Download A cytoarchitectonic and TH-immunohistochemistry

Journal of Chemical Neuroanatomy 55 (2014) 58–66
Contents lists available at ScienceDirect
Journal of Chemical Neuroanatomy
journal homepage: www.elsevier.com/locate/jchemneu
A cytoarchitectonic and TH-immunohistochemistry characterization
of the dopamine cell groups in the substantia nigra, ventral tegmental
area and retrorubral field in the rock cavy (Kerodon rupestris)
José R.L.P. Cavalcanti a,b,*, Joacil G. Soares b, Francisco G. Oliveira b,c, Fausto P. Guzen a,b,
André L.B. Pontes d, Twyla B. Sousa b, Jeferson S. Cavalcante d, Expedito S. Nascimento Jrb,
Judney C. Cavalcante b, Miriam S.M.O. Costa b
a
Department of Biomedical Sciences, Laboratory of Experimental Neurology, Health Science Center, University of State of Rio Grande do Norte, Mossoró, RN,
Brazil
b
Department of Morphology, Laboratory of Neuroanatomy, Biosciences Center, Federal University of Rio Grande do Norte, Natal, RN, Brazil
c
Department of Biological Sciences, Biological Sciences and Health Center, Regional University of Cariri, Crato, CE, Brazil
d
Department of Physiology, Laboratory of Neurochemical Studies, Biosciences Center, Federal University of Rio Grande do Norte, Natal, RN, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 10 July 2013
Received in revised form 5 November 2013
Accepted 6 January 2014
Available online 17 January 2014
The 3-hydroxytyramine/dopamine is a monoamine of the catecholamine group and it is a precursor of
the noradrenaline and adrenaline synthesis, in which the enzyme tyrosine hydroxylase acts as a ratelimiting enzyme. The dopaminergic nuclei retrorubral field (A8 group), substantia nigra pars compacta
(A9 group) and ventral tegmental area (A10 group) are involved in three complex circuitries named
mesostriatal, mesocortical and mesolimbic, which are directly related to various behavioral
manifestations such as motor control, reward signaling in behavioral learning, motivation and
pathological manifestations of Parkinson’s disease and schizophrenia. The aim of this study was to
describe the delimitation of A8, A9 and A10 groups and the morphology of their neurons in the brain of
the rock cavy (Kerodon rupestris), a typical Brazilian Northeast rodent belonging to the suborder
Hystricomorpha, family Caviidae. Coronal and sagittal sections of the rock cavy brains were submitted to
Nissl staining and TH immunohistochemistry. The organization of these dopaminergic nuclei in the rock
cavy brain is very similar to that found in other animals of the Rodentia order, except for the presence of
the tail of the substantia nigra, which is found only in the species under study. The results revealed that,
apart some morphological variations, A8, A9 and A10 groups are phylogenetically stable brain structures.
ß 2014 Elsevier B.V. All rights reserved.
Keywords:
Dopamine
Retrorubral field
Rock cavy
Substantia nigra
Tyrosine hydroxylase
Ventral tegmental area
1. Introduction
Abbreviations: 3N, oculomotor nucleus; Aq, cerebral aqueduct; Cli, caudal linear
nucleus of the raphe; cp, cerebral peduncle; csc, commissure of the superior
colliculus; fr, fasciculus retroflexus; Hb, habenular nucleus; IF, interfascicular
nucleus; IP, interpeduncular nucleus; ml, medial lemniscus; MN, mammilary
nucleus; ns, nigrostriatal bundle; PAG, periaqueductal gray; PBP, parabrachial
pigmented nucleus; pc, posterior commissure; PIF, parainterfascicular nucleus; PN,
paranigral nucleus; RLi, rostral linear nucleus; RN, red nucleus; RRF/A8, retrorubral
field; rs, rubrospinal tract; SN/A9, substantia nigra pars compacta (nuclear
complex); SNCD, substantia nigra dorsal tier; SNCL, substantia nigra lateral cluster;
SNCM, substantia nigra medial cluster; SNCV, substantia nigra ventral tier; SNR,
substantia nigra reticulate; STh, subthalamic nucleus; SuM, supramammilary
nucleus; tSN, tail of the substantia nigra; VTA/A10, ventral tegmental area (nuclear
complex); VTAR, ventral tegmental area rostral part; ZI, zona incerta.
* Corresponding author at: Department of Biomedical Sciences/Laboratory of
Experimental Neurology, Health Science Center, University State of Rio Grande do
Norte, 59607-360, Mossoró, RN, Brazil. Tel.: +55 84 33152248;
fax: +55 84 33152248.
E-mail addresses: [email protected], [email protected]
(José R.L.P. Cavalcanti).
0891-0618/$ – see front matter ß 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jchemneu.2014.01.002
In the 1950s, 3-hydroxytyramine/dopamine (DA) was described as a neurotransmitter in the central nervous system, in
addition to its role as a precursor of the noradrenaline and
adrenaline synthesis (Carlsson et al., 1958; Björklund and Dunnett,
2007a). As such, DA is a monoamine included in the catecholamine
group, is a major neurotransmitter in the modulation of brain
function and plays a crucial role in the adaptation of animal
behavior throughout evolution (Smeets and González, 2000; Jones
and Pilowski, 2002; Yamamoto and Vernier, 2011).
The first detailed description of the distribution of neurons
containing catecholamine substances identified 12 neuronal
groups, designated A1–A12 in caudorostral direction in the rat
encephalon (Dahlström and Fuxe, 1964). Subsequent studies
added five more cell groups, A13–A17 (Hökfelt et al., 1984). Of
all these, the caudal groups A1–A7 are mainly noradrenergic,
J.R.L.P. Cavalcanti et al. / Journal of Chemical Neuroanatomy 55 (2014) 58–66
A8–A11 cells are mainly dopaminergic, whereas the TH-positive
cells in A12, A14 and A15 indicate absence of aromatic amino acid
decarboxylase (AADC), the dopamine producing enzyme (Björklund
and Dunnett, 2007b). A8, A9 and A10 are developed from the
neuromere midbrain, with expansion to prosomere p1 (diencephalon), rostrally, and to isthmus-rhombomere 1 region (I-r1), caudally
(Marı́n et al., 2005). These groups are coincident with the retrorubral
field (RRF), the substantia nigra (SN), and the ventral tegmental area
(VTA), respectively. The neurons of these groups express tyrosine
hydroxylase (TH), but not dopamine beta-hydroxylase, which is an
enzyme active in the conversion step for noradrenaline/adrenaline.
Because of this, the A8, A9 and A10 groups are considered typically
dopaminergic groups (Björklund and Dunnett, 2007b). Beside their
DA content, these neuronal clusters can be divided on cytoarchitectonic and chemoarchitectonic grounds, as described in the mouse
(Fu et al., 2012). It is known that these nuclei are involved in three
complex circuitries, named mesostriatal, mesolimbic and mesocortical (François et al., 1999; Smith and Kieval, 2000; Björklund and
Dunnett, 2007b) which are involved with motor control, motivation,
cognition, reinforcement learning and some neurological/psychiatric disorders, such as Parkinson’s disease and schizophrenia
(Chudasama and Robbins, 2004; Nicola et al., 2005; Fields et al.,
2007; Cohen et al., 2012).
Given the functional and pathological relevance of dopamine
we believe it is necessary to expand studies on these neuronal
groups in order to reach the greatest number of species.
The rock cavy (Kerodon rupestris) is a rodent inhabiting the
semiarid Caatinga of the Brazilian Northeast, although it can be
also found in the Southeast region as far as the south state of Minas
Gerais (Cabrera, 1961). This species reaches adulthood at 200 days,
and can reach up to 50 cm in length and 1 kg in body weight
(Roberts et al., 1984). According to traditional taxonomy, the rock
cavy belongs to the order Rodentia (Carleton and Musser, 2005).
According to classifications using the skull shape as a primary
characteristic – Anomaluromorpha, Castorimorpha, Hystricomorpha, Myomorpha and Sciuromorpha (Carleton and Musser, 2005),
the rock cavy is part of the suborder Hystricomorpha, infraorder
Caviomorpha, superfamily Cavioidea, family Caviidae, subfamily
Caviinae. Morphological (Silva Neto, 2000) and molecular biology
(Rowe and Honeycutt, 2002) studies have connected the genus
Kerodon with the genus Hydrochaeris, which includes the capybara
(family Hydrochaeridae), and is closely related to the genus
Dolichotis of the subfamily Dolichotinae, whose representative in
South America is the Patagonian hare (Dolicothis patagonum). The
order Rodentia is the most diverse among placental mammals. A
new nuclear gene analysis supports the division of this order in
‘‘squirrel-related’’, ‘‘mouse-related’’ clades, as well as the ‘‘Ctenohystrica clade’’, in which the suborder Hystricomorpha is included
(Blanga-Kkanfi et al., 2009; Fabre et al., 2012).
The rock cavy has a predominantly crepuscular behavior (Sousa
and Menezes, 2006) and is adapted to the Brazilian Northeast
ecological conditions such as heat, water and food scarcity,
especially in periods of severe drought. It inhabits rocky places
with numerous crevices where it takes shelter from predators and
spends much of its time. Moreover, rock cavies are excellent jumpers
and can climb rocks and tree branches where they draw food,
consisting mainly of tree bark, unlike other terrestrial caviinaes that
eat grass (Carvalho, 1969; Lacher, 1981; Mendes, 1985).
For a while this species has been used as an experimental model
for studies on the nervous system, for example in research on
retinal projections to thalamic nuclei (Nascimento Jr. et al., 2008,
2010a) and circadian centers (Nascimento Jr. et al., 2010b) and the
serotonergic system in the brain (Soares et al., 2012).
The present study aimed to describe the morphology of A8, A9 and
A10 dopamine groups in the rock cavy by TH immunohistochemistry.
It provides a foundation for future research on hodological and
59
functional aspects of these neuronal groups in this species,
broadening the basis for understanding evolutionary processes
associated with the nuclear organization of this neuronal system.
2. Materials and methods
Four young adult rock cavies (two males and two females), weighing between
300 and 400 g, from rural municipalities in the state of Rio Grande do Norte, Brazil,
were used. Animal capture was authorized by the Brazilian Environmental Agency
(IBAMA, license 21440-1). Approval for the experiments was obtained from the
local Animal Experimentation Ethics Committee (Protocol 015/2009-addendum) in
compliance with National Institute of Health (NIH) guidelines. All efforts were made
to minimize the number of animals and their suffering.
Individuals were housed for a short adaptation period in 3.00 2.00 2.60 m
masonry cages consisting of four wire screen walls, ceramic tile ceilings and natural
soil floor, with creeping vegetation and rocks to simulate their natural habitat. The
animals were exposed to environmental temperature, air humidity and light, with
unlimited access to food and water. Each individual was pre-anesthetized with an
intramuscular injection of tramadol chloridrate and xylazine, both 5 mg/kg and
maintained with gas isofluoran and 100% oxygen. Upon deep anesthesia, they were
perfused through a cannula positioned in the ascending aorta, and connected to a
peristaltic pump (Cole-Parmer). After cutting the right auricula, 300 ml of 0.9%
saline solution in 0.1 M phosphate buffer, pH 7.4, containing heparin 5000 IU/ml
(Parinex, Hipolabor, Sabará, MG, Brazil, 2 ml/1000 ml of saline solution) were
injected for approximately 5 min. 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 De Martino, 1967) was administered. A flow rate of 70 ml/min was
applied for half the solution and 17.5 ml/min for the other half, totaling 30 min for
the entire procedure.
After perfusion, two animals were placed in the stereotaxic frame and the incisor
bar was adjusted until the lambda and bregma 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 coronal sections: one at the bregma level
and the other at the lambda level. Finally, the encephalon was removed from the
skull, stored in 30% sucrose solution in 0.1 M phosphate buffer, pH 7.4, for 24–48 h,
and then sectioned by dry ice freezing in a sliding microtome, obtaining coronal
sections of 30 mm. The brains of the other two animals were sectioned at the
sagittal plane. In both cases, the sections were collected sequentially into 6
compartments, each containing one of every 6 sections, thereby representing a
serial sequence with a distance of 180 mm between the sections.
Sections from one series were immediately mounted on gelatin coated glass
slides and Nissl stained with thionin, to visualize the cytoarchitectonic delimitation
of neuronal groups. Sections from another series were submitted to immunohistochemistry to reveal TH. All the immunohistochemical procedures were
performed at room temperature. Free-floating sections, previously submitted to
pre-treatment with sodium borohydride and hydrogen peroxide (H2O2), were
placed in contact with the mouse anti-TH antibody (Sigma, 1:10,000) and 2%
normal goat 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 donkey
anti-mouse (Jackson Immunoresearch Labs.) under gentle shaking in a rotator, for
90 min. In order to visualize the reaction, the sections underwent 90-min
incubation in an avidin–biotin–HRP complex (Vector Elite ABC kit), followed by
the final reaction in a medium containing H2O2 as substrate and 3,30 diaminobenzidine tetrahydrochloride 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 releases H2O2 (Itoh et al., 1979). The sections
were thoroughly washed with a 0.1 M phosphate buffer, pH 7.4, at the beginning,
between each step and at the end. Sections were mounted on previously gelatinized
glass slides, which, after drying at room temperature, were rapidly submerged in a
solution of 0.05% osmium tetroxide to enhance the visibility of the reaction product.
The sections were dehydrated in a battery of gradually increasing alcohols, cleared,
and then coverslipped in an Entellan1 mounting medium.
With respect to staining specificity, a number of sections were submitted to
immunohistochemical reactions omitting the primary or secondary antibodies. In
these cases, no TH-immunoreactivity was obtained.
TH-immunostained coronal sections of the rock cavy brain were analyzed using
an optical microscope (Olympus BX41) under bright field illumination. Digital
images were obtained from representative sections using a digital video camera
(Nikon DXM1200) coupled to the microscope. The digitized images were converted
to a gray scale, corrected minimally for brightness and contrast, and mounted using
Adobe Photoshop CS5 software (Adobe Systems, Mountain View, CA, USA).
Diagrams were obtained from images of Nissl-stained coronal and sagittal sections
with Adobe Illustrator CS5 software (Adobe Systems, Mountain View, CA, USA).
3. Results
In this study, TH immunohistochemistry was used to delimit
dopaminergic neuronal groups A8, A9 and A10 in the rock cavy
60
J.R.L.P. Cavalcanti et al. / Journal of Chemical Neuroanatomy 55 (2014) 58–66
retroflexus (Fig. 2 C-G). VTAR (Fig. 3A and B) and PBP (Fig. 4A and
B) TH-IR neurons are multipolar, rounded or triangular shaped,
and do not show dendritic organization. The IF was located
medially to the VTAR/PBP and dorsally to the interpeduncular
nucleus (Fig. 2C–F). This group is formed by multipolar and
bipolar, fusiform and rounded shaped neurons, which do not
show a dendritic organization pattern (Fig. 4A and D). The PIF is
located laterally to the IF and medially to PBP (Fig. 2C and D). This
cluster is formed by a moderate density of TH-IR rounded,
multipolar type neurons, whose dendrites are arranged in a
dorsoventral orientation (Fig. 3A and C). The PN is located
ventrally to the PBP and medially to the SNCM (Fig. 2D–F). This
cluster is formed by TH-IR multipolar and triangular neurons,
which do not show a dendritic organization pattern (Fig. 4A and
C). The RLi (Fig. 3A and D) and CLi (Fig. 7A and D) were located in
the brain midline, dorsally to the IF and ventrally to the
periaqueductal gray (Fig. 2C–G). They are formed by a moderate
density of TH-IR neurons, which are ovoid, bipolar and
multipolar, with dorsoventral dendritic orientation.
Sparse TH-IR neurons were seen spread in the ventral
periaqueductal gray in the territory of the dorsal raphe nucleus
(not shown).
3.2. Substantia nigra pars compacta (SNC/A9 complex)
Fig. 1. Photographs of the dorsal (A) and ventral (B) aspects of the brain of the rock
cavy. Scale bar = 6 mm.
brain. TH-immunoreactive (TH-IR) neurons are shown in photomicrographs of immunostained coronal sections taken from
several levels of a representative animal. The location of TH-IR
neurons was determined according to apparently corresponding
sections from the rat brain atlas (Paxinos and Watson, 2007). The
nomenclature we used to describe the cytoarchitectonic and
immunohistochemically defined groups conforms closely to that
adopted for the rat (Paxinos and Watson, 2007) and mouse (Fu
et al., 2012).
The rostrocaudal length of the rock cavy encephalon, from
the olfactory bulb to the bulb-spinal transition, was around
3.6 cm (Fig. 1). TH-immunostained and Nissl-stained sections
contributed to establish the anatomical boundaries, cytoarchitecture and possible subdivisions of the dopaminergic groups
in the territory from the caudal diencephalon to the isthmus.
To facilitate understanding, illustrative diagrams were made
(Fig. 2).
3.1. Ventral tegmental area (VTA/A10 complex)
The VTA/A10 complex could be subdivided in seven neuronal
clusters: the interfascicular (IF), rostral linear (Rli) and caudal
linear (Cli) nuclei, situated along the midline, the paranigral (PN)
and parainterfascicular (PIF) nuclei, forming an intermediate
group, and dorsolaterally, the rostral ventral tegmental area
(VTAR) and parabrachial pigmented nucleus (PBP).
VTAR and PBP were seen to be located laterally to the
mamillary and supramamillary nuclei, medially to the substantia
nigra medial cluster (SNCM) and substantia nigra reticulata
(SNR) and ventrally to the medial lemniscus, red nucleus and
nigro-striatal projections. At caudal levels, the PBP is located
dorso-laterally to the interpeduncular nucleus and fasciculus
Identification of the subunits along the entire extent of the
SNC was possible based on the density of distribution and
morphology of its neurons. It could be seen that the SNC
was divided into the following: substantia nigra, dorsal tier
(SNCD); substantia nigra, ventral tier (SNCV); substantia nigra,
lateral cluster (SNCL), SNCM; and tail of the substantia nigra
(tSN).
The SNCM was identified laterally to the mammillary and
supramammillary nuclei, medially to the subthalamic nucleus,
ventrally to the medial lemniscus, zona incerta, red nucleus and
nigro-striatal projections and dorsally to the cerebral peduncle
and SNR (Fig. 2C–E). SNCM neurons are bipolar or multipolar, and
ovoid or triangular shaped. Dendrites of rostral neurons are
arranged parallel to the edges of the cerebral peduncle and SNR
(Fig. 5A and B). However, at caudal levels, this pattern was
replaced by a random arborization (not shown). SNCD was found
laterally to the SNCM, ventrally to the medial lemniscus and
dorsally to the SNR (Fig. 2B–G). SNCD neurons are bipolar and
multipolar, with ovoid and triangular formats and their dendritic
organization have predominantly medial-lateral orientation
(Fig. 5A and C). SNCL was found laterally to the SNCD, dorsally
to the SNR, and, at caudal levels, ventrally to the RRF/A8 (Fig. 2C–
F). SNCL neurons are multipolar, ovoid or piriform shaped and
devoid of any dendritic organizational pattern (Fig. 5A and D). At
caudal level, a low density TH-IR neuronal group was found as a
vertical dorsal protrusion into the SNCD and SNCL, which was
called the tSN (Fig. 2D and E). The tSN contains fusiform and ovoid
shaped neurons, whose dendrites are arranged in a dorsoventral
orientation (Fig. 5A and E, and Fig. 6A and B). TH-IR neurons were
also found ventrally to SNCD and dorsally to cerebral peduncle,
inserted into the SNR, forming the SNCV division (Fig. 2D–G). This
group has low neuronal density, its neurons are ovoid and
multipolar shaped, with dorsoventral dendritic orientation
(Fig. 5A and F).
3.3. Retrorubral field (RRF/A8)
The A8 group is formed by numerous, sparsely distributed
neurons located in the lower half of the midbrain tegmentum
(Fig. 2F and G). These neurons are ovoid or fusiform shaped, and do
not show dendritic organization pattern (Fig. 7A–C).
J.R.L.P. Cavalcanti et al. / Journal of Chemical Neuroanatomy 55 (2014) 58–66
61
Fig. 2. Drawings of sagittal (A) and coronal (B-G) sections through the rock cavy brainstem depicting the location of the midbrain TH-immunoreactive neuronal groups (gray
shaded areas). Numbers on the right indicate distance from the bregma. See list for abbreviations.
4. Discussion
4.1. Technical and morphological considerations
The present investigation provides the first detailed description
of the distribution of catecholaminergic/dopaminergic neurons in
the A8, A9 and A10 in the brain of a Brazilian Northeast rodent, the
rock cavy, based on TH immunohistochemistry and Nissl staining.
TH is an enzyme common to the synthesis of all the
catecholamines, by which TH immunohistochemistry can reveal
dopaminergic, noradrenergic and adrenergic neurons. Evidence
from physiological, pharmacological, clinical and molecular
biology studies agree that TH-IR neurons in the midbrain are DA
producers, making TH a reliable dopamine marker in that region
(Grimm et al., 2004; Prakash and Wurst, 2006; Margolis et al.,
2006).
In the rat (German and Manaye, 1993; McRitchie et al., 1996;
Paxinos and Watson, 2007), mouse (Fu et al., 2012), human
(McRitchie et al., 1996), and baboon (Papio ursinus, McRitchie et al.,
1998), A10 neurons are distributed throughout a heterogeneous
62
J.R.L.P. Cavalcanti et al. / Journal of Chemical Neuroanatomy 55 (2014) 58–66
Fig. 3. Photomicrographs of TH-immunostained brainstem coronal sections illustrating (A) VTA/A10 complex. The boxed regions are shown in higher magnification (B–D): (B)
VTAR, (C) PIF, and (D) RLi. See list for abbreviations. Level of section: 4.32 mm p.b. Scale bar: A = 1 mm and B–D = 100 mm.
Fig. 4. Photomicrographs of TH-immunostained brainstem coronal sections illustrating (A) VTA/A10 complex. The boxed regions are shown in higher magnification (B–D): (B)
PBP, (C) PN and (D) IF. See list for abbreviations. Level of section: 5.22 mm p.b. Scale bar: A = 1 mm and B–D = 100 mm.
J.R.L.P. Cavalcanti et al. / Journal of Chemical Neuroanatomy 55 (2014) 58–66
63
Fig. 5. Photomicrographs of TH-immunostained brainstem coronal sections illustrating (A) SN/A9 complex. The boxed regions are shown in higher magnification (B–F): (B)
SNCM, (C) SNCD, (D) SNCL, (E) tSN and (F) SNCV. See list for abbreviations. Level of section: 5.04 mm p.b. Scale bar: A = 1 mm and B–F = 100 mm.
Fig. 6. Photomicrographs of TH-immunostained brainstem coronal sections illustrating the SN/A9 complex (A). The boxed region corresponds to tSN and is shown in higher
magnification in B. See list for abbreviations. Level of section: 5.22 mm p.b. Scale bar: A = 1 mm and B = 100 mm.
64
J.R.L.P. Cavalcanti et al. / Journal of Chemical Neuroanatomy 55 (2014) 58–66
Fig. 7. Photomicrographs of TH-immunostained brainstem coronal sections illustrating (A) RRF/A8 and VTA/A10 clusters. The boxed regions are shown in higher
magnification (B–D): (B) and (C) RRF and (D) CLi. See list for abbreviations. Level of section: 6.12 mm p.b. Scale bar: A = 1 mm and B–D = 100 mm.
complex of nuclei, where seven components can be distinguished.
In the midline, the interfascicular nucleus (IF), located in the rostral
pole of the interpeduncular nucleus, and immediately above, the
raphe caudal linear nucleus (CLi), which is replaced by the raphe
rostral linear nucleus (RLi) rostrally. Forming a medial group, there
are the paranigral nucleus (PN) and the parainterfascicular nucleus
(PIF). The more lateral clusters are the rostral ventral tegmental
area (VTAR) and the parabrachial pigmented nucleus (PBP).
Regarding the A9 complex, the following divisions can be
distinguished: substantia nigra compacta, dorsal tier (SNCD),
substantia nigra compacta, ventral tier (SNCV), substantia nigra
compacta, lateral cluster (SNCL), and substantia nigra compacta,
medial cluster (SNCM). The A8 dopamine cell cluster, contained in
the caudal midbrain reticular formation, corresponding to the
retrorubral field (RRF), does not permit any subdivisions.
Catecholamine groups were studied in several rodent African
species, such as Highveld gerbil (Tatera brantsii, Moon et al., 2007)
and African pigmy mouse (Mus minutoides, Kruger et al., 2012),
both belonging to Muridae family, a mouse-related clade. African
rodents of the Ctenohystrica clade, suborder Hystricomorpha were
also studied for catecholamine groups: the Highveld mole-rat
(Cryptomys hottentotus, Da Silva et al., 2006; Bhagwandin et al.,
2008) and Cape dune mole-rat (Bathyergus suillus, Bhagwandin
et al., 2008), both of family Bathyergidae, as well as greater canerat
(Thryonomys swinderianus, family Thryonomyidae, Dwarika et al.,
2008) and the Cape porcupine (Hystrix africaeaustralis, family
Hystricidae, Limacher et al., 2008). There is no register of such
studies of the Hystricomorpha Caviidae rodent species, beyond the
rock cavy (present study). Other African non-rodent species have
been used in the study of catecholamine groups, such as the rock
hyrax (Procavia capensis, Gravett et al., 2009), rock elephant shrew
(Elephantulus myurus, Pieters et al., 2010), giraffe (Giraffa
camelopardalis, Bux et al., 2010), among others. According to all
those studies, the A10 is divisible into A10, which would
correspond to the VTAR, PBP, PN and PIF; A10c (central), which
would correspond to the PF; and A10d (dorsal), which would
correspond to the CLi and RLi, and even the A10dc (dorsal caudal),
the latter being a dopamine neuronal cluster spread in the
periaqueductal gray in the territory of the dorsal raphe nucleus.
Those neurons are also considered a component of A10 group by
Smeets and González (2000). Although we have seen sparse TH-IR
neurons in that location in the rock cavy brain, however, we did not
consider them a component of A10, in compliance with the
terminology adopted in Paxinos and Watson (2007). In the abovementioned species, the A9 complex could be subdivided in: A9pc,
which would correspond to SNCD; A9l, which would correspond to
SNCL; A9m, which would correspond to SNCM; and A9v, which
would correspond to SNCV. The A8 dopamine cell cluster,
contained in the caudal midbrain reticular formation, corresponding to the retrorubral field (RRF), does not permit any subdivisions.
Among mammals, the Megachiropteria species, such as the
rousette flying fox (Rousettus aegyptiacus, Maseko et al., 2007); the
straw-coloured fruit bat (Eidolon helvum) and the Wahlberg’s
epauletted fruit Bat (Epomophorus wahlbergi) (Dell et al., 2010)
have been studied with respect to the catecolamine groups,
revealing all subdivisions found for the most mammals. However,
among microbats, such as the long-fingered bat (Miniopterus
schreibersii, Maseko and Manger, 2007) and Cardioderma cor,
Chaerophon pumilus, Coleura afra, Hipposideros commersoni and
Triaenops persicus (Kruger et al., 2010) it was noted a trend to
absence of some subdivisions.
Our proposal of subdivision of the A8, A9 and A10 dopaminergic
groups agrees with what has been described for other species,
although some discrepancies can be attributed to different
terminologies. The exception found refers to the presence of the
tSN as a component of A9 complex, described in our material in the
rock cavy, which, to date, has been described only in the Göttingen
minipig (Nielsen et al., 2009). One could guess that the tSN is part
J.R.L.P. Cavalcanti et al. / Journal of Chemical Neuroanatomy 55 (2014) 58–66
of the A9l or even an extension of the RRF. However, the
cytoarchitectonic aspects of the tSN, such as its dorsoventral
dendritic orientation, allow us to distinguish it as a distinct
subdivision of A9.
4.2. Evolutionary considerations
Based in previous studies in other animals, mainly referring to
those on the Rodentia order, it has been possible to deduce that,
although noticing differences relative to phenotypic characteristics, the nuclear complexity of the dopamine centers in the brain
seems be different among the different orders, but not within the
same order. Thus, it has been suggested that the phenotypic
variations, lifestyle, and evolutionary characteristics in the
Rodentia order do not lead to a significant variation in the brain
nuclei (Manger, 2005).
The results revealed that the rock cavy A8, A9 and A10
dopaminergic nuclei are, in general, similar to what has already
been described in other species of mammals, suggesting that these
nuclei are phylogenetically stable brain structures in the species.
An exception exists with respect to the presence of the tSN, which,
besides in the rock cavy brain (present study) has been described
only in a non-rodent species, the Göttingen minipig (Susscrofa
domesticus), a laboratory-developed ungulate derived from the
domestic pig (Nielsen et al., 2009). In this point it is worth
emphasizing that, although the dopaminergic groups have been
studied in many rodent species, to date, the rock cavy (present
study) is the first species of the Ctenohystrica clade, Hystricomorpha suborder, among the Caviomorpha superfamily to be
studied in this particular way. To our knowledge, there is no
register of studies involving other species, such as agouti, capybara
or paca, animals of the suborder Hystricomorpha, characteristic of
South America. This raises the need for more detailed studies about
these neuronal clusters, with particular reference to tSN. We
recommend an expansion of the comparative analysis of brain
samples from animals of the same and different orders, suborders
and families, or even different classes of vertebrates, in order to
test the consistency of such nuclear division.
This work reported on the organization of the A8, A9 and A10
dopaminergic neurons of the rock cavy brain and discussed this in
light of the features of other rodent or non-rodent mammalian
species. Further studies are needed to clarify some variations, as
well as to expand our understanding about the many different
functions of this system.
Acknowledgments
This study was financially supported by the National Council for
Scientific and Technological Development (CNPq), Coordination for
High Level Staff Improvement (CAPES) and Research and Projects
Financing (FINEP), Brazil.
References
Bhagwandin, A., Fuxe, K., Bennett, N.C., Manger, P.R., 2008. Nuclear organization and
morphology of cholinergic, putative catecholaminergic and serotonergic neurons in the brains of two species of African mole-rats. J. Chem. Neuroanat. 35,
371–387.
Björklund, A., Dunnett, S., 2007a. Fifty years of dopamine research. Trends Neurosci.
30, 185–187.
Björklund, A., Dunnett, S., 2007b. Dopamine neuron systems in the brain: an update.
Trends Neurosci. 30, 194–202.
Blanga-Kkanfi, S., Miranda, H., Penn, O., Pupko, T., DeBry, R.W., Huchon, D., 2009.
Rodent phylogeny revised: analysis of six nuclear genes from all major rodent
clades. BMC Evol. Biol. 9, 71–82.
Bux, F., Bhagwandin, A., Fuxe, K., Manger, P.R., 2010. Organization of cholinergic,
putative catecholaminergic and serotonergic nuclei in the diencephalon, midbrain and pons of sub-adult male giraffes. J. Chem. Neuroanat. 39, 189–203.
65
Cabrera, A., 1961. Catálogo de los mamı́feros de America del Sur. Rev. Mus.
Argentino Cien. Nat. 4, 1–732.
Carleton, M.D., Musser, G.G., 2005. Order Rodentia In: Wilson, D.E., Reeder, D.M.
(Eds.), Mammal Species of the World: A Taxonomic and Geographic Reference.
John Hopkins University Press, Baltimore, MD, pp. 745–752.
Carlsson, A., Lindqvist, M., Magnusson, T., Waldeck, B., 1958. On the presence of 3hydroxytyramine in brain. Science 127, 471.
Carvalho, J.C.M., 1969. Notas de viagem de um zoólogo à região das caatingas e áreas
limı́trofes. Imprensa universitária do Ceará, Fortaleza.
Chudasama, Y., Robbins, T.W., 2004. Psychopharmacological approaches to modulating attention in the five-choice serial reaction time task: implications for
schizophrenia. Psychopharmacology (Berl) 174, 86–98.
Cohen, J.Y., Haesler, S., Vong, L., Lowell, B.B., Uchida, N., 2012. Neuron-type-specific
signals for reward and punishment in the ventral tegmental area. Nature 482,
85–88.
Dahlström, A., Fuxe, K., 1964. Evidence for the existence of monoamine containing
neurons in the central nervous system. I. Demonstration of monoamines in the
cell bodies of brain stem neurons. Acta Physiol. Scand. Suppl. 232, 1–55.
Da Silva, J.N., Fuxe, K., Manger, P.R., 2006. Nuclear parcellation of certain immunohistochemically identifiable neuronal systems in the midbrain and pons of
the Highveld molerat (Cryptomys hottentotus). J. Chem. Neuroanat. 31, 37–50.
Dell, L.A., Kruger, J.L., Bhagwandin, A., Jillani, N.E., Pettigrew, J.D., Manger, P.R., 2010.
Nuclear organization of cholinergic, putative catecholaminergic and serotonergic systems in the brains of two megachiropteran species. J. Chem. Neuroanat.
40, 177–195.
Dwarika, S., Maseko, B.C., Ihunwo, A.O., Fuxe, K., Manger, P.R., 2008. Distribution and
morphology of putative catecholaminergic and serotoninergic neurons in the
brain of the greater canerat, Thryonomys swinderianus. J. Chem. Neuroanat. 35,
108–122.
Fabre, P.-H., Hautier, L., Dimitrov, D., Douzery, E.J.P., 2012. A glimpse on the pattern
of rodent diversification: a phylogenetic approach. BMC Evol. Biol. 12, 88–106.
Fields, H.L., Hjelmstad, G.O., Margolis, E.B., Nicola, S.M., 2007. Ventral tegmental
area neurons in learned appetitive behavior and positive reinforcement. Annu.
Rev. Neurosci. 30, 289–316.
François, C., Yelnik, D., Tandé, D., Agid, Y., Hirsh, E.C., 1999. Dopaminergic cell group
A8 in the monkey: anatomical organization and projections to the striatum. J.
Comp. Neurol. 414, 334–347.
Fu, Y., Yuan, Y., Halliday, G., Rusznák, Z., Watson, C., Paxinos, G., 2012. A cytoarchitetonic and chemoarchitetonic analysis of the dopamine cell groups in the
susbstantia nigra, ventral tegmental area, and retrorubral field in the mouse.
Brain Struct. Funct. 217, 591–612.
German, D.C., Manaye, K.F., 1993. Midbrain dopaminergic neurons (Nuclei A8, A9,
and A10): three-dimensional reconstruction in the rat. J. Comp. Neurol. 331,
297–309.
Gravett, N., Bhagwandin, A., Fuxe, K., Manger, P.R., 2009. Nuclear organization and
morphology of cholinergic, putative catecholaminergic and serotonergic neurons in the brains of the rock hyrax, Procavia capensis. J. Chem. Neuroanat. 38,
57–74.
Grimm, J., Mueller, A., Hefti, F., Rosenthal, A., 2004. Molecular basis for catecholaminergic neuron diversity. Proc. Natl. Acad. Sci. U.S.A. 38, 11389–13896.
Hökfelt, T., Matensson, R., Björklund, A., Kleinau, S., Goldstein, M., 1984. Distributional maps of tyrosine-hydroxylase-immunoreactive neurons in the rat brain.
In: Björklund, A., Hökfelt, T. (Eds.), Classical Transmitters in the CNS, part I.
Handbook of Chemical Neuroanatomy, vol. 2. Elsevier, Amsterdam, pp. 227–
379.
Itoh, K., Konish, A., Nomura, S., Mizuno, N., Nakamura, Y., Sugimoto, T., 1979.
Application of coupled oxidation reaction to electron microscopic demonstration of horseradish peroxidase: cobalt-glucose oxidase method. Brain Res. 175,
341–346.
Jones, H.M., Pilowski, L.S., 2002. Dopamine and antipsychotic drug action revisited.
Br. J. Psychiatry 181, 271–275.
Kruger, J.L., Dell, L.A., Bhagwandin, A., Jillani, N.E., Pettigrew, J.D., Manger, P.R., 2010.
Nuclear organization of cholinergic, putative catecholaminergic and serotonergic systems in the brains of five microchiropteran species. J. Chem. Neuroanat.
40, 210–222.
Kruger, J.L., Patzke, N., Fuxe, K., Bennett, N.C., Manger, P.R., 2012. Nuclear organization of cholinergic, putative catecholaminergic, serotonergic and orexinergic systems in the brain of the African pygmy mouse (Mus minutoides):
organizational complexity is preserved in small brains. J. Chem. Neuroanat.
44, 45–56.
Lacher Jr., T.E., 1981. The comparative social behavior of Kerodon rupestris and Galea
spixii and the evolution of behavior in the cavidiae. Bull. Carnegie Mus. Nat. Hist.
17, 5–71.
Limacher, A.M., Bhagwandin, A., Fuxe, K., Manger, P.R., 2008. Nuclear organization
and morphology of cholinergic, putative catecholaminergic and serotonergic
neurons in the brain of the Cape porcupine (Hystrix africaeaustralis): increased
brain size does not lead to increased organizational complexity. J. Chem.
Neuroanat. 36, 33–52.
Manger, P.R., 2005. Stablishing order at the systems level in mammalian brain
evolution. Brain Res. Bull. 66, 282–289.
Marı́n, F., Herrero, M.T., Vyas, S., Puelles, L., 2005. Ontogeny of tyrosine hydroxylase
mRNA expression in mid- and forebrain: neuromeric pattern and novel positive
regions. Dev. Dyn. 234, 709–717.
Margolis, E.B., Lock, H., Hjelmstad, G.O., Fields, H.L., 2006. The ventral tegmental
area revisited: is there an electrophysiological marker for dopaminergic
neurons? J. Physiol. 577, 907–924.
66
J.R.L.P. Cavalcanti et al. / Journal of Chemical Neuroanatomy 55 (2014) 58–66
Maseko, B.C., Bourne, J.A., Manger, P.R., 2007. Distribution and morphology of
cholinergic, putative catecholaminergic and serotonergic neurons in the brain
of the Egyptian rousette flying fox (Rousettus aegyptiacus). J. Chem. Neuroanat.
34, 108–127.
Maseko, B.C., Manger, P.R., 2007. Distribution and morphology of cholinergic,
catecholaminergic and serotonergic neurons in the brain of Schreiber’s longfingered bat, Miniopterus schreibersii. J. Chem. Neuroanat. 34, 80–94.
McRitchie, D.A., Cartwright, H., Pond, S.M., Van der Schyf, C.J., Castagnoli Jr., N., Van
der Nest, D.G., Halliday, G.M., 1998. The midbrain dopaminergic cell groups in
the baboon Papio ursinus. Brain Res. Bull. 47, 611–623.
McRitchie, D.A., Hardman, C.D., Halliday, G.M., 1996. Cytoarchitectural distribution
of calcium binding proteins in midbrain dopaminergic regions of rats and
humans. J. Comp. Neurol. 364, 121–150.
Mendes, B.V., 1985. Alternativas tecnológicas para a agropecuária do semi-árido.
Nobel, São Paulo, pp. 171.
Moon, D.J., Maseko, B.C., Ihunwo, A.O., Fuxe, K., Manger, P.R., 2007. Distribution and
morphology of catecholaminergic and serotonergic neurons in the brain of the
highveld gerbil, Tatera braintsii. J. Chem. Neuroanat. 34, 134–144.
Nascimento Jr., E.S., Duarte, R.B., Silva, S.F., Engelberth, R.C.G.J., Toledo, C.A.B., Cavalcante, J.S., Costa, M.S.M.O., 2008. Retinal projections to the thalamic paraventricular nucleus in the rock cavy (Kerodon rupestris). Brain Res. 1241, 56–61.
Nascimento Jr., E.S., Cavalcante, J.S., Cavalcante, J.C., Costa, M.S.M.O., 2010a. Retinal
afferents to the thalamic mediodorsal nucleus in the rock cavy (Kerodon
rupestris). Neurosci. Lett. 475, 38–43.
Nascimento Jr., E.S., Sousa, A.P., Duarte, R.B., Magalhães, M.A., Silva, S.F., Cavalcante,
J.C., Cavalcante, J.S., Costa, M.S.M.O., 2010b. The suprachiasmatic nucleus and
intergeniculate leaflet in the rock cavy (Kerodon rupestris): retinal projections
and immunohistochemical characterization. Brain Res. 1320, 34–46.
Nicola, S.M., Taha, S.A., Kim, S.W., Fields, H.L., 2005. Nucleus accumbens dopamine
release is necessary and sufficient to promote the behavioral response to
reward-predictive cues. Neuroscience 135, 1025–1033.
Nielsen, M.S., Sørensen, J.C., Bjarkam, C.R., 2009. The substantia nigra pars compacta
of Göttingen minipig: an anatomical and stereological study. Brain Struct.
Funct. 213, 481–488.
Paxinos, G., Watson, C., 2007. The Rat Brain in Stereotaxic Coordinates. Academic
Press, San Diego.
Pieters, R.P., Gravett, N., Fuxe, K., Manger, P.R., 2010. Nuclear organization of
cholinergic, putative catecholaminergic and serotonergic nuclei in the brain
of the eastern rock elephant shrew, Elephantulus myurus. J. Chem. Neuroanat.
39, 175–188.
Prakash, N., Wurst, W., 2006. Development of dopaminergic neurons in the mammalian brain. Cell. Mol. Life Sci. 63, 187–206.
Roberts, M.E., Maliniak, E., Deal, M., 1984. The reproductive biology of the rock cavy,
Kerodon rupestris in captivity: a study of reproductive adaptation in a tropic
specialist. Mammalia 48, 253–266.
Rowe, D.L., Honeycutt, R.L., 2002. Phylogenetic relationships, ecological correlates,
and molecular evolution within the Cavioidea (Mammalia, Rodentia). Mol. Biol.
Evol. 19, 263–277.
Silva Neto, E.J., 2000. Morphology of the regions ethmoidalis and orbitotemporalis
in Galea musteloides Meyen 1832 and Kerodon rupestris (Wied-Neuwied 1820)
(Rodentia: Caviidae) with comments on the phylogenetic systematics of the
Caviidae. J. Zool. Syst. Evol. Res. 38, 219–229.
Smeets, W.J.A.J., González, A., 2000. Catecholamine systems in the brain of vertebrates: new perspectives through a comparative approach. Brain Res. Rev. 33,
308–379.
Smith, Y., Kieval, J.Z., 2000. Anatomy of the dopamine in the basal ganglia. Trends
Neurosci. 23 (Suppl. 10) S28–S33.
Soares, J.G., Cavalcanti, J.R.L.P., Oliveira, F.G., Pontes, A.L.B., Sousa, T.B., Freitas, L.M.,
Cavalcante, J.S., Nascimento Jr., E.S., Cavalcante, J.C., Costa, M.S.M.O., 2012.
Nuclear organization of the serotonergic system in the brain of the rock cavy
(Kerodon rupestris). J. Chem. Neuroanat. 43, 112–119.
Sousa, R.A., Menezes, A.A.L., 2006. Circadian rhythms of motor activity of the
Brazilian rock cavy (Kerodon rupestris) under artificial photoperiod. Biol.
Rhythm Res. 3, 443–450.
Yamamoto, K., Vernier, P., 2011. The evolution of dopamine systems in chordates.
Front. Neuroanat. 5, 1–21.
Zamboni, L., De Martino, L., 1967. Buffered picric acid formaldehyde: a new rapid
fixative for electron microscopy. J. Cell Biol. 35, 148A.