Distribution of NADPH-diaphorase activity in the hypothalamo-hypophysial system of the frog, Rana esculenta

Neuroscience Letters 235 (1997) 61–64

Distribution of NADPH-diaphorase activity in the hypothalamohypophysial system of the frog, Rana esculenta P.D. Prasada Rao a, T. Sato b, M. Ueck c ,* a

Department of Zoology, Nagpur University, Nagpur 440010, India Department of Anatomy, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo 113, Japan c Department of Anatomy and Cell Biology, Justus Liebig University, Aulweg 123, D-35385 Giessen, Germany b

Received 4 September 1997; received in revised form 17 September 1997; accepted 17 September 1997

Abstract Using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase (ND) histochemistry, this study reports a wide distribution of ND activity in the hypothalamus, and for the first time in the median eminence (ME), the neural lobe (NL) and in the pars distalis (PD) of the frog, Rana esculenta. Perikarya are ND-active within the nucleus preopticus (NPO), the nucleus preopticus periventricularis (NPP), located around the preoptic recess (PR), the suprachiasmatic nucleus (SCN) and within five infundibular nuclei. Several ND-positive neurons of the nucleus infundibularis ventralis are cerebrospinal fluid-contacting in nature, while a few occupy a subependymal region. The infundibulum shows a thick sheet-like fiber plexus which receives fibers not only from its ND-active neurons, but also from the anterior and central thalamic nuclei. The ME, NL and most cells of the posterodorsal PD are ND-positive. The pituitary function may be mediated by nitric oxide through modulating the secretion of hormone-releasing factors of the hypothalamus. Possible functional significance of the ND-stained hypothalamic areas is discussed.  1997 Elsevier Science Ireland Ltd.

Keywords: Nicotinamide adenine dinucleotide phosphate-diaphorase; Histochemistry; Hypothalamus; Pituitary gland; Amphibia; Rana esculenta

Nicotinamide adenine dinucleotide phosphate (NADPH)diaphorase (ND) histochemistry is known to be a selective marker for a specific population of neurons both in the central (CNS) [16] and the peripheral [6] nervous system. The available data support the assumption that the brain ND is a nitric oxide synthase (NOS) [7]. However, there exist discrepancies between the results obtained after employing ND histochemistry and immunocytochemical methods, as reported in the olfactory bulb and the adrenocortical cells of different species, indicating that the specificity of ND histochemistry for NOS is limited [4,10]. Several studies demonstrated ND activity in the mammalian CNS [16], but among the non-mammalian vertebrates, the information generated using ND histochemistry and/or NOS-immunocytochemistry is restricted to a cyclostome, a few teleosts, a turtle and a few avian species [9]. To our * Corresponding author. Tel.: +49 641 9947024; fax: +49 641 9947159.

knowledge, there are only two investigations on the distribution of ND activity in the brain of anurans, viz. Xenopus laevis [4] and Rana perezi [9]. Nitric oxide (NO) is considered to be a messenger molecule in the anterior pituitary gland [3,17]; NOS was demonstrated in the rat [5,17] and human [8] anterior pituitary, and ND activity was visualized in the rat posterior pituitary [14]. Nevertheless, there is no information on the occurrence of NOS/ND in the amphibian hypophysis. Recent studies have demonstrated that NO may influence anterior pituitary hormone secretion [3]. In view of the presence of several hypophysiotropic hormones in the amphibian hypothalamus [2], and lack of information on the ND activity in anuran pituitary, we undertook this study on the frog, Rana esculenta. Ten adult frogs, Rana esculenta, maintained under natural photoperiod, were sacrificed in the month of September. After anesthetizing with Ketavet (Parke-Davis; 20 mg/ kg body weight), the frogs were perfused with 25–50 ml of ice-cold saline followed by 100–150 ml of ice-cold fixative

0304-3940/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(97) 00711- 8

62

P.D. Prasada Rao et al. / Neuroscience Letters 235 (1997) 61–64

containing 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). After perfusion, the brains were dissected out, transferred to the same fixative for 2 h at 4°C and then transferred overnight to 0.1 M phosphate buffer containing 15% sucrose at 4°C. Transverse, sagittal and horizontal sections were cut at 20 mm on a cryostat and mounted serially on gelatin-chrome alum-subbed slides and stored overnight at −20°C. The sections were incubated in ND incubation medium (15 mM sodium malate, 1 mM MnCl2, 1 mM NADP, 0.2 mM nitroblue tetrazolium and 0.2% Triton X-100 in 0.1 M Tris–HCl, pH 8.0–8.2) for 90 min at 37°C according to the method of Scherer-Singler et al. [15]. Control sections were incubated

in the medium by omission of NADP. Subsequently, the sections were washed in 0.1 M phosphate buffer (pH 7.4), dehydrated in graded series of alcohol, cleared in xylene and mounted in malinol. Our study visualizes a wider distribution of ND activity in the hypothalamus than reported previously [4,9], and for the first time in the pituitary gland and median eminence (EM) among amphibians. Nevertheless, the differences we report may be attributed to species-specificity, physiological state of the individuals and/or differences in the modification of the method. The ND-active perikarya are subdivided into large, strongly reactive cells showing ‘Golgi-like’ images (Type I), into perikarya with granular appearance, short

Fig. 1. Photomicrographs of sagittal (A–C; E–I) and transverse (D) sections of the brain and pituitary gland of Rana esculenta. ND-positive cells in the nucleus preopticus periventricularis around the preoptic recess (pr) (A), the nucleus preopticus (B), the suprachiasmatic nucleus dorsal to the optic tract (ot) (C). (D) Arc-shaped ND-positive fiber plexus (dark area) and neurons (large arrows) of the rostral hypothalamic nucleus. (E) The infundibulum (inf) shows a thick, large fiber plexus (dark area) which surrounds infundibular cells (arrowheads); neurons of the tuberculum posterior (small arrows). (F) Neurons in the tuberculum posterior (large arrows) and in the dorsal hypothalamic nucleus (arrowheads). (G) Cerebrospinal fluid-contacting (large arrows) and subependymal (arrowheads) neurons in the caudal infundibulum. (H) Neural lobe encompassing blood capillaries. (I) Pars distalis of the pituitary showing ND-active (dark) cells. *Blood capillaries; v, third ventricle. Scale bar, (A–F) 100 mm; (G–I) 50 mm.

P.D. Prasada Rao et al. / Neuroscience Letters 235 (1997) 61–64

processes and clear nuclei (Type II) and into weakly reactive cells with a stained area like a rim around the prominent nucleus (Type III). A few Type I and several Type II neurons are present in the nucleus preopticus periventricularis (NPP) around the preoptic recess (PR) (Figs. 1A and 2); some neurons in the rostral area of the PR are multipolar in nature. In a comparable nucleus of R. perezia few somata possess club-like processes which extend into the PR [9]. However, in R. esculenta, even though some perikarya are located adjacent to the PR, projections into the ventricle are not seen. Nevertheless, the anuran PR organ which is composed of monoaminergic perikarya and occupies the area around the PR, shows club-like projections into the recess and represents a separate nucleus [13]. The second, relatively large, neuronal group that shows ND activity is the nucleus preopticus (NPO) (Figs. 1B and 2). In the entire NPO of R. esculenta Type II and III cells occur dispersed, besides a few Type I cells, whereas in R. perezi, the anterior NPO shows only very few pale cells [9]. Similarly, the NPO of X. laevis has only a few weakly ND-positive cells [4]. The neuronal processes extend into different directions; this result confirms some immunocytochemical observations on this nucleus [2]; the amphibian NPO mainly projects to the ME and neural lobe (NL) and contains several neuropeptides [2]. Experimental studies and a knowledge about the coexistence of different neuropeptides and ND activity in the NPO and NL would be useful to elucidate the functional significance of ND. The suprachiasmatic nucleus (SCN) is located dorsal to the optic tract (OT) (Figs. 1C and 2); the entire nucleus, commencing from the area above the optic chiasma and extending posterodorsally to the region dorsal to the rostral infundibular recess (IR), shows several Type II and a few Type III ND-stained perikarya, besides a few Type I cells. In R. perezi only the caudal portion of the SCN reveals ND-positive cells [9]. In the rat, NO partici-

63

pates in the mechanism which mediates circadian light signalling in the SCN [1] and a similar role may be attributed to NO in the SCN of R. esculenta. Our results reveal that the hypothalamic infundibulum of R. esculenta has a wider distribution of ND-positive neurons than it is seen in X. laevis [4] and R. perezi [9]. ND-activity is found in three infundibular nuclei (Fig. 1D–G), in isolated perikarya of two other neuronal groups (DMH and VMH in Fig. 2) and in a thick fiber plexus (Fig. 1E). In the rostral hypothalamic nucleus (RHN) (RH in Fig. 2), located in the anterior infundibular area caudal to the OT, several large, strongly ND-positive Type I and II cells occur on either side of the third ventricle; they project with thick processes into the fiber plexus (Fig. 1D,E). The infundibular fiber plexus appears arc-shaped in transverse sections, (Fig. 1D), but in sagittal sections it is in the form of a large sheet of strongly ND active fibers occupying the central area of the infundibulum (Figs. 1E and 2). The dorsal hypothalamic nucleus (DH), located ventral to the tuberculum posterior (TP), reveals several small, scattered Type III neurons adjacent to the third ventricle (Figs. 1F and 2). The nucleus infundibularis ventralis (NIV) shows numerous, large, ND-active Type II cerebrospinal fluid (CSF)-contacting neurons adjacent to the third ventricle (Fig. 1G) in the caudal part of the infundibulum (Fig. 2). CSF-contacting neurons are reported in a comparable area in X. laevis [4] but not in R. perezi [9]. Several large Type I and II neurons are located in the subependymal region below the CSF-contacting neurons (Fisg. 1G and 2); these neurons possess thick processes which extend to the infundibular fiber plexus. Additionally, ND-positive fibers of the caudal infundibulum extend to the ME (Figs. 1G and 2). Scattered, weakly NDactive Type III perikarya are visualized in the dorsomedial (DMH) and ventromedial (VMH) hypothalamic nuclei (Fig. 2); their processes extend to the infundibular fiber plexus.

Fig. 2. Schematic drawing of a sagittal view of the hypothalamo-hypophysial system showing the topography of the ND-positive structures in Rana esculenta. BC, Blood capillary; DH, dorsal hypothalamic nucleus; DMH, dorsomedial hypothalamic nucleus; IC, infundibular cells; ME, median eminence; NIV, nucleus infundibularis ventralis; NL, neural lobe; NPO, nucleus preopticus; NPP, nucleus preopticus periventricularis; OT, optic tract; PD, pars distalis; PI, pars intermedia; PR, preoptic recess; RH, rostral hypothalamic nucleus; SCN, suprachiasmatic nucleus; TP, neurons in the tuberculum posterior; V, third ventricle; VMH, ventromedial hypothalamic nucleus. Type I (large closed circles), Type II (small closed circles) and Type III (small open circles) cells; dots and broken lines represent fibers.

64

P.D. Prasada Rao et al. / Neuroscience Letters 235 (1997) 61–64

In R. perezi, lightly stained CSF-contacting cells are reported [9] in the nucleus of the periventricular organ (previously called paraventricular organ, PVO [13]). However, comparable cells were neither reported in X. laevis [4] nor in R. esculenta; generally, the anuran PVO contains monoaminergic CSF-contacting neurons [13]. Thus, our study confirms the presence of one ND-positive nucleus (RH of our study) which has been reported also in the infundibulum of R. perezi [9]; the additional four infundibular nuclei, we report in R. esculenta, are new to this genus. The infundibular fiber plexus receives not only fine-caliber fibers from infundibular neurons, but also a few thick processes from the anterior and central thalamic ND-active nuclei. The contribution of thalamic nuclei to the formation of the fiber plexus is supported by retrograde labelling of these nuclei following application of wheat germ agglutinin-horseradish peroxidase into the infundibulum of the bullfrog, R. catesbeiana [11]. The fibers of this plexus surround groups of unstained infundibular perikarya, and they may have synaptic contacts to regulate neuronal activity. A few small ND-active Type II and III perikarya are stained in the tuberculum posterior (TP) of the hypothalamus (Figs. 1F and 2); the same is reported in X. laevis [4] and R. perezi [9]. In the ME, there are numerous ND-positive granules and punctuated structures (Fig. 2); several neuropeptidergic fiber terminals are found in the amphibian ME [2] and a functional relationship may exist among these structures. Granular reaction product is discerned in the NL and the constituent fibers encompass blood capillaries (Fig. 1H); apparently these fibers originate in the NIV, although contribution from other neuronal entities cannot be ruled out. In the rat, salt-loading for 8 days increases markedly the ND activity in the posterior pituitary [14], and previous studies have demonstrated the role of neurohypophysial hormones in osmoregulatory function of amphibians [12]. Several cells in the posterodorsal area of the pars distalis (PD) show fine ND-positive granules (Fig. 1I). Possibly NO controls certain hormone-producing cells in the frog PD; this suggestion receives support from the report that NO potentially stimulates gonadotropin-releasing hormone secretion from the hypothalamus, which in turn regulates luteinizing hormone [3]. It has been suggested that NO provides a novel mechanism for rapid regulation of hypothalamic neuronal networks regardless of their cytoarchitecture or may even exert direct effects at the level of the pituitary to modulate its hormone secretion [3] such as luteinizing hormone [17], growth hormone and prolactin [3]. Studies aimed for identifying the nature of pituitary cell types that posses ND activity would be useful in anurans. This work was supported by fellowships of the Deutscher Akademischer Austauschdienst (P.D.P.R.) and Alexander

von Humboldt Foundation (T.S.). The authors are thankful to Mrs. A. Hach for technical assistance and Mrs. K. Michael for skilful drawing assistance. [1] Amir, S., Blocking NMDA receptors or nitric oxide production disrupts light transmission to the suprachiasmatic nucleus, Brain Res., 586 (1992) 336–339. [2] Andersen, A.C., Tonon, M., Pelletier, G., Conlon, J.M., Fasolo, A. and Vaudry, H., Neuropeptides in the amphibian brain, Int. Rev. Cytol., 138 (1992) 89–188. [3] Brann, D.W., Bhat, G.K., Lamar, C.A. and Mahesh, V.B., Gaseous transmitters and neuroendocrine regulation, Neuroendocrinology, 65 (1997) 385–395. [4] Bru¨ning, G. and Mayer, B., Localization of nitric oxide synthase in the brain of the frog, Xenopus laevis, Brain Res., 741 (1996) 331–343. [5] Ceccatelli, S., Hulting, A., Zhuang, X., Gustafsson, L., Villar, M. and Ho¨kfelt, T., Nitric oxide synthase in the rat anterior pituitary gland and a role of nitric oxide in regulation of luteinizing hormone secretion, Proc. Natl. Acad. Sci. USA, 90 (1993) 11292– 11296. [6] Grozdanovic, Z., Baumgarten, H.G. and Bru¨ning, G., Histochemistry of NADPH-diaphorase, a marker of neuronal nitric oxide synthase, in the peripheral autonomic nervous system of the mouse, Neuroscience, 48 (1992) 225–235. [7] Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neuronal NADPH-diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811–2814. [8] Lloyd, R.V., Jin, L., Quian, S. and Scheithauer, B.W., Nitric oxide synthase in the human pituitary gland, Am. J. Pathol., 146 (1995) 86–94. [9] Mun˜oz, M., Mun˜oz, A., Marı´n, O., Alonso, J.R., Are´valo, R., Porteros, A. and Gonza´lez, A., Topographical distribution of NADPH-diaphorase activity in the central nervous system of the frog, Rana perezi, J. Comp. Neurol., 367 (1996) 54–69. [10] Nathan, C. and Xie, Q.W., Regulation of biosynthesis of nitric oxide, J. Biol. Chem., 269 (1994) 13725–13728. [11] Neary, T.J., Afferent projections to the hypothalamus in ranid frogs, Brain Behav. Evol., 46 (1995) 1–13. [12] Pang, P.K.T., Osmoregulatory functions of neurohypophysial hormones in fishes and amphibians, Am. Zool., 17 (1977) 739–749. [13] Prasada Rao, P.D. and Hartwig, H.G., Monoaminergic tracts of the diencephalon and innervation of the pars intermedia in Rana temporaria. A fluorescence and microspectrofluorimetric study, Cell Tissue Res., 151 (1974) 1–26. [14] Sagar, S.M. and Ferriero, D., NADPH diaphorase activity in the posterior pituitary: relation to neuronal function, Brain Res., 400 (1987) 348–352. [15] Scherer-Singler, U., Vincent, S.R., Kimura, H. and McGeer, M.C., Demonstration of a unique population of neurons with NADPH diaphorase histochemistry, J. Neurosci. Methods, 9 (1983) 229–234. [16] Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience, 46 (1992) 755– 784. [17] Yamada, K., Xu, Z.-Q., Zhang, X., Gustafsson, L., Hulting, A.L., Vente, J.D., Steinbusch, H.W.M. and Ho¨kfelt, T., Nitric oxide synthase and cGMP in the anterior pituitary gland: effect of a GnRH antagonist and nitric oxide donors, Neuroendocrinology, 65 (1997) 147–156.