Increases in mature brain-derived neurotrophic factor protein in the frontal cortex and basal forebrain during chronic sleep restriction in rats: possible role in initiating allostatic adaptation.

Chronic sleep restriction (CSR) has various negative consequences on cognitive performance and health. Using a rat model of CSR that uses alternating cycles of 3h of sleep deprivation (using slowly rotating activity wheels) and 1h of sleep opportunity continuously for 4 days ('3/1' protocol), we previously observed not only homeostatic but also allostatic (adaptive) sleep responses to CSR. In particular, non-rapid eye movement sleep (NREMS) electroencephalogram (EEG) delta power, an index of sleep intensity, increased initially and then declined gradually during CSR, with no rebound during a 2-day recovery period. To study underlying mechanisms of these allostatic responses, we examined the levels of brain-derived neurotrophic factor (BDNF), which is known to regulate NREMS EEG delta activity, during the same CSR protocol. Mature BDNF protein levels were measured in the frontal cortex and basal forebrain, two brain regions involved in sleep and EEG regulation, and the hippocampus, using Western blot analysis. Adult male Wistar rats were housed in motorized activity wheels, and underwent the 3/1 CSR protocol for 27 h, for 99 h, or for 99 h followed by 24h of recovery. Additional rats were housed in either locked wheels (locked wheel controls [LWCs]) or unlocked wheels that rats could rotate freely (wheel-running controls [WRCs]). BDNF levels did not differ between WRC and LWC groups. BDNF levels were increased, compared to the control levels, in all three brain regions after 27 h, and were increased less strongly after 99 h, of CSR. After 24h of recovery, BDNF levels were at the control levels. This time course of BDNF levels parallels the previously reported changes in NREMS delta power during the same CSR protocol. Changes in BDNF protein levels in the cortex and basal forebrain may be part of the molecular mechanisms underlying allostatic sleep responses to CSR.

Time-of-day modulation of homeostatic and allostatic sleep responses to chronic sleep restriction in rats.

To study sleep responses to chronic sleep restriction (CSR) and time-of-day influences on these responses, we developed a rat model of CSR that takes into account the polyphasic sleep patterns in rats. Adult male rats underwent cycles of 3 h of sleep deprivation (SD) and 1 h of sleep opportunity (SO) continuously for 4 days, beginning at the onset of the 12-h light phase ("3/1" protocol). Electroencephalogram (EEG) and electromyogram (EMG) recordings were made before, during, and after CSR. During CSR, total sleep time was reduced by ∼60% from baseline levels. Both rapid eye movement sleep (REMS) and non-rapid eye movement sleep (NREMS) during SO periods increased initially relative to baseline and remained elevated for the rest of the CSR period. In contrast, NREMS EEG delta power (a measure of sleep intensity) increased initially, but then declined gradually, in parallel with increases in high-frequency power in the NREMS EEG. The amplitude of daily rhythms in NREMS and REMS amounts was maintained during SO periods, whereas that of NREMS delta power was reduced. Compensatory responses during the 2-day post-CSR recovery period were either modest or negative and gated by time of day. NREMS, REMS, and EEG delta power lost during CSR were not recovered by the end of the second recovery day. Thus the "3/1" CSR protocol triggered both homeostatic responses (increased sleep amounts and intensity during SOs) and allostatic responses (gradual decline in sleep intensity during SOs and muted or negative post-CSR sleep recovery), and both responses were modulated by time of day.

Estradiol replacement enhances sleep deprivation-induced c-Fos immunoreactivity in forebrain arousal regions of ovariectomized rats.

To understand how female sex hormones influence homeostatic mechanisms of sleep, we studied the effects of estradiol (E(2)) replacement on c-Fos immunoreactivity in sleep/wake-regulatory brain areas after sleep deprivation (SD) in ovariectomized rats. Adult rats were ovariectomized and implanted subcutaneously with capsules containing 17beta-E(2) (10.5 microg; to mimic diestrous E(2) levels) or oil. After 2 wk, animals with E(2) capsules received a single subcutaneous injection of 17beta-E(2) (10 microg/kg; to achieve proestrous E(2) levels) or oil; control animals with oil capsules received an oil injection. Twenty-four hours later, animals were either left undisturbed or sleep deprived by "gentle handling" for 6 h during the early light phase, and killed. E(2) treatment increased serum E(2) levels and uterus weights dose dependently, while attenuating body weight gain. Regardless of hormonal conditions, SD increased c-Fos immunoreactivity in all four arousal-promoting areas and four limbic and neuroendocrine nuclei studied, whereas it decreased c-Fos labeling in the sleep-promoting ventrolateral preoptic nucleus (VLPO). Low and high E(2) treatments enhanced the SD-induced c-Fos immunoreactivity in the laterodorsal subnucleus of the bed nucleus of stria terminalis and the tuberomammillary nucleus, and in orexin-containing hypothalamic neurons, with no effect on the basal forebrain and locus coeruleus. The high E(2) treatment decreased c-Fos labeling in the VLPO under nondeprived conditions. These results indicate that E(2) replacement modulates SD-induced or spontaneous c-Fos expression in sleep/wake-regulatory and limbic forebrain nuclei. These modulatory effects of E(2) replacement on neuronal activity may be, in part, responsible for E(2)'s influence on sleep/wake behavior.

Indirect projections from the suprachiasmatic nucleus to major arousal-promoting cell groups in rat: implications for the circadian control of behavioural state.

The circadian clock housed in the suprachiasmatic nucleus (SCN) controls various circadian rhythms including daily sleep-wake cycles. Using dual tract-tracing, we recently showed that the medial preoptic area (MPA), subparaventricular zone (SPVZ) and dorsomedial hypothalamic nucleus (DMH) are well positioned to relay SCN output to two key sleep-promoting nuclei, namely, the ventrolateral and median preoptic nuclei. The present study examined the possibility that these three nuclei may link the SCN with wake-regulatory neuronal groups. Biotinylated dextran-amine with or without cholera toxin B subunit was injected into selected main targets of SCN efferents; the retrograde labeling in the SCN was previously analyzed. Here, anterograde labeling was analyzed in immunohistochemically identified cholinergic, orexin/hypocretin-containing and aminergic cell groups. Tracer injections into the MPA, SPVZ and DMH resulted in moderate to dense anterograde labeling of varicose fibers in the orexin field and the tuberomammillary nucleus. The locus coeruleus, particularly the dendritic field, contained moderate anterograde labeling from the MPA and DMH. The ventral tegmental area, dorsal raphe nucleus, and laterodorsal tegmental nucleus all showed moderate anterograde labeling from the DMH. The substantia innominata showed moderate anterograde labeling from the MPA. These results suggest that the MPA, SPVZ and DMH are possible relay nuclei for indirect SCN projections not only to sleep-promoting preoptic nuclei as previously shown, but also to wake-regulatory cell groups throughout the brain. In the absence of major direct SCN projections to most of these sleep/wake-regulatory regions, indirect neuronal pathways probably play an important role in the circadian control of sleep-wake cycles and other physiological functions.

Stimulant doses of caffeine induce c-FOS activation in orexin/hypocretin-containing neurons in rat.

Although caffeine is a commonly used CNS stimulant, neuronal mechanisms underlying its stimulatory effect are not fully understood. Orexin (hypocretin)-containing neurons play a critical role in arousal and might be activated by acute administration of caffeine. We examined this possibility by using dual-immunostaining for orexin B and c-Fos protein as a marker for neuronal activation. Rats were administered intraperitoneally with 10, 30 or 75 mg/kg caffeine, or saline. As previously reported, caffeine increased locomotion at 10 and 30 mg/kg, but not at 75 mg/kg. The numbers of orexin-immunoreactive and non-orexin-immunoreactive neurons expressing c-Fos were analysed using three counting boxes within the orexin field in the posterior hypothalamus. Compared with saline, all doses of caffeine increased the number of cells immunoreactive for both orexin and c-Fos. The average magnitude of this increase across doses in orexin neurons differed amongst regions; c-Fos expression increased by 343% in the perifornical area and by 158% in the more medial, dorsomedial nucleus. In the lateral hypothalamic area, c-Fos expression increased by 226% at 10 and 30 mg/kg but no change was seen at 75 mg/kg. In contrast, caffeine significantly increased the number of non-orexin-immunoreactive neurons expressing c-Fos only in the dorsomedial nucleus. These results indicate that systemically administered caffeine preferentially activates orexin neurons over non-orexin neurons in the same field, and that this activation is most pronounced in the perifornical region where orexin neurons are most concentrated. The activation of orexin neurons might play a role in the behavioural activation by caffeine.

Lesions of the pedunculopontine tegmental nucleus reduce paradoxical sleep (PS) propensity: evidence from a short-term PS deprivation study in rats.

Cholinergic neurons in the mesopontine tegmentum are thought to play a critical role in the generation of paradoxical sleep (PS). However, no study has yet examined whether lesions of these neurons cause deficits of PS in the rat. We describe here the effects of lesions of the pedunculopontine tegmental nucleus (PPT) on spontaneous PS and on PS propensity, expressed during and after a short period of PS deprivation. Lesions were induced by bilateral injections of ibotenate. PS deprivation was performed manually by gently waking rats each time they showed polygraphic signs of PS. Two weeks after lesions, an 8-h baseline recording was performed; the following day, rats were PS deprived for 6 h and polygraphic recordings were then continued for 2 h, to examine recovery sleep. The same protocol was repeated 1 week later. Compared with controls and with rats with limited PPT lesions, rats bearing > 60% NADPH-diaphorase-positive cell loss within the PPT showed unaffected PS under baseline conditions. However, they made fewer attempts to enter PS during deprivation and they exhibited an attenuated rebound increase in PS time after deprivation. The number of PS attempts and the magnitude of PS rebound were negatively correlated with the percent loss of diaphorase-positive neurons within the PPT. Thus, PS propensity that accumulated as a result of PS deprivation was reduced after extensive PPT lesions. In summary, although spontaneous PS was found to be unaltered, the PS deprivation procedure used in this study demonstrated the dysfunctioning of PS caused by PPT lesions.

Chronic, low-level exposure to the cholinesterase inhibitor DFP. I. Time course of neurochemical changes in the rat pontomesencephalic tegmentum.

Rats were repeatedly administered with a low dose of diisopropylfluorosphosphate (DFP; 0.2 mg/kg/day, SC, for 9 or 21 days), an irreversible cholinesterase (ChE) inhibitor. Control rats received a daily injection of oil vehicle. Neurochemical changes occurring in the pontomesencephalic tegmentum (PMT), a brain stem region critically involved in behavioral state control, were evaluated at various times of treatment and after DFP withdrawal. First, enzyme assay revealed a profile of ChE inhibition in the whole PMT which looked like that observed in the striatum; both the inhibition and recovery proceeded more slowly than they did in the plasma. Second, quantitative histochemistry indicated that ChE activity in the mesopontine cholinergic nuclei and the pontine reticular formation progressively decreased across the first days of DFP exposure, to reach an asymptotic level of inhibition after 6 days (74-82% inhibition). The inhibition was less pronounced in the locus coeruleus (49%). Third, [3H]QNB autoradiography showed that muscarinic receptor density was unchanged in any of the PMT areas selected. These results are discussed regarding the question of regional variation in susceptibility to anti-ChE agents. To what extent behavioral state alterations occur concomitantly with ChE activity changes is assessed in the companion article.

Chronic, low-level exposure to the cholinesterase inhibitor DFP. II. Time course of behavioral state changes in rats.

Rats were repeatedly administered with low doses of diisopropylfluorophosphate (DFP; 0.2 mg/kg/day, SC), an irreversible cholinesterase (ChE) inhibitor. Control rats received a daily injection of oil vehicle or of saline. Recordings of the sleep-wake states were obtained in the 6 h following 1, 3, 6, 9, 13, 17, and 21 injections, as well as 2, 4, and 19 days after 9-day treatment. DFP administration increased waking at the expense of slow-wave sleep (SWS), but not of paradoxical sleep (PS); as a result, the PS/SWS ratio was strongly enhanced. These changes developed across days, were maximal after six to nine injections, and were then maintained at that level until cessation of treatment. This time course of behavioral state alterations paralleled the time course of ChE inhibition in the mesopontine cholinergic nuclei and the pontine reticular formation described in the companion article. In contrast, after DFP withdrawal, behavioral states returned to control values more rapidly (in 2-4 days) than did ChE activity. These results are discussed regarding the promoting role of cholinergic neurotransmission in brain-activated states.

Pontine microinjection of carbachol does not reliably enhance paradoxical sleep in rats.

It has been repeatedly shown in cats that acute administration of carbachol into the pontine reticular formation (PRF) readily evokes a state that closely mimics natural paradoxical sleep (PS). Surprisingly, there are few corresponding studies in rats. In order to further characterize the effects of pontine carbachol in rats, 151 injections of different doses (from 3 micrograms to 0.005 microgram in 0.1 microliter saline) of carbachol were made at different sites within the PRF of 70 rats. Sleep-waking states obtained in the 4 hours following carbachol administration were compared to control values, obtained both under baseline condition (no injection) and following pontine injection of 0.1 microliter saline. On the one hand, from the whole set of carbachol injections, it appeared that: 1) most injections (112/151) did not significantly alter the sleep-wake states; 2) when carbachol was effective, it induced either increased PS (20 injections) or increased waking (19 injections); and 3) effective injection sites were intermingled with noneffective sites. Dose- or site-dependency effects can account in part, but not totally, for these discordant results. On the other hand, in accordance with previous rat studies, we found that: 1) the PRF medial and ventral to the motor trigeminal nucleus was the most effective region for carbachol to increase PS; 2) carbachol-induced PS enhancement was of moderate magnitude (+60% above control saline level over the 4-hour recording time); 3) latency to onset of the first PS episode was not shortened; and 4) only the number of PS episodes was increased, their duration was not prolonged. These characteristics of carbachol-induced PS enhancement strongly differ, both in terms of magnitude and timing, from those described in cats. We suggest that the less reliable and weaker effects of pontine carbachol injection in rats compared to cats can be due to methodological problems inherent in the intracerebral microinjection technique and also to species-related differences in the mechanisms controlling the PS state.