J. Aberdeen

Marked increases in calcitonin gene-related peptide-containing nerves in the developing rat following long-term sympathectomy with guanethidine

Changes in the innervation of the cardiovascular system, urinogenital tract and sympathetic and non-sympathetic ganglia have been examined following long-term sympathectomy. Patterns of innervation were investigated using histochemical and immunohistochemical techniques, while levels of noradrenaline and neuropeptides were measured by neurochemical assays. Large doses of guanethidine (50 mg/kg) were given daily for 3 weeks to 8-day-old rat pups, which were killed at 6 or 20 weeks of age. In both age groups noradrenergic nerves were severely depleted or absent, while in some regions dramatic increases of calcitonin gene-related peptide levels were demonstrated. This was revealed by an increase in the density of nerve fibres and in calcitonin gene-related peptide content (up to 18-fold), most notably in the right atrium and superior cervical ganglion. No changes in substance P- or vasoactive intestinal polypeptide-immunolabelled nerves were seen. Conversely, short-term sympathectomy by 6-hydroxy-dopamine treatment caused a depletion of noradrenaline which was not accompanied by an increase in the number or content of calcitonin gene-related peptide-immunolabelled nerves. The possibility that nerve growth factor is involved in the mechanism of hyperinnervation by calcitonin gene-related peptide-containing sensory nerves following long-term sympathectomy is discussed.

Relaxation of sheep cerebral arteries by vasoactive intestinal polypeptide and neurogenic stimulation: inhibition by l-NG-monomethyl arginine in endothelium-denuded vessels

1 Perivascular nerves of the sheep middle cerebral artery show immunoreactivity for both vasoactive intestinal polypeptide (VIP) and calcitonin gene‐related peptide (CGRP). 2 Rings of endothelium‐denuded sheep middle cerebral artery precontracted with 5‐hydroxytryptamine were relaxed by CGRP (maximum relaxation = 87.8 ± 8.1%, pD2 = 7.81 ± 0.12, n = 12) and by VIP (maximum relaxation = 55.1 ± 4.1%, pD2 = 7.65 ± 0.04, n = 18). Rings of endothelium‐denuded cat middle cerebral artery precontracted with U46619 were also relaxed by vasoactive intestinal polypeptide (maximum relaxation = 53.1 ± 6.1%, pD2 = 7.82 ± 0.11, n = 6). 3 Haemolysate (1 μl ml−1) inhibited VIP‐induced relaxation in endothelium‐denuded sheep and cat middle cerebral artery (n = 6) but had no effect on the CGRP‐induced relaxation of the sheep middle cerebral artery (n = 6). 4 The relaxant response to VIP in endothelium‐denuded sheep middle cerebral artery was inhibited by methylene blue (10 μm) and augmented by either M&B 22948 (10 μm) or superoxide dismutase (150 units ml−1). Indomethacin (1 μm) had no effect. 5 The addition of l‐NG‐monomethyl arginine (100 μm) inhibited both neurogenic and VIP‐induced relaxation of endothelium‐denuded sheep MCA by 56 ± 6% and 60 ± 6% (n = 5) respectively. The CGRP‐induced relaxation was unaffected. 6 It is concluded that neurally mediated vasodilatation in the sheep middle cerebral artery is mediated largely by VIP through a direct action on smooth muscle through a cyclic‐GMP‐mediated mechanism that appears to involve synthesis of nitric oxide from l‐arginine. Vasodilatation by CGRP, which is also contained in perivascular nerves, does not utilize this pathway.

NEURAL BASIS FOR REGULATION OF COCHLEAR BLOOD-FLOW - PEPTIDERGIC AND ADRENERGIC-INNERVATION OF THE SPIRAL MODIOLAR ARTERY OF THE GUINEA-PIG

The spiral modiolar artery is the terminal artery in the cochlea, and as such is expected to play a major role in the control of cochlear blood flow. In this study, we examined the distribution of adrenergic and peptidergic nerve fibres on the spiral modiolar artery of the guinea pig using histofluorescence and immunofluorescence techniques. The spiral modiolar artery was dissected from the modiolus so that the entire length of the vessel and its branches, could be observed. Noradrenaline was identified using the glyoxylic acid histofluorescence technique. The presence of the vasoactive peptides substance P, calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), was investigated using antibodies against these peptides. Each putative transmitter tested yielded labelled nerve fibres throughout the length of the spiral modiolar artery and its branches. Double-labelling experiments confirmed that CGRP and substance P are contained in the same fibres but that VIP and substance P appear to be contained in different populations of fibres. These results establish that nerve fibres containing vasoactive peptides and noradrenaline supply the spiral modiolar artery and suggest that they are involved in the regulation of cochlear blood flow.

Guanethidine sympathectomy of mature rats leads to increases in calcitonin gene-related peptide and vasoactive intestinal polypeptide-containing nerves

Changes in the innervation of the heart (right atrium), mesenteric blood vessels, vas deferens and superior cervical ganglia have been examined following long-term sympathectomy of the mature rat. Patterns of innervation were investigated by histochemical and immunohistochemical techniques, while levels of noradrenaline and neuropeptides were measured by neurochemical assays. Large doses of guanethidine (80 mg/kg) were given daily for four weeks to 12-14 week-old male rats which were killed at 18-20 weeks of age. Catecholamine-containing nerves were severely depleted or absent in all tissues, together with a reduction in noradrenaline content. Neuropeptide Y levels were depleted by 97% in vas deferens, 78% in mesenteric vein and 50% in right atrium and superior cervical ganglion. Increases in levels of calcitonin gene-related peptide were seen in the mesenteric vein (up seven-fold), superior cervical ganglia (up 11-fold) and vas deferens (prostatic portion up three-fold), which were also evident by assessment of immunolabelling of nerve fibres. Calcitonin gene-related peptide levels were not increased in the right atrium. In addition, an increase in vasoactive intestinal polypeptide-immunoreactive nerve fibre density was seen in the mesenteric artery and vas deferens, although no significant differences were observed in assays of vasoactive intestinal peptide levels in any tissue. No changes were seen in the innervation of any of the tissues by substance P-immunoreactive nerve fibres either by immunohistochemical or immunochemical assay assessment. This study indicates that there are selective changes in the mature nervous system in response to the loss of sympathetic nerves. Differences between these changes and the response of the developing nervous system to long-term sympathectomy are discussed.

Sympathetic and nonsympathetic neuropeptide Y-containing nerves in the rat myocardium and coronary arteries.

We have examined the neuropeptide Y-containing intrinsic nerves of the heart in young (6-week-old) and adult (4-month-old) rats to determine whether they project to the coronary arteries or are capable of doing so if the neuropeptide Y-containing extrinsic nerves are removed. Chronic treatment of neonates with guanethidine was used to permanently destroy the sympathetic nerves. In the young treated animals, 33-54% of the neuropeptide Y remained in the heart despite a 90-99% reduction in norepinephrine; these proportions did not change in the animals that were allowed to develop to adulthood. The level of neuropeptide Y in the right atrium of young animals was unexpectedly high (252 +/- 28.7 pmol/g) compared with adults (75.4 +/- 18.8 pmol/g). The coronary arteries in the control rats received a moderately dense supply of neuropeptide Y-containing nerves; after guanethidine, all neuropeptide Y-containing nerves innervating the large coronary arteries disappeared, but some were still seen in association with small resistance vessels. No compensatory proliferation of the intrinsic neuropeptide Y-containing neurons occurred in the adult sympathectomized animals, and the intrinsic nerves did not reinnervate the large coronary arteries. These results are discussed in relation to the clinical syndrome of coronary artery spasm.

Increases in NPY in non-sympathetic nerve fibres supplying rat mesenteric vessels after immunosympathectomy

The effect of nerve growth factor (NGF) deprivation on developing peripheral peptide-containing nerves has been examined in Wistar rats. Animals were treated from birth for 7 days with antibodies to NGF (10 microliters/g body weight) and killed at 4 or 8 weeks of age. The nerves of the mesenteric and femoral blood vessels, vas deferns and bladder were viewed with histochemical and immunohistochemical techniques. The effectiveness of anti-NGF treatment was monitored by viewing catecholamine (CA)-containing nerves, which were virtually absent from the blood vessels, but were little affected in the vas deferens and bladder in both age groups. Immunoreactivity for substance P and calcitonin gene-related peptide was slightly reduced in the blood vessels. Immunoreactivity for neuropeptide Y (NPY) was reduced in the femoral blood vessels by 88% at both ages, but reductions in NPY immunoreactivity (NPY-IR) in the mesenteric vessels varied with age. In the mesenteric artery at 4 weeks, NPY-IR was reduced by 96% from control values, but at 8 weeks it was reduced by only 37%. Acute sympathectomy with 6-OHDA treatment reduced NPY-IR in the mesenteric artery by 98% at 4 weeks and 93% at 8 weeks. It is proposed that the increase in NPY-IR but not CA-containing nerves in the mesenteric artery between 4 and 8 weeks after immunosympathectomy is due to compensatory innervation from a non-sympathetic source (probably enteric neurons) that is available to mesenteric, but not to femoral blood vessels.

Differential effect of immunosympathectomy on the expression of rat enteric neurotransmitters

The effect of immunosympathectomy on the pattern of distribution of catecholamine- and peptide-containing nerve fibres and neurones in the myenteric and submucous plexuses of rat ileum was investigated. There was an increase in vasoactive intestinal polypeptide (VIP)-, galanin (GAL)- and substance P-like immunoreactivity in the myenteric plexus of ileum from rats treated with nerve growth factor (NGF) antiserum compared with controls. A similar increase in immunoreactivity was observed in VIP-, GAL- and neuropeptide Y (NPY)-containing submucous neurones and nerve fibres. In contrast, the immunosympathectomy had no effect on the pattern of distribution of catecholamine-, calcitonin gene-related peptide (CGRP)- and NPY-containing nerve fibres in the myenteric plexus or on substance P- and CGRP-containing neurones and nerve fibres of the submucous plexus. The findings of the present study suggest that NGF may differentially regulate the expression of enteric neuropeptides at a postnatal stage of development.

Selective Damage to Sensorimotor Perivascular Nerves in the Mesenteric Vessels of Diabetic Rats

The perivascular innervation of the superior mesenteric artery and vein was examined using immunohistochemical and immunoassay techniques in rats 8 weeks after induction of diabetes with streptozotocin (STZ). Increased density of innervation and fluorescence intensity was noted for substance P– and calcitonin gene-related peptide–immunoreactive nerves in the diabetic vessels. A slight increase in the density of vasoactive intestinal polypeptide-immunoreactive nerve fibers innervating the mesenteric artery was also noted. However, there was no change in the density of neuropeptide Y– and dopamine β-hydroxylase–immunoreactive nerve fibers, although the fluorescence intensity of neuropeptide Y–immunoreactive nerve fibers was reduced in diabetic rat vessels. Immunoassays showed that the levels of substance P– and calcitonin gene–related peptide were increased > 10-fold in the diabetic mesenteric vein, while levels of neuropeptide Y and vasoactive intestinal polypeptide were unchanged. In summary, there is a marked increase in nerve fibers containing sensory neuropeptides in mesenteric vessels of STZ-induced diabetic rats, which, in view of the reported impaired sensorimotor function in these vessels, is likely to reflect a neuropathic change.

The effect of atherosclerosis on neuromodulation of sympathetic neurotransmission by neuropeptide Y and calcitonin gene-related peptide in the rabbit mesenteric artery.

The neuromodulatory actions of neuropeptide Y (NPY) (0.1 microM) and calcitonin gene-related peptide (CGRP) (0.01 microM) on nerve-evoked contractions have been studied in the Watanabe heritable hyperlipidemic (WHHL) rabbit mesenteric artery from 4-, 6- and 12-month-old animals with New Zealand white (NZW) rabbits being used as age- and sex-matched controls. Nerve-evoked contractions in 12-month-old rabbits were smaller in WHHL in comparison to NZW rabbits, with no difference between the two strains of rabbit at 4 and 6 months of age. Both the potentiating effect of NPY and the inhibitory effect of CGRP on nerve-evoked contractions increased significantly at 12 months of age compared with responses measured in younger WHHL rabbits, and were greater than in 12-month-old control NZW rabbits. In contrast, the direct smooth muscle relaxant response of CGRP on raised-tone preparations was not different between the two strains of rabbit at any age. Both NPY-immunoreactive and CGRP-immunoreactive nerve fibres were less varicose in 6- and 12-month-old WHHL rabbits when compared with younger WHHL rabbits and NZW controls. In conclusion, this study shows that while nerve-evoked contractions are reduced, in the 12-month-old WHHL rabbit mesenteric artery, the neuromodulatory actions of NPY and CGRP are augmented.

Neuropeptide Y in non-sympathetic nerves of the rat: Changes during maturation but not after guanethidine sympathectomy

Non-sympathetic neuropeptide Y-containing nerves were demonstrated by their persistence after destruction of sympathetic nerve terminals by acute 6-hydroxydopamine treatment for 48 h. In order to examine whether these neuropeptide Y-containing nerves reinnervate tissues following the loss of sympathetic nerves we administered guanethidine sulphate to one-week-old rat pups for three weeks to produce a complete and long-lasting sympathectomy and we monitored the innervation of the superior cervical ganglion, mesenteric vein, vas deferens and urinary bladder by noradrenaline- and neuropeptide Y-containing nerves two and 16 weeks later (assay and histochemical observations). By two weeks the reduction in neuropeptide Y content of tissues was similar to the reduction after acute sympathectomy with 6-hydroxydopamine treatment, indicating that there was no early reinnervation by non-sympathetic neuropeptide Y-containing nerve fibres at a time when sensory transmitters increase. Furthermore, there was no reinnervation by neuropeptide Y-containing nerve fibres by the time these sympathectomized animals had reached maturity, 16 weeks after cessation of treatment. Neuropeptide Y levels increased in the superior cervical ganglion with normal maturation but decreased in the prostatic end of the vas deferens. A non-sympathetic source of neuropeptide Y demonstrated in the immature rat vas deferens was no longer evident in the mature animal.