Whey protein plus bicarbonate supplement has little effects on structural atrophy and proteolysis marker immunopatterns in skeletal muscle disuse during 21 days of bed rest.

OBJECTIVES: To investigate the effect of whey protein plus potassium bicarbonate supplement on disused skeletal muscle structure and proteolysis after bed rest (BR). METHODS: Soleus (SOL) and vastus lateralis (VL) biopsies were sampled from ten (n=10) healthy male subjects (aged 31±6 years) who did BR once with and once without protein supplement as a dietary countermeasure (cross-over study design). The structural changes (myofibre size and type distribution) were analysed by histological sections, and muscle protein breakdown indirectly via the proteolysis markers, calpain 1 and 3, calpastatin, MuRF1 and 2, both in muscle homogenates and by immunohistochemistry. RESULTS: BR caused size-changes in myofiber cross-sectional area (FCSA, SOL, p=0,004; VL, p=0.03), and myofiber slow-to-fast type transition with increased hybrids (SOL, p=0.043; VL, p=0.037) however with campaign differences in SOL (p<0.033). No significant effect of BR and supplement was found by any of the key proteolysis markers. CONCLUSIONS: Campaign differences in structural muscle adaptation may be an issue in cross-over design BR studies. The whey protein plus potassium bicarbonate supplement did not attenuate atrophy and fibre type transition during medium term bed rest. Alkaline whey protein supplements may however be beneficial as adjuncts to exercise countermeasures in disuse.

Bone and muscle structure and quality preserved by active versus passive muscle exercise on a new stepper device in 21 days tail-suspended rats.

Human performance in microgravity is characterized by reversed skeletal muscle actions in terms of active vs. passive mode contractions of agonist/antagonist groups that may challenge principal biodynamics (biomechanical forces translated from muscle to bone) of the skeletal muscle-bone unit. We investigated active vs. passive muscle motions of the unloaded hindlimb skeletal muscle-bone unit in the 21 days tail-suspended (TS) rat using a newly designed stepper exercise device. The regimen included both active mode motions (TSA) and passive mode motions (TSP). A TS-only group and a normal cage group (CON) served as positive or negative controls. The muscle and bone decrements observed in TS-only group were not seen in the other groups except TSP. Active mode motions supported femur and tibia bone quality (5% BMD, 10% microtrabecular BV/TV, Tb.Th., Tb.N. parameters), whole soleus muscle/myofiber size and type II distribution, 20% increased sarcolemma NOS1 immunosignals vs. CON, with 25% more hybrid fiber formation (remodeling sign) for all TS groups. We propose a new custom-made stepper device to be used in the TS rat model that allows for detailed investigations of the unique biodynamic properties of the muscle-bone unit during resistive-load exercise countermeasure trials on the ground or in microgravity.

The 2nd Berlin BedRest Study: protocol and implementation.

Long-term bed-rest is used to simulate the effect of spaceflight on the human body and test different kinds of countermeasures. The 2nd Berlin BedRest Study (BBR2-2) tested the efficacy of whole-body vibration in addition to high-load resisitance exercise in preventing bone loss during bed-rest. Here we present the protocol of the study and discuss its implementation. Twenty-four male subjects underwent 60-days of six-degree head down tilt bed-rest and were randomised to an inactive control group (CTR), a high-load resistive exercise group (RE) or a high-load resistive exercise with whole-body vibration group (RVE). Subsequent to events in the course of the study (e.g. subject withdrawal), 9 subjects participated in the CTR-group, 7 in the RVE-group and 8 (7 beyond bed-rest day-30) in the RE-group. Fluid intake, urine output and axiallary temperature increased during bed-rest (p < .0001), though similarly in all groups (p > or = .17). Body weight changes differed between groups (p < .0001) with decreases in the CTR-group, marginal decreases in the RE-group and the RVE-group displaying significant decreases in body-weight beyond bed-rest day-51 only. In light of events and experiences of the current study, recommendations on various aspects of bed-rest methodology are also discussed.

Molecular biomarkers monitoring human skeletal muscle fibres and microvasculature following long-term bed rest with and without countermeasures.

The cellular mechanisms of human skeletal muscle adaptation to disuse are largely unknown. The aim of this study was to determine the morphological and biochemical changes of the lower limb soleus and vastus lateralis muscles following 60 days of head-down tilt bed rest in women with and without exercise countermeasure using molecular biomarkers monitoring functional cell compartments. Muscle biopsies were taken before (pre) and after bed rest (post) from a bed rest-only and a bed rest exercise group (n = 8, each). NOS1 and NOS3/PECAM, markers of myofibre 'activity' and capillary density, and MuRF1 (E3 ubiquitin-ligase), a marker of proteolysis, were documented by confocal immunofluorescence and immunoblot analyses. Morphometrical parameters (myofibre cross-sectional area, type I/II distribution) were largely preserved in muscles from the exercise group with a robust trend for type II hypertrophy in vastus lateralis. In the bed rest-only group, the relative NOS1 immunostaining intensity was decreased at type I and II myofibre membranes, while the bed rest plus exercise group compensated for this loss particularly in soleus. In the microvascular network, NOS3 expression and the capillary-to-fibre ratio were both increased in the exercise group. Elevated MuRF1 immunosignals found in subgroups of atrophic myofibres probably reflected accelerated proteolysis. Immunoblots revealed overexpression of the MuRF1 protein in the soleus of the bed rest-only group (> 35% vs. pre). We conclude that exercise countermeasure during bed rest affected both NOS/NO signalling and proteolysis in female skeletal muscle. Maintenance of NO signalling mechanisms and normal protein turnover by exercise countermeasure may be crucial steps to attenuate human skeletal muscle atrophy and to maintain cell function following chronic disuse.

Nitric oxide and fibroblast growth factor in autonomic nervous system: short- and long-term messengers in autonomic pathway and target-organ control.

The freely diffusible messenger nitric oxide (NO), generated by NO synthase (NOS)-containing "nitroxergic" (NO-ergic) neurons, is unique among classical synaptic chemical transmitters because of its "non-specificity", molecular "NO-receptors" (e.g. guanylyl cyclase, iron complexes, nitrosylated proteins or DNA) in target cells, intracellular targeting, regulated biosynthesis, and growth factor/cytokine-dependence. In the nervous system, expression of NOS is particularly intriguing in central and peripheral autonomic pathways and their targets. Here, anatomical and functional links appear to exist between NOS, its associated catalytic NADPH-diaphorase enzyme activity (NOSaD) and fibroblast growth factor-2 (FGF-2), a pleiotropic cytokine with mitogenic actions, suggesting mutual "short- and long-term" actions. Several recent studies performed in the rat sympathoadrenal system, an anatomically and neurochemically well-defined autonomic pathway with target-specific functional units of sympathetic preganglionic neurons (SPNs) in the spinal cord, provide evidence for this hypothesis. The NO and cytokine signals may interact at the level of gene expression, transcription factors, post-transcriptional control or second messenger cross-talk. Thus, unique biological roles of FGF-2 and the NO system are likely to exist in neuroendocrine actions, vasomotory perfusion control as well as in neurotrophic actions in sympathetic innervation of the adrenal gland. In view of their anatomical co-existence, functional interplay and synchronizing effects on neuronal networks, multiple roles are suggested for both "short- and long-term" signalling molecules in neuroendocrine functions and integrated autonomic target organ control.

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)

Neuroprotective fibroblast growth factor type-2 down-regulates the c-Jun transcription factor in axotomized sympathetic preganglionic neurons of adult rat.

The immediate-early gene encoded transcription factor c-Jun is highly inducible following axotomy and therefore serves as a valuable marker in neuronal de- and regeneration. As the signals that may trigger c-Jun expression are still obscure, molecules derived from lesioned neurons and/or their targets such as growth factors or cytokines have been proposed as candidates for interneuronal transcriptional regulation in vivo. We therefore tested whether local administration of the neuroprotective cytokine fibroblast growth factor type-2 in vivo has an effect on the axotomy-induced nuclear expression patterns of the activator protein-1 transcription factors c-Fos and JunB, or c-Jun in the spinal cord-intermedolateral nucleus-adrenal axis lesion paradigm in the rat. Partial axotomy of preganglionic nerve fibres by selective unilateral removal of the adrenal medulla resulted in strong staining patterns of c-Jun in the nuclei of preganglionic cell bodies located in the spinal intermediolateral cell column identified by in vivo retrograde prelabelling with the fluorescent tracer Fast Blue prior to lesion. Axotomy-induced nuclear c-Jun expression was highly increased when compared with the moderate baseline expression in normal or sham-operated animals. In animals treated with fibroblast growth factor-2 gelfoams implanted to the lesioned adrenal gland the nuclear c-Jun staining pattern is reduced or even absent from these neurons. By contrast, c-Fos and JunB induction did not occur in the intermediolateral nucleus in the lesion paradigm investigated. These results support the idea of functional links between neurotrophic cytokines such as fibroblast growth factor-2 and transcriptional effectors such as c-Jun. The target derived fibroblast growth factor-2 thus may signal the intactness of the neuron-target axis resulting in suppression of central extrinsic neurons and promotion of neuroprotective gene activation. Neuronal survival in absence of c-Jun indicates that c-Jun exerts negative actions in vulnerated neurons.

Co-existence of NADPH-diaphorase, fibroblast growth factor-2 and fibroblast growth factor receptor in spinal autonomic system suggests target-specific actions.

In the rat spinal cord, we found substantial co-existence of fibroblast growth factor-2, fibroblast growth factor receptor (type-1 or flg) immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity (a histochemical marker for neuronal nitric oxide synthase) in preganglionic autonomic cell groups of intermediate layers VI, VII and X. Anti-fibroblast growth factor-2 and anti-nitric oxide synthase binding sites were confined to the cytoplasm of reactive neurons as judged by immunogold electron microscopy. Within the major autonomic nucleus, i.e. intermediolateral column, three different populations were identified: (i) fibroblast growth factor and fibroblast growth factor receptor, (ii) fibroblast growth factor/NADPH-diaphorase and (iii) NADPH-diaphorase-only stained cell groups. Sympathoadrenal neurons were prelabelled with fluorescent tracer Fast Blue and co-stained for fibroblast growth factor-like protein and NADPH-diaphorase, suggesting heterologous diversification of neuronal phenotypes and functional organization in the spinal autonomic system. Our findings suggest intriguing roles for nitric oxide and fibroblast growth factor-2 cytokine in the preganglionic sympathetic spinal cord system: The "short-term" diffusible messenger nitric oxide may act as "tonic" and/or "phasic" signal within rostrocaudally oriented function-specific preganglionic units necessary for integrated target control. The "long-term" messenger fibroblast growth factor-2 may be involved in, for example, cytokine-dependent regulation of neuronal NADPH-diaphorase/nitric oxide synthase. Furthermore, co-existence of NADPH-diaphorase, fibroblast growth factor-2 and receptor in sympathoadrenal neurons suggest mutual target-specific regulatory functions, e.g. hormone release and blood perfusion or maintenance of phenotype and plasticity responsiveness of adrenal medullary tissue.

Just in time and place: NOS/NO system assembly in neuromuscular junction formation.

Recent advances in the molecular, biochemical, and anatomical aspects of postsynaptic membrane components at the neuromuscular junction (NMJ) are briefly reviewed focussing on assembly, architecture, and function of the multi-subunit dystrophin-protein complex (DPC) and its associated nitric oxide (NO)-signaling complex. Elucidation of unique structural binding motifs of NO-synthases (NOS), and microscopical codistribution of neuronal NOS (nNOS), the major isoform of NOS expressed at the NMJ, with known synaptic proteins, i.e., family members of the DPC, nicotinic acetylcholine receptor (AChR), NMDA-receptor, type-1 sodium and Shaker K(+)-channel proteins, and linker proteins (e.g., PSD-95, 43K-rapsyn), suggests targeting and assembly of the NO-signaling pathway at postsynaptic membrane components. NO mediates agrin-induced AChR-aggregation and downstream signal transduction in C2 skeletal myotubes while administration of L-arginine, the limiting substrate for NO-biosynthesis, enhances aggregation of synapse-specific components such as utrophin. At the NMJ, NO appears to be a mediator of (1) early synaptic protein clustering, (2) synaptic receptor activity and transmitter release, or (3) downstream signaling for transcriptional control. Multidisciplinary data obtained from cellular and molecular studies and from immunolocalization investigations have led us to propose a working model for step-by-step binding of nNOS, e.g., to subunit domains of targeted and/or preexisting membrane components. Formation of NOS-membrane complexes appears to be governed by agrin-signaling as well as by NO-signaling, supporting the idea that parallel signaling pathways may account for the spatiotemporally defined postsynaptic assembly thereby linking the NOS/NO-signaling cascade to early membrane aggregations and at the right places nearby preexisting targets (e.g., juxtaposition of NO source and target) in synapse formation.

Matrix metalloproteinases MMP-2, MMP-7 and MMP-9 in denervated human muscle.

Proteolytic enzyme expression was studied by matrix metalloproteinases (MMP) immunoreactivity (-IR) in muscle biopsies from patients with amyotrophic lateral sclerosis (ALS), spinal muscle atrophy (SMA) and chronic axonal neuropathies (CANP). In normal muscle MMP-2-, MMP-7-, and MMP-9-IR was localized at neuromuscular junctions, in vessels and nerve branches. ALS biopsies of clinically non-affected muscles revealed neither MMP-2, -7-IR nor MMP-9-IR in atrophied myofibers. ALS-affected biopsies revealed MMP-9-IR, and to lesser extent MMP-2- and MMP-7-IR at normal sized and atrophied myofibers. Biopsies of SMA showed MMP-9- and MMP-7-IR at normal sized and atrophic myofibers, while MMP-2-IR was undetectable. In CANP MMP-9-IR was found at normal sized and atrophied myofibers. Distinct expression patterns of MMPs may thus reflect different stages of muscle denervation atrophy.

NOS type-1 mRNA expression and protein localization in spinal autonomic neurons.

Autonomic neurons of the rat spinal cord show strong NADPH diaphorase activity and immunoreactivity for nitric oxide synthase (NOS). Here we show mRNA expression of NOS type-1 (neuronal or brain NOS) transcripts in cell bodies of sympathetic preganglionic neurons (SPNs) of the intermediolateral (IML) cell column by non-radioactive in situ hybridization using NOS-I riboprobes. Hybridization signals occurred only in neuronal cell bodies and not outside, in what appeared to be fibers and/or terminals. In preganglionic fibers of SPNs, however, dense axoplasmic immunogold labeling was detected with a monoclonal anti-NOS-I antibody. Expression of NOS-I mRNA in SPN cell bodies and axoplasmic immunolocalization of NOS-I protein suggest that protein translocation is involved in NO-mediated preganglionic control of peripheral targets.

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.

Spatial and temporal patterns of neurotrophic activities in rat adrenal medulla and cortex.

We have studied the spatial distribution and temporal pattern of expression of neurotrophic factor (NTF) activity present in the rat adrenal gland. Tissue extracts of the densely innervated medulla and sparsely innervated cortex from adult and various developmental stages were assayed for their ability to promote the in vitro survival of embryonic chick ciliary (CG), dorsal root ganglia (DRG) and ventral spinal cord (SC) neurons. NTF activity was found in medulla but not in cortex extracts. NTF activity became first detectable at postnatal day (P) 12. At this developmental stage the cholinergic adrenomedullary innervation becomes functional. Specific activity successively increased in medulla extracts from P16 to P30 as revealed by the CG-assay. No further changes occurred during adulthood. In contrast, activity addressing SC neurons present in P16 and P30 medulla extracts could not be detected in adult stages P90 or P120. In DRG-assays, NTF activity could not be blocked by the addition of anti-NGF antibodies to medulla extracts. The activity was sensitive to heat and protease treatment suggesting its proteinaceous nature. At high concentrations cortex extracts had neurotoxic effects that were also seen when 10 microM of dexamethasone were added to saturated amounts of medulla extract. However, gel filtration of cortex material to remove low molecular components including corticosteroid hormones failed to reveal any NTF activity in these preparations. Adrenal NTF activity therefore appears to be restricted to the densely innervated medullary tissue. Moreover, our results also suggest a distinct temporal pattern of NTF activity in the adrenal gland.

Ciliary neurotrophic factor supports target-deprived preganglionic sympathetic spinal cord neurons.

Spinal cord neurons in the intermediolateral column (IML) that innervate the adrenal medulla require target-derived factor(s) for their maintenance in vivo. Selective destruction of the adult rat adrenal medulla causes an approximate 25% loss of Nissl-stained IML neurons between spinal cord levels Th 4 weeks after surgery. We have previously suggested that basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF) or closely related molecules are present in adrenal chromaffin cells and granules. Basic FGF supplemented in gelfoams to the medullectomized adrenal gland fully prevented IML neuron losses. These results are now extended by demonstrating that (i) CNTF administered in vivo (7.2 micrograms/gelfoam) also rescues IML neurons, and (ii) the rescue effect is abolished by splanchnicotomy, i.e. interruption of axonal pathway from the spinal cord to the adrenal gland. These data would be consistent with CNTF being a retrograde trophic messenger to the spinal cord, but do not exclude the possibility that CNTF mimics or induces the formation of an endogenous trophic factor.

In situ identification of neuronal nitric oxide synthase (NOS-I) mRNA in mouse and rat skeletal muscle.

Skeletal muscle provides a major source of the signaling molecule nitric oxide (NO) however in situ identification of NO-synthase (NOS) mRNA has not been verified. We have used NOS-I (neuronal NOS) probes prepared from plasmid DNA by reverse transcription-polymerase chain reaction (RT-PCR) to detect mRNA transcripts in skeletal muscle cells and myofibers of rat and mouse. Mouse C2C12 myoblasts and myotubes reveal strong cytosolic in situ hybridization (ISH) signals in vitro. In adult animals, ISH signals are detectable in striated myofibers at subsarcolemmal and perinuclear regions whilst the myofibrillar compartment is devoid of signals. Expression of NOS-I mRNA in fusion-competent myoblasts suggests that the NOS/NO system is of relevance to myogenic differentiation. Compartmentalization of NOS-I mRNA may reflect spatiofunctional actions between NOS message and protein and the putative subcellular NO targets.

Partial loss of NADPH-diaphorase/nitric oxide synthase-complex in amyotrophic lateral sclerosis and human type-II myofiber atrophy.

To substantiate the role of nitric oxide synthase type-I (NOS-I) in neurogenic muscular disorders we investigated human biopsy samples of type-II fiber atrophy and amyotrophic lateral sclerosis (ALS) by NOS-I immunoreactivity (-IR), NOS-associated NADPH-dependent diaphorase activity (NOSaD) and Western blot analysis. In type-II atrophy, loss of NOSaD and reduced NOS-I-IR was apparent in atrophic myofibers. In atrophic fiber groups lacking NOSaD, both NOS-I and dystrophin-IR was decreased while sarcolemmal beta-dystroglycan- and adhalin-IR (markers of the sarcolemmal dystrophin-glycoprotein complex) was normal. In ALS, groups of scattered angulated atrophic fibers revealed partial loss of NOS-I-IR/NOSaD. Atrophied fibers of either type-I or type-II thus revealed differential sarcolemmal NOS/NOSaD pattern thereby reflecting myopathological alterations of the NO-system in human type-II atrophy and ALS.

Light-microscopic studies on spatial and temporal binding of the lectins concanavalin A, wheat-germ agglutinin and peanut agglutinin in early rat odontogenesis.

The spatial distribution and temporal expression of alpha-D-mannosyl(glucosyl)-, N-acetyl-D-glucosaminyl- and beta-D-galactosyl residues as detected by peroxidase-conjugated lectins correlated with early odontogenic events in six principal developmental stages (fetal days 13.5, 14, 15, 17, 18.5 and 19.5). The odontogenic epithelium of 13.5- and 14-day-old fetuses was characterized by strong concanavalin A (Con A) binding and between days 17 and 19.5, the stellate reticulum displayed strong peanut agglutinin (PNA) binding. Between 15 and 19.5, differentiation of dental ectomesenchyme was characterized by a rhythmic expression of terminal galactosyl residues shown by PNA-binding. At the developing dental basement membrane, there were various carbohydrate-specific regions. At days 13.5 and 14, the odontogenic basement membrane was specific for N-acetyl-D-glucosamines detected by wheat-germ agglutinin (WGA). The results suggest that the carbohydrates present at the inner dental basement membrane at days 17 to 19.5 may be involved in cell-matrix interactions during cytodifferentiation.

Adhalin (alpha-sarcoglycan) is not required for anchoring of nitric oxide synthase I (NOS I) to the sarcolemma in non-mammalian skeletal (striated) muscle fibers.

Previous studies have shown the association of NOS I with the sarcolemma in mammalian striated muscle fibers, implicating the dystrophin complex (DC) as a major anchor for the enzyme. The potential role of the sarcoglycan subcomplex, especially of alpha-sarcoglycan (adhalin), as part of the DC in holding of NOS I in the sarcolemmal position was examined by carrying out a comparative study on the distribution of NOS I, dystrophin, dystrophin-associated glycoproteins (DAG) and alpha-sarcoglycan in various skeletal muscles of non-mammals. Rat muscles were included since they reflect the situation in mammals. Catalytic NOS-associated diaphorase (NOSaD) activity as well as NOS I and DAG immunoreactivities were positive in the saracolemma region of skeletal muscle fibers of rats, chicken, and turtles. Adhalin immunoreactivity was present in the rat but absent in the chicken and turtle muscle surface membrane. These data suggest that alpha-sarcoglycan and therefore the entire sarcoglycan subcomplex may not be needed for localizing NOS I to the sarcolemma in these non-mammalian species. This may hold for skeletal muscle fibers in general.

Nitric oxide and target-organ control in the autonomic nervous system: anatomical distribution, spatiotemporal signaling, and neuroeffector maintenance.

Recent neuroanatomical studies, neurochemical coding and physiological findings of multiple cotransmitter actions and/or receptor patterns, and the characterization of synaptic molecules and nitrergic (NOergic) signaling mechanisms may help for a better understanding of target-organ control in the autonomic nervous system. Thus, nitric oxide (NO) synthase, which generates the freely diffusible and short-lived messenger NO and expression of neurotrophic proteins (e.g., neurotrophins, glial cell-line-derived neurotrophic factor, fibroblast growth factors) in autonomic neural pathways or target organs suggest unique actions in autonomic neurotransmission. In central NOergic pathways, NO may serve as spatial (volume) messenger within hierarchically ordered autonomic neuron pools and convergent/divergent pathways for synchronized autonomic outflow. Likewise, NO modulates intraganglionic and interaxonal transmission and postganglionic activity including long-term potentiation. In the visceral targets, NO appears to be a spatial modulator in local intrinsic networks or at varicose terminals. In endocrine glands, NO possibly acts as synaptic coactivator or inhibitor, as a cotransmitter affecting stimulus-coupled exocytosis, or as a local vasoactive signal. The short-term neural messenger NO may also induce diffusible target-derived long-term neurotrophic signals, thereby supporting neuroeffector maintenance and plasticity, if not synaptic efficacy, in autonomic target-organ control.