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Two bioactive products of pituitary adenylate cyclase-activating polypeptide (PACAP) prohormone have been isolated from ovine hypothalamus: PACAP38 with 38 residues and PACAP27 corresponding to the N-terminal 27 residues of PACAP38. Immunocytochemical and RIA results support the existence of PACAP in the rat brain, posterior pituitary, and various peripheral tissues. Furthermore, high affinity PACAP-binding sites have been detected in the rat brain, anterior pituitary, and cultured astrocytes which differ from those in lung, liver, and cultured mouse splenocytes. In the present study additional rat tissues were examined to elucidate the location and characteristics of PACAP-binding sites using [125I] PACAP27 with conventional methods of receptor autoradiography and RRA. Binding specificity was established by displacement with unlabeled PACAP27 or a related peptide, vasoactive intestinal polypeptide (VIP). PACAP27-binding sites were localized autoradiographically in the testis, epididymis, adrenal gland, lung, liver, prostate gland, and seminal vesicle; binding sites were not detected in the heart, kidney, or thymus. In the testis and epididymis, a PACAP27-binding site was localized on germinal cells and in the adrenal gland on medullary chromaffin cells. Excess VIP did not displace PACAP27 binding localized in these three tissues. A site with a greater affinity for PACAP27 than for VIP was detected in adrenal gland and epididymis, characteristic of a site recognized previously in hypothalamus, anterior pituitary, and cultured astrocytes. The PACAP-specific site was more abundant in these tissues than a second site to which PACAP27 and VIP bound with similar affinities. Accordingly, the first site has been named type I. In lung, liver, prostate, and seminal vesicle, VIP displaced PACAP27 binding localized autoradiographically. Lung and liver contained an abundant site to which PACAP27 and VIP bound with similar affinities. This binding site, measured previously in lung, liver, and cultured splenocytes, may be shared by PACAP and VIP and has been named type II. Taken together, these data support the existence of two high affinity binding sites for PACAP with different tissue distribution.
Assuntos
Neuropeptídeos/metabolismo , Glândulas Suprarrenais/metabolismo , Animais , Autorradiografia/métodos , Sítios de Ligação , Genitália Masculina/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Masculino , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Ratos , Distribuição Tecidual , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
Recent evidence suggests that substances derived from the hypophyseal intermediate lobe (IL) play a crucial role in the regulation of suckling-induced PRL secretion. The purpose of the present study was to explore this possibility further by determining whether the suckling stimulus acutely increases the secretory activity of the IL and whether alpha MSH, a major secretory product of the IL, plays a specific role in suckling-induced PRL release. Light microscopic morphometric analysis of serial pituitary sections obtained from lactating rats revealed that as little as 1 min of suckling caused a significant increase in the proportion of the IL that was in secretory configuration (11.8 +/- 0.7% vs. 6.7 +/- 0.5%; 1-min suckled vs. nonsuckled control; mean +/- SE). Moreover, the fraction of the IL in secretory configuration continued to increase after 5 and 10 min of nursing (to 16.0 +/- 0.8% at 5 min and 18.2 +/- 0.7% at 10 min). In contrast, serum PRL was not significantly elevated above the control level after 1 min of suckling (18.1 +/- 13.5 vs. 9.9 +/- 6.5 ng/ml, 1-min suckled vs. control). In fact, a significant rise in PRL levels (to 314.4 +/- 19.4 ng/ml) could be detected only after 10 min of nursing. Thus, secretion by the IL in response to suckling preceded the release of adenohypophyseal PRL, suggesting that a secretory product(s) from the pars intermedia is involved in the modulation of nursing-induced PRL release. Having established a sequential temporal relationship between these two phenomena, we next investigated whether alpha MSH was the IL factor involved in the regulation of suckling-induced PRL secretion. To this end, lactating rats were injected either with antiserum to alpha MSH or preimmune serum and then allowed to nurse their pups. Serial blood samples were taken from the mothers 15, 30, 60, and 90 min after the litters were returned, and serum PRL was measured by RIA. We found that the suckling-induced rise in serum PRL was severely attenuated in animals that received anti-alpha MSH serum. This suppression was most evident at 15 min (70.1 +/- 13.4 vs. 323.5 +/- 127.0 ng/ml, antibody treated vs. preimmune serum control) and persisted throughout the entire 90-min test period. When taken together, our results suggest that suckling-induced PRL secretion is mediated at least in part by alpha MSH released from the hypophyseal IL.
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Lactação/metabolismo , Hipófise/metabolismo , Prolactina/metabolismo , alfa-MSH/fisiologia , Animais , Feminino , Soros Imunes/imunologia , Soros Imunes/farmacologia , Microscopia Eletrônica , Hipófise/ultraestrutura , Prolactina/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , alfa-MSH/imunologiaRESUMO
The presence and synthesis of vasoactive intestinal polypeptide (VIP) has been demonstrated in the rat anterior pituitary. It was recently confirmed that immunoreactive VIP is present in the anterior pituitary, and in a thyroid-deficient state, VIP could be detected by light microscopic immunohistochemistry. It has also been suggested that VIP plays a stimulatory role in PRL secretion. To gain more detailed information on the localization of VIP and the conditions that alter the synthesis of VIP, we examined VIP immunoreactivity using immunohistochemistry in pituitaries of normal male and cycling female rats and in those states in which PRL secretion was enhanced (pregnancy, lactation, long term estrogen treatment, and pituitary implanted under kidney capsule of normal or estrogen-treated female rats). In situ, implanted and cultured pituitary cells were stained for VIP immunoreactivity using the peroxidase-antiperoxidase method. VIP immunoreactivity was observed in about 45% of the male rats, in all estrogen-treated female rats, in the implanted pituitaries under the kidney capsule (three of five from estrogen-treated and one of five from intact females, respectively), and in the pituitary cell cultures derived from estrogen-treated rats. Using a double labeling procedure we have also observed PRL immunoreactivity in a small population of the VIP-positive cells. These results suggest a positive regulatory role of estrogen in expression of the VIP gene. The physiological and pathophysiological significance of VIP in PRL secretion, however, remains to be clarified.
Assuntos
Dietilestilbestrol/farmacologia , Adeno-Hipófise/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Células Cultivadas , Implantes de Medicamento , Feminino , Imuno-Histoquímica , Rim , Masculino , Hipófise/transplante , Adeno-Hipófise/citologia , Prolactina/metabolismo , Ratos , Ratos Endogâmicos , Valores de Referência , Coloração e RotulagemRESUMO
In order to examine the morphological substrates for neuronal connections between cells of the hypothalamic suprachiasmatic nucleus (SCN) that contain immunoreactivity for different neurotransmitters, a double ultrastructural immunocytochemical analysis was used. For double immunostaining, the first neuroactive substance antigen was labeled with gold-substituted silver-intensified peroxidase (GSSP), which results in a granular gold deposit of high electron and light opacity. The second antigen was labeled with peroxidase and a diaminobenzidine chromagen. The GSSP reaction product greatly increased the visibility of immunoreactive structures, with both light and electron microscopy. Intensification with the GSSP method worked at all depths of thick tissue sections as determined with analysis of immunostained sections cut perpendicular to their flat surface, and with analysis of thick 80-micron sections of brain tissue into which horseradish peroxidase (HRP) has been microinjected. On a nitrocellulose dot-blot comparison of different substrates for HRP, the GSSP intensification compared favorably with tetramethylbenzidine, but unlike tetramethylbenzidine, the GSSP was stable in a wide range of buffers. In addition to diaminobenzidine, the GSSP reaction was used to intensify and stabilize both the Hanker-Yates reagent and tetramethylbenzidine on the nitrocellulose model system. Through the use of the GSSP reaction, five new synaptic relationships in the suprachiasmatic nucleus were revealed. By increasing the sensitivity of the peroxidase method by silver-gold intensification, immunoreaction product could be found in dendrites at a greater distance from the perikaryon than in nonintensified material. Because of this greater sensitivity, the neuroactive substance contained in the cell of origin of a dendrite could sometimes be identified. Boutons immunoreactive for vasopressin-associated neurophysin were found to make synaptic contact with postsynaptic dendrites that also contained vasopressin-neurophysin immunoreactivity. Similarly, boutons containing gastrin-releasing peptide immunoreactivity made synaptic contact with cells also exhibiting gastrin-releasing peptide immunoreactivity. Neurons stained with GSSP reaction product could be easily discriminated from those containing only HRP-precipitated diaminobenzidine, allowing the simultaneous use of these two markers in the same 30-micron tissue section and subsequently in ultrathin sections for electron microscopy.(ABSTRACT TRUNCATED AT 400 WORDS)
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Glutamato Descarboxilase/metabolismo , Peptídeos/metabolismo , Núcleo Supraquiasmático/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Vasopressinas/metabolismo , Animais , Feminino , Peptídeo Liberador de Gastrina , Técnicas Imunoenzimáticas , Masculino , Microscopia Eletrônica , Neurotransmissores/metabolismo , Ratos , Núcleo Supraquiasmático/ultraestrutura , Sinapses/metabolismo , Sinapses/ultraestruturaRESUMO
The cellular and subcellular localization of the mGluR5 metabotropic glutamate receptor subtype was studied in the rat cerebellar cortex, by using the preembedding immunoperoxidase and immunogold techniques. Light microscopic observations revealed an abundant, intense labeling of neurons in the granular layer as well as in the molecular layer. Lugaro and Golgi cells exhibited an intense mGluR5 immunoreactivity, while only a fraction of the neurons in the molecular layer were found to be mGluR5 immunopositive. In addition to a dense plexus of immunoreactive dendrites in the molecular layer of the cerebellar cortex, the mGluR5 immunopositive Golgi cell dendrites resembling axons at the light microscopic level were also labeled in the granular layer. At the ultrastructural level, mGluR5 immunoreactivity was present in neuronal elements postsynaptic to axon terminals of different morphology. By using a pre-embedding immunogold method, it was found that mGluR5 immunoreactivity is accumulated at the plasma membranes extrasynaptically as well as at the periphery of the postsynaptic specializations, mainly of the parallel fiber synaptic contacts. These findings provide morphological evidence that mGluR5 is expressed by a population of neurons in the cerebellar cortex and can synaptically be activated via the parallel fiber system.
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Córtex Cerebelar/metabolismo , Neurônios/metabolismo , Ratos/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Western Blotting , Linhagem Celular , Córtex Cerebelar/citologia , Córtex Cerebelar/ultraestrutura , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Microscopia Eletrônica , Neurônios/ultraestruturaRESUMO
The topographical distribution of neuropeptide-containing cell bodies, fibers and terminals was studied in human parabrachial nuclei and the pontine tegmentum with immunohistochemical stainings. Brains of seven adult human subjects of 35-72 years were fixed within 2 h post mortem. Serial sections were immunostained by antisera of 14 different neuropeptides--oxytocin, vasopressin, thyrotropin-releasing hormone, angiotensin II, calcitonin gene-related peptide, beta-endorphin, dynorphin A, dynorphin B, leucine-enkephalin, alpha-melanocyte stimulating hormone, substance P, neuropeptide Y, cholecystokinin and galanin--alternately. All of these peptides were found to be present in nerve fibers and terminals, but only two, angiotensin II and dynorphin B, in cell bodies of the parabrachial nuclei. Calcitonin gene-related peptide-, neuropeptide Y-, cholecystokinin- and galanin-immunoreactive cells were present in other areas of the pontine tegmentum, like the motor trigeminal nucleus, locus coeruleus, periventricular gray matter but not in the parabrachial nuclei. Peptidergic fibers were distributed unevenly throughout the pontine tegmentum having unique, individual distribution patterns. In the parabrachial nuclei, substance P, neuropeptide Y, cholecystokinin and galanin showed the highest density of immunoreactive neuronal networks. Moderate to low concentrations of immunoreactive processes were detected by calcitonin gene-related peptide, alpha-melanocyte stimulating hormone, dynorphin B, thyrotropin releasing hormone, leucine-enkephalin, dynorphin A, angiotensin II, beta-endorphin, vasopressin and oxytocin antisera, respectively. Other pontine tegmental areas, like the locus coeruleus, dorsal tegmental, pontine raphe and motor trigeminal nuclei as well as the central gray of the tegmental region exhibited a varying assortment of neuropeptides with distinct, individual localization patterns. Their detailed topographical distributions are mapped and given in coronal sections.
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Locus Cerúleo/citologia , Neuropeptídeos/análise , Ponte/citologia , Adulto , Idoso , Feminino , Humanos , Imuno-Histoquímica , Locus Cerúleo/anatomia & histologia , Masculino , Pessoa de Meia-Idade , Terminações Nervosas/ultraestrutura , Fibras Nervosas/ultraestrutura , Especificidade de Órgãos , Ponte/anatomia & histologiaRESUMO
The postsynaptic targets of three vasoactive intestinal polypeptide-containing GABAergic interneuron types were examined in the rat hippocampus. Two of them showed remarkable target selectivity for other GABAergic neurons, while the third contacted the somata and proximal dendrites of pyramidal cells. Vasoactive intestinal polypeptide-positive interneurons innervating the stratum oriens/alveus border in the CA1 region were shown to establish multiple contacts with horizontal GABAergic interneurons immunoreactive for type 1 metabotropic glutamate receptor. Similarly, identified axons of vasoactive intestinal polypeptide-positive interneurons projecting to stratum radiatum were found to establish symmetrical synapses largely on GABAergic dendrites. The majority of these postsynaptic GABAergic neurons were shown to contain calbindin or vasoactive intestinal polypeptide. In contrast to the first two vasoactive intestinal polypeptide-containing cell populations, vasoactive intestinal polypeptide-positive interneurons arborizing in stratum pyramidale formed baskets around pyramidal cells. These results revealed a new element in cortical microcircuits, interneurons which are specialized to innervate other GABAergic interneurons. The role of this new component may be the synchronization of dendritic inhibition, or an input-specific disinhibition of pyramidal cells in various dendritic domains. In contrast, vasoactive intestinal polypeptide-containing basket cells are likely to be involved in perisomatic inhibition of pyramidal neurons, and represents a new basket cell type different from that containing parvalbumin.
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Hipocampo/citologia , Hipocampo/fisiologia , Interneurônios/citologia , Interneurônios/fisiologia , Células Piramidais/fisiologia , Peptídeo Intestinal Vasoativo/análise , Animais , Corpo Estriado/fisiologia , Dendritos/fisiologia , Dendritos/ultraestrutura , Imuno-Histoquímica , Interneurônios/ultraestrutura , Masculino , Microscopia Imunoeletrônica , Células Piramidais/citologia , Ratos , Ratos Wistar , Sinapses/fisiologia , Sinapses/ultraestrutura , Ácido gama-Aminobutírico/análiseRESUMO
The termination pattern of septohippocampal axons visualized by anterograde transport of Phaseolus vulgaris leucoagglutinin was studied in the hippocampal formation in the rat, with special reference to the innervation of neurons immunoreactive for the neuroactive peptides cholecystokinin, somatostatin or vasoactive intestinal polypeptide. The type I, GABAergic, septohippocampal afferents were shown to terminate on neurons immunoreactive for each of the three peptides. The cholecystokinin-like immunoreactive neurons in all regions, and the somatostatin-immunoreactive cells in stratum oriens of CA1 region were the most preferred targets. Cholecystokinin-immunoreactive cells, especially those in the granule cell layer of the dentate gyrus, were often seen to be contacted by type II (presumed cholinergic) axons as well. The somatostatin-immunoreactive cells in the hilus were also innervated by type I septohippocampal axons, although less frequently than those in stratum oriens of the CA1 subfield. Each type of peptidergic neuron received multiple symmetrical synaptic input from the Phaseolus vulgaris leucoagglutinin-labelled septal afferents, as confirmed by correlated electron microscopy. The majority of these neuropeptide-containing cells are known to be GABAergic, and to have distinct input and output relationships. Thus, the present results demonstrate that the GABAergic septohippocampal pathway can control a wide range of putative inhibitory circuits, and thereby influence the pattern of electrical activity in the hippocampal formation.
Assuntos
Hipocampo/fisiologia , Neurônios Aferentes/fisiologia , Neurônios/fisiologia , Peptídeos/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Axônios/efeitos dos fármacos , Colecistocinina/fisiologia , Ensaio de Imunoadsorção Enzimática , Hipocampo/citologia , Imuno-Histoquímica , Masculino , Fito-Hemaglutininas , Ratos , Ratos Endogâmicos , Somatostatina/fisiologia , Peptídeo Intestinal Vasoativo/fisiologiaRESUMO
A simple and reliable method is described for the intensification of the end-product of the diaminobenzidine reaction demonstrating peroxidase activity. After completing the diaminobenzidine reaction, the preparations to be intensified are immersed first in thioglycolic acid solution, then in distilled water, and finally in a special physical developer containing silver nitrate.
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3,3'-Diaminobenzidina , Benzidinas , Peroxidases/análise , 3,3'-Diaminobenzidina/metabolismo , Histocitoquímica , Humanos , Polímeros/metabolismoRESUMO
Serotonin immunoreactive structures of the rat central nervous system (CNS) were detected by the recently developed silver-intensified peroxidase-antiperoxidase complex (SI-PAP) method at both the light and electron microscopic levels. The silver postintensification of the diaminobenzidine (DAB) chromogen increased the sensitivity of the original PAP method, resulting in a very Golgi-like appearance of serotonin-immunopositive neuronal elements. The metallic silver and gold deposited onto DAB-labeled organelles, filling out the whole immunoreactive neuron, assures the easy tracing of thin neuronal processes far from the cell body. At the ultrastructural level, metallic grains were seen over immunolabeled structures only, proving the specificity of the silver method. In neurons of the dorsal raphe nucleus, free ribosomes, endoplasmic reticulum, and granules (80-100 nm in diameter) were labeled. Immunoreactive, e.g., serotoninergic, dendrites were seen to receive afferent terminals. The increased electron density of the intensified immunolabel facilitates the ultrastructural recognition of even weakly labeled profiles, while its metallic components (Ag and Au) provide a base for X-ray analysis of the immunolabeled biological specimen.
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3,3'-Diaminobenzidina , Benzidinas , Neurônios/análise , Núcleos da Rafe/análise , Serotonina/análise , Animais , Compostos Cromogênicos , Microanálise por Sonda Eletrônica , Ouro , Histocitoquímica , Técnicas Imunoenzimáticas , Masculino , Neurônios/ultraestrutura , Ratos , Ratos Endogâmicos , PrataRESUMO
An improved gold-substituted silver intensification procedure for the peroxidase-diaminobenzidine (DAB) reaction product was developed. The method was applied in the rat medial preoptic area to label tyrosine hydroxylase (TH)-immunoreactive profiles. Following the gold toning, the same sections were immunostained for glutamic acid decarboxylase (GAD) immunoreactivity with non-intensified peroxidase-DAB. Single DAB-labeled GAD axons were found in symmetric synaptic connection with unlabeled dendrites as well as with gold-toned immunoperoxidase-containing TH neurons.
Assuntos
3,3'-Diaminobenzidina , Benzidinas , Glutamato Descarboxilase/análise , Ouro , Área Pré-Óptica/enzimologia , Prata , Tirosina 3-Mono-Oxigenase/análise , Animais , Feminino , Glutamato Descarboxilase/imunologia , Técnicas Imunoenzimáticas , Microscopia Eletrônica , Prolactina/metabolismo , Ratos , Ratos Endogâmicos , Tirosina 3-Mono-Oxigenase/imunologiaRESUMO
The distribution of twelve biologically active neuropeptides, i.e., thyrotropin-releasing hormone, corticotropin-releasing factor, pro-opiomelanocortin-derived peptides (adrenocorticotropic hormone, beta-endorphin, alpha-melanocyte-stimulating hormone), leucine-enkephalin, dynorphin A, dynorphin B, cholecystokinin, substance P, galanin and calcitonin gene-related peptide, was examined by immunohistochemistry in the human dorsal vagal complex including the nucleus of the solitary tract, the dorsal motor nucleus of the vagus and the area postrema. Immunoreactivity of all the twelve neuropeptides was found widely distributed in the various subdivisions of the nucleus of the solitary tract, showing a unique distribution for every peptide. Neuronal cell bodies immunostained with leucine-enkephalin, galanin and dynorphin B were found in this region. There were no immunopositive perikarya for any of the peptides in the other structures studied. Fibers containing galanin, corticotropin-releasing factor, substance P, dynorphin B, thyrotropin-releasing hormone and calcitonin gene-related peptide were observed at a relatively high density in the nucleus of the solitary tract. In the same structure, a moderately dense network of fibers immunostained with dynorphin A, cholecystokinin and leucine-enkephalin, but only solitary pro-opiomelanocortin-derived peptides-containing fiber fragments were observed. In the dorsal motor nucleus of the vagus the most prominent network of fibers was found to contain thyrotropin-releasing hormone, galanin and substance P. In contrast to these, no beta-endorphin immunoreactivity was detected. The area postrema contained only moderate to low densities of galanin-, substance P-, calcitonin gene-related peptide-, dynorphin B- and cholecystokinin-immunoreactive fibers.
Assuntos
Rede Nervosa/química , Neuropeptídeos/análise , Nervo Vago/química , Adulto , Idoso , Mapeamento Encefálico , Ventrículos Cerebrais/química , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Terminações Nervosas/química , Fibras Nervosas/química , Perfusão , Núcleo Solitário/químicaRESUMO
A variety of histochemical findings have contributed to a more differentiated architectonical description of the bed nucleus of the stria terminalis (BNST) in the mammalian brain. However, in the human brain investigations of the chemoarchitecture of this nucleus have been rare. Therefore we chose this region in six human autopsy brains in order to map the distribution patterns of 13 immunohistochemical markers for neurotensin (NT), neuropeptide Y (NPY), somatostatin (SOM), enkephalins (ENK), vasoactive intestinal polypeptide (VIP), substance P (SP), neurophysins (NPH), glial fibrillary acid protein, 3-fucosyl-N-acetyl-lactosamine epitope, myelin basic protein (MBP), calbindin (CAB), synaptophysin (SYN) and chromogranin-A (CHR-A). Three chemoarchitectonically distinct areas could be defined. The lateral subdivision of the BNST contained high amounts of NPY and SP-fibre immunoreactivity and was further characterized by the occurrence of neurons labelled for NPY. The central subdivision of the BNST appeared as a histochemically clearly circumscribed compartment with massive fibre immunoreactivity for SOM, ENK, VIP, SYN, CHR-A, CAB as well as SOM, ENK, NT and CAB positive cells but lacked cytosolic or fibre-like immunolabel for NPY and SP. This structure was also ensheathed by myelinated fibres identified by means of MBP immunohistochemistry. The medial subdivision of the BNST showed moderate to high SP and NPY fibre immunoreactivity but lacked immunolabelled neurons and was only scarcely supplied with varicose or punctiform ENK immunoproduct. In the most posterior levels of our sections a cell group labelled for NPH was located lateral to the fornix columns. The lateral subdivision of the BNST (with NPY, SYN) and mainly the central BNST (with SOM, ENK, VIP, SYN and CHR-A) contributed to ventrolateral extensions of dense patchy fibre immunoreactivity throughout the basal forebrain region.
Assuntos
Biomarcadores , Neuropeptídeos/análise , Tálamo/química , Adulto , Calbindinas , Cromogranina A , Cromograninas/análise , Feminino , Proteína Glial Fibrilar Ácida/análise , Humanos , Técnicas Imunoenzimáticas , Antígenos CD15/análise , Masculino , Pessoa de Meia-Idade , Proteína Básica da Mielina/análise , Proteína G de Ligação ao Cálcio S100/análise , Sinaptofisina/análise , Tálamo/anatomia & histologiaRESUMO
The presence of PACAP was revealed in the anterior pituitary with RIA, HPLC, and with the demonstration of its mRNA. The level of PACAP mRNA in the anterior pituitary is the highest during the proestrous LH surge. In our immunohistochemical studies we were able to demonstrate PACAP immunoreactive cells in the anterior pituitary. The shape and the distribution of PACAP immunoreactive cells were very similar to that of the gonadotropes; however, the number of PACAP cells was less than that of LH cells. Additionally, another PACAP-positive cell population with small diameter appeared in the proestrous stage, during pregnancy and lactation. Double labeling revealed that the major part of large PACAP cells exhibited LH immunoreactivity and those with a small diameter contained PRL. It is not clear whether the pituitary- or the hypothalamic-born PACAP, or both, influence pituitary LH and PRL secretion. I.c.v. administration of PACAP just prior to the critical period in the proestrous stage inhibited the expected ovulation and blocked the proestrus LH and PRL surge, although i.v. administration of PACAP had no effect. PACAP antiserum did not interfere with ovulation when i.c.v. or i.v. injection was used. Our results support the view that PACAP has a role in the control of LH and PRL secretion during the estrous cycle, pregnancy, and lactation. The inhibitory effect of PACAP on ovulation is mediated through the hypothalamus.
Assuntos
Ventrículos Cerebrais/fisiologia , Hormônio Luteinizante/metabolismo , Neuropeptídeos/farmacologia , Ovulação/efeitos dos fármacos , Adeno-Hipófise/fisiologia , Prolactina/metabolismo , Animais , Ventrículos Cerebrais/efeitos dos fármacos , Estro , Feminino , Soros Imunes , Injeções Intravenosas , Injeções Intraventriculares , Lactação , Hormônio Luteinizante/sangue , Masculino , Fibras Nervosas/ultraestrutura , Neuropeptídeos/administração & dosagem , Neuropeptídeos/fisiologia , Neurotransmissores/farmacologia , Neurotransmissores/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Adeno-Hipófise/citologia , Gravidez , Prolactina/sangue , Ratos , Transcrição GênicaRESUMO
The excitatory amino acid glutamate, acting via ionotropic and metabotropic glutamate receptors, appears to play an important role in the control of neuroendocrine functions. The aim of the present investigations was to determine whether hypothalamic neurones which synthesize arginin-vasopressin (AVP), CRF and VIP express metabotropic glutamate receptor (mGluR). Double-label immunocytochemistry and the mirror technique were used. We found that AVP immunoreactive neurones of the paraventricular, supraoptic and suprachiasmatic nuclei contain mGluR1a, but the number of double-labelled neurones was different in the three cell groups. mGluR1a was present in a significant number of paraventricular CRF nerve cells, and in almost all VIP neurones of the SCHN. These results support the view that the excitatory transmitter glutamate may directly influence AVP, CRF and VIP neurones of the three hypothalamic cell groups.
Assuntos
Arginina Vasopressina/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Hipotálamo/metabolismo , Neurônios/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Hipotálamo/citologia , Imuno-Histoquímica , Masculino , Núcleo Hipotalâmico Paraventricular/metabolismo , Ratos , Ratos Sprague-Dawley , Núcleo Supraquiasmático/metabolismo , Núcleo Supraóptico/metabolismoRESUMO
The immunocytochemical localization of the recently cloned metabotropic glutamate receptor 1 alpha (mGluR1 alpha) was demonstrated with a C-terminus specific antibody in rat cerebellar cortex. This antibody detects a 138-140 kDa major, and a 46 kDa minor band in membrane preparations of rat cortex and cerebellum. mGluR1 alpha immunoreactivity (mGRi) was present in Purkinje and basket cells. Purkinje cell dendritic spines and their postsynaptic membranes showed selective labelling. Presynaptic membranes, parallel fibres and glial processes were devoid of mGRi. It is suggested that the selective postsynaptic localization of this receptor at the dendritic spines of Purkinje cells serves as the morphological basis for long term depression processes in the molecular layer of the cerebellar cortex.
Assuntos
Córtex Cerebelar/metabolismo , Receptores de Glutamato/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Córtex Cerebelar/anatomia & histologia , Córtex Cerebelar/imunologia , Dendritos/imunologia , Dendritos/metabolismo , Epitopos , Hemocianinas/imunologia , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Dados de Sequência Molecular , Células de Purkinje/imunologia , Células de Purkinje/metabolismo , Ratos , Ratos Wistar , Receptores de Glutamato/imunologia , Membranas Sinápticas/imunologia , Membranas Sinápticas/metabolismo , Sinaptossomos/imunologia , Sinaptossomos/metabolismoRESUMO
Growth hormone-releasing hormone (GHRH) and beta-endorphin are mainly synthesized in neurones of the hypothalamic arcuate nucleus. Arcuate neurones also contain both ionotropic and metabotropic glutamate receptors. The aim of present study was to investigate whether glutamate receptors are present in GHRH and beta-endorphin containing nerve cells of this hypothalamic area. Using double-label immunocytochemistry as well as the mirror technique, we found that almost all GHRH and beta-endorphin immunoreactive arcuate neurones contain the metabotropic glutamate receptor la. The observations provide morphological evidence for the view that glutamate, which appears to be a major excitatory neurotransmitter in the hypothalamus, may directly stimulate GHRH and beta-endorphin neurones of the medial hypothalamus.
Assuntos
Núcleo Arqueado do Hipotálamo/citologia , Hormônio Liberador de Hormônio do Crescimento/análise , Neurônios/citologia , Receptores de Glutamato Metabotrópico/análise , beta-Endorfina/análise , Animais , Anticorpos Monoclonais , Núcleo Arqueado do Hipotálamo/fisiologia , Hipotálamo/fisiologia , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The cellular and subcellular distribution of the mGluR5a metabotropic glutamate receptor was studied in the spinal cord of the rat using an antibody raised against a mGluR5a-specific carboxy-terminal peptide. Strong mGluR5a-immunoreactivity (mGluR5a-ir) was found in the laminae I-II of the dorsal horn, which gradually decreased towards the deeper layers. At the electron microscopical level, mGluR5a-ir was present exclusively in neuronal somata and dendrites. Immunometal labelling revealed that mGluR5a-ir is concentrated at the periphery of postsynaptic densities of asymmetrical synapses or localized extrasynaptically at dendritic and somatic membranes. The mGluR5a-immunoreactive dendritic profiles were often targeted by synaptic boutons with the morphological characteristics of C-fibre terminals. These observations provide evidence for mGluR5a being involved in the nociceptive transmission at the dorsal horn.
Assuntos
Neurônios/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Medula Espinal/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Dados de Sequência Molecular , Neurônios/ultraestrutura , Ratos , Ratos Wistar , Receptores de Glutamato Metabotrópico/genética , Medula Espinal/citologia , Medula Espinal/ultraestrutura , Distribuição TecidualRESUMO
Borna disease virus (BDV)-induced meningoencephalitis is associated with the dysfunction of the cholinergic system. Temporal development of this cholinergic decline during pre-encephalitic and encephalitic stages of BDV infection remains however elusive. Changes in choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were therefore determined in the cerebral cortex, hippocampus, striatum, amygdala and cholinergic basal forebrain nuclei (ChBFN) of rats infected with BDV. Immunocytochemistry for ChAT and vesicular acetylcholine transporter (VAChT) was employed to identify morphological consequences of BDV infection on cholinergic neurons. Whereas both ChAT and AChE activities changed only slightly under pre-encephalitic conditions, the encephalitic stage was characterized by a significant decrease of ChAT activity in the cerebral cortex, horizontal diagonal band of Broca (hDBB), hippocampus and amygdala concomitant with a marked reduction of AChE activity in the cerebral cortex, hDBB and hippocampus. The striatum and medial septum remained unaffected. ChAT and VAChT immunocytochemistry revealed prominent axonal degeneration in affected cortical and limbic projection areas of ChBFN. In summary, our data indicate progressive deterioration of forebrain cholinergic systems that parallels the progression of BDV encephalitis.