DAB; Sigma), 0.003% H₂O₂ and 0.6% nickel ammonium sulphate. Two washes of 30 min were performed between each step.

The Fos stained sections were incubated the following day in a rabbit antiserum to either hypocretin (1:10,000; Phoenix Pharmaceutical) or MCH (1:100,000; Phoenix Pharmaceutical) in PBST-Az over 3 days at 4°C. Amplification steps were similar to those above but revelation was performed in DAB solution without nickel. Finally, the sections were mounted on slides, dried and coverslipped with DePex.

Drawings of double-immunostained hemi-sections were made with a Leitz Orthoplan microscope equipped with an X/Y sensitive stage and a video camera connected to a computerized image analysis system (Historag, BIOCOM). Single and double-labeled neurons were counted in each hypothalamic structure present on one side of 6 MCH/Fos double-labeled sections and 4 hypocretin/Fos double-labeled sections.

For all rats, a guide cannula was implanted into the left lateral ventricle. Animals were allowed to recover for 3 days and were handled daily during 5 days prior to injection to minimize non-specific stress. Five microliters of MCH (0.02 μg, n = 4; 0.2 μg, n = 7; 1 μg, n = 7, 5 μg, n = 9 and 10 μg, n = 5 dissolved in 0.9% saline) or saline (n = 13) were administered at the rate of 1 μl/min at the beginning of the dark period (6:00 PM), with a stainless steel injector placed in the cannula and connected to an Hamilton syringe (Hamilton, Bonaduz, AG) by polythene tubing (id, 0.5 mm; od, 1 mm). All animals received at least one saline injection and one dose of MCH. After injection, animals returned to their jars and vigilance states were recorded for 8 h. The placement of the cannula was verified at the end of the study by microscopic examination of coronal counterstained brain sections.

ANOVA were performed on the different vigilance states across experimental conditions. A post hoc PLSD Fisher test was used to identify significant pairwise differences. The same tests were performed to compare cell counts between the animals groups in the different hypothalamic structures. Cell counts were correlated with physiological variables using a simple linear regression analysis. All statistics were performed using Statview 5.0.