Introduction

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Cannabis and its derivative compounds, collectively known as cannabinoids, produce an array of pharmacological symptoms in animals and humans (Chaperon and Thiebot, 1999), including hypothermia (Fitton and Pertwee, 1982; Ovadia et al., 1995). Two subtypes of receptors, CB₁ and CB₂, mediate cannabinoid-induced effects (Howlett, 1995; Felder et al., 1998). CB₁ receptors are located primarily in the central nervous system and are thought to mediate the central effects of cannabinoids (Howlett, 1995). In contrast, CB₂ receptors are expressed almost exclusively by peripheral immune cells (Dragic et al., 1996).

The development of synthetic cannabinoid agonists has provided remarkable advances in cannabis research. One such ligand is the aminoalkylindole, (+)-WIN 55,212-2 [(4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one] (WIN 55212-2), which displays high selectivity for cannabinoid receptors. Previous studies have demonstrated that WIN 55212-2 is highly potent and efficacious in vivo and in vitro. WIN 55212-2 prevents intravenous cocaine self-administration, increases tail-flick reflexes, exerts antihyperalgesic effects, and induces hypothermia in rats, indicating that WIN 55212-2 is pharmacologically active in vivo (Compton et al., 1992; Martin et al., 1998; Fox et al., 2001). WIN 55212-2 undergoes less nonspecific binding than classical cannabinoids and interacts negligibly with other neurotransmitter systems and ion channels (Jansen et al., 1992; Shire et al., 1996; Showalter et al., 1996). In contrast, ⁹-tetrahydrocannabinol (⁹-THC) has been reported to produce hypothermia by interacting with other neurotransmitters, including serotonin (Davies and Graham, 1980). WIN 55212-2 is also more readily soluble than the extremely hydrophobic ⁹-THC. The actions of ⁹-THC in vivo have been investigated by suspending the ⁹-THC in solution using a suspending agent such as polyvinylpyrrolidone (Davies and Graham, 1980). In contrast, WIN 55212-2 is soluble in a Cremophor/saline vehicle, which has better dissolution characteristics than a suspension because it remains a homogenous solution even upon standing for extended periods of time. In addition, Cremophor/saline does not alter body temperature or require the use of ethanol, which has been commonly used to administer ⁹-THC (Compton et al., 1992, 1996; Aceto et al., 1995). This is crucial, since ethanol is a centrally acting depressant that may alter the neurochemical and behavioral effects of ⁹-THC. Thus, we chose WIN 55212-2 to induce hypothermia in the present study.

The anterior nucleus of the hypothalamus (POAH) is thought to play a major role in thermoregulation (Boulant, 1981; Xin et al., 1997). A large population of thermosensitive neurons in the POAH receives and integrates information from central and peripheral sensors. In turn, these intra-POAH neurons send the modulating signal to thermoregulatory effectors that maintain body temperature around a given point, or set-point temperature. The destruction or inactivation of the POAH disrupts thermoregulation.

Pharmacological evidence supports a role for the POAH in cannabinoid-induced hypothermia. ⁹-THC injected directly into the POAH produced dose-dependent hypothermia (Fitton and Pertwee, 1982). More recently, Ovadia et al. (1995) reported that HU-210, [(BaR)-trans-3-(1,1-dimethylheptyl)-6,7,10,10-tetrahydro-1-hydroxy-6,5-dimethyl-H-dibenzo[b,d]pyran-9-methanol], a synthetic cannabinoid agonist, induced hypothermia after intra-POAH injection. Those studies indicate that cannabinoids act centrally, probably within the POAH, to produce hypothermia.

An accumulating body of evidence suggests that cannabinoid receptors are located in the POAH. In particular, cells expressing CB₁ receptor mRNA are densely distributed in hypothalamic regions where autoradiographic binding studies have demonstrated dense CB₁ receptor binding (Herkenham et al., 1991; Mailleux and Vanderhaeghen, 1992). Recently, CB₁ receptor immunoreactivity was detected in the hypothalamus, with especially robust levels reported in the POAH (Moldrich and Wenger, 2000). Thus, it is likely that cells in the POAH express CB₁ receptor protein.

Although these data indicate that the POAH is a central locus of cannabinoid-induced hypothermia, the lack of selective cannabinoid receptor antagonists has precluded the investigation of the role that cannabinoid receptor subtypes play in mediating the hypothermic response. The recent development of the selective CB₁ antagonist, [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A), and the selective CB₂ antagonist, N-{(1S)-endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide} (SR144528), has provided insight into the receptor-mediated mechanisms that underlie the physiological effects of cannabinoids (Rinaldi-Carmona et al., 1994, 1998; Wiley et al., 1995; Compton et al., 1996).

Thus, in the present study we used SR141716A and SR144528 to ascertain the roles of the POAH and of CB₁ and CB₂ receptors in cannabinoid-induced hypothermia. We investigated the 1) hypothermic effects of WIN 55212-2 after systemic or intra-POAH injection, 2) effects of SR141716A or SR144528 alone on hypothermia, and 3) effects of SR141716A or SR144528 on WIN 55212-2 hypothermia.

	Materials and Methods

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Animals. All animal use procedures were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (Zivic-Miller, Pittsburgh, PA) weighing 250 to 350 g were used in the present study. Rats were housed three per cage for a minimum of 5 days before experimental use. They were fed rat chow and water ad libitum. The temperature and relative humidity of the animal room were kept at 22 ± 2°C and 50 ± 2%, respectively. Rats were maintained on a 12-h light/dark cycle.

Surgery and Cannula Implantation. Rats were anesthetized with an intraperitoneal injection of a solution of ketamine hydrochloride (100 mg/kg) and acepromazine maleate (0.2 mg/kg) and placed into a stereotaxic apparatus. The skull was exposed and a 1-mm hole was bored dorsal to the POAH. The dura was carefully penetrated with a drill, leaving the adjacent meninges intact. A 21-gauge, stainless steel guide cannula (Small Parts Inc., Hialeah, FL) measuring 14 cm in length was implanted unilaterally into the POAH. Stereotaxic coordinates were as follows: 2 mm anterior to bregma, 1 mm lateral to the midline suture, and 7.5 mm ventral to the dural surface (Pellegrino and Cushman, 1967). The cannula was affixed to the skull with dental acrylic (Durelon, Seefeld, Germany) and stabilized by inserting self-tapping bond screws (Plastics One Inc., Roanoke, VA) adjacent to the cannula site. The cannula extended 6.5 mm above the dural surface. After solidification of the dental cement, a 25-gauge, 14-cm, stainless steel stylet (Small Parts Inc.) was inserted into the guide cannula to minimize blood coagulation in the cannula tract and prevent infection. After a recovery interval of at least 5 days, stylets were removed and drugs were injected into the POAH with a #10 µl airtight syringe (Hamilton Co., Reno, NV).

Drug Preparation and Administration. The cannabinoid agonist, WIN 55212-2, was obtained from Sigma-Aldrich (St. Louis, MO). The selective CB₁ receptor antagonist, SR141716A, and the selective CB₂ receptor antagonist, SR144528, were obtained from the National Institute on Drug Abuse (Rockville, MD). Cremophor EL, a derivative of castor oil and ethylene oxide, was purchased from Sigma-Aldrich. In all cases, the vehicle and control solution was 10% Cremophor/saline. All drugs were administered intramuscularly, except for the injection of WIN 55212-2 into the POAH. When injected into the POAH, WIN 55212-2 was injected in a volume of 1 µl. In the systemic experiments, WIN 55212-2 was injected into the right hind leg of each rat. Conversely, all other drugs were injected into the left hind leg.

Experimental Protocol. Body temperature experiments were started between 9:00 and 10:00 AM. On the morning of the experiment, rats were placed in an environmental room which was maintained at a constant temperature of 21 ± 0.3°C and relative humidity of 52 ± 2%. After a 1-h acclimation interval, baseline temperature measurements were taken (Xin et al., 1997). A thermistor probe (YSI series 400; Yellow Springs Instrument Co., Yellow Springs, OH) was lubricated and inserted approximately 7 cm into the rectum. A digital thermometer (model 49 TA, YSI) was used to record body temperature. Rats were unrestrained during the temperature readings, with only the tail being held gently between two fingers. Body temperature was taken every 30 min during a 90-min baseline interval. After the baseline interval, either WIN 55212-2 (1, 2.5, 5, or 10 mg/kg, i.m.) or vehicle was injected. Body temperature was measured for 5 h.

Data and Histological Analysis. To allow the rat to adapt to the technique, the first temperature reading was discarded in all experiments. Two consecutive body temperature readings were then recorded and averaged to establish a baseline temperature prior to drug injection. Data are presented as the mean ± S.E. of body temperature. Statistical analysis of differences between groups was determined by a one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A value of p  0.05 was considered to be statistically significant.

	Results

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Intramuscular Injection of WIN 55212-2 Produces Dose-Dependent Hypothermia. The intramuscular injection of WIN 55212-2 at doses of 1 to 10 mg/kg produced hypothermia in a dose-dependent manner (Fig. 1). Body temperature was measured 15 min after the injection of WIN 55212-2. The lowest dose did not produce a significant fall in body temperature. The hypothermia produced by a dose of 10 mg/kg was not greater than that produced by a dose of 5 mg/kg. Accordingly, a dose of 5 mg/kg was chosen for subsequent experiments. The fall in body temperature began 15 min after the injection of WIN 55212-2. The peak hypothermia (3.4 ± 0.4°C) produced by 5 mg/kg WIN 55212-2 occurred 90 min postinjection. Temperatures recovered gradually and reached predrug levels 5 h postinjection.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   WIN 55212-2 (1-10 mg/kg, i.m.) produces hypothermia in a dose-dependent manner. WIN 55212-2 was injected at 0 min, as indicated by the arrow. Data are expressed as the mean (±S.E.) of body temperature. n, number of rats. Tb, change in body temperature.

CB₁, but not CB₂, Receptors Mediate WIN 55212-2-Induced Hypothermia. In separate experiments, we determined whether CB₁ or CB₂ receptors contributed to the WIN 55212-2-induced hypothermia. SR141716A or SR144528 was injected 30 min before WIN 55212-2. SR141716A abolished the hypothermia. SR141716A blocked the WIN 55212-2-induced hypothermia (Fig. 2). The effect of SR141716A was dose-dependent. A dose of 1 mg/kg partially reversed the WIN 55212-2-induced hypothermia. Doses of 5 or 10 mg/kg SR141716A completely abolished the hypothermia produced by WIN 55212-2. In contrast to SR141716A, SR144528 (5 or 10 mg/kg) did not alter WIN 55212-2-evoked hypothermia (Fig. 3). These data suggest that WIN 55212-2 produces hypothermia by activating CB₁ receptors.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   SR141716A (1-10 mg/kg, i.m.) blocks WIN 55212-2-induced (5 mg/kg, i.m.) hypothermia. WIN 55212-2 was injected at 0 min. SR141716A was injected 30 min prior to WIN 55212-2. Data are expressed as the mean (±S.E.) of body temperature. n, number of rats. Tb, change in body temperature.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   SR144528 (5 or 10 mg/kg, i.m.) does not alter WIN 55212-2-induced (5 mg/kg, i.m.) hypothermia. WIN 55212-2 was injected at 0 min. SR144528 was injected 30 min before WIN 55212-2. Data are expressed as the mean (±S.E.) of body temperature. n, number of rats. Tb, change in body temperature.

Endocannabinoid System Does Not Tonically Regulate Body Temperature. To determine whether the endocannabinoid system tonically regulates body temperature, the effects of SR141716A or SR144528 on rectal temperature were measured. Neither SR141716A nor SR144528 significantly affected baseline temperatures (Fig. 4). Moreover, SR141716A or SR144528 elicited no visible behavioral effects. The present data suggest that endogenous cannabinoids do not tonically affect body temperature.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   SR141716A (10 mg/kg, i.m.) or SR144528 (5 mg/kg, i.m.) does not affect baseline body temperature. SR141716A or SR144528 was injected at 0 min, as indicated by the arrow. Data are expressed as the mean (±S.E.) of body temperature. n, number of rats. Tb, change in body temperature.

Intra-POAH Injection of WIN 55212-2 Produces Hypothermia in a Dose-Dependent Manner. To verify the central effect of WIN 55212-2 on the major thermoregulatory center, we injected WIN 55212-2 directly into the POAH. WIN 55212-2, at doses of 5 to 30 µg/µl, produced dose-dependent hypothermia (Fig. 5). The lowest dose did not alter body temperature. The highest dose, 30 µg/µl, produced a peak hypothermia of 1.3 ± 0.1°C 15 to 30 min postinjection. A dose of 15 µg/µl produced a similar transient hypothermic response, with peak hypothermia (1.0 ± 0.1°C) occurring 30 to 45 min postinjection. Rectal temperatures returned to predrug levels by 60 min after the injection of 30 µg/µl WIN 55212-2. 

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Intra-POAH injection of WIN 55212-2 (5-30 µg/µl) induces hypothermia in a dose-dependent manner. Either WIN 55212-2 or 10% Cremophor/saline was injected in a volume of 1 µl at 0 min, as indicated by the arrow. Data are expressed as the mean (±S.E.) of body temperature. n, number of rats. Tb, change in body temperature.

CB₁ Receptors Mediate the Hypothermia Induced by the Intra-POAH Injection of WIN 55212-2. To determine whether CB₁ receptors in the POAH mediated WIN 55212-2-induced hypothermia, SR141716A was injected intramuscularly 30 min before the intra-POAH injection of WIN 55212-2. SR141716A (5 mg/kg) abolished WIN 55212-2-evoked hypothermia (Fig. 6). The injection of SR141716A alone did not affect body temperature.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   SR141716A (5 mg/kg, i.m.) blocks WIN 55212-2-induced (15 µg/µl) hypothermia. Either 15 µg/µl WIN 55212-2 or 10% Cremophor/saline was injected into the POAH in a volume of 1 µl at 0 min. Either SR141716A or 10% Cremophor/saline was injected 30 min before WIN 55212-2. Data are expressed as the mean (±S.E.) of body temperature. n, number of rats. Tb, change in body temperature.

	Discussion

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The acute administration of cannabinoid agonists produces hypothermia in rats, mice, and primates (Compton et al., 1992; Fan et al., 1994; Spina et al., 1998; Fox et al., 2001). Similarly, in the present study, the intramuscular injection of WIN 55212-2 induced hypothermia in rats. Three aspects of the present data correlate well with the previous studies (Compton et al., 1992; Spina et al., 1998; Fox et al., 2001). First, WIN 55212-2 elicited hypothermia in a dose-dependent manner, thus revealing a receptor-mediated mechanism. Second, the onset of action of WIN 55212-2-induced hypothermia was rapid, with significant hypothermia observed 15 min postinjection. Third, the duration of the hypothermic response evoked by WIN 55212-2 persisted for at least 3 h. In fact, the highest dose, 10 mg/kg, elicited hypothermia that was still present 5 h postinjection.

SR141716A prevented the WIN 55212-2-evoked hypothermia, confirming that a CB₁ receptor mechanism mediated the hypothermic effect. Blockade by SR141716A of cannabinoid-induced hypothermia in the present study is consistent with previous reports. For example, SR141716A blocked cannabinoid agonist-induced hypothermia in rats (McGregor et al., 1996; Nava et al., 2000; Fox et al., 2001) and mice (Wiley et al., 1995). Recent studies demonstrated that cannabinoid agonists were unable to evoke hypothermia in mice that lacked the CB₁ receptor, providing still further evidence that CB₁ receptors regulate cannabinoid-evoked hypothermia (Ledent et al., 1999; Zimmer et al., 1999). The present data and the fact that CB₁ receptors are located primarily in the CNS confirm the idea that the hypothermic effect of cannabinoids is mediated centrally.

SR144528 did not affect WIN 55212-2-induced hypothermia, indicating that the hypothermia is insensitive to CB₂ receptor activation. These results suggest that a CB₂ receptor mechanism does not play a role in mediating the hypothermic response to cannabinoids, underscoring the differing roles of CB₁ and CB₂ receptors in pharmacoregulation.

Neither SR141716A nor SR144528 by itself altered body temperature, suggesting that the endocannabinoid system does not tonically modulate body temperature. Previous studies have also demonstrated that cannabinoid antagonists do not affect thermoregulation (Compton et al., 1996; McGregor et al., 1996; Nava et al., 2000). This is unlike the opioid system, where the µ and  receptors mediate hyperthermia and hypothermia, respectively, and are in tonic balance (Xin et al., 1997). The lack of cannabimimetic activity exhibited by SR141716A or SR144528 suggests that these antagonists bind selectively to CB₁ and CB₂ receptors, respectively, and that the recognition and activation of cannabinoid receptors are separable events. These data also argue against SR141716A and SR144528 acting as inverse agonists (MacLennan et al., 1998; Rinaldi-Carmona et al., 1998). Although it is unlikely that the endocannabinoid system tonically suppresses body temperature, other pharmacological endpoints, particularly inflammatory hyperalgesia and antinociception, are thought to be influenced by a cannabinergic tone (Calignano et al., 1998; Welch et al., 1998).

The injection of WIN 55212-2 into the POAH evoked dose-dependent hypothermia. The onset of WIN 55212-2-induced hypothermia was rapid, with the peak hypothermia occurring 15 to 45 min postinjection. The duration of hypothermia was brief. Our results provide further evidence that the POAH is a central locus of thermoregulation (Boulant, 1981; Xin et al., 1997) and indicate that the POAH plays a role in the regulation of cannabinoid-induced hypothermia (Fitton and Pertwee, 1982; Ovadia et al., 1995). The dose-dependent nature of the WIN 55212-2-induced hypothermia suggests the involvement of a receptor-sensitive mechanism.

SR141716A abolished the hypothermic response to the intra-POAH injection of WIN 55212-2. The present data are the first direct evidence that the activation of CB₁ receptors in the POAH mediates cannabinoid-induced hypothermia. Prior to the discovery of cannabinoid receptors and development of cannabinoid antagonists, Fitton and Pertwee (1982) demonstrated that the injection of ⁹-THC into the POAH produced hypothermia in a dose-dependent manner (Fitton and Pertwee, 1982). In addition, Ovadia et al. (1995) demonstrated that the injection of HU-210, a synthetic cannabinoid agonist, into the POAH induced hypothermia. However, the authors did not determine whether the hypothermic response to HU-210 was dose-dependent or cannabinoid receptor-dependent. Because they did not attempt to reverse the HU-210-induced hypothermia with a cannabinoid receptor antagonist, the possibility exists that HU-210 acted through mechanisms other than cannabinoid receptor activation to produce hypothermia.

An accumulating body of evidence suggests that CB₁ receptors are located in the POAH. CB₁ receptor mRNA is expressed in hypothalamic nuclei where CB₁ receptor binding has been demonstrated (Herkenham et al., 1991; Mailleux and Vanderhaeghen, 1992). Recently, CB₁ receptor immunoreactivity has been detected in the hypothalamus (Pettit et al., 1998; Tsou et al., 1998), including the lateral hypothalamic area and the POAH (Moldrich and Wenger, 2000). The presence of CB₁ immunoreactivity indicates that neurons in the POAH express the CB₁ receptor. It is still unclear whether the CB₁ receptor-containing neurons are intrinsic or extrinsic to the POAH. Romero et al. (1998) suggested that cannabinoid receptor-containing neurons are all intrinsic to the hypothalamic nuclei, since hypothalamic deafferentation did not alter cannabinoid receptor binding. Although the mechanism that mediates WIN 55212-2-induced hypothermia is not entirely known, it is clear that CB₁ receptors in the POAH play a major role.

Several aspects of the WIN 55212-2-evoked hypothermia were dependent on the injection route. The most striking differences were the duration and magnitude of the hypothermia. When injected intramuscularly, WIN 55212-2 produced a hypothermia that lasted for at least 3 h. However, the intra-POAH injection of WIN 55212-2 produced a brief hypothermia, with body temperature returning to baseline within 60 to 90 min. The administration route also affected the peak hypothermia. WIN 55212-2 produced maximal hypothermia 60 to 120 min after intramuscular injection. Not surprisingly, the intra-POAH injection of WIN 55212-2 produced peak hypothermia more rapidly, with the maximal reduction in body temperature occurring 30 min postinjection. In addition, the systemic injection of WIN 55212-2 produced a greater hypothermic response than intra-POAH injection.

It is unclear why the magnitude and duration of the WIN 55212-2-induced hypothermia are sensitive to the injection route. One explanation is that cannabinoid systems outside the POAH may play a role in mediating the hypothermia produced by the systemic, but not intra-POAH, injection of WIN 55212-2. Indeed, a high density of CB₁ receptors are located in extrahypothalamic sites where cannabinoid systems have been reported to interact with other neurotransmitter systems (Herkenham et al., 1991; Chaperon and Thiebot, 1999). Furthermore, an involvement of extrahypothalamic sites in cannabinoid-evoked hypothermia has been reported previously (Schmeling and Hosko, 1980; Fitton and Pertwee, 1982). Other neurotransmitters have also been reported to contribute to the hypothermic effects of systemically administered cannabinoids (Malone and Taylor, 1998; Chaperon and Thiebot, 1999; Nava et al., 2000). Thus, the POAH, as well as sites outside of the hypothalamus, appear to mediate cannabinoid-induced hypothermia.

Although structurally dissimilar, WIN 55212-2 and ⁹-THC appear to act at the same site and produce similar effects, such as catalepsy, hypothermia, antinociception, and hypolocomotion (Devane et al., 1988; Compton et al., 1992; Chaperon and Thiebot, 1999). Still, the lack of a consistent relationship between the potencies of these compounds across a spectrum of pharmacological endpoints, including hypothermia (Compton et al., 1992), does suggest that WIN 55212-2 and ⁹-THC may not reduce body temperature through identical mechanisms. In comparing rat data, we found that a dose of 1 mg/kg SR141716A, which prevented hypothermia evoked by 5 mg/kg ⁹-THC (Nava et al., 2000), did not completely block the hypothermic response to 5 mg/kg WIN 55212-2. Higher doses of SR141716A (2.5 and 5 mg/kg) were effective in our hands. Previous studies using mice have demonstrated that WIN 55212-2, in comparison with ⁹-THC, was 3-fold less potent in producing hypothermia, 3-fold more potent in producing antinociception, and 11-fold more potent in depressing spontaneous activity (Compton et al., 1992). Moreover, rat data suggest dissimilar mechanisms for discrimination and the behavioral disruption exemplified by response rate suppression. Therefore, receptor subtypes may exist for which WIN 55212-2 has different affinity or efficacy from ⁹-THC. In addition, actions on differing neuropeptide and/or neurotransmitter systems involved in thermoregulation cannot be ruled out. Differences in metabolism and disposition may also account for some of the dissimilar effects. It should be noted, however, that cross-tolerance develops to the hypothermic effects of WIN 55212-2 in ⁹-THC-treated mice (Pertwee et al., 1993; Fan et al., 1994).

In conclusion, the present experiments reveal that CB₁ receptors in the POAH regulate WIN 55212-2-induced hypothermia. These data are the first demonstration that the blockade of intra-POAH CB₁ receptors prevents the hypothermic effect of cannabinoids. These results also confirm that the POAH is a central locus of thermoregulation and provide further evidence that SR141716A and SR144528 are valuable tools for characterizing the pharmacological effects of cannabinoids in vivo.