Gonadotropin-releasing hormone-immunoreactive neurons and associated nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons in the brain of a teleost, Rhodeus amarus.

Using combined nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) histochemistry and salmon gonadotropin-releasing hormone (sGnRH) immunocytochemistry, it is reported for the first time that possible potential contacts occur between the nitric oxide (NO)- and the GnRH-containing neurons in the brain of a freshwater teleost, Rhodeus amarus. GnRH-immunoreactive (ir) neurons were observed in the olfactory nerve (OLN), olfactory bulb (OB), medial olfactory tract (MOT), ventral telencephalon (VT), nucleus preopticus periventricularis (NPP), nucleus lateralis tuberis (NLT), and midbrain tegmentum (MT). Although NADPHd neurons were widely distributed in the brain, only those having an association with GnRH-ir neurons are described. Based on the nature of the association between the GnRH and the NADPHd neurons, the former were classified into three types. The Type I GnRH neurons were characterized by the presence of NADPHd-positive granules in the perikarya and processes and occurred in the OLN, OB, MOT, and VT. The Type II GnRH neurons, having soma-soma or soma-process contacts with the NADPHd neurons, were restricted to the MT; the long processes of NADPHd cells crossed over either the perikarya or the thick processes of GnRH cells. However, the Type III GnRH neurons, found in the NPP and NLT, did not show direct contact, but a few NADPHd fibers were present in the vicinity. The terminal-soma contacts in the olfactory system and the VT and the soma-soma contacts in the MT represent the sites of possible potential contacts indicating a direct NO involvement in GnRH function, although NO action by diffusion remains possible. NO may influence the NPP and NLT GnRH cells by diffusion only, since a direct contact was not observed.

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

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.

Immunocytochemical localization of taurine in the pineal organ and retina of an anadromous fish, Plecoglossus altivelis.

Light and electron microscopic immunolocalization of taurine, a sulfur-containing free amino acid, was investigated in the photoneuroendocrine pineal organ and the retinal photoreceptor and pigment epithelial layer of the ayu Plecoglossus altivelis, an anadromous fish. Intense immunostaining was found in the outer segments of pineal photoreceptors and retinal cone-like cells. Moderate but definite immunostaining was found in the cytoplasmic processes of pineal supporting cells, the outer segments of retinal rod-like cells, and the apical processes of pigment epithelial cells. Although the electron microscopic immunogold labeling was not completely coincident with the results of light microscopic immunostaining, concentrated immunogold particles appeared in the inner segments of photoreceptor cells, the cytoplasmic processes of pineal supporting cells, and the apical processes of pigment epithelial cells. These light and electron microscopic findings in taurine immunolocalization were discussed in relation to the functions of taurine known mainly from retinal physiology. It was suggested that the abundant taurine localization may be involved at least in the protection of photoreceptor outer segments against harmful factors, and in the transportation of nutrients and metabolites. The immunostaining for taurine is useful for the discrimination of different types of photoreceptor cells in the pineal organ and retina of fish.

Immunocytochemical demonstration of taurine in the retina and pineal organ of the pigeon.

Taurine-like immunoreactivity (TLI) is found in the pigeon retina at high levels within cones, in select cells of the inner nuclear layer and in sublaminae of the inner plexiform layer. At the ultrastructural level a high density of immuno-gold particles in visualized in the ellipsoid, but not in the paraboloid area of the inner segment. It is present in perikarya and the presynaptic endings of cones, but not in postsynaptic cell processes. The immuno-gold particles are not associated directly with the synaptic vesicles. In the pineal organ TLI was confined to the so-called dark pinealocytes which are surrounded by only family stained light pinealocytes. Clusters of protein A-gold particles are located in the ergastoplasm between polyribosomes adjacent to fibrous proteins and in chains along filamentous structures. Granules, vesicles and membrane whorls, which originate from modified cilia remain unlabeled. The results are discussed on the basis of previous findings; a direct or indirect functional relationship of taurine to ATP-dependent processes is suggested.

Immunohistological localization of melatonin in the pineal gland and retina of the rat.

An incubation procedure for the demonstration of melatonin is described using freeze-dried, unfixed material embedded in vacuum directly in glycolmethacrylate. The results indicate the binding of melatonin in the nucleus of pineal parenchymal cells and in the outer nuclear layer of the retina of rats; binding is greater at night than during the day.

The neural organization of the pineal complex in the frog: stratification and regional differences.

Plexiform areas are functionally important units of the pineal complex. The silver impregnation technique by Matsuyama (1972) reveals in the frog that nerve fibres branch and terminate in these areas and that they are presynaptic to nerve cells which are part of the plexiform areas. Histochemically, the nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) method according to Scherer-Singler et al. (1983) indicates that pinealocytes and nerve cells are both stained, a strong stratification of different cell types can be visualized. Plexiform areas of different size exist at the dorsal and ventral surface of the organ; the biggest are located rostrodorsally. Approximately twenty large multipolar nerve cells are localized at the ventral surface of the pineal organ mainly at the rostral and lateral borders. They send long processes mainly in caudal direction. These processes are post-synaptic to the boutons of the endfeet belonging to photosensitive pinealocytes. Processes of several multipolar nerve cells converge to one plexiform area, branch and obviously terminate there. As in horizontal cells the processes of the multipolar cells seem to be capable of both receiving and transmitting signals. On the postsynaptic side of the processes of the multipolar cells are smaller nerve cells juxtaposed to the plexiform areas. They are known as pseudounipolar, unipolar or bipolar cells (Wake et al., 1974); the latter are free of boutons of the pinealocytes, their axons forming the pineal tract. Three different types of photosensitive pinealocytes can be distinguished: a slender, a spherical and a double-cone type. Also, the number of boutons varies in the endfeet: boutons occur alone, in groups of two or three, and in clusters.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochemical demonstration of acid phosphatase activity in the pineal organ of the rainbow trout, Salmo gairdneri.

Activity of acid phosphatase (ACP) was investigated cytochemically in the pineal organ of the rainbow trout, Salmo gairdneri. Intense reaction product for ACP activity was observed in lysosomes varying in size and shape and in endoplasmic reticulum associated with the Golgi complex of the pineal photoreceptor and supporting cells, vascular endothelial cells, and macrophages inhabiting pineal lumen, parenchymal epithelium and perivascular spaces. This localization of ACP is discussed with particular reference to the capacity for lysosomal digestion in a pineal organ combining photoreceptive and secretory functions, and lacking a blood-brain barrier, as holds true for the pineal of the rainbow trout. Taking advantage of its capacity for endocytotic uptake and lysosomal digestion, the pineal organ of the rainbow trout may serve as a barrier between the blood circulation and the cerebrospinal-fluid compartment. Furthermore, the macrophages may be considered as an essential component in pineal function of fish.

Histochemical and cytochemical demonstration of Ca++-ATPase activity in the stellate cells of the adenohypophysis of the guinea pig.

The histo- and cytochemical localization of Ca++-ATPase activity in the adenohypophysis of the guinea pig was studied utilizing a newly developed method (Ando et al. 1981). An intense reaction was observed in the wall of the blood vessels and between non-secretory cells (stellate cells) and endocrine cells of the pars distalis. Under the electron microscope the Ca++-ATPase reaction product was located extracellularly in relation to the plasmalemma of the stellate cells. This reaction was dependent on Ca++ and the substrate, ATP, and reduced by the addition of 0,1 mM quercetin to the standard incubation medium. Preheating of the sections before incubation completely inhibited the enzyme activity. When Mg++ in different concentrations were substituted for Ca++ in the incubation medium the reaction was always reduced. Both Ca++ and Mg++ in the incubation medium also reduced the reaction. The plasmalemma of the endocrine cells contains no demonstrable amount of Ca++-ATPase activity. The function of the Ca++-ATPase activity is discussed in relation to the regulation of the extracellular Ca++ concentration which seems to be important with respect not only to the secretory process of the endocrine cells but also to the metabolism of the adenohypophysis.

Localization and function of cyclic guanosine monophosphate-phosphodiesterase activity in the retinal rods of the rat by means of a newly developed cytochemical method.

Cyclic guanosine monophosphate-phosphodiesterase (cGMP-PDEase) activity was studied histo- and cytochemically in the retinal rods of the rat with the use of a newly developed technique. Intense activity of cGMP-PDEase was evenly distributed over the outer segments of the rods. Reaction product was observed on the plasmalemma and on the disk membranes of the outer segments. A weak reaction product occurred also on the plasmalemma of the inner segments; however, no precipitate was found in the perinuclear and synaptic portions of the rod cells. The enzyme activity was strongly inhibited by 2 mM theophilline and by 2 mM 3-isobutyl-1-methylxanthine (IBMX). To confirm the specificity of this new cGMP-PDEase method, the localization of 5' nucleotidase (5'GMPase) was also studied. In contrast to the activity of cGMP-PDEase, the activity of 5'GMPase was distributed on the plasma membrane of the photoreceptor cells extending over a wide range from the synaptic endings in the outer plexiform layer to the tip of the outer segments.

A new histo- and cytochemical method for demonstration of cyclic 3',5'-nucleotide phosphodiesterase activity in retinal rod photoreceptor cells of the rat.

Cyclic 3',5'-mononucleotide phosphodiesterase (cyclic nucleotide PDEase) activity was studied histo- and cytochemically in the retinal rod photoreceptor cells of the rat by means of a newly developed technique utilizing the intrinsic 5' nucleotidase activity instead of an exogenous 5' nucleotidase source (snake venom). Cyclic GMP and was used as a substrate, the intense activity of phosphodiesterase (PDEase) was distributed over the entire rod outer segments; reaction product was observed on the plasmalemma and on the disk membranes of the outer segments. A slight reaction was also observed on the plasmalemma of the inner segments. However, no precipitate was found in the perinuclear and synaptic regions of the rod photoreceptors. In contrast, when cyclic AMP was utilized as a substrate, a moderate reaction was seen in the synaptic region of the plexiform layer. The intensity of the reaction in the outer segments was much reduced in comparison to the results with cyclic GMP. The enzyme activity was almost completely inhibited by 2 mM 3-isobutyl-1-methylxanthine (IBMX) or 2 mM theophylline, which were potent inhibitors of PDEase. To confirm the propriety of our new cytochemical method, the localization of 5' nucleotidase was also studied utilizing 5' AMP or 5' GMP as substrates. In contrast to the activity of cyclic nucleotide PDEase, the activity of 5' nucleotidase was distributed on all membranes of the photoreceptors from the synaptic outer plexiform layer to the tip of outer segments.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultracytochemical localization of Ca++-ATPase activity in pituicytes of the neurohypophysis of the guinea pig.

Ca++-ATPase activity (cf. Ando et al. 1981) was examined both light- and electron-microscopically in the neurohypophysis of the guinea pig. Apart from a strong activity within the walls of the blood vessels, in the parenchyma of the neurohypophysis the reaction product of the Ca++-ATPase activity was restricted to the plasmalemma of the pituicytes. This reaction was completely dependent upon Ca++ and the substrate, ATP; the reaction was inhibited by 0.1 mM quercetin, an inhibitor of Ca++-ATPase. A reduction of the enzyme activity occurred by 1) adding Mg++ to the standard incubation medium, and 2) substituting Ca++ with Mg++ at varying concentrations. In all experiments the neurosecretory fibers were devoid of Ca++-ATPase activity. The function of the Ca++-ATPase activity in the plasmalemma of the pituicytes is discussed in connection with the regulation of the extracellular Ca++ concentration, which seems to be important with respect to the discharge of secretory material from the neurosecretory fibers.

Ultracytochemical localization of Ca++-ATPase activity in the paraphyseal epithelial cells of the frog, Rana esculenta.

Ca++-ATPase activity was studied ultracytochemically (cf. Ando et al. 1981) in the paraphysis cerebri of the frog. An intense reaction was demonstrated on the plasmalemma of the microvilli at the apical pole of paraphyseal cells; in contrast, the basolateral plasmalemma showed only a slight staining. In addition, mitochondria, gap junctions, cilia, and cytoplasmic elements (e.g., microfilaments) displayed Ca++-ATPase activity. Variation of the Ca++-concentration in the incubation medium from 0.1 mM to 100 mM altered the Ca++-ATPase activity of the cell organelles. The substitution of Ca- by Mg-ions resulted in a conspicuous decrease in the enzyme activity, especially on the apical plasmalemma. Ca++-ATPase activity is claimed to be involved in a number of extra- and intracellular functions. In comparison to the epithelium of the adjacent choroid plexus the paraphyseal epithelial cell is thought to be a principal Ca-ion regulator of the cerebrospinal fluid in frogs.

Ultracytochemical study of Ca++-ATPase and K+-NPPase activities in retinal photoreceptors of the guinea pig.

Ca++-ATPase activity was demonstrated histochemically at light- and electron-microscopic levels in inner and outer segments of retinal photoreceptor cells of the guinea pig with the use of a newly developed one-step lead-citrate method (Ando et al. 1981). The localization of ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity, which represents the second dephosphorylative step of the Na+-K+-ATPase system, was studied by use of the one-step method newly adapted for ultracytochemistry (Mayahara et al. 1980). In retinal photoreceptor cells fixed for 15 min in 2% paraformaldehyde the electron-dense Ca++-ATPase reaction product accumulated significantly on the inner membranes of the mitochondria but not on the plasmalemma or other cytoplasmic elements of the inner segments. The membranes of the outer segments remained unstained except the membrane arrays in close apposition to the retinal pigment epithelium. The cytochemical reaction was Ca++- and substrate-dependent and showed sensitivity to oligomycin. When Mg++-ions were used instead of Ca++-ions, a distinct reaction was also found on mitochondrial inner membranes. In contrast to the localization of the Ca++-ATPase activity, the K+-NPPase activity was demonstrated only on the plasmalemma of the inner segments, but not on the mitochondria, other cytoplasmic elements or the outer segment membranes. This reaction was almost completely abolished by ouabain or by elimination of K+ from the incubation medium.

Localization of ATPases in retinal receptor cells.

Ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K- NPPase ) activity of the Na-K-ATPase complex and the Ca-ATPase activity were studied ultracytochemically in the inner and outer segments of guinea pig photoreceptor cells by means of newly developed methods. The K- NPPase activity was demonstrated on the plasmalemma of the inner segments, whereas the Ca-ATPase activity was restricted to the matrix side of the inner membrane of the mitochondria which were accumulated in the inner segments. In contrast to the enzyme activities in the inner segments no such activity could be detected on the plasma and disk membranes of the outer segments. The functional meaning of the enzyme activities is discussed.

Ultracytochemical localization of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity in the lacrimal gland of the rat.

The electron-microscopic localization of ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity of the Na - K-ATPase complex was studied in the exorbital lacrimal gland of the untreated rat with the use of a newly developed one-step lead-citrate method (Mayahara and Ogawa 1980; Mayahara et al. 1980). In the rat lacrimal gland fixed for 15 min in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde, an electron-dense reaction product was observed on the plasma membrane of the basal infoldings and the lateral interdigitations of the ductal cells. The most intense reaction product - and thus the major site of the Na - K-ATPase activity - was evident on the basolateral membranes of the cells of the large interlobular ducts; a weak reaction was seen on the basolateral, extensively folded plasma membranes of the small intercalated ducts; no reaction product was observed on the plasma membranes of the acinar cells. Addition of 1) 10 mM ouabain, 2) p-chloromercuri-phenyl-sulfonic acid (PCMB-S), 3) elimination of K-ions from the incubation medium, or 4) preheating abolished completely the K-NPPase reaction. The activity was also substrate-dependent. Mg-ATPase-activity was observed not only in the basolateral membranes of all ductal cells but also in the basal part of the acinar cells and on the walls of blood vessels. This reaction was neither inhibited by ouabain nor activated by K-ions. The precipitate of the Mg-ATPase-activity was localized at the extracellular side of the plasma membrane, whereas the K-NPPase-reaction product was restricted to the cytoplasmic side of the plasmalemma. In contrast, non-specific alkaline-phosphatase (ALPase) activity was missing in cells of the large interlobular ducts, but obvious on the apical plasmalemma of cells lining the small intercalated ducts. With respect to its localization and reactivity pattern the activity of the K-NPPase (member of the Na - K-ATase complex) differs markedly from the Mg-ATPase- and ALPase-activity.

Histo- and cytochemical demonstration of guanosine triphosphatase activity in the outer segments of rods in the rat retina by means of a newly developed method.

Guanosine triphosphatase (GTPase) activity was studied histo- and cytochemically in the rod outer segments of the rat retina by means of a newly developed method. Differences in the distribution pattern of the enzyme activity exist within the outer segment: the activity is more intense at the tip of the rod outer segments near the pigment epithelium than in their proximal portion. Ultracytochemically, the new procedure reveals the reaction product of GTPase activity partly (i) on the extradisk membrane side and (ii) on the disk membranes. This result is in contrast to the cytochemical localization of guanylate cyclase (GCLase), an enzyme also localized at the tip of the rod outer segments: GCLase activity is restricted to the intradisk membrane area of the rod outer segments. The functional role of GTPase activity in the outer segments of rods is discussed.

Cerebrospinal fluid-contacting neurons and other somatostatin-immunoreactive perikarya in brains of tadpoles of Xenopus laevis.

In untreated tadpoles of Xenopus laevis, stage 60 (Nieuwkoop and Faber 1956), somatostatin-immunoreactive perikarya and fiber networks are already discernible in the pallium and the septum, as well as in the anterior and posterior hypothalamus. In addition, somatostatin-immunoreactive cerebrospinal fluid (CSF)-contacting neurons were found in the periventricular gray of the most caudal division of the ventral tuber cinereum. The results are discussed with respect to the inhibitory influence of the system of CSF-contacting and other somatostatin-immunoreactive neurons on the anterior pituitary and other target sites, especially during the climax of metamorphosis.