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Haloperidol reduces the sympathetic and thermogenic activation induced by orexin A
Neuroscience Research 45 (2003) 17 /23 www.elsevier.com/locate/neures
Haloperidol reduces the sympathetic and thermogenic activation induced by orexin A M. Monda , A. Viggiano, V. De Luca Department of Experimental Medicine, Section of Human Physiology, Second University of Naples, via Costantinopoli 16, 80138 Naples, Italy Received 5 June 2002; accepted 12 September 2002
Abstract This experiment tested the effect of haloperidol on the sympathetic and thermogenic effects induced by orexin A. The firing rates of the sympathetic nerves to interscapular brown adipose tissue (IBAT), along with IBAT and colonic temperatures and heart rate were monitored in urethane-anesthetized male Sprague /Dawley rats before and 5 h after an injection of orexin A (1.5 nmol) into the lateral cerebral ventricle. The same variables were monitored in rats with an intraperitoneal administration of haloperidol (1 mg/kg bw), a D2 receptor antagonist. The results show that orexin A increases the sympathetic firing rate, IBAT and colonic temperatures and heart rate. This increase is reduced by the haloperidol. These findings suggest that dopaminergic system is activated during the orexin A-induced hyperthermia. # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Body temperature; Haloperidol; Orexin A; Rat; Sympathetic activity
1. Introduction Orexin A (Sakurai et al., 1998), hypothalamic peptide so-called for its stimulation on food intake (Sweet et al., 1999), causes vegetative responses, when this peptide is intracerebroventricularly (icv) injected (Lubkin and Stricker-Krongrad, 1998; Shirasaka et al., 1999). The autonomic reactions include the sympathetic and thermogenic changes. Indeed, an icv administration of orexin A induces an increase in firing rate of the sympathetic nerves to interscapular brown adipose tissue (IBAT), accompanied with a rise in IBAT and colonic temperatures (Monda et al., 2001). A role of orexins in sleep regulation has been demonstrated (Beuckmann and Yanagisawa, 2002). Deficiency in orexin neurotransmission results in the sleep disorder narcolepsy in mice, dogs, and humans (Taheri et al., 2002). Intraperitonel administration of haloperidol produces hypothermic effects in the rats (Lin et al., 1979), and in
Corresponding author. Tel.: /39-81-566-5833; fax: /39-81-5665846 E-mail address: [email protected] (M. Monda).
the rabbits (Vybiral and Vesela, 1994). Oral administration of haloperidol in normal volunteers causes significant attenuation of the normal daytime increase of body temperature (De Koning and De Vries, 1995). This indicates that dopaminergic system is involved in the regulation of body temperature. Furthermore, an intraperitoneal administration of haloperidol reduces the hyperlocomotion induced by an icv injection of orexin A, suggesting an involvement of dopaminergic system in the motor reactions due to orexin A (Nakamura et al., 2000). The aim of this experiment was to test the effects of haloperidol on the sympathetic and thermogenic reactions induced by orexin A, so that a possible involvement of the dopaminergic system could be considered.
2. Materials and methods 2.1. Animals Male Sprague /Dawley rats (n /24), 3 months old and weighing 270/320 g were used in the experiments. The rats were housed in pairs at controlled temperature (229/1 8C) and humidity (70%) with a 12:12 h light /
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dark /light cycle from 07:00 to 19:00 h. The experiments were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). 2.2. Apparatus A pair of silver wire electrodes recorded the firing rate of nerves to IBAT. The electrical pulses were amplified by a condenser-coupled amplifier and were filtered by band-pass filters (NeuroLog System, Digitimer). The raw pulses were displayed on an oscilloscope (Tektronix) and sent to a window discriminator. Square waves from the discriminator were sent to an analog-digital converter (DAS system, Keithley) and stored on a computer (Personal Computer AT, IBM) every 5 s. A rate meter with a reset time of 5 s was also used to observe the time course of the nerve activity recorded by pen recorder (Dynograph, Beckman). Because signal-tonoise ratio depended on the number of nerve filaments and the condition of contact between nerve and electrodes, the basal burst rates were different for each rat. The threshold level of the event detector was fixed during the experiment at 50% of the peaks of the largest pulses and above background noise. Thermocouples (Ellab) were used to monitor colonic and IBAT temperatures (TC and TIBAT) and the values were stored on a chart recorder. Two electrodes applied to the forelegs monitored the heart rate (beats/min). Electrical signals were addressed to a polygraph (Dynograph, Beckman) to record the electrocardiographic activity on the card and on a computer disk. 2.3. Drugs and doses Orexin A and haloperidol were purchased respectively from Peninsula, England and Lusofarmaco, Italy. We used a dose of 1.5 nmol of orexin A, after a preliminary experiment with doses of 0.15, 1.5 and 15 nmol in three groups of two rats. This test showed a dose/effect curve on TC. The temperature modifications were: (a) from 37.209/0.13 to 37.759/0.17 with dose of 0.15 nmol; (b) from 37.129/0.08 to 38.429/0.12 with 1.5 nmol; (c) from 37.159/0.11 to 38.669/0.16 with 15 nmol. We chose the sub-maximal dose of 1.5 nmol. A similar preliminary test was carried out with doses of 0.5, 1 and 2 mg/kg bw of haloperidol in three groups of two rats injected with 1.5 nmol of orexin A. The temperature modifications were: (a) from 37.129/0.11 to 38.059/0.19 with dose of 0.5 mg/kg bw; (b) from 37.079/0.14 to 37.529/0.15 with 1 mg/kg bw; (c) from 37.119/0.13 to 37.459/0.18 with 2 mg/kg bw. The dose of 1 mg/kg was utilized. Since the low number of animals did not permit a statistical analysis, the preliminary experiments were merely indicative to determine the opportune dose. The purity of orexin is about 98% measured by HPLC, so that the
possibility of pyrogenic effects due to contaminants [for example prostaglandin (PG) of E series] is very unlikely. Indeed, the quantity of PG of E series should be only 10 ng. This dose does not induce hyperthermia when icv injected (Malkinson et al., 1998; Stitt, 1999). The same reasoning should also be valid for other pyrogens. 2.4. Surgery All animals were anesthetized with ip pentobarbital (50 mg/kg bw) and a 20-gauge stainless guide cannula was positioned stereotaxically (Pellegrino et al., 1979) above a lateral cerebral ventricle at the following coordinates: 1.7 mm lateral to the midline, 0.4 mm posterior to the bregma, 3.0 mm from the cranial theca. The rats were given 7/10 days to recover from surgery judged by recovery of preoperative body weight. 2.5. Procedure After recovery, six animals (1st group) were anesthetized with ethyl-urethane (1.2 g/kg bw ip) and mounted in a stereotaxic instrument (Stoelting). The level of anesthesia was kept constant and evaluated by skeletal muscle relaxation, eye and palpebral responses to stimuli. Nerve activity was recorded by small nerve bundles dissected from the intercostal nerves supplying the right side of IBAT. These nerve bundles (only innervating IBAT) do not contain motor fibers discharging with respiratory rhythm. Nerve filaments were isolated from the central cut end of these nerve bundles under a dissecting microscope; the efferent activity was recorded with a pair of silver wire electrodes. The nerve filaments were covered with a mixture of vaseline and liquid petroleum at 37 8C to avoid dehydration. The firing rate was recorded over 60 min before and 300 min after the injection of orexin A (1.5 nmol dissolved in 5 ml of 0.9% NaCl sterile solution). The orexin A was delivered into the left cerebral ventricle by gravity flow over 2 min. The cannula for the injection was 0.4 mm longer than the guide cannula. Furthermore, the heart rate, TC and TIBAT were monitored at the same time as the nerve activity was recorded. TC was measured by inserting the thermocouple into the colon 4 cm from the anus, while TIBAT was monitored by inserting the thermocouple in the left side of IBAT. The same variables were recorded in the other six animals (2nd group), but haloperidol (1 mg/kg bw dissolved in 2 ml of 0.9% NaCl sterile solution) was injected ip 30 min before the icv injection of orexin A. In the other six rats treated with an ip injection of haloperidol (3rd group), saline was injected into the cerebral ventricle and the same variables were monitored. In six control rats (4th group), saline was injected both in the peritoneal cavity and in the lateral ventricle. The baseline values of TC from all animals used were maintained constant by a
M. Monda et al. / Neuroscience Research 45 (2003) 17 /23
heating pad. The electrical energy supplied to pad was not altered during the experimental period. 2.6. Histology At the end of the experiment, the location of the cannula was identified. A stain (bromophenol blue) was injected in the lateral ventricle; the volume of the stain is the same as the volume used for drug administration. The rats were then injected with an overdose of pentobarbital (150 mg/kg bw) and were perfused with 0.9% NaCl followed by 10% formalin solution. The brain was removed and stored in formalin solution. After a few days, 50 mm coronal sections of the fixed brain were cut and stained with neutral red. 2.7. Statistical analysis The values are presented as means9/S.E.M. Statistical analysis was performed using analysis of variance. Multiple comparisons were performed by Newman / Keuls post hoc test.
3. Results Fig. 1 shows the percentage changes in the firing rate of the nerves to IBAT. The icv injection of orexin increased the firing rate and this rise was reduced in the
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animals with ip administration of haloperidol. In the other rats, neither saline nor haloperidol caused any modification. An analysis of variance showed significant effects for orexin [F (1, 20) /235.9, P B/0.01], for haloperidol [F (1, 20) /66.5, P B/0.01], for time [F (11, 220) /63.6, P B/0.01], for interaction orexin /haloperidol [F (1, 20) /80.2, P B/0.01], orexin /time [F (11, 220) /30.6, P B/0.01], haloperidol /time [F (11, 220) /7.7, P B/0.01], and orexin /haloperidol /time [F (11, 220) /23.2, P B/0.01]. The post hoc test showed that the orexin/saline group was different from the orexin/haloperidol group at 1.5 /5 h. The baseline absolute values were respectively of 44.19/8.8 spikes/5 s in the 1st group, 42.49/8.2 in the 2nd group, 45.79/6.8 in the 3rd group and 44.89/9.3 in the 4th group. There were no differences in the baseline absolute values of all groups. Examples of the changes in firing rate are shown in Fig. 2. Fig. 3 illustrates TIBAT changes. The icv injection of orexin caused a rise. This increase was reduced in the animals ip injected with haloperidol. In the other rats, neither saline nor haloperidol caused any modification. An analysis of variance showed significant effects for orexin [F (1, 20) /925.1, P B/0.01], for haloperidol [F(1, 20) /156.9, P B/0.01], for time [F (11, 220) /267.5, P B/ 0.01], for interaction orexin /haloperidol [F (1, 20) / 455.3, P B/0.01], orexin /time [F (11, 220) /90.1, P B/ 0.01], haloperidol /time [F (11, 220) /13.1, P B/0.01], and orexin /haloperidol /time [F (11, 220) /71.6, P B/
Fig. 1. Means9/S.E.M. of changes in firing rate of nerves to interscapular brown adipose tissue. Intraperitoneal (ip) injection of saline or haloperidol (1 mg/kg bw) was made at /0.5 h. Intracerebroventricular (icv) injection of saline or orexin A (1.5 nmol) was made at time 0. Statistical significance (P B/0.05) between the haloperidol/orexin group and the saline/orexin group.
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peridol [F (1, 20) /5.0, P B/0.01], for time [F (11, 220) / 13.3, P B/0.01], for interaction orexin /time [F (11, 220) /11.5, P B/0.01], and orexin /haloperidol /time [F (11, 220) /2.4, P B/0.01]. The post hoc test showed that orexin/saline group was different from orexin/ haloperidol group at 0.5 /5 h. The figures do not illustrate when the effects of orexin disappear, because the registrations of this experiment were interrupted at 5 h. Our unpublished observations showed that the effects of orexin disappear 7 /9 h after icv injection.
4. Discussion
Fig. 2. Actual firing rate changes in a rat receiving intraperitoneal (ip) injection of saline plus intracerebroventricular (icv) injection of orexin A (panel C) or saline (panel D), and in a rat receiving ip injection of haloperidol plus icv injection of orexin A (panel A) or saline (panel B). The arrow indicates the time of icv injection. Ip administrations was made 30 min before the icv injection.
0.01]. Newman /Keuls post hoc test showed that the orexin/saline group was different from orexin/haloperidol group at 1.5 /5 h. Fig. 4 illustrates TC changes. The icv injection of orexin induced a rise in the rats ip injected with saline. Haloperidol reduced the increase due to orexin. No changes resulted in the animals with saline or haloperidol administration. An analysis of variance showed significant effects for orexin [F(1, 20) /808.1, P B/0.01], for haloperidol [F(1, 20) /131.9, P B/0.01], for time [F (11, 220) /278.6, P B/0.01], for interaction orexin / time [F (11, 220) /93.0, P B/0.01], haloperidol /time P B/0.01], and orexin / [F (11, 220) /13.9, haloperidol /time [F (11, 220) /73.1, P B/0.01]. Newman /Keuls post hoc test showed that the orexin/saline group was different from orexin/haloperidol group at 1.5 /5 h. Fig. 5 shows changes in heart rate. The orexin injection induced an increase, which was reduced by haloperidol. Neither the administration of saline nor haloperidol caused any change in the rats without orexin injection. An analysis of variance showed significant effects for orexin [F (1, 20) /13.4, P B/0.01], for halo-
To our knowledge, these findings are the first to demonstrate that an ip injection of haloperidol reduces the sympathetic and thermogenic effects due to an icv administration of orexin A. The effects of orexin A on the sympathetic discharge of the nerves to IBAT corroborate recent evidences demonstrating the role played by this neuropeptide in the control of the autonomic nervous system. Shirasaka et al. (1999) illustrated that icv injections of orexins increase activity of the renal sympathetic nerves, which play an important role in the homeostasis of body fluids and the circulatory system. We only used orexin A because Shirasaka et al. (1999) demonstrated that plasma norepinephrine increases with a high dose of orexin A, but not with orexin B. Since the increase in the sympathetic activity induced by orexin A is reduced by haloperidol, an involvement of the D2 receptors of the dopaminergic system can be hypothesized. Because an injection of quinpirole, a D2 receptor agonist, induces an increase in the sympathetic discharge (Damase-Michel et al., 1990), an activation of dopaminergic system could be a possible mechanism of sympathetic stimulation induced by orexin A. In this mechanism, an involvement of the D2 receptors could play a key role. Our experiment considered the activity of sympathetic nerves to IBAT. This tissue is the principal effector of non-shivering thermogenesis (Cannon et al., 1998) and plays a relevant role in the thermoregulation. We have significantly demonstrated that icv injections of PGE1 induce an increase in the firing rate of sympathetic nerves to IBAT (Monda et al., 1998, 2000), and lysine acetylsalicylate reduces the sympathetic activation induced by orexin A, suggesting that prostaglandins have an implication in the mediation of this phenomenon (Monda et al., 2001). Since the haloperidol reduces the fever due to PGE synthesis (Vybiral and Vesela, 1994), we can hypothesize an involvement of the dopaminergic system in the thermogenic activation due to prostaglandin synthesis by orexin A. In other words, orexin A induces a synthesis of the prostaglandins (Monda et al.,
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Fig. 3. Means9/S.E.M. of changes in temperature of interscapular brown adipose tissue (IBAT). Intraperitoneal (ip) injection of saline or haloperidol (1 mg/kg bw) was made at /0.5 h. Intracerebroventricular (icv) injection of saline or orexin A (1.5nmol) was made at time 0. Statistical significance (P B/0.05) between haloperidol/orexin group and saline/orexin group.
2001), which, in turn, utilize the dopaminergic activation to produce hyperthermia. The rise of the sympathetic discharge induced by orexin A is corroborated by the increase in heart rate, although a possible reduction on the vagal tone cannot
be excluded. Since a possible modification of the vagal tone cannot be evidenced by a registration of the sympathetic nerves to IBAT, the vagal tone reduction could explain the sustained increase in heart rate, which is quicker than the increase in firing rate of nerves to
Fig. 4. Means9/S.E.M. of changes in colonic temperature. Intraperitoneal (ip) injection of saline or haloperidol (1 mg/kg bw) was made at /0.5 h. Intracerebroventricular (icv) injection of saline or orexin A (1.5 nmol) was made at time 0. Statistical significance (P B/0.05) between haloperidol/ orexin group and saline/orexin group.
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Fig. 5. Means9/S.E.M. of changes in heart rate. Intraperitoneal (ip) injection of saline or haloperidol (1 mg/kg bw) was made at /0.5 h. Intracerebroventricular (icv) injection of saline or orexin A (1.5 nmol) was made at time 0. Statistical significance (P B/0.05) between haloperidol/ orexin group and saline/orexin group.
IBAT. Nevertheless, the injection of haloperidol reduced the increase in heart rate showing that a D2 receptor antagonist affects the variation of cardiac functions induced by orexin A. Since a D2 agonist bromocriptine causes increases in heart rate (Lahlou et al., 1993), orexin A could induce tachycardia through an activation of D2 receptors. Then, haloperidol reduces the orexininduced tachycardia through an effect on the D2 receptors. The haloperidol did not completely abolish the positive chronotropic action of icv orexin A. Since Van Den Pol (1999) demonstrated a direct innervation of the intermediolateral column of spinal cord by orexin-fibers, there might be another direct pathway of the orexin-induced activation of the sympathetic nervous system. The icv injection of orexin A stimulates thermogenesis and increases body temperature in anesthetized rats. These evidences corroborate findings showing that orexin A is involved in thermoregulation independently in eating behavior. Hara et al. (2001) demonstrated that orexin-neurons ablated mice showed late-onset obesity along with narcoleptic phenotype. This obesity might be partly attributable to impairment of thermogenic effect of orexins. Because food intake activates thermogenesis (Bray, 2000; Monda, et al., 1997), substances affecting food consumption induce a secondary influence on body temperature. Since our experiment is carried out in anesthetized animals, the rise in body temperature induced by orexin A is a primary effect of this peptide. Since anesthesia with pentobarbital is well known to affect the autonomic nervous system (Watkins and
Maixner, 1991), we used urethane in the present study. This anesthetic does not significantly modify the sympathetic (DiMicco et al., 1986; DiMicco and Abshire, 1987) and thermogenic changes induced by various substances (Malkinson et al., 1998). Moreover, rats anesthetized with urethane are useful for experiments on the thermoregulation, when the behavioral influences (as food intake) must be excluded. The orexin A affects the temperature of IBAT, which is the most important effector of non shivering thermogenesis in the rat, illustrating that the rise in heat production is also due to the activation of thermogenesis unrelated to muscle activity. The increase in colonic temperature emphasizes the effect of orexin A on ‘core’ temperature suggesting the inclusion of orexin A among the peptides controlling body temperature. Haloperidol injection reduced both temperatures, indicating that these thermic reactions involve an activation of D2 receptors of the dopaminergic system. In conclusion, this experiment strongly demonstrates that haloperidol reduces the sympathetic and thermogenic activation induced by orexin A. Furthermore, these findings emphasize the role played by orexin A in the thermoregulation.
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M. Monda et al. / Neuroscience Research 45 (2003) 17 /23 Beuckmann, C.T., Yanagisawa, M., 2002. Orexins: from neuropeptides to energy homeostasis and sleep/wake regulation. J. Mol. Med. 80, 329 /342. Cannon, B.J., Houstek, J., Nedergaard, J., 1998. Brown adipose tissue. More than an effector of thermogenesis. Ann. N. Y. Acad. Sci. 856, 171 /187. Damase-Michel, C., Montastruc, J.L., Gharib, C., Geelen, G., De Saint-Blanquat, G., Tran, M.A., 1990. Effect of quinpirole, a specific dopamine DA2 receptor agonist on the sympathoadrenal system in dogs. J. Pharmacol. Exp. Ther. 252, 770 /777. De Koning, P., De Vries, M.H., 1995. A comparisons of the neuroendocrinological and temperature effects of DU 29894, flesinoxan, sulpiride and haloperidol in normal volunteers. Br. J. Clin. Pharmacol. 39, 7 /14. DiMicco, J.A., Abshire, V.M., 1987. Evidence for GABA-ergic inhibition of a hypothalamic sympathoexcitatory mechanism in anesthetized rats. Brain Res. 402, 1 /10. DiMicco, J.A., Abshire, V.M., Hankins, K.D., Sample, R.H.B., Wible, J.H., 1986. Microinjection of GABA antagonists into the posterior hypothalamus elevates heart rate in anesthetized rats. Neuropharmacology 25, 1063 /1066. Hara, J., Beuckmann, C.T., Nambu, T., Willie, J.T., Chemelli, R.M., Sinton, C.M., Sugiyama, F., Yagami, K., Goto, K., Yanagisawa, M., Sakurai, T., 2001. Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity. Neuron 30, 345 / 354. Lin, M.T., Wang, H.S., Wang, Z., Chern, Y.F., 1979. Haloperidol produces hypothermic effects in the rats. Experientia 35, 1469 / 1470. Lahlou, S., Duarte, G.P., Demenge, P., 1993. Central dopaminergic origin of bromocriptine induced tachycardia in normotensive rats. Cardivasc. Res. 27, 2022 /2027. Lubkin, A., Stricker-Krongrad, A., 1998. Independent feeding and metabolic action of orexins in mice. Biochem. Biophys. Res. Commun. 253, 241 /245. Malkinson, T.J., Cooper, K.E., Veale, W.L., 1998. Physiological changes during thermoregulation and fever in urethan-anaesthetized rats. Am. J. Physiol. 255, R73 /R81. Monda, M., Sullo, A., De Luca, B., 1997. Lesions of ventromedial hypothalamus reduce postingestional thermogenesis. Physiol. Behav. 61, 687 /691.
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Monda, M., Viggiano, A., Sullo, A., De Luca, V., 1998. Aspartic and glutamic acids increase in the frontal cortex during prostaglandin E1 hyperthermia. Neuroscience 83, 1239 /1243. Monda, M., Viggiano, A., De Luca, V., 2000. Intracerebroventricular injection of prostaglandin E1 changes concentration of biogenic amines in the posterior hypothalamus of the rat. Brain Res. 873, 197 /202. Monda, M., Viggiano, A., Mondola, P., De Luca, V., 2001. Inhibition of prostaglandin synthesis reduces hyperthermic reactions induced by hypocretin-1/orexin A. Brain Res. 909, 68 /74. Nakamura, T., Uramura, K., Nambu, T., Yada, T., Goto, K., Yanagisawa, M., Sakurai, T., 2000. Orexin-induced hyperlocomotion and stereotypy are mediated by the dopaminergic system. Brain Res. 873, 181 /187. Pellegrino, L.J., Pellegrino, A.S., Cushman, A.J., 1979. A Stereotaxic Atlas of the Rat Brain. Plenum Press, New York. Sakurai, T., Amemiya, A., Ishii, M., Matsuzaki, I., Chemelli, R.M., Tanara, H., Williams, S.C., Richardson, J.A., Kozlowski, G.P., Wilson, S., Arch, J.R.S., Buckingham, R.E., Haynes, A.C., Carr, S.A., Annan, R.S., McNulty, D.E., Liu, W.S., Terrett, J.A., Elshourbary, N.A., Bergsma, D.J., Yanagisawa, M., 1998. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92, 573 /585. Shirasaka, T., Nakazato, M., Matsukura, S., Takasaki, M., Kannan, H., 1999. Sympathetic and cardiovascular actions of orexins in conscious rats. Am. J. Physiol. 277, R1780 /R1785. Stitt, J.T., 1999. Prostaglandin E as the neural mediator of the febrile response. Yale J. Biol. Med. 59, 137 /149. Sweet, D.C., Levine, A.S., Billington, C.J., Kotz, C.M., 1999. Feeding response to central orexins. Brain Res. 821, 535 /538. Taheri, S., Zeitzer, J.M., Mignot, E., 2002. The role of hypocretins (orexins) in sleep regulation and narcolepsy. Annu. Rev. Neurosci. 25, 283 /313. Van Den Pol, A.N., 1999. Hypothalamic hypocretin (orexin): robust innervation of the spinal cord. J. Neurosci. 19, 3171 /3182. Vybiral, S., Vesela, R., 1994. Can haloperidol disguise fever. Physiol. Res. 43, 359 /363. Watkins, L., Maixner, W., 1991. The effect of pentobarbital anesthesia on the autonomic nervous system control of heart rate during baroreceptor activation. J. Auton. Nerv. Syst. 36, 107 /114.
Haloperidol reduces the sympathetic and thermogenic activation induced by orexin A M. Monda , A. Viggiano, V. De Luca Department of Experimental Medicine, Section of Human Physiology, Second University of Naples, via Costantinopoli 16, 80138 Naples, Italy Received 5 June 2002; accepted 12 September 2002
Abstract This experiment tested the effect of haloperidol on the sympathetic and thermogenic effects induced by orexin A. The firing rates of the sympathetic nerves to interscapular brown adipose tissue (IBAT), along with IBAT and colonic temperatures and heart rate were monitored in urethane-anesthetized male Sprague /Dawley rats before and 5 h after an injection of orexin A (1.5 nmol) into the lateral cerebral ventricle. The same variables were monitored in rats with an intraperitoneal administration of haloperidol (1 mg/kg bw), a D2 receptor antagonist. The results show that orexin A increases the sympathetic firing rate, IBAT and colonic temperatures and heart rate. This increase is reduced by the haloperidol. These findings suggest that dopaminergic system is activated during the orexin A-induced hyperthermia. # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Body temperature; Haloperidol; Orexin A; Rat; Sympathetic activity
1. Introduction Orexin A (Sakurai et al., 1998), hypothalamic peptide so-called for its stimulation on food intake (Sweet et al., 1999), causes vegetative responses, when this peptide is intracerebroventricularly (icv) injected (Lubkin and Stricker-Krongrad, 1998; Shirasaka et al., 1999). The autonomic reactions include the sympathetic and thermogenic changes. Indeed, an icv administration of orexin A induces an increase in firing rate of the sympathetic nerves to interscapular brown adipose tissue (IBAT), accompanied with a rise in IBAT and colonic temperatures (Monda et al., 2001). A role of orexins in sleep regulation has been demonstrated (Beuckmann and Yanagisawa, 2002). Deficiency in orexin neurotransmission results in the sleep disorder narcolepsy in mice, dogs, and humans (Taheri et al., 2002). Intraperitonel administration of haloperidol produces hypothermic effects in the rats (Lin et al., 1979), and in
Corresponding author. Tel.: /39-81-566-5833; fax: /39-81-5665846 E-mail address: [email protected] (M. Monda).
the rabbits (Vybiral and Vesela, 1994). Oral administration of haloperidol in normal volunteers causes significant attenuation of the normal daytime increase of body temperature (De Koning and De Vries, 1995). This indicates that dopaminergic system is involved in the regulation of body temperature. Furthermore, an intraperitoneal administration of haloperidol reduces the hyperlocomotion induced by an icv injection of orexin A, suggesting an involvement of dopaminergic system in the motor reactions due to orexin A (Nakamura et al., 2000). The aim of this experiment was to test the effects of haloperidol on the sympathetic and thermogenic reactions induced by orexin A, so that a possible involvement of the dopaminergic system could be considered.
2. Materials and methods 2.1. Animals Male Sprague /Dawley rats (n /24), 3 months old and weighing 270/320 g were used in the experiments. The rats were housed in pairs at controlled temperature (229/1 8C) and humidity (70%) with a 12:12 h light /
0168-0102/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 2 ) 0 0 1 9 1 - 8
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dark /light cycle from 07:00 to 19:00 h. The experiments were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). 2.2. Apparatus A pair of silver wire electrodes recorded the firing rate of nerves to IBAT. The electrical pulses were amplified by a condenser-coupled amplifier and were filtered by band-pass filters (NeuroLog System, Digitimer). The raw pulses were displayed on an oscilloscope (Tektronix) and sent to a window discriminator. Square waves from the discriminator were sent to an analog-digital converter (DAS system, Keithley) and stored on a computer (Personal Computer AT, IBM) every 5 s. A rate meter with a reset time of 5 s was also used to observe the time course of the nerve activity recorded by pen recorder (Dynograph, Beckman). Because signal-tonoise ratio depended on the number of nerve filaments and the condition of contact between nerve and electrodes, the basal burst rates were different for each rat. The threshold level of the event detector was fixed during the experiment at 50% of the peaks of the largest pulses and above background noise. Thermocouples (Ellab) were used to monitor colonic and IBAT temperatures (TC and TIBAT) and the values were stored on a chart recorder. Two electrodes applied to the forelegs monitored the heart rate (beats/min). Electrical signals were addressed to a polygraph (Dynograph, Beckman) to record the electrocardiographic activity on the card and on a computer disk. 2.3. Drugs and doses Orexin A and haloperidol were purchased respectively from Peninsula, England and Lusofarmaco, Italy. We used a dose of 1.5 nmol of orexin A, after a preliminary experiment with doses of 0.15, 1.5 and 15 nmol in three groups of two rats. This test showed a dose/effect curve on TC. The temperature modifications were: (a) from 37.209/0.13 to 37.759/0.17 with dose of 0.15 nmol; (b) from 37.129/0.08 to 38.429/0.12 with 1.5 nmol; (c) from 37.159/0.11 to 38.669/0.16 with 15 nmol. We chose the sub-maximal dose of 1.5 nmol. A similar preliminary test was carried out with doses of 0.5, 1 and 2 mg/kg bw of haloperidol in three groups of two rats injected with 1.5 nmol of orexin A. The temperature modifications were: (a) from 37.129/0.11 to 38.059/0.19 with dose of 0.5 mg/kg bw; (b) from 37.079/0.14 to 37.529/0.15 with 1 mg/kg bw; (c) from 37.119/0.13 to 37.459/0.18 with 2 mg/kg bw. The dose of 1 mg/kg was utilized. Since the low number of animals did not permit a statistical analysis, the preliminary experiments were merely indicative to determine the opportune dose. The purity of orexin is about 98% measured by HPLC, so that the
possibility of pyrogenic effects due to contaminants [for example prostaglandin (PG) of E series] is very unlikely. Indeed, the quantity of PG of E series should be only 10 ng. This dose does not induce hyperthermia when icv injected (Malkinson et al., 1998; Stitt, 1999). The same reasoning should also be valid for other pyrogens. 2.4. Surgery All animals were anesthetized with ip pentobarbital (50 mg/kg bw) and a 20-gauge stainless guide cannula was positioned stereotaxically (Pellegrino et al., 1979) above a lateral cerebral ventricle at the following coordinates: 1.7 mm lateral to the midline, 0.4 mm posterior to the bregma, 3.0 mm from the cranial theca. The rats were given 7/10 days to recover from surgery judged by recovery of preoperative body weight. 2.5. Procedure After recovery, six animals (1st group) were anesthetized with ethyl-urethane (1.2 g/kg bw ip) and mounted in a stereotaxic instrument (Stoelting). The level of anesthesia was kept constant and evaluated by skeletal muscle relaxation, eye and palpebral responses to stimuli. Nerve activity was recorded by small nerve bundles dissected from the intercostal nerves supplying the right side of IBAT. These nerve bundles (only innervating IBAT) do not contain motor fibers discharging with respiratory rhythm. Nerve filaments were isolated from the central cut end of these nerve bundles under a dissecting microscope; the efferent activity was recorded with a pair of silver wire electrodes. The nerve filaments were covered with a mixture of vaseline and liquid petroleum at 37 8C to avoid dehydration. The firing rate was recorded over 60 min before and 300 min after the injection of orexin A (1.5 nmol dissolved in 5 ml of 0.9% NaCl sterile solution). The orexin A was delivered into the left cerebral ventricle by gravity flow over 2 min. The cannula for the injection was 0.4 mm longer than the guide cannula. Furthermore, the heart rate, TC and TIBAT were monitored at the same time as the nerve activity was recorded. TC was measured by inserting the thermocouple into the colon 4 cm from the anus, while TIBAT was monitored by inserting the thermocouple in the left side of IBAT. The same variables were recorded in the other six animals (2nd group), but haloperidol (1 mg/kg bw dissolved in 2 ml of 0.9% NaCl sterile solution) was injected ip 30 min before the icv injection of orexin A. In the other six rats treated with an ip injection of haloperidol (3rd group), saline was injected into the cerebral ventricle and the same variables were monitored. In six control rats (4th group), saline was injected both in the peritoneal cavity and in the lateral ventricle. The baseline values of TC from all animals used were maintained constant by a
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heating pad. The electrical energy supplied to pad was not altered during the experimental period. 2.6. Histology At the end of the experiment, the location of the cannula was identified. A stain (bromophenol blue) was injected in the lateral ventricle; the volume of the stain is the same as the volume used for drug administration. The rats were then injected with an overdose of pentobarbital (150 mg/kg bw) and were perfused with 0.9% NaCl followed by 10% formalin solution. The brain was removed and stored in formalin solution. After a few days, 50 mm coronal sections of the fixed brain were cut and stained with neutral red. 2.7. Statistical analysis The values are presented as means9/S.E.M. Statistical analysis was performed using analysis of variance. Multiple comparisons were performed by Newman / Keuls post hoc test.
3. Results Fig. 1 shows the percentage changes in the firing rate of the nerves to IBAT. The icv injection of orexin increased the firing rate and this rise was reduced in the
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animals with ip administration of haloperidol. In the other rats, neither saline nor haloperidol caused any modification. An analysis of variance showed significant effects for orexin [F (1, 20) /235.9, P B/0.01], for haloperidol [F (1, 20) /66.5, P B/0.01], for time [F (11, 220) /63.6, P B/0.01], for interaction orexin /haloperidol [F (1, 20) /80.2, P B/0.01], orexin /time [F (11, 220) /30.6, P B/0.01], haloperidol /time [F (11, 220) /7.7, P B/0.01], and orexin /haloperidol /time [F (11, 220) /23.2, P B/0.01]. The post hoc test showed that the orexin/saline group was different from the orexin/haloperidol group at 1.5 /5 h. The baseline absolute values were respectively of 44.19/8.8 spikes/5 s in the 1st group, 42.49/8.2 in the 2nd group, 45.79/6.8 in the 3rd group and 44.89/9.3 in the 4th group. There were no differences in the baseline absolute values of all groups. Examples of the changes in firing rate are shown in Fig. 2. Fig. 3 illustrates TIBAT changes. The icv injection of orexin caused a rise. This increase was reduced in the animals ip injected with haloperidol. In the other rats, neither saline nor haloperidol caused any modification. An analysis of variance showed significant effects for orexin [F (1, 20) /925.1, P B/0.01], for haloperidol [F(1, 20) /156.9, P B/0.01], for time [F (11, 220) /267.5, P B/ 0.01], for interaction orexin /haloperidol [F (1, 20) / 455.3, P B/0.01], orexin /time [F (11, 220) /90.1, P B/ 0.01], haloperidol /time [F (11, 220) /13.1, P B/0.01], and orexin /haloperidol /time [F (11, 220) /71.6, P B/
Fig. 1. Means9/S.E.M. of changes in firing rate of nerves to interscapular brown adipose tissue. Intraperitoneal (ip) injection of saline or haloperidol (1 mg/kg bw) was made at /0.5 h. Intracerebroventricular (icv) injection of saline or orexin A (1.5 nmol) was made at time 0. Statistical significance (P B/0.05) between the haloperidol/orexin group and the saline/orexin group.
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peridol [F (1, 20) /5.0, P B/0.01], for time [F (11, 220) / 13.3, P B/0.01], for interaction orexin /time [F (11, 220) /11.5, P B/0.01], and orexin /haloperidol /time [F (11, 220) /2.4, P B/0.01]. The post hoc test showed that orexin/saline group was different from orexin/ haloperidol group at 0.5 /5 h. The figures do not illustrate when the effects of orexin disappear, because the registrations of this experiment were interrupted at 5 h. Our unpublished observations showed that the effects of orexin disappear 7 /9 h after icv injection.
4. Discussion
Fig. 2. Actual firing rate changes in a rat receiving intraperitoneal (ip) injection of saline plus intracerebroventricular (icv) injection of orexin A (panel C) or saline (panel D), and in a rat receiving ip injection of haloperidol plus icv injection of orexin A (panel A) or saline (panel B). The arrow indicates the time of icv injection. Ip administrations was made 30 min before the icv injection.
0.01]. Newman /Keuls post hoc test showed that the orexin/saline group was different from orexin/haloperidol group at 1.5 /5 h. Fig. 4 illustrates TC changes. The icv injection of orexin induced a rise in the rats ip injected with saline. Haloperidol reduced the increase due to orexin. No changes resulted in the animals with saline or haloperidol administration. An analysis of variance showed significant effects for orexin [F(1, 20) /808.1, P B/0.01], for haloperidol [F(1, 20) /131.9, P B/0.01], for time [F (11, 220) /278.6, P B/0.01], for interaction orexin / time [F (11, 220) /93.0, P B/0.01], haloperidol /time P B/0.01], and orexin / [F (11, 220) /13.9, haloperidol /time [F (11, 220) /73.1, P B/0.01]. Newman /Keuls post hoc test showed that the orexin/saline group was different from orexin/haloperidol group at 1.5 /5 h. Fig. 5 shows changes in heart rate. The orexin injection induced an increase, which was reduced by haloperidol. Neither the administration of saline nor haloperidol caused any change in the rats without orexin injection. An analysis of variance showed significant effects for orexin [F (1, 20) /13.4, P B/0.01], for halo-
To our knowledge, these findings are the first to demonstrate that an ip injection of haloperidol reduces the sympathetic and thermogenic effects due to an icv administration of orexin A. The effects of orexin A on the sympathetic discharge of the nerves to IBAT corroborate recent evidences demonstrating the role played by this neuropeptide in the control of the autonomic nervous system. Shirasaka et al. (1999) illustrated that icv injections of orexins increase activity of the renal sympathetic nerves, which play an important role in the homeostasis of body fluids and the circulatory system. We only used orexin A because Shirasaka et al. (1999) demonstrated that plasma norepinephrine increases with a high dose of orexin A, but not with orexin B. Since the increase in the sympathetic activity induced by orexin A is reduced by haloperidol, an involvement of the D2 receptors of the dopaminergic system can be hypothesized. Because an injection of quinpirole, a D2 receptor agonist, induces an increase in the sympathetic discharge (Damase-Michel et al., 1990), an activation of dopaminergic system could be a possible mechanism of sympathetic stimulation induced by orexin A. In this mechanism, an involvement of the D2 receptors could play a key role. Our experiment considered the activity of sympathetic nerves to IBAT. This tissue is the principal effector of non-shivering thermogenesis (Cannon et al., 1998) and plays a relevant role in the thermoregulation. We have significantly demonstrated that icv injections of PGE1 induce an increase in the firing rate of sympathetic nerves to IBAT (Monda et al., 1998, 2000), and lysine acetylsalicylate reduces the sympathetic activation induced by orexin A, suggesting that prostaglandins have an implication in the mediation of this phenomenon (Monda et al., 2001). Since the haloperidol reduces the fever due to PGE synthesis (Vybiral and Vesela, 1994), we can hypothesize an involvement of the dopaminergic system in the thermogenic activation due to prostaglandin synthesis by orexin A. In other words, orexin A induces a synthesis of the prostaglandins (Monda et al.,
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Fig. 3. Means9/S.E.M. of changes in temperature of interscapular brown adipose tissue (IBAT). Intraperitoneal (ip) injection of saline or haloperidol (1 mg/kg bw) was made at /0.5 h. Intracerebroventricular (icv) injection of saline or orexin A (1.5nmol) was made at time 0. Statistical significance (P B/0.05) between haloperidol/orexin group and saline/orexin group.
2001), which, in turn, utilize the dopaminergic activation to produce hyperthermia. The rise of the sympathetic discharge induced by orexin A is corroborated by the increase in heart rate, although a possible reduction on the vagal tone cannot
be excluded. Since a possible modification of the vagal tone cannot be evidenced by a registration of the sympathetic nerves to IBAT, the vagal tone reduction could explain the sustained increase in heart rate, which is quicker than the increase in firing rate of nerves to
Fig. 4. Means9/S.E.M. of changes in colonic temperature. Intraperitoneal (ip) injection of saline or haloperidol (1 mg/kg bw) was made at /0.5 h. Intracerebroventricular (icv) injection of saline or orexin A (1.5 nmol) was made at time 0. Statistical significance (P B/0.05) between haloperidol/ orexin group and saline/orexin group.
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Fig. 5. Means9/S.E.M. of changes in heart rate. Intraperitoneal (ip) injection of saline or haloperidol (1 mg/kg bw) was made at /0.5 h. Intracerebroventricular (icv) injection of saline or orexin A (1.5 nmol) was made at time 0. Statistical significance (P B/0.05) between haloperidol/ orexin group and saline/orexin group.
IBAT. Nevertheless, the injection of haloperidol reduced the increase in heart rate showing that a D2 receptor antagonist affects the variation of cardiac functions induced by orexin A. Since a D2 agonist bromocriptine causes increases in heart rate (Lahlou et al., 1993), orexin A could induce tachycardia through an activation of D2 receptors. Then, haloperidol reduces the orexininduced tachycardia through an effect on the D2 receptors. The haloperidol did not completely abolish the positive chronotropic action of icv orexin A. Since Van Den Pol (1999) demonstrated a direct innervation of the intermediolateral column of spinal cord by orexin-fibers, there might be another direct pathway of the orexin-induced activation of the sympathetic nervous system. The icv injection of orexin A stimulates thermogenesis and increases body temperature in anesthetized rats. These evidences corroborate findings showing that orexin A is involved in thermoregulation independently in eating behavior. Hara et al. (2001) demonstrated that orexin-neurons ablated mice showed late-onset obesity along with narcoleptic phenotype. This obesity might be partly attributable to impairment of thermogenic effect of orexins. Because food intake activates thermogenesis (Bray, 2000; Monda, et al., 1997), substances affecting food consumption induce a secondary influence on body temperature. Since our experiment is carried out in anesthetized animals, the rise in body temperature induced by orexin A is a primary effect of this peptide. Since anesthesia with pentobarbital is well known to affect the autonomic nervous system (Watkins and
Maixner, 1991), we used urethane in the present study. This anesthetic does not significantly modify the sympathetic (DiMicco et al., 1986; DiMicco and Abshire, 1987) and thermogenic changes induced by various substances (Malkinson et al., 1998). Moreover, rats anesthetized with urethane are useful for experiments on the thermoregulation, when the behavioral influences (as food intake) must be excluded. The orexin A affects the temperature of IBAT, which is the most important effector of non shivering thermogenesis in the rat, illustrating that the rise in heat production is also due to the activation of thermogenesis unrelated to muscle activity. The increase in colonic temperature emphasizes the effect of orexin A on ‘core’ temperature suggesting the inclusion of orexin A among the peptides controlling body temperature. Haloperidol injection reduced both temperatures, indicating that these thermic reactions involve an activation of D2 receptors of the dopaminergic system. In conclusion, this experiment strongly demonstrates that haloperidol reduces the sympathetic and thermogenic activation induced by orexin A. Furthermore, these findings emphasize the role played by orexin A in the thermoregulation.
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