Occurrence of neuropeptide Y (NPY)-like immunoreactivity in catecholamine neurons in the human medulla oblongata

We report here the coexistence of a neuropeptide and catecholamines in neurons of the human brain. Using indirect immunofluorescence histochemistry, combined with elution and restaining experiments, neurons in the medulla oblongata of man were demonstrated to contain both a neuropeptide Y-like peptide and the catecholamine synthesizing enzyme tyrosine hydroxylase.     

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.     

Co-localization of neurotensin- and cholecystokinin-like immunoreactivities in catecholamine neurons in the rat dorsomedial medulla

Co-localization of neurotensin and cholecystokinin in tyrosine hydroxylase-containing neurons in the nucleus tractus solitarius of the rat was demonstrated by immunocytochemistry with fluorescent double-staining combined with the peroxidase-antiperoxidase method. Co-localization of neurotensin/tyrosine hydroxylase or cholecystokinin/tyrosine hydroxylase was consistently found in small neurons in the region dorsomedial to the tractus solitarius at the level of the area postrema with high percentages of co-existence: 91.0% tyrosine hydroxylase-immunoreactive neurons contained neurotensin and 91.1% cholecystokinin, suggesting that they represent the same neurons. Accordingly, co-localization of neurotensin and cholecystokinin was assessed on tyrosine hydroxylase-containing neurons bisected into two adjacent sections, and then identified in a certain number of the catecholamine neurons in this region. Furthermore these catecholamine neurons exhibited immunoreactivity for an adrenaline-synthesizing enzyme, phenylethanolamine N-methyltransferase. It was concluded that catecholamine, in particular adrenaline, neurons, characterized by co-localization of neurotensin and cholecystokinin, established a distinct subpopulation in the catecholaminergic system in the dorsomedial medulla of the rat.     

Expression of phenylethanolamine N-methyltransferase (PNMT) and neuropeptide Y (NPY) in embryonic rat medulla oblongata grown in Oculo

Expression and development of specific markers of the adrenergic phenotype were studied in central neurons grown in transplant system. Medulla oblongata from embryonic day 12.5 (E12.5) or E18 rat was grafted into the anterior chamber of the eye of adult rat hosts. After two months, grafts were examined for the presence of immunoreactivity (IR) and catalytic activity to the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28), a specific adrenergic marker. In addition, grafts were examined for immunoreactivity to neuropeptide Y (NPY) and tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. In E12.5 grafts, PNMT was expressed de novo, enzyme activity developed to levels similar to those in adult rat brainstem and PNMT-IR neurons were observed. TH-IR and NPY-IR neurons were also observed. In contrast, PNMT-IR was not observed in E18 grafts even though these already contained PNMT-IR neurons at the time of grafting. This was not due to poor growth of E18 grafts, in general, since TH-IR neurons were present and the protein content of the grafts was similar to that of E12.5 grafts. These studies suggest that adrenergic neurons survive well in oculo if they are transplanted prior to the age when neuroblasts have initially expressed the adrenergic phenotype, migrated to their final positions and elaborated processes. In addition, these studies establish a transplant system in which factors required for the development of central adrenergic neurons can be more easily studied than in situ.     

Neuropeptide Y (NPY) in human nasal mucosa

Neuropeptide Y (NPY), a potent vasoconstrictor peptide found in sympathetic neurons, was analyzed in human inferior turbinate nasal mucosal tissue. NPY content determined by radioimmunoassay was 3.13 +/- 0.79 pmol/g tissue (n = 6) in mucosa extracted with ethanol-acetic acid. NPY-immunoreactive nerves were found around small muscular arteries, arterioles, arteriovenous anastomoses, and as free fibers near arteriolar and venous vessels. They formed a plexus around the arterial vessels, and were also present between vascular smooth muscle cells. Few NPY fibers were present near glands or the epithelium. [125I]NPY binding sites were localized by autoradiography to small muscular arteries, arterioles, and a few venous sinusoids. In explant culture experiments, 4 microM NPY did not stimulate release of [3H]glucosamine-labeled glycoconjugates or lactoferrin (a product of serous cells) from nasal mucosal fragments. Degradation of NPY by a tissue homogenate was rapid (t1/2 = 13.5 +/- 2.3 min). The degradation was inhibited by thiorphan and phosphoramidon, inhibitors of neutral endopeptidase activity. NPY released from sympathetic neurons may play a role as a constrictor of arterial vessels and regulate vasomotor tone in the human nasal mucosa.     

Hypotension activates neuropeptide Y-containing neurons in the rat medulla oblongata.

The present study was designed to determine whether neurons within cardiovascular control nuclei of the rat brainstem that become activated following a hypotensive insult also possess the capacity to utilize neuropeptide Y. Adult male Wistar-Kyoto rats were injected with glyceryl trinitrate (10 mg/kg, i.p.) or vehicle, and 4 h later anaesthetized (pentobarbitone, 60 mg/kg, i.p.) and transcardially perfused. The brains were removed and processed by standard two-colour peroxidase immunohistochemistry. Activated cells were determined by incubation with a primary antibody to Fos protein, which was followed by a second incubation with a primary antibody to neuropeptide Y for double labelling of Fos-positive cells. Compared to vehicle, glyceryl trinitrate-induced hypotension caused a marked induction of Fos protein in the caudal one-third of the nucleus tractus solitarius (bregma -14 to -13.3 mm), which tailed off rapidly in more rostral sections. Following hypotension, significant populations of activated cells were also observed in the rostral and caudal ventrolateral medulla. In the caudal nucleus tractus solitarius and the posterior part of the medial nucleus tractus solitarius, respectively, 15 of 104 and 40 of 120 Fos-positive cells exhibited cytoplasmic neuropeptide Y immunoreactivity following hypotension, compared to seven of 40 and 15 of 40 in vehicle-treated rats, indicating a significant (two- to three-fold) increase in double-labelled cells following systemic glyceryl trinitrate (P < 0.05, unpaired t-test). In contrast, in the anterior part of the medial nucleus tractus solitarius, the number of double-labelled cells did not change following hypotension. An increase in double-labelled cells was also observed in the rostral ventrolateral medulla (2.5-fold increase compared to vehicle) and caudal ventrolateral medulla (5.8-fold increase compared to vehicle) following hypotension. These data indicate that, in the rat, neuropeptide Y-containing neurons are involved in the central response to a hypotensive challenge. The primary regions where neuropeptide Y-containing neurons appear to be activated are the caudal one-third of the nucleus tractus solitarius and the caudal ventrolateral medulla/rostral ventrolateral medulla, which are key nuclei associated with the integration of the baroreceptor heart rate reflex and sympathetic vasomotor outflow.     

Neuropeptide Y (NPY) inhibits dimethylphenylpiperazinium (DMPP)-induced gastric acid secretion in isolated rat stomach

The effect of neuropeptide Y (NPY) on gastric acid secretion was investigated on an everted preparation of isolated rat stomach. Perfusion with synthetic NPY did not modify the basal secretion of gastric acid. However, NPY reduced dimethylphenylpiperazinium (DMPP)-stimulated acid secretion at concentrations insufficient to affect acid secretion stimulated by muscarine, histamine or gastrin. The decrease in acetylcholine (ACh) release from postganglionic cholinergic neurons by NPY is therefore considered to be responsible. Determination of NPY content by radioimmunoassay, in mucosal and muscular layers of the stomach, indicates that NPY possibly produces cholinergic inhibition under physiological levels. The present study suggests, therefore, that NPY has the ability to inhibit the release of ACh from postganglionic cholinergic neurons, thus producing a decrease in gastric acid secretion.     

Neuropeptide Y does not reset the circadian clock in NPY Y2-/- mice

Mammalian circadian rhythms are modulated by neuropeptide Y (NPY), a peptide contained in the projection from the intergeniculate leaflet to the suprachiasmatic nuclei of the circadian pacemaker. NPY resets the circadian clock during the subjective day, mediating non-photic inputs. Previous studies using receptor-selective agonists have indicated that this action of NPY is mediated by the Y2 receptor in hamsters. The present study determined if NPY applied to the suprachiasmatic nuclei in the mid-subjective day can phase-advance the rhythm of spontaneous firing rate of Y2-/- mice. We observed that NPY did reset the rhythm of control mice but did not significantly shift the phase of this rhythm in the Y2-/- mice. These results provide strong evidence for the role of the Y2 receptor mediating neuropeptide Y subjective day phase-advance shifts in mice.     

Late vagal inhibition in neurons of the ventrolateral medulla oblongata in the rat.

Stimulation of cervical vagal afferents elicits long-lasting inhibitory effects in a variety of neuronal populations, although little is known concerning the cellular mechanisms that are involved in these effects. In the present study, the electrophysiological characteristics of responses elicited by cumulative activation of vagal afferents were examined in neurons of the rostral ventrolateral medulla oblongata, which play an important role in the coordination of cardiovascular and other visceral activities. The study has focused on the late-onset, slow inhibitory component of vagal responses, which is likely to affect the temporal modulation of postsynaptic effects. Vagal stimulation elicited four distinct response patterns in intracellularly penetrated neurons (n = 78): excitation, inhibition, excitation-inhibition and inhibition-inhibition. The late inhibitory component was encountered in 43 (55%) of the cells, including five putative medullospinal neurons. It was due to a postsynaptic hyperpolarization which reversed at potentials more negative than -83 mV. The voltage dependency, as well as the average onset latency (93+/-3.0 ms), duration (270+/-16.5 ms) and amplitude (1.3+/-0.2 mV as measured at resting membrane potentials), of late inhibition were clearly different from those of the short-latency inhibitory response. The differences in the voltage dependency and time-course of the short-latency responses and the late inhibition indicate that they are mediated by different central relays. In the majority of neurons, late inhibition could be elicited by stimulating only myelinated vagal afferents. The magnitude of the response was, however, significantly enhanced in 63% of the examined cells when the intensity of stimulation was raised to recruit further myelinated and non-myelinated fibres. This indicates that late vagal inhibition is often elicited by a cumulative activation of convergent afferent inputs. The intracellularly labelled vagally responsive neurons were present at all rostrocaudal levels of the rostral ventrolateral medulla, with an accumulation in the region of the lateral paragigantocellular nucleus. Neurons that exhibited late vagal inhibition were dominant in the juxtafacial region of this nucleus. Due to its slow time-course, late vagal inhibition may contribute to a tonic modulation of the activity of neurons in the rostral ventrolateral medulla oblongata. It is proposed that late vagal inhibition plays an important role in the temporal integration of sensory inputs in neurons of the rostral ventrolateral medulla oblongata. The time-course and strength of this modulatory effect are related to the level of activity in those visceral sensory inputs that converge onto the inhibitory interneurons that mediate late inhibition to rostral ventrolateral medulla oblongata neurons.     

Neuropeptide Y (NPY) induced inhibition of preganglionic nerve stimulation evoked release of adrenalin and noradrenaline in the pithed rat

NPY, a peptide with 36 amino acid residues, is co-stored together with noradrenaline (NA) in cardiac and sympathetic perivascular nerves as well as with adrenalin (A) in adrenal chromaffin cells. NPY is released together with NA from sympathetic nerves and with A from the adrenal glands and appears to be involved in the control of sympathetic neurotransmission. The aim of the present study was to analyse the effect of NPY on the preganglionic nerve stimulation (PNS) evoked increases in plasma A and NA concentrations in pithed rats. In the first part of the study (I) only one PSN period (2 Hz for 45 s) was performed in each rat and the control group was compared to the NPY treated group. In the second part of the study (II) two PNS periods (1 Hz for 45 s) were performed in each rat, which either received saline or NPY before the second PNS. Thus, interindividual changes between the responses to the first and second PNS in control and NPY rats could be compared. In both study I and II, systemic infusion of NPY (2 micrograms kg-1 min-1 i.v.) significantly reduced the PNS-induced increase in plasma A by 26% and 42%, respectively (P less than 0.05). However, the increase in plasma NA elicited by PNS was significantly reduced only in study II by 23% (P less than 0.05). Infusion of NPY did not affect basal heart rate in either of the studies, but significantly increased basal blood pressure by about 10 mmHg. The blood pressure responses to PNS were significantly greater in NPY treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuropeptide Y (NPY)-like immunoreactive nerve fibers in the human and monkey (Macaca fascicularis) kidney

Nerve fibers immunoreactive for neuropeptide Y (NPY) are demonstrated for the first time by the indirect immunofluorescence technique in the human and monkey kidney. NPY-like immunoactivity (NPY-LI) is shown in a bundle of nerve fibers in the surrounding connective tissue of arteries and to a lesser extent, veins, mainly at the juxtamedullary region. Varicose nerve terminals are shown associated with blood vessels and passing between tubules in the mid and lower cortex. NPY-LI nerve fibers are also seen surrounding afferent and occasionally efferent arterioles at the vascular pole of the glomeruli. The distribution of NPY-LI nerve fibers in the monkey and human kidneys is similar to that of other species, only the quantity of the nerve fibers varies.     

Increased neuropeptide Y (NPY)-like immunoreactivity in rat sensory neurons following peripheral axotomy.

The effects of peripheral axotomy (sciatic nerve transection) on the presence and distribution of neuropeptide Y (NPY) in rat dorsal root ganglion (DRG) and spinal grey matter were examined using immunocytochemistry. In normal rats and on the sham-operated side of experimental rats, NPY-like immunoreactivity (NPYir) was observed in all laminae of the lumbar spinal cord, with an especially dense concentration of immunostained axons and axonal varicosities in laminae I-II of the dorsal horn. There was no detectable NPYir in L4-L5 DRG cells from normal rats or from the sham-operated side of experimental rats. At 14 days after axotomy, there was a large ipsilateral increase in the density of NPYir axons and varicosities in the lumbar spinal cord on the side of the nerve injury; this was especially apparent in laminae III-V. In the same rats, NPYir was observed in many small, medium, and large neurons in the L4-L5 DRGs on the side of the severed nerve.     

Neuropeptide Y afferents have synaptic interactions with histaminergic (histidine decarboxylase-immunoreactive) neurons in the rat brain

The synaptic interaction was identified between histaminergic cells and neuropeptide Y (NPY)-immunoreactive axon terminals in the caudal magnocellular nucleus, by means of the immunoelectron microscopic mirror method. This observation suggests that NPY afferents exert monosynaptic influences upon the functioning of the central histaminergic neuronal system.     

Neurochemistry of bulbospinal presympathetic neurons of the medulla oblongata

This review focuses on presympathetic neurons in the medulla oblongata including the adrenergic cell groups C1–C3 in the rostral ventrolateral medulla and the serotonergic, GABAergic and glycinergic neurons in the ventromedial medulla. The phenotypes of these neurons including colocalized neuropeptides (e.g., neuropeptide Y, enkephalin, thyrotropin-releasing hormone, substance P) as well as their relative anatomical location are considered in relation to predicting their function in control of sympathetic outflow, in particular the sympathetic outflows controlling blood pressure and thermoregulation. Several explanations are considered for how the neuroeffectors coexisting in these neurons might be functioning, although their exact purpose remains unknown. Although there is abundant data on potential neurotransmitters and neuropeptides contained in the presympathetic neurons, we are still unable to predict function and physiology based solely on the phenotype of these neurons.     

Simultaneous demonstration of phenylethanolamine N-methyltransferase immunofluorescent and catecholamine fluorescent nerve cell bodies in the rat medulla oblongata

The distribution of phenylethanolamine N-methlytransferase-immunoreactive nerve cell bodies was investigated in the rat medulla using an antiserum to bovine phenylethanolamine N-methyltransferase raised in rabbits. A procedure that combines immunohistochemistry and catecholamine fluorescence histochemistry was developed with a formaldehyde/glutaraldehyde mixture as a fixative. Three groups of immunoreactive nerve cell bodies were found in the medulla: a ventrolateral group, C1, a dorsal group, C2, in the nucleus of the tractus solitarius and a smaller medial group of cells, C3, scattered in the medial longitudinal fasciculus. Most of the phenylethanolamine N-methyltransferase positive nerve cells did not show catecholamine fluorescence and did not correspond to the catecholamine cell groups A1 and A2. Both groups C1 and C2 of immunoreactive nerve cells extended further rostrally than A1 and A2. Group C3 has not previously been described as a distinct group of catecholamine fluorescent nerve cell bodies.Inhibition of phenylethanolamineN-methyltransferase and monoamine oxidase results in the appearance of catecholamine fluorescence in the immunoreactive cell bodies suggesting that they usually store adrenaline which reacts poorly with the formaldehyde/glutaraldehyde mixture or other aldehydes which induce catecholamine fluorescence and it is for this reason that they are not normally identified in maps of catecholamine fluorescent cells.     

Neuropeptide Y (NPY): a vasoconstrictor in the eye,brain and other tissues in the rabbit

The effect of neuropeptide Y (NPY) on uveal vascular resistance was studied in rabbits by direct determination of uveal blood flow from a cannulated vortex vein. Regional blood flows, in the eye, the brain and several other tissues, were measured, with radioactive microspheres, during neuropeptide Y-infusion in rabbits with and without α-adrenoceptor blockade. Intravenous infusion of increasing doses of neuropeptide Y caused a dose-dependent increase in the total uveal vascular resistance. Maximal effect, a 70% increase, was achieved with 120 pmol kg^-1 min^-1. In the microsphere experiments, this dose rate was given i.v. over 10 minutes and blood flow determinations were made before and at 2 and 10 minutes after the start of the infusion. After 2 minutes of neuropeptide Y, there were marked blood flow reductions in the spleen, kidneys, adrenal glands, gastro-intestinal tract, choroid plexus and pineal and pituitary gland. The effect in the eye was small at 2 minutes, but at 10 minutes local blood flows in the choroid and the ciliary body were decreased by 50% and the iridal blood flow by 30%. Retinal blood flow was not affected by neuropeptide Y. At 10 minutes there were also significant blood flow reductions in the brain, tongue, masseter muscle and several glandular tissues. The effects of neuropeptide Y on local blood flow in rabbits that had been subjected to α-adrenoceptor blockade were very similar to the effects in the animals without α-adrenoceptor blockade. The results show that, in the rabbit, neuropeptide Y has marked effects on local blood flows in several tissues, including the eye, and suggest that neuropeptide Y may significantly contribute to the uveal vasoconstriction during sympathetic nerve stimulation.