The circadian clock in the mouse habenula is set by catecholamines.

Circadian rhythms are those variations in behavioral and molecular processes of organisms that follow roughly 24 h cycles in the absence of any external cue. The hypothalamic suprachiasmatic nucleus (SCN) harbors the principal brain pacemaker driving circadian rhythms. The epithalamic habenula (Hb) contains a self-sustained circadian clock functionally coupled to the SCN. Anatomically, the Hb projects to the midbrain dopamine (DA) and serotonin (5-HT) systems, and it receives inputs from the forebrain, midbrain, and brainstem. The SCN is set by internal signals such as 5-HT or melatonin from the raphe nuclei and pineal gland, respectively. However, how the Hb clock is set by internal cues is not well characterized. Hence, in the present study, we determined whether DA, noradrenaline (NA), 5-HT, and the neuropeptides orexin (ORX) and vasopressin influence the Hb circadian clock. Using PER2::Luciferase transgenic mice, we found that the amplitude of the PER2 protein circadian oscillations from Hb explants was strongly affected by DA and NA. Importantly, these effects were dose-and region (rostral vs. caudal) dependent for NA, with a main effect in the caudal part of the Hb. Furthermore, ORX also induced a significant change in the amplitude of PER2 protein oscillations in the caudal Hb. In conclusion, catecholaminergic (DA, NA) and ORXergic transmission impacts the clock properties of the Hb clock likely contributing to the circadian regulation of motivated behaviors. Accordingly, pathological conditions that lead in alterations of catecholamine or ORX activity (drug intake, compulsive feeding) might affect the Hb clock and conduct to circadian disturbances.

Rev-erbα modulates the hypothalamic orexinergic system to influence pleasurable feeding behaviour in mice.

The drive to eat is regulated by two compensatory brain pathways termed as homeostatic and hedonic. Hypothalamic orexinergic (ORX) neurons regulate metabolism, feeding and reward, thus controlling physiological and hedonic appetite. Circadian regulation of feeding, metabolism and rhythmic activity of ORX cells are driven by the brain suprachiasmatic clock. How the circadian clock impacts on ORX signalling and feeding-reward rhythms is, however, unknown. Here we used mice lacking the nuclear receptor REV-ERBα, a transcription repressor and a key component of the molecular clockwork, to study food-reward behaviour. Rev-Erbα mutant mice showed highly motivated behaviours to obtain palatable food, an increase in the intake and preference for tasty diets, and in the expression of the ORX protein in the hypothalamus. Palatable food intake was inhibited in animals treated with the ORX1R antagonist. Analyzing the Orx promoter, we found Retinoic acid-related Orphan receptor Response Element binding sites for Rev-Erbα. Furthermore, Rev-Erbα dampened the activation of Orx in vitro and in vivo. Our data provide evidence for a possible repressive role of Rev-Erbα in the regulation of ORX signalling, highlighting an implication of the circadian clockwork in modulating food-reward behaviours with an important impact for the central regulation of overeating.

A suprachiasmatic-independent circadian clock(s) in the habenula is affected by Per gene mutations and housing light conditions in mice.

For many years, the suprachiasmatic nucleus (SCN) was considered as the unique circadian pacemaker in the mammalian brain. Currently, it is known that other brain areas are able to oscillate in a circadian manner. However, many of them are dependent on, or synchronized by, the SCN. The Habenula (Hb), localized in the epithalamus, is a key nucleus for the regulation of monoamine activity (dopamine, serotonin) and presents circadian features; nonetheless, the clock properties of the Hb are not fully described. Here, we report, first, circadian expression of clock genes in the lateral habenula (LHb) under constant darkness (DD) condition in wild-type mice which is disturbed in double Per1-/--Per2Brdm1 clock-mutant mice. Second, using Per2::luciferase transgenic mice, we observed a self-sustained oscillatory ability (PER2::LUCIFERASE bioluminescence rhythmicity) in the rostral and caudal part of the Hb of arrhythmic SCN-ablated animals. Finally, in Per2::luciferase mice exposed to different lighting conditions (light-dark, constant darkness or constant light), the period or amplitude of PER2 oscillations, in both the rostral and caudal Hb, were similar. However, under DD condition or from SCN-lesioned mice, these two Hb regions were out of phase, suggesting an uncoupling of two putative Hb oscillators. Altogether, these results suggest that an autonomous clock in the rostral and caudal part of the Hb requires integrity of circadian genes to tick, and light information or SCN innervation to keep synchrony. The relevance of the Hb timing might reside in the regulation of circadian functions linked to motivational (reward) and emotional (mood) processes.

Brain Tissue Culture of Per2::Luciferase Transgenic Mice for ex vivo Bioluminescence.

In circadian research, it is essential to be able to track a biological rhythm for several days with the minimum perturbation for the organisms or tissues. The use of transgenic mice lines, in which the luciferase reporter is coupled to a molecular clock protein (here PERIOD2), gives us the opportunity to follow the circadian activity in different tissues or even single clock cells for days without manipulation. This method creates sections using a mouse brain matrix, which allows us to obtain several brain samples quickly at a single time point.

The Clock Gene Rev-Erbα Regulates Methamphetamine Actions on Circadian Timekeeping in the Mouse Brain.

Circadian rhythms are strongly affected by drugs. In rodents, chronic methamphetamine (METH) intake changes circadian activity rhythms, mainly by altering light synchronization that generates the expression of a free-running rhythm with a period longer than 24 h and a second behavioral component that is independent of the main suprachiasmatic (SCN) clock. Although a number of clock genes do not appear to be involved in the effects of METH on circadian behavior, the molecular clockwork controlling these changes is still unclear. Therefore, we investigated the role of the clock gene Rev-Erbα in METH-induced behavioral and molecular responses using knockout mice and their wild-type littermates. Chronic intake of METH alters period circadian behavior of wild-type mice. However, in mice lacking the clock gene Rev-Erbα METH had no effect on their behavioral rhythms. Furthermore, PER2 bioluminescence rhythms in two extra-SCN brain oscillators, the dorsomedial hypothalamus and the habenula, were altered by METH in wild type but not in KO mice. Together, the present results implicate Rev-Erbα in the modulation of the circadian responses to METH and may provide a better comprehension into the mechanisms underlying circadian alterations provoked by drug addiction.

Insights into the Role of the Habenular Circadian Clock in Addiction.

Drug addiction is a brain disease involving alterations in anatomy and functional neural communication. Drug intake and toxicity show daily rhythms in both humans and rodents. Evidence concerning the role of clock genes in drug intake has been previously reported. However, the implication of a timekeeping brain locus is much less known. The epithalamic lateral habenula (LHb) is now emerging as a key nucleus in drug intake and addiction. This brain structure modulates the activity of dopaminergic neurons from the ventral tegmental area, a central part of the reward system. Moreover, the LHb has circadian properties: LHb cellular activity (i.e., firing rate and clock genes expression) oscillates in a 24-h range, and the nucleus is affected by photic stimulation and has anatomical connections with the main circadian pacemaker, the suprachiasmatic nucleus. Here, we describe the current insights on the role of the LHb as a circadian oscillator and its possible implications on the rhythmic regulation of the dopaminergic activity and drug intake. These data could inspire new strategies to treat drug addiction, considering circadian timing as a principal factor.