Neuropeptide Y in the adrenal gland: characterization, distribution and drug effects

The occurrence of neuropeptide Y-like immunoreactivity in the adrenal gland was investigated by means of radioimmunoassay, chromatography and immunohistochemistry. The adrenal levels of neuropeptide Y-like immunoreactivity varied considerably between species with lowest amounts in the rat and highest in the cow where immunoreactivity was observed in chromaffin cells and nerve fibres in the capsule and cortex. The distribution of neuropeptide Y-like immunoreactivity in the cow medulla overlapped that of enkephalin-like immunoreactivity. Chromatographic characterisation of rat and cow adrenal extracts showed that the majority of the neuropeptide Y-like immunoreactivity was similar in molecular weight and solubility properties to porcine neuropeptide Y. Rat adrenal contained additional material some of which may represent oxidised neuropeptide Y. The administration of insulin and reserpine to rats in vivo showed that the turnover of adrenal neuropeptide Y-like immunoreactivity is regulated by the splanchnic nerve. Splanchnic activation following insulin-induced hypoglycaemia elicited a 60% depletion of neuropeptide Y-like immunoreactivity 2 h post insulin injection. A smaller (maximum 40%) depletion of neuropeptide Y-like immunoreactivity was measured 24 h after reserpine injections which may be explained by splanchnic activation. Five days after reserpine injections the levels of adrenal neuropeptide Y-like immunoreactivity were increased to 200% of control levels and remained slightly elevated at 25 days. Adrenal enkephalin-like immunoreactivity showed similar but not identical changes following reserpine. The reserpine-induced elevation in neuropeptide Y-like immunoreactivity at the 5-day time point was abolished in rats with a chronic bilateral splanchnectomy. This evidence indicates that neuropeptide Y may be considered as a new adrenal medullary hormone.     

Mechanisms underlying changes in the contents of neuropeptide Y in cardiovascular nerves and adrenal gland induced by sympatholytic drugs

Neuropeptide Y (NPY) is a recently isolated vasoactive peptide, which is present, together with catecholamines, in sympathetic nerves and in the adrenal medulla. In the present study, we report that pretreatment with sympatholytic agents influences the tissue levels of NPY-like immunoreactivity (NPY-LI) in the guinea-pig. Thus, 24 h after reserpine not only noradrenaline (NA), but also NPY-LI, was depleted in the heart, spleen and the adrenal gland. The levels of NPY-LI in the vas deferens and stellate ganglia, however, were unaffected by reserpine in spite of marked depletions of NA. The reserpine-induced depletion of NPY-LI was probably caused by enhanced nerve-impulse flow and subsequent release from cardiovascular nerves in excess of resupply, since it could be prevented by the ganglionic-blocking agent chlorisondamine. Long-term (6 days) treatment with chlorisondamine reduced the levels of NPY-LI in the stellate ganglion. Short-term treatment (48 h) with guanethidine partially prevented the reserpine-induced depletion of NPY-LI, probably due to inhibition of NPY release. Long-term guanethidine treatment depleted not only NA, but also NPY-LI from the spleen. Pretreatment with the alpha-receptor antagonist phenoxybenzamine did not influence the NA levels but reduced the content of NPY-LI in the spleen via a mechanism that was dependent on intact ganglionic transmission. Since NPY has several cardiovascular actions, changes in NPY mechanisms may contribute to the pharmacological and therapeutical effects of sympatholytic agents.     

Neuropeptide tyrosine in the rat adrenal gland--immunohistochemical and in situ hybridization studies

The adrenal gland of the rat was analysed with immunohistochemistry and antisera to neuropeptide tyrosine, to the catecholamine-synthesizing enzymes tyrosine hydroxylase, phenyl-ethanolamine-N-methyltransferase, and to acetylcholinesterase and with in situ hybridization using a nick-translated 280 base pair deoxyribonucleic acid probe coding for exon 2 of the rat neuropeptide tyrosine gene. Neuropeptide tyrosine-like immunoreactivity was observed in three structures: chromaffin cells, medullary ganglion cells and nerve fibers. The chromaffin cells were of both the noradrenaline- and adrenaline-type. The ganglion cells did not seem to contain any catecholamine-synthesizing enzymes but exhibited a strong immunoreaction for acetylcholinesterase. They were thus in all probability cholinergic neurons. In situ hybridization using the nick-translated deoxyribonucleic acid probe to rat neuropeptide tyrosine messenger ribonucleic acid revealed a very high-grain density over the ganglion cells, a moderate density over the chromaffin cells and a low background over cortex, in agreement with the immuno-histochemical demonstration of neuropeptide tyrosine-like immunoreactivity both in chromaffin and ganglion cells. The intense neuropeptide tyrosine-like immunoreactivity and low content of neuropeptide tyrosine messenger ribonucleic acid suggest that the chromaffin cells have fairly large peptide stores but that the peptide turnover is low. In contrast, the ganglion cell bodies seem to contain low amounts of neuropeptide tyrosine-like immunoreactivity but exhibit a high neuropeptide tyrosine synthesis rate. Preliminary studies with the amine-depleting drug reserpine revealed an increase in messenger ribonucleic acid both in ganglion cells and medullary cells. In the chromaffin cells the highest activity was seen 3 and 4 days after injection, and the levels were down to normal after 8 days. The present findings demonstrate neuropeptide tyrosine synthesis and storage in two cell populations in the adrenal medulla. In situ hybridization with its cellular resolution can provide information on possible differential effects of drugs and experimental procedures on these two neuropeptide tyrosine stores.     

Effects of vinblastine on neuropeptide Y levels in the sympathoadrenal system,bone marrow and thrombocytes of the rat

The dose-related and time-related effects of vinblastine on tissue, platelet and plasma content of neuropeptide Y (NPY) were investigated in the rat and compared to the effects on catecholamine (CA) content. CA was quantified by HPLC with electrochemical detection and NPY-like immunoreactivity (LI) was analyzed by radioimmunoassay (RIA). Vinblastine (3.0 mg/kg, i.v.) decreased levels of both NPY-LI and CA after 48 h in the kidney, vas deferens and adrenal gland, whereas in the coeliac ganglion and bone marrow vinblastine induced an increase of NPY-LI which occurred already at a dose of 0.3 mg/kg. Also the content of NPY-LI in platelet-poor plasma and platelets as well as the decapitation-induced increase of plasma levels of noradrenaline (NA) and adrenaline (A) were attenuated by vinblastine (3.0 mg/kg). The elevation of NPY-LI content in the kidney, coeliac ganglion and bone marrow as well as the reduced levels of NPY-LI in platelets and platelet-poor plasma was observed already after 24 hours, whereas the reduction of NPY-LI and CA in the kidney and adrenal gland was present after 2 days. Vinblastine caused a biphasic effect on the content of NPY-LI in the sympathetic nerves of the kidney with an initial increase (by 120% at 24 h) followed by a decrease (by 79% at 4 days). The effect on NA-levels, however, was only a decrease. The axonal transport of NPY-LI as revealed by accumulation above a ligation of the sciatic nerve was reduced by 27% 2 days after vinblastine 3 mg/kg. The vinblastine-evoked depletion of NPY-LI and catecholamines in the kidney as well as in the adrenal was largely prevented by chlorisondamine, a nicotinic ganglionic blocking agent, suggesting that preganglionic neuronal activity was a key factor for this effect, in contrast to the influence on the coeliac ganglion cells and the megakaryocytes in the bone marrow. Furthermore, the delayed vinblastine depletion of NPY-LI in the kidney resembled the influence of surgical axotomy while reserpine caused a more rapid and complete depletion of both NPY-LI and NA. It is concluded that the multiple effects of vinblastine on sympathetic nerves, adrenal gland and megakaryocytes/thrombocytes can be monitored by analysis of NPY and be related to interference with microbutuli function and/or neuronal activation.     

Effect of reserpine, phenoxybenzamine and cold stress on the neuropeptide Y content of the rat peripheral nervous system

The effect of reserpine treatment on the neuropeptide Y content of the rat adrenal gland, heart, kidney and vasculature was studied using a specific radioimmunoassay. One hour after reserpine administration (5 mg/kg) the neuropeptide Y concentration in the adrenal gland was significantly reduced and after 4 h a similar reduction was seen in the heart and kidney. After 48 h, neuropeptide Y concentrations were reduced in all tissues. The greatest reduction occurred in the cardiac septum (77%) and the least in the inferior vena cava (25%). Phenoxybenzamine (2 mg/kg) also caused a reduction in neuropeptide Y concentrations which was less marked than after reserpine, except in the adrenal gland where it was similar. Cold stress caused no change in neuropeptide Y concentrations. The neuropeptide Y depletion induced by reserpine was compared to that following 6-hydroxydopamine. In the heart and pial arteries both drugs caused a similar neuropeptide Y depletion whilst in the pineal gland and renal artery 6-hydroxydopamine had more effect than reserpine. The implications of these results on NPY storage sites are discussed.     

Tyrosine hydroxylase and neuropeptide Y are increased in ciliary ganglia of sympathectomized rats.

We have examined the expression of tyrosine hydroxylase and neuropeptide Y in ciliary ganglia of normal adult rats and of adult rats in which the environment of these neurons was altered by sympathectomy at birth. Following neonatal 6-hydroxydopamine treatment, the proportion of tyrosine hydroxylase-immunoreactive and neuropeptide Y-immunoreactive neurons in ciliary ganglia was significantly increased. In ciliary neurons of both control and sympathectomized rats, neuropeptide Y immunoreactivity was preferentially co-localized with tyrosine hydroxylase. Immunoblot analysis confirmed the presence of tyrosine hydroxylase and its increase following sympathectomy. In situ hybridization studies revealed that many ciliary neurons contain mRNA for tyrosine hydroxylase and for neuropeptide Y. Like tyrosine hydroxylase immunoreactivity, the number of ciliary neurons containing tyrosine hydroxylase mRNA and the amount of mRNA per cell were increased in 6-hydroxydopamine-treated rats. In contrast, neuropeptide Y mRNA levels were the same in control and 6-hydroxydopamine-treated rats. Nerve growth factor is a candidate for mediating the effects of sympathectomy and most ciliary neurons in control and sympathectomized rats expressed immunoreactivity for the low-affinity nerve growth factor receptor. In addition, ciliary neurons from 6-hydroxydopamine-treated animals possessed increased nerve growth factor receptor immunoreactivity. These studies indicate that both tyrosine hydroxylase and neuropeptide Y in the ciliary ganglion are regulated by alterations in their environment. Their expression was enhanced by chemical sympathectomy which does not affect ciliary neurons directly but, rather, removes sympathetic innervation of shared targets, including the iris. In situ hybridization analysis suggests that the increased tyrosine hydroxylase and neuropeptide Y levels result from different mechanisms and provides evidence that neuropeptide levels can be regulated without changes in mRNA levels.     

Neuropeptide Y mRNA regulation in rat sympathetic ganglia: effect of reserpine.

To study the mechanism underlying trans-synaptic neuropeptide regulation, mRNA levels of neuropeptide Y were examined in the rat stellate and superior cervical ganglia using a specific neuropeptide Y cRNA probe. Basal levels of neuropeptide Y mRNA were detectable in total RNA extracts from single ganglia. Reserpine induced a large rise in ganglion neuropeptide Y mRNA. Decentralization prevented the increase of neuropeptide Y mRNA content in the ganglia. This suggests that the reserpine induced increase in neuropeptide Y mRNA was dependent on transsynaptic stimulation. Consequently, neuropeptide mRNA levels in sympathetic ganglia may be under the control of preganglionic impulse flow.     

Differential co-existence of neuropeptide Y (NPY)-like immunoreactivity with catecholamines in the central nervous system of the rat

The distribution of neuropeptide Y immunoreactive cell bodies in relation to various types of catecholamine-containing cell bodies in the rat brain was analyzed immunohistochemically using antisera to tyrosine hydroxylase, dopamine β-hydroxylase and phenylethanolamine N-methyltransferase. Coexistence of the peptide in catecholamine cell bodies was established by using an elution-restaining procedure.Neuropeptide Y-like immunoreactivity was observed in most noradrenergic cell bodies of the Al/Cl cell groups in the ventro lateral medulla oblongata. Similarly this peptide immunoreactivity was also observed in the majority of the adrenergic cell bodies of the C2 group. In the dorsal and dorsal-lateral part of the nucleus of the solitary tract, where a group of small adrenergic cells is present, several small neuropeptide Y immunoreactive cells were also observed. The possibility of coexistence of adrenaline and neuropeptide Y in these cells remains to be established. The majority of the noradrenergic cell bodies of the A2 group, as well as the presumptive dopaminergic cells within its ventromedial part, seemed to lack neuropeptide Y-like immunoreactivity. Many noradrenergic cell bodies of the A6 group in the locus coeruleus proper were neuropeptide Y-immunoreactive, whereas the peptide could not be observed in the subcoeruleus group. Neither the A5 and A7 noradrenergic cells in the pons, nor any of the dopaminergic cell groups in the mesencephalon and forebrain (A8–A15) seemed to contain a neuropeptide Y-like peptide.The findings indicate that central catecholamine neurons can be subdivided into distinct sub-groups based upon the coexistence of a specific peptide.     

Renal sympathetic nerve activation in relation to reserpine-induced depletion of neuropeptide Y in the kidney of the rat

The effect of reserpine treatment on renal sympathetic nerve activity and tissue levels of neuropeptide Y (NPY)-like immunoreactivity (LI) and noradrenaline (NA) were studied in rats. Injection of reserpine (1 mg kg-1 i.v.) caused a clear-cut (about 50%) increase in rectified activity of the post-ganglionic sympathetic nerves to the kidney within 15 min in chloralose-anaesthetized rats compared to a saline-treated control group. This increase in nerve activity was still maintained 120 min after the reserpine injection. The renal nerve activation was accompanied by a progressive fall in mean arterial blood pressure and an initial tachycardia. In a separate group of conscious rats, the levels of NPY-LI (1.3 +/- 0.06 pmol g-1) and NA (1.6 +/- 0.07 nmol g-1) in the kidney were significantly reduced (by 74 and 83%, respectively) 24 h after reserpine treatment (1 mg kg-1 i.v.). The reserpine-induced depletion of NPY-LI, but not that of NA, was inhibited by pretreatment with the ganglionic blocking agent chlorisondamine or the alpha 2-adrenoceptor agonist clonidine, both of which are known to decrease renal sympathetic nerve activity. The tissue content of NPY-LI in the right atrium (16.3 +/- 0.7 pmol g-1) was not reduced by reserpine. Arterial plasma NPY-LI in the rat was high (222 +/- 5 pmol l-1), and this value did not change after pretreatment with reserpine, chlorisondamine or clonidine, indicating that, in the rat, circulating NPY-LI is not a good indicator of sympatho-adrenal activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-release of neuropeptide Y and noradrenaline from pig spleen in vivo: importance of subcellular storage, nerve impulse frequency and pattern, feedback regulation and resupply by axonal transport

The importance of subcellular storage, nerve impulse rate and pattern, and feedback regulation, as well as resupply by axonal transport for the release of noradrenaline and neuropeptide Y-like immunoreactivity, was studied in the blood perfused pig spleen in vivo. Vasoconstrictor responses were recorded as perfusion pressure changes. Subcellular fractionation experiments using sucrose density gradients showed a bimodal distribution of noradrenaline (peak concentrations at 0.8 and 1.1 M sucrose) while only one main peak of neuropeptide Y was present (at 1.1 M sucrose). Overflow suggesting release of noradrenaline and neuropeptide Y-like immunoreactivity could be detected after 10 s stimulation at 10 Hz. The ratio for the output of noradrenaline and neuropeptide Y upon continuous nerve stimulation in control animals decreased with frequency. After inhibition of noradrenaline reuptake by desipramine the vasoconstrictor response and noradrenaline output were enhanced while the corresponding overflow of neuropeptide Y was reduced by 50% at 0.5 Hz. Stimulation with the irregular or regular bursting patterns at high frequencies caused larger perfusion pressure increase and relative enhancement of neuropeptide Y output compared to noradrenaline than a continuous stimulation both before and after desipramine treatment. A similar fractional release per nerve impulse was calculated both for [3H]noradrenaline (5.6 +/- 1.0 x 10(-5) and neuropeptide Y (7.3 +/- 0.3 x 10(-5). After reserpine treatment combined with preganglionic denervation the vasoconstrictor responses were more long-lasting, neuropeptide Y release was enhanced while noradrenaline content and release were reduced by 99%. The difference in neuropeptide Y overflow between continuous and bursting types of stimulation was smaller after reserpine treatment. After prolonged intermittent stimulation with regular bursts (20 Hz) for 1 h the splenic content of neuropeptide Y was reduced by 58%, while no change was observed for noradrenaline. The maximal perfusion pressure increase upon prolonged nerve stimulation after reserpine was similar in control and reserpine-treated animals, but after reserpine the vasoconstrictor response and neuropeptide Y release were subjected to fatigue. Ligation experiments of the splenic nerves revealed the splenic neuropeptide Y content was resupplied by axonal transport with a calculated total tissue turnover time of 11 days. In contrast, axonal transport contributed only to a marginal extent for the resupply of noradrenaline.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of the inhibitory roles of neuropeptide Y and galanin on cardiac vagal action in the dog.

Prolonged attenuation of cardiac vagal action occurs following cardiac sympathetic nerve stimulation or intravenous neuropeptide Y (NPY) injections in anaesthetised dogs. Equimolar intravenous injections of galanin (GAL) had no effect on cardiac vagal action in this species. Immunohistochemical analysis of dog stellate ganglia and cardiac muscle showed that most nerve cell bodies showing tyrosine hydroxylase immunoreactivity (TH-IR) also showed immunoreactivity to both NPY and GAL. The results are consistent with the proposal that NPY released from cardiac sympathetic nerves is responsible for the prolonged inhibition of cardiac vagal action known to be caused by such stimulation. A role for GAL, shown here to exist in cardiac sympathetic nerves in the dog, has yet to be determined.     

Changes in the expression of neuropeptide Y (NPY) during maturation of human sympathetic ganglionic neurons: correlation with tyrosine hydroxylase immunoreactivity

Developmental patterns of neuropeptide (NPY) and tyrosine hydroxylase (TH)-immunoreactivities (IR) were investigated using the method of indirect immunohistochemistry in the stellate and thoracic sympathetic ganglia of human neonates ranging in gestational age from 24 to 27 weeks (premature group) and from 38 to 41 weeks (mature group). In the paravertebral ganglia of premature neonates a small (up to 7%) population of NPY-IR nerve cells was revealed. With the gestational age increase (a mature group), a marked elevation of the number of NPY-IR ganglionic neurons (up to 41%) was noted. In contrast, in the sympathetic ganglia of premature neonates almost all the neurons were tyrosine hydroxylase immunoreactive and any change in pattern during maturation was insignificant.

The results demonstrate an age-related increase of neuropeptide Y-immunoreactivity in human paravertebral ganglia during maturation, and suggest that peptidergic co-transmission arises later in development than do the classical autonomic messengers. Adaptability of the fetus to a new external environment at birth demands a qualitatively new activity level of the autonomic nervous system, and this is provided side by side with the classical messengers noradrenaline and acetylcholine by the cotransmitter and modulating role of the neuropeptides. The appearance of neuropeptide Y in the principal sympathetic ganglionic neurons defines not only a qualitatively new level in the functional regulation of target organs at birth, but serves as an index of neonatal maturity.     

The sensory and sympathetic innervation of guinea-pig lung and trachea as studied by retrograde neuronal tracing and double-labelling immunohistochemistry.

The sympathetic and sensory innervation of guinea-pig trachea and lung were studied by means of retrograde neuronal tracing using fluorescent dyes, and double-labelling immunofluorescence. Sympathetic neurons supplying the lung were located in stellate ganglia and in thoracic sympathetic chain ganglia T2-T4; those supplying the trachea resided in the superior cervical and stellate ganglia. Retrogradely labelled sympathetic neurons were usually immunoreactive to tyrosine hydroxylase; the majority also contained neuropeptide Y immunoreactivity. However, a small number were non-catecholaminergic (i.e. tyrosine hydroxylase negative), but neuropeptide Y immunoreactive. Within the airways, tyrosine hydroxylase/neuropeptide Y-immunoreactive axons were found in the smooth muscle layer, around blood vessels including the pulmonary artery and vein, and to a lesser extent in the lamina propria. Periarterial axons contained in addition dynorphin immunoreactivity. Sensory neurons supplying the lung were located in jugular and nodose vagal ganglia as well as in upper thoracic dorsal root ganglia; those supplying the trachea were most frequently found bilaterally in the nodose ganglia and less frequently in the jugular ganglia. A spinal origin of tracheal sensory fibres could not be consistently demonstrated. With regard to their immunoreactivity to peptides, three types of sensory neurons projecting to the airways could be distinguished: (i) substance P/dynorphin immunoreactive; (ii) substance P immunoreactive but dynorphin negative; and (iii) negative to all peptides tested. Substance P-immunoreactive neurons innervating the airways invariably contained immunoreactivity to neurokinin A and calcitonin gene-related peptide. Retrogradely labelled neurons located in the nodose ganglia belonged almost exclusively (greater than or equal to 99%) to the peptide-negative group, whereas the three neuron types each represented about one-third of retrogradely labelled neurons in jugular and dorsal root ganglia. Within the airways, axons immunoreactive to substance P/neurokinin A and substance P/calcitonin gene-related peptide were distributed within the respiratory epithelium of trachea and large bronchi, in the lamina propria and smooth muscle from the trachea down to the smallest bronchioli (highest density at the bronchial level), in the alveolar walls, around systemic and pulmonary blood vessels, and within airway ganglia. Those axons also containing dynorphin immunoreactivity were restricted to the lamina propria and smooth muscle. The origin of nerve fibres immunoreactive for vasoactive intestinal polypeptide, of which a part were also neuropeptide Y immunoreactive, could not be determined by retrograde tracing experiments. Vasoactive intestinal polypeptide-immunoreactive fibres terminating within airway ganglia may be of preganglionic parasympathetic origin, whereas others (e.g. those found in smooth muscle) may arise from intrinsic ganglia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dendritic morphology of presumptive vasoconstrictor and pilomotor neurons and their relations with neuropeptide-containing preganglionic fibres in lumbar sympathetic ganglia of guinea-pigs

We have used intracellular dye-filling combined with multiple-labelling immunofluorescence to examine the dendritic morphology of neurons and their relations with neuropeptide-containing preganglionic terminals in the lumbar sympathetic chain of guinea-pigs. Presumptive vasoconstrictor neurons with immunoreactivity for both tyrosine hydroxylase and neuropeptide Y dendritic fields that were significantly smaller, on average, than those of presumptive pilomotor neurons containing immunoreactivity to tyrosine hydroxylase but not to neuropeptide Y. However, there was considerable variation in the sizes of the dendritic fields of the vasoconstrictor neurons. Preganglionic nerve terminals containing immunoreactivity to calcitonin gene-related peptide, but not to substance P, only surrounded cell bodies of vasoconstrictor neurons containing immunoreactivity to tyrosine hydroxylase and neuropeptide Y. In most cases, the neuropeptide-containing preganglionic terminals were not associated closely with the distal dendrites of these neurons. Few neuropeptide-containing terminals were associated closely with either the cell bodies or dendrites of the pilomotor neurons.

These results show that there is a considerable range in the size of dendritic trees of sympathetic final motor neurons. Some of this variation is related to the pathways within which the neurons lie, so that presumptive pilomotor neurons generally are larger than presumptive vasoconstrictor neurons. The marked variation in size of vasoconstrictor neurons raises the possibility that there may be a size dependent recruitment of these neurons, similar to that seen in pools of spinal motor neurons. The distribution of the peptide-containing preganglionic endings suggests that they would act predominantly at the cell body and proximal dendrites of the final motor neurons.     

Neuropeptide Y (NPY)-like immunoreactivity in rat sympathetic neurons and small granule-containing cells

The distribution of neuropeptide Y-like immunoreactivity (NPY-LI) was examined in the rat superior cervical and hypogastric ganglia. NPY-LI was localized in the majority of the sympathetic neurons, a few small granule-containing (SGC) cells and nerve terminals. Most of the NPY-immunoreactive sympathetic neurons were also tyrosine hydroxylase (TH)-immunoreactive but in hypogastric ganglia few neurons with NPY-LI were devoid of TH-immunoreactivity. Electron microscopically NPY-LI was found in the Golgi complexes of sympathetic neurons, in large cytoplasmic granules (100-150 nm in diameter) of the SGC cells and in large dense-cored vesicles (80-100 nm in diameter) of the nerve terminals. NPY-LI coexists mainly with noradrenaline in sympathetic neurons, and may have regulatory functions in sympathetic ganglia and in target organs.     

Plasticity in the phenotypic expression of catecholamines and vasoactive intestinal peptide in adult rat superior cervical and stellate ganglia after long-term hypoxia in vivo.

Sympathetic ganglia in the adult rat contain various populations of nerve cells which demonstrate plasticity with respect to their transmitter phenotype. The plasticity of the neuronal cell bodies and of the small intensely fluorescent cells in the superior cervical and stellate ganglia in response to hypoxia in vivo (10% O2 for seven days) was assessed by studying the expression of catecholamines and vasoactive intestinal peptide. The levels of norepinephrine, dopamine, 3,4-dihydroxyphenylacetic acid and vasoactive intestinal peptide immunoreactivity were determined. In addition, the density of the immunohistochemical staining of cells for tyrosine hydroxylase and vasoactive intestinal peptide was evaluated. In the intact superior cervical ganglion, hypoxia increased the dopamine level as well as the density of small intensely fluorescent cells immunolabelled for tyrosine hydroxylase and vasoactive intestinal peptide. In the axotomized ganglion, hypoxia elicited a twofold rise in the level of the vasoactive intestinal peptide as well as enhancing the density of neuronal cell bodies immunostained for this peptide. Thus, the effect of hypoxia on the expression of vasoactive intestinal peptide expression in neurons was dependent on neural interactions. In the intact stellate ganglion, hypoxia alone induced a 1.5-fold increase in the density of neuronal cell bodies immunostained for vasoactive intestinal peptide. Thus, ganglia-specific factors appeared to play a role in determining changes in neuronal phenotype in response to hypoxia. The present study provides evidence for the involvement of dopamine and vasoactive intestinal peptide in ganglionic responses to long-term hypoxia as well as for differential responses by the two ganglionic cell populations, i.e. neuronal cell bodies and small intensely fluorescent cells. Changes in the expression of the vasoactive intestinal peptide during long-term hypoxia may be of energetic, trophic and/or synaptic significance. Hypoxia may be considered to be a vasoactive intestinal peptide-inducing factor in sympathetic ganglia.     

Early and widespread normalization of dopamine-neuropeptide Y interactions in the rat striatum after transplantation of fetal mesencephalon cells.

Graft-to-host interactions were examined at cellular level, by measuring changes in the immunoreactivity of striatal interneurons expressing neuropeptide Y after dopamine denervation and transplantation of fetal mesencephalon neurons into the striatum of adult rats. Mesencephalic cell suspensions were implanted unilaterally into the dorsal part of the striatum in rats two weeks after intranigral injection of 6-hydroxydopamine. One month and three to four months later, rats showing abolition of amphetamine-induced turning were perfused. Serial brain sections containing intrastriatal grafts were treated for tyrosine hydroxylase and neuropeptide Y immunocytochemistry, and neuropeptide Y-immunoreactive neurons were quantified in various parts of the striatal surface and compared with the striatum of controls and age-matched rats with lesions. Biochemical analyses of dopamine and dihydroxyphenyl acetic acid tissue levels and [3H]dopamine uptake were also performed on striatal samples from similar groups of normal, lesioned and transplanted rats. As early as one month post-grafting, a complete reversal of the increase in the number of neuropeptide Y-immunoreactive neurons occurring after 6-hydroxydopamine lesion was observed in dopamine-grafted animals, although a partial restoration of the tyrosine hydroxylase immunostaining and a recovery of 8% dopamine tissue level were observed in the striata of grafted as compared to normal rats. This effect on the host immunoreactivity was found to be specific to dopamine grafts, since no reversal was observed in sham-spinal cord-transplanted rats. Moreover, similar degrees of normalization were recorded either in the total striatum, or in the area immediately adjacent to the graft, or even in the zone most sensitive to dopamine denervation in terms of neuropeptide Y immunoreactivity. No more pronounced functional effects were observed three to four months after transplantation. These data suggest that grafted dopamine neurons are able to induce rapid and extensive host responsiveness, possibly by means of mechanisms involving synaptic and diffuse release of dopamine and adaptive changes in the host brain. These data may provide a cellular basis for interpreting larger behavioural recoveries than those expected to occur with dopamine grafts in view of the partial restoration of the dopaminergic innervation.     

Neuropeptide Y and differential sympathetic control of splenic blood flow and capacitance function in the pig and dog

The roles of different mediators in the sympathetic regulation of the pig and dog spleens were investigated using a preparation with intact vascular perfusion in vivo. Sympathetic nerve stimulation caused overflow of neuropeptide Y-like immunoreactivity (NPY-LI) and noradrenaline (NA), arterial vasoconstriction, increase in venous blood flow and haematocrit. The dog spleen responded to single impulse stimulation, whereas more prolonged stimulation was required to elicit vascular responses in the pig spleen. Furthermore, the maximal splenic capacitance response was about 10 times larger in the dog than in the pig. After depletion of neuronal NA content by reserpine combined with preganglionic denervation, about 70% of the splenic arterial vasoconstrictor responses in the dog and pig still remained at 5 Hz stimulation. Fifty per cent of the capacitance response evoked by nerve stimulation still remained in the pig while in the dog spleen the capacitance response was virtually abolished after reserpine. The stimulation-evoked overflow of NPY-LI in pig spleen was increased several fold after reserpine treatment as compared to controls reaching levels in the venous effluent where exogenous NPY evokes vasoconstriction. In the dog spleen, overflow of NPY-LI was only observed after reserpine. Administration of NA caused arterial vasoconstriction with an initial increase in venous blood flow while NPY mainly reduced arterial blood flow. It is concluded that NA is involved in both the splenic arterial vasoconstriction and the capacitance responses while a non-adrenergic splenic vasoconstriction at least in the pig may be mediated by NPY.     

Long-term chemical sympathectomy leads to an increase of neuropeptide Y immunoreactivity in cerebrovascular nerves and iris of the developing rat

Short-term (surgical) and long-term (chemical) sympathectomy have revealed the presence of a population of neuropeptide Y-like immunoreactive nerve fibres which do not degenerate in parallel with noradrenaline-containing nerves supplying cerebral vessels and the iris of the rat. Two days after bilateral removal of the superior and middle cervical ganglia of 7-week-old rats, noradrenaline-containing nerves could not be detected along any of the arteries of the rat circle of Willis or of the iris, but 18-32% of neuropeptide Y-like immunoreactive nerves remained. Long-term treatment (6 weeks) with guanethidine commencing in developing 1-week-old rats caused degeneration of the sympathetic neurons in cervical ganglia and disappearance of 5-hydroxydopamine-labelled nerves (that showed dense-cored vesicles at the electron microscope level) from rat cerebral vessels, but did not significantly change the density of neuropeptide Y-like immunoreactive axons on the vessels. Furthermore, whilst in control rats neuropeptide Y-like immunoreactivity was localized largely within 5-hydroxydopamine-labelled cerebrovascular nerves, after long-term sympathectomy with guanethidine, neuropeptide Y-like immunoreactivity was seen only in nerves lacking small dense-cored vesicles. A small number of catecholamine-containing nerves appeared along the internal carotid and anterior cerebral arteries after long-term sympathectomy; these may arise from neurons of central origin. These results suggest that as a consequence of long-term sympathectomy with guanethidine, compensatory changes occur, involving an increase in the expression of neuropeptide Y-like immunoreactivity in non-sympathetic axons in cerebrovascular nerves and iris of the rat. In contrast, the neuropeptide Y-like immunoreactive nerves in the dura mater appear to be entirely sympathetic, since none were present after short-term sympathectomy and none appeared after long-term sympathectomy.     

Immunohistochemical localization of epinephrine, norepinephrine, catecholamine-synthesizing enzymes, and chromogranin in neuroendocrine cells and tumors.

The immunohistochemical localization of epinephrine (E), norepinephrine (NE), and chromogranin was analyzed in normal and neoplastic neuroendocrine cells. The immunohistochemical detection of tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) was used to distinguish between uptake and biosynthesis of catecholamines. E, NE, chromogranin, TH, DBH, and PNMT were found in the normal human adrenal medulla and in pheochromocytomas. Although many neuroendocrine tissues outside of the adrenal gland contained immunoreactive NE, only a small percentage of these tissues contained DBH. E was found in a few neuroendocrine tissues outside of the adrenal, including cardiac paragangliomas, and the enzyme PNMT was localized in some of these neoplasms. There was very close agreement between the localization of chromogranin and of catecholamines in normal and neoplastic neuroendocrine tissues. These results indicate that the presence of catecholamines and chromogranin in neuroendocrine cells and tumors within the adrenal medulla and in many other sites may be closely related.