Synaptic regulation of paraventricular arginine vasopressin-containing neurons by neuropeptide Y-containing monoaminergic neurons in rats

Summary Synaptic regulation of arginine vasopressin (AVP)-containing neurons by neuropeptide Y (NPY)-containing monoaminergic neurons was demonstrated in the paraventricular nucleus of the rat hypothalamus. NPY and AVP were immunolabeled in the pre- and the post-embedding procedures, respectively, and monoaminergic fibers were marked by incorporating 5-hydroxydopamine (5-OHDA), a false neurotransmitter. The immunoreaction for NPY was expressed by diaminobenzidine (DAB) chromogen, and that for AVP by gold particles. The DAB chromogen was localized on the surface of the membrane structures, such as vesicles or mitochondria, and on the core of large cored vesicles. Gold particles were located on the core of the secretory granules within the AVP cell bodies and processes. The incorporated 5-OHDA was found as dense cores within small or large vesicular structures. From these data, three types of nerve terminals were discernible: NPY-containing monoaminergic, NPY-containing non-aminergic, and monoaminergic fibers. The AVP cell bodies appeared to have synaptic junctions formed by these nerve terminals as well as by the unlabeled nerve terminals which have small clear vesicles and large cored vesicles. These different types of nerve terminals were frequently observed in a closely apposed position on the same AVP cell bodies. The functional relationships of these three types of neuronal terminals are discussed.     

Simultaneous immunogold labeling of GABAergic terminals and vasopressin-containing neurons in the rat paraventricular nucleus

Summary The GABAergic innervation of vasopressin-containing cells in the magnocellular part of the paraventricular nucleus was studied at the electron-microscope level using antibodies against GABA and vasopressin. The detection of both GABA and vasopressin on the same ultrathin section, performed with a double-labeling immunogold method, revealed GABAergic terminals in symmetrical synaptic contact with vasopressin-containing neurons. These GABAergic terminals displayed mitochondria, clear synaptic vesicles and varying numbers of electron-dense vesicles. Vasopressin-immunoreactivity was associated with neurosecretory granules, whereas GABA-immunoreactivity was found above mitochondria, clear synaptic vesicles and some electron-dense vesicles. This study, demonstrating the extensive participation of GABA in the innervation of magnocellular vasopressin-secreting neurons, suggests that this inhibitory neurotransmitter regulates vasopressin secretion at the level of the paraventricular nucleus.     

Intragranular co-storage of neuropeptide Y and arginine vasopressin in the paraventricular magnocellular neurons of the rat hypothalamus

Summary Certain populations of arginine vasopressin (AVP) neurons in the magnocellular paraventricular nucleus became immunoreactive for neuropeptide Y (NPY) when rats were treated with colchicine or monosodium glutamate (MSG). The co-storage of these peptides was examined by empooying a post-embedding electron-microscopic immunohistochemistry technique using goldlabeled antibodies to the two peptides. In colchicinetreated rats, the neuronal perikarya contained numerous secretory granules showing co-storage of the two peptides. The cells of the MSG-treated rats were characterized by having well-developed Golgi bodies with the granular structures also co-storing the two peptides, although the secretory granules in the perikarya were rather fewer than in the colchicine-treated rats. It is concluded that the destruction of the arcuate nucleus by MSG-treatment may potentiate the synthesis of NPY in AVP neurons, the synthesis of which is latent in intact animals.     

Catecholaminergic innervation of neurons containing corticotropin-releasing factor in the paraventricular nucleus of the rat hypothalamus

Catecholaminergic synaptic input to neurons containing corticotropin-releasing factor (CRF) in the parvocellular portion of the paraventricular nucleus (PVN) in the rat hypothalamus was observed. The experimental techniques used combine autoradiography after 3H-noradrenaline (3H-NA) injection or uptake of 5-hydroxydopamine (5-OHDA) with immunocytochemistry using CRF antiserum. CRF-like immunoreactive cell bodies and fibers in the PVN received synaptic inputs from the axon terminals in which a selective accumulation of 3H-NA or 5-OHDA was found. This finding suggests that the secretion of CRF neurons may be regulated via synapses by catecholaminergic neurons.     

Tumor anorexia: effects on neuropeptide Y and monoamines in paraventricular nucleus

Paraventricular (PVN) concentrations of neuropeptide Y (NPY), serotonin (5-HT) and dopamine (DA) in anorectic tumor-bearing (TB) rats were measured before and after tumor resection. At onset of anorexia in TB versus non-tumor bearing (NTB) Controls 5-HT increased from 12.19+/-0.49 pg/microg to 14.89+/-0.81 pg/microg ( P<0.05 ) while DA and NPY decreased from 7.34+/-0.42 pg/microg to 4.97+/-0.56 pg/microg and 23.47+/-4.27 pg/microg to 13.64+/-1.44 pg/microg, respectively ( P<0.05 ). After tumor resection, these neuromediators normalized when compared to sham-operated NTB rats. NTB pair-fed Controls were also studied. We conclude that the increased 5-HT and the decreased DA and NPY concentrations in PVN are associated with cancer anorexia and that the NPY food stimulatory effect is linked to serotoninergic and dopaminergic systems in hypothalamus.     

Neuropeptide FF and neuropeptide VF inhibit GABAergic neurotransmission in parvocellular neurons of the rat hypothalamic paraventricular nucleus

Neuropeptide FF (NPFF) and neuropeptide VF (NPVF) are octapeptides belonging to the RFamide family of peptides that have been implicated in a wide variety of physiological functions in the brain, including central autonomic and neuroendocrine regulation. The effects of these peptides are mediated via NPFF1 and NPFF2 receptors that are abundantly expressed in the rat brain, including the hypothalamic paraventricular nucleus (PVN), an autonomic nucleus critical for the secretion of neurohormones and the regulation of sympathetic outflow. In this study, we examined, using whole cell patch-clamp recordings in the brain slice, the effects of NPFF and NPVF on inhibitory GABAergic synaptic input to parvocellular PVN neurons. Under voltage-clamp conditions, NPFF and NPVF reversibly and in a concentration-dependent manner reduced the evoked bicuculline-sensitive inhibitory postsynaptic currents (IPSCs) in parvocellular PVN neurons by 25 and 31%, respectively. RF9, a potent and selective NPFF receptor antagonist, blocked NPFF-induced reduction of IPSCs. Recordings of miniature IPSCs in these neurons following NPFF and NPVF applications showed a reduction in frequency but not amplitude, indicating a presynaptic locus of action for these peptides. Under current-clamp conditions, NPVF and NPFF caused depolarization (6-9 mV) of neurons that persisted in the presence of TTX but was abolished in the presence of bicuculline. Collectively, these data provide evidence for a disinhibitory role of NPFF and NPVF in the hypothalamic PVN via an attenuation of GABAergic inhibitory input to parvocellular neurons of this nucleus and explain the central autonomic effects of NPFF.     

Ultrastructural identification of noradrenergic nerve terminals and vasopressin-containing neurons of the paraventricular nucleus in the same thin section

Since many peptidergic cell groups receive a diverse and complex monoaminergic innervation, we have developed a double-label procedure to visualize a peptide and a catecholamine in the same ultrathin section. Radiolabeled norepinephrine (NE) is applied locally and its reuptake into NE terminals is demonstrated by ultrastructural radioautography. Controls in this and other studies demonstrate that the NE labels only NE (and possibly epinephrine) terminals and not dopaminergic or serotonergic terminals. In the same tissue, vasopressin is localized by immunocytochemistry on unembedded sections that are subsequently embedded in epoxy resins for thin sectioning. The procedure as described here shows that NE terminals in the periventricular zone of the paraventricular nucleus of the hypothalamus innervate both vasopressin-positive and vasopressin-negative structures. This technique is useful in determining the chemical connectivity of the hypothalamus.     

Developmental switch in neuropeptide Y and melanocortin effects in the paraventricular nucleus of the hypothalamus

Homeostatic regulation of energy balance in rodents changes dramatically during the first 3 postnatal weeks. Neuropeptide Y (NPY) and melanocortin neurons in the arcuate nucleus, a primary energy homeostatic center in adults, do not fully innervate the paraventricular nucleus (PVN) until the third postnatal week. We have identified two classes of PVN neurons responsive to these neuropeptides, tonically firing neurosecretory (NS) and burst-firing preautonomic (PA) cells. In neonates, NPY could inhibit GABAergic inputs to nearly all NS and PA neurons, while melanocortin regulation was minimal. However, there was a dramatic, age-dependent decrease in NPY responses specifically in the PA neurons, and a 3-fold increase in melanocortin responses in NS cells. These age-dependent changes were accompanied by changes in spontaneous GABAergic currents onto these neurons. This primarily NPYergic regulation in the neonates likely promotes the positive energy balance necessary for growth, while the developmental switch correlates with maturation of homeostatic regulation of energy balance.     

Differential effects of galanin and neuropeptide Y on extracellular norepinephrine levels in the paraventricular hypothalamic nucleus of the rat: a microdialysis study.

Evidence suggests that the peptides galanin (GAL) and neuropeptide Y (NPY) interact with the amine norepinephrine (NE) in the hypothalamic paraventricular nucleus (PVN) to stimulate feeding behavior. To directly investigate the nature of these interactions, extracellular levels of PVN NE were monitored in freely-moving rats using the microdialysis/HPLC technique. Following PVN administration of GAL (0.3 nmol), NPY (78 pmol) or Ringer's solution, local NE levels were measured at 20-min intervals for 2 hrs postinjection, under two feeding conditions, namely, in the presence or absence of food. The results demonstrate different effects of these peptides on endogenous NE levels. Following GAL administration, PVN NE levels were enhanced by 80 to 90%, up to 40 min postinjection, independent of food availability. In contrast, following NPY injection, NE levels were significantly reduced 20 min postinjection with food absent, and when food was available, NE levels tended to be enhanced. These results, consistent with pharmacological and biochemical studies, reveal different patterns of peptide-amine interactions in the PVN.     

Adrenergic innervation of corticotropin releasing factor (CRF)-synthesizing neurons in the hypothalamic paraventricular nucleus of the rat. A combined light and electron microscopic immunocytochemical study

Corticotropin releasing factor (CRF), a neuropeptide synthesized in the parvocellular subnuclei of the hypothalamic paraventricular nucleus (PVN), takes part in the regulation of different stress evoked responses of the organism. In order to elucidate the role of the central adrenergic system in the regulation of these CRF-synthesizing neurons, a novel ultrastructural immunocytochemical dual localization technique was utilized. Phenylethanolamine-N-methyltransferase (PNMT), a specific enzyme marker for the central adrenaline system, and CRF-immunoreactive elements were simultaneously visualized in hypothalamic sections. PNMT-immunoreactive axon terminals established synaptic connections with somata, dendrites and spinous structures of CRF-producing neurons. This morphological finding indicates that the central adrenergic system directly influences CRF-synthesizing neurons in the PVN and provides basis for a more definitive pharmacological manipulation of this system.     

Coerulospinal cells containing neuropeptide Y in the cat.

Spinally projecting neuropeptide Y (NPY)-immunoreactive cells were sought in the feline locus coeruleus (LC) nuclear complex after horseradish peroxidase (HRP) injection into the lumbar cord; HRP injection was followed by intracerebroventricular colchicine administration. Our results revealed that a significant number (approximately 20% of all descending cells from the LC complex) of spinally projecting NPY-immunoreactive neurons arise from the LC alpha, the subcoeruleus and the K?lliker-Fuse nuclei. Other nonspinally projecting NPY-containing cells were also evident in the laterodorsal tegmental nucleus and the LCd, in addition to those occurring in the aforementioned LC nuclear complex.     

HRP-labeled masticatory neurons in the rat trigeminal mesencephalic nucleus: a light and electron microscopic study

The neurons innervating the muscles of mastication were labeled retrogradely with horseradish peroxidase (HRP) which was injected into each muscle of mastication of the rats. The TMB-HRP labeled neurons were for light microscopic and DAB-HRP labeled neurons for electron microscopic study. Many HRP-labeled mesencephalic neurons were observed in the trigeminal mesencephalic nucleus (TMEN) after HRP injection in jaw-closing muscles (JCM). On the other hand, no labeled neurons were found following the application of HRP to the lateral pterygoid and the anterior belly of the digastric muscles, with the exception of a very few from the mylohyoid muscle. The latter three muscles were jaw-opening muscles (JOM). The mesencephalic neurons of each JCM in the TMEN were rather randomly distributed, although they were concentrated more in the caudal region of this nucleus. These neurons were typically unipolar, with spherical to oval perikarya. Each neuron had a single process which coursed caudolaterally to join the mesencephalic tract of the trigeminal nerve. Ultrastructurally, mesencephalic masticatory neurons had a rather regular nucleus locating either centrally or eccentrically in the perikaryon, which is rather plump. The cytoplasm was endowed with very well developed Golgi apparatus and rough endoplasmic reticulum. Neurofilaments, varying in number, intermingled mostly with the Golgi apparatus in the cytoplasm. Somatic spines were frequently observed; however, synapses abutting upon the soma were few. Macula adherens-like structures were occassionally encountered in the contact zone between two cells.     

Synaptic interaction of serotonergic axons and corticotropin releasing factor (CRF) synthesizing neurons in the hypothalamic paraventricular nucleus of the rat. A light and electron microscopic immunocytochemical study

The morphological interrelationship between the central serotonergic and hypothalamic corticotropin-releasing factor (CRF) synthesizing systems was studied in the hypothalamic paraventricular nucleus (PVN) of colchicine pretreated male rats. The simultaneous immunocytochemical localization of the transmitter and peptide employed the peroxidase-antiperoxidase complex (PAP) technique using the silver-gold intensified (SGI) and non-intensified forms of the oxidized 3,3'-diaminobenzidine (DAB) chromogen. The paraventricular nucleus received a moderate serotonergic innervation as compared with other diencephalic structures. The distribution and arborization of serotonergic axons were more prominent in the parvocellular subnuclei than in the magnocellular units of the nucleus. Serotonin containing axons formed terminal bouton and en passant type synapses with dendrites and somata of parvocellular neurons. The immunocytochemical double labelling technique revealed the overlapping of serotonergic axons and CRF-immunoreactive neurons. Vibratome (40 micron) and semithin (1 micron) sections indicated that the interneuronal communication may take place on both dendrites and cell bodies of CRF-immunoreactive neurons. Ultrastructural analysis demonstrated that serotonin-containing terminals formed axo-dendritic and axo-somatic synapses with CRF-immunoreactive neurons. These findings indicate that the central serotonergic neuronal system can influence the function of the pituitary-adrenal endocrine axis via a direct action upon the hypophysiotrophic CRF synthesizing neurons.     

Age-related change of neuropeptide Y-immunoreactive neurons in the rat anterior olfactory nucleus

Neuropeptide Y (NPY) is located in the olfactory system, including the olfactory bulb, and is thought to be one of the main neurotransmitters for olfaction. Thus, we examined age-related changes of NPY-immunoreactive (IR) neurons in the rat anterior olfactory nucleus (AON) at various aging stages over a period of 2 years; postnatal months 1 (PM 1), PM 6, PM 12 and PM 24. NPY-IR neurons in the AON were present in the lateral and medial subdivisions at PM 1 and at PM 6 were distributed in all subdivisions of the AON. Prior to PM 12, the NPY-IR neurons showed a tendency to change from bipolar cells with short processes into multipolar cells with long processes. Moreover, the population of NPY-IR neurons and nerve fibers in the AON increased in proportion to age. In particular, the number of NPY-IR neurons increased about 6-fold between PM 1 and PM 3. At PM 24, the number of NPY-IR neurons was much smaller than that at PM 12 and somal size had decreased. It is therefore suggested that the dramatic increase in the number and size of the NPY-IR neurons between PM 1 and PM 3 may be associated with sexual maturation and that the decrease in the number and cell size of the NPY-IR neurons at PM 24 may underlie age-related changes in the olfactory process.     

白藜芦醇抑制大鼠下丘脑脑片室旁核神经元放电

本文旨在研究白藜芦醇(resveratrol)对下丘脑脑片室旁核神经元放电的影响.应用玻璃微电极细胞外记录单位放电技术,在下丘脑脑片上观察白藜芦醇对静息状态下室旁核神经元放电的影响.结果如下:(1)在29张下丘脑脑片室旁核神经元放电单位给予白藜芦醇(O.05,0.5,5.0 μmol/L)2 min,有28张脑片(96.6%)放电频率显著降低,且呈剂量依赖性;(2)预先用0.2mmol/L的L.glutamate灌流8张下丘脑脑片,8张脑片(100%)放电频率显著增加,表现为癫痫样放电,该放电可被白藜芦醇(5.0 μmol/L)灌流2 min抑制:(3)预先用L型钙通道开放剂Bay K8644(0.1μmol/L)灌流8张下丘脑脑片,8张脑片(100%)放电频率显著增加,该放电可被白藜芦醇(5.0 μmol/L)灌流2 min抑制;(4)用一氧化氮合酶抑制剂Nω-nitro.L-arginine methyl ester(L-NAME)50μmol/L灌流8张下丘脑脑片,7张脑片(87.5%)放电频率显著增加,该放电可被白藜芦醇(5.0 μmol/L)灌流2 min抑制.以上结果提示,白藜芦醇抑制下丘脑室旁核神经元自发放电,可能通过降低心血管中枢的活动性而产生中枢保护作用.这种抑制作用可能与白藜芦醇抑制L型钙通道、减少钙内流有关,与NO释放无关.     

Catecholamine distribution and relationship to magnocellular neurons in the paraventricular nucleus of the rat

Summary The distribution of catecholamine synthesizing enzymes within the paraventricular nucleus of the rat hypothalamus is elucidated immunocytochemically by use of antibodies to tyrosine hydroxylase, dopamine -hydroxylase, and phenylethanolamine-N-methyltransferase. Tyrosine hydroxylase-immunostained cell bodies are localized in the periventricular stratum and adjacent parvocellular regions, but rarely in magnocellular subnuclei of the paraventricular nucleus. Tyrosine hydroxylase-immunostained fibers are present in greatest density in the periventricular zone, and moderate density in the parvocellular and magnocellular subnuclei. Dopamine -hydroxylase-immunostained fibers are remarkably dense in the posterior magnocellular division of the paraventricular nucleus, especially in the dorso-lateral portion where vasopressin-containing cells predominate. Noradrenergic fiber input to these magnocellular neurons is likely since phenylethanolamine-N-methyltransferase-immunostained fibers are sparse in magnocellular subnuclei of the paraventricular nucleus. Dual immunocytochemical staining of thick and thin tissue sections demonstrates with clarity an anatomical association of dopamine -hydroxylase-immunostained fibers and magnocellular neurons. Dopamine -hydroxylase-immunostained and phenylethanolamine-N-methyltransferase-immunostained fibers are dense in the medial parvocellular component of the paraventricular nucleus; distinct features of both antisera are presented.     

Relationship of CRF-immunostained cells and magnocellular neurons in the paraventricular nucleus of rat hypothalamus

The distribution of corticotropin-releasing factor (CRF), vasopressin (VP) and oxytocin (OXY) containing neurons within the magnocellular and parvocellular divisions in the paraventricular nucleus (PVN) of rat hypothalamus is described in brains from normal untreated, colchicine treated and adrenalectomized animals. Double immunostained preparations using glucose oxidase-antiglucose oxidase (GAG) complex combined with PAP complex to visualize two antigens with contrasting colors in the same tissue sections were employed. Separate and distinct populations of cells containing the immunoreactive (ir) elements were seen. Immunostained CRF neurons present in the ventral medial portion of the posterior magnocellular division were juxtaposed to oxytocin-ir perikarya in colchicine treated and adrenalectomized animals. CRF-ir cells were for the most part concentrated in the medial parvocellular component of PVN. An intimate anatomical proximity between CRF-ir and VP-ir perikarya was evident in this medial parvocellular division in brains of adrenalectomized animals; this area is normally VP-ir poor except in the adrenalectomized rats. This extension of VP-ir cells into this CRF rich region and the very close approximation between the two cell bodies suggests potential cell to cell communication following perturbation of the brain-pituitary-adrenal axis. No evidence for the co-existence of two peptidergic systems in the same neuron was apparent in the present study.     

Interrelation between alpha 2-adrenoreceptor system and neuropeptide Y in rat nucleus tractus solitarii.

In several systems, alpha 2-adrenoreceptor agonists and neuropeptide Y (NPY) potentiate one another. We reported recently that NPY is a potent depressor agent in the nucleus tractus solitarii (NTS). The purpose of this study is to investigate the possible modulation of the agonist effect by NPY in this site. Microinjection (60nl) of NPY, anti-NPY antiserum, the alpha 2 agonist alpha-methylnorepinephrine (alpha-MNE), clonidine, and the alpha 2 antagonists idazoxan and yohimbine were made into the NTS. Administration of idazoxan (0.2 nmol) prior to the injection of NPY (2.3 pmol) attenuated the potent depressor and bradycardic effect of NPY. There was a similar attenuation of yohimbine's effect. Similarly, prior administration of the anti-NPY antiserum attenuated the depressor effect of the central antihypertensive agents, alpha-MNE and clonidine, whereas inactivated antiserum or control normal rabbit serum were not able to attenuate these effects. Even a subdepressor dose of NPY (47 fmol) could potentiate the effect of alpha-MNE. These results demonstrate a reciprocal potentiation of NPY and alpha 2 agonists in the brainstem, and suggest that NPY and catecholamines interact in central cardiovascular regulation.     

Light- and electron microscopic localization of vasopressin or a vasopressin-like substance in the neurons of the rat suprachiasmatic nucleus

Summary In the suprachiasmatic nucleus of the rat light microscopic immunostaining for vasopressin reveals a distribution pattern of the immunoreactive material different from that known for the supraoptic nucleus. Among non-stained neurons positive-reacting perikarya display a cap- or tiplike labeling. The area of the suprachiasmatic nucleus is marked by delicate vasopressin-positive fibers. At the ultrastructural level the reaction product, after incubation with anti-vasopressin, is localized in small elementary granules unevenly distributed over the cytoplasm. Groups of axons containing specifically labeled granules contact non-reacting fibers.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr. 569/2) and Stiftung Volkswagenwerk     

Synaptic interactions between ghrelin- and neuropeptide Y-containing neurons in the rat arcuate nucleus

Morphological relationships between neuropeptide Y- (NPY) like and ghrelin-like immunoreactive neurons in the arcuate nucleus (ARC) were examined using light and electron microscopy techniques. At the light microscope level, both neuron types were found distributed in the ARC and could be observed making contact with each other. Using a preembedding double immunostaining technique, some NPY-immunoreactive axon terminals were observed at the electron microscope level to make synapses on ghrelin-immunoreactive cell bodies and dendrites. While the axo-somatic synapses were mostly symmetric in nature, the axo-dendritic synapses were both symmetric and asymmetric. In contrast, ghrelin-like immunoreactive (ghrelin-LI) axon terminals were found to make synapses on NPY-like immunoreactive (NPY-LI) dendrites although no NPY-like immunoreactive perikarya were identified receiving synapses from ghrelin-LI axon terminals. NPY-like axon terminals were also found making synapses on NPY-like neurons. Axo-axonic synapses were also identified between NPY- and ghrelin-like axon terminals. The present study shows that NPY- and ghrelin-LI neurons could influence each other by synaptic transmission through axo-somatic, axo-dendritic and even axo-axonic synapses, and suggests that they participate in a common effort to regulate the food-intake behavior through complex synaptic relationships.