Melatonin MT1 and MT2 Receptors Exhibit Distinct Effects in the Modulation of Body Temperature across the Light/Dark Cycle

The maintenance of body temperature (T_b) in mammalians is critical for survival and internal homeostasis. The main brain structure involved in controlling T_b is the hypothalamus, which receives inputs from the thermoreceptors located in both the brain and the periphery. Depending on these inputs, homeostatic changes are subsequently induced, causing sweating or shivering [1]. In particular, the preoptic area (POA) and dorsomedial hypothalamus (DMH) are critical hypothalamic areas for thermoregulation. In these areas, based on the firing rate responses to changes in local brain temperature, electrophysiological recordings have shown three different neuronal populations: warm-sensitive neurons (~30%), cold-sensitive neurons (~6%), and insensitive neurons (~60%), [2,3]. Activation of the thermally-responsive GABAergic and glutamatergic neurons in the ventral part of the lateral preoptic nucleus (vLPO) and the dorsal part of the dorsomedial hypothalamus (DMD), respectively, decreases temperature, physical activity, and metabolic rate. On the contrary, GABAergic neurons in the DMD promote the increase of T_b, energy expenditure, and physical activity [4].

Recently, it has also been found that T_b can be internally regulated in a circadian manner by endogenous signals from other parts of the hypothalamus [4,5], specifically in the suprachiasmatic nucleus (SCN) [1,6,7]. The SCN regulates the circadian rhythmicity of several physiological responses [1], including the oscillatory decrease of the thermoregulatory threshold of heat production during day time and heat loss during night time in diurnal species [1,8,9]. However, the mechanisms underlying this circadian modulation of T_b remain unclear. Interestingly, the hypothalamus, including the SCN, DMH, and POA, are rich in melatonin (MLT) MT₁ and MT₂ receptors [7,10,11,12,13], and the activation of these MLT receptors modulates numerous physiological effects including the control of T_b [14].

MLT is produced in the pineal gland, mostly during the dark phase in both diurnal and nocturnal species [12,15]. The circadian production and release of MLT are controlled by the SCN [16]. Most of the physiological effects of MLT result from the activation of two high-affinity G-protein coupled receptors (GPCRs), MT₁ (pK_i = 10.09) and MT₂ (pK_i = 9.42), both of which are widely expressed in the mammalian brain [7,10,17]. The specific localization of the two MLT receptor subtypes in different regions of the brain and/or neuronal populations [11,18,19,20] partially explains the selective and differential functional activity of the two MLT receptor subtypes, such as in sleep [18,21,22,23], anxiety [24], pain [25,26], circadian rhythms [27], and depression [28]. In addition to these high-affinity MLT receptors, another low-affinity MLT binding site, termed MT₃ (pKi = 6.0), has been reported [29,30]. Given its both hydrophilic and lipophilic nature, MLT can easily pass through the cell membrane and bind nuclear receptors, including retinoic acid receptor-related orphan receptors (RORs) [31].

It is known that MLT decreases T_b during the night in diurnal species [1]. Clinical studies have shown that exogenous administration of MLT suppresses the physiological increase in T_b observed during daytime [32,33] and has hypothermic properties at the dose of 5 mg/kg [34,35,36,37,38]. Similar results have been demonstrated in preclinical studies in diurnal animals in which administration of MLT acted as a hypothermic agent in the active/light phase in fat sand rats and Marshall broiler chickens [39,40]. However, the neurobiological mechanism through which MLT exerts this hypothermic effect, as well as the selective contribution of the three MLT receptor subtypes, are yet to be investigated.

Therefore, we investigated modifications in T_b produced by selectively activating the three MLT receptor subtypes across the light–dark cycle in rats. To achieve this aim, we tested the effects of the selective MT₂ receptor partial agonist N-{2-[(3-bromophenyl)-(4-fluorophenyl)amino] ethyl}acetamide (UCM924) (pK_iMT1 = 6.76; pK_iMT2 = 9.27) [41], the selective MT₁ receptor partial agonist N-(2-{Methyl-[3-(4-phenylbutoxy)phenyl]amino}ethyl) acetamide (UCM871) (pK_iMT1 = 8.93; pK_iMT2 = 7.04) [42], and the MT₃ receptor agonist 5-Methoxycarbonylamino-N-acetyltryptamine (GR135531↗) (pK_iMT3 = 29.5) on T_b. The effects of UCM924, UCM871, and GR135531↗ were compared to those of MLT. In addition, selective and non-selective MLT receptor antagonists were also tested together with MLT and the other MLT receptor agonists/partial agonists to further dissect the MLT receptor subtypes involved in their thermoregulatory effects.

In physiological conditions, as already known [1,9], there are changes in T_b between the light and the dark phase; in particular, T_b oscillations mostly occur during the phase shift (Figure 1). During the shift from the dark to the light phase, the T_b drops from an average of 38.45 to 37.5 °C after the light turns on (Figure 1A). The opposite occurs during the shift from the light to the dark phase when the light is turned off (Figure 1B).

In this study, we investigated the effects of MLT and its three receptors on T_b during the light and the dark phase for the first time. To achieve this aim, we used a pharmacological approach employing MLT, the selective MT₁ receptor partial agonist UCM871, the selective MT₂ receptor partial agonist UCM924, the MT₃ receptor agonist GR135531↗, and selective/non-selective MLT receptor antagonists, including the MT₂ selective antagonist 4P-PDOT, the MT₁/MT₂ non-selective antagonist luzindole, and the MT₃/α₁ antagonist prazosin. The exogenous administration of MLT during the light phase decreased T_b immediately after the administration and before the light–dark phase shift, an effect blocked by both 4P-PDOT and luzindole. Interestingly, unlike MLT, neither UCM924 nor UCM871 produced a change in T_b during the light phase. In contrast, the selective MT₂ partial agonist UCM924 administered at the end of the dark phase decreased T_b during the dark phase, just prior to the dark–light switch, whereas the selective MT₁ partial agonist UCM871 injected at the end of the light phase increased T_b during the following dark phase. On the other hand, MT₃ receptors did not seem to be involved in the regulation of T_b, since the MT₃ receptor agonist GR135531↗ and the MT₃/α₁ antagonist prazosin alone had not produced any effect on T_b.

The rat circadian body temperature displays a cosine wave [39], showing a temperature that oscillates around the 35.6–36 °C during the light phase and around 37.8–38 °C during the dark phase [43]. The daily change in T_b shows a characteristic deviation at two different times, consistent with the switch between day (light phase) and night (dark phase) hours [44]. The present study replicates the same physiological T_b deviation that was previously reported in nocturnal rodents [43,44], with a daily T_b peak during the night, which is concomitant with the increase in the activity of the animals [45].

In nocturnal rodents, T_b peaks during night time when MLT levels are high, and decreases during the light phase when MLT levels are low. In contrast, in diurnal species, T_b regulation follows the reverse direction in relation to MLT levels, showing a T_b peak during the light phase [45] when circulating levels of MLT are very low (~10 pg/mL) [12,15,46]. However, the mechanism by which MLT regulates T_b has not been established.

The neuronal circuit controlling the regulation of T_b involves several structures including the SCN, SON, mPOA, and DMH [4,5]. Notably, the hypothalamus is an area rich in MLT receptors [11], and their expression may vary according to the phase and the time of the day [47,48,49,50,51].

Previous reports have shown that exogenous MLT administration during the light phase induced a decrease in T_b in both humans and rodents [32,35,36,37,39,52], although the active doses in humans are lower than those in rodents due to a significantly faster metabolism and very short half-life of MLT in the latter [53]. Our findings confirm that exogenous MLT influences T_b, and its effects are strictly dependent on the time of day: MLT reduces T_b only towards the end of the light phase and if administered during the light phase. In regard to its possible mechanism of action during the light phase, we found that both the selective MT₂ antagonist 4P-PDOT and the non-selective MT₁/MT₂ antagonist luzindole blocked the T_b reduction due to MLT, yet neither the selective MT₁ partial agonist UCM871 nor the selective MT₂ partial agonist UCM924 recapitulated the effects of MLT on T_b. These findings suggest that during the light phase, MLT needs to simultaneously activate both MT₁ and MT₂ receptors to modulate T_b. Indeed, the selective activation/inhibition of only MT₁ or MT₂ receptors did not affect T_b during the day. In contrast, UCM871 and UCM924 produced changes in T_b at different times of the dark phase and of opposite magnitude: UCM871 enhanced T_b just after the light–dark transition, whereas UCM924 decreased T_b just before the dark–light transition. Importantly, unlike UCM871 and UCM924, MLT did not induce any change in T_b during the dark phase. We previously observed a similar time-of-day-dependent effect of MLT on sleep [23]. However, it is interesting that when α₁ receptors/MT₃ receptors were blocked by prazosin, MLT decreased T_b also during the dark phase. These complex findings observed during the dark phase are likely dependent on the fact that during the dark phase there is a significant increase in the endogenous levels of MLT, and thus the expression of the two MLT receptors [19], as well as the involvement of other receptors implicated in thermoregulation, such as α₁ adrenoceptors [54], probably vary.

Interestingly, it is now well recognized that MLT receptors can form MT₁/MT₂ hetero-oligomers and also heteromers with other receptors, and from a functional point of view, their properties are different from those of the corresponding homomers [55,56]. Since MLT receptors as well as other receptors including α₁-adrenoceptors are highly expressed in brain regions/nuclei involved in thermoregulation, we cannot exclude that some of the effects of MLT on T_b described here were mediated by these oligomers/heteromers. Future studies are needed to investigate the possible circadian variability in the formation and role of oligomers and/or heteromers of MLT receptors in hypothalamic nuclei regulating T_b. Similarly, the potential contribution of nuclear receptors, among which RORs that are also activated by MLT [31], is worth investigating.

The phase-dependent response of T_b to exogenous MLT may depend not only on the changes in the density of MLT receptors across the light–dark cycle, but also on the relative distribution and function of MT₁ and MT₂ receptors that control unique physiological responses in the brain, for example in sleep [21,22,23], anxiety [18,24], pain [25,26,57], and depression [28,58,59], and in the periphery, for example at the cardiovascular level [60,61].

In conclusion, we have investigated the role of MLT receptors in thermoregulation, and found that during the light phase T_b is affected only if both MT₁ and MT₂ receptors are simultaneously activated. Further, during the dark phase, a time-dependent effect was found in that the activation of MT₁ and MT₂ produces an increase and decrease of T_b respectively. No effects on T_b of the MLT MT₃ receptor subtype were evidenced. However, MT₁ and MT₂ receptors control T_b in synergy with other receptors including α₁ adrenoceptors. These data further support the recent findings showing that the MT₁ and MT₂ receptors modulate physio-pathological functions in different and sometimes opposing ways, and in a time-of-day dependent manner. In particular, MT₁ or MT₂ agonists may be further tested for hypothermia or hyperthermia, respectively.

Conceptualization, G.G.; Data curation, M.L.-C., S.H.M., and S.C.; Formal analysis, M.L.-C. and S.C.; Investigation, M.L.-C., S.H.M., L.P., and D.D.G.; Project administration, G.G.; Resources, A.B. and G.S.; Supervision, G.G. and S.C.; Writing—original draft, M.L.-C. and S.H.M.; Writing—review & editing, G.G. and S.C.

This work was supported by grants from the Ministère de l’Économie, Science et Développement du Québec (MESI-PSR-SIIRI) and the Canadian Institutes of Health Research (CIHR, MOP-130285) to G.G. M.L.C. is supported by the CONACyT (272351) and the Faculty of Medicine, McGill University, Ferring postdoctoral fellowship. L.P. received a PhD fellowship from the the Louise and Alan Edwards Foundation. D.D.G. is supported by a post-doctoral fellowship from the Fonds de la Recherché du Quebec en Santé (FRQS). S.C. is supported in part by a 2017 NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation.

Dr. Gabriella Gobbi is an inventor and assignee in patents regarding selective melatonin MT₂ agonists. The other authors declare no conflicts of interest.