nadph-diaphorase in the central nervous system of the ...the journal of comparative neurology...

THE JOURNAL OF COMPARATIVE NEUROLOGY 352:398420 (1995)

NADPH-Diaphorase in the Central Nervous System of the Tench

(Tinca tinca L., 1758)

R. AREVALO, J.R. ALONSO, E. GARC~A-OJEDA, J.G. BRINON, c. CRESPO, AND J. AIJON

Departamento de Biologia Celular y Patologia, Universidad de Salamanca, E-37007 Salamanca, Spain

ABSTRACT The distribution and morphological characterization of nicotinamide adenine dinucleotide

phosphate-diaphorase (ND)-positive cells and fibers in the tench central nervous system was mapped by using a direct histochemical method. This enzyme was observed in specific cell populations throughout all main divisions of the tench brain.

In the telencephalon, we found strongly labeled olfactory fibers, as well as positive cells and fibers in the area ventralis of the telencephalic lobes. Positive staining was observed in the following diencephalic nuclei: nucleus preopticus magnocellularis pars magnocellularis, nucleus recessus lateralis, nucleus recessus posterioris, nucleus posterior tuberis, and nucleus diffusus torus lateralis, as well as small cells with a diffuse distribution surrounding the diencephalic ventricle. In the mesencephalon, heavily stained ND-positive neurons were observed in the nucleus fasciculi longitudinalis medialis, nucleus nervi oculomotorius, and nucleus nervi trochlearis. In the hindbrain the most evident staining was observed as large neurons located in the nuclei of the cranial nerves, scattered positive cells located between the negative fibers of the cranial nerves, and in the nucleus fasciculi solitari. Finally, in the spinal cord, ND-positive cells and fibers were mainly located in the ventral horn.

This distribution of ND labeling in the brain of the tench is significantly different from previous data on ND activity in the brain of terrestrial vertebrates and does not correlate with the presence and distribution patterns of several neurotransmitters and neuroactive substances in the teleost brain. o 199.5 Wiley-Liss, Inc.

Indexing terms: brain mapping, fish, histochemistry

Neuronal reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase is an enzyme capable of transferring electrons between NADPH and various electron acceptors (Scherer-Singler et al., 1983; Hope and Vincent, 1989). I t can be identified in a histochemical reaction by its ability to reduce chromogens such as tetrazolium salts to insoluble dark formazan reaction products (Thomas and Pearse, 1961). Recent biochemical and immu- nochemical assays have shown that nitric oxide synthase and NADPH-diaphorase (ND) activities can be immunopre- cipitated with an antibody recognizing neuronal ND and that the nitric oxide synthase is competitively inhibited by the ND substrate nitroblue tetrazolium. These data support the assumption that the brain ND is a nitric oxide synthase (Hope et al., 1991). Although the distributions of ND labeling and nitric oxide synthase are generally coincident, there are some exceptions. In the adrenal gland, adrenal cortical cells demonstrate ND staining but are nitric oxide synthase-immunonegative (Afework et al.,

1992). Kishimoto et al. (1993) have reported the coinci- dence in the neuronal labeling in the olfactory bulb between nitric oxide synthase protein and mRNA and ND activity, but no evidence for nitric oxide synthase expression was found in the main olfactory epithelium or in the vomeronasal organ, despite their strong ND labeling. In the olfactory system of the Atlantic salmon as in other brain regions, the distributions of ND staining and nitric oxide synthase immunoreactivity are not coincident (Holmqvist et al., 1994). In addition, the correspondence between ND and nitric oxide synthase is not yet established in other groups of vertebrates, including teleosts.

The ND histochemical technique selectively stains distinct populations of neurons in the peripheral (Grozdanovic

Accepted August 10, 1994. Address reprint requests to Dr. J .R. Alonso, Dpto. Biologia Celular y

Patologia, Facultad de Medicina, Avda. Campo Charro sin, 37007 Sala- manca, Spain.

0 1995 WILEY-LISS, INC.

NADPH-DIAPHORASE IN THE TENCH BRAIN 399

al., 1989; rat: Kowall et al., 1985; Vincent and Kimura, 1992; and mouse: Wallace and Fredens, 19891, and pri- mates (squirrel monkey: Mufson et al., 1990; rhesus macaque, owl monkey: Sandell, 1985; and human: Ellison et al., 1987). In birds, reports from our group and others (Sato, 1990a; Montagnese et al., 1991; Panzica et al., 1994) indicate important differences with the distribution of ND activity reported in mammals. In anamniotes, available data are fragmentary (retina and pineal organ of frog: Sato, 1990a,b; diencephalon of the rainbow trout: Schober et al., 1993; and retina and brain of the Atlantic salmon: Ostholm and Holmqvist, 1994; Holmqvist et al., 19941, and there is no comprehensive study on the distribution of ND activity in the different subdivisions of the brain. After adapting this method for use in teleosts (ArBvalo et al., 19931, we describe in the present work the morphological characteristics of ND-positive neurons and fibers in the central nervous system of a cyprinoid, the tench Tinca tinca.

et al., 1992) and central nervous systems (Scherer-Singler et al., 1983; Vincent, 1986; Mizukawa et al., 1989; Sims and Williams, 1990; Vincent and Kimura, 1992, among others). This simple technique provides useful morphological information since it gives a Golgi-like image of particular groups of neurons, delineating cell bodies, dendritic trees, and axonal networks. It has been employed in different regions of the nervous system, such as the olfactory bulb (Croul- Ottmann and Brunjes, 19881, retina (Sato, 1990a1, pineal complex (Ueck et al., 19891, medial septal complex (Kinjo et al., 19891, striatum and nucleus accumbens (Kowall et al., 19851, hippocampal formation (Mufson et al., 19901, cerebral cortex (Mizukawa et al., 19881, substantia innominata (Ellison et al., 19871, nucleus locus coeruleus (Kauffman et al., 19741, hypothalamus (Sagar and Ferriero, 19871, amygdala (Sims and Williams, 1990), caudate nucleus and putamen (Sandell et al., 19861, superior colliculus (Wallace and Fredens, 19891, pedunculopontine nucleus (Skinner et al., 19891, pontomesencephalic region (Nakamura et al., 19881, motor nucleus of the vagus (Gonziilez et al., 19871, reticular system (Vincent et al., 19861, and spinal cord (Valtschanoff et al., 19921, among others.

Most studies on the distribution of this enzyme have been carried out in amniotes, mainly in mammals. There are studies in the cat (Sandell et al., 1986; Mizukawa et al., 19891, rabbit (Sagar, 19861, rodents (guinea pig: Cobcroft et

MATERIALS AND METHODS Twelve adult tenches, Tinca tinca L. (Cyprinidae, Teleos-

tei), weighing 200-280 g and obtained from a local breeder (“Vegas del Guadiana,” Badajoz) were used in the present study. The animals were deeply anesthetized with 0.03% tricaine methanesulfonate (MS-222, Sandoz) and perfused

AP hc CA CAns cc CH cmsp cnd CNP cnv co CP CT CTc dbc Dc Dd D1 Dm DT emgr Fr fll flm frf G H HY LF NCP ND NDLI

NDTL NE Nfl NI NLmes NLV NP NPC NPMm NPPd

NDP

NPPp

Abbreviations

area pretectalis brachium conjunctivum commissura anterior commissura ansulata corpus cerebelli commissura habenularum columna motoria spinalis cornu dorsale complex nuclearis preglomerularis cornu ventrale chiasma opticum commissura posterior commissura transversa commissura tecti decussatio brachium conjunctivum area dorsalis telencephali, pars centralis area dorsalis telencephali, pars dorsalis area dorsalis telencephali, pars lateralis area dorsalis telencephali, pars medialis tractus descendens nervi trigemini eminentia granularis formatio reticularis fasciculus longitudinalis lateralis fasciculus longitudinalis medialis fasciculus retroflexus glomeruli habenula hypothalamus lobus facialis nucleus commissura posterioris NADPH-diaphorase nucleus diffusus lobi inferioris nucleus dorsalis posterioris nucleus diffusus torus lateralis nucleus entopeduncularis nucleus fasciculi longitudinalis nucleus interpeduncularis nucleus lateralis mesencephali nucleus lateralis valvulae nucleus pretectalis nucleus posterioris centralis nucleus preopticus magnocellularis pars magnocellularis nucleus pretectalis periventricularis pars dorsalis nucleus preopticus parvocellularis posterioris

NPPv NPSm NPSp NPT NPTp NRL NRP nufsol NIII NlY N v NVI NVII NVIII NIX Nx nI nIII nIV nV nVII nx PB R rdV ST Tel TL tmca tmcp TO tr Tr. op TS ttbc ttbr V vc Vd Vi VL vl v s vv

nucleus pretectalis periventricularis pars ventralis nucleus pretectalis superficialis pars magnocellularis nucleus pretectalis superficialis pars parvocellularis nucleus posterior tuberis nucleus periventricularis tuberculum posterioris nucleus recessus lateralis nucleus recessus posterioris nucleus fasciculi solitari nucleus nervi oculomotorius nucleus nervi trocblearis nucleus nervi trigemini nucleus nervi abducentis nucleus nervi facialis nucleus octavolateralis eferentis nucleus nervi glossopharyngei nucleus nervi vagi nervus olfactorius nervus oculomotorius nervus trochlearis nervus trigeminus nervus facialis nervus vagalis 0.1 M phosphate buffer, pH 7.4 nucleus raphes radix descendens nervi trigemini tractus gustatorius secundarius telencephalon torus longitudinalis tractus mesencephalocerebellaris anterior tractus mesencephalocerebellaris posterior tectum opticum tractus rotundus tractus opticus torus semicircularis tractus tectobulbaris cruciatus tractus tectobulbaris rectus ventriculus valvula cerebelli area ventralis telencephali, pars dorsalis area ventralis telencephali, pars intermedia lobus vagalis area ventralis telencephali, pars lateralis area ventralis telencephali, pars supracommissuralis area ventralis telencephali, pars ventralis

400 R. AREVALO ET AL.

transcardially with 50 ml of 0.63% saline followed by 200 ml of a fixative containing 4% paraformaldehyde and 15% saturated picric acid in 0.1 M phosphate buffer, pH 7.4 (PB). The brains were then removed from the skull, postfixed in the same fixative for 6 hours, washed in PB at 4°C overnight, and then placed in a 30% (viv) sucrose solution for cryoprotection.

Thirty-micron-thick transverse sections were cut on a cryostat and collected in cold PB and, after several washes in PB, processed for NADPH-diaphorase histochemistry.

Free-floating sections were incubated in a medium made up of 1 mM P-NADPH, 0.8 mM nitro blue tetrazolium, and 0.06% Triton X-100 in 0.1 M phosphate buffer (pH 7.6), at 37°C for 1-2 hours. All chemicals were purchased from Sigma.

After incubation, the sections were rinsed in PB, mounted on gelatin-coated glass slides, and air-dried overnight. The following day they were dehydrated, cleared in xylene, and coverslipped with Entellan.

In order to determine the specificity of the histochemical reaction, the following controls were carried out: 1) incubation without the substrate P-NADPH, 2) incubation without the chromogen nitro blue tetrazolium in order to rule out possible nonspecific formation of reaction product, 3) heat denaturation of the enzyme activity by heating the tissue at 84°C for 5 minutes, 4) overfixation of tissue (2 weeks in 10% formalin), and 5 ) incubation in a medium with nicotinamide adenine dinucleotide phosphate (NADP) instead of P-NADPH. In all cases, no residual reaction was observed.

A selected set of stained sections was counterstained with cresyl violet (1% in distilled water) to confirm the position of labeled cells and fibers.

RESULTS General comments and technical

considerations Following the histochemical procedure, ND activity was

detected in all divisions of the brain of the tench. The reaction end-product of diaphorase histochemistry is a blue precipitate (formazan) scattered through the cytoplasm of positive cells.

The demonstration of ND activity was performed using a direct method with exogenous NADPH (Arevalo et al., 1993). In this variant, the activity of endogenous ND reduces NADPH in the presence of the dye nitro blue tetrazolium to form a blue insoluble reaction product. Other authors (e.g., Sagar, 1986) have used NADP, malic acid, and magnesium chloride to synthesize endogenous NADPH. We consider our method to be at least equally effective and easier to perform.

ND-positive fibers were mainly located in the telencephalon. They were less frequently observed in the myelencephalon and spinal cord. ND-active cells were distributed throughout all main divisions of the brain, and they constituted a very heterogeneous group. Three morphological types of ND-active cells can be differentiated:

Type I: Large and medium-sized cells with one to three or more dendrites completely filled with formazan reaction product, in a “Golgi-like” way. Portions of the axons were frequently stained.

Type 11: Medium-size, moderately stained cells with one to three prominent nonvaricose processes. The negative cell nucleus could be distinguished.

Type 111: Small, weakly stained cells where only, at most, the proximal dendrites were labeled.

Most ND-active cells were clearly identified as neurons. In different brain regions, no other elements (including astrocytes, oligodendrocytes, ependymocytes, and microglia) were stained. However, in fibrous bundles such as the chiasma opticum (CO), ND-stained cells whose morphological aspect resembled that of oligodendrocytes were labeled.

The nomenclature and boundaries for the nuclear masses and fibers in the teleostean brain remain controversial. The following description is based on a nomenclature consistent with both teleost neuroanatomy in general and with previous works on cypriniform fishes (Peter and Gill, 1975; Northcutt and Braford, 1984; Arevalo et al., 1992b). Al- though most elements were clearly identified according to their location and morphological characteristics, in some cases it would be necessary to be completely certain of the identification of ND-positive elements to combine the ND histochemical technique with other markers or techniques (e.g., with retrograde tract-tracing for the nuclei of the cranial nerves).

Telencephalon Two large divisions may be distinguished in the telen-

cephalon of the tench: the olfactory bulbs and the telencephalic hemispheres.

ND-positive structures in the olfactory bulb of the tench were identified as olfactory fibers forming the olfactory nerve and the first stratum in the lamination of the olfactory bulb. The olfactory fibers were very strongly labeled, and it was possible to follow their courses when they entered into the second stratum of the olfactory bulb, the glomerular layer (Fig. 7a). All olfactory fibers seemed to be stained. Other components of the olfactory nerve fiber layer as well as all the intrinsic neurons of the olfactory bulb and the centrifugal fibers, axons coursing from more caudal brain areas, were ND-negative.

The telencephalic lobes in the tench are two solid masses separated from each other by a T-shaped medial ventricle. Two main regions may be distinguished: the area ventralis telencephali and the area dorsalis telencephali. Within both areas several nuclei can be differentiated (Fig. la-c): The dorsal area is divided into medialis (Dm), dorsalis (Dd), centralis (Dc), and lateralis (Dl) nuclei; the ventral area is divided into dorsalis (Vd), ventralis (Vv), supracommissuralis (Vs), intermedia (Vi), and lateralis nuclei (Vl) and the nucleus entopeduncularis (NE).

In Vl and Vv, ND-active type I11 cells were observed (Fig. 7d). In both regions, the stained cells showed similar morphological characteristics. They were small in size (5-7 Fm), and only occasionally was a proximal dendrite moderately stained. Some stained cells extended also into Dc. The regions where these cells were found corresponded with the entering region in the telencephalic hemispheres of the tractus olfactorius medialis and tractus olfactorius lateralis, originating in the olfactory bulb.

Some scarce neurons were found in the area ventralis telencephali surrounding the ventricle. These medium- sized cells showed a stained process oriented laterally.

In Vv, some ND-positive fibers were also observed (Fig. 7c). These labeled fibers crossed from one telencephalic lobe to the other, via the commissura anterior (CA). However, most fibers in this commissura were ND-negative. At the caudalmost levels of telencephalon, there were scarce positive cells in Vi and Dc.

NADPH-DIAPHORASE IN THE TENCH BRAIN

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C

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Fig. 1. a-d Schematic drawings at different coronal section levels of the tench telencephalon showing on the left side the ND-positive staining (solid circles, neuronal bodies; small dotted areas, fibers) and

on the right, the main nuclei and fascicles identified at this level. The section level is shown in the small drawing in the upper portion. Scale bar = 1 mm.

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The chiasma opticum (CO) showed a population of stained cells with different shapes (normally round or ovoid) appearing between unstained fibers (Fig. Id). They were small (6-7 pm) and had one or two stained prolongations, or, more rarely, they were multipolar.

Diencephalon The precise boundaries of the diencephalon are not easily

determined in the tench. We have located the rostra1 boundary at the beginning of the preoptic area, and the caudal boundary between the commissura posterior (CP) and the commissura tecti (CTc), dorsally, and between the nucleus fasciculi longitudinalis (Nfl) and the nucleus nervi oculomotorius (NIII), ventrally.

In the epithalamus, no ND-positive structures could be detected. In the thalamus and hypothalamus we observed ND-stained cells surrounding the diencephalic ventricle (Figs. 2a-q 3a). There were numerous type I11 cells without visible processes. These cells were located in the following diencephalic nuclei: nucleus preopticus parvocellularis posterioris (NPPp), nucleus pretectalis periventricularis pars dorsalis and ventralis (NPPd and NPPv), nucleus posterioris centralis (NPC), nucleus periventricularis tubercullum posterioris (NPTp), and nucleus dorsalis posterioris (NDp).

In addition, some positive cells were occasionally observed ventrolateral to the diencephalic ventricle. These cells were classified as type I1 and were medium-sized, round, or piriform, and had a short process oriented toward the ventral region.

In the thalamus, we also observed ND-positive staining in the nucleus posterior tuberis (NPT) (Figs. 2c, 3a,b). This region showed a scarce number of weakly stained cells of medium or small size (8-10 km), oval or round in shape. Weakly stained dendrites with undulated courses were also observed.

In the nucleus diffusus torus lateralis (NDTL), located in the ventral thalamus, immediately under the tectum opticum (TO) (Figs. 2, 3a,b), ND-active cells were also present. This nucleus showed a moderate density of small stained cells (type 11) with rounded or elongated somata and a scarce number (one or two by cell) of positively stained dendrites (Fig. 7e). The complex nuclearis preglomerularis (CNP) was negative.

In the hypothalamus, ND-positive cells were observed in the nucleus preopticus magnocellularis pars magnocellularis (NPMm) (Fig. Id). These cells were intensely stained, large, and showed round somata. Despite the intense soma1 staining, only some ND-positive proximal dendrites were observed (Fig. 70.

ND-positive cells were observed in the nucleus recessus lateralis (NRL) and nucleus recessus posterioris (NRP). Both nuclei are situated surrounding the diverticles of the diencephalic ventricle, the former in the lateral part and the latter in the caudalmost ventricular diverticle of the hypothalamic lobes (Figs. 2c, 3a,b). In both nuclei we saw small, moderately labeled cells which were round or oval in shape. The positive cells showed one to three dendritic processes, which were relatively thick in relation to the cell size. The background staining in these nuclei was intense.

The portion of the brain between the diencephalon and the mesencephalon is identified as synencephalon (Braford and Northcutt, 1983). This region is composed of the regio commissura posterior, dorsally, and the nucleus fasciculi longitudinalis, ventrally. Several ND-labeled cell types were observed in the commissura posterior (CP) (Figs. 2c, 3a,

R. AREVALO ET AL.

8a): large globular cells with a short, fine cell process; large piriform cells with one branched dendrite (Fig. 8b); fusiform neurons with two prolongations arising from opposite poles, and coursing parallel to the commissural fibers (Fig. 8b); and very small round neurons (type 111) demonstrating occasionally stained dendrites.

In the nucleus fasciculi longitudinalis (Nfl) intensely stained fusiform neurons (type I) with one or two labeled prolongations were observed. Round cells without stained prolongations could be also seen (Fig. 8c). ND-active cells located within unstained fibers were observed lateral to the Nfl.

Mesencephalon The mesencephalon is divided into two regions: tectum

opticum (TO) and tegmentum. In the TO, small ND-positive cells (type 11) were observed

in the stratum album centrale. Furthermore, there were labeled structures in the tractus opticus (Tr. op) (Figs. 2, 3a). The Tr. op is divided into two parts by the nucleus pretectalis superficialis pars magnocellularis (NPSm) and the nucleus pretectalis superficialis pars parvocellularis (NPSp): tractus opticus dorsomedialis and tractus opticus ventromedialis. The ND staining was similar in both parts. Fusiform or round stained cells of small size (6-7 km) with some cell processes were observed. These prolongations were generally short and rarely branched (Fig. 7b). These stained cells showed similar characteristics to those previously described in the CO. The NPSm and NPSp were negative for ND activity. The area pretectalis (AP) and the nucleus pretectalis (NP) which are situated close to the NPSm and NPSp (Fig. 2a,b) were also ND-negative.

Both hemitecta are connected by the commissura tecti (CTc) (Figs. 3b,c, 4a). In this commissura a very low number of small ND-positive cells (type II), round in shape and weakly labeled, were observed. The commissural fibers cross the torus longitudinalis (TL), where no ND-positive structures (cells or fibers) were found.

The torus semicircularis (TS) and the nucleus of the torus semicircularis (nucleus lateralis mesencephali) (NLmes) are located under the mesencephalic ventricle (Figs. 3b,c, 4a). This region contained moderately ND- stained small cells which showed round or fusiform somata with thin, short, ND-labeled prolongations (Fig. 9c). The commissura transversa (CT) is located ventral to the TS (Figs. 2b,c, 3). We observed a similar staining in this commissura and in the TS. The staining for ND in the CT and the NLmes, as well as in the region between this commissura and the Tr. op, was very similar.

The nucleus lateralis valvulae (NLV) is associated with the NLmes and situated medially to it (Fig. 3b). This nucleus is formed by a group of cells extending in a position medial to the TS, and it is included in the cerebellar peduncle, intermingled with fibers of the tractus mesen- cephalo-cerebellaris anterior (tmca). ND-positive cells were found in this region of the cerebellar peduncle. The stained cells were large, spherical, and had fine, short, labeled

Fig. 2. a-c: Schematic drawings at different coronal section levels of the tench diencephalon and mesencephalon showing on the left side the ND-positive staining (solid circles, neuronal bodies) and on the right, the main nuclei and fascicles identified at this level. The section level is shown in the small drawing in the upper portion. Scale bar = 1 mm.

NAI

Figure 2

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processes. A scarce number of small ND-labeled cells was found scattered among the unstained fibers of the tmca.

In the tegmentum, the most intense staining was observed in the nucleus nervi oculomotorius (NIII) and the nucleus nervi trochlearis (NIV). The NIII showed numerous large ND-positive type I neurons situated immediately caudal to the Nfl (Fig. 3c). This group of stained neurons was found in the rostralmost sections, dorsal to the flm, and in the caudalmost sections, between the flm of both sides (Fig. 8d,e). The cells were intensely stained, globular or piriform, with a single thick prolongation, laterally directed, and with several branching points.

We found stained neurons in the NIV. This nucleus is situated in the dorsal mesencephalic tegmentum, immediately caudal to the NIII (Fig. 4a). The ND-active neuronal somata bordered the dorsolateral aspect of the flm. The ND-positive neurons in this nuclei were classified as type I and showed the following morphological characteristics: large size (40-50 pm), rounded or oval shapes, and strong staining. Their labeled dendrites extended ventrolaterally, initially following the outer curvature of the flm (Fig. 9a). The staining in NIII and NIV was very similar.

In the roots of the oculomotorius (nIII) and nervi trochlearis (nIV), scarce small to medium ND positive cells were observed located among the negative fibers (Fig. 8d).

Dorsal to the NIV and lateral to the ventricle, small stained cells could be seen. These cells had spherical shape and no visible cell process. The cells located close to the ventricle were smaller than those more laterally situated. The lateralmost neurons normally had two processes running in opposite directions (Figs. 4a, 9e). At caudalmost levels, similar ND-positive staining was found in the brachium conjunctivum (bc), located laterally to the ventricle (Fig. 4b).

The fasciculus longitudinalis medialis (flm) extends from the rostra1 part of the mesencephalon, caudally to the commissura posterior (CP), through the brainstem and continues caudally under the fourth ventricle (Figs. 3c, 4,5, 6a). Throughout all this fascicle, a scarce number of ND-labeled cells was observed situated between the unstained fibers. In the rostralmost levels of the fascicle, labeled cells were small and round, without stained prolongations or only one labeled dendrite. In the caudalmost levels, there were also large, normally multipolar, stained cells (type I) with oval or fusiform shapes.

ND-positive staining was also seen in the zones adjacent to the flm. Round or piriform medium-sized cells were situated between both flm. These cells showed one or two stained prolongations arising from different poles. Dorsal to the flm there were large positive cells (type I) with different morphologies, as well as smaller (5-10 pm), round or piriform cells.

Ventral to the flm and the NIV, and dorsal to the nucleus interpeduncularis (NI) (negative for ND activity), we observed a region crossed by unstained fibers (Fig. 4a). A large number of positively stained neurons were observed among these nerve fibers: heavily stained, large, elongated type I cells with two short intensely labeled dendrites arising from opposite poles and a ND-stained axon initial portion; medium-sized, heavily stained neurons with globular or stel- late shapes, normally multipolar; and small, round, moderately stained neurons, with short positive processes.

In the fasciculus longitudinalis lateralis (fll) (Figs. 3b,c, 4a,b) a reduced number of small oval or round weakly stained cells without prominent prolongations were ob-

R. AREVALO ET AL.

served. The formatio reticularis (Fr) mesencephali showed scattered large labeled neurons, situated ventrolateral to the flm. The nucleus raphes (R), included in the rhomben- cephalon (Fig. 4b), is situated in the medial zone. The boundary between this nucleus and the Fr could not be determined in the ND-stained sections. We observed in all of this region a small number of large fusiform or triangular weakly labeled neurons with one or two thin positive dendrites. Another population of smaller neurons, spherical and weakly stained, was also seen among the large neurons. A relatively high background staining was observed in this zone.

Within the different tectal connections, in addition to the CTc, we found ND staining in the tractus tectobulbaris rectus (ttbr) and in the tractus tectobulbaris cruciatus (ttbc) or commissura ansulata (CAns) (Figs. 3c, 4). Both tracts showed unstained fibers and scattered labeled cells (Fig. 9b). Morphologically heterogeneous cell types normally heavily stained were observed: small round neurons and with no visible cell processes or sometimes with a single prolongation arising from the cell body (type I cells); medium-sized globular or irregular neurons with three or four normally unbranched dendrites running in variable directions; and medium-sized (10-20 pm) piriform cells with one or two branched prolongations.

In the fasciculus retroflexus (fin the ND staining was similar to that described in the CAns.

Metencephalon The cerebellum in the tench consists of three regions:

valvula cerebelli (VC), corpus cerebelli (CC), and lobus vestibulo-lateralis (Figs. 3,4,5a,b).

We observed a scarce ND staining in the cerebellum. Occasionally, some labeled type I11 cells were seen in the molecular layer of the VC (Fig. 9d). These cells were fusiform, weakly stained, and showed one to two stained dendrites. There were also round cells with one or two dendrites. A population of ND-positive cells could also be seen surrounding the ventricle in the zone ventral to VC. In the eminentia granularis (emgr) (Fig. 5a), a small population of ND-positive neurons was observed. These cells were round, weakly stained, of medium or small size, and had no stained processes (type I11 cells).

In fiber tracts establishing connections between the cerebellum and mesencephalic nuclei, such as the tmca, the tmcp, and the bc, positive cells were seen with similar characteristics to those previously described in the mesencephalon (Figs. 2c, 3, 4a).

Myelencephalon The greatest number of ND-positive cells in the brain of

the tench was observed within the myelencephalon. In the rostralmost levels of the myelencephalon, ND-

active staining was found in the nucleus nervi trigemini (NV), situated ventral to the fourth ventricle (Fig. 4c). There were piriform or elongated type I neurons with a thick labeled process normally unbranched, and ventrolaterally directed (Fig. 10a).

We also saw ND staining in the nervus trigeminus (nV) (Fig. 5a). The nV contained small weakly labeled cells with round shapes and no stained processes or, occasionally, one unbranched process (Fig. lob#.

Close to the nV is the tractus gustatorius secundarius (ST) where the ND staining was very similar to that described in the nV: small and weakly labeled cells. At the

NADPH-DIAPHORASE IN THE TENCH BRAIN

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Fig. 5. a-c: Schematic drawings at different coronal section levels of the tench metencephalon and myelencephalon showing on the left side the ND-positive staining (solid circles, neuronal bodies) and on the

right, the main nuclei and fascicles identified at this level. The section level is shown in the small drawing in the upper portion. Scale bar = 1 mm.


Fig. 6. a-e: Schematic drawings at different coronal section levels of the tench myelencephalon and medulla spinalis showing on the left side the ND-positive staining (solid circles, neuronal bodies) and on the

right, the main nuclei and fascicles identified at this level. The section level is shown in the small drawing in the upper portion. Scale bar = 1 mm.

level of the NV, lateral to the ventricle, a region with two different stained cell types was observed: medium-sized cells with some stained dendritic process and small cells normally without labeled prolongations (Fig. 90. In the

nucleus nervi abducentis (NVI) (Fig. 5a), the morphological characteristics of the ND-active cells were similar to those described in NV. There were large, intensely labeled type I neurons, in most cases with globular shape although other


Fig. 7. a: ND-positive fibers in the olfactory bulb showing their branching and termination in the glomerular layer (GI. b: ND-positive labeling in an intermediate coronal section of the Tr. op. c: ND-active fibers in the CA. d: ND activity in V1. Note positive cells without stained prolongations and the high background staining which is not observed in other telencephalic nuclei. e: ND active neurons in the

NDTL. Small, round, and elongated neurons with long processes (arrows) and relatively high background staining are observed. f: ND- positive neurons in the NPMm. Cellular diversity and some neurons with positive prolongations are observed. Scale bars = 100 km in a,c, e,f, 30 pm in b, 40 Fm in d.


Figure 8


ones were piriform. The cells presented a large dendritic process directed ventrolaterally (Fig. 1Oc).

Below the ventricle, ventromedially, and between both flm, we found a large number of cells with positive enzy- matic activity. There were strongly labeled neurons with different sizes and morphologies (piriform, fusiform, triangular, or globular). When the cells were large (25-30 pm), they showed long and prominent dendrites (type I cells). On the contrary, the smaller neurons (5-10 pm) only occasionally had one stained proximal dendrite (Fig. 10d,e). Further- more, some ND-positive fibers could be seen (Fig. l ld ) . These fibers might correspond to axons of the positive cells located in adjacent sections.

More ventral at the same section level, we observed the R and the Fr (Figs. 3b,c, 5 , 6a). In this region, we found positive cells with similar characteristics to those described in the Fr mesencephali (Fig. l le) .

The nucleus nervi facialis (NVII) and the nucleus octavolateralis eferentis (NVIII) are located caudal to NVI, ventrolateral to the ventricle, and dorsolateral to the flm (Fig. 5b). We found two groups of stained cells in this area: one more medially located, identified as NVIII, and a second one, with a more lateral situation, identified as NVII. The positive neurons in these nuclei were of type I, medium to large size (30-40 pm), piriform, and round or elongated in shape. The cells were intensely stained, and they showed a thick prolongation, occasionally branched, which was oriented toward the lateral region (Fig. l la,b).

Positive cells were also seen in the n e w s facialis (nVII). We found a small number of medium-sized, moderately labeled cells with two thick positive dendrites and another population of smaller cells without stained prolongations (Fig. 110. Both the nucleus nervi glossopharyngei (NIX) and the nucleus nervi vagi (NX) constitute a continuous ND-stained cell group (Figs. 5c, 6a,b). At this level, two groups of strongly labeled neurons were observed: medium- sized piriform or rounded cells, dorsally located, with one or two dendrites, and larger neurons with similar shapes but more ventrally situated (Fig. 12a,c,d). These larger neurons could be seen in the caudalmost sections of the myelencephalon. Although the rostral portion of the complex is probably glossopharyngeal and the caudal portion vagal, the exact boundary between them could not be easily determined.

Close to the NX we found the nucleus fasciculi solitari (nufsol) (Fig. 6c,d) with labeled cells very similar to those observed in the NX (Fig. 12b). In the lobus vagalis (VL), two weakly stained cell types could be seen. There were small, rounded cells without positive prolongations (type I), and other larger rounded cells with a short labeled dendrite. In the nervus vagalis (IS) we found small, rounded or elongated cells with one or two dendrites and intensely labeled medium-sized cells (Fig. l lc) .

Fig. 8. a: Transverse section of the CP. Note positive neurons all along the commissura, but more abundant in the lateral regions. b: ND-stained neuron in the CP (large arrow). Two cells belonging to the fusiform type with more weakly stained prolongations are also observed (small arrows). c: ND-stained neurons in the Nfl. Two populations of positive cells are observed: large fusiform neurons, strongly labeled with one or two stained prolongations (solid arrows), and smaller, more weakly stained cells (open arrows). d Transverse section at the level of the NIII. Note positive labeling in NIII, the flm, and in nIII. e: Higher magnification of the ventral part of the NIII showing large neurons with long processes coursing toward the dorsolateral zone. Scale bars = 200 pm in a, 50 Fm in b,c, 500 p m in d, 100 pm in e.

In the caudalmost sections of the myelencephalon we have observed ND staining in the columna motoria spinalis (cmsp) (Fig. 6c,d). Several labeled cell types were observed in this region: large piriform or fusiform cells with one to three cell processes, and small round neurons with, sometimes, stained proximal dendrites (Fig. 12a,b). In the lateral zone of the caudal sections, presumably identified as the radix descendens nervi trigemini (rdV), there were scattered small stained cells.

Medulla spinalis ND staining was observed in the cornu ventrale (cnv)

(Fig. 6e). The intensely labeled cells were large and piriform with one to three stained dendrites. Occasionally, cells with a thick branched prolongation could be seen. Another population of smaller neurons was situated among the large neurons. These small positive neurons showed the following morphologies: round or elongated, with piriform or fusiform shapes, from which arose one to three branched prolongations (Fig. 12e,D.

In the lateral zone of the dorsal spinal cord, scattered small labeled cells with round somata and scarce stained dendrites were seen. Furthermore, some ND-positive short fibers were observed in the cornu ventrale.

DISCUSSION The present study reports the topographical localization

and morphological characteristics of cells and fibers containing NADPH-diaphorase in the different subdivisions of the brain of a teleost, the tench Tinca tinca. Three different populations of active cells were differentiated according to their size and staining intensity (weakly, moderately, and strongly labeled). These differences have been similarly reported in previous classifications of ND-positive cells in the central nervous system of mammals (Mizukawa et al., 1989). In teleosts, ND-positive cells were very heterogeneous, widely distributed, and included neurons with different morphological, neurochemical, and physiological features. In some brain regions and nuclei (e.g., Vl and Vv of telencephalon), it seemed that only subpopulations of a previously considered homogeneous neuronal group were ND stained. Although the functional significance of ND expression is still controversial, the selective appearance of ND activity in certain morphologically distinct classes of neurons, or subsets of neurons within the same class, may reflect physiological differences (Vincent and Johansson, 1983; Vaney and Young, 1988).

Most ND-stained elements were undoubtedly identified as neurons. Other elements such as astrocytes, microglia, and ependymocytes were not stained in our material. How- ever, we found ND-stained cells in the CO, Tr. op, nV, nVII, and nX, whose aspect resembled those of oligodendrocytes. A similar staining has not been previously reported in any class of vertebrates, ND being described as an exclusively neuronal marker, not detectable in glia (Bredt and Snyder, 1992; Vincent and Kimura, 1992). However, nitric oxide synthase activity with similar properties to that of the brain enzyme has been reported in astrocytes following a lesion (Wallace and Fredens, 1992) and in cell culture (Murphy et al., 1990).

Comparative distribution of ND activity in teleosts and tetrapods

ND histochemistry has been used for the study of particular cell groups mainly in mammals: rats (Kowall et al.,


Figure 9

NADPH-DIAF'HORASE IN THE TENCH BRAIN 413

19851, guinea pigs (Cobcroft et al., 19891, cats (Sandell et al., 19861, monkeys (Mufson et al., 1990), and humans (Ellison et al., 1987). There are also some studies in birds (Columba liuia: Sato, 1990a; Taenopygia guttata: Montag- nese et al., 1991; Coturnixjaponica: Panzica et al., 1994) and in frogs (Sato, 1990a,b). Hitherto, the only studies available on the presence of ND activity in the brain of teleosts are restricted to the diencephalon of the rainbow trout, Oncorhynchus mickiss (Schober et al., 1993) and a comparative study on the distribution of nitric oxide synthase and ND activity in the brain of the Atlantic salmon (Holmqvist et al., 1994). Important differences can be observed between the study of Schober et al. (1993) and the present report both in the method used (they have to incubate the sections for more than 5 hours to obtain sufficient staining) and in the location of the labeling: Schober et al. (1993) only report ND-positive staining in the paraventricular organ and in the wall of the third ventricle, whereas in the diencephalon we also observed ND activity in NPT, NDTL, NPMm, NRL, NRP, CP, and Nfl. The pattern of staining is also different in the Atlantic salmon and the tench. Regions that in the tench brain showed ND (Nfl, CP, NIII, NIV, among others), in the Atlantic salmon were ND-negative. On the contrary, ND staining was located in brain regions of the Atlantic salmon (optic tectum, Purkinje cells of the cerebellum) which are ND- negative in the tench.

One of the most notable results of the present work is that the pattern of staining in amniotes and anamniotes is completely different. In the tench, the most abundant ND staining was found in the caudalmost regions of the brain (mesencephalon and myelencephalon). In mammals, on the contrary, the largest number of stained neuronal elements is observed in the rostra1 regions, mainly in the telencephalon (Vincent, 1986; Mizukawa et al., 1989). Additionally, some brain regions which are relatively conserved during evolution (i.e., spinal cord or myelencephalon) showed very different staining patterns, including variations in the size, shape, dendritic, and axonal branching patterns of the ND-stained cell populations, in each philogenetic group.

We discuss the results of the ND labeling in the tench with previous results in other groups of vertebrates. These comparisons are outlined in the same sequence as the results were described, following the main brain subdivisions.

Telencephalon In the bulbus olfactorius of the tench, all olfactory fibers

and glomeruli were positive for ND. In the rat, ND staining

Fig. 9. a: ND activity at the level of the NIV. Stained elements are located close to the ventricle (V) and in the lateralmost regions (ttbc). b: Histochemically stained neurons in the ttbr. Intermediate fusiform or piriform neurons with labeled dendrites (solid arrows) and a group of smaller neurons without stained prolongations (open arrows) are observed. Some stained dendrites are oriented parallel to the tract fascicles, whereas others course transversely. c: Positive neurons in the TS. Labeled neurons and their prolongations are oriented parallel to the negative fascicles of the TS. d Labeled neurons in the VC. Two populations: large (open arrow) and small (large solid arrows) of stained elements are distinguished. Note the negative profiles of the Purkinje cells (small arrows). e: Medium-sized neurons with stained prolongations (solid arrows) and smaller cells without visible processes (open arrows) are observed at the level of NIV, in the zone lateral to the ventricle. f: Zone between the ventricle and the NV with abundant small labeled neurons. Scale bars = 50 pm in a,e, 30 pm in b, 40 pm in c,d, 100 km in f.

is restricted to a spatially segregated subpopulation of olfactory fibers and glomeruli (Scott et al., 1987; Villalba et al., 1989; Davis, 1991; Alonso et al., 1993). On the other hand, in the rat olfactory bulb, ND-positive short axon cells and periglomerular cells have been seen (Davis, 1991). Short axon cells have been described in the teleost bulbus olfactorius (Ichikawa, 1976; Alonso, 19871, but it is not evident if these cells are assimilable to those of higher vertebrates (Alonso, 1987). In any case, in the tench they were ND-negative. Periglomerular cells are not present in the teleost olfactory bulb (Allison, 1953; Andres, 1970; Alonso, 1987).

ND-labeled fibers were observed in the area ventralis telencephali crossing from one telencephalic hemisphere to the other through the CA, indicating the existence of an interhemispheric ND-positive connection in teleosts. By contrast, Ellison et al. (1987) observed ND staining in neurons but not in fibers in the human CA.

The scarce number of ND-active neurons observed on the tench telencephalon contrasts with the abundant positive neurons observed in mammalian brain regions supposedly homologous to those in fish. For example, the area dorsalis telencephali has been suggested as homologous to the pallium and striatum of the tetrapod vertebrates according to immunocytochemical markers such as acetylcholine and catecholamines (Northcutt and Davis, 1983). These telencephalic areas and related brain regions are characterized by the presence of abundant ND-positive cells (Kowall et al., 1985; Mizukawa et al., 1989) which have been described in the cerebral cortex (Mizukawa et al., 1989; Hedlich et al., 1990) and in the striatum of rat (Villalba et al., 1988), macaque (Sandell et al., 19861, and human (Kowall et al., 1987). In the tench, however, we observed only a scarce number of small stained cells located in Dc. In addition, the morphological characteristics of the telencephalic cells described in mammals are not similar to those observed by us in the tench in supposedly homologous regions.

In the ventral division, we have observed scarce stained cells in Vl, which has been related to the olfactory tubercle (Northcutt and Braford, 1980). The Vv and Vd are considered homologous to the septum, whereas the remainder of the area ventralis telencephali has been related with the amygdala of land vertebrates (Northcutt and Braford, 1980). The septum is another telencephalic region with ND-positive structures in mammals: the lateral septal nucleus (Mizukawa et al., 19891, medial septum (Vincent, 1986), diagonal band of Broca (Vincent, 19861, and islands of Calleja (Mizukawa et al., 1989). In the tench, we observed labeling in the Vv but not in the Vd, indicating further interspecies differences in the ND staining patterns. Simi- lar interspecies differences are obtained with other telencephalic regions of mammals such as the hippocampal formation and entorhinal cortex (Mizukawa et al., 1989; Mufson et al., 1990), and previous studies on birds (Montagnese et al., 1991; Panzica et al., 1994). We can conclude that the quantity and distribution of ND neurons in the telencephalon of the tench, as well as their morphological features, differ profoundly from those reported in mammals and birds.

Diencephalon The comparison in this brain region is difficult due to the

large structural variability among different species of teleosts, the paucity of available data on the distribution of ND in the diencephalon of other vertebrates, and the different

4 14 R. AREVALO ET AL.

Fig. 10. a: Piriform neurons of NV with one stained process coursing ventrolaterally toward the nerve root. b: Stained cells in the rdV. c: Labeled neurons in the NVI. Densely grouped large cells with piriform of fusiform somata are observed. d. ND active neurons in the zone dorsal to the flm. Very large (open arrows) and small cells (arrows) can

be distinguished. e: Positive neurons located between both flm. Stained neurons are located in the boundaries between the fascicles (arrows). fi Stained elements in the nV, illustrating the presence of two distinct populations of positive cells identifiable by their sizes (solid and open arrows). Scale bars = 100 pm in a,c, 50 pm in b,d, 80 wm in e,f.


Fig. 11. a: Transverse section at the level of the NVII and the NVIII. b: Higher magnification of the positive neurons in the NVIII. c: Positive neurons in the root of the nX. Some medium-sized neurons with stained prolongations are located in the lateral boundary (solid arrows). There is another group of smaller cells (open arrows) located between the axonal fascicles. d: Stained elements at the caudalmost

level of the flm. e: Rostral section of the Fr showing large, triangular or fusiform neurons with two stained prolongations. f: nVII. Medium- sized neurons with long prolongations (open arrows) and small neurons without stained prolongations (solid arrows) can be distinguished. Scale bars = 300 bm in a, 50 pm in b,c,f, 100 pm in d,e.

Fig. 12. a: Transverse section showing positive neurons in the NIX, in the NX, and in the cmsp. b: ND labeling at a level just anterior to the spinal cord. We observed stained cells in the nufsol and the cmsp. c: Stained neurons in the ventral part of the NIX. d Magnification of the neurons in the NX. These labeled neurons show a clear polarization: thick prolongations coursing toward the lateral zone, whereas none arise from the zone close to the ventricle (solid arrow). Another smaller

neuron possesses long prolongations coursing toward the lateral zone (open arrow). e: ND activity in cnv of spinal cord. Note two different populations of reactive neurons: large cells showing labeled prolongations (solid arrow) and smaller neurons without long visible processes (open arrow). f: Stained fusiform neurons at rostra1 levels of the spinal cord. Scale bars = 150 Fm in a, 200 Fm in b, 90 I*.m in c, 100 km in d-f.

NADPH-DIAF'HORASE IN THE TENCH BRAIN 417

nomenclatures and interpretations of the boundaries of the teleostean diencephalic nuclei (Billard and Peter, 1982; Braford and Northcutt, 1983; Fernald and Shelton, 1985; Keller et al., 1990).

In the tench diencephalon, ND staining was scarce. We found labeled elements in the thalamus and hypothalamus although in most cases they were not located in well- delimited nuclei.

In the rat hypothalamus, ND-stained cells have been described in, among other nuclei, the paraventricular and supraopticus nuclei (Vincent, 1986; Ar6valo et al., 1992a). These nuclei are considered homologous to the NPMm of teleosts (Johnston and Maler, 19921, where we found ND-active cells. In addition, ND staining was detected in the NRL and NRP in the tench hypothalamus.

Mesencephalon The TO is the largest part of the teleostean mesencepha-

lon. Occasionally, we observed some small ND-positive cells in the stratum album centrale, where the neurons which originate the tectotectal projection are located (Vanegas and Ito, 1983). The superior colliculus, the supposed homo- l o p e of the TO, showed a more abundant ND-positive labeling formed by intrinsic neurons and intrinsic and extrinsic fibers in the mouse, rat, cat, monkey, and human (Wallace and Fredens, 1989). In addition, we found ND- positive neurons in NIII, NIV, nIII, nIV, flm, fll, and fr. In the cat, medium-sized neurons with stained dendrites can be also seen in NIII, as well as in the flm (Mizukawa et al., 1989).

Metencephalon Occasional labeled cells were observed in the molecular

layer of VC. In the mammalian cerebellum, histochemical ND staining has not been described. However, Bredt et al. (1990) have demonstrated positive immunoreactivity in the molecular and granular layers of cerebellum with an anti- nitric oxide synthase serum. The immunoreactivity was located in granule cells processes, as well as in basket cells.

Myelencephalon ND staining was mainly located in the nuclei of the

cranial nerves, where we have seen large, strongly labeled neurons with stained dendrites. In mammals, there are labeled structures in NVII and NX of cat, as well as in the nucleus praepositus hypoglossi, vestibularis, cochlearis, trigeminospinalis, infratrigeminus, reticularis paramedi- anus, oliva inferior, and trapezoid body (Mizukawa et al., 1989). However, in the rat no labeled neurons or fibers have been described in these nuclei. On the contrary, the nucleus praepositus hypoglossi, raphe obscurus, reticularis parame- dianus, nucleus tractus solitari, and nucleus reticularis paragigantocelularis are positive for ND in the rat (Vincent, 1986). We have found ND labeling in some of these nuclei such as NX, NVII, and nufsol and, additionally, in some other nuclei which were ND-negative in land vertebrates (e.g., NVI and NIX).

Medulla spinalis ND staining was observed in the cnv of the tench medulla

spinalis and in the region lateral to the cnd. In the cat, Mizukawa et al. (1989) described labeled neurons in cnv and stained fibers in the region lateral to the cnd. In the rat, by contrast, Valtschanoff et al. (1992) found stained neu-

rons mainly located in the cnd. They also demonstrated scattered ND-positive cells in the cnv, intermediolateral column, and in the white matter.

Colocalization between ND and neurotransmitters and neuroactive substances

In previous studies, ND histochemistry has been combined with immunohistochemical staining for various peptides and neurotransmitters. In some regions such as the cerebral cortex, striatum, and bulbus olfactorius, ND enzyme appears in neurons that contain somatostatin and neuropeptide Y (Vincent et al., 1985). However, these three substances do not always occur together in the same group of neurons, and there are other brain regions (e.g., the hypothalamus) where somatostatin and ND show a low degree of coexistence (Alonso et al., 1992). Substance P (Vincent et al., 1983a), gastrin-releasing peptide, and corti- cotropin-releasing factor (Vincent et al., 1986) are colocal- ized with ND in the tegmental cholinergic cell group. However, in other brain regions the positive neurons for these peptides do not stain for ND.

In the tegmental fields of the mesencephalon and pons of the rat, all cholinergic neurons display intense ND activity, and all neurons showing intense ND activity in this area are also positive for choline acetyltransferase (Vincent et al., 1983b). However, many cholinergic cells in the striatum and motor nuclei did not seem to contain ND.

The existence of ND activity in some neurons synthesiz- ing y-aminobutyric acid, cholecystokinin, enkephalin, vasoactive intestinal peptide, or noradrenaline has been suggested (Kauffman et al., 1974; Moss and Basbaum, 1983; Nagai et al., 1983). Little is known about the distribution of these neuropeptides and neuroactive substances in the tench central nervous system. ND-positive structures were located in regions which showed positive immunoreactivity for some neurotransmitters and neuroactive substances in other species of teleosts. Thus, in Salmo gairdneri, the NPMm, NRL, NRP, NIII, TS, nufsol, cmsp, cnd, and cnv were substance P-positive (Vecino et al., 1989). Some of these structures (e.g., NRL, TS, cmsp, and nufsol) were also immunoreactive for enkephalin (Vecino, 1989). Other regions that in the tench brain showed ND were positive for enkephalin but not for substance P in the trout brain: olfactory fibers of the olfactory bulb, ventral area of the telencephalon, fibers in the CA, NPT, NLV, R, and Fr (Vecino, 1989).

The NPMm, which in the tench showed ND activity, is positively labeled after enkephalin immunostaining in Car- assius (Reaves and Hayward, 1979). In the same species, colocalization of neuropeptide Y and somatostatin has been found in some ND-positive regions: the ventrolateral area of the telencephalon, nucleus entopeduncularis (NE), dorso- centralis area of the telencephalon, NX, and TS (Pickavance et al., 1992), but other nuclei positive for neuropeptide Y and somatostatin such as the nucleus entopeduncularis did not contain ND. In some diencephalic nuclei of Apteronotus leptorhynchus where we found ND in the tench, such as NRL and NRP, serotonin and substance P immunoreactivity have been detected (Johnston et al., 1990; Weld and Maler, 1992). In addition, Sas and Maler (1991) have observed somatostatin in other nuclei of Apteronotus leptorhynchus which were ND-positive in the tench: the ventral area of the telencephalon, CA, TS, R, and Fr. In another cyprinoid, Phoxinus phoxinus, in some regions demonstrating ND activity in the tench such as the NLV and the

418

cranial nerve motor nuclei there were choline acetyltransferase-positive neurons (Ekstrom, 1987).

All these neuroactive substances are also located in other regions that in the tench are ND-negative. We can conclude that the coexistence of ND with other neuroactive substances, as is the case in mammals, is not a general feature, but a regional-specific characteristic with only partial over- lap.

R. AREVALO ET AL.

NADPH-diaphorase activity Hope et al. (1991) have demonstrated that ND is a nitric

oxide synthase. Nitric oxide synthase is an enzyme that uses arginine as its substrate to form citrulline and nitric oxide and requires NADPH as an electron donor. Most studies on nitric oxide have been carried out in mammalian cells; however, Radomski et al. (1991) found that a nitric oxide synthetic pathway from L-arginine, although using a calcium-independent enzyme, exists in an arthropod, the horseshoe crab (Limulus polyphemus). The finding of the nitric oxide pathway in this arthropod, an animal which has remained without substantial changes for the last 500 million years, indicates that a biosynthetic pathway for nitric oxide is highly conserved during evolution. However, as shown in this study, the distribution of ND, or of nitric oxide synthase if this parallelism can also be established in anamniotes, differs profoundly among different groups of vertebrates. It suggests that although nitric oxide synthesis is a mechanism biochemically conserved, it is expressed by different and unrelated cell populations. A recent study in the hypothalamic supraoptic nucleus (Pow, 1992) indicates that ND staining is activity related. Thus, the interspecies variability might correspond to differences in the relative activity of the diverse brain areas in each class of vertebrates.

This high interspecies variability indicates that ND- histochemical staining alone is not a sufficient tool to establish or support homological comparisons between teleosts and land vertebrates. However, the “Golgi-like” quality of the staining provides valuable information about the morphological characteristics of specific populations of neurons throughout the brain.

ACKNOWLEDGMENTS The authors are greatly indebted to Dr. Ann B. Butler

(University of Maryland) for helpful comments and to L. Stefanacci and W. Suzuki for revising the manuscript. This work was supported by DGICyT (PB91-0424) and Junta de Castilla y Leon projects (SA 55/09/92).

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