RESUMO
The spatiotemporal activities of astrocyte Ca²âº signaling in mature neuronal circuits remain unclear. We used genetically encoded Ca²âº and glutamate indicators as well as pharmacogenetic and electrical control of neurotransmitter release to explore astrocyte activity in the hippocampal mossy fiber pathway. Our data revealed numerous localized, spontaneous Ca²âº signals in astrocyte branches and territories, but these were not driven by neuronal activity or glutamate. Moreover, evoked astrocyte Ca²âº signaling changed linearly with the number of mossy fiber action potentials. Under these settings, astrocyte responses were global, suppressed by neurotransmitter clearance, and mediated by glutamate and GABA. Thus, astrocyte engagement in the fully developed mossy fiber pathway was slow and territorial, contrary to that frequently proposed for astrocytes within microcircuits. We show that astrocyte Ca²âº signaling functionally segregates large volumes of neuropil and that these transients are not suited for responding to, or regulating, single synapses in the mossy fiber pathway.
Assuntos
Astrócitos/metabolismo , Sinalização do Cálcio/fisiologia , Hipocampo/citologia , Fibras Musgosas Hipocampais/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/ultraestrutura , Sinalização do Cálcio/efeitos dos fármacos , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , GABAérgicos/farmacologia , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fibras Musgosas Hipocampais/efeitos dos fármacos , Receptores de GABA-B/genética , Receptores de Glutamato Metabotrópico/genética , Bloqueadores dos Canais de Sódio/farmacologia , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
Intracellular Ca(2+) transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca(2+) has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca(2+) indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca(2+) signals in acute hippocampal slices in vitro from adult mice (aged â¼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca(2+) microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca(2+) signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function.
Assuntos
Astrócitos/fisiologia , Cálcio/metabolismo , Microdomínios da Membrana/metabolismo , Animais , Astrócitos/metabolismo , Sinalização do Cálcio , Citosol/metabolismo , Citosol/fisiologia , Dependovirus/genética , Dependovirus/metabolismo , Feminino , Hipocampo/metabolismo , Hipocampo/fisiologia , Masculino , Microdomínios da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções/métodosRESUMO
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
Assuntos
Sinalização do Cálcio , Corantes Fluorescentes/química , Fluorometria/métodos , Proteínas de Fluorescência Verde/química , Neuroimagem/métodos , Neurônios/química , Peptídeos/química , Transmissão Sináptica , Animais , Astrócitos/química , Astrócitos/ultraestrutura , Caenorhabditis elegans , Cristalografia por Raios X , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Corantes Fluorescentes/análise , Genes Sintéticos , Vetores Genéticos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/isolamento & purificação , Células HEK293/química , Células HEK293/ultraestrutura , Hipocampo/química , Hipocampo/citologia , Humanos , Larva , Lasers , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Junção Neuromuscular/química , Junção Neuromuscular/ultraestrutura , Neurônios/fisiologia , Neurônios/ultraestrutura , Neurópilo/química , Neurópilo/fisiologia , Neurópilo/ultraestrutura , Neurônios Receptores Olfatórios/química , Neurônios Receptores Olfatórios/fisiologia , Neurônios Receptores Olfatórios/ultraestrutura , Peptídeos/análise , Peptídeos/genética , Estimulação Luminosa , Conformação Proteica , Ratos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Células Bipolares da Retina/química , Células Bipolares da Retina/fisiologia , Células Bipolares da Retina/ultraestrutura , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
BACKGROUND: The neuronal nicotinic receptors that mediate excitatory transmission in autonomic ganglia are thought to be formed mainly by the α3 and ß4 subunits. Expressing this composition in oocytes fails to reproduce the properties of ganglionic receptors, which may also incorporate the α5 and/or ß2 subunits. We compared the properties of human α3ß4 neuronal nicotinic receptors expressed in Human embryonic kidney cells (HEK293) and in Xenopus oocytes, to examine the effect of the expression system and α:ß subunit ratio. METHODOLOGY/PRINCIPAL FINDINGS: Two distinct channel forms were observed: these are likely to correspond to different stoichiometries of the receptor, with two or three copies of the α subunit, as reported for α4ß2 channels. This interpretation is supported by the pattern of change in acetylcholine (ACh) sensitivity observed when a hydrophilic Leu to Thr mutation was inserted in position 9' of the second transmembrane domain, as the effect of mutating the more abundant subunit is greater. Unlike α4ß2 channels, for α3ß4 receptors the putative two-α form is the predominant one in oocytes (at 1:1 α:ß cRNA ratio). This two-α form has a slightly higher ACh sensitivity (about 3-fold in oocytes), and displays potentiation by zinc. The putative three-α form is the predominant one in HEK cells transfected with a 1:1 α:ß DNA ratio or in oocytes at 9:1 α:ß RNA ratio, and is more sensitive to dimethylphenylpiperazinium (DMPP) than to ACh. In outside-out single-channel recordings, the putative two-α form opened to distinctive long bursts (100 ms or more) with low conductance (26 pS), whereas the three-α form gave rise to short bursts (14 ms) of high conductance (39 pS). CONCLUSIONS/SIGNIFICANCE: Like other neuronal nicotinic receptors, the α3ß4 receptor can exist in two different stoichiometries, depending on whether it is expressed in oocytes or in mammalian cell lines and on the ratio of subunits transfected.
Assuntos
Oócitos/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Linhagem Celular , Humanos , Técnicas de Patch-Clamp , Receptores Nicotínicos/química , XenopusRESUMO
Calcium signaling is studied as a potential form of astrocyte excitability that may control astrocyte involvement in synaptic and cerebrovascular regulation. Fundamental questions remain unanswered about astrocyte calcium signaling, as current methods can not resolve calcium in small volume compartments, such as near the cell membrane and in distal cell processes. We modified the genetically encoded calcium sensor GCaMP2 with a membrane-tethering domain, Lck, increasing the level of Lck-GCaMP2 near the plasma membrane tenfold as compared with conventional GCaMP2. Using Lck-GCaMP2 in rat hippocampal astrocyte-neuron cocultures, we measured near-membrane calcium signals that were evoked pharmacologically or by single action potential-mediated neurotransmitter release. Moreover, we identified highly localized and frequent spontaneous calcium signals in astrocyte somata and processes that conventional GCaMP2 failed to detect. Lck-GCaMP2 acts as a genetically targeted calcium sensor for monitoring calcium signals in previously inaccessible parts of astrocytes, including fine processes.
Assuntos
Astrócitos/fisiologia , Sinalização do Cálcio/fisiologia , Potenciais de Ação/fisiologia , Animais , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/fisiologia , Técnicas de Cocultura , Hipocampo/citologia , Hipocampo/fisiologia , Humanos , Neurônios/fisiologia , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Fatores de TempoRESUMO
P2X receptors are ligand-gated cation channels that transition from closed to open states upon binding ATP. The crystal structure of the closed zebrafish P2X4.1 receptor directly reveals that the ion-conducting pathway is formed by three transmembrane domain 2 (TM2) alpha-helices, each being provided by the three subunits of the trimer. However, the transitions in TM2 that accompany channel opening are incompletely understood and remain unresolved. In this study, we quantified gated access to Cd(2+) at substituted cysteines in TM2 of P2X2 receptors in the open and closed states. Our data for the closed state are consistent with the zebrafish P2X4.1 structure, with isoleucines and threonines (Ile-332 and Thr-336) positioned one helical turn apart lining the channel wall on approach to the gate. Our data for the open state reveal gated access to deeper parts of the pore (Thr-339, Val-343, Asp-349, and Leu-353), suggesting the closed channel gate is between Thr-336 and Thr-339. We also found unexpected interactions between native Cys-348 and D349C that result in tight Cd(2+) binding deep within the intracellular vestibule in the open state. Interpreted with a P2X2 receptor structural model of the closed state, our data suggest that the channel gate opens near Thr-336/Thr-339 and is accompanied by movement of the pore-lining regions, which narrow toward the cytosolic end of TM2 in the open state. Such transitions would relieve the barrier to ion flow and render the intracellular vestibule less splayed during channel opening in the presence of ATP.
Assuntos
Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/fisiologia , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Animais , Cádmio/química , Cristalografia por Raios X/métodos , Cisteína/química , Cisteína/genética , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Ratos , Receptores Purinérgicos P2X , Peixe-ZebraRESUMO
Astrocytes are involved in synaptic and cerebrovascular regulation in the brain. These functions are regulated by intracellular calcium signalling that is thought to reflect a form of astrocyte excitability. In a recent study, we reported modification of the genetically encoded calcium indicator (GECI) GCaMP2 with a membrane-tethering domain, Lck, to generate Lck-GCaMP2. This GECI allowed us to detect novel microdomain calcium signals. The microdomains were random and 'spotty' in nature. In order to detect such signals more reliably, in the present study we further modified Lck-GCaMP2 to carry three mutations in the GCaMP2 moiety (M153K, T203V within EGFP and N60D in the CaM domain) to generate Lck-GCaMP3. We directly compared Lck-GCaMP2 and Lck-GCaMP3 by assessing their ability to monitor several types of astrocyte calcium signals with a focus on spotty microdomains. Our data show that Lck-GCaMP3 is between two- and four-times better than Lck-GCaMP2 in terms of its basal fluorescence intensity, signal-to-noise and its ability to detect microdomains. The use of Lck-GCaMP3 thus represents a significantly improved way to monitor astrocyte calcium signals, including microdomains, and will facilitate detailed exploration of their molecular mechanisms and physiological roles.
Assuntos
Astrócitos/metabolismo , Sinalização do Cálcio/fisiologia , Genes Reporter , Microdomínios da Membrana , Neurofisiologia/métodos , Animais , Células HEK293 , Humanos , RatosRESUMO
In this study we report the effects of neramexane, a novel amino-alkyl-cyclohexane derivative that is a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, on recombinant rat alpha9alpha10 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes. We compared its effects with those of memantine, a well-studied pore blocker of NMDA receptors, currently used in therapeutics for the treatment of Alzheimer's disease. Our results indicate that both compounds block acetylcholine-evoked responses at micromolar concentrations with a rank order of potency of neramexane>memantine, P<0.05. Block by neramexane of acetylcholine responses was not overcome at high concentrations of the agonist, indicative of a non-competitive inhibition. The lack of interaction of neramexane with the ligand binding domain was confirmed by radioligand binding experiments in transfected tsA201 cells. Moreover, block did not involve an increase in desensitization kinetics, it was independent of the resting potential of the membrane at low concentrations of neramexane and slightly voltage-dependent at concentrations higher than 1 microM. Finally, clinically-relevant concentrations of neramexane blocked native alpha9alpha10-containing nicotinic acetylcholine receptors of rat inner hair cells, thus demonstrating a possible in vivo relevance in potentially unexplored therapeutic areas.
Assuntos
Ciclopentanos/farmacologia , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Antagonistas Nicotínicos/farmacologia , Subunidades Proteicas/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Animais , Linhagem Celular , Células Ciliadas Auditivas Internas/fisiologia , Humanos , Memantina/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Subunidades Proteicas/genética , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
In this study, we report the effects of the quinoline derivatives quinine, its optical isomer quinidine, and chloroquine on alpha9alpha10-containing nicotinic acetylcholine receptors (nAChRs). The compounds blocked acetylcholine (ACh)-evoked responses in alpha9alpha10-injected Xenopus laevis oocytes in a concentration-dependent manner, with a rank order of potency of chloroquine (IC50 = 0.39 microM) > quinine (IC50 = 0.97 microM) approximately quinidine (IC50= 1.37 microM). Moreover, chloroquine blocked ACh-evoked responses on rat cochlear inner hair cells with an IC50 value of 0.13 microM, which is within the same range as that observed for recombinant receptors. Block by chloroquine was purely competitive, whereas quinine inhibited ACh currents in a mixed competitive and noncompetitive manner. The competitive nature of the blockage produced by the three compounds was confirmed by equilibrium binding experiments using [3H]methyllycaconitine. Binding affinities (Ki values) were 2.3, 5.5, and 13.0 microM for chloroquine, quinine, and quinidine, respectively. Block by quinine was found to be only slightly voltage-dependent, thus precluding open-channel block as the main mechanism of interaction of quinine with alpha9alpha10 nAChRs. The present results add to the pharmacological characterization of alpha9alpha10-containing nicotinic receptors and indicate that the efferent olivocochlear system that innervates the cochlear hair cells is a target of these ototoxic antimalarial compounds.