Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

b r a i n r e s e a r c h 1 5 8 6 ( 2 0 1 4 ) 9 9 – 1 0 8

http://dx.doi.org/100006-8993/& 2014 El

Abbreviations: 3V

AM, anteromedial

nucleus; DAB, diam

nucleus; IAM, inte

mt, mammillotha

nucleus; PV, parav

nucleus, posterior p

medullaris of the thnCorresponding autE-mail address: e1Present address

Norte, 59072-970 Na

Research Report

Serotonergic fibers distribution in the midline andintralaminar thalamic nuclei in the rock cavy (Kerodonrupestris)

Alane de Medeiros Silvaa, Melquisedec Abiare Dantas de Santanaa,Paulo Leonardo Araujo de Gois Moraisa, Twyla Barros de Sousaa,Rovena Clara Galvao Januario Engelberthb, Eudes Euler de Souza Lucenab,Clarissa Loureiro das Chagas Campeloc, Jeferson Sousa Cavalcanteb,Judney Cley Cavalcantea, Miriam Stela Maris de Oliveira Costaa,Expedito Silva do Nascimento Jra,n,1

aDepartment of Morphology/Laboratory of Neuroanatomy, Biosciences Center, Federal University of Rio Grande do Norte,59072-970 Natal, RN, BrazilbDepartment of Physiology/Laboratory of Neurochemical Studies, Biosciences Center, Federal University of Rio Grandedo Norte, 59072-970 Natal, RN, BrazilcDepartment of Physiology/Memory Studies Laboratory, Biosciences Center, Federal University of Rio Grande do Norte,59072-970 Natal, RN, Brazil

a r t i c l e i n f o

Article history:

Accepted 16 August 2014

The thalamic midline/intralaminar complex is part of the higher-order thalamus, which

receives little sensory input, and instead forms extensive cortico-thalamo-cortical pathways.

Available online 23 August 2014

Keywords:

Serotonergic system

Midline/intralaminar nuclei

Thalamus

Rock cavy

.1016/j.brainres.2014.08.04sevier B.V. All rights res

, 3rd ventricle; 5-HT,

thalamic nucleus; AOI,

inobenzidine; f, fornix

ranteromedial thalamic

lamic tract; PC, paracen

entricular thalamic nucl

art; Re, reuniens thalam

alamus; Sub, submediuhor. Fax: þ55 84 32119207xpeditojr@cb.ufrn.br (E.S: Department of Morphotal, RN, Brazil.

a b s t r a c t

The midline thalamic nuclei connect with the medial prefrontal cortex and the medial

temporal lobe. On the other hand, the intralaminar nuclei connect with the fronto-parietal

cortex. Taking into account this connectivity pattern, it is not surprising that the midline/

intralaminar complex has been implicated in a broad variety of cognitive functions, including

memory process, attention and orientation, and also reward-based behavior. Serotonin (5-HT)

is a neurotransmitter that exerts different post-synaptic roles. Serotonergic neurons are

almost entirely restricted to the raphe nuclei and the 5-HT fibers are distributed widely

7erved.

serotonin; 5-HT-IR, 5-HT immunoreactive fibers; AD, anterodorsal thalamic nucleus;

area of interest; CL, centrolateral thalamic nucleus; CM, central medial thalamic

; fr, fasciculus retroflexus; Hb, habenular nucleus; IAD, interanterodorsal thalamic

nucleus; IMD, intermediodorsal thalamic nucleus; MD, mediodorsal thalamic nucleus;

tral thalamic nucleus; PF, parafascicular thalamic nucleus; PT, paratenial thalamic

eus; PVA, paraventricular thalamic nucleus, anterior part; PVP, paraventricular thalamic

ic nucleus; Rh, rhomboid thalamic nucleus; ROD, relative optical density; sm, stria

s thalamic nucleus; VRe, ventral renuens thalamic nucleus; ZI, zona incerta..d. Nascimento Jr).logy/Laboratory of Neuroanatomy, Biosciences Center, Federal University of Rio Grande do

b r a i n r e s e a r c h 1 5 8 6 ( 2 0 1 4 ) 9 9 – 1 0 8100

Optical density

Fiber morphology

throughout the brain, including the midline/intralaminar complex. The present study

comprises a detailed description of the morphologic features and semiquantitative analysis

of 5-HT fibers distribution in the midline/intralaminar complex in the rock cavy, a typical

rodent of the Northeast region of Brazil, which has been used by our group as an anatomical

model to expand the comprehension about phylogeny on the nervous system. The 5-HT fibers

in the midline/intralaminar nuclei of the rock cavy were classified into three distinct

categories: (1) beaded fibers, which are relatively fine and endowed with large varicosities;

(2) fine fibers, with thin axons and small varicosities uniformly distributed in whole axon; and

(3) stem axons, showing thick non-varicose axons. Moreover, the density of 5-HT fibers is

variable among the analyzed nuclei. On the basis of this diversity of the morphological fibers

and the differential profile of optical density among the midline/intralaminar nuclei of the

rock cavy, we conclude that the serotonergic system uses a diverse morphologic apparatus to

exert a large functional repertory in the midline/intralaminar thalamic nuclei.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

The rock cavy (Kerodon rupestris), a typical rodent in theNortheast region of Brazil, dwells in the mountainous regionsof the Caatinga in the Brazilian semi-arid interior. Morpho-logical (Silva Neto, 2000) and molecular biology studies (Roweand Honeycutt, 2002) put the genus Kerodon in the familyHydrochaeridae, in which the capybara (Hydrochoerus hydro-chaeris) is also included. The rock cavy has a crepuscular habit(Sousa and Menezes, 2006) and has been used in severalneuroanatomical studies in our laboratory (Cavalcante et al.,2008; Nascimento et al., 2008, 2010a, 2010b; Soares et al.,2012). The rock cavy has advanced our understanding aboutthe nervous system, providing a framework for the interpre-tation of evolutionary patterns, allowing inferences to bedrawn about the brain and its phylogenetic congruenceamong diverse biological features.

The structural organization of the raphe has been exten-sively studied in various species (Azmitia and Gannon, 1983;Dahlstrom and Fuxe, 1964; Dwarika et al., 2008; Fuxe et al.,1969; Harding et al., 2004; Hornung and Fritschy, 1988;Hornung, 2010; Limacher et al., 2008; Moon et al., 2007;Paxinos and Watson, 2007). The nuclear organization of theraphe serotonergic system in the rock cavy is similar to thatdescribed in other mammals with small anatomical varia-tions (Soares et al., 2012). Moreover, subsequent reports usingtracing techniques have pointed out the dorsal and medianraphe nuclei as the main sources of serotonergic projections(Azmitia and Segal, 1978; Moore et al., 1978; Morin and Meyer-Bernstein, 1999; Vertes and Martin, 1988; Vertes, 1991; Verteset al., 1999), although the majority of the serotonergic fibersare concentrated in limbic regions of the forebrain (Jacobsand Azmitia, 1992; Lowry et al., 2008; Steinbusch, 1981; Vertesand Linley, 2007, 2008). Serotonin (5-HT) exerts excitatorymodulatory activity on some of its targets (Curtis and Davis,1962; Kayama et al., 1989; Marks et al., 1987; Monckton andMcCormick, 2001; Rogawski and Aghajanian, 1980; Yoshidaet al., 1984). In other areas, however, it produces changes inthe conductance and permeability of cell membranes, caus-ing hyperpolarization of neurons (Mccormick and Pape, 1990;Lee and Mccormick, 1996). The functional duality of the 5-HT

in the thalamus has been widely associated with the varioustypes of receptors present in the different thalamic nuclei(Chapin and Andrade, 2001a, b; Lopez-Gimenez et al., 1998,2001; Kia et al., 1996; Mengod et al., 1996; Pompeiano et al.,1994), as well as in differences in fibers morphology, origin,and preference in area of innervation (Wilson, 1989). The5-HT axons are generally divided into three morphologicaldifferent fiber types. Thin axons with small fusiform orgranular boutons, named fine fibers, are supposed to origi-nated from the dorsal raphe nucleus; the beaded fibers withlarge spherical boutons; and non-varicose stem-axons origi-nated from the beaded fibers, supposed to originated fromthe median raphe nucleus (Hornung and Fritschy, 1990;Wilson and Molliver, 1991).

A low-frequency stimulation of the midline/intralaminarthalamic nuclei induces a low synchronous activity in differentregions of the cortex (Dempsey and Morison, 1943). Thisthalamic nuclear complex is classically viewed as nonspecificthalamus, exerting a global influence on the cortical layers(Groenewegen and Berendse, 1994). Nowadays, the notion ofthe nonspecific functions has been changed as a result ofseveral anatomical studies which have demonstrated, that themidline/intralaminar complex projects to specific areas of thecerebral cortex, mainly to the prefrontal cortex (Groenewegenand Witter, 2004; Van der Werf et al., 2002; Vertes, 2006).Furthermore, the electrical stimulation of individual nucleiproduces selective effects on their cortical targets (Viana diPrisco and Vertes, 2006). Descriptions of the serotonergicprojections in the thalamus have been shown in monkey andrat studies (Cropper et al., 1984; Lavoie and Parent, 1991; Vertes,2002; Vertes et al., 2010) and suggest that the serotonergic fibersexert a fundamental role on modulation in the midline/intra-laminar thalamic nuclei. Furthermore, it is widely agreed thatcell types and morphological fiber pattern serve as the buildingblocks of nervous systems and that exploring their diversityand determining how cells are assembled into circuits isessential for understanding brain function.

In the present study we aim describe and compare thedistribution of serotonergic fibers in the rock cavy midline/intralaminar thalamic nuclei. We also assess the relativeabundance of serotonergic fibers throughout the various

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nuclei of the midline/intralaminar complex in the rock cavyand supply a morphological description of the 5-HT fibers andtheir differential distribution within this nuclear group.

2. Results

2.1. Cytoarchitectonic analysis

The midline nuclei in the rock cavy include the paraventri-cular (PV), paratenial (PT), intermediodorsal (IMD), reuniens(Re), and rhomboid (Rh) nuclei. On the other hand, theintralaminar nuclei contain the central medial (CM), centrallateral (CL), paracentral (PC), and parafascicular nuclei (PF).

2.1.1. Midline thalamic nucleiThe PV in the rock cavy is a conspicuous nucleus, lyingimmediately ventral to the third ventricle in the thalamicmidline. On coronal sections, it appears first as a triangularlyshaped nucleus. It extends through almost all the rostro-caudal area of the thalamus and is clearly divided inthree cytoarchitectonical divisions (PV anterior, PV middle,and PV posterior). Its lateral border is limited by the PT inrostral levels and by the mediodorsal nucleus (MD) in themiddle and caudal ones, while ventrally it borders on the CM(Fig. 1).

The PT is a rounded nucleus located in the dorsal thala-mus. In the rostral levels of the thalamus, it constitutes thelateral and inferior borders of the PV (Fig. 1A and B). In itscaudal extremity it merges with the MD. The IMD, as its namesuggests, is located between the right and left MD thalamicnucleus and is more easily visualized in the middle coronalsections of the thalamus with the PV (Fig. 1C–F). The Re islocated in the ventral part of the thalamic midline, immedi-ately dorsal to the third ventricle. Two cell clusters constituteit; situated laterally to the third ventricle in the rostral levels,they fuse together along thalamic rostrocaudal length(Fig. 1). Finally, the Rh is found just below the internalmedullary lamina. In rostral thalamic sections, the Rh isimmediately dorsal to the Re and in caudal ones it is easilydistinguished by its conspicuous shape and strongly stainedcells (Fig. 1C–F).

2.1.2. Intralaminar thalamic nucleiIn the rock cavy thalamus we have identified a CM nucleus asa conspicuous cell cluster located centrally in the internalmedullary lamina, distinct from the midline nuclei. Thisnucleus shows large stained cells along the length of thethalamic rostrocaudal. Laterally, the CM is continuous withPC and CL. PC appears laterally to the CM and its cells aredifficult to distinguish from those in the CM. The CM lies atthe curved portion of the internal medullary lamina, imme-diately lateral to the MD and medially to the ventral thalamicnucleus. Dorsally it is continuous with the CL nucleus. The CLis the most dorsal part of the intralaminar nuclei. It is difficultto identify its boundary with the PC. The CL is larger than theCM, PC, and PF and contains strongly stained cells (Fig. 1E andF). Finally, the PF is located in the caudal part of thethalamus. It can be clearly detected where the midbrainstructures start to appear. The PF is comprised of a large

round mass of cells, lying in close proximity to the fasciculusretroflexus and dorsally bordered by the CL and lateral to themedial habenula (Fig. 1G and H).

2.2. Relative optical density analysis

The ROD values were higher in the midline nuclei comparedwith the intralaminar ones [t(3)¼7.553, p¼0.005] (Fig. 2).Furthermore, several differences in the ROD values werefound by comparing nuclei from the midline and intralami-nar each other (Fig. 3). Comparisons among midline nucleieach other revealed significant increase ROD values in the Re[p¼0.003] and PV [p¼0.022] compared with PT. Although nodifferences in any other midline thalamic nuclei could bedetected, the photomicrographs suggest variations in theircontents (Fig. 3). When we used the one-way ANOVA [F(4,19)¼6.301, p¼0.002] and Tukey’s multiple comparisontests, the Re shows higher ROD values, followed by PV, Rh,IMD, and PT (Fig. 4). Comparisons among intralaminar nucleieach other revealed significant increase ROD values in the CM[p¼0.038]; PC [p¼0.005], and PF [p¼0.004] compared with theCL. The One-way ANOVA [F(3,15)¼8.506, p¼0.003] andTukey’s multiple comparison tests revealed a significantincreased ROD values among intralaminar thalamic nuclei,with the CL showing a higher ROD value followed by CM, PC,and PF (Fig. 5). There was no significant difference in RODvalues between male and female brains across the midline/intralaminar nuclei.

2.3. Descriptions of serotonergic fibers

The midline/intralaminar complex in the rock cavy receiveda multitude of fibers that demonstrated strong and distinctanatomical variations (Fig. 6). These fibers could be classifiedinto three distinct groups, based on morphological criteria:beaded fibers, fines fibers and stem-axons. In the midlinethalamic nucleus, the PV was amply supplied by the beadedfibers (Fig. 6D) with thin axons that had large varicosities,probably corresponding to the same beaded fiber (BF) typedescribed in other species (Hornung, Fritschy, 1990; Wilsonand Molliver, 1991). The thin axons with small fusiform andgranular varicosities, corresponding to the fine fiber (FF)(Fig. 6E), and thick non-varicose axons, probably the stem-axons (SA) (Fig. 6F), were seen uniformly distributed through-out the midline/intralaminar nuclei, except in PV.

3. Discussion

The rock cavy midline/intralaminar complex is denselyinnervated by serotonin immunoreactive fibers and thisinnervation has a complex pattern. Substantial variation inROD values and morphology of 5-HT fibers is detected amongmidline/intralaminar nuclei. The highest ROD value isobserved in the Re among the midline nuclei. In the intrala-minar nuclei, however, the highest ROD values is observed inthe CL. The medium ROD values are found in the PV, IMD,and Rh in the midline, while among intralaminar nuclei theCM and PC show medium ROD values. The lowest fiberdensities are observed in the PT and PF. The present work

Fig. 1 – Drawings of coronal sections through the rock cavy brain illustrating the morphology of the midline and intralaminarthalamic nuclei (A), (C), (E), and G), and digital images of Nissl stained coronal sections at corresponding levels (B), (D), (F), and(H). Rostral level representation in (A) and (B) and caudal level (G) and (H). Numbers on the right indicate distance from thebregma. See list of abbreviations. Bar 280 lm.

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is the first to analyze the thalamic midline/intralaminarcomplex organization using a Relative Optical Density toolto quantify serotonergic fibers in these nuclei.

Although the serotonergic system shows a firmly estab-lished morphological organization among numerous species(Bjarkam et al., 1997; Harding et al., 2004; Hornung, 2010;

Fig. 2 – ROD values across the midline and intralaminarthalamic nuclei of rock cavy (n¼8). The bars represent themeans of the mean ROD values in the midline andintralaminar nuclei of individual animals (across allrostrocaudal levels and nuclei). The paired t-test revealeda significant difference between the midline andintralaminar nuclei. Values are expressed as mean andstandard error of the mean. npo0.05—[t(3)¼7.553, p¼0.005].

Fig. 3 – Semiquantitative analysis showing the densities of5-HT-IR fibers in the different midline/intralaminar nuclei.To compare across these nuclei, the Relative Optical Densityvalues in each nucleus are displayed as levels of gray in thetable at the left. In the table, nuclei are listed accordingrostracaudal levels. See list of abbreviations.

Fig. 4 – ROD values in the midline thalamic nuclei of rockcavy (n¼8). The bars represent the means of the mean RODvalues of individual animals (across all rostrocaudal levels).One-way ANOVA revealed ROD significant differencebetween the nuclei analyzed. Values are expressed as meanand standard error of the mean. np¼0.003 nnp¼0.022—Tukey’s multiple comparison test [F(4,19)¼6.301, p¼0.002].See list for abbreviations.

Fig. 5 – ROD values in the intralaminar thalamic nuclei ofrock cavy (n¼8). The bars represent the means of the meanROD values of individual animals (across all rostrocaudallevels). One-way ANOVA revealed ROD significant differencebetween the nuclei analyzed. Values are expressed as meanand standard error of the mean. np¼0.038 nnp¼0.005nnnp¼0.004—Tukey’s multiple comparison test [F(3,15)¼8.506, p¼0.003]. See list for abbreviations.

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Jacobs et al., 1984), we observed several differences in theserotonergic innervation of the rock cavy midline/intralami-nar nuclei compared with rodents and primates. In albinorats, the PV, Rh and Re show a high fiber density, while the PThas a low fiber density (Cropper et al., 1984). Another studywith rats using SERT (serotonin transporter), has reported thehighest fiber density in the PV, followed by Rh, Re, and IMD(Vertes et al., 2010). In contrary, in the present study, the RODvalues shown suggest higher density in the Re. In our results,based on ROD values, the intralaminar nuclei show moderateor low fiber innervation when compared with other midlinenuclei, contrary to data in rats, in which dense serotonergicinnervation in the CM and CL was found (Vertes et al., 2010).In the hamster (Mesocricetus auratus), the highest density isdescribed in the PV and Re, while in the intralaminar nucleithe highest density is observed in the CM (Morin and Meyer-Bernstein, 1999). In a primate species, the squirrel monkey

(Saimiri sciureus), a highest density of 5-HT fibers can beobserved in the PV and Re among the midline nuclei. Other-wise, in the intralaminar nuclei the highest density isobserved in the CM (Lavoie and Parent, 1991). Contrary toour results, in another primate species (Macaca Mulatta), theserotonergic fibers are more densely concentrated in the PVcompared to other midline/intralaminar nuclei (Hsu andPrice, 2009).

Through extensive dendritic and axonal arborizations,neurons define ample domain of influence. Morphologicalcharacterization, such as shaft caliber, tapering or branching,determines action potential propagation in axons (Goldsteinand Rall, 1974). Spines and dendritic segments are alsoimportant in defining biochemical compartments in neurons,which are dependent on their size, length, and shape(Helmchen, 1999; Korkotian and Segal, 2000). Overall, differ-ent patterns in density and spatial distribution of axonalor dendritic branches create a wide spectrum of wiring

Fig. 6 – Photomicrograph of 5-HT-immunostained brain coronal sections at the level of approximately 2.30 mm p.b. (A);(B) magnification of box homonymous in A, with an area corresponding to the dorsal midline, including PV and IMD; (C)magnification of box homonymous in A, with an area corresponding to the intralaminar region, including the PC; (D)magnification of superior box homonymous in B, with an area corresponding to the nucleus PV; (E) magnification of inferiorbox homonymous in B, with an area corresponding to the nucleus IMD; and (F) magnification of box homonymous in C, withan area corresponding to the PC nucleus. Black arrows in D correspond to the BF-like fibers; arrowheads in E correspond to theFF-like fibers; and White arrows in F correspond to the SA-like fibers. See list of abbreviations. Bar: 230 lm A; 140 lm B, and C;15 lm D, E, and F.

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configurations, which are currently subject to intensiveexperimental and theoretical research (Chklovskii et al.,2002; Petrof and Sherman, 2013; Rubio-Garrido et al., 2009;Sousa et al., 2013). The present work is the first to describemorphological differences in the pattern of distribution ofserotonergic fibers into midline/intralaminar complex. Wefound the BF-like type fibers more frequently inside the PV.Otherwise, the FF-like fibers were distributed throughout theremainder midline/intralaminar nuclei merged with SA-likefibers. The morphological descriptions of the serotonergicfibers have been made only in the cerebral cortex of rats(Kosofsky and Molliver, 1987) and primates (Wilson andMolliver, 1991), as well as in rabbit hippocampus (Bjarkamand Sorensen, 2005). These pronounced differences in fiberdensity as well as in morphological features of 5-HT fibers inthe midline/intralaminar complex indicate a possible speciesdifferences in serotonergic innervation between rodents andnonhuman primates. As is well known, the main source ofserotonergic fibers throughout the brain comes from thedorsal and median raphe nuclei. Two classes of serotonergicfibers have been described for rodents and primates, and arethought to arise from separated raphe nuclei (Kosofsky andMolliver, 1987; Wilson, 1989; Wilson and Molliver, 1991). Fineaxons with granular or fusiform varicosities originate fromthe dorsal raphe nucleus, while larger beaded axons arise

from median raphe nucleus (Kosofsky and Molliver, 1987).These morphological findings suggest that the rock cavymidline/intralaminar complex receives different classes ofserotonergic fibers arising from different sources in theraphe. In addition, it has been shown that psychoactive drug,such as 3,4 methyledioxymetamphetamine (Ecstasy) and p-chloroamphetamine, selectively destroy the FF in the centralnervous system, but leave the BF unharmed (Wilson, 1989;Haring et al., 1992). One may differentiate among systems ofascending serotonergic fibers, with clear differences in fiberorigin, fiber morphology, preference in area of innervation,and sensitive to psychoactive drugs.

In accordance with our findings, the midline nuclei of therock cavy contained more 5-HT fibers than the intralaminarones. Furthermore, there is a differential density of 5-HT-IRfiber among them. For example, a moderate density wasobserved in the nuclei associated with feeding (PV), learningand memory (PT), arousal and awareness (Rh), seizure reg-ulation (CM), and motor regulation (CL) (Bentivoglio et al.,1991; Ichinohe et al., 2001; Miller and Ferrendelli, 1990;Stratford and Wrtshafter, 2013; Van der Werf et al., 2002).The specific effects of 5-HT release depend on the class ofreceptor involved (Goodwin, et al., 1985; Larsson andAhlenius, 1999; Meltzer, 1990; Nordberg, 1992; Pazos et al.,1987a, b; Stuart et al., 1986), and the type of neuron received

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in the synaptic contact. It is clear that a more precisedescription of the 5-HT fiber ultrastructure and serotoninreceptor distribution is needed to provide the basis for furtherevaluation of the functional role of the 5-HT in the midline/intralaminar thalamic nuclei.

The above descriptions of differences in the density andmorphological distribution of serotonergic fibers in the mid-line/intralaminar nuclei of the rock cavy are an attempt tounderstand the functional and evolutionary pathway bywhich the 5-HT acts in the thalamus through the indispen-sable anatomical background, as well as understand thefundamental importance of identification, characterization,and comparative analysis of the great diversity of cell typespresent in complex nervous system. Although functionalstudies are strictly necessary to understand the real inter-ference of 5-HT in the midline/intralaminar nuclei, thisanatomical study suggests that the serotonergic systemexerts a fine control over thalamic function to modulatebehavior.

4. Experimental procedures

4.1. Animals and housing

Eight young adult rock cavies (four males and four females),weighing 300 and 400 g, from the rural municipalities in RioGrande do Norte state, Brazil, were used. Animal capture wasauthorized by the Brazilian Environmental Agency (IBAMA,licenses 21440-1). Approval for the experiments was obtainedfrom local Animal Experimentations Ethics Committee incompliance with National Institute of Health (NIH) guidelines.All efforts were made to minimize the number of animals andtheir suffering.

Individuals were housed in 3.00 m�2.00 m�2.60 mmasonry cages consisting of four wire screen walls, ceramictile ceilings and natural soil floor, with creeping vegetationand rocks to simulate their natural habitat. The animals wereexposed to environmental temperature, air humidity andlight, with unlimited access to food and water.

4.2. Perfusion and microtomy

Each individual was pre-anesthetized with an intramuscularinjection of tramadol chloridrate and xylazine, both 5 mg/kg,and maintained with gas isoflurane and 100% oxygen. Upondeep anesthesia, they were perfused for approximately 5 minusing a cannula positioned in the ascending aorta andconnected to a peristaltic pump (Cole-Parmer), with 300 mlof 0.9% saline solution in an 0.1 M phosphate buffer, pH 7.4,containing heparin (Parinex, Hipolabor, Sabará, MG, Brazil,2 ml/1000 ml of saline solution). Next, 700 ml of a 4% paraf-ormaldehyde, 2% picric acid and 0.05% glutaraldehyde fixa-tive solution in 0.1 M phosphate buffer, pH 7.4 (Zamboni andDe Martino, 1967) was administered. A flow rate of 70 ml/minwas established for half the solution and 17.5 ml/min for theother half, with the entire procedure taking 30 min.

After perfusion, the animals were placed in the stereotaxicframe and the incisor bar was adjusted until the lambda andbregma were at the same height. The skull bones were

removed to expose the dorsal surface of the encephalon,which was sectioned into 3 blocks by means of two coronalsections one at the bregma level and the other at the lambdalevel. Finally, each brain was removed from the skull, storedin 30% sucrose solution in 0.1 M phosphate buffer, pH 7.4, for24–48 h, frozen by dry ice, and serially cut in the coronalplane into 30 mm thick sections in a sliding microtome.

4.3. Nissl staining and immunohistochemistry

Sections from one series were immediately mounted ongelatin coated glass slides and Nissl stained with thionin, tovisualize the cytoarchitectonic delimitation of neuronalgroups. Sections from another series were submitted toimmunohistochemistry to reveal 5-HT. All the immunohis-tochemical procedures were performed at room temperature.The sections, previously submitted to a pre-treatment withsodium borohydride and hydrogen peroxide (H2O2), wereplaced in contact with the rabbit anti-5-HT antibody (Sigma,1:5000) and 2% normal goat serum in 0.4% Triton X-100 for18 h, in 0.1 M phosphate buffer, pH 7.4, in a rotator. This wasfollowed by incubation in a secondary antibody, consisting of1:1000 biotinylated goat anti-rabbit (Jackson ImmunoresearchLabs.) under gentle shaking in a rotator, for 90 min. In order tovisualize the reaction, the sections underwent 90 min incu-bation in an avidin–biotin–HRP complex (Vector Elite ABC kit),followed by a final reaction in a medium containing H2O2 assubstrate and 3,30-diaminobenzidine tetrahydrochloride(DAB) as chromogen. H2O2 was added indirectly, by mixingoxidase glucose and ß-D glucose, causing a reaction in whichthe former acting on the latter releases H2O2. The sectionswere thoroughly washed with 0.1 M phosphate buffer, pH 7.4,at the beginning, between each step, and at the end. Sectionswere mounted on previously gelatinized glass slides, whichafter drying at room temperature were rapidly submerged ina solution of 0.05% osmium tetroxide to enhance the visibilityof the reaction product.

The immunostainings were performed concomitantly,minimizing possible differences in background between theanimals. With respect to staining specificity, a number ofsections were submitted to immunohistochemical reactionsomitting the primary or secondary antibodies. In these cases,no 5-HT immnoreactivity was obtained. Diagrams wereobtained from image of Nissl-Stained sections with AdobeIllustrator software (Adobe Systems, Mountain View, CA,USA). The anatomical location of the brain structures wasdetermined using the rat brain atlas of Paxinos and Watson(2007), and our experience in the anatomy of the rock cavybrain (Nascimento et al., 2008, 2010a, 2010b; Soares et al.,2012).

4.4. Qualitative and quantitative analysis

5-HT-IR was identified as a black-purple precipitate. Thetissue was analyzed with an optic microscope (BX41 Olym-pus) under brightfield illumination. Digital images werecaptured using a digital video camera (Nikon DXM1200)coupled to the microscope. In order to identify morphologicaldifferences among 5-HT fibers/terminal in the midline/intra-laminar nuclei, we performed qualitative analysis under high

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magnification. The digitized images were corrected mini-mally for brightness and contrast, and mounted using AdobePhotoshop 7.0 software (Adobe Systems, Mountain View,CA, USA).

In order to accomplish the quantitative analysis, allimages were obtained in brightfield illumination at a fixedintensity for each of 16 rostro-caudal levels per individual.The resulting brightfield images were 3840�3072 pixel, witha resolution of 0.59 pixel/mm (with 4� objective). The RelativeOptical Density (ROD) analysis was accomplished usingImage J software (Version 1.46i, NIH). The images wereconverted to gray scale images (8-bit). The next stage con-sisted to the binarization of them, adjusting the contrast to100%. After this procedure, the images presented only twovalues that could be observed in the histogram, the zero thatcorresponds to black and the 255 that corresponds to white.Finally, the program provided the number of black pixels inthe sampled areas (Santos et al., 2013). The mean gray valueof each sampled area was measure using a square(0.3�0.3 mm) in the area of interest (AOI). This AOI waslocated 3–4 times on a well-defined DAB stained in eachnucleus throughout rostro-caudal levels and the mean grayvalues were taken. The medium number of black pixels in thetarget area was subtracted from the medium values ofa control region (areas that should not have specific 5-HTstaining). The value of OD (optical density) of the AOI wasrelated to the background value by the formula: [(OD AOI�ODbackground)/OD background]�100, thus eliminating thevariability in background staining among sections (Krugerset al., 1996). The data were presented to each target area asthe mean of pixels in the AOI. A manual selection of thestained positively for DAB chromogen was performed (Cuestaet al., 2013). All results are expressed as mean and standarderror of the mean (S.E.M). Statistical analysis was performedusing a computer program (Statistical Package for SocialSciences—IBM SPSS, version 21). The ROD data was subjectedto t-test to analyze differences between the mean number ofblack pixels in the midline and intralaminar nuclear complex.One-way ANOVA followed by post-hoc Tukey’s multiplecomparisons was accomplished to verify the differencesamong nuclei in the midline and intralaminar nucleieach other.

Conflict of interest statement

The authors declare no conflict of interest.

Author contributions

AMS: Participated in the design of the research and dataanalysis, and manuscript writing.

MADS: Participated in the analysis of the results.PLG: Participated in the analysis of the results.TBS: Participated in the analysis of the results.RCGE: Participated in the research.EESL: Participated in the analysis of the results.CLCC: Participated in the data analysis.

JSC: Participated in the design of the research and dataanalysis.

JCC: Participated in the design of the research, and manu-script writing.

MSMOC: Participated in the design of the research anddata analysis, and manuscript writing.

ESNJ: Designed, supervised studies, interpreted results,and prepare the manuscript.

Acknowledgments

We thank Miriam Regina Celi de Oliveira Costa for assistancewith experiments, histological preparations and animal sur-gery. This study was supported by funding from the NationalCouncil of Technological and Scientific Development (CNPq)and by Coordination for Improvement of High Level Staff(CAPES). The English version of this text was revised bySidney Pratt, Canadian, BA, MAT (The Johns HopkinsUniversity), RSAdip (TEFL).

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