Intrinsic organization of the suprachiasmatic nucleus in the capuchin monkey

brain research 1543 (2014) 65–72

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Research Report

Intrinsic organization of the suprachiasmatic nucleus in the capuchin monkey V.A. Rochaa, R. Fraza˜oa, L.M.G. Camposa, P. Melloa, J. Donato Jr.c, R.J. Cruz-Rizzolod, M.I. Nogueiraa,1, L. Pinatob,n,1 a

Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, SP, Brazil Department of Speech Language and Hearing Therapy, São Paulo State University, Marília, SP, Brazil c Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, SP, Brazil d Department of Basic Sciences, São Paulo State University Araçatuba, SP, Brazil b

art i cle i nfo

ab st rac t

Article history:

The suprachiasmatic nucleus (SCN), which is the main circadian biological clock in mammals, is

Accepted 20 October 2013

composed of multiple cells that function individually as independent oscillators to express the

Available online 24 October 2013

self-sustained mRNA and protein rhythms of the so-called clock genes. Knowledge regarding

Keywords:

the presence and localization of the proteins and neuroactive substances of the SCN are

Biological rhythms

essential for understanding this nucleus and for its successful manipulation. Although there

Diurnal monkey

have been advances in the investigation of the intrinsic organization of the SCN in rodents, little

Neuroanatomy

information is available in diurnal species, especially in primates. This study, which explores

Per2

the pattern of expression and localization of PER2 protein in the SCN of capuchin monkey, evaluates aspects of the circadian system that are common to both primates and rodents. Here, we showed that PER2 protein immunoreactivity is higher during the light phase. Additionally, the complex organization of cells that express vasopressin, vasoactive intestinal polypeptide, neuron-specific nuclear protein, calbindin and calretinin in the SCN, as demonstrated by their immunoreactivity, reveals an intricate network that may be related to the similarities and differences reported between rodents and primates in the literature. & 2013 Elsevier B.V. All rights reserved.

1.

Introduction

In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the master circadian pacemaker, as it controls daily variations in physiology and behavior (Moore and Eichler, 1972; Welsh et al., 1995). As well as other cells that show rhythmicity, SCN cells have a feedback loop mechanism in which proteins of the so-called “clock genes” downregulate the synthesis of their own n

messenger RNA (mRNA) (Aton and Herzog, 2005; Bass and Takahashi, 2010; Lowrey and Takahashi, 2011; Reppert and Weaver, 2001). Each loop takes approximately 24 h to be completed, which results in circadian oscillations of mRNA and protein levels (Darlington et al., 1998; Shearman et al., 2000). At the center of this auto-regulatory mechanism, the aryl hydrocarbon receptor nuclear translocator-like (ARNTL) (also known as BMAL1) protein and the circadian locomotor output cycles kaput (CLOCK) protein dimerize to activate the

Correspondence to: Department of Speech Language and Hearing Therapy, São Paulo State University, Av. Vicente Ferreira, 1278–zip code: 17515-901, Marília, SP, Brazil, fax: þ55 11 30917366. E-mail address: [email protected] (L. Pinato). 1 These authors are joint senior authors. 0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.10.037

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transcription of the period (Per) and cryptochrome (Cry) genes. The PER and CRY proteins form a repressive complex that accumulates to inhibit the trans-activating activity of the BMAL-CLOCK complex (Lowrey and Takahashi, 2011; Reppert and Weaver, 2002). To entrain the cells of the master clock to the light–dark cycle, the retinal ganglion cells project to the SCN through the retinohypothalamic tract (RHT), which conveys photic information to the nucleus (Morin and Allen, 2006). Therefore, the SCN perceives light information from the environment and subsequently synchronizes all of the peripheral clocks present in different oscillatory tissues (Yamazaki et al., 2000; Yamazaki et al., 2002) to generate coherent systemic rhythms for the entire organism (Liu et al., 2007). In several mammalian species, mainly rodents, the SCN was demonstrated to be anatomically and functionally divided into two distinct subdivisions: the ventrolateral region (vlSCN) and the dorsomedial region (dmSCN), which are also called the core and the shell of the SCN, respectively (van den Pol, 1980). The vlSCN and dmSCN have different functional roles, both in decoding photic information and in their independent regulation on the circadian rhythmic outputs (de la Iglesia et al., 2004; Lee et al., 2009; Moore et al., 2002). The vlSCN is densely innervated by the RHT and by projections from other retinorecipient areas outside the SCN. The dmSCN, in contrast, receives sparse retinal input and is densely innervated by projections from the vlSCN. As a chemical intrinsic feature, vasopressin (AVP) is typically expressed by dmSCN neurons, whereas vasoactive intestinal peptide (VIP) is predominantly expressed by vlSCN neurons (Cassone et al., 1988; Sofroniew and Weindl, 1980). Studies of the intrinsic organization of the human SCN have demonstrated

that, as in rodent, the human SCN is also segregated into anatomical subdivisions (Moore, 1992; Dai et al., 1997). Several other neuroactive substances, such as calcium-binding proteins, calretinin (CalR) and calbindin (CalB), also have specific patterns of distribution in the SCN among different species (Leak and Moore, 2001; Silver and Schwartz, 2005) Therefore, SCN cells do not comprise a homogeneous group but can instead be differentiated by their neurotransmitter phenotype, their pattern of efferent and afferent projections and their pattern of gene expression and electrical activity (Hamada et al., 2004; Saeb-Parsy and Dyball, 2003; Schaap et al., 2003). In addition, some differences between rodents and primates have already been shown regarding afferent projections to the SCN (Cavalcante et al., 2002; Moore and Speh, 2004; Pinato et al., 2007; Pinato et al., 2009). However, the morphological organization of the SCN at the neurochemical and molecular levels, which may predict similar functions among species, is still unclear in primates. Given the functional importance of these anatomical aspects and the known differences between diurnal and nocturnal species, this study addresses inter-species differences in phenotypes related to the intrinsic organization of SCN cells in relation to the expression of the clock gene PER2 protein within a 24-h cycle and the topography of several neuroactive substances in the SCN of a diurnal primate.

2.

Results

Expression of the PER2 protein in the SCN of the capuchin monkey was evaluated six times, in a period of 24 h, and showed rhythmicity in this diurnal primate species.

Fig. 1 – Rhythmic PER2 protein expression in the SCN of capuchin monkey. Brightfield photomicrographs of coronal monkey (Sapajus apella) brain sections through the SCN showing PER2 protein immunoreactivity at different ZTs (A ZT2, B ZT5, C ZT10, D ZT14, E ZT18, and F ZT22). Scale bar¼200 lm; 3 V: third ventricle; OX: optic chiasm.

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The PER2 protein immunoreactivity revealed rhythmic expression during the 24 h cycle, with higher expression during most of the day (ZT2¼ 321720; ZT5¼ 371735 and ZT10¼ 295750) (Fig. 1A-C) and low expression during the night (ZT14¼247779 and ZT22¼234727) (Fig. 1D and F). At ZT2, ZT5 PER2-immunoreactive cells were found throughout the SCN (Fig. 1A and B). At ZT10 and ZT22, a cluster of imunorreactive cells was found in the ventromedial region (Fig. 1C and F). At ZT18, few immunoreactive cells were found in the SCN; these were mostly in the central SCN region (Fig. 1E). The analysis of the neurochemical identity of the SCN neurons showed that one of the main neuroactive substances, VIP, but not AVP, maintained a conserved pattern of distribution, as previously reported in rodents. The distribution of VIP-IR cells revealed a dense population of cell bodies and fibers concentrated in the ventral region of the SCN (number of cells: 5771.4) at the rostral level and in the vlSCN at the caudal level (number of cells: 6172.8).VIP-IR fibers were also found in the ventral and dorsal regions of the SCN (Figs. 2 and 3, A–B). Two clusters of AVP-IR cell bodies and fibers were observed, one in the medial SCN and the other in the ventral SCN at the rostral level (number of cells: 4.571.8) (Fig. 2C), whereas at the caudal level, the AVP-IR cell bodies and fibers were observed in the dmSCN and in the central region of the SCN (number of cells: 16.573.5) (Fig. 3B). Similarly, a cluster of the NeuN-IR neurons was observed in the central region at the rostral level of the SCN (number of cells: 9775) (Fig. 2E). In contrast, two clusters of NeuN-IR neurons were detected at the caudal level of the SCN, one in the ventral region and the other in the dmSCN (number of cells: 3571.5) (Fig. 3B). In contrast to rodents, capuchin monkey had abundant CalB-IR cells in the SCN throughout the rostrocaudal extension, and these cells filled the dorsal, ventral, medial and distal regions of the SCN (number of cells: 152722). Thus, staining for this neuropeptide could be used for the delimitation of the SCN (Fig. 2G). A few CalR-IR cells were distributed at the rostral level of the SCN (number of cells: 5.570.5), whereas a higher density was observed at the caudal level in the dorsal and medial regions of the SCN (number of cells: 26.670.5) (Fig. 2I, Fig. 3).

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The images for the staining of serotonergic projections (as previous published, Pinato et al., 2007) and other neuroactive substances (studied in the present work), were overlapped to investigate how circadian information could be integrated in the SCN in this diurnal primate (Fig. 3). An intricate distribution of internally heterogeneous multi-phenotype cells was observed that revealed the complex structure of the SCN. At least three regions were visualized in the coronal sections of the SCN: the first region was the vlSCN, which was occupied by VIP-IR and AVP-IR cell bodies and fibers and by NeuN-IR cell bodies; the second was the dorsal region, which was occupied by CalR-IR and CalB-IR as well as a few NeuN-IR cell bodies and AVP-IR fibers; and the third was a central region that was occupied mainly by NeuN-IR cell bodies and AVP-IR fibers (Fig. 3).

3.

Discussion

A data set on the circadian timing system of the capuchin monkey, including the morphology of the SCN and its major afferent inputs, has been assembled by our group (Frazão et al., 2008; Pinato et al., 2007; Pinato et al., 2009). The present study expands the current knowledge on this particular primate's molecular circadian oscillator concerning the spatial and temporal patterns of PER2 protein. Thereby, also a description of the intrinsic neurochemical organization of the capuchin monkey SCN, showing the spatial pattern of neuroactive substances within this nucleus, is also described. The circadian system, when entrained to the day–night cycle, allows organisms to anticipate and adapt to the 24-h daily cycles of the environment, thus ensuring that behavioral and physiological responses occur during the appropriate temporal niche. Similarly to other core clock genes, Per2 is regulated by a combination of enhancer elements including E Boxes (recognized by BMAL1/CLOCK) and D Boxes (recognized by DBP/E4BP4) (Ueda et al., 2005). However, these transcription factors are themselves regulated by Per2 and other clock gene components, which adds complexity to the circadian transcriptional machinery because of the implicated dependencies between these circadian pathways.

Fig. 2 – Immunohistochemical characterization of the SCN of capuchin monkey. Brightfield photomicrographs of coronal sections at the rostral level of the SCN of capuchin monkey. The specific neurotransmitter and NeuN labels are indicated in the top panels of A and B (VIP), C and D (AVP), E and F (NeuN), G and H (Calb) I and J (CalR). 3V: third ventricle; OX: optic chiasm. Scale bar: 200 lm (A, C, E, G and I), 100 lm (B, D, F, H and J).

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Fig. 3 – Neurochemical organization of the SCN. Schematic drawings along the anterior–posterior axis of the SCN of capuchin monkey demonstrating the distribution of different groups of cells immunoreactive for vasoactive intestinal polypeptide (VIP), nuclear protein calbindin (CalB), calretinin (CalR), neuron specific (NeuN) and serotonin (5-HT). The rostral (A) and caudal (B) levels of the nucleus are drawn from the original material shown in Fig. 2 and previously published material (Pinato et al., 2009). Dashed lines represent the perimeter of the SCN as indicated by the Nissl staining; 3V: third ventricle; OX: optic chiasm.

The present results suggest similarity between the phases of PER2 protein higher expression between the primate studied and rats, since in both PER2 expression was higher during most of the day and low during the night (Jiang et al., 2011). Nevertheless, there was a difference in the time of peak expression of this protein in the SCN between primates and rodents, from ZT2 and ZT5 in the Sapajus to ZT9 in rats (Jiang et al., 2011). It should be remembered that the environmental light cycle is the most important factor for synchronization of clock genes in most organisms (Aschoff, 1960; Daan and Pittendrigh, 1976; Meijer and Schwartz, 2003; Moore and Lenn, 1972). Therefore, it is important to consider that the results presented here were obtained from animals that were kept under natural light–dark cycles and exposed to moonlight as well as differences in lighting and other seasonal differences, whereas most studies thus far were conducted in rodents that were kept in an artificial light cycle. Thus, a possible synchronizer for the difference in peak expression time observed between primates and the rodents could be the respective environmental light–dark cycle, which may contribute to the up-regulation of the two putative clock genes, Per1 and Per2 and consequently PER1 and PER2 proteins (Yan, 2009). In addition, several other factors may contribute to the synchronization of PER2 expression, including behavioral factors, differences in activity and feeding or evolutionary factors (Bouchard-Cannon and Cheng, 2012; Chong et al., 2012; Jiang et al., 2011). Behavioral and physiological adaptations that allow animals to predict the timing of food availability are essential for their survival (Mistlberger, 2011). In the housing conditions used in this and other published studies, feeding of capuchin monkey occurs at specific times; however, laboratory rodent feeding occurs ad libitum. In the present work there were different patterns of PER2 regional expression. These differences were time dependent since at ZT2 and ZT5 PER2-immunoreactive cells were found throughout the SCN, at ZT10 and ZT22, a cluster of

imunorreactive cells was found in the ventromedial region and at ZT18 few immunoreactive cells were found in the central SCN region. In rats, regional differences were visualized, and both the ventral and dorsal regions showed temporal changes in PER2 expression. There is substantial evidence that the SCN can be divided into two distinct regions based on the evaluation of peptide expression, cytoarchitecture, efferent projections and gene expression (Hamada et al., 2001; Silver et al., 1996a). Furthermore, in hamsters and mice, some neurons in the core exhibit circadian rhythmicity in clock gene expression and electrical activity, whereas many cells in the shell express such rhythms (Hamada et al., 2001; Karatsoreos et al., 2004; LeSauter et al., 2003). It was previously demonstrated that the SCN could be subdivided into a rhythmic region delineated by cells containing AVP and a non-rhythmic region delineated by a population of CalB-IR neurons that receive direct synaptic retinal input (Bryant et al., 2000; Hamada et al., 2001). These findings were incorporated into a formal model in which the SCN comprises two distinct cell types; the core gate cells provide a daily organizing signal that maintains phase coherence in the ensemble of shell oscillator cells, and the output of this ensemble regulates the activity of the gate (Antle et al., 2003). The predominant distribution of VIP-IR neurons in the ventral portion of the SCN of capuchin monkey is similar to that observed in most species studied thus far, including humans (Dai et al., 1997), rats (Moore et al., 2002; Morin et al., 2006; van den Pol and Tsujimoto, 1985), rock cavy (Nascimento et al., 2010), hedgehogs (Antonopoulos et al., 1987), mice (Abrahamson and Moore, 2001; Cassone et al., 1988; Morin et al., 2006), moles (Kudo et al., 1991), degu (Goel et al., 1999) and grass rats (Smale and Boverhof, 1999). In contrast, in the opossum, VIP-IR cells are localized in the dmSCN (Cassone et al., 1988). Thus, it is known that VIP neurons participate in local connections within the ventral region and connect with

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AVP-IR neurons in the dorsal region (Ibata et al., 1993). This characteristic seems to be partially preserved in the SCN of capuchin monkey, as we observed a high density of VIP-IR fibers in the dorsal region, especially at the caudal level of the nucleus. The similar expression pattern observed in most rodents and humans suggests that this organization is a common property of the mammalian SCN. The present study shows that AVP, which is a molecule that relays timing information regarding activity from the SCN to other parts of the brain (Buijs et al., 1995; Bult et al., 1993), is expressed in the dmSCN and ventral SCN of capuchin monkey. This characteristic is different from what was described for most mammalian species studied to date, including the golden hamster (Card and Moore, 1984), rat (Buijs et al., 1995; Moore et al., 2002; Morin et al., 2006; van den Pol and Tsujimoto, 1985), mouse (Abrahamson and Moore, 2001; Cassone et al., 1988; Morin et al., 2006), ground squirrel (Reuss et al., 1989), grass rat (Smale and Boverhof, 1999) and degu (Goel et al., 1999). However, AVP-IR cells may be absent or may have different distributions in some species (Martinet et al., 1995; Tominaga et al., 1992), and the pattern of distribution of AVPIR in the dmSCN is frequently considered to be a standard feature of the mammalian SCN (Moore et al., 2002), which seems to differ in the SCN of capuchin monkey. The expression of NeuN in the SCN of capuchin monkey agrees with the results reported in adult rats (Geoghegan and Carter, 2008). NeuN is used extensively to identify neurons in the adult nervous system of various vertebrate species, including rodents and humans. Many of the SCN cells in capuchin monkey were NeuN-negative, These data agree with previously obtained data indicating that several rodent brain regions, including the SCN, are NeuN-negative (Weyer and Schilling, 2003). The significance of the lack of expression of this protein in neurons of specific regions and the cause of the interspecies differences in certain regions are still unclear. In several species, abundant CalB-IR was detected throughout the SCN. The results obtained in the SCN of capuchin monkey are similar to those found in marmosets (Costa and Britto, 1997), Cryptomys (Negroni et al., 2003) and humans (Mai et al., 1991). In rats, CalB-IR cells are concentrated mainly in the peripheral portions of the SCN (Celio, 1990). In mice, CalBIR cells are also abundant in the SCN, but their density is higher in the dorsal part of the nucleus, and these cells are relatively sparse or weakly stained in a fairly central region of the SCN (Morin et al., 2006). In hamsters, where calbidinexpressing neurons are necessary for the maintenance of circadian rhythms (LeSauter and Silver, 1999), a population of CalB-IR cells is located in a central sub-region of the caudal portion of the nucleus (Morin et al., 1992; Silver et al., 1996b). Few CalR-IR cells were observed in the SCN of capuchin monkey; these cells were scattered throughout the rostrocaudal extension of the nucleus. This pattern differs from that observed in other species. Studies in mice have shown an abundance of CalR-IR cells, mainly in the ventral part of the SCN, which indicates that the distribution of CalR complements that of CalB in this species (Morin et al., 2006). In rats, CalR-IR cells are one of the main populations of the SCN, as they account for 14% of SCN cells, and they are located in the dorsal and lateral portions of the SCN but extend beyond the borders of the nucleus (Moore et al., 2002).

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Among the neuroactive substances analyzed in this investigation, immunoreactivity for substance P and neurotensin was absent in the SCN of the capuchin monkey, in contrast to rats and marmosets, where SP-IR terminals branch out into a dense plexus in the ventral SCN (Takatsuji et al., 1991; van den Pol and Tsujimoto, 1985), and in contrast to hamsters, where staining for these substances is detected in the peripheral portion of the SCN (Morin et al., 1992). However, SP-IR cells and fibers are sparse in the degu (Goel et al., 1999), ground squirrel (Smale et al., 1991) and mouse (Abrahamson and Moore, 2001). SP-IR perikarya are also rare in the SCN of hamsters (Morin et al., 1992; Reuss and Bürger, 1994) and grass rats (Smale and Boverhof, 1999), and they are very scarce in mice (Abrahamson and Moore, 2001). It is interesting to note that pretreatment with colchicine was used in all species in which SP-IR perikarya were identified. SP fibers have been suggested to convey light information to the SCN (Abe et al., 1996; Takatsuji et al., 1991). However, this observation was not confirmed in subsequent studies in rodents (Hannibal and Fahrenkrug, 2002). Another aspect that should be considered for all of the analyzed peptides is the possibility of rhythmic expression, as has already been demonstrated for CalB (Hamada et al., 2003). In summary, the present study represents the first description of spatial and temporal oscillatory expression of PER2 protein in a diurnal primate. Investigation of the peptide constituents in the SCN of capuchin monkey revealed substantial heterogeneity in the expression of VIP, AVP, NeuN, CalB and CalR in each rostrocaudal level. A comparison of the present findings with data in the literature led us to conclude that the SCN of capuchin monkey shares many characteristics with the SCN of other species; however, the expression of some peptides can be species-specific. These data agree with recent studies that propose more than two subdivisions within the SCN because the pattern observed here does not match the classical scheme, i.e., core-shell organization. Further studies are needed to completely understand the function of the various sectors of the SCN. Such studies should especially use primates raised under multiple environmental conditions to characterize the evolutionary aspects of the circadian timing system and its differences between diurnal and nocturnal species.

4.

Conclusion

We present, for the first time, the spatial and temporal patterns of PER2 protein, showing a similar phase of PER2 expression and an advance in the time of peak expression of this protein between primates and rodents. The analysis of the neurochemical identity of the SCN neurons showed that one of the main neuroactive substances, VIP, but not AVP, maintained a conserved pattern of distribution. In addition, an intricate distribution of internally heterogeneous multiphenotype cells revels at least three regions visualized in the coronal sections of the capuchin monkey SCN's: the vlSCN, which was occupied by VIP-IR and AVP-IR cell bodies and fibers and by NeuN-IR cell bodies; the dorsal region, which was occupied by CalR-IR and CalB-IR as well as a few NeuN-IR cell bodies and AVP-IR fibers; and a central region that

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was occupied mainly by NeuN-IR cell bodies and AVP-IR fibers.

5.

Materials and methods

5.1.

Animals

Brain sections of adult male Sapajus apella monkeys weighing 2 to 3 kg were obtained from the Núcleo de Procriação de Macacos Prego of the Universidade Estadual Paulista “Júlio de Mesquita Filho”, Araçatuba, São Paulo, Brazil. The animals were kept in individual cages and in natural conditions of humidity, temperature and light–dark cycles. The sunrise time during the experiments was approximately at 06:00 h and was considered the Zeitgeber time 0 (ZT0) as a reference; the sunset time started at approximately 18:00 h. The animals received a standard diet consisting of protein, fruits and vegetables administered twice a day (the first one around 10:00 h and the second around 17:00 h). The procedures for animal handling and confinement were in accordance with the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care as well as the norms of the IBAMA Decree 016/94. The study was approved by the local ethics committee on the use of laboratory animals. For the gene expression study, a group of animals was anesthetized and perfused transcardially at ZT2, 5, 10, 14, 18 or 22 (n¼ 2 per ZT). The brains were exposed and cut into blocks using a stereotaxic apparatus. The blocks were then removed from the skull and placed in a cryoprotective solution containing 10% glycerol and 2% dimethylsulfoxide in 0.1 M borate buffer, pH 9.0, at 4 1C. After three days, the blocks were transferred to a similar solution containing an increased concentration of glycerol (20%), and they were incubated for four additional days, as previously described (Rosene et al., 1986). Next, the blocks were cut into 35-μm coronal sections using a cryomicrotome and collected in an antifreeze solution (ten series), one series per time was used for immunohistochemistry.

5.2.

Immunohistochemistry

For immunohistochemistry, the sections were incubated for 1 h in a solution of Triton X-100 (Amresco, Solon, OH, USA) and 0.4% normal donkey serum (Chemicon International, Inc., Temecula, CA, USA) in 0.1 M PBS. Next, the sections were incubated with the following primary antibodies for 48 h: antiCalB (Sigma, St. Louis, MO, USA; 1:1000), anti-CalR (Chemicon, Temecula, CA, USA; 1:1000), anti-AVP (Chemicon, Temecula, CA, USA; 1:1000), anti-VIP (Chemicon, Temecula, CA, USA; 1:1000), anti-neuron-specific nuclear protein (NeuN) (Millipore Corporation, Billerica, MA, USA; 1:20,000), anti-5HT (Protos Biotech, New York, NY, USA; 1:5000), anti-substance P (Millipore Corporation, Billerica, MA, USA; from 1:100 to 1:1000), antineurotensin (Chemicon, Temecula, CA, USA; from 1:100 to 1:1000) and anti-PER2 (Thermo Scientific, Rockford, IL, USA; 1:500). After being incubated in the primary antibodies, the sections were washed in 0.1 M PBS (3  10 min) and incubated in a 0.1 M PBS solution containing 0.1 M Triton X-100 and the secondary antibodies conjugated with rhodamine (1:200) or

fluorescein isothiocyanate (FITC) (1:500) (Jackson Immunoresearch Labs) for 2 h. Then, they were rinsed in 0.1 M PBS (3  10 min), mounted on gelatinized slides, coverslipped using glycerol buffer (Sigma Chemical, St. Louis, MO, USA) as the mounting medium and stored in the dark at 4 1C. The negative staining controls lacked the primary antibodies. Under these conditions, the staining was completely abolished. For each animal, two coronal sections corresponding to the rostral and caudal portions of the SCN were cut at a distance of 350 mm and analyzed using each antibody. The slides were analyzed under light or fluorescence microscopy (Nikon Eclipse E1000, USA). The digitalized images were captured, and the distribution of cell bodies and fibers was mapped with the aid of the Canvas computer-drawing program (version 9.0).

Acknowledgments The authors thank FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo, FAPESP-2008/53689-8 research grant), PRPq-USP, the PIBIC fellowship to P Mello, the CAPES master fellowship to VA ROCHA, Prof. Dr. José Américo de Oliveira, Núcleo de Procriação de Macacos Prego, Universidade Estadual Paulista “Júlio de Mesquita” for providing capuchin monkey tissues and Dr. Horácio de la Iglesia for helpful critical comments on this manuscript.

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