Microscopic Study of Nervous System Plasticity: Interactions of Sympathetic Nerves with Neurons of Intraocular Hippocampal Transplants.

Functional interactions of sympathetic fibers innervating the iris with the neurons of central origin in intraocular transplants of the rat hippocampus were studied by optic, confocal, and electron microscopy. After formaldehyde fixation, fluorescent dye Dil was applied to the upper cervical ganglion; the dye migrated to the transplant by lateral diffusion via axons. Sympathetic nerves labeled with fluorescent dye grew into the neurotransplants along perivascular membranes of blood vessels. In addition, some fluorescent axons were identified in the transplant parenchyma. Electron microscopy showed large bundles of the peripheral type axons in the vascular adventitia and Schwann-axonal complexes in the transplant neuropil. Autonomic axons formed synaptic contacts with transplanted neurons.

Differential toxicities of intraneurally injected mercuric chloride for sympathetic and somatic motor fibers: an ultrastructural study.

BACKGROUND/PURPOSE: Mercury is a well-known neurotoxin but the susceptibility of autonomic nerves to mercury poisoning in vivo has seldom been studied. Our previous studies have shown that the hypoglossal nerve in hamsters contains somatic motor and postganglionic sympathetic fibers. The aim of this study was to investigate the ultrastructural changes in the nervous system following intraneural injection of mercuric chloride into the hypoglossal nerve in hamsters. METHODS: Six adult hamsters were used in this study. After anesthesia, the digastric muscle on the right side was removed and the trunk of the hypoglossal nerve was exposed. Two microliters of mercuric chloride aqueous solution was injected into the main trunk of the hypoglossal nerve at the bifurcation. The contralateral hypoglossal nerve was kept intact and used as the normal control. Animals were allowed to survive for 1 or 3 days and were prepared for ammonium sulfide histochemistry and electron microscopy. RESULTS: Three days after injection of mercuric chloride solution, almost all unmyelinated sympathetic fibers in the hypoglossal nerve trunk were lost, whereas myelinated somatic axons were spared. Although mercury deposition in the myelin sheaths of neuronal processes was observed in the hypoglossal nucleus, the neuronal somas were intact. By contrast, degenerated neuronal processes and mercury deposition in neuronal somas were frequently found in the superior cervical ganglia. CONCLUSION: This study demonstrated an undue susceptibility of sympathetic fibers to mercury intoxication. The mechanisms that underlie the selective reaction of sympathetic fibers to mercury warrant further investigation.

Ultrastructural changes of posterior lingual glands after hypoglossal denervation in hamsters.

Posterior lingual glands consist of two sets of minor salivary glands that serve important functions in oral physiology. To investigate the hypothesis that the hypoglossal nerve provides sympathetic innervation to the posterior lingual glands, we examined ultrastructural changes in the glands following hypoglossal denervation. In the posterior deep lingual glands (of von Ebner), the serous acinar cells showed a decrease in the number of secretory granules and an increase in lipofuscin accumulation. The ratios of cells containing lipofuscin granules were 11.39, 36.49 and 50.46%, respectively, of the control, 3- and 7-day post-axotomy glands (P < 0.001). Intraepithelial phagocytotic activity was increased. The mucous acinar cells in the posterior superficial lingual glands (of Weber) also showed degenerative changes after hypoglossal denervation. One week after nerve transection, marked cytoplasmic vacuolation and fragmentation of organelles were frequently observed. Degenerative changes were also found in unmyelinated axons associated with the glands. We provide the first evidence of the structural and functional connections between the sympathetic component of the hypoglossal nerve and posterior lingual glands.

Hepatocyte innervation in primates.

The efferent innervation and some characteristics of nerve fibers of the liver lobule in the tree shrew, a primate, are described. Nerve endings on hepatocytes were encountered regularly and were determined to be efferent adrenergic nerves. Transmission electron microscopy revealed nerve endings and varicosities in close apposition to the hepatocytes adjacent to the connective tissue of the triads as well as within the liver lobule in the space of Disse. Fluorescence microscopy indicated the existence of adrenergic nerves with a similar distribution. Autoradiography of the avid uptake of exogenous [3H]norepinephrine indicated that all intralobular nerves are potentially norepinephrinergic (adrenergic). Chemical sympathectomy with 6-OH-dopamine resulted in the degeneration of all intralobular liver nerve fibers as revealed by fluorescence microscopy and electron microscopy. Substantial regeneration occurred after 60-90 days but was not completed by that time. Some nerves were also observed in close association with von Kupffer cells and endothelial cells. The functional significance of the efferent liver innervation is discussed.

Spatial organization of axonal microtubules.

Several workers have found that axonal microtubules have a uniform polarity orientation. It is the "+" end of the polymer that is distal to the cell body. The experiments reported here investigate whether this high degree of organization can be accounted for on the basis of structures or mechanisms within the axon. Substantial depolymerization of axonal microtubules was observed in isolated, postganglionic sympathetic nerve fibers of the cat subjected to cold treatment; generally less than 10% of the original number of microtubules/micron 2 remained in cross section. The number of cold stable MTs that remained was not correlated with axonal area and they were also found within Schwann cells. Microtubules were allowed to repolymerize and the polarity orientation of the reassembled microtubules was determined. In fibers from four cats, a majority of reassembled microtubules returned with the original polarity orientation. However, in no case was the polarity orientation as uniform as the original organization. The degree to which the original orientation returned in a fiber was correlated with the number of cold-stable microtubules in the fiber. We suggest that stable microtubule fragments serve as nucleating elements for microtubule assembly and play a role in the spatial organization of neuronal microtubules. The extremely rapid reassembly of microtubules that we observed, returning to near control levels within the first 5 min, supports microtubule elongation from a nucleus. However, in three of four fibers examined this initial assembly was followed by an equally rapid, but transient decline in microtubule number to a value that was significantly different than the initial peak. This observation is difficult to interpret; however, a similar transient peak has been reported upon repolymerization of spindle microtubules after pressure induced depolymerization.

Biochemical and immunological analyses of cytoskeletal domains of neurons.

We have used cultured sympathetic neurons to identify microtubule proteins (tubulin and microtubule-associated proteins [MAPs]) and neurofilament (NF) proteins in pure preparations of axons and also to examine the distribution of these proteins between axons and cell bodies + dendrites. Pieces of sympathetic ganglia containing thousands of neurons were plated onto culture dishes and allowed to extend neurites. Dendrites remained confined to the ganglionic explant or cell body mass (CBM), while axons extended away from the CBM for several millimeters. Axons were separated from cell bodies and dendrites by dissecting the CBM away from cultures, and the resulting axonal and CBM preparations were analyzed using biochemical, immunoblotting, and immunoprecipitation methods. Cultures were used after 17 d in vitro, when 40-60% of total protein was in the axons. The 68,000-mol-wt NF subunit is present in both axons and CBM in roughly equal amounts. The 145,000- and 200,000-mol-wt NF subunits each consist of several variants which differ in phosphorylation state; poorly and nonphosphorylated species are present only in the CBM, whereas more heavily phosphorylated forms are present in axons and, to a lesser extent, the CBM. One 145,000-mol-wt NF variant was axon specific. Tubulin is roughly equally distributed between CBM and axon-like neurites of explant cultures. MAP-1a, MAP-1b, MAP-3, and the 60,000-mol-wt MAP are also present in the CBM and axon-like neurites and show distribution patterns similar to that of tubulin. In contrast, MAP-2 was detected only in the CBM, while tau and the 210,000-mol-wt MAP were greatly enriched in axons compared to the CBM. In immunostaining analyses, MAP-2 localized to cell bodies and dendrite-like neurites, but not to axon-like neurites, whereas antibodies to tubulin and MAP-1b localized to all regions of the neurons. The regional differences in composition of the neuronal cytoskeleton presumably generate corresponding differences in its structure, which may, in turn, contribute to the morphological differences between axons and dendrites.

Growth of sympathetic nerve fibers in culture does not require extracellular calcium.

A compartmented culture system in which distal neurites from newborn rat sympathetic neurons entered a fluid environment separate from that bathing the cell bodies and proximal neurites was used to investigate effects of extracellular Ca2+ deprivation on nerve fiber growth. Neurites readily grew into, elongated for many days within, and regenerated after neuritotomy within distal compartments substantially deprived of Ca2+ (0 added Ca2+, 0.5-5 mM EGTA), provided Ca2+ was supplied to the cell bodies. The Ca2(+)-deprived neurites generally extended at rates 20%-35% slower than controls. Growth of neurites did, however, cease within 2 days when the cell bodies were deprived of Ca2+, and the neurites and cell bodies eventually degenerated. These results show that neither extracellular Ca2+ nor the influx of Ca2+ at or near the growth cone is required for sustained neurite growth. They also rule out the possibility that the promotion of neurite growth by nerve growth factor is mediated, by the influx of extracellular Ca2+.

Rapid effects of laminin on the growth cone.

To gain insight into how laminin promotes neurite growth, high resolution video microscopy was used to determine the rapid effects of laminin on growth cone structure. Sympathetic growth cones in serum-free medium on polylysine substrate displayed extensive motility and protrusive activity and often had large lamellipodia. However, their neurites grew slowly because membranous organelles from the central region advanced into the lamellipodium only slowly. Acute addition of laminin accelerated growth severalfold and had visible effects on the growth cone within minutes. Laminin dramatically accelerated the advance of membranous organelles, which, with microtubules, rapidly filled the lamellipodium. Retraction of individual protrusions (filopodia and veils) was rapidly reduced. These effects of laminin are important in accelerating growth and suggest a mechanism for pathway selection by growing neurites.

Investigation of close interactions between sympathetic neural fibres and the follicular dendritic cells network in the mouse spleen.

In this study, co-localization between sympathetic neural fibres and the follicular dendritic cells (FDCs) network was observed within the mouse spleen by confocal technology. Immunohistochemical techniques were used to reveal the rare interactions between the FDCs network and sympathetic neural fibres. We estimated the frequency of three kinds of close interactions which could be defined as overlaps, contacts or neural fibres closer than 10 microm from a FDCs network. Using these estimates, a comparison was made between five uninfected mouse strains exhibiting the same Prnpa genotype but showing different incubation periods when inoculated with primary bovine spongiform encephalopathy (BSE)-infected brain. In prion disease, infectivity is generally detected in the spleen much earlier than in the brain, especially after peripheral inoculation. The way by which the infectious agent reaches the central nervous system is still unclear. From the five mouse strains, we obtained differences in the proportion of splenic FDCs networks with close interactions. Our work suggests that the percentage of splenic FDCs networks with at least one sympathetic neural fibre in close vicinity may influence the length of incubation period.

[Comparative analysis of vasomotor responses and sympathetic innervation of feed arteries from locomotor and respiratory muscles in a rat].

The characteristics of feeding arteries of diaphragm and medial gastrocnemius (with a diameter of 200-250 micron) were studied. The registration of the mechanical activity of ring preparations under isometric conditions revealed that diaphragm arteries, like arteries of other muscles with a high content of slow muscle fibers, are highly sensitive to adrenoceptor agonists and acetylcholine. The differences in endothelium-dependent relaxation between diaphragm and gastrocnemius arteries are preserved in the presence of L-NAME and diclofenac. Responses to serotonin in diaphragm and gastrocnemius arteries are similar. At the same time, the high density of innervation is characteristic of diaphragm artery only, while in other slow muscles it is low. The density of adrenergic fibers plexus in the diaphragm artery is much higher than in the gastrocnemius artery. The results suggest that the properties of small arteries of diaphragm are determined not only by the oxidative capacity of diaphragm muscle fibers but also by the belonging of the diaphragm to respiratory musculature.

An intrinsic adrenergic system in mammalian heart.

We have identified a previously undescribed intrinsic cardiac adrenergic (ICA) cell type in rodent and human heart. Northern and Western blot analyses demonstrated that ICA cell isolates contain mRNA and protein of enzymes involved in catecholamine biosynthesis. Radioenzymatic catecholamine assays also revealed that the catecholamine profile of adult rat ICA cell isolates differed from that of sympathetic neurons. Unlike sympathetic neuronal cells, isolated ICA cells have abundant clear vesicles on electron microscopy. Endogenous norepinephrine and epinephrine constitutively released by ICA cells in vitro affect the spontaneous beating rate of neonatal rat cardiac myocytes in culture. Finally, ICA cells could be identified in human fetal hearts at a developmental stage before sympathetic innervation of the heart has been documented to occur. These findings support the concept that these cells constitute an ICA signaling system capable of participating in cardiac regulation that appears to be independent of sympathetic innervation.

Wide distribution and subcellular localization of histamine in sympathetic nervous systems of different species.

Previous studies have demonstrated that histamine (HA) acts as a neurotransmitter in the cardiac sympathetic nervous system of the guinea pig. The aim of the current study was to examine whether HA widely exists in the sympathetic nervous systems of other species and the subcellular localization of HA in sympathetic terminals. An immunofluorescence histochemical multiple-staining technique and anterograde tracing method were employed to visualize the colocalization of HA and norepinephrine (NE) in sympathetic ganglion and nerve fibers in different species. Pre-embedding immunoelectron microscopy was used to observe the subcellular distribution of HA in sympathetic nerve terminals. Under the confocal microscope, coexistence of NE and HA was displayed in the superior cervical ganglion and celiac ganglion neurons of the mouse and dog as well as in the vas deferens, mesenteric artery axon, and varicosities of the mouse and guinea pig. Furthermore, colocalization of NE and HA in cardiac sympathetic axons and varicosities was labeled by biotinylated dextranamine injected into the superior cervical ganglion of the guinea pig. By electron microscopy, HA-like high-density immunoreactive products were seen in the small vesicles of the guinea pig vas deferens. These results provide direct cellular and subcellular morphological evidence for the colocalization of HA and NE in sympathetic ganglion and nerve fibers, and support that HA is classified as a neurotransmitter in sympathetic neurons.

Morphology and ultrastructure of the sympathetic celiac ganglion neurons projecting to the cardia and pylorus of the rat stomach.

The stomach receives sympathetic projections from the celiac ganglion. To determine what kinds of neurons in the celiac ganglion project to the cardia or the pylorus of the stomach, we injected the retrograde tracer Fluoro-Gold into the cardia and the retrograde tracer cholera toxin subunit b into the pylorus of the same animal. A few neurons (about 10%) innervating the cardia sent collateral projections to the pylorus. Ultrastructural observations revealed that the celiac ganglion contained oval, medium-sized to large neurons. They had a dark cytoplasm containing numerous free ribosomes, rough endoplasmic reticulum, mitochondria, lysosomes, several Golgi apparatuses, and an oval nucleus. The axon terminals were small and usually contacted thin processes extending from the dendrites or the soma. About half of the terminals contained round vesicles, while the rest contained pleomorphic vesicles. Both types of terminals made asymmetric synaptic contacts. We then retrogradely labeled the neurons projecting to the cardia and the pylorus with wheat germ agglutinin conjugated horseradish peroxidase to examine their ultrastructural characteristics. The neurons projecting to the cardia (33.3x22.4 microm) were similar to the neurons projecting to the pylorus (33.4x24.7 microm) in their size and ultrastructural appearance. The neurons not projecting to the stomach (40.4x28.0 microm) were significantly larger than the neurons projecting to the cardia or the pylorus. Only a few axosomatic terminals were found on the neurons projecting to the cardia (1.6 per somatic profile), the pylorus (1.3) or the neurons not projecting to the stomach (0.9). These results provide morphological bases for the sympathetic motor neurons innervating the stomach.

Ultrastructural evidence for selective noradrenergic innervation of CNS vagal projections to the fundus of the rat.

We reported pharmacological data suggesting that stimulation of the vago-vagal reflex activates noradrenergic neurons in the hindbrain that inhibit dorsal motor nucleus of the vagus (DMV) neurons projecting to the fundus, but not to the antrum [Ferreira Jr., M., Sahibzada, N., Shi, M., Panico, W., Neidringhaus, M., Wasserman, A., Kellar, K.J., Verbalis, J., Gillis, R.A., 2002. CNS site of action and brainstem circuitry responsible for the intravenous effects of nicotine on gastric tone. J. Neurosci. 22, 2764-2779.]. The purpose of this study was to use an ultrastructural approach to test the hypothesis that noradrenergic terminals form synapses with DMV fundus-projecting neurons, but not with DMV antrum-projecting neurons. A retrograde tracer, CTbeta-HRP, was injected into the gastric smooth muscle of either the fundus or the antrum of rats. Animals were re-anesthetized 48 h later and perfusion-fixed with acrolein and paraformaldehyde. Brainstems were processed histochemically for CTbeta-HRP, and immunocytochemically for either DbetaH or PNMT by dual-labeling electron microscopic methods. Most cell bodies and dendrites of neurons that were retrogradely labeled from the stomach occurred at the level of the area postrema. Examination of 482 synapses on 238 neurons that projected to the fundus revealed that 17.4+/-2.7% (n=4) of synaptic contacts were with DbetaH-IR terminals. Of 165 fundus-projecting neurons, 4.4+/-1.5% (n=4) formed synaptic contacts with PNMT-IR terminals. In contrast, the examination of 384 synapses on 223 antrum-projecting neurons revealed no synaptic contact with DbetaH-IR terminals. These data provide proof that norepinephrine containing nerve terminals synapse with DMV fundus-projecting neurons but not with DMV antrum-projecting neurons. These data also suggest that brainstem circuitry controlling the fundus differs from circuitry controlling the antrum.

[Sympathetic and parasympathetic centers of the brain and spinal marrow of rats during exposure to +Gz].

Structural transformations in the dorsal vagal complex and intermediolateral nucleus due to +G, loads were studied in white outbred male rats centrifuged according to the standard procedure (P.S.Paschenko, 1995). Methods of investigation included light and electron microscopy, morphometric analysis and statistical analysis. Acute exposure to +Gz loads resulted in essentially reactive changes in the centers under study. At the same time, regular exposure to this extreme factor led to cumulation of destructive changes. The peculiar structure of the centers governs uniqueness of disorders which may unbalance the autonomous regulation of organism functions.

Dense Intra-adipose Sympathetic Arborizations Are Essential for Cold-Induced Beiging of Mouse White Adipose Tissue.

Efferent signals from the central nervous system represent a key layer of regulation of white adipose tissue (WAT). However, the mechanism by which efferent neural signals control WAT metabolism remains to be better understood. Here, we exploit the volume fluorescence-imaging technique to visualize the neural arborizations in mouse inguinal WAT at single-fiber resolution. The imaging reveals a dense network of sympathetic arborizations that had been previously undetected by conventional methods, with sympathetic fibers being in close apposition to > 90% of adipocytes. We demonstrate that these sympathetic fibers originate from the celiac ganglia, which are activated by cold challenge. Sympathetic-specific deletion of TrkA receptor or pharmacologic ablation by 6-hydroxydopamine abolishes these intra-adipose arborizations and, as a result, cold-induced beiging of inguinal WAT. Furthermore, we find that local sympathetic arborizations function through beta-adrenergic receptors in this beiging process. These findings uncover an essential link connecting efferent neural signals with metabolism of individual adipocytes.

Overexpression of Sarcoendoplasmic Reticulum Calcium ATPase 2a Promotes Cardiac Sympathetic Neurotransmission via Abnormal Endoplasmic Reticulum and Mitochondria Ca2+ Regulation.

Reduced cardiomyocyte excitation-contraction coupling and downregulation of the SERCA2a (sarcoendoplasmic reticulum calcium ATPase 2a) is associated with heart failure. This has led to viral transgene upregulation of SERCA2a in cardiomyocytes as a treatment. We hypothesized that SERCA2a gene therapy expressed under a similar promiscuous cytomegalovirus promoter could also affect the cardiac sympathetic neural axis and promote sympathoexcitation. Stellate neurons were isolated from 90 to 120 g male, Sprague-Dawley, Wistar Kyoto, and spontaneously hypertensive rats. Neurons were infected with Ad-mCherry or Ad-mCherry-hATP2Aa (SERCA2a). Intracellular Ca2+ changes were measured using fura-2AM in response to KCl, caffeine, thapsigargin, and carbonylcyanide-p-trifluoromethoxyphenylhydrazine to mobilize intracellular Ca2+ stores. The effect of SERCA2a on neurotransmitter release was measured using [3H]-norepinephrine overflow from 340 to 360 g Sprague-Dawley rat atria in response to right stellate ganglia stimulation. Upregulation of SERCA2a resulted in greater neurotransmitter release in response to stellate stimulation compared with control (empty: 98.7±20.5 cpm, n=7; SERCA: 186.5±28.41 cpm, n=8; P<0.05). In isolated Sprague-Dawley rat stellate neurons, SERCA2a overexpression facilitated greater depolarization-induced Ca2+ transients (empty: 0.64±0.03 au, n=57; SERCA: 0.75±0.03 au, n=68; P<0.05), along with increased endoplasmic reticulum and mitochondria Ca2+ load. Similar results were observed in Wistar Kyoto and age-matched spontaneously hypertensive rats, despite no further increase in endoplasmic reticulum load being observed in the spontaneously hypertensive rat (spontaneously hypertensive rats: empty, 0.16±0.04 au, n=18; SERCA: 0.17±0.02 au, n=25). In conclusion, SERCA2a upregulation in cardiac sympathetic neurons resulted in increased neurotransmission and increased Ca2+ loading into intracellular stores. Whether the increased Ca2+ transient and neurotransmission after SERCA2A overexpression contributes to enhanced sympathoexcitation in heart failure patients remains to be determined.

Cocaine- and amphetamine-regulated transcript peptide and sympatho-adrenal axis.

Cocaine- and amphetamine-regulated transcript peptide (CART) is constitutively expressed in discrete regions of the mammalian central and peripheral nervous system. Immunohistochemical studies reveal a well-defined network of CART-immunoreactive (irCART) neurons organized along the sympatho-adrenal axis. Sympathetic preganglionic neurons, but not parasympathetic preganglionic neurons, in the lateral horn area are CART-positive; which in turn innervate postganglionic neurons in the paravertebral and prevertebral sympathetic ganglia as well as the adrenal medulla. A population of chromaffin cells in the adrenal medulla is CART-positive; whereas, postganglionic neurons are not. Sympathetic preganglionic neurons themselves are contacted by irCART cell processes arising from neurons in the arcuate nucleus, the retrochiasmatic nucleus and the rostral ventrolateral medulla. Results from several recent studies suggest CART directly excites neurons along the sympathetic neural axis or indirectly by potentiating the action of glutamate on NMDA receptors, as evidenced by an elevation of blood pressure and heart rate following intracerebroventricular, intracisternal or intrathecal administration of the peptide to anesthetized rats or conscious rabbits.

Evidence that estrogen directly and indirectly modulates C1 adrenergic bulbospinal neurons in the rostral ventrolateral medulla.

Blood pressure in women increases after menopause, and sympathetic tone in female rats decreases with estrogen injections in the rostral ventrolateral medulla (RVLM) region that contains bulbospinal C1 adrenergic neurons and is involved in blood pressure control. We investigated the anatomical and physiological basis for estrogen effects in the RVLM. Neurons with alpha- or beta-subtypes of estrogen receptor (ER) immunoreactivity (-ir) overlapped in distribution with tyrosine hydroxylase (TH)-containing C1 neurons. Immunoelectron microscopy revealed that ERalpha- and ERbeta-ir had distinct cellular and subcellular distributions. ERalpha-ir was most commonly in TH-lacking profiles, many of which were axons and peptide-containing afferents that contacted TH-containing dendrites. ERalpha-ir was also in some TH-containing dendrites. ERbeta-ir was most frequently in TH-containing somata and dendrites, particularly on endoplasmic reticula, mitochondria, and plasma membranes. In whole-cell patch clamp recordings from isolated bulbospinal RVLM neurons, 17beta-estradiol dose-dependently reduced voltage-gated Ca(++) currents, especially the long-lasting (L-type) component. This inhibition was reversed by washing or prevented by adding the non-subtype-selective ER antagonist ICI182780. An ERbeta-selective agonist, but not an ERalpha-selective agonist, reproduced the Ca(++) current inhibition. The data indicate that estrogens can modulate the function of RVLM C1 bulbospinal neurons either directly, through extranuclear ERbeta, or indirectly through extranuclear ERalpha in selected afferents. Moreover, Ca(++) current inhibition may underlie the decrease in sympathetic tone evoked by local 17beta-estradiol application. These findings provide a structural and functional basis for the effects of estrogens on blood pressure control and suggest a mechanism for the modulation of cardiovascular function by estrogen in women.

Geometry, kinetics and plasticity of release and clearance of ATP and noradrenaline as sympathetic cotransmitters: roles for the neurogenic contraction.

The paper compares the microphysiology of sympathetic neuromuscular transmission in three model preparations: the guinea-pig and mouse vas deferens and rat tail artery. The first section describes the quantal release of ATP and noradrenaline from individual sites. The data are proposed to support a string model in which: (i) most sites (> or = 99%) ignore the nerve impulse and a few (< or = 1%) release a single quantum of ATP and noradrenaline; (ii) the probability of monoquantal release is extremely non-uniform; (iii) high probability varicosities form 'active' strings; and (iv) an impulse train causes repeated quantal release from these sites. Analogy with molecular mechanisms regulating transmitter exocytosis in other systems is proposed to imply that coincidence of at least two factors at the active zone, Ca2+ and specific cytosolic protein(s), may be required to remove a 'fusion clamp', form a 'fusion complex' and trigger exocytosis of a sympathetic transmitter quantum, and that the availability of these proteins may regulate the release probability. The second section shows that clearance of noradrenaline in rat tail artery is basically > or = 30-fold slower than of co-released ATP, and that saturation of local reuptake and binding to local buffering sites maintain the noradrenaline concentration at the receptors, in spite of a profound decline in per pulse release during high frequency trains. The third section describes differences in the strategies by which mouse vas deferens and rat tail artery use ATP and noradrenaline to trigger and maintain the neurogenic contraction.