Sympathetic Varicosities Research Articles

Adrenoceptors Modulate Cholinergic Synaptic Transmission at the Neuromuscular Junction.

Adrenoceptor activators and blockers are widely used clinically for the treatment of cardiovascular and pulmonary disorders. More recently, adrenergic agents have also been used to treat neurodegenerative diseases. Recent studies indicate a location of sympathetic varicosities in close proximity to neuromuscular junctions. The pressing question is whether there could be any effects of endo- or exogenous catecholamines on cholinergic neuromuscular transmission. It was shown that the pharmacological stimulation of adrenoceptors, as well as sympathectomy, can affect both acetylcholine release from motor nerve terminals and the functioning of postsynaptic acetylcholine receptors. In this review, we discuss the recent data regarding the effects of adrenergic drugs on neurotransmission at the neuromuscular junction. The elucidation of the molecular mechanisms by which the clinically relevant adrenomimetics and adrenoblockers regulate quantal acetylcholine release from the presynaptic nerve terminals and postsynaptic sensitivity may help in the design of highly effective and well-tolerated sympathomimetics for treating a number of neurodegenerative diseases accompanied by synaptic defects.

Sympathomimetics regulate quantal acetylcholine release at neuromuscular junctions through various types of adrenoreceptors

The studies of the interaction between the sympathetic and motor nervous systems are extremely relevant due to therapy for many neurodegenerative and cardiovascular disorders involving adrenergic compounds. Evidences indicate close contact between sympathetic varicosities and neuromuscular synapses. This raises questions about the effects of catecholamines on synaptic transmission. The currently available information is contradictory, and the types of adrenoreceptors responsible for modulation of neurotransmitter release have not been identified in mammalian neuromuscular synapses. Our results have shown that the α1A, α1B, α2A, α2B, α2C, and β1 adrenoreceptor subtypes are expressed in mouse diaphragm muscle containing neuromuscular synapses and sympathetic varicosities. Pharmacological stimulation of adrenoreceptors affects both spontaneous and evoked acetylcholine quantal secretion. Agonists of the α1, α2 and β1 adrenoreceptors decrease spontaneous release. Activation of the α2 and β1 adrenoreceptors reduces the number of acetylcholine quanta released in response to a nerve stimulus (quantal content), but an agonist of the β2 receptors increases quantal content. Activation of α2 and β2 adrenoreceptors alters the kinetics of acetylcholine quantal release by desynchronizing the neurosecretory process. Specific blockers of these receptors eliminate the effects of the specific agonists. The action of blockers on quantal acetylcholine secretion indicates possible action of endogenous catecholamines on neuromuscular transmission. Elucidating the molecular mechanisms by which clinically utilized adrenomimetics and adrenoblockers regulate synaptic vesicle release at the motor axon terminal will lead to the creation of improved and safer sympathomimetics for the treatment of various neurodegenerative diseases with synaptic defects.

Spinal cord injury alters purinergic neurotransmission to mesenteric arteries in rats.

Complications associated with spinal cord injury (SCI) result from unregulated reflexes below the lesion level. Understanding neurotransmission distal to the SCI could improve quality of life by mitigating complications. The long-term impact of SCI on neurovascular transmission is poorly understood, but reduced sympathetic activity below the site of SCI enhances arterial neurotransmission (1). We studied sympathetic neurovascular transmission using a rat model of long-term paraplegia (T2-3) and tetraplegia (C6-7). Sixteen weeks after SCI, T2-3 and C6-7 rats had lower blood pressure (BP) than sham rats (103 ± 2 and 97 ± 4 vs. 117 ± 6 mmHg, P < 0.05). T2-3 rats had tachycardia (410 ± 6 beats/min), and C6-7 rats had bradycardia (299 ± 10 beats/min) compared with intact rats (321 ± 4 beats/min, P < 0.05). Purinergic excitatory junction potentials (EJPs) were measured in mesenteric arteries (MA) using microlectrodes, and norepinephrine (NE) release was measured using amperometry. NE release was similar in all groups, while EJP frequency-response curves from T2-3 and C6-7 rats were left-shifted vs. sham rats. EJPs in T2-3 and C6-7 rats showed facilitation followed by run-down during stimulation trains (10 Hz, 50 stimuli). MA reactivity to exogenous NE and ATP was similar in all rats. In T2-3 and C6-7 rats, NE content was increased in left cardiac ventricles compared with intact rats, but was not changed in MA, kidney, or spleen. Our data indicate that peripheral purinergic, but not adrenergic, neurotransmission increases following SCI via enhanced ATP release from periarterial nerves. Sympathetic BP support is reduced after SCI, but improving neurotransmitter release might maintain cardiovascular stability in individuals living with SCI.NEW & NOTEWORTHY This study revealed increased purinergic, but not noradrenergic, neurotransmission to mesenteric arteries in rats with spinal cord injury (SCI). An increased releasable pool of ATP in periarterial sympathetic nerves may contribute to autonomic dysreflexia following SCI, suggesting that purinergic neurotransmission may be a therapeutic target for maintaining stable blood pressure in individuals living with SCI. The selective increase in ATP release suggests that ATP and norepinephrine may be stored in separate synaptic vesicles in periarterial sympathetic varicosities.

Ca2+ ] changes in sympathetic varicosities and Schwann cells in rat mesenteric arteries-Relation to noradrenaline release and contraction.

This study aimed to assess intracellular Ca2+ dynamics in nerve cells and Schwann cells in isolated rat resistance arteries and determine how these dynamics modify noradrenaline release from the nerves and consequent force development. Ca2+ in nerves was assessed with confocal imaging, noradrenaline release with amperometry and artery tone with wire myography. Ca2+ in axons was assessed after loading with Oregon Green 488 BAPTA-1 dextran. In other experiments, arteries were incubated with Calcium Green-1-AM which loads both axons and Schwann cells. Schwann cells but not axons responded with a Ca2+ increase to ATP. Electrical field stimulation of nerves caused a frequency-dependent increase in varicose [Ca2+ ] ([Ca2+ ]v ). ω-conotoxin-GVIA (100nmol/L) reduced the [Ca2+ ]v transient to 2 and 16Hz by 60% and 27%, respectively; in contrast ω-conotoxin GVIA inhibited more than 80% of the noradrenaline release and force development at 2 and 16Hz. The KV channel blocker, 4-aminopyridine (10µmol/L), increased [Ca2+ ]v , noradrenaline release and force development both in the absence and presence of ω-conotoxin-GVIA. Yohimbine (1µmol/L) increased both [Ca2+ ]v and noradrenaline release but reduced force development. Acetylcholine (10µmol/L) caused atropine-sensitive inhibition of [Ca2+ ]v , noradrenaline release and force. In the presence of ω-conotoxin-GVIA, acetylcholine caused a further inhibition of all parameters. Modification of [Ca2+ ] in arterial sympathetic axons and Schwann cells was assessed separately. KV 3.1 channels may be important regulators of [Ca2+ ]v , noradrenaline release and force development. Presynaptic adrenoceptor and muscarinic receptor activation modify transmitter release through modification of [Ca2+ ]v .

VNUT and VMAT2 segregate within sympathetic varicosities and localize near preferred Cav2 isoforms in the rat tail artery.

ATP and norepinephrine (NE) are coreleased from peripheral sympathetic nerve terminals. Whether they are stored in the same vesicles has been debated for decades. Preferential dependence of NE or ATP release on Ca2+ influx through specific voltage-gated Ca2+ channel (Cav2) isoforms suggests that NE and ATP are stored in separate vesicle pools, but simultaneous imaging of NE and ATP containing vesicles within single varicosities has not been reported. We conducted an immunohistochemical study of vesicular monoamine transporter 2 (VMAT2/SLC18A2) and vesicular nucleotide translocase (VNUT/SLC17A9) as markers of vesicles containing NE and ATP in sympathetic nerves of the rat tail artery. A large fraction of varicosities exhibited neighboring, rather than overlapping, VNUT and VMAT2 fluorescent puncta. VMAT2, but not VNUT, colocalized with synaptotagmin 1. Cav2.1, Cav2.2, and Cav2.3 are expressed in nerves in the tunica adventitia. VMAT2 preferentially localized adjacent to Cav2.2 and Cav2.3 rather than Cav2.1. VNUT preferentially localized adjacent to Cav2.3 > Cav2.2 >> Cav2.1. With the use of wire myography, inhibition of field-stimulated vasoconstriction with the Cav2.3 blocker SNX-482 (0.25 µM) mimicked the effects of the P2X inhibitor suramin (100 µM) rather than the α-adrenergic inhibitor phentolamine (10 µM). Variable sensitivity to SNX-482 and suramin between animals closely correlated with Cav2.3 staining. We concluded that a majority of ATP and NE stores localize to separate vesicle pools that use different synaptotagmin isoforms and that localize near different Cav2 isoforms to mediate vesicle release. Cav2.3 appears to play a previously unrecognized role in mediating ATP release in the rat tail artery. NEW & NOTEWORTHY Immunofluorescence imaging of vesicular nucleotide translocase and vesicular monoamine transporter 2 in rat tail arteries revealed that ATP and norepinephrine, classical cotransmitters, localize to well-segregated vesicle pools. Furthermore, vesicular nucleotide translocase and vesicular monoamine transporter 2 exhibit preferential localization with specific Cav2 isoforms. These novel observations address long-standing debates regarding the mechanism(s) of sympathetic neurotransmitter corelease.

Advillin Is Expressed in All Adult Neural Crest-Derived Neurons.

Promoter-based genetic recombination (via, e.g., Cre-lox) is most useful when all cells of interest express a particular gene. The discovery that the actin-binding protein advillin is expressed in all somatic sensory neurons has been exploited repeatedly to drive DNA recombination therein, yet specificity of expression has not been well demonstrated. Here, we characterize advillin expression amongst sensory neurons and in several other neural and non-neural tissues. We first validate an advillin antibody against advillin knock-out tissue, advillin promoter-driven EGFP, and advillin mRNA expression. In the dorsal root ganglion (DRG), advillin is enriched in non-peptidergic nociceptors. We also show that advillin expression, and advillin promotor-driven EGFP and Cre-recombinase expression, occurs in multiple tissues including the dorsal habenula of the epithalamus, endocrine cells of the gut, Merkel cells in the skin, and most strikingly, throughout the autonomic nervous system (sympathetic, parasympathetic, and enteric neurons) in mice, rats, and non-human primates. In the mouse pelvic ganglion, advillin immunoreactivity is most intense in pairs of small neurons, and concentrated in spine-like structures on the axon initial segment contacted by sympathetic preganglionic axons. In autonomic targets (iris and blood vessels), advillin is distributed along cholinergic parasympathetic axons and in sympathetic varicosities. Developmentally, advillin expression is absent from sympathetics at postnatal day 4 but begins to emerge by day 7, accounting for previous reports (based on embryonic expression) of advillin’s specificity to sensory neurons. These results indicate that caution is warranted in interpreting previous studies in which advillin-driven genomic editing is either constitutive or performed after postnatal day 4.

A Practical Approach to Quantitative Processing and Analysis of Small Biological Structures by Fluorescent Imaging.

Standards in quantitative fluorescent imaging are vaguely recognized and receive insufficient discussion. A common best practice is to acquire images at Nyquist rate, where highest signal frequency is assumed to be the highest obtainable resolution of the imaging system. However, this particular standard is set to insure that all obtainable information is being collected. The objective of the current study was to demonstrate that for quantification purposes, these correctly set acquisition rates can be redundant; instead, linear size of the objects of interest can be used to calculate sufficient information density in the image. We describe optimized image acquisition parameters and unbiased methods for processing and quantification of medium-size cellular structures. Sections of rabbit aortas were immunohistochemically stained to identify and quantify sympathetic varicosities, >2 μm in diameter. Images were processed to reduce background noise and segment objects using free, open-access software. Calculations of the optimal sampling rate for the experiment were based on the size of the objects of interest. The effect of differing sampling rates and processing techniques on object quantification was demonstrated. Oversampling led to a substantial increase in file size, whereas undersampling hindered reliable quantification. Quantification of raw and incorrectly processed images generated false structures, misrepresenting the underlying data. The current study emphasizes the importance of defining image-acquisition parameters based on the structure(s) of interest. The proposed postacquisition processing steps effectively removed background and noise, allowed for reliable quantification, and eliminated user bias. This customizable, reliable method for background subtraction and structure quantification provides a reproducible tool for researchers across biologic disciplines.

Sympathetic Nervous System Moves Toward Center Stage in Cardiovascular Medicine

In 1664, the first anatomically correct depiction of the sympathetic nervous system came from Thomas Willis and his circle of London anatomists,1 included in The Anatomy of the Brain and Nerves , 1664 (Figure 1). This, the first work dedicated completely to the nervous system, also described the arterial loops at the base of the brain, which we now know as the Circle of Willis.1 Christopher Wren, an anatomist member of the group, was the principal illustrator1 before being asked by the City Fathers to turn his talents to town planning, architecture, and cathedral building after the 1666 Great Fire of London. Figure 1. An illustration of the human sympathetic nerves of the neck and thorax, from The Anatomy of the Brain and Sympathetic Nerves , published in 1664 by Thomas Willis and reproduced in Soul Made Flesh .1 Almost 2 centuries later, subsequent microscopic examination demonstrated that blood vessel walls were densely innervated, leading Stelling in 18402 to correctly conclude that these vasomotor fibers were in fact sympathetic nerves that were carried from the central nervous system to the blood vessels. In the mid-19th century, celebrated European physiologists, including Brown-Sequard, Waller, and Bernard,2 built on these observations, demonstrating vasoconstriction with electrical stimulation of the cut nerves and vasodilatation on nerve section, which indicated that the sympathetic fibers exerted a tonic, vasoconstrictor influence. The pressor nerves had gained recognition. Identification of the sympathetic neurotransmitter proved to be difficult. Claims for epinephrine3,4 and the hypothetical sympathins I and E confused the picture. Ulf von Euler compared bioassay responses of epinephrine, norepinephrine, and dihydroxy norephedrine with those of cattle splenic nerve extract, by testing blood pressure (BP) responses in the anesthetized cat and contractile responses in the isolated pregnant rabbit uterus, to definitively demonstrate the …

A Model of Cellular Cardiac-Neural Coupling That Captures the Sympathetic Control of Sinoatrial Node Excitability in Normotensive and Hypertensive Rats

Hypertension is associated with sympathetic hyperactivity. To represent this neural-myocyte coupling, and to elucidate the mechanisms underlying sympathetic control of the cardiac pacemaker, we developed a new (to our knowledge) cellular mathematical model that incorporates signaling information from cell-to-cell communications between the sympathetic varicosity and sinoatrial node (SAN) in both normotensive (WKY) and hypertensive (SHR) rats. Features of the model include 1), a description of pacemaker activity with specific ion-channel functions and Ca 2+ handling elements; 2), dynamic β-adrenergic modulation of the excitation of the SAN; 3), representation of ionic activity of sympathetic varicosity with NE release dynamics; and 4), coupling of the varicosity model to the SAN model to simulate presynaptic transmitter release driving postsynaptic excitability. This framework captures neural-myocyte coupling and the modulation of pacemaking by nitric oxide and cyclic GMP. It also reproduces the chronotropic response to brief sympathetic stimulations. Finally, the SHR model quantitatively suggests that the impairment of cyclic GMP regulation at both sides of the sympathetic cleft is crucial for development of the autonomic phenotype observed in hypertension.

Hypoxic fetal programming of the sympathetic nervous system

The link between intra uterine growth restriction (IUGR) and increased risk of adult cardiovascular disease in the offspring is well established. Chronic hypoxia in utero (CHU) is a well established cause of IUGR, and using in vitro methods, has been shown to cause endothelial dysfunction and sympathetic hyperinnervation in the offspring. Our aim is to assess the impact of CHU upon sympathetic nerve activity. We have made direct recordings of MSNA from arteries supplying the Spinotrapezius muscle under Alfaxan anaesthesia in Normal (N, n=7) and CHU rats (n=6), discriminating single unit activity from multiunit recordings. Arterial blood pressure, measured from the femoral artery, was similar in N and CHU rats (N:123±3, CHU: 119±5, mmHg). All single units (N=17, CHU=26) had cardiac and respiratory rhythmicity. Mean firing rates were similar (N: 0.48±0.09, CHU: 0.52±0.1, Hz). Further analysis showed that high frequency doublets and triplets of activity (>20hz) were more common in CHU. This may suggest increased release of Neuropeptide Y from sympathetic varicosities, and hence, impact upon cardiovascular health. Recordings during baro- and chemoreceptor stimulation are under analysis.

Presynaptic inhibition of neural vasodilator pathways to submucosal arterioles by release of purines from sympathetic nerves

Capsaicin-sensitive extrinsic sensory nerves and submucosal vasodilator neurons provide important vasodilator input to submucosal arterioles, but relatively little is known about the signaling between these populations and the sympathetic vasoconstrictor innervation. This study examined whether release of sympathetic purines can modulate dilator nerves. In vitro submucosal preparations from guinea pig ileum were modified to leave the parent mesenteric artery intact so that perivascular sympathetic and extrinsic afferent nerves could be activated by a bipolar stimulating electrode placed on the parent artery, and submucosal vasodilator neurons were activated using focal electrodes placed on submucosal ganglia. The outside diameter of submucosal arterioles was monitored using videomicroscopy, and dilator responses were examined after preconstricting vessels 80-95% with prostaglandin F(2alpha) (400 nM). Mesenteric nerve stimulation evoked a frequency-dependent dilation, with suramin (100 microM) present throughout to inhibit P(2X) receptor-mediated vasoconstrictions. In the presence of guanethidine (10 microM) to inhibit sympathetic purine release, superfusion of ATP (200 nM-6 microM) caused a concentration-dependent inhibition of nerve-evoked dilations. Vasodilations to substance P (10 nM) were not inhibited by ATP in the presence of guanethidine, implicating a presynaptic effect of ATP on neurotransmitter release. The inhibitory effect of ATP was blocked by the adenosine receptor antagonist 8-phenyltheophylline (8-PT; 10 microM). In addition, 8-PT increased the amplitude of nerve-evoked dilations, suggesting a tonic inhibitory effect of adenosine receptors on vasodilator release. Dilations evoked by electrical stimulation of submucosal ganglia were also inhibited almost 50% by ATP (2 microM) and its nonhydrolyzable analog, alpha,beta-methylene-ATP (10 microM). These data suggest that sympathetic varicosities release ATP or a related purine that can act at presynaptic adenosine receptors on extrinsic sensory and submucosal vasodilator neurons to inhibit neurotransmitter release.

Clinical and experimental evidence of sympathetic neural dysfunction during inflammatory bowel disease

1. Inflammatory bowel diseases (IBD) alter the function of the enteric nervous system and the sensory innervation of the gastrointestinal (GI) tract. Less is known about whether IBD also affects the sympathetic nervous system (SNS). Given the importance of the SNS in regulating GI function and possibly immune system activation, the present review examines the evidence of sympathetic dysfunction during IBD and its possible consequences. 2. Sympathetic axons within the GI tract innervate several cell types, including vascular myocytes, enteric neurons and immune cells. The major neurotransmitters released from sympathetic varicosities are noradrenaline, neuropeptide Y and ATP or a related purine. 3. Clinical studies of IBD patients have provided evidence of an association between IBD and axonal or demyelinating neuropathy. Assays of autonomic function suggest that ulcerative colitis and Crohn's disease, the two major forms of IBD, have contrasting effects on sympathetic neural activity. 4. Animal models of IBD have been used to examine the effects of these diseases on sympathetic neurophysiology. A decrease in the release of noradrenaline from sympathetic varicosities in inflamed and uninflamed regions of the GI tract has consistently been reported. Recent findings suggest that the decrease in neurotransmitter release may be due to inhibition of N-type voltage-gated Ca(2+) current in post-ganglionic sympathetic neurons. 5. Interest in the role of the SNS in IBD is rapidly increasing. However, much work needs to be done to enhance understanding of how SNS function is altered during IBD and what contribution, if any, these changes make to pathogenesis.

Inhibition of sympathetic N-type voltage-gated Ca2+ current underlies the reduction in norepinephrine release during colitis

Inflammatory bowel diseases (IBD) are associated with altered neuronal regulation of the gastrointestinal (GI) tract and impairment of norepinephrine release from sympathetic varicosities. The sympathetic innervation of the GI tract modulates motility, blood flow, and secretion, and therefore defective norepinephrine release may contribute to symptom generation in IBD. Accordingly, our aim here was to utilize the mouse model of dextran sulfate sodium (DSS; 5% wt/vol) of IBD to determine how norepinephrine release is reduced during GI inflammation. We hypothesized that the inflammation-induced reduction in norepinephrine release was due to inhibition of voltage-gated Ca(2+) current (I(Ca)) in prevertebral sympathetic neurons. We compared [(3)H]norepinephrine release in the colon and jejunum and I(Ca) amplitude in superior mesenteric ganglion (SMG) neurons from control mice and mice with DSS-induced colitis. Changes to voltage-gated Ca(2+) channels were investigated by fura 2-AM Ca(2+) imaging, perforated patch-clamp electrophysiology, and real-time PCR. Depolarization-induced norepinephrine release from the inflamed colon and uninflamed jejunum was significantly impaired in mice treated with DSS, as was depolarization-induced Ca(2+) influx in SMG neurons. Colitis reduced I(Ca) in SMG neurons by inhibiting omega-conotoxin GVIA (300 nM)-sensitive N-type Ca(2+) channels. The omega-conotoxin GVIA-sensitive component of norepinephrine release was significantly smaller in the colon during colitis. The inhibition of I(Ca) was accompanied by a decrease in mRNA encoding the Ca(2+) channel alpha subunit (CaV 2.2) and a rightward shift in the voltage dependence of activation of I(Ca). These findings suggest that DSS-induced colitis attenuates norepinephrine release in the colon and jejunum due to inhibition of N-type voltage-gated Ca(2+) channels. This may contribute to functional alterations in both inflamed and uninflamed regions of the GI tract during inflammation.

Neuroeffector Ca2+ transients for the direct measurement of purine release and indirect measurement of cotransmitters in rodents

Determining whether ATP and noradrenaline are released from the same vesicle at mature autonomic neuroeffector junctions is challenging because of the difficulty of simultaneously detecting the packeted release of these neurotransmitters. Contraction, overflow and electrophysiology experiments all show that both ATP and noradrenaline are released following field stimulation (although the ratio might vary) from autonomic nerves in tissues including the vas deferens, rat tail artery and mesenteric artery. The occurrence of purinergic neuroeffector Ca(2+) transients (NCTs) has been used to detect the packeted release of the neurotransmitter ATP acting on postjunctional P2X receptors to cause Ca(2+) influx. Neuroeffector Ca(2+) transients can also be used to detect the local effects of noradrenaline through its alpha(2)-adrenoceptor-mediated prejunctional autoinhibitory effects on nerve terminal Ca(2+) concentration and the probability of exocytosis (measured by counting NCTs). Evidence is presented that exocytosis from sympathetic varicosities does not occur in a manner independent of the history of that varicosity, but rather that the release of a packet of ATP transiently suppresses (or predicts the transient suppression of) subsequent release. This could arise by autoinhibition (by the prejunctional action of noradrenaline or purines) or due to a transient shortage of vesicles readily available for release. In summary, two high-resolution approaches are proposed to measure the intermittent release of packets of neurotransmitter: (1) local transient suppression of nerve terminal Ca(2+) transients; and (2) the local and transient inhibition of NCTs to infer local autoinhibition, hence transmitter release. Such approaches may allow the packeted corelease of ATP and noradrenaline to be investigated without the need to measure both neurotransmitters directly.

Depletion of noradrenaline inhibits electrically-evoked pain in the skin of the human forearm

Guanethidine displaces noradrenaline from sympathetic varicosities, and blocks sympathetic noradrenergic neurotransmission by inhibiting the release of noradrenaline from depleted neural stores. The aim of this study was to determine whether depletion of noradrenaline with guanethidine would oppose thermal hyperalgesia and/or electrically-evoked pain in mildly-burnt skin. Guanethidine was transferred by iontophoresis into a small patch of skin on the forearm of 35 healthy human subjects. The heat-pain threshold to a temperature gradient that increased at 0.5 °C/s was then measured at the guanethidine site, a nearby saline-control iontophoresis site, and in untreated skin. In addition, participants rated pain intensity to a 47 °C stimulus that was applied to each site for 7 s. Shortly after the iontophoreses, sensitivity to heat was greater at the guanethidine site than the two control sites, suggesting that ejection of noradrenaline from sympathetic varicosities increased sensitivity to heat. One day later, when neural stores of noradrenaline were depleted, sensitivity to heat did not differ between the guanethidine and control sites. The guanethidine pretreatment did not influence thermal hyperalgesia induced by a mild burn, but inhibited pain evoked by electrical stimulation of the skin (0.2 mA direct current for 4 min). These findings indicate that ongoing sympathetic neural discharge does not normally influence thermal hyperalgesia in inflamed skin, because depleting noradrenergic stores had no effect. However, electrically-evoked release of noradrenaline may increase nociceptive sensations. Further clarification of this human pain model could provide insights into the mechanism of adrenergic hyperalgesia in certain neuropathic pain syndromes.

Botulinum toxin potentiates cancer radiotherapy and chemotherapy.

Structural and functional abnormalities in the tumor vascular network are considered factors of resistance of solid tumors to cytotoxic treatments. To increase the efficacy of anticancer treatments, efforts must be made to find new strategies for transiently opening the tumor vascular bed to alleviate tumor hypoxia (source of resistance to radiotherapy) and improve the delivery of chemotherapeutic agents. We hypothesized that Botulinum neurotoxin type A (BoNT-A) could interfere with neurotransmitter release at the perivascular sympathetic varicosities, leading to inhibition of the neurogenic contractions of tumor vessels and therefore improving tumor perfusion and oxygenation. To test this hypothesis, BoNT-A was injected locally into mouse tumors (fibrosarcoma FSaII, hepatocarcinoma transplantable liver tumor), and electron paramagnetic resonance oximetry was used to monitor pO(2) in vivo repeatedly for 4 days. Additionally, contrast-enhanced magnetic resonance imaging was used to measure tumor perfusion in vivo. Finally, isolated arteries were mounted in wire myograph to monitor specifically the neurogenic tone developed by arterioles that were co-opted by the surrounding growing tumor cells. Using these tumor models, we showed that local administration of BoNT-A (two sites; dose, 29 units/kg) substantially increases tumor oxygenation and perfusion, leading to a substantial improvement in the tumor response to radiotherapy (20 Gy of 250-kV radiation) and chemotherapy (cyclophosphamide, 50 mg/kg). This observed therapeutic gain results from an opening of the tumor vascular bed by BoNT-A because we showed that BoNT-A could inhibit neurogenic tone in the tumor vasculature. The opening of the vascular bed induced by BoNT-A offers a way to significantly increase the response of tumors to radiotherapy and chemotherapy.

Hyperinnervation of Mesenteric Arteries in Spontaneously Hypertensive Rats by Sympathetic but Not Primary Afferent Axons

Hypertrophy of the perivascular plexus is thought to play a role in the development of hypertension in spontaneously hypertensive rats (SHR). However, it is not known whether the sympathetic varicosities are more numerous or larger, or form more neurovascular junctions. Further, a parallel hypertrophy of primary afferent terminals around the vessels might modulate any effects of hypertrophied sympathetic terminals. We have investigated the perivascular plexus around second-order mesenteric arteries of SHR and Wistar-Kyoto (WKY) rats by electron microscopy. Noradrenergic terminals were identified by the presence of small granular vesicles after chromaffin fixation, and substance P (SP+) afferent axons were identified by immunohistochemistry. The numbers of noradrenergic axon and varicosity profiles were higher (48 and 25%, respectively) in SHR than in WKY rats, and the majority lay closer to the medio-adventitial border. In contrast, there was no difference in the numbers of SP+ axons. Sympathetic and SP+ varicosities were indistinguishable in size, shape, vesicle content and mitochondrion content between each other and between the strains. However, both the number of neuromuscular junctions and the proportion of varicosities that formed them in SHR arteries were more than double those in WKY vessels. The data clearly show that hyperinnervation in SHR is specific for noradrenergic axons.

A quantitative description of the contraction of blood vessels following the release of noradrenaline from sympathetic varicosities

A model is presented that highlights the principal factors determining the form and extent of contraction in arteries upon stimulation of their sympathetic nerve supply. This model incorporates a previous quantitative model of the process of noradrenaline (NAd) diffusion into the vascular media and reuptake into sympathetic varicosities during nerve stimulation (J. Theor. Biol. 226 (2004) 359). It is also dependent on a model of how the subsequent activation of metabotropic receptors initiates a G-protein cascade, resulting in the production of inositol trisphosphate ( IP 3 ) and an increase in intracellular calcium concentration, [ Ca 2 + ] i , in the smooth muscle cells (J. Theor. Biol. 223 (2003) 93). In the present work we couple this rise in [ Ca 2 + ] i to the increase in phosphorylated myosin bound to actin in the cells and hence determine the force development in arteries due to nerve stimulation. The model accounts for force development as a function of [ Ca 2 + ] i and for the rate of change of force as a function of the rate of change of [ Ca 2 + ] i in single smooth muscle cells. It also accounts for the characteristic time course of the force developed by the media of the rat-tail artery upon nerve stimulation. This consists of a rapid rise to a transient peak followed by a sustained plateau of contraction during the stimulation period, after which the contraction slowly decays back to baseline at a rate dependent on the strength of the stimulation. The model indicates that the transient peak is primarily due to the partial block of the IP 3 receptor by the rise in [ Ca 2 + ] i and that the main determinant of the equilibrium condition indicated by the plateau phase is the rate of pumping of calcium into the sarcoplasmic reticulum. The relatively slow decline of contraction at the end of nerve stimulation is primarily a consequence of the slow rates of removal of NAd from the media by diffusion and reuptake into the sympathetic varicosities. The model thus provides a quantitative account of vascular smooth muscle contraction upon sympathetic nerve stimulation.

Correlation of non-uniform protein expression with variation in transmitter release probability

The strength of synaptic transmission is highly variable between different synapses. The present study examined some factors that may contribute to this variation in the strength of neurotransmission in sympathetic varicosities of the mouse vas deferens. Transmitter release was measured using a focal macropatch electrode placed over pairs of visualised varicosities. By regulating the calcium concentration of the solutions inside the recording electrode and in the bath independently of each other, transmitter release was restricted to one or two surface varicosities at each recording site. Using this technique, transmitter release probability was shown to be highly variable, even between adjacent varicosities on single axon branches. Very little variation was observed in the calcium influx following single impulse nerve stimulation between adjacent Oregon Green BAPTA-1 loaded varicosities. However, the staining intensities of three vesicular proteins, SV2, synaptophysin, and synaptotagmin 1, showed considerable variation between adjacent varicosities on single axon branches. This variation in staining intensity may be partly explained by variation in the density of synaptic vesicles. However, double staining experiments using two vesicular antigens showed some varicosities staining for one vesicular antigen, but not for the second, suggesting that the expression of these release machinery proteins is regulated locally within the varicosities. The results of the present study strengthen suggestions that synaptic strength is at least in part, regulated by variation in the expression of vesicular proteins.

A quantitative description of the diffusion of noradrenaline in the media of blood vessels following its release from sympathetic varicosities

A quantitative model is provided which describes how noradrenaline (NAd), released from varicosities at the adventitial surface of an artery, either diffuses into the media of the vessel to reach the intimal surface, diffuses into the volume of solution surrounding the artery, or is removed by the uptake 1 process in the varicosities. These predictions are then compared with experimental evaluations of the extent of changes in NAd to be found at the adventitial and intimal surfaces of the rat-tail artery, during and after trains of impulses, as determined using amperometry. In the model of the blood vessel there is a sequential decrease in the diffusion constant of NAd from the surrounding solution, to the adventitia, to the media, to the endothelium, to rise again in the lumen of the vessel; there is also an uptake 1 NAd pump in the varicosities described by Michaelis–Menten kinetics. This model is shown to provide a quantitative account of the spatial and temporal changes in NAd observed following trains of impulses at different frequencies of stimulation (5– 40 Hz ) for different periods of times (10– 40 s ). Changes in the spatio-temporal distribution of NAd observed following block of the uptake 1 NAd pump were also successfully predicted by the model. It is concluded that, within the context of the model, there is no need to evoke special mechanisms of buffering at the sympathetic varicosities, nor distinctions on the basis that only secreting varicosities utilize the uptake 1 mechanism, in order to describe the dynamics of NAd distribution in arteries during nerve activity.