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Journal of Chemical Neuroanatomy 43 (2012) 112–119

Contents lists available at SciVerse ScienceDirect

Journal of Chemical Neuroanatomy

jo urnal homepage: www.elsevier.com/locate/jchemneu

Nuclear organization of the serotonergic system in the of the rock cavy

(Kerodon rupestris)

a,b a,b a,b a,b

Joacil G. Soares , Jose´ R.L.P. Cavalcanti , Francisco G. Oliveira , Andre´ L.B. Pontes ,

a,b a,b a,b a,b

Twyla B. Sousa , Leandro M. Freitas , Jeferson S. Cavalcante , Expedito S. Nascimento Jr ,

a,b a,b,

Judney C. Cavalcante , Miriam S.M.O. Costa *

a

Departments of Morphology, Laboratory of Neuroanatomy, Biosciences Center, Federal University of Rio Grande do Norte, Natal, RN, Brazil

b

Department of Physiology, Laboratory of Neuroanatomy, 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: , or 5-hydroxytryptamine (5-HT), is a substance found in many tissues of the body, including as

Received 23 August 2011

a in the nervous system, where it can exert different post-synaptic actions. Inside the

Received in revised form 12 March 2012

neuro-axis, 5-HT neurons are almost entirely restricted to the of the . As such, 5-

Accepted 12 March 2012

HT-immunoreactivity has been considered a marker of the raphe nuclei, which are located in the

Available online 20 March 2012

brainstem, at or near the midline. The present study investigated distribution of serotonergic neurons in

the brain of the rock cavy (Kerodon rupestris), a rodent species inhabiting the Brazilian Northeast. The

Keywords:

cytoarchitectonic location of serotonergic neurons was established through a series of 5-HT

Brainstem

immunostained sections, compared with diagrams obtained from adjacent coronal and sagittal sections

Immunohistochemistry

stained by the Nissl method. The following nuclei were defined: the rostral group, consisting of rostral

Raphe nuclei

Rock cavy linear raphe, caudal linear raphe, median and paramedian raphe, dorsal raphe, and pontine raphe nuclei,

Rodent and the caudal group composed of raphe magnus, raphe pallidus and raphe obscurus nuclei. Other

Serotonin serotonergic neuronal clusters, such as the supralemniscal group and the rostral and caudal ventrolateral

clusters, were found outside the midline. Rare 5-HT-producing neurons were

identified in the lateral parabrachial and in the pontine , mostly along fibers

of the . Despite exhibiting some specializations, the picture outlined for serotonergic

groups in the rock cavy brain is comparable to that described for other mammalian species.

ß 2012 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: 3N, oculomotor nerve nucleus; 4N, trochlear nerve nucleus; 4V, 4th

ventricle; 7N, facial nerve nucleus; 10N, vagal nerve nucleus; 12N, hypoglossal

Interest in the morphology and functions of raphe nuclei was

nerve nucleus; AP, ; Aq, ; cp, ; Cu,

cuneatus nucleus; DTg, dorsal tegmental nucleus; ECu, external cuneate nucleus; triggered following the description of a system of monoaminergic

g7, genu of the facial nerve; Gi, gigantocellular reticular nucleus; Gr, gracilis neurons in the rat brainstem, using formaldehyde-induced fluores-

nucleus; IC, ; IO, inferior olive; IP, ; LC,

cence (Falck et al., 1962; Dahlstrom and Fuxe, 1964). Although a

; lfp, longitudinal fibers ; ll, lateral lemniscus; LL, lateral

large overlap was observed between putatively producing serotonin

lemniscus nuclei; LPB, lateral parabrachial nucleus; M5, motor trigeminal nucleus;

(5-HT) neurons and the raphe nuclei, serotonergic groups were

mcp, middle cerebelar peduncle; MdD, medullary reticular nucleus dorsal; MdV,

medullary reticular nucleus ventral; MG, medial geniculate nucleus; ml, medial designated as B1–B9 groups in a caudal to rostral direction, given

lemniscus; mlf, medial longitudinal fasciculus; MPB, medial parabrachial nucleus; that the overlap is imprecise (Dahlstrom and Fuxe, 1964). With the

PAG, ; Pn, ; PnC, pontine reticular nucleus caudal;

introduction of immunohistochemical techniques, this classification

PnO, pontine reticular nucleus oral; Pr5, principal sensory 5 nucleus; Pr, prepositus

was integrated with the cytoarchitectonic nomenclature of the

nucleus; py, pyramid; R, ; SC, ; scp, superior cerebelar

raphe system (Steinbusch et al., 1978; Steinbusch, 1981; To¨rk,

peduncle; scpx, superior cerebelar peduncle decussation; SN, ; Sol,

nucleus; Sp5, spinal trigeminal nucleus; sp5, spinal trigeminal tract; 1985). Even though the main cell groups in the serotonin system

SubB, subbrachial nucleus; VTA, ; vtgx, ventral tegmental

follow cytoarchitectonic divisions of the raphe nuclei, many 5-HT-

decussation.

immunoreactive (5-HT-IR) neurons are present in other areas of the

* Corresponding author at: Department of Morphology/Laboratory of Neuro-

brainstem beyond raphe nuclei boundaries, for example in the

anatomy, Biosciences Center, Federal University of Rio Grande do Norte, 59072-970,

Natal, RN, Brazil. Tel.: +55 84 32153431; fax: +55 84 32119207. vicinity of the or sectors of the reticular formation

E-mail address: [email protected] (Miriam S.M.O. Costa). (Jacobs and Azmitia, 1992; Vertes and Crane, 1997). On the other

0891-0618/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2012.03.001

Author's personal copy

J.G. Soares et al. / Journal of Chemical Neuroanatomy 43 (2012) 112–119 113

hand, other are also found in the neurons of raphe et al., 2008), nociceptive sensory processing (Bardin et al., 2000;

nuclei, including , thyrotropin-releasing hormone (TRH), Zeitz et al., 2002), circadian regulation (Pontes et al., 2010;

and gamma-aminobutyric acid (GABA), co-localized Cavalcante et al., 2011), among others. Moreover, 5-HT dysfunction

or not with 5-HT (To¨rk, 1985; Jacobs and Azmitia, 1992; Charara and has been associated with several neuropathological processes, such

Parent, 1998; Harding et al., 2004). Nevertheless, the long ascending as sleep disturbances (Neylan et al., 2001), anxiety, aggression (Van

and descending projections from raphe neurons are recognized as Praag, 1996; Ramboz et al., 1998; Lowry et al., 2008b), depression

serotonergic (Jacobs and Azmitia, 1992; Charara and Parent, 1998; (Jacobs, 2002; Michelsen et al., 2007; Lowry et al., 2008b), anorexia

Halberstadt and Balaban, 2006). and bulimia (Kaye et al., 2005), as well as neurodegenerative

Thus, serotonergic groups originally classified as B1–B9 from the disorders such as Alzheimer’s (Meltzer et al., 1998), Parkinson’s

medulla to the in the rat (Dahlstrom and Fuxe, 1964), may (Nicholson and Brotchie, 2002) and Huntington’s (Waeber and

be approximately correlated with raphe nuclei, generally divided Palacios, 1989) diseases (see also Verge´ and Calas, 2000).

into superior or rostral, and inferior or caudal, groups. In a classical The present study provides a foundation for future research on

description, the superior group consists of five main nuclei: the the hodological and functional aspects of these neuronal groups in

caudal linear raphe nucleus (CLi, B8); (MnR, this species, broadening the basis for understanding evolutionary

B8 and B5, previously referred to as the central superior nucleus); processes associated with the nuclear organization of this neuronal

(DR, B7 and B6), pontine raphe nucleus (caudal system.

end of B5, sometimes included in the median raphe nucleus), and

lateral neurons of the B9 group located just dorsal to the medial

2. Materials and methods

lemniscus and laterally displaced cells in the pontine nucleus

centralis oralis. The most rostral of the raphe nuclei, as cytoarch- Four young adult rock cavies (1 male and 3 females), weighing between 300 and

400 g, from rural municipalities in Rio Grande do Norte state, Brazil, were used.

itectonically defined (Taber et al., 1960), is the rostral linear raphe

Animal capture was authorized by the Brazilian Environmental Agency (IBAMA,

nucleus, which contains only rare serotonergic neurons and is

licenses 21440-1). Approval for the experiments was obtained from the local Animal

predominantly a dopaminergic nucleus (To¨rk, 1985).

Experimentation Ethics Committee in compliance with National Institute of Health

The inferior group consists of four main nuclei: the raphe (NIH) guidelines. All efforts were made to minimize the number of animals and their

obscurus nucleus (ROb, B2), raphe pallidus nucleus (RPa, B1 and suffering.

Individuals were housed in 3.00 m 2.00 m 2.60 m masonry cages consisting of

B4), raphe magnus nucleus (RMg, B3) and neurons in the

four wire screen walls, ceramic tile ceilings and natural soil floor, with creeping

ventrolateral medulla including lateral paragigantocellular and

vegetation and rocks to simulate their natural habitat. The animals were exposed to

intermediate reticular nuclei (B1/B3) (Brodal and Walberg, 1960;

environmental temperature, air humidity and light, with unlimited access to food and

Jacobs and Azmitia, 1992; Harding et al., 2004). The anatomy of water. Each individual was pre-anesthetized with an intramuscular injection of

these groups has been reviewed in many species, including rats tramadol chloridrate and xylazine, both 5 mg/kg and maintained with gas isofluoran

and 100% oxygen. Upon deep anesthesia, they were perfused for approximately 5 min

(Lidov and Molliver, 1982; Takeuchi et al., 1982; To¨rk, 1985;

using a cannula positioned in the ascending aorta and connected to a peristaltic pump

Harding et al., 2004), rabbits (Bjarkam et al., 1997), cats (Takeuchi

(Cole-Parmer), with 300 ml of 0.9% saline solution in 0.1 M phosphate buffer, pH 7.4,

et al., 1982; Jacobs et al., 1984), New and Old World monkeys containing heparin (Parinex, Hipolabor, Sabara´, MG, Brazil, 2 ml/1000 ml of saline

(Felten et al., 1974; Felten and Sladek, 1983; Azmitia and Gannon, solution). Next, 700 ml of a 4% paraformaldehyde, 2% picric acid and 0.05%

glutaraldehyde fixative solution in 0.1 M phosphate buffer, pH 7.4 (Zamboni and

1986; Hornung and Fritschy, 1988) and humans (To¨rk, 1990;

De Martino, 1967) was administered. A flow rate of 70 ml/min was applied for half the

Hornung, 2003). For a review see Hornung (2010).

solution and 17.5 ml/min for the other half, totaling 30 min for the entire procedure.

The rock cavy (K. rupestris) is a rodent species inhabiting the

After perfusion, two animals were placed in the stereotaxic frame and the incisor

semi-arid Caatinga of the Brazilian Northeast. According to bar was adjusted until the lambda and bregma were at the same height. The skull

traditional taxonomic classification based on morphological and bones were removed to expose the dorsal surface of the encephalon, which was

sectioned into 3 blocks by means of two coronal sections: one at the bregma level

behavioral aspects, the rock cavy is classified in the superfamily

and the other at the lambda level. Finally, the encephalon was removed from the

Cavioidea, family Caviidae, subfamily Caviinae, genus Kerodon,

skull, stored in 30% sucrose solution in 0.1 M phosphate buffer, pH 7.4, for 24–48 h,

along with Cavia, Galea and Microcavia (Cabrera, 1961; Lacher, and then sectioned by dry ice freezing in a sliding microtome, obtaining coronal

1981; Silva Neto, 2000). Morphological (Silva Neto, 2000) and sections of 30 mm. The of the other two animals were sectioned at the

sagittal plane. In both cases, the sections were collected sequentially into 6

molecular biology (Rowe and Honeycutt, 2002) studies placed the

compartments, each containing one of every 6 sections, thereby representing a

genus Kerodon sister to the Hydrochaeridae family, which also

serial sequence with a distance of 0.18 mm between the sections.

includes capybara (Hydrochoerus hydrochaeris) and is closely

Sections from one series were immediately mounted on gelatin coated glass

aligned with the subfamily Dolichotinae. slides and Nissl stained with thionin, to visualize the cytoarchitectonic delimitation

With regard to motor activity pattern, records of field of neuronal groups. Sections from another series were submitted to immunohis-

tochemistry to reveal 5-HT. All the immunohistochemical procedures were

observations show that on dark days rock cavies go out to feed

performed at room temperature. The sections, previously submitted to pre-

in the morning and afternoon, while on bright days their activity is

treatment with sodium borohydride and hydrogen peroxide (H2O2), were placed in

concentrated at night. Other observations indicate foraging contact with the rabbit anti-5-HT antibody (Sigma, 1:5000) and 2% normal goat

activity throughout the day and night (Lacher, 1981). In an serum in 0.4% Triton X-100 for 18 h, in a rotator. This was followed by incubation in

the secondary antibody, consisting of 1:1000 biotinylated goat anti-rabbit (Jackson

investigation using controlled laboratory conditions, the rock cavy

Immunoresearch Labs.) under gentle shaking in a rotator, for 90 min. In order to

was active throughout the 24-h day, although its activity increased

visualize the reaction, the sections underwent 90-min incubation in avidin–biotin–

during sunrise and sunset phases, suggesting a predominantly

HRP complex (Vector Elite ABC kit), followed by the final reaction in a medium

0

crepuscular behavior (Sousa and Menezes, 2006). containing H2O2 as substrate and 3,3 -diaminobenzidine tetrahydrochloride (DAB)

This study aimed to identify the serotonergic neuronal groups in as chromogen. H2O2 was offered indirectly, by mixing oxidase glucose and b-D

glucose into the solution, causing a reaction in which the former acting on the latter

the brain of the rock cavy by 5-HT immunohistochemistry. It is well

releases H2O2 (Itoh et al., 1979). The sections were thoroughly washed with 0.1 M

known that the serotonergic system is associated with several

phosphate buffer, pH 7.4, at the beginning, between each step and at the end.

functions, including regulation of the sleep-wake cycle (Portas et al.,

Sections were mounted on previously gelatinized glass slides, which, after drying at

2000), dream control and hallucinogenic states (Fischman, 1983), room temperature, were rapidly submersed in a solution of 0.05% osmium tetroxide

to enhance the visibility of the reaction product.

and attention (Robbins, 1997), memory and learning

With respect to staining specificity, a number of sections were submitted to

(McEntee and Crook, 1991), mood control (Cools et al., 2007),

immunohistochemical reactions omitting the primary or secondary antibodies. In

reward processing (Kranz et al., 2010), feeding behavior (Takase and

these cases, no 5-HT-immunoreactivity was obtained.

Nogueira, 2008; Magalha˜es et al., 2010), sexual behavior (Uphouse The 5-HT-immunostained coronal sections of the rock cavy brainstem were

and Guptarak, 2010), thermoregulation (Lin et al., 1998; Hodges analyzed using an optical microscope (Olympus BX41) under bright field

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114 J.G. Soares et al. / Journal of Chemical Neuroanatomy 43 (2012) 112–119

illumination. Digital images were obtained from representative sections using a

, extending from a level coinciding with the presence of

digital video camera (Nikon DXM1200) coupled to the microscope. The digitized

the oculomotor nerve nucleus to the motor trigeminal (cranial

images were converted to gray scale, corrected minimally for brightness and

nerve V) nucleus, along approximately 3.3 mm rostrocaudal. The

contrast, and mounted using Adobe Photoshop 7.0 software (Adobe Systems,

Mountain View, CA, USA). Diagrams were obtained from images of Nissl-stained DR begins as a small collection of neurons embedded in the ventral

coronal and sagittal sections with Adobe Illustrator software (Adobe Systems, periaqueductal gray (Fig. 1A) which then grow and exhibit dorsal

Mountain View, CA, USA).

(DRD), ventral (DRV) and interfascicular (DRIF) portions (Fig. 1B).

At a middle level, besides DRD, DRV and DRIF, some neurons

3. Results

expand laterally in the form of a wing-shaped neuronal cluster,

constituting the lateral (DRL) portion (Figs. 1C and 3A). The DR

This study used 5-HT immunohistochemistry to delimit

ends at the upper pons level, where the dorsal tegmental nucleus is

serotonergic nuclei in the rock cavy brainstem. 5-HT-immunore-

visible, constituting the caudal portion (DRC) (Figs. 1E and 3B).

active (5-HT-IR) neurons are shown in photomicrographs of

immunostained coronal sections taken from several levels of a

3.3. Pontine raphe (PnR)

representative animal. The location of 5-HT-IR neurons was

determined according to apparently corresponding sections from

At pontine level, where the motor trigeminal nucleus and

the rat brain atlas of Paxinos and Watson (2007). 5-HT-IR neurons

parabrachial complex are visible, a cluster of 5-HT-IR neurons was

were identified throughout the brainstem, predominantly in the

observed at the midline, identified as the pontine raphe nucleus

midline, from the level of the middle portion of the interpedun-

(PnR). This extended to the level of the suprageniculate nucleus

cular nucleus to the spinomedullary transition. According to the

and emergence of the facial nerve (cranial nerve VII), for around

current systematic in mammals, we grouped the 5-HT neuronal

1.3 mm (Figs. 1E and 3C).

collections into a rostral and a caudal cluster. The serotonergic

groups in the rock cavy brainstem are outlined in charts obtained

3.4. Caudal cluster

from adjacent coronal Nissl-stained sections (Fig. 1).

3.4.1. Raphe magnus (RMg)

3.1. Rostral cluster

The ensemble of 5-HT-IR neurons comprising the RMg were

noted from the pontine-bulbar transition to the end of the opened

3.1.1. Rostral linear nucleus (RLi)

portion of the medulla, as a pyramidal-shaped cluster of neurons,

In the rostrocaudal direction, the first 5-HT-IR neurons in the rock

located over the dorsal contours of the medullary pyramids, along

cavy brain appear in the midbrain, at a level marked by the presence

a rostrocaudal extension of about 2.8 mm (Figs. 1F and 4).

of the superior colliculus, medial geniculate nucleus, substantia

nigra, red nucleus and interpeduncular nucleus. These 5-HT-IR

3.5. Raphe pallidus (RPa)

neurons were seen forming a small cluster located ventral to the

periaqueductal gray, nucleus of the oculomotor nerve (cranial nerve

The scarce serotonergic neurons comprising the raphe pallidus

III) and medial longitudinal fasciculus, more precisely situated

nucleus (RPa) were observed in the ventral midline between the

between the dorsal tegmental decussation, dorsally, and the ventral

medullary pyramids, along the entire extension of the inferior

tegmental decussation, ventrally. Its rostrocaudal extension was

olivary complex, around 2.0 mm rostrocaudally (Figs. 1F and 4).

estimated at approximately 0.2 mm. Based on location and relation-

ships, we identified this neuronal agglomerate as the caudal end of

3.6. Raphe obscurus (ROb)

the rostral linear raphe nucleus (RLi) (Figs. 1A and 2A).

At the approximate level of the facial nerve nucleus, the midline

3.1.2. Caudal linear nucleus (CLi)

of the medulla oblongata is occupied by a double vertical band of 5-

More caudally, the RLi is replaced by a bulkier ensemble of 5-HT-

HT-IR neurons, corresponding to the raphe obscurus nucleus (ROb).

IR cells, the caudal linear raphe nucleus (CLi). These cells are

This extends to the spinomedullary transition, with a rostrocaudal

distributed along the midline between the medial longitudinal

extension of approximately 4.0 mm (Figs. 1F and G, and 4).

fasciculus and the interpeduncular nucleus. They are traversed by

the fibers of the decussation of the superior cerebellar peduncle,

3.7. Non-midline 5-HT-IR neuronal clusters

extending for around 0.6 mm caudally. Ventrally, 5-HT-IR neurons

of the CLi appear to invade the interpeduncular nucleus (Figs. 1B and

3.7.1. Supralemniscal nucleus (B9)

C, and 2B).

An arc-shaped neuronal group of 5-HT-IR cells was seen

immersed among the fibers of the medial lemniscus, laterally to

3.2. Median and paramedian raphe (MnR-PMnR) the interpeduncular nucleus, extending for around 2.0 mm in a

rostrocaudal direction. This group was recognized as the supra-

Proceeding caudally, at a level where the nucleus of the trochlear lemniscal nucleus or B9 group (B9, Figs. 1A–C and 5A).

nerve (cranial nerve IV) is present and the pontine nuclei are seen

ventrally, limiting the caudal portion of the interpeduncular nucleus, 3.7.2. Rostral ventrolateral (rVLMO) and caudal ventrolateral

all space in the midline ventral to the DR and dorsal to the pontine (cVLMO) medulla oblongata clusters

nuclei is occupied by the caudal end of the CLi, and ventrally by the A cluster of 5-HT-IR neurons emerges in the ventrolateral

median raphe nucleus (MnR), which is accompanied bilaterally by a region, between the pyramids and olivary superior complex,

row of cells constituting the paramedian raphe nucleus (PMnR). The rostrally, or the nucleus of the facial nerve caudally. This neuronal

set MnR-PMnR extends to a level in which the inferior colliculus set was identified as the rostral ventrolateral medulla oblongata

reaches its maximum size, measuring approximately 1.3 mm along cluster (rVLMO, Figs. 1F and 5B). More caudally, at the closed

the rostrocaudal extension (Figs. 1C and D, and 2C). portion of the medulla, these neurons were found laterally to the

inferior olivary complex, referred to here as the caudal ventrolat-

3.2.1. Dorsal raphe (DR) eral medulla oblongata cluster (cVLMO) (Figs. 1G and 5).

The 5-HT-IR neurons comprising the dorsal raphe complex Besides the raphe neuronal nuclei and the classical B9 and

were mostly located within the periaqueductal and periventricular VLMO groups, rare 5-HT-IR neurons were also found within the

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J.G. Soares et al. / Journal of Chemical Neuroanatomy 43 (2012) 112–119 115

Fig. 1. Drawings of coronal sections through the rock cavy brainstem depicting the location of the main 5-HT-immunoreactive neuronal groups (gray shaded areas). Numbers

on the right indicate distance from the bregma. See list for abbreviations.

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116 J.G. Soares et al. / Journal of Chemical Neuroanatomy 43 (2012) 112–119

Fig. 2. Photomicrographs of 5-HT-immunostained brainstem coronal sections

illustrating (A) the raphe rostral linear nucleus (RLi), (B) caudal linear nucleus (CLi),

and (C) the raphe median (MnR) and paramedian (PMnR) nuclei. See list for other

abbreviations. Bar: 400 mm for all figures.

lateral parabrachial nucleus in the pontine reticular territory,

bordering the ventral contour of the medial parabrachial nucleus,

and along the lateral lemniscus or above the medial lemniscus

(Figs. 1D and 5D).

Distribution of the serotonergic neuronal groups along the

midline of the brainstem is also demonstrated in a diagram

obtained from a 5-HT immunostained sagittal section overlapped

to an adjacent Nissl-stained sagittal section (Fig. 6).

4. Discussion

Fig. 4. Composed photomicrograph of 5-HT-immunostained brainstem coronal

The present investigation provides the first detailed description sections, showing the location of the raphe caudal cluster: the raphe magnus

of the distribution of serotonergic neuronal cell groups in the nucleus (MgR), raphe pallidus nucleus (RPa) and raphe obscurus nucleus (Rob). See

list for other abbreviations. Bar: 400 mm.

brainstem of a crepuscular rodent, the rock cavy, based on 5-HT

immunohistochemical staining. Moreover, direct comparison with

Nissl-stained material in alternating sections allowed association

of 5-HT-IR cells with respective cytoarchitectonic territories,

related or not to the raphe nuclei in the rock cavy brainstem. By

comparison with the organization of the rat brain (Dahlstrom and

Fuxe, 1964; Fuxe et al., 1969; Steinbusch, 1981; Harding et al.,

2004; Paxinos and Watson, 2007; Hornung, 2010), based on

cytoarchitectonic references, we identified the following nuclei in

the rock cavy brainstem along the midline, in a rostrocaudal

direction: the rostral linear raphe, caudal linear raphe, dorsal

raphe, median and paramedian raphe, pontine raphe, raphe

pallidus, raphe magnus and raphe obscurus nuclei. 5-HT-IR

neurons were also observed in some lateral clusters, such as the

supralemniscal group (B9) and ventrolateral medulla oblongata

region. Several neurons were also detected in the lateral

and in the pontine reticular formation, mostly

along the medial border of the lateral lemniscus.

The distribution, morphology and nuclear subdivisions of the

serotonergic system have also recently been studied using 5-HT

immunohistochemistry in the brains of a large number of African

species, such as the cane rat, a large nocturnal rodent (Greater

canerat, Dwarika et al., 2008), the Cape porcupine (Hystrix

africaeaustralis, Limacher et al., 2008), highveld gerbil (Tatera

brantsii, Moon et al., 2007), highveld mole rat (Cryptomys

Fig. 3. Photomicrographs of 5-HT-immunostained brainstem coronal sections,

hottentotus pretoriae, Da Silva et al., 2006), Cape dune mole rat

indicating (A) the dorsal raphe nucleus with its dorsal (DRD), ventral (DRV), lateral

(Bathyergus suillus, Bhagwandin et al., 2008), and other mammali-

(DRL) and interfascicular (DRIF) divisions, (B) the caudal portion of the dorsal raphe

nucleus (DRC) and (C) the pontine raphe nucleus (PnR). See list for other an species including the giraffe (Badlangana et al., 2007; Bux et al.,

abbreviations. Bar: 400 mm for all figures. 2010), several species of microchiroptera bats (Maseko and

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J.G. Soares et al. / Journal of Chemical Neuroanatomy 43 (2012) 112–119 117

the brain of some primate species. In the Japanese monkey (Macaca

fascicularis, Azmitia and Gannon, 1986), common marmoset

(Callithrix jacchus, Hornung and Fritschy, 1988) and among humans

(Azmitia and Gannon, 1986; Hornung, 2003), serotonergic neurons

formed a rostral group, consisting of the dorsal raphe nucleus (B6

and B7), median or superior central nucleus (B8, B5 and B7 in part),

pontine raphe nucleus (B5), and supralemniscal nucleus (B9), and a

caudal group composed of the raphe magnus (B3), raphe obscurus

(B2) and raphe pallidus nuclei (B1). Additionally, the common

marmoset brain exhibited extra-raphe serotonergic groups such as

the supralemniscal B9 group, several serotonergic neurons in the

interpeduncular nucleus, a substantial number of cells located

laterally to the median raphe nucleus in the reticular formation of

the caudal midbrain and rostral pons, in the bulbar reticular

formation and , among others (Hornung and

Fritschy, 1988).

The serotonergic system was also studied in two species of

marsupials: the opossum (Didelphis virginiana, Martin et al., 1985)

and the wallaby (Macropus eugenii, Ferguson et al., 1999). As

observed in eutherian mammals, the brainstem of these species

contains groups of 5-HT-IR neurons in the midline: the rostral

cluster, comprising the caudal linear raphe nucleus, median raphe

nucleus, pontine raphe nucleus and dorsal raphe nucleus, neurons in

the interpeduncular nucleus, and the caudal cluster, including the

raphe magnus nucleus, the raphe pallidus nucleus and the raphe

obscurus nucleus, as well as cells located more laterally in the B9

Fig. 5. Photomicrographs of 5-HT-immunostained brainstem coronal sections, group and others located near the pedunculopontine tegmental

demonstrating the location of non-midline 5-HT immunoreactive neuronal nucleus and ventrolateral medulla (Ferguson et al., 1999).

clusters. (A) The supralemniscal group (B9), (B) the rostral ventrolateral medulla

A significantly different pattern was only described in mono-

oblongata cluster (rVLMO), (C) the caudal ventrolateral medulla oblongata cluster

treme species since they exhibit serotonergic neurons in the

(cVLMO) and (D) neurons in the lateral parabrachial nucleus (LPB), and sectors of

, but do not retain neither the linear caudal nucleus

the reticular pontine formation (PnO). See list for other abbreviations. Bar: 400 mm

for all figures. in the rostral cluster, nor the rostral and caudal ventrolateral

medulla clusters (Manger et al., 2002).

In general, rock cavy serotonergic raphe nuclei resemble those

Manger, 2007; Kruger et al., 2010) and megachiroptera bats already found in other rodents studied, except the RLi, which is not

(Maseko et al., 2007; Dell et al., 2010), the rock hyrax (Gravett et al., mentioned as a serotonergic nucleus. The RLi is considered a midline

2009) and elephant shrew, a mammal of the order Macroscelidea low-dense dopaminergic component of the ventral tegmental area

(Pieters et al., 2010). In the brains of these species, serotonergic (VTA-A10) system (Oades and Halliday, 1987) and, as such, plays a

raphe neurons were grouped in a rostral cluster, mesencephalic- role in drug addiction mechanisms (Flores et al., 2006). According to

pontine and a caudal cluster, essentially bulbar. In the rostral group, a connectional study, projections of the RLi reflect the prevalence of a

the caudal linear raphe nucleus is considered the most rostral cluster non-dopaminergic phenotype and suggest that this nucleus may

of 5-HT-IR cells, since the presence of 5-HT-containing neurons was have a role in olfactory-guided exploratory behaviors (Del-Fava

not reported in the rostral linear raphe nucleus. The dorsal raphe et al., 2007). In the present study, we believe that the most rostral 5-

nucleus (with dorsal, ventral, interfascicular, lateral, and peripheral HT-IR neurons in the rock cavy midbrain, due to their location and

subnuclei) and median raphe nucleus complete the rostral group. relationships, belong to the RLi. Furthermore, the dorsal raphe

The raphe magnus nucleus, the raphe pallidus nucleus and the raphe nucleus can be divided into dorsal, lateral, ventral, interfascicular

obscurus nucleus were identified in the caudal group. Extra-raphe, and caudal parts through analogy with established divisions of the

the B9 (supralemniscal) group, at mesencephalic level, and the dorsal raphe nucleus of rats and other mammalian species

rostral and caudal ventrolateral medullary groupings were found to (Hornung, 2003; Lowry et al., 2008a; Vertes and Linley, 2008). As

contain 5-HT-IR neurons. opposed to peripheral division for the dorsal raphe nucleus,

Research on the serotonergic system correlated with the raphe considered for a variety of other species (Da Silva et al., 2006;

nuclei was also reviewed using 5-HT-immunohistochemistry in Maseko and Manger, 2007; Maseko et al., 2007; Moon et al., 2007;

Bhagwandin et al., 2008; Dwarika et al., 2008; Limacher et al., 2008;

Gravett et al., 2009; Bux et al., 2010; Kruger et al., 2010; Dell et al.,

2010; Pieters et al., 2010), we selected a lateral expansion of the

lateral division of the dorsal raphe nucleus for the rock cavy.

The lateral location of some 5-HT-IR neuron clusters in the rock

cavy brain (present study), as in other non-primate mammals

(Dwarika et al., 2008; Badlangana et al., 2007; Moon et al., 2007; Da

Silva et al., 2006; Bhagwandin et al., 2008; Bux et al., 2010; Maseko

and Manger, 2007; Maseko et al., 2007; Kruger et al., 2010; Dell

et al., 2010; Pieters et al., 2010), indicates that the lateralization of

serotonergic neurons is not a unique characteristic of primates as

previously thought (Azmitia and Gannon, 1986; Jacobs and

Fig. 6. Diagrammatic sagittal section of the rock cavy brain showing the location of Azmitia, 1992). Among extra-raphe serotonergic neurons, the

the midline serotonergic groups (gray shaded areas). See list for abbreviations. so-called supralemniscal nucleus, or B9 group, is the most

Author's personal copy

118 J.G. Soares et al. / Journal of Chemical Neuroanatomy 43 (2012) 112–119

Bhagwandin, A., Fuxe, K., Bennet, N.C., Manger, P.R., 2008. Nuclear organization and

consistent and has been described in all species studied, including

morphology of cholinergic, putative catecholaminergic and serotonergic neu-

monotremes. Although, to our knowledge, no hodological or

rons in the brains of two species of African mole-rat. J. Chem. Neuroanat. 35,

functional research has been conducted for this group, a 371–387.

Bjarkam, C.S., Sorensen, J.C., Geneser, F.A., 1997. Distribution and morphology of

quantitative study in rats shows that the B9 group contains more

serotonin-immunoreactive neurons in the brainstem of the New Zealand white

serotonergic neurons than any raphe group, except for the DR

rabbit. J. Comp. Neurol. 380, 507–519.

(Vertes and Crane, 1997). A substantial amount of serotonergic Brodal, E.T.A., Walberg, F., 1960. The nuclei of brain stem in the cat. II. Efferent

connections. J. Comp. Neurol. 114, 239–259.

neurons was also noted in the pontomesencephalic reticular

Bux, F., Bhagwandin, A., Fuxe, K., Manger, P.R., 2010. Organization of cholinergic,

formation in rats (Vertes and Crane, 1997). In the rock cavy

putative catecholaminergic and serotonergic nuclei in the diencephalon, mid-

(present study) these neurons are restricted to peripheral areas, brain and pons of sub-adult male giraffes. J. Chem. Neuroanat. 39, 189–203.

bordering the medial parabrachial nucleus ventrally, lateral Cabrera, A., 1961. Catalogo de los mamı´feros de Ame´rica del Sur. II. (Sirenia

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de Ciencias Naturales Bernardino Rivadavia, Ciencias Zoologicas 4, 309–732.

dorsally.

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The presence of serotonergic neurons in a ventrolateral district E.S., Borda, J.S., Pinato, L., Costa, M.S.M.O., Toledo, C.A.B., 2011. 5-HT1B receptor

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Dwarika, S., Maseko, B.C., Ihunwo, A.O., Fuxe, K., Manger, P.R., 2008. Distribution and

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Even when considering the detection of small variations, many

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the rewarding and sensitizing properties of heroin. Neuropsychopharmacology

cavy is very similar to that found in other rodent species and

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among mammals. This reinforces the suggestion that the

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serotonergic system is an ancient system of neurotransmitters mammalian central nervous system by histochemical studies. In: Hooper, G.

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and evolutionarily well conserved.

Gravett, N., Bhagwandin, A., Fuxe, K., Manger, P.R., 2009. Nuclear organization and

Finally, the present study provides foundations for future

morphology of cholinergic, putative catecholaminergic and serotonergic neu-

investigations using the rock cavy as an experimental model, in rons in the brain of the rock hyrax, Procavia capensis. J. Chem. Neuroanat. 38, 57–

74.

order to increase understanding regarding the functional role of

Halberstadt, A.L., Balaban, C.D., 2006. Serotonergic and nonserotonergic neurons in

the serotonergic system.

the dorsal raphe nucleus send collateralized projections to both the vestibular

nuclei and the central amygdaloid nucleus. Neuroscience 140, 1067–1077.

Acknowledgments Harding, A., Paxinos, G., Halliday, G., 2004. The serotonin and tachykinin systems.

In: Paxinos, G. (Ed.), The Rat Nervous System. Elsevier, Amsterdam, pp. 1205–

1256.

This study was financially supported by the National Council for

Hodges, M.R., Tattersal, G.J., Harris, M.B., McEvoy, S.D., Richerson, D.N., Deneris, E.S.,

Scientific and Technological Development (CNPq), Coordination for Johnson, R.L., Chen, Z.-F., Richerson, G.B., 2008. Defects in breathing and

High Level Staff Improvement (CAPES) and Research and Projects thermoregulation in mice with near-complete absence of central serotonin

neurons. J. Neurosci. 28, 2495–2505.

Financing (FINEP), Brazil.

Hornung, J.-P., 2003. The human raphe nuclei and the serotonergic system. J. Chem.

Neuroanat. 26, 331–343.

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