- Email: [email protected]
5-HT1A receptor antagonist p-MPPI attenuates acute ethanol effects in mice and rats
Neuroscience Letters 322 (2002) 1–4 www.elsevier.com/locate/neulet
5-HT1A receptor antagonist p-MPPI attenuates acute ethanol effects in mice and rats Nina K. Popova*, Elena A. Ivanova Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, Lavrentyeva 10, 630090 Novosibirsk, Russia Received 13 August 2001; received in revised form 24 October 2001; accepted 26 November 2001
Abstract The effect of a selective 5-HT1A antagonist, 4-(2 0 -methoxy-)phenyl-1-[2 0 -(N-2 0 0 -pyridinyl)-p-iodobenzamino-]ethylpiperazine (p-MPPI), on acute ethanol-induced hypothermia, sleep and suppression of acoustic startle reflex in C3H/ He mice and Wistar rats was studied. Administration of p-MPPI at the doses of 0.4, 0.7 and 1.0 mg/kg reduced in a dosedependent manner the ethanol-induced hypothermia and the sleep time and attenuated the ethanol-induced decrease of acoustic startle reflex magnitude in mice. Similar p-MPPI (0.4 mg/kg) effects on ethanol-induced sleep and hypothermia were obtained in rats. It was concluded that 5-HT1A receptors were involved in the mechanisms of the ethanol-induced hypothermia and sleep, and that 5-HT1A antagonist increased acute ethanol tolerance. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acute ethanol effects; 5-HT1A receptors; 5-HT1A antagonist; Ethanol-induced hypothermia; Hypnotic sleep
There are some, not numerous, data which suggest that changes in the brain serotonin (5-HT) system bring about an alteration of ethanol tolerance. It has been demonstrated that administration of a 5-HT synthesis precursor, l-tryptophan increases the rate of development of tolerance to the hypothermic ethanol effect and attenuates the motor disorders caused by ethanol [13], whereas an electrolytic lesion of medial raphe nuclei, as well as a decrease in the brain 5HT level provoked by the neurotoxin 5,7-dihydroxytryptamine [9], delay the development of ethanol tolerance. Elucidation of the mechanisms of the 5-HTergic system effects on ethanol tolerance is complicated due to polymorphism of 5-HT receptors. At present, seven types and 14 subtypes of 5-HT receptors are known [8], and so far, it has not been established which of these types are involved in the mechanisms of ethanol sensitivity. There are some data suggesting 5-HT3 receptor involvement in acute ethanol effects [11]. At the same time, the binding density of 5HT1A sites in the hippocampus and cerebral cortex is higher in alcohol-preferring P rat strain than in non-preferring animals [20]. 5-HT1A receptors are of special interest, since there are data on their involvement in the mechanisms of hypother* Corresponding author. Tel.: 17-383-2-32-3101; fax: 17-383-233-1278. E-mail address: [email protected] (N.K. Popova).
mia [7] and sleep [15,18]. The hypothermia and sleep produced by ethanol administration gives reasons to hypothesize that 5-HT1A receptors may be involved in these ethanol-induced effects, whose expressions are just the basic indicators of acute ethanol tolerance [11]. The goal of the present work was to study the effect of 5HT1A blockade in mice and rats with the selective 5-HT1A antagonist 4-(2 0 -methoxy-)phenyl-1-[2 0 -(N-2 0 0 -pyridinyl)p-iodobenzamino-]ethyl-piperazine (p-MPPI) [1,2,12] on acute ethanol effects, i.e. hypothermia, sleep and suppressed acoustic startle reflex. Piperazine derivative p-MPPI is a ‘silent’ 5-HT1A antagonist with a high affinity to 5-HT1A receptors. p-MPPI blocked the ability of selective 5-HT1A agonist 8-hydroxy-2-(di-N-propylamino) tetralin (8-OHDPAT) to inhibit forskolin-stimulated adenylyl cyclase activity without demonstrating significant effects of its own [12]. The experiments were carried out on mature male 2month-old, 23–25 g C3H/He mice (C3H) and 3-monthold, 230–250 g Wistar rats. The animals were kept under standard conditions of the animal house of the Institute of Cytology and Genetics, under natural illumination; water and food were given ad libitum. Mice were kept in groups of eight, and rats in groups of four animals per standard cage. Two to three days before the experiment, to remove the possible effect of social interactions, animals were placed in individual cages. Testing was carried out in winter
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 51 9- 8
2
N.K. Popova, E.A. Ivanova / Neuroscience Letters 322 (2002) 1–4
at 14:00–16:00 h at room temperature 21–22 8C. All experimental procedures were in compliance with the European Communities Council Directive of November 24, 1986 (86/ 609/EEC). The animals were injected intraperitoneally with a 20% v/ v ethanol solution: mice received a dose of 5.0 g/kg; rats
received 3.0 g/kg. Recorded were such indicators of alcohol sensitivity as ethanol-induced hypothermia and sleep duration measured by the difference between the time of loss and recovery of the righting reflex. In a separate series of experiments, the change of acoustic startle reflex magnitude was studied after ethanol administration in smaller doses, i.e. 2.0 g/kg to mice and 1.0 g/kg to rats. The acoustic startle reflex was tested in a device SR-Pilot (San Diego Instruments) which represents a Plexiglas chamber with dimensions of 15 £ 19 £ 25 cm. On the bottom of the chamber, there is a Plexiglas platform connected to a highly sensitive piezotransducer; on the ceiling, a microphone is fixed. After switching on, a ‘white noise’ of 65 dB was sent to the chamber. A 115 dB acoustic stimulus that provoked the startle reflex was switched on simultaneously with recording the animals’ movements on the platform. The movement of the platform was recorded automatically by means of microcomputers, the numerical values were displayed on the front panel of the device. Fifteen minutes after ethanol administration, the animal was placed into the device for 3 min for adaptation, whereupon at 30 s intervals, four consecutive acoustic stimuli were given. The initial body temperature was measured and that at 20 min time point intervals for 1 h after ethanol injection by means of a KJT thermocouple (Hanna Instruments, Singapore) with copper–constantan rectal probes for rats and for mice (Physitemp Instruments, USA). The selective 5-HT1A antagonist p-MPPI HCl (Research Biochemicals, Inc., USA) was administered i.p. in doses of 0.4, 0.7 and 1.0 mg/kg to mice and 0.4 mg/kg to rats 15 min prior to ethanol administration. p-MPPI was dissolved in a drop of concentrated hydrochloric acid, diluted with bidistilled water, and the pH was adjusted with 10 N NaOH solution to 6.0. Control animals were administered the vehicle. Statistical treatment of data was performed using oneway analysis of variance followed by Duncan’s test using ‘Microcal Origin’ ver.5.0 and STATISTICA ver. 5.0. It was demonstrated that ethanol administration resulted, in mice and rats, in a loss of the righting reflex, a drastic decrease of body temperature (P , 0:001) and a decrease of acoustic startle reflex magnitude (P , 0:001). The 5-HT1A
Fig. 1. Effect of 5-HT1A receptor antagonist p-MPPI on ethanolinduced hypothermia, hypnotic sleep and diminution of acoustic startle reflex in mice. (A,B) p-MPPI was administered i.p. 15 min before 20% v/v ethanol injection (5.0 g/kg i.p.). The initial body temperature and that every 20 min after ethanol injection was measured. (C) The duration of ethanol-induced sleep was determined by the differences between the time of loss and recovery of righting reflex. (D) p-MPPI was administered i.p. at the dose 0.4 mg/kg 15 min before 20% v/v ethanol injection (2.0 g/kg). The magnitude of acoustic startle reflex was determined in 15 min after ethanol injection. Data are shown as mean values ^ SEM from ten mice in each group. *P , 0:05,**P , 0:01,*** P , 0:001 vs. vehicle (A–C); *P , 0:05 vs. vehicle, ###P , 0:001 vs. vehicle without ethanol (D).
N.K. Popova, E.A. Ivanova / Neuroscience Letters 322 (2002) 1–4
receptor blockade diminished these ethanol-induced effects in a clearly dose-dependent manner. Administration of the 5-HT1A antagonist p-MPPI decreased the expression of ethanol-induced hypothermia in mice as compared with animals to which the vehicle was injected. In p-MPPI-pretreated mice, the maximal ethanol-induced decrease of body temperature was less expressed and the recovery of normal temperature set on earlier than in vehicle-treated animals (Fig. 1A,B). In p-MPPI-pretreated mice, the time of ethanol-induced hypnotic sleep was reduced, which was manifested in a more rapid restitution of the righting reflex (by more than two times at a dose of 1.0 mg/kg as compared with control animals). This effect was also dose-dependent (Fig. 1C). The ethanol-induced suppression of the acoustic startle reflex was also attenuated in p-MPPI-pretreated mice (P , 0:05; Fig. 1D). In rats, p-MPPI administration also attenuated ethanolinduced hypothermia and sleep. Rats exhibited a higher sensitivity to the 5-HT1A antagonist than mice did, and pMPPI in a dose of 0.4 mg/kg brought about a clear-cut reduction of the hypothermic effect of ethanol (Fig. 2A,B). The effect of the 5-HT1A antagonist was the most manifested with respect to ethanol-induced sleep. Ethanol produced hypnotic sleep in none of the eight p-MPPI-treated rats, but in six out of the eight control rats (x2 ¼ 6:67, P , 0:01) with the time of sleep 7.5 ^ 1.41 min. Like in mice, in rats, ethanol administration brought about a decrease of acoustic startle reflex. Administration of pMPPI resulted in a tendency towards diminution of the ethanol-induced attenuation of the startle reflex (Fig. 2C). p-MPPI is considered to be an effective pharmacological tool as a 5-HT1A receptor antagonist because it was shown to be effective at blocking responses mediated by 5-HT1A receptors without agonistic activity [1]. Therefore, blockade of 5-HT1A receptors resulted in an increase of acute alcohol tolerance. The p-MPPI-induced increase of tolerance was observed with respect to all of the three effects of alcohol—hypnotic, hypothermic and reflex-suppressing. It is noteworthy that the 5-HT1A antagonist attenuated the effects of ethanol both in mice and in rats, which suggests more general than purely species-specific effects. The influence of 5-HT1A blockade was manifested to the highest degree in the hypothermic and hypnotic effects of ethanol and less in the ethanol-induced suppression of the acoustic startle reflex. One may suppose that the rather weak effect of p-MPPI on ethanol suppressed startle is associated with a smaller involvement of 5-HT1A receptors located in the ponto-medullar brainstem neuronal circuit mediating the acoustic startle reflex [10]. 5-HT1A receptors have been implicated in the control of both thermoregulation [7] and sleep [15,18]. Concomitantly, it is known that the sleep and thermoregulation mechanisms are closely associated [14]. Most probably, this association is based on the partially coinciding neurochemical control of sleep and temperature regulation. The data obtained give reasons to suppose that 5-HT1A receptors
3
represent just the receptor type which participates both in sleep and temperature control not only in natural but also in ethanol-induced sleep and hypothermia. 5-HT1A receptors belong to the superfamily of receptors bound to G-proteins and are located both presynaptically
Fig. 2. Effect of 5-HT1A receptor antagonist p-MPPI on ethanolinduced hypothermia and diminution of acoustic startle reflex in rats. (A,B) p-MPPI (0.4 mg/kg) was administered 15 min before 20% v/v ethanol injection (3.0 g/kg i.p.). Initial body temperature and that every 20 min after ethanol injection was measured. (C) p-MPPI was administered i.p. at the dose 0.4 mg/kg 15 min before 20% v/v ethanol injection (1.0 g/kg). The magnitude of acoustic startle reflex was determined 15 min after ethanol injection (1.0 g/kg). Data are shown as mean values ^ SEM from eight rats in each group. 11P , 0:01, 111P , 0:001 vs. initial body temperature (A); **P , 0:01 vs. vehicle (B); ###P , 0:001 vs. vehicle without ethanol (C).
4
N.K. Popova, E.A. Ivanova / Neuroscience Letters 322 (2002) 1–4
and postsynaptically [16]. The highest 5-HT1A receptor density has been found in the main aggregation of pericarya of 5-HT neurons—in midbrain raphe nuclei where they play the role of autoreceptors whose activation decreases the functional activity of the 5-HT system [3]. It has been demonstrated that 5-HT1A agonists decrease the regional glucose metabolism rate in brain structures [4] and decrease cell firing [5,19], these effects being manifested most clearly in raphe nuclei neurons. At the same time, a systemic pMPPI administration increased the firing rate of serotonergic dorsal raphe nuclei neurons, enhanced the neuronal activity during wakefulness [2], and suppressed the rapid eye movement sleep [18]. In this respect, noteworthy is the fact that mice with a highly excitable nervous system are more tolerant to the effects of acute alcohol administration [17]. The idea that the hypothermic and the hypnotic effects of ethanol are associated with 5-HT1A receptor activation is corroborated to some extent by the data witnessing that alcohol inhibits the specific binding of the agonist 8-OHDPAT and antagonist p-MPPF to the 5-HT1A receptors [6]. On the basis of the data obtained, one may hypothesize that 5-HT1A receptors are involved in the mechanisms of acute ethanol tolerance and that the ethanol sensitivity may depend on the functional state and on the density (probably genetically determined) of brain 5-HT1A receptors. The study was supported by grant number 99-04-49409 of the Russian Foundation for Basic Investigation. [1] Allen, A.R., Singh, A., Zhuang, Z.-P., Kung, M., Kung, H.F. and Lucki, I., The 5-HT1A receptor antagonist p-MPPI blocks responses mediated by postsynaptic and presynaptic 5HT1A receptors, Pharmacol. Biochem. Behav., 57 (1997) 301–307. [2] Bjorvatn, B., Fornal, C.A., Martin, F.J., Metzler, C.W. and Jacobs, B.L., The 5-HT1A receptor antagonist p-MPPI blocks 5-HT1A autoreceptors and increases dorsal raphe unit activity in awake cats, Eur. J. Pharmacol., 356 (1998) 167–178. [3] Evrard, A., Laporte, A.M., Chastanet, M., Hen, R., Hamon, M. and Adrien, J., 5-HT1A and 5-HT1B receptors control the firing of serotoninergic neurons in the dorsal raphe nucleus of the mouse: studies in 5-HT1B knock-out mice, Eur. J. Neurosci., 11 (1999) 3823–3831. [4] Freo, U., Pietrini, G., Furey-Kurkjian, M., Merico, A., Ruggero, S., Dam, M. and Battistini, L., Dose-dependent effects of buspirone on behavior and cerebral glucose metabolism in rats, Brain Res., 622 (1995) 213–220.
[5] Gobert, A., Lejeune, F., Rivet, J.M., Audinot, V., NewmanTancredi, A. and Millan, M.J., Modulation of the activity of central serotoninergic neurons by novel serotonin1A receptor agonists and antagonists: a comparison to adrenergic and dopaminergic neurons in rats, J. Pharmacol. Exp. Ther., 273 (1995) 1032–1046. [6] Harikumar, K.G. and Chattopadhyay, A., Modulation of agonist and antagonist interactions in serotonin 1A receptors by alcohols, FEBS Lett., 438 (1998) 96–100. [7] Hjorth, S., Hypothermia in the rat induced by the potent serotoninergic agent 8-OH-DPAT, J. Neural Transm., 61 (1985) 131–135. [8] Hoyer, D., Clarke, D.-E., Fozard, J.R. and Hartig, P.R., International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin), Pharmacol. Rev., 46 (1994) 157–200. [9] Khanna, J.M., Le, A.D., Kalant, H. and LeBlanc, A.E., Role of serotonin (5-HT) in tolerance to ethanol and barbiturates, Adv. Exp. Med. Biol., 126 (1980) 181–195. [10] Koch, M., The neurobiology of startle, Prog. Neurobiol., 59 (1999) 107–128. [11] Kostowsky, W., 5-HT3 receptors and central effects of ethanol, Pol. J. Pharmacol., 48 (1996) 243–254. [12] Kung, H.E., Kung, M.-P., Clarke, W., Maayani, S. and Zhuang, Z.P., A potential 5-HT1A receptor antagonist: pMPPI, Life Sci., 55 (1994) 1459–1462. [13] Le, A.D., Khanna, J.M., Kalant, H. and LeBlanc, A.E., The effect of lesions in the dorsal, median and magnus raphe nuclei on the development of tolerance to ethanol, J. Pharmacol. Exp. Ther., 218 (1981) 525–529. [14] McGinty, D. and Szymusiak, R., Keeping cool: a hypothesis about the mechanisms and functions of slow-wave sleep, Trends Neurosci., 13 (1990) 480–487. [15] Portas, C.M., Bjorvatn, B. and Ursin, R., Serotonin and the sleep/wake cycle: special emphasis on microdialysis studies, Prog. Neurobiol., 60 (2000) 13–35. [16] Saudou, F. and Hen, R., 5-Hydroxytryptamine receptor subtypes: molecular and functional diversity, Adv. Pharmacol., 30 (1994) 327–380. [17] Shen, E.H., Dorow, J.D., Huson, M. and Phillips, T.J., Correlated responses to selection in FAST and SLOW mice: effects of ethanol on ataxia, temperature, sedation, and withdrawal, Alcohol. Clin. Exp. Res., 20 (1996) 688–696. [18] Sorensen, E., Bjorvatn, B. and Ursin, R., Sleep–wake effects following the selective 5-HT(1A) receptor antagonist pMPPI in the freely moving rat, Behav. Brain Res., 114 (2000) 31–38. [19] Stamford, J.A., Davidson, C., McLaughlin, D.P. and Hopwood, S.E., Control of dorsal raphe 5-HT function by multiple 5-HT(1) autoreceptors: parallel purposes or pointless plurality? Trends Neurosci., 23 (2000) 459–465. [20] Wong, D.T., Threlkeld, P.G., Lumeng, L. and Li, T.K., Higher density of serotonin-1A receptors in the hippocampus and cerebral cortex of alcohol-preferring P rats, Life Sci., 46 (1990) 231–235.
5-HT1A receptor antagonist p-MPPI attenuates acute ethanol effects in mice and rats Nina K. Popova*, Elena A. Ivanova Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, Lavrentyeva 10, 630090 Novosibirsk, Russia Received 13 August 2001; received in revised form 24 October 2001; accepted 26 November 2001
Abstract The effect of a selective 5-HT1A antagonist, 4-(2 0 -methoxy-)phenyl-1-[2 0 -(N-2 0 0 -pyridinyl)-p-iodobenzamino-]ethylpiperazine (p-MPPI), on acute ethanol-induced hypothermia, sleep and suppression of acoustic startle reflex in C3H/ He mice and Wistar rats was studied. Administration of p-MPPI at the doses of 0.4, 0.7 and 1.0 mg/kg reduced in a dosedependent manner the ethanol-induced hypothermia and the sleep time and attenuated the ethanol-induced decrease of acoustic startle reflex magnitude in mice. Similar p-MPPI (0.4 mg/kg) effects on ethanol-induced sleep and hypothermia were obtained in rats. It was concluded that 5-HT1A receptors were involved in the mechanisms of the ethanol-induced hypothermia and sleep, and that 5-HT1A antagonist increased acute ethanol tolerance. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acute ethanol effects; 5-HT1A receptors; 5-HT1A antagonist; Ethanol-induced hypothermia; Hypnotic sleep
There are some, not numerous, data which suggest that changes in the brain serotonin (5-HT) system bring about an alteration of ethanol tolerance. It has been demonstrated that administration of a 5-HT synthesis precursor, l-tryptophan increases the rate of development of tolerance to the hypothermic ethanol effect and attenuates the motor disorders caused by ethanol [13], whereas an electrolytic lesion of medial raphe nuclei, as well as a decrease in the brain 5HT level provoked by the neurotoxin 5,7-dihydroxytryptamine [9], delay the development of ethanol tolerance. Elucidation of the mechanisms of the 5-HTergic system effects on ethanol tolerance is complicated due to polymorphism of 5-HT receptors. At present, seven types and 14 subtypes of 5-HT receptors are known [8], and so far, it has not been established which of these types are involved in the mechanisms of ethanol sensitivity. There are some data suggesting 5-HT3 receptor involvement in acute ethanol effects [11]. At the same time, the binding density of 5HT1A sites in the hippocampus and cerebral cortex is higher in alcohol-preferring P rat strain than in non-preferring animals [20]. 5-HT1A receptors are of special interest, since there are data on their involvement in the mechanisms of hypother* Corresponding author. Tel.: 17-383-2-32-3101; fax: 17-383-233-1278. E-mail address: [email protected] (N.K. Popova).
mia [7] and sleep [15,18]. The hypothermia and sleep produced by ethanol administration gives reasons to hypothesize that 5-HT1A receptors may be involved in these ethanol-induced effects, whose expressions are just the basic indicators of acute ethanol tolerance [11]. The goal of the present work was to study the effect of 5HT1A blockade in mice and rats with the selective 5-HT1A antagonist 4-(2 0 -methoxy-)phenyl-1-[2 0 -(N-2 0 0 -pyridinyl)p-iodobenzamino-]ethyl-piperazine (p-MPPI) [1,2,12] on acute ethanol effects, i.e. hypothermia, sleep and suppressed acoustic startle reflex. Piperazine derivative p-MPPI is a ‘silent’ 5-HT1A antagonist with a high affinity to 5-HT1A receptors. p-MPPI blocked the ability of selective 5-HT1A agonist 8-hydroxy-2-(di-N-propylamino) tetralin (8-OHDPAT) to inhibit forskolin-stimulated adenylyl cyclase activity without demonstrating significant effects of its own [12]. The experiments were carried out on mature male 2month-old, 23–25 g C3H/He mice (C3H) and 3-monthold, 230–250 g Wistar rats. The animals were kept under standard conditions of the animal house of the Institute of Cytology and Genetics, under natural illumination; water and food were given ad libitum. Mice were kept in groups of eight, and rats in groups of four animals per standard cage. Two to three days before the experiment, to remove the possible effect of social interactions, animals were placed in individual cages. Testing was carried out in winter
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 51 9- 8
2
N.K. Popova, E.A. Ivanova / Neuroscience Letters 322 (2002) 1–4
at 14:00–16:00 h at room temperature 21–22 8C. All experimental procedures were in compliance with the European Communities Council Directive of November 24, 1986 (86/ 609/EEC). The animals were injected intraperitoneally with a 20% v/ v ethanol solution: mice received a dose of 5.0 g/kg; rats
received 3.0 g/kg. Recorded were such indicators of alcohol sensitivity as ethanol-induced hypothermia and sleep duration measured by the difference between the time of loss and recovery of the righting reflex. In a separate series of experiments, the change of acoustic startle reflex magnitude was studied after ethanol administration in smaller doses, i.e. 2.0 g/kg to mice and 1.0 g/kg to rats. The acoustic startle reflex was tested in a device SR-Pilot (San Diego Instruments) which represents a Plexiglas chamber with dimensions of 15 £ 19 £ 25 cm. On the bottom of the chamber, there is a Plexiglas platform connected to a highly sensitive piezotransducer; on the ceiling, a microphone is fixed. After switching on, a ‘white noise’ of 65 dB was sent to the chamber. A 115 dB acoustic stimulus that provoked the startle reflex was switched on simultaneously with recording the animals’ movements on the platform. The movement of the platform was recorded automatically by means of microcomputers, the numerical values were displayed on the front panel of the device. Fifteen minutes after ethanol administration, the animal was placed into the device for 3 min for adaptation, whereupon at 30 s intervals, four consecutive acoustic stimuli were given. The initial body temperature was measured and that at 20 min time point intervals for 1 h after ethanol injection by means of a KJT thermocouple (Hanna Instruments, Singapore) with copper–constantan rectal probes for rats and for mice (Physitemp Instruments, USA). The selective 5-HT1A antagonist p-MPPI HCl (Research Biochemicals, Inc., USA) was administered i.p. in doses of 0.4, 0.7 and 1.0 mg/kg to mice and 0.4 mg/kg to rats 15 min prior to ethanol administration. p-MPPI was dissolved in a drop of concentrated hydrochloric acid, diluted with bidistilled water, and the pH was adjusted with 10 N NaOH solution to 6.0. Control animals were administered the vehicle. Statistical treatment of data was performed using oneway analysis of variance followed by Duncan’s test using ‘Microcal Origin’ ver.5.0 and STATISTICA ver. 5.0. It was demonstrated that ethanol administration resulted, in mice and rats, in a loss of the righting reflex, a drastic decrease of body temperature (P , 0:001) and a decrease of acoustic startle reflex magnitude (P , 0:001). The 5-HT1A
Fig. 1. Effect of 5-HT1A receptor antagonist p-MPPI on ethanolinduced hypothermia, hypnotic sleep and diminution of acoustic startle reflex in mice. (A,B) p-MPPI was administered i.p. 15 min before 20% v/v ethanol injection (5.0 g/kg i.p.). The initial body temperature and that every 20 min after ethanol injection was measured. (C) The duration of ethanol-induced sleep was determined by the differences between the time of loss and recovery of righting reflex. (D) p-MPPI was administered i.p. at the dose 0.4 mg/kg 15 min before 20% v/v ethanol injection (2.0 g/kg). The magnitude of acoustic startle reflex was determined in 15 min after ethanol injection. Data are shown as mean values ^ SEM from ten mice in each group. *P , 0:05,**P , 0:01,*** P , 0:001 vs. vehicle (A–C); *P , 0:05 vs. vehicle, ###P , 0:001 vs. vehicle without ethanol (D).
N.K. Popova, E.A. Ivanova / Neuroscience Letters 322 (2002) 1–4
receptor blockade diminished these ethanol-induced effects in a clearly dose-dependent manner. Administration of the 5-HT1A antagonist p-MPPI decreased the expression of ethanol-induced hypothermia in mice as compared with animals to which the vehicle was injected. In p-MPPI-pretreated mice, the maximal ethanol-induced decrease of body temperature was less expressed and the recovery of normal temperature set on earlier than in vehicle-treated animals (Fig. 1A,B). In p-MPPI-pretreated mice, the time of ethanol-induced hypnotic sleep was reduced, which was manifested in a more rapid restitution of the righting reflex (by more than two times at a dose of 1.0 mg/kg as compared with control animals). This effect was also dose-dependent (Fig. 1C). The ethanol-induced suppression of the acoustic startle reflex was also attenuated in p-MPPI-pretreated mice (P , 0:05; Fig. 1D). In rats, p-MPPI administration also attenuated ethanolinduced hypothermia and sleep. Rats exhibited a higher sensitivity to the 5-HT1A antagonist than mice did, and pMPPI in a dose of 0.4 mg/kg brought about a clear-cut reduction of the hypothermic effect of ethanol (Fig. 2A,B). The effect of the 5-HT1A antagonist was the most manifested with respect to ethanol-induced sleep. Ethanol produced hypnotic sleep in none of the eight p-MPPI-treated rats, but in six out of the eight control rats (x2 ¼ 6:67, P , 0:01) with the time of sleep 7.5 ^ 1.41 min. Like in mice, in rats, ethanol administration brought about a decrease of acoustic startle reflex. Administration of pMPPI resulted in a tendency towards diminution of the ethanol-induced attenuation of the startle reflex (Fig. 2C). p-MPPI is considered to be an effective pharmacological tool as a 5-HT1A receptor antagonist because it was shown to be effective at blocking responses mediated by 5-HT1A receptors without agonistic activity [1]. Therefore, blockade of 5-HT1A receptors resulted in an increase of acute alcohol tolerance. The p-MPPI-induced increase of tolerance was observed with respect to all of the three effects of alcohol—hypnotic, hypothermic and reflex-suppressing. It is noteworthy that the 5-HT1A antagonist attenuated the effects of ethanol both in mice and in rats, which suggests more general than purely species-specific effects. The influence of 5-HT1A blockade was manifested to the highest degree in the hypothermic and hypnotic effects of ethanol and less in the ethanol-induced suppression of the acoustic startle reflex. One may suppose that the rather weak effect of p-MPPI on ethanol suppressed startle is associated with a smaller involvement of 5-HT1A receptors located in the ponto-medullar brainstem neuronal circuit mediating the acoustic startle reflex [10]. 5-HT1A receptors have been implicated in the control of both thermoregulation [7] and sleep [15,18]. Concomitantly, it is known that the sleep and thermoregulation mechanisms are closely associated [14]. Most probably, this association is based on the partially coinciding neurochemical control of sleep and temperature regulation. The data obtained give reasons to suppose that 5-HT1A receptors
3
represent just the receptor type which participates both in sleep and temperature control not only in natural but also in ethanol-induced sleep and hypothermia. 5-HT1A receptors belong to the superfamily of receptors bound to G-proteins and are located both presynaptically
Fig. 2. Effect of 5-HT1A receptor antagonist p-MPPI on ethanolinduced hypothermia and diminution of acoustic startle reflex in rats. (A,B) p-MPPI (0.4 mg/kg) was administered 15 min before 20% v/v ethanol injection (3.0 g/kg i.p.). Initial body temperature and that every 20 min after ethanol injection was measured. (C) p-MPPI was administered i.p. at the dose 0.4 mg/kg 15 min before 20% v/v ethanol injection (1.0 g/kg). The magnitude of acoustic startle reflex was determined 15 min after ethanol injection (1.0 g/kg). Data are shown as mean values ^ SEM from eight rats in each group. 11P , 0:01, 111P , 0:001 vs. initial body temperature (A); **P , 0:01 vs. vehicle (B); ###P , 0:001 vs. vehicle without ethanol (C).
4
N.K. Popova, E.A. Ivanova / Neuroscience Letters 322 (2002) 1–4
and postsynaptically [16]. The highest 5-HT1A receptor density has been found in the main aggregation of pericarya of 5-HT neurons—in midbrain raphe nuclei where they play the role of autoreceptors whose activation decreases the functional activity of the 5-HT system [3]. It has been demonstrated that 5-HT1A agonists decrease the regional glucose metabolism rate in brain structures [4] and decrease cell firing [5,19], these effects being manifested most clearly in raphe nuclei neurons. At the same time, a systemic pMPPI administration increased the firing rate of serotonergic dorsal raphe nuclei neurons, enhanced the neuronal activity during wakefulness [2], and suppressed the rapid eye movement sleep [18]. In this respect, noteworthy is the fact that mice with a highly excitable nervous system are more tolerant to the effects of acute alcohol administration [17]. The idea that the hypothermic and the hypnotic effects of ethanol are associated with 5-HT1A receptor activation is corroborated to some extent by the data witnessing that alcohol inhibits the specific binding of the agonist 8-OHDPAT and antagonist p-MPPF to the 5-HT1A receptors [6]. On the basis of the data obtained, one may hypothesize that 5-HT1A receptors are involved in the mechanisms of acute ethanol tolerance and that the ethanol sensitivity may depend on the functional state and on the density (probably genetically determined) of brain 5-HT1A receptors. The study was supported by grant number 99-04-49409 of the Russian Foundation for Basic Investigation. [1] Allen, A.R., Singh, A., Zhuang, Z.-P., Kung, M., Kung, H.F. and Lucki, I., The 5-HT1A receptor antagonist p-MPPI blocks responses mediated by postsynaptic and presynaptic 5HT1A receptors, Pharmacol. Biochem. Behav., 57 (1997) 301–307. [2] Bjorvatn, B., Fornal, C.A., Martin, F.J., Metzler, C.W. and Jacobs, B.L., The 5-HT1A receptor antagonist p-MPPI blocks 5-HT1A autoreceptors and increases dorsal raphe unit activity in awake cats, Eur. J. Pharmacol., 356 (1998) 167–178. [3] Evrard, A., Laporte, A.M., Chastanet, M., Hen, R., Hamon, M. and Adrien, J., 5-HT1A and 5-HT1B receptors control the firing of serotoninergic neurons in the dorsal raphe nucleus of the mouse: studies in 5-HT1B knock-out mice, Eur. J. Neurosci., 11 (1999) 3823–3831. [4] Freo, U., Pietrini, G., Furey-Kurkjian, M., Merico, A., Ruggero, S., Dam, M. and Battistini, L., Dose-dependent effects of buspirone on behavior and cerebral glucose metabolism in rats, Brain Res., 622 (1995) 213–220.
[5] Gobert, A., Lejeune, F., Rivet, J.M., Audinot, V., NewmanTancredi, A. and Millan, M.J., Modulation of the activity of central serotoninergic neurons by novel serotonin1A receptor agonists and antagonists: a comparison to adrenergic and dopaminergic neurons in rats, J. Pharmacol. Exp. Ther., 273 (1995) 1032–1046. [6] Harikumar, K.G. and Chattopadhyay, A., Modulation of agonist and antagonist interactions in serotonin 1A receptors by alcohols, FEBS Lett., 438 (1998) 96–100. [7] Hjorth, S., Hypothermia in the rat induced by the potent serotoninergic agent 8-OH-DPAT, J. Neural Transm., 61 (1985) 131–135. [8] Hoyer, D., Clarke, D.-E., Fozard, J.R. and Hartig, P.R., International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin), Pharmacol. Rev., 46 (1994) 157–200. [9] Khanna, J.M., Le, A.D., Kalant, H. and LeBlanc, A.E., Role of serotonin (5-HT) in tolerance to ethanol and barbiturates, Adv. Exp. Med. Biol., 126 (1980) 181–195. [10] Koch, M., The neurobiology of startle, Prog. Neurobiol., 59 (1999) 107–128. [11] Kostowsky, W., 5-HT3 receptors and central effects of ethanol, Pol. J. Pharmacol., 48 (1996) 243–254. [12] Kung, H.E., Kung, M.-P., Clarke, W., Maayani, S. and Zhuang, Z.P., A potential 5-HT1A receptor antagonist: pMPPI, Life Sci., 55 (1994) 1459–1462. [13] Le, A.D., Khanna, J.M., Kalant, H. and LeBlanc, A.E., The effect of lesions in the dorsal, median and magnus raphe nuclei on the development of tolerance to ethanol, J. Pharmacol. Exp. Ther., 218 (1981) 525–529. [14] McGinty, D. and Szymusiak, R., Keeping cool: a hypothesis about the mechanisms and functions of slow-wave sleep, Trends Neurosci., 13 (1990) 480–487. [15] Portas, C.M., Bjorvatn, B. and Ursin, R., Serotonin and the sleep/wake cycle: special emphasis on microdialysis studies, Prog. Neurobiol., 60 (2000) 13–35. [16] Saudou, F. and Hen, R., 5-Hydroxytryptamine receptor subtypes: molecular and functional diversity, Adv. Pharmacol., 30 (1994) 327–380. [17] Shen, E.H., Dorow, J.D., Huson, M. and Phillips, T.J., Correlated responses to selection in FAST and SLOW mice: effects of ethanol on ataxia, temperature, sedation, and withdrawal, Alcohol. Clin. Exp. Res., 20 (1996) 688–696. [18] Sorensen, E., Bjorvatn, B. and Ursin, R., Sleep–wake effects following the selective 5-HT(1A) receptor antagonist pMPPI in the freely moving rat, Behav. Brain Res., 114 (2000) 31–38. [19] Stamford, J.A., Davidson, C., McLaughlin, D.P. and Hopwood, S.E., Control of dorsal raphe 5-HT function by multiple 5-HT(1) autoreceptors: parallel purposes or pointless plurality? Trends Neurosci., 23 (2000) 459–465. [20] Wong, D.T., Threlkeld, P.G., Lumeng, L. and Li, T.K., Higher density of serotonin-1A receptors in the hippocampus and cerebral cortex of alcohol-preferring P rats, Life Sci., 46 (1990) 231–235.