Characterization of calretinin-immunoreactive structures in the striatum of the rat

Previous observations have shown that the striatum contains a population of neurones that display immunoreactivity for calretinin. In order to morphologically characterize these neurones, sections of the rat striatum were immunostained to reveal calretinin and examined at both light and electron microscopic levels. The striatum contained a small population of calretinin-immunoreactive neurones, which were of medium-size (9–17 μm) and possessed few aspiny, infrequently branching dendrites which tapered to become very thin processes in their most distal portions. Although the calretinin-immunoreactive neurones were homogeneously distributed in the frontal plane, there was a marked rostrocaudal gradient with a much greater density of cells in the rostral than in the caudal parts of the striatum. At the ultrastructural level, calretinin-immunoreactive neurones were seen to possess an indented nucleus and to receive synaptic input from at least three types of boutons. In addition to the calretinin-immunoreactive neurones, the striatum also contained axons and terminal boutons that displayed immunoreactivity for calretinin. At least two types of immunoreactive terminals were identified, those forming symmetrical synaptic specialisations and those forming asymmetrical synaptic specialisations. Approximately 50% formed asymmetrical contacts with spines and 30% formed symmetrical synaptic contact with dendritic shafts. In an attempt to further chemically characterize the calretinin-containing neurones, double pre-embedding immunocytochemistry for calretinin and parvalbumin or choline acetyltransferase was carried out and calretinin immunocytochemistry was combined with histochemistry for NADPH-diaphorase. Analysis of these double-stained sections revealed that the population of calretinin-immunoreactive neurones was distinct from the populations of neurones containing parvalbumin, choline acetyltransferase or NADPH-diaphorase. It is concluded that: (1) on the basis of distribution, morphology, chemistry, ultrastructure and afferent synaptic input, the calretinin-immunoreactive neurones are distinct from the major classes of neurones that have been previously recognised in the striatum; (2) calretinin-immunoreactive terminals are heterogeneous and are probably derived from local calretinin-containing neurones and possibly other sources.     

Cholinergic somata and terminals in the rat substantia nigra: an immunocytochemical study with optical and electron microscopic techniques

The topographical distribution, histochemical characteristics, and anatomical relationships of the cellular elements containing choline acetyltransferase (ChAT) immunoreactivity, demonstrated with specific monoclonal antibodies to ChAT following the unlabelled antibody peroxidase-antiperoxidase (PAP) procedure at the optical and electron microscopic levels, were investigated in the rat substantia nigra (SN). Scarce, large (20-30 microns in maximum soma extent) cholinergic cell bodies and processes were found within the boundaries of the SN, in the borders of the pars compacta and pars reticulata, principally at caudal levels. Occasionally, cholinergic neurons were also found at intermediate levels of the SN, in the borders of the pars reticulata and pars lateralis. Cytologically, these large cells resembled ChAT-positive neurons localized in other areas of the central nervous system (CNS) of the rat--for example, the pontomesencephalotegmental (PMT) cholinergic complex (Ch5-Ch6) and the nucleus basalis of Meynert (nbM) (Ch4). Histochemically, ChAT-positive cells in the SN were characterized by their ability to utilize the reduced cofactor nicotinamide adenine dinucleotide phosphate (NADPH). Identified ChAT-positive neurons in the light microscope were subsequently studied in the electron microscope. All cholinergic neurons in the SN share essentially the same ultrastructural characteristics. The copious cytoplasm was rich in organelles with large lipofuscin granules. The synaptic input onto cell bodies and their dendrites was studied in serial sections. Synaptic contacts onto the perikarya and proximal dendrites were sparse and of asymmetric type. Both symmetric and asymmetric synaptic specializations onto ChAT-positive distal dendrites were detected. Asymmetric synaptic contacts onto cell bodies and dendrites were often defined by the presence of subjunctional dense bodies associated with the postsynaptic membrane. The pattern of the synaptic input to these cells differs strikingly from that onto unlabelled neighboring neurons. The perikarya and dendrites of the latter were characteristically covered with synaptic boutons. Scarce immunoreactive terminals in asymmetric synaptic contact with unlabelled dendritic profiles were also detected in portions of SN compacta with no ChAT-positive cells. Extranigrally located ChAT-positive cells of the PMT cholinergic complex were also examined in the electron microscope for comparison purposes. These cells exhibited, on the basis of their morphology and synaptic input pattern, very similar characteristics to those shown by SN cholinergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunocytochemical distribution of nitric oxide synthase in the human seminal vesicle: a light and electron microscopical study

Although nitric oxide (NO) has been proven to be one of the most important non-adrenergic, non-cholinergic mediators in the control of human reproductive tract organs, to date information on the significance of NO-mediated signal transduction in the control of human seminal vesicle (SV) function is still sparse. Recent investigations have underlined the significance of NO in the maintenance of sperm capacitation and viscosity of the seminal plasma as well as in the control of mammalian seminal vesicle smooth muscle tone. In order to further investigate the functional impact of NO on the regulation of normal SV function, we examined the distribution of NADPH-diaphorase (NADPH-d), endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) in the cellular anatomy of human SV by means of light and electron microscopical immunocytochemistry (LM, EM) in combination with the tyramide signal amplification technique. Human SV were obtained from 15 patients who had undergone surgery for pelvic malignancies (carcinoma of the prostate or urinary bladder). SV specimens were fixed, sectioned and examined by LM and EM for the presence of NAPDH-d, eNOS and nNOS using specific antibodies and advanced staining procedures. LM revealed a dense NADPH-d reaction in glandular epithelial structures, whereas no substantial labeling was detected in the fibromuscular stroma. EM showed that the NADPH-d reaction product was abundantly detectable attached to membranes of the endoplasmic reticulum, mitochondria and the nuclei of glandular epithelial cells. nNOS staining was found in nerve fibers branching within the SV tissue. eNOS staining was present in small vessels but was only observed to a minor degree in glandular and subglandular structures and the smooth muscle stroma. Our results support the hypothesis that human SV is a site of NO production. The distribution of NADPH-d may give rise to the speculation that NO is mainly involved in the regulation of SV secretory activity. The sparse correlation between NADPH-d-, eNOS- and nNOS-staining might hint at the existence of a previously unidentified NOS isoform in human SV.     

Vasoactive intestinal and calcitonin gene-related peptides,tyrosine hydroxylase and nitrergic markers in the innervation of the rat central retinal artery

The vascular supply of the optic nerve has been studied with different methods including corrosion casts both in humans and in other mammals. In man, primates and some other mammals, such as the rat, a distinct central retinal artery accompanies the optic nerve, and runs through the lamina cribosa to reach the optic nerve head. Similarities between human and rat central retinal artery could serve to understand changes in the autonomic perivascular innervation in glaucoma using the rat as an animal model. Nitric oxide, calcitonin gene-related peptide, neuropeptide Y, substance P and vasoactive intestinal peptide have been identified around the monkey central retina artery. Innervation of the rat central artery, however, has not been described in detail. Using immuno- and histochemical methods, the present study investigates the peptidergic, adrenergic and nitrergic innervation of the rat posterior ciliary artery as well as the central retina artery. Numerous nitric oxide positive nerve fibers were visualized posterior and anterior to the lamina cribosa of the optic nerve. They colocalized with NADPH-diaphorase positive fibers, which could also be observed in two of six specimens studied at the level of the optic nerve head. Calcitonin gene-related peptide, tyrosine hydroxylase, and VIP positive fibers were also observed surrounding the vessels of the rat optic nerve. The presence of neuronal nitric oxide/NADPH-diaphorase and vasoactive intestinal peptide positive nerve fibers surrounding the posterior ciliary and central retinal arteries indicates a vasodilator effect in the rat optic nerve. Tyrosine hydroxylase positive innervation indicates the presence of sympathetic activity, and calcitonin gene-related peptide positive fibers indicate sensory innervation by trigeminal primary efferents.     

NADPH-diaphorase Histochemical Labeling Patterns in the Hippocampal Neuropil and Visual Cortical Neurons in Weaned Rats Reared during Lactation on Different Litter Sizes

Abstract

Tissue distribution of nitric oxide-synthases was investigated in the rat hippocampus and visual cortex under nutritional changes induced by modification of the litter size. Young (30-45-days-old) rats, suckled in litters formed by 3, 6 or 12 pups (called small, medium and large litters, respectively), were studied by using nicotine-adenine-dinucleotide phosphate-diaphorase histochemistry (shortly, diaphorase), a simple and robust procedure to characterize tissue distribution of nitric oxide-synthases. We assessed morphometric features of the diaphorase-positive cells in visual cortex, and the neuropil histochemical activity in hippocampal CA1 and dentate gyrus using densitometry analysis. In the large-litter group, the labeled-cell density in white matter of area 17 was higher, as compared to the small-litter group. There was a clear trend, in the large-litter group, to lower values of soma area, dendritic field and branches per neuron, but the differences were not significant. Densitometry analysis of hippocampus revealed a significant increase in the relative neuropil histochemical activity of the dentate gyrus molecular layer in the larger litters, which may be associated to increased compensatory blood flow in the hippocampus. The pathophysiological mechanisms of the observed changes remain to be investigated.     

不完全性脑缺血大鼠海马CA1区一氧化氮合酶的变化

用双侧颈总动脉夹闭加放血方法造成大鼠一过性不完全性脑缺血及再灌注模型 ,以还原型尼克酰胺腺嘌呤二核苷酸脱氢酶 ( NADPH-d)组织化学方法对缺血再灌注期间海马 CA1 区内一氧化氮合酶阳性神经细胞变化进行研究。结果发现 ,海马 CA1 区神经细胞受损 ,在缺血 3 0 min时一氧化氮阳性细胞数最高 ( 44 .5± 7.42 ) ,为空白对照组 2倍 ,再灌注 2 h、12 h、2 4h、3 d后逐渐下降 ,5 d时恢复正常水平 ( 2 1.12± 3 .5 0 )。研究表明 ,不完全性脑梗塞时一氧化氮合酶表达与海马 CA1 区神经细胞损伤及迟发性死亡有关     

Enzyme histochemical profile of immunohistochemically identified Renshaw cells in rat lumbar spinal cord

Activity levels of cytochrome oxidase, acid phosphatase, and NADPH diaphorase were examined in the perikarya of immunohistochemically identified Renshaw cells from sections of rat lumbar spinal cord. Renshaw cell profiles were identified on the basis of their characteristic anti-gephyrin-immunofluorescent labelling. Intrasomatic densities of enzyme histochemical reaction product were employed as indicators of relative mitochondrial activity (cytochrome oxidase), intracytoplasmic digestion (acid phosphatase), or putative nitrergic signalling (NAPDH-diaphorase). Approximately half of the Renshaw cell somata examined displayed moderate levels of cytochrome oxidase reaction product (142 of 262 Renshaw cells) or low levels of acid phosphatase activity (156 of 243 Renshaw cells). A majority (160 of 202 cells) of Renshaw cells contained low intrasomatic levels of NADPH-diaphorase activity but most of these cells were closely apposed by at least one NADPH-diaphorase reactive axonal varicosity. Our findings suggest that moderate levels of perikaryal oxidative metabolism and low levels of intracytoplasmic digestion are sufficient for, and support, the unique physiological capabilities of Renshaw cells. The presence of NADPH-diaphorase containing somatic close contacts indicate that nitric oxide may have at least a minor role in the regulation of Renshaw cell activity. These results are complementary and consistent with previous morphological and pharmacological demonstrations of Renshaw cell heterogeneity.     

急性高眼压大鼠外侧膝状体一氧化氮合酶的表达

目的:探讨高眼压视神经损伤中NO的作用。方法:SD大鼠20只,随机分为空白对照组4只,另16只大鼠右眼为实验组,施行高压前房灌注维持时间60min,按照高眼压后再灌注时间不同又随机分为4小组,每组4只,即高眼压后0.5,1,3和7d组。各组应用NADPH-黄递酶法检测外侧膝状体一氧化氮合酶(nitric oxide synthase,NOS)的表达及分布情况。结果:空白对照组及实验各组外侧膝状体均有NOS表达,NOS阳性细胞在外侧膝状体主要分布于腹外侧核。各实验组外侧膝状体的NOS阳性细胞数明显高于空白对照组(P<0.01);实验各组中高眼压后1d组的NOS阳性细胞数明显高于其它3组实验组(P<0.01);其它3组实验组之间比较无显著性差异。结论:NO既参与眼正常生理功能的调节,也在高眼压视神经及其上行视觉通路的损伤中发挥重要作用。     

Neuronal nitric oxide synthase is localized in extrinsic nerves regulating perireceptor processes in the chemosensory nasal mucosae of rats and humans

Nitric oxide synthase is the enzyme responsible for the production of the free radical gas nitric oxide, which has been implicated as an intercellular messenger in both the central and peripheral nervous systems. Immunoreactivity for nitric oxide synthase is often coincident with the histochemical demonstration of NADPH-diaphorase activity. Using an antibody to the neuronal form of nitric oxide synthase and a histochemical technique for NADPH-diaphorase, we have compared the localization of immunoreactivity and histochemical reaction product in the nasal mucosae of rats and humans. Immunoreactivity for neuronal nitric oxide synthase was localized in the extrinsic perivascular innervation of the olfactory and vomeronasal mucosae of rats and in the olfactory mucosa of humans. In the rat nasal mucosa, specific groups of glands were also innervated; the density of nitrinergic innervation varied among them, with vomeronasal glands and posterior glands of the nasal septum being the most densely innervated. In contrast, NADPH-diaphorase activity was present in olfactory, vomeronasal, and septal organ receptor neurons in rats and in olfactory receptor neurons in humans as well as in numerous nerve fibers, glands, and surface epithelial cells. The localization of neuronal nitric oxide synthase in extrinsic perivascular and periglandular nerve fibers suggests that nitric oxide may modulate the perireceptor processes of local blood flow and mucus secretion that influence the access to and clearance of chemical stimuli from rat and human chemosensory mucosae. © Wiley-Liss, Inc.     

Angiotensin-(1–7) and nitric oxide synthase in the hypothalamo-neurohypophysial system

The organization of angiotensin (Ang)-(l-7) immunoreactivity with respect to neurons containing the nitric oxide synthase, nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), in the hypothalamo-neurohypophysial (HNS) system was examined. The majority of neurons in this system were single labeled with Ang-(1–7) or NADPH-d and were found localized in two separate populations of neurons within the HNS. A small number of double-labeled neurons were observed in the supraoptic (SO) and paraventricular (PV) nuclei. Double-labeled accessory neurosecretory neurons were also found distributed between the SO and PV. Although a well-defined function for nitric oxide in the magnocellular hypothalamic system has not been determined, the codistribution and limited coexistence of Ang-(1–7), a heptapeptide involved in antidiuresis, and NADPH-d in the HNS suggests a potential role for these substances in mechanisms modulating fluid homeostasis.