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.     

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.     

Immunohistochemical features of substance P-immunoreactive chromaffin cells and nerve fibers in the rat adrenal gland

The distribution of substance P (SP) immunoreactivity and the colocalization of SP with other bioactive substances in chromaffin cells and nerve fibers were investigated in the rat adrenal gland at the light microscopic level. In the capsule and cortex, SP immunoreactivity was seen in some nerve fibers around blood vessels and in thick nerve bundles passing through the cortex directly into the medulla. In the medulla, the SP immunoreactivity was observed in a small number of chromaffin cells; these SP-immunoreactive chromaffin cells were either phenylethanolamine N-methyltransferase (PNMT) immunoreactive or immunonegative, indicating that they were either adrenaline cells or noradrenaline (NA) cells. SP-immunoreactive varicose nerve fibers were also found in the medulla and were in contact with a cluster of the NA cells showing catecholamine fluorescence, which suggests that SP from medullary nerve fibers may regulate the secretory activity of the NA cells. Because no SP-immunoreactive ganglion cell was present in the rat adrenal gland, the intra-adrenal nerve fibers were considered to be extrinsic in origin. The double-immunostaining method further revealed that the SP-immunoreactive chromaffin cells also exhibit immunoreactivities for calcitonin gene-related peptide (CGRP), and neuropeptide tyrosine (NPY), suggesting that these peptides can also be released from the chromaffin cells by certain stimuli. The intra-adrenal nerve fibers in the medulla were composed of SP-single immunoreactive, and SP/CGRP-, SP/choline acetyltransferase (ChAT)-, SP/nitric oxide synthase (NOS)-, SP/pituitary adenylate cyclase activating polypeptide (PACAP)-, ChAT/NOS-, and ChAT/PACAP-immunoreactive nerve fibers, which may affect the secretory activity of the NA cells. In the adrenal capsule, the nerve fibers were present around blood vessels and showed immunoreactivities for SP/ CGRP, SP/NPY, SP/NOS, and SP/vasoactive intestinal polypeptide, suggesting that the origin of nerve fibers in the capsule may differ from those in the medulla.     

Demonstration of a neuropeptide Y (NPY)-like immunoreactivity in the pigeon retina

The distribution of neuropeptide Y (NPY)-like immunoreactivity in the pigeon retina was investigated by fluorescence immunohistochemistry. NPY-positive cells were found in central and peripheral retina. NPY somata were located in the proximal portion of the inner nuclear layer and their processes directed to the inner plexiform layer where they ramified in 3 immunoreactive bands. NPY might play a role as a neurotransmitter or neuromodulator in the pigeon retina.     

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.     

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.     

Immunoelectron microscopic study of tyrosine hydroxylase immunoreactive nerve fibers and ganglion cells in the rat adrenal gland

The present immunocytochemical study used an antiserum to tyrosine hydroxylase (TH), the first enzyme in the biosynthetic pathway of catecholamines, and revealed TH immunoreactivity in the ganglion cells and in the varicose nerve fibers of the cortex and medulla in the rat adrenal gland. TH immunoreactive nerve fibers in the cortex and medulla contained large and small granular vesicles, and also small clear vesicles. The immunoreactive nerve fibers were in close apposition to cortical cells in the cortex and in apposition to smooth muscle cells of blood vessels in both the cortex and medulla. Furthermore, TH immunoreactive nerve fibers were sometimes in close apposition to pericytes of blood vessels in the cortex and chromaffin cells in the medulla. The present results suggest that the catecholaminergic nerve fibers in the rat adrenal gland may be both intrinsic and extrinsic in origin.     

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.     

Immunoelectron microscopic study of tyrosine hydroxylase immunoreactive nerve fibers and ganglion cells in the rat adrenal gland.

The present immunocytochemical study used an antiserum to tyrosine hydroxylase (TH), the first enzyme in the biosynthetic pathway of catecholamines, and revealed TH immunoreactivity in the ganglion cells and in the varicose nerve fibers of the cortex and medulla in the rat adrenal gland. TH immunoreactive nerve fibers in the cortex and medulla contained large and small granular vesicles, and also small clear vesicles. The immunoreactive nerve fibers were in close apposition to cortical cells in the cortex and in apposition to smooth muscle cells of blood vessels in both the cortex and medulla. Furthermore, TH immunoreactive nerve fibers were sometimes in close apposition to pericytes of blood vessels in the cortex and chromaffin cells in the medulla. The present results suggest that the catecholaminergic nerve fibers in the rat adrenal gland may be both intrinsic and extrinsic in origin.     

Glial fibrillary acidic protein (GFAP)-like immunoreactivity in normal and transected rat olfactory nerve

Summary Normal and transected rat olfactory nerves were stained immunohistochemically using a monoclonal antibody previously shown to selectively detect GFAP-like immunoreactivity in central astrocytes but not in peripheral Schwann cells. Low levels of central type GFAP were found in the olfactory nerves, presumably in ensheathing cells. The levels of GFAP increased dramatically after nerve transection. A population of strongly GFAP-positive cells was detected at the junction between the olfactory epithelium and initial part of the nerves, of possible relevance to the regenerative abilities of this pathway.     

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.     

Enkephalin-like immunoreactivity in gland cells and nerve terminals of the adrenal medulla.

Met-enkephalin-like immunoreactivity has been observed in adrenal medullary gland cells of the rat, guinea-pig and cat. There were marked quantitative differences in untreated animals. Most medullary gland cells were positive in the cat, a large proportion in the guinea-pig but only a few in the rat. After sectioning of the splanchnic nerve, however, a large proportion of the gland cells were met-enkephalin positive also in the rat. Occasionally the met-enkephalin-like immunoreactivity had a granular appearance suggesting that the storage sites could be vesicular.In the guinea-pig a moderate number of met-enkephalin immunoreactive nerve fibers were observed in the adrenal medulla. They disappeared to a large extent after sectioning of the splanchnic nerve. A few fibers were also seen in the rat adrenal medulla, and in the cat some fibers could be seen in areas with few immunoreactive cells.The present findings suggest that in the adrenal medulla an enkephalin-like peptide(s) is present both in gland cells and in nerve terminals arising mainly from fibers in the splanchnic nerve. Thus, the possibility exists that in the adrenal medulla opioid peptides may be released from the gland cells into the blood as hormones as well as from nerve terminals to act as a modulator or transmitter. It should, however, be emphasized that the well-documented metabolic instability of met-enkephalin may be somewhat difficult to reconcile with a hormonal role of this peptide.     

Reserpine increases opiate-like peptide content and tyrosine hydroxylase activity in adrenal medullary chromaffin cells in culture

Primary cultures of adrenal medullary chromaffin cells maintained in a serum-free medium retain high levels of both catecholamines and opiate-like peptides. Addition of reserpine (100nM) to the culture medium results in the exponential loss of cellular catecholamines (t_½ = 1.5days) and in an elevation of opiate-like peptide content and tyrosine hydroxylase activity without altering total cell protein content. The maximum increase in opioid activity as a result of reserpine treatment averaged 230% of untreated cell levels and was reached by 2–3 days after initiation of treatment. A similar time-course was observed for the elevation of tyrosine hydroxylase activity. The increases of opiate-like peptides and tyrosine hydroxylase induced by reserpine are blocked by inclusion of actinomycin D or cycloheximide in the culture medium, suggesting that both messenger ribonucleic acid and protein synthesis are required for the induction.These data suggest that synaptic activation is not the only long-term regulator of opiate-like peptide and catecholamine biosynthesis in the adrenal medulla and that the biosynthesis of opiate-like peptides, which are components of chromaffin vesicles, and tyrosine hydroxylase, the cytoplasmic, rate-limiting enzyme in catecholamine biosynthesis, are coordinately regulated.     

Simultaneous ultrastructural localization of cholecystokinin- and tyrosine hydroxylase-like immunoreactivity in nerve fibers of the rat nucleus accumbens

Ultrathin frozen sections were used to study the localization of cholecystokinin (CCK) in dopaminergic systems in the rat nucleus accumbens. Antibodies against CCK and tyrosine hydroxylase (TH), a synthetic enzyme of dopamine, were differentially visualized using protein A conjugated to colloidal gold particles of different sizes. Nerve processes were observed to be immunocytochemically labelled for either CCK or TH but also in some cases for both CCK and TH. CCK-like immunoreactivity was localized in vesicles with a diameter of 70-160 nm, whereas TH-like immunoreactivity was primarily localized in the axoplasm. Most of the double-labelled nerve processes did not show pre- or postsynaptic specializations and most likely represent preterminal elements.     

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.