THE JOURNAL OF COMPARATIVE NEUROLOGY 367:5449 (1996)

Topographical Distribution of NADPH-Diaphorase Activity in the Central

Nervous System of the Frog, Ranaperezi

M. MUNOZ, A. MUNOZ, 0. MARIN, J.R. ALONSO, K. AREVALO, A. PORTEROS, AND A. GONZALEZ

Department of Cell Biology, Faculty of Biology, University Complutense of Madrid, Spain (M.M., A.M., O.M., A.G.), and Department of Cell Biology and Pathology, Faculty of Biology,

University of Salamanca, Spain (J.R.A., R.A., A.P.)

ABSTRACT The distribution of NADPH-diaphorase (ND) activity was histochemically investigated in

the brain of the frog Ranaperezi. This technique provides a highly selective labeling of neurons and tracts. In the telencephalon, labeled cells are present in the olfactory bulb, pallial regions, septal area, nucleus of the diagonal band, striatum, and amygdala. Positive neurons surround the preoptic and infundibular recesses of the third ventricle. The magnocellular and suprachias- matic hypothalamic nuclei contain stained cells. Numerous neurons are present in the anterior, lateral anterior, central, and lateral posteroventral thalamic nuclei. Positive terminal fields are organized in the same thalamic areas but most conspicuously in the visual recipient plexus of Bellonci, corpus geniculatum of the thalamus, and the superficial ventral thalamic nucleus. Labeled fibers and cell groups are observed in the pretectal area, the mesencephalic optic tectum, and the torus semicircularis. The nuclei of the mesencephalic tegmentum contain abundant labeled cells and a conspicuous cell population is localized medial and caudal to the isthmic nucleus. Numerous cells in the rhombencephalon are distributed in the octaval area, raphe nucleus, reticular nuclei, sensory trigeminal nuclei, nucleus of the solitary tract, and, at the obex levels, the dorsal column nucleus. Positive fibers are abundant in the superior olivary nucleus, the descending trigeminal, and the solitary tracts. In the spinal cord, a large population of intensely labeled neurons is present in all fields of the gray matter throughout its rostrocaudal extent. Several sensory pathways were heavily stained including part of the olfactory, visual, auditory, and somatosensory pathways. The distribution of ND-positive cells did not correspond to any single known neurotransmitter or neuroactive molecule system. In particular, abundant codistribution of ND and catecholamines is found in the anuran brain. Double labeling techniques have revealed restricted colocalization in the same neurons and only in the posterior tubercle and locus coeruleus. If ND is in amphibians a selective marker for neurons containing nitric oxide synthase, as generally proposed, with this method the neurons that may synthesize nitric oxide would be identified. This study provides evidence that nitric oxide may be involved in novel tasks, primarily related to forebrain functions, that are already present in amphibians.

Indexing terms: nitric oxide synthase, amphihians, histuchemistry, catecholamines, evolution

o IXX WiIey-Liss, Inc.

Numerous experiments have demonstrated that the po- tent vasodilator endothelium-derived relaxing factor (EDRF), described by Furchgott and Zawadzki (19801, is identical to nitric oxide (NO, Ignarro et al., 1987; Palmer et al., 1987, 1988). Subsequently, the possible roles of EDRF (Garthwaite et al., 1988) and NO (Bredt and Snyder, 1989; Garthwaite et al., 1989) as intracellular messengers in the nervous system were proposed and several recent reviews outline our knowledge accumulated on this subject (Garth- waite, 1991; Moncada et al., 1991; Snyder and Bredt, 1991;

Bredt and Snyder, 1992; Snyder, 1992; Vincent and Hope, 1992). Although there are diverse tissue specific forms of NO synthase (NOS, see Bredt and Snyder, 19921, the neuronal NOS is a calcium/calmodulin-dependent nicotin- amide adenine dinucleotide phosphate (NADPH)-depen-

Accepted September 1, 1995. Address reprint requests to Dr. Agustin Gonzdez, Department of Cell

Biology, Faculty of Biology, University Complutense, 28040-Madnd, Spain. Email: ilgustin~r'eucmax.sim.ucm.es

G 1996 WILEY-LISS, INC.

NADPH-DIAPHORASE IN THE FROG BRAIS 55

dent enzyme that converts arginine to citrulline, generating NO (Bredt and Snyder, 1990; Garthwaite, 1991; Hope et al., 1991; Schmidt and Murad, 1991). Moreover, it has been reported that NOS is identical to neuronal NADPH- diaphorase (ND, Dawson et al., 1991; Hope et al., 19911, which makes it possible to localize NOS with a specific histochemical method using p-NADPH as substrate and tetrazolium dyes as chromogen (Thomas and Pearse, 1964). The activity of ND produces an insoluble dark-blue forma- zan precipitate (Kuonen et al., 1988) that labels the neu- rons that contain NOS and, most likely, use NO as a neuronal messenger molecule (Dawson et al., 1991). This technique stains distinct populations of neurons selectively both in the peripheral (Grozdanovic et al., 1992) and central nervous systems (see e.g., Vincent et al., 1983a,b, 1986; Vincent and Kimura, 1992).

Recently, some controversial data have been reported as to whether NOS and ND are always the same in the central nervous system (CNS; Schmidt et al., 1992; Valtschanoff et al., 1993). However, a one-to-one correlation between ND- positive neurons and NOS-immunoreactive neurons has been described in various sites in the central (Bredt et al., 1991; Dawson et al., 1991) and peripheral nervous systems moung et al., 1992), and even the relative staining intensi- ties obtained were similar with each technique. Therefore, it appears that ND histochemistry provides a useful marker for neurons producing NO. With this simple technique, Golgi-like images of particular neurons are obtained giving detailed information on the morphology of cell bodies and, frequently, of dendritic arborizations and axons. Thus, the complete distribution of the NO neuronal system has been analyzed in a number of mammalian species by ND histo- chemistry (e.g., Mizukawa et al., 1989; Vincent and Kimura, 1992). Subsequent studies of colocalization demonstrate

that, with the exception of citrulline, a side product in NO synthesis, the distributions of ND partially overlaps that of several other neuroactive substances (Vincent et al., 1983a,b; Reiner and Vincent, 1987; Villalba et al., 1988; Schober et al., 1989; Mizukawa et al., 1989; Caballero-Bleda et al., 1992; Alonso et al., 1992, 1993). In particular, a selective distribution of ND activity within a subpopulation of monoaminergic neurons has been observed in the rat brain (Johnson and Ma, 1993) and a role of NO on the metabo- lism of catecholamines was suggested (Rokoski et al., 1987).

Although NO synthases were originally identified in mammalian tissues, there is recent evidence that NO might be a neuronal messenger of early phylogenetic origin and conserved throughout evolution since NOS activities have been demonstrated in different tissues of arthropods, gastro- pods, and echinoderms (Radomski et al., 1991; Elphick et al., 1993a,b; Martinez et al., 1994). The evolutionary his- tory of the NO system in the CNS of vertebrates has received increasing attention in the last few years. Thus, the early fragmentary data of NO activity in the brain of several nonmammalian species (Regidor and Poch, 1988; Sato, 1990a,b; Luebke et al., 1992; Briining, 1994; Schober et al., 1994b) have become more complete with comprehen- sive studies on the overall distribution of ND activity in the brain of a cyclostome (Schober et al., 1994a1, several teleosts (Schober et al., 1993; Arevalo et al., 19951, the turtle (Briining et al., 1994), and some birds (Briining, 1993; Panzica et al., 1994).

NADPH-diaphorase activity has been confirmed in the retina, pineal organ, brain, and peripheral nervous system of anuran amphibians (Sato, 1990a,b; Li et al., 1992; Pitzer and Wirtshafter, 1993, Crowe et al., 1994). The goal of this study is to map the distribution of ND activity in the brain ofthe frog Ranaperezi. Since the protocol for ND histochem-

A Acc Ad Aob APl APm B ct Cb CP D DB DCN DP d Hd Hv igl IP Is L L a Lc lfb LH LM LP LPd Lpv Ls Mg ml mot MP

anterior thalamic nucleus nucleus accumbens anterodorsal tegmental nucleus accessory olfactory bulb amygdala, pars lateralis arnygdala, pars medialis neuropil of Bellonci central thalamic nucleus cerebellum corpus geniculatum thalamicum dorsal field ofthe spinal cord nucleus of the diagonal band of Broca dorsal column nucleus dorsal pallium glomerular layer nucleus habenulac, pars dorsalis nucleus habenulae, pars ventralis internal granular layer nucleus interpeduncularis nucleus isthmi lateral field of the spinal cord latcral thalamic nucleus, pars anterior locus coeruleus lateral forebrain bundle latcral hypothalamic nucleus lateral motor field of the spinal cord lateral pallium lateral thalamic nucleus, pars posterodorsalis lateral thalamic nucleus, pars posternventralis lateral septum magnocellular preoptic nucleus mitral cell layer medial olfactory tract medial pallium

Abbreviations

Ms NMLF NPC NPM NPv

OlS P PC POa NPM PtG Rai IzaS Ri Rm sc sol Str Tc Tect T1 Tmg T P

Vd VH VL VM Vm vs I11 VIIId VIIIV

OC

V

medial septum nucleus of the medial longitudinal fasciculus nucleus of the posterior commissure nucleus profundus meseucepali nucleus of the periventricular organ optic chiasm superior olivary nucleus posterior thalamic nucleus posterior commissure anterior preoptic area nucleus profundu s mesencephali pretectal y a y nucleus raphe inferior nucleus raphe superior nucleus reticularis inferior nucleus reticularis medius nucleus suprachiasmaticus tractus solitarius striaturn commissural nucleus of the torus semicircularis mesencephalic tectum laminar nucleus of the torus semicircularis magnocellular nucleus of the torus semicircularis nucleus tuberculi posterioris ventricle descending trigeminal tract ventral hypothalamic nucleus ventrolateral field nfthe spinal cord ventromedial field of the spinal cord ventromedial thalamic nucleus superficial ventral thalamic nucleus oculomotor nucleus dorsal octaval nucleus ventral octaval nucleus

56 M. MUNOZ ET AL.

istry used in this study was applied previously to the brain of a teleost (Arevalo et al., 1995) and a bird (Panzica et al., 1994), direct comparison of the results can be made. In addition, in this study we have combined histochemistry with immunohistochemical localization of tyrosine hydroxy- lase (TH) to examine the distribution of ND activity within the catecholaminergic nuclei, and to determine whether ND activity is colocalized with catecholamines in neurons of these nuclei.

MATERIALS 4ND METHODS For the present study, the brains of 13 adult Spanish

green frogs, Rana perezi (formerly Rana ridibunda), were used. The animals were obtained from the laboratory stock of the Department of Cell Biology, University Complutense of Madrid, where they were kept at 22°C with a lightidark cycle of 12:12 hours. The animals were anesthetized in a 0.3% solution of tricaine methanesulfonate (MS-222; San- doz Basel, SW), and were then perfused transcardially with saline followed by a fixative mixture of 4% paraformalde- hyde and 15%' saturated picric acid in 0.1 M phosphate buffer, pH 7.4 (PB). The brains were removed and placed in the same fixative for 5-6 hours. They were subsequently immersed in a solution of 30% sucrose in PB for 5-8 hours at 4°C (until they sank). The brains were then blocked in a solution of 15% gelatin and 30% sucrose in PB and were stored for 5 hours in a 4% formaldehyde solution with 30% sucrose, at room temperature. Sections were cut on a freezing microtome at 40 km in the frontal plane, collected in cold PB and rinsed in the same buffer.

Free-floating sections were incubated in a medium of 1 mM 0-NADPH, 0.8 mM nitro blue tetrazolium, and 0.06% Triton X-100 in 0.1 M phosphate (pH 7.6), at 37°C for 1-2 hours. All chemicals were purchased from Sigma (St. Louis, MO).

In four cases, after the NADPH-diaphorase reaction the sections were incubated in mouse anti-TH antiserum (Inc- star Stillwater, MN) diluted 1:1000, for 48-60 hours. Subsequently, the indirect peroxidase anti-peroxidase tech- nique (Sternberger, 1979) was used with diaminobenzidine (DAB) as chromogen (see Gonzalez et al., 1993).

After incubation, the sections were rinsed in PB and mounted on glass slides (mounting medium: 0.25% gelatin in Tris buffer, pH 7.6) and dried overnight. The sections were then coverslipped with DePeX, either directly or after ethanol dehydration and xylene cleaning. A selected set of stained sections were counterstained with cresyl violet (1% in distilled water). Some sections from each brain were incubated in a medium without P-NADPH. A second group of control sections was heated in PB to 70°C for 10 minutes. In both cases, no reaction was observed.

Evaluation and presentation of the results The demonstration of ND activity was achieved by means

of a direct method using exogenous NADPH (Arevalo et al., 1993). In this variant, the activity of endogenous ND reduced NADPH in the presence of the dye nitro blue tetrazolium formed a blue insoluble reaction product.

The pattern of histochemical reaction in the brain of adult frogs was consistent from animal to animal. Cell bodies, dendritic processes, and thin axon arborizations with varicosities or terminal swellings were clearly revealed in all the divisions of the brain and in the spinal cord. The positive blue reaction was easily identified but the intensity of the staining varied among the labeled elements. How-

ever, the intensity of the labeling was consistent from animal to animal.

In the results, we first describe the distribution of ND-positive cell bodies in the CNS. Three cell types were distinguished, according to the intensity of the staining. These types have been previously demonstrated in fish (Arevalo et al., 1995). avian (Panzica et al., 19941, and mammalian (Mizukawa et al., 1989; Vincent and Kimura, 1992) brains. Thus, type I cells that are heavily stained with a Golgi-like appearance appear homogeneously dark with long, branched dendrites. A dense precipitate occurs in the perikarya of type I1 neurons but the nuclear area appears as a pale circle; only short dendritic processes are present. Type 111 cells are labeled weakly in the perinuclear cytoplas- mic rim and, only occasionally, some details of the proximal dendrites can be observed. In a second section, we describe the distribution of reactive fibers and axon terminal fields in the brain.

The distribution of ND-positive cell bodies and fibers in the brain of Rana perezi was charted in Figure 1 using representative transverse sections at indicated levels. Draw- ings were made by means of a camera lucida. Sections counterstained with cresyl violet facilitated the localization of reactive structures. The nomenclature used in the pre- sent study is essentially the same as that used in previous studies in amphibians (Gonzalez and Smeets, 1991, 1992a).

RESULTS In the brain of Rana perezi, ND activity was always

observed in neuronal cell bodies and their processes but never in glial cells, blood vessels, meninges, or ependymo- cytes, as reported previously for mammals (see e.g., Sestan and Kostovic, 1994).

NADPH-diaphorase positive cell bodies The present work identified several distinct populations

of neurons likely to release nitric oxide in the anuran central nervous system. As in Golgi-stained preparations, the dark reaction product and low background provided outstandingly clear details of neuronal morphologies.

The most rostrally located ND-positive cell bodies in the frog brain are in the olfactory bulbs (Fig. la). Small type I11 cells lay in the internal granular layer, close to the rostral extent of the ventricles. Scattered, lightly stained cells are also found more peripherally in the granular layer. These cells have oval-to-round perikarya with primary processes directed ventrally. In the accessory bulbs, labeled cells were also present close to the ventricle in the granular cell layer.

In the telencephalon proper, ND-positive cells were ob- served both in the pallial and subpallial regions. Rostrally, type I1 cells are scattered in the dorsal and medial pallial areas (Figs. lb,c, 2a). They are present in the periventricu- lar cell layer and have fine processes that extend into the external fibrous zone. Among the Golgi-like stained cells, weakly stained type I11 neurons are also seen (Fig. 2b). At caudal hemispheric levels, type I1 cells are abundant in the lateral pallium, primarily in its ventral portion (Fig. ld,e), whereas only pale, type I11 cells are found in the medial (Fig. 2c) and dorsal pallia. In the ventromedial hemispheric wall, rostrally, a conspicuous group of labeled type I1 (with fewer type 111) cells is observed in the nucleus of the diagonal band and in the lateral septum (Figs. lc, 2d). Some type I11 cells are also present in the nucleus accumbens. More caudally, weakly labeled cells (type 111) are found in

Fig. 1. a-n: Diagrams nf transverse sections through the brain of the frogRanaperezi at the levels indicated in the schematic dorsal view of the frog brain. NADPH-diaphorase (ND)-positive cell bodies are indicated as large, black dots (type I), medium-sized, black dots (type

11), and empty dots (type I l l ) , whereas fibers are drawn as small dots or wavy lines. The labeling is represented on the right side of every section. Scale bar, 1 mm.

58 M. MUNOZ ET AL.

Fig. 2. Photomicrographs of ND-positive cells and fibers in the dorsal pallium (a,b 1, medial pallium (c ) , and nucleus of the diagonal band (d ). Arrows in e point to weak type I11 cells as compared to type I1 and I in the other pictures. Scale bars, 100 pm (a); 50 pm (b,c,d).

the lateral septum, just beneath the zona limitans medialis. However, at caudal telencephalic levels, stained cell bodies (type I1 and 111) are only found in the medial septal nucleus (Fig. Id).

In the ventrolateral wall of the hemispheres, scattered and well-labeled cells (type I and 11) are located both in the dorsal and ventral striatal portions (Figs. lc,d, 3a). They are abundant in periventricular positions with long pro- cesses that extend laterally. Only few cells are found out of the periventricular cell layer. More caudally, in the lateral amygdala, type I multipolar cells are larger and have thick processes branching among the fibers of the lateral fore- brain bundle (Figs. le, 3b). In the caudal lateral amygdala, the morpholom and labeling of the cells differ (type I1 and 111); they are more scattered and smaller, with processes that extend toward the interior of the amygdaloid neuropil.

Numerous perikarya are recognized in the preoptic area. Rostrally, pale type 111 cells are distributed around the preoptic recess (Fig. 3c), and some of them have a short club-like process that extends into the ventricle. More caudally, the cells form a bilateral group clustered dorsolat- erally at the preoptic recess, some distance from the ventricle (Fig. le). Only very few pale cells are found more ventrally in the anterior preoptic area. In the caudal portion of the preoptic area, numerous type 111 cells are observed in the magnocellular nucleus, separated from the ventricle.

They are arranged in rows parallel to the ventricular lining and extend caudally to form a band above the suprachias- matic nucleus. Their processes are directed dorsorostrally to reach the lateral amygdala (Figs. lf, 4a).

In the caudal hypothalamus, large type I and I1 cells appear in the infundibular region. They are located in rows in the periventricular cell layer; some of them lie very close to the ventricle. These are round cells whose long processes extend into the lateral aspect of the ventral hypothalamus, within a dense ND-positive neuropil (Figs. lg, 4b). Also in the hypothalamus, type I11 cells are found in the caudal portion of the suprachiasmatic nucleus and in the posterior tubercle (Figs. lg,h). Occasionally, very lightly stained CSF-contacting cells are observed in the nucleus of the periventricular organ (Fig. lh) .

Dorsally in the diencephalon, numerous groups of ND- positive cells are recognized. Thus, type 111 cells are located in the ventral habenula, nucleus of Bellonci, and corpus geniculatum of the thalamus (Fig. 10. More darkly stained type I1 cells are present in the medial portion of the anterior, and particularly the lateral anterior thalamic nuclei. Darkly labeled type I (and also 11) cell bodies are found more caudally in the central and lateral nuclei (Figs. lg, 4c). More caudally, large type I cells are found in the posteroventral division of the lateral thalamic nucleus (Fig. Ih). A few moderate to weak (type I1 and 111) cells are

NADPH-DIAPHORASE I N THE FROG BRAIN 59

Fig. 3. Photomicrographs of ND-positive cells and fibers in the lateral wall of the hemisphere (a), the area of the lateral amygdala and lateral forebrain bundle (b), and the anterior preoptic area (c) . Note the different staining between type I and I1 cells in a and b and type 111 in c. Scale bars, 200 pm (a); 100 pm (b,c).

present in the ventrolateral and superficial ventral tha- lamic nuclei (Fig. 10.

Conspicuous groups of type I1 and 111 cells are found in the pretectal area in the nucleus of the posterior commis- sure and the so-called pretectal gray (Figs. l i , 4d). In the latter, scattered large multipolar type-I cells are also pre- sent.

In the mesencephalon, a large population of ND-positive cells in the optic tectum is distributed preferentially in layer 6 with only few cells in layers 2, 4, and 8 (after Szekely and Lazar, 1976). Generally, the cell bodies are pear-shaped with a main dendritic trunk directed toward the meningeal surface (Figs. li ,j , 4e). In the torus semicircularis, all five subnuclei (as described by Potter, 1965) possess type I1 and 111 cell bodies whereas type I cells are only found in the ventromedial portion of the principal nucleus and the magnocellular toral nucleus (Fig. lj). But it is in the mesencephalic, isthmic, and rhombencephalic tegmental regions where the most strongly labeled cells are located. Conspicuous Golgi-like scattered cells are found in the anteroventral, anterodorsal, posteroventral, and posterodor- sal tegmental nuclei, as well as in the nucleus profundus mesencephalicus and the reticular isthmic nucleus. Only moderately labeled cells (type 11) are found in the nucleus of the fasciculus longitudinalis medialis (Fig. li , j).

In the isthmic region, a strikingly well-stained cell group is located medial to the prominent isthmic nucleus (Figs. lk, 4f). The latter group is composed of large type I cells in a thin band between the isthmic nucleus and the ventricle and extends ventrally into the reticular formation. In the cerebellum, only scattered light, type I11 cell bodies are located both in the molecular and granular layers.

In the rhombencephalon the majority of labeled cells (type I and 11) are found in the reticular nuclei. However, other sensory nuclei possess labeled cells (Fig. l l m ) . Thus, the trigeminal principal and descendmg nuclei have type I

and I1 somata throughout their rostrocaudal extent. The anterior, ventral, and dorsal octaval nuclei also have stained (types I and 11) cell bodies, while in the caudal nucleus only very weakly labeled cell bodies are observed. Rostrally in the raphe nucleus, type 111 cell bodies are present, while at caudal rhombencephalic levels very darkly labeled cells are located in the inferior raphe (Fig. 5a,b). From midrhomben- cephalic levels to the obex, a conspicuous population of type I1 cells are found around the solitary tract (Fig. 5c). These cells have thick dendrites which extend occasionally into the reticular formation. At the obex, medium-sized, type I and I1 labeled cells are situated in the area of the dorsal column nuclear complex (Fig. 5d).

In the spinal cord, a striking population of ND-positive neurons is located at all spinal segments (Figs. In, 6a-c). These cells are distributed in the dorsal, central, lateral, ventromedial, ventrolateral, and lateral motor fields of the gray matter (nomenclature according to Ebbesson, 1976). Since they are stained Golgi-like (type I), their morphology will be described in some detail. In transverse section, numerous round and fusiform, and a few stellate, somata are seen in the dorsal field, primarily at cervical levels. Their dendrites enter a densely stained neuropil that caps the dorsal horn. Labeled cells are more abundant in the lateral field at thoracic and lumbar segments. They are round and fusiform, multipolar cells with main dendrites that extend into the lateral funiculus among the underlying motoneurons (Fig. 6b). At the border between the gray and white matter in this lateral field, labeled elongated cells (intermediolateral neurons) send their dendrites primarily towards the ventrolaiaal spinal aspect, close to the emer- gence of the ventral roots. Within the lateral motor field, type I1 cells and some multipolar type I neurons are observed (Fig. 6c). The latter cells have dendritic trees that invade the lateral funiculus and, to a lesser extent, the ventral funiculus.

M. MUNOZ ET AL.

Fig. 4. Photomicrographs of ND-positive cells and fibers in the rostra1 diencephalon (a), infundibular hypothalamus (b), ccntral tha- lamic nucleus (c) , pretectal area (d), optic tectum (.e), and isthmic

region (0. Arrows in a and b point to the ND positive tract originating from the lateral wall of the hemisphere. Scale bars, 200 p m (a,b); 100 Frn (d,fl; 50 pm (c,ei.

KADPH-diaphorase positive fibers The present study revealed extensive networks of ND-

positive fibers throughout the brain of Rana perezi. They form distinct tracts in recognizable positions of the brain but widespread fibers occur in almost all brain areas. The rostralmost fibers were observed in the olfactory bulb where intense reaction in the glomeruli covering the ventro- lateral aspect of the bulb, is clearly observed (Fig. la). Only scarce labeling of fibers is present in other layers but an impresively labeled neuropil is present just medial to the

unstained glomeruli of' the accessory olfactory bulb. Fine, varicose fibers, loosely arranged, occupy the medial pallium (Fig. 2c), whereas only few fibers course in the dorsal pallium and in the dorsal portion of the lateral pallium. In contrast, a dense neuropil is found in the ventral portion of the lateral pallium including the area of the lateral olfactory tract (Figs. lb,c, 3a). Caudally in the hemispheres, the lateral area of the medial pallium, just beneath the lateral ventricle, is richly innervated with ND positive axon termi- nals (Fig. lc,d). Both medial and lateral septal subdivisions

NADPH-DIAPHORASE IK THE FROG BRAIN 61

receive dense terminals. In addition, the medial olfactory tract has ND activity (Fig. lc). The innervation of the lateral septa1 nucleus continues caudally into the medial amygdala. The innervation is conspicuous in the striatal cell layer, while the striatal neuropil is only poorly inner- vated by reactive terminals (Figs. Id, 3a). Similarly, the innervation of the lateral amygdala is moderate; only its lateralmost portion above the lateral forebrain bundle is well stained.

Abundant cell processes penetrate the preoptic area and caudally, in the hypothalamus. The most conspicuous feature is found at infundibular levels where a ND-positive fiber tract extends from the lateral wall of the hemispheres to form a concise band parallel to the ventricular surface (Figs. l f g , 4a,b). Most subdivisions of the thalamus contain ND-positive fibers and terminals. The densest distribution is found in the nucleus of Bellonci, corpus geniculatum and superficial ventral thalamic nucleus (Figs. lf, 4a). A very dense innervation of the central, lateral anterior, and ventromedial thalamic nuclei is found but the fibers in the anterior and posterior nuclei are moderately dense (Fig. If-h). Noteworthy is the asymmetrical innervation of the hahenula where only the right side is labeled in the dorsal bahenular division. Both sides of the ventral habenula are sparsely innervated (Fig. lf).

In the pretectal area, the nucleus lentiformis and the area above the nucleus of the posterior commissure are inner- vated by ND-positive fibers. Numerous smooth fibers cross in the tectal commissure but only few fibers cross in the posterior commissure. In the optic tectum, abundant fibers are distributed in layers 3, 5 , and 7 with only scattered fibers in more superficial layers. The torus semicircularis, in turn, is completely filled with varicose fibers and axon terminals. Its ventral and lateral portions are the most densely innervated.

NU-positive fibers are widespread in the reticular forma- tion of the brainstem and form longitudinal tracts that reach the spinal cord. At caudal mesencephalic levels, the interpeduncular nucleus is densely innervated. Within the prominent isthmic nucleus, reactive fibers enter its lateral aspect and form a concise terminal field in the medial band of the nucleus (Figs. lk, 4f). Dorsally, in the cerebellum, scattered fibers are distributed primarily in the granular cell layer where they arborize profusely and have varicose swellings.

In the rhombencephalon among the numerous labeled fibers, the area of the superior olivary nucleus is particu- larly well stained (Fig. 5a). ND-positive fibers course in the descending trigeminal and solitary tracts (Figs. l l m , 5a,c,d). Disperse fibers course caudally in the ventrolateral aspect oi'the rhombencephalic tegmentum (Fig. lm).

At spinal cord levels, a dense ND-positive neuropil is formed in the dorsal field of the gray matter, principally in cervical segments (Figs. In, 6a). More caudally, abundant fibers course in the ventral and lateral funiculi.

Fig. 5. Photomicrographs of ND-positive cells and fibcrs in the rhombencephalon. a corresponds to a level similar to L in Figure 1; b corresponds to the middle portion of a section equivalent to the one represented in level M of Figure 1, while c illustrates the dorsolateral aspect of the same section; d illustrates cells and fibers with ND activity at the obex region in the dorsal column nucleus and the descending trigcminal nucleus. Scale bars, 200 pm (a); 100 pm (b,c,d).

62 M. MUNOZ ET AL.

Fig. 6 Photomicrographs of ND-positive cells in the spinal cord at cervical (a ) and thoracic (b,c) levels. The neurons in b are in the lateral field with their main dendrites directed latcrally. Scale bars, 200 pm (a); 50 pm (b); 100 pm (c).

Double labeling experiments The comparative analysis of the distribution of ND-

positive cells and catecholaminergic cells (as revealed by TH immunoreactivity) shows high codistribution in the nucleus of the solitary tract, locus coeruleus, posterior tubercle, caudal dorsal thalamus, anterior preoptic area, and olfac- tory bulb. However, when sections were stained sequen-

tially for ND activity and TH immunoreactivity only a very small number of blue-brown, doubly labeled neurons was observed.

In the nucleus of the solitary tract no colocalization is observed, although ND-positive and TH-immunoreactive cells are strongly intermingled (Fig. 7a). In the locus coeruleus (a catecholaminergic cell group in the isthmic region, see Gonzalez and Smeets, 1994) a small population of cells in the caudal portion are labeled for TH and ND (Fig. 7b). The hypothalamic posterior tubercle has a large population of TH-positive cells but the ND-positive cells in this area seem to be slightly more medially or laterally placed with respect to the catecholaminergic cells (Fig. 7c) and only a few cells were doubly labeled. In the dorsal thalamus the separation is complete since TH-immunoreac- tive cells are restricted to the posterior nucleus, whereas ND-positive cells in these levels are located more laterally, in particular in the posteroventral division of the lateral thalamic nucleus (Fig. 7d). The anterior preoptic area possesses abundant ND-positive cells in close relation with the dorsal aspect of the ventricular recess. However, the catecholaminergic cells in this area are located more medi- ally, lining the ventricle, and no colocalization is present (Fig. 7e). Finally, in the olfactory bulbs numerous TH- immunoreactive cells are distributed in all bulbar layers while ND-positive cells are found only in the internal granular layer where colocalization was not observed. How- ever, in the accessory olfactory bulb the internal portion is so densely labeled for ND (Fig. 70 that colocalization in cells of these areas could not be discerned.

DISCUSSION General considerations

The histochemical method employed in the present study has proven useful for revealing ND-positive neurons in the CNS of Rana perezi. We identified labeled neurons, fine processes and puncta throughout the CNS. As noted in the Introduction, the presence of ND-positive structures most likely indicates the presence of a NO converting enzyme, NOS (Hope et al., 1991). Thus, the present study in the frog would show that neurons and their processes in all major brain areas generate the putative messenger molecule NO. The intensity of the ND reaction product has been used to classify labeled cells into three types. These types are largely intermingled in most areas of the CNS. These differences have been reported previously in topographical studies of different vertebrate brains (Mizukawa et al., 1989; Vincent and Kimura, 1992; Arevalo et al., 1995; Panzica et al., 1994). Until now there has been no convinc- ing explanation for this particular feature of staining. I t seems that the intensity of the staining reflects the degree of ND activity (Alonso et al., 1994).

The present study shows that the distribution of ND- positive neurons and fibers in the anuran brain is distinct from that of any other previously described for any neuro- transmitter or neuroactive substance. However, partial overlap in certain areas can be easily postulated. In fact, this would not be a novel finding since in other species of vertebrates, where ND histochemistry has been combined with immunohistochemistry for various neurotransmitters or neuroactive substances, partial colocalization has been observed (e.g., Kauffman et al., 1974; Nagai et al., 1983; Vincent et al., 1983a,b; Reiner and Vincent, 1987; Alonso et al., 1992, 1993).

NADPH-DIAF'HORASE IN THE FROG BRAIN 63

Fig. 7. Photomicrographs of doubly stained sections for ND histo- chemistry (blue reaction) and TH immunohistochemistry (brown reac- tion) in a) the caudal rhombencephalon at the nucleus of the solitary t rad ; b) the caudal portion of the locus coeruleus; c) the hypothalamic

nucleus of the posterior tubercle; d) the caudal dorsal thalamus; e) the anterior preoptic area; and 0 the lateral aspect of the olfactory bulb. Arrows in b point to doubly labeled cells. Scale bar, 100 pm (a-0.

Comparison with other vertebrates histochemical technique to reveal ND activity as applied in the present study, direct comparisons can be made.

In the last few years there have been numerous studies dealing with the topographic distribution of ND in the CNS of a wide range of vertebrates, from cyclostomes through mammals. Since many studies have used the same ND

TdencephaZon.. The presence of ND activity in the glomeruli of the main olfactory bulb seems to be a general feature in anamniotes (Ar6valo et al., 1995; Schober et al., 1994a,b); however, in rodents only a subset of glomeruli are

61 M. MUGOZ ET AL.

ND-positive (Alonso et al., 1993; Davis, 1991). ND-positive cells in the olfactory bulb have been reported in periglomeru- lar positions in mammals (Davis, 1991) and in the tubercu- lum olfactorium of a bird (Panzica et al., 1994) but labeled cells seem to be absent in the olfactory bulb of anamniotes. The lack of ND activity in the glomerular layer of the accessory olfactory bulb is shared with birds (Panzica et al., 19941, while maximal ND activity is present in the rat accessory olfactory bulb (Vincent and Kimura, 1992; Por- teros et al., 1994). Therefore, it seems that although the proportion differs between vertebrate classes, the olfactory pathway seems to be influenced by KO.

The ventral aspect of the lateral pallium was densely labeled in the present study. In another anuran species, Rana pipiens (Pitzer and Wirtshafter, 1993), intense label- ing was found in a similar place but which they called the lateral prominence and relationated with the amygdala. All pallial regions of the anuran telencephalon possess a large population of ND-positive cells. This situation resembles that of the reptilian cortex (Regidor and Poch, 1988; Powers and Giusti, 1993). However, the equivalent areas of the teleost brain lack ND-labeled cells (Arevalo et al., 1995). Widely distributed ND-positive cells in several areas of mammalian cerebral cortex have been described (Mizukawa et al., 1989; Hedlich et al., 1990; Mufson et al., 1990). Another feature of the frog brain which is shared with mammals is the intense labeling of cells in the diagonal band of Broca and lateral and medial septa1 areas (Vincent et al., 1986; Mizukawa et al., 1989; Vincent and Kimura, 1992; Kitchener and Diamond, 1993). A more restricted presence of labeled structures is observed in equivalent areas of the ventromedial hemispheric wall in the avian (Panzica et al., 19941, teleostean (Arevalo et al., 1995) and reptilian (Powers and Giusti, 1993; Sarrafizadeh et a]., 1993) brains.

A common feature of amniotes and anurans (present results) is the presence of ND-positive neurons in the striatum and amygdala. ND cells in the human striatum have been described as interneurons distributed predomi- nantly in the matrix compartment (Kowal et al., 1987; Sajin et al., 1992). Here they colocalize with somatostatin and neuropeptide Y (NPY). The same is true for rat (Kowal et al., 1987; Vincent et a]., 1983a). In the frog, however, NPY-immunoreactive cells have not been observed (Lazar et al., 1993) and the distribution of somatostatin in the striatum seems different from the ND distribution (Inagaki et al., 1981).

A strikingly high ND activity has been found throughout the diencephalon of the frog, both in its dorsal and ventral subdivisions. Dorsally, in the habenular region, the generally moderate staining of the ventral and dorsal habenular nuclei contrasts with the dense fibers found only in the right dorsal habenular nucleus. ND activity was previously demonstrated in pinealocytes and nerve cells of the frog but was not found in the pineal organ of the rat (Sato, 1990a). The densely ND-reactive fiber bundle in the right dorsal habenula of the frog could arise from the pineal organ which organizes asymmetrical projec- tions to the habenula (Eldred et al., 1980; Kemali and De Santis, 1983).

The thalamus of the frog possesses a large population of ND-positive neurons whose locations are in the majority of the delineated nuclei (Neary and Northcutt, 1983). In the diencephalon of a teleost (Tench, Arevalo et al., 1995), ND staining was scarce in the thalamus, but in contrast in

Diencephalon.

another species, Atlantic salmon (Holmqvist et al., 1994), distinct NOS-immunoreactive neurons are present in the dorsomedial, dorsolateral, and ventromedial thalamic nu- clei. ND-positive cells are widespread in the avian thalamus (Panzica et al., 1994). In contrast, the mammalian thala- mus seems to be devoid of ND-reactive neurons (Eg- berongbe et al., 1994) with the exception of the lateral geniculate nucleus (Agarwala et al., 1992a,b; Mitrofanis, 1992). The anterior, central, lateral anterior, and lateral posterior (ventral part) thalamic nuclei of the frog have many ND-positive neurons. whereas sparse cells are found in the ventromedial and ventrolateral thalamic nuclei. Hodologcal studies showed that the anterior nucleus re- ceives visual information from the tectum and that struc- ture in turn, projects to the medial pallium (Northcutt and Ronan, 1992). This could therefore be the positive fiber network in this pallial area found in our study. Most cells in the central nucleus have been shown to receive auditory information from the torus semicircularis (Feng and Lin, 1991) and project to the striatum Wilczynski and North- cutt, 1983). The ventromedial and ventrolateral nuclei receive somatosensory information from the spinal cord and the dorsal column nucleus (A. Munoz et al., 1994, 1995). In addition, most thalamic nuclei with abundant ND-positive cells (anterior, central, lateral posterior, and ventromedial) have descending projections to the optic tectum or the torus semicircularis (Neary and Wilczynski, 1980; Wilczynski, 1981; Masino and Grobstein, 1990). The ND-positive fibers in the striatum leave almost empty the central neuropil formed by the thalamic afferents (Wilczyn- ski and Northcutt, 1983) and, therefore, the fibers origi- nated from labeled cells in the thalamus most likely contrib- ute to the innervation of the optic tectum and torus semicircularis observed in this study.

ND-positive fibers in the thalamus of the frog are also abundant. Significantly, strongly labeled fibers coming from the retina or the optic tectum are present in visual centers, i.e., the neuropil of Bellonci, corpus geniculatum and superficial ventral thalamic nucleus (Scalia and Greg- ory, 1970; Levine, 1980; Masino and Grobstein, 1990). Caudally, in the pretectal area, labeled fibers, together with cell groups have been found in the present study. In the rabbit divisions of the pretectal complex are readily delin- eated with ND histochemistry (Caballero-Bleda et al., 1992).

The hypothalamus of the frog contains numerous cells with ND activity. Interestingly, ND-positive cells have been found in the region of the magnocellular nucleus (Neary and Northcutt, 1983) which contains a completely inter- mingled population of vasotocinergic and mesotocinergic cells (Conway and Gainer, 1987; Gonzalez and Smeets, 1992a,b). The hypothalamic magnocellular secretory nuclei of mammals are known to be positive for ND (Sagar and Ferreiro, 1987; Arevalo et al., 1992; Pow, 1992). Further- more, in a recent study in the rat (Miyagawa et al., 19941, most oxytocinergic neurons in the paraventricular and supraoptic nuclei had ND activity but virtually no vaso- pressinergic neurons were ND-reactive. However in other studies, some paraventricular neurons in the rat hypothala- mus are colocalized with vasopressin and NOS (Sanchez et al., 1994; Villar et al., 1994). In the quail. no staining for ND was observed within the paraventricular and lateral groups containing vasotocin-positive cells (Viglietti-Pan- zica, 1986; Panzica et al., 1994) and no labeled fibers were present in the eminentia media. In the frog, the eminentia media did not have ND-positive fibers what would suggest,

NADPH-DIAPHORASE IN THE FROG BRAIN 65

as in birds, that there is not co-existence of ND with neuropeptides.

Also in the present study, the hypothalamus is almost completely devoid of ND-positive cells in the nucleus of t.he periventricular organ. This fact contrasts with the situa- tion in other anamniotes (Schober et al., 1993; Holmqvist et al., 1994) where the perivent,ricular organ shows abundant ND-positive cells, some of which have protrusions into the ventricle, i.e., cells of CSF-contacting type. Although the periventricular organ is well developed in amphibians, reptiles, and birds (Smeets and Gonzalez, 19901, significant ND labeling is present only in fish. A shared character between the frog and the teleost is the presence of ND- reactive cells in the posterior tubercle and around the infundibular recess of the diencephalic ventricle (Holm- qvist et al., 1994; Ar6valo et al., 1995). In the suprachias- matic nucleus, ND-positive neurons and processes are found in its caudal portion, which is a retinocipient area (Tuinhof et al., 1994). Recently, the avian homologue of the suprachiasmatic nucleus have been shown to possess weakly labeled ND cells (Panzica et al., 1994).

The optic tectum of the frog has numer- ous ND-positive cells in layer 6, principally. Some efferent pathways from labeled cells in the tectum are also positive for NO, i.e., tectotegmental, tectodiencephalic, and tectoisth- mal pathways (Masino and Grobstein, 1990). In other vertebrates a small population of cells has been found in the periventricular gray (Powers and Giusti, 1993; Holmqvist et al., 1994).

Other mesencephalic centers of the frog that possess ND-positive cells include the torus semicircularis and the tegmental nuclei. In the rat mesencephalon, the NOS- containing cells are located predominantly in a longitudi- nally organized column in the dorsolateral periaqueductal gray (Onstott et al., 1993). Even though in the frog there is not a clear equivalent to the periaqueductal gray, the disposition of the ND-positive cells, dorsolateral to the ventricle in the anterodorsal and posterodorsal tegmental nuclei (named after Potter, 1965), resembles the situation in the rat.

In the isthmic region of the frog a peculiar feature is the concise group of labeled terminals in the medial region of the isthmic nucleus. This can be related to the tecto-isthmal projection that arises from cells in tectal layer 6 which have the same morphology as ND-positive cells observed in this study (Gruberget al., 1994).

The cerebellum of the frog has only moder- ately stained cells in the molecular and granular layers, whereas fibers were present primarily in the granular layer. Similar observations have been made in other non- mammalian vertebrates (Arevalo et al., 1995; Holmqvist et al., 1994; Panzica et al., 1994). However, controversial results have been reported for mammals. Thus, biochemical evidence in rats suggests that the bulk of NOS activity is in the granule cells (Forstermann et al., 1991) and immunohis- tochemistry demonstrates the presence of NOS in basket i stellate cells (Schmidt et al., 1992). Purkinje cell degenera- tion in mutant mice were observed to have at least 50% less NOS activity than controls (Ikeda et al., 1993). Recently, the finding of NOS immunoreactivity in Purkinje cells of human cerebellum is contrary to observations made with similar approaches in other species studied to date (Eg- berongbe et al., 1994).

Rhombencephalon. As in other anamniotes, the popula- tion of ND-positive cells in the frog rhombencephalon is

Mesencephalon.

Cerebellum.

very large. In contrast in mammals, there is generally weak staining of structures in the rhombencephalon. Recently, it was found that cytosolic levels of NOS are lowest. in the medulla (Forstermann et al., 1991).

The motor nuclei of cranial nerves in the rhombencepha- lon of the frog did not stain for ND. These nuclei are strongly ND-positive in the tench (Arbvalo et al., 1995), whereas they were not labeled in reptiles and birds (Luebke et a]., 1992; Panzica et al., 1994). Only in the cat were a few labeled cells found in the facial and vagal motor nuclei (Mizukawa et al., 1989). However, the general amniotic feature seems to be the lack of ND activity in motor nuclei. Transient ND activity in motor nuclei of foetal human brainstem has been demonstrated (Gonzalez-Hernandez et al., 1994) but no other developmental studies are available to confirm this fact in other species.

In the frog, as in the lizard (Luebke et al., 1992), a remarkable number of ND-positive cells are found in the reticular nuclei of the brainstem. Similarly, abundant reticdospinal cells have been found in tracing studies (ten Donkelaar, 1982; Toth et al., 1985). The recent finding in the lamprey of giant reticulospinal ND-positive cells located in the floor of the third and fourth ventricles (Schober et al., 1994a) would indicate that early in phylogeny, the reticulo- spinal systems are influenced by NO.

Other structures with ND-positive neurons in the brain- stem of the frog include the octaval nuclei, raphe complex, trigeminal sensory nuclei, and the nucleus of the solitary tract. ND activity in cells in equivalent places have been reported for amniotes. Some controversial results note the presence/absence of ND-positive neurons in the spinal trigeminal nucleus (Vsp). In humans there was no NOS immunostaining of either fibers or cells in the Vsp (Eg- berongbe et a]., 1994). However, NOS-immunoreactive structures have been demonstrated in the rat (Vincent and Kimura, 1992; Dohrn et al., 1994) where a population of ND-positive intertrigeminal interneurons in the Vsp were found. In a recent work on the frog, an elaborated system of intertrigeminal neurons in the brainstem which may reflect the presence of ND-positive cells in the trigeminal sensory nuclei, was found (M. Munoz et al., 1994).

In the transition zone between the rhomb- encephalon and the spinal cord, a population of ND-positive cells was found in the dorsal column nucleus of the frog (A. Munoz et al., 1995). The presence of labeling for ND in this location has been previously reported in the turtle (Briin- ing, 1994; Briining et al., 1994) and in humans (Egberongbe et al., 1994) implying a role for NO in the somatosensory system. In the spinal cord from cervical to lumbar levels, a large population of densely labeled cells has been found. The distribution of cells varies along the spinal cord but almost all fields of the gray matter (as defined by Ebbesson, 1976) possess cells with ND activity. Only in mammals, where detailed information exists about the distribution of ND-positive cells in the spinal cord, have significant differ- ences between species been reported (Mizukawa et al., 1989; Leight et al., 1990; Anderson, 1992; Blottner and Baumgarten, 1992; Valtschanoff et al., 1992). However, a recent study with NOS immunohistochemical techniques in rat, cat, mouse and monkey (Dun et al., 1993) has demonstrated that the distribution of NOS-immunoreac- tive neurons in the spinal cord was similar across the four species. Although a comparative study between the frog and mammalian spinal cord at this point would be an ardous task it is worth mentioning that the extremely high activity

Spinal cord.

M. MUNOZ ET AL. 66

of ND in dorsal field of the frog spinal cord, as in laminae I, 11, and X of mammals, underscores a prominent role of this neurons in visceral and sensory functions.

Colocalization of NADPH-diaphorase and catecholamines

Recently, the localization of catecholaminergic cell groups in the brain of anuran amphibians has been established by means of immunohistochemical techniques (for review, see Gonzalez and Smeets, 1994). Numerous ND-positive cells are located within the catecholaminergic nuclei of the frog brain (present study). However, when sections were stained for both ND activity and TH immunoreactivity, only a small subpopulation of catecholamine neurons were doubly la- beled, These few TH-immunoreactive cells that contained ND activity were located in the posterior tubercle and the locus coeruleus.

In mammals, ND activity was suggested for dopaminer- gic cells of the substantia nigra (Mizukawa et al., 1989). However, in a recent study using double labeling tech- niques (Johnson and Ma, 1993), it was found that most dopaminergic neurons in the midbrain lacked ND activity. Only a few TH/ND neurons were observed in the rostrome- dial portion of the ventral tegmental area in cells that reportly project to the anterior limbic areas (Swanson, 1982). In anurans the only catecholaminergic cell group found in the mesencephalic tegmentum is located rostrally in the midline between the oculomotor nuclei (Gonzalez and Smeets, 1991). The present study shows that ND- positive cells are not present in this area. Noteworthy, in a recent study using tract tracing and immunohistochemical techniques (Marin et al., 1995), it has been found that the limbic areas of the anuran brain receive a remarkable dopaminergic innervation from the posterior tubercle.

ND-positive cells have been found in the region of the locus coeruleus of reptiles (Luebke et al., 1992), birds (Panzica et al., 19941, and mammals (Mizukawa et al., 1989). However, the ND-positive cells colocalize with acetyl- choline in the neurons of this area rather than with noradrenergic cells (Vincent et al., 198313; Reiner and Vincent, 1987; Luebke et al., 1992; Johnson and Ma, 1993). In the frog there is a large population of ND-positive cells medial to the isthmic region, in the area where the noradren- ergic cells of the locus coeruleus are present (Gonzalez and Smeets, 1994). The results of the present study show that just a few cells in this area are doubly labeled for TH and ND. Cholinergic cells in the same region could be also present (Ciani et al., 1988) but a double labeling approach should be accomplished to conclude colocalization with ND.

In the rat brain, using double labeling techniques, it has been found that the distribution of catecholaminergic cells with ND activity is restricted to the anterior ventral tegmental area and the periaqueductal gray (Johnson and Ma, 1993). In the anuran brain, by means of similar techniques, we have found very limited distribution of TH/ND cells. Double labeling methods should be applied in those species where the possibility of higher degree of colocalization has been postulated. A role of NO on the metabolism of catecholamines was suggested since it was found that cGMP activates tyrosine hydroxylase, the rate- limiting enzyme in catecholamine synthesis (Rokoski et al., 1987). If this is true, certainly most of the catecholaminer- gic cells in the brain of the frog, as in the rat brain, would lack the possibility of this activation.

Possible significance of NOS in the CNS Nitric oxide is presently regarded as a novel transcellular

messenger, apparently not requiring synapses to exert its effects. The best-documented effect of NO in the brain is the activation of cytoplasmic guanylate cyclase (Ignarro, 1990; Garthwaite, 1991; Schmidt and Murad, 1991). Cyclic guano- sine monophosphate (cGMP) is known to have numerous important effects on intracellular regulatory enzymes (Briine and Lapetina, 1989; Duman et al., 1991; Williams et al., 1991) and directly in neurotransmitter receptors (Fuji- mori and Pan-Hou, 1991).

Several possible roles for NO have been identified, includ- ing control of cerebral blood flow (Macrae et a]., 1991; Iadecola, 1992; Kozniewska et al., 1992), as a transmitter that may modulate synaptic transmission (Schuman and Madison, 1991; Haley ct al., 1992; Shibuki and Okada, 1991; Vincent, 1994) or as a neurotoxic agent (Koh et al., 1986; Koh and Choi, 1988; Dawson eta]., 1991).

The places in the CNS where NO could affect function have been infered by ND histochemistry. In fact in many studies, the distribution of ND may reflect the localization of NOS and, therefore the neurons that produce NO. However, we must be cautious about interpretation of these results in light of recent evidence suggesting that ND might be a form of NOS that does not require argmine as a substrate and is not calcium/calmodulin-dependent (Schmidt et al., 1992). Furthermore, Dun et al. (1993) have suggested that most of the ND activity in intact cells is in the particulate fraction and is not NOS. Therefore, it cannot be ruled out that neuronal localization of ND may not reflect the presence of neurons that produce NO. However, most of the NU histochemical and KOS immuno- histochemical studies carried out point to a codistribution of both substances in the same regions (Bredt et al., 1991; Dawson et al., 1991; Young et al., 1992). In particular, in the anuran peripheral system a complete match between ND and NOS has been reported using histochemical and immunohistochemical methods, respectively (Liet al., 1992). Therefore, although the controversy is still open, the use of ND histochemistry seems to be a powerful procedure to give insights into the organization of NO systems in the CNS.

Practically all the available data on NO function have been obtained from mammalian material. NOS is generally present in all vertebrates, and even in invertebrates, which leads us to assume that corresponding functions would occur in non-mammalian cells. However, more experimen- tal work is required to fully characterize NO actions in non-mammalian tissues.

ACKNOWLEDGMENTS This research was supported by grants of the Spanish

DGICYT (PB91-0424 and PB93-0083) and the Junta de Castilla y Leon (SA 55/09/92). We are grateful to Dr. W.J.A.J. Smeets for his valuable suggestions to improve the manuscript. The critical evaluation and linguistic correc- tions of the manuscript made by Dr. G.E. Meredith are most appreciated.

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