Splenic sympathetic signaling contributes to acute neutrophil infiltration of the injured spinal cord.

BACKGROUND: Alterations in the immune system are a complication of spinal cord injury (SCI) and have been linked to an excessive sympathetic outflow to lymphoid organs. Still unknown is whether these peripheral immune changes also contribute for the deleterious inflammatory response mounted at the injured spinal cord. METHODS: We analyzed different molecular outputs of the splenic sympathetic signaling for the first 24 h after a thoracic compression SCI. We also analyzed the effect of ablating the splenic sympathetic signaling to the innate immune and inflammatory response at the spleen and spinal cord 24 h after injury. RESULTS: We found that norepinephrine (NE) levels were already raised at this time-point. Low doses of NE stimulation of splenocytes in vitro mainly affected the neutrophils' population promoting an increase in both frequency and numbers. Interestingly, the interruption of the sympathetic communication to the spleen, by ablating the splenic nerve, resulted in reduced frequencies and numbers of neutrophils both at the spleen and spinal cord 1 day post-injury. CONCLUSION: Collectively, our data demonstrates that the splenic sympathetic signaling is involved in the infiltration of neutrophils after spinal cord injury. Our findings give new mechanistic insights into the dysfunctional regulation of the inflammatory response mounted at the injured spinal cord.

Denervation of gastroepiploic artery graft can reduce vasospasm.

BACKGROUND: The right gastroepiploic artery is useful as an in situ arterial graft for coronary artery bypass grafting. However, the gastroepiploic artery is more likely to cause vasospasms compared with the internal thoracic artery. We hypothesized that the cause of the spasms is the stimulation of the periarterial sympathetic nerve, because the gastroepiploic artery is classified as a muscular artery. In this study, we examined whether the spasm is reduced by removing the periarterial sympathetic nerve. METHODS: Unused parts of the gastroepiploic artery were obtained from patients who underwent coronary artery bypass grafting. The vessel was cut into 2 segments, and they were assigned to control (N+) and denervation (N-) groups. The periarterial nerve was microscopically removed from the vessels of the N- group. The vessels in both groups were investigated by hematoxylin-eosin or immunohistochemical staining, and they were stimulated by electrical field stimulation with serial frequency for isometric tension measurement. RESULTS: Histologic analyses revealed that periarterial connective tissues including neuropeptide Y were removed to expose the external elastic membrane in the N- vessel, whereas they were preserved in N+. The mean contraction by electrical field stimulation with serial frequency was consistently lower in N- than in N+ (P < .05 at 20 and 50 Hz; n = 8 each). Endothelium-dependent relaxation and contractile function of the smooth muscle were similar in both groups. CONCLUSIONS: The removal of the periarterial sympathetic nerve from the human gastroepiploic artery reduced vascular contraction, elicited by peripheral nerve stimulation, without disturbing endothelial and smooth muscle contractile functions. This reduction may contribute to the prevention of vasospasms.

Chemical coding for cardiovascular sympathetic preganglionic neurons in rats.

Cocaine and amphetamine-regulated transcript peptide (CART) is present in a subset of sympathetic preganglionic neurons in the rat. We examined the distribution of CART-immunoreactive terminals in rat stellate and superior cervical ganglia and adrenal gland and found that they surround neuropeptide Y-immunoreactive postganglionic neurons and noradrenergic chromaffin cells. The targets of CART-immunoreactive preganglionic neurons in the stellate and superior cervical ganglia were shown to be vasoconstrictor neurons supplying muscle and skin and cardiac-projecting postganglionic neurons: they did not target non-vasoconstrictor neurons innervating salivary glands, piloerector muscle, brown fat, or adrenergic chromaffin cells. Transneuronal tracing using pseudorabies virus demonstrated that many, but not all, preganglionic neurons in the vasoconstrictor pathway to forelimb skeletal muscle were CART immunoreactive. Similarly, analysis with the confocal microscope confirmed that 70% of boutons in contact with vasoconstrictor ganglion cells contained CART, whereas 30% did not. Finally, we show that CART-immunoreactive cells represented 69% of the preganglionic neuron population expressing c-Fos after systemic hypoxia. We conclude that CART is present in most, although not all, cardiovascular preganglionic neurons but not thoracic preganglionic neurons with non-cardiovascular targets. We suggest that CART immunoreactivity may identify the postulated "accessory" preganglionic neurons, whose actions may amplify vasomotor ganglionic transmission.

Muscleblind-like 2: circadian expression in the mammalian pineal gland is controlled by an adrenergic-cAMP mechanism.

Muscleblind-like 2 (Mbnl2) is a zinc finger protein first identified in Drosophila. It appears to be essential for photoreceptor development and to be involved in RNA splicing. Here we report that Mbnl2 is strongly expressed in the rat pineal gland. The abundance of pineal Mbnl2 transcripts follows a marked circadian rhythm with peak levels approximately sevenfold higher at night than day levels. Mbnl2 protein exhibits a similar rhythm. In vitro studies indicate that the abundance of Mbnl2 transcripts and protein are controlled by an adrenergic/cAMP mechanism.

Adrenergic and noradrenergic innervation of the midbrain ventral tegmental area and retrorubral field: prominent inputs from medullary homeostatic centers.

Adrenergic agents modulate the activity of midbrain ventral tegmental area (VTA) neurons. However, the sources of noradrenergic and adrenergic inputs are not well characterized. Immunostaining for dopamine beta-hydroxylase revealed fibers within dopamine (DA) neuron areas, with the highest density in the retrorubral field (A8 cell group), followed by the VTA (A10 cell group), and very few fibers within substantia nigra compacta. A less dense, but a similar pattern of fibers was also found for the epinephrine marker, phenylethanolamine N-methyl transferase. Injection of the retrograde tracer wheat germ agglutinin-apo (inactivated) horseradish peroxidase conjugated to colloidal gold, or cholera toxin subunit b, revealed that the noradrenergic innervation of the A10 and A8 regions arise primarily from A1, A2, A5, and locus ceruleus neurons. Selective lesions of the ventral noradrenergic bundle confirmed a prominent innervation from A1 and A2 areas. Retrogradely labeled epinephrine neurons were found mainly in the C1 area. The identification of medullary noradrenergic and adrenergic afferents to DA neuron areas indicates new pathways for visceral-related inputs to reward-related areas in the midbrain.

The rat pineal gland comprises an endocannabinoid system.

In the mammalian pineal gland, the rhythm in melatonin biosynthesis depends on the norepinephrine (NE)-driven regulation of arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme of melatonin biosynthesis. A recent study showed that phytocannabinoids like tetrahydrocannabinol reduce AANAT activity and attenuate NE-induced melatonin biosynthesis in rat pineal glands, raising the possibility that an endocannabinoid system is present in the pineal gland. To test this hypothesis, we analyzed cannabinoid (CB) receptors and specific enzymes for endocannabinoid biosynthesis or catabolism in rat pineal glands and cultured pinealocytes. Immunohistochemical and immunoblot analyses revealed the presence of CB1 and CB2 receptor proteins, of N-acyl phosphatidyl ethanolamine hydrolyzing phospholipase D (NAPE-PLD), an enzyme catalyzing endocannabinoid biosynthesis and of fatty acid amide hydrolase (FAAH), an endocannabinoid catabolizing enzyme, in pinealocytes, and in pineal sympathetic nerve fibers identified by double immunofluorescence with an antibody against tyrosine hydroxylase. The immunosignals for the CB2 receptor, NAPE-PLD, and FAAH found in pinealocytes did not vary under a 12 hr light:12 hr dark cycle. The CB1 receptor immunoreaction in pinealocytes was significantly reduced at the end of the light phase [zeitgeber time (ZT) 12]. The immunosignal for NAPE-PLD found in pineal sympathetic nerve fibers was reduced in the middle of the dark phase (ZT 18). Stimulation of cultured pinealocytes with NE affected neither the subcellular distribution nor the intensity of the immunosignals for the investigated CB receptors and enzymes. In summary, the pineal gland comprises indispensable compounds of the endocannabinoid system indicating that endocannabinoids may be involved in the control of pineal physiology.

Chronic hyperinsulinemia enhances adrenergic vasoconstriction and decreases calcitonin gene-related peptide-containing nerve-mediated vasodilation in pithed rats.

The present study investigated the influence of chronic hyperinsulinemia on vascular responsiveness induced by adrenergic nerves and calcitonin gene-related peptide-containing (CGRPergic) nerves in pithed rats with insulin resistance. Male Wistar rats (6 weeks old) received 15% fructose solution in drinking fluid for 10 weeks (fructose-drinking rats: FDR), which resulted in significant increases in plasma levels of insulin, total cholesterol and triglyceride, and systolic blood pressure, as compared with control rats. Pithed FDR showed greater adrenergic nerve-mediated pressor response to spinal cord stimulation (SCS) at the lower thoracic vertebra (Th 9-12) and pressor response to exogenous noradrenaline than control rats. In pithed FDR with blood pressure artificially increased by continuous infusion of methoxamine and blockade of autonomic ganglia by hexamethonium, CGRPergic nerve-mediated depressor responses to SCS were significantly smaller than those in control rats, but depressor responses to other vasodilators such as acetylcholine, CGRP and sodium nitroprusside were similar to those in control rats. These results suggest that chronic hyperinsulinemia in FDR facilitates adrenergic nerve-mediated vasoconstriction, which is associated with attenuated CGRPergic nerve-mediated vasodilation.

Metabolic alterations in rat myocardium in experimental acute atrial fibrillation.

Experimental atrial fibrillation in intact rats significantly decreased the content of catecholamines in atrial adrenergic fibers and phosphorylase activity, which attests to enhanced glycogen consumption in the heart. These changes were specific of the fibrillating myocardium and atria, but were absent in the ventricles. Induced atrial fibrillation did not modify activities of SDH and monoamine oxidase in cardiac subdivisions. It was hypothesized that increased energy requirements in the atria during myocardial fibrillation led to activation of anaerobic metabolism.

Interaction of Mash1 and Phox2b in sympathetic neuron development.

The transcription factors Mash1 and Phox2b are both essential for sympathetic neuron development. To understand in more detail their function and interaction, Phox2b and Mash1 were ectopically expressed in vivo, in peripheral nerve precursors. Here, we demonstrate that the Phox2b-induced generation of ectopic noradrenergic neurons in chick peripheral nerve involves the induction of Cash1, the chick homolog of Mash1. All Phox2-induced neurons coexpress the noradrenergic marker genes TH and DBH. Conversely, Mash1 induces neuronal differentiation characterized by the expression of generic neuronal genes SCG10, Hu and NF160; however, only a subpopulation of these neurons also displays an autonomic, noradrenergic phenotype. This context-dependent action of Mash1 implicates autonomic codeterminants, required for noradrenergic differentiation in response to Mash1. In contrast, Phox2b coordinates generic and noradrenergic gene expression, recruiting Mash1/Cash1, which may have a major function in the control of pan-neuronal gene expression during noradrenergic neuron development.

Orexin neurons project to diverse sympathetic outflow systems.

The viral transneuronal labeling method was used to demonstrate that orexin-containing neurons of the lateral hypothalamic area (LHA) are linked via multisynaptic connections to different sympathetic outflow systems. Two different types of transneuronal tracing experiments were performed: single- and double-virus studies. In the first series of experiments, Bartha pseudorabies virus (PRV), a retrograde transneuronal tracer, was injected into single sympathetic targets, viz., stellate ganglion, adrenal gland, celiac ganglion, and kidney. Six to 7 days post-injection, orexin (hypocretin) neurons were transneuronally labeled. In a second set of experiments, the double-virus tracing method was used to determine whether single orexin LHA neurons are linked to two different sympathetic outflow systems. Two isogenic forms of Bartha PRV were used that differed by a single gene. beta-Galactosidase Bartha PRV was injected into the stellate ganglion and green fluorescent protein Bartha PRV into the adrenal gland of the same rat. The reverse placement of viral injections was made in another set of rats. In both paradigms, some orexin LHA neurons were transneuronally labeled with both viruses, indicating that they are capable of modulating multiple sympathetic outflow systems. These findings raise the possibility that orexin LHA neurons regulate general sympathetic functions, such as those that occur during arousal or the fight-or-flight response.

No barrier to diffusion between cell soma and neurite membranes in sympathetic neurons for a GPI-anchored glycoprotein.

As neurons extend their axons, it is thought that newly synthesised membrane components travel in vesicles along the axon, fuse with the growth cone membrane, and diffuse back along the axonal membrane. However, it is difficult to explain how axons continue to be populated with membrane proteins as they extend in length. To investigate this problem, we have used a CEPU-green fluorescent protein (GFP) chimeric protein to study the site of insertion of new glycosyl phosphatidyl inositol (GPI)-anchored glycoproteins and their subsequent behaviour in chick dorsal root ganglia (DRG) neurons. Infection of cultures grown for 24 h revealed rapid expression of CEPU-GFP over the whole surface of the neuron, more rapidly than could be accounted for by diffusion from the growth cone, and fluorescence intensity was uniform along the length of the neurite. Photobleaching experiments of neurite membrane revealed that recovery of fluorescence was due to diffusion from adjacent membranes and there was no evidence for membrane flow in either direction. Photobleaching of membrane adjacent to the cell body also showed rapid recovery, with chimera diffusing both from cell body membrane and the distal neurite membrane into the bleached area. These results suggest there is no barrier to diffusion between the cell body and neurite membrane in DRG and sympathetic neurons cultured for 1 or 2 days in vitro. We propose that the neurite is populated by newly synthesised chimera by diffusion from both regions. This situation may also occur in neurons in the early stages of extending axons in vivo prior to polarisation and the development of the dendritic field.

Immunohistochemical localization of nNOS and VIP in the mantle integument of the mussel, Mytilus galloprovincialis.

The phylogeny and functional roles of many bioactive compounds in the invertebrate integument are still unclear. In order to deal with this issue, we performed an immunohistochemical investigation of the integument of the mussel, Mytilus galloprovincialis, to demonstrate the presence of nNOS- and VIP-positive nerve fibers in subepidermal connective tissue of the mantle. Positive nerve cell bodies were detected in this tissue as well as in cortex of sperm follicles, and adjacent to maturating oocytes and spermatocytes located in the thickness of the mantle. These results indicate involvement of a local inhibitory non-adrenergic-non-cholinergic (NANC) regulatory mechanism of epidermal functions, such as mucous secretion and ciliary beating. At the gonadic level, this mechanism probably regulates the cycle of maturation and release of the gametes in both sexes.

NANC nerves in the respiratory air sac and branchial vasculature of the Indian catfish, Heteropneustes fossilis.

Gill and air sac of the Indian catfish Heteropneustes fossilis harbour a nerve network comprising an innervated system of neuroepithelial endocrine cells; the latter cells are found especially in the gill. A series of antibodies was used for the immunohistochemical detection of neurotransmitters of the neural non-adrenergic, non-cholinergic (NANC) systems such as the sensory neuropeptides (enkephalins), the inhibitory neuropeptide VIP and neuronal nitric oxide synthase (nNOS) responsible for nitric oxide (NO) production which is an inhibitory NANC neurotransmitter. NADPH-diaphorase (NADPH-d) histochemistry was used as marker of nNOS although it is not a specific indicator of constitutively-expressed NOS in gill and air sac tissues. A tyrosine hydroxylase antibody was used to investigate adrenergic innervation. Nitrergic and VIP-positive sensory innervation was found to be shared by gill and air sac. Immunohistochemistry revealed the presence of enkephalins, VIP, NOS and NADPH-d in nerves associated with branchial and air sac vasculature, and in the neuroendocrine cell systems of the gill. Adrenergic nerve fibers were found in some parts of the air sac vasculature. The origin of the nerve fibers remains unclear despite previous findings showing the presence of both NADPH-d and nNOS in the sensory system of the glossopharyngeal and vagus nerves including the branchial structure. Scarce faintly stained nNOS-positive neurons were located in the gill but were never detected in the air sac. These findings lead to the conclusion that a postganglionic innervation of the airways is absent. Mucous goblet cells in the gill were found to express nNOS and those located in the non-respiratory interlamellar areas of the air sac were densely innervated by nNOS-positive and VIP-positive nerve fibers. Our immunohistochemical studies demonstrate that most arteries of the gill and air sac share a NANC (basically nitrergic) innervation which strongly suggests that they are homologous structures.

Anatomical substrates for the central control of sympathetic outflow to interscapular adipose tissue during cold exposure.

The thermogenic activity of interscapular brown adipose tissue (IBAT) in response to physiologic stimuli, such as cold exposure, is controlled by its sympathetic innervation. To determine which brain regions might be involved in the regulation of cold-evoked increases in sympathetic outflow to IBAT, the present study compared central nervous system (CNS) areas activated by cold exposure with brain regions anatomically linked to the sympathetic innervation of IBAT. Immunocytochemical localization of Fos was examined in the brains of rats exposed to 4 degrees C for 4 hours. In a separate group of rats, the neural circuit involved in IBAT control, including the location of sympathetic preganglionic neurons in the spinal cord, was characterized with pseudorabies virus, a retrograde transynaptic tracer. Central noradrenergic and serotonergic groups related to the sympathetic outflow to IBAT also were identified. Localization of viral antigens at different survival times (66-96 hours) revealed infection in circumscribed CNS populations, but only a subset of the regions comprising this circuitry showed cold-evoked Fos expression. The raphe pallidus and the ventromedial parvicellular subdivision of the paraventricular hypothalamic nucleus (PVH), both infected at early survival times, were the main areas containing sympathetic premotor neurons activated by cold exposure. Major cold-sensitive areas projecting to spinal interneurons or to regions containing sympathetic premotor neurons, which became infected at intermediate intervals, included lateral hypothalamic, perifornical, and retrochiasmatic areas, anterior and posterior PVH, ventrolateral periaqueductal gray, and Barrington's nucleus. Areas infected later, most likely related to reception of cold-related signals, comprised the lateral preoptic area, parastrial nucleus, dorsomedial hypothalamic nucleus, lateral parabrachial nucleus, and nucleus of the solitary tract. These interconnected areas, identified by combining functional and retrograde anatomic approaches, likely constitute the central circuitry responsible for the increase in sympathetic outflow to IBAT during cold-evoked thermogenesis.

Heterogeneous expression of SNAP-25 and synaptic vesicle proteins by central and peripheral inputs to sympathetic neurons.

Neurons in prevertebral sympathetic ganglia receive convergent synaptic inputs from peripheral enteric neurons in addition to inputs from spinal preganglionic neurons. Although all inputs are functionally cholinergic, inputs from these two sources have distinctive neurochemical and functional profiles. We used multiple-labeling immunofluorescence, quantitative confocal microscopy, ultrastructural immunocytochemistry, and intracellular electrophysiologic recordings to examine whether populations of inputs to the guinea pig coeliac ganglion express different levels of synaptic proteins that could influence synaptic strength. Boutons of enteric intestinofugal inputs, identified by immunoreactivity to vasoactive intestinal peptide, showed considerable heterogeneity in their immunoreactivity to synaptosome-associated protein of 25 kDa (SNAP-25), synapsin, synaptophysin, choline acetyltransferase, and vesicular acetylcholine transporter. Mean levels of immunoreactivity to these proteins were significantly lower in terminals of intestinofugal inputs compared with terminals of spinal preganglionic inputs. Nevertheless, many boutons with undetectable levels of SNAP-25 immunoreactivity formed morphologically normal synapses with target neurons. Treatment with botulinum neurotoxin type A (20-50 nM for 2 hours in vitro) generated significant cleavage of SNAP-25 and produced similar dose- and time-dependent inhibitions of synaptic transmission from all classes of inputs, regardless of their mean level of SNAP-25 expression. The simplest interpretation of these results is that only synaptic boutons with detectable levels of SNAP-25 immunoreactivity contribute significantly to fast cholinergic transmission. Consequently, the low synaptic strength of intestinofugal inputs to final motor neurons in sympathetic pathways may be due in part to the low proportion of their boutons that express SNAP-25 and other synaptic proteins.

Pontine sources of norepinephrine in the cat cochlear nucleus.

In the current study, the distribution of noradrenergic neurons in the pontine tegmentum that project to the cochlear nucleus was determined with retrograde tract tracing combined with neurotransmitter immunohistochemistry in the cat. Double-labeled neurons were observed in all noradrenergic cell groups, in both the dorsolateral and the ventrolateral tegmentum. Half of the double-labeled cells were located in the locus coeruleus complex. Most of these were situated in its ventral division. Most other double-labeled cells were located in peribrachial regions, especially lateral to the brachium conjunctivum. Relatively few double-labeled cells were observed in both the A4 and the A5 cell groups, 2% and 0.4%, respectively, of the total. Except for neurons in A5, which projected only contralaterally, the projections were bilateral, with an ipsilateral preponderance. The results indicate that neurons located in the ipsilateral dorsolateral tegmentum, namely, in the locus coeruleus complex and the peribrachial region, are the primary source of pontine noradrenergic afferents to the cochlear nucleus of the cat.

Desipramine attenuates loss of cardiac sympathetic neurotransmitters produced by congestive heart failure and NE infusion.

We reported recently that inhibition of neuronal reuptake of norepinephrine (NE) by desipramine prevented the reduction of sympathetic neurotransmitters in the failing right ventricle of right heart failure animals. In this study, we studied whether desipramine also reduced the sympathetic neurotransmitter loss in animals with left heart failure induced by rapid ventricular pacing (225 beats/min) or after chronic NE infusion (0.5 microg. kg(-1). min(-1)). Desipramine was given to the animals for 8 wk beginning with rapid ventricular pacing or NE infusion. Animals receiving no desipramine were studied as controls. We measured myocardial NE content, NE uptake activity, and sympathetic NE, tyrosine hydroxylase, and neuropeptide Y profiles by histofluorescence and immunocytochemical techniques. Effects of desipramine on NE uptake inhibition were evidenced by potentiation of the pressor response to exogenous NE and reduction of myocardial NE uptake activity. Desipramine treatment had no effect in sham or saline control animals but attenuated the reduction of sympathetic neurotransmitter profiles in the left ventricles of animals with rapid cardiac pacing and NE infusion. In contrast, the panneuronal marker protein gene product 9.5 profile was not affected by either rapid pacing or NE infusion, nor was it changed by desipramine treatment in the heart failure animals. The study confirms that excess NE contributes to the reduction of cardiac sympathetic neurotransmitters in heart failure. In addition, it shows that the anatomic integrity of the sympathetic nerves is relatively intact and that the neuronal damaging effect of NE involves the uptake of NE or its metabolites into the sympathetic nerves.

Chemical sympathectomy-induced changes in TH-, VIP-, and CGRP-immunoreactive fibers in the rat mandible periosteum: influence on bone resorption.

The expression of neurotransmitter receptors by bone cells supports the concept that the nervous system is a regulator of bone metabolism. The discrimination of the respective roles of the sensory and sympathetic nervous systems requires evidence of topographic relationships between the corresponding fibers and the cells involved in bone turnover, in vivo. In this study, the influence of the sympathetic system on bone resorption was assessed by using a synchronized model of cortical resorption along the mandible. The sympathetic system was destroyed by daily injections of guanethidine (40 mg/kg) for 25 days; a resorption wave was induced on day 21. The distribution of periosteal tyrosine-hydroxylase (TH)-, vasoactive intestinal polypeptide (VIP)-, and calcitonin gene-related peptide (CGRP)-immunoreactive (IR) fibers was studied by compartmentalizing the periosteum. Most fibers were located in the distal, non-osteogenic compartment. TH-IR fibers were located perivascularly, VIP-IR fibers were gathered at the boundary with the osteogenic compartment, and CGRP-IR fibers were scattered. Sympathectomy decreased the number of TH- and VIP-IR fibers and increased the number of CGRP-IR fibers, without changing their topography. After the injection of Fast blue, a retrograde fluorescent marker, over the periosteum, fluorescent neuronal cell bodies were found in the superior cervical ganglion (SCG). Many neurons were TH-IR and very few were VIP-IR. Sympathectomy decreased the numbers of fluorescent and TH-IR cell bodies. It also decreased the number of preosteoclasts and osteoclasts, which had a drastic effect on the cortical bone surface, as assessed by scanning electron microscopy. These data indicate that VIP-IR fibers have a strategic position close to the most peripheral and less differentiated, osteogenic cells, pointing to a functional relationship. As poorly differentiated osteogenic cells support preosteoclast differentiation, VIP-IR fibers may be involved in this process, as suggested by the smaller number of preosteoclasts in sympathectomized rats. Although VIP is predominantly a parasympathetic mediator, it seemed to be conveyed by sympathetic fibers, as shown by the marked effect of guanethidine treatment. Nevertheless, these fibers did not originate from the SCG, contrary to TH-IR fibers.

Enhanced tissue plasminogen activator synthesis by the sympathetic neurons that innervate aging vessels.

We investigated the source of the increased release of tissue plasminogen activator (t-PA) into the circulation that occurs during natural aging. Both the basal release and the acute stress-associated release induced by sympathetic stimulations are greater in older subjects. It is widely assumed that the source of these increases is vascular endothelium. However, the sympathetic neurons that densely innervate resistance vessel walls were recently shown to synthesize and transport active t-PA to axon terminals in vascular smooth muscle, suggesting an alternative source. These fine t-PA-bearing axons lie in the seldom-studied deep adventitia of vessel walls, where they are less visible than endothelium in tissue sections. Using Northern blot analysis, we observed that t-PAmRNA synthesis is increased 54% in the ganglion parent neuron cell bodies that innervate aged vessels. The t-PA release from isolated, aged ganglia in cultures was twofold greater than that from younger controls. In addition, aged whole-artery explants showed a 20% greater basal and a 50% greater acute release of stored t-PA in vitro. In vivo levels of active t-PA were 33% greater in the blood and 40% greater in the aqueous humor. These results are consistent with an increased infusion of the active t-PA protease from sympathetic axon terminals into the vessel wall extracellular matrix and the blood during natural aging, in addition to the basal endothelial release. We suggest that the cumulative impact of an accelerated plasmin production and matrix degradation within vessel walls, especially during repetitive stress, may play an unrecognized role in the pathogenesis of vascular aging. The possibility that increased sympathetic nervous system plasminogenesis influences the aging process in nonvascular tissues also deserves further investigation.