Nuclear organization and morphology of cholinergic neurons in the brain of the rock cavy (Kerodon rupestris) (Wied, 1820)

Journal of Chemical Neuroanatomy 94 (2018) 63–74

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Nuclear organization and morphology of cholinergic neurons in the brain of the rock cavy (Kerodon rupestris) (Wied, 1820)

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N.R. Resendea, P.L. Soares Filhoa, P.P.A. Peixotoa, A.M. Silvaa, S.F. Silvaa, J.G. Soaresa, ⁎ E.S. do Nascimento Jr.a, J.C. Cavalcantea, J.S. Cavalcanteb, M.S.M.O. Costaa, a b

Department of Morphology, Laboratory of Neuroanatomy, Biosciences Center, Federal University of Rio Grande do Norte, Natal, RN, Brazil Department of Physiology, Laboratory of Neurochemical Studies, Biosciences Center, Federal University of Rio Grande do Norte, Natal, RN, Brazil

ARTICLE INFO

ABSTRACT

Keywords: Acetylcholine Choline acetyltransferase Cholinergic system Kerodon rupestris Neurotransmitters

The aim of this study was to conduct cytoarchitectonic studies and choline acetyltransferase (ChAT) immunohistochemical analysis to delimit the cholinergic groups in the encephalon of the rock cavy (Kerodon rupestris), a crepuscular Caviidae rodent native to the Brazilian Northeast. Three young adult animals were anesthetized and transcardially perfused. The encephala were cut in the coronal plane using a cryostat. We obtained 6 series of 30-μm-thick sections. The sections from one series were subjected to Nissl staining. Those from another series were subjected to immunohistochemistry for the enzyme ChAT, which is used in acetylcholine synthesis, to visualize the different cholinergic neural centers of the rock cavy. The slides were analyzed using a light microscope and the results were documented by description and digital photomicrographs. ChAT-immunoreactive neurons were identified in the telencephalon (nucleus accumbens, caudate-putamen, globus pallidus, entopeduncular nucleus and ventral globus pallidus, olfactory tubercle and islands of Calleja, diagonal band of Broca nucleus, nucleus basalis, and medial septal nucleus), diencephalon (ventrolateral preoptic, hypothalamic ventrolateral, and medial habenular nuclei), and brainstem (parabigeminal, laterodorsal tegmental, and pedunculopontine tegmental nuclei). These findings are discussed through both a functional and phylogenetic perspective.

Abbreviations: 3N, oculomotor nucleus; 3V, third ventricle; A, amygdaloid area; ac, anterior commissure; aca, anterior commissure, ant; Acb, accumbens nucleus; ACh, acetylcholine; acp, anterior commissure, post; AD, anterodorsal thalamic nucleus; AHA, anterior hypothalamic area; APT, anterior pretectal nucleus; Aq, aqueduct; Arc, arcuate hypothalamic nucleus; B, basal nucleus; BIC, nucleus brachium inferior colliculus; bic, brachium inferior colliculus; cc, corpus callosum; ChAT, cholineacetyltransferase; Cl, claustrum; CLi, caudal linear nucleus of the raphe; Co, cortical amygdaloid nucleus; cp, cerebral peduncle; CPu, caudate putamen; csc, commissure superior colliculus; CxA, cortex-amygdala transition; DLG, dorsal lateral geniculate nucleus; DM, dorsomedial hypothalamic nucleus; DR, dorsal raphe nucleus; ec, external capsule; En, endopiriform nucleus; EP, entopeduncular nucleus; f, fornix; fr, fasciculus retroflexus; GP, globus pallidus; hbc, habenular commissure; HDB, diagonal nucleus of Broca, horizontal limb; IC, inferior colliculus; ic, internal capsule; ICj, islands of Calleja; IGL, intergeniculate leaflet; IP, interpeduncular nucleus; LD, laterodorsal thalamic nucleus; LDTg, laterodorsal tegmental nucleus; lfp, longitudinal fasciculus pons; LH, lateral hypothalamic area; LHb, lateral habenular nucleus; lo, lateral olfactory tract; LP, lateral posterior thalamic nucleus; LPO, lateral preoptic area; LV, lateral ventricle; M, mammillary nucleus; Me5, mesencephalic trigeminal nucleus; MG, medial geniculate nucleus; MHb, medial habenular nucleus; ml, medial lemniscos; mlf, medial longitudinal fasciculus; MnPO, median preoptic nucleus; MnR, median raphe nucleus; MPA, medial preoptic area; MS, medial septal nucleus; mt, mammillothalamic tract; och, optic chiasm; opt, optic tract; Pa, paraventricular hypothalamic nucleus; PAG, periaqueductal gray; PBG, parabigeminal nucleus; PF, parafascicular thalamic nucleus; Pir, piriform cortex; PMnR, paramedian raphe nucleus; Pn, pontine nuclei; PT, paratenial thalamic nucleus; PTg, pedunculopontine tegmental nucleus; PV, paraventricular thalamic nucleus; PVP, paraventricular thalamic nucleus, posterior; RCh, retrochiasmatic area; Rt, reticular thalamic nucleus; SC, superior colliculus; SCh, suprachiasmatic nucleus; sm, stria medullaris thalamus; SNC, substantia nigra, compacta; SNR, substantia nigra, reticular; SO, supraoptic nucleus; ST, bed nucleus stria terminalis; st, stria terminalis; STh, subthalamic nucleus; tfp, transverse fibers pons; TT, tenia tecta; Tu, olfactory tubercle; VDB, diagonal nucleus of Broca, vertical limb; VLG, ventral lateral geniculate nucleus; VLH, ventrolateral hypothalamic nucleus; VLPO, ventrolateral preoptic nucleus; VMH, ventromedial hypothalamic nucleus; VP, ventral pallidum; xscp, decussation superior cerebellar peduncle; ZI, zona incerta ⁎ Corresponding author at: Department of Morphology, Laboratory of Neuroanatomy, Biosciences Center, Federal University of Rio Grande do Norte, 59072-970, Natal, RN, Brazil. E-mail address: [email protected] (M.S.M.O. Costa). https://doi.org/10.1016/j.jchemneu.2018.09.001 Received 22 February 2018; Received in revised form 20 September 2018; Accepted 20 September 2018 0891-0618/ © 2018 Elsevier B.V. All rights reserved.

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1. Introduction Acetylcholine (ACh) was the first neurotransmitter that was discovered as the neurotransmitter used by somatic motor and autonomic neurons. It was subsequently identified in several neuronal clusters in the central nervous system, such as interneurons and large projection neurons (Von Bohlen et al., 2006). Neurons that synthesize and release acetylcholine for neurotransmission are referred to as cholinergic neurons (Oda and Nakanishi, 2000). ACh is a fast-acting, point-to-point neurotransmitter at the neuromuscular junction and in the autonomic ganglia. ACh, however, also appears to act as a neuromodulator in the brain despite its role as the primary excitatory neurotransmitter at the periphery. In the brain, ACh modifies neuronal excitability, alters the presynaptic release of other neurotransmitters, and coordinates the firing of neuronal groups (Picciotto et al., 2012). In this sense, in addition to its role in known motor regulation (Calabresi et al., 2000), central cholinergic modulation participates in functions such as synaptic plasticity (Drever et al., 2011; Giocomo and Hasselmo, 2007; McKay et al., 2007) and neuronal development (Role and Berg, 1996). ACh also participates in the modulation of brain systems such as the mesolimbic dopaminergic system, which is associated with addiction and reward (Omeichenko and Sesack, 2006). It is also involved in the regulation of cortical activity (Hasselmo and Sarter, 2011). Cholinergic signaling may also alter hypothalamic functions, such as thermoregulation (Myers and Waller, 1973), sleep patterns (Steriade, 2004), food intake (Mineur et al., 2011), pancreatic insulin and glucagon release (Ishikawa et al., 1982). Increasing evidence suggests that ACh signaling in a number of brain cells is important for stress response (Mark et al., 1996), and in learning and memory processes (Gais and Born, 2004; Gold, 2003; Hasselmo, 1999, 2006). In the central nervous system, ACh is found in some populations of cholinergic interneurons that may be found in the prosencephalon and brainstem, as well as so-called large cholinergic neurons in the basal prosencephalon and the mesopontine tegment, which result in large ascending projections. This is in addition to its presence in cholinergic somatic and autonomic motor neurons in the spinal cord and brainstem. The most representative interneurons are those in the striatum. These interneurons interact with the dopaminergic terminals of neurons that project to the striatum from the substantia nigra. There are also cholinergic interneurons sparsely distributed in the cerebral cortex, hippocampus, and olfactory bulb (Von Bohlen et al., 2006). Among the projection neurons, cholinergic groups of neurons in the basal prosencephalon include those in the medial septal nucleus (Ch1), the horizontal and vertical limbs of the diagonal band nucleus (Ch2–Ch3), and the basal nucleus of Meynert (Ch4). These neurons are responsible for large ascending projections and topographically innervate neurons throughout the cerebral cortex, hippocampus, and amygdala. The mesopontine cholinergic neurons are divided into a ventrolateral column (cell group Ch6, or the pedunculopontine nucleus) close to the lateral border of the superior cerebellar peduncle, and a dorsomedial column (cell group Ch5, or the laterodorsal tegmental nucleus), which is a component of the periaqueductal gray located just rostral to the locus coeruleus. Both these nuclei send important descending projections to the pontobulbar reticular formation, vestibular nuclei, locus coeruleus, and several raphe nuclei in addition to providing extensive ascending cholinergic innervation to the thalamus and hypothalamus. It is believed that these projections have a prominent role in regulating the sleep-wake cycle. Ch7 neurons are present in the habenula and project to the interpeduncular nucleus. Finally, Ch8 neurons are located in the parabigeminal nucleus and send projections to the superior colliculus (Mesulam et al., 1983a; Mufson et al., 1986; Von Bohlen et al., 2006). Cholinergic nuclei are immunohistochemically delimited by ChAT or ACh vesicular transporters in the brain of several species of mammals, such as rat (Armstrong et al., 1983; Ichikawa et al., 1997; Roghani et al., 1998; Schäfer et al., 1998); monotremes (Manger et al., 2002), mole rat (Bhagwandin et al., 2008; Da Silva et al., 2006), bats (Dell

Fig. 1. Photograph of the rock cavy in captivity.

et al., 2010; Kruger et al., 2010; Maseko and Manger, 2007; Maseko et al., 2007), porcupine (Limacher et al., 2008), guinea pig (Motts et al., 2008), rock hyrax (Gravett et al., 2009), giraffe (Bux et al., 2010), elephant shrew (Pieters et al., 2010), African pygmy mouse (Kruger et al., 2012), three Afrotherian species (Calvey et al., 2013), Tasmanian devil (Patzke et al., 2014), two species of Euarchontoglires (Calvey et al., 2015a), five species of insectivore (Calvey et al., 2016), the river hippopotamus (Dell et al., 2016), the Goettingen miniature pig (Mahady et al., 2017), non-human primates (Benzing et al., 1993; Calvey et al., 2015b; Kus et al., 2003; Satoh and Fibiger, 1985a,b), and human (Oda and Nakanishi, 2000). Considering the importance of studying neural systems from a comparative evolutionary point of view, it is imperative to extend such studies to the greatest number of species. This is why we chose to undertake this study in the rock cavy. The rock cavy (Kerodon rupestris) (Fig. 1) is classified taxonomically as a representative of the phylum Chordata, class Mammalia, superorder Glires, order Rodentia, suborder Hystricomorpha, family Caviidae, and subfamily Caviinae (Silva Neto, 2000). The suborder Hystricomorpha includes several families with a number of species found in Brazil. In addition to the rock cavy, these species include the agouti (family Dasyproctidae), the paca (family Cuniculidae), and the capybara (Hydrochaeridae). All of these animals are used as experimental models by Brazilian researchers (see for example, Freire et al., 2010; Picanço-Diniz et al., 1991, 2011; Rocha et al., 2009, 2012; Silveira, 1985; Silveira et al., 1989). Phylogenetic studies using a molecular approach have connected the genus Kerodon with the genus Hydrochaeris, which includes the capybara (family Hydrochaeridae) and is closely related to the genus Dolicotis of the subfamily Dolicothinae, whose representative in South America is the Patagonian hare (Dolichotis patagonum) (Rowe and Honeycutt, 2002). The rock cavy inhabits the semiarid Caatinga region of the Brazilian Northeast, although it can be found in the Southeast region as far as the state of Minas Gerais. Colonies of rock cavies usually live in cracks and crevices of granitic rocks, which serve as refuge and shelter from predators (Lacher, 1981). 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). Behavioral studies conducted in the field have reported that this rodent species emerges to forage throughout both the day and night, but most of the activity occurs during the day, with peaks of activity at dawn and dusk (Carvalho, 1969; Lacher, 1981). In concordance with these observations, an investigation performed under controlled laboratory conditions showed that the rock cavy was active throughout the day, with peaks during sunrise and sunset. The animals were observed to have a predominantly crepuscular behavior (Sousa and Menezes, 2006). Neuroanatomical studies in the rock cavy were started in our laboratory when this species was adopted as a regional rodent model in circadian rhythm studies. In addition to the characterization of the activity rhythm and other circadian responses (Sousa and Menezes, 2006), the structures controlling circadian rhythmicity–the suprachiasmatic nucleus and the intergeniculate leaflet–have been 64

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identified for their neurochemical content and retinal projection (Cavalcante et al., 2008; Nascimento et al., 2010a). Direct retinal projections to the paraventricular (Nascimento et al., 2008) and mediodorsal (Nascimento et al., 2010b) thalamic nuclei, as well as those to the caudal zona incerta (Morais et al., 2014) were identified. Investigation of the serotonergic system enabled the identification of the raphe nuclei and other extra-raphe nuclei (Soares et al., 2012), as well as the distribution of serotonergic terminals on the thalamic midline and intralaminar nuclei (Silva et al., 2014). The midbrain dopaminergic (Cavalcanti et al., 2014) and diencephalon nitrergic (Reis et al., 2018) groups were also outlined. The anatomy of the eye and the structures of the retina (Oliveira et al., 2014) and other retinal specializations (Oliveira et al., 2018) were also described. Studies of the targets of retinal projections constituting the primary visual and accessory optic systems, as well as projections to other hypothalamic targets, are ongoing. The present study aimed to outline the cholinergic cell groups in the brain of the rock cavy using ChAT immunohistochemistry, as well as to describe the neuronal morphology in each nucleus.

mounted on silanized glass slides and Nissl-stained with thionin to visualize the cytoarchitectonic delimitation of neuronal groups. Sections from another series underwent immunohistochemistry for ACh using an antibody against ChAT, which is the synthesizing enzyme for ACh. Sections were pretreated with hydrogen peroxide (H2O2) and placed in a solution containing a goat anti-ChAT polyclonal antibody (Millipore, cat. #AB144P, lot #NG1780580) at 1:1000 dilution and 2% normal rabbit serum in 0.4% Triton X-100 overnight on a rotator (Fisher, 099ARD524) at a speed of 3 rpm. The sections were then immersed in rabbit biotinylated anti-goat secondary antibody (Jackson Immunoresearch Labs; West Grove, PA, USA) diluted at 1:1000 in the same vehicle as above. The sections were placed on the same rotator and were incubated at the same speed for 90 min. To visualize the reaction, the sections were placed in a solution containing an avidinbiotin-horseradish peroxidase complex (Vector Elite ABC kit). This was followed by the final reaction in a medium containing H2O2 as substrate and tetrahydrochloride diaminobenzidine as chromogen. The sections were thoroughly washed with 0.1 M phosphate buffer, pH 7.4 between experimental steps. The sections were mounted on silanized glass slides, which, after drying at room temperature, were submersed rapidly in a 0.05% osmium tetroxide solution to intensify the reaction. The sections were then dehydrated in an ethanol series with increasing concentration, cleared with xylene, and cover-slipped with ERV-mount® (Erviegas, Brazil). All immunohistochemical procedures were performed at room temperature. As control for staining specificity, some sections underwent immunohistochemistry without the primary antibody. In these cases, no ChAT immunoreactivity was observed. Sections in the remaining compartments were stored in an antifreeze solution consisting of ethylene glycol-sucrose in 0.1 M phosphate buffer, pH 7.4, at −20 °C for eventual repetition of immunohistochemistry for ChAT or other substances. Brightfield microscopy using a Nikon Eclipse NI microscope was used to examine Nissl- and ChAT-immunostained sections. Digital images were obtained from representative sections using a digital video camera (Nikon DS Ri1) attached to the microscope and the software NIS-Elements AR. The digitized images were converted to gray scale, corrected minimally for brightness and contrast, and composed using Adobe Photoshop CC2017 software (Adobe Systems; Mountain View, CA, USA). Diagrams were obtained from images of Nissl-stained coronal sections using Canvas version 12 software (ACD Systems of America, Inc.). The corresponding immunostained sections were then matched to the drawings and the immunopositive cell bodies were plotted in the ratio of approximately 1:1 using the Canvas 12 drawing program.

2. Materials and methods Three young adult rock cavies, one female and two males, weighing 300–470 g with brain masses ranging between 3.6 and 4.6 g were used in our study. The rock cavies were obtained from countryside municipalities in the state of Rio Grande do Norte, Brazil. The animals were captured after permission from the Brazilian Environmental Agency (IBAMA, license SISBIO 42960-1, 10/03/2014) and housed in a large room with ceramic tile ceiling and natural soil floor with creeping vegetation and rocks simulating the animal’s natural habitat. The animals were exposed to environmental temperature, air humidity, and light, with unlimited access to food and water. All efforts were made to minimize the number of animals and their suffering, and the procedures strictly followed the norms established by the National Research Council of the National Academy published in the “Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research” and those recommended by the Brazilian Society of Neuroscience and Behavior (SBNeC), http://www.sbnec.gov.br/links. The project was approved by the local Ethics Committee (CEUA-UFRN, protocol 004/ 2014). All experimental procedures were performed in the Neuroanatomy Laboratory, Department of Morphology, UFRN. Each animal was anesthetized with an intramuscular injection of 10% ketamine hydrochloride (Agener União, Brazil, 1 ml) and 2% xylazine hydrochloride (Agener União, Brazil, 0.1 ml), both per kilogram of animal weight. The animals were perfused using a cannula positioned in the aorta and connected to a peristaltic pump (Cole-Parmer) with 300 ml of 0.9% saline solution in 0.1 M phosphate buffer, pH 7.4, containing heparin (Parinex, Hipolabor, Sabará, MG, Brazil, 2 ml/ 1000 ml of saline solution) at a 60 ml/minute flow rate. Next, 700 ml of a 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 fixative solution was pumped into the animal. Half of the solution was pumped at a 70 ml/minute flow rate and the other half was pumped at a 17.5 ml/ minute flow rate. The entire procedure lasted 30 min. After perfusion, the animals were kept unhandled on the perfusion grid in the supine position for 15–60 min. The animals were then placed in a stereotaxic frame, in which 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 to 48 h, and then sectioned following freezing in dry ice in a sliding microtome. We thus obtained coronal sections 30 μm in thickness. The sections were grouped sequentially into 6 compartments. Each compartment contained one of every 6 sections. We thus obtained serial sequences with an interval of 180 μm between adjacent sections. Sections from one series were immediately

3. Results ChAT-immunoreactive (ChAT-IR) neurons were found in the telencephalon, diencephalon, and brainstem (midbrain-pons). We did not consider cholinergic neurons found in cranial nerve nuclei present in the brainstem, whose axons enter from the peripheral nervous system and compose the respective cranial nerves. 3.1. Telencephalic groups 3.1.1. Nucleus accumbens The nucleus accumbens (Acb) is located ventromedially to the caudate-putamen nucleus (CPu) and extends from the anterior end of the anterior horn of the lateral ventricle rostrally to the anterior commissure. ChAT-IR neurons were found sparsely distributed throughout the Acb in both the core and the shell without distinction (Figs. 2A–C and 3 A) and exhibited a predominantly fusiform shape, although we also observed some triangular neurons (Fig. 3B). 3.1.2. Caudate-putamen nucleus The CPu is located laterally to the lateral ventricle and extends approximately from the corpus callosum to the middle level of the 65

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Fig. 2. (continued)

habenular nuclei. ChAT-IR neurons were found sparsely distributed throughout this nucleus (Figs. 2A–I and 3 C) and exhibited predominantly triangular and rounded shapes, although some fusiform neurons were also observed (Fig. 3D). 3.1.3. Olfactory tubercle and islands of Calleja These nuclei are located ventrally to the Acb and extend from the level of the anterior horn of the lateral ventricle to the level of the anterior commissure. ChAT-IR neurons formed clusters in regions of the islands of Calleja (ICj) (Figs. 2A–D, 3 E, and F) and the granule cell layer of the olfactory tubercle (Tu). These neurons exhibited a predominantly triangular shape (Fig. 3F and G).

Fig. 2. Canvas serial drawings of Nissl-stained coronal sections through the rock cavy brain. ChAT-immunoreactive neurons (dots) were superimposed on the right half and the acronyms on the left half of the figure. Numbers on the right indicate the distance of the section to the previous section. For the meaning of each acronym, see the list of abbreviations.

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hemisphere. ChAT-IR neurons filled this nucleus almost completely (Figs. 2C–F and 4 D). These neurons were fusiform or triangular (Fig. 4E). 3.1.6. Ventral globus pallidus The ventral globus pallidus (VP) is a neuronal cluster located ventrally to the Acb and the anterior commissure. This nucleus contains widely distributed ChAT-IR neurons in its caudal portion (Fig. 2C–F). 3.1.7. Basal nucleus The basal nucleus (B) is found in the vicinity of the VP ventral to the globus pallidus. This nucleus is strongly labeled by the presence of ChAT-IR neurons (Figs. 2E, F and 5A) exhibiting triangular or fusiform shapes (Fig. 5B). 3.1.8. Globus pallidus The globus pallidus (GP) is located ventromedially to the CPu. It starts rostrally together with the posterior part of the anterior commissure and extends to the end of the habenular nuclei. The GP is characterized by its poor dye affinity in Nissl staining. ChAT-IR neurons were seen in this nucleus mainly concentrated in its lateral contour bordering the CPu (Figs. 2E–H and 5 C). These neurons were characterized by their triangular or fusiform shapes (Fig. 5D). 3.1.9. Entopeduncular nucleus The entopeduncular nucleus (EP) is a cluster of neurons within the ventromedial end of the internal capsule and is considered equivalent to the inner segment of the globus pallidus of primates in carnivores and rodents. This nucleus contained sparse ChAT-IR neurons (Figs. 2H, I and 5 E) exhibiting an elongated shape (Fig. 5F). 3.2. Diencephalic groups 3.2.1. Ventrolateral preoptic nucleus The ventrolateral preoptic nucleus (VLPO) is a small cell cluster in the region of the lateral preoptic nucleus located dorsally to the supraoptic nucleus. ChAT-IR neurons were seen in this locus (Figs. 2F and 6 A). These neurons exhibited a predominantly triangular shape (Fig. 6B). 3.2.2. Ventrolateral hypothalamic nucleus The ventrolateral hypothalamic nucleus (VLH) is a small cell cluster in the region of the lateral hypothalamus located dorsally to the supraoptic nucleus and caudally to the VLPO, where ChAT-IR neurons were detected (Figs. 2G and 6 C). These neurons were predominantly triangular (Fig. 6D). 3.2.3. Medial habenular nucleus The medial habenular nucleus (MHb) is the medial subdivision of the habenula, which is part of the epithalamus. It is found in the dorsal part of the diencephalon on the floor of the third ventricle. The MHb is strongly labeled by the massive presence of ChAT-IR neurons, mainly in its ventral part (Figs. 2I, J and 6 E). These neurons were characterized by their fusiform shape (Fig. 6F).

Fig. 2. (continued)

3.1.4. Diagonal band nucleus The diagonal band nucleus (DB) is located at the medial and ventral contours of the telencephalon at rostral levels. ChAT-IR neurons were seen in both the horizontal (HDB) and vertical (VDB) portions of that nucleus (Fig. 2C and 4A) and formed a continuum. These neurons exhibited a fusiform shape in the VDB (Fig. 4B) and triangular and rounded shapes in the HDB (Fig. 4C).

3.3. Pontomesencephalic nuclei 3.3.1. Parabigeminal nucleus The parabigeminal nucleus (PBG) is a small oval neuronal collection located in the pontomesencephalic region in the lateral aspect of the tegmentum ventral to the inferior colliculus. This nucleus is strongly labeled by the massive presence of ChAT-IR neurons (Figs. 2K–M and 7 A). These neurons exhibited a predominantly fusiform shape, with their longer axis aligned with the longer axis of the nucleus, although some triangular neurons were also present (Fig. 7B).

3.1.5. Medial septal nucleus The medial septal nucleus (MS) is the most medial nucleus of the septal complex and is located in the medial wall of the cerebral 67

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Fig. 3. Photomicrographs of coronal ChAT-immunostained sections showing (A) ChAT-IR neurons in the nucleus accumbens (Acb), (B) higher magnification of the ChAT-IR neurons within the boxed area in A, (C) ChAT-IR neurons in the caudate-putamen nucleus (CPu), (D) higher magnification of the ChAT-IR neurons within the boxed area in C, (E) ChAT-IR neurons in the olfactory tubercle/islands of Calleja (Tu/ICj), (F) higher magnification of the ChATIR neurons within the boxed area of the ICj in E, and (G) higher magnification of the ChAT-IR neurons within the Tu in the boxed area in E. Arrow points to triangular neurons. Open arrow points to fusiform neurons. Bar: 500 μm in A, C, and E, and 100 μm in B, D, F, and G.

3.3.2. Laterodorsal tegmental nucleus The laterodorsal tegmental nucleus (LDTg) is incrusted in the dorsal portion of the periaqueductal gray lateral to the dorsal nucleus of raphe. This nucleus is characterized by a considerable presence of ChAT-IR neurons (Figs. 2L, M and 7 C) exhibiting a predominantly triangular shape (Fig. 7D).

morphologies, with fusiform or elongated, triangular, and round shapes varying very little from one region to another. Our findings are similar to descriptions in previous publications. 4.1. Telencephalic groups Consistent with our findings, the presence of cholinergic neurons in the striatal complex comprising nucleus accumbens, ventral pallidum, caudate-putamen, and globus pallidus, as well as the olfactory tubercle and islands of Calleja, diagonal band nucleus, medial septal nucleus, and basal nucleus has been reported in all previously studied species, such as rat (Armstrong et al., 1983; Ichikawa et al., 1997; Roghani et al., 1998), monotremes (Manger et al., 2002), mole rat (Bhagwandin et al., 2008), bats (Dell et al., 2010; Kruger et al., 2010; Maseko and Manger, 2007; Maseko et al., 2007), Cape porcupine (Limacher et al., 2008), rock hyrax (Gravett et al., 2009), elephant shrew (Pieters et al., 2010), African pygmy mouse (Kruger et al., 2012), Afrotherian species (Potomogale velox, Amblysomus hottetotus, and Petrodromus tetradactylus) (Calvey et al., 2013), Tasmanian devil (Patzke et al., 2014), a lagomorph (Lepus capensis), a Scadentia (Tupaia belangeri) (Calvey et al., 2015a), insectivores (Crocidura cyanea, Crocidura olivieri, Sylvisorex ollula, Paraechinus aethiopicus, and Atelerix frontalis) (Calvey et al., 2016),

3.3.3. Pedunculopontine tegmental nucleus The pedunculopontine tegmental nucleus (PTg) is found in the pontomesencephalic tegmentum ventromedial to the superior cerebellar peduncle and ventrolateral to the LDTg. A large number of ChATIR neurons was present in this nucleus (Figs. 2L, M and 7 C). These neurons exhibited fusiform or triangular shapes (Fig. 7E). 4. Discussion ChAT-IR neurons were found in the telencephalon, diencephalon, and brainstem (midbrain-pons) of the rock cavy. The results indicate that the distribution of cholinergic neurons is similar in this species to what has previously been described in other mammals, with some minor differences. In terms of neuronal morphology, we characterized the cholinergic neurons in the rock cavy brain as having variable 68

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Fig. 4. Photomicrographs of coronal ChAT-immunostained sections showing (A) ChAT-IR neurons in the vertical (VDB) and horizontal (HDB) portions of the diagonal band nucleus, (B) higher magnification of the ChAT-IR neurons within the boxed area in the VDB in A, (C) higher magnification of the ChAT-IR neurons within the boxed area in the HDB in A, (D) ChAT-IR neurons in the medial septal nucleus (MS), and (E) higher magnification of the ChAT-IR neurons within the boxed area in D. Arrow points to triangular neurons. Open arrow points to fusiform neurons. Bar: 500 μm in A and D, and 100 μm in B, C, and E.

river hippopotamus (Dell et al., 2016), Goettingen miniature pig (Mahady et al., 2017), non-human primates (Calvey et al., 2015b; Kus et al., 2003; Satoh and Fibiger, 1985a, 1985b), and human (Oda and Nakanishi, 2000). We also detected cholinergic neurons in the entopeduncular nucleus, which has only been reported in rat by Armstrong et al. (1983). The Acb is a crucial component of the so-called reward circuit, receiving dopaminergic inputs from the ventral tegmental area, signaling predictive reward (Rodriguez-López et al., 2017; Schultz, 1998), and projecting to regions that modulate motor output, such as the VP and the substantia nigra (Heimer et al., 1991). Several studies have confirmed the participation of Acb cholinergic interneurons in several processes, such as memory, modulation of appetitive learning, performance, and regulation of mood and motivation (Pratt and Blackstone, 2009; Pratt and Kelley, 2004; Warner-Schmidt et al., 2012). Cholinergic neurons in the CP and GP represent the main source of ACh in the striatum, which plays a crucial role in the control of voluntary movements. Thus, it has been proposed that a functional imbalance between dopaminergic and cholinergic systems due to the loss of dopaminergic neurons is the basis for the motor symptoms present in Parkinson’s disease (Barbeau, 1962; Calabresi et al., 2000). Experimental evidence has implicated the Tu in reward actions related to cocaine, morphine, and the rewarding effects of brain stimulation (Kornetsky et al., 1991). The ICj are clusters of granule cells located near the Tu close to the septum and Acb. Connectional studies have shown that these cells are related olfactory and non-olfactory components of the basal forebrain and are afferented by dopaminergic neurons of the ventral tegmental area and medial substantia nigra (Fallon, 1983; Fallon et al., 1978). Cytoarchitectonic, hodological, histochemical, and immunohistochemical studies also situate the ICj into the striatopallidal system (Fallon et al., 1983; Meyer et al., 1989; Millhouse, 1987). The cholinergic neurons in the basal forebrain are interspersed with non-cholinergic neurons and distributed in a series of nuclei, including the DB, which has vertical and horizontal portions, the MS, and the B (Woolf, 1991). Cholinergic neurons in these regions collectively serve as the major sources of cholinergic projection to the neocortex,

hippocampus, and amygdala (Mesulam et al., 1983b). Cholinergic signaling from these neurons appears to provide important control over circuit dynamics underlying cognitive processing, with deficits in this signaling pathway linked to cognitive decline and subsequent neurodegeneration in diseases, such as Alzheimer’s disease and Parkinson’s disease (Ballinger et al., 2016). It is important to note that we did not find cholinergic neurons in any region of the cerebral cortex in the rock cavy. The presence of cholinergic neurons in the cerebral cortex has been a controversial subject. The description of ACh-producing neurons in the cerebral cortex of rat (Bayraktar et al., 1997; Eckenstein and Thoenen, 1983; Houser et al., 1983, 1985) and CD-1 mouse (Mufson and Cunningham, 1988) has led to the generalization that this is a characteristic of the brain in animals in the order Rodentia. This was mainly motivated by the hypothesis that subdivisions of a neural system exhibit the same complement of neurons in all species of a given order of mammals (Manger, 2005). However, the presence of cortical cholinergic neurons was confirmed in two strains (Long-Evans and Sprague-Dawley) of rats, three strains (AKR3, C3H, and wild-caught striped mice) of mice (Bhagwandin et al., 2006), and the African pygmy mouse (Kruger et al., 2012), but not in other rodent species studied, such as the wild bushveld gerbil, wild-caught greater canerats, and wild-caught common molerats (Bhagwandin et al., 2006). However, cortical cholinergic interneurons were described in the Hottentot golden mole (Calvey et al., 2013), Goettingen miniature pig (Mahady et al., 2017), and humans (Oda and Nakanishi, 2000). The great variability of cholinergic expression in cortical neurons among different species argues strongly that cortical cholinergic neurons are not integrated within the traditionally defined cholinergic system. 4.2. Diencephalic groups Cholinergic neurons have been reported to form dorsal, lateral, and ventral hypothalamic clusters in several species, such as African molerat (Bhagwandin et al., 2008), Afroterian species (Calvey et al., 2013), two Euarchontoglires (Calvey et al., 2015a), three strepsirrhine 69

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Fig. 5. Photomicrographs of coronal ChAT-immunostained sections showing (A) ChAT-IR neurons in the basal nucleus (B), (B) higher magnification of the ChAT-IR neurons within the boxed area in A, (C) ChAT-IR neurons in the globus pallidus (GP), (D) higher magnification of the ChAT-IR neurons within the boxed area in C, (E) ChAT-IR neurons in the entopeduncular nucleus (EP), and (F) higher magnification of the ChAT-IR neurons within the boxed area in E. Arrow points to triangular neurons. Open arrow points to fusiform neurons. Bar: 500 μm in A, C, and E, and 100 μm in B, D, and F.

primates (Calvey et al., 2015b), five insectivores (Calvey et al., 2016), bats (Dell et al., 2010; Kruger et al., 2010; Maseko and Manger, 2007; Maseko et al., 2007), rock hyrax (Gravett et al., 2009), African pygmy mouse (Kruger et al., 2012), Cape porcupine (Limacher et al., 2008), rock elephant shrew (Pieters et al., 2010), Tasmanian devil (Patzke et al., 2014), river hippopotamus (Dell et al., 2016) and baboon (Satoh and Fibiger, 1985a). Cholinergic neurons were also reported in the arcuate nucleus in the mouse (Jeong et al., 2016) and Goettingen miniature pig (Mahady et al., 2017). In the rock cavy (present study), cholinergic neurons were not visualized in these locations, although we detected cholinergic neuronal soma in the VLPO and VLH, a finding that has not been reported until now. The VLPO, which is identified in many species, is considered a critical component of the sleep circuitry (Saper et al., 2005; Szymusiak et al., 1998, 2007). However, the functional significance of the cholinergic neurons in this region is unknown. The MHb has been reported to contain cholinergic neurons in most of the mammalian species studied (Bhagwandin et al., 2008; Calvey et al., 2013, 2015a, 2015b, 2016; Dell et al., 2010; Gravett et al., 2009;

Kruger et al., 2010, 2012; Kus et al., 2003; Limacher et al., 2008; Mahady et al., 2017; Manger et al., 2002; Maseko and Manger, 2007; Maseko et al., 2007; Patzke et al., 2014; Pieters et al., 2010; Roghani et al., 1998). The cholinergic MHb neurons receive input from the septum via the stria medularis. Efferents from these neurons reach the single midline interpeduncular nucleus via the habenulo-interpeduncular tract, or fasciculus retroflexus. The fasciculus retroflexus is thus a key link between the limbic forebrain and the midbrain (Sutherland, 1982). Functionally, this system has been implicated in the control of emotional behaviors, including anxiety and fear responses (Yamaguchi et al., 2013), nicotine aversion and withdrawal (AntolinFontes et al., 2015; Pang et al., 2016), mood disorders, schizophrenia, and substance use disorder (Fakhoury, 2017), as well as cocaine, methamphetamine, and alcohol addiction (Viswanath et al., 2014). Cholinergic neurons were detected in diverse thalamic nuclei, such as, the anterodorsal and anteroventral dorsal in the rock hyrax (Gravett et al., 2009) and intralaminar in the river hippopotamus (Dell et al., 2016). However, we did not find cholinergic neurons in this region in our study. 70

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Fig. 6. Photomicrographs of coronal ChAT-immunostained sections showing (A) ChAT-IR neurons in the ventrolateral preoptic nucleus (VLPO), (B) higher magnification of the ChAT-IR neurons within the boxed area in A, (C) ChAT-IR neurons in the ventrolateral hypothalamic nucleus (VLH), (D) higher magnification of the ChAT-IR neurons within the boxed area in C, (E) ChAT-IR neurons in the medial habenular nucleus (MHb), and (F) higher magnification of the ChAT-IR neurons within the boxed area in E. Bar: 500 μm in A, C, and E, and 100 μm in B, D, and F.

4.3. Pontomesencephalic nuclei

Consistent with findings in several other mammalian species studied, we detected ACh-producing neurons in two cell clusters in the rock cavy pontine tegmentum, the LDTg and PTg (Armstrong et al., 1983; Bhagwandin et al., 2008; Bux et al., 2010; Calvey et al., 2013, 2015a,b, 2016; Da Silva et al., 2006; Dell et al., 2010, 2016; Gravett et al., 2009; Kruger et al., 2010, 2012; Kus et al., 2003; Limacher et al., 2008; Mahady et al., 2017; Manger et al., 2002; Maseko and Manger, 2007; Maseko et al., 2007; Motts et al., 2008; Oda and Nakanishi, 2000; Patzke et al., 2014; Pieters et al., 2010; Roghani et al., 1998; Satoh and Fibiger, 1985a,b). Classically, the PTg has been identified by its population of cholinergic neurons, the labeling of which is still the best way to define it. Both the LDTg and PTg have been traditionally associated with functions of the reticular activating system due to the presence of the ascending cholinergic projections to the thalamus that modulate cortical activation (Garcia-Rill et al., 2013; Steriade, 2004) and with the functions of the mesencephalic locomotor region through the modulation of goal-directed locomotion (Mena-Segovia, 2016). Furthermore, considering their interconnections, it has been suggested that the PTg should be considered a part of the basal ganglia (Mena-

In concordance with several studies in many mammalian species (Bhagwandin et al., 2008; Bux et al., 2010; Calvey et al., 2013, 2015a,b, 2016; Da Silva et al., 2006; Dell et al., 2010; Gravett et al., 2009; Kruger et al., 2010, 2012; Kus et al., 2003; Limacher et al., 2008; Mahady et al., 2017; Manger et al., 2002; Maseko and Manger, 2007; Maseko et al., 2007; Motts et al., 2008; Mufson et al., 1986; Oda and Nakanishi, 2000; Patzke et al., 2014; Pieters et al., 2010; Roghani et al., 1998), the rock cavy PBG was found to be heavily marked by the presence of AChproducing neurons. The PBG is strongly interconnected with the superior colliculus, of which it has been characterized as a satellite (Graybiel, 1978; Henderson, 1989; Mufson et al., 1986), and projects to other brain structures such as the dorsal lateral geniculate nucleus (Harting et al., 1986; Hashikawa et al., 1986) and the amygdala (Usunoff et al., 2007). Recordings of PBG neuronal activity suggest that the PBG is an integral part of the mesencephalic circuit that generates target location information (Cui and Malpeli, 2003), with indications of spontaneous activity (Goddard et al., 2007). 71

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Fig. 7. Photomicrographs of coronal ChAT-immunostained sections showing (A) ChAT-IR neurons in the parabigeminal nucleus (PBG), (B) higher magnification of ChAT-IR neurons within the boxed area in A, (C) ChAT-IR neurons in the laterodorsal tegmental nucleus (LDTg) and the pedunculopontine tegmental nucleus (PTg), (D) higher magnification of the ChAT-IR neurons within the boxed area in the LDTg, and (E) higher magnification of the ChAT-IR neurons within the boxed area in the PTg in C. Bar: 500 μm in A and C, and 100 μm in B, D, and E.

Behavioral Research” and recommended by the Brazilian Society of Neuroscience and Behavior (SBNeC), http://www.sbnec.gov.br/links. The project was approved by the local Ethics Committee (CEUA-UFRN, protocol 004/2014).

Segovia et al., 2004). Thus, dysfunction of the PTg may be associated with movement disorders, such as Parkinson’s disease (Hamani et al., 2007; Lee et al., 2000; Pahapill and Lozano, 2000; Wen et al., 2015; Winn, 2008). In summary, the present findings indicate that, although there are some differences, the overall distribution and morphological characteristics of cholinergic neurons identified in the rock cavy are similar to those previously found in other species, as above reported. Notably, at the telencephalic level, the presence of ChAT-immunopositive neurons in the nucleus accumbens, caudate-putamen, ventral pallidum, globus pallidum, diagonal band nucleus, medial septal nucleus, olfactory tubercle/islands of Calleja, and basal nucleus appears to be a constant in all species. In the diencephalon, the presence of a significant population of cholinergic neurons in the medial habenular nucleus also appears to be a constant. However, our findings are discrepant with previous reports regarding cholinergic neurons in the hypothalamus. We found cholinergic neurons at a rather different location in the rock cavy than that reported in other species. In fact, monotremes have been reported to lack cholinergic neurons in this region. Although cholinergic neurons were reported in some thalamic nuclei, we did not find cholinergic neurons in this region in our study. In the rock cavy, we found cholinergic neurons in the parabigeminal, laterodorsal tegmental, and pedunculopontine tegmental nuclei at the midbrain pons level. The presence of these neurons in the laterodorsal tegmental and pedunculopontine tegmental nuclei also appears to be a constant. However, in some species studied, there is an absence of cholinergic neurons in the parabigeminal nucleus, as previously described. Thus, it can be concluded that, for the most part, the cholinergic system is largely maintained in mammalian species.

Conflict of interest All authors declare that they have no conflict of interest. Acknowledgments We would like to thank Miriam Regina Celi de Escala Oliveira for expert technical assistance. This study was supported by grants from the National Council for Scientific and Technological Development (CNPq), grant no.474623/2013-0, Coordination for the Improvement of Higher Education Personnel (CAPES), Rio Grande do Norte Research Support Foundation (FAPERN) and Research and Projects Financing (FINEP) in Brazil. References Antolin-Fontes, B., Ables, J., Görlich, A., Ibañes-Tallon, I., 2015. The habenulo-interpeduncular pathway in nicotine aversion and withdrawal. Neuropharmacology 96, 213–222. Armstrong, D.M., Saper, C.B., Levey, A.I., Wainer, B.H., Terry, R.D., 1983. Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of cholineacetyltransferase. J. Comp. Neurol. 216, 53–68. Ballinger, E.C., Ananth, M., Talmage, D.A., Role, L.W., 2016. Basal forebrain cholinergic circuits and signaling in cognition and cognitive decline. Neuron 91, 1199–1218. Barbeau, A., 1962. The pathogenesis of Parkinson´s disease: a new hypothesis. Can. Med. Assoc. J. 87, 802–807. Bayraktar, T., Staiger, J.F., Acsady, L., Cozzari, C., Freund, T.F., Zilles, K., 1997. Colocalization of vasoactive intestinal polypeptide, gamma-amino-butyric acid and choline acetyltransferase in the neocortical interneurons of the adult rat. Brain Res. 757, 209–217. Benzing, W.C., Kordower, J.H., Mufson, E.J., 1993. Galanin immunoreactivity within the primate basal forebrain: evolutionary change between monkeys and apes. J. Comp. Neurol. 336, 31–39. Bhagwandin, A., Fuxe, K., Manger, P.R., 2006. Choline acetyltransferase immunoreactive cortical interneurons do not occur in all rodents: a study of the phylogenetic occurrence of this neural characteristic. J. Chem. Neuroanat. 32, 208–216. Bhagwandin, A., Fuxe, K., Bennet, N.C., Manger, P.R., 2008. Nuclear organization and

Ethical statement All efforts were made to minimize the number of animals and their suffering, the procedures following strictly the norms established by the National Research Council of the National Academy published in the “Guidelines for the Care and Use of Mammals in Neuroscience and 72

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