RESUMEN
The retrotrapezoid nucleus (RTN) is a hub for respiratory chemoregulation in the mammal brainstem that integrates chemosensory information from peripheral sites and central relays. Chemosensitive neurons of the RTN express specific genetic and molecular determinants, which have been used to identify RTN precise location within the brainstem of rodents and nonhuman primates. Based on a comparative approach, we hypothesized that among mammals, neurons exhibiting the same specific molecular and genetic signature would have the same function. The co-expression of preprogalanin (PPGAL) and SLC17A6 (VGluT2) mRNAs with duplex in situ hybridization has been studied in formalin fixed paraffin-embedded postmortem human brainstems. Two specimens were processed and analyzed in line with RTN descriptions in adult rats and macaques. Double-labeled PPGAL+/SLC17A6+ neurons were only identified in the parafacial region of the brainstem. These neurons were found surrounding the nucleus of the facial nerve, located ventrally to the nucleus VII on caudal sections, and slightly more dorsally on rostral sections. The expression of neuromedin B (NMB) mRNA as a single marker of chemosensitive RTN neurons has not been confirmed in humans. The location of the RTN in human adults is provided. This should help to develop investigation tools combining anatomic high-resolution imaging and respiratory functional investigations to explore the pathogenic role of the RTN in congenital or acquired neurodegenerative diseases.
Asunto(s)
Tronco Encefálico/metabolismo , Tronco Encefálico/patología , Galanina/biosíntesis , Neuronas/metabolismo , Neuronas/patología , Proteína 2 de Transporte Vesicular de Glutamato/biosíntesis , Biomarcadores/metabolismo , Núcleo Motor del Nervio Facial/metabolismo , Núcleo Motor del Nervio Facial/patología , Galanina/genética , Expresión Génica , Humanos , Cuerpo Trapezoide/metabolismo , Cuerpo Trapezoide/patología , Proteína 2 de Transporte Vesicular de Glutamato/genéticaRESUMEN
After injury, peripheral axons usually re-extend toward their target, and neuronal functions recover. Previous studies have reported that expression of various molecules are transiently altered in motor neurons after nerve injury, but the time course of these changes and their relationship with functional recovery have not been clearly demonstrated. We used the mouse facial nerve transection and suturing model, and examined the changes in expression of five molecules, choline acetyl transferase (ChAT), galanin, calcitonin gene-related protein (CGRP), gephyrin, and potassium chloride co-transporter 2 (KCC2) in the facial motor neurons after surgery until recovery. Number of ChAT-positive neurons was markedly decreased at days 3 and 7, and recovered to the normal level by day 60, when facial motor functions recovered. Localization of two neuropeptides, CGRP and galanin, was increased in the perikarya and axons during regeneration, and returned to the normal levels by days 60 and 28, respectively. Expression of two postsynaptic elements of γ-amino butyric acid synapses, gephyrin and KCC2, was decreased at days 3 and 7, and recovered by day 60. These results suggest that ChAT, CGRP, and KCC2 may be objective indicators of regeneration, and altering their expression may be related to the functional recovery and axonal re-extension.
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Péptido Relacionado con Gen de Calcitonina/biosíntesis , Colina O-Acetiltransferasa/biosíntesis , Nervio Facial/fisiología , Neuronas Motoras/fisiología , Regeneración Nerviosa/fisiología , Simportadores/biosíntesis , Animales , Biomarcadores/análisis , Proteínas Portadoras/biosíntesis , Traumatismos del Nervio Facial/metabolismo , Galanina/biosíntesis , Masculino , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Cotransportadores de K ClRESUMEN
The lateral hypothalamus (LHA) integrates reward and appetitive behavior and is composed of many overlapping neuronal populations. Recent studies associated LHA GABAergic neurons (LHA GABA ), which densely innervate the ventral tegmental area (VTA), with modulation of food reward and consumption; yet, LHA GABA projections to the VTA exclusively modulated food consumption, not reward. We identified a subpopulation of LHA GABA neurons that coexpress the neuropeptide galanin (LHA Gal ). These LHA Gal neurons also modulate food reward, but lack direct VTA innervation. We hypothesized that LHA Gal neurons may represent a subpopulation of LHA GABA neurons that mediates food reward independent of direct VTA innervation. We used chemogenetic activation of LHA Gal or LHA GABA neurons in mice to compare their role in feeding behavior. We further analyzed locomotor behavior to understand how differential VTA connectivity and transmitter release in these LHA neurons influences this behavior. LHA Gal or LHA GABA neuronal activation both increased operant food-seeking behavior, but only activation of LHA GABA neurons increased overall chow consumption. Additionally, LHA Gal or LHA GABA neuronal activation similarly induced locomotor activity, but with striking differences in modality. Activation of LHA GABA neurons induced compulsive-like locomotor behavior; while LHA Gal neurons induced locomotor activity without compulsivity. Thus, LHA Gal neurons define a subpopulation of LHA GABA neurons without direct VTA innervation that mediate noncompulsive food-seeking behavior. We speculate that the striking difference in compulsive-like locomotor behavior is also based on differential VTA innervation. The downstream neural network responsible for this behavior and a potential role for galanin as neuromodulator remains to be identified.SIGNIFICANCE STATEMENT The lateral hypothalamus (LHA) regulates motivated feeding behavior via GABAergic LHA neurons. The molecular identity of LHA GABA neurons is heterogeneous and largely undefined. Here we introduce LHA Gal neurons as a subset of LHA GABA neurons that lack direct innervation of the ventral tegmental area (VTA). LHA Gal neurons are sufficient to drive motivated feeding and locomotor activity similar to LHA GABA neurons, but without inducing compulsive-like behaviors, which we propose to require direct VTA innervation. Our study integrates galanin-expressing LHA neurons into our current understanding of the neuronal circuits and molecular mechanisms of the LHA that contribute to motivated feeding behaviors.
Asunto(s)
Galanina/biosíntesis , Área Hipotalámica Lateral/fisiología , Actividad Motora/fisiología , Neuronas/fisiología , Recompensa , Ácido gamma-Aminobutírico/fisiología , Animales , Antipsicóticos/farmacología , Clozapina/farmacología , Conducta Compulsiva , Condicionamiento Operante/efectos de los fármacos , Condicionamiento Operante/fisiología , Metabolismo Energético/fisiología , Alimentos , Área Hipotalámica Lateral/citología , Área Hipotalámica Lateral/metabolismo , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Red Nerviosa/citología , Red Nerviosa/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotransmisores/metabolismoRESUMEN
Redes neurales distribuidas en el encéfalo sustentan la generación de la vigilia y dos estados de sueño: sueño no REM y sueño REM. Estos tres estados comportamentales se engranan conjuntamente en una secuencia regular que constituye el ciclo vigilia-sueño. Este trabajo revisa y actualiza el conocimiento sobre la implicación del sistema histaminérgico en la organización del ciclo vigilia-sueño. Las neuronas histaminérgicas se localizan exclusivamente en el núcleo tuberomamilar hipotalámico, pero son el origen de proyecciones extensas a numerosas regiones encefálicas. Las neuronas histaminérgicas están activas durante la vigilia, especialmente con alta demanda atencional, y permanecen silentes en sueño no REM y sueño REM. Se han descrito cuatro receptores histaminérgicos metabotrópicos, de los cuales H1R, H2R y H3R están presentes en el sistema nervioso. H1R y H2R son fundamentalmente heterorreceptores postsinápticos, mientras que se piensa que H3R es mayormente un auto y heterorreceptor presináptico. Las neuronas histaminérgicas son activadas por las neuronas hipocretinérgicas y se cree que muchos de los efectos activadores de las hipocretinas se deben a acciones histaminérgicas. Las interacciones entre los axones histaminérgicos y los núcleos colinérgicos en el prosencéfalo y el troncoencéfalo son particularmente importantes para la activación cortical. Por el contrario, las neuronas histaminérgicas tuberomamilares, al igual que otras neuronas aminérgicas del locus coeruleus o del núcleo dorsal del rafe, son inhibidas por las neuronas del área preóptica promotoras de sueño no REM. Acciones inhibidoras adicionales sobre las neuronas histaminérgicas proceden de la liberación de adenosina en la región tuberomamilar. Finalmente, las neuronas histaminérgicas inhiben a las neuronas hipotalámicas REM-on que contienen hormona concentradora de melanina, apoyando así un papel permisivo del núcleo tuberomamilar en el sueño REM. De hecho, ratones deficientes para descarboxilasa de histidina, la enzima de síntesis de la histamina, muestran un aumento significativo de sueño REM (AU)
Distributed neural networks in the brain sustain generation of wakefulness and two sleep states: non-REM sleep and REM sleep. These three behavioral states are jointly ingrained in a rhythmic sequence that constitutes the sleepwakefulness cycle. This paper reviews and updates knowledge about the involvement of the histaminergic system in sleep-wakefulness cycle organization. Histaminergic neurons are exclusively located in the hypothalamic tuberomammillary nucleus, but are the source of a widespread projection system to many brain regions. Histamine neurons are active during waking, especially with high attention need, and remain silent in both non-REM and REM sleep. There have been described four metabotropic histamine receptors, of which H1R, H2R and H3R are present in the nervous system. H1R and H2R are mainly postsynaptic heteroreceptors, whereas H3R is thought to be mostly a presynaptic auto- and hetero-receptor. Histaminergic neurons are excited by hypocretinergic neurons and most of the arousing hypocretin effects are thought to depend on histaminergic actions. Interactions among histaminergic axons and cholinergic nuclei within forebrain and brainstem are particularly important for cortical activation. In contrast, histaminergic tuberomammillary neurons, similarly to other aminergic neurons in locus coeruleus or dorsal raphe nucleus, are inhibited by non-REM sleep-promoting neurons of the preoptic region. Further inhibitory actions on histamine neurons come from adenosine release on tuberomammillary region. Finally, histaminergic neurons inhibit REM-on hypothalamic neurons containing melanine-concentrating hormone, thus supporting a permissive role of tuberomammillary nucleus in REM sleep. Actually, knockout mice for histidine decarboxylase, the enzyme synthetizing histamine, show a significant REM sleep increase (AU)
Asunto(s)
Humanos , Masculino , Femenino , Trastornos del Sueño-Vigilia/tratamiento farmacológico , Promotores de la Vigilia/uso terapéutico , Receptores Histamínicos/uso terapéutico , Neuronas , Conducción Nerviosa , Privación de Sueño/tratamiento farmacológico , Neuronas Colinérgicas , Galanina/biosíntesis , DescarboxilaciónRESUMEN
The symptomatology, mood and cognitive disturbances seen in post-traumatic stress disorder (PTSD) and mild blast-induced traumatic brain injury (mbTBI) overlap considerably. However the pathological mechanisms underlying the two conditions are currently unknown. The neuropeptide galanin has been suggested to play a role in the development of stress and mood disorders. Here we applied bio- and histochemical methods with the aim to elucidate the nature of any changes in the expression of galanin and its receptors in a rodent model of mbTBI. In situ hybridization and quantitative polymerase chain reaction studies revealed significant, injury-induced changes, in some cases lasting at least for one week, in the mRNA levels of galanin and/or its three receptors, galanin receptor 1-3 (GalR1-3). Such changes were seen in several forebrain regions, and the locus coeruleus. In the ventral periaqueductal gray GalR1 mRNA levels were increased, while GalR2 were decreased. Analysis of galanin peptide levels using radioimmunoassay demonstrated an increase in several brain regions including the locus coeruleus, dorsal hippocampal formation and amygdala. These findings suggest a role for the galanin system in the endogenous response to mbTBI, and that pharmacological studies of the effects of activation or inhibition of different galanin receptors in combination with functional assays of behavioral recovery may reveal promising targets for new therapeutic strategies in mbTBI.
Asunto(s)
Traumatismos por Explosión/metabolismo , Lesiones Encefálicas/metabolismo , Galanina/biosíntesis , Precursores de Proteínas/biosíntesis , Receptores de Galanina/biosíntesis , Animales , Traumatismos por Explosión/patología , Lesiones Encefálicas/patología , Locus Coeruleus/metabolismo , Masculino , Prosencéfalo/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Núcleos del Rafe/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Galanina Tipo 1/biosíntesis , Receptor de Galanina Tipo 1/genética , Receptor de Galanina Tipo 2/biosíntesis , Receptor de Galanina Tipo 2/genética , Receptor de Galanina Tipo 3/biosíntesis , Receptor de Galanina Tipo 3/genéticaRESUMEN
We previously found that daidzein decreased food intake in female rats. The present study aimed to elucidate the relationship between dynamics of appetite-mediated neuropeptides and the anorectic effect of daidzein. We examined appetite-mediated gene expression in the hypothalamus and small intestine during the 3 meals per day feeding method. Daidzein had an anorectic effect specifically at the second feeding. Neuropeptide-Y (NPY) and galanin mRNA levels in the hypothalamus were significantly higher after feeding in the control but not in the daidzein group, suggesting that daidzein attenuated the postprandial increase in NPY and galanin expression. The daidzein group had higher corticotrophin-releasing hormone (CRH) mRNA levels in the hypothalamus after feeding, and increased cholelcystokinin (CCK) mRNA levels in the small intestine, suggesting that CCK is involved in the hypothalamic regulation of thisãanorectic effect. Therefore, daidzein may induce anorexia by suppressing expression of NPY and galanin and increasing expression of CRH in the hypothalamus.
Asunto(s)
Anorexia/genética , Apetito/genética , Ingestión de Alimentos/genética , Galanina/biosíntesis , Neuropéptido Y/biosíntesis , Animales , Anorexia/patología , Apetito/fisiología , Peso Corporal , Ingestión de Alimentos/efectos de los fármacos , Métodos de Alimentación , Femenino , Galanina/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hipotálamo/metabolismo , Hipotálamo/fisiología , Isoflavonas/administración & dosificación , Neuropéptido Y/genética , ARN Mensajero/biosíntesis , Ratas , Receptores de Colecistoquinina/biosíntesis , Receptores de Hormona Liberadora de Corticotropina/biosíntesisRESUMEN
Although galanin has been shown to increase glucose transporter 4 (GLUT4) expression in skeletal muscle and adipocytes of rats, there is no literature available about the effect of galanin on GLUT4 expression in cardiac muscle of type 2 diabetic rats. In this study, we investigated the relationship between intracerebroventricular administration of M35, a galanin receptor antagonist, and GLUT4 expression in cardiac muscle of type 2 diabetic rats. The rats tested were divided into four groups: rats from healthy and type 2 diabetic drug groups were injected with 2 µM M35 for three weeks, while both control groups with 2 µl vehicle control. The euglycemic-hyperinsulinemic clamp test was conducted for an index of glucose infusion rates. The cardiac muscle was processed for determination of GLUT4 expression levels. The present study showed that the plasma insulin and retinol binding protein 4 (RBP4) levels were higher in both drug groups than controls respectively. Moreover, the results showed the inhibitive effect of central M35 treatment on glucose infusion rates in the euglycemic-hyperinsulinemic clamp test and GLUT4 expression levels in the cardiac muscle. These results demonstrate that endogenous galanin, acting through its central receptor, has an important attribute to increase GLUT4 expression, leading to enhance insulin sensitivity and glucose uptake in cardiac muscle of type 2 diabetic rats. Galanin and its fragment can play a significant role in regulation of glucose metabolic homeostasis in cardiac muscle and galanin is an important hormone relative to diabetic heart.
Asunto(s)
Bradiquinina/análogos & derivados , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Galanina/metabolismo , Transportador de Glucosa de Tipo 4/biosíntesis , Fragmentos de Péptidos/administración & dosificación , Animales , Glucemia , Bradiquinina/administración & dosificación , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/patología , Galanina/administración & dosificación , Galanina/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Insulina/sangre , Miocardio/metabolismo , Miocardio/patología , Ratas , Receptores de Galanina/antagonistas & inhibidores , Proteínas Plasmáticas de Unión al Retinol/metabolismoRESUMEN
Obesity rates continue to rise throughout the world. Recent evidence has suggested that environmental factors contribute to altered energy balance regulation. However, the role of epigenetic modifications to the central control of energy homeostasis remains unknown. To investigate the role of DNA methylation in the regulation of energy balance, we investigated the role of the de novo DNA methyltransferase, Dnmt3a, in Single-minded 1 (Sim1) cells, including neurons in the paraventricular nucleus of the hypothalamus (PVH). Dnmt3a expression levels were decreased in the PVH of high-fat-fed mice. Mice lacking Dnmt3a specifically in the Sim1 neurons, which are expressed in the forebrain, including PVH, became obese with increased amounts of abdominal and subcutaneous fat. The mice were also found to have hyperphagia, decreased energy expenditure, and glucose intolerance with increased serum insulin and leptin. Furthermore, these mice developed hyper-LDL cholesterolemia when fed a high-fat diet. Gene expression profiling and DNA methylation analysis revealed that the expression of tyrosine hydroxylase and galanin were highly upregulated in the PVH of Sim1-specific Dnmt3a deletion mice. DNA methylation levels of the tyrosine hydroxylase promoter were decreased in the PVH of the deletion mice. These results suggest that Dnmt3a in the PVH is necessary for the normal control of body weight and energy homeostasis and that tyrosine hydroxylase is a putative target of Dnmt3a in the PVH. These results provide evidence for a role for Dnmt3a in the PVH to link environmental conditions to altered energy homeostasis.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , ADN (Citosina-5-)-Metiltransferasas/fisiología , Metabolismo Energético/fisiología , Homeostasis , Neuronas/metabolismo , Proteínas Represoras/metabolismo , Tejido Adiposo/fisiología , Animales , LDL-Colesterol/sangre , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ADN Metiltransferasa 3A , Dieta Alta en Grasa , Femenino , Galanina/biosíntesis , Perfilación de la Expresión Génica , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/fisiopatología , Hiperfagia/complicaciones , Hiperfagia/genética , Hiperfagia/fisiopatología , Insulina/sangre , Leptina/sangre , Masculino , Ratones , Ratones Noqueados , Obesidad/sangre , Obesidad/complicaciones , Obesidad/genética , Obesidad/fisiopatología , Núcleo Hipotalámico Paraventricular/metabolismo , Tirosina 3-Monooxigenasa/biosíntesis , Regulación hacia ArribaRESUMEN
Galanin is a neuropeptide expressed in the central and peripheral nervous systems. Galanin is known to be biosynthesized in neural and endocrine cells, but little evidence exists for its synthesis in other cells. In this study, we explored galanin-releasing nonneural cells using radioimmunoassay, finding that some fibroblasts produced and released the galanin-like immunoreactive component (galanin-LI). The molecular weight of the galanin-LI obtained from the fibroblasts, as measured by gel filtration chromatography and Western blotting, was 14 kDa and suggested that the compound was progalanin. Peptide mass fingerprinting analysis identified the large form of galanin-LI as progalanin without its signal sequence. In addition, galanin-LI was located in the Golgi bodies and vesicle-like structures of the fibroblasts. Furthermore, the addition of brefeldin A, an inhibitor of transport from the ER, decreased the release of galanin-LI. In this study, we showed that the fibroblast, a nonneural and nonendocrine cell type, produced and released a galanin precursor, progalanin, without processing via Golgi bodies or secretory vesicles.
Asunto(s)
Fibroblastos/metabolismo , Galanina/biosíntesis , Galanina/metabolismo , Animales , Células Cultivadas , Pollos , Cricetulus , Galanina/química , Humanos , RatonesRESUMEN
Cocaine- and amphetamine-regulated transcript (CART) is a recently discovered peptide inducing strong anxiogenic-like effect. CART distribution and its role(s) at periphery are not well understood. Immunohistochemisty was utilized to investigate the distribution patterns of CART in the stomach of the pig and wild boar. Double immunohistochemisty was applied to elucidate whether CART-immunoreactive (IR) neuronal elements coexpress galanin, substance P (SP) and neuropeptide Y (NPY). In the pig stomach, different proportions of CART-IR myenteric neurons were found in the antrum (42.3 ± 3.5%), corpus (18.0 ± 1.9%) and pylorus (33.2 ± 3.0%). CART-IR myeneric neurons were also found in the antrum, corpus and pylorus of the wild boar stomach (41.7 ± 3.2, 36.0 ± 2.2 and 35.8 ± 3.5%; respectively). In both species, none of gastric submucous neurons were CART-IR; however, CART-IR nerve fibres encircled submucous perikarya. In all portions of the pig and wild boar stomach, CART-IR nerve fibres were frequently found in the smooth muscle layer as well as in the lamina muscularis mucosae. In all regions of the pig and wild boar stomach, the expression of galanin and SP was found in CART-IR myenteric neurons and smooth muscle-supplying nerve fibres. CART/NPY coexpression was not found in the porcine stomach; however, in different regions of the wild boar stomach, subpopulations of CART-IR/NPY-IR myenteric neurons were noted. In conclusion, in this study, the existence and distribution patterns of CART in discrete regions of the pig and wild boar stomach were described in details. Colocalization studies revealed that in both animal species, a functional cooperation of CART with several neuropeptides is likely.
Asunto(s)
Sistema Nervioso Entérico/inmunología , Proteínas del Tejido Nervioso/inmunología , Estómago/inervación , Sus scrofa/inmunología , Animales , Femenino , Galanina/biosíntesis , Inmunohistoquímica , Masculino , Membrana Mucosa/inmunología , Membrana Mucosa/inervación , Músculo Liso/inmunología , Músculo Liso/inervación , Neuropéptido Y/biosíntesis , Neurotransmisores/inmunología , Antro Pilórico/inmunología , Antro Pilórico/inervación , Estómago/inmunología , Sustancia P/biosíntesisRESUMEN
OBJECTIVE: To construct an N-2a cell line stably expressing PcDNA 3.1-platelet derived growth factor-galanin (GAL) and explore the effect of over-expressed GAL on proliferation and apoptosis of N-2a cell in vitro. METHODS: The vector containing the target gene was transfected into N-2a cells by liposome, and cell clones stably over-expressing GAL was obtained via G418 screening. GAL mRNA and protein levels were determined by reverse transcriotion-polymerase chain reaction (RT-PCR) and Western blot. The proliferation of N-2a cells was detected by MTT.The cell cycle and apoptosis were detected by flow cytometry. RESULTS: RT-PCT and Western blot indicated that GAL genes were highly expressed in the transfected N-2a cells (i.e.GAL-N-2a). As shown by MTT, the proliferation of the N-2a cells transfected with PcDNA 3.1-PDGF-GAL was significantly slower than the control group(P<0.05). Compared with the non-transfected cells in the control group, the N-2a cells with endogenously overexpressed GAL were arrested at the G0/G1 phases, and the over-expressed GAL protein significantly induced the N-2a cell apoptosis in a concentration-dependent fashion. CONCLUSION: Eukaryotic expression vector PcDNA 3.1-PDGF-GAL can encode the expression of GAL in N-2a cells. Aslo, it can inhibit cell proliferation and promote the cell apoptosis.
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Apoptosis , Línea Celular Tumoral , Proliferación Celular , Galanina/biosíntesis , Animales , Ratones , ARN Mensajero/biosíntesisRESUMEN
One of the major pathological characteristics of Alzheimer's disease (AD) is the presence of enhanced deposits of beta-amyloid peptide (Aß). The neuropeptide galanin (GAL) and its receptors are overexpressed in degenerating brain regions in AD. The functional consequences of galaninergic systems plasticity in AD are unclear. The objective of the present study was to investigate whether exogenous galanin could attenuate spatial memory impairment and hippocampal Aß aggregation in rat model of AD. The effects of Aß, galanin, galanin receptor 1 agonist M617 and galanin receptor 2 agonist AR-M1896 on spatial memory were tested by Morris water maze. The effects of Aß, galanin, M617 and AR-M1896 on hippocampal Aß protein expression were evaluated by western blot assay. The expression of galanin, galanin receptors 1 and 2 in rats' hippocampus were detected by real time PCR and western blot assay. The results showed that (1) Galanin administration was effective in improving the spatial memory and decreasing hippocampal Aß levels after intracerebroventricular injection of Aß; (2) AR-M1896 rather than M617 could imitate these effects of galanin; (3) GAL and GALR2 mRNA and protein levels increased significantly in hippocampus after Aß administration, while GALR1 mRNA and protein levels did not change; (4) GAL, AR-M1896 and M617 administration did not show significant effect on GAL, GalR1 and GalR2 mRNA and protein levels in hippocampus after Aß administration. These results implied that galanin receptor 2, but not receptor 1 was involved in the protective effects against spatial memory impairment and hippocampal Aß aggregation.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/toxicidad , Modelos Animales de Enfermedad , Galanina/biosíntesis , Hipocampo/metabolismo , Trastornos de la Memoria/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Reacción de Fuga/efectos de los fármacos , Reacción de Fuga/fisiología , Galanina/farmacología , Galanina/uso terapéutico , Hipocampo/efectos de los fármacos , Masculino , Trastornos de la Memoria/tratamiento farmacológico , ARN Mensajero/biosíntesis , Distribución Aleatoria , Ratas , Ratas Wistar , Conducta Espacial/efectos de los fármacos , Conducta Espacial/fisiologíaRESUMEN
In order to understand how nociceptive information is processed in the spinal dorsal horn we need to unravel the complex synaptic circuits involving interneurons, which constitute the vast majority of the neurons in laminae I-III. The main limitation has been the difficulty in defining functional populations among these cells. We have recently identified 4 non-overlapping classes of inhibitory interneuron, defined by expression of galanin, neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) and parvalbumin, in the rat spinal cord. In this study we demonstrate that these form distinct functional populations that differ in terms of sst(2A) receptor expression and in their responses to painful stimulation. The sst(2A) receptor was expressed by nearly all of the nNOS- and galanin-containing inhibitory interneurons but by few of those with NPY and none of the parvalbumin cells. Many galanin- and NPY-containing cells exhibited phosphorylated extracellular signal-regulated kinases (pERK) after mechanical, thermal or chemical noxious stimuli, but very few nNOS-containing cells expressed pERK after any of these stimuli. However, many nNOS-positive inhibitory interneurons up-regulated Fos after noxious thermal stimulation or injection of formalin, but not after capsaicin injection. Parvalbumin cells did not express either activity-dependent marker following any of these stimuli. These results suggest that interneurons belonging to the NPY, nNOS and galanin populations are involved in attenuating pain, and for NPY and nNOS cells this is likely to result from direct inhibition of nociceptive projection neurons. They also suggest that the nociceptive inputs to the nNOS cells differ from those to the galanin and NPY populations.
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Galanina/biosíntesis , Interneuronas/fisiología , Inhibición Neural/fisiología , Neuropéptido Y/biosíntesis , Óxido Nítrico Sintasa de Tipo I/biosíntesis , Células del Asta Posterior/fisiología , Animales , Galanina/análisis , Interneuronas/química , Masculino , Neuropéptido Y/análisis , Óxido Nítrico Sintasa de Tipo I/análisis , Células del Asta Posterior/química , Ratas , Ratas WistarRESUMEN
BACKGROUND: Alcoholism is a heterogeneous disease, with subjects possibly differing both in the best measure that predicts their excess consumption and in their most effective pharmacotherapy. Two different measures, high novelty-induced activity and high-fat-induced triglycerides (TGs), are known to identify subgroups of animals prone to consuming higher amounts of ethanol (EtOH). The question investigated here is whether these subgroups are, in fact, similar in their neurochemical phenotype that may contribute to their overconsumption. METHODS: EtOH-naïve, Sprague-Dawley rats were subgrouped based on the 2 predictor measures of activity or TG levels, and then quantitative real-time polymerase chain reaction and digoxigenin-labeled in situ hybridization were used to measure their expression of hypothalamic peptides that affect EtOH intake. In additional subgroups subsequently trained to drink 9% EtOH, the opioid antagonist and alcoholism medication, naltrexone, was tested at a low dose (0.02 mg/kg, s.c.) to determine the rats' sensitivity to its effects. RESULTS: The 2 measures, while both effective in predicting amount of EtOH intake, were found to identify distinctive subgroups. Rats with high compared to low activity exhibited significantly greater expression of galanin and enkephalin in the paraventricular nucleus (PVN) and of orexin in the perifornical lateral hypothalamus (PFLH), but no difference in melanin-concentrating hormone in PFLH or neuropeptide Y in arcuate nucleus. This contrasts with rats having high TG, which exhibited greater expression only of PVN galanin, along with reduced PFLH orexin. The high-activity rats with elevated enkephalin, but not high-TG rats, were also unusually sensitive to naltrexone, which significantly reduced their alcohol intake. CONCLUSIONS: In addition to revealing differences in endogenous peptides and drug responsiveness in predicted high EtOH drinkers, this study demonstrates that these disturbances differ markedly between the 2 at-risk subgroups. This indicates that simple tests may be effective in identifying subjects most responsive to a specific pharmacotherapy.
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Consumo de Bebidas Alcohólicas/genética , Consumo de Bebidas Alcohólicas/fisiopatología , Química Encefálica/genética , Etanol/administración & dosificación , Consumo de Bebidas Alcohólicas/metabolismo , Animales , Química Encefálica/efectos de los fármacos , Encefalinas/biosíntesis , Predicción , Galanina/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Neuropéptidos/biosíntesis , Neuropéptidos/genética , Orexinas , Valor Predictivo de las Pruebas , Ratas , Ratas Sprague-Dawley , Triglicéridos/metabolismoRESUMEN
Exposure to ethanol during the prenatal period contributes to increased alcohol consumption and preference in rodents and increased risk for alcoholism in humans. With studies in adult animals showing the orexigenic peptides, enkephalin (ENK), galanin (GAL) and orexin (OX), to stimulate ethanol consumption, the question addressed here is whether prenatal ethanol alters the development in utero of specific neurons that express these peptides. With reports describing suppressive effects of high doses of ethanol, we examined the offspring of dams gavaged from embryonic day 9 to parturition with a control solution or lower ethanol doses, 1 and 3g/kg/day, known to promote ethanol consumption in the offspring. To understand underlying mechanisms, measurements were taken in postnatal offspring of the expression of ENK in the hypothalamic paraventricular nucleus (PVN) and nucleus accumbens (NAc), GAL in the PVN, and OX in the perifornical lateral hypothalamus (PFLH) using real-time qPCR and in situ hybridization, and also of the cell proliferation marker, 5-bromo-2-deoxyuridine (BrdU), and its double-labeling with either neuronal nuclei (NeuN), a marker of mature neurons, or the peptides. On postnatal day 15 (P15), after two weeks without ethanol, the offspring showed increased expression of ENK in the PVN and NAc core but not shell, GAL in the PVN, and OX in the PFLH. In these same areas, prenatal ethanol compared to control increased the density at birth (P0) of neurons expressing these peptides and at P0 and P15 of neurons double-labeling BrdU and NeuN, indicating increased neurogenesis. These BrdU-positive neurons were found to express ENK, GAL and OX, indicating that prenatal ethanol promotes neurogenesis in these specific peptide systems. There were no changes in gliogenesis or apoptosis. This increase in neurogenesis and density of peptide-expressing neurons suggests the involvement of these hypothalamic and accumbal peptide systems in mediating the increased alcohol consumption observed in prenatal ethanol-exposed offspring.
Asunto(s)
Alcoholismo/etiología , Depresores del Sistema Nervioso Central/efectos adversos , Etanol/efectos adversos , Hipotálamo/metabolismo , Sistema Límbico/metabolismo , Efectos Tardíos de la Exposición Prenatal/patología , Efectos Tardíos de la Exposición Prenatal/psicología , Alcoholismo/psicología , Animales , Antimetabolitos , Encéfalo/patología , Bromodesoxiuridina , Depresores del Sistema Nervioso Central/sangre , Digoxigenina , Encefalinas/biosíntesis , Etanol/sangre , Femenino , Técnica del Anticuerpo Fluorescente , Galanina/biosíntesis , Hipotálamo/efectos de los fármacos , Inmunohistoquímica , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Sistema Límbico/efectos de los fármacos , Neuropéptidos/biosíntesis , Neuropéptidos/fisiología , Orexinas , Embarazo , Efectos Tardíos de la Exposición Prenatal/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
We have previously demonstrated nerve growth factor (NGF) regulation of pituitary adenylate cyclase-activating polypeptide (PACAP)/receptors in bladder reflex pathways using a transgenic mouse model of chronic NGF overexpression in the bladder using the urothelial-specific uroplakin II promoter. We have now explored the contribution of target-derived NGF in combination with cyclophosphamide (CYP)-induced cystitis to determine whether additional changes in neuropeptides/receptors are observed in micturition reflex pathways due to the presence of additional inflammatory mediators in the urinary bladder. Quantitative PCR was used to determine PACAP/vasoactive intestinal polypeptide (VIP), substance P, galanin, and receptor transcript expression in the urinary bladder (urothelium, detrusor) in mice with overexpression of NGF in the urothelium (NGF-OE) and wild-type (WT) mice with CYP-induced cystitis (4 h, 48 h, and chronic). With CYP-induced cystitis (4 h), WT and NGF-OE mice exhibited similar changes in galanin transcript expression in the urothelium (30-fold increase) and detrusor (threefold increase). In contrast, PACAP, VIP, and substance P transcripts exhibited differential changes in WT and NGF-OE with CYP-induced cystitis. PAC1, VPAC1, and VPAC2 transcript expression also exhibited differential responses in NGF-OE mice that were tissue (urothelium vs. detrusor) and CYP-induced cystitis duration-dependent. Using conscious cystometry, NGF-OE mice treated with CYP exhibited significant (p ≤ 0.01) increases in voiding frequency above that observed in control NGF-OE mice. In addition, no changes in the electrical properties of the major pelvic ganglia neurons of NGF-OE mice were detected using intracellular recording, suggesting that the urinary bladder phenotype in NGF-OE mice is not influenced by changes in the efferent limb of the micturition reflex. These studies are consistent with target-derived NGF and other inflammatory mediators affecting neurochemical plasticity and the reflex function of micturition pathways.
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Cistitis/metabolismo , Regulación de la Expresión Génica/fisiología , Músculo Liso/metabolismo , Factor de Crecimiento Nervioso/fisiología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/biosíntesis , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/biosíntesis , Receptores de Tipo II del Péptido Intestinal Vasoactivo/biosíntesis , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/biosíntesis , Vejiga Urinaria/metabolismo , Micción/fisiología , Urotelio/metabolismo , Péptido Intestinal Vasoactivo/biosíntesis , Animales , Ciclofosfamida/administración & dosificación , Ciclofosfamida/toxicidad , Cistitis/inducido químicamente , Cistitis/genética , Cistitis/fisiopatología , Progresión de la Enfermedad , Galanina/biosíntesis , Galanina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor de Crecimiento Nervioso/biosíntesis , Factor de Crecimiento Nervioso/genética , Especificidad de Órganos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/genética , Receptores de Tipo II del Péptido Intestinal Vasoactivo/genética , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Reflejo Anormal , Sustancia P/biosíntesis , Sustancia P/genética , Péptido Intestinal Vasoactivo/genéticaRESUMEN
Autonomic neurons commonly respond to injury/axotomy with an increased expression of neuropeptides including galanin and pituitary adenylyl cyclase-activating polypeptide (PACAP). The increased peptide expression may enhance neuronal survival and axonal regeneration. Using quantitative (Q) PCR and immunocytochemistry, the present study tested whether galanin expression increased in male mouse major pelvic ganglia (MPG) neurons in response to injury. Galanin transcript expression increased significantly in MPG neurons following 72 h in explant culture and 72 h after unilateral transection of the cavernous nerve. Under both conditions, the increase in galanin transcript levels was greater than the increase in PACAP transcript levels. In control MPG, galanin-IR nerve fibers formed pericellular arrangements around MPG neurons although few galanin-IR cells were evident and many of the galanin-IR cells may be small intensely fluorescent (SIF) cells. In 3-day-cultured MPGs, many more galanin-IR cells and nerve fibers were noted. The increased galanin expression was most apparent in neurons that were also immunoreactive for neuronal nitric oxide synthase, rather than tyrosine hydroxylase. Some explant-cultured MPG neurons exhibited immunoreactivity to galanin and PACAP. As reported previously for PACAP, there is an injury-induced increase in MPG galanin expression, which occurs preferentially in the parasympathetic postganglionic neurons.
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Galanina/biosíntesis , Ganglios Parasimpáticos/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Fibras Parasimpáticas Posganglionares/lesiones , Pene/inervación , Traumatismos de los Nervios Periféricos/metabolismo , Animales , Axotomía , Técnica del Anticuerpo Fluorescente Directa , Galanina/genética , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Óxido Nítrico Sintasa de Tipo I/análisis , Técnicas de Cultivo de Órganos , Fibras Parasimpáticas Posganglionares/metabolismo , Traumatismos de los Nervios Periféricos/genética , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/biosíntesis , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , ARN Mensajero/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Galanina Tipo 1/biosíntesis , Receptor de Galanina Tipo 1/genética , Receptor de Galanina Tipo 2/biosíntesis , Receptor de Galanina Tipo 2/genética , Receptor de Galanina Tipo 3/biosíntesis , Receptor de Galanina Tipo 3/genética , Factores de Tiempo , Tirosina 3-Monooxigenasa/análisisRESUMEN
Despite the known importance of galanin in the nervous system of vertebrates, the galanin gene structure and expression and the consequences of galanin deficiency in developing zebrafish are unknown. We cloned the galanin gene and analyzed its expression by using in situ hybridization, PCR, and immunocytochemistry throughout the early development of zebrafish until the end of the first week of life. The single zebrafish galanin gene encoded for a single amidated galanin peptide and a galanin message-associated peptide. Two forms resulting from alternative processing were identified. Galanin mRNA was maternally expressed and found in developing fish throughout early development. In situ hybridization showed the first positive neurons in three groups in the brain at 28 hours postfertilization. At 2 days postfertilization, three prosencephalic neuron groups were seen in the preoptic area and in rostral and caudal periventricular hypothalamus. In addition, two other groups of weakly stained neurons were visible, one in the midbrain and another in the hindbrain. Translation inhibition of galanin mRNA with morpholino oligonucleotides caused complete disappearance of galanin immunoreactivity in the brain until 7 dpf and did not induce known cascades of nonspecific pathways or morphological abnormalities. A minor disturbance of sensory ganglia was found. Galanin knockdown did not alter the expression of tyrosine hydroxylases 1 and 2, choline acetyltransferase, histidine decarboxylase, or orexin mRNA. The results suggest that galanin does not regulate the development of these key markers of specific neurons, although galanin-expressing fibers were in a close spatial proximity to several neurons of these neuronal populations.
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Galanina/genética , Neurogénesis/genética , Neuronas/metabolismo , Pez Cebra , Animales , Encéfalo/metabolismo , Galanina/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Inmunohistoquímica , Hibridación in Situ , Larva , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TranscriptomaRESUMEN
AIM: This study aims to identify new genes not described previously that may be relevant in the etiology or pathophysiology of patients with Hirschsprung disease (HD). This was done by identifying differences in gene expression between normal and abnormal segments of bowel in HD patients compared with controls. METHODS: Full-thickness colonic tissue samples were taken from HD patients, both from the diseased (Ds) and normal segment of the colon (Nr), and from controls (Ct). Samples were further dissected into mucosa (MUC) and muscle (MUS). RNA was extracted and analyzed on Affymetrix Gene Chip Human Gene 1.0 ST arrays. Statistical analyses using ANOVA with a fold change cut off of 2 was applied to detect a number of differentially expressed genes. Selected genes were revalidated by quantitative real-time reverse transcriptase polymerase chain reaction. RESULTS: Thirty-four samples (18 MUS and 16 MUC) were analyzed. MUC (1.64 ± 0.46 µg/mg) and MUS (0.83 ± 0.48 µg/mg) showed good RNA extraction yield and quality. Of the 24,987 filtered on expression genes, MUS showed 220 genes with expression difference of 2-fold, out of which 120 genes were significant with P ≤ .05. Similarly, MUC demonstrated 206 genes with 2-fold changes and 9 had P ≤ .05. Some genes showing differential expression between groups and therefore subject to further analysis were RELN, GAL, GAP43, NRSN1, and GABRG2. CONCLUSION: Analyzed data showed significant differences in expression of above sets of genes with up- and down-regulation, which has not been described before in HD and could have a role in pathogenesis of this condition.