Abstract
Recreational drugs such as 3,4-methylenedioxymethamphetamine and cocaine induce hyperthermia, which is affected by ambient temperature. 2-(4-Bromo-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25B-NBOMe), a selective agonist of 5-HT2A receptor used as a recreational drug, reportedly induces hyperthermia. This study aimed to verify whether 25B-NBOMe induces ambient temperature-dependent hyperthermia and to clarify its mechanism. Eight-week-old male Sprague-Dawley rats were administered intraperitoneal injection of 25B-NBOMe at an ambient temperature of 23°C or 29°C. 25B-NBOMe administration at 23°C did not change the core body temperature of the rats, whereas administration at 29°C induced significant hyperthermia 30–120 minutes postadministration. Tail surface temperature temporarily decreased 30 minutes postadministration, indicating heat storage by peripheral vasoconstriction despite a high ambient temperature. Because 25B-NBOMe-induced-hyperthermia was suppressed by sarpogrelate, but not by destruction of central noradrenaline or serotonin neurons, peripheral 5-HT2A receptors were considered contributors to the development of hyperthermia at a high ambient temperature, independently from central neurons. The temperature of brown adipose tissue (BAT) increased 60–120 minutes postadministration of 25B-NBOMe at 29°C, indicating thermogenesis. Previous studies have reported that peripheral serotonin contributes to the inhibition of BAT thermogenesis. Decreased plasma serotonin levels were observed at 29°C, and serotonin administration partially suppressed 25B-NBOMe-induced hyperthermia at a high ambient temperature, suggesting that decreased levels of peripheral serotonin induced BAT thermogenesis. Our findings indicate that 25B-NBOMe induces hyperthermia at a high ambient temperature via vasoconstriction regulated by 5-HT2A receptors and BAT thermogenesis mediated by decreased levels of plasma serotonin. Thus, peripheral serotonin plays a partial but important role in thermoregulation.
Introduction
Recreational drugs such as 3,4-methylenedioxymethamphetamine (MDMA) or cocaine are known to induce hyperthermia, which is affected by ambient temperatures (Parrott, 2012; Auger et al., 2017). Freedman et al. (2005) verified that ambient temperature-dependent hyperthermia is induced by MDMA administration in 10 participants, and reported that body temperature among the participants following drug-administration at 30°C was significantly higher than that at 18°C. Many studies have reported ambient temperature-dependent drug-induced hyperthermia in animal models. Gonzalez (1993) demonstrated that hyperthermia occurred in rats administered cocaine at 27°C, whereas cocaine administered at 20°C induced hypothermia. Malberg and Seiden (1998) studied MDMA-induced changes in the body temperature of rats at every 2°C change in a strictly controlled ambient temperature and demonstrated that ambient temperatures of ≥24°C and ≤22°C induce hyperthermia and hypothermia, respectively, suggesting that MDMA attenuates thermoregulation. However, the precise mechanism underlying ambient temperature-dependent hyperthermia remains unknown.
Thermal information is perceived by the transient receptor potential family in the skin. At high ambient temperatures, signals from the thermoregulatory central neural pathways of the preoptic area, medial preoptic area, and dorsal hypothalamic area, as well as the rostral raphe pallidus nucleus, are transmitted to the peripheral thermoregulatory organs, resulting in heat loss through vasodilation and evaporative cooling by sweating or through reduced thermogenesis in skeletal muscles and brown adipose tissue (BAT) (Morrison and Nakamura, 2011). Certain serotonin neurons have been reported to play specific roles in the central neural thermoregulation system (Hodges and Richerson, 2010), and central serotonin neurons in the thermoregulatory system have been the key focus of drug-induced hyperthermia studies, because many drugs affecting serotonergic neurons induce hyperthermia (Musselman and Saely, 2013). Lin et al. (1998) have reported that the 5-HT2 receptor agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) injected into the rat hypothalamus increased intracellular serotonin levels, leading to hyperthermia, whereas 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) injections exerted an adverse hypothermic effect. Some studies have reported that DOI induced peripheral vasoconstriction and nonshivering thermogenesis in the intrascapular BAT (iBAT) in rabbits and rats via sympathetic neural stimulation (Blessing and Seaman, 2003; Ootsuka and Blessing, 2006).
Serotonin is a neurotransmitter, but at the same time it also acts as a peripheral hormone in various organs (Gamoh et al., 2013; Herr et al., 2017). Separated by the blood-brain barrier (BBB), central serotonin is synthesized by tryptophan hydroxylase (TPH) 2 in the brain stem, whereas peripheral serotonin is synthesized by TPH1 in the gut (Walther and Bader, 2003); hence, central and peripheral serotonins are considered to be independent from each other. Currently, the roles of peripheral serotonin and its receptors in the thermoregulatory system are receiving increased attention.
5-HT2A receptors are distributed in cardiovascular smooth muscle cells and platelets and contribute to vasoconstriction and aggregation, respectively (Kaumann and Levy, 2006). Rat adipose tissue has also been reported to bear 5-HT2A receptors, which suppress lipolysis by β-adrenergic stimulation (Hansson et al., 2016). Recently, Crane et al. (2015) formulated a novel theory regarding the association between BAT thermogenesis and peripheral serotonin levels. They demonstrated that peripheral serotonin reduced by TPH1 inhibition increases the expression of uncoupling protein-1 (UCP1) in the mitochondrial membrane and iBAT thermogenesis induced by noradrenergic stimulation, indicating a peripheral serotonin thermoregulatory system independent from central signaling; however, the association of the theory with drug-induced hyperthermia or whether it is affected by ambient temperature remains unclear.
Drug-induced hyperthermia has also been reported in cases using 2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25B-NBOMe), a substance abused as a recreational drug (Poklis et al., 2014; Yoshida et al., 2015). NBOMes are compounds formed by the addition of a 2-methoxybenzyl group to the 2C family of phenethylamine and act as agonist of 5-HT2A and 5-HT2C receptors. In particular, 25B-NBOMe has been reported to show a high affinity for 5-HT2A receptors (Juncosa et al., 2013). Intravenous (Ettrup et al., 2013) or intraperitoneal (Shintani-Ishida et al., 2018) 25B-NBOMe injections show that the compound is heavily localized in the lungs and multiple peripheral organs, as well as the brain.
In this study, we confirmed that intraperitoneal 25B-NBOMe administration induces hyperthermia in rats at a high ambient temperature, which is similar to the effects of MDMA and cocaine. Moreover, we aimed to verify the contribution of the peripheral thermoregulatory system to ambient temperature-dependent 25B-NBOMe-induced hyperthermia in an animal model.
Materials and Methods
Eight-week-old male Sprague-Dawley rats (n = 131) corresponding to young adults in human (Sengupta, 2013) were purchased from Shimizu Laboratory Supplies Company (Kyoto, Japan). Postoperatively, the rats were individually housed in cages with wood-chip bedding at 23 ± 1°C ambient temperature, 12-hour light/dark cycle, with free access to water and food. The experiments were performed with the rats freely moving in the same cage.
All experiments were started at 2:30 PM in consideration of the influence of the circadian rhythm. The ambient temperature was controlled by a room air conditioner and was maintained (23°C or 29°C) from 1 hour before the start of the experiment.
The core body temperature was measured by implanting a logger, DST milli-T (Star-Oddi, Gardabaer, Iceland), which was set up to gather samples at 5-minute intervals during the experiment, into the abdominal cavity. Implantation was performed under general anesthesia (isoflurane inhalation, 2.0% to 2.5%) at least 4 days before the start of the experiment for recovery. Data were retrieved after the experiment and analyzed using Mercury software (Star-Oddi). The same method was used to measure BAT temperature with the logger implanted into the intrascapular area (Cannon and Nedergaard, 2004). Tail surface temperature was measured using an infrared thermometer FHT-P2 Avantek (Claybox Ltd., Hong Kong) at 10-minute intervals during the experiment. Before the experiment, the accuracy of the infrared thermometer was confirmed to be less than 1.76% at 23°C to 29°C ambient temperatures by measuring water at 34°C to 50°C.
25B-NBOMe hydrochloride (PubChem CID: 76965389; Kim et al., 2016) was obtained from Lipomed AG (Arlesheim, Switzerland), and 1 mg of the compound was dissolved in 10 μl of dimethyl sulfoxide and 90 μl of methanol and subsequently diluted to 0.25 mg/ml with phosphate-buffered saline (PBS) for intraperitoneal administration. Serotonin hydrochloride (CID: 160436) was purchased from Nakalai Tesque (Kyoto, Japan) and was dissolved in PBS to achieve a concentration of 0.05 or 0.1 mg/ml. Sarpogrelate hydrochloride (CID: 444005) was obtained from Wako Pure Chemical Industries, Ltd (Osaka, Japan), and 1.0 mg of the compound was dissolved in 10 μl of dimethyl sulfoxide and subsequently diluted to 5.0 mg/ml with PBS. Sarpogrelate hydrochloride concentration was determined via a previously described method (Rajesh et al., 2006). 6-Hydroxydopamine (6-OHDA) hydrobromide (CID: 176170) and 5,7-dihidroxytryptamine (5,7-DHT) hydrobromide (CID: 35781) were obtained from MilliporeSigma (St. Louis, MO) and Adipogen Life Sciences, Inc. (San Diego, CA), respectively. Ten milligram of each compound was diluted in 400 μl of saline containing 0.1% ascorbic acid. Escitalopram oxalate (CID: 146571) and desipramine hydrochloride (CID: 65327) were obtained from MilliporeSigma and Wako Pure Chemical Industries, Ltd, respectively. Escitalopram was diluted to 5.0 mg/ml with PBS and desipramine was diluted to 25.0 mg/ml with PBS.
Intraventricular administration of 6-OHDA or 5,7-DHT was carried out as previously described (Reader and Gauthier, 1984; Tanaka et al., 2017). Rats were pretreated intraperitoneally with 5.0 mg/kg of escitalopram 30 minutes before 6-OHDA infusion, or 25.0 mg/kg of desipramine likewise before 5,7-DHT infusion. Under general anesthesia (isoflurane inhalation, 2.5% to 3.0%), 6-OHDA (200 μg/8 μl) or 5,7-DHT (200 μg/8 μl) was infused into the bilateral ventricle (0.80 mm posterior, 1.5 mm lateral, 4.0 mm deep from bregma and skull; Paxinos and Watson, 1986) using a 23-gauge needle and a microsyringe infusion pump over a period of 10 minutes. The rats had at least 5 days to recover before 25B-NBOMe was administered. The effects of 6-OHDA and 5,7-DHT were confirmed by measuring catecholamine and serotonin levels in brain tissue using high-performance liquid chromatography (Loftis et al., 2010).
Platelet-poor plasma was prepared from rats 1 hour post-25B-NBOMe administration to measure noradrenaline, adrenaline, dopamine, and serotonin levels. The rats were killed by collecting total blood by cardiac centesis under general anesthesia. Blood was transferred into a plastic tube containing Na2EGTA and centrifuged within 30 minutes of collection to separate the plasma: Plasma levels of three different catecholamines and serotonin were measured at LSI Medience Corporation (Tokyo, Japan) using high-performance liquid chromatography and an enzyme-linked immunosorbent assay, respectively.
Temperature data are presented as the mean ± S.E. Biochemical levels are shown as the median with 75% confidence intervals. Statistical analyses were performed using EZR (Easy R) on R Commander version 1.31 (based on R Commander version 2.2-3) (Kanda, 2013).
Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals, 2011) and were approved by the Animal Experiments Committee of Kyoto Prefectural University of Medicine (approval number: M28-417).
Results
25B-NBOMe Induced Hyperthermia at a High Ambient Temperature.
Intraperitoneal and tail surface temperatures of rats were monitored following the administration of either 0.25 mg/kg of 25B-NBOMe or vehicle at 23°C or 29°C ambient temperature. The administration of 25B-NBOMe or vehicle at 23°C did not increase core body temperature (Fig. 1). However, at 29°C, the administration of 25B-NBOMe significantly increased the core body temperature 30–120 minutes postadministration compared with the administration of vehicle [one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison tests, P < 0.05] (Fig. 1).
Core body temperatures of rats following the administration of 0.25 mg/kg of 25B-NBOMe or vehicle at 23°C or 29°C ambient temperature. Values = means, error bar = S.E.; one-way ANOVA, followed by Tukey’s multiple comparison test, *P < 0.05, 29°C vehicle vs. 29°C 25B-NBOMe; n = 6 for 23°C vehicle and 23°C 25B-NBOMe, n = 5 for 29°C vehicle and 29°C 25B-NBOMe.
25B-NBOMe Impaired Heat Loss through Vasodilation via 5-HT2A Receptors, Resulting in Hyperthermia at a High Ambient Temperature.
The tail surface temperature of the rats reflects peripheral vasoconstriction or vasodilation (El Bitar et al., 2014). Before the experiments, the tail temperatures of rats at 29°C were higher than those at 23°C (Fig. 2), indicating vasodilation, as previously reported (El Bitar et al., 2014). At 29°C ambient temperature, 25B-NBOMe administration temporarily decreased tail surface temperature with a peak at 30 minutes postadministration compared with the vehicle group (one-way ANOVA, followed by Tukey’s multiple comparison test, P < 0.05). At 23°C ambient temperature, the tail surface temperature was not significantly different between the 25B-NBOMe and vehicle groups (Fig. 2).
Tail surface temperature of rats following the administration of 0.25 mg/kg of 25B-NBOMe or vehicle at 23°C or 29°C ambient temperature. Values = means, error bar = S.E.; one-way ANOVA, followed by Tukey’s multiple comparison test, *P < 0.05; 29°C vehicle vs. 29°C 25B-NBOMe; n = 5.
To investigate whether hyperthermia during the early phase of 30 minutes postadministration resulted from vasoconstriction induced by 25B-NBOMe, 5.0 mg/kg of sarpogrelate was intraperitoneally administered 30 minutes before the administration of 0.25 mg/kg of 25B-NBOMe to the animals at 29°C. Sarpogrelate hydrochloride is a selective antagonist of 5-HT2A receptors (Pertz and Elz, 1995) that cannot pass the BBB (Nitanda et al., 2005). The 25B-NBOMe-induced decrease in tail surface temperature was controlled in sarpogrelate-preadministered animals to an insignificant level compared with that in the vehicle group (Fig. 3A). Thus, sarpogrelate preadministration restrained 25B-NBOMe-induced hyperthermia at a high ambient temperature (Welch’s t test, P < 0.05) (Fig. 3B).
(A) Change in tail temperature of rats following the administration of vehicle, 0.25 mg/kg of 25B-NBOMe alone, or with the preinfusion of 5.0 mg/kg of sarpogrelate hydrochloride at 29°C ambient temperature. Values = means, error bar = S.E.; One-way ANOVA, followed by Tukey’s multiple comparison test, *P < 0.05; 29°C vehicle vs. 29°C 25B-NBOMe, n.s. 29°C vehicle vs. 29°C 25B-NBOMe + sarpogrelate; n = 5. (B) Change in Core temperature of rats following the administration of 0.25 mg/kg of 25B-NBOMe alone or with the preinfusion of 5.0 mg/kg of sarpogrelate hydrochloride at 29°C ambient temperature. Values = means, error bar = S.E.; Welch’s t test, *P < 0.05; **P < 0.01; n = 6.
25B-NBOMe Induced BAT Thermogenesis during the Late Phase at a High Ambient Temperature.
The tail surface temperatures of animals administered 25B-NBOMe at 29°C ambient temperature returned to baseline 60 minutes postadministration, but the core body temperature remained higher until 120 minutes postadministration. The temperature of the intrascapular area, where BAT is distributed in rats (Crane et al., 2015; Al-Noori et al., 2016), was monitored to evaluate BAT thermogenesis as another indicator of hyperthermia. The BAT temperature was elevated at 30 minutes postadministration, which was similar in both the vehicle and 25B-NBOMe groups (Fig. 4). BAT temperature in the vehicle group gradually returned to the baseline after 30 minutes (Fig. 4). In contrast, the BAT temperature in the 25B-NBOMe group continued to increase at 60–90 minutes postadministration (Fig. 4), showing a trend similar to the changes in core body temperature (Fig. 1).
Change in iBAT temperature of rats following the administration of 0.25 mg/kg of 25B-NBOMe or vehicle at 29°C ambient temperature. Values = means, error bar = S.E.; Welch’s t test, *P < 0.05; n = 8.
Plasma Serotonin Level Decreased at a High Ambient Temperature.
Plasma catecholamine and serotonin levels were measured 1 hour postadministration, when the BAT temperature significantly increased in the 25B-NBOMe group at 29°C ambient temperature (Fig. 4). Both the 25B-NBOMe and vehicle groups showed lower plasma serotonin levels at 29°C than those at 23°C (Kruskal-Wallis rank sum test followed by Steel Dwass test, P < 0.05; 23°C vehicle vs. 29°C vehicle, 23°C vehicle vs. 29°C 25B-NBOMe, 23°C 25B-NBOMe vs. 29°C vehicle, and 23°C 25B-NBOMe vs. 29°C 25B-NBOMe) (Fig. 5). Noradrenaline, adrenaline, and dopamine levels tended to be higher in the 25B-NBOMe group than in the vehicle group at both 23°C and 29°C ambient temperatures although the difference was not statistically significant (data not shown).
Serum serotonin levels 1 hour postadministration of 0.25 mg/kg of 25B-NBOMe or vehicle at 23°C or 29°C ambient temperature. Dots = median, error bar = 75% confidence interval; Kruskal-Wallis rank sum test, followed by Steel Dwass test, *P < 0.05 for 23°C vehicle and †P < 0.05 for 23°C 25B-NBOMe, respectively. n = 4 for 23°C vehicle, n = 5 for 23°C 25B-NBOMe, n = 7 for 29°C vehicle, and n = 6 for 29°C 25B-NBOMe.
Preinjection of Serotonin Prevented the Prolongation of 25B-NBOMe-Induced Hyperthermia at a High Ambient Temperature.
To investigate whether decreased serotonin levels contribute to 25B-NBOMe-induced hyperthermia, 0.05 or 0.1 mg/kg of serotonin was intraperitoneally administered to the animals at 29°C 30 minutes before the administration of 0.25 mg/kg of 25B-NBOMe to compensate for the decreased plasma serotonin. In a pretreatment with 0.1 mg/kg of serotonin, the core body temperature started to increase post-25B-NBOMe administration but began to decrease after 60 minutes, and was significantly lower at 90 minutes than that in the 25B-NBOMe group alone (Welch’s t test, P < 0.05) (Fig. 6A). Pretreatment with 0.05 mg/kg of serotonin slightly suppressed 25B-NBOMe-induced hyperthermia at 60–120 minutes, indicating a dose-dependent effect (Fig. 6A). BAT hyperthermia was also diminished by pretreatment with 0.1 mg/kg of serotonin although this was not statistically significant (Fig. 6B).
(A) Change in core temperature of rats following the administration of 0.25 mg/kg of 25B-NBOMe alone or with the preinfusion of 0.05 or 0.1 mg/kg of serotonin hydrochloride at 29°C ambient temperature. Values = means, error bar = S.E.; Welch’s t test, *P = 0.05; 25B-NBOMe alone vs. preinfusion of 1.0 mg/kg of serotonin hydrochloride; n = 6. (B) Change in iBAT temperature of rats following the administration of 0.25 mg/kg of 25B-NBOMe alone or with the preinfusion of 1.0 mg/kg of serotonin hydrochloride at 29°C ambient temperature. Values = means, error bar = S.E.; Welch’s t test, n.s.
Destruction of Central 5-HT Neurons or Central Noradrenaline Neurons Did Not Attenuate 25B-NBOMe-Induced Hyperthermia at a High Ambient Temperature.
Intraventricular administration of 6-OHDA, a selective neurotoxin that destroys noradrenaline neurons prior to dopamine neurons depending on the dose (Uretsky and Iversen, 1970), decreased brain noradrenaline levels (Supplemental Data). On the other hand, intraventricular administration of 5,7-DHT, which destroys serotonin neurons (Reader and Gauthier, 1984), decreased brain serotonin levels (Supplemental Data). Rats were given 0.25 mg/kg of 25B-NBOMe at 29°C ambient temperature. The administration of 25B-NBOMe induced hyperthermia in the animals pretreated with 6-OHDA as well as in sham-operated animals. The core body temperatures in those pretreated with 5,7-DHT showed significant elevation 30–150 minutes postadministration of 25B-NBOMe compared with the sham group, resulting in three fatalities: two rats died 90 minutes postadministration; another died after 150 minutes (Fig. 7).
Core body temperatures of rats pretreated with 5,7-DHT, 6-OHDA, or sham-operated following the administration of 0.25 mg/kg of 25B-NBOMe at 29°C ambient temperature. Values = means, error bar = S.E.; one-way ANOVA, followed by Tukey’s multiple comparison test, *P < 0.05, 5,7-DHT group vs. sham-operated group; n = 5, †a, two rats of 5,7-DHT-pretreated group died 90 minutes postadministration; †b, one rat of 5,7-DHT-pretreated group died 150 minutes postadministration.
Discussion
In this study, we successfully demonstrated that 25B-NBOMe induced hyperthermia in rats, which has been observed among patients abusing 25B-NBOMe compounds. Our study was conducted with 0.25 mg/kg of 25B-NBOMe intraperitoneal administration, which corresponds to a moderate-to-high dose in humans (Ettrup et al., 2013; Papoutsis et al., 2015). However, hyperthermia was induced only at a high ambient temperature (Fig. 1). High ambient temperatures have been reported to enhance hyperthermia induced by adrenergic substances such as MDMA and cocaine (Gonzalez, 1993; Parrott, 2012); however, our study showed that other drugs, including serotonergic agents, can mediate a similar phenomenon.
25B-NBOMe-induced hyperthermia in rats at 29°C lasted 120 minutes, with two failures in peripheral thermoregulation. The first failure was observed during the early phase of hyperthermia, at 30 minutes post-25B-NBOMe administration. Increased tail surface temperature owing to vasodilation at a high ambient temperature was temporarily suppressed by 25B-NBOMe (Fig. 2). A previous report indicated that 5-HT2A receptor agonist induces rat tail vasoconstriction and increases core body temperature (Blessing and Seaman, 2003). This mechanism has been recognized as a contributor to the maintenance of thermoneutral conditions at a low ambient temperature or to be an action toward pain (El Bitar et al., 2014). In our study, temporal vasoconstriction was not observed in the vehicle group; therefore, vasoconstriction may result from 5-HT2A receptor activation by 25B-NBOMe rather than an action toward pain caused by the intraperitoneal injection. 25B-NBOMe administration at 23°C did not show significant vasoconstriction, perhaps because ambient temperature affects vasoconstriction to a certain extent (Ootsuka and Blessing, 2006). Sarpogrelate hydrochloride, a selective 5-HT2A receptor blocker, inhibited 25B-NBOMe-induced vasoconstriction at 29°C (Fig. 3A), resulting in the control of the early-phase hyperthermia (Fig. 3B). Blessing and Seaman (2003) reported that the administration of DOI reduces cutaneous blood flow in rabbits and rats and simultaneously increases their core body temperatures. Because DOI-induced vasoconstriction was reduced in rabbits with cervical sympathetic trunk cuts, they posited that peripheral vasoconstriction by DOI is regulated by sympathetic neural stimulation. However, because sarpogrelate cannot pass the BBB (Nitanda et al., 2005), 5-HT2A receptors in the peripheral organs, such as vascular smooth muscle, must contribute to thermoregulation, in addition to the central or sympathetic nerves.
We considered another mechanism for the prolongation of hyperthermia in the late phase, because the decrease in tail surface temperature was transient. High iBAT temperature was observed 90 minutes post-25B-NBOMe administration, indicating iBAT thermogenesis, only in the 25B-NBOMe-administered rats at a high ambient temperature (Fig. 4). Crane et al. (2015) demonstrated that serotonin attenuates isoproterenol-stimulated cyclic AMP in iBAT cells, thus suggesting that peripheral serotonin controls BAT thermogenesis triggered by β-adrenergic stimulation. Moreover, Oh et al. (2015) demonstrated that 5-HT3 receptor activation by 1-(m-chlorophenyl)-biguanide (m-CPBG) in brown adipocytes suppresses β-adrenergic thermogenesis. In our study, a high ambient temperature reduced plasma serotonin level in rats despite 25B-NBOMe administration (Fig. 5), and compensation for the decreased serotonin level via preinjection with a 0.1 mg/kg dose controlled the elevation of core body temperature in the late phase and shortened the duration of hyperthermia (Fig. 6A). Because the supplied serotonin neither controls early-phase hyperthermia nor passes the BBB (Gershon and Tack, 2007), we propose that the decreased plasma serotonin levels contributed to the prolongation of hyperthermia in the late phase. BAT thermogenesis in the study by Crane et al. might have contributed to late-phase hyperthermia, and the supplied serotonin might have controlled BAT thermogenesis via 5-HT3 receptors. However, the contribution made by the peripheral regulation of BAT thermogenesis during drug-induced hyperthermia may have occurred in part because serotonin preinjections only slightly restrained elevated iBAT temperature (Fig. 6B). Although previous reports have shown that central 5-HT2A stimulation increases central dopamine or peripheral adrenaline levels (Bagdy et al., 1989; Gobert and Millan, 1999), we could not increase peripheral catecholamine levels through 25B-NBOMe administration. This result also supported the theory of the partial contribution of β-adrenergic thermogenesis in iBAT during drug-induced hyperthermia at a high ambient temperature.
Surprisingly, the destruction of central noradrenergic pathways by 6-OHDA and that of 5-HT neurons by 5,7-DHT did not attenuate 25B-NBOMe-induced hyperthermia at 29°C. On the contrary, 25B-NBOMe administration at a high ambient temperature induced further hyperthermia in animals pretreated with 5,7-DHT compared with the sham-operated group (Fig. 7), and several rats died after their core body temperatures exceeded 42°C. Myers (1975) reported previously that microinjections of 5,6-dihydroxytryptamine to the anterior hypothalamus caused impaired thermoregulation in exposure to warm (35°C) or cold (8°C) temperatures in rats. In our study, neurotoxin-pretreated animals had the same core body temperatures at 29°C before 25B-NBOMe administration as per the sham-operated group; however, 25B-NBOMe-induced hyperthermia was higher among the animals. Our results may indicate that the destruction of serotonin neurons by 5,7-DHT reduces the control of 25B-NBOMe-induced hyperthermia at high ambient temperatures, which is evoked by peripheral reaction.
In conclusion, we showed that 25B-NBOMe-induced hyperthermia was affected by ambient temperatures. Hyperthermia at a high ambient temperature was induced by vasoconstriction mediated by peripheral 5-HT2A receptors, which generally dilate for heat loss. However, because vasoconstriction was only transient, long-lasting hyperthermia may be associated with decreased peripheral serotonin levels at high ambient temperature, which allow further thermogenesis in the peripheral organs, including BAT. Importantly, the peripheral administration of both sarpogrelate and serotonin successfully controlled drug-induced hyperthermia, and the destruction of central noradrenergic or serotonergic neurons did not prevent drug-induced hyperthermia. Our findings may be useful for the development of novel peripheral treatments for drug-induced hyperthermia.
Acknowledgments
We thank Dr. Tsuchimochi and Dr. Shirai of the National Cerebral and Cardiovascular Center for advice on physiological measurements. We also thank Dr. Harada and Dr. Yoshida of Department of Legal Medicine, Tokyo Medical University, for their technical assistance in the administration of the intracerebral drug.
Authorship Contributions
Participated in research design: Nakamura, Shintani-Ishida.
Conducted experiments: Nakamura, Shintani-Ishida.
Contributed new reagents or analytic tools: Nakamura, Shintani-Ishida.
Performed data analysis: Nakamura.
Wrote or contributed to the writing of the manuscript: Nakamura, Shintani-Ishida, Ikegaya.
Footnotes
- Received April 26, 2018.
- Accepted September 6, 2018.
This work was supported by the Japan Society for the Promotion of Science KAKENHI [Grant Number 16K09215].
↵
This article has supplemental material available at jpet.aspetjournals.org.
Abbreviations
- 25B-NBOMe
- 2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine
- 5-HT
- 5-hydroxytryptamine
- 5,7-DHT
- 5,7-dihidroxytryptamine
- 6-OHDA
- 6-hydroxydopamine
- ANOVA
- analysis of variance
- BAT
- brown adipose tissue
- BBB
- blood brain barrier
- iBAT
- intrascapular brown adipose tissue
- DOI
- (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane
- MDMA
- 3,4-methylenedioxymethamphetamine
- PBS
- phosphate-buffered saline
- TPH
- tryptophan hydroxylase
- Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics
