Cerebrospinal fluid 5-HIAA concentrations correlate with cardiac uptake of 123I-MIBG during myocardial scintigraphy in drug naïve Parkinson's disease.

We examined the correlations between cerebrospinal fluid (CSF) concentrations of homovanillic acid (HVA) and 5-hydroxyindole acetic acid (5-HIAA) and imaging assessment scores, using 123I-Ioflupane SPECT and 123I-MIBG myocardial scintigraphy in 23 drug naïve PD patients. The CSF 5-HIAA concentration correlated with the H/M ratio of the delayed image (r = 0.458, p < 0.05) and the washout rate (r = - 0.642, p < 0.01) of 123I-MIBG myocardial scintigraphy. These correlations suggest some unclarified pathophysiological links between the central serotonergic and cardiac sympathetic systems.

Immunohistochemical distribution of cocaine and amphetamine regulatory peptide-like immunoreactive (CART-LI) nerve fibers in the circular muscle layer and their relationship to other peptides in the human caecum.

Motor activity of the gastrointestinal tract is extensively controlled by the enteric nervous system (ENS). Numerous neurotransmitters and neuromodulators are responsible for this regulation. One of them is cocaine- and amphetamine-regulated transcript peptide (CART). So far, there are few reports available concerning the distribution, functions, and co-localization of CART in the human gastrointestinal tract. The aim of the present investigation was to study the distribution and degree of co-localization of CART with substances taking part in conducting sensory stimuli, such as: substance P (SP), neurokinin A (NKA), calcitonin gene related peptide (CGRP) and Leu 5 enkephalin (L-ENK) in the circular muscle layer of the human caecum. CART-like immunoreactive (CART-LI) nerve fibers formed a very dense meshwork in the circular muscle layer of the caecum in all patients studied. Moreover, all neuronal substances tested during the present investigation were observed in CART-LI processes, but the degree of co-localization depended on the type of substance. The highest number of CART-positive nerves also contained L-ENK. A slightly lower level of co-localization was observed in the case of CART and SP or NKA, while only single nerve fibers were simultaneously CART- and CGRP-positive.

Early life stress sensitizes the renal and systemic sympathetic system in rats.

We hypothesized that maternal separation (MS), an early life stress model, induces a sensitization of the sympathetic system. To test this hypothesis, we evaluated the renal and systemic sympathetic system in 12- to 14-wk-old male control or MS rats with the following parameters: 1) effect of renal denervation on conscious renal filtration capacity, 2) norepinephrine (NE) content in key organs involved in blood pressure control, and 3) acute systemic pressor responses to adrenergic stimulation or ganglion blockade. MS was performed by separating pups from their mothers for 3 h/day from day 2 to 14; controls were nonhandled littermates. Glomerular filtration rate (GFR) was examined in renal denervated (DnX; within 2 wk) or sham rats using I¹²5-iothalamate plasma clearance. MS-DnX rats showed significantly increased GFR compared with MS-SHAM rats (3.8 ± 0.4 vs. 2.4 ± 0.2 ml/min, respectively, P < 0.05), whereas DnX had no effect in controls, indicating that renal nerves regulate GFR in MS rats. NE content was significantly increased in organ tissues from MS rats (P < 0.05, n = 6-8), suggesting a sensitization of the renal and systemic sympathetic system. Conscious MS rats displayed a significantly greater increase in mean arterial pressure (MAP) in response to NE (2 µg/kg ip) and a greater reduction in MAP in response to mecamylamine (2 mg/kg ip, P < 0.05, n = 4) monitored by telemetry, indicating that MS rats exhibit exaggerated responses to sympathetic stimulation. In conclusion, these data indicate that MS sensitizes the renal and systemic sympathetic system ultimately impairing blood pressure regulation.

Macrophages are unsuccessful in clearing aggregated alpha-synuclein from the gastrointestinal tract of healthy aged Fischer 344 rats.

With age, alpha-synuclein (α-SYNC) misfolds and forms insoluble deposits of protein in the myenteric plexus, leading presumably to dystrophy and degeneration in the circuitry controlling gastrointestinal (GI) function. The present experiment examined aggregates of α-SYNC in the aging small intestine and investigated how macrophages in the wall of the GI tract respond to these aberrant deposits. Groups of adult and aged Fisher 344 rats were studied. Whole mounts of duodenal, jejunal, and ileal smooth muscle wall, including the myenteric plexus, were prepared. Double labeling immunohistochemistry was used to stain α-SYNC protein and the phenotypic macrophage antigens CD163 and MHCII. Alpha-synuclein accumulated in dense aggregates in axons of both postganglionic and preganglionic neurons throughout the small intestine. Staining patterns suggested that deposits of protein occur initially in axonal terminals and then spread retrogradely toward the somata. Macrophages that were adjacent to dystrophic terminal processes were swollen and contained vacuoles filled with insoluble α-SYNC, and these macrophages commonly had the phenotype of alternatively activated phagocytes. The present results suggest that macrophages play an active phagocytotic role in removing α-SYNC aggregates that accumulate with age in the neural circuitry of the gut. Our observations further indicate that this housekeeping response does not clear the protein sufficiently to eliminate all synucleinopathies or their precursor aggregates from the healthy aging GI tract. Thus, accumulating deposits of insoluble α-SYNC in the wall of the GI tract may contribute, especially when compounded by disease or inflammation, to the age-associated neuropathies in the gut that compromise GI function.

Cyclophosphamide induced cystitis alters neurotrophin and receptor tyrosine kinase expression in pelvic ganglia and bladder.

PURPOSE: We examined neurotrophin and receptor tyrosine kinase (Trk) expression in the bladder and major pelvic ganglia (MPG) after cyclophosphamide induced cystitis in rats. MATERIALS AND METHODS: The bladder and MPG were used in immunohistochemical studies, enzyme-linked immunoassays and Western blots for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), TrkA and TrkB. Bladder postganglionic MPG cells were labeled by tracing techniques. RESULTS: NGF and BDNF expression was decreased in the bladder of all rats after cystitis (p < or =0.001). NGF and BDNF expression was increased in the MPG in male rats with cystitis (p < or =0.01). Cells expressing TrkA and TrkB immunoreactivity (IR) increased 78% to 81% in the MPG in male rats with cystitis. TrkA-IR or TrkB-IR bladder postganglionic cells increased 50% to 74% with cystitis. Cystitis increased TrkA-IR 5 to 10-fold and TrkB-IR 10 to 12-fold in detrusor muscle. TrkA-IR and TrkB-IR were prominent in control urothelium but decreased with cystitis. After cystitis TrkB-IR nerve fibers and TrkA-IR cellular infiltrates were more apparent compared to controls. CONCLUSIONS: Cystitis decreases bladder NGF and BDNF expression, whereas MPG expression is increased. This change may reflect neurotrophin release at the bladder and retrograde transport to the MPG. TrkA-IR and TrkB-IR are increased in bladder postganglionic cells and bladders with cystitis. This increase may reflect a shift in Trk staining from urothelium to detrusor muscle and nerve fibers with cystitis. Neurotrophin/Trk interactions in the bladder and MPG may contribute to bladder overactivity with cystitis.

Synthesis of nitric oxide in postganglionic myenteric neurons during endotoxemia: implications for gastric motor function in rats.

We have investigated the mechanisms underlying acute changes in gastric motor function triggered by endotoxemia. In fundal strips from rats pre-treated with endotoxin (40 microg/kg, i.p. 30 min), mechanical activity was analyzed and the source of nitric oxide (NO) was visualized by confocal microscopy of tissue loaded with the fluorescent dye DAF-FM. NOS expression was determined by quantitative RT-PCR and Western blot, and enzyme activity by the citrulline assay. Strips from endotoxin-treated rats were hypo-contractile. This was prevented by pre-incubation with the neurotoxin tetrodotoxin, the gangliar blocker hexamethonium, or non-selective and neuronal-specific NOS inhibitors (L-NOARG and TRIM, respectively). The soluble guanylyl cyclase (sGC) inhibitor ODQ and the inhibitor of small conductance Ca2+-activated K+ channels apamin prevented relaxation induced by endotoxin, nicotine, exogenous NO (DETA-NONOate), and the NO-independent sGC activator BAY 41-2272. NO synthesis was observed in neuronal soma, axons, and nerve endings of the myenteric plexus in the fundus of endotoxin-treated rats and was prevented by L-NAME, tetrodotoxin, and hexamethonium. nNOS and iNOS mRNA and protein contents were unchanged. Our findings demonstrate synthesis of NO in post-ganglionic myenteric neurons during early endotoxemia that mediates gastric hypo-contractility. The effect of NO is mediated via sGC and small conductance Ca2+-activated K+channels.

A possible indirect sympathomimetic action of metformin in the arterial vessel wall of spontanously hypertensive rats.

The antidiabetic drug metformin (MF) typically achieves only micromolar levels in plasma with normal therapeutic use. However, it is also known to accumulate in various tissues up to several times higher after standard oral dosing and we now have evidence from both in vivo and in vitro experiments with spontaneously hypertensive rats (SHR) that millimolar levels stimulate release of norepinephrine (NE) from vascular sympathetic nerve endings (SNEs). As shown in the present work with SHR tail arterial tissue (rich in SNEs), the known vasodilator effect of millimolar levels of MF on the smooth muscle (even if contracted with a nonadrenergic agonist), is attenuated by the presence of the SNEs unless phentolamine (an alpha receptor blocker) is present. We reasoned that the mechanism for this apparent NE-releasing action of MF is not exocytotic release as that would require depolarization of the neuronal cell membranes in SNEs, and MF at millimolar levels is known to repolarize (not depolarize) membranes of other cells. Thus, we tested the possibility that MF releases NE by an indirect sympathomimetic-like action. Such an action should be amplified by monoamine oxidase inhibitors (e.g. iproniazid) and blocked by NE-carrier inhibitors (e.g. desipramine). Accordingly, we found that the abovementioned attenuating effect of intact SNEs on MF's relaxation of SHR tail arterial tissue (compared to tissues in which SNEs were experimentally removed with 6-hydroxydopamine) was amplified nearly 3-fold by iproniazid (p<0.05) and blocked by desipramine (p<0.05). These results support an indirect sympathomimetic action of MF and raise the question whether commonly used antidepressants with properties similar to iproniazid and desipramine might alter MF's beneficial vasodilatory (and thus antihypertensive) effectiveness in diabetic patients with hypertension.

[Autonomic innervation of feline laryngeal glands: an immuno-electronmicroscopic study].

Autonomic innervation of feline laryngeal glands was investigated immunoelectronmicroscopically using antisera for vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), and tyrosine hydroxylase (TH). A relatively dense population of immunoreactive (ir) VIP fibers was found in the vicinity of the basement membranes of glandular and myoepithelial cells. Some VIP -ir fibers contacted the basement membrane and some of the fibers penetrated the adjoining glandular cells intercellularly without making synaptic contacts with them. Ir-NPY and -TH fibers were less abundant than VIP-fibers and were distributed beneath the basement membranes of myoepithelial cells. These fibers were not encountered between glandular cells. The estimated ratio of ir-VIP, -TH and -NPY fibers was 20:4:1, based on the density of fibers in glandular tissues. Because this value was equal between serous and mucous glandular cell, these two tissues may receive the same pattern of autonomic innervation.

Nitric oxide and arachidonic acid modulation of calcium currents in postganglionic neurones of avian cultured ciliary ganglia.

1. A study has been made of the modulation of high-voltage activated transient and sustained calcium currents in cultured neurones of avian ciliary ganglia by nitric oxide (NO) and arachidonic acid. 2. Sodium nitroprusside (100 microM) reduced the transient calcium current (ICa) on average by 31% and the sustained ICa by 32% during a test depolarization to +20 mV from a holding potential of -100 mV. This reduction was maintained for at least 30 min following a single application of sodium nitroprusside. 3. L-Arginine (270 microM) reduced the transient ICa on average by 28% and the sustained ICa by 22% and these effects were prevented by the presence of the NO-synthase competitive blocker NG-nitro-L-arginine methylester (L-NAME; 100 microM) in the bathing solution. 4. Arachidonic acid (50 microM) reduced the transient ICa on average by 28% and the sustained ICa by 33%. When added together, arachidonic acid (50 microM) and L-arginine (270 microM) produced the same effects as arachidonic acid alone. 5. Blocking the conversion of arachidonic acid to prostaglandins by addition of indomethacin (20 microM) to the bathing solution did not prevent the depression of either the transient or the sustained calcium current during application of arachidonic acid (50 microM). The effects of arachidonic acid were also not occluded by L-NAME (100 microM) when present in the bathing solution. 6. Inhibiting the biosynthesis of leukotrienes by applying L-663,536 (MK-886; 3 microM) to the bathing solution prevented the depression of both components of ICa during application of arachidonic acid (50 microM). 7. These results indicate that endogenous NO and arachidonic acid pathways are present in parasympathetic ciliary neurones, and that both act to depress high-voltage, gated, calcium channel activity.

Degeneration of motor nerve fibers enhances the expression of calcitonin gene-related peptide in rat sensory neurons.

Calcitonin gene-related peptide (CGRP) in skeletal muscle is of neural origin. After degeneration of sensory fibers induced by removal of the lumbar dorsal root ganglia (DRG), the CGRP content in the rat soleus muscle was reduced to about 5% of the normal level. In contrast, degeneration of motor fibers induced by sectioning of the lumbar ventral roots markedly increased the CGRP content in the soleus muscle and CGRP levels in the lumbar DRG. It is suggested that the expression of CGRP in sensory neurons is up-regulated by some humoral factor induced by degeneration of motor nerve fibers.

Prejunctional opioid mu-receptors and adenosine A1-receptors on the sympathetic nerve endings of the rat tail artery interact with the alpha 2-adrenoceptors.

Experiments were designed to study the interaction between prejunctional alpha 2-adrenoceptors and both adenosine and opioid receptors at the postganglionic sympathetic nerve endings innervating the tail artery of the rat. Segments of this vessel were preincubated with [3H]-noradrenaline and then perfused/superfused with [3H]-noradrenaline-free medium. Their perivascular nerves were field stimulated with standard stimulation parameters: 24 pulses at 0.4 Hz, 0.3 ms, 200 mA. In some experiments, the stimulation parameters were adjusted in order to obtain similar reference release values despite the presence of a first release-modulating drug. The adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA; 0.3-10 mumol/l) and [D-Ala2,MePhe4,Glyol5]enkephalin (DAGO; 0.3-10 mumol/l) depressed the stimulation-evoked overflow of tritium in a concentration dependent manner. The release-inhibiting effect of both NECA and DAGO was enhanced in the presence of the alpha 2-adrenoceptor antagonist rauwolscine (3 mumol/1) while it was attenuated in the presence of the alpha 2-adrenoceptor agonist 5-bromo-6-[2-imidazolin-2yl-amino]-quinoxaline (UK-14,304; 0.1 mumol/l). These changes occurred both at standard and adjusted stimulation parameters. These results demonstrate that the prejunctional adenosine A1- and opioid mu-receptors interact with the prejunctional alpha 2-adrenoceptors. The level at which these interactions take place (receptors themselves or transduction mechanisms) as well as the physiological significance of the phenomenon remain to be determined.

Lack of presynaptic modulation by isoprenaline of 3H-noradrenaline release from rabbit isolated ear artery.

The aim of the present investigation was to examine whether or not presynaptic facilitatory beta-adrenoceptors are detectable on the postganglionic nerves in the rabbit isolated ear artery. Strips of rabbit central ear artery were incubated with 3H-noradrenaline (10(-7) mol/l; 30 min or 10(-6) mol/l; 60 min). Subsequently, they were washed repeatedly with physiological salt solution. The strips were subjected to electrical-field stimulation (S1-S8) and the resultant 3H-overflow was determined. When the ear artery was stimulated with 150 pulses (0.5 ms; 3 Hz; 225 mA), isoprenaline (10(-9)-10(-6) mol/l) either alone or in the presence of either rauwolscine (10(-6) mol/l) or phentolamine (10(-6) mol/l) did not alter the stimulation-evoked 3H-overflow. This was also the case in the presence of rauwolscine (10(-6) mol/l) plus either the selective phosphodiesterase inhibitor ICI 63 197 (3 x 10(-5) mol/l) or forskolin (10(-6) mol/l). When the ear artery was stimulated with 300 pulses (1 ms; 5 Hz; 225 mA), isoprenaline had no effect on the stimulation-evoked 3H-overflow. This was also the case when phentolamine (10(-6) mol/l) was present. Propranolol (10(-7)-10(-5) mol/l) did not alter the stimulation-evoked 3H-overflow. In some experiments, the stimulation current was reduced to 175 mA in order to obtain similar reference release (S3) values despite the presence of rauwolscine (150 pulses; 0.5 ms; 3 Hz). Even then, isoprenaline (10(-9)-10(-6) mol/l) did not change stimulation-evoked 3H-overflow. The results suggest that postganglionic sympathetic nerves in rabbit central ear artery do not possess presynaptic facilitatory beta-adrenoceptors.

A presynaptic excitatory M1 muscarine receptor at postganglionic cardiac noradrenergic nerve fibres that is activated by endogenous acetylcholine.

Rabbit atria were isolated with the extrinsic right vagus and sympathetic nerves intact and perfused with Tyrode solution. Noradrenaline overflow evoked by sympathetic nerve stimulation (SNS) at 3 Hz for 3 min was determined before, during, and after vagus nerve stimulation (VNS), also at 3 Hz and for 3 min. The VNS pulses preceded the SNS pulses by 3, 100 and 233 ms. Acetylcholine overflow was determined after labelling of the transmitter stores with [14C]choline. Pirenzepine 80 nmol/l failed to alter the muscarinic inhibition of noradrenaline overflow when the vago-sympathetic impulse intervals were 3 and 233 ms. At an interval of 100 ms VNS did not significantly inhibit noradrenaline overflow in the absence of pirenzepine but produced an inhibition in the presence of the drug. When the pirenzepine concentration was varied (0.4-300 nmol/l) the largest inhibition of noradrenaline overflow was observed at 5.7 nmol/l whereas 300 nmol/l fully antagonized the inhibition. Acetylcholine overflow evoked by VNS was not altered by pirenzepine 0.4-300 nmol/l. AF-DX 116 (11-[(2[(diethylamino)methyl]-1-piperidinyl)-acetyl]-5, 11-dihydro-6H-pyrido-[2,3-b]-[1,4]benzodiazepine-6-one), an M2 receptor selective antagonist, concentration-dependently (100-800 nmol/l) inhibited the decrease of tension development elicited by VNS. At the 100 ms vago-sympathetic impulse interval noradrenaline overflow was enhanced in the presence of AF-DX 116 400 and 800 nmol/l. However, already 100 nmol/l of the drug caused a maximum (fourfold) increase of acetylcholine overflow. It is concluded that acetylcholine released onto noradrenergic nerve fibres causes a small facilitation of noradrenaline overflow at a vago-sympathetic impulse interval of 100 ms.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of neuropeptide Y in neuroendocrine stress responses. Central and peripheral studies.

Neuropeptide Y (NPY) is closely associated to stress-reactive structures in the central and peripheral nervous system. In the periphery, the peptide is colocalized with catecholamines in postganglionic sympathetic fibres and the adrenal medulla. In the brain, the paraventricular nucleus of the hypothalamus receives a dense innervation of NPYergic neurons, some of which also contain monoamines. With the use of a specific immunoradiometric assay, we have demonstrated that NPY is released into the peripheral circulation during psychological stress together with catecholamines. The postganglionic origin of the peptide was demonstrated by the activity of the nicotinic antagonist hexamethonium to attenuate the response. Adrenalectomy or insulin-induced hypoglycemia did not alter basal or stimulated NPY plasma levels, showing that the adrenal is not a major source of circulating NPY in the rat. Although NPY and noradrenaline are frequently released in parallel in various experimental conditions, a clear dissociation can be found in several cases, such as cold stress or the response to phentolamine, where no change can be seen in plasma NPY despite a large activation of noradrenergic terminals. Furthermore, the neuropeptide may play a role in stress-induced pathological states such as hypertension, since its release is greater in animals previously submitted to chronic stress and high-sodium diet. On the other hand, its role in the central nervous system control mechanisms of the stress response is far from being clear, but to understand the interaction of NPY we need a better knowledge of the role of noradrenergic neurons in the central control of the adrenocortical axis or sympathetic nervous system activity.

Organization of the sympathetic postganglionic innervation of the rat heart.

The origins and organization of cardiac sympathetic postganglionic nerves in the rat were identified in the present investigation. The retrograde tracer, Diamidino Yellow, was injected into the right or left ventricles to label somata in the sympathetic chain. Analysis of all sympathetic ganglia from superior cervical ganglion through the 10th thoracic ganglion indicated that the postganglionic innervation of the rat cardiac ventricles originates bilaterally. The majority of these somata were located in the middle and inferior cervical ganglia (middle cervical-stellate ganglion complex) (approximately 92% of all labelled cells), with lesser contributions from the superior cervical and 4th through 6th thoracic ganglia. To confirm and further quantitate these findings, the middle cervical-stellate ganglion complex was removed (MC-S ganglionectomy) bilaterally or ipsilaterally from the left or right sides, and regional cardiac norepinephrine concentration (left and right atrial appendages and left and right ventricles) was analysed 7 or 28 days later. At both times after bilateral MC-S ganglionectomy, regional cardiac norepinephrine was reduced by 89% to 100%, indicating the removal of almost all cardiac noradrenergic cells of origin and possibly fibers of passage. The results of unilateral MC-S ganglionectomy experiments indicated that the atrial appendages and the left ventricle receive bilateral innervation from the middle cervical-stellate ganglion complex. However, the left middle cervical-stellate ganglion complex appears to contribute a majority of the norepinephrine to the right ventricle. Furthermore, between 7 and 28 days after contralateral MC-S ganglionectomy, atrial appendages, but not ventricles, display significant recovery of norepinephrine content. The present data demonstrate: (1) a bilateral locus of origin of cardiac sympathetic postganglionic neurons, limited longitudinally to cervical through mid-thoracic ganglia, and (2) the ability of the cardiac postganglionic innervation to regenerate after partial denervation. These results demonstrate anatomical evidence for significant bilateral integration of cardiac sympathetic activity at the level of the sympathetic ganglion in the rat.

Substance P mRNA is present in a population of CGRP-immunoreactive cholinergic postganglionic sympathetic neurons of the cat: evidence from combined in situ hybridization and immunohistochemistry.

A synthetic oligonucleotide probe, complementary to a sequence of the rat beta-preprotachykinin mRNA coding for part of the mature substance P (SP), was used to localize, by in situ hybridization, SP mRNA to individual paravertebral sympathetic ganglion cells of the cat. Subsequent immunohistochemical analysis revealed that most of these neurons contained immunoreactivity to calcitonin gene-related peptide (CGRP), suggesting that they belong to the cholinergic cell population of the scattered type. These cells contain, in addition to SP and CGRP, vasoactive intestinal polypeptide and peptide histidine isoleucine.

Innervation of the gastrointestinal canal of the toad Bufo marinus by neurons containing 5-hydroxytryptamine-like immunoreactivity.

The gut of the toad, Bufo marinus, was examined for evidence of enteric neurons containing 5-hydroxytryptamine-like immunoreactivity. Such neurons were absent from the stomach. They were present in the small intestine, with processes confined to the myenteric plexus. Immunoreactive nerve cell bodies lay on branches of the pelvic nerves supplying the large intestine; fibres were found in the submucosa of the posterior large intestine and in the muscularis external of the anterior large intestine. It is concluded, on morphological grounds, that the neurons in the small intestine are interneurons, whereas those in the large intestine are postganglionic parasympathetic motoneurons.

Vasoactive intestinal polypeptide-immunoreactive nerves in the pulmonary vasculature of the aquatic file snake Acrochordus granulatus.

The indirect immunofluorescence technique was used to determine the distribution of vasoactive intestinal polypeptide-immunoreactive and somatostatin-immunoreactive axons in the pulmonary vasculature of the aquatic file snake Acrochordus granulatus. A dense distribution of vasoactive intestinal polypeptide-immunoreactive axons was found on the common pulmonary artery, the anterior and posterior pulmonary arteries, and the smaller arteries branching to the lung. The density of these axons appeared greater in arterial preparations taken from more distal regions of the lung. The densest distribution of vasoactive intestinal polypeptide-immunoreactive axons was observed on the larger pulmonary veins in all regions of the lung. These axons were observed on the larger veins within the lung parenchyma but not on the smaller veins. Axons and cell bodies were observed in the vagal nerve trunks which run parallel to the pulmonary arteries and veins. In contrast, no somatostatin-immunoreactive axons were observed in any region of the pulmonary vasculature. It is proposed that the perivascular plexus of vasoactive intestinal polypeptide-immunoreactive axons may represent part or all of the vagal postganglionic innervation of the pulmonary vasculature.

The presence and possible role of a galanin-like peptide in the mudpuppy heart.

A correlated histochemical and pharmacological study was undertaken to establish the presence, origin, and possible function of nerve fibers containing a galanin-like peptide in the mudpuppy (Necturus maculosus) heart. Whole mount preparations of septum-sinus venosus or atria and sections of ventricular muscle were prepared for immunocytochemistry. Galanin-immunoreactive fibers were found coursing diffusely across the septum-sinus venosus to form complex networks over cardiac muscle strands. Individual atrial muscle strands were densely innervated by galanin-immunoreactive fibers and galanin-immunoreactive fibers were also observed in the epicardial and myocardial layers of the ventricle. Most of the parasympathetic postganglionic neurons in the cardiac ganglion and many of the small intensely fluorescent-like cells exhibited galanin immunoreactivity. Galanin-immunoreactive fibers were present in the nerve trunks connecting clusters of parasympathetic postganglionic neurons. Close associations between galanin-positive fibers and individual parasympathetic postganglionic neurons were also observed. The presence of the galanin-immunoreactive fibers was similar in preparations taken from animals pretreated with 6-hydroxydopamine to that seen in preparations taken from control animals, indicating that the galanin-positive fibers were not sympathetic postganglionic axons. Moreover, the galanin-immunoreactive nerve fibers were separate from fibers containing substance P and/or calcitonin gene-related peptide that have previously been shown to be processes of afferent fibers. In twitch-tension experiments, galanin in the range 1 x 10(-7) to 1 x 10(-6) M caused cardioinhibition of spontaneously beating isolated septal-sinus venosus preparations. Galanin also produced a concentration-dependent (1 x 10(-7) to 1 x 10(-6) M) decrease in the twitch-tension development of electrically stimulated atrial or ventricular preparations. Local application of galanin produced hyperpolarization of cardiac muscle fibers in both isolated septal-sinus venosus preparations and atrial preparations. The response of individual parasympathetic ganglion cells to local application of galanin varied between neurons; some neurons were depolarized whereas others were hyperpolarized. We conclude that a galanin-like peptide is contained in both the parasympathetic postganglionic neurons and small intensely fluorescent-like cells and their processes. Further, we hypothesize that in the case of the parasympathetic postganglionic neurons, the galanin-like peptide may work in conjunction with acetylcholine to regulate cardiac activity.

Localization of calcitonin gene-related peptide (CGRP) at a neuronal nicotinic synapse.

The localization of calcitonin gene-related peptide (CGRP) in the parasympathetic cardiac ganglion of the frog was investigated with an immunofluorescence method. By doubly staining the whole ganglion with antibodies against a synaptic-vesicle antigen and anti-CGRP antibodies, we found that all of the boutons at the nicotinic synapses on the postganglionic neurons contained immunoreactivity against this peptide. In contrast, neither the muscarinic nor the adrenergic terminals of the postganglionic fibers on the cardiac muscle contained CGRP immunoreactivity. This suggests that CGRP is involved in the functions of the neuronal nicotinic synapses.