Differential effects of acute and repeat dosing with the H3 antagonist GSK189254 on the sleep-wake cycle and narcoleptic episodes in Ox-/- mice.

BACKGROUND AND PURPOSE: Histamine H3 receptor antagonists are currently being evaluated in clinical trials for a number of central nervous system disorders including narcolepsy. These agents can increase wakefulness (W) in cats and rodents following acute administration, but their effects after repeat dosing have not been reported previously. EXPERIMENTAL APPROACH: EEG and EMG recordings were used to investigate the effects of acute and repeat administration of the novel H3 antagonist GSK189254 on the sleep-wake cycle in wild-type (Ox+/+) and orexin knockout (Ox-/-) mice, the latter being genetically susceptible to narcoleptic episodes. In addition, we investigated H3 and H1 receptor expression in this model using radioligand binding and autoradiography. KEY RESULTS: In Ox+/+ and Ox-/- mice, acute administration of GSK189254 (3 and 10 mg x kg(-1) p.o.) increased W and decreased slow wave and paradoxical sleep to a similar degree to modafinil (64 mg x kg(-1)), while it reduced narcoleptic episodes in Ox-/- mice. After twice daily dosing for 8 days, the effect of GSK189254 (10 mg x kg(-1)) on W in both Ox+/+ and Ox-/- mice was significantly reduced, while the effect on narcoleptic episodes in Ox-/- mice was significantly increased. Binding studies revealed no significant differences in H3 or H1 receptor expression between Ox+/+ and Ox-/- mice. CONCLUSIONS AND IMPLICATIONS: These studies provide further evidence to support the potential use of H3 antagonists in the treatment of narcolepsy and excessive daytime sleepiness. Moreover, the differential effects observed on W and narcoleptic episodes following repeat dosing could have important implications in clinical studies.

Differential c-fos expression in the rhinencephalon and striatum after enhanced sleep-wake states in the cat.

In order to delimit the supra-brainstem structures that are activated during the sleep-waking cycle, we have examined c-fos immunoreactivity in four groups of polygraphically recorded cats killed after 3 h of prolonged waking (W), slow-wave sleep (SWS), or paradoxical sleep (PS), following microinjection of muscimol (a gamma-aminobutyric acid, GABA agonist) into the periaqueductal grey matter and adjacent areas [Sastre et al. (1996), Neuroscience, 74, 415-426]. Our results demonstrate that there was a direct relationship between a significant increase in c-fos labelling and the amount of PS in the laterodorsalis tegmenti in the pons, supramamillary nucleus, septum, hippocampus, gyrus cingulate, amygdala, stria terminalis and the accumbens nuclei. Moreover, in all these structures, the number of Fos-like immunoreactive neurons in the PS group was significantly higher (three to 30-fold) than in the SWS and W groups. We suggest that the dense expression of the immediate-early gene c-fos in the rhinencephalon and striatum may be considered as a tonic component of PS at the molecular level and that, during PS, the rhinencephalon and striatum are the main targets of an excitatory system originating in the pons.

Hyperoxia increases paradoxical sleep rhythm in the pontine cat.

Pontine cat is an ectothermic preparation, whose central temperature can artificially be lowered from 36 degrees C to 26 degrees C; this gradual hypothermia is accompanied by a dramatic increase in paradoxical sleep (PS). Two main hypotheses might explain this result: executive systems of PS might be switched on gradually by cold-sensitive thermodetectors, whereas inhibitory monoaminergic mechanisms appear to be warm-sensitive. On the other hand, energy saving mechanisms peculiar to hypothermia might promote PS appearance. Indeed, in normal animals, PS is selectively suppressed both by hyperthermia and hypoxia. The inhibitory effect of hypoxia might explain why hypothermia, which protects the brain against hypoxic alterations, might facilitate PS. If this last hypothesis is correct, the putative increase in cerebral oxygen supply might increase PS. For this reason, we submitted eight pontine carotid-deafferented cats, kept at the same central temperature (34 +/- 0.5 degrees C: temperature clamp) to periodic hyperoxia (PaO2 = 58 +/- 7 kPa) or room air (PaO2 = 17 +/- 2 kPa) alternatively during 4- or 12-h periods. Hyperoxia induced an 85% increase in PS, mainly due to an increase in PS rhythm (PS cycle duration was 65 +/- 4 min in normoxia and 45 +/- 4 min in hyperoxia, p<0.0001). In five animals, after hyperoxia, PS cycle returned gradually back to control values in 4 to 12 h. These findings show that PS is exquisitely sensitive to conditions that impair oxidative metabolism. The role of cholinergic executive PS systems as putative metabolic-sensitive neurons remains to be established.

Importance of the ventrolateral region of the periaqueductal gray and adjacent tegmentum in the control of paradoxical sleep as studied by muscimol microinjections in the cat.

It has been demonstrated that coagulation in the area of the periaqueductal gray induces a marked increase in paradoxical sleep in the cat [Petitjean F. et al, (1975) brain Res. 88, 439-453]. This effect was obtained either by the destruction of ascending or descending fibres or by the lesion of a specific group of local neurons. To assess the role of these neurons, muscimol (0.5 microgram/0.5 microliter) was injected bilaterally in 31 cats in this area of the periaqueductal gray. Polygraphic recordings were performed before and after injections. Following muscimol (GABAA agonist) injection, there was a consistent increase in paradoxical sleep lasting 269 +/- 8 min (mean +/- S.E.M.), with a latency of 31 +/- 2 min. The increase varied from small (20-30%) to medium (30-50%) to large (50-100% of the recording time), depending on the injection site. The intensity of hypersomnia was correlated with the site of the injection. That is, the most profound hypersomnia was obtained when muscimol was injected in the vicinity of a target area which lies in the ventrolateral periaqueductal gray (at the level of the fourth nucleus) and in the reticular formation situated immediately below. Similar effects were also obtained in insomniac cats pretreated with p-chlorophenylalanine and in cats whose brainstem was transected 3 mm rostral to the injection site. Injections of baclofen, a GABAB agonist (0.25-5 micrograms), did not alter the quantity of paradoxical sleep, whereas injections of bicuculline, a GABAA antagonist, significantly decreased the quantity of paradoxical sleep at the doses of 0.2-2 micrograms. It was concluded that inactivation of ventrolateral periaqueductal gray neurons induces a very important increase in paradoxical sleep. The exact mechanisms of this effect remain to be investigated.

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

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.

[Is there a bulbar pacemaker responsible for the ultradian rhythm of paradoxical sleep?]. / Existe-T-IL un pacemaker bulbaire responsable du rythme ultradien du sommeil paradoxal?

Since there are complex regulations of paradoxical sleep at the supra-pontine level, the chronic pontine preparation appears to be the best model for studying the mechanisms of the ultradian rhythm of PS (tau'). In these preparations, which are ectothermic, tau' is considerably dependent upon temperature conditions. a) PS never occurs above a central temperature (Tc) of 36 degrees C which constitutes the absolute threshold for PS. b) If Tc is regulated at a plateau between 34.5 degrees C and 35.5 degrees C, the duration of tau' corresponds to about 60 min (circhoral) whereas the duration of PS is 5 min, thus the cyclic ratio: tau'/duration of PS is 12. During deep hypothermia (from 35 degrees C to 25 degrees C), tau' of PS is temperature-compensated. It remains close to 60 min, so that its Q10 is about 1. c) However, in the same conditions, the duration of PS episodes increases from 5 min to 55 min, so that the Q10 of PS is 0.1 (8% at 35 degrees C - 80% at 25 degrees C). These data are discussed in the light of the present theories explaining tau' (i.e., the reciprocal inhibition between monoaminergic permissive systems and cholinergic executive systems). An increase in PS during hypothermia might be possible provided that it should be proved that permissive mechanisms are excited by heat while executive mechanisms would be cold-sensitive. But there are no data on this point. However, even this "differential thermosensitivity hypothesis" would not explain the striking fixity of tau' between 35 degrees C and 25 degrees C. For this reason, one should hypothetize that there is a temperature-compensated oscillator or pacemaker which would act upon both executive and permissive mechanisms. This oscillator would also be controlled by metabolic factors as shown by the effect of O2 and prolactin.

Suppression of Ottoson waves in the isolated olfactory bulb during sleep in the pontine cat.

In the chronic pontine cat, in which the olfactory bulbs are isolated from the brain stem, the Ottoson and/or Adrian-induced waves are suppressed during paradoxical sleep. This phenomenon cannot be explained by a central centrifugal control. The suppression of olfactory electrical activity was found to correlate with vasodilatation of the nasal mucosa. It is likely, therefore that control of the olfactory bulb activity might be mediated through the vasomotor innervation of the nasal mucosa originating from the medulla.

Dopaminergic neurons in the cat dorsal motor nucleus of the vagus, demonstrated by dopamine, AADC and TH immunohistochemistry.

In the rostral part of the dorsal motor nucleus of the vagus of the cat, neurons do not contain histochemically detectable catecholamines, even though many perikarya contain both intense aromatic L-amino acid decarboxylase (AADC) immunoreactivity and strong monoamine oxidase enzymatic activity. Similarly located perikarya have distinct immunoreactivities to tyrosine hydroxylase (TH) and dopamine after treatment with colchicine. Since inhibition of monoamine oxidase fails to reveal dopamine in these cells, its absence in non-colchicine-treated animals cannot be due to rapid deamination. It appears that dopamine is synthesized by TH and AADC in dorsal motor vagal cells and is then rapidly transported from the perikarya.

Localization of CRF-immunoreactive neurons in the cat medulla oblongata: their presence in the inferior olive.

Corticotropin releasing factor (CRF)-immunoreactive (IR) perikarya, visualized by the indirect immunoperoxidase method in colchicine-pretreated cats, were localized in many discrete regions of the medulla oblongata. They were found mainly in the dorsal aspect and midline of the medulla oblongata, and more rostrally in the ventrolateral portion. Our results also demonstrated CRF-IR neurons in the rostrocaudal extent of the inferior olive, probably projecting to the cerebellar cortex via thick axons visualized along the lateral edge of the medulla. CRF-IR olivary cells were also found in the pontine cat from which the forebrain was removed, but neither in hypophysectomized nor adrenalectomized cats.

[Hypothermia and paradoxical sleep. I. Pontile cats without hypothalamo-hypophysis]. / Hypothermie et sommeil paradoxal. I. Chat pontique sans îlot hypothalamo-hypophysaire.

Chronic pontile cats (without hypothalamo-hypophysis) were kept during 4 days at central T (TC) between 37.5 and 30.8 degrees C at stable ambiant T (TA) between 28.5 and 23 degrees C. The vasomotor index of the forepaw was chosen for studying change in vasomotricity. Small and slow variations of TA (+1.5 degrees C) around 27 degrees C were followed by thermoregulatory response since a progressive decrease of TA under 27 degrees C led to vasoconstriction and increase of TC while progressive increase of TA above 27 degrees C led to vasodilatation and decrease of TC. However rapid and large decrease of TA under 27 degrees C (24-23 degrees C) led to the expected hypothermia with decrease of TC but without vasoconstriction. Paradoxical sleep (PS) amounts were strongly correlated with TC. At TC above 35.5 degrees C PS was almost totally suppressed while it increased significantly under 35 degrees C (Q10 = 0.10). Under 35 degrees C at stable TC and TA, PS occurred with an endogenous circahoral rhythm which did not vary significantly between 35 and 32 degrees C. These results strongly suggest that in pontile cats, PS is both gated and regulated by TC, while TC is regulated by pontobulbar vasomotor systems in response to TA. The putative role of the ventro-lateral medulla, in controlling both vasomotricity, TC and the excitability of the locus coeruleus is discussed in relation with PS.

[Central temperature is the principal factor of regulation of paradoxical sleep in pontile cats]. / La température centrale est le facteur principal de régulation du sommeil paradoxal chez le chat pontique.

In chronic poïkilothermic pontile cats whose central temperature is artificially regulated, the quantity of paradoxical sleep (PS), its ultradian periodicity and the duration of PS episodes are dependent upon central temperature level and periodicity.

[Systemic or intracerebral injection of bovine neurointermediate lobe extracts may induce paradoxical sleep in insomnia induced by p-chlorophenylalanine in cats]. / L'injection systémique ou intracérébrale d'extraits de lobe neuro-intermédiaire de Boeuf peut induire le sommeil paradoxal au cours de l'insomnie provoquée par la P. chlorophénylalanine chez le chat.

Injections of Bovine neuro-intermediate lobe extracts, either subcutaneously (5 U), intraventricularly (20 mU) or directly in the vicinity of the nucleus magnocellularis of the medulla (1,3 mU) may induce paradoxical sleep in totally insomniac cats pretreated with P. chlorophenylalanine.

[Is the pars intermedia of the hypophysis responsible for provoking paradoxical sleep in pontine cats with an isolated hypophysis?]. / La pars intermedia de l'hypophyse est-elle responsable du déclenchement du sommeil paradoxal chez le chat pontique avec "hypophyse isolée"?

In chronic pontine cats without hypothalamo-hypophysis, daily subcutaneous or intraventricular injection of total extracts of Bovine neuro-intermediate lobe increases both survival and quantity of paradoxical sleep, while synthetic Lysine vasopressin, Oxytocin or ACTH have no effect. Chronic pontine cats with "isolated hypophysis" have a longer survival and a more significant amount of paradoxical sleep than those without hypothalamo-hypophysis. Factor(s) liberated by pars intermedia are very likely to be responsible for the increase of PS since only pars intermedia remains functional in the "isolated hypophysis" as shown by immunocytochemical controls.

[Bacitracin, a peptidase inhibitor, increases paradoxical sleep in the cat]. / La bacitracine, un inhibiteur des peptidases, augmente le sommeil paradoxal chez le chat.

In the Cat, subcutaneous injections of 2,000 u/kg of Bacitracin, a peptidase inhibitor, induce an important and significant increase of paradoxical sleep. This increase begins after about 3 hrs. following the injection, and lasts for 18th hour.

Are the gigantocellular tegmental field neurons responsible for paradoxical sleep?

We have compared the effects of electrolytic and kainic acid lesions of the pontine gigantocellular tegmental field (FTG) upon paradoxical sleep (PS). Following bilateral electrolytic destruction of the ventrolateral part of the FTG, there was an almost total suppression of PS which lasted at least for 5 weeks. Muscular atonia was absent and ponto-geniculo-occipital (PGO) activity was reduced by 80% in the few remaining episodes of PS. Contrary to to these effects, total neuronal cell loss of the FTG induced by bilateral kainic acid injection was not followed by a significant quantitative and qualitative alteration of PS. These results indicate that the neurons located within the FTG are not critical for the generation of both phasic and tonic components of PS. Elimination of this state of sleep after electrolytic destruction of the ventrolateral pontine reticular formation can be explained by interruption of fibers connecting the region of the locus coeruleus complex and the bulbar reticular formation.

Tegmentoreticular projections with special reference to the muscular atonia during paradoxical sleep in the cat: an HRP study.

Using the retrograde tracer technique with horseradish peroxidase (HRP), attempts were made to determine the cells of origin and the descending pathway of the tegmentoreticular projections in order to give an anatomical substrate for the physiological phenomenon of the postural atonia observed during paradoxical sleep (PS) in the cat. The HRP was injected into various parts of the pontomedullary reticular formation (RF) including the caudal raphe nuclei, nucleus (n.) reticularis gigantocellularis (Gc), n. reticularis magnocellularis (Mc), and other pontomedullary structures adjacent to the Mc. The results indicated that the HRP injection into the Mc, particularly its caudal and lateral two-thirds, resulted in specific labeling of cells located in an area just medial to the LCa together with those in the most medial part of the LCa. Bilateral lesions of these pontine structures have been reported to suppress the atonia otherwise observed during PS in the normal cat. In addition to the HRP labeled cells, we have also observed HRP filled fiber bundles directed to labeled cells in the medial part of the LCa and immediately adjacent tegmental RF area. The same course of HRP labeled fiber bundles was also observed together with HRP labeled cells in the Mc after HRP injections into the medial part of the LCa area, indicating the existence of an interconnection between the LCa area and the Mc. The location of the tegmentoreticular pathway corresponded to that of the lesions effective to suppress the muscular atonia during PS. HRP injections into the caudal medullar caudal to the Mc, on the other hand, resulted in no or almost no HRP labeled cells in the area medial to the LCa, in spite of the presence of HRP containing neurons in other parts of the pontomedullary RF areas, showing that the tegmentoreticular projections as described above terminate almost exclusively in the Mc.

[Persistence of paradoxical sleep in the cat after destruction of the pontine gagantocellular tegmental field with kainic acid]. / Persistance du sommeil paradoxal chez le chat après destruction de l'aire gigantocellulaire du tegmentum pontique par l'acide kaïnique.

The pontine gigantocellular tegmental field (FTG) does not play any role in paradoxical sleep (PS) since the destruction of the cell bodies of the FTG with kaïnic acid does not alter PS in the Cat. Elimination of PS following electrolytic destruction of the ventrolateral pontine reticular formation can be explained by lesions of fibers connecting the region of the locus caeruleus and the bulbar reticular formation.

[Supppression of paradoxical sleep by chloramphenicol. Absence of effect by thiamphenicol]. / Suppression du sommeil paradoxal par le chloramphénicol chez le chat. Absence d'effet du thiamphénicol

The oral administration of 160-250 mg/kg of chloramphenicol selectively suppresses Paradoxical Sleep in the cat for a duration of 10-20 hours. Larger doses (330 mg/kg) suppress Paradoxical Sleep for 27 hours, but aslso decrease Slow Wave Sleep. At the same dose, Thiamphenicol has no effect.