Effect of Sustained Physiological Hyperinsulinaemia on Hypothalamic Neuropeptide Y and NPY mRNA Levels in the Rat

Neuropeptide Y (NPY) synthesized in the arcuato-paraventricular projection in the rat hypothalamus is thought to play an important role in controlling energy homeostasis. The factors that regulate hypothalamic NPY are not known but, amongst others, insulin has been postulated as an inhibitory modulatory agent. To test this hypothesis, normal male rats were given either insulin (2 units/day) or saline via subcutaneous osmotic minipumps for 3 days. Euglycaemia was maintained by a concomitant glucose infusion in insulin-infused rats which had peripheral insulin levels 5–8 times higher than saline-infused controls. Hyperinsulinaernic rats ate 42% less than controls, but their total energy intake (food intake plus glucose infusion) was higher than that of controls, and they gained more weight than controls during the experimental period. Hyperinsulinaemia had no significant effect on hypothalamic NPY mRNA or NPY levels in the arcuate nucleus. NPY concentrations in the paraventricular nucleus were, however, significantly increased by 73% in hyperinsulinaemic rats, but were closely similar to controls in all other areas. Insulin may act as a satiety factor in that hyperinsulinaemic rats ate less, but the fact that these animals had increased total energy intake and gained excessive weight suggests that insulin may not function as an overall regulator of energy balance. In addition, physiological hyperinsulinaemia does not apparently inhibit NPY gene expression in the arcuate nucleus. Due to the lack of effect of hyperinsulinaemia on NPY synthesis in the arcuate nucleus, the elevated NPY concentrations in the paraventricular nucleus could result from a reduction of its release, which would be in keeping with the reduction in food intake.     

Striatal NPY-Containing Neurons Receive GABAergic Afferents and may also Contain GABA: An Electron Microscopic Study in the Rat

Dual labelling methods were applied to localize simultaneously neuropeptide Y (NPY) and glutamate decarboxylase (GAD) immunoreactivities on ultrathin sections of the rat caudate-putamen (CP). By means of a double peroxidase-anti-peroxidase technique, using 3,3'-diaminobenzidine and benzidine dihydrochloride as chromogens in animals with no colchicine pretreatment, GAD immunoreactivity was found to be present in terminals only whereas NPY immunoreactivity was detected in neurons displaying the features of aspiny type cells and processes. With this approach, we observed numerous synaptic associations of the symmetrical type between GAD-immunoreactive (-Ir) axonal boutons and NPY-Ir cell bodies and dendrites. By combining immunoperoxidase and radioimmunocytochemical labelling in animals pretreated with colchicine, NPY was again detected in a single population of aspiny type neurons whereas GAD immunoreactivity was observed in neurons which could be classified as aspiny and spiny on the basis of their ultrastructural characteristics. All the cells of the aspiny type displaying clear-cut NPY immunoreactivity were also found to be GAD-positive. Some other neurons of both the aspiny and the spiny type were found to be immunoreactive to GAD alone. GAD/NPY dually labelled terminals were also observed and some axo-axonic appositions between GAD- and NPY-Ir terminals were also detected. All in all, these data show that NPY aspiny type neurons of the rat CP receive GABAergic afferents and provide morphological support for two hypotheses: that NPY is co-localized with GABA in some cell bodies, dendrites and axons, and that presynaptic interactions may occur between NPY and GABAergic neuronal systems.     

Neuropeptide Y: regional distribution chromatographic characterization and immunohistochemical demonstration in post-mortem human brain

Using a neuropeptide Y (NPY)-directed radioimmunoassay the post-mortem stability of NPY was assessed in both rat and human brain samples. The regional distribution of NPY-like immunoreactivity in human brain was determined. The NPY-like immunoreactivity in human brain separated on Sephadex G-50 columns and ¹⁸C reverse phase at the position expected for NPY. Immunohistochemical staining using the NPY-directed antiserum revealed a characteristic population of cortical and striatal neurons containing NPY-like immunoreactivity.     

Localization of Neuropeptide Y Y1 mRNA in the Human Brain: Abundant Expression in Cerebral Cortex and Striatum

Many neurobiological functions have been ascribed to the NPY Y1 receptor subtype, but autoradiographic analysis has failed to detect Y1 binding sites in most human brain areas, in contrast to the rat. We examined the regional distribution of Y1 mRNA-containing cells in the post-mortem human brain to clarify if there is a major species difference in terms of the existence of Y1 receptors in the human telencephalon, in particular the striatum and cortex. In situ hybridization experiments revealed widespread distribution of Y1 mRNA signals in all layers of most limbic and neocortical regions, predominantly in layer IV (most cortical regions) and layer VI. The striatum showed moderate Y1 receptor mRNA expression levels with intensely expressing cells localized to the nucleus accumbens. The highest Y1 receptor mRNA expression was apparent within the dentate gyrus, and the lowest in the subiculum, parahippocampal gyrus, cerebellum, and thalamus. In vitro autoradiography using [¹²⁵I]Leu³¹Pro³⁴-PYY and [¹²⁵I]PYY with NPY (13–36) or Leu³¹ Pro³⁴ NPY; confirmed the presence of low Y1–like binding in the human brain despite abundant Y1 mRNA expression. However, using a rat model of the human autopsy process, it was apparent that the inability to reveal high Y1– versus Y2–like receptors in the human brain was related in part to marked reductions of Y1–like, but not Y2–like, receptors within a 4 h post-mortem delay. Altogether, the results indicate that the Y1 receptor gene is abundant in the human brain and this receptor may have important roles in cognitive, limbic and motor function.     

Ontogeny of a novel peptide, neuropeptide Y (NPY) in rat brain

Changes in the concentration of a newly discovered peptide, neuropeptide Y (NPY) have been determined in the developing rat brain using a recently developed radioimmunoassay and chromatographic analysis. NPY was present in the brain stem (14.8 ± 5.6 pmol/g) and diencephalon (12.1 ± 12.1 pmol/g) in the earliest embryos studied (14 days postconception), but appeared only on the 19th day postconception in the cerebral cortex. The concentrations of NPY showed a rapid postnatal rise in all regions examined. The finding of NPY early in the development of the embryonic rat brain and particularly in caudal regions has some similarities to the pattern of development of the catecholaminergic system.     

Frontal decortication and adaptive changes in striatal cholinergic neurons in the rat

Interruption of the corticostriatal pathway by undercutting the cortex resulted in a reduction of glutamate uptake by 55% and in a depression of acetylcholine (ACh) synthesis by 30% in striatum after two postlesion weeks without affecting the content of ACh and choline, the specific binding of [3H]dexetimide to muscarinic receptors, the activity of choline acetyltransferase and the levels of noradrenaline, serotonin, dopamine and 3,4-dihydroxyphenylacetic acid. The influence of this excitatory pathway on striatal cholinergic neuropharmacology was investigated. It was found that the effect of a number of agonists (R-apomorphine, bromocriptine, lisuride, quinpirole, JL-14389, 2-chloroadenosine, oxotremorine and methadone), capable of depressing cholinergic activity in the striatum through receptor-mediated responses--reflected as an increase in ACh content--is operative only when the corticostriatal pathway is intact. By contrast, antagonists capable of decreasing ACh content, i.e. the typical neuroleptics pimozide, haloperidol and the atypical ones clozapine, L-sulpiride, as well as the anti-muscarinic agent scopolamine, were not influenced by the lesion. The possibility that the lesion non-specifically damaged striatal cells on which the agonists, but not the antagonists acted was excluded by results showing, firstly, that the increase in striatal ACh elicited by the ACh precursor, choline, was not blocked by decortication, and secondly, that the degeneration of the corticostriatal neurons did not prevent the ACh-increasing effect of bromocriptine, a long-acting ergot alkaloid, when sufficient time was allowed for the drug to act. It was furthermore possible to restore the inhibitory action of apomorphine on cholinergic neurons either by short-term chemical lesion of the nigrostriatal dopaminergic input or by the administration of choline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuropeptide Y in Rat Spiral Ganglion Neurons and Inner Hair Cells of Organ of Corti and Effects of a Nontraumatic Acoustic Stimulation

Neuropeptide Y (NPY) is an important neuromodulator found in central and peripheral neurons. NPY was investigated in the peripheral auditory pathway of conventional housed rats and after nontraumatic sound stimulation in order to localize the molecule and also to describe its response to sound stimulus. Rats from the stimulation experiment were housed in monitored sound-proofed rooms. Stimulated animals received sound stimuli (pure tone bursts of 8 kHz, 50 ms duration presented at a rate of 2 per second) at an intensity of 80 dB sound pressure level for 1 hr per day during 7 days. After euthanizing, rat cochleae were processed for one-color immunohistochemistry. The NPY immunoreactivity was detected in inner hair cells (IHC) and also in pillar and Deiters’ cells of organ of Corti, and in the spiral ganglion putative type I (≥1,009 μm³) and type II (≤225 μm³) neurons. Outer hair cells (OHC) showed light immunoreaction product. Quantitative microdensitometry showed strong and moderate immunoreactions in IHC and spiral ganglion neurons, respectively, without differences among cochlear turns. One week of acoustic stimulation was not able to induce changes in the NPY immunoreactivity intensity in the IHC of cochlea. However, stimulated rats showed an overall increase in the number of putative type I and type II NPY immunoreactive spiral ganglion neurons with strong, moderate, and weak immunolabeling. Localization and responses of NPY to acoustic stimulus suggest an involvement of the neuropeptide in the neuromodulation of afferent transmission in the rat peripheral auditory pathway.     

Distribution of neuropeptide Y in the lower brainstem: an immunohistochemical analysis

The distribution of neuropeptide Y (NPY)-like immunoreactive (NPYI) structures in the rat lower brainstem was examined by means of indirect immunofluorescence or peroxidase-antiperoxidase methods. In addition to the well known immunoreactive NPYI containing cell groups, the present study demonstrated a much wider distribution of immunoreactive NPYI cells in the lower brainstem, i.e. substantia nigra pars lateralis, interpeduncular nucleus, inferior colliculus, nucleus cuneiformis, dorsal tegmental nucleus of Gudden, nucleus laterodorsalis tegmenti, nucleus vestibularis medialis, nucleus vestibularis inferioris, in the fasciculus longitudinalis medialis and nucleus parvocellularis compacta. We also demonstrated an extensive network of NPYI fibers in various areas of the lower brainstem including the auditory system, viscerosensory system, visceromotor system, raphe nuclei, reticular formation, parabrachial area, locus coeruleus and interpeduncular nucleus, etc. The possible importance of NPY is briefly discussed.     

Phenotype Specification of Cortical Neurons During a Period of Molecular Plasticity

The transient expression of neuropeptide transmitters is a common feature of the developing cortex. We have now analysed the role of cortical afferents in shaping the neurochemical architecture of rat visual cortex using organotypic cultures. Deafferented cortex monocultures prepared from newborn rats reveal a constant NPY mRNA expression in 6–8% of all cortical neurons up to 90 days in vitro (DIV). In contrast, afferent thalamocortical and corticocortical axonal innervation elicits a progressive reduction in the percentage of NPY mRNA expressing neurons from initially 6–8% in 30DIV cocultures to 2–3% and 3–4% respectively in 60DIV cocultures, which is maintained for up to 90DIV. This phenotype restriction is not observed in only efferently connected corticocollicular cocultures. Further, axonal innervation does not change the percentage of GAD mRNA-expressing neurons, which remains at 13% in mono- and cocultures. When feeding thalamocortical cocultures with monoculture-conditioned medium between 3–20DIV followed by normal medium up to 60DIV, the phenotype restriction fails to occur in the cocultured cortex. We conclude that cortex-derived factors secreted into the medium by a monoculture suppress the phenotype-restricting capacity of the afferents, but only when present within the first 14DIV during the period of formation of axonal connections. To elucidate the nature of the cortex-derived factors, brain-derived neurotrophic factor was applied to the medium. When applied for the first 14DIV, it does not prevent the phenotype restriction from occurring. This suggests that epigenetic factors such as axonal innervation and cortex-derived factors other than brain-derived neurotrophic factor govern a phenotype decision in neocortical neurons during a period of molecular plasticity.     

Neuropeptide Levels after Pentylenetetrazol Kindling in the Rat

Levels of several neuropeptides were measured in the frontal cortex, dorsal hippocampus, striatum, and amygdala/pyriform cortex in rats kindled for 5 weeks by daily injection of pentylenetetrazol (30 mg/kg, i.p.). Significantly increased concentrations (by 30 - 140%) were found in all examined brain areas for neuropeptide Y, somatostatin (except hippocampus) and neurokinin-like immunoreactivity 10 days after the last kindling session. Similar but less pronounced changes were also found 24 h after the last seizure. The increase in total neurokinin-like immunoreactivity was due to a marked increase in neurokinin B as revealed by HPLC analysis. Increases in peptide levels, however, were restricted to fully kindled animals. At the same time no changes in levels of substance P, vasoactive intestinal polypeptide and calcitonin gene-regulated peptide were observed. Cholecystokinin octapeptide was enhanced only in the hippocampus (by 46%). The increases in neuropeptide Y, somatostatin, and neurokinin-like immunoreactivity subsided after 3 months. A markedly decreased seizure threshold was observed 10 days and 2 months after the final kindling session. No nerve cell degeneration was observed in kindled rats 24 h or 10 days after the last pentylenetetrazol injection. Some animals (2 of 4), however, exhibited signs of blood - brain barrier damage when examined 24 h after the last kindling session which may reflect the preceding convulsions. No such changes were detected after 10 days. The increases in peptide levels may suggest increased activity of respective neurons which, at least to some degree, may be associated with gamma-aminobutyric acid. The changes in peptide levels may be more closely related to the kindling procedure itself than to the decreased seizure threshold of the animals.     

Neuropeptide Y-immunoreactive neurons in the striatum of cat and monkey: Morphological characteristics, intrinsic organization and co-localization with somatostatin

A detailed study of the distribution of neuropeptide Y (NPY) in the striatum of squirrel monkey (Saimiri sciureus) and cat was undertaken by means of indirect immunofluorescence and peroxidase-antiperoxidase (PAP) methods. In monkey, the NPY-immunoreactivity is homogeneously distributed along the entire extent of the caudate nucleus (CD) and putamen (PUT), while in cat marked heterogeneities are noted. In the CD of cat, the NPY-immunoreactive fibers and cell bodies are concentrated in numerous patches of various sizes, which can be readily distinguished from zones of poor NPY-immunostaining. In the CD and PUT of squirrel monkey the NPY-positive neurons are either triangular, fusiform or globular, with long and smooth dendrites branching infrequently. The numerical density of NPY-immunoreactive cell bodies is greater in the CD than in the PUT, and it increases markedly along the rostrocaudal extent of the striatum. In the rostral CD and PUT the densities are 23 cells/mm2 and 14 cells/mm2, respectively, whereas the values for caudal CD and PUT are 35 cells/mm2 and 20 cells/mm2, respectively. Quantitative measurements reveal that these NPY-immunoreactive cells belong to a single subset of striatal neurons having a maximum diameter of 19.2 +/- 0.1 micron and a cross-sectional area of 145.5 +/- 0.6 micron2 (mean +/- S.E.M.; n = 1238 CD cells and 1169 PUT cells). Furthermore, experiments combining the use of lectin-conjugated HRP as retrograde tracer with PAP immunohistochemical method demonstrate that striatal NPY-immunoreactive neurons in squirrel monkey and cat do not project outside the striatum. Finally, co-localization studies in monkey reveal that the vast majority of striatal NPY-positive neurons also contains somatostatin. These results show that the NPY-immunoreactive neurons in mammalian striatum form a subpopulation of medium-sized interneurons containing somatostatin.     

神经肽Y受体在人垂体腺瘤中的基因表达

目的研究NPY受体在人垂体腺瘤中表达及其规律。方法收集2004年1月~2005年8月期间我院神经外科手术切除垂体腺瘤获取标本57例。通过巢式聚会酶链反应(N-PCR)测定NPY的Y1R和Y2R受体。结果不同类型垂体腺瘤中均有NPY的Y1R、Y2R mRNA表达。Y1R基因表达的差异无统计学意义(F=1.97,P=0.098);Y2R表达的差异有显著统计学意义(F=2.703,P=0.03)。NPY与Y2R呈正相关(r=0.414,P=0.003),但与Y1R无相关性(r=-0.123,P=0.405),且Y1R与Y2R之间也无相关性(r=0.158,P=0.284)。PRL腺瘤中Y2R的表达明显低于GH腺瘤和促性腺瘤。GH腺瘤中NPY与Y2R表达呈正相关(r=0.558,P=0.025)。结论垂体腺瘤中存在NPY和Y1R及Y2R表达;Y2R表达的差异有显著统计学意义。Y2R在GH腺瘤和促性腺细胞腺瘤中的表达水平明显高于PRL腺瘤,而Y1R的表达无显著性差异。NPY受体的表达差异可能与不同类型垂体腺瘤的发生、发展及其内分泌行为有关。     

Some Acute Effects of Intracerebroventricular Neuropeptide Y on Insulin Secretion and Glucose Metabolism in the Rat

Acute administration of neuropeptide Y(NPY) into the hypothalamus and cerebral ventricles can stimulate insulin secretion in the absence of available food. However, the relationship of this effect to blood glucose and other hormones which regulate glucose metabolism remains unclear. The purpose of this study was to compare the effects of NPY injected into the third ventricle (ICV) on serum insulin, glucose, glucagon, corticosterone and non-esterified fatty acids. Studies were performed on conscious, unrestrained female rats, not given access to food. ICV NPY, 2 and 5  μg produced an increase in serum insulin and glucagon, while the 5  μg dose only increased plasma glucose transiently and increased non-esterified fatty acids for a longer period. Corticosterone was not affected by ICV NPY. The insulinaemic response to i.v. glucose, 0.5 g/kg was doubled by ICV NPY, 4  μg. The maximal insulin levels were 113±18 for ICV NPY versus 67±8  μU/ml for ICV saline-treated animals. The glycaemic response was not altered. The hypoglycaemic response to i.v. insulin, 0.15 U/kg was significantly attenuated by ICV NPY, 5  μg. We concluded that ICV NPY promotes insulin secretion in the absence of available food and may potentiate the insulinaemic response to hyperglycaemia. Furthermore, possibly through its effects on glucagon and non-esterified fatty acids, ICV NPY may decrease the ability of insulin to control glucose metabolism.     

Neuropeptide Y (NPY) and C-flanking peptide of NPY in the pineal gland of normal and ganglionectomized sheep.

The present immunohistochemical study describes the presence and distribution of nerve fibers containing neuropeptide Y (NPY), and C-Flanking Peptide Of NPY (CPON) in the pineal gland of the sheep. Nerve fibers were detected by using a series of antisera directed against NPY or against CPON. Many positive immunoreactive nerve fibers were identified in the pial capsule of the pineal, in connective septae and in the parenchyma between pinealocytes. The intraparenchymal fibers were particularly evident and created an extensive network throughout the gland. Nerve fibers immunoreactive for all the peptides were also observed in the posterior commissure and in the stria medullaris thalami. No NPY- or CPON-positive neurons were found in the pineal gland. In order to study the site of origin of NPY- and CPON-immunoreactive nerve fibers, the superior cervical ganglia were bilaterally removed in a series of animals. Sympathetic denervation was checked by using an antiserum against tyrosine hydroxylase (TH). Nearly all TH-immunoreactive elements disappeared in the pineal glands of animals sacrificed 15 days after surgery. Also the density of NPY- and CPON-immunoreactive nerve fibers decreased in the animals after the ganglionectomy. However, a number of nerve fibers still remained in the gland. These data indicate that some NPY- and CPON-immunoreactive nerve fibers of the sheep pineal gland derive from an extrasympathetic origin. The very dense innervation of the sheep pineal gland with nerve fibers containing NPY and CPON strongly indicates a functional role for this family of peptides in the pineal gland of this species.     

The effects of acute phencyclidine treatment on neuropeptide Y (NPY) neuronal system in the rat arcuate nucleus studied by immunocytochemistry and in situ hybridization

Summary Phencyclidine (PCP) is a dissociative drug and an antagonist of N-methyl-D-aspartate (NMDA) receptor. The effects of PCP treatment on neuropeptide Y (NPY) system in the arcuate nucleus of the rat hypothalamus were examined both by immunocytochemistry and in situ hybridization. In acute PCP-treated rats, the NPY-immunoreactive perikarya appeared in the arcuate nucleus but no perikarya were detected in controls, without colchicine pretreatment. The signals of NPY mRNA by in situ hybridization increased in the PCP-treated rats than those of controls. These results suggest that the NPY system in the arcuate nucleus might be partly controlled by glutamatergic neurons.     

Distribution of neuropeptide Y in the forebrain and diencephalon: an immunohistochemical analysis

The distribution of neuropeptide Y (NPY)-like immunoreactive (NPYI) structures in the rat forebrain and upper brainstem was examined by means of indirect immunofluorescence and peroxidase-antiperoxidase methods. The present study has demonstrated a much wider and more abundant distribution of NPYI structures in the forebrain and diencephalon than earlier studies in which antisera against avian pancreatic polypeptide or bovine pancreatic polypeptide were used, suggesting that NPY is involved in a variety of brain functions.     

Role of Neuropeptide Y in the Regulation of Tyrosine Hydroxylase Messenger Ribonucleic Acid Levels in the Male Rat Arcuate Nucleus as Evaluated by in situ Hybridization

Neuroanatomical data have clearly demonstrated the existence of synaptic contacts between neuropeptide Y (NPY) endings and tuberoinfundibular dopaminergic (TIDA) neurons in the rat arcuate nucleus. In order to determine the influence of NPY in the biosynthesis of dopamine, we have studied the effects of NPY and some NPY analogs on tyrosine hydroxylase (TH) gene expression in TlDA neurons in the male rat. The following peptides: NPY, PYY, [Leu³¹, Pro³⁴]-NPY (a Y, receptor agonist) and NPY_13–36 (a Y₂ receptor agonist) were injected into the left lateral ventricle of adult male rats. All the animals were perfused with 4% paraformaldehyde 4 h after injection. Cryostat sections through the arcuate nucleus were processed for quantitative in situ hybridization. The intracerebroventricular injection of NPY, PYY and [Leu³¹, Pro³⁴]-NPY induced an increased of 43, 33 and 42%, respectively, in the number of grains overlying TH neurons. On the other hand, the Y₂ receptor agonist NPY_13–36 did not influence mRNA levels. These data then strongly suggest that NPY positively regulates the genetic expression of TH in rat TlDA neurons via the Y, NPY receptor subtype.     

Endocrine Actions of Central Neuropeptide Y in the Ewe: Activation of the Hypothalamo-Pituitary-Adrenal Axis by Exogenous Neuropeptide Y and Role of Endogenous Neuropeptide Y in the Secretion of Luteinizing Hormone During the Oestrous Cycle

Neurons immunoreactive for neuropeptide Y (NPY) are abundant in the hypophysiotrophic areas of the brain. In particular, there is considerable anatomical evidence for the influence of this neuropeptide on the reproductive and hypothalamo-pituitary-adrenal axes. We therefore investigated whether central administration of NPY can alter the activities of the reproductive and hypothalamo-pituitary-adrenal axes in the ewe, and whether ovarian steroids are involved in the modulation of these events. We also attempted to investigate whether endogenous NPY is important in the control of luteinizing hormone-releasing hormone/luteinizing hormone (LHRH/LH) secretion in the sheep oestrous cycle. Central injection of NPY (0.15 and 1.5 nmol in 50 μl saline), delivered by gravity flow into the third cerebral ventricle, had no effect on LH levels in ovariectomized (OVX) ewes (n = 6) or OVX ewes implanted with oestradiol (OVX/E₂) (n = 7), nor was LH secretion altered by central NPY (1.5 nmol) in intact cycling animals in either the follicular or the luteal phase (n = 5). However, central administration of 1.5 nmol NPY to intact ewes during both the follicular (P> 0.05) and the luteal phase (P> 0.01), and in OVX/E₂ ewes (P<0.5) caused a large and significant increase in plasma cortisol levels. High litre antibodies were raised to NPY in sheep and the effects of peripheral and central (intracerebroventricular) administration of anti-NPY antibodies on the timing and/or characteristics of the E₂-induced LH surge in anoestrous ewes and of the preovulatory surge of LH in cycling ewes were determined. Intravenous administration of anti-NPY antibodies (n = 6) had no effect on the oestradiol benzoate-induced LH surge, compared with the control injection of non-immune plasma (n = 6). Likewise, passive systemic immunization against NPY (n = 10) was without effect on the characteristics of the preovulatory LH surge, compared with the control group (n = 10). However, central (intracerebroventricular) administration of anti-NPY antibodies (n = 4) delayed or abolished the preovulatory LH surge when compared with non-immune plasma treatment in the same animals. In summary, tonic LHRH/LH secretion is unaffected by centrally administered NPY at the doses used in this study. However, the same doses of NPY activate the hypothalamo-pituitary-adrenal axis, thus lending clear support to the hypothesis that NPY is involved in the multifactorial regulation of adrenocorticotrophin and cortisol secretion in this species, probably by stimulating corticotrophin-releasing hormone and/or arginine vasopressin secretion within the hypothalamus. The results of the immunization experiments further suggest that hypothalamic NPY may play a part in the modulation of the tinning of the LHRH/LH surge in the ewe. In contrast to the role of NPY in this regard in the rat, this only occurs at the level of the hypothalamus. The function of NPY in the regulation of reproduction in the sheep may therefore be to provide a means whereby the efficient generation of a preovulatory LHRH/LH surge is ensured.     

Characterization of NPY Y2 receptor protein expression in the mouse brain. II. Coexistence with NPY, the Y1 receptor, and other neurotransmitter-related molecules

Neuropeptide Y (NPY) is widely expressed in the brain and its biological effects are mediated through a variety of receptors. We examined, using immunohistochemistry, expression of the Y2 receptor (R) protein in the adult mouse brain and its association with NPY and the Y1R, as well as a range of additional neurotransmitters and signaling-related molecules, which previously have not been defined. Our main focus was on the hippocampal formation (HiFo), amygdaloid complex, and hypothalamus, considering the known functions of NPY and the wide expression of NPY, Y1R, and Y2R in these regions. Y2R-like immunoreactivity (-LI) was distributed in nerve fibers/terminal endings throughout the brain axis, without apparent colocalization with NPY or the Y1R. Occasional coexistence between NPY- and Y1R-LI was found in the HiFo. Following colchicine treatment, Y2R-LI accumulated in cell bodies that coexpressed γ-aminobutyric acid (GABA) in a population of cells in the amygdaloid complex and lateral septal nucleus, but not in the HiFo. Instead, Y2R-positive nerve terminals appeared to surround GABA-immunoreactive (ir) cells in the HiFo and other neuronal populations, e.g., NPY-ir cells in HiFo and tyrosine hydroxylase-ir cells in the hypothalamus. In the HiFo, Y2R-ir mossy fibers coexpressed GABA, glutamic acid decarboxylase 67 and calbindin, and Y2R-LI was found in the same fibers that contained the presynaptic metabotropic glutamate receptor 2, but not together with any of the three vesicular glutamate transporters. Our findings provide further support that Y2R is mostly presynaptic, and that Y2Rs thus have a modulatory role in mediating presynaptic neurotransmitter release.     

Dynamic Changes in Neuropeptide Y Concentrations in the Median Eminence in Association with Preovulatory Luteinizing Hormone Release in the Rat

Neuropeptide Y (NPY) stimulates luteinizing hormone (LH) release in the rat by a dual action, one in the hypothalamus to excite LH-releasing hormone (LHRH) release and the other at the level of pituitary gonadotrophs to activate and/or potentiate LH release induced by LHRH. Because NPY produces effects similar to norepinephrine with which it may comprise an excitatory hypothalamic circuit, it was hypothesized that NPY concentrations in the hypothalamus would change in a time- and site-specific manner in association with the preovulatory LH surge on proestrus. Concentrations of NPY in individual nuclei of the preoptic-tuberal pathway and serum LH levels were estimated by radioimmunoassays in rats during diestrus 2 and proestrus. On proestrus, serum LH levels were basal between 1000 and 1400 h, rose significantly at 1500 h and plateaued between 1600 and 1800 h. Of the five neural sites examined, only NPY in the median eminence displayed marked fluctuations in close association with the LH surge. NPY concentrations were low between 1000 and 1300 h, and rose abruptly at 1400 h (P<0.05) preceding the onset of LH rise at 1500 h. These elevated levels were maintained until 1600 h, during which time serum LH rose to a plateau and then fell at 1800 h to the low range seen between 1000 and 1300 h. In contrast, the pattern of changes in NPY levels in the arcuate nucleus, suprachiasmatic nucleus and medial preoptic area, three additional sites in the preoptic-tuberal pathway known to participate in the preovulatory LH surge, were markedly different from that seen in the median eminence. In each of these three sites, NPY levels rose significantly at 1800 h from the values at 1000 to 1200 h with a slightly different time-course of increment. None of these areas exhibited changes in NPY concentrations on diestrus 2; NPY concentrations also were unaltered on diestrus 2 or proestrus in the ventromedial nucleus. The present observations of site-specific, dynamic changes in NPY levels on proestrus, in a manner previously documented for LHRH, support the hypothesis that a subset of NPY neurons terminating in the median eminence may be a component of excitatory neural circuitry that either independently or in co-action with the adrenergic system is responsible for the induction of preovulatory LH release.