Immunocytochemical characterization of the pregeniculate nucleus and distribution of retinal and neuropeptide Y terminals in the suprachiasmatic nucleus of the Cebus monkey

Journal of Chemical Neuroanatomy 37 (2009) 207–213

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Immunocytochemical characterization of the pregeniculate nucleus and distribution of retinal and neuropeptide Y terminals in the suprachiasmatic nucleus of the Cebus monkey L. Pinato a,1,*, R. Fraza˜o a,1, R.J. Cruz-Rizzolo b, J.S. Cavalcante c, M.I. Nogueira a a b c

Department of Anatomy, Institute of Biomedical Sciences, University of Sa˜o Paulo, SP, Brazil Department of Basic Sciences, Arac¸atuba Campus, Sa˜o Paulo State University - UNESP, SP, Brazil Department of Physiology, Biosciences Center, Federal University of Rio Grande do Norte, Natal, RN, Brazil

A R T I C L E I N F O

A B S T R A C T

Article history: Received 20 August 2008 Received in revised form 18 December 2008 Accepted 30 January 2009 Available online 10 February 2009

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are modulated by photic and nonphotic stimuli. In rodents, direct photic stimuli reach the SCN mainly through the retinohypothalamic tract (RHT), whereas indirect photic stimuli are mainly conveyed by the geniculohypothalamic tract (GHT). In rodents, retinal cells form a pathway that reaches the intergeniculate leaflet (IGL) where they establish synapses with neurons that express neuropeptide Y (NPY), hence forming the GHT projecting to the SCN. In contrast to the RHT, which has been well described in primates, data regarding the presence or absence of the IGL and GHT in primates are contradictory. Some studies have suggested that an area of the pregeniculate nucleus (PGN) of primates might be homologous to the IGL of rodents, but additional anatomical and functional studies on primate species are necessary to confirm this hypothesis. Therefore, this study investigated the main histochemical characteristics of the PGN and the possible existence of the GHT in the SCN of the primate Cebus, comparing the distribution of NPY immunoreactivity, serotonin (5-HT) immunoreactivity and retinal terminal fibers in these two structures. The results show that a collection of cell bodies containing NPY and serotonergic immunoreactivity and retinal innervations are present within a zone that might be homologous to the IGL of rodents. The SCN also receives dense retinal innervations and we observed an atypical distribution of NPY- and 5-HT-immunoreactive fibers without regionalization in the ventral part of the nucleus as described for other species. These data may reflect morphological differences in the structures involved in the regulation of circadian rhythms among species and support the hypothesis that the GHT is present in some higher primates (diurnal animals). ß 2009 Elsevier B.V. All rights reserved.

Keywords: Biological rhythms Retinohypothalamic tract Geniculohypothalamic tract Primate Circadian rhythms

1. Introduction Ubiquitously expressed in the central nervous system of different species, neuropeptide Y (NPY) is related to a variety of physiological processes, including the modulation of circadian rhythms and hormone release (Allen et al., 1983; Everitt et al., 1984; Hokfelt et al., 1998; Ueda et al., 1986). Investigations on the neural circuitry of the circadian timing system conducted on rodents have revealed a population of NPY-immunoreactive (NPYIR) neurons in the intergeniculate leaflet (IGL), which is a thin lamina of cells interposed between the dorsal lateral geniculate

* Corresponding author at: Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes, 2415, 05508-900 Sa˜o Paulo, SP, Brazil. Tel.: +55 11 3091 7401; fax: +55 11 3091 7366. E-mail address: [email protected] (L. Pinato). 1 These authors contributed equally to this work. 0891-0618/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2009.01.005

nucleus (LGN) and the ventral LGN (Hickey and Spear, 1976). These neurons receive projections from retinal ganglion cells and project as the geniculohypothalamic tract (GHT) mainly to the ventral part of the suprachiasmatic nucleus (SCN) (Allen et al., 1983; Card and Moore, 1989; Harrington et al., 1987; Moore and Card, 1985, 1994; Ueda et al., 1986). The SCN, the main circadian pacemaker in mammals, is able to generate physiological and behavioral rhythms synchronized by the environmental light–dark cycle through photic and non-photic stimuli. The cytoarchitecture, afferents, efferents, neurotransmitters and the molecular mechanism of the SCN have been extensively studied, especially in rodents (Morin and Allen, 2006). It is known that impulses act on the SCN through different neurotransmitters released by various pathways that reach different regions of the SCN. Photic impulses, the main cue that synchronizes biological rhythms to the environment in most vertebrates, reach the SCN through terminals of the retinohypothalamic tract (RHT), a direct pathway that originates from

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retinal ganglion cells (Hendrickson et al., 1972; Moore and Lenn, 1972; Moore, 1973). In rodents, RHT terminals are located mainly in the ventrolateral region of the SCN, coexisting with NPY-IR terminals of the GHT and also with serotonergic terminals arising from the median raphe nucleus (Hendrickson et al., 1972; MeyerBernstein and Morin, 1996; Moore and Lenn, 1972; Moore et al., 1978, 2002; Moore, 1983). These two neuromodulators, NPY and serotonin (5-HT), are involved in the modulation of photic information to the SCN since 5-HT can reduce retinal input to the circadian system by acting at presynaptic 5-HT1B receptors located on retinal axons in the SCN (Lall and Biello, 2002; Pickard et al., 1999; Rea et al., 1994), as well as in the process of non-photic synchronization of endogenous rhythms generated by the SCN (Biello et al., 1994; Glass et al., 2003). The importance of the GHT in the modulation of the circadian system in rodents has been well documented (Gamble et al., 2006; Menet et al., 2001; Yannielli et al., 2004). In primates the picture is controversial, with some authors suggesting the absence of this system or that NPY might be replaced with other neuroactive substances in humans (Moore, 1989; Pelletier et al., 1984) and in some non-human primates (Chevassus-au-Louis and Cooper, 1998; Ueda et al., 1986). In the case of nocturnal and diurnal primates studies have reported a distribution of NPY-IR terminals in the ventral region of the SCN similar to that observed in rodents (Cavalcante et al., 2002; Chevassus-au-Louis and Cooper, 1998; Moore, 1989, 1993). In primates, the lateral geniculate complex differs substantially in organization from that of rodents, containing a large, laminated dorsal lateral geniculate nucleus (DLG). This nucleus is mainly surrounded by cell groups that have been designated the pregeniculate nucleus (PGN) (Jones, 1985). These cell groups include a large division located medial and, to a limited extent, dorsal to the DLG. The large group medial to the DLG is continuous with the zona incerta and, as described previously, contains NPY-IR neurons which project to the SCN (Moore, 1989). This group appears to be the primate homolog of the IGL but its connections and role remain unknown (Chevassus-au-Louis and Cooper, 1998; Costa and Britto, 1997; Moore, 1989, 1993). The position of the IGL homolog has changed during evolution within the primate group. In the more primitive species, Microcebus, this structure assumes a position ventral to the dorsal LGN and roughly similar to that observed in rodents. In contrast, in New and Old World monkeys, the homolog of the IGL is shifted medially or even dorsomedially to the dorsal LGN (Chevassus-auLouis and Cooper, 1998). The existence of a GHT in non-human primates therefore is a crucial issue in terms of the appropriateness of primate models for the study of human circadian physiology. Moreover, the conservation of this pathway during phylogeny is a question of basic interest for comparative anatomy. To explore this challenging picture and because of the relative lack of information about the neuroanatomy of the circadian system in diurnal mammals, we explored the existence and characteristics of the PGN and SCN in the primate Cebus apella. These nuclei were evaluated with respect to their retinal afferents, serotonin-immunoreactive (5-HT-IR) terminals and the presence and distribution of NPY-IR perikarya and terminals. We consider the primate C. apella to be an appropriate model for this study since it is a diurnal species and is comparable to the Old World monkey Macaca in terms of brain size and sulcal pattern, as well as the relative position of homologous neuroanatomical and functional areas (Gattass and Gross, 1981; Gattass et al., 1981, 1987, 2005; Rosa et al., 1988; Fiorani et al., 1989; Padberg et al., 2007). In contrast, these characteristics are not observed in smaller New World monkeys. It has been suggested that differences in the chemoarchitectural pattern exist even between members of the

same order and that they are important for tracing evolutionary adaptations and phylogenetic relationships (Bourne et al., 2007). Thus, the present study conducted on Cebus will permit comparisons of the cytochemical organization of structures of the circadian timing system between primates and rodents, between New World monkeys, and between New and Old World monkeys. 2. Materials and methods For this study we used four young adult male C. apella monkeys obtained from the Primate Center of ‘‘Ju´lio de Mesquita Filho’’ Sa˜o Paulo State University, Sa˜o Paulo, Brazil. Experimental procedures were conducted according to the Guidelines for the care and use of mammals in neuroscience and behavioral research (2003) and were approved by the local laboratory animal care and use committee. All efforts were made to reduce the number of animals and to minimize suffering. The monkeys were housed in individual cages under natural conditions of humidity and temperature on a natural day-night cycle (start of the day at about 6:00 h and nightfall at about 18:00 h). The animals received a standard diet consisting of fruits and vegetables. The technique of intraocular injection of cholera toxin B subunit (CTb), a tracer with retrograde and anterograde properties, was used here. This technique has been successfully applied in primates to trace retinal projections to several targets (Angelucci et al., 1996; Cavalcante et al., 2005; Costa et al., 1999; Fraza˜o et al., 2008). The animals were anesthetized with sodium thiopental (30 mg/kg, i.p.) and then injected unilaterally with 100 ml of a solution containing 1.0% CTb (low salt; List Biological Laboratories, Campbell, CA, USA) in 10% dimethylsulfoxide. Fifteen days after injection, between 9:00 and 11:00 h, the animals were anesthetized and perfused transcardially with 800 ml 0.9% saline, followed by 1500 ml 4% paraformaldehyde in 0.1 M acetate buffer, pH 6.5, and subsequently 1500 ml 4% paraformaldehyde in 0.1 M borate buffer, pH 9.0. 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 8C. After 3 days, the blocks were transferred to a similar solution but containing an increased concentration of glycerol (20%) and were incubated for four additional days according to the method described by Rosene et al. (1986). Next, the blocks were cut into 40-mm coronal sections with a cryomicrotome and collected in an antifreeze solution. As described in a previous study analyzing coronal thionine-stained sections (Nissl method), the SCN of C. apella measures about 1.05 mm in length from the rostral to the caudal pole (Pinato et al., 2007). Thus, in the present study coronal sections at an interval of 400 mm were analyzed per series, corresponding to the portions of the anteroposterior axis of this nucleus. One series of brain sections was incubated for 48 h with primary anti-NPY antibody (Chemicon International Inc., Temecula, CA, USA) diluted 1:1000 in 0.1 M PBS containing 0.3% Triton X-100, followed by incubation with the secondary fluorescent antibody (1:200) (Rhodamine, Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 120 min, or was detected by the avidin–biotin immunoperoxidase method. Another series was incubated with primary antibody against CTb (1:4000) (anti-CTb, List Biological Laboratories), and one-third of brain sections were treated immunohistochemically overnight with a primary antiserotonin antibody (Protos Biotech NT-102, New York, NY, USA) diluted 1:1000 in 0.01 M PBS containing 0.3% Triton X-100. Both series were detected by the avidin– biotin immunoperoxidase method according to the protocol of Fite et al. (1999). Brain sections of non-injected animals used in another study served as controls for CTb. Reactions in which the primary antibody was omitted were performed for the control of nonspecific labeling. The efficacy of the intraocular injections was confirmed by the demonstration of CTb-IR retinal terminals in several central visual areas such as the superior colliculus and was as expected (data not shown).

3. Results 3.1. Suprachiasmatic nucleus Neuropeptide Y: The density of small NPY-IR axons and terminals in the C. apella SCN ranged from moderate to sparse along the anteroposterior axis, with the observation of a small plexus of small IR axons and terminals centrally and ventrally overlapping the distribution of RHT axons and contrasting with a dense network of larger caliber NPY-IR fibers in adjacent hypothalamic structures (Fig. 1A and B). In the rostral portion where the nucleus has a triangular shape, NPY-IR fibers were sparsely distributed at the periphery of the nucleus (Fig. 1A). Its

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Fig. 1. Organization of CTb- and NPY-immunoreactive fibers in the suprachiasmatic nucleus (SCN) of Cebus apella. Photomicrographs of coronal sections cut in the rostral (A, C, E) to intermediate-caudal (B, D, F) direction and submitted to neuropeptide Y (NPY) (A and B) and cholera toxin B subunit (CTb) (C, D, E and F) immunohistochemistry using fluorescence and bright-field microscopy, respectively. The lines indicate the SCN. C and D correspond to the contralateral side of intraocular CTb injection, and E and F correspond to the ipsilateral side. 3V = third ventricle; ox = optic chiasm. Scale bar: 200 mm.

typical ventral regionalization, described for other species, was not noted (Fig. 1A and B). In the intermediate-caudal portion where the boundaries of the nucleus extend along the dorsoventral axis and decline in the middle-lateral axis assuming an oval shape, NPY terminal fibers became more densely clustered, especially in the central and lateral portions of the SCN (Fig. 1B). Few terminals were observed in the ventral region (Fig. 1B). Retinal afferents: Four injected brains presented terminals containing almost identical amounts of the reaction product in the targets described in this study. Dense retinal terminals were observed in the contra- (Fig. 1C and D) and ipsi-lateral SCN (Fig. 1E and F). Innervations arose as a more concentrated ventral plexus in the rostral half of the nucleus which was enlarged to fill the oval profile of the intermediate-caudal part of the nucleus (Fig. 1C, D and E, F, respectively). Serotonin afferents: In agreement with previous results (Pinato et al., 2007), serotonergic terminals were observed mainly at the periphery of the SCN, with few fibers being distributed in the central and lateral region of the nucleus (Fig. 3). 3.2. Pregeniculate nucleus Neuropeptide Y: A collection of cell bodies presenting NPY immunoreactivity were observed within a zone with a wedge shape that might be homologous to the IGL of rodents. The highest concentration of cells was observed in the medial portion (Fig. 2A).

This region is part of the PGN that is located dorsomedial to the parvocellular layer of the LGN (Fig. 2A). Retinal afferents: CTb-labeled terminals were observed throughout the PGN, mainly contralateral to the site of CTb injection (Fig. 2B). The distribution of CTb terminals was compared to the distribution of NPY-IR cells and fibers in adjacent sections throughout the PGN and also formed an area with a wedge shape on the contralateral side. Serotonin: The immunohistochemical distribution of 5-HT fibers was compared to the distribution of NPY and CTb immunoreactivity in adjacent sections to evaluate their presence in the PGN. A dense plexus of 5-HT fibers and terminals was observed throughout this nucleus, coinciding with the location of NPY-IR cells and CTb immunoreactivity (Fig. 2C). 4. Discussion 4.1. Suprachiasmatic nucleus The presence of NPY fibers in the SCN of C. apella and their divergent distribution within this nucleus among different species raise questions regarding the uniform organization of the circadian timing system in mammals. This subject has been addressed in several studies (Cavalcante et al., 2002; Morin and Allen, 2006; Morin, 2007; Ueda et al., 1986) which like the present investigation, question the generalization of the characteristics found in studies on rats to all species.

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Fig. 2. Immunohistochemical characterization of the Cebus apella pregeniculate nucleus (PGN). Photomicrographs showing immunoreactivity to neuropeptide Y (NPY) (A), cholera toxin B subunit (CTb) (B) and serotonin (5-HT) (C). The area characterized as PGN (underlined) is located above the lateral geniculate nucleus in this primate species. The PGN comprises an area characterized by the presence of a population of NPY-immunoreactive neurons (A, higher magnification on the right). This region also receives projections from retinal ganglion cells, with predominance on the contralateral side (B, higher magnification on the right). In addition, the retinal projections are also distributed contralaterally (B) and ipsilaterally (not shown) through the layers of the lateral geniculate nucleus. The PGN is also characterized by dense innervation of serotonergic terminals (C, higher magnification on the right). Scale bar: 500 mm (A–C), 20 mm (higher magnification).

In most studies, two areas have been defined in diurnal and nocturnal rodents: (1) the ventrolateral portion of the SCN, which receives glutamatergic afferents from the retina, serotonergic terminals from raphe and projections from the IGL containing NPY (Hendrickson et al., 1972; Moore and Lenn, 1972; Moore et al., 2002); (2) the dorsomedial portion of the SCN that receives projections from the hypothalamus, limbic cortical areas, thalamus

and brainstem (Moga and Moore, 1997). The modulatory circadian signals leaving these different regions of the SCN certainly differ since efferents of the ventrolateral portion are mainly influenced by photic stimuli, whereas the dorsomedial portion, in addition to receiving projections from the ventrolateral SCN, is also influenced by nonvisual impulses arising from various areas. Thus, the circadian signal generated by the SCN results from complex

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interactions and varies with respect to the subdivision of origin (Moore et al., 2002; Ueda et al., 1986). Although similarity exists among some species, many of these characteristics may vary among mammals, such as the pattern of NPY immunoreactivity, with dense concentrations of NPY-IR fibers being found mainly in the ventral part of the SCN in rats, or might be absent or located in the central region of the nucleus in some rodents and primates. NPY immunoreactivity also may vary in SCN of diurnal and nocturnal rodents euthanasied at specific times of the day. In the diurnal rodent SCN NPY-IR levels peak during the middle of the night, whereas in nocturnal rodents NPY-like immunoreactive innervation of the ventrolateral part of the SCN shows a 24-h rhythm, with values rising gradually during the light phase and falling during the dark phase (Vidal and Lugo, 2006; Calza´ et al., 1990). The differences in the pattern of fluctuation of NPY in the SCN between diurnal and nocturnal specie suggest that this peptide may play distinct roles in the circadian system of diurnal and nocturnal species. In C. apella, the diurnal primate studied here, NPY-IR fibers were sparsely distributed in the SCN around 10:00 AM and did not show the typical ventral regionalization inside the nucleus demonstrated in the SCN of rats (Moore et al., 2002; Morin et al., 2006; Ueda et al., 1986), marmosets (Cavalcante et al., 2002; Chevassus-au-Louis and Cooper, 1998), Macaca mulatta (Moore, 1989), and Microcebus murinus, a nocturnal primate species (Chevassus-au-Louis and Cooper, 1998). In squirrel monkey (Saimiri sciureus), few NPY-IR fibers were found throughout the subdivisions of the SCN, with the concentration of these fibers being higher in the ventral part along the middle to caudal portion (Smith et al., 1985). Some rodent species such as mice show a less concentrated distribution in the ventral portion, in which the densest part of the NPY-IR terminal plexus was described in the central SCN (Morin et al., 2006). In hamsters, Ueda et al. (1986) reported a moderate density of NPY-IR fibers in the ventral region, with the observation of few fibers in the dorsal part of the rostral half of the SCN and dense labeling throughout the nucleus in the caudal half. In cats, the density of NPY-IR fibers is moderate in the ventral part of the SCN but low when compared to other hypothalamic areas (Ueda et al., 1986). A sparse plexus of NPY-IR axons and a large number of NPY-IR neurons confined ventrally have been identified in the human SCN (Moore, 1989). Other studies carried out on humans and the non-human primate species Saguinis oedipus and Macaca fuscata did not detect NPY immunoreactivity in the SCN or LGN (Schwartzberg et al., 1990; Ueda et al., 1986). These results suggest morphological differences in the neuronal connections of the SCN among mammals, a fact requiring reanalysis of the subdivisions (core and shell) classically determined for this nucleus or at least restricting their use to some species. This reanalysis has been suggested by Morin et al. (2006) and Morin (2007). The pattern of distribution of retinal projections shows that retinal cells project to both the contra- and ipsi-lateral SCN in C. apella monkeys, with a contralateral predominance as described for rodents and other primates (Costa et al., 1999; Hendrickson et al., 1972; Johnson et al., 1988; Levine et al., 1991; Major et al., 2003; Moore and Lenn, 1972; Moore, 1973), although a wide distribution also exists on the ipsilateral side. In the diurnal ground squirrel (Spermophilus lateralis or Citellus tridecemlineatus), retinal projections within the hypothalamus appear to be restricted to the contralateral SCN, with no labeled fibers being observed in the ipsilateral SCN (Agarwala et al., 1989). The absence of an ipsilateral projection is unusual since the SCN of most mammals is heavily innervated by both eyes (Cassone et al., 1988; Johnson et al., 1988; Magnin et al., 1989; Moore, 1973). In contrast, the retino-SCN projection in the house musk shrew (Suncus murinus) has been reported to exhibit a slight ipsilateral predominance (Tokunaga

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et al., 1992). In hamsters there are studies describing an ipsilateral predominance at the rostral level and symmetry at the caudal level (Pickard and Silverman, 1981), a symmetrical projection (Johnson et al., 1988; Youngstrom et al., 1991), or even a contralateral asymmetry (Yellon et al., 1993). The significance of this variation in the bilateral distribution of the RHT in the SCN is not completely understood. It is possible that these differences merely reflect individual variations and methodological drawbacks such as the higher sensitivity of the CTb tracing technique used here (Major et al., 2003). Costa et al. (1999) suggested that contralateral or ipsilateral asymmetry might be due to selective uptake of the tracer by specific groups of ganglion cells in the retina of different species. Therefore, as observed for other species, in C. apella retinal projections are mainly located in the ventral region of the nucleus in the rostral part of its anteroposterior axis. However, in the intermediate-caudal portions of this nucleus the retinal terminals fill out the whole nucleus, a finding also observed in hamster in which the RHT innervates almost the entire SCN (Johnson et al., 1988; Morin et al., 2003), and contrary to the marmoset in which retinal projections form a dense plexus in the ventral part of the SCN throughout its anteroposterior axis, with sparse labeling being seen in the dorsal portion at its intermediate and caudal levels (Costa et al., 1998, 1999). In contrast to other species, comparison of the distribution of retinal terminals and of NPY-IR and 5-HT-IR terminals in the C. apella SCN only revealed small overlaps of these terminals in the ventral portion of the nucleus. However, overlapping of NPY-IR and retinal terminals was observed in the central portion of the nucleus, and some 5-HT-IR fibers overlapped with retinal terminals at the periphery of the SCN (Fig. 3).

Fig. 3. Chartings of coronal sections of the Cebus apella suprachiasmatic nucleus (SCN), showing cholera toxin B subunit-immunoreactive (CTb-IR) fibers/terminals in the nucleus (blue), neuropeptide Y-immunoreactive (NPY-IR) fibers/terminals (green) and serotonin-immunoreactive (5-HT-IR) fibers/terminals (pink) arranged at the intermediate-caudal level of the SCN. 3v = third ventricle. Scale bar: 200 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

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4.2. Pregeniculate nucleus (primate homolog of IGL + VLG) In rodents, the IGL is a structure with a distinctive immunohistochemical profile and a characteristic pattern of efferents and afferents (Card and Moore, 1989; Harrington et al., 1987; Hickey and Spear, 1976; Morin and Blanchard, 2005). In rats and hamsters, the IGL can be identified as the region that divides the LGN into its dorsal and ventral parts, contains NPY-IR cells, receives bilateral retinal input (Morin et al., 1992, 2003; Muscat et al., 2003; Pickard, 1985), and projects to the SCN and the contralateral IGL (Card and Moore, 1989). In diurnal mammals, the anatomy and function of the IGL, and consequently of the GHT, and even its existence have been questioned by some authors. The SCN and LGN of monkeys (S. sciureus and M. fuscata) were almost devoid of NPY immunoreactivity (Smith et al., 1985; Ueda et al., 1986). The present study demonstrated the presence of NPY-IR cells and 5-HT and retinal terminals in a region dorsomedial of the dorsal LGN, suggesting that this region is the primate homolog of the rodent IGL (Costa and Britto, 1997; Costa et al., 1998; Moore, 1989, 1993). The distribution of NPY-IR cells within the PGN of C. apella is similar to that described for other primates (Chevassus-au-Louis and Cooper, 1998; Moore, 1989). Comparison of the distribution of NPY-IR cells with that of CTb-labeled terminals in adjacent sections shows that, as in rodents, each retina tends to project to the NPY-IR regions of both the ipsi- and contra-lateral PGN. We refer to the dorsomedial portion of the geniculate complex, in which bilateral retinal afferents overlap with NPY-IR cells and 5-HT-IR terminals, as the part of the PGN that might be homologous to the rodent IGL. The present results do not permit to determine whether the other part of the PGN, the VLG, is present in this species, but studies using antisera against substances known to display distinct patterns of localization in the VGL may provide further insights. In conclusion, our results support the existence of differences in the mechanism involved in the regulation of circadian rhythms among different mammalian species (diurnal and nocturnal primates, rodents and carnivores). In fact, the quantity and distribution of NPY terminals differ between diurnal and nocturnal primates (Chevassus-au-Louis and Cooper, 1998; Moore, 1989). In view of the importance of the peptidergic system and of the GHT in the circadian timing system, new studies investigating this pathway in primates are necessary in order to establish an appropriate primate model for the study of the circadian timing system. Moreover, these findings demonstrate the importance of interspecies differences for the intrinsic organization of neurons and nerve terminals in the SCN and PGN despite the conservative tendency of evolutionary processes. Acknowledgments The authors thank FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo) and CAPES (coordenac¸a˜o de aperfeic¸oamento de pessoal de nı´vel superior) for financial support, and Prof. Dr. Jose´ Ame´rico de Oliveira, Nu´cleo de Procriac¸a˜o de Macacos Prego, Universidade Estadual Paulista ‘‘Ju´lio de Mesquita Filho’’, for providing the Capuchin monkeys. The authors also thank Lorenzo Morales (Department of Neurology and Anatomy, University of Texas Medical School at Houston, TX, USA), for reviewing the artwork. References Agarwala, S., Petry, H.M., May 3rd, J.G., 1989. Retinal projections in the ground squirrel (Citellus tridecemlineatus). Vis. Neurosci. 3, 537–549. Allen, Y.S., Adrian, T.E., Allen, J.M., Tatemoto, K., Crow, T.J., Bloom, S.R., Polak, J.M., 1983. Neuropeptide Y distribution in the rat brain. Science 221, 877–879.

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