Judy L. Morris

Co-localization of neuropeptide Y, vasoactive intestinal polypeptide and dynorphin in non-noradrenergic axons of the guinea pig uterine artery

Two major populations of perivascular axons containing immunoreactivity to neuropeptide Y (NPY) have been revealed in the main uterine artery of the guinea pig by immunohistochemical procedures which allow the simultaneous visualization of two antigens. One population contained immunoreactivity to dopamine-beta-hydroxylase (D beta H) and was presumably noradrenergic. The other main population of axons with NPY-like immunoreactivity (NPY-LI) did not have D beta H-like immunoreactivity (D beta H-LI) and was presumably non-noradrenergic. These non-noradrenergic axons also contained immunoreactivity to vasoactive intestinal polypeptide (VIP) and dynorphin (DYN). Indeed, nearly all axons with VIP-LI also contained NPY-LI and DYN-like immunoreactivity (DYN-LI). NPY constricted the uterine artery perfused in vitro, whilst VIP dilated uterine arteries preconstricted with noradrenaline or NPY. Thus, we have evidence for the coexistence of a vasoconstrictor peptide and a vasodilator peptide in the same non-noradrenergic perivascular axons, which also contain an opioid peptide, dynorphin.

Innervation of the large arteries and heart of the toad (Bufo marinus) by adrenergic and peptide-containing neurons

SummaryThe innervation of the major arteries and heart of the toad (Bufo marinus) was examined by use of glyoxylic acid-induced catecholamine fluorescence and peptide immunohistochemistry. All arteries possessed a moderate to dense plexus of adrenergic axons, which also showed neuropeptide Y-like immunoreactivity (NPY-LI). Some adrenergic axons in the intracardiac vagal trunks showed NPY-LI, but the varicose adrenergic axons innervating the cardiac muscle of the atria and ventricle, and the coronary blood vessels did not display NPY-LI. About half of the nerve cell bodies in the anterior sympathetic chain ganglia with dopamine-β-hydroxylase-LI (DBH-LI) also contained NPY-LI. The nerve cell bodies with DBH-LI alone were generally larger (median diameter 30 μm) than those with both DBH-LI and NPY-LI (median diameter 20 μm). Some cell bodies showing DBH-LI alone were surrounded by boutons with NPY-LI but not DBH-LI. Axons that displayed simultaneously both substance P-LI (SP-LI) and calcitonin gene-related peptide-LI (CGRP-LI) also formed a plexus around all arteries studied, being particularly dense around the mesenteric and pulmonary arteries. These axons are most likely sensory since SP-LI was reduced by capsaicin treatment, and nerve cell bodies with both SP-LI and CGRP-LI were found in dorsal root ganglia and the vagal ganglion. A dense plexus of axons showing somatostatin-LI was located around the pulmonary artery and its main intrapulmonary branches. A few nerves with vasoactive intestinal polypeptide-LI were found around the dorsal aorta and pulmonary artery. No perivascular nerves with enkephalin-LI were observed. Reversed-phase, high-pressure liquid chromatography of acid extracts of the large arteries showed that the major peaks of NPY-LI and SP-LI coeluted with porcine NPY (1–36) and synthetic SP (1–11), respectively. Thus, the location and structure of these peptides in perivascular nerves has been highly conserved during vertebrate evolution.

Multiple populations of neuropeptide-containing intrinsic neurons in the guinea-pig heart

Recent studies of autonomic ganglia have shown that specific combinations of neuropeptides and other potential neurotransmitters distinguish different functional types of neurons. In the present paper the patterns of coexistence of neurochemicals in guinea-pig cardiac ganglion cells was examined, using multiple-labelling immunohistochemistry. Many neurons were found to contain somatostatin immunoreactivity with various combinations of immunoreactivity for dynorphin B, substance P, neuropeptide Y and nitric oxide synthase. There were several small populations of neurons without somatostatin immunoreactivity, which contained combinations of immunoreactivity for vasoactive intestinal peptide, neuropeptide Y, dynorphin B, substance P and nitric oxide synthase. Possible synaptic inputs to these populations of ganglion cells were identified using multiple-labelling immunohistochemistry combined with long-term organ culture. These experiments demonstrated that cardiac ganglia contain prominent pericellular baskets of varicose nerve terminals of sympathetic and sensory origin. In addition, populations of intrinsic intraganglionic nerve terminals were identified which were immunoreactive for vasoactive intestinal peptide, neuropeptide Y or both peptides. These terminals presumably originate from intrinsic neurons, with the same combinations of neuropeptides, located in other cardiac ganglia. These results have demonstrated that there are diverse populations of cardiac ganglion cells in the guinea-pig and that some of these neurons may act as interneurons within the intrinsic cardiac plexuses. Therefore it is highly likely that vagal transmission in the heart is modified by sympathetic, sensory and intrinsic neurons and that cardiac ganglia are complex integrators of convergent neuronal activity rather than simple relays.

Sympathetic noradrenergic neurons containing dynorphin but not neuropeptide Y innervate small cutaneous blood vessels of guinea-pigs.

We have used double-labelling immunofluorescence techniques and retrograde axonal transport of Fast Blue to characterize three populations of sympathetic noradrenergic neurons innervating blood vessels in the hairless skin of the ears and paws of guinea-pigs. Each population of neurons innervated a specific level of the vascular bed, and had a distinctive content of neuropeptides. Sympathetic noradrenergic neurons innervating large distributing arteries contained immunoreactivity to neuropeptide Y. Neurons innervating smaller cutaneous arteries contained immunoreactivity to prodynorphin-derived peptides in addition to neuropeptide Y. Finally, sympathetic neurons innervating the smallest arterioles, and arterio-venous anastomoses, contained immunoreactivity to prodynorphin-derived peptides, but had no detectable neuropeptide Y. Although the major form of dynorphin immunoreactivity in perivascular sympathetic axons was dynorphin A(1-8), immunoreactivity to both dynorphin A(1-8) and dynorphin A(1-17) occurred in the cell bodies of these neurons, suggesting that dynorphin A is processed during axonal transport to the terminals. The perivascular sympathetic neurons containing prodynorphin-derived peptides but not neuropeptide Y are most likely to be involved in the regulation of thermoregulatory cutaneous vascular circuits.

Most peptide-containing sensory neurons lack proteins for exocytotic release and vesicular transport of glutamate

We used multiple‐labeling immunohistochemistry and confocal microscopy to examine co‐expression of immunoreactivity for vesicular glutamate transporters (VGluTs), synaptic vesicle proteins, and soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins in peptide‐containing sensory neurons of guinea pigs, mice, and toads. Axon terminals in the superficial layers of the dorsal horn of the spinal cord with immunoreactivity (IR) for both substance P (SP) and calcitonin gene‐related peptide (CGRP) lacked IR for synaptosome‐associated protein of 25 kDa (SNAP‐25), syntaxin, synaptotagmin, synaptophysin, and synapsin, although adjacent varicosities without neuropeptides had IR for these synaptic proteins. Similarly, peptide‐containing axon terminals in the superficial dorsal horn lacked IR for VGluT1 and VGluT2, despite the presence of VGluT2‐IR in nearby nonpeptide varicosities. VGluT3‐IR was sparse in the dorsal horn of the mouse spinal cord and was not present in peptide‐containing axons. Most peripheral terminals of sensory neurons with both SP‐IR and CGRP‐IR in the skin, viscera, and autonomic ganglia of guinea pigs and mice also lacked IR for synaptic vesicle proteins, SNARE proteins, VGluT1, and VGluT2. In dorsal root ganglia from guinea pigs and mice, most small neurons with IR for both SP and CGRP lacked IR for SNAP‐25, VGluT1, and VGluT2. Thus, proteins considered essential for vesicular uptake and exocytotic release of glutamate are not expressed at detectable levels by most sensory neurons containing SP and CGRP in rodents and toads. These data raise the possibility that most peptide‐containing sensory neurons may not normally release glutamate as a transmitter. J. Comp. Neurol. 483:1–16, 2005.

Pathway-specific connections between peptide-containing preganglionic and postganglionic neurons in the vagus nerve of the toad (Bufo marinus)

We have examined immunohistochemically the distribution of postganglionic nerve cell bodies and their preganglionic inputs in the vagus nerve of the toad, Bufo marinus. Nerve cell bodies containing immunoreactivity (IR) to somatostatin (SOM) were found at the origin of the oesophago-gastric ramus; these neurons projected to the lung. Cell bodies with SOM-IR also occurred in the intracardiac branches of the vagus, but were absent from the distal segments of the pulmonary and oesophageal rami of the vagus. Cell bodies with IR to vasoactive intestinal peptide (VIP) also occurred at the origin of the oesophago-gastric ramus, but most of these neurons projected to the oesophagus. Most neurons in the distal pulmonary and oesophageal rami were VIP-IR. Some nerve cell bodies in the vagosympathetic trunk and in the intracardiac rami contained both SOM-IR and VIP-IR. Vagal preganglionic nerve fibres with IR both to a somatostatin-like peptide and to substance P were associated exclusively with those postganglionic VIP-IR neurons that projected to the oesophagus. These results provide evidence for highly specific connections between immunohistochemically defined populations of preganglionic and postganglionic neurons in the vagus nerve.

Galanin-like immunoreactivity in sympathetic and parasympathetic neurons of the toad Bufo marinus

Immunoreactivity (IR) to galanin (GAL) was detected in a wide range of peripheral autonomic neurons in the toad Bufo marinus. Forty percent of adrenergic nerve cell bodies in paravertebral sympathetic ganglia had GAL-IR in addition to neuropeptide Y (NPY)-IR. Some of these neurons projected to systemic arteries. GAL-IR was localized in parasympathetic neurons supplying the heart, lung, pulmonary artery, bladder, rectum and tongue. Eighty-two percent of intracardiac vagal nerve cell bodies had both GAL-IR and somatostatin (SOM)-IR. GAL-IR and SOM-IR were also co-localized in cholinergic post-ganglionic vagal neurons supplying the lung musculature and the pulmonary artery, and in neurons intrinsic to the bladder. Many postganglionic glossopharyngeal neurons in the tongue contained both GAL-IR and vasoactive intestinal peptide (VIP)-IR. Therefore, in Bufo marinus, a GAL-like peptide, in combination with other peptides or with adrenaline or acetylcholine, may be involved in neurotransmission in several different functional classes of autonomic neurons.

Partial depletion of neuropeptide Y from noradrenergic perivascular and cardiac axons by 6-hydroxydopamine and reserpine

The effects of 6-hydroxydopamine (6-OHDA) and reserpine on the storage of neuropeptide Y (NPY) in noradrenergic cardiovascular nerves were examined with both immunohistochemistry and radioimmunoassay (RIA). Immunohistochemical double-labelling techniques demonstrated that NPY was located only in noradrenergic axons in the guinea-pig carotid artery, mitral valve, thoracic inferior vena cava, thoracic aorta, superior mesenteric artery and small saphenous vein. Treatment with 6-OHDA in vivo eliminated noradrenergic, NPY-containing axon terminals from all tissues, but preterminal axons were still prominent in the superior mesenteric artery. The greatest depletion of NPY detected by RIA after 6-OHDA treatment was found in tissues with a predominance of terminal noradrenergic axons, such as the small saphenous vein, whereas NPY accumulating in preterminal axons masked the loss of NPY from terminal axons in the superior mesenteric artery. After treatment with doses of reserpine that led to a rapid depletion of noradrenaline (NA) from perivascular nerves, NPY was still detected histochemically at all times although levels sometimes appeared to be reduced. RIA demonstrated that the partial depletion of NPY after reserpine consisted of a rapid phase seen in the vena cava and saphenous vein after the highest doses, and a slower phase of NPY depletion from all tissues after all doses of reserpine. The greatest depletion of NPY from terminal axons by reserpine (in small saphenous vein) was 85-90%. These results demonstrate that some NPY can be stored in noradrenergic perivascular axons in the absence of noradrenaline, but that partial depletion of NPY from axon terminals results when NA stores are depleted by reserpine. The variation in extent of NPY depletion between tissues after drug treatments can be explained by variation in the ratio of preterminal to terminal axons.

Co-transmission from autonomic vasodilator neurons supplying the guinea pig uterine artery

This study set out to identify the neurotransmitters involved in autonomic vasodilatation of the guinea pig uterine artery. Non-noradrenergic, paracervical neurons supplying this artery contain at least four neuropeptides: vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), dynorphin A (1-17) and somatostatin, probably in addition to acetylcholine. Transmural nerve stimulation of arterial segments precontracted with phenylephrine (3 x 10(-7) mol l-1 and treated with guanethidine (10(-6) mol l-1), produced relaxations which varied in form with the frequency of stimulation and the length of the pulse train. The relaxations were monophasic at low frequencies (< 2 Hz), and were biphasic at higher frequencies (> 5 Hz) and with longer pulse trains (> 50 pulses). Neither phase of the relaxations was reduced by hyoscine (10(-6) mol l-1), or by removal of the endothelium. The faster phase of the relaxations was selectively reduced (by 61%) during treatment with L-nitro-arginine methyl ester (L-NAME; up to 3 x 10(-5) mol l-1). This reduction was reversed by an excess of L-arginine, indicating that the fast relaxation was mediated by nitric oxide, possibly acting as a neurotransmitter. The slower phase of the neurogenic relaxation was preferentially reduced (by 43%) by the endopeptidase, trypsin (1-3 micrograms.ml-1). As VIP is the only currently identified peptide present in the paracervical neurons which causes vasodilatation, it is likely that VIP, or a closely-related peptide, is the transmitter responsible for the slow relaxation. Acetylcholine and an opioid peptide also seem to be released from the vasodilator neurons, but their effects were small, and may have been restricted to pre-synaptic sites. The slower neurogenic relaxations were inhibited by exogenous neuropeptide Y (68% reduction in amplitude), and were slightly potentiated by somatostatin (21% increase in amplitude). Therefore, endogenous stores of these peptides may also contribute to the sum effect of stimulating the paracervical vasodilator neurons. In conclusion, many different substances may act as autonomic co-transmitters from these pelvic vasodilator neurons.

Evidence that neuropeptide Y released from noradrenergic axons causes prolonged contraction of the guinea-pig uterine artery

The participation of neuropeptide Y (NPY) in transmission from sympathetic vasoconstrictor neurons was investigated in the guinea-pig uterine artery, where NPY has been demonstrated immunohistochemically in noradrenergic axons. Exogenous NPY produced long-lasting contractions of isolated arterial segments at low resting tone. Low concentrations of NPY (10(-8)-3 X 10(-7) mol.l-1) were more potent than equimolar concentrations of noradrenaline (NA). NPY produced concentration-dependent desensitization to further application of NPY, but did not affect the magnitude of NA contractions. Trypsin (1.4-2 micrograms.ml-1) reduced NPY-induced contractions by 80-100%, but did not alter NA-induced contractions. Transmural electrical stimulation of arterial segments, after surgical removal of vasodilator axons, produced biphasic contractions which were abolished by guanethidine. Prazosin abolished the fast phase of the neurogenic contraction, leaving a slow contraction with a time course similar to that produced by a low concentration of NPY. The slow contraction was more pronounced at higher frequencies of stimulation (15-20 Hz) than at lower frequencies, and was selectively reduced after desensitization produced by NPY (10(-5) mol.l-1), or after exposure to trypsin. These results suggest that sympathetic vasoconstriction of the guinea-pig uterine artery is produced by release of both NA and NPY from noradrenergic axons.