RESUMEN
α3ß4 nicotinic acetylcholine receptors (nARs) are pentameric ligand-gated cation channels that function in peripheral tissue and in the peripheral and central nervous systems, where they are critical mediators of ganglionic synaptic transmission and modulators of reward-related behaviours. In the pentamer, two α3ß4 subunit couples provide ligand-binding sites, and the fifth single (accessory) subunit (α3 or ß4) regulates receptor trafficking from the endoplasmic reticulum to the cell surface. A number of rare missense variants of the human ß4 subunit have recently been linked to nicotine dependence and/or sporadic amyotrophic lateral sclerosis, and altered responses to nicotine have been reported for these variants; however, it is unknown whether the effects of mutations depend on the subunit within the ligand-binding couples and/or on the fifth subunit. Here, by expressing single populations of pentameric receptors with fixed stoichiometry in cultured cells, we investigated the effect of ß4 variants in the fifth position on the assembly and surface exposure of α3ß4 nAChRs. The results demonstrate that the missense mutations in the accessory subunit alone, despite not affecting the assembly of α3ß4 receptors, alter their trafficking and surface localisation. Thus, altered trafficking of an otherwise functional nAChR may underlie the pathogenic effects of these mutations.
Asunto(s)
Mutación Missense , Receptores Nicotínicos , Humanos , Ligandos , Receptores Nicotínicos/metabolismo , Nicotina/metabolismo , Membrana Celular/metabolismoRESUMEN
Microglia cells are active players in regulating synaptic development and plasticity in the brain. However, how they influence the normal functioning of synapses is largely unknown. In this study, we characterized the effects of pharmacological microglia depletion, achieved by administration of PLX5622, on hippocampal CA3-CA1 synapses of adult wild type mice. Following microglial depletion, we observed a reduction of spontaneous and evoked glutamatergic activity associated with a decrease of dendritic spine density. We also observed the appearance of immature synaptic features and higher levels of plasticity. Microglia depleted mice showed a deficit in the acquisition of the Novel Object Recognition task. These events were accompanied by hippocampal astrogliosis, although in the absence ofneuroinflammatory condition. PLX-induced synaptic changes were absent in Cx3cr1-/- mice, highlighting the role of CX3CL1/CX3CR1 axis in microglia control of synaptic functioning. Remarkably, microglia repopulation after PLX5622 withdrawal was associated with the recovery of hippocampal synapses and learning functions. Altogether, these data demonstrate that microglia contribute to normal synaptic functioning in the adult brain and that their removal induces reversible changes in organization and activity of glutamatergic synapses.
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Microglía , Neuronas , Animales , Encéfalo , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Hipocampo , Ratones , Compuestos Orgánicos/farmacología , Sinapsis/fisiologíaRESUMEN
Glioblastomas (GBMs), the most frequent brain tumours, are highly invasive and their prognosis is still poor despite the use of combination treatment. MG624 is a 4-oxystilbene derivative that is active on α7- and α9-containing neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Hybridisation of MG624 with a non-nicotinic resveratrol-derived pro-oxidant mitocan has led to two novel compounds (StN-4 and StN-8) that are more potent than MG624 in reducing the viability of GBM cells, but less potent in reducing the viability of mouse astrocytes. Functional analysis of their activity on α7 receptors showed that StN-4 is a silent agonist, whereas StN-8 is a full antagonist, and neither alters intracellular [Ca2+] levels when acutely applied to U87MG cells. After 72 h of exposure, both compounds decreased U87MG cell proliferation, and pAKT and oxphos ATP levels, but only StN-4 led to a significant accumulation of cells in phase G1/G0 and increased apoptosis. One hour of exposure to either compound also decreased the mitochondrial and cytoplasmic ATP production of U87MG cells, and this was not paralleled by any increase in the production of reactive oxygen species. Knocking down the α9 subunit (which is expressed at relatively high levels in U87MG cells) decreased the potency of the effects of both compounds on cell viability, but cell proliferation, ATP production, pAKT levels were unaffected by the presence of the noncell-permeable α7/α9-selective antagonist αBungarotoxin. These last findings suggest that the anti-tumoral effects of StN-4 and StN-8 on GBM cells are not only due to their action on nAChRs, but also to other non-nicotinic mechanisms.
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Compuestos de Amonio/farmacología , Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Estilbenos/farmacología , Adenosina Trifosfato/metabolismo , Animales , Astrocitos/efectos de los fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Fenómenos Fisiológicos Celulares/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Ligandos , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores Nicotínicos/genética , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
The kinetics of IPSCs influence many neuronal processes, such as the frequencies of oscillations and the duration of shunting inhibition. The subunit composition of recombinant GABA(A) receptors (GABA(A)Rs) strongly affects the deactivation kinetics of GABA-evoked currents. However, for GABAergic synapses, the relationship between subunit composition and IPSC decay is less clear. Here we addressed this by combining whole-cell recordings of miniature IPSCs (mIPSCs) and quantitative immunolocalization of synaptic GABA(A)R subunits. In cerebellar stellate, thalamic relay, and main olfactory bulb (MOB) deep short-axon cells of Wistar rats, the only synaptic α subunit was α1, and zolpidem-sensitive mIPSCs had weighted decay time constants (τ(w)) of 4-6 ms. Nucleus reticularis thalami neurons expressed only α3 as the synaptic α subunit and exhibited slow (τ(w) = 28 ms), zolpidem-insensitive mIPSCs. By contrast, MOB external tufted cells contained two α subunit types (α1 and α3) at their synapses. Quantitative analysis of multiple immunolabeled images revealed small within-cell, but large between-cell, variability in synaptic α1/α3 ratios. This corresponded to large cell-to-cell variability in the decay (τ(w) = 3-30 ms) and zolpidem sensitivity of mIPSCs. Currents evoked by rapid application of GABA to patches excised from HEK cells expressing different mixtures of α1 and α3 subunits displayed highly variable deactivation times that correlated with the α1/α3 cDNA ratio. Our results demonstrate that diversity in the decay of IPSCs can be generated by varying the expression of different GABA(A)R subunits that alone confer different decay kinetics, allowing the time course of inhibition to be tuned to individual cellular requirements.
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Potenciales Postsinápticos Inhibidores/fisiología , Inhibición Neural/fisiología , Neuronas/fisiología , Receptores de GABA-A/metabolismo , Sinapsis/fisiología , Animales , Animales Recién Nacidos , Proteínas Portadoras/metabolismo , Cerebelo/citología , Cerebelo/metabolismo , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , GABAérgicos/farmacología , Expresión Génica/fisiología , Humanos , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Masculino , Proteínas de la Membrana/metabolismo , Inhibición Neural/efectos de los fármacos , Vías Nerviosas/fisiología , Bulbo Olfatorio/citología , Bulbo Olfatorio/metabolismo , Técnicas de Placa-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Ratas Wistar , Receptores de GABA-A/genética , Sinapsis/efectos de los fármacos , Tálamo/citología , Tálamo/metabolismo , Factores de Tiempo , Transfección , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Ionotropic glutamate receptors, which underlie a majority of excitatory synaptic transmission in the CNS, associate with transmembrane proteins that modify their intracellular trafficking and channel gating. Significant advances have been made in our understanding of AMPA-type glutamate receptor (AMPAR) regulation by transmembrane AMPAR regulatory proteins. Less is known about the functional influence of cornichons-unrelated AMPAR-interacting proteins, identified by proteomic analysis. Here we confirm that cornichon homologs 2 and 3 (CNIH-2 and CNIH-3), but not CNIH-1, slow the deactivation and desensitization of both GluA2-containing calcium-impermeable and GluA2-lacking calcium-permeable (CP) AMPARs expressed in tsA201 cells. CNIH-2 and -3 also enhanced the glutamate sensitivity, single-channel conductance, and calcium permeability of CP-AMPARs while decreasing their block by intracellular polyamines. We examined the potential effects of CNIHs on native AMPARs by recording from rat optic nerve oligodendrocyte precursor cells (OPCs), known to express a significant population of CP-AMPARs. These glial cells exhibited surface labeling with an anti-CNIH-2/3 antibody. Two features of their AMPAR-mediated currents-the relative efficacy of the partial agonist kainate (I(KA)/I(Glu) ratio 0.4) and a greater than fivefold potentiation of kainate responses by cyclothiazide-suggest AMPAR association with CNIHs. Additionally, overexpression of CNIH-3 in OPCs markedly slowed AMPAR desensitization. Together, our experiments support the view that CNIHs are capable of altering key properties of AMPARs and suggest that they may do so in glia.
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Proteínas del Huevo/metabolismo , Proteínas de la Membrana/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Receptores AMPA/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Células Cultivadas , Proteínas del Huevo/genética , Agonistas de Aminoácidos Excitadores/farmacología , Femenino , Ácido Glutámico/farmacología , Humanos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/genética , Ácido Kaínico/farmacología , Masculino , Proteínas de la Membrana/genética , Neuroglía/citología , Neuroglía/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Nervio Óptico/citología , Nervio Óptico/efectos de los fármacos , Nervio Óptico/metabolismo , Ratas , Receptores AMPA/genética , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genética , TransfecciónRESUMEN
The mechanisms of communication between the brain and the immune cells are still largely unclear. Here, we characterize the populations of resident natural killer (NK) cells and innate lymphoid cells (ILC) 1 in the meningeal dura layer of adult mice. We describe that ILC1/NK cell-derived interferon-γ and acetylcholine can contribute to the modulation of brain homeostatic functions, shaping synaptic neuronal transmission and neurotransmitter levels with effects on mice behavior. In detail, the interferon-γ plays a role in the formation of non-spatial memory, tuning the frequency of GABAergic neurotransmission on cortical pyramidal neurons, while the acetylcholine is a mediator involved in the modulation of brain circuitries that regulate anxiety-like behavior. These findings disclose mechanisms of immune-to-brain communication that modulate brain functions under physiological conditions.
Asunto(s)
Acetilcolina , Interferón gamma , Animales , Ratones , Linfocitos , Inmunidad Innata , Células Asesinas Naturales , AnsiedadRESUMEN
High-affinity extrasynaptic GABA(A) receptors (GABA(A)Rs) are a prominent feature of cerebellar granule neurons and thalamic relay neurons. In both cell types, the presence of synaptic glomeruli would be expected to promote activation of these GABA(A)Rs, contributing to phasic spillover-mediated currents and tonic inhibition. However, the precise role of different receptor subtypes in these two phenomena is unclear. To address this question, we made recordings from neurons in acute brain slices from mice, and from tsA201 cells expressing recombinant GABA(A)Rs. We found that δ subunit-containing GABA(A)Rs of both cerebellar granule neurons and thalamic relay neurons of the lateral geniculate nucleus contributed to tonic conductance caused by ambient GABA but not to spillover-mediated currents. In the presence of a low "ambient" GABA concentration, recombinant "extrasynaptic" δ subunit-containing GABA(A)Rs exhibited profound desensitization, rendering them insensitive to brief synaptic- or spillover-like GABA transients. Together, our results demonstrate that phasic spillover and tonic inhibition reflect the activation of distinct receptor populations.
Asunto(s)
Receptores de GABA-A/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Línea Celular , Cerebelo/citología , Cerebelo/fisiología , Humanos , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/fisiología , Técnicas de Placa-Clamp , Subunidades de Proteína/fisiología , Ratas , Receptores de GABA-A/genética , Sinapsis/fisiología , Tálamo/citología , Tálamo/fisiología , TransfecciónRESUMEN
Nano- or microdevices, enabling simultaneous, long-term, multisite, cellular recording and stimulation from many excitable cells, are expected to make a strategic turn in basic and applied cardiology (particularly tissue engineering) and neuroscience. We propose an innovative approach aiming to elicit bioelectrical information from the cell membrane using an integrated circuit (IC) bearing a coating of nanowires on the chip surface. Nanowires grow directly on the backend of the ICs, thus allowing on-site amplification of bioelectric signals with uniform and controlled morphology and growth of the NWs on templates. To implement this technology, we evaluated the biocompatibility of silicon and zinc oxide nanowires (NWs), used as a seeding substrate for cells in culture, on two different primary cell lines. Human cardiac stromal cells were used to evaluate the effects of ZnO NWs of different lengths on cell behavior, morphology and growth, while BV-2 microglial-like cells and GH4-C1 neuroendocrine-like cell lines were used to evaluate cell membrane-NW interaction and contact when cultured on Si NWs. As the optimization of the contact between integrated microelectronics circuits and cellular membranes represents a long-standing issue, our technological approach may lay the basis for a new era of devices exploiting the microelectronics' sensitivity and "smartness" to both improve investigation of biological systems and to develop suitable NW-based systems available for tissue engineering and regenerative medicine.
RESUMEN
Gradual decline in cognitive and non-cognitive functions are considered clinical hallmarks of Alzheimer's Disease (AD). Post-mortem autoptic analysis shows the presence of amyloid ß deposits, neuroinflammation and severe brain atrophy. However, brain circuit alterations and cellular derailments, assessed in very early stages of AD, still remain elusive. The understanding of these early alterations is crucial to tackle defective mechanisms. In a previous study we proved that the Tg2576 mouse model of AD displays functional deficits in the dorsal hippocampus and relevant behavioural AD-related alterations. We had shown that these deficits in Tg2576 mice correlate with the precocious degeneration of dopamine (DA) neurons in the Ventral Tegmental Area (VTA) and can be restored by L-DOPA treatment. Due to the distinct functionality and connectivity of dorsal versus ventral hippocampus, here we investigated neuronal excitability and synaptic functionality in the ventral CA1 hippocampal sub-region of Tg2576 mice. We found an age-dependent alteration of cell excitability and firing in pyramidal neurons starting at 3 months of age, that correlates with reduced levels in the ventral CA1 of tyrosine hydroxylase - the rate-limiting enzyme of DA synthesis. Additionally, at odds with the dorsal hippocampus, we found no alterations in basal glutamatergic transmission and long-term plasticity of ventral neurons in 8-month old Tg2576 mice compared to age-matched controls. Last, we used computational analysis to model the early derailments of firing properties observed and hypothesize that the neuronal alterations found could depend on dysfunctional sodium and potassium conductances, leading to anticipated depolarization-block of action potential firing. The present study depicts that impairment of cell excitability and homeostatic control of firing in ventral CA1 pyramidal neurons is a prodromal feature in Tg2576 AD mice.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Región CA1 Hipocampal/fisiopatología , Fenómenos Electrofisiológicos , Células Piramidales , Potenciales de Acción , Envejecimiento , Animales , Dopaminérgicos/farmacología , Neuronas Dopaminérgicas , Femenino , Levodopa/farmacología , Masculino , Ratones , Ratones Transgénicos , Canales de Potasio , Canales de Sodio , Tirosina 3-Monooxigenasa/metabolismo , Área Tegmental Ventral/fisiopatologíaRESUMEN
In mammals, identifying the contribution of specific neurons or networks to behavior is a key challenge. Here we describe an approach that facilitates this process by enabling the rapid modulation of synaptic inhibition in defined cell populations. Binding of zolpidem, a systemically active allosteric modulator that enhances the function of the GABAA receptor, requires a phenylalanine residue (Phe77) in the gamma2 subunit. Mice in which this residue is changed to isoleucine are insensitive to zolpidem. By Cre recombinase-induced swapping of the gamma2 subunit (that is, exchanging Ile77 for Phe77), zolpidem sensitivity can be restored to GABAA receptors in chosen cell types. We demonstrate the power of this method in the cerebellum, where zolpidem rapidly induces significant motor deficits when Purkinje cells are made uniquely sensitive to its action. This combined molecular and pharmacological technique has demonstrable advantages over targeted cell ablation and will be invaluable for investigating many neuronal circuits.
Asunto(s)
Conducta Animal/efectos de los fármacos , Neuronas/fisiología , Receptores de GABA-A/fisiología , Sinapsis/fisiología , Animales , Autorradiografía , Electrofisiología , Femenino , Agonistas del GABA/farmacología , Genotipo , Proteínas Fluorescentes Verdes/genética , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Noqueados , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Equilibrio Postural/efectos de los fármacos , Equilibrio Postural/fisiología , Ingeniería de Proteínas , Células de Purkinje/efectos de los fármacos , Células de Purkinje/fisiología , Piridinas/farmacología , Receptores de GABA-A/genética , Receptores de GABA-B/genética , Receptores de GABA-B/fisiología , Sinapsis/efectos de los fármacos , ZolpidemRESUMEN
Exposure to aversive events during sensitive developmental periods can affect the preferential coping strategy adopted by individuals later in life, leading to either stress-related psychiatric disorders, including depression, or to well-adaptation to future adversity and sources of stress, a behavior phenotype termed "resilience". We have previously shown that interfering with the development of mother-pups bond with the Repeated Cross Fostering (RCF) stress protocol can induce resilience to depression-like phenotype in adult C57BL/6J female mice. Here, we used patch-clamp recording in midbrain slice combined with both in vivo and ex vivo pharmacology to test our hypothesis of a link between electrophysiological modifications of dopaminergic neurons in the intermediate Ventral Tegmental Area (VTA) of RCF animals and behavioral resilience. We found reduced hyperpolarization-activated (Ih) cation current amplitude and evoked firing in VTA dopaminergic neurons from both young and adult RCF female mice. In vivo, VTA-specific pharmacological manipulation of the Ih current reverted the pro-resilient phenotype in adult early-stressed mice or mimicked behavioral resilience in adult control animals. This is the first evidence showing how pro-resilience behavior induced by early events is linked to a long-lasting reduction of Ih current and excitability in VTA dopaminergic neurons.
RESUMEN
A series of diastereomeric 2-(2-pyrrolidinyl)-1,4-benzodioxanes bearing a small, hydrogen-bonding substituent at the 7-, 6-, or 5-position of benzodioxane have been studied for α4ß2 and α3ß4 nicotinic acetylcholine receptor affinity and activity. Analogous to C(5)H replacement with N and to a much greater extent than decoration at C(7), substitution at benzodioxane C(5) confers very high α4ß2/α3ß4 selectivity to the α4ß2 partial agonism. Docking into the two receptor structures recently determined by cryo-electron microscopy and site-directed mutagenesis at the minus ß2 side converge in indicating that the limited accommodation capacity of the ß2 pocket, compared to that of the ß4 pocket, makes substitution at C(5) rather than at more projecting C(7) position determinant for this pursued subtype selectivity.
Asunto(s)
Dioxanos/química , Agonistas Nicotínicos/química , Receptores Nicotínicos/química , Sitios de Unión , Microscopía por Crioelectrón , Dioxanos/síntesis química , Dioxanos/metabolismo , Humanos , Enlace de Hidrógeno , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Agonistas Nicotínicos/síntesis química , Agonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/metabolismo , Pirrolidinas/química , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
There is a growing market demand for small-scale biomass gasifiers that is driven by the economic incentives and the legislative framework. Small-scale gasifiers produce a gaseous fuel, commonly referred to as producer gas, with relatively low heating value. Thus, the most common energy conversion systems that are coupled with small-scale gasifiers are internal combustion engines. In order to increase the electrical efficiency, the operators choose dual fuel engines and mix the producer gas with diesel. The Wiebe function has been a valuable tool for assessing the efficiency of dual fuel internal combustion engines. This study introduces a thermodynamic model that works in parallel with the Wiebe function and calculates the emissions of the engines. This "vis-à-vis" approach takes into consideration the actual conditions inside the cylinders-as they are returned by the Wiebe function-and calculates the final thermodynamic equilibrium of the flue gases mixture. This approach aims to enhance the operation of the dual fuel internal combustion engines by identifying the optimal operating conditions and-at the same time-advance pollution control and minimize the environmental impact.
Asunto(s)
Biocombustibles/análisis , Fuentes Generadoras de Energía , Gases/análisis , Modelos Teóricos , Madera/química , Biomasa , TermodinámicaRESUMEN
INTRODUCTION: The Italian Heat Health Watch Warning System (HHWWS) was set up following the over 3000 excess deaths which occurred in Italy during the heat wave of 2003, In June 2005 the warning system issued a heat warning in various Italian cities. METHODS: A case control study was performed in one of these cities (Bari) in order to identify individual and environmental risk factors as well as preventive strategies for reducing mortality during future heat waves. Cases were defined as subjects aged <65 years who had died during the heat wave and whose death certificate listed one of the following as the cause of death: heat, cardiovascular or cerebrovascular disorders, neurocognitive disorders, dehydration or fever/infection not otherwise specified. For each case, three age-matched controls were randomly selected among individuals followed by the same general practitioner as the case. All variables significantly associated with mortality (<0.1) in the univariate analysis were entered into a conditional logistic regression model and the population attributable fraction (PAF) was calculated for significant variables (at p<0.05). RESULTS: Twenty cases and sixty controls were included in the study. In 17 cases (89%) death had occurred at home and 11(55%) of these were cardiovascular- related deaths. At the multivariate analysis, the factors significantly associated with mortality risk during the heat wave were: having a functioning air conditioner at home [OR:0.09(95% CI 0.01-1.00)], having an Activities of Daily Living score <2 [OR:21.0(95%CI 1.81-242.47)] and having been hospitalized the year preceding death [OR:18.1(95%CI 2.04-160.51)]. CONCLUSIONS: Public health interventions during heat waves should include the provision of access to an air conditioned environment. Subjects with impaired health (especially if recently hospitalized) and with significant limitations in their activities of daily living are probably at higher risk during heat waves.
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Actividades Cotidianas , Calor , Estudios de Casos y Controles , Humanos , Italia/epidemiología , Factores de RiesgoRESUMEN
Noise can enhance perception of tactile and proprioceptive stimuli by stochastic resonance processes. However, the mechanisms underlying this general phenomenon remain to be characterized. Here we studied how externally applied noise influences action potential firing in mouse primary sensory neurons of dorsal root ganglia, modelling a basic process in sensory perception. Since noisy mechanical stimuli may cause stochastic fluctuations in receptor potential, we examined the effects of sub-threshold depolarizing current steps with superimposed random fluctuations. We performed whole cell patch clamp recordings in cultured neurons of mouse dorsal root ganglia. Noise was added either before and during the step, or during the depolarizing step only, to focus onto the specific effects of external noise on action potential generation. In both cases, step + noise stimuli triggered significantly more action potentials than steps alone. The normalized power norm had a clear peak at intermediate noise levels, demonstrating that the phenomenon is driven by stochastic resonance. Spikes evoked in step + noise trials occur earlier and show faster rise time as compared to the occasional ones elicited by steps alone. These data suggest that external noise enhances, via stochastic resonance, the recruitment of transient voltage-gated Na channels, responsible for action potential firing in response to rapid step-wise depolarizing currents.
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Potenciales de Acción , Ruido , Células Receptoras Sensoriales/citología , Animales , Femenino , Humanos , Masculino , Ratones , Procesos EstocásticosRESUMEN
The neuronal alpha7 nicotinic acetylcholine (ACh) receptor is believed to be a highly Ca(2+) permeable ligand-gated receptor-channel. However, the contribution of Ca(2+) to cationic current generated by ACh has not yet been directly measured to date. Simultaneous fluorescence and whole-cell current measurements using the Ca(2+) indicator dye fura-2 were made in GH4C1 pituitary cells stably expressing human alpha7 receptors and the fractional Ca(2+) current (the proportion of whole-cell current carried by Ca(2+); P(f)) was determined. We report that the P(f) value was 11.4+/-1.3%. This value was significantly larger than P(f) of human L248Talpha7 receptor mutant (P(f)=6.3+/-1.0%) and of rat alpha7 receptor (P(f)=8.8+/-1.5%) both determined in transiently transfected GH4C1 cells. In our knowledge, the findings here reported indicate the human alpha7 receptors are the most Ca(2+) conductive homomeric ligand-gated receptor-channels expressed in a heterologous cell system.
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Calcio/metabolismo , Neuronas/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Línea Celular , Permeabilidad de la Membrana Celular , Fura-2 , Humanos , Técnicas de Placa-Clamp , Hipófisis , Ratas , Transfección , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
A large body of evidence indicates that ligand-gated channels may open spontaneously, exhibiting a basal activity in the absence of the neurotransmitter. In the present work, we were interested in studying the Ca(2+)-induced modulation of the basal channel activity of unliganded human L248Talpha7 receptors expressed in Xenopus oocytes. While the basal channel activity was blocked by either the nicotinic antagonist methyllycaconitine or the superfusion with a Ca(2+)-free medium, it was enhanced by increasing external Ca2+ concentrations. External Ca2+ significantly influenced the channel properties lengthening the channel duration and reducing the channel conductance, in a dose dependent manner. Furthermore, the basal channel activity in standard medium was blocked by N,N,N',N'-tetrakis-2-pyridylmethyl-ethylenediamine, the chelator of divalent cations with very high affinity for Zn2+, and was induced by Zn2+ when Ca2+ was present in the external medium. We conclude that basal activity of alpha7 mutant receptor-channels is caused by divalent cation contaminants present in the external medium, namely Zn2+; is positively modulated by the external Ca2+; and is inhibited when Ca2+ is absent from the medium. The patho-physiological consequences of these findings are discussed.
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Calcio/farmacología , Activación del Canal Iónico/efectos de los fármacos , Mutación/fisiología , Receptores Nicotínicos/fisiología , Zinc/farmacología , Animales , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Activación del Canal Iónico/fisiología , Mutación/genética , Receptores Nicotínicos/genética , Xenopus , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
In the present work, we studied the effects of the stimulation of the chemokine CXC receptor 4 (CXCR4) by the stromal-derived cell growth factor-1alpha (SDF-1alpha) on the evoked excitatory postsynaptic current. This was generated in Purkinje neurons (PN) from mouse cerebellar slices by the stimulation of parallel fibers. It was found that the amplitude of EPSC was reversibly reduced by SDF-1alpha application. This effect was dose-dependent (IC(50)=0.34 nM) and was abolished by the anti-CXCR4 monoclonal antibody (mAb) 12G5. This SDF-1alpha-induced synaptic depression was caused by a decrease of evoked glutamate release, rather than a decrease in the postsynaptic glutamate receptor (GluR) sensitivity, as the mean amplitude of the spontaneous EPSCs was not influenced by chemokine application. Moreover, NMDA receptors (NMDARs) are involved in EPSC depression being inhibited by the NMDAR blocker 2-amino-5-phosphonopentanoic acid (AP-5). The mechanisms by which SDF-1alpha modulates neurotransmission in the cerebellar cortex are discussed.
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Células de Purkinje/fisiología , Receptores CXCR4/metabolismo , Sinapsis/fisiología , Transmisión Sináptica/inmunología , 2-Amino-5-fosfonovalerato/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/inmunología , Ratones , Ratones Endogámicos BALB C , Técnicas de Placa-Clamp , Receptores AMPA/metabolismo , Receptores CXCR4/inmunología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/efectos de los fármacosRESUMEN
Oligodendrocyte precursor cells (OPCs), a major glial cell type that gives rise to myelinating oligodendrocytes in the CNS, express calcium-permeable AMPA receptors (CP-AMPARs). Although CP-AMPARs are important for OPC proliferation and neuron-glia signaling, they render OPCs susceptible to ischemic damage in early development. We identified factors controlling the dynamic regulation of AMPAR subtypes in OPCs from rat optic nerve and mouse cerebellar cortex. We found that activation of group 1 mGluRs drove an increase in the proportion of CP-AMPARs, reflected by an increase in single-channel conductance and inward rectification. This plasticity required the elevation of intracellular calcium and used PI3K, PICK-1 and the JNK pathway. In white matter, neurons and astrocytes release both ATP and glutamate. Unexpectedly, activation of purinergic receptors in OPCs decreased CP-AMPAR expression, suggesting a capacity for homeostatic regulation. Finally, we found that stargazin-related transmembrane AMPAR regulatory proteins, which are critical for AMPAR surface expression in neurons, regulate CP-AMPAR plasticity in OPCs.