RESUMO
In this brief review we summarize the current fndings relative to the discovery of a small peptide ligand, phoenixin (PNX). Using a bioinformatic approach, two novel peptides PNX-14 and PNX-20 containing 14 and 20 amino acids, respectively, were isolated from diverse tissues including the brain, heart, lung and stomach. Mass spectrometry analysis identified a major and minor peak corresponding to PNX-14 and PNX-20, in rat or mouse spinal cord extracts. With the use of a rabbit polyclonal antiserum, phoenixin immunoreactivity (irPNX) was detected in discrete areas of the rodent brain including several hypothalamic subnuclei and dorsal motor nucleus of the vagus. In addition, irPNX was detected in a population of sensory ganglion cells including dorsal root ganglion, nodose ganglion and trigeminal ganglion, and in cell processes densely distributed to the superficial layers of the dorsal horn, nucleus of the solitary tract and spinal trigeminal tract. irPNX cell processes were also detected in the skin and myenteric plexus, suggesting a brain-gut and/or brain-skin connection. Pharmacological studies show that PNX-14 injected subcutaneously to the nape of the neck of mice provoked dose-dependent repetitive scratching bouts directed to the back of the neck with the hindpaws. Our result suggests that the peptide PNX-14 and/or PNX-20, may serve as one of the endogenous signal molecules transducing itch sensation. Additionally, results from other laboratories show that exogenous PNX may affect a number of diverse behaviors such as memory formation, depression, reproduction, food-intake and anxiolytic-like behaviors.
Assuntos
Hormônios Hipotalâmicos/fisiologia , Hormônios Peptídicos/fisiologia , Peptídeos/fisiologia , Sequência de Aminoácidos , Animais , Humanos , Hormônios Hipotalâmicos/administração & dosagem , Hormônios Hipotalâmicos/química , Hipotálamo/metabolismo , Memória/fisiologia , Plexo Mientérico/metabolismo , Hormônios Peptídicos/administração & dosagem , Hormônios Peptídicos/química , Peptídeos/administração & dosagem , Peptídeos/química , Prurido/metabolismo , Medula Espinal/metabolismoRESUMO
This study tested the hypothesis that repetitive scratching provoked by two known pruritogens, compound 48/80 and 5'-guanidinonaltrindole (GNTI), is accompanied by activation of microglial cells in the mouse spinal cord. Immunohistochemical studies revealed that the complement receptor 3, also known as cluster determinant 11b (CD11b), a cell surface marker of microglial cells, was upregulated in the spinal cord 10-30 min after a subcutaneous (s.c.) injection of compound 48/80 (50 µg/100 µl) or GNTI (0.3 mg/kg) to the back of the mouse neck. Numerous intensely labeled CD11b-immunoreactive (CD11b-ir) cells, with the appearance of hypertrophic reactive microglia, were distributed throughout the gray and white matter. In contrast, weakly labeled CD11b-ir cells were distributed in the spinal cord from mice injected with saline. Western blots showed that CD11b expression levels were significantly increased in spinal cords of mice injected s.c. with either pruritogen, reached a peak response in about 30 min, and declined to about the basal level in the ensuing 60 min. In addition, phospho-p38 (p-p38) but not p38 levels were upregulated in spinal cords from mice injected with compound 48/80 or GNTI, with a time course parallel to that of CD11b expression. Pretreatment of the mice with nalfurafine (20 µg/kg; s.c.), a κ-opioid receptor agonist that has been shown to suppress scratching, reduced CD11b and p-p38 expression induced by either pruritogen. The results demonstrate, for the first time, that scratch behavior induced by the pruritogens GNTI and compound 48/80 is accompanied by a parallel activation of microglial cells in the spinal cord.
Assuntos
Comportamento Animal/fisiologia , Antígeno CD11b/metabolismo , Microglia/metabolismo , Prurido/metabolismo , Medula Espinal/metabolismo , Animais , Guanidinas , Masculino , Camundongos , Morfinanos , Fosforilação , Prurido/induzido quimicamente , Regulação para Cima , p-Metoxi-N-metilfenetilamina , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The G protein-coupled receptor GPR30 has recently been identified as a nonnuclear estrogen receptor. Reverse transcriptase-polymerase chain reaction revealed expression of GPR30 mRNA in varying quantities in the rat spinal cord, dorsal root ganglia, nodose ganglia, trigeminal ganglia, hippocampus, brain stem, and hypothalamus. Immunohistochemical studies that used a rabbit polyclonal antiserum against the human GPR30 C-terminus revealed a fine network of GPR30-immunoreactive (irGPR30) cell processes in the superficial layers of the spinal cord; some of which extended into deeper laminae. A population of neurons in the dorsal horn and ventral horn were irGPR30. Dorsal root, nodose, and trigeminal ganglionic neurons displayed varying intensities of irGPR30. Positively labeled neurons were detected in the major pelvic ganglion, but not in the superior cervical ganglion. A population of chromaffin cells in the adrenal medulla was irGPR30, so were cells of the zona glomerulosa. Double-labeling the adrenal medulla with GPR30 antiserum and tyrosine hydroxylase antibody or phenylethanolamine-N-methyltransferase antiserum revealed that irGPR30 is expressed in the majority of tyrosine hydroxylase-positive chromaffin cells. Last, some of the myenteric ganglion cells were irGPR30. Tissues processed with preimmune serum resulted in no staining. Voltage-sensitive dye imaging studies showed that the selective GPR30 agonist G-1 (1, 10, and 100 nM) depolarized cultured spinal neurons in a concentration-dependent manner. Collectively, our result provides the first evidence that GPR30 is expressed in neurons of the dorsal and ventral horn as well as in sensory and autonomic neurons, and activation of GPR30 by the selective agonist G-1 depolarizes cultured spinal neurons.
Assuntos
Gânglios Autônomos/metabolismo , Gânglios Sensitivos/metabolismo , Neurônios/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Medula Espinal/metabolismo , Animais , Tronco Encefálico/metabolismo , Células Cultivadas , Células Cromafins/metabolismo , Ciclopentanos/farmacologia , Relação Dose-Resposta a Droga , Feminino , Expressão Gênica , Hipocampo/metabolismo , Hipotálamo/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Feniletanolamina N-Metiltransferase/metabolismo , Quinolinas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Nesfatin-1 is a recently identified satiety molecule detectable in neurons of the hypothalamus and nucleus of solitary tract (NTS). Immunohistochemical studies revealed nesfatin-1-immunoreactive (irNEF) cells in the Edinger-Westphal nucleus, dorsal motor nucleus of vagus, and caudal raphe nuclei of the rats, in addition to the hypothalamus and NTS reported in the initial study. Double-labeling immunohistochemistry showed that irNEF cells were vasopressin or oxytocin positive in the paraventricular and supraoptic nucleus; cocaine-amphetamine-regulated transcript or tyrosine hydroxylase positive in arcuate nucleus; cocaine-amphetamine-regulated transcript or melanin concentrating hormone positive in the lateral hypothalamus. In the brainstem, irNEF neurons were choline acetyltransferase positive in the Edinger-Westphal nucleus and dorsal motor nucleus of vagus; tyrosine hydroxylase positive in the NTS; and 5-hydroxytryptamine positive in the caudal raphe nucleus. The biological activity of rat nesfatin-1 (1-82) (100 nm) was assessed by the Ca(2+) microfluorometric method. Nesfatin-1 elevated intracellular Ca(2+) concentrations [Ca(2+)](i) in dissociated and cultured hypothalamic neurons. The response was prevented by pretreating the cells with pertussis toxin (100 nm) or Ca(2+)-free solution and by a combination of the L-type and P/Q-type calcium channel blocker verapamil (1 microm) and omega-conotoxin MVIIC (100 nm). The protein kinase A inhibitor KT 5720 (1 microm) suppressed nesfatin-1-induced rise in [Ca(2+)](i). The result shows that irNEF is distributed to several discrete nuclei in the brainstem, in addition to the hypothalamus and NTS reported earlier, and that the peptide interacts with a G protein-coupled receptor, leading to an increase of [Ca(2+)](i), which is linked to protein kinase A activation in cultured rat hypothalamic neurons.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA , Ativação Enzimática , Feminino , Hipotálamo/citologia , Hipotálamo/metabolismo , Imuno-Histoquímica , Membranas Intracelulares/metabolismo , Masculino , Bulbo/metabolismo , Neurônios/metabolismo , Nucleobindinas , Concentração Osmolar , Ponte/metabolismo , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
The G protein-coupled receptor 30 (GPR 30) has been identified as the non-genomic estrogen receptor, and G-1, the specific ligand for GPR30. With the use of a polyclonal antiserum directed against the human C-terminus of GPR30, immunohistochemical studies revealed GPR30-immunoreactivity (irGPR30) in the brain of adult male and non-pregnant female rats. A high density of irGPR30 was noted in the Islands of Calleja and striatum. In the hypothalamus, irGPR30 was detected in the paraventricular nucleus and supraoptic nucleus. The anterior and posterior pituitary contained numerous irGPR30 cells and terminal-like endings. Cells in the hippocampal formation as well as the substantia nigra were irGPR30. In the brainstem, irGPR30 cells were noted in the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus; a cluster of cells were prominently labeled in the nucleus ambiguus. Tissue sections processed with pre-immune serum showed no irGPR30, affirming the specificity of the antiserum. G-1 (100 nM) caused a large increase of intracellular calcium concentrations [Ca(2+) ](i) in dissociated and cultured rat hypothalamic neurons, as assessed by microfluorometric Fura-2 imaging. The calcium response to a second application of G-1 showed a marked homologous desensitization. Our result shows a high expression of irGPR30 in the hypothalamic-pituitary axis, hippocampal formation, and brainstem autonomic nuclei; and the activation of GPR30 by G-1 is associated with a mobilization of calcium in dissociated and cultured rat hypothalamic neurons.
Assuntos
Sistema Nervoso Central/química , Receptores Acoplados a Proteínas G/análise , Animais , Cálcio/análise , Cálcio/metabolismo , Células Cultivadas , Sistema Nervoso Central/metabolismo , Ciclopentanos/farmacologia , Citosol/química , Feminino , Hipocampo/química , Hipocampo/metabolismo , Hipotálamo/química , Hipotálamo/metabolismo , Soros Imunes/farmacologia , Imuno-Histoquímica , Ligantes , Masculino , Bulbo/química , Bulbo/metabolismo , Mesencéfalo/química , Mesencéfalo/metabolismo , Hipófise/química , Hipófise/metabolismo , Prosencéfalo/química , Prosencéfalo/metabolismo , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/imunologia , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Cocaine- and amphetamine-regulated transcript (CART) peptide consists of a family of peptides. Expression of the peptide fragment CART(1-39) was explored in the rat using an antiserum directed against CART(1-39) of the short form of the human CART prohormone. CART(1-39)-immunoreactivity, herein referred to as irCART, was detected in the rat central and peripheral nervous tissues with a pattern similar to that labeled with the antiserum CART(55-102) or CART(79-102). For example, irCART cells were detected in the hypothalamus, pons, medulla oblongata, spinal cord, and adrenal medulla. In urethane-anesthetized rats, CART(1-39) (0.05 to 2 nmol) by intrathecal injection did not cause a significant change of blood pressure or heart rate, but potentiated the pressor effects of glutamate injected intrathecally. Lastly, the effect of CART(1-39) on intracellular calcium concentrations [Ca2+]i was assessed and compared to that caused by CART(55-102) in cultured rat cortical neurons using the microfluorimetric method. CART(1-39) (100 nM) induced two types of responses in a population of cortical neurons: 1) a slowly rising increase in [Ca2+]i superimposed with oscillations, and 2) a fast increase followed by a sustained increase of [Ca2+]i. CART(55-102) caused only a slowly rising increase in [Ca2+]i in cortical neurons. Our result shows that the expression pattern of irCART in the rat nervous system and the potentiating action of CART(1-39) on glutamate-induced pressor response is similar to that reported for CART(55-102); but the calcium mobilizing action of CART(1-39) differs from that of CART(55-102), suggesting the possible existence of multiple CART receptors coupled to different calcium signaling pathways.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Glândulas Suprarrenais/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Tronco Encefálico/metabolismo , Cálcio/metabolismo , Células Cultivadas , Gânglios/metabolismo , Ácido Glutâmico/administração & dosagem , Frequência Cardíaca/efeitos dos fármacos , Humanos , Hipotálamo/metabolismo , Imuno-Histoquímica , Injeções Espinhais , Líquido Intracelular/metabolismo , Masculino , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/química , Neurônios/citologia , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Sistema Nervoso Simpático/metabolismoRESUMO
Insulin-like peptide 5 (INSL5) mRNA was detected in the mouse hypothalamus by RT-PCR. Immunohistochemical studies using an antiserum against the mouse INSL5 peptide revealed INSL5-immunoreactive (irINSL5) neurons in the paraventricular, supraoptic, accessory secretory, and supraoptic retrochiasmatic nuclei and immunoreactive cell processes in the internal layer of the median eminence. In the pituitary, irINSL5 was detected in terminal-like elements of the posterior lobe and in cells of the anterior lobe. Double-labeling experiments showed that irINSL5 is expressed in vasopressin-, but not oxytocin-containing neurons. INSL5 (100 nm) administered to dissociated and cultured mouse hypothalamic neurons elevated cytosolic calcium concentrations [Ca(2+)](i), as assessed by the microfluorimetric fura-2 method. In a Ca(2+)-free medium, INSL5 induced in dissociated neurons an increase of [Ca(2+)](i), which was sensitive to the endoplasmic reticulum calcium pump inhibitor thapsigargin (1 microm) and the IP(3) receptor blocker 2-aminoethoxydiphenyl borate (100 microm) or xestospongin C (5 microm). Our result provides the first evidence that INSL5 is expressed in a population of cells in the mouse hypothalamus and pituitary and that it elevates [Ca(2+)](i) by a mechanism involving both Ca(2+) influx and Ca(2+) release from intracellular stores. The concentration of irINSL5 in the hypothalamic-pituitary axis suggests a neuroendocrine function of this insulin superfamily member.
Assuntos
Encéfalo/metabolismo , Cálcio/metabolismo , Insulina/biossíntese , Animais , Células Cultivadas , Citosol/metabolismo , Hipotálamo/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Microscopia Confocal , Neurônios/metabolismo , Hipófise/metabolismo , Proteínas , RNA Mensageiro/metabolismoRESUMO
Obestatin, a 23 amino acid peptide recently isolated from the rat stomach, is encoded by the same gene that encodes ghrelin. With the use of an antiserum directed against the mouse/rat obestatin, obestatin immunoreactivity (irOBS) was detected in cells of the gastric mucosa, myenteric plexus, and in Leydig cells of the testis in Sprague-Dawley rats. Double labeling the myenteric plexus with obestatin antiserum and choline acetyltransferase (ChAT) antiserum revealed that nearly all irOBS neurons were ChAT positive and vice versa. For comparative purposes, myenteric ganglion cells, cells in the gastric mucosa, and Leydig cells of the testis were shown to be immunoreactive to preproghrelin. The biological activity of obestatin on rat central neurons was assessed by the calcium microfluorimetric Fura-2 method. Obestatin (100 nM) administered to dissociated and cultured rat cerebral cortical neurons elevated cytosolic calcium concentrations [Ca2+]i in a population of cortical neurons. The result provides the first immunohistochemical evidence that obestatin is expressed in cells of the gastric mucosa and myenteric ganglion cells, and also in Leydig cells of the testis; the peptide is biologically active on central neurons.
Assuntos
Mucosa Gástrica/química , Células Intersticiais do Testículo/química , Plexo Mientérico/química , Neurônios/química , Hormônios Peptídicos/análise , Animais , Cálcio/metabolismo , Células Cultivadas , Colina O-Acetiltransferase/análise , Fluorometria , Mucosa Gástrica/metabolismo , Grelina , Hormônio do Crescimento/metabolismo , Imuno-Histoquímica/métodos , Células Intersticiais do Testículo/metabolismo , Masculino , Microscopia Confocal , Plexo Mientérico/metabolismo , Neurônios/metabolismo , Hormônios Peptídicos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Cocaine- and amphetamine-regulated transcript peptide (CART) is constitutively expressed in discrete regions of the mammalian central and peripheral nervous system. Immunohistochemical studies reveal a well-defined network of CART-immunoreactive (irCART) neurons organized along the sympatho-adrenal axis. Sympathetic preganglionic neurons, but not parasympathetic preganglionic neurons, in the lateral horn area are CART-positive; which in turn innervate postganglionic neurons in the paravertebral and prevertebral sympathetic ganglia as well as the adrenal medulla. A population of chromaffin cells in the adrenal medulla is CART-positive; whereas, postganglionic neurons are not. Sympathetic preganglionic neurons themselves are contacted by irCART cell processes arising from neurons in the arcuate nucleus, the retrochiasmatic nucleus and the rostral ventrolateral medulla. Results from several recent studies suggest CART directly excites neurons along the sympathetic neural axis or indirectly by potentiating the action of glutamate on NMDA receptors, as evidenced by an elevation of blood pressure and heart rate following intracerebroventricular, intracisternal or intrathecal administration of the peptide to anesthetized rats or conscious rabbits.
Assuntos
Glândulas Suprarrenais/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Sistema Nervoso Simpático/fisiologia , Glândulas Suprarrenais/química , Glândulas Suprarrenais/ultraestrutura , Animais , Humanos , Microscopia Eletrônica , Modelos Biológicos , Fibras Nervosas/química , Fibras Nervosas/ultraestrutura , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso Simpático/química , Sistema Nervoso Simpático/ultraestruturaRESUMO
Metastin, the gene product of metastasis suppressor gene KiSS-1, is the endogenous ligand for the G-protein-coupled receptor GPR54 (or AXOR12, or OT7T175). The expression of KiSS-1 gene and peptide and the distribution of metastin were studied in the rat central nervous system by reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemical methods. KiSS-1 gene and peptide expression was higher in the hypothalamus than in the brainstem and spinal cord. In the brain, metastin-like immunoreactivity (irMT) was found mainly in three groups of cells: dorsomedial hypothalamic nucleus, nucleus of the solitary tract, and caudal ventrolateral medulla. Immunoreactive fibers of varying density were noted in bed nucleus of stria terminalis, septal nuclei, nucleus accumbens, caudate putamen, diagonal band, amygdala, hypothalamus, zona incerta, thalamus, periaqueductal gray, raphe nuclei, lateral parabrachial nucleus, locus coeruleus, spinal trigeminal tract, rostral ventrolateral medulla, and medullary reticular nucleus. Preabsorption of the antiserum with metastin peptide fragment (45-54)-NH2 (1 microg/ml) resulted in no staining in any of the sections. The biological activity of metastin was assessed by monitoring intracellular calcium [Ca2+]i in cultured hippocampal neurons, which are known to express GPR54. Metastin increased [Ca2+]i in a population of cultured hippocampal neurons. The results show that metastin is biologically active in rat central neurons, and its anatomical distribution suggests a possible role in nociception and autonomic and neuroendocrine functions.
Assuntos
Encéfalo/metabolismo , Proteínas/metabolismo , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Encéfalo/anatomia & histologia , Mapeamento Encefálico , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Núcleo Hipotalâmico Dorsomedial/citologia , Núcleo Hipotalâmico Dorsomedial/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Imuno-Histoquímica , Kisspeptinas , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas/genética , Proteínas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G , Receptores de Kisspeptina-1 , Receptores de Neuropeptídeos/metabolismo , Formação Reticular/citologia , Formação Reticular/metabolismo , Núcleo Solitário/citologia , Núcleo Solitário/metabolismoRESUMO
Insulin-like 6 immunoreactivity (irINSL6) was detected in Leydig cells of the mouse testis. In the brain, labeled somata were detected mainly in the caudal hypothalamus and midbrain. Double labeling the brainstem sections revealed that irINSL6 somata were 5-hydroxytryptamine (5-HT) positive. The presence of irINSL6 in discrete populations of hypothalamic and brainstem neurons and in Leydig cells of the testis suggests a diverse biological function of this novel peptide.
Assuntos
Química Encefálica/fisiologia , Insulina/análogos & derivados , Insulina/análise , Testículo/química , Animais , Encéfalo/metabolismo , Feminino , Imuno-Histoquímica , Insulina/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Endogâmicos ICR , Testículo/metabolismoRESUMO
Neuropeptide B (NPB) is a recently identified endogenous ligand for the orphan G protein-coupled receptors GPR7 and GPR8. NPB mRNA is expressed in the human, rat, and mouse brain. With the use of an antiserum directed against the rat NPB, immunoreactivity to NPB (irNPB) was detected in several discrete areas of the hypothalamus and midbrain. In the hypothalamus, irNPB cells were present in the medial preoptic area and nucleus, ventromedial preoptic nucleus, retrochiasmatic nucleus, paraventricular hypothalamic nucleus, supraoptic nucleus, accessory neurosecretory nuclei, periventricular hypothalamic nucleus, dorsomedial hypothalamic nucleus, supraoptic retrochiasmatic nucleus, lateral hypothalamic area, posterior hypothalamic area, dorsal hypothalamic area, and zona incerta. A few irNPB perikarya were noted in the arcuate nucleus, whereas a dense network of nerve fibers was present in the median eminence. In the midbrain, irNPB somata were noted in the substantia nigra (compact, reticular, medial, and lateral parts), paranigral nucleus, ventral tegmental area, interfascicular nucleus, and dorsal raphe nucleus. Neurons in the Edinger-Westphal were strongly labeled. Labeled cells were not detected in the cortex, medulla oblongata, and spinal cord; few lightly labeled cells were occasionally seen in the hippocampus. Double labeling the hypothalamic sections with NPB antiserum and vasopressin or oxytocin antibody revealed that a population of vasopressin- but not oxytocin-immunoreactive cells was irNPB. Tyrosine hydroxylase-positive neurons in the midbrain, presumably dopaminergic, were irNPB. The distribution of irNPB neurons in several areas of the hypothalamus and midbrain together with the colocalization with vasopressin or tyrosine hydroxylase suggests that the peptide may subserve neuroendocrine, autonomic, and motor functions.
Assuntos
Sistema Nervoso Central/metabolismo , Neurônios/metabolismo , Neuropeptídeos/biossíntese , Animais , Sistema Nervoso Autônomo/anatomia & histologia , Sistema Nervoso Autônomo/metabolismo , Mapeamento Encefálico , Sistema Nervoso Central/anatomia & histologia , Dopamina/metabolismo , Hipotálamo/anatomia & histologia , Hipotálamo/metabolismo , Imuno-Histoquímica , Masculino , Mesencéfalo/anatomia & histologia , Mesencéfalo/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Neuropeptídeos/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Vasopressinas/metabolismoRESUMO
Cocaine- and amphetamine-regulated transcript (CART) peptide-immunoreactivity was detected in neurons of the rostral ventrolateral medulla (RVLM), but few in the caudal ventrolateral medulla (CVLM). Double-labeling the medullary sections with sheep polyclonal phenylethanolamine N-methyltransferase-antiserum (PNMT) or monoclonal tyrosine hydroxylase-antibody and rabbit polyclonal CART peptide-antiserum revealed that nearly all adrenergic cells in the C1 area were CART peptide-positive and vice versa; tyrosine hydroxylase-positive cells in the A1 area were not. In the thoracolumbar spinal cord, neurons in the intermediolateral cell column (IML) and other sympathetic autonomic nuclei were CART peptide-positive; some of these were contacted by immunoreactive fibers arising from the lateral funiculus. By immuno-electron microscopy, axon terminals containing closely packed agranular CART peptide-immunoreactive vesicles appeared to make synaptic contacts with immunoreactive dendrites and soma in the IML, albeit the incidence of such contacts was low. Microinjection of the retrograde tracer Fluorogold into the lateral horn area of the T1-T3 spinal segments labeled a population of neurons in the C1 area, many of which were also CART peptide-positive. The results indicate that CART peptide-immunoreactivity is expressed in C1 adrenergic neurons, some of which project to the thoracolumbar spinal cord. The presence of this novel peptide in C1 adrenergic neurons underscores the multiplicity of putative transmitters that may be involved in signaling between putative cardiovascular neurons in the medulla oblongata and sympathetic preganglionic neurons (SPNs) in the spinal cord.
Assuntos
Bulbo/citologia , Proteínas do Tecido Nervoso/análise , Neurônios/química , Receptores Adrenérgicos/fisiologia , Estilbamidinas , Sistema Nervoso Simpático/citologia , Animais , Corantes Fluorescentes , Bulbo/química , Bulbo/enzimologia , Microscopia Eletrônica , Proteínas do Tecido Nervoso/imunologia , Vias Neurais , Neurônios/enzimologia , Neurônios/ultraestrutura , Feniletanolamina N-Metiltransferase/análise , Feniletanolamina N-Metiltransferase/imunologia , Ratos , Medula Espinal/química , Medula Espinal/citologia , Medula Espinal/enzimologia , Sistema Nervoso Simpático/química , Sistema Nervoso Simpático/enzimologia , Tirosina 3-Mono-Oxigenase/análise , Tirosina 3-Mono-Oxigenase/imunologiaRESUMO
Orexin A (or hypocretin 1)-immunoreactive neurons in the rat lateral hypothalamus project to several areas of the medulla oblongata that are closely associated with cardiovascular regulation. The present study was undertaken to further strengthen the hypothesis that orexin A accelerates cardiovascular response by activating sympathoexcitatory neurons in the rat rostral ventrolateral medulla (RVLM). First, immunohistochemical studies revealed the presence of orexin A-immunoreactive fibers in the RVLM. Double labeling the sections with orexin A- and tyrosine hydroxylase (TH)-antisera further showed that orexin A-immunoreactive fibers are in close proximity with TH-immunoreactive neurons, some of which may be barosensitive, bulbospinal neurons in the RVLM. Second, microinjection of orexin A (6.35, 12.7 and 38.1 microM) into the RVLM, which was verified later by histological examination, caused a significant increase of mean arterial pressure (MAP) and a moderate increase of heart rate (HR) in awake rats. L-glutamate (33.3 mM) injected into the same sites, caused a larger increase in MAP, but a decrease in HR; whereas, saline injection was without significant effect. Results from this study suggest that orexin A, which may be released from the nerve fibers originating from the neurons in the lateral hypothalamus, acting on RVLM neurons in the medulla, increases sympathetic outflow targeted to the heart and blood vessels in awake animals.
Assuntos
Proteínas de Transporte/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Bulbo/efeitos dos fármacos , Neuropeptídeos/farmacologia , Pressorreceptores/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Sistema Cardiovascular , Estado de Consciência/fisiologia , Ácido Glutâmico/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Injeções Intraventriculares/métodos , Masculino , Bulbo/anatomia & histologia , Bulbo/fisiologia , Microinjeções/métodos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Orexinas , Pressorreceptores/fisiologia , Ratos , Ratos Wistar , Tirosina 3-Mono-Oxigenase/fisiologiaRESUMO
Beacon is a 73-amino acid peptide encoded by a novel gene in the hypothalamus of Israeli sand rat Psammomys obesus. Reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical techniques were used to investigate the presence of beacon mRNA and the distribution of beacon-immunoreactivity (irBC) in the hypothalamus of ICR mice. RT-PCR experiments revealed beacon mRNA in the mouse hypothalamus. Using a rabbit polyclonal antiserum directed against the synthetic C-terminal peptide fragment (47-73), irBC was detected in the mouse hypothalamus and pituitary. In the hypothalamus, irBC was concentrated in perikarya of the supraoptic (SO), paraventricular (PVH) and accessory neurosecretory nuclei and in cell processes of the median eminence and pituitary stalk. In the pituitary, irBC was noted mainly in the posterior lobe. Double-labeling the hypothalamic sections with guinea-pig vasopressin-antiserum or mouse monoclonal oxytocin-antibody and beacon-antiserum revealed that <30% of vasopressin-immunoreactive neurons and nearly all oxytocin-immunoreactive neurons in the PVH and SO were irBC. The result shows the presence of beacon mRNA in the mouse hypothalamus, and the distribution of irBC is distinctively different from that reported in the hypothalamus of Psammomys obesus, but similar to that of the Sprague-Dawley rats described in our earlier study. More interestingly, Blast search uncovered a 73-amino acid peptide, human ubiquitin-like 5, which has the same exact sequence as beacon. Thus, irBC observed in the mouse brain could be that of ubiquitin-like 5.
Assuntos
Proteínas do Olho/análise , Hipotálamo/química , Proteínas do Tecido Nervoso/análise , Hipófise/química , Ubiquitinas/análise , Animais , Proteínas do Olho/biossíntese , Feminino , Humanos , Hipotálamo/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/biossíntese , Hipófise/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ubiquitinas/biossínteseRESUMO
Neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30) are 23- and 30-amino acid peptides recently isolated from the porcine hypothalamus. Immunohistochemical studies using a rabbit polyclonal antiserum against the rat NPW23 peptide revealed a limited distribution in the rat brain. NPW23-immunoreactive (irNPW) cells were detected in the paraventricular nucleus (PVH), mainly in the parvocellular division, supraoptic nucleus (SO), accessory neurosecretory nuclei, dorsal and lateral hypothalamic areas, perifornical nucleus, arcuate nucleus, and anterior and posterior pituitary; whereas, irNPW fibers were noted in the PVH and SO, retrochiasmatic nucleus, dorsal and lateral hypothalamic areas, median eminence, amygdala, and posterior pituitary. The pattern of distribution of irNPW in the hypothalamus corroborates a possible role of NPW on prolactin release and feeding behavior reported by others.
Assuntos
Hipotálamo/metabolismo , Neuropeptídeos/metabolismo , Hipófise/metabolismo , Animais , Feminino , Imuno-Histoquímica , Masculino , Neurônios/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
With the use of an antiserum against human apelin-36, apelin-immunoreactivity (irAP) was detected in neurons and cell processes of the supraoptic nucleus (SO), paraventricular nucleus (PVH), accessory neurosecretory nuclei (Acc) and suprachiasmatic nucleus. Strongly labeled cells/processes were noted in the internal layer of the median eminence, infundibular stem, anterior and posterior pituitary. Double-labeling the sections with goat polyclonal neurophysin I-antiserum and rabbit polyclonal apelin-antiserum revealed a population of magnocellular neurons in the PVH, SO and Acc expressing both irAP and neurophysin I-immunoreactivity (irNP), the latter being a marker of oxytocin-containing neurons. By inference, the AP-positive but irNP-negative magnocellular neurons could be vasopressin-containing. The presence of irAP in certain hypothalamic nuclei and pituitary suggests that the peptide may be a signaling molecule released from the hypothalamic-hypophysial axis.
Assuntos
Proteínas de Transporte/análise , Hipotálamo/química , Hipófise/química , Animais , Apelina , Feminino , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Microscopia Confocal , Neurofisinas/análise , Ratos , Ratos Sprague-DawleyRESUMO
Distribution of the novel peptide beacon in the hypothalamus of Sprague-Dawley rats was examined by immunohistochemical methods. Beacon-immunoreactive (irBC) neurons were found in the paraventricular, supraoptic, and accessory neurosecretory nuclei, and intensely labeled fibers in the median eminence and infundibulo-pituitary stalk. Scattered cells and/or fibers were noted in the suprachiasmatic nucleus, arcuate nucleus, retrochiasmatic area, lateral and medial preoptic area, as well as anterior and lateral hypothalamic area. The wide distribution of irBC in the hypothalamus of Sprague-Dawley rats suggests that the peptide may influence, in addition to a proposed role in feeding, a multitude of biological activities associated with the hypothalamic-pituitary axis.
Assuntos
Regulação do Apetite/fisiologia , Hipotálamo/metabolismo , Proteínas do Tecido Nervoso , Neurônios/metabolismo , Proteínas/metabolismo , Animais , Especificidade de Anticorpos/imunologia , Feminino , Sistema Hipotálamo-Hipofisário/citologia , Sistema Hipotálamo-Hipofisário/metabolismo , Hipotálamo/citologia , Imuno-Histoquímica , Masculino , Neurônios/citologia , Neurossecreção/fisiologia , Núcleo Hipotalâmico Paraventricular/citologia , Núcleo Hipotalâmico Paraventricular/metabolismo , Ratos , Ratos Sprague-Dawley , Núcleo Supraóptico/citologia , Núcleo Supraóptico/metabolismo , UbiquitinasRESUMO
Metastin, the product of metastasis suppressor gene KiSS-1, is proposed to be the natural ligand for the G-protein-coupled receptor GPR54, known also as AXOR12. This immunohistochemical study, using a rabbit polyclonal antiserum against the human metastin fragment (45-54)-NH(2), showed that in rats metastin-like immunoreactivity (MTS-LI) was present in neurons of the nucleus of the solitary tract and caudoventrolateral reticular nucleus, and in cell processes of the spinal trigeminal tract and lateral reticular nucleus. MTS-LI was confined mainly to neurons and fibers at or caudal to the area postrema. In the spinal cord, MTS-LI cell processes formed a dense plexus in superficial layers I and II of the dorsal horn. The pattern of distribution of MTS-LI in the medulla and spinal cord suggests that this novel peptide may participate in autonomic and sensory neural signaling.
Assuntos
Bulbo/química , Proteínas/análise , Medula Espinal/química , Animais , Feminino , Humanos , Soros Imunes , Imuno-Histoquímica , Kisspeptinas , Masculino , Células do Corno Posterior/química , Proteínas/imunologia , Coelhos , Ratos , Ratos Sprague-Dawley , Núcleo Solitário/química , Proteínas Supressoras de TumorRESUMO
Immunohistochemical studies were conducted to assess the subunits of ionotropic and metabotropic glutamate receptor present in the rostral ventrolateral medulla (RVLM) of the rat. Double labeling the medullary sections with polyclonal GluR1, GluR2/3, GluR4, NMDAR1, NMDAR2A/B, mGluR1alpha, and mGluR2/3 antiserum and monoclonal tyrosine hydroxylase (TH) antiserum revealed nearly all TH immunoreactive (irTH) cells and many TH-negative neurons were immunoreactive to GluR2/3 (irGluR2/3), NMDAR1 (irNMDAR1), and NMDAR2A/B (irNMDAR2A/B). A few RVLM neurons were immunoreactive to GluR1 (irGluR1) and GluR4 (irGluR4), but they were generally TH-negative. Immunoreactivity to mGluR1alpha (irmGluR1alpha) appeared to be localized exclusively to fiber-like elements in the RVLM area. Our results show that neurons in the RVLM, including irTH, are endowed mainly with GluR2/3 and NMDAR1 or NMDAR2A/B ionotropic receptor subunits, and that irmGluR1alpha splice variant appears to be located on nerve fibers ramifying within the RVLM. Moreover, TH-negative neurons in the RVLM appear to bear similar subunits of ionotropic glutamate receptors.