1 ORIGINALMicroinjection of ARTICLESmelanin-concentrating hormone (MCH) into the median raphe nucleus promotes REM sleep in rats Microinjection of melanin-concentrating hormone (MCH) into the median raphe nucleus promotes REM sleep in rats Claudia Pascovich l ABSTRACT Sofia Niño 1 Alejandra Mondino1 Melanin concentrating hormone (MCH) is a sleep-promoting neuromodulator synthesized by Ximena Lopez-Hill 2 neurons located in the postero-lateral hypothalamus and incerto-hypothalamic area. MCHergic Jessika Urbanavicius 2 neurons have widespread projections including the serotonergic dorsal (DR) and median (MnR) Jaime Monti3 raphe nuclei, both involved in the control of wakefulness and sleep. In the present study, we Patricia Lagos1 explored in rats the presence of the MCH receptor type 1 (MCHR-1) in serotonergic neurons of Pablo Torterolo1* the MnR by double immunofluorescence. Additionally, we analyzed the effect on sleep of MCH microinjections into the MnR. We found that MCHR-1 protein was present in MnR serotonergic and non-serotonergic neurons. In this respect, the receptor was localized in the primary cilia of 1 Facultad de Medicina, Universidad de la these neurons. Compared with saline, microinjections of MCH into the MnR induced a dose- República, Fisiología, Montevideo - Uruguay. related increase in REM sleep time, which was related to a rise in the number of REM sleep 2 Instituto de Investigaciones Biológicas Clemente episodes, associated with a reduction in the time spent in W. No significant changes were observed Estable, Neurofarmacología Experimental, in non-REM (NREM) sleep time. Our data strongly suggest that MCH projections towards the Montevideo - Uruguay. MnR, acting through the MCHR-1 located in the primary cilia, promote REM sleep. 3 Hospital de Clínicas, Farmacología y Terapéutica, Montevideo - Uruguay. Keywords: Serotonin; Hypothalamus; Sleep, REM; Peptide; Paradoxical Sleep; Slow Wave Sleep. * Corresponding author: Pablo Torterolo E-mail: [email protected] / [email protected] Received: , July 15, 2020; Accepted: , October 30, 2020. DOI: 10.5935/1984-0063.20200075 Sleep Sci. 2020; Ahead of Print Pascovich C, et al. 2 INTRODUCTION Reagents and antibodies The melanin-concentrating hormone (MCH) is a The reagents to prepare the solutions were obtained neuropeptide synthesized by neurons whose cell bodies are from Sigma-Aldrich (Eugene, OR, USA). Biotinylated secondary located in the postero-lateral hypothalamus and incerto- antibodies, fluorophore-conjugated secondary antibodies and hypothalamic area; these neurons project to various regions of normal donkey serum (NDS) were obtained from Jackson 1-5 the central nervous system (CNS) . MCH exerts its biological ImmunoResearch (West Grove, PA, USA). Streptavidin function through two receptors, but only the MCH receptor type conjugated with fluorophores were obtained from Molecular 6 1 (MCHR-1) is functional in rodents . Probes- ThermoFischer Scientific (Eugene, OR, USA). MCHergic neurons are involved in physiological processes such as energy homeostasis, mood regulation and sleep7-10. A high Immunohistochemical procedures density of MCHergic fibers and receptors has been demonstrated The perfusion of three animals was performed during to be present in specific regions of the CNS, such as the dorsal the light phase of the light/dark cycle. The animals were (DR) and median (MnR) raphe nuclei, which are involved in the anaesthetized with urethane (1.5g/kg) and perfused with 0.9% 1,2,4,5,11-14 generation of wakefulness (W) and sleep . NaCl followed by a 4% paraformaldehyde (PFA) solution. The The serotonergic neurons of the mesopontine raphe brains were immediately dissected out and fixed by immersion 12,15,16 nuclei play an important role in the control of REM sleep . in 4% PFA overnight. Thereafter, they were cryoprotected in Previous studies have shown that microinjections of MCH 30% sucrose solution in 0.1M PB for 48h and frozen. Coronal into the DR produce a significant increase in REM sleep and sections (30μm) were obtained by a cryostat (Leica CM 1900, 17 a moderate increment in slow wave sleep (SWS) . Moreover, Leica Microsystems, Nussloch, Germany). Sections containing microinjections of MCH into the DR promote a depressive- the MnR were between the levels AP -7.2-8.28mm (from 9 like behavior . These effects were probably related to the fact Bregma) according to the atlas of Paxinos and Watson (2005)20. that MCH decreases the firing rate of putative DR serotonergic The sections were collected and stored in an anti-freeze solution 11,18 neurons and the synaptic release of serotonin . at -20 °C until immunostaining procedures were performed. Recent reports in rodents have described that the MCHR- Single and double immunofluorescence procedures were 14,19 1 is localized in neuronal primary cilia . In this regards, Niño- performed to detect MCHR-1 protein (goat anti-MCHR-1, 19 Rivero et al. (2019) have shown that 4% of serotonergic and 12% sc-5534, Santa Cruz Biotechnologies; or rabbit anti-MCHR1, of GABAergic DR neurons express the MCHR-1 in their primary 702618, ThermoFisher Scientific, Il, USA; 1:1000) and serotonin cilia. With respect to the MnR, by means of intracerebroventricular (goat anti-serotonin, 20079, ImmunoStar, WI, USA; 1:500). (i.c.v.) microinjections of MCH conjugated with the fluorophore Negative controls in all procedures consisted of omission of the rhodamine (R-MCH), we demonstrated that serotonergic and primary antibodies. The identification of MCHR-1 in primary GABAergic neurons of the MnR internalized R-MCH both in rats cilia and MCHR-1 in serotonergic neurons were described in and in cats, suggesting that such neurons were MCH-receptive and Niño-Rivero et al. (2019)19. probably express functional MCHR-113. In addition, we also found that i.c.v. and juxtacellular administration of MCH decreases the Histological data analyses firing rate of presumed serotonergic MnR neurons. Hence, the We examined six coronal sections per rat assayed by aims of the present study were: 1. to demonstrate the presence double immunofluorescence against MCHR-1 and serotonin of MCHR-1 in MnR neurons, and to confirm if the receptor is obtained from three rats. In each coronal section, five 20x located in their primary cilia; 2. to determine whether the receptor is microphotographs were taken along the dorso-ventral axis of the present in serotonergic neurons; 3. to find out if, as in the DR, MCH serotonergic (central) region of the MnR, with a 20x objective microinjections into the MnR promote sleep. lens on a Moticam Pro 282B camera (Motic, Hong Kong, China), coupled to an Eclipse 50i epifluorescence microscope MATERIAL AND METHODS (Nikon, Tokio, Japan). Photoshop and Image J softwares were Animals used to quantify the number of serotonergic neurons in each microphotograph, as well as the number of MCHR-1 positive Nine male Wistar rats (250-310g) were used in this study. signals in structures recognized as primary cilia in our previous All the experimental procedures were conducted in accordance study19. The number of counted cells were averaged per animal; th with the Guide for the Care and Use of Laboratory Animals (8 thereafter, a grand average was performed for all the animals. edition, National Academy Press, Washington, DC, 2010) and the The values are presented as mean±S.E.M. (standard error of national law on animal experimentation (Law N° 18.611). The the mean). experimental protocols were approved by the School of Medicine Bioethics Committee (Protocols Nº 070153-00515-13 and Nº Surgical procedures for sleep recordings 070153- 000841-18). Adequate measures were taken to minimize Six rats were anesthetized with a mixture of ketamine- pain, discomfort or stress of the animals, and all efforts were made xylazine-acepromazine (90mg/kg, 5mg/kg and 2mg/kg, i.p., to use the minimal number of animals necessary to produce respectively), positioned in a stereotaxic frame (David Kopf reliable scientific data. Sleep Sci. 2020; Ahead of Print 3 Microinjection of melanin-concentrating hormone (MCH) into the median raphe nucleus promotes REM sleep in rats Instruments, USA) and prepared for standard polysomnography. epochs; the predominant activity of each epoch was assigned Following scalp incision, skull landmarks were visualized and to the following categories based in standard criteria: W, light screw electrodes (1mm diameter) were implanted in the skull for sleep (LS), SWS, REM sleep and non- REM (NREM) sleep recording the frontal, parietal and occipital electroencephalogram (LS+SWS). Latencies to SWS (from the beginning of the (EEG); a referential electrode was implanted in the cerebellum. recording to the first of two consecutive epochs of SWS) and A bipolar electrode was inserted in the neck muscles to monitor REM sleep (from the beginning of the recording to REM sleep the electromyogram (EMG). All the electrodes were soldered to onset), as well as the number and mean duration of W and sleep a connector. In addition, a guide cannula (gauge 26) was inserted episodes were also determined. into the MnR (AP -7.8, L 2.6 and H 6.7mm from Bregma)20. All values are presented as mean±S.E.M. (standard Guide cannulas were lowered at an angle of 20º to avoid the error of the mean). The statistical significance of the difference sagittal vein and were placed 2mm above the MnR to minimize between controls vs. MCH effects was evaluated using one way cellular damage at the injection site. The connector and cannula analysis of variance (ANOVA) and Dunnett post hoc test (one- were cemented to the skull with dental acrylic. At the end of tailed analysis). The criterion used to discard the null hypothesis the surgical procedures, an analgesic (Ketoprofen, 1mg/kg, was p<0.05. subcutaneously) was administered. The animals were treated postoperatively for 24hs with antibiotics RESULTS (Cefradine, 50mg/kg i.m.). A topical antibiotic cream (Neomycin) was Figure 1A shows a topographic view of the location of applied to the skin margins surrounding the implant. the serotonergic neurons within the MnR.