5 ( J J L a b c a A R R A K S S C P C S T f C o c t n e c i g l [ s [ i [ p U T 0 d Neuroscience Letters 488 (2011) 6–10 Contents lists available at ScienceDirect Neuroscience Letters journa l homepage: www.e lsev ier .com/ locate /neule t -HT1B receptor in the suprachiasmatic nucleus of the common marmoset Callithrix jacchus) eferson S. Cavalcantea,∗, André L.B. de Pontesa, Rovena C.G.J. Engelbertha, udney C. Cavalcantea, Expedito S. Nascimento Jr. a, Janaína S. Bordaa, uciana Pinatob, Miriam S.M.O. Costaa, Claudio A.B. de Toledoc Laboratory of Chronobiology, Biosciences Center, Federal University of Rio Grande do Norte, 59072-970 Natal, RN, Brazil Speech-Language and Hearing Therapy Department, São Paulo State University, 17525-900, Marilia, SP, Brazil Laboratory of Neurosciences, Neuroscience Research Nucleus, City University of São Paulo, 03071-000 São Paulo, SP, Brazil r t i c l e i n f o rticle history: eceived 30 April 2010 eceived in revised form 2 October 2010 ccepted 28 October 2010 a b s t r a c t Serotonin (5-HT) is involved in the fine adjustments at several brain centers including the core of the mammal circadian timing system (CTS) and the hypothalamic suprachiasmatic nucleus (SCN). The SCN receives massive serotonergic projections from the midbrain raphe nuclei, whose inputs are described in rats as ramifying at its ventral portion overlapping the retinohypothalamic and geniculohypothala- eywords: uprachiasmatic nucleus erotonin receptor allithrix jacchus rimate mic fibers. In the SCN, the 5-HT actions are reported as being primarily mediated by the 5-HT1 type receptor with noted emphasis for 5-HT1B subtype, supposedly modulating the retinal input in a presy- naptic way. In this study in a New World primate species, the common marmoset (Callithrix jacchus), we showed the 5-HT1B receptor distribution at the dorsal SCN concurrent with a distinctive location of 5-HT- immunoreactive fibers. This finding addresses to a new discussion on the regulation and synchronization in rec ircadian timing system erotonin of the circadian rhythms he circadian timing system (CTS) is a neural network responsible or the generation and modulation of the circadian rhythms. The TS of mammals comprises an encephalic circuitry, in the center f which, the hypothalamic suprachiasmatic nucleus (SCN) is con- eived as the main circadian pacemaker. Along the last decades, he SCN has been investigated in respect to its major hodological, eurochemical, and molecular features [34,37]. The rhythms generated by the SCN are synchronized by nvironmental cues, of which the light/dark cycle is the major syn- hronizing agent. Three main pathways reach the SCN influencing ts activity: (1) the retinohypothalamic tract (RHT), from the retinal lutamatergic ganglion cells [7,17,20,26]; (2) the geniculohypotha- amic tract (GHT) [18], formed by neuropeptide Y-positive fibers 14] from the thalamic intergeniculate leaflet [15]; and (3) the erotonergic midbrain projections from the median raphe nucleus 16,22,25]. In rats, all of these pathways are described as ram- fying in an overlapping way at the ventral portion of the SCN 16,26,25,35], even though that in a previous study of a New World rimate the common marmoset (Callithrix jacchus), serotonergic ∗ Corresponding author at: Department of Physiology, Biosciences Center, Federal niversity of Rio Grande do Norte, 59072-970 Natal, RN, Brazil. el.: +55 84 3215 3409; fax: +55 84 3211 9206. E-mail address: jefsc@uol.com.br (J.S. Cavalcante). 304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. oi:10.1016/j.neulet.2010.10.070 ent primates. © 2010 Elsevier Ireland Ltd. All rights reserved. terminal plexus was described as reaching the most dorsal portion of the SCN in a complementary manner to the ventral RHT and GHT projections [5,31]. The serotonergic pathway is functionally implicated in both photic and non-photic modulation of the SCN [13,23,27,25,28,29]. The 5-HT actions are dependent on several serotonergic receptor subtypes, which can be pre or post-synaptically positioned. To date, the main subtypes of 5-HT receptors found in the SCN, character- ized by biochemical and pharmacological approaches, belong to the 5-HT1 family [26]. Also, in different species the subtype 5-HT1B seems to prevail [12,30,33]. 5-HT1B receptors can be found in the axon terminals of neurons (acting as a presynaptic modulator) or in the postsynaptic cellular membrane [21]. The presence of 5-HT fibers in the dorsal SCN of the marmoset [31] motivated us to search the presence of serotonergic receptor subtypes in the same species, so that, the aim of this study was to identify and map the 5-HT1B receptor distribution comparing with serotonergic and retinal innervations. Four adult male marmosets (257–378 g) from the Primatology Center of the Federal University of Rio Grande do Norte (IBAMA register 1/24/92/0039-0), Natal, Rio Grande do Norte, Brazil, were used in this study. The animals were housed under natural lighting, temperature, and humidity conditions, with food and water freely available. The maintenance and minimal use of the experimental animals followed the guidelines of the Brazilian Society of Neuro- dx.doi.org/10.1016/j.neulet.2010.10.070 http://www.sciencedirect.com/science/journal/03043940 http://www.elsevier.com/locate/neulet mailto:jefsc@uol.com.br dx.doi.org/10.1016/j.neulet.2010.10.070 oscien s C t t s h i o a s c r t t s a t a w o p b f t s S r s a c u T t w H r b r i a i o P q E t d H e C a L c D a 1 n t ( u c d i J.S. Cavalcante et al. / Neur cience and Behavior under recommendations of the Guide for the are and Use of Laboratory Animals in Research (in accordance with he National Institutes of Health, NIH, USA). The marmosets were deeply anesthetized with sodium thiopen- al (Cristália, São Paulo, SP, Brazil, 40 mg/kg, i.p.), placed on a urgical table and received a topical application of tetracaine ydrochloride in the cornea followed by a unilateral intraocular njection of cholera toxin subunit B (CTb) (List Biological Lab- ratories, Inc., Campbell, CA, USA). A total of 80 �l of 1 mg/ml queous CTb solution containing 10% dimethylsulfoxide was pres- ure injected into the vitreous humor through a 30-gauge needle atheter attached to a micropump, introducing the solution at a ate of 1 �l/min. To minimize reflux and spread of the tracer to he extraocular muscles and to avoid postoperative local infection, he ocular surface was continuously cleansed with saline during urgery. The ocular surface was then rewashed with saline and n antibiotic ointment (Dexafenicol, Alergon, Brazil) was applied opically. After 5–7 post-injection days, the marmosets were re- nesthetized with sodium thiopental and transcardially perfused ith 500 ml of phosphate-buffered saline, pH 7.4, containing 500 IU f heparine (Parinex, Hipolabor, Brazil) followed by 700 ml of 4% araformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. The rains were removed from the skull, postfixed for 2–4 h and trans- erred to a solution containing 30% sucrose in PB. Brain blocks from a point of a few millimeters anterior to he optic chiasm through the midbrain were frozen and serially ectioned in the coronal plane (30 �m) on a sliding microtome. ections were sequentially collected and kept in PB in six sepa- ate compartments. To establish the limits of the SCN, one of the ix series of sections from each animal was stained with thionin nd another was submitted to immunohistochemical reaction to albindin (CB), a calcium-binding protein, which is reported to be a seful marker for identifying the SCN boundaries in this species [6]. hree other series were used for the immunohistochemical detec- ion of the CTb, 5-HT and 5-HT1B receptor. The remaining series ere used to perform double labeling experiments combining 5- T1B receptor and CB labels to analyze the arrangement of this eceptor in relation to the SCN cells and nuclear architecture. For the immunohistochemical detection of CTb, free floating rain sections of the injected animal were incubated for 18 h at oom temperature (ca. 24 ◦C) using goat anti-CTb IgG (List Biolog- cal Labs, Inc., Campbell, CA, USA), diluted at 1:5000, as primary ntibody and 2% of normal donkey serum in 0.4% Triton X-100 n PB. The sections were then incubated with a biotinylated sec- ndary antibody (donkey anti-goat IgG, Jackson Labs, Westgrove, A, USA) diluted at 1:1000 for 90 min. The sections were subse- uently incubated with an avidin–biotin–peroxidase solution (ABC lite kit, Vector Labs., Burlingame, CA, USA) for 90 min. They were hen reacted for peroxidase activity in a solution of diaminobenzi- ine tetrahydrochloride (DAB, Sigma, St Louis, MO, USA) and 0.03% 2O2 in PB. The sections were washed with PB (5 × 5 min) between ach step and at the end of the procedure. For detection of the B, 5-HT and 5-HT1B receptor, the same general procedure was dopted using as primary antibodies a mouse anti-CB (Sigma, St ouis, 1:5000), a rabbit anti-5HT (Immunostar, Hudson, WI, USA, at # 20079, 1:5000), a guinea-pig anti-5HT1B (PharMinger, San iego, CA, USA, cat # 550470, 1:500) and the respective secondary ntibodies all raising in goat (Jackson Labs, Westgrove, PA, USA, :1000). All of the incubations with primary antibody included 2% ormal goat serum in 0.4% Triton X-100 in PB. Specificity tests and he controls for the 5-HT label can be found at the company site www.immunostar.com). The anti-5-HT1B was made in guinea pig sing a synthetic peptide conjugated with bovine thyroglobulin orresponding to amino acids 273–287 with homologue correspon- ence in rodents, canines and primates. As for control procedures, n some sections all the antibodies were used except the primary ce Letters 488 (2011) 6–10 7 antibody, which was substituted by normal serum or by incubation of the secondary antibody only. For the double labeling experiments, the tissue was simultane- ously incubated with an antibody guinea pig anti-5-HT1B receptor (PharMinger, San Diego, CA, USA) and a mouse-anti-CB (Sigma Chemicals, St Louis, MO, USA), both at a dilution of 1:250 during 24 h. The labeling was developed using secondary anti-guinea pig fluorescein-labeled and anti-mouse rhodamine-labeled antibodies all made in donkey (Sigma, St Louis, MO, USA) at a 1:100 dilution in PB with 0.4% Triton X-100. Proper immunofluorescence con- trols were performed by the omission of the primary and produced lack of label. Material was examined under bright-field illumina- tion in an Olympus microscope for the single-label experiments and in a Nikon E-800 epifluorescence microscope for the double- label experiments. The images were captured using a CCD camera (Optronics Magnafire, Goleta, CA, USA) and the composite images were mounted with the aid of Adobe Photoshop (Adobe Software). The distribution of labeled elements and staining intensity were subjectively evaluated. The SCN of the marmoset displays a triangular shape located over the optic chiasm bilateral to the third ventricle as evidenced in Nissl-stained coronal sections (Fig. 1A). The SCN CB-containing neurons exhibit a clear label with visible and well delimited cell bodies containing some apparent neuropil contrasting with adja- cent hypothalamic areas (Fig. 1B). Retinal CTb-immunoreactive fibers in the common marmoset SCN were visualized in an unam- biguous placement at its entire ventral portion with a noticeable enlargement in the middle portion (Fig. 1C). This pattern appears to be consistent from rostral to caudal levels showing a slight decrease in intensity as the SCN extends toward the caudal direc- tion. Fine 5-HT-immunoreactive fibers/terminals were identified in an apparently scattered distribution in the SCN of the mar- moset (Fig. 1D). However, a more detailed observation shows that these 5-HT positive fibers exhibit a preferential distribution medi- ally around the ventricle wall at the upper dorsal limit of the SCN (Fig. 1D). In general, the disposition of the CTb-containing fibers overlap the 5-HT containing fibers on the ventral portion of the SCN, but the site of CTb exuberance coincides with the absence of 5-HT in an apparent complementary position in relation to RHT. As for CTb, the 5-HT immunopattern appears to be constant from rostral to caudal levels showing a slight decrease in intensity as the SCN moves toward the caudal position. 5-HT1B immunoreactive (5-HT1B-IR) neurons show a broad and apparent and homogeneous distribution in the hypothalamus of the marmoset (Fig. 2A). In the entire rostro-caudal SCN the distri- bution for the 5-HT1B-IR was more evident dorsally concentrated in the medial contour near the ventricle wall, corresponding to higher concentration of 5-HT innervation and letting an immunoreactive free zone, which corresponds to the location of the bulk of RHT fibers (Fig. 2B and C). Most of the CB-positive neurons of the dorsal district appear to be encircled by the dotted 5-HT1B label (Fig. 2D and E). The 5-HT1B immunoreactivity pattern in the SCN differs from that of midbrain, which resembles postsynaptic neurons or autore- ceptors since they appear to immunolabel the entire cell in the dorsal raphe nucleus used now as a positive control label (Fig. 3A and B). No label was observed in the control experiments (Fig. 3C and D). For all parameters evaluated the results were similar in all cases with only minor variations. Evidences implicating 5-HT as a key neurotransmitter modu- lating the effects of light on circadian rhythmicity is recurrent. For example, the depletion of serotonergic input to the SCN of rodents provokes a change of the circadian rhythms with an elongation of the activity phase and disruption in constant light [22] and it was proposed that this effect was mainly mediated by the 5-HT1B receptor [30]. This subtype was observed to be associated with the http://www.immunostar.com/ 8 J.S. Cavalcante et al. / Neuroscience Letters 488 (2011) 6–10 Fig. 1. Digital images of coronal sections at hypothalamic level of the marmoset brain showing (A) the triangular shaped SCN at Nissl stained sections; (B) CB-immunostained neurons filling all sectional area of the SCN; (C) distribution of CTb-retinal projections in the ventral portion (arrows) of the SCN; (D) 5-HT positive fibers ramifying in the medial and dorsal SCN, depriving the ventral area (arrows). The upper image in B (B′) is a detail of the CB-positive labeling in the SCN. Note the rounded nature of the small neurons (average of 10 �m). 3v, third ventricle; oc, optic chiasm. Scale bar: 180 �m in A, 100 �m in B–D, 50 �m in B′ . Fig. 2. Digital images of coronal sections of the marmoset brain at the SCN level showing (A) 5-HT1B-immunoexpression in all anterior hypothalamus, including the SCN; (B) higher magnification of A showing 5-HT1B-positive structures with the dorsal portion containing the richest labeling (arrows); (C) higher magnification of the region delimited by arrows in B showing the typical dot-like label pattern of 5-HT1B; (D) the merged exposure of CB-containing neurons visualized by TRITC-conjugated secondary antiserum (red) and 5-HT1B-positive structures detected by FITC-conjugated secondary anti-serum (green); (E) higher magnification of the neurons indicated by arrows in D. APL, preoptic lateral area; APM, preoptic medial area. Scale bar: 200 �m in A, 100 �m in B, 70 �m in C and D, 15 �m in E. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) J.S. Cavalcante et al. / Neuroscience Letters 488 (2011) 6–10 9 F l in DR ( ). AM m p t i m r T v l a s o c t d o u a t p p 5 i a g m r A t m c p i p a t ig. 3. Digital images of coronal sections of the marmoset brain showing 5-HT1B labe B). Absence of labeling was evidenced in control experiments at DRN (C) and SCN (D embrane of retinal terminals in the SCN suggesting that by this resynaptic mechanism 5-HT can modify the response of the SCN o light [30,29]. More recently, 5-HT1B binding sites were massively dentified in nonretinal terminals in the SCN, and combined phar- acological and electrophysiological assays indicated that these eceptors could be actually located on GABAergic terminals [3]. hus, the 5-HT1B influence on SCN neurons activity might be pro- ided by inhibition of a GABA release in an intrinsic circuitry, most ikely in the vicinity of afferent retinal fibers. Currently it is assumed that there be a correlation between 5-HT nd RHT innervations with 5-HT1B in the ventral SCN, but in this tudy we identified the occurrence of 5-HT1B receptors in the SCN f the common marmoset more concentrated in the dorsal portion oncurrent with the predominant location of 5-HT immunoreactive erminals. Our present experimental approach does not allow us to etermine the precise elemental location of 5-HT1B receptors, but ur data indicate that they could be acting as a presynaptic mod- lator like in rodents. Thus, 5-HT1B immunoreactivity seemingly round the cell body together with the lack of cytosolic label and he absence of membrane contour strongly suggest its presynaptic osition. Furthermore, the dot-like appearance and the topogra- hy of the 5-HT1B immunoreactivity coincide with the presence of -HT terminal fibers. We observed that, although scarce, 5-HT1B- mmunoreactivity was also detected in the marmoset ventral SCN, nd we speculate that they are connected with putative GABAer- ic ventral interneurons as reported in rodents [3]. However, it ust be pointed out that the predominant labeling of the non- etinorecipient dorsal SCN be a remarkable difference in our results. t this time we are unable to provide a definitive explanation for his finding, however, it is possible that in the marmoset the 5-HT odulates the SCN in an alternative way, in that we cannot dis- ard a non-photic role. Pharmacological manipulation suggests the articipation of the 5-HT1A and 7 subtypes in the phase advance n behavioral circadian rhythms in hamster [8,24] that was inde- endent of light inputs. This also reinforces the participation of dditional subtypes conveying the 5-HT transmission [2]. In addition to the median raphe nucleus (MRN)–SCN pathway, he dorsal raphe nucleus (DRN) projects to the IGL (DRN-IGL path- N neurons (A) delimiting the neuronal body better evidenced at high magnification e, mesencephalic aqueduct. Scale bar: 200 �m in A and C, 30 �m in B, 100 �m in D. way) and these two pathways appear to exhibit different functional attributes in the mammal CTS. 5-HT fibers from the MRN facilitating the synchronization of the animal to the light–dark cycle and the disruption of the DRN-IGL do not influence this response [22]. The reception of photic information by the raphe mesencephalic com- plex through the retinal projections is well characterized and fully recognized in some species [9–11] attributing to this system a piv- otal role for 5-HT in circadian rhythmicity by indirect modulation in response to light [23,27,28]. Furthermore, some data show that the function of 5-HT in circadian rhythmicity goes beyond the light response. This includes participation in sleep–wake cycle control and food synchronization suggesting that the serotonergic system takes part in the general organization of the photic-independent mechanism [13,29]. The well-known rhythmic discharge rate of the raphe neurons may have a tonic influence on the CTS, besides the light influence. We also cannot visualize if the double shaped 5-HT innervation in the marmoset SCN (present study) comes from dis- tinct raphe nuclei. Whereas we cannot provide a prompt response for that, it is already known that 5-HT receptors immunoreactivity show a circadian rhythm in its expression in different rat brain areas [1], as well as the 5-HT levels as a neurotransmitter, verified both in the raphe complex [32] and in the innervation over the SCN [4]. The SCN also exhibits an inherent capability to respond to the light–dark cycle [36], which reinforces the power of intrinsic SCN circuitries in order to maintain and adjust endogenous rhythmicity according to environmental clues, not always exclusively light-dependent. Lesions in the MRN and DRN or the use of serotonergic blockers produce similar alterations in their targets, such as a loss of inhibi- tion and increased discharge of SCN neurons [3,30]. In agreement with this, 5-HT1B knockout mice display altered photic synchro- nization and a reduced but not abolished light-induced phase shift [33]. Until now, the occurrence of 5-HT1B has been more linked to ventral SCN serotonergic innervation, and in our data we show that in the marmoset this receptor appears to be preferentially dis- tributed on CB-enriched neurons in the medio-dorsal portion of the SCN. 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