Title: DISTRIBUTION OF NITRIC OXIDE IN THE ROCK CAVY

(Kerodon rupestris) BRAIN II: THE BRAINSTEM

Lucimário Thiago Félix de Araújo,Maria Emanuela Martins dos Reis, Wylqui

Mikael Gomes de Andrade, Nayra da Silva Resende, Ruthnaldo Rodrigues Melo de
Lima, Expedito Silva do Nascimento Jr, Miriam Stela Maris de Oliveira Costa, Judney
Cley Cavalcante

Laboratory of Neuroanatomy, Department of Morphology, Biosciences Center,

Federal University of Rio Grande do Norte, Natal, RN, Brazil.

Correspondence to: Dr. Judney C. Cavalcante, Laboratory of Neuroanatomy,

Dept. of Morphology, Biosciences Center, Federal University of Rio Grande do Norte,
59072-970 Natal, RN, Brazil. Phone: +55 (84) 98829 7484. E-mail: judney@cb.ufrn.br.
 Abstract

In a recent paper, we described the distribution of Nitric oxide (NO) in the

diencephalon of the rock cavy (Kerodon rupestris). This present paper follows it
showing the distribution of NO synthesizing neurons in the rock cavy's brainstem. For
this, we used immunohistochemistry against the neuronal form of nitric oxide synthase
(NOS) and NADPH diaphorase histochemistry. In contrast to the diencephalon in the
rock cavy, where NOS neurons were seen to be limited to some nuclei in the thalamus
and hypothalamus, the distribution of NOS in the brainstem is wide. Neurons
immunoreactive to NOS (NOS-ir) were seen as rostral asthe pretectal nuclei and as far
as the caudal and gelatinous parts of the spinal trigeminal nucleus. Places such as the
raphe nuclei, trigeminal complex, superior and inferior colliculus, oculomotor complex,
periaqueductal grey matter, solitary tract nucleus, laterodorsal tegmental nucleus,
pedunculopontine tegmental and other nuclei of the reticular formation are among the
locations with most NOS-ir neurons. This distribution is similar, but with a number of
differences from those described for other rodents, indicating that NO also has an
important role in rock cavy's physiology.

Keywords: rock cavy; nitric oxide synthase; brainstem; nitric oxide

 1. Introduction

The rock cavy (Kerodon rupestris) is an endemic Brazilian rodent. It inhabits the
dry semi-arid Caatinga of the Brazilian Northeast (Cabrera, 1961), a place where this
species faces extreme conditions such as high heat and scarcity of water, and food. To
adapt to this environment, the rock cavy inhabits rocky places with numerous crevices
where it takes shelter from predators and spends much of its time. As grass is scarce, the
rock cavies eat mainly tree bark. To do this, they have developed the ability to jump and
to climb rocks, and tree branches (Carvalho, 1969; Lacher Jr, 1981; Mendes, 1985).
Also to avoid predators and the extreme environmental condition, the rock cavy has a
predominantly crepuscular behavior (Sousa and Menezes, 2006).

Nitric oxide (NO) is a peculiar neurotransmitter with a wide distribution in the

brain of many vertebrate species. In a previous paper we described the distribution of its
synthesis enzyme, the nitric oxide synthase (NOS), within the rock cavy diencephalon
(Reis et al., 2018). NOS is highly expressed in many hypothalamic nuclei, such as the
supraoptic, paraventricular, ventromedial, ventral, and dorsal premammillary nucleus. It
follows the same distribution pattern in the hypothalamus of rats and mice (Vincent and
Kimura, 1992; Rodrigo et al., 1994; Gotti et al., 2005). The distribution of NOS
immunoreactive neurons in the rock cavy thalamus, however, has been seen to be far
more restricted than reported in other rodents (Vincent and Kimura, 1992; Rodrigo et
al., 1994; Bertini and Bentivoglio, 1997; Gotti et al., 2005).

This work aimed to map the distribution of the nitrergic neurons in the brainstem
of the rock cavy. To do this we used the immunoperoxidase technique to mark NOS and
histochemistry to map NADPH-diaphorase activity, as we have also done in previous
work (Reis et al., 2018).

2. Results
NOS immunoreactive (NOS-ir) neurons were widely distributed throughout the
brainstem with almost every nucleus with some NOS-ir cells. The same pattern of
staining was seen using NADPH-diaphorase (NADPHd) histochemistry.
2.1 Distribution of neurons NOS-ir
The distribution of the NOS-ir neurons and the intensity of staining are
presented in Table 1.
 At the mesodiencephalic level, the NOS-ir neurons appear in the precommissural
nucleus (PrC), at the level of the mammillary nuclei. The NOS-ir neurons were strongly
stained, with high density throughout the PrC. The pretectal nuclei showed
heterogeneous staining. The medial pretectal nucleus (MPT) showed a low density of
strongly stained NOS-ir neurons. The anterior pretectal nucleus (APT) presented a low
density of NOS-ir neurons with medium intensity of staining in both subdivisions,
dorsal (APTD), and ventral (APTV). The posterior pretectal nucleus (PPT) and the
olivary pretectal (OPT) presented a moderate density of medium stained NOS-ir cells.
Both the medial (MT) and lateral (LT) terminal nucleus of the accessory optic tract
showed just a few strongly labeled NOS-ir neurons. The dorsal terminal nucleus of the
accessory optic tract (DT) presented high density of strongly labeled NOS-ir neurons.
The reticular part of substantia nigra (SNR) showed a very low density of NOS-
ir neurons with weak intensity throughout the whole nucleus. The lateral part of the
substantia nigra (SNL) presented a moderate density of medium labeled NOS-ir
neurons. The compact part, dorsal tier of the substantia nigra (SNCD) showed a low
density of NOS-ir neurons with strong staining.
The periaqueductal grey matter (PAG) showed NOS-ir neurons in all the
divisions. In the anterior PAG, the pleomorphic subdivision (PAGp) presented a
moderate density of medium stained NOS-ir neurons, more concentrated in the medial
aspect of the PAG. Caudal to the PAGp, the PAG is formed by columns. The lateral
(LPAG) and the ventrolateral (VLPAG) columns presented a high density of medium
labeled NOS-ir neurons. The dorsomedial (DMPAG) and the dorsolateral columns
(DLPAG) presented a high density of strongly stained NOS-ir neurons. The
supraoculomotor (Su3) and the supraoculomotor cap (Su3C) are small subdivisions of
the PAG dorsal to the oculomotor nucleus(XXX). Both showed few strongly stained
NOS-ir neurons.
We were able to identify 7 layers in the superior colliculus. The zonal layer (Zo)
and the deep gray layer (DpG) showed moderate density and strongly labeled NOS-ir
neurons. The superficial grey layer (SuG), the optic nerve layer (Op), and the
intermediate gray layer (InG) had a moderate density of medium stained NOS-ir cells.
The nucleus of the optic tract (OT) and the nucleus of the posterior commissure (PCom)
showed a low density of medium stained cells. The paracommissural nucleus of the
posterior commissure (PaC) showed a low density of strongly stained NOS-ir neurons.
 The external (ECIC), the central (CIC) and the dorsal (DCIC) cortex of the
inferior colliculus presented low density while the nucleus of the brachium of the
inferior colliculus (BIC) had a high density of medium stained NOS-ir neurons.
The nucleus of the optic tract (OT) showed low density and medium labeled
NOS-ir neurons. The magnocellular nucleus of the posterior commissure (MCPC) and
the retroparafascicular nucleus (RPF) presented high density, strongly labeled NOS-ir
cells. The interstitial nucleus of Cajal (InC) presented just a few medium stained NOS-ir
neurons. The lithoid nucleus (Lth) showed few strongly stained neurons. A low density
of NOS-ir neurons were seen in the nucleus of the posterior commissure (PCom) and
the paracommissural nucleus of the posterior commissure (PaC).
The parabrachial showed low density and medium labeled NOS-ir neurons in the
medial nucleus (MPB), the internal (LPBI), and ventral (LPBV) part of the lateral
nucleus (LPB). The subbrachial nucleus (SubB) showed just a few NOS-ir neurons but
these were strongly stained.

In the ventral tegmental area (VTA), we found NOS-ir neurons in the

parabrachial pigmented nucleus (PBP) and the rostral part of the VTA (VTAR), both
with a moderate density of strongly labeled cells. The paranigral nucleus (PN) presented
low density, medium labeled NOS-ir neurons, while the Darkschewitsch nucleus (DK)
showed moderate density, strongly stained cells. The medial terminal (MT) and lateral
terminal (LT) nuclei of the accessory optic tract presented a very low density of strongly
labeled NOS-ir cells, while the dorsal terminal nuclei of the accessory optic tract (DT)
presented high density of NOS-ir cells.

A low density of strongly stained NOS-ir neurons is seen throughout the

prerubral field (PR). The pararubral nucleus (PaR), retrorubral field (RRF), and the red
nucleus showed just a few medium labeled NOS-ir neurons, while the retrorubral
nucleus (RR) presented moderate density medium stained cells. The pararubral nucleus
(PaR), the retrorubral field (RRF), the prerubral field (PR), the parvicellular (RPC) and
magnocellular (RMC) part of the red nucleus, the interfascicular (IF), and the
peripeduncular nucleus (PP) showed a very low density of NOS-ir cells with medium to
strong intensity of staining.

The subbrachial nucleus (SubB) presented a very low density of strongly labeled
NOS-ir cells. On the other hand, the nucleus of the brachium of the inferior colliculus
(BIC) presented medium stained NOS-ir neurons in high density.
 The oculomotor complex presented NOS-ir neurons in the pre-Edinger-Westphal
nucleus (PrEW) and the medial accessory oculomotor nucleus (MA3) when the
trochlear nucleus (4N) showed low density strongly labeled NOS-ir neurons.

In the interpeduncular nucleus (IP), the rock cavy presented a great collection of
NOS-ir neurons. The rostral subnucleus (IPR) showed a high density, while the caudal
(IPC), intermediary (IPI), and lateral (IPL) subnuclei showed moderate density. All of
them with strongly stained cells. The dorsolateral subnucleus (IPDL), the smallest
division, showed a low density of medium labeled NOS-ir neurons.

The pedunculopontine tegmental nucleus (PTg) presented a high density of

strongly labeled NOS-ir neurons. The pontine nucleus (Pn) and the oral (PnO), ventral
(PnV), and caudal (PnC) divisions of the pontine reticular nucleus showed a low density
of medium stained NOS-ir cells. The parabigeminal nucleus (PBG), the pre-cuneiforme
area (PrCnF), and the reticulotegmental nucleus of the pons (RtTg) presented a low
density of NOS-ir neurons.
Many NOS-ir neurons were found in the raphe nuclei. The rostral linear raphe
nucleus (RLi) presented a moderate density of strongly labeled NOS-ir cells. The dorsal
raphe nucleus (DR) many subnuclei showed NOS-ir cells. The lateral (DRL) and the
posterolateral (PDR) parts of the DR showed strongly stained NOS-ir cells with high
density. On the other side, the dorsal (DRD) and the ventral (DRV) parts of the DR
showed a very low density of medium labeled NOS-ir neurons similar to the median
raphe nucleus (MnR). The paramedian raphe nucleus (PMnR) showed low density and
strong staining. The raphe magnus nucleus (RMg) presented low density and medium
staining, while the raphe obscurus nucleus (ROb) showed a moderate density of strong
labeled NOS-ir neurons.
We found a low density of medium labeled NOS-ir in the pontine nucleus (Pn),
oral (PnO), ventral (PnV) and caudal (PnC) parts of the pontine reticular nucleus,
parabigeminal nucleus (PBG), and the reticulotegmental nucleus of the pons (RtTg)
Many nuclei in the pontine tegument, such as the subpeduncular tegmental
nucleus (SPTg), microcellular tegmental nucleus (MiTg), posterodorsal tegmental
nucleus (PDTg), triangular (TrLL), ventral (VLL) and lateral (ILL) nuclei of the lateral
lemniscus, medial paralemniscal (MPL), ventral part of perilemniscal nucleus (PLV),
medioventral periolivary nucleus (MVPO), lateroventral periolivary nucleus (LVPO),
laterodorsal tegmental nucleus (LDTg), and the dorsomedial tegmental area (DMTg)
showed just a few NOS-ir neurons with different intensities of staining. A low or very
low density of NOS-ir neurons is also seen in the nucleus of the trapezoid body (Tz),
alpha (SubCA), dorsal (SubCD) and ventral (SubCV) parts of the subcoeruleus nucleus,
anterior (VCA) and posterior (VCP) parts of the ventral cochlear nucleus. The locus
coeruleus (LC) showed a moderate density of NOS-ir neurons strongly stained.

The trigeminal complex showed heterogeneous distribution of NOS-ir neurons.

The dorsomedial (Pr5DM) and ventrolateral (Pr5VL) parts of the principal sensory
trigeminal nucleus showed a low density of medium labeled NOS-ir cells. The
mesencephalic trigeminal nucleus (Me5) showed the same density but strongly stained
neurons. The dorsomedial (Sp5DM), oral (Sp5O), and interpolar (PS5I) parts of the
spinal trigeminal nucleus (Sp5) also showed a low density of medium labeled NOS-ir
neurons, while the caudal part (Sp5C) presented moderate density of strongly labeled
cells. The gelatinous layer of the caudal spinal trigeminal nucleus (Ge5) showed a high
density of strongly labeled NOS-ir cells.

The alpha (CGA) and beta (CGB) parts of the central grey showed low density
and medium stained NOS-ir neurons, while the central gray of the pons (CGPn) showed
a very low density and weakly stained cells. The superior vestibular nucleus (SuVe)
showed a low density of strongly labeled cells. The paraabducens (Pa6) and the
abducens (6N) nuclei expressed a moderate density of strongly labeled NOS-ir neurons.

The gigantocellular reticular nucleus (Gi) presented a great cluster of NOS-ir

neurons spread over its extension with medium staining in general, but strongly labeled
in its alpha part (GiA). The intermediate reticular nucleus (IRt) presented low density
strongly stained NOS-ir neurons. The parvicellular reticular nucleus (PCRt) showed a
low density of medium stained cells. The magnocellular and parvicellular parts of the
vestibular nucleus (MVeMC and MVePC) showed the same density and intensity of
NOS-ir neurons as the IRt.

The prepositus nucleus (Pr) showed a moderate density of strongly labeled cells.
The same was seen in the alpha (LPGiA) and external (LPGiE) parts of the lateral
paragigantocelullar nucleus (LPGi) and the dorsal paragigantocelullar nucleus (DPGi).
In the vestibular nucleus, NOS-ir neurons were seen in the lateral (LVe) and spinal
subnuclei (SpVe). The X nucleus (X) showed a moderated density of strongly labeled
cells. The inferior salivatory nucleus (IS) and the matrix region of the medulla (Mx)
showed a low density of NOS-ir neurons with medium and strong labeling, respectively.

The solitary tract nucleus (Sol) has many subdivisions and all of them showed
low density and medium labeling, except the medial (SolM) subdivision with high
intensity. The dorsal motor of the nucleus vagus (10N) showed the same staining
pattern as the most Sol subdivisions.

Many other nuclei of the medulla, such as the lateral reticular nucleus (LRt),
medullary reticular (Md), cuneate nucleus (Cu), gracile nucleus (Gr), inferior olive (IO),
retroambiguus nucleus and the interstitial nucleus of the medulla (IB) showed strongly
labeled NOS-ir neurons with low to moderate density.

2.2 NADPH diaphorase. 3:25

The same as in our previous work in the hypothalamus (Reis et al., 2018), the
NADPH diaphorase (NADPHd) activity stained the brainstem with a bluish color with
heterogeneous intensity. In general, the stronger immunolabeled areas showed the
deepest blue color with neurons easily located, while some areas or nuclei with just a
few or weakly stained NOS-ir neurons presented a blue staining above the background
but with no neurons distinguished. Figure X shows NADPHd staining in the IP, superior
colliculus, MiTG, LC, Me5, Ge5, and Sp5C.
 3. Discussion
NO is a small molecule with the ability to pass through cell membranes to
perform its biological activity. In the nervous system, this ability makes the NO an
economic and versatile neurotransmitter working in the modulation of various
neurological systems and functions. This also contributes to its evolutionary success,
making it a well-preserved molecule among the species.
The technical considerations have been discussed in a previous work (Reis et al.,
2018).
3.1 Comparative neurochemistry
Inconsistencies and differences can be seen among the works describing the
distribution of nitrergic neurons even when using the same rodent species, such as rat
(Vincent and Kimura, 1992; Rodrigo et al., 1994) or mouse (Matsushida et al., 2001;
Gotti et al., 2005). A comparison of our work with these descriptions showed the
distribution of nitrergic neurons in the rock cavy brainstem to be wider, with virtually
every nucleus showing NOS-ir cells. The differences were more pronounced in the rock
cavy brainstem than those seen in the hypothalamus (Reis et al., 2018).
3.1.1 NO in the rock cavy's brainstem
The most rostral NOS-ir cells in the rock cavy’s brainstem were in the PrC and
the pretectal nuclei, which was reported to be more evident in mouse than in rat
(Rodrigo et al., 1994; Matsushida et al., 2001). The superior colliculus is the most
rostral structure demonstrating NO cells in the human brainstem (Egberongbe et al.,
1994) and is a great source of Nitrergic cells in the rat (Vincent and Kimura, 1992;
Rodrigo et al., 1994; Chong et al., 2019), mouse (Matsushida et al., 2001; Gotti et al.,
2005) and rock cavy (present work). Still, in the rostral mesencephalon, the rock cavy
presented few NOS-ir neurons in all the divisions of the substantia nigra, as also
reported in previous works (Rodrigo et al., 1994; Chong et al., 2019). Matsushida and
co-workers (2001) described only very few sparse NADPH diaphorase positive cells in
the compact part of the substantia nigra of mouse, while other works only reported
fibers in rat, mouse and human (Vincent and Kimura, 1992; Egberongbe et al., 1994;
Gotti et al., 2005). The VTA complex, which includes the IP and IF, has a consistent
distribution of NOS-ir neurons among the rodent species (Rodrigo et al., 1994; Gotti et
al., 2005) including the rock cavy. This is also true in other locations such as the inferior
colliculus, DK, and MCPM (Rodrigo et al., 1994; Gotti et al., 2005; Hinova-Palova et
al., 2017).
 A high density of strong and medium stained NOS-ir neurons characterize the
whole rock cavy PAG. In the rat, NOS is a known marker of the PAG dorsolateral
column (Onstott et al., 1993), although nitrergic neurons have also been described in
other columns (Rodrigo et al., 1994; Chong et al., 2019), as we found in the rock cavy.
Other differences can be seen in the rubral complex, including the red nucleus, where
few NOS-ir neurons were seen in the rock cavy, while only nitrergic fibers have been
seen in the rat, mouse and human (Vincent and Kimura, 1992; Egberongbe et al., 1994;
Rodrigo et al., 1994; Gotti et al., 2005). Rodrigo and co-workers (1994) described NOS-
ir neurons in the EW in rat, what is not consistent with has been described by other
studies with rats and mice (Vincent and Kimura, 1992; Matsushida et al., 2001; Gotti et
al., 2005). We found very few NOS-ir neurons in the PrEW.
Seen as a group, the distribution of nitrergic neurons in the rock cavy's raphe
nuclei is similar to previous descriptions in rodents (Vincent and Kimura, 1992;
Rodrigo et al., 1994; Matsushida et al., 2001; Gotti et al., 2005; Okere and Waterhouse,
2006), such as the trigeminal complex, parabrachial nuclei and Sol (Vincent and
Kimura, 1992; Rodrigo et al., 1994; Matsushida et al., 2001; Gotti et al., 2005; Fan et
al., 2009; Pajolla et al., 2009).
Most of the nitrergic nuclei of the pons of the rock cavy presented few NOS-ir
neurons, except the PTg whose high density of strongly stained NOS-ir neurons was
similar to other rodents. Like other rodents, the rock cavy has NOS-ir neurons in the Pn,
LC, LPGi, Gi, and GiA. The LDTg neurons in rat and mouse, however, showed an
intense and rich NOS-ir staining (Vincent and Kimura, 1992; Rodrigo et al., 1994;
Matsushida et al., 2001; Gotti et al., 2005), different from our data with the rock cavy.
Other differences among species can be seen in the distribution of Nitrergic neurons in
the vestibular and cochlear nuclei. While the rock cavy shows NOS-ir neurons in all the
vestibular subnuclei and the ventral cochlear nucleus, there is no description of NO in
mouse cochlear nuclei and only a few neurons in the spinal vestibular nucleus (Gotti et
al., 2005). Vincent and Kimura (1992) described neurons NADPH diaphorase in
neurons only in the medial vestibular nucleus and posterior cochlear nucleus of rat,
while Rodrigo and co-workers (1994) described NOS-ir neurons in the medial and
spinal vestibular nuclei and dorsal cochlear nucleus. Chong and co-workers (2019)
described the absence of NOS in the neurons of the vestibular nuclei. However, a
comparative study in different species of mammals, including humans and non-human
primates, showed that NOS-ir neurons were present in the ventral cochlear nucleus
(Baizer et al., 2018), as we found for the rock cavy.
In the medulla, in addition to some nuclei described above, the presence of
Nitrergic neurons in nuclei such as the Pr, IB, Cu, and 10N are consistent with previous
descriptions (Rodrigo et al, 1994; Matsushida et al, 2001).

3.2 Functional aspects

Because of its molecular characteristics and its wide distribution, NO seems to
have an important role in almost every neural function (Philippu, 2016). This is even
more accentuated when we consider brainstem functions. Looking to NOS distribution
in the rock cavy’s brainstem we can say that NO has this same feature. It is important,
however, to highlight some points that we believe are more relevant when we consider
the functions of the NO neurons in the brainstem.
NO plays a major role in the visual system (Cramer et al., 1998; Cudeiro e
Rivadulla, 1999). The pretectal nuclei and the superior colliculus belong to the visual
system and both express abundant NOS in the rock cavy. The pretectal nuclei belong to
the accessory optic system and are involved with pupillary reflex and eye movement
(Simpson, 1984). The pretectal nuclei are also involved with analgesia in rats.
Microinjections of L-arginine in the rat pretectum produced an analgesic response,
which was blocked by injections of L-NAME, a NOS antagonist, or by a cyclic GMC
inhibitor, suggesting that L-arginine causes production of NO, which in turn activates
the pretectal analgesic system involving cyclic GMP (Kumar et al., 1993).
Although the superior colliculus belongs to the main optic system, it is a place
where signals from the different senses are combined and used to guide adaptive motor
responses (King, 2004). NOS is expressed in neurons of the superior colliculus of many
species of mammals, such as the cat (Scheiner et al., 2001), ferret (Behan et al, 2002),
opossum (Giraldi-Guimarães et al., 1999), rat (Rodrigo et al., 1994), mouse (Matsushida
et al., 2001) and human (Egberongbe et al., 1994). In the superior colliculus, NO has
been related to the development, axonal plasticity, and refinement of the neuronal
connections (Wu et al., 2000; Chung et al, 2004; Chacur et al., 2006).
NO neurons in the PAG modulate many functions in rat, such as defensive/panic
behavior, antinociception, cardiovascular and respiratory changes in responses to
external stressors (Miguel and Nunes-de-Souza, 2006; Dampney et al., 2013; Fernandes
et al., 2019). Since the rock cavy’s PAG has a great number of NOS neurons, we
believe that same type of modulation happens in this species.
NOS is expressed in neurons of all the raphe nuclei of the rock cavy. The raphe
nuclei are the main source of serotonin in the brain of virtually every species, including
the rock cavy (Soares et al, 2012). In rat, NO and serotonin colocalize only in the
mesopontine raphe nuclei, from 40% to more than 70% of colocalization, depending on
which study is being consulted (Johnson and Ma, 1993; Lu et al., 2010; Gartside et al.,
2020); and the NOS donor inhibits about 70% and excites 10% of the DR neurons
(Gartside et al., 2020). As an example of NO and serotonin functional interaction, a
NOS knockout male mouse has elevated aggression (Chiavegato et al., 2001). This
excessive aggressive behavior is caused by reductions in serotonin turnover and
deficient 5-HT1a and 5-HT1b receptor functions in brain regions regulating emotion.
This could be reversed by serotonin precursors and by treatment with specific 5-HT1a
and 5-HT1b receptor agonists (Chiavegatto and Nelson, 2003). Although we cannot say
if NOS and serotonin colocalize in rock cavy’s brainstem, our data strongly indicates a
relation between those neurotransmitters.
Nitrergic cells are located in the auditory system from the periphery to central
(Fessenden et al, 1999). NOS is detected throughout the whole rat superior olivary
complex and some of those Nitrergic neurons project to the inferior colliculus and the
cochlea (Reuss e Riemann, 2000). NO in the inferior colliculus has been involved in the
control of sound-evoked electrocortical arousal (Nisrticò et al., 1994; Iannone et al.,
1996) and the processing and transmission of the acoustic input to the auditory cortex
(Grassi et al., 1995). NO also regulates the firing rate of neurons in the ventral cochlear
nucleus of the guinea pig (Hockley et al, 2019). The rock cavy inhabits an open field
environment with low ground cover and short grass, where audition is very important to
detect a predator. Although the rock cavy's superior olivary nucleus does not express
NOS, the inferior colliculus and other nuclei of the superior olivary complex, such as
trapezoid, cochlear and periolivary nuclei, presented NOS-ir neurons. So, we believe
that NO has an important role in the auditory system of rock cavy.
NO is involved with brain pathological mechanisms. Karolewicz and co-workers
(2004) detected a lower concentration of NOS in the LC of humans diagnosed with
major depression. This decrease in NO can disrupt the glutamatergic signaling affecting
the input to this important noradrenergic nucleus. Agmatine is a biologically active
substance that produces several central and peripheral effects. It potentiates morphine
analgesia, blocks opiate tolerance development (Kolesnikov et al., 1996; Aricioglu-
Kartal and Uzbay, 1997) and stimulates pituitary hormone release (Kalra et al., 1995). A
study using a NOS inhibitor shows that agmatine stimulates the firing rate of locus
coeruleus neurons via a nitric oxide synthase-dependent mechanism (Ruiz-Durantéz et
al., 2002).
Liver cirrhosis can evolve and affect other organs including the brain, causing
hepatic encephalopathy. It causes motor disturbance in response to striatal dopaminergic
alteration. Chen and co-workers (2007) showed that rats with liver cirrhosis had a
decreased number of NADPHd neurons in the SN and that those neurons had less
NADPHd activity. NOS-ir and dopamine do not co-exist in mouse SN neurons, but are
in close apposition (Matthews et al, 1997). Injection of 1-methyl-4-phenylpyridinium
(MPP+), a drug that induces idiopathic Parkinson’s disease, in the anterior striatum of
mice results in less damage in the SN of NOS knockout mice than in wild mice
(Matthews et al, 1997). A NOS inhibitor had the same result in the rat brain (Matthews
et al., 1997). Oral administration of a NO donor reduced motor impairment
significantly, the decreasing of dopamine fibers in the striatum, and the loss of
dopamine neurons in the SN induced by MTPT, another Parkinson's disease-inducing
drug (L’Episcopo et al., 2010). The roles of NO in the SN and dopaminergic systems
that control motricity must be also true in the rock cavy nervous system since it has
NOS-ir neurons in the SN. Another source of dopamine, the VTA, has NOS-ir neurons
in the rock cavy brainstem. Those NOS-ir neurons co-express dopamine in rat (Klejbor
et al., 2004). The dopaminergic neurons in the nuclei of the VTA are involved in the
regulation of motor and motivational aspects of behavior as well as being implicated in
emotional and cognitive disturbances associated with schizophrenia.
LDTg and PTg show NOS-ir neurons in the rock cavy. These are classic
cholinergic nuclei, part of the reticular activating ascending system and NO are in
virtually all of these acetylcholine neurons in the mouse brainstem (Veleanu et al.,
2016). Stimulation of these nuclei promotes NO increases in the somatosensory cortex
of the cat and systemic blockade of NO production abolishes the sleep-awake transition
after stimulation of the reticular brainstem (Espinosa et al., 2015).
Central NO has been implicated in many autonomic functions, mainly in
sympathetic control of the cardiovascular system (Zanzinger, 1999; Hirooka et al,
2011). In fact, many brainstem nuclei that control the autonomic nervous system
produce NO in different species (Zanzinger and Seller, 1997; Maisky et al., 2013). In
agreement, here we report NOS-ir neurons in the LPB, 10N, nuclei of the rostral and
caudal ventrolateral medulla, and Sol of the rock cavy, showing that NO can have an
important role in the motor and sensorial aspect of the autonomic system also in the
rock cavy.

4. Conclusion
The distribution of nitrergic neurons in the rock cavy's brainstem was similar to
that of other rodents, although differences have been reported, mainly due to the
variations among studies. The distribution seen indicates conservation in the evolution
of this neurotransmitter but also points to differences in its function inside the brain of a
species that inhabits a specific environment.
Here we conclude that NO inside the rock cavy's brainstem seems to participate
in many circuits modulating virtually every brainstem function and that NOS and
NADPHd staining was found to be a valuable tool in the study of rock cavy
neuroanatomy.

5. Experimental Procedures

The experimental procedures were the same as previously published (Reis et al.,
2018). To summarize, seven young adult rock cavies were used in this study. The
capture and handling of the animals were authorized by IBAMA (license 214440-1).
Individuals were housed in 3.00m x 2.00m x 2.60m masonry cages and exposed to
environmental temperature, air humidity, and light, with unlimited access to food and
water.

Anesthetized animals were perfused with 700ml of a 4% paraformaldehyde

solution in 0.1M phosphate buffer, pH 7.4 (PB). After perfusion, the 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 and the brains were
sectioned into 3 blocks employing two coronal sections one at the bregma level and the
other at the lambda level. Each brain was removed from the skull and stored in a 30%
sucrose solution in PB for 24–48 h. The brains were cut in the coronal plane (30m
sections) using a sliding freezing microtome. Six series were collected in antifreeze
solution and stored in a freezer.
 Sections from one series were Nissl stained. A second series was submitted to
immunohistochemistry to reveal NOS. All the immunohistochemical procedures were
performed as previously described (Reis at al., 2018). A third series was submitted to
reveal NADPHd as described in a previous work (Reis et al., 2018)
The analysis and production of images were made using microscopy (Nikon
Eclipse Ci-L). The nuclei containing NOS-ir neurons were classified according to the
density and intensity of staining. The photomicrographs were captured using a digital
camera (Nikon DS-Ri1) adapted to the same microscope and the software NIS (Nikon).
The software Canvas 10 was used to edit the photomicrographs (brightness, sharpness,
and contrast), integrate them into plates, and make the illustrations. These sketches were
based on the Nissl and immunostained sections. The nuclei and areas were identified
based on the Nissl staining and the brain atlas of rat and mouse (Paxinos and Watson,
1998; Paxinos and Franklin, 2008).

Acknowledgment: We would like to thank Miriam Regina Celi de Escala

Oliveira for expert technical assistance. The English version of this text was revised by
Sidney Pratt, Canadian, BA, MAT (The Johns Hopkins University), RSAdip (TEFL).

Funding: Financial support was provided in the form of grants and fellowships
from the National Council of Technological and Scientific Development(CNPq -
474623/2013-0).
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