RESUMEN
In obstacle-filled media, such as extracellular or intracellular lumen of brain tissue, effective ion-diffusion permeability is a key determinant of electrogenic reactions. Although this diffusion permeability is thought to depend entirely on structural features of the medium, such as porosity and tortuosity, brain tissue shows prominent nonohmic properties, the origins of which remain poorly understood. Here, we explore Monte Carlo simulations of ion diffusion in a space filled with overlapping spheres to predict that diffusion permeability of such media decreases with stronger external electric fields. This dependence increases with lower medium porosity while decreasing with radial (two-dimensional or three-dimensional) compared with homogenous (one-dimensional) fields. We test our predictions empirically in an electrolyte chamber filled with microscopic glass spheres and find good correspondence with our predictions. A theoretical insight relates this phenomenon to a disproportionately increased dwell time of diffusing ions at potential barriers (or traps) representing geometric obstacles when the field strength increases. The dependence of medium ion-diffusion permeability on electric field could be important for understanding conductivity properties of porous materials, in particular for the accurate interpretation of electric activity recordings in brain tissue.
Asunto(s)
Porosidad , Difusión , Conductividad Eléctrica , Método de Montecarlo , PermeabilidadRESUMEN
Brain function relies on vesicular release of neurotransmitters at chemical synapses. The release probability depends on action potential-evoked presynaptic Ca2+ entry, but also on the resting Ca2+ level. Whether these basic aspects of presynaptic calcium homeostasis show any consistent trend along the axonal path, and how they are controlled by local network activity, remains poorly understood. Here, we take advantage of the recently advanced FLIM-based method to monitor presynaptic Ca2+ with nanomolar sensitivity. We find that, in cortical pyramidal neurons, action potential-evoked calcium entry (range 10-300 nM), but not the resting Ca2+ level (range 10-100 nM), tends to increase with higher order of axonal branches. Blocking astroglial glutamate uptake reduces evoked Ca2+ entry but has little effect on resting Ca2+ whereas both appear boosted by the constitutive activation of group 1/2 metabotropic glutamate receptors. We find no consistent effect of transient somatic depolarization or hyperpolarization on presynaptic Ca2+ entry or its basal level. The results unveil some key aspects of presynaptic machinery in cortical circuits, shedding light on basic principles of synaptic connectivity in the brain.
Asunto(s)
Calcio/metabolismo , Ácido Glutámico/metabolismo , Neuronas/metabolismo , Imagen Óptica/métodos , Transmisión Sináptica/fisiología , Animales , Corteza Cerebral/metabolismo , Ratones , Ratones Endogámicos C57BL , Neurotransmisores/metabolismo , Terminales Presinápticos/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sinapsis/metabolismoRESUMEN
Astroglia are vital facilitators of brain development, homeostasis, and metabolic support. In addition, they are also essential to the formation and regulation of synaptic circuits. Due to the extraordinary complex, nanoscopic morphology of astrocytes, the underlying cellular mechanisms have been poorly understood. In particular, fine astrocytic processes that can be found in the vicinity of synapses have been difficult to study using traditional imaging techniques. Here, we describe a 3D three-colour super-resolution microscopy approach to unravel the nanostructure of tripartite synapses. The method is based on the SMLM technique direct stochastic optical reconstruction microscopy (dSTORM) which uses conventional fluorophore-labelled antibodies. This approach enables reconstructing the nanoscale localisation of individual astrocytic glutamate transporter (GLT-1) molecules surrounding presynaptic (bassoon) and postsynaptic (Homer1) protein localisations in fixed mouse brain sections. However, the technique is readily adaptable to other types of targets and tissues.
Asunto(s)
Astrocitos/citología , Encéfalo/diagnóstico por imagen , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Imagen Individual de Molécula/métodos , Sinapsis/metabolismo , Animales , Astrocitos/metabolismo , Encéfalo/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Proteínas de Andamiaje Homer/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/instrumentación , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía/métodos , Microscopía Fluorescente/instrumentación , Proteínas del Tejido Nervioso/metabolismo , Imagen Individual de Molécula/instrumentaciónRESUMEN
Creating and running realistic models of neural networks has hitherto been a task for computing professionals rather than experimental neuroscientists. This is mainly because such networks usually engage substantial computational resources, the handling of which requires specific programing skills. Here we put forward a newly developed simulation environment ARACHNE: it enables an investigator to build and explore cellular networks of arbitrary biophysical and architectural complexity using the logic of NEURON and a simple interface on a local computer or a mobile device. The interface can control, through the internet, an optimized computational kernel installed on a remote computer cluster. ARACHNE can combine neuronal (wired) and astroglial (extracellular volume-transmission driven) network types and adopt realistic cell models from the NEURON library. The program and documentation (current version) are available at GitHub repository https://github.com/LeonidSavtchenko/Arachne under the MIT License (MIT).
Asunto(s)
Modelos Neurológicos , Red Nerviosa/fisiología , Redes Neurales de la Computación , Programas Informáticos , Comunicación Celular/fisiología , Biología Computacional , Simulación por Computador , Humanos , Neuroglía/fisiología , Neuronas/fisiología , Interfaz Usuario-ComputadorRESUMEN
Whilst astrocytes in culture invariably respond to dopamine with cytosolic Ca2+ rises, the dopamine sensitivity of astroglia in situ and its physiological roles remain unknown. To minimize effects of experimental manipulations on astroglial physiology, here we monitored Ca2+ in cells connected via gap junctions to astrocytes loaded whole-cell with cytosolic indicators in area CA1 of acute hippocampal slices. Aiming at high sensitivity of [Ca2+ ] measurements, we also employed life-time imaging of the Ca2+ indicator Oregon Green BAPTA-1. We found that dopamine triggered a dose-dependent, bidirectional Ca2+ response in stratum radiatum astroglia, a jagged elevation accompanied and followed by below-baseline decreases. The elevation depended on D1/D2 receptors and engaged intracellular Ca2+ storage and removal whereas the dopamine-induced [Ca2+ ] decrease involved D2 receptors only and was sensitive to Ca2+ channel blockade. In contrast, the stratum lacunosum moleculare astroglia generated higher-threshold dopamine-induced Ca2+ responses which did not depend on dopamine receptors and were uncoupled from the prominent inhibitory action of dopamine on local perforant path synapses. Our findings thus suggest that a single neurotransmitter-dopamine-could either elevate or decrease astrocyte [Ca2+ ] depending on the receptors involved, that such actions are specific to the regional neural circuitry and that they may be causally uncoupled from dopamine actions on local synapses. The results also indicate that [Ca2+ ] elevations commonly detected in astroglia can represent the variety of distinct mechanisms acting on the microscopic scale. GLIA 2017;65:447-459.
Asunto(s)
Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Calcio/metabolismo , Dopamina/farmacología , Hipocampo/citología , Sinapsis/efectos de los fármacos , Animales , Astrocitos/citología , Dopamina/metabolismo , Dopaminérgicos/farmacología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Técnicas In Vitro , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Masculino , Red Nerviosa/efectos de los fármacos , Red Nerviosa/fisiología , Neurotransmisores/farmacología , Imagen Óptica , Técnicas de Placa-Clamp , RatasRESUMEN
Sustained activation of NMDA receptors (NMDARs) plays an important role in controlling activity of neural circuits in the brain. However, whether this activation reflects the ambient level of excitatory neurotransmitter glutamate in brain tissue or whether it depends mainly on local synaptic discharges remains poorly understood. To shed light on the underlying biophysics here we developed and explored a detailed Monte Carlo model of a realistic three-dimensional neuropil fragment containing 54 excitatory synapses. To trace individual molecules and their individual receptor interactions on this scale, we have designed and implemented a dedicated computer cluster and the appropriate software environment. Our simulations have suggested that sparse synaptic discharges are 20-30 times more efficient than nonsynaptic (stationary, leaky) supply of glutamate in controlling sustained NMDAR occupancy in the brain. This mechanism could explain how the brain circuits provide substantial background activation of NMDARs while maintaining a negligible ambient glutamate level in the extracellular space. Thus the background NMDAR occupancy, rather than the background glutamate level, is likely to reflect the ongoing activity in local excitatory networks.
Asunto(s)
Modelos Neurológicos , Neurópilo/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Animales , Potenciales Postsinápticos Excitadores , Ácido Glutámico/metabolismo , RatasRESUMEN
Melatonin (N-acetyl-5-methoxytryptamine) is reported to improve mood disorders in perimenopausal women and gut microbiome composition is altered during menopausal period. The possible role of microbiome in the treatment effect of melatonin on menopausal depression remains unknown. Here, it is shown that melatonin treatment reverses the gut microbiota dysbiosis and depressive-like behaviors in ovariectomy (OVX) operated mice. This effect of melatonin is prevented by antibiotic cocktails (ABX) treatment. Transferring microbiota harvested from adolescent female mice to OVX-operated mice is sufficient to ameliorate depressive-like behaviors. Conversely, microbiota transplantation from OVX-operated mice or melatonin-treated OVX-operated mice to naïve recipient mice exhibits similar phenotypes to donors. The colonization of Alistipes Inops, which is abundant in OVX-operated mice, confers the recipient with depressive-like behaviors. Further investigation indicates that the expansion of Alistipes Inops induced by OVX leads to the degradation of intestinal tryptophan, which destroys systemic tryptophan availability. Melatonin supplementation restores systemic tryptophan metabolic disorders by suppressing the growth of Alistipes Inops, which ameliorates depressive-like behaviors. These results highlight the previously unrecognized role of Alistipes Inops in the modulation of OVX-induced behavioral disorders and suggest that the application of melatonin to inhibit Alistipes Inops may serve as a potential strategy for preventing menopausal depressive symptoms.
RESUMEN
The surface of astrocyte processes that often surround excitatory synapses is packed with high-affinity glutamate transporters, largely preventing extrasynaptic glutamate escape. The shape and prevalence of perisynaptic astroglia vary among brain regions, in some cases providing a complete isolation of synaptic connections from the surrounding tissue. The perception has been that the geometry of perisynaptic environment is therefore essential to preventing extrasynaptic glutamate escape. To understand to what degree this notion holds, we modelled brain neuropil as a space filled with a scatter of randomly sized, overlapping spheres representing randomly shaped cellular elements and intercellular lumen. Simulating release and diffusion of glutamate molecules inside the interstitial gaps in this medium showed that high-affinity transporters would efficiently constrain extrasynaptic spread of glutamate even when diffusion passages are relatively open. We thus estimate that, in the hippocampal or cerebellar neuropil, the bulk of glutamate released by a synaptic vesicle is rapidly bound by transporters (or high-affinity target receptors) mainly in close proximity of the synaptic cleft, whether or not certain physiological or pathological events change local tissue geometry.
RESUMEN
Information processing and memory formation in the brain relies on release of the main excitatory neurotransmitter glutamate from presynaptic axonal specialisations. The classical Hebbian paradigm of synaptic memory, long-term potentiation (LTP) of transmission, has been widely associated with an increase in the postsynaptic receptor current. Whether and to what degree LTP induction also enhances presynaptic glutamate release has been the subject of debate. Here, we took advantage of the recently developed genetically encoded optical sensors of glutamate (iGluSnFR) to monitor its release at CA3-CA1 synapses in acute hippocampal slices, before and after the induction of LTP. We attempted to trace release events at multiple synapses simultaneously, by using two-photon excitation imaging in fast frame-scanning mode. We thus detected a significant increase in the average iGluSnFR signal during potentiation, which lasted for up to 90 min. This increase may reflect an increased amount of released glutamate or, alternatively, reduced glutamate binding to high-affinity glutamate transporters that compete with iGluSnFR.
Asunto(s)
Ácido Glutámico/metabolismo , Potenciación a Largo Plazo , Imagen Óptica , Sinapsis/metabolismo , Animales , Axones/metabolismo , Transporte Biológico , Dependovirus/metabolismo , Femenino , Hipocampo/metabolismo , Masculino , Ratones Endogámicos C57BLRESUMEN
In the original publication of this article [1], text has been introduced erroneously to Figs. 4a and 5d due to a typesetting mistake.
RESUMEN
Astroglia regulate neurovascular coupling while engaging in signal exchange with neurons. The underlying cellular machinery is thought to rely on astrocytic Ca2+ signals, but what controls their amplitude and waveform is poorly understood. Here, we employ time-resolved two-photon excitation fluorescence imaging in acute hippocampal slices and in cortex in vivo to find that resting [Ca2+] predicts the scale (amplitude) and the maximum (peak) of astroglial Ca2+ elevations. We bidirectionally manipulate resting [Ca2+] by uncaging intracellular Ca2+ or Ca2+ buffers and use ratiometric imaging of a genetically encoded Ca2+ indicator to establish that alterations in resting [Ca2+] change co-directionally the peak level and anti-directionally the amplitude of local Ca2+ transients. This relationship holds for spontaneous and for induced (for instance by locomotion) Ca2+ signals. Our findings uncover a basic generic rule of Ca2+ signal formation in astrocytes, thus also associating the resting Ca2+ level with the physiological "excitability" state of astroglia.
Asunto(s)
Astrocitos/metabolismo , Señalización del Calcio , Calcio/metabolismo , Animales , Fluorescencia , Locomoción , Ratones , Fracciones SubcelularesRESUMEN
Extrasynaptic actions of glutamate are limited by high-affinity transporters expressed by perisynaptic astroglial processes (PAPs): this helps maintain point-to-point transmission in excitatory circuits. Memory formation in the brain is associated with synaptic remodeling, but how this affects PAPs and therefore extrasynaptic glutamate actions is poorly understood. Here, we used advanced imaging methods, in situ and in vivo, to find that a classical synaptic memory mechanism, long-term potentiation (LTP), triggers withdrawal of PAPs from potentiated synapses. Optical glutamate sensors combined with patch-clamp and 3D molecular localization reveal that LTP induction thus prompts spatial retreat of astroglial glutamate transporters, boosting glutamate spillover and NMDA-receptor-mediated inter-synaptic cross-talk. The LTP-triggered PAP withdrawal involves NKCC1 transporters and the actin-controlling protein cofilin but does not depend on major Ca2+-dependent cascades in astrocytes. We have therefore uncovered a mechanism by which a memory trace at one synapse could alter signal handling by multiple neighboring connections.
Asunto(s)
Astrocitos/metabolismo , Ácido Glutámico/metabolismo , Potenciación a Largo Plazo/fisiología , Sinapsis/metabolismo , Animales , Astrocitos/ultraestructura , Femenino , Imagenología Tridimensional/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Sinapsis/ultraestructuraRESUMEN
All-optical registration of neuronal and astrocytic activities within the intact mammalian brain has improved significantly with recent advances in optical sensors and biophotonics. However, relating single-synapse release events and local astroglial responses to sensory stimuli in an intact animal has not hitherto been feasible. Here, we present a multiplexed multiphoton excitation imaging approach for assessing the relationship between presynaptic Ca2+ entry at thalamocortical axonal boutons and perisynaptic astrocytic Ca2+ elevations, induced by whisker stimulation in the barrel cortex of C57BL/6 mice. We find that, unexpectedly, Ca2+ elevations in the perisynaptic astrocytic regions consistently precede local presynaptic Ca2+ signals during spontaneous brain activity associated with anaesthesia. The methods described here can be adapted to a variety of optical sensors and are compatible with experimental designs that might necessitate repeated sampling of single synapses over a longitudinal behavioural paradigm.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , Calcio/metabolismo , Sinapsis/metabolismo , Animales , Astrocitos/citología , Axones/metabolismo , Encéfalo/citología , Señalización del Calcio , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Neuronas/metabolismo , Terminales Presinápticos/metabolismo , Corteza Somatosensorial/fisiologíaRESUMEN
Information processing by brain circuits depends on Ca2+-dependent, stochastic release of the excitatory neurotransmitter glutamate. Whilst optical glutamate sensors have enabled detection of synaptic discharges, understanding presynaptic machinery requires simultaneous readout of glutamate release and nanomolar presynaptic Ca2+ in situ. Here, we find that the fluorescence lifetime of the red-shifted Ca2+ indicator Cal-590 is Ca2+-sensitive in the nanomolar range, and employ it in combination with green glutamate sensors to relate quantal neurotransmission to presynaptic Ca2+ kinetics. Multiplexed imaging of individual and multiple synapses in identified axonal circuits reveals that glutamate release efficacy, but not its short-term plasticity, varies with time-dependent fluctuations in presynaptic resting Ca2+ or spike-evoked Ca2+ entry. Within individual presynaptic boutons, we find no nanoscopic co-localisation of evoked presynaptic Ca2+ entry with the prevalent glutamate release site, suggesting loose coupling between the two. The approach enables a better understanding of release machinery at central synapses.
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Calcio/metabolismo , Ácido Glutámico/metabolismo , Homeostasis , Imagenología Tridimensional , Terminales Presinápticos/metabolismo , Animales , Axones/metabolismo , Microscopía Fluorescente , Nanopartículas/química , Plasticidad Neuronal , Fotones , Ratas Sprague-DawleyRESUMEN
Actions of the excitatory neurotransmitter glutamate inside and outside the synaptic cleft determine the activity of neural circuits in the brain. However, to what degree local glutamate transporters affect these actions on a submicron scale remains poorly understood. Here we focus on hippocampal area CA1, a common subject of synaptic physiology studies. First, we use a two-photon excitation technique to obtain an estimate of the apparent (macroscopic) extracellular diffusion coefficient for glutamate, approximately 0.32 mum(2)/ms. Second, we incorporate this measurement into a Monte Carlo model of the typical excitatory synapse and examine the influence of distributed glutamate transporter molecules on signal transmission. Combined with the results of whole-cell recordings, such simulations argue that, although glutamate transporters have little effect on the activation of synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, this does not rule out the occurrence of up to several dozens of transporters inside the cleft. We further evaluate how the expression pattern of transporter molecules (on the 10-100 nm scale) affects the activation of N-methyl-D-aspartic acid or metabotropic glutamate receptors in the synaptic vicinity. Finally, we extend our simulations to the macroscopic scale, estimating that synaptic activity sufficient to excite principal neurons could intermittently raise extracellular glutamate to approximately 1 muM only at sparse (microns apart) hotspots. Greater rises of glutamate occur only when <5% of transporters are available (for instance, when an astrocyte fails). The results provide a quantitative framework for a better understanding of the relationship between glutamate transporters and glutamate receptor signaling.
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Sistema de Transporte de Aminoácidos X-AG/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/química , Hipocampo/fisiología , Modelos Neurológicos , Receptores de Glutamato/metabolismo , Transmisión Sináptica/fisiología , Sistema de Transporte de Aminoácidos X-AG/química , Animales , Células Cultivadas , Simulación por Computador , Ácido Glutámico/química , Masculino , Modelos Estadísticos , Nanoestructuras/química , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato/químicaRESUMEN
Nanomolar-range fluctuations of intracellular [Ca2+] are critical for brain cell function but remain difficult to measure. We have advanced a microscopy technique to monitor intracellular [Ca2+] in individual cells in acute brain slices (also applicable in vivo) using fluorescence lifetime imaging (FLIM) of the Ca2+-sensitive fluorescent indicator Oregon Green BAPTA1 (OGB-1). The OGB-1 fluorescence lifetime is sensitive to [Ca2+] within the 10-500 nM range but not to other factors such as viscosity, temperature, or pH. This protocol describes the requirements, setup, and calibration of the FLIM system required for OGB-1 imaging. We provide a step-by-step procedure for whole-cell OGB-1 loading and two-photon FLIM. We also describe how to analyze the obtained FLIM data using total photon count and gated-intensity record, a ratiometric photon-counting approach that provides a highly improved signal-to-noise ratio and greater sensitivity of absolute [Ca2+] readout. We demonstrate our technique in nerve cells in situ, and it is adaptable to other cell types and fluorescent indicators. This protocol requires a basic understanding of FLIM and experience in single-cell electrophysiology and cell imaging. Setting up the FLIM system takes â¼2 d, and OGB-1 loading, imaging, and data analysis take 2 d.
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Calcio/análisis , Líquido Intracelular/diagnóstico por imagen , Imagen Óptica/métodos , Encéfalo , Calcio/metabolismo , Calcio/fisiología , Citoplasma , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes , Proteínas Sensoras del Calcio Intracelular , Transporte Iónico , Microscopía Fluorescente/métodos , Neuronas , Análisis de la Célula Individual/instrumentación , Análisis de la Célula Individual/métodosRESUMEN
The pathogenesis of Alzheimer's disease (AD) is thought to involve acute neurotoxic effects exerted by oligomeric forms of amyloid-ß 1-42 (Aß). Application of Aß oligomers in physiological concentrations have been shown to transiently elevate internal Ca2+ in cultured astroglia. While the cellular machinery involved has been extensively explored, to what degree this important signalling cascade occurs in organised brain tissue has remained unclear. Here we adapted two-photon excitation microscopy and calibrated time-resolved imaging (FLIM), coupled with patch-clamp electrophysiology, to monitor Ca2+ concentration ([Ca2+]) inside individual astrocytes and principal neurons in acute brain slices. Inside the slice tissue local micro-ejection of Aß in sub-micromolar concentrations triggered prominent [Ca2+] elevations in an adjacent astrocyte translated as an approximately two-fold increase (averaged over â¼5min) in basal [Ca2+]. This elevation did not spread to neighbouring cells and appeared comparable in amplitude with commonly documented spontaneous [Ca2+] rises in astroglia. Principal nerve cells (pyramidal neurons) also showed Ca2+ sensitivity, albeit to a lesser degree. These observations shed light on the extent and dynamics of the acute physiological effects of Aß on brain cells in situ, in the context of AD.
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Péptidos beta-Amiloides/metabolismo , Astrocitos/metabolismo , Calcio/metabolismo , Hipocampo/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos beta-Amiloides/administración & dosificación , Animales , Astrocitos/efectos de los fármacos , Cationes Bivalentes/metabolismo , Fármacos del Sistema Nervioso Central/administración & dosificación , Hipocampo/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microscopía Fluorescente , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Placa-Clamp , Fragmentos de Péptidos/administración & dosificación , Ratas Sprague-Dawley , Análisis de la Célula Individual , Técnicas de Cultivo de TejidosRESUMEN
Neural activity relies on molecular diffusion within nanoscopic spaces outside and inside nerve cells, such as synaptic clefts or dendritic spines. Measuring diffusion on this small scale in situ has not hitherto been possible, yet this knowledge is critical for understanding the dynamics of molecular events and electric currents that shape physiological signals throughout the brain. Here we advance time-resolved fluorescence anisotropy imaging combined with two-photon excitation microscopy to map nanoscale diffusivity in ex vivo brain slices. We find that in the brain interstitial gaps small molecules move on average ~30% slower than in a free medium whereas inside neuronal dendrites this retardation is ~70%. In the synaptic cleft free nanodiffusion is decelerated by ~46%. These quantities provide previously unattainable basic constrains for the receptor actions of released neurotransmitters, the electrical conductance of the brain interstitial space and the limiting rate of molecular interactions or conformational changes in the synaptic microenvironment.
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Química Encefálica , Difusión , Polarización de Fluorescencia , Fluoroinmunoensayo , Neurotransmisores/análisis , Imagen Óptica , Sinapsis/metabolismo , Animales , Ratas Sprague-Dawley , Sinapsis/químicaRESUMEN
Brain function relies in large part on Ca2+-dependent release of the excitatory neurotransmitter glutamate from neuronal axons. Establishing the causal relationship between presynaptic Ca2+ dynamics and probabilistic glutamate release is therefore a fundamental quest across neurosciences. Its progress, however, has hitherto depended primarily on the exploration of either cultured nerve cells or giant central synapses accessible to direct experimental probing in situ. Here we show that combining patch-clamp with time-resolved imaging of Ca2+ -sensitive fluorescence lifetime of Oregon Green BAPTA-1 (Tornado-FLIM) enables readout of single spike-evoked presynaptic Ca2+ concentration dynamics, with nanomolar sensitivity, in individual neuronal axons in acute brain slices. In parallel, intensity Tornado imaging of a locally expressed extracellular optical glutamate sensor iGluSnFr provides direct monitoring of single-quantum, single-synapse glutamate releases in situ. These two methods pave the way for simultaneous registration of presynaptic Ca2+ dynamics and transmitter release in an intact brain at the level of individual synapses.
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Encéfalo/metabolismo , Calcio/metabolismo , Ácido Glutámico/metabolismo , Terminales Presinápticos/metabolismo , Sinapsis/metabolismo , Potenciales de Acción/fisiología , Compuestos de Anilina/metabolismo , Animales , Axones/metabolismo , Fluoresceínas/metabolismo , Hipocampo/metabolismo , Ratones Endogámicos C57BL , Ratas Sprague-DawleyRESUMEN
Maintaining low intracellular calcium is essential to the functioning of brain cells, yet the phenomenology and mechanisms involved remain an enigma. We have advanced a two-photon excitation time-resolved imaging technique, which exploits high sensitivity of the OGB-1 fluorescence lifetime to nanomolar Ca(2+) concentration ([Ca(2+)]) and enables a high data acquisition rate in situ. The [Ca(2+)] readout is not affected by dye concentration, light scattering, photobleaching, micro-viscosity, temperature, or the main known concomitants of cellular activity. In quiescent tissue, standard whole-cell configuration has little effect on resting [Ca(2+)] inside neuronal dendrites or inside astroglia dye-filled via gap junctions. Mapping basal [Ca(2+)] in neurons and astrocytes with submicron resolution unveils heterogeneous concentration landscapes that depend on age and preceding activity. The rich information content represented by such landscapes in acute slices and in vivo promises to unveil the hitherto unexplored, potentially fundamental aspects of brain cell physiology.