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Research Report

The suprachiasmatic nucleus and the intergeniculate leaflet inthe rock cavy (Kerodon rupestris): Retinal projections andimmunohistochemical characterization

Expedito S. Nascimento Jr.⁎, Adriana P.M. Souza, Renata B. Duarte,Márcia A.F. Magalhães, Sebastião F. Silva, Judney C. Cavalcante,Jeferson S. Cavalcante, Miriam S.M.O. CostaHealth Science Center of the Trairi, Laboratory of Chronobiology, Federal University of Rio Grande do Norte, 59200-000, Santa Cruz-RN, Brazil

A R T I C L E I N F O

⁎ Corresponding author. Fax: +55 84 32119207E-mail address: expeditojunior@click21.co

0006-8993/$ – see front matter © 2010 Elsevidoi:10.1016/j.brainres.2010.01.034

A B S T R A C T

Article history:Accepted 13 January 2010Available online 21 January 2010

In this study, two circadian related centers, the suprachiasmatic nucleus (SCN) and theintergeniculate leaflet (IGL) were evaluated in respect to their cytoarchitecture, retinalafferents and chemical content of major cells and axon terminals in the rock cavy (Kerodonrupestris), a Brazilian rodent species. The rock cavy SCN is innervated in its ventral portion byterminals from the predominantly contralateral retina. It also contains vasopressin,vasoactive intestinal polypeptide and glutamic acid decarboxilase immunoreactive cellbodies and neuropeptide Y, serotonin and enkephalin immunopositive fibers and terminalsand is marked by intense glial fibrillary acidic protein immunoreactivity. The IGL receives apredominantly contralateral retinal projection, contains neuropeptide Y and nitric oxidesynthase-producing neurons and enkephalin immunopositive terminals and ischaracterized by dense GFAP immunoreactivity. This is the first report examining theneural circadian system in a crepuscular rodent species for which circadian properties havebeen described. The results are discussed comparing with what has been described for otherspecies and in the context of the functional significance of these centers.

© 2010 Elsevier B.V. All rights reserved.

Keywords:Cholera toxin subunit BCircadian rhythmImmunohistochemistryKerodon rupestrisNeurotransmitterRetinohypothalamic tract

1. Introduction

Awide variety of behavioral and physiological processes showcircadian rhythms which are generated by a time-keepingsystem, also called circadian timing system. This system inmammals is built from a neural network consisting of threemajor functional components: (1) a central pacemaker, whichgenerates rhythmicity even in the absence of external stimuli;(2) input pathways, including retinal afferents to allow thesynchronization of the rhythms to the environmental cycles;

.m.br (E.S. Nascimento).

er B.V. All rights reserved

and (3) output pathways connecting the pacemaker to thebrain and body's effectors.

Since the discovery of an unequivocal retinohypothalamicprojection (Hendrickson et al., 1972; Moore and Lenn, 1972),other experimental evidence have emerged to support the roleof the circadian pacemaker ascribed to the suprachiasmaticnucleus (SCN) of the hypothalamus (see Klein et al., 1991; vanEsseveldt et al., 2000; Reuss, 2003; Morin and Allen, 2006).

The SCN is a paired nucleus located at the anteriorhypothalamus, on either side of the third ventricle immediately

.

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dorsal to the optic chiasm. The neurochemical characterizationof theSCN, as studiedby immunohistochemical techniques, hasrevealed both consistencies and variations among the species.For example, in the SCN of virtually all mammals studied, twomajor cell populations have been identified, one consisting ofvasopressin (VP)- and another formed by vasoactive intestinalpolypeptide (VIP)-producing neurons. As a rule, VP cells arelocated in a dorsomedial position and VIP cells in a ventral orventrolateral position, as observed in coronal sections of theSCN (Card et al., 1981; Ueda et al., 1983; Card and Moore, 1984;Van den Pol and Tsujimoto, 1985; Cassone et al., 1988; Smale etal., 1991; Mai et al., 1991; Morin et al., 1992; Tessoneaud et al.,1994; Negroni et al., 1997, 2003; Goel et al., 1999; Smale andBoverhof, 1999), although thereare someexceptions (Cassoneetal., 1988; Martinet et al., 1995; Wang et al., 1997; Abrahamsonand Moore, 2001). The VIP cell territory is the preferable site forthe arborization of dense retinal afferents, neuropeptide Y(NPY)-containing terminals of the geniculohypothalamic tract(GHT) and serotonin (5-HT) terminals from the midbrain raphe(Moore et al., 2002). The differential arrangement of VP and VIPcells allied with the pattern of distribution of its afferentsprovided the basis for the division of the SCN into dorsomedialandventrolateral portions in the rat (VandenPolandTsujimoto,1985; Card and Moore, 1991). These terms were replaced by“shell” and “core”, based on the configuration observed in thehamster (Moore et al., 2002). Although it is difficult to generalizeto all species, considering the great variability, a compartmen-talization of the SCN stems in the neurochemical phenotype(Van den Pol and Tsujimoto, 1985), the pattern of distribution oftheir afferent terminals (Moga and Moore, 1997), the organiza-tion of their outputs (Leak and Moore, 2001), and even theirmolecular basis (Dardente et al., 2002).

Many other substances acting as neurotransmitters, neuro-modulators or related enzymes were found in both perikaryaand terminals of the SCN, such as NPY, 5-HT, gamma-amino-butyric acid (GABA), glutamate (GLU), bombesin (BBS), gastrin-releasing peptide (GRP), cholecystokinin (CCK), substance P (SP),angiotensin II, enkephalin (ENK), neurotensin (NT), somatostatin(SS), thyrotrophin-releasing hormone (TRH), tyrosine hydroxy-lase (TH), and nitric oxide synthase (NOS), among others (seeReuss, 2003).

A secondary componentof the circadian timingsystemis theintergeniculate leaflet (IGL), a thin retinorecipient cell layerwhich, in rodents, is intercalated between the dorsal (DLG) andventral (VLG) lateral geniculate nuclei, over the entire rostro-caudal lengthof the thalamic lateral geniculate complex (Hickeyand Spear, 1976; Moore and Card, 1994). The geniculohypotha-lamic tract (GHT) derives mainly from NPY-producing cellswhich project from the IGL ending in the SCN (Harrington et al.,1985, 1987; Card and Moore, 1989; Morin et al., 1992). As long asNPY cells are present in the lateral geniculate complex, theyhave beenusedas amarker of the IGL. In the rodent IGL, theNPYcolocalizes with GABA in most of the cells (Moore and Speh,1993; Moore and Card, 1994) and with unidentified neurotrans-mitter cells to compose the geniculohypothalamic projection(Card and Moore, 1989). Many interspecific variations are foundin the organization of the IGL/GHT. For example, the GHT alsooriginates from enkephalin (ENK) cells in the IGL of hamsters(Morin et al., 1992; Morin and Blanchard, 1995, 2001), but not ofrats (Card and Moore, 1989).

Although the IGL is not essential to photic synchronization(Klein and Moore, 1979), it has been shown to be involved inthe modulation of photic and non-photic synchronization ofthe circadian rhythms (Harrington and Rusak, 1986; Mro-sovsky, 1995; Muscat and Morin, 2006).

The rock cavy (Kerodon rupestris) is a rodent which accordingto the traditional taxonomy is classified in the superfamilyCavioidea, family Caviidae, subfamily Caviinae, genus Kerodon,together with Cavia, Galea andMicrocavia (Cabrera, 1961; Lacher,1981). However, after a molecular phylogeny of the superfamilyCavioidea using two nuclear sequences and one mitochondrialgene, Kerodon is placed sister to the family Hydrochaeridae, towhich belongs also the capybara (Hydrochaeris) and closelyaligned with the subfamily Dolichotinae (Rowe and Honeycutt,2002). Rock cavies are found in the Brazilian Northeast regionwhere they inhabit rocky areas, inwhich they usually shelter inits fissures or cracks. Based in reports of local hunters, thisspecies is seen during the day and the night, although is moreexposed and easily captured at dusk (Cabrera, 1961; Lacher,1981). These observationswere confirmed in captivity, since inalaboratory controlled condition study it was registered that therock cavy is active along all 24-hours per day, although itsactivity is intensified around sunset and dawn phases, suggest-ing a predominantly crepuscular behavior (Sousa andMenezes,2006).

Having as a goal to establish a regional model to circadianresearch, the aimof this studywas to identify and characterizethe circadian system of the rock cavy using immunohisto-chemical techniques. The SCN and the IGL were evaluated asto their cytoarchitecture, retinal afferents, and the presence ofneuroactive substances and neuronal or glial markers. Thecontent in calcium binding proteins of these centers in thisspecies has already been described in a study of our laboratory(Cavalcante et al., 2008). Likewise, retinal projections to thethalamic paraventricular nucleus, a putative circadian centerwas also described (Nascimento et al., 2008).

2. Results

2.1. Suprachiasmatic nucleus

In Nissl-stained coronal sections, the rock cavy SCN was seenas paired cluster cells located in the anterior hypothalamus,dorsal to the optic chiasm, on each side of the third ventricle.At rostral levels (Figs. 1 and 2A), the SCN appeared as atriangular shaped nucleus and at mid (Figs. 1 and 2B) andcaudal (Figs. 1 and 2C) levels, the nucleus assumed a pear-shaped contour, with its larger axis directed dorsoventrally,having medial and ventral boundaries more precise thandorsal and lateral ones. In the entire extent, the nuclei wereseen to be separated each other partially by the third ventricle,being contacted by only its most dorsal portions. Apparently,there was an agglomerate of compact and darkly stained cellsin the central region, surrounded by an area of sparser and lessstained cells. Within this cell cluster, mostly in the caudalsections, it was also possible to visualize a set of cells closelycompacted forming a ventral portion, and another one ofloosely collected cells, constituting a dorsal portion (Figs. 2Band C). Each SCN was around 600 µm along its rostrocaudal

Fig. 1 – Schematic representation of the cytoarchitecture, retinal projections, cells and fiber distribution of 5 neurochemicals atthree rostrocaudal levels through the SCN. Dots represent perikarya and lines with small dots represent fibers and terminals.oc, optic chiasm; 3v, third ventricle; SCN, suprachiasmatic nucleus.

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length and measured approximately 350 µm in width and400 µm in height at mid level.

In coronal sections from animals intraocularly injectedwith CTb, both SCNswere filled with CTb-immunoreactive (IR)terminals, with evident contralateral predominance. At rostraland mid levels CTb-IR terminals ramified forming a compactplexus in the ventral portion, expanding laterally andmedially, shaping a crescent of dorsal concavity only sparselyfilled by CTb-IR terminals (Figs. 1 and 2E). In the caudalsections,mainly in the contralateral side, the CTb-IR terminalsfill densely and uniformly all nucleus, extending dorsallybeyond their cytoarchitectonic boundaries (Figs. 1 and 2F).

VP-IR perikarya and fibers/terminals were observed only atrostral levels of the SCN, being more concentrated in thedorsal portion (Figs. 1 and 3A).

VIP-IR perikarya immersed in a dense neuropil were foundin a ventromedial position in the SCN atmid and caudal levels.The remainder of the nucleus is only sparsely occupied by VIP-IR terminals and rare cell bodies. None VIP-IR elements wereseen at rostral levels (Figs. 1 and 3B).

A moderate plexus of NPY-IR fibers/terminals was ob-served within the SCN throughout its rostrocaudal length andmore concentrated in the ventral part of the nucleus (Figs. 1and 3C). No NPY-IR cell bodies were found in the SCN.

Fig. 2 – Photomicrographs of coronal sections of the SCN at rostral (A and D), middle (B and E) and caudal (C and F) levels,illustrating its cytoarchitectonic characteristics by Nissl staining (A–C) and the distribution pattern of retinal projections (D–F).Dashed lines in A–C represent the delimitation of the SCN based on a combination of cytoarchitecture, retinal projections andneurochemical content. oc, optic chiasm; 3v, third ventricle; SCN, suprachiasmatic nucleus. Scale bar: 220 μm.

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The rock cavy SCN showed a dense plexus of 5-HT-IRfibers/terminals at mid and caudal sections, seemingly fillingall cytoarchitectonic limits, but it was possible to visualize ahigher density at the dorsomedial contour, appearing toencompass medially the VIP-immunoreactivity area (Figs. 1and 3D).

GAD-IR perikarya and fibers/terminals were visualized inall rostrocaudal extensions of the SCN, on a dorsal position atrostral levels and in the periphery of the nucleus around alittle centrolateral region devoid of GAD immunoreactivity atmid and caudal levels (Figs. 1 and 3E).

The rock cavy SCN was completely devoid of any GALimmunoreactivity, incontrast to thesurroundinghypothalamus,which contained a dense network of GAL positive fibers andterminals (Fig. 3F).

The rock cavy SCN was also completely devoid of SPimmunopositive elements, contrasting with the surroundinghypothalamus, which contained a dense mesh of SP positivefibers and terminals (Fig. 4A).

ENK-IR fibers/terminals were also observed in the rostralsections of the SCN, being more concentrated in the lateralpart of the nucleus (Fig. 4B).

Fig. 3 – Photomicrographs of SCN coronal sections showing the immunoreactivity pattern against VP (A), VIP (B), NPY (C), 5-HT(D), GAD (E) and GAL (F). The section is at rostral level in A and at middle level in B–F. Abbreviations as in Fig. 2. Scale bar:210 μm.

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GFAP-immunoreactivity was also detected in the wholeSCN, although without great contrast with surroundinghypothalamic areas (Fig. 4C).

2.2. Intergeniculate leaflet

The rock cavy lateral geniculate complex in the Nissl-stainedsections appeared as containing two distinct structures: theconspicuous DLG and the VLG. Less distinctly, it could benoticed as a less dense cell population, as a thin leaflet locatedbetween the DLG and the VLG, which, by its location, could beconsidered to be the IGL (Figs. 5 and 6A–C).

In coronal sections from rock cavies intraocularly injectedwith CTb, CTb-IR terminals were seen bilaterally in everydivision of the lateral geniculate complex, including the IGL.However, it was observed as a strong contralateral predomi-nance related toGLDandGLV, andanalmostat symmetry to the

IGL. By the distribution of the CTb-retinal terminals, it could beseen that the rostral and middle IGL assumes its classic leafletshape between the DLG and VLG (Figs. 5 and 6D–E, onlycontralateral side shown). At the caudal level it exhibits adescending portion which outlines the VLG laterally and themedial geniculate nucleus (MG) medially (Figs. 5 and 6F).

A fewNPY immunopositive cells, fibers and terminals weredetected in the mid and caudal IGL. These neurons paralleledthe latero-lateral axis of the nucleus. No NPY-IR cell body wasvisualized in the DLG and VLG (Figs. 5 and 7A).

Scarce NOS-IR neurons were found in the IGL, contrastingwith the VLG, which contains a dense distribution of neuronscontaining this substance (Fig. 7B).

A few immunopositive ENK fibers and terminals were alsoobserved in all latero-lateral extension of the IGL, althoughdenser medially, contrasting with the complete absence ofthese elements in the DLG and VLG (Fig. 7C).

Fig. 4 – Photomicrographs of SCN coronal sections showingthe immunoreactivity pattern against SP (A), ENK (B), andGFAP (C). Abbreviations as in Fig. 2. Scale bar: 210 μm.

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GFAP-IR was abundant within the IGL, throughout itsrostrocaudal extent, contrastingwith a lesser density observedin the adjacent DLG and VLG (Fig. 7D).

3. Discussion

3.1. Suprachiasmatic nucleus

The rock cavy SCN, like that of other mammals, is distin-guished from the adjacent hypothalamus, since it is identifiedby the presence of a darkly Nissl-stained cell group located ateach side of the third ventricle and dorsal to the optic chiasm.The shape and relation to the third ventricle is similar to thatfound in hamster, in that both nuclei are only partiallyseparated by the third ventricle at mid and caudal levels(Card and Moore, 1984). The topography conforms to thepattern described for all mammals studied (see, for example,

Lydic et al., 1982; Card and Moore, 1984; Cassone et al., 1988;Smale et al., 1991; Tessoneaud et al., 1994; Costa et al., 1998;Goel et al., 1999; Smale and Boverhof, 1999; Abrahamson andMoore, 2001), what is consistent with the suggestion that themammalian SCN is a phylogenetically stable neural structure.

According toour results, the rockcavySCNreceivesabilateral,predominantly contralateral retinal projection,which ramifies ina dense arched plexus in the ventral region. Considering thepattern of distribution of terminals in the sectional area of theSCN, this is similar to that found in the rat (Levine et al., 1991) andcommon marmoset (Costa et al., 1998, 1999). The RHT ramifiesfilling practically all cytoarchitectonic limits of the SCN in thehamster (Johnsonetal., 1988b; Lingetal., 1998;Muscatetal., 2003)and mouse (Abrahamson and Moore, 2001).

A common feature of the SCN of all mammalian speciesstudied is the bilateral innervation from the retina, except forsome squirrel species, in which that innervation is describedto be exclusively contralateral (Agarwala et al., 1989; Smale etal., 1991). The pattern of the retino-SCN innervation has beendescribed as almost completely contralateral, predominantlyipsilateral, or with almost complete bilateral symmetry(Magnin et al., 1989; see comments in Costa et al., 1999).

The functional significance of variability in the pattern ofRHT innervation in theSCN remainsunknown. Inanearly study(Magnin et al., 1989) an evolutive theory was proposed. It wassuggested that the retino-SCN innervation has evolved from acontralateral predominance or bilateral equivalence in rodentsto an ipsilateral predominance in insectivores and primates(Magnin et al., 1989). However, there are many conflictingfindings around this theory. For example, the retino-SCNprojection is described to be ipsilateral in someprimate species,such as the chimpanzee (Tigges et al., 1977), bush baby, potto,gibbon and macaque (Macaca fascicularis) (Magnin et al., 1989).However, that projection exhibits a contralateral pattern inother primate species, such as Rhesusmonkey (Hendrickson etal., 1972; Moore, 1973), squirrel monkey (Tigges and O'Steen,1974) and commonmarmoset (Costa et al., 1999; see commentsin Costa et al., 1999). It is also clear that variations are notassociated to neither photoperiodism (Youngstron and Nunez,1986`) nor aspects of a temporal niche of the animals. Forexample, when comparing two diurnal rodent species, the RHTis predominantly contralateral in the grass rat (Smale andBoverhof, 1999) and bilateral and symmetrical in the degu (Goelet al., 1999). The contralateral pattern found in the retino-SCNprojection in the rock cavy (present results) is also found indiurnal species, suchas thegrass rat (Smale andBoverhof, 1999),the common marmoset (Costa et al., 1999), or nocturnal ones,such as the rat (Levine et al., 1991), to cite some examples. Thus,that is not an exclusive feature of a crepuscular species. It is alsopossible that differences merely reflect individual variationsand methodological drawbacks. We cannot exclude, for exam-ple, the possibility that the contralateral or ipsilateral asymme-try could be due to selective uptake of the tracer by specificgroups of ganglion cells in the retina of different species. Whatappears to be undisputable is that each SCN receives a denseretinal input necessary and sufficient for synchronizing SCNpacemaker cells to the environmental light/dark cycle (Johnsonet al., 1988a).

The presence of twomajor cell groups, one VP and the otherVIP, is considered to be a typical feature of themammalian SCN.

Fig. 5 – Schematic representation of the cytoarchitecture, retinal projections (contralateral side to the injected eye) and NPYcells and fiber distribution at three rostrocaudal levels through the IGL. Dots represent perikarya and lines with small dotsrepresent fibers and terminals. DLG, dorsal lateral geniculate nucleus; VLG, ventral lateral geniculate nucleus; IGL,intergeniculate leaflet.

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VP-IR neurons located in the dorsal part of the rock cavy SCN(current results) are similar to those found in several mamma-lian species studied, such as the golden hamster (Card andMoore, 1984), rat (Van den Pol and Tsujimoto, 1985; Buijs et al.,1995;Moore et al., 2002;Morin et al., 2006),mouse (Cassone et al.,1988; Abrahamson and Moore, 2001; Morin et al., 2006), groundsquirrel (Reuss et al., 1989), grass rat (Smale andBoverhof, 1999),and degu (Goel et al., 1999). At present, in only two species, themusk shrew (Tokunagaetal., 1992) and themink (Martinet et al.,1995), no VP-IR cells have been identified in the SCN. Thedistribution of VIP-IR neurons in the predominantly ventralposition in the rock cavy SCN is similar to that found inmost ofthe species studied, such as the rat (Van den Pol and Tsujimoto,1985; Moore et al., 2002; Morin et al., 2006), hedgehog (Antono-poulos et al., 1987), mouse (Cassone et al., 1988; AbrahamsonandMoore, 2001;Morin et al., 2006),mole (Kudoetal., 1991), degu(Goel et al., 1999), and grass rat (Smale and Boverhof, 1999). Incontrast, opossums (Cassone et al., 1988) have VIP cells in thedorsomedial region of theSCN.Asaunique characteristic of thisspecies, VP-IR neurons were found only at rostral SCN, andthose VIP-IR were found only at mid and caudal levels,suggesting that besides a dorsal–ventral organization, the rockcavy SCN exhibits a rostral–caudal organization in respect to VPand VIP neuron distribution. Under light–dark conditions, VPlevels reveal a peak at early light phase and a trough during thedarkphase, a pattern that ismaintainedunder constant dark, or

constant light, characterizing a circadian rhythm in the rat SCN(Tominaga et al., 1992). Furthermore, there is evidence that VPcells are involved in relaying timing information about activityfrom the SCN to other parts of the brain (Bult et al., 1993; Buijs etal., 1995). Electron microscopic studies have shown that mostafferents to the SCN, such as glutamatergic retinal afferents,NPY fibers from the IGL and 5-HT fibers from the raphe, makesynaptic contacts with VIP cells (Hisano et al., 1988a,b; Ibata etal., 1989). Besides that, it was evidenced that VIP content in therat SCN does not show circadian rhythm in constant darkness,but under light–dark conditions, it decreases over the course ofthe light phase, recovering gradually during the dark phase(Shinohara et al., 1993). Taken together, these data corroboratethe hypothesis that VIP neurons are involved in the synchro-nization of circadian rhythms. A more recent study providesevidence supporting a role in promoting rhythmicity for VIPneurons (Aton et al., 2005). VIP neurons participate in localconnectionswithin theventral regionandwithVP-IRneurons inthedorsal region (Ibataetal., 1993). Furthermore,VPcells projectto the VIP cells, providing an anatomical support for a feedbackmechanism, throughwhichmessages related to environmentallighting conditions may be constantly modulated by temporalinformation on a circadian basis (Jacomy et al., 1999).

In line with the findings in the SCN of all widely-studiedspecies, except humans (Mai et al., 1991), the rock cavy SCNdoes not contain NPY-IR cells, but instead contains a plexus of

Fig. 6 – Photomicrographs of the lateral geniculate complex coronal sections at rostral (A and D), middle (B and E) and caudal(C and F) levels, illustrating its cytoarchitectonic characteristics by Nissl staining (A–C) and the distribution pattern of retinalprojections in the contralateral side to the injected eye (D–F). Dotted lines in A–C represent the delimitation of the IGL based in acombination of cytoarchitecture, retinal projections and neurochemical content. DLG, dorsal lateral geniculate nucleus; VLG,ventral lateral geniculate nucleus; IGL, intergeniculate leaflet; MG, medial geniculate nucleus. Scale bar: 180 μm.

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NPY-IR fibers and terminals restricted to the ventral SCN. Thispattern is similar to that found in some species such as the rat(Van den Pol and Tsujimoto, 1985; Moore et al., 2002), commonmarmoset (Costa et al., 1998; Cavalcante et al., 2002), grass rat(Smale and Boverhof, 1999), and degu (Goel et al., 1999), but inother species NPY-IR terminals fill its cytoarchitectonic limits,as for example, the hamster (Card and Moore, 1984) andmouse (Cassone et al., 1988; Abrahamson and Moore, 2001).NPY is thought to modulate photic and certain kinds of non-photic information to the SCN via the IGL/GHT (Mrosovsky,1995; Shinohara and Inouye, 1995; Muscat and Morin, 2006).

A dense plexus of 5-HT-IR fibers and terminals is presentat the middle and caudal levels of the rock cavy SCN, seem-ingly overlapping the VIP-IR cell area. Although species differ-ences exist with respect to the density and pattern ofdistribution, 5-HT-IR fibers and terminals were found in theSCNofall studied species, suchas rat, goldenhamster, squirrels,

andcat SCN(Uedaetal., 1983), sheep (Tilletet al., 1989), commonmarmoset (Costa et al., 1998; Cavalcante et al., 2002; Pinato et al.,2007), cynomolgusmonkey and human (Moore and Speh, 2004)and capuchin monkey (Cebus apella) (Pinato et al., 2007). It hasbeen suggested that 5-HT from the raphe nuclei cells modulatephotic responses (Cagampang and Inouye, 1994; Meyer-Bern-stein and Morin, 1996) as well as non-photic information ingolden hamsters (Bobrzynska et al., 1996).

According to our results, the rock cavy SCN contains GABA-producing neurons, evidenced by GAD immunoreactivity.GABA is also present in rodent SCN neurons (Van den Pol andTsujimoto, 1985; Moore and Speh, 1993; Buijs et al., 1995;Tanaka et al., 1997; Castel and Morris, 2000; Abrahamson andMoore, 2001), with varying degrees of colocalization with VPandVIP (Buijs et al., 1995, Tanaka et al., 1997, Castel andMorris,2000). There is evidence that GABA controls the amplitude ofcircadian rhythms in SCN neurons (Aton et al., 2006).

Fig. 7 – Photomicrographs of the lateral geniculate complex coronal sections showing the immunoreactivity pattern againstNPY (A), NOS (B), ENK (C), and GFAP (D). Abbreviations as in Fig. 6. Scale bar: 170 μm.

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GAL-IR cells or fibers are absent in the rock cavy SCN, incontrast to the adjacent hypothalamic regions. GAL-IR fibersand terminals were observed at periphery, but extending intothe shell subdivision at caudal levels of the mouse SCN(Abrahamson and Moore, 2001). GAL-IR neurons colocalizedwith VP have been reported in the human SCN (Gai et al., 1990).GAL receptor subtype R2 expression has also been detected inthe rat SCN (Mitchell et al., 1999). However, the role of GAL incircadian rhythm regulation is not well understood.

SP immunoreactivity is absent in fibers or cells in the rockcavy SCN. However, the distribution of this neurotransmitterin the SCN is a strong example of species variation. Thus, SP-IRterminals branch out into a dense plexus in the ventral SCN ofthe rat (Van den Pol and Tsujimoto, 1985; Takatsuji et al., 1991;Larsen, 1992) and marmoset (Costa et al., 1998), and in theperipheral portion of the golden hamster SCN (Morin et al.,1992), but are sparse in the degu (Goel et al., 1999), groundsquirrel (Smale et al., 1991) and mouse (Abrahamson andMoore, 2001). Rare SP positive perikaryawere found in the SCNof the hamster (Reuss and Burger, 1994; Reuss et al., 1994,Morin et al., 1992) and grass rat (Smale and Boverhof, 1999) butwere very scarce in themouse (Abrahamson andMoore, 2001).It is interesting to note that in all species in which SP-IRperikarya were identified, it used the pre-treatment withcolchicine. It has been suggested that SP fibers convey lightinformation to the SCN (Takatsuji et al., 1991; Piggins et al.,1995; Abe et al., 1996). However, this observation has not beenconfirmed in later studies (Hannibal and Fahrenkrug, 2002).

ENK-IR fibers and terminals, but not cells in the rock cavySCN is a result that corroborates what is found in some speciessuch as humans (Mai et al., 1991) and sheep (Tessoneaud et al.,

1994), but differs from what is found in others such as thehamster (Card and Moore, 1984; Morin et al., 1992), rat (Cardand Moore, 1989), ground squirrel (Smale et al., 1991), degu(Goel et al., 1999) and mouse (Abrahamson and Moore, 2001),in that ENK positive cells are also present in the SCN of thesespecies, although with a variable distribution pattern.

Intense GFAP-immunoreactivity is found in the rock cavySCN, although with the same density as that of surroundinghypothalamic areas. In rats and hamsters (Morin et al., 1989)and mice (Moriya et al., 2000; Santos et al., 2005) the SCN ismarked by dense GFAP-immunoreactivity, which is signifi-cantly higher than in adjacent hypothalamic areas. The GFAPexpression exhibits a circadian rhythm in the hamster(Lavialle and Servière, 1993; Lavialle et al., 2001) and mouse(Santos et al., 2005) and it appears to be under a seasonalinfluence in the rat (Gerics et al., 2006), suggesting that thisprotein plays an essential role in the clock mechanisms.

3.2. Intergeniculate leaflet

As inmostmammals, the rock cavy IGL is located between theDLG and VLG as a thin leaflet. It is delimited by retinal projec-tions and neuropeptidergic content.

Oneof criteriaused todefine the IGL is thepresenceofbilateralretinal afferents (Morin et al., 1992). The rock cavy IGL receives amassive bilateral retinal projection with a discrete contralateralpredominance. This pattern is very similar to that found in thedegu (Goel et al., 1999), musk shrew (Mizuno et al., 1991), rat(Hickey and Spear, 1976; Moore and Card, 1994), golden hamster(Morin et al., 1992), rabbit (Takahashi et al., 1977), hedgehog(Dinopoulos et al., 1987) and squirrel (Agarwala et al., 1989).

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Another criteria used to delineate the IGL is the presence ofNPY immunoreactivity. In the rock cavy IGL, NPY-IR scatteredcells immersed in a mesh of fibers and terminals weredistributed in the nucleus. This was also found in thesubterranean mole rat (Negroni et al., 2003). In species suchas the degu (Goel et al., 1999), hamster (Morin et al., 1992), andrat (Moore and Card, 1994), the IGL is easily identified by itsabundant content of NPY-IR cells and fibers. The NPY cells ofthe IGL are the origin of the GHT in rodents (Morin andBlanchard, 1995; Moore and Card, 1994). The presence of NPYin the rock cavy IGL, although in small quantity, as well as thepresence of NPY terminals in the SCN, suggests the existenceof the pathway in this species.

We have identified ENK terminals in the IGL of the rockcavy. Our results are similar to those found in other rodents(Moore and Card, 1994) and in contrast to those found in thesubterraneous mole rat (Negroni et al., 2003). ENK positivecells are absent in the rock cavy IGL. In the rat, ENK positivecells project only to the contralateral IGL (Card and Moore,1989), but in the hamster they also project to the SCN (Morin etal., 1992). These projections are probably lacking in the rockcavy.

Like rats and hamster (Morin et al., 1989), GFAP-IR allows usto delimitate the rock cavy IGL in all its rostrocaudal extent.GFAP-IR was shown to be increased in the IGL of micesubmitted to constant light conditions (Moriya et al., 2000).However, the role of the GFAP in the IGL related to circadianrhythm regulation is not well understood.

4. Conclusion

In summary, this paper provides the characterization of theretinal innervation and chemical phenotypic organization ofthe SCN and IGL in a crepuscular active rodent, the rock cavy.From the analysis of the data in comparison with that fromthe literature, it is possible to conclude that the rock cavy SCNand IGL sharemany characteristics with several other species,but reveal some singularities, such as a rostrocaudal organi-zation related to VP-VIP cell distribution. Although thecytoarchitecture, the retinal innervation and the chemoarch-itecture may suggest an organizational division of the rockcavy SCN, it does not match classical schemes proposed, suchas dorsal–ventral or core–shell organization. Additional hodo-logical and molecular studies are needed to establish acoherent division of the SCN in this species. Furthermore, noapparent relationship between lifestyles or temporal nichesand structures emerges, with respect to either the pattern ofretinal innervation to the SCN and IGL, or the neurotransmit-ter content and distribution in these structures. However, thepresent results mean an important contribution that allows toestablish the rock cavy as a regional experimental model tothe study of biological rhythms in mammals.

5. Experimental procedures

Ten adult male rock cavies (body weight range, 300–500 g)captured fromrural citiesof thestate of theRioGrandedoNorte,Brazil, were used in this study. The capture and handling of the

animals were authorized by IBAMA (no. 007/05). The mainte-nance and use of all experimental animals adhered to theethical requirements approved by the Brazilian Society ofNeuroscience and Behavior, which follows the recommenda-tions of the Society for Neuroscience (USA). The animals werehousedundernatural light (sunriseat05:20 h±1 minandsunsetat 17:15 h±1min), temperature (25.9±1 °C, mean±SEM; mini-mum 22.3 °C; maximum 29.8 °C) and humidity (82.4±2.1%;minimum 72.0%; maximum 90.0%) conditions, with food andwater freely available. All surgical procedures, such as injectionand perfusion, were performed in the morning around 9:00 am.

The rock cavies were anesthetized with ketamine (40 mg/kg i.m.), and xylazine (4 mg/kg i.m.), placed on a surgical table,and restrained in a headholder. After topical application oftetracaine hydrochloride to the cornea, they received aunilateral intraocular injection of cholera toxin subunit B(CTb) (List Biological Laboratories, Inc., Campbell, CA). A totalof 80 µl of a 1 mg/ml aqueous CTb, containing 5% dimethyl-sulfoxide, was injected into the vitreous humor through a 30-gauge needle catheter attached to a micropump, whichpumped the solution at a rate of 1 µl/min. To minimize refluxand the spread of the tracer to the extraocular muscles as wellas to avoid postoperatory local infection, the ocular surfacewas cleaned with saline during the surgical procedure. Theneedle was left in the site until 15 min post-injection and thenwithdrawn. The ocular surface was then rewashed with salineand antibiotic ointment was topically applied. After 5–7 dayspost-injection, the rock cavies were reanesthetized with thesame anesthetic drugs and perfused transcardially with salinein 0.1 M phosphate-buffer pH 7.4 (PB) followed by 4%paraformaldehyde in PB solution. After perfusions, theanimals were positioned in the stereotaxic frame and theincisor bar was adjusted until the lambda and bregma heightswere equal. The skull bone was then removed and the brainsexposed. The brains were divided into three blocks by twocoronal sections: one at a plane at 2.00 mm anterior to thebregma and another at the lambda level. The brains wereremoved and, after post-fixation in the same fixative for 2–4 h,stored in a 30% sucrose in PB solution.

The frozen brains were cut into 30 µm coronal sections,which were obtained parallel to the stereotaxic planes bymounting each block on the stereotaxic plane of a horizontalsliding microtome. The sections were collected in PB and aone-in-six series was subjected to immunohistochemistry todetect CTb. Floating sections were incubated with a goat anti-CTb antiserum (List Biological Labs, Campbell, CA, USA)diluted at 1:5000 in PB containing 0.4% Triton X-100 and 5%normal donkey serum for 18–24 h. The sections were incubat-ed with a secondary antiserum (biotinylated donkey anti-goatIgG; Jackson Labs, Westgrove, PA, USA) diluted at 1:1000 in thesame medium as above for 90 min. The sections were reactedwith ABC reagent (Elite ABC kit, Vector Labs, Burlingame, CA,USA) for 90 min and then reacted for peroxidase activity in asolution of diaminobenzidine (DAB) tetrahydrochloride and0.01% H2O2 in PB. After three rinses in PB, the sections weremounted on precleaned and gelatin-chrome alumen-coatedslides and allowed to dry. The sections were then treatedwith osmium tetroxide to enhance the visibility of the reac-tion product. The sections were then dehydrated, delipidatedand coverslipped with DPX. One of the remaining series was

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Nissl-stained to identify the cytoarchitectonic boundaries ofthe regions under examination. The remaining series wereimmunostained for vasopressin (rabbit anti-VP antiserum),vasoactive intestinal polypeptide (rabbit anti-VIP antiserum),neuropeptide Y (rabbit anti-NPY antiserum), serotonin (mouseanti-5-HT antiserum), glutamic acid descarboxilase (rat anti-GAD antiserum), galanin (rabbit anti-GAL antiserum), sub-stance P (rat anti-SP antiserum), enkephalin (mouse anti-ENKantiserum), or glial fibrillary acidic protein (mouse anti-GFAPantiserum). The sections were washed in PB and incubatedwith their respective primary antibodies (VP, Chemicon,1:2000; VIP, Chemicon, 1:3000; NPY, Sigma, 1:5000; 5-HT,Chemicon, 1:3000; GAD, Sigma, 1:3000; GAL, Chemicon,1:1000; ENK, SP, Sigma, 1:3000; Chemicon, 1:3000; GFAP,Sigma, 1:2000) for 18–24 h. The ABC protocol was usedfor immunodetection and subsequent procedures wereperformed as described for CTb. Each antigen was examinedin 4 animals on average. All immunohistochemical proce-dures were performed at room temperature. As a control forthe specificity of staining, some sections were submitted tothe immunohistochemical reaction without the primaryantiserum.

The sections were examined under brightfield or darkfieldillumination in an Olympus microscope, and digital images ofrepresentative sectionswere takenwith a digital video camera(Nikon, DXM1200). The images were minimally processed forbrightness and contrast using Adobe Photoshop 7.0. Measuresof the brain sections were taken using Image Tool 3.0 software(available at the site ddsdx.uthscsa.edu/dig/itdesc.ht).

Acknowledgments

This study was supported by funding from the NationalCounsel of Technological and Scientific Development (CNPq),Coordination for Improvement of High Level Staff (CAPES), andFoundation for the Support of Research of the State of SãoPaulo (FAPESP).

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