Paul Klosen

Sarawut Lapmanee

Carole Schuster

Béatrice Guardiola

David Hicks

Paul Pevet
email: pevet@inci-cnrs.unistra.fr

Marie Paule Felder-Schmittbuhl

MT1 and MT2 melatonin receptors are expressed in nonoverlapping

Keywords: Melatonin, Melatonin receptor subtypes (MT1, MT2), transgenic mice, immunohistochemistry, tissue distribution Melatonin, Melatonin receptor subtypes (MT1, MT2), transgenic mice, immunohistochemistry, tissue distribution

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Introduction

Experiments in animals as well as clinical or epidemiological studies in humans demonstrate that disorders of rhythmicity can cause a variety of pathologies, and are known to impair processes involved in metabolism and can lead to cardiovascular disease and cancer [START_REF] Schernhammer | Night work and risk of breast cancer[END_REF][START_REF] Dochi | Shift work is a risk factor for increased total cholesterol level: a 14-year prospective cohort study in 6886 male workers[END_REF][START_REF] Bechtold | Circadian dysfunction in disease[END_REF][START_REF] Schroeder | How to fix a broken clock[END_REF][START_REF] Zelinski | The trouble with circadian clock dysfunction: multiple deleterious effects on the brain and body[END_REF]. It is now known that a complex multi-oscillatory circadian network, orchestrated by a master clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus, governs optimal and anticipatory temporal organization of functions in mammals [START_REF] Barclay | Interaction of central and peripheral clocks in physiological regulation[END_REF][START_REF] Dibner | The mammalian circadian timing system: organization and coordination of central and peripheral clocks[END_REF]. Nervous and endocrine effectors receive signals from the master clock via nervous and endocrine pathways and translate them into hormonal, physiological or behavioral responses in specialized structures [START_REF] Buijs | Hypothalamic integration of central and peripheral clocks[END_REF][START_REF] Pevet | Melatonin: both master clock output and internal time-giver in the circadian clocks network[END_REF]. Among these structures is the pineal gland, which secretes the hormone melatonin (MLT)*. Being tightly controlled by the SCN through a well-characterized multisynaptic neural pathway, MLT is synthesized by the pineal gland during the night [START_REF] Perreau-Lenz | In vivo evidence for a controlled offset of melatonin synthesis at dawn by the suprachiasmatic nucleus in the rat[END_REF][START_REF] Arendt | Melatonin: characteristics, concerns, and prospects[END_REF]. MLT is immediately released into the general circulation after its synthesis, hence circulating MLT is an endocrine signal from the SCN providing a nocturnal/ circadian message to the organism. Although MLT is not the only endocrine output of the circadian clock it constitutes the most stable one, directly measurable in saliva and plasma, and thus provides a robust and reliable indicator of SCN clock function. Furthermore, the duration of the nocturnal peak of MLT secretion also reflects the length of the night, and changes in MLT duration are integrated by the brain to measure photoperiodic time [START_REF] Reiter | The melatonin rhythms: both a clock and a calendar[END_REF][START_REF] Malpaux | Melatonin and seasonal reproduction: understanding the neuroendocrine mechanisms using the sheep as a model[END_REF][START_REF] Pevet | Melatonin: from seasonal to circadian signal[END_REF][START_REF] Morgan | Photoperiodic signalling through the melatonin receptor turns full circle[END_REF][START_REF] Revel | Melatonin controls seasonal breeding by a network of hypothalamic targets[END_REF][START_REF] Barrett | Molecular pathways involved in seasonal body weight and reproductive responses governed by melatonin[END_REF]. The daily profile of MLT secretion conveys internal information used for both circadian and seasonal temporal organization. Although MLT (N-Acetyl-5-Methoxytryptamine) has been credited with many properties since its identification [START_REF] Lerner | Isolation of melatonin and 5-methoxyindole-3acetic acid from bovine pineal glands[END_REF], it is the finding that MLT acts as both SCN clock output and internal synchronizer within the circadian clock network [START_REF] Pevet | Melatonin: both master clock output and internal time-giver in the circadian clocks network[END_REF] which strongly suggests that its major function is to act as a 'time-giver". MLT acts through several mechanisms and understanding its physiological function depends on how and where its actions are exerted. In view of its amphiphilic nature, MLT is able to enter any tissue and cellular compartment. At higher concentrations (μM and above), the pharmacological actions of MLT are thought to be mediated via interactions with intracellular proteins such as calmodulin or quinone reductase (QR2) [START_REF] Benítez-King | Melatonin as a cytoskeletal modulator: implications for cell physiology and disease[END_REF][START_REF] Boutin | Quinone reductase 2 as a promising target of melatonin therapeutic actions[END_REF]. Also, at very high concentrations MLT appears to be a potent free radical scavenger [START_REF] Galano | On the free radical scavenging activities of melatonin's metabolites AFMK and AMK[END_REF]. These antioxidant properties, of which the physiological significance still remains to be established, are independent from the 'timegiving' action of the hormone.

As for other hormones, MLT exerts its physiological effects principally through highaffinity receptors. Since 1987, with the use of 2-[ 125 I] MLT as a highly specific ligand, binding sites have been reported in a large number of structures within the brain and periphery, with a great variability in number, localization and density among species [START_REF] Ng | Melatonin receptors: distribution in mammalian brain and their respective putative functions[END_REF][START_REF] Weaver | Localization and characterization of melatonin receptors in rodent brain by in vitro autoradiography[END_REF][START_REF] Masson-Pevet | An attempt to correlate brain areas containing melatonin binding sites with rhythmic functions: a study in five hibernator species[END_REF][START_REF] Maywood | Regional distribution of iodomelatonin binding sites within the suprachiasmatic nucleus of the Syrian hamster and the Siberian hamster[END_REF][START_REF] Vanecek | Hypothalamic melatonin receptor sites revealed by autoradiography[END_REF]. Different subtypes of mammalian MLT receptors have been cloned [START_REF] Reppert | Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses[END_REF][START_REF] Dubocovich | International Union of Basic and Clinical Pharmacology. LXXV Nomenclature, classification, and pharmacology of G protein-coupled melatonin receptors[END_REF] and characterized pharmacologically [START_REF] Dubocovich | International Union of Basic and Clinical Pharmacology. LXXV Nomenclature, classification, and pharmacology of G protein-coupled melatonin receptors[END_REF][START_REF] Jockers | Update on melatonin receptors: IUPHAR Review 20[END_REF]. The MT1 and the MT2 subtypes are members of the seventransmembrane G-protein-coupled receptor family and exhibit sub-nanomolar affinity for MLT [START_REF] Dubocovich | International Union of Basic and Clinical Pharmacology. LXXV Nomenclature, classification, and pharmacology of G protein-coupled melatonin receptors[END_REF][START_REF] Jockers | Update on melatonin receptors: IUPHAR Review 20[END_REF][START_REF] Jockers | Melatonin receptors, heterodimerization, signal transduction and binding sites: What's new?[END_REF]. The MT3 subtype does not correspond to a membrane receptor but to a low affinity binding site on QR2 [START_REF] Boutin | Quinone reductase 2 as a promising target of melatonin therapeutic actions[END_REF]. The two different cloned MLT membrane receptor subtypes possess intrinsic differences, evidenced by different signal transduction pathways as well as their differential tissue distribution. The widespread experimental use of _______________________________________________________________________ *Foot-note : Numerous other MLT sources have been identified beside the pineal gland (retina, Harderian gland, gastrointestinal tract, red blood cells, platelets, mononuclear cells, etc. [START_REF] Hardeland | Melatonin--a pleiotropic, orchestrating regulator molecule[END_REF][START_REF] Acuña-Castroviejo | Extrapineal melatonin: sources, regulation, and potential functions[END_REF][START_REF] Pevet | The melatonin[END_REF][START_REF] Tosini | Melatonin: an underappreciated player in retinal physiology and pathophysiology[END_REF]). The pineal gland, however, is the main source of the nocturnal peak of circulating MLT, as evidenced by the lack of detectable levels in the blood after pinealectomy. Whether MLT in these other organs has autocrine or paracrine effects is also an important question. For that, identification of MLT receptors at the cellular level would also be of interest.

2-[ 125 I]-iodo-melato-nin receptor binding in rodents, monkey and human, as well as the great number of structurally different MLT receptor ligands developed, have already provided great progress in understanding the action and function of MLT. However, understanding the links between specific target sites for MLT, receptor subtypes, and particular physiological actions is still challenging. The continuing search for alternative radioligands [START_REF] Legros | Alternative radioligands for investigating the molecular pharmacology of melatonin receptors[END_REF] and the development of MT1-or MT2-selective ligands [START_REF] Mor | Recent advances in the development of melatonin MT1 and MT2 receptor agonists[END_REF][START_REF] Spadoni | Highly Potent and Selective MT2 Melatonin Receptor Full Agonists from Conformational Analysis of 1-Benzyl-2-acylaminomethyl-tetrahydroquinolines[END_REF][START_REF] Rivara | Therapeutic uses of melatonin and melatonin derivatives: a patent review (2012 -2014)[END_REF] will be of great help in the future. Knockout animals [START_REF] Liu | Molecular dissection of two distinct actions of melatonin on the suprachiasmatic circadian clock[END_REF] have also allowed the functional characterization of MT1 and MT2 receptors that, even if far from being complete, has highlighted some differential roles for the two subtypes.

In situ hybridization and reverse transcription polymerase chain reaction (RT-PCR) analysis have also been used to analyze the distribution of mRNA coding for the two receptor subtypes. Most expression sites have only been described at the macroscopical level [START_REF] Masana | Circadian rhythm of mt1 melatonin receptor expression in the surprachiasmatic nucleus of the C3H/H/HeN mouse[END_REF][START_REF] Weaver | The Mel1a melatonin receptor gene is expressed in human suprochiasmatic nuclei[END_REF][START_REF] Soares | Functional melatonin receptors in rat ovaries at various stages of the estrous cycle[END_REF][START_REF] Uz | The regional and cellular expression profile of the melatonin receptor MT1 in the central dopaminergic system[END_REF][START_REF] Schuster | Photic regulation of mt1 melatonin receptors in the siberian hamster pars tuberalis and suprachiasmatic nuclei: Involvement of the circadian clock and intergeniculate leaflet[END_REF][START_REF] Poirel | MT1 melatonin receptor mRNA tissular localization by PCR amplification[END_REF]. With the exception of the pars tuberalis [START_REF] Klosen | The mt1 melatonin receptor and RORbeta receptor are co-localized in specific TSHimmunoreactive cells in the pars tuberalis of the rat pituitary[END_REF], and the SCN in Siberian Hamster [START_REF] Song | Co-expression of melatonin (MEL1a) receptor and arginine vasopressin mRNAs in the Siberian hamster suprachiasmatic nucleus[END_REF], none of these techniques achieve sufficient cellular resolution to identify the individual cells/neurons concerned. Immunohistochemical approaches have generated detailed mapping at the cellular level of both MLT receptors in the rat [START_REF] Lacoste | Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain[END_REF][START_REF] Waly | Circadian Pattern of Melatonin MT1 and MT2 Receptor Localization in the Rat Suprachiasmatic Nucleus[END_REF]. However the questionable specificity of most MLT receptor antibodies has seriously hampered obtaining convincing data. Knowledge of the cell types that contain MT1 and/or MT2 receptors is critical in defining their potential differential roles and to revive long-standing interests regarding possible therapeutic applications for MLT. Transgenic mice expressing marker enzymes under control of the endogenous MT1 or MT2 gene promoters should allow precise phenotyping of the cells containing MLT receptors. Previous attempts using bacterial artificial chromosome transgenesis did not provide convincing data [START_REF] Adamah-Biassi | Distribution ofMT1melatonin receptor promoter-driven RFP expression in the brains of BAC C3H/HeN transgenic mice[END_REF]. We have thus used a specific reporter "knock-in" strategy replacing the MT1 or MT2 coding sequence with the LacZ reporter enzyme in the endogenous loci without modifying the endogenous upstream and downstream regulatory elements

Materials and Methods

Generation of Mtnr1a and Mtnr1b reporter mice

Targeted ES cells generated in the KnockOut Mouse Project (KOMP), were derived from the C57BL/6NTac mouse strain following the deletion strategy already described by [START_REF] Valenzuela | Highthroughput engineering of the mouse genome coupled with high-resolution expression analysis[END_REF]. In the recombinant alleles (Mtnr1a tm1(KOMP)Vlcg and Mtnr1b tm1(KOMP)Vlcg ) of both Mtnr1a and Mtnr1b, a cassette (ZEN_Ubl) containing a modified E. coli β-galactosidase open reading frame in tandem with a selection gene (Neomycine resistance) replaces the coding sequence of the melatonin receptor (including intron 1-2). This cassette is inserted precisely at the original endogenous start codon and ends after the original stop codon (respectively 45 and 40 bp downstream of the stop) (Fig. 1). Mice carrying the recombinant allele were generated by classical ES cell-based/germline transmission procedure at the Institut Clinique de la Souris, Illkirch, France for Mtnr1a and at the KOMP Repository (UC Davis, US) for Mtrn1b (MGI ID: 4399541). In both cases, the selection gene was eliminated by breeding with mice expressing the CRE recombinase. The 5' and 3' junctions between the Mtnr1a/Mtnr1b genes and the inserted reporter cassettes were confirmed by PCR amplification and sequencing.

Animals and Housing

Mice carrying the recombinant alleles were backcrossed on a C57BL/6J background and segregation of the rd8 mutation (initially present in the C57BL/6NTac ES cells) was verified by genotyping [START_REF] Mattapallil | The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes[END_REF]. All mice processed for histological analysis were from the 5 th generation backcross and beyond. Mice homozygous for the knockin allele were generated on an ad hoc basis by heterozygote breeding. Likewise, heterozygote mice on a F1 C57BL/6J x C3H/HeN or a F1 C57BL/6J x CBA/J background were generated by crossing B6 heterozygotes to C3H melatonin proficient, rd1/rd1 mice. All backcross mice were purchased from Charles River, France.

Mouse Genotyping

For both lines, genotyping was performed on DNA isolated from tail biopsies by the Hot Shot technique, by using primers and PCR conditions optimized by the suppliers (ICS for Mtnr1a, KOMP for Mtnr1b). For both genes, DNA samples were amplified separately for the wild type (wt) and the knock-in (ki) allele. For Mtnr1a primer sequences were: Xf 5'-CAAGTTGCTGGGCAGTGGACAGCAG and Wr 5'-CTTCTTGTTGCGGTACACAGACAGG for wt (425 bp amplicon) and Xf2 5'-GCGGGAGGGCCATAAAAAGTGGC and Sr 5'-CTAGTCTGTTCAGCTGTGTCACACC for ki (196 bp amplicon). Both PCR reactions were performed for 35 cycles of 30 s at 95°C, 30 s at 59 °C, 1 min at 72°C using Taq polymerase (Euromedex). For Mtnr1b primer sequences were: Mtnr1b-wtF 5'-AAGCACCAGCAATTTCCCATTCTCC and Mtnr1b-wtR 5'-TCTTGCCTGAGGAAGGTCAGAATCC for wt (143 bp amplicon) and Mtnr1b-LacF 5'-ACTTGCTTTAAAAAACCTCCCACA and Mtnr1bR 5'-ATGGGTCCCTGGAAGTCACTCACC for ki (666 bp amplicon). PCR was performed for 35 cycles of 30 s at 95°C, 30 s at 55 °C, 1 min at 72°C using Taq polymerase (Euromedex). Wild type and recombinant mice were generated and housed in the Chronobiotron facility (UMS3415 CNRS-University of Strasbourg) as described above (1.5-18 months, 20-60g) with a 12:12-h light:dark cycle and with food and water ad libitum. During all experiments, all efforts were made to minimize the number of animals used. Experimental procedures were approved by the Animal Care and Use Committee of Institute of Cellular and Integrative Neurosciences, University of Strasbourg, France

Tissue processing;

Mice were euthanized during the light phase (9.00-12.00 a.m.) by CO2 inhalation. The animals were fixed by transcardiac perfusion with either 4% phosphate-buffered formaldehyde for LacZ enzymology or Periodate-Lysine-Paraformaldehyde (PLP) fixative for immunohistochemistry (IHC) and in situ hybridization (ISH) [START_REF] Sekerková | Visualization of beta-galactosidase by enzyme and immunohistochemistry in the olfactory bulb of transgenic mice carrying the LacZ transgene[END_REF] The brains were removed from the skulls and post-fixed for 24 hours with 4% formaldehyde for LacZ staining or 12 hours in PLP fixative for IHC/ISH. Formaldehyde and PLP-fixed brains were cryoprotected in 30% sucrose and snap-frozen in isopentane chilled to -78°C with a dry ice-ethanol mix. Some PLP-fixed brains were dehydrated and embedded in polyethylene-glycol (PEG) for IHC and ISH according to Klosen [START_REF] Klosen | PEG embedding for immunocytochemistry: application to the analysis of immunoreactivity loss during histological processing[END_REF]. Brains were then processed either as free floating (LacZ staining or IHC) or PEG sections (IHC/ISH): serial 40 μm sections were cut on a cryotome and collected in PBS containing 0.02% sodium azide before either LacZ enzymology or IHC.

LacZ histoenzymological staining

The LacZ beta-galactosidase enzyme activity was visualized using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) [START_REF] Hopman | Rapid synthesis of biotin-, digixigenin-, trinitrophenyl-, and flurochrome-labeled tyramides and their application for In situ hybridization using CARD application[END_REF]. After washing the sections 3 times for 15 min each in LacZ wash buffer (PBS, 2 mM MgCl 2 , 0.01% sodium deoxycholate, 0.02% Nonidet-P40) at room temperature, they were stained overnight at 37 °C with the X-gal development solution (5mM K 4 Fe(CN) 6 , 5 mM K 3 Fe(CN) 6 , and 1mg/ml X-gal in LacZ wash buffer). On the following day, the sections were washed for 3 times 5 min each in LacZ wash buffer and mounted on gelatinized slides. Slides were dehydrated by 5 min bath in 70%, 95%, and 100% (twice) ethanol, cleared in toluene (twice) and coverslipped with Eukitt medium.

Immunohistochemistry (IHC)

Immunohistochemistry was performed on free floating sections after a 30 min permeabilization with 0.3% Triton X-100 in PBS. For immunohistochemitry on PEG sections, the sections were subjected to an antigen reactivation procedure for 1 hour at 95°C in 10 mM Tris-Citrate buffer. For single label IHC, LacZ was detected using a chicken anti-LacZ antibody (ab9361, Abcam, UK), followed by a biotinylated donkey anti-chicken antibody (Jackson ImmunoResearch, PA, USA) and Neutravidin Peroxidase (ThermoFisher). Peroxidase activity was visualized with Diaminobenzidine as a chromogen. For sequential double label IHC, LacZ was detected with the chicken anti-LacZ antibody followed by a peroxidase conjugated donkey anti chicken antibody (Jackson ImmunoResearch, PA, USA) and diaminobenzidine development. The second immunostaining was then performed using biotinylated secondary antibodies (Jackson ImmunoResaech, PA, USA) and Neutravidin peroxidase (ThermoFisher), followed by visualization of peroxidase activity using tyramide signal amplification with fluorescent tyramides [START_REF] Klosen | PEG embedding for immunocytochemistry: application to the analysis of immunoreactivity loss during histological processing[END_REF]. Vasopressinergic neurons were visualized using a rabbit antiserum against Neurophysin 2 (Sigma N0744, St Quentin Fallavier, France). Vasoactive Intestinal Peptide (VIP) was detected using a rabbit antiserum against VIP (Viper, Netherlands, Institute for Brain Research, Amsterdam, Netherlands) and Calretinin using a monoclonal mouse antibody (clone 6B3, Swant, Marly, Switzerland)

Combined ISH with IHC

Combined non radioactive ISH and IHC was performed on PLP-fixed PEG sections. Riboprobes were transcribed from linearized plasmids in the presence of digoxigenin-labelled nucleotides in accordance with standard procedures. We used Mus musculus probes for Gastrin Releasing Peptide (GRP 763 bp of Mus musculus NM 175012.4), Corticotropin Releasing Hormone (CRH 999 bp of Mus musculus NM 205769.3) and Glutamate Decarboxylase 1 and 2 (GAD1 983 bp of Mus musculus NM 008077.5, GAD2 879bp of Mus musculus NM 008078.2). The length of the probes was confirmed by formaldehyde-3-(Nmorpholino) propanesulphonic acid agarose gel electrophoresis and Northern blotting. The in situ hybridization was performed as reported previously [START_REF] Klosen | The mt1 melatonin receptor and RORbeta receptor are co-localized in specific TSHimmunoreactive cells in the pars tuberalis of the rat pituitary[END_REF]. Briefly, polyethylene glycol sections were postfixed, digested for 30 min at 37°C with 0.5µg/ml Proteinase K (Roche) and acetylated twice for 10 min in 100 mM triethanolamine and 0.25% acetic anhydride. Hybridization was performed for 40 h at 60°C with 200 ng/ml labelled sense or antisense probes in 50% formamide, 5×SSC, 5×Denhardt's solution and 1 mg/ml salmon sperm DNA. Stringency rinses were performed for 6×10 min in 0.1×SSC at 72°C. Digoxigenin-labelled bound probes were detected with alkaline phosphatase-labelled anti-digoxigenin antibodies (Roche). Alkealine phosphatase activity was visualized using Nitroblue Tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP). The sections were then processed for LacZ IHC as described above using a peroxidase conjugated donkey anti-chicken antibody (Jackson ImmunoResearch, PA, USA) and fluorescent tyramides for the visualization of peroxidase activity [START_REF] Hopman | Rapid synthesis of biotin-, digixigenin-, trinitrophenyl-, and flurochrome-labeled tyramides and their application for In situ hybridization using CARD application[END_REF].

Image acquisition and processing for visualization of double labelings

Micrographs were taken on a Leica DMRB microscope (Leica Microsystems, Rueil-Malmaison, France) equipped with an Olympus DP50 digital camera (Olympus France, Rungis, France). The images were then processed with Adobe Photoshop CS6 (Adobe Systems, Mountain View, CA, USA). Bright field images of diaminobenzidine IHC or NBT/BCIP ISH stains were converted to "virtual" fluorescence images by inverting the images to negative images. The colors in these negative images were then converted to either red or green to be combined with the fluorescence images of the second stain.

Results

Transgenic reporter mice

Sequencing of the LacZ transgene sequence confirmed that the LacZ coding sequence started at the original ATG start codon of both the MT1 (Mtnr1a) and MT2 (Mtnr1b) receptor sequence and ended a few bases after the original stop codons (Fig. 1). Thus all upstream and downstream regulatory elements were conserved. Only potential regulatory elements located in intron 1-2 of the MLT receptor genes were lost by this approach.

All the different mouse strains bearing heterozygous or homozygous combinations of the different constructs (Mtnr1a +/+ , Mtnr1a +/ki , Mtnr1a ki/ki , Mtnr1b +/+ , Mtnr1b +/ki , Mtnr1b ki/ki ) generated in this study were viable and healthy with normal reproductive cycles. We verified that Mtnr1a +/ki F1 mice generated by breeding with the MLT-competent C3H strain also produced melatonin, by assaying melatonin concentrations during the night. These heterozygous F1 crosses had 179.27±24.42 (mean±s.e.m.) pg of melatonin per pineal at night, and 23.28±9.54 pg per pineal during the day, within the range of MLT concentrations observed in the C3H mouse strain. These Mtnr1a +/ki and Mtnr1b +/ki F1 C57/C3H mice developed and bred normally.

LacZ enzyme and immunocytochemical staining

X-Gal LacZ histochemical staining was visible as dots in almost all brain areas (Fig. 2, SI Table 1). When LacZ expression was visualized by immunohistochemistry, in some neurons labelling could be seen in the nucleus together with lighter cytoplasmic staining sometimes extending into the dendrites. Only a few neurons displayed LacZ immunostaining throughout their cell body (Fig. 2), but even in these neurons one or two strongly stained dotlike structures were visible (Fig 2D). A similar difference between the LacZ enzyme stain and immunostain has previously been described in a number of papers [START_REF] Mattapallil | The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes[END_REF]. The strongly stained dot-like structures are most likely high density lysosomal accumulations of the E.coli LacZ, appearing larger in enzyme-stained sections because of increased diffusion of the reaction product.

In all regions we investigated, both the LacZ enzyme stain and immunostain could be detected. We used the LacZ enzyme stain for mapping studies because of the better contrast with this method. For double labelling we used immunostaining because it was easier to combine with either neuronal markers or in situ hybridization.

Validation of the transgenic approach for MT1

To validate the transgenic reporter strategy and check for faithful expression patterns of LacZ under the control of the MLT receptor regulatory elements, we compared the MT1-LacZ transgene expression pattern with 125 I-iodomelatonin binding in the mouse as described by Liu et al. [START_REF] Liu | Molecular dissection of two distinct actions of melatonin on the suprachiasmatic circadian clock[END_REF]. A similar comparative approach for the MT2-LacZ transgene was not feasible because 125 I-iodomelatonin binding reflects uniquely MT1 receptor expression; since it is absent on tissue sections from the MT1 gene knock-out mice [START_REF] Liu | Molecular dissection of two distinct actions of melatonin on the suprachiasmatic circadian clock[END_REF].

In MT1-LacZ mice, the strongest reporter expression was observed in cells of the pars tuberalis of the adenohypophysis (Figs. 3 and5). Other strongly labeled structures were the suprachiasmatic nucleus (SCN), the paraventricular nucleus of the thalamus (PVT) and the paratenial thalamic nucleus (PT) (Fig. 3). The anterior hypothalamic area (AHA) adjacent to the SCN displayed slightly lighter staining than the previously mentioned structures (Fig. 3). This expression pattern, particularly in the anterior region of the PVT-PT complex and the SCN-AHA area, was perfectly coincident to the 125 I-iodomelatonin binding described by Liu et al. [START_REF] Liu | Molecular dissection of two distinct actions of melatonin on the suprachiasmatic circadian clock[END_REF].

MT1 and MT2 expression mapping in the mouse brain (Table 1 and Fig 3)

We analyzed the brains of adult male and female C57BL/6J mice between 1-5 and 18 months of age in all genotypes; wild type, heterozygous and homozygous LacZ knock-in (MT1 or MT2 knockout). Furthermore, as the C57BL/6J mice do not produce MLT, we also analyzed the brains of F1 C57BL/6J x C3H mice to investigate the influence of MLT on its own receptor's expression. Only mice carrying the LacZ transgene showed either LacZ enzyme labeling or LacZ immunolabeling -as expected, both stains were completely absent from wild type mice. Furthermore, we did not see any obvious differences in either MT1-LacZ or MT2-LacZ expression pattern related to age, sex or presence of MLT in the F1 crosses with C3H mice. The only obvious difference was between heterozygous and homozygous LacZ knock-in animals, the latter showing stronger enzyme and antibody staining than the former (data not shown), revealing a gene dosage effect as previously shown for MT1 in mice [START_REF] Liu | Molecular dissection of two distinct actions of melatonin on the suprachiasmatic circadian clock[END_REF]. Strong MT1-LacZ reporter expression could be detected in only a few structures in the mouse brain: the SCN, the PT and the PVT. Below the median eminence, the pars tuberalis of the pituitary contained by far the strongest labeled cells (Fig. 3, Table 1). In the SCN, MT1-LacZ expressing cells were seen in all quadrants with an apparent higher density in the core region (Fig. 3). In the olfactory bulb, preoptic area and some subnuclei of the geniculate nucleus, the density of MT1-LacZ expressing neurons was lower but still generalized (Figs 2,3,4). Finally, isolated dispersed cells could be detected in a number of structures throughout the brain (SI Appendix). Of note, no MT1-LacZ expression was detected in the telencephalic cortex, and only very few faintly labelled cells could be seen in the hippocampus.

On the contrary, MT2-LacZ expression in the mouse brain was far more widespread (Fig. 3, Table 1). MT2-LacZ could be seen from the olfactory bulb down to the brain stem, with the notable exception of the pars tuberalis, where only MT1-LacZ was detected. The highest density of MT2-LacZ-expressing cells was seen in the Islets of Calleja, but many other structures also contained robustly labeled cells. In the SCN, MT2-LacZ-expressing cells were less dense than those expressing MT1-LacZ (Fig. 4). The intensity of MT2-LacZexpressing cells surrounding the SCN was higher than that of MT1-LacZ. One of the highest densities of MT2-LacZ expressing cells was seen in the paraventricular nucleus of the hypothalamus PVN, where labeled cells were concentrated in the dorso-lateral part of this structure (Fig. 3). MT2-LacZ expression was also quite widespread in the basal forebrain, the bed nucleus of the stria terminalis and the amydaloid complex, and showed a layered appearance in the optic tectum (Fig. 3).

A major difference between MT1-LacZ and MT2-LacZ expression was the presence of MT2-LacZ in the telencephalic area, most notably in the hippocampus. The strongest MT2-LacZ expression in the neocortex was observed in layer 4 neurons of the somatosensory cortex. In motor areas, visual areas, auditory areas and cingulate cortex, the density of MT2-LacZ expressing cells was reduced, becoming virtually undetectable in associative areas and in the entorhinal cortex. In the piriform cortex, MT2-LacZ was present in layer 2 neurons (Fig. 3). The hippocampus presented a very interesting pattern with very strong staining in the pyramidal neurons of the CA2 region and complete absence from the CA1 and CA3 zones. MT2-LacZ expression could also be seen in the hypothalamus and thalamus. In the hypothalamus, three structures displayed a particularly high density of MT2-LacZ expressing cells: the PVN as previously mentioned, but also the arcuate nucleus and the most medial part of the medial tuberal nucleus (Fig. 5). Next to the rostral arcuate nucleus, a few MT2-LacZ expressing cells were located in the ependymal layer, and could be identified as tanycytes by immunocytochemistry (Fig. 5). The thalamic expression pattern for MT1 and MT2 was quite interesting (Fig. 6). While MT1-LacZ expression was strong in the PVT, only very few cells expressed MT2-LacZ in this structure. MT2-LacZ staining was present in an arc-shaped area encompassing the centro-medial and centro-lateral thalamic nuclei. MT1-LacZ expression within this thalamic complex was more restricted. Another notable site of MT2-LacZ expression was the dorsal raphe nucleus (Fig. 7). In this structure, only a very few isolated cells expressed MT1-LacZ reporter whereas MT2-LacZ positive cells clearly delineated the boundaries of the central dorsal raphe nucleus. These cells were visualized both with enzyme histochemistry and immunohistochemistry for the LacZ protein. The distribution of the MT2-LacZ expressing cells is highly reminiscent of serotoninergic neurons as visualized by the presence of serotonin itself, tryptophan hydroxylase or the serotonin transporter [START_REF] Dougherty | The disruption of Celf6, a gene identified by translational profiling of serotonergic neurons, results in autism-related behaviors[END_REF].

Few structures contained both MT1-LacZ-and MT2-LacZ-expressing cells. When this was the case, the expression of MT1-LacZ was usually limited to a more discrete area within these structures (see central thalamic complex). Finally, only a few structures seemed to express comparable levels of the two MLT receptors, most notably the SCN, the mitral cell layer of the olfactory bulb, the subfornical organ and the median and ventro-median preoptic nuclei (Table 1).

MT1-LacZ expression in the SCN (Figs. 4 and 8)

In the SCN, whereas MT2-LacZ expression encompassed the entire region, MT1-LacZ expression was stronger and more restricted (Fig. 4), suggesting localization within a specific cell type. We tested the 3 classical markers of neuronal subpopulations of the SCN, arginine vasopressin (AVP), vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) (Figs. 7 and8). AVP did not co-distribute with MT1-LacZ, which was consistent with the general aspect of staining (Fig. 8A). On the other hand, double immunostaining for VIP and MT1-LacZ showed that a very few VIP neurons in the ventrolateral quadrant of the SCN also stained for MT1-LacZ (Fig. 8B). Double immunostaining for MT1-LacZ and GRP proved inconclusive, because the GRP antibody stained only fibers and not cell bodies. We therefore used non-radioactive in situ hybridization to visualize GRP cell bodies. Again, some GRP cells expressed the MT1-LacZ transgene, but constituted only a minority of GRP neurons (Fig. 8C). As we failed to identify the majority of MT1-LacZ neurons, we examined Calretinin, which in the mouse is a marker of SCN core cells similar to Calbindin in the Syrian hamster. Double immunostaining for MT1-LacZ and Calretinin showed all possible scenarios:

Calretinin-positive/MT1-positive, Calretinin-positive/MT1-negative, and Calretinin-negative/MT1-positive cells (Fig. 8D). Therefore it appears that MT1-LacZ is expressed in a subpopulation of VIP neurons and some SCN core cells, but the majority of MT1-LacZ expressing SCN cells remain to be identified. We could exclude astrocytes, as a GFAP/MT1-LacZ stain did not show any co-localisation of these two markers.

MT2-LacZ expression in the paraventricular nucleus of the hypothalamus

Much like MT1-LacZ expression in the SCN, MT2-LacZ expression in the PVN was restricted to a subpopulation of PVN neurons. An AVP/MT2-LacZ double immunostain showed that MT2-LacZ was clearly not expressed either in the magnocellular or the parvocellular AVP neurons of the PVN (Fig. 9A). The MT2-LacZ expression pattern in the dorsolateral domain of the PVN suggested that the MT2-LacZ expressing cells might be CRH neurons [START_REF] Dougherty | The disruption of Celf6, a gene identified by translational profiling of serotonergic neurons, results in autism-related behaviors[END_REF]. As for GRP in the SCN, immunostaining for CRH only stained the CRH fibers and not the cell bodies. Therefore, we stained CRH neurons by non-radioactive in situ hybridization for the CRH mRNA. In these double label studies, both CRH mRNA and MT2-LacZ immunolabel were clearly located in the medial parvicellular part of the dorsal PVN. In some cells, MT2-LacZ was clearly co-localized with CRH, but there were also single labeled cells for either MT2-LacZ or CRH (Fig. 9B).

MT2-LacZ expression in the hippocampus (Fig. 10

The MT2-LacZ expression pattern in the hippocampus proved interesting in several aspects. First, while MT2-LacZ expression was clearly present in the hippocampus with strong immunostaining in some cells, MT1-LacZ expression appeared to be completely absent. Second, MT2-LacZ expression was present in two very distinct zones and two completely different neuronal types. Firstly, MT2-LacZ was expressed in the pyramidal cell layer of the CA2 but was completely absent from either CA1 or CA3 (Fig. 2, Fig. 10A). We identified GABAergic cells in the hippocampus by combining immunolabeling for MT2-LacZ with non-radioactive in situ hybridization for either GAD1 or GAD2. While we could find a few GABAergic interneurons also expressing MT2-LacZ, the vast majority of the MT2-LacZ expressing neurons in the CA2 contained neither GAD1 nor GAD2, and thus most probably constitute excitatory glutamatergic pyramidal neurons (Fig. 10B). In the hilus of the dentate gyrus, MT2-LacZ expression was also sometimes co-localized with GAD1 or GAD2 mRNA (Fig. 10C). Secondly, there was a population of small interneurons located mainly in the outer molecular layer of the dentate gyrus (Fig. 10A andD). This cell type was also seen in the most ventral parts of the subiculum and the outer layer 1 of the most ventral areas of the entorhinal cortex, the pyriform cortex and the cortical amygdalar areas. These were among the most strongly MT2-LacZ-stained cells, particularly within the cytoplasm. They were also one of the few cell types where immunostaining extended into the dendrites allowing clear visualization of the dendritic structure of these cells. Based on this staining, these cells were qualified as "small horizontal interneurons". We were unable to find any description of similar cells in the literature, and our attempts to phenotype them were unsuccessful: we screened a large battery of neuropeptide antibodies and mRNA probes to look for staining in this population of MT2-LacZ neurons but were unable to find any neuropeptide marker that stained such "small horizontal interneurons". Although the outer molecular layer contained some GABAergic neurons, most of these MT2-LacZ neurons expressed neither GAD1 nor GAD2 (Fig. 10E).

MT1-and MT2-LacZ expression in the retina (Fig. 11)

Both MT1-LacZ and MT2-LacZ expression were seen in the retina. Weak MT1-LacZ enzyme staining was detected in numerous photoreceptor inner segments, indicating the majority of labeled cells were rods (Fig. 11A). MT1-LacZ expression could also be detected in isolated cells located in the vitreal-most part of the inner nuclear layer (INL), likely a subset of amacrine cells, and in a few isolated cells in the ganglion cell layer (GCL) (Fig. 11A andC). MT2-LacZ expression was absent from the photoreceptor layer, but could be detected in some cells in the inner part of the INL and GCL (Fig. 11B andD). MT2-LacZ stained cells were more numerous than MT1-LacZ in these inner layers, but MT1-LacZ expression in putative amacrine cells was stronger than MT2-LacZ expression. Also, MT1-LacZ and MT2-LacZ expression was observed in distinctly separate subsets of amacrine cells: MT2-LacZ positive amacrine cells were smaller and located closer to the boundary of the inner nuclear and plexiform layers, whereas MT1-LacZ positive cells were more internally positioned within the INL. Furthermore, the strong MT1-LacZ immunostaining extended into their dendrites (Fig. 11E). These MT1-LacZ positive cells do not contain calretinin (Fig. 11F) and their dendrites ramified at the interface of strata S1 and S2 (Fig. 11F). In tyrosine hydroxylase -LacZ double immunostains, the dendrites of MT1-LacZ positive neurons clearly ramified below the layer of tyrosine hydroxylase-positive dendrites (Fig. 11G). We detected only a single cell double labeled for both MT1-LacZ and tyrosine hydroxylase in all the animals that we analyzed (Fig. 11H).

Discussion

The present "knock-in" strategy which replaces the MT1 or MT2 coding sequences with the LacZ reporter inserted into the endogenous loci has for the first time allowed to clearly differentiate cells/neurons containing MT1 and MT2 receptors in the mouse brain. Striking differences in the distribution of MT1 and MT2 receptors were observed. Compared with the MT1 subtype which was expressed in only a few structures, MT2 subtype receptors were identified in numerous brain structures. Targeted disruption of MT1 receptors results in the total disappearance of 2-iodomelatonin binding sites in the mouse brain, while such disappearance was not observed following disruption of MT2 receptors [START_REF] Liu | Molecular dissection of two distinct actions of melatonin on the suprachiasmatic circadian clock[END_REF]. It seems thus that in the mouse, the well described 2-iodomelatonin binding sites on tissue sections are exclusively due to binding to the MT1 receptor. The expression of MT2 has not been previously clearly detectable by in situ hybridization. Consequently, and contrary to the situation for MT1-LacZ, there are no experimental data to allow comparative validation of the present results for MT2. Nevertheless, independent validation of the results obtained with MT1-LacZ, and the fact that the MT2-LacZ knock-in was generated with an identical strategy, lead us to conclude that the LacZ staining faithfully reports MT2 expression as well. Notably, overlapping expression of the 2 receptor subtypes was observed in only very few structures (see Table 1). Moreover, even in these structures co-expression was rarely if ever present in the same individual neurons/cells, as exemplified within the SCN (Fig. 4) or retina (Fig. 11). This suggests that the two receptor subtypes are cell/neuron-specific for almost all brain structures. The difference in the distribution pattern of MT1 and MT2 receptors thus not only indicates the compartmentalization of several physiological functions by MLT, but also raises several fundamental questions about the mechanism of action of these receptors.

It has been suggested that MT1 and MT2 receptor subtypes form heterodimers in situ (58, 59,). However this heterodimerization, has only been demonstrated in in vitro studies. With the exception of the work of Baba et al [START_REF] Baba | Heteromeric MT1/MT2 melatonin receptors modulate photoreceptor function[END_REF] which used transgenic mice, full validation is lacking. An absolute minimal requirement for MT1/MT2 heterodimer formation is that they be expressed in the same cell/neuron. There are no clear data available from the literature to support such a heterodimer hypothesis. Thus, the bona fide presence of MT1/MT2 heterodimers in the brain and their physiological relevance remains a matter of debate.

It has also been reported that MLT receptors have the capacity to heterodimerize with other GPCRs [START_REF] Bodinat | Agomelatine, the first melatonergic antidepressant: Discovery, characterization and development[END_REF]. In this context the expression of MT2-LacZ in the dorsal raphe (Fig. 7) is of particular interest. The antidepressant effect of agomelatine ( 61) is considered to be due to the "synergistic" MLT agonist/5-HT2C antagonist profile of the drug. Heterodimerization of MT2 with serotonin receptor subtypes (5HT2C) might explain such data [START_REF] Kamal | Convergence of melatonin and serotonin (5-HT) signaling at MT2/5-HT2C receptor heteromers[END_REF]. The existence of functional MT2/5-HT2C heterodimers has been demonstrated but again only in vitro [START_REF] Ayoub | Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer[END_REF]. In the present work the pattern of MT2-lacZ expression in the dorsal raphe is quite similar to the distribution of serotoninergic neurons but not that of GABAergic interneurons [START_REF] Gocho | Electrophysiological and pharmacological properties of GABAergic cells in the dorsal raphe nucleus[END_REF]. Previous studies have demonstrated that 5HT2C-mediated negative feedback on serotoninergic neuron activity occurs through 5-HT2C receptors on GABAergic neurons of the dorsal raphe [START_REF] Serrats | Expression of serotonin 5-HT2C receptors in GABAergic cells of the anterior raphe nuclei[END_REF][START_REF] Quérée | Further pharmacological characterization of 5-HT(2C) receptor agonist-induced inhibition of 5-HT neuronal activity in the dorsal raphe nucleus in vivo[END_REF][START_REF] Boothman | In vivo evidence that 5-HT(2C) receptors inhibit 5-HT neuronal activity via a GABAergic mechanism[END_REF]. The GABAergic neurons then inhibit the activity of the L-type voltage dependent calcium channels responsible for the spontaneous activity of serotoninergic neurons [START_REF] Asaoka | Chronic antidepressant potentiates spontaneous activity of dorsal raphe serotonergic neurons by decreasing GABAB receptor-mediated inhibition of Ltype calcium channels[END_REF]. Consequently, the cell type mediating the melatoninergic agonistic action of agomelatine appears to be distinct from the cell type responsible for the 5-HT2C antagonist action. Therefore in the dorsal raphe, MT2/5-HT2C heterodimerization is probably not an active phenomenon. Nevertheless, it might be present in some other brain structures. According to the Allen Mouse Brain reference Atlas (mouse brain-map.org), 5-HT2C containing neurons are present in layer 4 of the cortex, a structure which displays MT2-lacZ staining. In the central thalamus (Fig. 6) and BNST (Fig. 3) the presence of both MT2 and 5-HT2C receptors might allow heterodimerization of these two receptors. Clearly, further studies are essential to address the exact role of receptor heterodimerization in the field of MLT biology.

The distinct distribution patterns of the two MLT receptor subtypes shown in the present work indicate that they may mediate different/divergent functions. Such functions will only be clearly distinguished once highly selective MT1 and MT2 agonists or antagonists are developed. Even if extensive work to develop such compounds has been performed in the last decades [START_REF] Mor | Recent advances in the development of melatonin MT1 and MT2 receptor agonists[END_REF][START_REF] Spadoni | Highly Potent and Selective MT2 Melatonin Receptor Full Agonists from Conformational Analysis of 1-Benzyl-2-acylaminomethyl-tetrahydroquinolines[END_REF][START_REF] Rivara | Therapeutic uses of melatonin and melatonin derivatives: a patent review (2012 -2014)[END_REF][START_REF] Dubocovich | Melatonin receptor antagonists that differentiate between the human Mel1a and Mel1b recombinant subtypes are used to assess the pharmacological profile of the rabbit retina ML1 presynaptic heteroreceptor[END_REF][START_REF] Zlotos | Recent progress in the development of agonists and antagonists for melatonin receptors[END_REF], most of these ligands have weak selectivity which limits their use in vivo, meaning that current knowledge of the fundamental mechanisms involved is still very poor. Until now, MLT or MLT agonists beside action for the seasonal level have been mostly demonstrated to be useful in modulating clock (SCN) activity in vivo (chronobiotic effect, [START_REF] Arendt | Melatonin as a chronobiotic Sleep[END_REF][START_REF] Pevet | The chronobiotic properties of melatonin[END_REF][START_REF] Pevet | Melatonin receptors as therapeutic targets in the suprachiasmatic nucleus[END_REF]. MLT or drugs have proven effective in improving sleep latency, sleep efficiency and subjective total sleep time in individuals with perturbed sleep/wake cycles, as well as for non-24h sleep-wake disorders in blind people [START_REF] Emens | Effect of light and melatonin and other melatonin receptor agonists on human circadian physiology[END_REF][START_REF] Skene | Circadian rhythms disorders in the blind and their treatment with melatonin[END_REF][START_REF] Lockley | Alertness, mood and performance rhythm disturbances associated with circadian sleep disorders in the blind[END_REF][START_REF] Fitzgerald | Melatonin agonists for the treatment of sleep disorders and major depression[END_REF]. Since these compounds (including authorized drugs, Agomelatine, Ramelteon, Tasimelteon and Circadin) are all non-specific MT1/MT2 agonists, they (as well as MLT) will act on both receptors when administered. The development of selective agonists to stimulate a given MLT receptor without affecting the other is thus necessary to understand the exact mechanism of action. As noted in the introduction, precise phenotyping of the neurons containing MLT receptor(s) is a prerequisite to understanding the physiological mechanisms involved. In the Siberian hamster which expresses only the MT1 subtype, receptors were observed in AVP-positive neurons of the SCN [START_REF] Song | Co-expression of melatonin (MEL1a) receptor and arginine vasopressin mRNAs in the Siberian hamster suprachiasmatic nucleus[END_REF]. We have not confirmed this observation in mice, but this may be due to species differences.

MLT does not work exclusively through action upon the SCN (chronobiotic effect). MLT has been described to affect almost all the main physiological functions of the body and to be useful in a vast array of experimental models reproducing different pathological conditions. Some of these observations are based on the use of pharmacological doses (1 mM and above) [START_REF] Reiter | The universal nature, unequal distribution and antioxidant functions of melatonin and its derivatives[END_REF], as opposed to physiological doses (below the nanomolar range) [START_REF] Dubocovich | International Union of Basic and Clinical Pharmacology. LXXV Nomenclature, classification, and pharmacology of G protein-coupled melatonin receptors[END_REF]. Looking at the large inter-species differences in the distribution of MLT binding sites in the brain [START_REF] Masson-Pevet | An attempt to correlate brain areas containing melatonin binding sites with rhythmic functions: a study in five hibernator species[END_REF][START_REF] Maywood | Regional distribution of iodomelatonin binding sites within the suprachiasmatic nucleus of the Syrian hamster and the Siberian hamster[END_REF][START_REF] Vanecek | Hypothalamic melatonin receptor sites revealed by autoradiography[END_REF] we cannot exclude that MLT acts very differently from one species to another [START_REF] Liu | MT1 and MT2 Melatonin Receptors: A Therapeutic Perspective[END_REF].

Use of a "knock-in" strategy has allowed us to detect a large number of brain sites expressing the MT2 receptor gene. Interestingly, these structures have been described as containing MLT receptors in different mammalian species, even if the techniques used (e.g. immunocytochemistry) were not optimal. The precise identification of cells/neurons in these structures will open new perspectives for the identification of links between MLT brain targets and specific MLT receptor subtypes. For example, MLT receptors are expressed in key brain regions involved in memory [START_REF] Lacoste | Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain[END_REF]. Co-expression of MT1 and MT2 in pyramidal neurons of the hippocampus (as shown by RT-PCR) (80) has been reported. Immunohistochemical analysis showed that MT1 and MT2 expression patterns partially overlap, with MT1 being expressed predominantly in the CA1 hippocampal subfield and MT2 in the CA3/CA4 subfields [START_REF] Lacoste | Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain[END_REF]. Our present results allow unambiguous identification of MT2 expressing cells in the hippocampus, and apparent absence of MT1, and should facilitate experimental studies on the mechanism of MLT action on memory [START_REF] Savaskan | Reduced hippocampal MT2 melatonin receptor expression in Alzheimer's disease[END_REF][START_REF] Wang | Melatonin inhibits hippocampal long-term potentiation[END_REF][START_REF] Collins | Melatonin blocks the induction of long-term potentiation in an N-methyl-D-aspartate independent manner[END_REF][START_REF] El-Sherif | The actions of a charged melatonin receptor ligand, TMEPI, and an irreversible MT2 receptor agonist, BMNEP, on mouse hippocampal evoked potentials in vitro[END_REF][START_REF] Larson | Impaired hippocampal long-term potentiation in melatonin MT2 receptor-deficient mice[END_REF][START_REF] Jilg | Melatonin modulates daytime-dependent synaptic plasticity and learning efficiency[END_REF].

The moderate MLT receptor immunoreactivity in the rat dorsal raphe nucleus suggested the possible involvement of MT1 receptors in the pathogenesis of depression [START_REF] Lacoste | Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain[END_REF]. This is supported by a study which demonstrated behavioral changes mimicking the symptoms of melancholic depression in MLT receptor knockout mice [START_REF] Comai | Melancholic-Like behaviors and circadian neurobiological abnormalities in melatonin MT1 receptor knockout mice[END_REF]. There is evidence of MLT imposing tonic inhibitory control over a subpopulation of serotonin-containing neurons in the dorsal raphe [START_REF] Comai | Melancholic-Like behaviors and circadian neurobiological abnormalities in melatonin MT1 receptor knockout mice[END_REF]. Our study demonstrates a link between MLT and serotonin neurotransmission through MT2 receptors.

Finally the results obtained in the retina deserve some comments. Functional MLT receptors were first characterized in the retina [START_REF] Dubocovich | Melatonin is a potent modulator of dopamine release in the retina[END_REF], and immunohistochemical and in situ studies in rodent and human retina [START_REF] Fujieda | Expression of mt1 melatonin receptor in rat retina: evidence for multiple cell targets for melatonin[END_REF][START_REF] Scher | AII amacrine cells express the MT1 melatonin receptor in human and macaque retina[END_REF][START_REF] Savaskan | The MT2 melatonin receptor subtype is present in human retina and decreases in Alzheimer's disease[END_REF] have shown that both receptors are detected in virtually every cell [START_REF] Yang | Melatonin inhibits tetraethylammonium-sensitive potassium channels of rod ON type bipolar cells via MT2 receptors in rat retina[END_REF](94)(95). This observation was used as strong evidence in favor of MLT receptor heterodimerization (96). The hypothetical physiological concept is that MLT secreted from photoreceptors (or from the general circulation) binds to MLT receptors on amacrine cells thereby inhibiting dopamine release, and conversely that dopamine binding to D2 receptors on photoreceptor cells suppresses MLT synthesis (97). MT1 receptors are detected in the photoreceptor layer, and both MT1 and MT2 receptors are present in scattered but distinct amacrine and ganglion cells. Our present data argue against heterodimerization of the two receptors, at least in mouse photoreceptors. Again, we will have to await further experimental work using specific agonists or antagonists in order to determine the mechanism of action of MLT within the retina. This line of reasoning can be extended to nearly all brain structures (see above and in the figures) identified in the present work.

In conclusion, the present work using a "knock-in" strategy has allowed us to determine fundamental differences in the distribution of MT1 and MT2 receptors in the mouse brain. The abundance of MT2 receptors was observed to be much greater than previously thought. Clearly, discovery of new pharmaco logical tools selective for each receptor subtype is critical for a clearer understanding of MLT action, and in order to develop innovative therapeutic approaches for pathologies such as diabetes, cancer, depression, memory failure, etc. regions of these structures (A). MT2-LacZ expression in the mediobasal hypothalamus is much more widespread than MT1-LacZ expression (B), with the notable exception of the pars tuberalis, which is completely devoid of MT2-LacZ staining (double arrow heads). As with all structures where both MT1-LacZ and MT2-LacZ expression can be detected, MT1-LacZ expression is present in a more reduced and discrete subset of cells than MT2-LacZ, as seen here for both the arcuate nucleus and the medial tuberal nucleus. MT2-LacZ expression is also detected in some cells of the ependymal wall, which are identified as tanycytes (arrowheads in C) in LacZ immunohistochemical staining by the processes (C). Bar in B = 200 µm for A/B Bar in C = 100 µm Figure 6. MT1-LacZ and MT2-LacZ expression in the thalamus. Both MT1-LacZ and MT2-LacZ expression can be seen in various nuclei of the central and dorsal thalamus, as well as the habenula (*). In the paraventricular nucleus of the thalamus, MT1-LacZ expression is particularly high (black arrowhead), while MT2-LacZ expression is reduced to a few cells. (open arrowhead). In almost all other structures, MT1-LacZ expression is reduced to a much smaller subset of cells than MT2-LacZ expression. In the habenula (*), almost no MT1-LacZ expression can be seen. Bar = 250 µm (C) In the central part of the SCN, MT1-LacZ (green) can also be seen in some neurons expressing the mRNA for gastrin releasing peptide (red, non-radioactive in situ hybridization). After in situ hybridization, the LacZ immunostaining can only be seen in the dot like structures (arrowheads). (D) In the central part of the SCN, calretinin (red) can be seen in a number of MT1-LacZ (green) expressing cells (arrowheads). However, there are also cells expressing only either calretinin (red) or MT1-LacZ (green). Bar in D = 50 µm for A, B and D Bar in C = 20 µm (A) Weak MT1-LacZ enzyme histochemical stain can be seen in the photoreceptor layer (PR). Much stronger enzyme activity is detected in some amacrine cells (fleche SVP) in the inner nuclear layer (INL) and some ganglion cells.(GCL). (B) MT2-LacZ expression is absent from the photoreceptor layer, but present in some amacrine cells and some ganglion cells. (C) MT1-LacZ expression in amacrine cells and ganglion cells. The cell bodies of the amacrine cells expressing MT1-LacZ are located within the inner nuclear layer (INL) away from the boundary of the inner nuclear layer with the inner plexiform layer (IPL). (D) The cell bodies MT2-LacZ positive amacrine cells are mostly located at the interface between the inner nuclear and plexiform layers. (E) In the MT1-LacZ amacrine cells, the LacZ immunostaining extends into a neurite ramifying in the upper strata of the inner plexiform layer. (F) Double immunostaining for MT1-LacZ (red) and calretinin (green) shows that the MT1-LacZ amacrine cells are calretinin negative, and that their neurite ramifies in the upper calretinin layer between strata 1 and 2 (G) Most MT1-LacZ (green) amacrine cells are located higher than the dopaminergic amacrine cells (red, tyrosine hydroxylase). (H) Only one MT1-LacZ cell (green) colocalized with tyrosine hydroxylase (red) identified Bar in A = 50 µm for A and C Bar in B = 20 µm for all other micrographs PR photoreceptor layer, ONI outer nuclear layer, OPL outer plexiform layer, INL, Inner nuclear layer, IPL, inner plexiform layer, GCL, ganglion cell layer.

  SM, Godson C, Mahle CD, Weaver DR, Slaugenhaupt SA, Gusella JF (1995) Molecular characterization of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor. Proc Natl Acad Sci U S A. 92:8734-8738. 95. Pack W, Hill DD, Wong KY (2015) Melatonin modulates M4-type ganglion-cell photoreceptors. Neuroscience 303:178-188. 96. Piano I, Baba K, Gargini C, Tosini G 2018) Heteromeric MT 1 /MT 2 melatonin receptors modulate the scotopic electroretinogram via PKCζ in mice. Exp Eye Res 177:50-54. 97. Fujieda H, Scher J, Hamadanizadeh SA, Wankiewicz E, Pang SF, Brown GM. (2000) Dopaminergic and GABAergic amacrine cells are direct targets of melatonin: immunocytochemical study of mt1 melatonin receptor in guinea pig retina. 17:63-70.

Figure 1 .

 1 Figure 1. Targeting strategy for the generation of Mtnr1a and Mtnr1b LacZ knock in mice: the wild-type allele is representative of Mtnr1a gene but exon/intron structure and strategy are similar for Mtnr1b. A reporter-selection cassette containing a a) LacZ open reading frame, poly-A site and b) a floxed human Ubiquitin promoter, Neomycine resistance gene, poly-A site sequence, was inserted between the ATG and the stop codon region of the target gene by using a BAC strategy. Mice carrying this targeted allele were generated by ES cellbased/germline transmission. The final knock in mice were derived from the preceding ones by breeding with CRE-deleter mice to remove the selection gene and final segregation of the Cre gene.

Figure 2 .Figure 3 .

 23 Figure 2. MT1-LacZ expression in the suprachiasmatic nucleus detected by LacZ enzyme histochemistry (A, C) or by LacZ immunohistochemistry (B,D). The LacZ enzyme activity is visualized as strongly stained dots, which most probably are lysosomal accumulations of the transgenic LacZ enzyme (A,C). Immunohistochemical detection of the LacZ protein visualizes LacZ in these same dot-like structures, as well as in the nucleus and cytoplasm of most SCN neurons (B,D). The dot-like structures are bigger in enzyme histochemistry than in immunohistochemistry probably because of the higher diffusion of the X-Gal reaction product compared to the peroxidase diaminobenzidine precipitate. MT1-LacZ expressing neurons are unevenly distributed in the SCN, with the highest density of MT1-LacZ expressing cells located in the medio-ventral quadrant of the SCN. Bar in A = 100 µm for A/C, Bar = 50 µm for B/D

Figure 4 .Figure 5 .

 45 Figure 4. MT1-LacZ (A) and MT2-LacZ (B) expression in the SCN. MT1-LacZ expression in the SCN is higher than MT2-LacZ expression and delineates the boundaries of the SCN. Furthermore, MT2-LacZ expression also is more diffuse in the anterior hypothalamic area. Bar = 50 µm

Figure 7 .Figure 8 .

 78 Figure 7. MT2-LacZ expression in the dorsal raphe nucleus by LacZ enzyme histochemistry (A, B) or immunohistochemistry (C). MT2-LacZ expression can be seen in cells delineating an inverted triangular area in the most central part of the dorsal raphe, similar to that observed for serotonergic neurons. Bar = 100 µm

Figure 9 .Figure 10 .Figure 11 .

 91011 Figure 9. Colocalization of MT2-LacZ immunohistochemistry with neuropeptide markers in the paraventricular nucleus of the hypothalamus. (A) Double immunostaining for vasopressin-neurophysin2 (red) and MT2-LacZ (green) show no colocalisation. MT2-LacZ expression is present in cells located in the dorsal part of the medial parvicellular subdivision of the paraventricular nucleus. (B) Double staining for MT2-LacZ (green) and the mRNA of corticotropin releasing hormone (CRH, red) shows that the MT2-LacZ neurons are mostly CRH neurons, although some MT2-

Figure 1 .

 1 Figure 1. Targeting strategy for the generation of Mtnr1a and Mtnr1b LacZ knock in mice: the wild-type allele is representative of Mtnr1a gene but exon/intron structure and strategy are similar for Mtnr1b. A reporter-selection cassette containing a a) LacZ open reading frame, poly-A site and b) a floxed human Ubiquitin promoter, Neomycine resistance gene, poly-A site sequence, was inserted between the ATG and the stop codon region of the target gene by using a BAC strategy. Mice carrying this targeted allele were generated by ES cell-based/germline transmission. The final knock in mice were derived from the preceding ones by breeding with CRE-deleter mice to remove the selection gene and final segregation of the Cre gene. 254x190mm (96 x 96 DPI)

Figure 2 .Figure 3 .

 23 Figure 2. MT1-LacZ expression in the suprachiasmatic nucleus detected by LacZ enzyme histochemistry (A, C) or by LacZ immunohistochemistry (B,D). The LacZ enzyme activity is visualized as strongly stained dots, which most probably are lysosomal accumulations of the transgenic LacZ enzyme (A,C). Immunohistochemical detection of the LacZ protein visualizes LacZ in these same dot-like structures, as well as in the nucleus and cytoplasm of most SCN neurons (B,D). The dot-like structures are bigger in enzyme histochemistry than in immunohistochemistry probably because of the higher diffusion of the X-Gal reaction product compared to the peroxidase diaminobenzidine precipitate. MT1-LacZ expressing neurons are unevenly distributed in the SCN, with the highest density of MT1-LacZ expressing cells located in the medio-ventral quadrant of the SCN. Bar in A = 100 µm for A/C, Bar = 50 µm for B/D

Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .Figure 9 .

 456789 Figure 4. MT1-LacZ (A) and MT2-LacZ (B) expression in the SCN. MT1-LacZ expression in the SCN is higher than MT2-LacZ expression and delineates the boundaries of the SCN. Furthermore, MT2-LacZ expression also is more diffuse in the anterior hypothalamic area. Bar = 50 µm

Figure 10 .Figure 11 .

 1011 Figure 10. MT2-LacZ expression in the hippocampus. (A) Particularly dense MT2-LacZ dot-like immunostaining is seen in the CA2 region of the hippocampus (rectangle B). In the hilus of the dentate gyrus, (rectangle C) some lightly stained neurons are present. The strongest staining for MT2-LacZ however is detected in small neurons located in the outer molecular layer of the dentate gyrus (rectangle D). The rectangles show the areas selected for figures 9B, C and D. (B) Only a few MT2-LacZ positive neurons in the CA2 area co localize with GAD1 or GAD2. (C) In the hilus of the dentate gyrus, GAD1 or GAD2 can be detected in MT2-LacZ positive neurons (arrowheads). (D) The small neurons located in the outer molecular layer of the dentate gyrus display a particularly strong immunostaining for LacZ, extending into their neurites. (E) The MT2-LacZ positive neurons in the outer molecular layer mostly did not express either GAD1 or GAD2. Bar in A = 250 µm

Table 1 :

 1 Mapping of MT1-LacZ and MT2-LacZ in the mouse brain. Abbreviations of the structures according to Franklin & Paxinos (2007) The Mouse Brain in Stereotaxic Coordinates Third Edition. Elsevier.

	~scattered isolated lightly labelled cells
	±	scattered lightly labelled cells
	+	scattered cells not delineating the structure
	++	labelled cells delineating the structure
	+++ high density of labelled cells
	++++ very high density of labelled cells completely filling the structure
		F
		o r
		P
		e e r
		R e v
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Acknowledgements

The authors are grateful to the Chronobiotron platform, UMS3415, CNRS, University of Strasbourg) for help with animals management. This work was supported by the Centre National de la Recherche Scientifique, University of Strasbourg (EC, DH, MPF, PK, PP and S Lapmaneya) and the Institut de Recherches Internationales Servier (CSK and BGL). The authors have no potential conflict of interest with respect to the research, authorship, and/or publication of this article. Recombinant ES cells and the Mtnr1b +/ki mice were generated in the Knock Out Mouse Project (KOMP). The Mtnr1a +/ki strain was generated in the Institut Clinique de la Souris (Illkirch, France).