Rafael Ochoa-Sanchez

Promotion of Non-Rapid Eye Movement Sleep and Activation of Reticular Thalamic Neurons by a Novel MT2 Melatonin Receptor Ligand

Melatonin activates two brain G-protein coupled receptors, MT1 and MT2, whose differential roles in the sleep–wake cycle remain to be defined. The novel MT2 receptor partial agonist, N-{2-[(3-methoxyphenyl) phenylamino] ethyl} acetamide (UCM765), is here shown to selectively promote non-rapid eye movement sleep (NREMS) in rats and mice. The enhancement of NREMS by UCM765 is nullified by the pharmacological blockade or genetic deletion of MT2 receptors. MT2, but not MT1, knock-out mice show a decrease in NREMS compared to the wild strain. Immunohistochemical labeling reveals that MT2 receptors are localized in sleep-related brain regions, and notably the reticular thalamic nucleus (Rt). Microinfusion of UCM765 in the Rt promotes NREMS, and its systemic administration induces an increase in firing and rhythmic burst activity of Rt neurons, which is blocked by the MT2 antagonist 4-phenyl-2-propionamidotetralin. Since developing hypnotics that increase NREMS without altering sleep architecture remains a medical challenge, MT2 receptors may represent a novel target for the treatment of sleep disorders.

Sleep–wake characterization of double MT1/MT2 receptor knockout mice and comparison with MT1 and MT2 receptor knockout mice

The neurohormone melatonin activates two G-protein coupled receptors, MT1 and MT2. Melatonin is implicated in circadian rhythms and sleep regulation, but the role of its receptors remains to be defined. We have therefore characterized the spontaneous vigilance states in wild-type (WT) mice and in three different types of transgenic mice: mice with genetic inactivation of MT1 (MT1(-/-)), MT2 (MT2(-/-)) and both MT1/MT2 (MT1(-/-)/MT2(-/-)) receptors. Electroencephalographic (EEG) and electromyographic sleep-wake patterns were recorded across the 24-h light-dark cycle. MT1(-/-)mice displayed a decrease (-37.3%) of the 24-h rapid eye movement sleep (REMS) time whereas MT2(-/-)mice showed a decrease (-17.3%) of the 24-h non rapid eye movement sleep (NREMS) time and an increase in wakefulness time (14.8%). These differences were the result of changes occurring in particular during the light/inactive phase. Surprisingly, MT1(-/-)/MT2(-/-) mice showed only an increase (8.9%) of the time spent awake during the 24-h. These changes were correlated to a decrease of the REMS EEG theta power in MT1(-/-)mice, of the NREMS EEG delta power in MT2(-/-)mice, and an increase of the REMS and wakefulness EEG theta power in MT1(-/-)/MT2(-/-) mice. Our results show that the genetic inactivation of both MT1 and MT2 receptors produces an increase of wakefulness, likely as a result of reduced NREMS due to the lack of MT2 receptors, and reduced REMS induced by the lack of MT1 receptors. Therefore, each melatonin receptor subtype differently regulates the vigilance states: MT2 receptors mainly NREMS, whereas MT1 receptors REMS.

Anxiolytic effects of the melatonin MT2 receptor partial agonist UCM765: Comparison with melatonin and diazepam☆

Melatonin (MLT) is a neurohormone known to be involved in the regulation of anxiety. Most of the physiological actions of MLT in the brain are mediated by two high-affinity G-protein-coupled receptors, denoted MT(1) and MT(2). However, the particular role of these receptors in anxiety remains to be defined. Here we used a novel MT(2)-selective partial agonist, UCM765 to evaluate the involvement of MT(2) receptors in anxiety. Adult male rats were acutely injected with UCM765 (5-10-20mg/kg), MLT (20mg/kg) or diazepam (DZ, 1mg/kg). Anxiety-related behaviors were assessed in the elevated plus maze test (EPMT), novelty suppressed feeding test (NSFT) and open field test (OFT). UCM765 at the dose of 10mg/kg showed anxiolytic-like properties by increasing the time spent in the open arm of the EPMT, and by reducing the latency to eat in a novel environment in the NSFT. In the EPMT, animals treated with UCM765 (10mg/kg) or MLT (20mg/kg) spent more time in the open arms compared to vehicle-treated animals, but to a lesser extent compared to DZ (1mg/kg). In the NSFT, all treatments similarly decreased the latency to eat in a novel environment compared to vehicle. UCM765 and MLT did not affect the total time and the number of entries into the central area of the OFT, but unlike DZ, did not impair locomotion. The anxiolytic effects of UCM765 and MLT in the EPMT and the NSFT were blocked using a pre-treatment with the MT(1)/MT(2) antagonist luzindole (10mg/kg) or the MT(2) antagonist 4P-PDOT (10mg/kg). These results demonstrated, for the first time, the anxiolytic properties of UCM765 and suggest that MT(2)-receptors may be considered a novel target for the development of anxiolytic drugs.

Adolescent amphetamine exposure elicits dose-specific effects on monoaminergic neurotransmission and behaviour in adulthood

Despite the growing non-medical consumption of amphetamine (Amph) during adolescence, its long-term neurobiological and behavioural effects have remained largely unexplored. The present research sought to characterize the behavioural profile and electrophysiological properties of midbrain monoaminergic neurons in adult rodents after Amph exposure during adolescence. Adolescent rats were administered vehicle, 0.5, 1.5, or 5.0 mg/kg.d Amph from postnatal day (PND) 30-50. At adulthood (PND 70), rats were tested in an open-field test (OFT) and elevated plus maze (EPM), paralleled by in-vivo extracellular recordings of serotonin (5-HT), dopamine (DA) and norepinephrine (NE) neurons from the dorsal raphe nucleus, ventral tegmental area, and locus coeruleus, respectively. 5-HT firing in adulthood was increased in rats that had received Amph (1.5 mg/kg.d) during adolescence. At this regimen, DA firing activity was increased, but not NE firing. Conversely, the highest Amph dose regimen (5.0 mg/kg.d) enhanced NE firing, but not DA or 5-HT firing rates. In the OFT, Amph (1.5 mg/kg.d) significantly increased the total distance travelled, while the other doses were ineffective. In the EPM, all three Amph doses increased time spent in the open arms and central platform, as well as the number of stretch-attend postures made. Repeated adolescent exposure to Amph differentially augments monoaminergic neuronal firing in a dose-specific fashion in adulthood, with corresponding alterations in locomotion, risk assessment (stretch-attend postures and central platform occupancy) and risk-taking behaviours (open-arm exploration). Thus, adolescent Amph exposure induces long-lasting neurophysiological alterations that may have implications for drug-seeking behaviour in the future.

Short-term effects of melatonin and pinealectomy on serotonergic neuronal activity across the light–dark cycle

Melatonin (MLT) and serotonin (5-HT) are two biosynthetically related compounds implicated in several common physiological functions and the etiology of mood disorders. How they interact, though, is not yet fully understood. In this study, single-unit extracellular recordings were used to monitor dorsal raphe nucleus (DR) 5-HT neuronal activity in anesthetized rats, under basal conditions (CTRL), in response to MLT administration, and after pinealectomy (PX) across the light–dark cycle. Under basal conditions, the number of spontaneously active 5-HT neurons and their firing rate were both significantly lower in the dark phase. In the light phase, administration of MLT at low doses (0.5–1 mg/kg, i.v.) decreased 5-HT firing activity. This inhibitory effect of MLT was completely blocked by the MT1/MT2 receptor antagonist luzindole, but not by the selective MT2 receptor antagonist 4P-PDOT, the selective 5-HT1A receptor antagonist WAY100635, or by the α2 adrenoceptor antagonist idazoxan. In the opposite experiment, PX increased 5-HT firing activity in the dark phase, and this was reversed by MLT administration (1 mg/kg, i.v.). Finally, in a forced swim test, MLT (1 mg/kg, i.p.) increased immobility time and decreased swimming behavior. Together, these results suggest that nocturnal MLT secretion imposes tonic inhibitory control over a sub-population of DR 5-HT neurons. This MLT-induced decrease in 5-HT neurotransmission may represent a biological mechanism underlying mood disorders characterized by increased MLT secretion, such as seasonal affective disorder.

Melancholic-Like behaviors and circadian neurobiological abnormalities in melatonin MT1 receptor knockout mice.

Background: Melancholic depression, described also as endogenous depression, is a mood disorder with distinctive specific psychopathological features and biological homogeneity, including anhedonia, circadian variation of mood, psychomotor activation, weight loss, diurnal cortisol changes, and sleep disturbances. Although several hypotheses have been proposed, the etiology of this disorder is still unknown. Methods: Behavioral, electrophysiological and biochemical approaches were used to characterize the emotional phenotype, serotonergic and noradrenergic electrical activity, and corticosterone in melatonin MT1 receptor knockout mice and their wild type counterparts, during both light and dark phases. Results: Melatonin MT1 receptor knockout mice have decreased mobility in the forced swim and tail suspension tests as well as decreased sucrose consumption, mostly during the dark/inactive phase. These mood variations are reversed by chronic treatment with the tricyclic antidepressant desipramine. In addition, MT1 receptor knockout mice exhibit psychomotor disturbances, higher serum levels of corticosterone the dark phase, and a blunted circadian variation of corticosterone levels. In vivo electrophysiological recordings show a decreased burst-firing activity of locus coeruleus norepinephrine neurons during the dark phase. The circadian physiological variation in the spontaneous firing activity of high-firing neuronal subpopulations of both norepinephrine neurons and dorsal raphe serotonin neurons are abolished in MT1 knockout mice. Conclusions: These data demonstrate that melatonin MT1 receptor knockout mice recapitulate several behavioral and neurobiological circadian changes of human melancholic depression and, for the first time, suggest that the MT1 receptor may be implicated in the pathogenesis of melancholic depression and is a potential pharmacological target for this mental condition.

Melatonin, selective and non-selective MT1/MT2 receptors agonists: Differential effects on the 24-h vigilance states

Melatonin (MLT) is a neurohormone implicated in several physiological processes such as sleep. Contrasting results have been produced on whether or not it may act as a hypnotic agent, and the neurobiological mechanism through which it controls the vigilance states has not yet been elucidated. In this study we investigated the effect of MLT (40 mg/kg), a non-selective MT1/MT2 receptor agonist (UCM793, 40 mg/kg), and a selective MT2 partial agonist (UCM924, 40 mg/kg) on the 24-h vigilance states. EEG and EMG sleep-wake patterns were registered across the 24-h light-dark cycle in adult Sprague-Dawley male rats. MLT decreased (-37%) the latency to the first episode of non rapid eye movement sleep (NREMS), enhanced the power of NREMS delta band (+33%), but did not alter the duration of any of the three vigilance states. Differently, UCM793 increased the number of episodes (+52%) and decreased the length of the episodes (-38%) of wakefulness but did not alter the 24-h duration of wakefulness, NREMS and REMS. UCM924 instead reduced the latency (-56%) and increased (+31%) the duration of NREMS. Moreover, it raised the number of REMS episodes (+57%) but did not affect REMS duration. Taken together, these findings show that MLT and non-selective MT1/MT2 receptor agonists do not increase the quantity of sleep but differently influence the three vigilance states. In addition, they support the evidence that selective MT2 receptor agonists increase NREMS duration compared to MLT and non-selective MT1/MT2 agonists.

Reduction in cholinergic interneuron density in the nucleus accumbens attenuates local extracellular dopamine release in response to stress or amphetamine.

Depletion of cholinergic interneurons in the ventral striatum (nucleus accumbens or N.Acc.) in adult rats increases the locomotor activating effects of amphetamine. It also impairs sensorimotor gating processes, an effect reversed by the antipsychotic haloperidol. These behavioral effects are suggestive of pronounced hyper‐responsiveness of the mesolimbic dopamine (DA) projection to the N.Acc. However, it is unclear whether local cholinergic depletion results predominantly in exaggerated presynaptic DA release or a postsynaptic upregulation of DAergic function. The purpose of the present study is to test the former possibility by employing in vivo voltammetry to examine changes in the levels of extracellular DA within the N.Acc. in response to either mild tail pinch stress or amphetamine administration. While both cholinergic‐lesioned and control rats showed reliable stress‐induced increases in extracellular DA on two consecutive test days, those in the lesioned rats were significantly less pronounced. In response to amphetamine, a separate cohort of lesioned rats also exhibited smaller increases in extracellular DA release than controls, despite showing greater locomotor activity. Moreover, the increased behavioral response to amphetamine in lesioned rats coincided temporally with decreasing levels of DA in the N.Acc. The results confirm that cholinergic depletion within the N.Acc. suppresses presynaptic DA release and suggest that lesion‐induced behavioral effects are more likely due to postsynaptic DA receptor upregulation. The results are also discussed in the context of schizophrenia, where post mortem studies have revealed a selective loss of cholinergic interneurons within the ventral striatum. Synapse, 2013.

Cannabidiol modulates serotonergic transmission and reverses both allodynia and anxiety-like behavior in a model of neuropathic pain

Abstract Clinical studies indicate that cannabidiol (CBD), the primary nonaddictive component of cannabis that interacts with the serotonin (5-HT)1A receptor, may possess analgesic and anxiolytic effects. However, its effects on 5-HT neuronal activity, as well as its impact on models of neuropathic pain are unknown. First, using in vivo single-unit extracellular recordings in rats, we demonstrated that acute intravenous (i.v.) increasing doses of CBD (0.1-1.0 mg/kg) decreased the firing rate of 5-HT neurons in the dorsal raphe nucleus, which was prevented by administration of the 5-HT1A antagonist WAY 100635 (0.3 mg/kg, i.v.) and the TRPV1 antagonist capsazepine (1 mg/kg, i.v.) but not by the CB1 receptor antagonist AM 251 (1 mg/kg, i.v.). Repeated treatment with CBD (5 mg/kg/day, subcutaneously [s.c.], for 7 days) increased 5-HT firing through desensitization of 5-HT1A receptors. Rats subjected to the spared nerve injury model for 24 days showed decreased 5-HT firing activity, mechanical allodynia, and increased anxiety-like behavior in the elevated plus maze test, open-field test, and novelty-suppressed feeding test. Seven days of treatment with CBD reduced mechanical allodynia, decreased anxiety-like behavior, and normalized 5-HT activity. Antiallodynic effects of CBD were fully prevented by capsazepine (10 mg/kg/day, s.c., for 7 days) and partially prevented by WAY 100635 (2 mg/kg/day, s.c., for 7 days), whereas the anxiolytic effect was blocked only by WAY. Overall, repeated treatment with low-dose CBD induces analgesia predominantly through TRPV1 activation, reduces anxiety through 5-HT1A receptor activation, and rescues impaired 5-HT neurotransmission under neuropathic pain conditions.