Distribution of NADPH-diaphorase and nitric oxide synthase reactivity in the central nervous system of the goldfish (Carassius auratus)

Journal of Chemical Neuroanatomy 35 (2008) 12–32 www.elsevier.com/locate/jchemneu

Distribution of NADPH-diaphorase and nitric oxide synthase reactivity in the central nervous system of the goldfish (Carassius auratus) Rosa M. Giraldez-Perez a, Susana P. Gaytan a, Diego Ruano b, Blas Torres a, Rosario Pasaro a,* b

a Department of Fisiologı´a y Zoologı´a, Facultad de Biologı´a, Universidad de Sevilla, Sevilla 41012, Spain Department of Bioquı´mica, Bromatologı´a, Toxicologı´a y Medicina Legal, Facultad de Farmacia, Universidad de Sevilla, Sevilla 41012, Spain

Received 26 March 2007; received in revised form 3 May 2007; accepted 22 May 2007 Available online 31 May 2007

Abstract The nitrergic system has been inferred from cells positive to nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and/or to the neuronal isoform of nitric oxide synthase (nNOS) immunohistochemistry in different species of vertebrates. The aim of the present work was to systematically study the distribution of cell producing nitric oxide in the goldfish (Carassius auratus) brain. To reach this goal, we firstly studied co-localization for NADPHd and nNOS techniques and demonstrated an extensive double labeling. Then, we studied the distribution through the brain by the two separate methods and found labeled cells widely distributed in brain and spinal cord. In the telencephalon, such cells were in both dorsal and ventral areas. In the diencephalon, the cells were found in some nuclei of the preoptic area and hypothalamus, habenula, pretectum, and dorsal and ventral thalamic regions. In the midbrain, cells were observed in the optic tectum, torus longitudinalis, and tegmental nuclei. In the rhombencephalon, cells were found in the cerebellum, the reticular formation, the locus coeruleus, the raphe nuclei, and the nuclei of the cranial nerves. Labeled cells were also observed in the gray area of the spinal cord. Cognizing that a direct comparison of the present results with those reported in other vertebrates is not clear-cut because of homologies; we conclude that the nitrergic system is roughly similar from fish to mammals. # 2007 Elsevier B.V. All rights reserved. Keywords: Nitric oxide; nNOS immunohistochemistry; NADPH-diaphorase histochemistry; Teleost; Fish

1. Introduction Nitric oxide (NO) acts as a neuronal paracrine intercellular/ intracellular messenger molecule in the nervous system (Torreilles, 2001). The NO is produced from L-arginine in the brain by the constitutive neuronal isoform of nitric oxide synthase (nNOS); the activity of this enzyme is dependent on several cofactors, including nicotine adenine dinucleotide phosphate (NADPH) (Knowles et al., 1989; Mayer et al., 1990). Because the NO cannot be visualized directly in the tissue, the capacity of a neuron to produce NO has been inferred by histochemistry for NADPH-diaphorase (NADPHd) and/or

* Corresponding author at: Departamento de Fisiologı´a y Zoologı´a, Facultad de Biologı´a, Avda. Reina Mercedes 6, 41012 Sevilla, Spain. Tel.: +34 954556572; fax: +34 954233480. E-mail address: [email protected] (R. Pasaro). 0891-0618/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2007.05.007

immunohistochemistry against nNOS from fish (Holmqvist et al., 1994; Arevalo et al., 1995; Bru¨ning et al., 1995; Cuoghi et al., 2002; Jadhao and Malz, 2004) to mammals (Vincent and Kimura, 1992; Gotti et al., 2005). An extensive co-localization of nNOS and NADPHd has been reported in mammals (Dawson et al., 1991), while some discrepancies have been found in non-mammalian vertebrates, including fish (Holmqvist et al., 1994; Bru¨ning et al., 1995; Virgili et al., 2001). Because there is controversy on whether the distribution of NADPHd is identical to that of nNOS, and whether such differences could depend on the species studied (Alonso et al., 2000; Moreno et al., 2002), a first goal of this work was to study the co-localization of NADPHd and nNOS in the goldfish brain. It is widely accepted that NO plays a major role in sensory and motor systems (Funakoshi et al., 1999), learning and memory (Susswein et al., 2004), neurogenesis (Estrada and MurilloCarretero, 2005), and neuroendocrine and autonomic nervous activities (Gerstberger, 1999; Guo and Longhurst, 2003). On the

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basis of these important functions, the distribution of the neurons producing NO has been studied in mammals (Vincent and Kimura, 1992; Gotti et al., 2005), birds (Bru¨ning, 1993; Panzica et al., 1994), reptiles (Bru¨ning et al., 1994; Smeets et al., 1997), amphibians (Munoz et al., 1996; Gonzalez et al., 2002), and fish (Arevalo et al., 1995; Bru¨ning et al., 1995; Ando et al., 2004). Although, the localization of cells positive for NADPHd and nNOS has previously been reported in goldfish (Bru¨ning et al., 1995), that work did not identify neurons in the dorsal telencephalon, pretectum, optic tectum, mesencephalic reticular formation, nucleus isthmi, locus coeruleus, cerebellum, and motor nuclei of the cranial nerves, which have been included in the nitrergic system of vertebrates (see Section 4). Thus, this work investigates the localization of the cells producing NO in goldfish, emphasizing in those already cited areas. With this aim, we used some modifications to the method of study (see Section 4.1). Finally, the distribution of cells positive for NADPHd and nNOS reported here was compared with those observed in other species, to provide new insights about conservative features of the nitrergic system in the vertebrate brain. 2. Materials and methods For the present study, 25 goldfish (Carassius auratus) both males and females, obtained from a local supplier, and with a body length of 10–15 cm, and 1 rat were used. The Animal Ethics Committee of the University of Seville approved the experimental protocol, which followed the European Communities Council Directive (86/609/ECC) on animal care and experimentation. The animals were deeply anesthetized with 0.4% of 3-aminobenzoic acid ethyl ester methane sulfonate salt (MS-222, Sigma, St. Louis, MO) in freshwater.

2.1. Western blot Western blots were performed as previously described (Ruano et al., 2006). The brains of five goldfish and one rat were extracted in fresh after deep anesthesia. Briefly, proteins from nervous tissue were loaded on a 12% polyacrylamide gel for electrophoresis (SDS-P, Bio-Rad, USA) and transferred to a nitrocellulose membrane (Hybond-C Extra, Amersham, Sweden). After blocking, the membranes were incubated overnight at 4 8C with the primary polyclonal antibody rabbit anti-neuronal nitric oxide synthase (AB5380, Chemicon International; dilution 1:1000). Then, the membranes were incubated with a peroxidase-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Lab, USA; dilution of 1:6000). The blots were developed using the ECL-plus detection method (Amersham, Sweden).

2.2. Tissue processing Twenty fishes were perfused transcardially with 4% paraformaldehyde in 0.1 M cold phosphate-buffered saline (PBS, pH 7.4), after anesthesia. The brains were removed, and post-fixed for 4 h. Then, they were immersed in cold phosphate buffer with 30% of sucrose for 24 h. The brains were sectioned on a cryostat (Leica CM1850) at 30 mm in transverse plane, and mounted onto double-gelatinized slides. Two groups of experiments were carried out: first, to test if NADPHd and nNOS-positive neurons are identical, we performed double labeling experiments (n = 10). In this case, we determined nNOS cells by fluorescence immunolabeling and then NADPHd activity. Second, to determine the distribution of NADPHd and nNOS-positive cells (n = 10), the brains were cut in three parallel series of transverse sections: one was reacted for NADPHd histochemistry, another was treated with nNOS antibodies, and the last was counterstained with 0.1% neutral red solution for anatomical references. To test the idea that NADPHd and nNOS-positive neurons are identical, we carried out double labeling experiments. The sections were: treated with a solution of 5% donkey serum for 1 h in PBS plus 0.3% Triton X-100 (PBS+),

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and rinsed three times for 10 min in PBS; incubated with a cold solution of rabbit anti-nNOS (1:1000 AB5380, polyclonal antibody, Chemicon, Temecula, CA) in PBS+ for 72 h at 4 8C, and rinsed three times in PBS (10 min each); incubated at room temperature in a dark chamber with mouse anti-rabbit (1:100, TRITC-conjugated (H + L) 211-025-109, Jackson ImmunoResearch) for 1 h and a half, and rinsed three times in PBS (10 min each). Thereafter, the sections were mounted on glass slides with 50% glycerol without drying, studied and photographed; then, they were collected in PBS and rinsed three times (15 min each). The histochemical technique for NADPHd was subsequently performed. The present study used a variation of the method of Sims et al. (1974) for NADPHd histochemistry to determine the diaphorase activity. Sections were first incubated in a solution of 0.1 M Tris buffer solution (pH 8.0), 0.05% Triton X-100 (Sigma–Aldrich), plus 1 mM b-NADPH (Sigma–Aldrich, Steinheim, Germany) and 0.8 mM nitroblue tetrazolium (Sigma–Aldrich), at 37 8C for 1– 2 h, under microscope observation. Following incubation, the sections were rinsed three times in PBS solution for 5 min. Finally, all sections were dried overnight, dehydrated, and coverslipped with DPX mounting medium (Panreac, Barcelona, Spain). To test the accuracy of the histochemical protocol, controls were carried out by omission of the b-NADPH or nitroblue tetrazolium. In both cases, no positive cell for NADPHd was observed. To determine the distribution of NOS-positive cells we carried out separate labeling of NADPHd activity and nNOS immunohistochemistry and the results were studied by bright-field microscopy. NADPHd activity was revealed as above mentioned. To reveal nNOS cells, the sections were pretreated with 2% H2O2 and 5% methanol in Tris-buffered saline (TBS 0.05 M, pH 7.4) for 10 min, and rinsed three times in TBS; incubated with a cold solution of rabbit anti-nNOS (1:3000 AB5380, polyclonal antibody, Chemicon, Temecula, CA) in PBS+ for 72 h at 4 8C, and rinsed three times in PBS (10 min each); incubated at room temperature with mouse anti-rabbit (1:2000 biotin-SP-conjugated (H + L) 211-065-109, Jackson ImmunoResearch) secondary antibody in PBS+ and 5% donkey serum for 2 h, and rinsed three times in TBS (10 min each); incubated with the ABC kit (Vector Labs., Burlingame, CA) at 35 mL of avidin and 35 mL of biotin in 10 mL TBS plus 0.3% Triton X-100 (TBS+, pH 8.8) for 1.5 h at room temperature, and rinsed sequentially in TBS (10 min) and Tris–HCl buffer 0.05 M (pH 7.6) for 10 min; stained with 0.05% 3,30 -diaminobenzidine tetrahydrochloride (DAB) in Tris–HCl with 0.033% H2O2 for 30 min, and rinsed three times in Tris–HCl (10 min each). Finally, sections were dried overnight, dehydrated, and coverslipped with DPX. To verify specificity of the antibody used, two routine control procedures were carried out: omission of primary and secondary antibodies, and replacement of the two antisera with PBS. In both cases, any labeled cell was observed. The specificity of the antiserum used here has also been tested previously in mammals (Kruger et al., 2002; Rodrigo et al., 2006).

2.3. Analysis Sections were analyzed and photographed using an Olympus BX61 microscope (Olympus, Tokyo, Japan). Bright-field photomicrographs were captured with a digital camera (DP70, Olympus, Japan). Photomicrographs were subsequently processed with the CorelDRAW 12.0 program for adjustment of brightness and contrast. Measurement of the neurons was performed directly on the screen of a monitor coupled to the microscope using the Scion Image Beta 4.02 software program (Scion Corp., Maryland, USA). A sample of 40 labeled

Fig. 1. Expression of the neuronal nitric oxide synthase protein in the rat (R) and goldfish brain. Western blot analysis was performed loading increasing amounts of goldfish brain proteins (lanes 20, 40, and 80 mg). Membranes were immunostained with a rabbit anti-neuronal nitric oxide synthase antibody showing the presence of a band around 150 kDa, which was evident in the sample with the higher protein content (asterisk). A similar band was detected in rat brain used as a control. Molecular weight standards are indicated on the left.

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R.M. Giraldez-Perez et al. / Journal of Chemical Neuroanatomy 35 (2008) 12–32 cells was measured (maximum diameter) in each of the nuclei. All data were expressed as mean  S.D. We measured the nNOS-immunoreactive cells, with the exception of the ones that were only NADPH-diaphorase positive, such as the Mauthner cell. The nomenclature and arrangement for brain structures were taken from Meek and Nieuwenhuys (1998).

3. Results Western blot analysis of brain protein extracts with the polyclonal antiserum against rabbit nNOS in the goldfish and rat brains showed the presence of a single band in both cases of ffi150 kDa (Fig. 1). The immunoreaction product increased in function of the amount of protein (20, 40, and 80 mg) in the goldfish lanes. This result indicated that the antibody was accurate enough to be used in the goldfish brain. In order to investigate if NADPHd activity and nNOS immunoreactivity is co-localized in the same neuron of distinct populations of the goldfish brain, double labeling experiments were carried out in the same section. The results showed an extensive co-localization irrespective of the studied nuclei (Fig. 2). However, a number of NADPHd-positive cells were not positive against nNOS. Taken together these data we plotted the distribution of the NO producing cells using both procedures (Fig. 3). Thus, Fig. 3 illustrates the distribution of NADPHd- and nNOS-labeled cells per structure (each dot represents 10 cells) from the olfactory bulb to the spinal cord. As shown, the cells were found in the majority of brain structures. A direct comparison of the number of positive cells for the two techniques can be scored from the dots represented in the chartings in either the same nucleus or different nuclei. We will describe below the distribution of the labeled cells, the course of fibers when observable (not illustrated in Fig. 3), and the cases in which positive staining was found for one of the two procedures. The intensity of labeling for NADPHd activity varied from pale to very dark. 3.1. Olfactory bulb and telencephalon Labeling in the olfactory bulb was positive only for NADPHd activity (Fig. 3A). Many labeled fibers, densely packed as a braided cord, were observed penetrating into the olfactory bulb. These fibers coursed through the glomerular layer and ended in the glomeruli. Within the granular layer, the cells found were small (5.4  0.5 mm), rounded, and sparse, while they were large (18.9  0.2 mm) and packed in the glomerular layer. In addition, a few gigantocellular (33.4  0.2 mm) pyriform neurons, with one or two dendrites, were observed close to the fibers that entered the olfactory bulb. These latter cells were surrounded by very small cells. Cells labeled for NADPHd and nNOS were found in the dorsal and ventral areas of the telencephalon (Fig. 3B–D). A high number of neurons were found in the different dorsal

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telencephalic areas (Figs. 3B–D and 4A–D). These cells were small in the posterior (5.5  0.9 mm) and medial (8.1  0.7 mm) parts and rounded or pyramidal with few dendrites (Fig. 4A–D), except those in the dorsocentral area, which had medium-sized somata (15.0  0.7 mm) (Fig. 4C). Fibers positive for nNOS coursed with a funnel shape towards the dorsal areas (Fig. 4B), where thin axons with boutons showing en passant morphologies were observed in the different areas (Fig. 4C). In the ventral telencephalic areas, the labeling was different for NADPHd and nNOS. NADPHd-labeled neurons were found in all ventral telencephalic areas, while nNOS-labeled cells were confined to supracommissural- and posterior-ventral areas (Fig. 3B–D). These neurons were small (7.5  0.7 mm) in the ventrodorsal, (5.7  0.8 mm) ventroventral, (8.9  0.6 mm) ventrolateral, and (6.8  0.8 mm) ventroposterior parts, and rounded, with dendrites oriented dorsolaterally. Within the ventral telencephalic area, supracommissural part, the cells showed mediumsized (12.4  0.9 mm) rounded somata. Finally, small (9.2  0.7 mm) pyriform neurons were found within the entopeduncular nucleus. 3.2. Diencephalon 3.2.1. Preoptic area Labeled neurons were found within the preoptic nucleus, including its parvocellular and magnocellular subdivisions, as well as in the suprachiasmatic nucleus (Fig. 3C–E). Within the parvocellular region, the neurons found were small (9.6  0.9 mm) and pyriform, with one or two dendrites oriented laterally. Within the magnocellular part, two different types of cell were observed: a first type had large (24.0  1.4 mm) pyramidal cell bodies and the second type were gigantocellular (52.0  1.2 mm) neurons that were positive only for NADPHd (Figs. 3E and 5A). Neurons in the suprachiasmatic nucleus were small (8.2  0.7 mm) and rounded. Finally, some small (6.1  0.2 and 10  0.3 mm) cells, intermingled with labeled fibers, were found in the optic chiasm (Fig. 3D). 3.2.2. Posterior tubercle and hypothalamus The preglomerular complex showed positive large (16.9  0.9 mm) pyramidal cells, with long dendrites, along its whole rostrocaudal extent (Fig. 3E–H). Within anterior and posterior tuberal nuclei of the hypothalamus, there was a high number of densely packed small (6.9  0.6 mm, anterior) (8.4  0.8 mm, posterior) pyramidal cells, with ventrolaterally oriented dendrites (Figs. 3F–H and 5B). In addition, a few small (6.5  1.2 mm) labeled cells appeared within the periventricular organ. Ventral to the anterior tuberal nucleus, a few small (8.3  0.7 mm) rounded cells with apical processes were located within the lateral hypothalamus (Fig. 3F and G). A high number of small (8.9  0.6 mm) rounded cells with multiple

Fig. 2. Photomicrographs showing NADPHd-labeled (A–C and G–H) and nNOS-labeled (D–F and I) cells. NADPHd-labeled (A) and nNOS-labeled (D) cells within the oculomotor nucleus. NADPHd-labeled (B) and nNOS-labeled (E) cells within the trigeminal motor nucleus. NADPHd-labeled (C) and nNOS-labeled (F) cells within the rhombencephalic reticular formation. (G) NADPHd labeling of a rhombencephalic section showing the location of labeled neurons within the tangential octaval nucleus location (boxed area) showed in (H) and (I) at higher magnification. NADPHd-labeled (G and H) and nNOS-labeled (I) cells within the tangential octaval nucleus. Arrows from (A) to (I) point to some double labeled neurons.

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Fig. 3. (A–R) Schematic drawings of transverse sections through the brain of goldfish (Carassius auratus) to illustrate the localization of nNOS- and NADPHdlabeled cells. At the top is shown the level of each drawing on the brain representation. Each dot represents 10 stained cells. Gray areas indicate a widespread staining. AT: anterior tuberal nucleus; AVIII: anterior octaval nucleus; CB: cerebellar body; CC: nucleus of the commissure of Cajal; CH: central nucleus of the inferior hypothalamic lobe; CLCB: caudal lobe of the cerebellar body; CV: cerebellar valvula; CX: caudal vagal motor nucleus; Dc: dorsal telencephalic area, central part; Dd: dorsal telencephalic area, dorsal part; DF: diffuse nucleus of the inferior hypothalamic lobe; Dl: dorsal telencephalic area, lateral part; Dm: dorsal telencephalic area, medial part; Dp: dorsal telencephalic area, posterior part; DT: dorsal thalamus; DTl: dorsal tegmental nucleus, lateral part; DVIII: descending octaval nucleus; EG: eminentia granularis; ENT: entopeduncular nucleus; FXL: fiber zone of the vagal lobe; G: preglomerular complex; Gl: glomerular layer of the olfactory bulb; Gr: granular layer of the olfactory bulb; H: habenula; Hb: hypobranchial nucleus; IRRF: inferior rhombencephalic reticular formation; I: interpeduncular nucleus; LC: locus coeruleus; LH: lateral hypothalamus; ll: lateral lemniscus; LR: nucleus of the lateral recess; LV: lateral nucleus of the cerebellar valvula; LX: lateral nucleus of the vagal complex; MB: mammillary body; MCX: motor zone of the vagal lobe; mlf: medial longitudinal fasciculus; MRF: mesencephalic reticular formation; MRRF: medial rhombencephalic reticular formation; MVIII: medial octaval nucleus; Nmlf: nucleus of the medial longitudinal fasciculus; OC: optic chiasm; OT: optic tectum; Pm: preoptic nucleus, magnocellular part; Pp: preoptic nucleus, parvocellular part; PPv: periventricular pretectal nucleus, ventral part; PT: posterior tuberal nucleus; PVIII: posterior octaval nucleus; R: raphe nucleus; SC: suprachiasmatic nucleus; SG: secondary gustatory nucleus; SPm: superficial pretectal nucleus; magnocellular part; SPMC: spinal motor column; SRRF: superior rhombencephalic reticular formation; TL: torus longitudinalis; TS: torus semicircularis; TVIII: tangential octaval nucleus; Vd: ventral telencephalic area, dorsal part; VIIL: facial lobe; Vl: ventral telencephalic area, lateral part; Vp: ventral telencephalic area, posterior part; Vs: ventral telencephalic area, supracommissural part; VT: ventral thalamus; Vv: ventral telencephalic area, ventral part; III: oculomotor nucleus; IIn: optic nerve; V: trigeminal motor nucleus; VIc: abducens nucleus, caudal part; VIr: abducens nucleus, rostral part; VII: facial motor nucleus; IX: glossopharyngeal motor nucleus; IX–X: glosso-pharyngeal and vagal motor nucleus; X: vagal motor nucleus; XL: vagal lobe.

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dorsolaterally oriented dendrites were found close to the lateral recess (Fig. 3G–I). Medium-sized (11.6  0.6 mm) labeled neurons were found within the central nucleus of the inferior hypothalamic lobe (Fig. 3I). The mammillary body showed small (6.1  0.7 mm) pyriform cells with several dendrites (Figs. 3I and 5C). Finally, a high number of small (5.6  0.7 mm) rounded cells were found in the diffuse nucleus of the inferior hypothalamic lobe. The cells were densely packed, and more abundant in the middle part of the nucleus (Fig. 3F–J).

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3.2.3. Epithalamus, pretectal nuclei, and thalamus The habenula displayed a widespread NADPHd activity (shaded area of Fig. 3E), and a few small (9.6  0.8 mm) cells positive only for nNOS were dorsally located (Fig. 3E). Among the pretectal nuclei, abundant small (8.7  0.7 mm) rounded cell bodies were found within the magnocellular region of the superficial pretectal nucleus (Figs. 3E and 5D), and a few small (9.6  0.9 mm) pyriform cells were found within the periventricular pretectal nucleus (Fig. 3F and G). As in the habenula, a widespread NADPHd activity was observed in the central

Fig. 3. (Continued ).

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and (in its parvocellular region) superficial pretectal nuclei (Fig. 3E). Labeled cells were found within the thalamus (Fig. 3E–G). Cells of the ventral thalamus were of different sizes depending on their location, being large (16.5  0.3 mm) in the lateral areas and medium-sized (11.3  0.9 mm) in the medial ones. Medium-sized (11.7  0.1 mm) rounded

unipolar neurons, with the processes extending laterally, were also present within the anterior nucleus of the dorsal thalamus (Fig. 3F and G). Finally, within the nucleus of the medial longitudinal fasciculus, there were medium-sized (12.9  0.2 mm) cells with fusiform and pyriform morphologies (Fig. 3H).

Fig. 3. (Continued ).

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3.3. Mesencephalon 3.3.1. Optic tectum Labeled cells were found mostly in the stratum periventriculare (Fig. 3E–J). These cells had small (4.9  0.2 mm)

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pyriform cell bodies with a very long apical dendrite, and were tightly packed (Fig. 6A). A few cells were also found in the following strata: album centrale, griseum centrale, fibrosum et griseum superficiale, opticum, and marginale. Cells in the intermediate layers had large fusiform or polygonal somata

Fig. 3. (Continued ).

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Fig. 3. (Continued ).

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Fig. 4. Photomicrographs showing NADPHd-labeled (A) and nNOS-labeled (B–D) neurons within medial (Dm), central (Dc), and posterior (Dp) dorsal parts of the telencephalon. Arrows, arrowheads, and asterisk point to some representative labeled cells, fibers, and axons with boutons (insert in C), respectively.

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(22.5  0.9 mm in the stratum album centrale and 23.2  0.7 mm in the griseum). Cells in the superficial layers had medium-sized (13.8  0.3 mm) rounded somata. Furthermore, some nNOS-labeled fibers showing en passant boutons were found within the different layers (Fig. 6A). The torus longitudinalis showed a widespread NADPHd-labeling (Fig. 3G–I) and some small (5.9  0.6 mm) rounded cell bodies positive only for nNOS (Figs. 3G–I and 6B). Within the tectal commissure, small, sparse nNOS-labeled cells were also found. 3.3.2. Tegmentum The torus semicircularis showed rounded and pyramidal small (8.9  0.5 mm) cells with several laterally oriented dendrites (Figs. 3G–I and 6C); below it, medium-sized (11.1  0.4 mm) clustered cells were observed in the dorsolateral tegmentum (Figs. 3H and 6C). A few medium-sized (23  1.9 mm) polygonal cells were found within the reticular formation (Fig. 3I and J). A high number of cells were also found in the lateral nucleus of the cerebellar valvula (Fig. 3G–I). They exhibited two morphologies: very small (3.2  0.2 mm) and rounded, or medium-sized (10.0  0.4 mm) and pyramidal with long dendrites. Ventrolaterally to the caudal part of the lateral nucleus of the cerebellar valvula, a population of NADPHdlabeled cells was found. This tightly packed group of cells has been termed the nucleus vermiformis (Bru¨ning et al., 1995). The cells of this nucleus had small (7.2  0.2 mm) rounded cell bodies, and a plexus of stained fibers filled its core. The interpeduncular nucleus was also labeled. This nucleus showed widespread NADPHd activity (Fig. 3J), and nNOS-positive cells having small-sized (7.6  0.3 mm) rounded somata with dendrites oriented laterally and ventrally. Most of the oculomotor nucleus neurons were labeled. They had large (17.8  1.4 mm) pyriform cell bodies (Figs. 2A and D, 3I and 6D). The EdingerWestphal nucleus, located dorsolateral to the oculomotor nucleus, harbored medium-sized (11.3  0.4 mm) neurons. The trochlear nucleus showed medium-sized (14.7  0.2 mm) pyriform cell bodies (Fig. 6E). Some gigantocellular (29.6  0.1 mm) NADPHd-labeled cells appeared within the mesencephalic portion of the trigeminal nucleus. 3.4. Rhombencephalon 3.4.1. The isthmic region A few large (21.5  0.4 mm) pyriform cells with long dendrites were found at the locus coeruleus (Figs. 3J and 7A). The secondary gustatory nucleus showed a widespread NADPHd activity and some sparsely distributed small (7.6  0.5 mm) rounded nNOS-labeled neurons (Fig. 3J). The nucleus isthmi showed densely packed labeled cells with medium-sized (11.9  1.0 mm) rounded somata. Finally, the external part of the lateral lemniscus was surrounded by

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spindle NADPHd- and nNOS-labeled cells (31.7  0.7 mm) (Fig. 3J). 3.4.2. Cerebellum Within the cerebellar valvula, large (12.7  0.7 mm) spindle cells were found in the ganglionic layer. In addition, some scattered cells (8.8  0.7 mm) were observed throughout the molecular layer (Figs. 3G–I and 7B). Within the cerebellar body and vestibulolateral area (eminentia granularis and caudal lobe), large (18.7  0.9 mm) spindle neurons were also found in the ganglionic layer (Figs. 3J–M and 7C and D). The granular layer showed a great number of NADPHd-positive puncta and a few cells (5.3  0.7 mm). Finally, some NADPHd fibers with en passant morphologies and puncta were observed in the molecular layer. 3.4.3. Dorsal and intermediodorsal zones Labeled neurons were found throughout the different octaval nuclei column: anterior, descending, tangential, medial, and posterior, with the exception of the magnocellular (Figs. 2G–I and 3K–N). These cells showed medium-sized somata: (10.0  0.8 mm) anterior, (12.5  0.7 mm) descending (Fig. 7E), (47.2  1.2 mm) tangential (Fig. 2G–I), (22.1  1.6 mm) medial (Fig. 7F), and (12.5  0.7 mm) posterior with pyriform and spindle morphologies. NADPHd-positive small (4.9  0.9 mm) rounded cells were found within the facial lobe (Fig. 3N and O); two groups of gigantocellular cells were found in the facial motor nucleus: in its dorsal aspects (50  0.4 mm) and only NADPHdpositive pyriform (31.7  0.9 mm) cells in the ventral portion (Fig. 7G). The vagal lobe showed labeled axons within the fiber layer, while the large pyriform neurons were restricted to the motor zone of this lobe (Figs. 3O and P and 7H). The glossopharyngeal and vagal motor nucleus showed a high number of large (22.2  0.9 mm) pyriform cell bodies (Figs. 3N–P, 7H and I and 8C). The caudal vagal motor nucleus harbored neurons (30.2  1.6 mm) with characteristics similar to those labeled in the rostral part (Fig. 3Q and R). The lateral nucleus of the vagal complex showed large (18.3  0.6 mm) pyriform cell bodies (Figs. 3P and 8C). 3.4.4. Intermedioventral and ventral zones Labeled neurons were found within the trigeminal motor nucleus (Fig. 3K). Two different morphologies were observed: gigantocellular (45.5  0.4 mm) pyriform and large (23.7  0.2 mm) pyramidal neurons (Figs. 2B and E and 8A). Labeled neurons located within superior, medial, and inferior rhombencephalic reticular formation were observed (Fig. 3K–P). Two groups of gigantocellular cells (54  1.2 and 31.2  1.2 mm) and one of medium (13.6  1.0 mm) somata size with different morphologies (Figs. 2C and F and 8B and C). The cell of Mauthner was only NADPHd-positive (Fig. 8D), with a measure of 135.7  0.2 mm largest and 13.3  0.2 mm

Fig. 5. (A) NADPHd-labeled neurons within the magnocellular part of the preoptic nucleus (Pm). Arrows denote two cell bodies with different sizes. Bottom-right is shown a magnification of the framed area in (A). (B) Densely packed neurons labeled for NADPHd within the anterior tuberal nucleus (AT). Bottom-left is shown a magnification of the framed area in (B). (C) nNOS-labeled neurons within the mammillary body (MB). (D) NADPHd-labeled neurons within the magnocellular part of the superficial pretectal nucleus (SPm). Arrows from (A) to (D) point to representative neurons.

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the shortest diameters, and the labeled axon could be observed along the rostrocaudal extent of the rhombencephalon, until it reached the spinal cord. Finally, there was a column of small (5.6  0.7 mm) cells close to the tectobulbar tract (Fig. 3J–P). The rostral and caudal abducens nucleus showed large (25.0  0.2 mm) multipolar labeled neurons (Figs. 3L and M and 8E and F). The raphe nuclei showed small-sized (6.1  0.2 mm) or medium-sized (12.7  0.2 mm) cells with polygonal or spindle somata morphologies along their entire rostrocaudal extent (Figs. 3K–P and 8B and C). The hypobranchial nucleus had gigantocellular (32.4  1.9 mm) multipolar labeled cells (Figs. 3P and 8C). Finally, a few labeled medium-sized (14.6  1.0 mm) pyriform neurons were found within the nucleus of the commissure of Cajal (Fig. 3Q). Clusters of small cells were observed close to the nuclei of the cranial nerves, from oculomotor (4.2  0.6 mm) to vagal (4.5  0.8 mm) nuclei. 3.5. Spinal cord Labeled cells were found in the spinal cord. They were more numerous in the dorsal horn, and showed medium-sized pyriform somata in the dorsal (11.2  0.7 mm), medial (13.3  1.5 mm), and lateral (14.3  0.8 mm) subdivisions (Fig. 3R). Medium-sized (14.3  0.8 mm) rounded cells were also identified in the intermediate gray matter. In the ventral horn, the neurons were very large (30.4  1.9 mm) and polygonal. Strongly NADPHd-labeled fibers of various thicknesses were observed in all divisions of the white matter. Finally, in the rostral aspects of the spinal cord, medium-sized (11.3  1.1 mm) pyriform neurons were also identified in the lateral reticular nucleus. 4. Discussion We report here the distribution of nitrergic cells in goldfish by the labeling of NADPHd activity and nNOS immunohistochemistry. The present study demonstrates that the nitrergic system is widely distributed throughout the brain and spinal cord of the goldfish. We will discuss these data with respect to those reported in other species, including fish and land vertebrates. 4.1. Methodological considerations By means of NADPHd histochemistry and nNOS immunohistochemistry it was demonstrated that positive neurons colocalized in almost all brain areas in numerous vertebrate species (Hope et al., 1991; Vincent and Kimura, 1992; Alonso et al., 2000). Such classical results support the hypothesis that

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nNOS and NADPHd activity are linked (Iadecola et al., 1993) and that diaphorase histochemistry marks cells producing NO (Dawson et al., 1991; Hope et al., 1991; Schmidt et al., 1992). The high correspondence between NADPHd and nNOS was further assessed by models of transgenic knockout mice for NOS which did not exhibit NADPHd staining (Huang et al., 1993). Furthermore, when a cell line was transfected with nNOS cDNA expressed de novo both the enzyme and NADPHd activity (Dawson et al., 1991). However, studies of colocalization and regional distribution of NADPHd and nNOSpositive cells have reported some discrepancies in nonmammalian vertebrates, including fish (Holmqvist et al., 1994; Arevalo et al., 1995; Bru¨ning et al., 1995; Pepperell and Levine, 1996; Virgili et al., 2001; Cuoghi et al., 2002). Present data demonstrated an extensive co-localization of NADPHd and nNOS in cells of the goldfish brain, but a number of NADPHd-positive cells were not positive against nNOS. In particular, the primary olfactory fibers and glomeruli were intensely labeled for NADPHd and nNOS-immunohistochemistry failed to label them. This difference could be due to the fact that the diaphorase activity in fish could be involved not only in the production of NO, but also in other functional processes (Matsumoto et al., 1993; Spessert et al., 1998; Ando et al., 2004). Otherwise, the various degrees of dissociation NADPHd activity and nNOS immunoreactivity detected in fishes have been attributed to the expression of different subtypes of nNOS in the forebrain (Bordieri et al., 2003). Comparison of the labeling in the present study with that previously reported in goldfish (Bru¨ning et al., 1995) reveals major differences. Thus, we have found abundant labeled cells in the dorsal telencephalon, pretectum, optic tectum, mesencephalic reticular formation, cerebellum, nucleus isthmi, locus coeruleus, and nuclei of the cranial nerves. In fact, these findings were the major discrepancies between the present work and previous experimental procedure, which used an alternative pre-incubation method and a non-commercial antibody (Bru¨ning et al., 1995). 4.2. Distribution of cells in the nitrergic system of goldfish 4.2.1. Olfactory bulb and telencephalon Present data show cells and fibers labeled only for diaphorase activity in the olfactory bulb. A positive staining for this technique in the olfactory bulb is a primitive character observable in cyclostomes (Schober et al., 1994), and maintained across vertebrates from fish (Holmqvist et al., 1994; Arevalo et al., 1995; Lema and Nevitt, 2001; Singru et al., 2003) to mammals (Alonso et al., 1998; Schoenfeld and Knott, 2002). Surprisingly, the available data support minor, if any, labeling with the nNOS technique in the olfactory bulb (Lema

Fig. 6. (A) Photomicrograph showing the labeling against nNOS in the different layers of the optic tectum: stratum marginale (SM); stratum opticum (SO); stratum fibrosum et griseum superficiale (SFGS); stratum griseum centrale (SGC); stratum album centrale (SAC); stratum periventriculare (SP). Upper-right is a magnified view of labeled axon with en passant boutons lying in the SGC. Bottom-left is a magnified view of labeled neurons lying in the SP and SGC. (B) nNOS-labeled neurons within the torus longitudinalis (TL). (C) NADPHd-labeled neurons within the torus semicircularis (TS) and the dorsal tegmental nucleus, lateral part (DTl). (D) nNOS-labeled neurons within the oculomotor nucleus (III). (E) NADPHd-labeled neurons within the trochlear nucleus (IV) surrounding the medial longitudinal fasciculus (mlf). Arrows from (A) to (E) point to some representative labeled neurons.

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Fig. 8. (A) nNOS-labeled neurons within the trigeminal motor nucleus (V). (B) NADPHd-labeled neurons within the medial rhombencephalic reticular formation (MRRF). (C) nNOS-labeled neurons within the lateral nucleus of the vagal complex (LX), the vagal motor nucleus (X), the raphe nucleus (R), the hypobranchial nucleus (Hb), and the spinal motor column (SPMC). (D) NADPHd-labeling of the cell of Mauthner (MA). (E and F) NADPHd-labeled neurons within the rostral (VIr) and caudal (Vic) abducens nucleus. Arrows from (A) to (F) point to some representative labeled neurons.

and Nevitt, 2001; Virgili et al., 2001). Therefore, considering what was stated in the methodological considerations, the question is raised as to whether the technique of labeling with NADPHd provides a reliable demonstration of cells producing NO in the olfactory bulb (Lopez and Gonzalez, 2002). Present data show a high number of labeled cells within all dorsal telencephalic areas. These data are in accord with those recently reported using in situ hybridization in the rainbow trout (Ando et al., 2004). In contrast, earlier works in goldfish

showed a low number of labeled cells in dorsocentral and dorsolateral areas, while the remaining dorsal zones were devoid of stained cells (Bru¨ning et al., 1995; Villani and Guarnieri, 1995). Furthermore, no labeled cells were found in the dorsal telencephalic areas of the tench (Arevalo et al., 1995). Although the homologies between the different dorsal telencephalic zones in the fish and cortical areas in mammals have not been resolved, similarities with the hippocampus and amygdala have recently been suggested by behavioral and

Fig. 7. (A) NADPHd-labeled neurons within the locus coeruleus (LC). (B) nNOS-labeled neurons within the cerebellar valvula (CV). Arrowhead indicates a fiber showing boutons with en passant morphology. (C and D) NADPHd-labeled neurons within the ganglionic layer of the cerebellar body (GLCB). The photomicrographs also show NADPHd staining in the granular layer of the cerebellar body (GRCB). (E and F) NADPHd-labeled neurons within the descending (DVIII) and medial (MVIII) octaval nuclei. (G) NADPHd-labeled neurons within the facial motor nucleus (VII). (H and I) NADPHd-labeled neurons within the glosso-pharyngeal and vagal motor nucleus (IX–X) and the motor zone of the vagal lobe (MCX). Arrows from (A) to (I) point to some representative labeled neurons.

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anatomical methods (Portavella et al., 2004; Wullimann and Mueller, 2004; Northcutt, 2006). Present results add that dorsolateral and dorsomedial telencephalic zones in goldfish are rich in neurons producing NO, which has been largely accepted as being involved in processes of spatial and emotional learning and memory in mammals (Susswein et al., 2004). In addition, the presence of abundant nitric oxide-producing neurons reported here in dorsal telencephalic areas is consistent with data reported in land vertebrates for pallial areas (Vincent and Kimura, 1992; Gonzalez et al., 2002). We have also found nitrergic cells in the ventral telencephalic areas and in the entopeduncular nucleus. These results agree with those previously reported in goldfish (Bru¨ning et al., 1995) and in other species of teleosts (Arevalo et al., 1995; Anken and Rahmann, 1996; Ando et al., 2004; Jadhao and Malz, 2004). Available information suggests that, as in fish, neurons of the subpallial nuclei in land vertebrates are nitrergic (Vincent and Kimura, 1992; Smeets et al., 1997; Gonzalez et al., 2002). 4.2.2. Diencephalon Present data show stained cells in the different nuclei of the preoptic area. These data are in accord with those previously reported in goldfish (Bru¨ning et al., 1995; Villani and Guarnieri, 1995) and other teleosts (Arevalo et al., 1995; Anken and Rahmann, 1996; Ando et al., 2004). The preoptic area in fish is related to the periventricular and tuberal hypothalamus, with which it shares the presence of many peptidergic and dopaminergic hypophysiotropic neurons and steroid and melatonin receptors (Meek and Nieuwenhuys, 1998; Teitsma et al., 1998; Mazurais et al., 2000; Gonzalez-Martinez et al., 2001), as reported for other vertebrates (Gerstberger, 1999). Furthermore, the co-localization of nNOS with argininevasotocin in the preoptic/hypothalamo/hypophysial system of teleosts suggests the involvement of NO in the modulation of hormone release as a primitive feature of vertebrates (Bordieri and Cioni, 2004; Ando et al., 2004). Finally, we report here positive cells in the optic chiasm, as in the tench (Arevalo et al., 1995), which could correspond with cells of glia (Perez et al., 1996). Present findings show that the habenula and the interpeduncular nucleus display a widespread dark NADPHd activity; furthermore, nNOS-labeled neurons are observed within different portions of the habenula and the interpeduncular nucleus. The habenulo-interpeduncular system is conserved across vertebrates, from lamprey to human (Andres et al., 1999). Present results show positive labeling in the pretectal superficial and periventricular nuclei. Previous studies in fish did not identify labeling in these nuclei (Arevalo et al., 1995; Bru¨ning et al., 1995; Villani and Guarnieri, 1995), disagreeing with observations in the pretectal nuclei of other vertebrates (Vincent and Kimura, 1992; Munoz et al., 1996; Smeets et al., 1997). The superficial and periventricular nuclei in teleosts have been reported as primary visual nuclei, related with the optic tectum, that contribute to the optokinetic reflex (Torres et al., 1995). We suggest that NO could be participating in both the habenulointerpeduncular system and the pretectal nuclei, parsing the sensorial signals from diencephalic to mesencephalic structures.

Labeled neurons are also located in both the dorsal and the ventral thalami in goldfish. These data agree with previous results in fish (Arevalo et al., 1995; Bru¨ning et al., 1995; Anken and Rahmann, 1996; Ando et al., 2004) and land vertebrates (Vincent and Kimura, 1992; Bru¨ning and Mayer, 1996; Lopez and Gonzalez, 2002; Soares et al., 2003; Gotti et al., 2005). The actual functional contributions of the thalamic nuclei in teleosts, in comparison with those suggested in other vertebrates, are controversial (Northcutt, 2006). The preglomerular complex is an important ascending thalamic relay center for visual, acoustic, gustatory, and lateral-line sensory signals to the dorsal telencephalic areas in cyprinids, and probably in other teleosts (Folgueira et al., 2005; Yamamoto and Ito, 2005). The high number of neurons producing NO in this complex suggests a major role in the processing of sensory signals as a character that appeared early in vertebrates. 4.2.3. Mesencephalon The labeled cells in the optic tectum are chiefly in the stratum periventriculare, and in a low number within the intermediate and superficial strata. Previous studies demonstrated few labeled cells, if any, in these layers of teleosts (Arevalo et al., 1995; Bru¨ning et al., 1995). In any case, present results show slight differences with that studies of in situ hybridization, which found cells restricted to the intermediate layer (Ando et al., 2004). However and consistent with our results, extensive labeling within intermediate and deep layers of the optic tectum has been found in land vertebrates (Smeets et al., 1997; Gonzalez et al., 2002; Diaz et al., 2006). Since intermediate and deep layers have been involved in the orienting response in goldfish (Herrero et al., 1998), further studies will be required to determine the participation of NO in the generation of this motor task. In addition, we report here cells in the torus longitudinalis, a structure unique to teleosts, involved in the dorsal light reflex (Gibbs and Northmore, 1996). Again, the role of NO in the sensorimotor transformations underlying this response should be studied. Present results show labeled cells within the torus semicircularis – which receives ascending auditory and lateral-line projections, and is accepted as homologous to the inferior colliculus (Meek and Nieuwenhuys, 1998) – and within the nucleus isthmi, which is functionally related with visual stimuli and projects to the optic tectum (Northmore, 1991). These findings are consistent with some previous studies in teleosts (Villani and Guarnieri, 1995), but not with others (Bru¨ning et al., 1995). Studies in land vertebrates also show that these structures are nitrergic (Smeets et al., 1997; Gonzalez et al., 2002; Lopez and Gonzalez, 2002; de Araujo Moreira et al., 2003). The presence of labeled cells in the torus semicircularis and nucleus isthmi could involve a role of NO in the modulation of sensory signals (Holmqvist et al., 1994; Ando et al., 2004). Present data show labeled cells within the dorsolateral tegmental nucleus and mesencephalic reticular formation. These results agree with those reported in the tench (Arevalo et al., 1995) and land vertebrates (Dun et al., 1994; Smeets et al., 1997; Gotti et al., 2005; Kodama and Koyama, 2006), but there has been no information in goldfish (Bru¨ning et al., 1995).

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As previously reported in other teleosts (Arevalo et al., 1995; Anken and Rahmann, 1996; Ando et al., 2004), labeled neurons are also found in the nucleus of the medial longitudinal fasciculus, and Edinger-Westphal, oculomotor, trochlear, and abducens nuclei. These latter data are in disagreement with the previous study in goldfish (Bru¨ning et al., 1995). In land vertebrates, labeling in these latter nuclei remains controversial: some studies report positive labeling (Mizukawa et al., 1989; Bennis et al., 1996; Spina et al., 2004), but not others (Gonzalez et al., 2002). These nuclei are involved in the motor control of eye and body movements (Pastor et al., 1991; Fetcho and Svoboda, 1993). In mammals, it has been proposed that NO modulates the extraocular muscle contraction (Kaminski and Richmonds, 2002); the extension of such proposal to other vertebrates would require further research. 4.2.4. Rhombencephalon Present findings show labeled neurons within the ganglionic layer of the cerebellum. Earlier studies either failed to identify these cells as nitrergic in the cerebellum of goldfish (Bru¨ning et al., 1995), or reported that they were restricted to the eminentia granularis (Arevalo et al., 1995). However, in situ hybridization found Purkinje-type cells in the cerebellar body of the Atlantic salmon (Oyan et al., 2000). Furthermore, labeled cells (in low number) were reported in other teleosts (Holmqvist et al., 1994; Villani and Guarnieri, 1995; Anken and Rahmann, 1996; Ando et al., 2004). Some studies in mammals have reported that both Purkinje and granule cells are nitrergic (Egberongbe et al., 1994; Shigeyoshi et al., 1997), while others indicated that the Purkinje cells were unstained (Vincent and Kimura, 1992; Iwase et al., 1998; Gotti et al., 2005). Finally, we have also found labeled cells within the precerebellar lateral nucleus of the cerebellar valvula. These latter data are in agreement with other studies in teleosts (Holmqvist et al., 1994; Arevalo et al., 1995; Ando et al., 2004). The cerebellum of goldfish, like that of other vertebrates (Rodrigo et al., 2006) showed nNOS immunopositivity, implying that NO could also be involved in motor coordination (Pastor et al., 1994), and fear conditioning (Yoshida et al., 2004); however, the physiology of NO contribution to such functions remains to be elucidated. The rhombencephalic reticular formation shows labeled neurons in goldfish. This is in agreement with previous reports in teleosts (Holmqvist et al., 1994; Arevalo et al., 1995; Bru¨ning et al., 1995; Ando et al., 2004; Bordieri et al., 2005). The labeling of cells within the brainstem reticular formation is a common characteristic across vertebrates (Munoz et al., 1996; Smeets et al., 1997; Atoji et al., 2001; Lopez and Gonzalez, 2002; Guo and Longhurst, 2003). As also found in other studies (Anken et al., 1996; Bell et al., 1997), the Mauthner cell showed an intense diaphorase activity, but failed a positive nNOS stain. Present results show labeled cells within the locus coeruleus, with a similar morphology to that described with dopamine bhydroxylase immunohistochemistry (Hornby and Piekut, 1990). Nitrergic neurons have not previously been reported within the locus coeruleus in teleosts (Arevalo et al., 1995; Bru¨ning et al., 1995), but they have been found in land vertebrates (Smeets et al., 1997; Fabris et al., 2000; Guo and

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Longhurst, 2003; Karolewicz et al., 2004; Kodama and Koyama, 2006). Consistent with previous studies in teleosts (Arevalo et al., 1995) and land vertebrates (Munoz et al., 1996; Smeets et al., 1997; Lopez et al., 2005), positive cells were also found within the different subdivisions of the raphe nucleus. Present data show labeled cells in the trigeminal, facial, hypoglossal, and vagal nuclei. These data disagree with those from the previous works in goldfish (Bru¨ning et al., 1995; Villani and Guarnieri, 1995), but are in accord with studies in other teleosts (Arevalo et al., 1995; Ando et al., 2004). In land vertebrates, some studies also showed nitric oxide-positive cells in these nuclei (Mizukawa et al., 1989; Bennis et al., 1996; Fessenden et al., 1999). In fish, the motor trigeminal, facial, hypoglossal, and vagal nuclei have been functionally involved in the movement for aquatic ventilation, including displacement of mouth and jaw (Hughes and Ballintjn, 1965; Ballintijn and Juch, 1984). Once again, the functional role of the neurons producing NO in the neural circuits that allow breathing in fish would require further physiological studies. In agreement with previous works (Bru¨ning et al., 1995; Arevalo et al., 1995), present data show a low number of nitrergic neurons in the sensorial areas of trigeminal, facial, hypoglossal, and vagal nuclei. These data, however, disagree with those from studies in other teleosts (Funakoshi et al., 1999). We have also found labeled cells in nuclei of the octaval column, with the exception of the magnocellular nucleus. These data are consistent with previous works in teleosts (Arevalo et al., 1995; Ando et al., 2004) and land vertebrates (Munoz et al., 1996; Fessenden et al., 1999; Burette et al., 2001). The sensorial portions of the facial, hypoglossal, and vagal nuclei, as well as the octaval column nuclei, participate in the gustatory, feeding, orientation, and navigation systems (Kanwal and Finger, 1992), but the contribution of NO neurons to these responses should be studied further. 4.2.5. Spinal cord The present findings, like those from previous works in teleosts (Arevalo et al., 1995; Bru¨ning et al., 1995; Virgili et al., 2001), show labeled cells in the spinal cord of goldfish. The presence of a great number of nitrergic cells is a feature shared with other vertebrates (Kitto et al., 1992; Pullen et al., 1997; Munoz et al., 2000; Smeets et al., 1997). 5. Conclusions Neurons producing NO are shown in the telencephalon, in both dorsal and ventral areas. In the diencephalon, the cells were found in some nuclei of the preoptic area and hypothalamus, habenula, pretectum, and dorsal and ventral thalamic regions. In the midbrain, cells were observed in the optic tectum, torus longitudinalis, and tegmental nuclei. In the rhombencephalon, cells were found in the cerebellum, the reticular formation, the locus coeruleus, the raphe nuclei, and the nuclei of the cranial nerves. Labeled cells were also observed in the gray area of the spinal cord. This distribution resembles that reported from fish to mammals, and suggests that the nitrergic system is essentially conserved across

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