RESUMEN
The striatum is the main input structure of the basal ganglia. Distinct striatal subfields are involved in voluntary movement generation and cognitive and emotional tasks, but little is known about the morphological and molecular differences of striatal subregions. The ventrolateral subfield of the striatum (VLS) is the orofacial projection field of the sensorimotor cortex and is involved in the development of orofacial dyskinesias, involuntary chewing-like movements that often accompany long-term neuroleptic treatment. The biological basis for this particular vulnerability of the VLS is not known. Potassium channels are known to be strategically localized within the striatum. In search of possible molecular correlates of the specific vulnerability of the VLS, we analyzed the expression of voltage-gated potassium channels in rodent and primate brains using qPCR, in situ hybridization, and immunocytochemical single and double staining. Here we describe a novel, giant, non-cholinergic interneuron within the VLS. This neuron coexpresses the vesicular GABA transporter, the calcium-binding protein parvalbumin (PV), and the Kv3.3 potassium channel subunit. This novel neuron is much larger than PV neurons in other striatal regions, displays characteristic electrophysiological properties, and, most importantly, is restricted to the VLS. Consequently, the giant striatal Kv3.3-expressing PV neuron may link compromised Kv3 channel function and VLS-based orofacial dyskinesias.
Asunto(s)
Discinesias , Parvalbúminas , Animales , Cuerpo Estriado/metabolismo , Discinesias/metabolismo , Interneuronas/metabolismo , Parvalbúminas/metabolismo , Canales de Potasio/metabolismo , Canales de Potasio Shaw/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos InhibidoresRESUMEN
The bed bug Cimex lectularius is a globally distributed human ectoparasite with fascinating biology. It has recently acquired resistance against a broad range of insecticides, causing a worldwide increase in bed bug infestations. The recent annotation of the bed bug genome revealed a full complement of neuropeptide and neuropeptide receptor genes in this species. With regard to the biology of C. lectularius, neuropeptide signaling is especially interesting because it regulates feeding, diuresis, digestion, as well as reproduction and also provides potential new targets for chemical control. To identify which neuropeptides are translated from the genome-predicted genes, we performed a comprehensive peptidomic analysis of the central nervous system of the bed bug. We identified in total 144 different peptides from 29 precursors, of which at least 67 likely present bioactive mature neuropeptides. C. lectularius corazonin and myosuppressin are unique and deviate considerably from the canonical insect consensus sequences. Several identified neuropeptides likely act as hormones, as evidenced by the occurrence of respective mass signals and immunoreactivity in neurohemal structures. Our data provide the most comprehensive peptidome of a Heteropteran species so far and in comparison suggest that a hematophageous life style does not require qualitative adaptations of the insect peptidome.
Asunto(s)
Chinches/química , Sistema Nervioso Central/química , Neuropéptidos/análisis , Animales , Infestaciones Ectoparasitarias , Genoma , Hormonas , Proteínas de Insectos , ProteómicaRESUMEN
BACKGROUND: Neuropeptides are key players in information transfer and act as important regulators of development, growth, metabolism, and reproduction within multi-cellular animal organisms (Metazoa). These short protein-like substances show a high degree of structural variability and are recognized as the most diverse group of messenger molecules. We used transcriptome sequences from the 1KITE (1K Insect Transcriptome Evolution) project to search for neuropeptide coding sequences in 24 species from the non-pterygote hexapod lineages Protura (coneheads), Collembola (springtails), Diplura (two-pronged bristletails), Archaeognatha (jumping bristletails), and Zygentoma (silverfish and firebrats), which are often referred to as "basal" hexapods. Phylogenetically, Protura, Collembola, Diplura, and Archaeognatha are currently placed between Remipedia and Pterygota (winged insects); Zygentoma is the sistergroup of Pterygota. The Remipedia are assumed to be among the closest relatives of all hexapods and belong to the crustaceans. RESULTS: We identified neuropeptide precursor sequences within whole-body transcriptome data from these five hexapod groups and complemented this dataset with homologous sequences from three crustaceans (including Daphnia pulex), three myriapods, and the fruit fly Drosophila melanogaster. Our results indicate that the reported loss of several neuropeptide genes in a number of winged insects, particularly holometabolous insects, is a trend that has occurred within Pterygota. The neuropeptide precursor sequences of the non-pterygote hexapods show numerous amino acid substitutions, gene duplications, variants following alternative splicing, and numbers of paracopies. Nevertheless, most of these features fall within the range of variation known from pterygote insects. However, the capa/pyrokinin genes of non-pterygote hexapods provide an interesting example of rapid evolution, including duplication of a neuropeptide gene encoding different ligands. CONCLUSIONS: Our findings delineate a basic pattern of neuropeptide sequences that existed before lineage-specific developments occurred during the evolution of pterygote insects.
Asunto(s)
Evolución Molecular , Proteínas de Insectos/genética , Insectos/genética , Neuropéptidos/genética , Secuencia de Aminoácidos , Animales , Artrópodos/clasificación , Artrópodos/genética , Proteínas de Insectos/química , Insectos/clasificación , Insectos/metabolismo , Datos de Secuencia Molecular , Neuropéptidos/química , Filogenia , TranscriptomaRESUMEN
Among all K2P channels, TASK-3 shows the most widespread expression in rat brain, regulating neuronal excitability and transmitter release. Using a recently purified and characterized polyclonal monospecific antibody against TASK-3, the entire rat brain was immunocytochemically analyzed for expression of TASK-3 protein. Besides its well-known strong expression in motoneurons and monoaminergic and cholinergic neurons, TASK-3 expression was found in most neurons throughout the brain. However, it was not detected in certain neuronal populations, and neuropil staining was restricted to few areas. Also, it was absent in adult glial cells. In hypothalamic areas, TASK-3 was particularly strongly expressed in the supraoptic and suprachiasmatic nuclei, whereas other hypothalamic nuclei showed lower protein levels. Immunostaining of hippocampal CA1 and CA3 pyramidal neurons showed strongest expression, together with clear staining of CA3 mossy fibers and marked staining also in the dentate gyrus granule cells. In neocortical areas, most neurons expressed TASK-3 with a somatodendritic localization, most obvious in layer V pyramidal neurons. In the cerebellum, TASK-3 protein was found mainly in neurons and neuropil of the granular cell layer, whereas Purkinje cells were only faintly positive. Particularly weak expression was demonstrated in the forebrain. This report provides a comprehensive overview of TASK-3 protein expression in the rat brain.
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Química Encefálica , Encéfalo/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Encéfalo/citología , Inmunohistoquímica , Masculino , Canales de Potasio de Dominio Poro en Tándem/análisis , Transporte de Proteínas , Ratas , Ratas WistarRESUMEN
Polyamines (PAs) including putrescine (PUT), spermidine (SPD) and spermine (SPM) are small, versatile molecules with two or more positively charged amino groups. Despite their importance for almost all forms of life, their specific roles in molecular and cellular biology remain partly unknown. The molecular structures of PAs suggest two presumable biological functions: (i) as potential buffer systems and (ii) as interactants with poly-negatively charged molecules like nucleic acids. The present report focuses on the question, whether the molecular structures of PAs are essential for such functions, or whether other simple molecules like small peptides with closely spaced positively charged side chains might be suitable as well. Consequently, we created titration curves for PUT, SPD, and SPM, as well as for oligolysines like tri-, tetra-, and penta-lysine. None of the molecules provided substantial buffering capacity at physiological intracellular pH values. Apparently, the most important mechanism for intracellular pH homeostasis in neurons is not a buffer system but is provided by the actions of the sodium-hydrogen and the bicarbonate-chloride antiporters. In a similar approach we investigated the interaction with DNA by following the extinction at 260 nm when titrating DNA with the above molecules. Again, PUT and tri-lysine were not able to interact with herring sperm DNA, while SPD and SPM were. Obviously, the presence of several positively charged groups on its own is not sufficient for the interaction with nucleic acids. Instead, the precise spacing of these groups is necessary for biological activity.
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ADN , Péptidos , Poliaminas , ARN , Concentración de Iones de Hidrógeno , ADN/química , ADN/metabolismo , Tampones (Química) , ARN/química , ARN/metabolismo , Poliaminas/química , Poliaminas/metabolismo , Péptidos/química , Péptidos/metabolismo , Animales , Espermidina/química , Espermidina/metabolismo , Espermina/química , Espermina/metabolismo , Putrescina/química , Putrescina/metabolismoRESUMEN
With a single gene encoding HV1 channel, proton channel diversity is particularly low in mammals compared to other members of the superfamily of voltage-gated ion channels. Nonetheless, mammalian HV1 channels are expressed in many different tissues and cell types where they exert various functions. In the first part of this review, we regard novel aspects of the functional expression of HV1 channels in mammals by differentially comparing their involvement in (1) close conjunction with the NADPH oxidase complex responsible for the respiratory burst of phagocytes, and (2) in respiratory burst independent functions such as pH homeostasis or acid extrusion. In the second part, we dissect expression of HV channels within the eukaryotic tree of life, revealing the immense diversity of the channel in other phylae, such as mollusks or dinoflagellates, where several genes encoding HV channels can be found within a single species. In the last part, a comprehensive overview of the biophysical properties of a set of twenty different HV channels characterized electrophysiologically, from Mammalia to unicellular protists, is given.
Asunto(s)
Canales Iónicos , Protones , Animales , Canales Iónicos/genética , Canales Iónicos/metabolismo , Membrana Celular/metabolismo , Estallido Respiratorio , Eucariontes/metabolismo , Mamíferos/metabolismoRESUMEN
Recently, three proton channels (HV ) have been identified and characterized in Aplysia californica (AcHV 1-3). Focusing on AcHV 1 and AcHV 2, analysis of Transcriptome Shotgun Assembly and genomic databases of 91 molluscs identified HV homologous channels in other molluscs: channels homologous to AcHV 1 and to AcHV 2 were found in 90 species (56 full-length sequences) and in 33 species (18 full-length sequences), respectively. Here, we report the discovery of a fourth distinct proton channel family, HV 4. This new family has high homology to AcHV 1 and AcHV 2 and was identified only in bivalvian molluscs (13 species, 12 full-length sequences). Typically, these channels possess an extracellular S1-S2 loop of intermediate size (~ 20 amino acids) compared to the shorter loops of molluscan HV 1 channels (~ 13 amino acids) and the much larger loops of molluscan HV 2 channels (> 65 amino acids). The characteristic voltage-sensor motif in S4 possesses only two arginine residues with the common third arginine being replaced by a lysine. Moreover, HV 4 channels are much smaller with only around 200 amino acids in total length. The smallest functional channel found so far in nature (189 amino acids) is expressed in the pacific oyster Crassostrea gigas (CgHV 4) and might be considered an archetypical minimal proton channel. Functional expression and electrophysiological characterization demonstrated that CgHV 4 shares distinctive hallmarks of other investigated proton channels as high proton selectivity, slow activation, and pH- and voltage-regulated gating. This work is the first description of a HV 4 type channel, adding a new member to the recently expanded family of proton channels.
Asunto(s)
Canales Iónicos , Protones , Animales , Canales Iónicos/metabolismo , Activación del Canal Iónico/fisiología , Aminoácidos , Arginina , Moluscos/genética , Moluscos/metabolismoRESUMEN
Voltage-gated ion channels, whose first identified function was to generate action potentials, are divided into subfamilies with numerous members. The family of voltage-gated proton channels (HV ) is tiny. To date, all species found to express HV have exclusively one gene that codes for this unique ion channel. Here we report the discovery and characterization of three proton channel genes in the classical model system of neural plasticity, Aplysia californica. The three channels (AcHV 1, AcHV 2, and AcHV 3) are distributed throughout the whole animal. Patch-clamp analysis confirmed proton selectivity of these channels but they all differed markedly in gating. AcHV 1 gating resembled HV in mammalian cells where it is responsible for proton extrusion and charge compensation. AcHV 2 activates more negatively and conducts extensive inward proton current, properties likely to acidify the cytosol. AcHV 3, which differs from AcHV 1 and AcHV 2 in lacking the first arginine in the S4 helix, exhibits proton selective leak currents and weak voltage dependence. We report the expansion of the proton channel family, demonstrating for the first time the expression of three functionally distinct proton channels in a single species.
Asunto(s)
Activación del Canal Iónico , Protones , Animales , Activación del Canal Iónico/fisiología , Canales Iónicos/metabolismo , Arginina , Citosol/metabolismo , Mamíferos/metabolismoRESUMEN
Motor neurons are large cholinergic neurons located in the brain stem and spinal cord. In recent years, a functional role for TASK channels in cellular excitability and vulnerability to anesthetics of motor neurons has been described. Using a polyclonal monospecific antibody against the tandem pore domain K(+) channel (K2P channel) TWIK-related acid-sensitive K(+) channel (TASK-3), we analyzed the expression of the TASK-3 protein in motor systems of the rat CNS. Immunocytochemical staining showed strong TASK-3 expression in motor neurons of the facial, trigeminal, ambiguus, and hypoglossal nuclei. Oculomotor nuclei (including trochlear and abducens nucleus) were also strongly positive for TASK-3. The parasympathetic Edinger-Westphal nucleus and dorsal vagal nucleus showed significant, but weaker expression compared with somato- and branchiomotoric neurons. In addition, motor neurons in the anterior horn of the spinal cord were also strongly labeled for TASK-3 immunoreactivity. Based on morphological criteria, TASK-3 was found in the somatodendritic compartment of motor neurons. Cellular staining using methyl green and immunofluorescence double-labeling with anti-vesicular acetylcholine transporter (anti-vAChT) indicated ubiquitous TASK-3 expression in motor neurons, whereas in other brain regions TASK-3 showed a widespread but not ubiquitous expression. In situ hybridization using a TASK-3 specific riboprobe verified the expression of TASK-3 in motor neurons at the mRNA level.
Asunto(s)
Neuronas Motoras/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Inmunohistoquímica , Masculino , Neuronas Motoras/citología , Transporte de Proteínas , Ratas , Ratas Wistar , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
Voltage-gated proton channels (HV 1) are expressed in eukaryotes, including basal hexapods and polyneopteran insects. However, currently, there is little known about HV 1 channels in insects. A characteristic aspartate (Asp) that functions as the proton selectivity filter (SF) and the RxWRxxR voltage-sensor motif are conserved structural elements in HV 1 channels. By analysing Transcriptome Shotgun Assembly (TSA) databases, we found 33 polyneopteran species meeting these structural requirements. Unexpectedly, an unusual natural variation Asp to glutamate (Glu) at SF was found in Phasmatodea and Mantophasmatodea. Additionally, we analysed the expression and function of HV 1 in the phasmatodean stick insect Extatosoma tiaratum (Et). EtHV 1 is strongly expressed in nervous tissue and shows pronounced inward proton conduction. This is the first study of a natural occurring Glu within the SF of a functional HV 1 and might be instrumental in uncovering the physiological function of HV 1 in insects.
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Canales Iónicos , Protones , Animales , Insectos/metabolismo , Canales Iónicos/genéticaRESUMEN
Polyamines (PAs) are small, versatile molecules with two or more nitrogen-containing positively charged groups and provide widespread biological functions. Most of these aspects are well known and covered by quite a number of excellent surveys. Here, the present review includes novel aspects and questions: (1) It summarizes the role of most natural and some important synthetic PAs. (2) It depicts PA uptake from nutrition and bacterial production in the intestinal system following loss of PAs via defecation. (3) It highlights the discrepancy between the high concentrations of PAs in the gut lumen and their low concentration in the blood plasma and cerebrospinal fluid, while concentrations in cellular cytoplasm are much higher. (4) The present review provides a novel and complete scheme for the biosynthesis of Pas, including glycine, glutamate, proline and others as PA precursors, and provides a hypothesis that the agmatine pathway may rescue putrescine production when ODC knockout seems to be lethal (solving the apparent contradiction in the literature). (5) It summarizes novel data on PA transport in brain glial cells explaining why these cells but not neurons preferentially accumulate PAs. (6) Finally, it provides a novel and complete scheme for PA interconversion, including hypusine, putreanine, and GABA (unique gliotransmitter) as end-products. Altogether, this review can serve as an updated contribution to understanding the PA mystery.
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Poliaminas , Espermina , Sistema Nervioso Central/metabolismo , Neuroglía/metabolismo , Poliaminas/metabolismo , Putrescina/metabolismo , Espermina/metabolismoRESUMEN
Large-conductance Ca(2+) -activated (BK) potassium channels are centrally involved in neurovascular coupling, immunity, and neural transmission. The ability to be synergistically activated by membrane depolarization, different ligands and intracellular Ca(2+) links intracellular signaling and membrane excitability. The diverse physiological functions of BK channels crucially depend on regulatory ß subunits. Although first studies characterized the neuronal distribution of BKß subunits in the rodent brain, it is largely unknown which ß subunit proteins are expressed in astrocytes and thus mediate these regulatory effects. We therefore analyzed the expression of BKß subunits in rat and mouse brain and glial cell cultures. A monospecific polyclonal antibody against the BKß4 channel subunit was raised, affinity-purified and extensively characterized. BKß4 and to a lesser degree BKß1 transcripts and protein were detected in several astrocytic populations and cultured cells. Particularly strong BKß4 immunostaining was detected in astrocytic progenitors derived from the subventricular zone. The overlapping expression of BKα and BKß4 in astrocytes implies a functional relationship and suggests that BKß4 is an important accessory ß subunit for astrocytic BK channels. In addition, BKß4 might exert effects independent of the α subunit as functional heterologous co-expression of Nav1.6 and BKß4 resulted in reduced Nav1.6 sodium currents. Thus, BKß4 expression in astrocytes likely participates in regulating astrocytic voltage gradients and maintaining K(+) homeostasis, hence enabling astrocytes to fulfill their complex regulatory influence on proper brain function.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Animales , Astrocitos/citología , Encéfalo/citología , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Células HEK293 , Homeostasis/genética , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Ratones , Ratones Endogámicos C57BL , Oocitos , Ratas , Ratas Wistar , Xenopus laevisRESUMEN
Monoaminergic and cholinergic systems are important regulators of cortical and subcortical systems, and a variety of vegetative functions are controlled by the respective neurotransmitters. Neuronal excitability and transmitter release of these neurons are strongly regulated by their potassium conductances carried by Kir and K(2P) channels. Here we describe the generation and characterization of a polyclonal monospecific antibody against rat TASK-3, a major brain K(2P) channel. After removal of cross-reactivities and affinity purification the antibody was characterized by ELISA, immunocytochemistry of TASK-3 transfected cells, and Western blots indicating that the antibody only detects TASK-3 protein, but not its paralogs TASK-1 and TASK-5. Western blot analysis of brain membrane fractions showed a single band around 45 kD, close to the predicted molecular weight of the TASK-3 protein. In addition, specific immunolabeling using the anti-TASK-3 antibody in Western blot analysis and immunocytochemistry was blocked in a concentration dependent manner by its cognate antigen only. Immunocytochemical analysis of rat brain revealed strong expression of TASK-3 channels in serotoninergic neurons of the dorsal and median raphe, noradrenergic neurons of the locus coeruleus, histaminergic neurons of the tuberomammillary nucleus and in the cholinergic neurons of the basal nucleus of Meynert. Immunofluorescence double-labeling experiments with appropriate marker enzymes confirmed the expression of TASK-3 in cholinergic, serotoninergic, and noradrenergic neurons. In the dopaminergic system strong TASK-3 expression was found in the ventral tegmental area, whereas TASK-3 immunoreactivity in the substantia nigra compacta was only weak. All immunocytochemical results were supported by in situ hybridization using TASK-3 specific riboprobes.
Asunto(s)
Colina/metabolismo , Neuronas/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Especificidad de Anticuerpos/inmunología , Núcleo Basal de Meynert/citología , Núcleo Basal de Meynert/metabolismo , Western Blotting , Encéfalo/metabolismo , Dopamina/metabolismo , Histamina/metabolismo , Área Hipotalámica Lateral/citología , Área Hipotalámica Lateral/metabolismo , Inmunohistoquímica , Locus Coeruleus/citología , Locus Coeruleus/metabolismo , Masculino , Neuronas/citología , Norepinefrina/metabolismo , Núcleos del Rafe/metabolismo , Ratas , Ratas Wistar , Serotonina/metabolismo , TransfecciónRESUMEN
In rat brain slices, the Kv channel blocker 4-aminopyridine (4-AP) induces seizure-like events. This effect is absent in slices from chronic epileptic rats generated using the kainic acid model. The reason for this phenomenon remained elusive as an altered expression level of Kv channels was ruled out as a mechanism. We recently described that the Ile400Val RNA editing of Kv1.1 generates 4-AP-insensitive Kv1 channels (Kv1.1(I400V)). We therefore hypothesized that altered RNA editing levels account for the reduced ictogenic potency of 4-AP in chronic epileptic rats. We found fourfold increased RNA editing ratios in the entorhinal cortex of chronic epileptic animals compared to healthy control animals. Electrophysiologic recordings in Xenopus oocytes revealed that the observed increased Kv1.1(I400V) editing level can in fact lead to significant loss of 4-AP sensitivity. Our data suggest that altered Kv1.1(I400V) RNA editing contributes to the reduced ictogenic potential of 4-AP in chronic epileptic rats.
Asunto(s)
4-Aminopiridina/farmacología , Convulsivantes/farmacología , Modelos Animales de Enfermedad , Epilepsia/inducido químicamente , Epilepsia/genética , Hipocampo/efectos de los fármacos , Canal de Potasio Kv.1.1/genética , Bloqueadores de los Canales de Potasio/farmacología , Edición de ARN/genética , Animales , Corteza Entorrinal/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Técnicas de Cultivo de TejidosRESUMEN
In insects, inhibitory neurotransmission is generally associated with members of the cys-loop ligand-gated anion channels, such as the glutamate-gated chloride channel (GluCl), the GABA-gated chloride channels (GABACl), and the histamine-gated chloride channels (HisCl). These ionotropic receptors are considered established target sites for the development of insecticides, and therefore it is necessary to obtain a better insight in their distribution, structure, and functional properties. Here, by combining electrophysiology and molecular biology techniques, we identified and characterized GluCl, GABACl, and HisCl in dorsal unpaired median (DUM) neurons of Locust migratoria. In whole cell patch-clamp recordings, application of glutamate, GABA, or histamine induced rapidly activating ionic currents. GluCls were sensitive to ibotenic acid and blocked by picrotoxin and fipronil. The pharmacological profile of the L. migratoria GABACl fitted neither the vertebrate GABA(A) nor GABA(C) receptor and was similar to the properties of the cloned Drosophila melanogaster GABA receptor subunit (Rdl). The expression of Rdl-like subunit-containing GABA receptors was shown at the molecular level using RT-PCR. Sequencing analysis indicated that the orthologous GABACl of D. melanogaster CG10357-A is expressed in DUM neurons of L. migratoria. Histamine-induced currents exhibited a fast onset and desensitized completely on continuous application of histamine. In conclusion, within the DUM neurons of L. migratoria, we identified three different cys-loop ligand-gated anion channels that use GABA, glutamate, or histamine as their neurotransmitter.
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Canales de Cloruro/metabolismo , Proteínas de Insectos/metabolismo , Neuronas/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Canales de Cloruro/genética , Ácido Glutámico/metabolismo , Histamina/metabolismo , Técnicas In Vitro , Proteínas de Insectos/genética , Locusta migratoria , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Neuronas/citología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia , Factores de Tiempo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Large conductance Ca(2+)-activated BK channels are important regulators of action potential duration and firing frequency in many neurons. As the pore-forming subunits of BK channels are encoded by a single gene, channel diversity is mainly generated by alternative splicing and interaction with auxiliary beta-subunits (BKbeta1-4). In hypothalamic neurons several BK channel subtypes have been described electrophysiologically; however, the distribution of BKbeta subunits is unknown so far. Therefore, an antibody against the large extracellular loop of the BKbeta1 subunit was raised, freed from cross-reactivity against BKbeta2-4 and affinity-purified. The resulting polyclonal monospecific BKbeta1 antibody was characterized by Western blot analysis, ELISA techniques and immunocytochemical staining of BKbeta1-4-transfected CHO and COS-1 cells. Regional and cellular distribution in the rat hypothalamus was analysed by immunocytochemistry and in situ hybridization experiments. Immunocytochemical staining of rat hypothalamic neurons indicates strong BKbeta1 expression in the supraoptic nucleus and the magno- and parvocellular parts of the paraventricular nucleus. Lower expression was found in periventricular nucleus, the arcuate nucleus and in the median eminence. Immunostaining was predominantly localized to somata. In addition, pericytes and ependymal epithelial cells showed BKbeta1 labelling. In all cases immunocytochemical results were supported by in situ hybridization.
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Hipotálamo/citología , Hipotálamo/metabolismo , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Neuronas/metabolismo , Animales , Especificidad de Anticuerpos , Western Blotting , Línea Celular , Masculino , Neuronas/citología , Especificidad de Órganos , Ratas , Ratas Wistar , Transducción de Señal , TransfecciónRESUMEN
Glutamatergic inputs to the ventral tegmental area (VTA), thought crucial to the capacity of the VTA to detect and signal stimulus salience, have been reported to arise in but a few structures. However, the afferent system of the VTA comprises very abundant neurons within a large formation extending from the prefrontal cortex to the caudal brainstem. Neurons in nearly all parts of this continuum may be glutamatergic and equivalently important to VTA function. Thus, we sought to identify the full range of glutamatergic inputs to the VTA by combining retrograde transport of wheat germ agglutinin-bound gold after injections into the VTA with nonisotopic in situ hybridization of the vesicular glutamate transporters (VGLUTs) 1, 2, and 3. We found glutamatergic neurons innervating the VTA in almost all structures projecting there and that a majority of these are subcortical and VGLUT2 mRNA positive. The tremendous convergence of glutamatergic afferents from many brain areas in the VTA suggests that (1) the function of the VTA requires integration of manifold and diverse bits of information and (2) the activity of the VTA reflects the ongoing activities of various combinations of its afferents.
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Vías Aferentes/fisiología , Área Tegmental Ventral/fisiología , Proteínas de Transporte Vesicular de Glutamato/fisiología , Vías Aferentes/química , Animales , Tronco Encefálico/química , Tronco Encefálico/fisiología , Masculino , Corteza Prefrontal/química , Corteza Prefrontal/fisiología , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Área Tegmental Ventral/química , Proteínas de Transporte Vesicular de Glutamato/análisisRESUMEN
The serotonergic system of the brainstem raphe is involved in mood control, the sleep-wake cycle, autonomic function, and stress response. The axons of certain dorsal raphe neurons form a dense serotonergic supraependymal plexus lining the brain ventricles, likely regulating ependymal metabolism and activity including ciliary movements and glucose homeostasis. In raphe neurons, serotonin exerts its function partly via 5-HT autoreceptors and G protein-gated inwardly rectifying potassium channels (Kir3/GIRK). To consider a similar mechanism in supraependymal fibres we examined immunocytochemically the distribution of Kir3 potassium channel subunits on supraependymal axons. In the present study, we show that the Kir3.3 subunit protein is expressed in raphe-derived axons at the light and electron microscopic level, but none of the other Kir3 subfamily members or the K(ATP) channel subunits Kir6.1 and Kir6.2. Thus, Kir3.3 containing potassium channels may be of functional importance in autoregulation and excitability of supraependymal fibres and the complex serotonergic regulation along the parenchyma/CSF border.
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Axones/metabolismo , Epéndimo/citología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Neuronas/citología , Núcleos del Rafe/citología , Animales , Axones/ultraestructura , Epéndimo/ultraestructura , Microscopía Inmunoelectrónica , Ratas , Ratas Wistar , Serotonina/metabolismoAsunto(s)
Autoanticuerpos/sangre , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Síndrome de Guillain-Barré/sangre , Factores de Crecimiento Nervioso/inmunología , Factores de Crecimiento Nervioso/metabolismo , Adulto , Anciano , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Síndrome de Guillain-Barré/inmunología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
UNLABELLED: The voltage-gated proton channel 1 (HV 1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. HV 1 has been identified in a wide range of eukaryotes throughout the animal kingdom, with the exception of insects. Therefore, it has been proposed that insects do not possess an HV 1 channel. In the present study, we report the existence of an HV 1-type proton channel in insects. We searched insect transcriptome shotgun assembly (TSA) sequence databases and found putative HV 1 orthologues in various polyneopteran insects. To confirm that these putative HV 1 orthologues were functional channels, we studied the HV 1 channel of Nicoletia phytophila (NpHV 1), an insect of the Zygentoma order, in more detail. NpHV 1 comprises 239 amino acids and is 33% identical to the human voltage-gated proton channel 1. Patch clamp measurements in a heterologous expression system showed proton selectivity, as well as pH- and voltage-dependent gating. Interestingly, NpHV 1 shows slightly enhanced pH-dependent gating compared to the human channel. Mutations in the first transmembrane segment at position 66 (Asp66), the presumed selectivity filter, lead to a loss of proton-selective conduction, confirming the importance of this aspartate residue in voltage-gated proton channels. DATABASE: Nucleotide sequence data have been deposited in the GenBank database under accession number KT780722.