Distributions of neuropeptide Y, vasoactive intestinal peptide and somatostatin in populations of postganglionic neurons innervating the rat kidney, spleen and intestine.

Some peripheral peptidergic nerves selectively innervate different types of tissue in abdominal organs. Neuropeptide Y- and vasoactive intestinal peptide-immunoreactive nerve terminals have been identified in the kidney, spleen and intestine and these peptides may have important physiological actions. Somatostatin has been found in sympathetic ganglia, and nerve terminals containing this peptide have been identified in the intestine. We have used fluorescent retrograde tracers to identify renal, splenic and mesenteric postganglionic neurons in rat sympathetic ganglia and then used immunocytochemistry to determine the proportions of these three identified groups of neurons displaying immunoreactivity for neuropeptide Y, vasoactive intestinal peptide and somatostatin. Most renal, splenic and mesenteric neurons were immunoreactive for neuropeptide Y and less than 1% of cells innervating these organs were immunoreactive for vasoactive intestinal peptide. Somatostatin immunoreactivity was present only in a small percentage of mesenteric neurons and not in renal or splenic neurons. The present study demonstrates that (i) the rat kidney, spleen and intestine do not differ in the proportion of innervation by neuropeptide Y-immunoreactive neurons, (ii) the solar plexus, splanchnic ganglion and chain ganglia (T12 and T13) provide very little vasoactive intestinal peptide-immunoreactive inputs to these organs, and (iii) somatostatin-immunoreactive neurons innervate the intestine but not the kidney or spleen.     

Ischemia changes the coexpression of somatostatin and neuropeptide Y in hippocampal interneurons

Transient cerebral ischemia causes extensive cell death in hippocampal CA1 pyramidal cells and selective loss of interneurons in the dentate hilus. Many hippocampal interneurons can be classified by their contents of somatostatin (SS) and/or neuropeptide Y (NPY). Following ischemia in the rat, most of the NPY immunoreactivity is permanently lost in hippocampus. Furthermore, SS interneurons in the dentate hilus die, whereas CA1 interneurons survive and their expression of SS mRNA and peptide returns to preischemic levels within 16 days after ischemia. We have addressed the following questions: (1) Does the loss of NPY involve a specific downregulation in surviving CA1 interneurons that preischemically expressed both SS and NPY? (2) Can the subpopulation of dying interneurons in hilus be identified from their preischemic coexpression of SS and NPY? We investigated the coexpression of SS mRNA and NPY peptide using combined in situ hybridization and immunocytochemistry. Cells containing one or both markers were counted in control sections and sections taken 2–16 days after ischemia from the hippocampal formation. In CA1, a decrease in the number of neurons containing NPY alone as well as a decrease in the number of neurons coexpressing NPY and SS was observed, whereas the number of neurons containing SS alone increased 16 days after ischemia. We conclude that neurons coexpressing SS and NPY before ischemia added to the number of neurons containing SS alone after ischemia, because NPY expression was selectively downregulated in the coexpressing population. In hilus, we demonstrated both survival and ischemic cell death of neurons expressing either SS, NPY or both, indicating that hilar interneurons dying from ischemia cannot unequivocally be identified from their preischemic colocalization of SS and NPY.     

Distribution of neuropeptide Y in the prosencephalon of man and Cotton-head Tamarin (Saguinus oedipus): colocalization with somatostatin in neurons of striatum and amygdala

Summary The presence, chromatographic properties and localization of neuropeptide Y was demonstrated in postmortem human brain areas of neurologically and neuropsychiatrically normative controls using immunocytochemistry and high performance liquid chromatography combined with radioimmunoassay. NPY-immunoreactivity was found in many regions of the prosencephalon. Numerous perikarya and fibers were present in the neocortex, basal ganglia and limbic-hypothalamic areas. A moderate number of neurons and fibers was observed in the basal forebrain, including the septal complex. A comparative immunohistochemical investigation in perfusion-fixed brains of the old-world ape Saguinus oedipus revealed an almost identical distribution of NPY-immunoreactivity with only minor differences. Colocalization experiments on 1–2 m thin consecutive paraffin sections revealed a large number of NPY neurons throughout the human neostriatum and amygdaloid complex that were also positive for somatostatin. Our findings indicate that detection of neuropeptides in fresh or fixed post-mortem human tissue by different immunochemical methods may actually reflect the in vivo conditions. In addition, the wide distribution of NPY throughout the human brain and its colocalization with other neurotransmitters suggests a physiological role as neuroactive substance, i.e. neuromodulator in the primate central nervous system.     

The regional distribution of somatostatin and neuropeptide Y in control and Alzheimer's disease striatum

Somatostatin-like immunoreactivity (SLI) and neuropeptide Y-like immunoreactivity (NPYLI) were measured in subdissections of both normal and Alzheimer's disease (AD) striatum at 5 coronal levels. Concentrations of both neuropeptides were relatively homogeneously distributed in the coronal and anterior-posterior planes except for a trend towards increased concentrations in the tail of the caudate and the posterior putamen. The nucleus accumbens showed 2-3-fold higher concentrations of both SLI and NPYLI than the rest of the striatum. There were no significant differences between control and AD brains. The high concentrations of SLI and NPYLI in the nucleus accumbens suggest that this region may receive somatostatin-neuropeptide Y afferents and that somatostatin and neuropeptide Y may play a role in the modulation of motor activity.     

Cellular localisation of somatostatin mRNA and neuropeptide Y mRNA in foetal striatal tissue grafts

Using in situ nucleic acid hybridisation histochemistry, we have studied the expression of somatostatin mRNA and neuropeptide Y mRNA in grafts of embryonic striatal neurones implanted into the ibotenic acid-lesioned rat neostriatum. Tissue sections of the grafted striatum were incubated with either 32P- or 35S-labelled complementary oligodeoxyribonucleotide probes specific for somatostatin mRNA and neuropeptide Y mRNA, exposed with X-ray film and dipped in Ilford K-5 emulsion. Neither somatostatin mRNA nor neuropeptide Y mRNA was detectable in the ibotenic acid-lesioned striatum indicating a pronounced degeneration of somatostatin- and neuropeptide Y-containing neurones. However, in the striatal grafts the levels of somatostatin mRNA and neuropeptide Y mRNA were substantially increased over those in the control intact striata. The results suggest that in the grafts, somatostatin mRNA and neuropeptide Y mRNA were expressed in a higher proportion of primordial striatal neurones and there was also an increased level of expression of each neuropeptide gene per individual neurone (reflecting a higher synthetic activity of such neurones) compared to the intact mature striatum. These data demonstrate the sensitivity of the in situ hybridisation technique to study patterns of gene expression in developing neuronal tissues after transplantation.     

Haloperidol-induced increase in neuropeptide Y immunoreactivity in the locus coeruleus of the rat brain.

The effect of haloperidol, a dopamine (preferably D2) receptor blocking agent on neuropeptide Y immunoreactivity was studied immunohistochemically in neurons of the locus coeruleus and striatum of rat brain. It was found that haloperidol given four times (5 and 2.5 mg/kg, i.p.) induced, after 24 h, a significant increase in the level of neuropeptide Y immunoreactivity in the locus coeruleus but not in the striatum. No changes in neuropeptide Y immunoreactivity in studied structures were observed after alpha-adrenergic receptor blocking agent phenoxybenzamine or serotonin-synthesis inhibitor D,L-p-chlorophenylalanine. The results suggest that the content of neuropeptide Y-immunoreactive material in nerve cell bodies of the locus coeruleus is inhibitorally controlled by monoaminergic (may be dopaminergic D2) receptors.     

Influence of imipramine on neuropeptide Y immunoreactivity in the rat brain

The effects of treatment with the antidepressant drug imipramine on neuropeptide Y immunoreactivity were studied immunocytochemically in the rat brain cortex and hypothalamus. It was found that the level of neuropeptide Y immunoreactivity in the cortex was significantly lowered three and 24 h after the last dose of chronic (14 days) imipramine administration as well as 3 h after acute administration. A tendency to decrease neuropeptide Y immunoreactivity was also found in the hypothalamus. The results obtained suggest an important role of the cortical neuropeptide Y in the action of the drug.     

Coexistence of neuropeptide Y and somatostatin in rat and human cortical and rat hypothalamic neurons

The distribution and coexistence of neuropeptide Y (NPY) and somatostatin (SOM) were evaluated in rat and human cerebral cortex and in the rat hypothalamic arcuate nucleus (n) using double immunofluorescent staining in which primary antisera were raised in different species. The results of the study indicate extensive coexistence of NPY and SOM in both rat and human cortex but only occasional coexistence in the rat arcuate n.     

Immunohistochemical expression and colocalization of somatostatin, carboxypeptidase-E and prohormone convertases 1 and 2 in rat brain

The processing of many peptides for their maturation in target tissue depends upon the presence of sorting receptor. Several previous studies have predicted that carboxypeptidase-E (CPE), prohormone convertase 1 (PC1) and prohormone convertase 2 (PC2) may function as sorting elements for somatostatin (SST) for its maturation and processing to appropriate targets. However, nothing is currently known about whether brain, neuronal culture or even endocrine cells express SST, CPE, PC1 and PC2 and exhibit colocalization. Accordingly, in the present study using peroxidase immunohistochemistry, double-labeled indirect immunofluorescence immunohistochemistry and Western blot analysis, we mapped the distributional pattern of SST, CPE, PC1 and PC2 in different rat brain regions. Additionally, we also determined the colocalization of SST with CPE, PC1 and PC2 as well as colocalization of CPE with PC1 and PC2. The localization of SST, CPE, PC1 and PC2 reveals a distinct and region specific distribution pattern in the rat brain. Using an indirect double-label immunofluorescence method we observed selective neuron specific colocalization in a region specific manner in cortex, striatum and hippocampus. These studies provide the first evidence for colocalization between SST, CPE, PC1 and PC2 as well as CPE with PC1 and PC2. SST in cerebral cortex colocalized in pyramidal and non-pyramidal neurons with CPE, PC1 and PC2. Most importantly, in striatum and hippocampus colocalization was mostly observed selectively and preferentially in interneurons. CPE is also colocalized with PC1 and PC2 in a region specific manner. The data presented here provide a new insight into the distribution and colocalization of SST, CPE, PC1 and PC2 in rat brain. Taken together, our data anticipate the possibility that CPE, PC1 and PC2 might be potential target for the maturation of SST.     

Pancreatic polypeptide immunoreactivity in rat brain is actually neuropeptide Y

Radioimmunoassay was combined with high pressure liquid chromatography and immunohistochemistry to establish the identity of pancreatic polypeptide-like immunoreactive material in the central nervous system of the rat. Antisera to avian pancreatic polypeptide, bovine pancreatic polypeptide, the invariant amidated carboxyterminal hexapeptide fragment of mammalian pancreatic polypeptides and the structurally related peptide, neuropeptide Y, were used immunocytochemically to localize neurons containing immunoreactive pancreatic polypeptide-like material in rat brain. Adjacent brain sections stained by the indirect immunofluorescent technique and single sections from double-staining experiments demonstrated that identical fibers and perikarya stained for pancreatic polypeptide-like immunoreactive material by antisera directed against each of the four peptides. Characterization of pancreatic polypeptide-like immunoreactive material in chromatographed rat brain extracts by radioimmunoassay using antisera to either neuropeptide Y or the carboxy-terminal portion of the pancreatic polypeptide molecule revealed that the major peak of immunoreactive material, as measured by either assay, appeared to co-elute with synthetic porcine neuropeptide Y. A minor peak of immunoreactive material co-eluting with peptide YY standard was indicated by the neuropeptide Y radioimmunoassay. This was contrasted by data obtained from chromatographic profiles of rat pancreas, which showed that the main immunoreactive peak, as measured by the neuropeptide Y assay, co-eluted with porcine peptide YY, with a minor peak co-eluting with porcine neuropeptide Y. The main peak of immunoreactive material in pancreas, as measured by the pancreatic polypeptide carboxy-terminal radioimmunoassay, eluted considerably earlier than standard peptide YY, neuropeptide Y and bovine pancreatic polypeptide, and is probably identical to rat pancreatic polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of enhanced silver staining combined with electron microscopical immunolabelling to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in cerebrovascular nerves.

The combination of immunolabelling at the electron microscope level and enhanced silver staining has been used to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in perivascular nerves supplying cerebral arteries of the rat. This has been shown in control tissue, but it is easier to demonstrate after long-term sympathectomy since that leads to an enhancement of neuropeptide Y in vasoactive intestinal polypeptide-containing parasympathetic nerves supplying these vessels. Immunolabelling of the antigens for these peptides was performed sequentially with the biotin streptavidin diaminobenzidine method, and the end product to the first antiserum was gold-silver intensified before the visualization of the second antigen. Using this technique, it was shown that all the neuropeptide Y immunoreactivity present in the rat cerebral vessels after long-term sympathectomy with guanethidine was localized in vasoactive intestinal polypeptide-containing nerves. Furthermore, an immunohistochemical analysis of the parasympathetic pterygopalatine ganglia in guanethidine-treated rats showed an increase in the percentage of neurons displaying neuropeptide Y immunoreactivity. In order to clarify if the pterygopalatine ganglion was the origin of those neuropeptide Y/vasoactive intestinal polypeptide-immunoreactive cerebrovascular nerves, which had increased in number after sympathectomy, a fluorescent neuronal tracer (Fast Blue) was applied to the right middle cerebral artery of rats which had undergone guanethidine treatment for six weeks. Immunohistochemical analysis of the ipsilateral ganglion 72 h after application of the tracer revealed the presence of immunoreactivity to both these peptides in retrogradely labelled neurons. It is concluded that neuropeptide Y and vasoactive intestinal polypeptide are colocalized in perivascular parasympathetic nerves supplying the middle cerebral artery of the rat, which have their origin in the pterygopalatine ganglion. Furthermore, long-term sympathectomy with guanethidine leads to an increase in the expression of neuropeptide Y in these vasoactive intestinal polypeptide-immunoreactive neurons.     

Immunohistochemical analysis of the early ontogeny of the neuropeptide Y system in rat brain

The distribution of neuropeptide Y in the developing rat brain was studied with immunocytochemistry, using the peroxidase-antiperoxidase method. Immunoreactive perikarya were first seen on embryonic day 13 and staining of fibres appeared from embryonic day 15 onwards: perikaryal staining was generally more intense prenatally than after birth. Areas rich in neuropeptide Y immunostaining included the monoaminergic regions of the brain stem from embryonic day 13 (especially the lateral reticular nucleus and the medullary reticular formation), the dorsal mesencephalon (with spots of immunoreactivity in the outer subventricular zone at embryonic days 13 or 14 and many cells and fibres in the inferior colliculus from embryonic days 16-20) and the olfactory tubercle/ventral striatum from embryonic day 15 until birth. The period of development of cortical neurones extended from embryonic day 19 until postnatal day 21. A hitherto unreported feature unique to neuropeptide Y was the presence in certain parts of the cerebral cortex of transient cells at the base of the cortical plate bearing radial processes which transverse its width. They were present from embryonic day 17 until postnatal day 4 and were maximally developed at embryonic days 20 or 21, contributing at this age a substantial fibre projection through the immature corpus callosum. The abundance of neuropeptide Y in the prenatal rat brain suggests it may play an important role in development.     

Galanin and neuropeptide Y in chromaffin granules from the guinea-pig.

We have examined the subcellular distribution of galanin-like immunoreactivity, neuropeptide Y-like immunoreactivity and the catecholamines noradrenaline and adrenaline in the adrenal medulla from guinea-pigs. By differential centrifugation of the adrenal medulla homogenate the neuropeptides as well as the catecholamines sedimented in a 10,000 g pellet. This pellet was resuspended and further examined in discontinuous and continuous density gradients. In the discontinuous gradient the catecholamines peaked in the heavy bottom fraction, assumed to contain chromaffin granules. Galanin-like immunoreactivity and neuropeptide Y-like immunoreactivity were also enriched in this fraction. However, both neuropeptides showed high levels of sedimentable material also in a fraction of intermediate density. In the continuous density gradient, the sum of sedimentable and soluble catecholamines showed peak values in two fractions corresponding to 1.07 and 1.47 M sucrose, respectively. The NA peak in the denser fraction was more pronounced than the corresponding A peak. Galanin-like immunoreactivity showed only one peak, in the fraction corresponding to 1.07 M sucrose. The data suggest that galanin-like immunoreactivity and neuropeptide Y-like immunoreactivity are partly stored with catecholamines in chromaffin granules. However, galanin-like immunoreactivity and neuropeptide Y-like immunoreactivity was also found in fractions lighter than those containing the bulk of the catecholamines.     

Decreased somatostatin immunoreactivity but not neuropeptide Y immunoreactivity in cerebral cortex in senile dementia of Alzheimer type

The content of two neuropeptides, somatostatin (SRIF) and neuropeptide Y (NPY) has been determined in two cerebral cortical areas of Alzheimer's disease brain and in age-matched control brains. The content of SRIF-like immunoreactivity (SRIF-LI) was found to be decreased in Alzheimer temporal cortex (Brodmann area 21) compared to control temporal cortex. The decreased content of SRIF was significantly correlated with the observed number of neuritic plaques and neurofibrillary tangles. No difference was observed in NPY-LI between Alzheimer cerebral cortex and control cortex. Furthermore, no correlations were observed between NPY content and plaque count, neurofibrillary tangle estimate or SRIF content despite widespread reports of NPY/SRIF coexistence.     

Concomitant increase of somatostatin, neuropeptide Y and glutamate decarboxylase in the frontal cortex of rats with decreased seizure threshold

The neuropeptides somatostatin and neuropeptide Y and the activity of glutamate decarboxylase were determined in the frontal cortex of rats subjected to experimental epilepsy. Two different animal models, (1) rats kindled for 4 weeks by daily injection of pentylenetetrazole, and (2) rats which had undergone strong limbic seizures induced by kainic acid, were used. Decreased seizure threshold, as shown by injection of a subconvulsive dose of pentylenetetrazole, was observed 10 days after the last kindling session and 1 month after injection of kainic acid, respectively. Significantly increased levels of somatostatin (by 60%), neuropeptide Y (135%) and increased activity of glutamate decarboxylase (22%) were found in the frontal cortex of rats previously treated with kainic acid. Separation of somatostatin-like immunoreactivity by size exclusion high-performance liquid chromatography showed a marked increase of immunoreactivity in fractions containing the somatostatin precursor (by 200%) and less prominently of somatostatin-14 and somatostatin-28 (by 60 and 80%, respectively). Michaelis-Menten kinetics of glutamate decarboxylase revealed an increased maximal velocity (Vmax) in the frontal cortex of kainic acid-treated rats, but no change in the Km value was found. Similar results were also obtained in pentylenetetrazole-kindled rats. Injection of cysteamine (100 mg/kg, i.p.) resulting in a 30% decrease of cortical somatostatin in kainic acid-pretreated rats markedly suppressed seizures induced by an otherwise subconvulsive dose of pentylenetetrazole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of reserpine administration on neuropeptide Y immunoreactivity in the locus coeruleus and caudate-putamen nucleus of the rat brain.

The effects of treatment with reserpine (10 mg/kg, i.p.) a monoamine depleting agent, on neuropeptide Y immunoreactivity were studied immunohistochemically in neurons of two rat brain structures: locus coeruleus and caudate-putamen nucleus. It was found that reserpine after 24 h increased neuropeptide Y immunoreactivity level but no significant changes were observed 4 and 72 h or 5 days after the injection. The results indicate that despite the known co-existence of neuropeptide Y and noradrenaline in some neurons of the locus coeruleus no concomitant decrease in neuropeptide Y immunoreactivity level was found after reserpine when noradrenaline was depleted from nerve cell bodies and terminals. The increase in neuropeptide Y immunoreactivity observed 24 h after reserpine injection may suggest that the neuropeptide Y-containing neuronal systems of the locus coeruleus and caudate-putamen nucleus are controlled by monoaminergic afferents.     

New insights on the neuropeptide Y system in the larval lamprey brain: neuropeptide Y immunoreactive neurons,descending spinal projections and comparison with tyrosine hydroxylase and GABA immunoreactivities

Lampreys are useful models for studying the evolution of the nervous system of vertebrates. Here we used immunofluorescence and tract-tracing methods to study new aspects of the neuropeptide Y-immunoreactive (NPY-ir) system in larval sea lampreys. NPY-ir neurons were observed in brain nuclei that contain NPY-ir cells in other lamprey species. Moreover, a group of NPY-ir cells that migrated away the periventricular layer was observed in the lateral part of the dorsal hypothalamus, which suggests a role for NPY in feeding behavior in lampreys. We also report NPY-ir cells in the dorsal column nucleus, which appears to be unique among vertebrates, and in the habenula. A combination of tract-tracing and immunohistochemical labeling demonstrated the presence of spinal projecting NPY-ir reticular cells in the anterior rhombencephalic reticular formation, and the relationships between the NPY-ir system and the reticulospinal nuclei and some afferent systems. The colocalization of catecholamines and GABA in lamprey NPY-ir neurons was investigated by double immunofluorescence methods. Colocalization of tyrosine hydroxylase (TH) and NPY immunoreactivities was not observed in any brain neuron, although reported in amphibians and mammals. The frequent presence of NPY-ir terminals on TH-ir cells suggests that NPY modulates the activity of some dopaminergic nuclei in lampreys. Colocalization of NPY and GABA immunoreactivities was frequently observed in neurons of different rhombencephalic and diencephalic NPY-ir populations. These results in lampreys suggest that the coexpression of NPY and GABA in neurons appeared early on in the brains of vertebrates.     

Effect of neuropeptide Y on behavior and on the time course of the brain noradrenalin level

Laboratory of General Physiology of Functional Systems, P. K. Anokhin Research Institute of Normal Physiology, Academy of Medical Sciences of the USSR, Moscow. Institute of Pathophysiology, Szeged Medical University, Hungary. (Presented by Academician of the Academy of Medical Sciences of the USSR I. P. Ashmarin). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 8, pp. 132–135, August, 1989.