Nitric oxide synthetase (NOS)-containing sympathoadrenal cholinergic neurons of the rat IML-cell column: evidence from histochemistry, immunohistochemistry, and retrograde labeling.

Nitric oxide synthetase (NOS) can be selectively stained in neurons by either NADPH-diaphorase (i.e., NOS)-histochemistry or immunohistochemistry with antibodies raised against NOS, which apparently label identical reactive sites (Hope, B.T., G.J. Michael, K.M. Knigge, and S.R. Vincent, Proc. Natl. Acad. Sci. USA 88:2811-2814, '91). We provide histochemical evidence for the existence of a neuron-specific NOS-activity in autonomic neurons of the thoracic spinal cord. Among the four main preganglionic cell clusters investigated at mid-thoracic levels, Th7-10, the intermediolateral (IML)-cell column was the most prominently stained cell group. The histochemical staining was absent in other spinal cord neurons and non-neuronal cells, e.g., GFAP-positive glial cells. Staining was completely blocked by N omega-nitro-L-arginine (L-NNA), a potent NOS-inhibitor for brain and peripheral autonomic neurons, but was still observed in the presence of another NOS-inhibitor, N omega-monomethyl-L-arginine (MeArg). The NOS-activity co-localized with nearly half of the ChAT-immunostained neurons located in the mid-thoracic IML-cell column as quantified by cell counts in single and double-stained tissue sections. We conclude that NOS-activity-containing neurons represent a distinct group among cholinergic IML-neurons, which suggests a more general function of this newly defined subpopulation of the spinal cord autonomic system. In vivo Fast blue retrograde labeling combined with histochemical staining and immunostaining revealed that sympathoadrenal projection neurons belong to the distinct NOS and ChAT-positive IML-cell group.(ABSTRACT TRUNCATED AT 250 WORDS)

Histochemistry of NADPH-diaphorase, a marker for neuronal nitric oxide synthase, in the peripheral autonomic nervous system of the mouse.

In order to identify possible sites of synthesis of nitric oxide in the peripheral nervous system, several mouse organs were investigated for the presence of NADPH-diaphorase activity. Diaphorase-positive neurons and fibers were localized in the tongue, submandibular salivary glands, gastrointestinal and biliary duct systems, lower urinary tract and pelvic ganglia. By thionin counterstaining it was found that a distinct subpopulation of neurons was labeled. The present study indicates that nitric oxide synthase may be present in intrinsic neurons of various organs, suggesting a widespread function of nitric oxide in the peripheral autonomic nervous system.

Quantitative autoradiography of [3H]norharman [( 3H]beta-carboline) binding sites in the rat brain.

The anatomical distribution of [3H]norharman binding sites was determined by quantitative autoradiography in rat brain slices. They are enriched in hypothalamic, thalamic, accumbens and amygdaloid nuclei as well as in hippocampal, neocortical and olfactory-related structures. The distribution pattern differs from that of other previously described receptors or binding sites (e.g. monoamine oxidase, benzodiazepine, tryptamine, 5-hydroxytryptamine receptors (5-HT1A, 5-HT1B, 5-HT1C, 5HT2], which suggests that a unique class of [3H]norharman binding sites exists in the rat brain. The findings are consistent with previous experiments which showed high affinity binding sites for [3H]norharman in rat brain membranes (KD 1.552 nM; autoradiography KD 5.5 nM). A correspondence in the displacing activity of drugs was found for both methods (crude membrane fraction: harman much greater than tryptamine much greater than 5-hydroxytryptamine greater than N-methyl-beta-carboline-3-carboxamide (FG 7142) = diazepam; autoradiography: harman much greater than tryptamine much greater than FG 7142 greater than 5-hydroxytryptamine greater than diazepam). Provided that the binding sites represent functional receptors, the present anatomical findings may explain the biological effects of norharman, e. g. pro-conflict behaviour (limbic-hypothalamic structures), tonic-clonic convulsions (limbic-cortical structures) and alterations of locomotor activity (accumbens nucleus).

Autoradiographic analysis of benzodiazepine receptors in mutant mice with cerebellar defects.

[3H]Flunitrazepam binding sites were analysed in the cerebellar cortex, deep cerebellar and vestibular nuclei of Purkinje cell deficit (pcd) mutant mice which have an almost complete postnatal loss of Purkinje cells, and weaver mutant mice, in which there is a postnatal degeneration of granule cells. Increases in [3H]flunitrazepam binding site densities were found in the molecular and granule cell layer of weaver mutant mice, whereas decreases were observed in pcd mutant mice when compared to wildtype 'control' mice. Apparently unaltered benzodiazepine receptor densities were found in the flocculus of pcd mutant mice, whereas increases were seen in the flocculus of weaver mutant mice. The densities of benzodiazepine binding sites in the medial and lateral deep cerebellar nuclei of both mutants significantly exceeded those in control mice. Significant increases in flunitrazepam binding sites were also found in the superior and spinal nucleus of the vestibular complex of pcd mice as compared to wildtype. In the weaver mutants the benzodiazepine receptor density is enhanced in the superior and medial vestibular nucleus. Apparently unaltered numbers of such receptors compared to the wildtype control group were found in the remaining vestibular nuclei of both mutants.

Nitric oxide synthase in skeletal muscle fibers: a signaling component of the dystrophin-glycoprotein complex.

The present review deals with the anatomical distribution, physiological importance, and pathological implications of the neuronal-type nitric oxide synthase (nNOS) in skeletal muscle. Throughout the body, nNOS is located beneath the sarcolemma of skeletal muscle fibers. In rodents, nNOS is enriched in type IIb muscle fibers, but is more homogenously distributed among type II and type I fibers in humans and subhuman primates. It is accumulated on the postsynaptic membrane at the neuromuscular junction. An increased concentration of nNOS is noted at the sarcolemma of muscle spindle fibers, in particular nuclear bag fibers, which belong to type I fibers. The association of nNOS with the sarcolemma is mediated by the dystrophin-glycoprotein complex. Specifically, nNOS is linked to alpha 1-syntrophin through PDZ domain interactions. Possibly, it also directly binds to dystrophin. The activity and expression of nNOS are regulated by both myogenic and neurogenic factors. Besides acetylcholine, glutamate has also been shown to stimulate nNOS, probably acting through N-methyl-D-aspartate receptors, which are colocalized with nNOS at the junctional sarcolemma. Functional studies have implicated nitric oxide as a modulator of skeletal muscle contractility, mitochondrial respiration, carbohydrate metabolism, and neuromuscular transmission. A clinically relevant aspect of nNOS is its absence from the skeletal muscle sarcolemma of patients with Duchenne muscular dystrophy (DMD). A concept is presented which suggests that, as a consequence of the disruption of the dystrophin-glyoprotein complex in DMD, nNOS fails to become attached to the sarcolemma and is subject to downregulation in the cytosol.

Nitroxergic autonomic neurones in rat spinal cord.

We have used a polyclonal monospecific antibody to rat cerebellum nitric oxide synthase type I (NOS-I, 160 kD) in combination with reduced NADPH-diaphorase histochemical reaction (NADPH-d) to verify colocalization of both NOS protein and NOS enzymatic activity in the rat spinal cord autonomic system. Strong immunoreactivity (IR) of NOS-I was clearly detected in the four main thoracolumbar autonomic nuclei in spinal cord layers of Rexed's laminae VI, VII and X. In all labelled neurones, NOS-I-IR colocalized with NADPH-d activity, suggesting coexistence of brain-specific NOS-I-like protein and its associated enzyme activity. For these neurones the new term 'nitroxergic' (i.e. NO-generating) neurones appears to be justified. Spinal cord nitroxergic neurones are part of a NO-mediated signal transduction pathway for control of the sympathetic 'outflow' to various peripheral target organs.

Nitric oxide synthase-containing nerve fibres and neurones in the gall bladder and biliary pathways of the guinea-pig.

We investigated the distribution pattern of nitric oxide (NO) synthesizing nerve cell bodies and axons in the biliary system of the guinea-pig using immunohistochemistry for nitric oxide synthase (NOS). Nerve fibres staining for NOS were found to contact non-vascular smooth myocytes and to course beneath the epithelium. No perivascular NOS fibres were observed. The innervation density varied in different parts of the biliary tree. The lower portion of the common bile duct was more richly innervated than the remaining parts of the duct system. NOS-containing neurones encompassed a subpopulation of intramural ganglion cells. Sympathetic neurones in the coeliac ganglion were not stained. It is suggested that intrinsic NOergic neurones are involved in inhibitory motor control of the biliary musculature, including the sphincter of Oddi.

Characterization and quantitative autoradiography of [3H]tryptamine binding sites in rat brain.

[3H]Tryptamine binds with high affinity (Kd = 9.1 nM, Bmax = 54 fmol/mg wet wt.) to tissue sections of rat brain. The binding occurs rapidly and is reversible. Low concentrations of the beta-carbolines harmaline (IC50 = 25 nM) and tetrahydronorharman (tetrahydro-beta-carboline, IC50 = 50 nM) inhibit [3H]tryptamine binding. Serotonin (5-HT, IC50 = 2600 nM) as well as the 5-HT receptor antagonists methysergide and metergoline displace [3H]tryptamine at much higher concentrations from brain slices. The distribution of [3H]tryptamine binding sites in sections of rat brain has been analyzed by quantitative autoradiography. The highest density of binding sites is found in the nucleus (n.) interpeduncularis, a slightly lower one in the locus coeruleus. Moderately labelled are the n. accumbens septi, n. septi lateralis, n. medialis habenulae, n. tractus olfactorii lateralis, the central region of the amygdala, n. caudatus/putamen, n. reuniens and the hippocampal formation. A low density of binding sites is detected in the cerebral cortex and the subiculum. Even less binding sites are found in the n. dorsalis raphe and the substantia nigra. The pattern of distribution of [3H]tryptamine binding sites differs from that of [3H]5-HT (5-HT1), [3H]ketanserin (5-HT2) as well as [3H]imipramine binding sites. These data suggest unique tryptamine binding sites.

Autoradiographic mapping of a selective cyclic adenosine monophosphate phosphodiesterase in rat brain with the antidepressant [3H]rolipram.

Rolipram is a clinically effective antidepressant with selective cAMP phosphodiesterase (PDE) inhibiting properties. (+/-)-[3H]Rolipram binds with high affinity (Kd = 2.52 +/- 0.47 nM) to sections of rat brain (Hill number = 0.90 +/- 0.05). Binding is stereospecific. Association of (+/-) [3H]rolipram to sections is rapid (47% of specific binding in the first minute, kobs = 0.52 min-1). Dissociation of (+/-)-[3H]rolipram exhibits non first order kinetics (3 component model; t1/2 = 2.5 min, 50 min and 6 h, respectively). A number of PDE inhibitors reduce (+/-)-[3H]rolipram binding to the level of nonspecific binding ((-)-rolipram, IC50 = 0.9 nM; (+/-)-rolipram, IC50 = 1.5 nM; Ro 20-1724, IC50 = 11 nM; ICI 63.197, IC50 = 35 nM; medazepam, IC50 = 240 nM; diazepam, IC50 = 1200 nM; IBMX, IC50 = 3800 nM). In vitro autoradiography reveals high binding site densities in the cerebellum, olfactory bulb, lateral septal nucleus, frontal cortex, subiculum and CA1 of hippocampus. Most of the labeled structures are part of the limbic system. In vivo autoradiography of (+/-)-[3H]rolipram binding shows much more nonspecific binding than in vitro, nevertheless the distribution pattern of (+/-)-[3H]rolipram binding sites is similar. A comparison of the distribution pattern of (+/-)-[3H]rolipram binding sites with that of an antidepressant (monoamine oxidase inhibitor, monoamine uptake inhibitor) reveals no overlap. Limited, though significant correlations exist with the distribution of beta 1-adrenergic, adenosine1 and glutamate/quisqualate receptors as well as protein kinase C, but not with beta 2-adrenergic receptors and forskolin binding sites.

Distribution of tritium label in the neonate rat brain following intracisternal or subcutaneous administration of [3H]6-OHDA. An autoradiographic study.

The present report describes the distribution of tritium label after injection of newborn rats with [3H]6-hydroxydopamine ([3H]6-OHDA). The animals were injected either intracisternally (i.c.) or subcutaneously (s.c.), with or without pretreatment with nomifensine, which blocks the high-affinity uptake of both noradrenaline (NA) and dopamine (DA), and sacrificed at intervals from 40 min to 24 h post-injection (p.i.). In i.c. injected animals, tritium label is demonstrable as early as 40 min p.i. in neurons of all known NA and DA cell groups. In NA neurons, it is taken up into cell body, dendrites, preterminal and terminal axons. The intensity of neuronal labeling is highest within the first 4 h p.i. and decreases in most neurons with longer postinjection intervals. A significant proportion of both NA and DA neurons degenerate beginning 6 h p.i., the majority show morphological signs of the axon reaction 24 h p.i. Uptake of [3H]6-OHDA into serotonergic and non-catecholaminergic neurons is not demonstrable. [3H]6-OHDA is accumulated by the following extraneuronal cells of the CNS: ependymal cells, epithelial cells of the choroid plexus, subependymal macrophages, smooth muscle cells in the wall of large intraparenchymal blood vessels, meningeal cells and glial cells. The time course of accumulation and disappearance of the label varies among these extraneuronal elements. The meningeal cells show the highest labeling intensity and degenerate within 24 h p.i. After pretreatment of the animals with nomifensine, the uptake of [3H]6-OHDA into NA and DA neurons is totally blocked; by contrast uptake of the labeled drug into extraneuronal cells is not prevented. These findings show that [3H]6-OHDA is not only accumulated by neurons possessing the high-affinity uptake for NA or DA, but by numerous other, extraneuronal cells which also participate in the metabolism of catecholamines.

Postnatal development of [3H]flunitrazepam and [3H]strychnine binding sites in rat spinal cord localized by quantitative autoradiography.

The development of inhibitory receptors in rat spinal cord was investigated by autoradiography using [3H]flunitrazepam as a ligand for benzodiazepine receptors and [3H]strychnine as a ligand for glycine receptors. The development of benzodiazepine receptors follows a similar pattern at all levels of the spinal cord. The density of [3H]flunitrazepam binding sites is already high at birth, increases 2-fold by days 3-7 and thereafter declines to levels already present at birth. In contrast, [3H]strychnine binding sites are weakly expressed at birth and increase up to 7-fold between days 4 and 21. A craniocaudal gradient in the development of glycine receptors is not apparent. However, maturation of [3H]strychnine binding in the ventral horn precedes that in the dorsal horn for 3-4 days. In summary, the developmental expression of these two inhibitory receptors in the spinal cord appears to be regulated differently.

Quantitative autoradiographic localization of alpha 2-antagonist binding sites in rat brain using [3H]idazoxan.

The distribution of alpha 2-receptors was determined by quantitative autoradiography with the antagonist [3H]idazoxan. Apart from regions which are known to be enriched in alpha 2-receptors, the highest silver grain densities were located over the subfornical organ and the area postrema. However, binding in these regions was not displaced by yohimbine and therefore, according to the present understanding, cannot be considered to represent alpha 2-receptors. Within the cerebellum, alpha 2-receptors were found to be arranged in 3 sagitally oriented strips within the molecular layer of lobules 9 and 10, suggesting a co-incidence with dopamine and substance P receptors in this structure.

Autoradiographic localization of [3H]buspirone binding sites in rat brain.

Binding sites for [3H]buspirone were identified on sections from rat brain by light microscopic autoradiography. Incubated sections were processed by the Ultrofilm as well as the coverslip method. Highest densities of [3H]buspirone binding were localized in the caudate/putamen, nucleus accumbens septi, olfactory tubercle and in the glomerula of the olfactory bulb. This distribution pattern is different from any serotonin receptor but fits well with that of dopamine receptors. Therefore, this study supports the hypothesis that buspirone acts, at least partially, on dopamine receptors.

Interbulbar axonal collateralization and morphology of anterior olfactory nucleus neurons in the rat.

The organizational patterns of the bilateral projections of the anterior olfactory nucleus (AON) to the main olfactory bulb (MOB) were defined in the rat with Golgi staining, HRP tracing-methods and fluorescent dyes. Three issues were addressed: (1) description of the morphology of the AON-neurons projecting to the MOB, (2) quantitative analysis of the bilateral pathways arising in different AON subdivisions and (3) ultrastructural identification of AON to MOB channels. The cytoarchitectural features of the AON as recognized in Golgi preparations were correlated with its neural architecture as revealed by retrograde HRP-tracing from the MOB. The following cell types were determined: (1) pyramidal like neurons typified by a lack of basal dendrites and a sparse covering with long spines (pars externa), (2) fusiform shaped cells with bipolar dendritic arborisations (pars medialis) and (3) densely spined fusiform, pyramidal, and polygonal neurons (pars ventroposterior, lateralis and dorsalis) with a tendency of radial orientation of their apical dendrites. In addition, in the more caudal parts of the pars ventroposterior there were neurons with tertiary dendritic processes oriented nearly parallel to the molecular layer. Quantitative analysis of AON neurons projecting to the MOB showed that the pars externa neurons project exclusively to the contralateral MOB while pars medialis neurons project almost exclusively to the ipsilateral MOB. All subdivisions of the AON which establish specific termination patterns within the MOB, participated in about equal portion in the ipsilateral projections to the MOB. The highest proportion of the bilaterally projecting neurons were found in the dorsal subdivision, followed by the lateral and ventroposterior subdivisions. The postsynaptic targets of the AON to MOB channel are the spinous processes and varicosities of the proximal and distal-most dendrites of granule cells. The boutons derived from AON projection neurons contained clear spherical vesicles and established exclusively asymmetric synaptic junctions.

Dihydroxytryptamines as tools to study the neurobiology of serotonin.

The neurotoxins 5,6- and 5,7-dihydroxytryptamine are accepted tools for "chemical degeneration" of serotonergic (5-HT) axons in the CNS (for reviews, see [11, 12, 15, 20] ). Optimum application of these substances requires knowledge of their chemical properties, disposition in the biophase and mechanism of action. Current knowledge and concepts on this issue are described and results of recent studies utilizing 5,7-DHT uptake as a tool for localizing 5-HT neurons neuroanatomically are reviewed.

Serotonin neurotoxins--past and present.

Autoxidation pathways and redox reactions of dihydroxytryptamines (5,6- and 5,7-DHT) and of 6-hydroxydopamine (6-OH-DA) are illustrated, and their potential role in aminergic neurotoxicity is discussed. It is proposed that certain aspects of the cytotoxicity of 6-OH-DA and of the DHTs, namely redox cycling of their quinone- and quinoneimine-intermediates as a source of free radicals, may also apply to quinoidal reactive intermediates and to glutathionyl- or cysteinyl conjugates ("thioether adducts") of o-dihydroxylated (catechol-like) metabolites of certain substituted amphetamines (of methylenedioxymethamphetamine (MDMA) and of methylenedioxyamphetamine (MDA)). Despite similarities in their primary interaction with the plasmalemmal (serotonergic transporter/dopamine transporter, SERT/DAT) and vesicular monoamine transporters (VMAT2), MDMA and fenfluramine (N-ethyl-meta-trifluoromethamphetamine, Fen) differ substantially in many aspects of their metabolism, pharmacokinetics, pharmacology, and neurotoxicology profile; the consequences of these differences for neuronal response patterns and long-term survival prospects are not yet fully understood. However, sustained hyperthermia appears to be a critical factor in these differences. Methodological requirements for adequate detection and description of pre- and postsynaptic forms of drug-induced neurotoxicity are exemplified using recently published accounts. The inclusion of microglial markers into research strategies has widened contemporary pathogenetic concepts on methamphetamine (MA)-induced neurotoxicity as an example of inflammatory neurodegeneration, thus complementing the traditional ROS and RNS-dependent stress models. Amphetamine-type neurotoxicity studies may assist in elaborating of preventive strategies for human neurodegenerative disorders.

Colocalisation of NADPH-diaphorase with neuropeptides in the ureterovesical ganglia of humans.

Neurones in the ureterovesical ganglion complex provide autonomic innervation to the pelvic ureter, the ureterovesical junction and the bladder trigone. We examined the distribution and peptide co-expression pattern of nitric oxide synthase (NOS) in the human ureterovesical ganglia by combining NADPH-diaphorase histochemistry with immunoreactivity for vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP). Less than 20% of nerve cells in the large ganglia of the ureterovesical complex were stained for NOS activity. In elderly individuals, ganglion cells regularly exhibited conspicuous morphological alterations suggestive of degenerative changes. Most of the NOS-positive cell bodies costained for VIP-immunoreactivity. A minority of NOS-expressing cells also reacted for NPY-immunoreactivity. CGRP-immunoreactivity was present in varicose terminal-like nerve fibres which were found to encircle NOS-containing perikarya. Occasionally, NOS-positive somata were surrounded by plexiform axon terminals which immunostained for VIP or NPY. We conclude that the passage of urine across the ureterovesical junction is under relaxatory control of a local nitric oxide/VIP(NPY) pathway which may be modulated by preganglionic efferent and/or primary afferent input.

Role of serotonin in obsessive-compulsive disorder.

BACKGROUND: Serotonin may play a role in the pathophysiology of obsessive-compulsive disorder (OCD) because of the anti-obsessional effect of selective serotonin reuptake inhibitors (SSRIs). METHOD: The literature is reviewed on knowledge of the role of serotonergic neurons in brain function, studies on monoamine metabolites in cerebrospinal fluid (CSF), various stress neuropeptides, neuroendocrine and behavioural challenge after administration of direct and indirect serotomimetic compounds, and neuroanatomical data on brain circuits organising behaviour. RESULTS: In most of the OCD cases analysed, CSF 5-hydroxyindoleacetic acid and homovanillic acid concentrations do not significantly differ from age-corrected controls. However, a relationship appears to exist between pre-treatment levels of these metabolites and clinical response to drugs acting on the serotonin transporter. Abnormalities in CSF arginine vasopressin, corticotropin-releasing hormone, oxytocin and somatostatin levels have been reported in OCD. Long-term treatment with high-doses of clomipramine, fluvoxamine, and fluoxetine tend to correct these neuropeptide abnormalities. CONCLUSIONS: We hypothesise that continuous treatment with SSRIs alters serotonin turnover and neuropeptide expression patterns in OCD-entertaining functional forebrain/midbrain circuits.