brain research 1586 (2014) 99–108
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Research Report
Serotonergic fibers distribution in the midline and intralaminar thalamic nuclei in the rock cavy (Kerodon rupestris) Alane de Medeiros Silvaa, Melquisedec Abiare´ Dantas de Santanaa, Paulo Leonardo Arau´jo de Go´is Moraisa, Twyla Barros de Sousaa, Rovena Clara Galva˜o Janua´rio Engelberthb, Eudes Euler de Souza Lucenab, Clarissa Loureiro das Chagas Campeˆloc, Jeferson Sousa Cavalcanteb, Judney Cley Cavalcantea, Miriam Stela Maris de Oliveira Costaa, Expedito Silva do Nascimento Jra,n,1 a
Department of Morphology/Laboratory of Neuroanatomy, Biosciences Center, Federal University of Rio Grande do Norte, 59072-970 Natal, RN, Brazil b Department of Physiology/Laboratory of Neurochemical Studies, Biosciences Center, Federal University of Rio Grande do Norte, 59072-970 Natal, RN, Brazil c Department of Physiology/Memory Studies Laboratory, Biosciences Center, Federal University of Rio Grande do Norte, 59072-970 Natal, RN, Brazil
art i cle i nfo
ab st rac t
Article history:
The thalamic midline/intralaminar complex is part of the higher-order thalamus, which
Accepted 16 August 2014
receives little sensory input, and instead forms extensive cortico-thalamo-cortical pathways.
Available online 23 August 2014
The midline thalamic nuclei connect with the medial prefrontal cortex and the medial temporal lobe. On the other hand, the intralaminar nuclei connect with the fronto-parietal
Keywords:
cortex. Taking into account this connectivity pattern, it is not surprising that the midline/
Serotonergic system
intralaminar complex has been implicated in a broad variety of cognitive functions, including
Midline/intralaminar nuclei
memory process, attention and orientation, and also reward-based behavior. Serotonin (5-HT)
Thalamus
is a neurotransmitter that exerts different post-synaptic roles. Serotonergic neurons are
Rock cavy
almost entirely restricted to the raphe nuclei and the 5-HT fibers are distributed widely
Abbreviations: 3V, AM,
3rd ventricle; 5-HT,
anteromedial thalamic nucleus; AOI,
serotonin; 5-HT-IR, area of interest; CL,
nucleus; DAB,
diaminobenzidine; f,
nucleus; IAM,
interanteromedial thalamic nucleus; IMD,
mt,
mammillothalamic tract; PC,
nucleus; PV,
anterodorsal thalamic nucleus;
centrolateral thalamic nucleus; CM,
fornix; fr, fasciculus retroflexus; Hb,
habenular nucleus; IAD,
intermediodorsal thalamic nucleus; MD,
paracentral thalamic nucleus; PF,
paraventricular thalamic nucleus; PVA,
nucleus, posterior part; Re,
5-HT immunoreactive fibers; AD,
central medial thalamic interanterodorsal thalamic
mediodorsal thalamic nucleus;
parafascicular thalamic nucleus; PT,
paraventricular thalamic nucleus, anterior part; PVP,
reuniens thalamic nucleus; Rh, rhomboid thalamic nucleus; ROD,
paratenial thalamic paraventricular thalamic
relative optical density; sm,
stria
medullaris of the thalamus; Sub, submedius thalamic nucleus; VRe, ventral renuens thalamic nucleus; ZI, zona incerta n Corresponding author. Fax: þ55 84 32119207. E-mail address: [email protected] (E.S.d. Nascimento Jr). 1 Present address: Department of Morphology/Laboratory of Neuroanatomy, Biosciences Center, Federal University of Rio Grande do Norte, 59072-970 Natal, RN, Brazil. http://dx.doi.org/10.1016/j.brainres.2014.08.047 0006-8993/& 2014 Elsevier B.V. All rights reserved.
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Optical density Fiber morphology
throughout the brain, including the midline/intralaminar complex. The present study comprises a detailed description of the morphologic features and semiquantitative analysis of 5-HT fibers distribution in the midline/intralaminar complex in the rock cavy, a typical rodent of the Northeast region of Brazil, which has been used by our group as an anatomical model to expand the comprehension about phylogeny on the nervous system. The 5-HT fibers in the midline/intralaminar nuclei of the rock cavy were classified into three distinct categories: (1) beaded fibers, which are relatively fine and endowed with large varicosities; (2) fine fibers, with thin axons and small varicosities uniformly distributed in whole axon; and (3) stem axons, showing thick non-varicose axons. Moreover, the density of 5-HT fibers is variable among the analyzed nuclei. On the basis of this diversity of the morphological fibers and the differential profile of optical density among the midline/intralaminar nuclei of the rock cavy, we conclude that the serotonergic system uses a diverse morphologic apparatus to exert a large functional repertory in the midline/intralaminar thalamic nuclei. & 2014 Elsevier B.V. All rights reserved.
1.
Introduction
The rock cavy (Kerodon rupestris), a typical rodent in the Northeast region of Brazil, dwells in the mountainous regions of the Caatinga in the Brazilian semi-arid interior. Morphological (Silva Neto, 2000) and molecular biology studies (Rowe and Honeycutt, 2002) put the genus Kerodon in the family Hydrochaeridae, in which the capybara (Hydrochoerus hydrochaeris) is also included. The rock cavy has a crepuscular habit (Sousa and Menezes, 2006) and has been used in several neuroanatomical studies in our laboratory (Cavalcante et al., 2008; Nascimento et al., 2008, 2010a, 2010b; Soares et al., 2012). The rock cavy has advanced our understanding about the nervous system, providing a framework for the interpretation of evolutionary patterns, allowing inferences to be drawn about the brain and its phylogenetic congruence among diverse biological features. The structural organization of the raphe has been extensively studied in various species (Azmitia and Gannon, 1983; Dahlstrom and Fuxe, 1964; Dwarika et al., 2008; Fuxe et al., 1969; Harding et al., 2004; Hornung and Fritschy, 1988; Hornung, 2010; Limacher et al., 2008; Moon et al., 2007; Paxinos and Watson, 2007). The nuclear organization of the raphe serotonergic system in the rock cavy is similar to that described in other mammals with small anatomical variations (Soares et al., 2012). Moreover, subsequent reports using tracing techniques have pointed out the dorsal and median raphe nuclei as the main sources of serotonergic projections (Azmitia and Segal, 1978; Moore et al., 1978; Morin and MeyerBernstein, 1999; Vertes and Martin, 1988; Vertes, 1991; Vertes et al., 1999), although the majority of the serotonergic fibers are concentrated in limbic regions of the forebrain (Jacobs and Azmitia, 1992; Lowry et al., 2008; Steinbusch, 1981; Vertes and Linley, 2007, 2008). Serotonin (5-HT) exerts excitatory modulatory activity on some of its targets (Curtis and Davis, 1962; Kayama et al., 1989; Marks et al., 1987; Monckton and McCormick, 2001; Rogawski and Aghajanian, 1980; Yoshida et al., 1984). In other areas, however, it produces changes in the conductance and permeability of cell membranes, causing hyperpolarization of neurons (Mccormick and Pape, 1990; Lee and Mccormick, 1996). The functional duality of the 5-HT
in the thalamus has been widely associated with the various types of receptors present in the different thalamic nuclei (Chapin and Andrade, 2001a, b; Lopez-Gimenez et al., 1998, 2001; Kia et al., 1996; Mengod et al., 1996; Pompeiano et al., 1994), as well as in differences in fibers morphology, origin, and preference in area of innervation (Wilson, 1989). The 5-HT axons are generally divided into three morphological different fiber types. Thin axons with small fusiform or granular boutons, named fine fibers, are supposed to originated from the dorsal raphe nucleus; the beaded fibers with large spherical boutons; and non-varicose stem-axons originated from the beaded fibers, supposed to originated from the median raphe nucleus (Hornung and Fritschy, 1990; Wilson and Molliver, 1991). A low-frequency stimulation of the midline/intralaminar thalamic nuclei induces a low synchronous activity in different regions of the cortex (Dempsey and Morison, 1943). This thalamic nuclear complex is classically viewed as nonspecific thalamus, exerting a global influence on the cortical layers (Groenewegen and Berendse, 1994). Nowadays, the notion of the nonspecific functions has been changed as a result of several anatomical studies which have demonstrated, that the midline/intralaminar complex projects to specific areas of the cerebral cortex, mainly to the prefrontal cortex (Groenewegen and Witter, 2004; Van der Werf et al., 2002; Vertes, 2006). Furthermore, the electrical stimulation of individual nuclei produces selective effects on their cortical targets (Viana di Prisco and Vertes, 2006). Descriptions of the serotonergic projections in the thalamus have been shown in monkey and rat studies (Cropper et al., 1984; Lavoie and Parent, 1991; Vertes, 2002; Vertes et al., 2010) and suggest that the serotonergic fibers exert a fundamental role on modulation in the midline/intralaminar thalamic nuclei. Furthermore, it is widely agreed that cell types and morphological fiber pattern serve as the building blocks of nervous systems and that exploring their diversity and determining how cells are assembled into circuits is essential for understanding brain function. In the present study we aim describe and compare the distribution of serotonergic fibers in the rock cavy midline/ intralaminar thalamic nuclei. We also assess the relative abundance of serotonergic fibers throughout the various
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nuclei of the midline/intralaminar complex in the rock cavy and supply a morphological description of the 5-HT fibers and their differential distribution within this nuclear group.
2.
Results
2.1.
Cytoarchitectonic analysis
The midline nuclei in the rock cavy include the paraventricular (PV), paratenial (PT), intermediodorsal (IMD), reuniens (Re), and rhomboid (Rh) nuclei. On the other hand, the intralaminar nuclei contain the central medial (CM), central lateral (CL), paracentral (PC), and parafascicular nuclei (PF).
2.1.1.
Midline thalamic nuclei
The PV in the rock cavy is a conspicuous nucleus, lying immediately ventral to the third ventricle in the thalamic midline. On coronal sections, it appears first as a triangularly shaped nucleus. It extends through almost all the rostrocaudal area of the thalamus and is clearly divided in three cytoarchitectonical divisions (PV anterior, PV middle, and PV posterior). Its lateral border is limited by the PT in rostral levels and by the mediodorsal nucleus (MD) in the middle and caudal ones, while ventrally it borders on the CM (Fig. 1). The PT is a rounded nucleus located in the dorsal thalamus. In the rostral levels of the thalamus, it constitutes the lateral and inferior borders of the PV (Fig. 1A and B). In its caudal extremity it merges with the MD. The IMD, as its name suggests, is located between the right and left MD thalamic nucleus and is more easily visualized in the middle coronal sections of the thalamus with the PV (Fig. 1C–F). The Re is located in the ventral part of the thalamic midline, immediately dorsal to the third ventricle. Two cell clusters constitute it; situated laterally to the third ventricle in the rostral levels, they fuse together along thalamic rostrocaudal length (Fig. 1). Finally, the Rh is found just below the internal medullary lamina. In rostral thalamic sections, the Rh is immediately dorsal to the Re and in caudal ones it is easily distinguished by its conspicuous shape and strongly stained cells (Fig. 1C–F).
2.1.2.
Intralaminar thalamic nuclei
In the rock cavy thalamus we have identified a CM nucleus as a conspicuous cell cluster located centrally in the internal medullary lamina, distinct from the midline nuclei. This nucleus shows large stained cells along the length of the thalamic rostrocaudal. Laterally, the CM is continuous with PC and CL. PC appears laterally to the CM and its cells are difficult to distinguish from those in the CM. The CM lies at the curved portion of the internal medullary lamina, immediately lateral to the MD and medially to the ventral thalamic nucleus. Dorsally it is continuous with the CL nucleus. The CL is the most dorsal part of the intralaminar nuclei. It is difficult to identify its boundary with the PC. The CL is larger than the CM, PC, and PF and contains strongly stained cells (Fig. 1E and F). Finally, the PF is located in the caudal part of the thalamus. It can be clearly detected where the midbrain structures start to appear. The PF is comprised of a large
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round mass of cells, lying in close proximity to the fasciculus retroflexus and dorsally bordered by the CL and lateral to the medial habenula (Fig. 1G and H).
2.2.
Relative optical density analysis
The ROD values were higher in the midline nuclei compared with the intralaminar ones [t(3)¼7.553, p¼ 0.005] (Fig. 2). Furthermore, several differences in the ROD values were found by comparing nuclei from the midline and intralaminar each other (Fig. 3). Comparisons among midline nuclei each other revealed significant increase ROD values in the Re [p¼ 0.003] and PV [p ¼0.022] compared with PT. Although no differences in any other midline thalamic nuclei could be detected, the photomicrographs suggest variations in their contents (Fig. 3). When we used the one-way ANOVA [F (4,19)¼6.301, p¼ 0.002] and Tukey’s multiple comparison tests, the Re shows higher ROD values, followed by PV, Rh, IMD, and PT (Fig. 4). Comparisons among intralaminar nuclei each other revealed significant increase ROD values in the CM [p¼ 0.038]; PC [p ¼0.005], and PF [p ¼0.004] compared with the CL. The One-way ANOVA [F(3,15)¼8.506, p¼ 0.003] and Tukey’s multiple comparison tests revealed a significant increased ROD values among intralaminar thalamic nuclei, with the CL showing a higher ROD value followed by CM, PC, and PF (Fig. 5). There was no significant difference in ROD values between male and female brains across the midline/ intralaminar nuclei.
2.3.
Descriptions of serotonergic fibers
The midline/intralaminar complex in the rock cavy received a multitude of fibers that demonstrated strong and distinct anatomical variations (Fig. 6). These fibers could be classified into three distinct groups, based on morphological criteria: beaded fibers, fines fibers and stem-axons. In the midline thalamic nucleus, the PV was amply supplied by the beaded fibers (Fig. 6D) with thin axons that had large varicosities, probably corresponding to the same beaded fiber (BF) type described in other species (Hornung, Fritschy, 1990; Wilson and Molliver, 1991). The thin axons with small fusiform and granular varicosities, corresponding to the fine fiber (FF) (Fig. 6E), and thick non-varicose axons, probably the stemaxons (SA) (Fig. 6F), were seen uniformly distributed throughout the midline/intralaminar nuclei, except in PV.
3.
Discussion
The rock cavy midline/intralaminar complex is densely innervated by serotonin immunoreactive fibers and this innervation has a complex pattern. Substantial variation in ROD values and morphology of 5-HT fibers is detected among midline/intralaminar nuclei. The highest ROD value is observed in the Re among the midline nuclei. In the intralaminar nuclei, however, the highest ROD values is observed in the CL. The medium ROD values are found in the PV, IMD, and Rh in the midline, while among intralaminar nuclei the CM and PC show medium ROD values. The lowest fiber densities are observed in the PT and PF. The present work
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Fig. 1 – Drawings of coronal sections through the rock cavy brain illustrating the morphology of the midline and intralaminar thalamic nuclei (A), (C), (E), and G), and digital images of Nissl stained coronal sections at corresponding levels (B), (D), (F), and (H). Rostral level representation in (A) and (B) and caudal level (G) and (H). Numbers on the right indicate distance from the bregma. See list of abbreviations. Bar 280 lm. is the first to analyze the thalamic midline/intralaminar complex organization using a Relative Optical Density tool to quantify serotonergic fibers in these nuclei.
Although the serotonergic system shows a firmly established morphological organization among numerous species (Bjarkam et al., 1997; Harding et al., 2004; Hornung, 2010;
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Fig. 2 – ROD values across the midline and intralaminar thalamic nuclei of rock cavy (n ¼8). The bars represent the means of the mean ROD values in the midline and intralaminar nuclei of individual animals (across all rostrocaudal levels and nuclei). The paired t-test revealed a significant difference between the midline and intralaminar nuclei. Values are expressed as mean and standard error of the mean. npo0.05—[t(3) ¼7.553, p¼ 0.005].
Fig. 3 – Semiquantitative analysis showing the densities of 5-HT-IR fibers in the different midline/intralaminar nuclei. To compare across these nuclei, the Relative Optical Density values in each nucleus are displayed as levels of gray in the table at the left. In the table, nuclei are listed according rostracaudal levels. See list of abbreviations.
Jacobs et al., 1984), we observed several differences in the serotonergic innervation of the rock cavy midline/intralaminar nuclei compared with rodents and primates. In albino rats, the PV, Rh and Re show a high fiber density, while the PT has a low fiber density (Cropper et al., 1984). Another study with rats using SERT (serotonin transporter), has reported the highest fiber density in the PV, followed by Rh, Re, and IMD (Vertes et al., 2010). In contrary, in the present study, the ROD values shown suggest higher density in the Re. In our results, based on ROD values, the intralaminar nuclei show moderate or low fiber innervation when compared with other midline nuclei, contrary to data in rats, in which dense serotonergic innervation in the CM and CL was found (Vertes et al., 2010). In the hamster (Mesocricetus auratus), the highest density is described in the PV and Re, while in the intralaminar nuclei the highest density is observed in the CM (Morin and MeyerBernstein, 1999). In a primate species, the squirrel monkey
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Fig. 4 – ROD values in the midline thalamic nuclei of rock cavy (n¼ 8). The bars represent the means of the mean ROD values of individual animals (across all rostrocaudal levels). One-way ANOVA revealed ROD significant difference between the nuclei analyzed. Values are expressed as mean and standard error of the mean. np¼ 0.003 nnp¼0.022— Tukey’s multiple comparison test [F(4,19)¼6.301, p ¼0.002]. See list for abbreviations.
Fig. 5 – ROD values in the intralaminar thalamic nuclei of rock cavy (n ¼8). The bars represent the means of the mean ROD values of individual animals (across all rostrocaudal levels). One-way ANOVA revealed ROD significant difference between the nuclei analyzed. Values are expressed as mean and standard error of the mean. np¼ 0.038 nnp¼0.005 nnn p¼ 0.004—Tukey’s multiple comparison test [F(3,15)¼ 8.506, p¼ 0.003]. See list for abbreviations.
(Saimiri sciureus), a highest density of 5-HT fibers can be observed in the PV and Re among the midline nuclei. Otherwise, in the intralaminar nuclei the highest density is observed in the CM (Lavoie and Parent, 1991). Contrary to our results, in another primate species (Macaca Mulatta), the serotonergic fibers are more densely concentrated in the PV compared to other midline/intralaminar nuclei (Hsu and Price, 2009). Through extensive dendritic and axonal arborizations, neurons define ample domain of influence. Morphological characterization, such as shaft caliber, tapering or branching, determines action potential propagation in axons (Goldstein and Rall, 1974). Spines and dendritic segments are also important in defining biochemical compartments in neurons, which are dependent on their size, length, and shape (Helmchen, 1999; Korkotian and Segal, 2000). Overall, different patterns in density and spatial distribution of axonal or dendritic branches create a wide spectrum of wiring
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Fig. 6 – Photomicrograph of 5-HT-immunostained brain coronal sections at the level of approximately 2.30 mm p.b. (A); (B) magnification of box homonymous in A, with an area corresponding to the dorsal midline, including PV and IMD; (C) magnification of box homonymous in A, with an area corresponding to the intralaminar region, including the PC; (D) magnification of superior box homonymous in B, with an area corresponding to the nucleus PV; (E) magnification of inferior box homonymous in B, with an area corresponding to the nucleus IMD; and (F) magnification of box homonymous in C, with an area corresponding to the PC nucleus. Black arrows in D correspond to the BF-like fibers; arrowheads in E correspond to the FF-like fibers; and White arrows in F correspond to the SA-like fibers. See list of abbreviations. Bar: 230 lm A; 140 lm B, and C; 15 lm D, E, and F.
configurations, which are currently subject to intensive experimental and theoretical research (Chklovskii et al., 2002; Petrof and Sherman, 2013; Rubio-Garrido et al., 2009; Sousa et al., 2013). The present work is the first to describe morphological differences in the pattern of distribution of serotonergic fibers into midline/intralaminar complex. We found the BF-like type fibers more frequently inside the PV. Otherwise, the FF-like fibers were distributed throughout the remainder midline/intralaminar nuclei merged with SA-like fibers. The morphological descriptions of the serotonergic fibers have been made only in the cerebral cortex of rats (Kosofsky and Molliver, 1987) and primates (Wilson and Molliver, 1991), as well as in rabbit hippocampus (Bjarkam and Sorensen, 2005). These pronounced differences in fiber density as well as in morphological features of 5-HT fibers in the midline/intralaminar complex indicate a possible species differences in serotonergic innervation between rodents and nonhuman primates. As is well known, the main source of serotonergic fibers throughout the brain comes from the dorsal and median raphe nuclei. Two classes of serotonergic fibers have been described for rodents and primates, and are thought to arise from separated raphe nuclei (Kosofsky and Molliver, 1987; Wilson, 1989; Wilson and Molliver, 1991). Fine axons with granular or fusiform varicosities originate from the dorsal raphe nucleus, while larger beaded axons arise
from median raphe nucleus (Kosofsky and Molliver, 1987). These morphological findings suggest that the rock cavy midline/intralaminar complex receives different classes of serotonergic fibers arising from different sources in the raphe. In addition, it has been shown that psychoactive drug, such as 3,4 methyledioxymetamphetamine (Ecstasy) and pchloroamphetamine, selectively destroy the FF in the central nervous system, but leave the BF unharmed (Wilson, 1989; Haring et al., 1992). One may differentiate among systems of ascending serotonergic fibers, with clear differences in fiber origin, fiber morphology, preference in area of innervation, and sensitive to psychoactive drugs. In accordance with our findings, the midline nuclei of the rock cavy contained more 5-HT fibers than the intralaminar ones. Furthermore, there is a differential density of 5-HT-IR fiber among them. For example, a moderate density was observed in the nuclei associated with feeding (PV), learning and memory (PT), arousal and awareness (Rh), seizure regulation (CM), and motor regulation (CL) (Bentivoglio et al., 1991; Ichinohe et al., 2001; Miller and Ferrendelli, 1990; Stratford and Wrtshafter, 2013; Van der Werf et al., 2002). The specific effects of 5-HT release depend on the class of receptor involved (Goodwin, et al., 1985; Larsson and Ahlenius, 1999; Meltzer, 1990; Nordberg, 1992; Pazos et al., 1987a, b; Stuart et al., 1986), and the type of neuron received
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in the synaptic contact. It is clear that a more precise description of the 5-HT fiber ultrastructure and serotonin receptor distribution is needed to provide the basis for further evaluation of the functional role of the 5-HT in the midline/ intralaminar thalamic nuclei. The above descriptions of differences in the density and morphological distribution of serotonergic fibers in the midline/intralaminar nuclei of the rock cavy are an attempt to understand the functional and evolutionary pathway by which the 5-HT acts in the thalamus through the indispensable anatomical background, as well as understand the fundamental importance of identification, characterization, and comparative analysis of the great diversity of cell types present in complex nervous system. Although functional studies are strictly necessary to understand the real interference of 5-HT in the midline/intralaminar nuclei, this anatomical study suggests that the serotonergic system exerts a fine control over thalamic function to modulate behavior.
4.
Experimental procedures
4.1.
Animals and housing
Eight young adult rock cavies (four males and four females), weighing 300 and 400 g, from the rural municipalities in Rio Grande do Norte state, Brazil, were used. Animal capture was authorized by the Brazilian Environmental Agency (IBAMA, licenses 21440-1). Approval for the experiments was obtained from local Animal Experimentations Ethics Committee 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 in 3.00 m 2.00 m 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.
4.2.
Perfusion and microtomy
Each individual was pre-anesthetized with an intramuscular injection of tramadol chloridrate and xylazine, both 5 mg/kg, and maintained with gas isoflurane and 100% oxygen. Upon deep anesthesia, they were perfused for approximately 5 min using a cannula positioned in the ascending aorta and connected to a peristaltic pump (Cole-Parmer), with 300 ml of 0.9% saline solution in an 0.1 M phosphate buffer, pH 7.4, containing heparin (Parinex, Hipolabor, Sabará, MG, Brazil, 2 ml/1000 ml of saline solution). Next, 700 ml of a 4% 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 established for half the solution and 17.5 ml/min for the other half, with the entire procedure taking 30 min. 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
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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, each brain was removed from the skull, stored in 30% sucrose solution in 0.1 M phosphate buffer, pH 7.4, for 24–48 h, frozen by dry ice, and serially cut in the coronal plane into 30 mm thick sections in a sliding microtome.
4.3.
Nissl staining and immunohistochemistry
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 5-HT. All the immunohistochemical procedures were performed at room temperature. The sections, previously submitted to a pre-treatment with sodium borohydride and hydrogen peroxide (H2O2), were placed in contact with the rabbit anti-5-HT antibody (Sigma, 1:5000) and 2% normal goat serum in 0.4% Triton X-100 for 18 h, in 0.1 M phosphate buffer, pH 7.4, in a rotator. This was followed by incubation in a secondary antibody, consisting of 1:1000 biotinylated goat anti-rabbit (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 a final reaction in a medium containing H2O2 as substrate and 3,30 -diaminobenzidine tetrahydrochloride (DAB) as chromogen. H2O2 was added indirectly, by mixing oxidase glucose and ß-D glucose, causing a reaction in which the former acting on the latter releases H2O2. The sections were thoroughly washed with 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 immunostainings were performed concomitantly, minimizing possible differences in background between the animals. With respect to staining specificity, a number of sections were submitted to immunohistochemical reactions omitting the primary or secondary antibodies. In these cases, no 5-HT immnoreactivity was obtained. Diagrams were obtained from image of Nissl-Stained sections with Adobe Illustrator software (Adobe Systems, Mountain View, CA, USA). The anatomical location of the brain structures was determined using the rat brain atlas of Paxinos and Watson (2007), and our experience in the anatomy of the rock cavy brain (Nascimento et al., 2008, 2010a, 2010b; Soares et al., 2012).
4.4.
Qualitative and quantitative analysis
5-HT-IR was identified as a black-purple precipitate. The tissue was analyzed with an optic microscope (BX41 Olympus) under brightfield illumination. Digital images were captured using a digital video camera (Nikon DXM1200) coupled to the microscope. In order to identify morphological differences among 5-HT fibers/terminal in the midline/intralaminar nuclei, we performed qualitative analysis under high
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magnification. The digitized images were corrected minimally for brightness and contrast, and mounted using Adobe Photoshop 7.0 software (Adobe Systems, Mountain View, CA, USA). In order to accomplish the quantitative analysis, all images were obtained in brightfield illumination at a fixed intensity for each of 16 rostro-caudal levels per individual. The resulting brightfield images were 3840 3072 pixel, with a resolution of 0.59 pixel/mm (with 4 objective). The Relative Optical Density (ROD) analysis was accomplished using Image J software (Version 1.46i, NIH). The images were converted to gray scale images (8-bit). The next stage consisted to the binarization of them, adjusting the contrast to 100%. After this procedure, the images presented only two values that could be observed in the histogram, the zero that corresponds to black and the 255 that corresponds to white. Finally, the program provided the number of black pixels in the sampled areas (Santos et al., 2013). The mean gray value of each sampled area was measure using a square (0.3 0.3 mm) in the area of interest (AOI). This AOI was located 3–4 times on a well-defined DAB stained in each nucleus throughout rostro-caudal levels and the mean gray values were taken. The medium number of black pixels in the target area was subtracted from the medium values of a control region (areas that should not have specific 5-HT staining). The value of OD (optical density) of the AOI was related to the background value by the formula: [(OD AOI OD background)/OD background] 100, thus eliminating the variability in background staining among sections (Krugers et al., 1996). The data were presented to each target area as the mean of pixels in the AOI. A manual selection of the stained positively for DAB chromogen was performed (Cuesta et al., 2013). All results are expressed as mean and standard error of the mean (S.E.M). Statistical analysis was performed using a computer program (Statistical Package for Social Sciences—IBM SPSS, version 21). The ROD data was subjected to t-test to analyze differences between the mean number of black pixels in the midline and intralaminar nuclear complex. One-way ANOVA followed by post-hoc Tukey’s multiple comparisons was accomplished to verify the differences among nuclei in the midline and intralaminar nuclei each other.
Conflict of interest statement The authors declare no conflict of interest.
Author contributions AMS: Participated in the design of the research and data analysis, and manuscript writing. MADS: Participated in the analysis of the results. PLG: Participated in the analysis of the results. TBS: Participated in the analysis of the results. RCGE: Participated in the research. EESL: Participated in the analysis of the results. CLCC: Participated in the data analysis.
JSC: Participated in the design of the research and data analysis. JCC: Participated in the design of the research, and manuscript writing. MSMOC: Participated in the design of the research and data analysis, and manuscript writing. ESNJ: Designed, supervised studies, interpreted results, and prepare the manuscript.
Acknowledgments We thank Miriam Regina Celi de Oliveira Costa for assistance with experiments, histological preparations and animal surgery. This study was supported by funding from the National Council of Technological and Scientific Development (CNPq) and by Coordination for Improvement of High Level Staff (CAPES). The English version of this text was revised by Sidney Pratt, Canadian, BA, MAT (The Johns Hopkins University), RSAdip (TEFL).
r e f e r e n c e s
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Sousa, T.B., Santana, M.A.D., Silva, A.M., Guzen, F.P., Oliveira, F.G., Cavalcante, J.C., Cavalcante, J.S., Costa, M.S.M.O., Nascimento Jr, E.S., 2013. Mediodorsal thalamic nucleus receives a direct retinal input in marmoset monkey (Callithrix jacchus): a subunit B cholera toxin study. Ann. Anat. 195, 32–38. Steinbusch, H.W., 1981. Distribution of serotoninimmunoreactivity in the central nervous system of the rat-cell bodies and terminals. Neuroscience 6, 557–618. Stratford, T.R., Wrtshafter, D., 2013. Injections of muscimol into the paraventricular thalamic nucleus, but not mediodorsal thalamic nucleus, induce feeding in rats. Brain Res. 1490, 128–133. Stuart, A.M., Mitchell, I.J., Unwin, H.L., Crossman, A.R., 1986. A semi-quantitative atlas of 5-hydroxytryptamine-1 receptors in the primate brain. Neuroscience 18 (3), 619–639. Van der Werf, Y.D., Witter, M.P., Groenewegen, H.J., 2002. The intralaminar and midline nuclei of the thalamus. Anatomical and functional evidence for participation in processes of arousal and awareness. Brain Res. Rev. 39, 107–140. Vertes, R.P., 1991. A PHA-L analysis of ascending projections of the dorsal raphe nucleus in the rat. J. Comp. Neurol. 313, 643–668. Vertes, R.P., 2002. Analysis of projections from the medial prefrontal cortex to the thalamus in the rat, with emphasis on nucleus reuniens. J. Comp. Neurol. 442, 163–187. Vertes, R.P., 2006. Interactions among the medial prefrontal cortex, hippocampus and midline thalamus in emotional and cognitive processing in the rat. Neuroscience 142, 1–20. Vertes, R.P., Fortin, W.J., Crane, A.M., 1999. Projections of the median raphe nucleus in the rat. J. Comp. Neurol. 407, 555–558. Vertes, R.P., Linley, S.B., 2007. Comparison of projections of the dorsal and median raphe nuclei, with some functional considerations. Int. Congr. Ser 1304, 98–120. Vertes, R.P., Linley, S.B., 2008. Efferent and afferent connections of the dorsal and median raphe nuclei in the rat. In: Monti, J.M., Pandi-Perumal, S.R., Jacobs, L., Nutt, D.J. (Eds.), Serotonin and Sleep: Molecular, Functional and Clinical Aspects. Birkhauser, Basel, pp. 69–102. Vertes, R.P., Linley, S.B., Hoover, W.B., 2010. Pattern of distribution of serotonergic fibers to the thalamus. Brain Struct. Funct. 215, 1–28. Vertes, R.P., Martin, G.F., 1988. Autoradiographic analysis of ascending projections from the pontine and mesencephalic reticular formation and the median raphe nucleus in the rat. J. Comp. Neurol. 175, 511–541. Viana di Prisco, G., Vertes, R.P., 2006. Excitatory actions of the ventral midline thalamus (rhomboid/reuniens) on the medial prefrontal cortex in the rat. Synapse 60, 45–55. Yoshida, M., Sasa, M., Takaori, S., 1984. Serotonin-mediated inhibition from dorsal raphe nucleus of neurons in dorsal lateral geniculate and thalamicreticular nuclei. Brain Res. 290, 95–105. Wilson, M.A., 1989. Distinct morphologic classes of serotonergic axons in the primates exhibit differential vulnerability to the psychotropic drug 3,4-methylenedioxymethamphetamine. Neuroscience 28 (1), 121–137. Wilson, M.A., Molliver, M.E., 1991. The organization of serotonergic projection to cerebral cortex in primates: regional distribution of axon terminals. Neuroscience 44 (3), 537–553. Zamboni, L., De Martino, L., 1967. Buffered picric acid formaldehyde: a new rapid fixative for electron microscopy. J. Cell Biol. 35, 148A.
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Research Report
Serotonergic fibers distribution in the midline and intralaminar thalamic nuclei in the rock cavy (Kerodon rupestris) Alane de Medeiros Silvaa, Melquisedec Abiare´ Dantas de Santanaa, Paulo Leonardo Arau´jo de Go´is Moraisa, Twyla Barros de Sousaa, Rovena Clara Galva˜o Janua´rio Engelberthb, Eudes Euler de Souza Lucenab, Clarissa Loureiro das Chagas Campeˆloc, Jeferson Sousa Cavalcanteb, Judney Cley Cavalcantea, Miriam Stela Maris de Oliveira Costaa, Expedito Silva do Nascimento Jra,n,1 a
Department of Morphology/Laboratory of Neuroanatomy, Biosciences Center, Federal University of Rio Grande do Norte, 59072-970 Natal, RN, Brazil b Department of Physiology/Laboratory of Neurochemical Studies, Biosciences Center, Federal University of Rio Grande do Norte, 59072-970 Natal, RN, Brazil c Department of Physiology/Memory Studies Laboratory, Biosciences Center, Federal University of Rio Grande do Norte, 59072-970 Natal, RN, Brazil
art i cle i nfo
ab st rac t
Article history:
The thalamic midline/intralaminar complex is part of the higher-order thalamus, which
Accepted 16 August 2014
receives little sensory input, and instead forms extensive cortico-thalamo-cortical pathways.
Available online 23 August 2014
The midline thalamic nuclei connect with the medial prefrontal cortex and the medial temporal lobe. On the other hand, the intralaminar nuclei connect with the fronto-parietal
Keywords:
cortex. Taking into account this connectivity pattern, it is not surprising that the midline/
Serotonergic system
intralaminar complex has been implicated in a broad variety of cognitive functions, including
Midline/intralaminar nuclei
memory process, attention and orientation, and also reward-based behavior. Serotonin (5-HT)
Thalamus
is a neurotransmitter that exerts different post-synaptic roles. Serotonergic neurons are
Rock cavy
almost entirely restricted to the raphe nuclei and the 5-HT fibers are distributed widely
Abbreviations: 3V, AM,
3rd ventricle; 5-HT,
anteromedial thalamic nucleus; AOI,
serotonin; 5-HT-IR, area of interest; CL,
nucleus; DAB,
diaminobenzidine; f,
nucleus; IAM,
interanteromedial thalamic nucleus; IMD,
mt,
mammillothalamic tract; PC,
nucleus; PV,
anterodorsal thalamic nucleus;
centrolateral thalamic nucleus; CM,
fornix; fr, fasciculus retroflexus; Hb,
habenular nucleus; IAD,
intermediodorsal thalamic nucleus; MD,
paracentral thalamic nucleus; PF,
paraventricular thalamic nucleus; PVA,
nucleus, posterior part; Re,
5-HT immunoreactive fibers; AD,
central medial thalamic interanterodorsal thalamic
mediodorsal thalamic nucleus;
parafascicular thalamic nucleus; PT,
paraventricular thalamic nucleus, anterior part; PVP,
reuniens thalamic nucleus; Rh, rhomboid thalamic nucleus; ROD,
paratenial thalamic paraventricular thalamic
relative optical density; sm,
stria
medullaris of the thalamus; Sub, submedius thalamic nucleus; VRe, ventral renuens thalamic nucleus; ZI, zona incerta n Corresponding author. Fax: þ55 84 32119207. E-mail address: [email protected] (E.S.d. Nascimento Jr). 1 Present address: Department of Morphology/Laboratory of Neuroanatomy, Biosciences Center, Federal University of Rio Grande do Norte, 59072-970 Natal, RN, Brazil. http://dx.doi.org/10.1016/j.brainres.2014.08.047 0006-8993/& 2014 Elsevier B.V. All rights reserved.
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Optical density Fiber morphology
throughout the brain, including the midline/intralaminar complex. The present study comprises a detailed description of the morphologic features and semiquantitative analysis of 5-HT fibers distribution in the midline/intralaminar complex in the rock cavy, a typical rodent of the Northeast region of Brazil, which has been used by our group as an anatomical model to expand the comprehension about phylogeny on the nervous system. The 5-HT fibers in the midline/intralaminar nuclei of the rock cavy were classified into three distinct categories: (1) beaded fibers, which are relatively fine and endowed with large varicosities; (2) fine fibers, with thin axons and small varicosities uniformly distributed in whole axon; and (3) stem axons, showing thick non-varicose axons. Moreover, the density of 5-HT fibers is variable among the analyzed nuclei. On the basis of this diversity of the morphological fibers and the differential profile of optical density among the midline/intralaminar nuclei of the rock cavy, we conclude that the serotonergic system uses a diverse morphologic apparatus to exert a large functional repertory in the midline/intralaminar thalamic nuclei. & 2014 Elsevier B.V. All rights reserved.
1.
Introduction
The rock cavy (Kerodon rupestris), a typical rodent in the Northeast region of Brazil, dwells in the mountainous regions of the Caatinga in the Brazilian semi-arid interior. Morphological (Silva Neto, 2000) and molecular biology studies (Rowe and Honeycutt, 2002) put the genus Kerodon in the family Hydrochaeridae, in which the capybara (Hydrochoerus hydrochaeris) is also included. The rock cavy has a crepuscular habit (Sousa and Menezes, 2006) and has been used in several neuroanatomical studies in our laboratory (Cavalcante et al., 2008; Nascimento et al., 2008, 2010a, 2010b; Soares et al., 2012). The rock cavy has advanced our understanding about the nervous system, providing a framework for the interpretation of evolutionary patterns, allowing inferences to be drawn about the brain and its phylogenetic congruence among diverse biological features. The structural organization of the raphe has been extensively studied in various species (Azmitia and Gannon, 1983; Dahlstrom and Fuxe, 1964; Dwarika et al., 2008; Fuxe et al., 1969; Harding et al., 2004; Hornung and Fritschy, 1988; Hornung, 2010; Limacher et al., 2008; Moon et al., 2007; Paxinos and Watson, 2007). The nuclear organization of the raphe serotonergic system in the rock cavy is similar to that described in other mammals with small anatomical variations (Soares et al., 2012). Moreover, subsequent reports using tracing techniques have pointed out the dorsal and median raphe nuclei as the main sources of serotonergic projections (Azmitia and Segal, 1978; Moore et al., 1978; Morin and MeyerBernstein, 1999; Vertes and Martin, 1988; Vertes, 1991; Vertes et al., 1999), although the majority of the serotonergic fibers are concentrated in limbic regions of the forebrain (Jacobs and Azmitia, 1992; Lowry et al., 2008; Steinbusch, 1981; Vertes and Linley, 2007, 2008). Serotonin (5-HT) exerts excitatory modulatory activity on some of its targets (Curtis and Davis, 1962; Kayama et al., 1989; Marks et al., 1987; Monckton and McCormick, 2001; Rogawski and Aghajanian, 1980; Yoshida et al., 1984). In other areas, however, it produces changes in the conductance and permeability of cell membranes, causing hyperpolarization of neurons (Mccormick and Pape, 1990; Lee and Mccormick, 1996). The functional duality of the 5-HT
in the thalamus has been widely associated with the various types of receptors present in the different thalamic nuclei (Chapin and Andrade, 2001a, b; Lopez-Gimenez et al., 1998, 2001; Kia et al., 1996; Mengod et al., 1996; Pompeiano et al., 1994), as well as in differences in fibers morphology, origin, and preference in area of innervation (Wilson, 1989). The 5-HT axons are generally divided into three morphological different fiber types. Thin axons with small fusiform or granular boutons, named fine fibers, are supposed to originated from the dorsal raphe nucleus; the beaded fibers with large spherical boutons; and non-varicose stem-axons originated from the beaded fibers, supposed to originated from the median raphe nucleus (Hornung and Fritschy, 1990; Wilson and Molliver, 1991). A low-frequency stimulation of the midline/intralaminar thalamic nuclei induces a low synchronous activity in different regions of the cortex (Dempsey and Morison, 1943). This thalamic nuclear complex is classically viewed as nonspecific thalamus, exerting a global influence on the cortical layers (Groenewegen and Berendse, 1994). Nowadays, the notion of the nonspecific functions has been changed as a result of several anatomical studies which have demonstrated, that the midline/intralaminar complex projects to specific areas of the cerebral cortex, mainly to the prefrontal cortex (Groenewegen and Witter, 2004; Van der Werf et al., 2002; Vertes, 2006). Furthermore, the electrical stimulation of individual nuclei produces selective effects on their cortical targets (Viana di Prisco and Vertes, 2006). Descriptions of the serotonergic projections in the thalamus have been shown in monkey and rat studies (Cropper et al., 1984; Lavoie and Parent, 1991; Vertes, 2002; Vertes et al., 2010) and suggest that the serotonergic fibers exert a fundamental role on modulation in the midline/intralaminar thalamic nuclei. Furthermore, it is widely agreed that cell types and morphological fiber pattern serve as the building blocks of nervous systems and that exploring their diversity and determining how cells are assembled into circuits is essential for understanding brain function. In the present study we aim describe and compare the distribution of serotonergic fibers in the rock cavy midline/ intralaminar thalamic nuclei. We also assess the relative abundance of serotonergic fibers throughout the various
brain research 1586 (2014) 99–108
nuclei of the midline/intralaminar complex in the rock cavy and supply a morphological description of the 5-HT fibers and their differential distribution within this nuclear group.
2.
Results
2.1.
Cytoarchitectonic analysis
The midline nuclei in the rock cavy include the paraventricular (PV), paratenial (PT), intermediodorsal (IMD), reuniens (Re), and rhomboid (Rh) nuclei. On the other hand, the intralaminar nuclei contain the central medial (CM), central lateral (CL), paracentral (PC), and parafascicular nuclei (PF).
2.1.1.
Midline thalamic nuclei
The PV in the rock cavy is a conspicuous nucleus, lying immediately ventral to the third ventricle in the thalamic midline. On coronal sections, it appears first as a triangularly shaped nucleus. It extends through almost all the rostrocaudal area of the thalamus and is clearly divided in three cytoarchitectonical divisions (PV anterior, PV middle, and PV posterior). Its lateral border is limited by the PT in rostral levels and by the mediodorsal nucleus (MD) in the middle and caudal ones, while ventrally it borders on the CM (Fig. 1). The PT is a rounded nucleus located in the dorsal thalamus. In the rostral levels of the thalamus, it constitutes the lateral and inferior borders of the PV (Fig. 1A and B). In its caudal extremity it merges with the MD. The IMD, as its name suggests, is located between the right and left MD thalamic nucleus and is more easily visualized in the middle coronal sections of the thalamus with the PV (Fig. 1C–F). The Re is located in the ventral part of the thalamic midline, immediately dorsal to the third ventricle. Two cell clusters constitute it; situated laterally to the third ventricle in the rostral levels, they fuse together along thalamic rostrocaudal length (Fig. 1). Finally, the Rh is found just below the internal medullary lamina. In rostral thalamic sections, the Rh is immediately dorsal to the Re and in caudal ones it is easily distinguished by its conspicuous shape and strongly stained cells (Fig. 1C–F).
2.1.2.
Intralaminar thalamic nuclei
In the rock cavy thalamus we have identified a CM nucleus as a conspicuous cell cluster located centrally in the internal medullary lamina, distinct from the midline nuclei. This nucleus shows large stained cells along the length of the thalamic rostrocaudal. Laterally, the CM is continuous with PC and CL. PC appears laterally to the CM and its cells are difficult to distinguish from those in the CM. The CM lies at the curved portion of the internal medullary lamina, immediately lateral to the MD and medially to the ventral thalamic nucleus. Dorsally it is continuous with the CL nucleus. The CL is the most dorsal part of the intralaminar nuclei. It is difficult to identify its boundary with the PC. The CL is larger than the CM, PC, and PF and contains strongly stained cells (Fig. 1E and F). Finally, the PF is located in the caudal part of the thalamus. It can be clearly detected where the midbrain structures start to appear. The PF is comprised of a large
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round mass of cells, lying in close proximity to the fasciculus retroflexus and dorsally bordered by the CL and lateral to the medial habenula (Fig. 1G and H).
2.2.
Relative optical density analysis
The ROD values were higher in the midline nuclei compared with the intralaminar ones [t(3)¼7.553, p¼ 0.005] (Fig. 2). Furthermore, several differences in the ROD values were found by comparing nuclei from the midline and intralaminar each other (Fig. 3). Comparisons among midline nuclei each other revealed significant increase ROD values in the Re [p¼ 0.003] and PV [p ¼0.022] compared with PT. Although no differences in any other midline thalamic nuclei could be detected, the photomicrographs suggest variations in their contents (Fig. 3). When we used the one-way ANOVA [F (4,19)¼6.301, p¼ 0.002] and Tukey’s multiple comparison tests, the Re shows higher ROD values, followed by PV, Rh, IMD, and PT (Fig. 4). Comparisons among intralaminar nuclei each other revealed significant increase ROD values in the CM [p¼ 0.038]; PC [p ¼0.005], and PF [p ¼0.004] compared with the CL. The One-way ANOVA [F(3,15)¼8.506, p¼ 0.003] and Tukey’s multiple comparison tests revealed a significant increased ROD values among intralaminar thalamic nuclei, with the CL showing a higher ROD value followed by CM, PC, and PF (Fig. 5). There was no significant difference in ROD values between male and female brains across the midline/ intralaminar nuclei.
2.3.
Descriptions of serotonergic fibers
The midline/intralaminar complex in the rock cavy received a multitude of fibers that demonstrated strong and distinct anatomical variations (Fig. 6). These fibers could be classified into three distinct groups, based on morphological criteria: beaded fibers, fines fibers and stem-axons. In the midline thalamic nucleus, the PV was amply supplied by the beaded fibers (Fig. 6D) with thin axons that had large varicosities, probably corresponding to the same beaded fiber (BF) type described in other species (Hornung, Fritschy, 1990; Wilson and Molliver, 1991). The thin axons with small fusiform and granular varicosities, corresponding to the fine fiber (FF) (Fig. 6E), and thick non-varicose axons, probably the stemaxons (SA) (Fig. 6F), were seen uniformly distributed throughout the midline/intralaminar nuclei, except in PV.
3.
Discussion
The rock cavy midline/intralaminar complex is densely innervated by serotonin immunoreactive fibers and this innervation has a complex pattern. Substantial variation in ROD values and morphology of 5-HT fibers is detected among midline/intralaminar nuclei. The highest ROD value is observed in the Re among the midline nuclei. In the intralaminar nuclei, however, the highest ROD values is observed in the CL. The medium ROD values are found in the PV, IMD, and Rh in the midline, while among intralaminar nuclei the CM and PC show medium ROD values. The lowest fiber densities are observed in the PT and PF. The present work
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Fig. 1 – Drawings of coronal sections through the rock cavy brain illustrating the morphology of the midline and intralaminar thalamic nuclei (A), (C), (E), and G), and digital images of Nissl stained coronal sections at corresponding levels (B), (D), (F), and (H). Rostral level representation in (A) and (B) and caudal level (G) and (H). Numbers on the right indicate distance from the bregma. See list of abbreviations. Bar 280 lm. is the first to analyze the thalamic midline/intralaminar complex organization using a Relative Optical Density tool to quantify serotonergic fibers in these nuclei.
Although the serotonergic system shows a firmly established morphological organization among numerous species (Bjarkam et al., 1997; Harding et al., 2004; Hornung, 2010;
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Fig. 2 – ROD values across the midline and intralaminar thalamic nuclei of rock cavy (n ¼8). The bars represent the means of the mean ROD values in the midline and intralaminar nuclei of individual animals (across all rostrocaudal levels and nuclei). The paired t-test revealed a significant difference between the midline and intralaminar nuclei. Values are expressed as mean and standard error of the mean. npo0.05—[t(3) ¼7.553, p¼ 0.005].
Fig. 3 – Semiquantitative analysis showing the densities of 5-HT-IR fibers in the different midline/intralaminar nuclei. To compare across these nuclei, the Relative Optical Density values in each nucleus are displayed as levels of gray in the table at the left. In the table, nuclei are listed according rostracaudal levels. See list of abbreviations.
Jacobs et al., 1984), we observed several differences in the serotonergic innervation of the rock cavy midline/intralaminar nuclei compared with rodents and primates. In albino rats, the PV, Rh and Re show a high fiber density, while the PT has a low fiber density (Cropper et al., 1984). Another study with rats using SERT (serotonin transporter), has reported the highest fiber density in the PV, followed by Rh, Re, and IMD (Vertes et al., 2010). In contrary, in the present study, the ROD values shown suggest higher density in the Re. In our results, based on ROD values, the intralaminar nuclei show moderate or low fiber innervation when compared with other midline nuclei, contrary to data in rats, in which dense serotonergic innervation in the CM and CL was found (Vertes et al., 2010). In the hamster (Mesocricetus auratus), the highest density is described in the PV and Re, while in the intralaminar nuclei the highest density is observed in the CM (Morin and MeyerBernstein, 1999). In a primate species, the squirrel monkey
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Fig. 4 – ROD values in the midline thalamic nuclei of rock cavy (n¼ 8). The bars represent the means of the mean ROD values of individual animals (across all rostrocaudal levels). One-way ANOVA revealed ROD significant difference between the nuclei analyzed. Values are expressed as mean and standard error of the mean. np¼ 0.003 nnp¼0.022— Tukey’s multiple comparison test [F(4,19)¼6.301, p ¼0.002]. See list for abbreviations.
Fig. 5 – ROD values in the intralaminar thalamic nuclei of rock cavy (n ¼8). The bars represent the means of the mean ROD values of individual animals (across all rostrocaudal levels). One-way ANOVA revealed ROD significant difference between the nuclei analyzed. Values are expressed as mean and standard error of the mean. np¼ 0.038 nnp¼0.005 nnn p¼ 0.004—Tukey’s multiple comparison test [F(3,15)¼ 8.506, p¼ 0.003]. See list for abbreviations.
(Saimiri sciureus), a highest density of 5-HT fibers can be observed in the PV and Re among the midline nuclei. Otherwise, in the intralaminar nuclei the highest density is observed in the CM (Lavoie and Parent, 1991). Contrary to our results, in another primate species (Macaca Mulatta), the serotonergic fibers are more densely concentrated in the PV compared to other midline/intralaminar nuclei (Hsu and Price, 2009). Through extensive dendritic and axonal arborizations, neurons define ample domain of influence. Morphological characterization, such as shaft caliber, tapering or branching, determines action potential propagation in axons (Goldstein and Rall, 1974). Spines and dendritic segments are also important in defining biochemical compartments in neurons, which are dependent on their size, length, and shape (Helmchen, 1999; Korkotian and Segal, 2000). Overall, different patterns in density and spatial distribution of axonal or dendritic branches create a wide spectrum of wiring
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Fig. 6 – Photomicrograph of 5-HT-immunostained brain coronal sections at the level of approximately 2.30 mm p.b. (A); (B) magnification of box homonymous in A, with an area corresponding to the dorsal midline, including PV and IMD; (C) magnification of box homonymous in A, with an area corresponding to the intralaminar region, including the PC; (D) magnification of superior box homonymous in B, with an area corresponding to the nucleus PV; (E) magnification of inferior box homonymous in B, with an area corresponding to the nucleus IMD; and (F) magnification of box homonymous in C, with an area corresponding to the PC nucleus. Black arrows in D correspond to the BF-like fibers; arrowheads in E correspond to the FF-like fibers; and White arrows in F correspond to the SA-like fibers. See list of abbreviations. Bar: 230 lm A; 140 lm B, and C; 15 lm D, E, and F.
configurations, which are currently subject to intensive experimental and theoretical research (Chklovskii et al., 2002; Petrof and Sherman, 2013; Rubio-Garrido et al., 2009; Sousa et al., 2013). The present work is the first to describe morphological differences in the pattern of distribution of serotonergic fibers into midline/intralaminar complex. We found the BF-like type fibers more frequently inside the PV. Otherwise, the FF-like fibers were distributed throughout the remainder midline/intralaminar nuclei merged with SA-like fibers. The morphological descriptions of the serotonergic fibers have been made only in the cerebral cortex of rats (Kosofsky and Molliver, 1987) and primates (Wilson and Molliver, 1991), as well as in rabbit hippocampus (Bjarkam and Sorensen, 2005). These pronounced differences in fiber density as well as in morphological features of 5-HT fibers in the midline/intralaminar complex indicate a possible species differences in serotonergic innervation between rodents and nonhuman primates. As is well known, the main source of serotonergic fibers throughout the brain comes from the dorsal and median raphe nuclei. Two classes of serotonergic fibers have been described for rodents and primates, and are thought to arise from separated raphe nuclei (Kosofsky and Molliver, 1987; Wilson, 1989; Wilson and Molliver, 1991). Fine axons with granular or fusiform varicosities originate from the dorsal raphe nucleus, while larger beaded axons arise
from median raphe nucleus (Kosofsky and Molliver, 1987). These morphological findings suggest that the rock cavy midline/intralaminar complex receives different classes of serotonergic fibers arising from different sources in the raphe. In addition, it has been shown that psychoactive drug, such as 3,4 methyledioxymetamphetamine (Ecstasy) and pchloroamphetamine, selectively destroy the FF in the central nervous system, but leave the BF unharmed (Wilson, 1989; Haring et al., 1992). One may differentiate among systems of ascending serotonergic fibers, with clear differences in fiber origin, fiber morphology, preference in area of innervation, and sensitive to psychoactive drugs. In accordance with our findings, the midline nuclei of the rock cavy contained more 5-HT fibers than the intralaminar ones. Furthermore, there is a differential density of 5-HT-IR fiber among them. For example, a moderate density was observed in the nuclei associated with feeding (PV), learning and memory (PT), arousal and awareness (Rh), seizure regulation (CM), and motor regulation (CL) (Bentivoglio et al., 1991; Ichinohe et al., 2001; Miller and Ferrendelli, 1990; Stratford and Wrtshafter, 2013; Van der Werf et al., 2002). The specific effects of 5-HT release depend on the class of receptor involved (Goodwin, et al., 1985; Larsson and Ahlenius, 1999; Meltzer, 1990; Nordberg, 1992; Pazos et al., 1987a, b; Stuart et al., 1986), and the type of neuron received
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in the synaptic contact. It is clear that a more precise description of the 5-HT fiber ultrastructure and serotonin receptor distribution is needed to provide the basis for further evaluation of the functional role of the 5-HT in the midline/ intralaminar thalamic nuclei. The above descriptions of differences in the density and morphological distribution of serotonergic fibers in the midline/intralaminar nuclei of the rock cavy are an attempt to understand the functional and evolutionary pathway by which the 5-HT acts in the thalamus through the indispensable anatomical background, as well as understand the fundamental importance of identification, characterization, and comparative analysis of the great diversity of cell types present in complex nervous system. Although functional studies are strictly necessary to understand the real interference of 5-HT in the midline/intralaminar nuclei, this anatomical study suggests that the serotonergic system exerts a fine control over thalamic function to modulate behavior.
4.
Experimental procedures
4.1.
Animals and housing
Eight young adult rock cavies (four males and four females), weighing 300 and 400 g, from the rural municipalities in Rio Grande do Norte state, Brazil, were used. Animal capture was authorized by the Brazilian Environmental Agency (IBAMA, licenses 21440-1). Approval for the experiments was obtained from local Animal Experimentations Ethics Committee 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 in 3.00 m 2.00 m 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.
4.2.
Perfusion and microtomy
Each individual was pre-anesthetized with an intramuscular injection of tramadol chloridrate and xylazine, both 5 mg/kg, and maintained with gas isoflurane and 100% oxygen. Upon deep anesthesia, they were perfused for approximately 5 min using a cannula positioned in the ascending aorta and connected to a peristaltic pump (Cole-Parmer), with 300 ml of 0.9% saline solution in an 0.1 M phosphate buffer, pH 7.4, containing heparin (Parinex, Hipolabor, Sabará, MG, Brazil, 2 ml/1000 ml of saline solution). Next, 700 ml of a 4% 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 established for half the solution and 17.5 ml/min for the other half, with the entire procedure taking 30 min. 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
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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, each brain was removed from the skull, stored in 30% sucrose solution in 0.1 M phosphate buffer, pH 7.4, for 24–48 h, frozen by dry ice, and serially cut in the coronal plane into 30 mm thick sections in a sliding microtome.
4.3.
Nissl staining and immunohistochemistry
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 5-HT. All the immunohistochemical procedures were performed at room temperature. The sections, previously submitted to a pre-treatment with sodium borohydride and hydrogen peroxide (H2O2), were placed in contact with the rabbit anti-5-HT antibody (Sigma, 1:5000) and 2% normal goat serum in 0.4% Triton X-100 for 18 h, in 0.1 M phosphate buffer, pH 7.4, in a rotator. This was followed by incubation in a secondary antibody, consisting of 1:1000 biotinylated goat anti-rabbit (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 a final reaction in a medium containing H2O2 as substrate and 3,30 -diaminobenzidine tetrahydrochloride (DAB) as chromogen. H2O2 was added indirectly, by mixing oxidase glucose and ß-D glucose, causing a reaction in which the former acting on the latter releases H2O2. The sections were thoroughly washed with 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 immunostainings were performed concomitantly, minimizing possible differences in background between the animals. With respect to staining specificity, a number of sections were submitted to immunohistochemical reactions omitting the primary or secondary antibodies. In these cases, no 5-HT immnoreactivity was obtained. Diagrams were obtained from image of Nissl-Stained sections with Adobe Illustrator software (Adobe Systems, Mountain View, CA, USA). The anatomical location of the brain structures was determined using the rat brain atlas of Paxinos and Watson (2007), and our experience in the anatomy of the rock cavy brain (Nascimento et al., 2008, 2010a, 2010b; Soares et al., 2012).
4.4.
Qualitative and quantitative analysis
5-HT-IR was identified as a black-purple precipitate. The tissue was analyzed with an optic microscope (BX41 Olympus) under brightfield illumination. Digital images were captured using a digital video camera (Nikon DXM1200) coupled to the microscope. In order to identify morphological differences among 5-HT fibers/terminal in the midline/intralaminar nuclei, we performed qualitative analysis under high
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magnification. The digitized images were corrected minimally for brightness and contrast, and mounted using Adobe Photoshop 7.0 software (Adobe Systems, Mountain View, CA, USA). In order to accomplish the quantitative analysis, all images were obtained in brightfield illumination at a fixed intensity for each of 16 rostro-caudal levels per individual. The resulting brightfield images were 3840 3072 pixel, with a resolution of 0.59 pixel/mm (with 4 objective). The Relative Optical Density (ROD) analysis was accomplished using Image J software (Version 1.46i, NIH). The images were converted to gray scale images (8-bit). The next stage consisted to the binarization of them, adjusting the contrast to 100%. After this procedure, the images presented only two values that could be observed in the histogram, the zero that corresponds to black and the 255 that corresponds to white. Finally, the program provided the number of black pixels in the sampled areas (Santos et al., 2013). The mean gray value of each sampled area was measure using a square (0.3 0.3 mm) in the area of interest (AOI). This AOI was located 3–4 times on a well-defined DAB stained in each nucleus throughout rostro-caudal levels and the mean gray values were taken. The medium number of black pixels in the target area was subtracted from the medium values of a control region (areas that should not have specific 5-HT staining). The value of OD (optical density) of the AOI was related to the background value by the formula: [(OD AOI OD background)/OD background] 100, thus eliminating the variability in background staining among sections (Krugers et al., 1996). The data were presented to each target area as the mean of pixels in the AOI. A manual selection of the stained positively for DAB chromogen was performed (Cuesta et al., 2013). All results are expressed as mean and standard error of the mean (S.E.M). Statistical analysis was performed using a computer program (Statistical Package for Social Sciences—IBM SPSS, version 21). The ROD data was subjected to t-test to analyze differences between the mean number of black pixels in the midline and intralaminar nuclear complex. One-way ANOVA followed by post-hoc Tukey’s multiple comparisons was accomplished to verify the differences among nuclei in the midline and intralaminar nuclei each other.
Conflict of interest statement The authors declare no conflict of interest.
Author contributions AMS: Participated in the design of the research and data analysis, and manuscript writing. MADS: Participated in the analysis of the results. PLG: Participated in the analysis of the results. TBS: Participated in the analysis of the results. RCGE: Participated in the research. EESL: Participated in the analysis of the results. CLCC: Participated in the data analysis.
JSC: Participated in the design of the research and data analysis. JCC: Participated in the design of the research, and manuscript writing. MSMOC: Participated in the design of the research and data analysis, and manuscript writing. ESNJ: Designed, supervised studies, interpreted results, and prepare the manuscript.
Acknowledgments We thank Miriam Regina Celi de Oliveira Costa for assistance with experiments, histological preparations and animal surgery. This study was supported by funding from the National Council of Technological and Scientific Development (CNPq) and by Coordination for Improvement of High Level Staff (CAPES). The English version of this text was revised by Sidney Pratt, Canadian, BA, MAT (The Johns Hopkins University), RSAdip (TEFL).
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