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Involvement of histaminergic neurons in arousal mechanisms demonstrated with H3-receptor ligands in the cat
Brain Research, 523 (1990) 325-330
325
Elsevier BRES 24189
Involvement of histaminergic neurons in arousal mechanisms demonstrated with H3-receptor ligands in the cat Jian-Sheng Lin 1, Kazuya Sakai 1, Giovanna Vanni-Mercier 1, Jean-Michel Arrang 2, Monique Garbarg z, Jean-Charles Schwartz 2 and Michel Jouvet 1 ID~partement de Mddecine Exp~rimentale, 1NSERM U52, CNRS UA 1195, Universit~ Claude Bernard, Lyon (France) and 2Unit~ 109 de Neurobiologie, Centre Paul Broca de I'INSERM, Paris (France)
(Accepted 3 April 1990) Key words: Histaminergic neuron; H3-receptor; Sleep-wakefulness; Thioperamide; a-Methylhistamine; Cat
The effects of histamine Ha-receptor ligands on sleep-waking parameters were studied in freely moving cats. Oral administration of (R)a-methylhistamine (aMHA), a Ha-agonist,caused a significantincrease in deep slow wave sleep while that of thioperamide, a Ha-antagonist, enhanced wakefulness in a marked and dose-dependent manner. The arousal effects of thioperamide were prevented by pretreatment with aMHA or mepyramine, a Hi-receptor antagonist. The findings support the hypothesis that the histaminergic neurons are critically involved in arousal mechanisms and suggest that Ha-receptors play an active part in these mechanisms by regulating histamine transmission. The sedative effects of 'antihistamines', i.e. histamine (HA) Hi-receptor antagonists, have been well known since their therapeutic application 6. However, the idea that H A might be a 'waking amine' still remains a matter of debate 11'2°, even though there is recent experimental evidence supporting the involvement of Hi-receptors in arousal 15A7'29'35. This is mainly because, until recently, effective drugs were not available to enhance histaminergic transmission in the brain. It is now known that the perikarya of histaminergic neurons are situated in the tuberomammillary nucleus of the posterior hypothalamus, a brain area involved in the maintenance of wakefulness 16A9'27, and project diffusely to almost all brain areas ~'14'23. Recently, the activity of such a neuronal system was demonstrated to be regulated through a presynaptic negative feedback process mediated by a novel class of H A receptors, namely H31-3. Highly potent, selective and brain-penetrating ligands to H3-receptors, i.e. (R)ct-methylhistamine (aMHA), a chiral agonist, and thioperamide, a competitive antagonist, were also developed more recently4. The agonist and antagonist were further shown to be able to decrease or increase, respectively, in a marked and long-lasting manner, the synthesis and release of H A in vitro 4'1° as well as in the brain of living animals 7,2~. It seems therefore that they represent novel and useful tools to clarify the functional role of histaminergic neurons in the brain. In the present study, we used the H3-receptor
agents to determine the role of histaminergic neurons and the involvement of Ha-receptors in the arousal mechanisms by examining their effects on sleep-waking parameters in freely moving cats. Sixteen adult cats of both sexes weighing 2.5-3.7 kg were chronically implanted, under pentobarbital anesthesia (25 mg/kg, i.v.), with electrodes for polygraphic recordings of pontogeniculooccipital (PGO) activity, neocortical electroencephalogram (EEG), hippocampal and olfactory bulb E E G , electromyogram (EMG), and electrooculogram (EOG). In addition, a small thermocouple was placed under deep neck muscles to record the body temperature. Experiments began 10 days after surgery. The animals were housed in a sound-attenuated and dimly illuminated cage at 24-27 °C and fed at 6 p.m. Oral administrations of lactose capsules alone (placebo), or containing (R)a-methylhistamine or thioperamide, were performed at 11 a.m. and subsequent polygraphic recordings were made for at least 24 h. There was an interval of 7 days between two administrations. The scoring of the polygraphic records was made minute by minute according to previously described criteria 24 for wakefulness (W), light slow wave sleep (S1), deep slow wave sleep ($2) and paradoxical sleep (PS). The paired Student's t-test was used to examine the difference between drug and placebo administration: individual animals served as their own controls. The dose and pretreatment effects of drugs were measured using
Correspondence: J.-S. Lin, Drpartement de M6decine Exprrimentale, INSERM U52, CNRS UA 1195, Universit6 Claude Bernard, 8 avenue Rockefeller, 69373 Lyon Cedex 2, France.
0006-8993/90/$03.50 O 1990 Elsevier Science Publishers B.V. (Biomedical Division)
326 one way analysis of variance (ANOVA). When administered orally, a M H A (10-20 mg/kg) induced a significant and dose-related increase in $2 (Figs. 1A and 2A). The maximal effect was found between the 2nd and the 6th hour after administration. During this period, the times spent in $2 were 124% and 158% of the respective control values at doses of 10 and 20 mg/kg (P < 0.02 and P < 0.01, n = 6). The decline in W, S1 and PS was, however, not statistically significant (Figs. 1A and 2A). The augmentation in $2 was characterized by an increase in the mean episode duration (4.9 _+ 0.3 min with a M H A 20 mg/kg, for example, vs 3.0 +
A
0.2 min with placebo, P < 0.001, t-test) while the mean episode frequency and the latency of both slow wave sleep (SWS) and PS onset were not significantly modified. No overt abnormality in behavior, nor in E E G , e.g. hypersynchronous cortical activity or high-voltage slow waves during behavioral arousal, could be observed. In contrast, thioperamide (2-10 mg/kg) elicited a marked, dose-related and long-lasting increase in W at the expense of both PS and SWS, especially $2 (Figs. 1A and 2A). These changes were not marked at a dose of 1 mg/kg (n = 4, not shown) but became significant at a dose of 2 mg/kg or more. When compared with the
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Fig. 1. Examples of 6-h hypnograms obtained following oral applications of placebo as well as of a-methylhistamine (aMHA) and thioperamide at different doses (A). Note that deep slow wave sleep ($2) increased by aMHA and wakefulness (I41) evoked by thioperamide were dose-related. In B, thioperamide was administered alone or combined with aMHA (per os, 15 min before) and mepyramine (i.p., 10 min before). Note the restoration of sleep from the hyposomnia induced by thioperamide after the pretreatments. Ordinate: PS, paradoxical sleep; S1, light slow wave sleep. Abscissa: time in hours.
327 occurred as early as the first h o u r after administration and the duration of the m a x i m a l effects d e p e n d e d on the doses: 6 - 7 , 7 - 9 and 9 - 1 0 h for doses of 2, 5 and 10 mg/kg, respectively. T h e increase in W was due to an a u g m e n t a t i o n of both waking episode d u r a t i o n and frequency (8.8 + 1.2 rain and 24.6 + 2.7 with 5 mg/kg,
respective control values, the m e a n a u g m e n t a t i o n of W b e t w e e n time 0 and the 6th hour was 56%, 150% and 160% at doses of 2 (Fig. 2, n = 8), 5 (n = 8) and 10 (n = 5) mg/kg, respectively. Analysis of variance revealed a significant difference a m o n g these changes elicited by the different doses used ( P < 0.005). T h e arousal effects
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Fig. 2. Effects of a-methylhistamine and thioperamide as well as thioperamide combined with aMHA or mepyramine pretreatments on cumulative values of sleep-waking stages in the cat. The curves show the evolution of wakefulness (W), light slow wave sleep (S1), deep slow wave sleep ($2) and paradoxical sleep (PS) over 22 h following the oral administrations at 11 a.m. The S.E.M. (vertical bars) of experimental groups is given when the mean values are out of the control zones (means +_ S.E.M. of placebo-tested cats). A: a group of cats receiving 8 oral administrations of placebo, aMHA (20 mg/kg) and thioperamide (2 mg/kg), alone and with aMHA pretreatment (20 mg/kg). Note that ctMHA increases $2 whereas thioperamide elicits arousal significantly and that the pretreatment with aMHA reverses almost completely the arousal effect of thioperamide and restores significantly $2 and PS. B: another group of cats receiving 8 oral administrations of thioperamide (2 mg/kg) following mepyramine pretreatment (1 mg/kg, i.p. 10 min before) and placebo. Note that, as compared with thioperamide alone in A, the H3-antagonist no longer suppresses $2, and its arousal effect is partially reduced. The PS-inhibiting effect of thioperamide is, however, enhanced by the pretreatment. Ordinate: quantities of sleep-waking stages in rain. Abscissa: clock time in 2-h periods. *P < 0.05; **P < 0.02; ***P < 0.01 (Student's t-test).
328 for example, vs 4.1 + 0.4 rain and 19.3 + 1.4 with placebo, P < 0.01 and P < 0.05, t-test). Significant delays in the latency of $2 and PS onsets were observed only with the largest dose (10 mg/kg): 158.2 + 44.2 rain and 82.8 + 10.5 min, respectively, vs 25.0 + 2.6 min and 43.6 + 5.3 min with placebo (P < 0.01 for both, t-test). In contrast with well-known effects of amphetamines 36, neither behavioral excitation nor disturbance in body temperature nor respiration could be noted during the thioperamide-induced arousal. To examine whether the effects of these ligands on sleep-waking cycle are mediated selectively by H 3receptors, a M H A at a dose of 20 mg/kg was administered 15 rain before thioperamide application (2 mg/kg) (n = 8). The pretreatment reversed almost completely the arousal effects of thioperamide (Figs. 1B and 2A). During 0-6 h recordings, W was even decreased by 7% when thioperamide was applied after a M H A pretreatment, instead of the 56% increase obtained with the antagonist alone. Figs. 1B and 2A show that simultaneously there is a significant restoration of SWS and PS. To evaluate if the thioperamide-induced W can be attributed to an increase in central HA release, another series of cats (n = 8) was pretreated with mepyramine, a HA antagonist at postsynaptic Hi-receptors, since the importance of the central Hi-receptors in the histaminergic arousal mechanisms is well evidenced 15'17'35. lntraperitoneal injections of mepyramine at a dose of 1 mg/kg, 10 min before thioperamide administration, completely prevented the S2-suppressing effect of thioperamide and significantly attenuated its arousing effects. Indeed, during 0-6 h recordings the 66% diminution in $2 evoked by thioperamide was converted to a 19% augmentation by the pretreatment while the W-increasing effect was reduced from 56% to 19%. The PS inhibitory effect of thioperamide was, however, enhanced by mepyramine (Figs. 1B and 2B). When comparing by ANOVA the results obtained with thioperamide alone and thioperamide with aMH A and mepyramine pretreatments, significant differences were observed for the W, $2 and PS amounts during 0-6 h recordings (P < 0.01, 0.001 and 0.01, respectively), confirming the pretreatment effects. There is now general agreement that the histaminergic neurons, at least in the cerebral cortex, are under the presynaptic auto-control of H3-receptors by a negative feedback process 1-4'7'10"21'32. Since a M H A and thioperamide were recently developed as H3-agonist and antagonist, respectively, several laboratories have confirmed their high pharmacological potency and selectivity7"1°'21. a M H A is about 15 times as potent as HA itself at H3-receptors and both aMHA and thioperamide are at least 104-fold more potent at H 3- than at Hj- or
H2-receptors in brain 4. In vivo, the ligands cross the blood-brain barrier at a low dose range after intraperitoneal or oral administrations and markedly modify the turnover of HA in a long-lasting and opposite manner 4' 7,21. In the present study, a M H A induced a significant increase in deep SWS, whereas thioperamide evoked arousal. The time course and dose dependence of these changes on the states of vigilance are consistent with those on HA release elicited by these drugs in rodent brain TM, suggesting that they are mediated by a decrease and increase in HA release, respectively. This interpretation is reinforced by our observations of pharmacological antagonisms of thioperamide effects by both a M H A and mepyramine. Indeed, the almost total prevention of thioperamide arousal effects by a M H A is in keeping with the demonstrations 4'7'21 that, both in vitro and in vivo, the increase in HA release elicited by thioperamide is antagonized by aMHA. This may mean that the effects of the two ligands on sleep-wakefulness are mediated by H3-receptors. Moreover, the arousal effect of the H 3antagonist was also attenuated by a blockade of postsynaptic Hi-receptors with mepyramine. This result further indicates a role of increased HA transmission in the mechanisms of thioperamide-induced arousal, and also suggests that the effects are due, at least in part, to a central action, since it is well evidenced that the central Hi-receptors are involved in the arousal mechanisms 15' 17,35 and that the Hi-antagonists induce sedation by a central action 26'31. However, it is noteworthy that the PS-inhibiting effect of thioperamide was actually enhanced by mepyramine, which, when administered alone, reduces PS 15'17'35. This effect, as well as the incomplete reversal of W by mepyramine may be explained by the fact that the postsynaptic actions of HA involve not only H 1- but also H2-receptors not blocked by mepyramine. A possible participation of H2-receptors in HA arousal mechanisms is suggested electrophysiologically by the selective potentiation of large excitatory inputs that they mediate on target neurons 8'9'13. In addition, some tuberomammillary neurons, at least in rats, contain not only HA but also a variety of co-transmitters such as GABA, substance P, enkephalins and galanin 11'3°. Moreover, H3-receptors are present not only on histaminergic neurons but also on neurons containing other neurotransmitters 12'28. The release of these transmitters might be, like that of HA, controlled by presynaptic H3-receptors but their actions are not antagonized by mepyramine. These features might also account for the fact that the pattern of action of a M H A on sleep-wakefulness was not superimposable to that of drugs interfering purely with HA metabolism or action e.g. mepyramine and a-fluoromethylhistidine, a selective inhibitor of HA synthesis. In addition to increasing $2,
329 mepyramine reduces both W and PS 15"17"35 and afluoromethylhistidine reduces W 15"~7'1s'34 whereas a M H A does not significantly affect either W or PS. It remains that, in spite of limited differences in their profile of actions, various agents impairing brain histaminergic transmission by distinct mechanisms all significantly increase deep S W S , whereas thioperamide, the first drug facilitating H A release, exerts a significant arousing effect. The critical role of histaminergic neurons in arousal mechanisms, demonstrated here by the use of Ha-ligands, is consistent with their anatomical disposition: the cell bodies are located in a brain area where the damage is accompanied by somnolence or transitory hypersomnia in various species including man16'19"27; they project in a highly divergent manner to the whole CNS, allowing them to regulate in a coordinated manner the sensitivity of large cerebral areas to excitatory signals. Furthermore, both neurochemica122'3°'34 and electrophysiologica133 studies indicate that the activity of putative histaminergic neurons is maximal during periods of W and is suppressed by barbiturates and other hypnotics 25.
The present findings provide further evidence for a role of H A in the sleep-waking control and indicate that H3-receptors are crucially involved in the histaminergic arousal mechanisms. The states of vigilance can be modified by the change in endogenous H A release through the activation or inhibition of H3-receptors. Recently, H3-receptors controlling H A release have been evidenced in human brain 5. Their stimulation or blockade may constitute novel approaches to the treatment of sleep or vigilance disorders. Hi-antagonists are still currently used as hypnotics, but they present several drawbacks such as prominent PS reduction 15"17'35, which may not occur or be less prominent with H3-agonists. Conversely, H3-antagonists like thioperamide, the first histaminergic drug capable of inducing arousal by systemic application, may provide a novel mean to promote W in vigilance deficits and sleep disorders such as hypersomnia or narcolepsy.
1 Arrang, J.-M., Garbarg, M. and Schwartz, J.-C., Autoinhibition of brain histamine release mediated by a novel class (H3) of histamine receptors, Nature, 302 (1983) 832-837. 2 Arrang, J.-M., Garbarg, M. and Schwartz, J.-C., Autoregulation of histamine release in brain by presynaptic Ha-receptors, Neuroscience, 15 (1985) 553-562. 3 Arrang, J.-M., Garbarg, M. and Schwartz, J.-C., Autoregulation of histamine synthesis mediated by presynaptic H3-receptors, Neuroscience, 23 (1987) 149-157. 4 Arrang, J.-M., Garbarg, M., Lancelot, J.-C., Lecomte, J.-M., Pollard, H., Robba, M., Schunack, W. and Schwartz, J.-C., Highly potent and selective ligands for histamine H3-receptors, Nature, 327 (1987) 117-123. 5 Arrang, J.-M., Devaux, B., Chodkiewicz, J.-P. and Schwartz, J.-C., H3-receptors control histamine release in human brain, J. Neurochem,, 51 (1988) 105-108. 6 Douglas, W.W., Histamine and 5-hydroxytryptamine (serotonin) and their antagonists. In A.G. Gilman, L.S. Goodman, T.W. Rail and E Murad (Eds.), The Pharmacological Basis of Therapeutics, Macmillan, New York, 1985, pp. 605-638. 7 Garbarg, M., Trung Tuong, M.D., Gros, C. and Schwartz, J.-C., Effects of histamine H3-receptor ligands on various biochemical indices of histaminergic neuron activity in rat brain, Eur. J. Pharmacol., 164 (1989) 1-11. 8 Haas, H.L. and Konnerth, A., Histamine and noradrenaline decrease calcium-activated potassium conductance in hippocampal pyramidal cells, Nature, 302 (1983) 432-434. 9 Haas, H.L. and Greene, R.W., Effects of histamine on hippocampal pyramidal cells of the rat in vitro, Exp. Brain Res., 62 (1986) 123-130. 10 Hill, S.J. and Straw, J.M., a2-Adrenoceptor-mediated inhibition of histamine release from rat cerebral cortical slices, Br. J. Pharmacol., 95 (1988) 1213-1219. 11 Hough, L.B., Cellular localization and possible functions for brain histamine: recent progress, Progr. Neurobiol., 30 (1988) 469-505. 12 Ishikawa, S. and Sperelakis, N., A novel class (H3) of histamine receptors on perivascular nerve terminals, Nature, 327 (1987) 158-160. 13 Jones, H., Bradlay, P.B. and Roberts, E, Histamine-induced excitation of spontaneously active medullary neurones in the rat
brain is mediated by H2-receptors, Neuropharmacology, 24 (1985) 1231-1239. Lin, J.-S., Luppi, P.-H., Salvert, D., Sakai, K. and Jouvet, M., Histamine-containing neurons in the cat hypothalamus, C.R. Acad. Sci., 303 (1986) 371-376. Lin, J.-S., Sakai, K. and Jouvet, M., Evidence for histaminergic arousal mechanisms in the hypothalamus of cats, Neuropharmacology, 27 (1988) 111-122. Lin, J.-S., Sakai, K., Vanni-Mercier, G. and Jouvet, M., A critical role of the posterior hypothalamus in the mechanisms of wakefulness determined by microinjection of muscimol in freely moving cats, Brain Research, 479 (1989) 225-240. Monti, J.M., Peilejero, T., Jantos, H. and Pazos, S., Role of histamine in the control of sleep and waking. In A. Wauquier, J.M. GaiUard, J.M, Monti and M. Radulovaski (Eds.), Sleep: Neurotransmitters and Neuromodulators, Raven Press, New York, 1985, pp. 197-209. Monti, J.M., D'Angeto, L., Jantos, H. and Pazos, S., Effects of a-fluoromethylhistidine on sleep and wakefulness in the rat, J. Neural. Transm., 72 (1988) 141-145. Moruzzi, G., The sleep-waking cycle, Ergeb. Physiol., 64 (1972) 1-165. Nicholson, A.N., Pasco, P.A. and Stone, B.M., Histaminergic system and sleep, studies in man with H 1 and H 2 antagonists, Neuropharmacology, 24 (1985) 245-250. Oishi, R., Itoh, Y., Nishibori, M. and Saeki, K., Effects of the histamine H3-agonist (R)-a-methylhistamine and the antagonist thioperamide on histamine metabolism in the mouse and rat brain, J. Neurochem., 52 (1989) 1388-1392. Orr, E. and Quay, W.B., Hypothalamic 24-hour rhythms in histamine, histidine decarboxylase and histamine-N-methyltransferase, Endocrinology, 96 (1975) 941-945. Panula, P., Histamine in the nervous system. In E Panula, H. P/iiv/irinta and S. Soinila (Eds.), Neurohistochemistry: Modern Methods and Applications, Liss, New York, 1986, pp. 425-442. Petitjean, E, Sakai, K., Blondaux, C. and Jouvet, M., Hypersomnie par lesion isthmique chez le chat. II. Etude neurophysiologique et neuropharmacologique, Brain Research, 88 (1975) 439-453. Pollard, H., Bischoff, S. and Schwartz, J.-C., Decreased histamine synthesis in the rat brain by hypnotics and anaesthe-
We thank Dr. G. Debilly for help with data analysis and Dr. J. Carew for the revision of the manuscript. This study was supported by INSERM (U52), CNRS (UAl195) and DRET (Grant 89-203).
14 15 16
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18 19 20 21
22 23 24
25
330 tics, J. Pharm. Pharmacol., 25 (1973) 920-922. 26 Quach, T.T., Duchemin, A.M., Rose, C. and Schwartz, J.-C., In vivo occupation of cerebral histamine H~-receptors evaluated with [3H]mepyramine may predict sedative properties of psychotropic drugs, Eur. J. Pharmacol., 60 (1979) 391-392. 27 Sallanon, M., Sakai, K., Buda, C., Puymartin, M. and Jouvet, M., Increase of paradoxical sleep induced by microinjections of ibotenic acid into the ventrolateral part of the posterior hypothalamus in the cat, Arch. ltal. Biol., 126 (1988) 87-97. 28 Schlicker, E., Betz, R. and G6thert, M., Histamine H3 receptormediated inhibition of serotonin release in the rat brain cortex, Naunyn-Schmiedeberg's Arch. Pharmacol., 337 (1988) 588-590. 29 Schwartz, J.-C., Histaminergic mechanisms in brain, Annu. Rev. Pharmacol. Toxicol., 17 (1977) 325-339. 30 Schwartz, J.-C., Arrang, J.-M., Garbarg, M., Pollard, H. and Ruat, M., Histaminergic transmission in the mammalian brain, Physiol. Rev., in press. 31 Uzan, A., Le Fur, G. and Malgouris, C., Are antihistamines sedative via a blockade of brain H~ receptors? J. Pharm. Pharmacol., 31 (1979) 701-702. 32 Van der Werf, J.E, Bast, A., Bijioo, G.J., Van der Vliet, A. and
33 34
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Timmerman, H., HA autoreceptor assay with superfused slices of rat brain cortex and electrical stimulation, Eur. J. Pharmacol., 138 (1987) 199-206. Vanni-Mercier, G., Sakai, K. and Jouvet, M., 'Waking-state specific' neurons in the caudal hypothalamus of the cat, C.R. Acad. Sci., 298 (1984) 195-200. Wada, H., Watanabe, T., Yamatodani, A., Maeyama, K., Itoi, N., Cacabelos, R., Seo, M.L., Kiyono, S., Nagai, K. and Nakagawa, T., Physiological functions of histamine in the brain. In C.R. Ganellin and J.-C. Schwartz (Eds.), Frontiers in Histamine Research: A Tribute to Heinz Schild. Advances in the Biosciences, Vol. 51, Pergamon, Oxford, 1985, pp. 225-236. Wauquier, A., Van den Broeck, W.A.E., Awouters, E and Janssen, P.A.J., A comparison between astemizole and other antihistamines on sleep-wakefulness cycles in dogs, Neuropharmacology, 20 (1981) 853-859. Weiner, N., Norepinephrine, epinephrine, and the sympathomimetic amines. In A.G. Gilman, L.S. Goodman, T.W. RaU and F. Murad (Eds.), The Pharmacological Basis of Therapeutics, Macmillan, New York, 1985, pp. 145-180.
325
Elsevier BRES 24189
Involvement of histaminergic neurons in arousal mechanisms demonstrated with H3-receptor ligands in the cat Jian-Sheng Lin 1, Kazuya Sakai 1, Giovanna Vanni-Mercier 1, Jean-Michel Arrang 2, Monique Garbarg z, Jean-Charles Schwartz 2 and Michel Jouvet 1 ID~partement de Mddecine Exp~rimentale, 1NSERM U52, CNRS UA 1195, Universit~ Claude Bernard, Lyon (France) and 2Unit~ 109 de Neurobiologie, Centre Paul Broca de I'INSERM, Paris (France)
(Accepted 3 April 1990) Key words: Histaminergic neuron; H3-receptor; Sleep-wakefulness; Thioperamide; a-Methylhistamine; Cat
The effects of histamine Ha-receptor ligands on sleep-waking parameters were studied in freely moving cats. Oral administration of (R)a-methylhistamine (aMHA), a Ha-agonist,caused a significantincrease in deep slow wave sleep while that of thioperamide, a Ha-antagonist, enhanced wakefulness in a marked and dose-dependent manner. The arousal effects of thioperamide were prevented by pretreatment with aMHA or mepyramine, a Hi-receptor antagonist. The findings support the hypothesis that the histaminergic neurons are critically involved in arousal mechanisms and suggest that Ha-receptors play an active part in these mechanisms by regulating histamine transmission. The sedative effects of 'antihistamines', i.e. histamine (HA) Hi-receptor antagonists, have been well known since their therapeutic application 6. However, the idea that H A might be a 'waking amine' still remains a matter of debate 11'2°, even though there is recent experimental evidence supporting the involvement of Hi-receptors in arousal 15A7'29'35. This is mainly because, until recently, effective drugs were not available to enhance histaminergic transmission in the brain. It is now known that the perikarya of histaminergic neurons are situated in the tuberomammillary nucleus of the posterior hypothalamus, a brain area involved in the maintenance of wakefulness 16A9'27, and project diffusely to almost all brain areas ~'14'23. Recently, the activity of such a neuronal system was demonstrated to be regulated through a presynaptic negative feedback process mediated by a novel class of H A receptors, namely H31-3. Highly potent, selective and brain-penetrating ligands to H3-receptors, i.e. (R)ct-methylhistamine (aMHA), a chiral agonist, and thioperamide, a competitive antagonist, were also developed more recently4. The agonist and antagonist were further shown to be able to decrease or increase, respectively, in a marked and long-lasting manner, the synthesis and release of H A in vitro 4'1° as well as in the brain of living animals 7,2~. It seems therefore that they represent novel and useful tools to clarify the functional role of histaminergic neurons in the brain. In the present study, we used the H3-receptor
agents to determine the role of histaminergic neurons and the involvement of Ha-receptors in the arousal mechanisms by examining their effects on sleep-waking parameters in freely moving cats. Sixteen adult cats of both sexes weighing 2.5-3.7 kg were chronically implanted, under pentobarbital anesthesia (25 mg/kg, i.v.), with electrodes for polygraphic recordings of pontogeniculooccipital (PGO) activity, neocortical electroencephalogram (EEG), hippocampal and olfactory bulb E E G , electromyogram (EMG), and electrooculogram (EOG). In addition, a small thermocouple was placed under deep neck muscles to record the body temperature. Experiments began 10 days after surgery. The animals were housed in a sound-attenuated and dimly illuminated cage at 24-27 °C and fed at 6 p.m. Oral administrations of lactose capsules alone (placebo), or containing (R)a-methylhistamine or thioperamide, were performed at 11 a.m. and subsequent polygraphic recordings were made for at least 24 h. There was an interval of 7 days between two administrations. The scoring of the polygraphic records was made minute by minute according to previously described criteria 24 for wakefulness (W), light slow wave sleep (S1), deep slow wave sleep ($2) and paradoxical sleep (PS). The paired Student's t-test was used to examine the difference between drug and placebo administration: individual animals served as their own controls. The dose and pretreatment effects of drugs were measured using
Correspondence: J.-S. Lin, Drpartement de M6decine Exprrimentale, INSERM U52, CNRS UA 1195, Universit6 Claude Bernard, 8 avenue Rockefeller, 69373 Lyon Cedex 2, France.
0006-8993/90/$03.50 O 1990 Elsevier Science Publishers B.V. (Biomedical Division)
326 one way analysis of variance (ANOVA). When administered orally, a M H A (10-20 mg/kg) induced a significant and dose-related increase in $2 (Figs. 1A and 2A). The maximal effect was found between the 2nd and the 6th hour after administration. During this period, the times spent in $2 were 124% and 158% of the respective control values at doses of 10 and 20 mg/kg (P < 0.02 and P < 0.01, n = 6). The decline in W, S1 and PS was, however, not statistically significant (Figs. 1A and 2A). The augmentation in $2 was characterized by an increase in the mean episode duration (4.9 _+ 0.3 min with a M H A 20 mg/kg, for example, vs 3.0 +
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0.2 min with placebo, P < 0.001, t-test) while the mean episode frequency and the latency of both slow wave sleep (SWS) and PS onset were not significantly modified. No overt abnormality in behavior, nor in E E G , e.g. hypersynchronous cortical activity or high-voltage slow waves during behavioral arousal, could be observed. In contrast, thioperamide (2-10 mg/kg) elicited a marked, dose-related and long-lasting increase in W at the expense of both PS and SWS, especially $2 (Figs. 1A and 2A). These changes were not marked at a dose of 1 mg/kg (n = 4, not shown) but became significant at a dose of 2 mg/kg or more. When compared with the
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327 occurred as early as the first h o u r after administration and the duration of the m a x i m a l effects d e p e n d e d on the doses: 6 - 7 , 7 - 9 and 9 - 1 0 h for doses of 2, 5 and 10 mg/kg, respectively. T h e increase in W was due to an a u g m e n t a t i o n of both waking episode d u r a t i o n and frequency (8.8 + 1.2 rain and 24.6 + 2.7 with 5 mg/kg,
respective control values, the m e a n a u g m e n t a t i o n of W b e t w e e n time 0 and the 6th hour was 56%, 150% and 160% at doses of 2 (Fig. 2, n = 8), 5 (n = 8) and 10 (n = 5) mg/kg, respectively. Analysis of variance revealed a significant difference a m o n g these changes elicited by the different doses used ( P < 0.005). T h e arousal effects
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Fig. 2. Effects of a-methylhistamine and thioperamide as well as thioperamide combined with aMHA or mepyramine pretreatments on cumulative values of sleep-waking stages in the cat. The curves show the evolution of wakefulness (W), light slow wave sleep (S1), deep slow wave sleep ($2) and paradoxical sleep (PS) over 22 h following the oral administrations at 11 a.m. The S.E.M. (vertical bars) of experimental groups is given when the mean values are out of the control zones (means +_ S.E.M. of placebo-tested cats). A: a group of cats receiving 8 oral administrations of placebo, aMHA (20 mg/kg) and thioperamide (2 mg/kg), alone and with aMHA pretreatment (20 mg/kg). Note that ctMHA increases $2 whereas thioperamide elicits arousal significantly and that the pretreatment with aMHA reverses almost completely the arousal effect of thioperamide and restores significantly $2 and PS. B: another group of cats receiving 8 oral administrations of thioperamide (2 mg/kg) following mepyramine pretreatment (1 mg/kg, i.p. 10 min before) and placebo. Note that, as compared with thioperamide alone in A, the H3-antagonist no longer suppresses $2, and its arousal effect is partially reduced. The PS-inhibiting effect of thioperamide is, however, enhanced by the pretreatment. Ordinate: quantities of sleep-waking stages in rain. Abscissa: clock time in 2-h periods. *P < 0.05; **P < 0.02; ***P < 0.01 (Student's t-test).
328 for example, vs 4.1 + 0.4 rain and 19.3 + 1.4 with placebo, P < 0.01 and P < 0.05, t-test). Significant delays in the latency of $2 and PS onsets were observed only with the largest dose (10 mg/kg): 158.2 + 44.2 rain and 82.8 + 10.5 min, respectively, vs 25.0 + 2.6 min and 43.6 + 5.3 min with placebo (P < 0.01 for both, t-test). In contrast with well-known effects of amphetamines 36, neither behavioral excitation nor disturbance in body temperature nor respiration could be noted during the thioperamide-induced arousal. To examine whether the effects of these ligands on sleep-waking cycle are mediated selectively by H 3receptors, a M H A at a dose of 20 mg/kg was administered 15 rain before thioperamide application (2 mg/kg) (n = 8). The pretreatment reversed almost completely the arousal effects of thioperamide (Figs. 1B and 2A). During 0-6 h recordings, W was even decreased by 7% when thioperamide was applied after a M H A pretreatment, instead of the 56% increase obtained with the antagonist alone. Figs. 1B and 2A show that simultaneously there is a significant restoration of SWS and PS. To evaluate if the thioperamide-induced W can be attributed to an increase in central HA release, another series of cats (n = 8) was pretreated with mepyramine, a HA antagonist at postsynaptic Hi-receptors, since the importance of the central Hi-receptors in the histaminergic arousal mechanisms is well evidenced 15'17'35. lntraperitoneal injections of mepyramine at a dose of 1 mg/kg, 10 min before thioperamide administration, completely prevented the S2-suppressing effect of thioperamide and significantly attenuated its arousing effects. Indeed, during 0-6 h recordings the 66% diminution in $2 evoked by thioperamide was converted to a 19% augmentation by the pretreatment while the W-increasing effect was reduced from 56% to 19%. The PS inhibitory effect of thioperamide was, however, enhanced by mepyramine (Figs. 1B and 2B). When comparing by ANOVA the results obtained with thioperamide alone and thioperamide with aMH A and mepyramine pretreatments, significant differences were observed for the W, $2 and PS amounts during 0-6 h recordings (P < 0.01, 0.001 and 0.01, respectively), confirming the pretreatment effects. There is now general agreement that the histaminergic neurons, at least in the cerebral cortex, are under the presynaptic auto-control of H3-receptors by a negative feedback process 1-4'7'10"21'32. Since a M H A and thioperamide were recently developed as H3-agonist and antagonist, respectively, several laboratories have confirmed their high pharmacological potency and selectivity7"1°'21. a M H A is about 15 times as potent as HA itself at H3-receptors and both aMHA and thioperamide are at least 104-fold more potent at H 3- than at Hj- or
H2-receptors in brain 4. In vivo, the ligands cross the blood-brain barrier at a low dose range after intraperitoneal or oral administrations and markedly modify the turnover of HA in a long-lasting and opposite manner 4' 7,21. In the present study, a M H A induced a significant increase in deep SWS, whereas thioperamide evoked arousal. The time course and dose dependence of these changes on the states of vigilance are consistent with those on HA release elicited by these drugs in rodent brain TM, suggesting that they are mediated by a decrease and increase in HA release, respectively. This interpretation is reinforced by our observations of pharmacological antagonisms of thioperamide effects by both a M H A and mepyramine. Indeed, the almost total prevention of thioperamide arousal effects by a M H A is in keeping with the demonstrations 4'7'21 that, both in vitro and in vivo, the increase in HA release elicited by thioperamide is antagonized by aMHA. This may mean that the effects of the two ligands on sleep-wakefulness are mediated by H3-receptors. Moreover, the arousal effect of the H 3antagonist was also attenuated by a blockade of postsynaptic Hi-receptors with mepyramine. This result further indicates a role of increased HA transmission in the mechanisms of thioperamide-induced arousal, and also suggests that the effects are due, at least in part, to a central action, since it is well evidenced that the central Hi-receptors are involved in the arousal mechanisms 15' 17,35 and that the Hi-antagonists induce sedation by a central action 26'31. However, it is noteworthy that the PS-inhibiting effect of thioperamide was actually enhanced by mepyramine, which, when administered alone, reduces PS 15'17'35. This effect, as well as the incomplete reversal of W by mepyramine may be explained by the fact that the postsynaptic actions of HA involve not only H 1- but also H2-receptors not blocked by mepyramine. A possible participation of H2-receptors in HA arousal mechanisms is suggested electrophysiologically by the selective potentiation of large excitatory inputs that they mediate on target neurons 8'9'13. In addition, some tuberomammillary neurons, at least in rats, contain not only HA but also a variety of co-transmitters such as GABA, substance P, enkephalins and galanin 11'3°. Moreover, H3-receptors are present not only on histaminergic neurons but also on neurons containing other neurotransmitters 12'28. The release of these transmitters might be, like that of HA, controlled by presynaptic H3-receptors but their actions are not antagonized by mepyramine. These features might also account for the fact that the pattern of action of a M H A on sleep-wakefulness was not superimposable to that of drugs interfering purely with HA metabolism or action e.g. mepyramine and a-fluoromethylhistidine, a selective inhibitor of HA synthesis. In addition to increasing $2,
329 mepyramine reduces both W and PS 15"17"35 and afluoromethylhistidine reduces W 15"~7'1s'34 whereas a M H A does not significantly affect either W or PS. It remains that, in spite of limited differences in their profile of actions, various agents impairing brain histaminergic transmission by distinct mechanisms all significantly increase deep S W S , whereas thioperamide, the first drug facilitating H A release, exerts a significant arousing effect. The critical role of histaminergic neurons in arousal mechanisms, demonstrated here by the use of Ha-ligands, is consistent with their anatomical disposition: the cell bodies are located in a brain area where the damage is accompanied by somnolence or transitory hypersomnia in various species including man16'19"27; they project in a highly divergent manner to the whole CNS, allowing them to regulate in a coordinated manner the sensitivity of large cerebral areas to excitatory signals. Furthermore, both neurochemica122'3°'34 and electrophysiologica133 studies indicate that the activity of putative histaminergic neurons is maximal during periods of W and is suppressed by barbiturates and other hypnotics 25.
The present findings provide further evidence for a role of H A in the sleep-waking control and indicate that H3-receptors are crucially involved in the histaminergic arousal mechanisms. The states of vigilance can be modified by the change in endogenous H A release through the activation or inhibition of H3-receptors. Recently, H3-receptors controlling H A release have been evidenced in human brain 5. Their stimulation or blockade may constitute novel approaches to the treatment of sleep or vigilance disorders. Hi-antagonists are still currently used as hypnotics, but they present several drawbacks such as prominent PS reduction 15"17'35, which may not occur or be less prominent with H3-agonists. Conversely, H3-antagonists like thioperamide, the first histaminergic drug capable of inducing arousal by systemic application, may provide a novel mean to promote W in vigilance deficits and sleep disorders such as hypersomnia or narcolepsy.
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We thank Dr. G. Debilly for help with data analysis and Dr. J. Carew for the revision of the manuscript. This study was supported by INSERM (U52), CNRS (UAl195) and DRET (Grant 89-203).
14 15 16
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18 19 20 21
22 23 24
25
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