Non-sympathetic Nerve Research Articles

Saliva secretion in engrafted mouse bioengineered salivary glands using taste stimulation

PurposeThe aim of this study was to compare saliva flow and protein composition induced using five basic taste stimulations between natural and bioengineered salivary glands. Materials and methodsWe developed a mouse saliva secretion model using taste stimulation and analyzed the saliva secretion from natural and bioengineered salivary glands using an assay. The protein components and alpha-amylase in the natural and bioengineered saliva were analyzed by gel electrophoresis and Western blotting. ResultsThe salivary flow responses induced by sour (citric acid) and bitter (quinine-HCl) stimuli were significantly high in the natural and bioengineered salivary glands. Although the protein concentrations in the natural and bioengineered saliva induced using five basic taste stimulations were similar, the protein composition and the amylase concentration in the natural saliva after taste stimulation had different profiles. Sympathetic and non-sympathetic nerves were observed around the acini and ducts in the natural and bioengineered salivary glands. However, the frequency of neuropeptide Y-positive sympathetic nerves in the bioengineered gland was relatively high compared to that in the natural gland. ConclusionsThese results suggest that the signal balance between the sympathetic and parasympathetic components of the efferent nerves in an engrafted bioengineered salivary gland may differ from that in a natural salivary gland.

Plasma neuropeptide Y (NPY) increases in humans in response to the alpha 2 antagonist yohimbine.

Previous studies have shown that the intravenous administration of yohimbine, an alpha 2 antagonist, increases norepinephrine turnover and has related anxiogenic effects in humans. We herein report that yohimbine also increases plasma neuropeptide Y (NPY) in healthy human subjects. This finding is consistent with previous reports in animals, but contrasts with a previously reported study in humans. NPY is a 36 amino acid peptide neurotransmitter located in sympathetic and nonsympathetic nerve fibers, as well as in brain structures such as the locus coeruleus, where it is colocalized with norepinephrine. NPY has been shown to inhibit locus coeruleus neuronal firing, decrease norepinephrine release, and increase postsynaptic noradrenergic signal transduction. When administered centrally, NPY also has anxiolytic properties. This study therefore suggests that yohimbine challenge may be useful in assessing NPY and noradrenergic system interactions in neuropsychiatric disorders such as panic disorder or post traumatic stress disorder in which noradrenergic system dysfunction has been observed.

Non-sympathetic Nerve Regulation of Daily Rhythms of Serine Dehydratase, D Site-binding Protein, and HMG-CoA Reductase mRNA Levels in Rat Liver

Rat liver serine dehydratase, D site-binding protein, HMG-CoA reductase mRNA levels are known to exhibit daily rhythms which are affected by lesions of the suprachiasmatic nuclei (Ogawa and Ansai (1995) Arch. Biochem. Biophys., 316: 844–850) but not by adrenalectomy (Ansai, Y. and Ogawa, H. (1996) Biol. Rhythm Res., 27: 175–184). Autonomic nerves have been reported to be important in some metabolic processes in the liver, but little is known about the role of innervation in the generation of these rhythms. To this end, rat sympathetic hepatic nerves were destroyed by surgical and chemical procedures, and after four days the animals were killed at 6-hour intervals. Concentrations of noradrenaline, a major catecholamine in the liver, decreased to below 5% of control levels, indicating the completeness of denervation. However, the above three mRNA levels at each time point were not substantially influenced by this treatment. These findings suggest that the daily rhythms of serine dehydratase, D site-binding protein, and HMG-CoA reductase mRNA levels are not under sympathetic nervous control.

Presence of nitric oxide synthase in the sheep pineal gland: an experimental immunohistochemical study.

By use of immunohistochemistry, a dense network of nerve fibres immunoreactive to the neuronal form of nitric oxide synthase (NOS, subtype I) was demonstrated in the pineal gland of sheep. The NOS-immunoreactive fibres were located in the pineal capsule and the connective tissue septae of the gland, but fibres were also present intraparenchymally between the pinealocytes. NOS-immunoreactive nerve fibres were still present in the gland 1 month after bilateral removal of the superior cervical ganglia. By use of an antibody directed against endothelial NOS (subtype III), only pineal blood vessels were stained. This staining was still present in the ganglionectomized animals. No difference was found in the staining between the control animals and the ganglionectomized ones. The pinealocytes were not stained, neither by the antibody against neuronal NOS nor by the antibody against endothelial NOS. By use of double immunohistochemical stainings, NOS was in many nerve fibres colocalized with vasoactive intestinal peptide. Western blot analysis of supernatant fractions of sheep pineal homogenates showed the presence of a band corresponding to the neuronal NOS. Thus, the present data show a prominent innervation of the sheep pineal gland with NOS-immunoreactive nerve fibres with their origin outside the sympathetic nervous system, indicating an influence of NO on the pinealocyte metabolism from non-sympathetic nerve fibres in this species. The presence of NOS in both perivascular nerve fibres and the endothelium of the blood vessels of the gland suggests a role of NO in the regulation of the circulation of the sheep pineal gland.

In vivo infusion of NGF induces the organotypic regrowth of perivascular nerves following their atrophy in aged rats

The aim of this study was to investigate whether NGF could reverse the nerve fiber atrophy exhibited by some neurons in old age. We used quantitative immunohistochemical techniques to investigate how the autonomic nerves that supply different blood vessels are affected by aging. Age changes in the nerve plexus were not widespread but were specific to particular vascular targets. Furthermore, where nerve fiber atrophy did occur, it affected different populations of nerves; specifically, sympathetic nerve fibers were lost from the middle cerebral artery while nonsympathetic nerves were lost from the tail vein. Peripheral target tissues have been shown to have a trophic influence on the pattern and density of their innervation, which declines in old age (Gavazzi et al., 1992), possibly as a result of decreased availability of neurotrophic factors such as NGF. Consequently, in an attempt to reverse nerve fiber atrophy, we used miniosmotic pumps to infuse NGF for 2 weeks over involuting nerve fibers in the middle cerebral artery of freely moving aged rats. Our results show that exogenous NGF can induce organotypic neurite outgrowth from aged neurons undergoing nerve fiber atrophy. Moreover, NGF can induce a change in the pattern of the old nerve plexus to one similar to that seen in young animals. NGF had different effects on the extent and manner of neurite outgrowth in young and aged nerves. NGF induced greater nerve growth in old compared to young nerves, causing old nerves to grow within existing nerve bundles and to sprout new nerve fibers, in contrast to young nerves, which tended to grow only within existing nerve bundles. In conclusion, this study shows the potential of exogenous NGF to reverse age-related changes in neuronal morphology.

Uptake of 5-hydroxydopamine into non-sympathetic nerves of guinea-pig uterine artery in late pregnancy.

Perivascular nerve fibres of the uterine artery of virgin and late pregnant guinea-pigs were examined under the electron microscope following loading with 5-hydroxydopamine, a marker for catecholamine uptake, and immunohistochemistry for dopamine beta hydroxylase, neuropeptide Y, vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide. Varicosities, loaded with 5-hydroxydopamine labelled vesicles, and immunoreactive axons were counted in whole transverse sections of uterine arteries. Localization of the immunoreactivities in 5-hydroxydopamine-labelled vesicles was also studied. Colocalization of substance P and dopamine beta hydroxylase immunoreactivities was investigated at the light microscopic level. Both total and relative number of varicosities with 5-hydroxydopamine-labelled vesicles in a whole section of the artery increased in late pregnancy (61.2 +/- 10.2 versus 24.5 +/- 3.2 in virgin, representing 35% and 27% respectively, of all varicosities). Also the number of neuropeptide Y, vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide-immunoreactive axons increased, but their relative proportion remained unchanged. In virgin guinea-pigs only calcitonin gene-related peptide and neuropeptide Y immunoreactivities were associated with varicosities loaded with small dense-cored vesicles, while in late pregnancy 5-hydroxydopamine-labelled vesicles were also seen in a number of vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide-immunoreactive axons. Double immunolabelling for dopamine beta hydroxylase and substance P immunoreactivity showed that substance P immunoreactivity was not present in dopamine beta hydroxylase-immunoreactive axons of the uterine artery, of neither virgin nor late pregnant guinea-pigs. It is concluded that vascular hypertrophy of the uterine artery in late pregnancy is associated with an increase in the number of perivascular nerve fibres, that involves many, if not all of the subpopulations of neurons supplying the uterine artery. Also 5-hydroxydopamine-labelled varicosities were increased, but the results of the present study indicate that some of the nerve fibres that are able to take up 5-hydroxydopamine in late pregnancy are not sympathetic (i.e. are sensory and/or parasympathetic in origin). The relevance of these findings in pregnancy is discussed.

Vasopressinergic Innervation of the Bovine Pineal Gland: Is There a Local Source for Arginine Vasopressin?

Although the presence of arginine vasopressin (AVP) in the mammalian pineal has been characterized biochemically, the source of this nonapeptide hormone remains enigmatic. Most earlier data pointed to an extrapineal origin, although some recent evidence suggests intrapineal synthesis of AVP. The present study examined this issue using a combination of immunohistochemistry with antibodies against both the AVP and neurophysin moieties of the AVP precursor polypeptide, together with polymerase chain reaction (PCR) assay for the specific mRNA. Furthermore, the effects of AVP on melatonin production by monolayer cultures of bovine pinealocytes were examined. Bovine pineal glands possessed numerous neurophysin- and AVP-immunopositive nerve fibers, mainly in the distal part of the gland. However, no positively stained perikarya were observed. As a positive control perikarya of AVP cells were easily identifiable in the magnocellular cells of the bovine hypothalamus. Nevertheless, a highly sensitive PCR assay specific for full-length AVP mRNA did indicate the presence of AVP gene transcripts in both bovine and ovine pineal glands, using two different primer combinations. This suggests either that there are AVP perikarya in the pineal whose peptide contents are below the level of detection by immunohistochemistry or that gene transcripts may be present in AVP nerve axons, as in the posterior pituitary. AVP had significant inhibitory effects on noradrenaline-provoked melatonin secretion in vitro. These results indicate that AVP released by nonsympathetic nerve fibers terminating in the bovine pineal gland may act to modulate melatonin production.

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 μl/g body weight) and killed at 4 or 8 weeks of age. The nerves of the mesenteric and femoral blood vessels, vas deferens 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.

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.

Neuropeptide Y-like immunoreactivity in relation to the distribution of sympathetic nerve fibers in the heart conduction system

The distribution of nerve fibers showing neuropeptide Y-like immunoreactivity (NPY-LI) was compared with the distribution of the sympathetic nerve fibers in all parts of the conduction system and adjacent atrial and ventricular tissue of the bovine heart. Tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH) served as markers for the sympathetic nerve fibers. NPY-LI was detected in most of the sympathetic nerve fibers that were present in nerve fascicles and that were associated with conduction cells and arterial walls in all regions examined. This phenomenon was more apparent when staining for NPY was compared with staining for DBH than with staining for TH. It was also found that some nerve varicosities exhibiting NPY-LI in association with arterial walls and local ganglia did not show DBH-LI. Furthermore, some of the ganglionic cells located in the regions of the conduction system showed NPY-LI but not DBH- or TH-LI. The observations are discussed in relation to what is known of the subcellular localization of NPY, TH and DBH. It is furthermore concluded that an NPY-like peptide is present in the sympathetic innervation of all parts of the conduction system and ordinary myocardial tissue, but that this peptide is also present in nerve fibers in the heart that do not represent sympathetic fibers. The observations raise important questions for further research aimed at determining the effects of NPY in relation to the function of the conduction system and in relation to the functions of not only sympathetic but also non-sympathetic nerve fibers in the heart.

Purinergic and non-purinergic innervation in the cerebral arteries of the dog.

1 Possible involvement of sympathetic purinergic transmission in the neurogenic response of dog cerebral and basilar arteries was examined with the use of alpha, beta-methylene ATP and adrenoceptor, cholinoceptor blocking agents. 2 In the isolated basilar arteries, electrical transmural stimulation produced a transient contraction which was frequently followed by a relaxation. This transient contraction was abolished after desensitization of P2-purinoceptors with alpha, beta-methylene ATP or by treatment with guanethidine. The relaxant response induced by electrical stimulation was also attenuated but was not abolished by such treatments. Prazosin, propranolol and atropine had no significant effect on the responses to electrical stimulation. Yohimbine augmented both the contractile and relaxant responses. 3 In most preparations of the dog middle cerebral arteries, electrical transmural stimulation produced only a relaxation. This relaxation was little affected after treatment with alpha, beta-methylene ATP or guanethidine, and was not inhibited by the other adrenoceptor and cholinoceptor blocking agents. 4 Tetrodotoxin abolished the responses induced by electrical transmural stimulation in both the basilar and middle cerebral arteries. 5 Exogenous ATP (10(-6) and 10(-5)M) produced a transient contraction followed by a relaxation of the basilar arteries and a relaxation of the middle cerebral arteries. Desensitization of P2-purinoceptors abolished the contractile response to ATP without affecting the amplitude of relaxation. 6. In the basilar and middle cerebral arteries preincubated with [3H]-noradrenaline, electrical transmural stimulation evoked an increase in 3H-efflux and this response was markedly inhibited by guanethidine or tetrodotoxin but was not affected by alpha, beta-methylene ATP. Yohimbine increased the evoked 3H-efflux. 7. These findings indicate that cerebral arteries of the dog are innervated by sympathetic purinergic nerves and non-sympathetic nerves which liberate unknown vasodilator substance(s), and that the former nerves are more dominant in the neurogenic response to electrical stimulation of the dog basilar artery than in the middle cerebral artery.

Degeneration of myelinated sympathetic nerve fibres following treatment with guanethidine.

The specificity and characteristics of the degeneration of myelinated axons after chronic guanethidine treatment have been investigated in sympathetic and non-sympathetic nerves. Adult male Sprague-Dawley rats aged approximately 43 weeks were treated with guanethidine sulphate (50 mg per kg body weight per day) for between ten days and six weeks. Tissues were examined by qualitative and quantitative light and electron microscopy. In the superior cervical (sympathetic) ganglion (SCG), guanethidine treatment produced a 78% decrease (P = 0.009) in the mean number of myelinated fibres at a standard level of section, compared to the contralateral control ganglion which was removed surgically prior to drug treatment. This reduction in the treated SCG was apparent after 10 days, though complete degeneration of nerve cell bodies was not widespread at this stage. Degeneration of unmyelinated axons was extensive. Degenerating myelinated fibres were consistently small in diameter (up to approximately 3 microns). In individual myelinated fibres the earliest signs of degeneration involved disruption of axonal organelles, particularly the cytoskeleton, and focal widening of the periaxonal space. Myelin breakdown followed these events; degeneration of myelin still associated with a structurally intact axon was not observed. Myelin breakdown appeared to take place initially within the Schwann cell, at least to the stage of 'loosened' membranes. However, infiltrating cells were also involved in myelin phagocytosis. At all stages of treatment some small diameter myelinated fibres remained intact, and there was no evidence of degeneration of the larger diameter fibres (up to approximately 15 microns) which are consistently present in small numbers in the SCG. In the cervical sympathetic trunk, which carries preganglionic axons to the SCG and the vagus and sciatic nerves, degeneration only of unmyelinated axons was detected. These results indicate that guanethidine does not exert a primary degenerative influence on myelin or myelinating Schwann cells and that the myelin degeneration observed in the SCG is a secondary result of the previously documented selectively destructive effect of guanethidine on postganglionic sympathetic neurons. Surviving, small diameter myelinated fibres in the SCG could be either preganglionic or processes of resistant postganglionic neurons, while the larger diameter fibres are likely to be somatic. While the cervical sympathetic trunk, vagus and sciatic nerves all contain postganglionic sympathetic fibres it appears that few of these are myelinated, at least at the levels sampled in this study.

Autonomic innervation of cerebral blood vessels decreases in renal hypertensive rats.

The ultrastructural distribution of the autonomic nerves of brain arteries was investigated in renal (one-kidney, one clip) hypertensive and normotensive Wistar-Kyoto rats. Sympathetic and nonsympathetic nerve terminals were found only in the adventitial layer of brain arteries of renal hypertensive and normotensive rats. In both normotensive and renal hypertensive rats the total nerve endings were dense in anterior cerebral artery, moderately dense in middle cerebral artery, and sparse in basilar artery. In normotensive rats, nonsympathetic nerves outnumbered sympathetic nerves in anterior cerebral, middle cerebral, and basilar arteries. In renal hypertensive rats these two types of nerve terminals in close apposition to smooth muscle decreased in anterior cerebral and basilar arteries, while those in middle cerebral arteries remained unchanged. These results suggest that the potential neurogenic control of cerebral blood vessels as well as the trophic effect of sympathetic nerves on brain blood vessels may decrease in renal hypertensive rats. As this finding contrasts with that in spontaneously hypertensive rats, the pattern of innervation in brain arteries may differ in different types of hypertension.

Altered cerebral vessel innervation in the spontaneously hypertensive rat.

Autonomic innervation in the cerebral arterial walls of adult male spontaneously hypertensive rats and of normotensive Wistar-Kyoto rats was studied. When examined by fluorescence microscopy, dense catecholamine fluorescence was observed in anterior cerebral and middle cerebral arteries of both Wistar-Kyoto and spontaneously hypertensive rats. However, vertebral and basilar arteries and small pial arteries of Wistar-Kyoto rats received extremely sparse or no catecholamine fluorescence, whereas, in the respective regions of spontaneously hypertensive rats, catecholamine fluorescence was found to be significantly elevated. The endogenous norepinephrine content was also higher in cerebral arteries of spontaneously hypertensive than of Wistar-Kyoto rats. When examined ultrastructurally (potassium permanganate fixation), the incidence of granular vesicle-containing nerves, indicative of sympathetic nerves, was found to be significantly elevated in all cerebral arteries of spontaneously hypertensive rats examined. In contrast, the agranular vesicle-containing nerve, indicative of nonsympathetic nerves, with close synaptic cleft distance (less than 2 micron) was found to decrease or remain unchanged in the cerebral arteries of spontaneously hypertensive rats. These results suggest that cerebral sympathetic vasoconstriction may become more prominent than nonsympathetic vasodilation in spontaneously hypertensive rats. This finding lends further credence to the previous in vivo findings that cerebral sympathetic vasoconstrictor nerves become more functional and exhibit a protective effect against brain lesions during hypertension. The potential roles of neurogenic components involved in cerebral blood flow autoregulation are also discussed.

Vasoactive intestinal polypeptide-like substance: the potential transmitter for cerebral vasodilation.

In vitro pharmacological studies demonstrated that exogenously applied vasoactive intestinal polypeptide (VIP) relaxes the smooth muscle cells of cat cerebral arteries, whereas substance P constricts them. Ultrastructural-immunocytochemical techniques show that a VIP-like substance is present in the large granular vesicles of nonsympathetic nerve axons and terminals in the cerebral arterial walls. These results provide strong evidence in favor of the hypothesis that a VIP-like substance is the transmitter for vasodilation in cerebral blood vessels.

Physiology of nerve growth factor.

Physiology of nerve growth factor.

Nerve growth factor.

Nerve growth factor.

CELLULAR LOCALIZATION OF MONOAMINES IN THE MEDIAN EMINENCE AND THE INFUNDIBULAR STEM OF SOME MAMMALS.

A histochemical analysis of the monoamines which are strongly accumulated in the median eminence and the proximal part of infundibular stem of all species examined (mouse, rat, guinea pig, hamster, rabbit, and cat) was performed with the help of a highly specific and sensitive fluorescence method. Strong evidence was obtained for the view that the monoamines are localized in very high concentrations to the terminal parts of non-sympathetic nerve fibres, which — mainly at least — converge to the primary plexus of the hypophyseal portal system. The capillaries are densely and closely surrounded by the nerve fibres. Pharmacological experiments, involving the administration of reserpine, nialamide, m-tyrosine and α-methyl-m-tyrosine, furnished good evidence for the view that primary catecholamines, probably mainly DA but also NA, are the predominant monoamines present. The experiments also revealed the existence of catecholamine-containing nerve cells in the arcuate nuclei and the ventral portion of the anterior periventricular nuclei. These nerve cells, situated in the regions where the tubero-infundibular tract arises, may be the cell bodies of adrenergic neurons to which the amine-containing nerve fibres at least partly belong. The findings indicate that primary catecholamines are released to the primary plexus of the hypophyseal portal system and thus transported to the anterior lobe. These amines may consequently act as neuro-humoral transmittors for the regulation of the activity of the anterior pituitary. — No direct adrenergic innervation of the cells in the pars tuberalis and anterior lobe was found. The portal vesstes in the pars tuberalis receive a very sparse adrenergic innervation and the vessels in the anterior lobe receive no or very few adrenergic nerves. Pars intermedia, on the other hand, may have a non-sympathetic adrenergic innervation.