Abstract:This unit provides a step-by-step protocol for constructing cell type- and mitochondria-targeted GCaMP genetically encoded Ca indicators (GECIs) for mitochondrial Ca imaging in astrocytes. Mitochondrial Ca plays a critical role in controlling cytosolic Ca buffering, energy metabolism, and cellular signal transduction. Mitochondrial Ca overload contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Live-cell mitochondrial Ca imaging is an i… Show more
“…GECIs such as GCaMPs are widely employed for calcium imaging as they have distinct advantages over traditional calcium dyes such as ease of use, relatively consistent expression, fluorescence stability, and the ability to tag specific neuronal populations and subcellular domains (de Juan‐Sanz et al, ; T. W. Chen et al, ; Xing & Wu, ; Zhang & Ding, ). However, expression at high levels may result in exogenous calcium chelation, affecting synaptic transmission in undesirable ways (Steinmetz et al, ).…”
Section: Resultsmentioning
confidence: 99%
“…The GCaMP family of proteins, in particular, is a widely utilized tool for calcium imaging (Akerboom et al, ; Badura, Sun, Giovannucci, Lynch, & Wang, ; Chen et al, ; T. W. Chen et al, ; Chisholm, Khovanov, Lopes, LaRussa, & McMahon, ; Xing & Wu, ). Targeted expression of these indicators has paved the way to more precisely explore the calcium activity in a variety of subcellular compartments like endoplasmic reticulum (ER; de Juan‐Sanz et al, ), mitochondria (Zhang & Ding, ), and in neuronal synapses (Xing & Wu, ). Though development of calcium indicators has been rapidly pursued and fluorescence readout is used to explain observed properties, little is known about the effects of GECIs on basal physiological function (Pérez Koldenkova & Nagai, ).…”
Synaptic vesicle (SV) exocytosis is intimately dependent on free local Ca2+ near active zones. Genetically encoded calcium indicators (GECIs) have become an indispensable tool to monitor calcium dynamics during physiological responses, and they are widely used as a proxy to monitor activity in neuronal ensembles and at synaptic terminals. However, GECIs’ ability to bind Ca2+ at physiologically relevant concentration makes them strong candidates to affect calcium homeostasis and alter synaptic transmission by exogenously increasing Ca2+ buffering. In the present study, we show that genetically expressed GCaMP6m modulates SV release probability at the mouse calyx of Held synapse. GCaMP6m expression for approximately three weeks decreased initial SV release for both low-frequency stimulation and high-frequency stimulation trains, and slowed presynaptic short-term depression. However, GCaMP6m does not affect quantal events during spontaneous activity at this synapse. This study emphasizes the careful use of GECIs as monitors of neuronal activity and inspects the role of these transgenic indicators which may alter calcium-dependent physiological responses.
“…GECIs such as GCaMPs are widely employed for calcium imaging as they have distinct advantages over traditional calcium dyes such as ease of use, relatively consistent expression, fluorescence stability, and the ability to tag specific neuronal populations and subcellular domains (de Juan‐Sanz et al, ; T. W. Chen et al, ; Xing & Wu, ; Zhang & Ding, ). However, expression at high levels may result in exogenous calcium chelation, affecting synaptic transmission in undesirable ways (Steinmetz et al, ).…”
Section: Resultsmentioning
confidence: 99%
“…The GCaMP family of proteins, in particular, is a widely utilized tool for calcium imaging (Akerboom et al, ; Badura, Sun, Giovannucci, Lynch, & Wang, ; Chen et al, ; T. W. Chen et al, ; Chisholm, Khovanov, Lopes, LaRussa, & McMahon, ; Xing & Wu, ). Targeted expression of these indicators has paved the way to more precisely explore the calcium activity in a variety of subcellular compartments like endoplasmic reticulum (ER; de Juan‐Sanz et al, ), mitochondria (Zhang & Ding, ), and in neuronal synapses (Xing & Wu, ). Though development of calcium indicators has been rapidly pursued and fluorescence readout is used to explain observed properties, little is known about the effects of GECIs on basal physiological function (Pérez Koldenkova & Nagai, ).…”
Synaptic vesicle (SV) exocytosis is intimately dependent on free local Ca2+ near active zones. Genetically encoded calcium indicators (GECIs) have become an indispensable tool to monitor calcium dynamics during physiological responses, and they are widely used as a proxy to monitor activity in neuronal ensembles and at synaptic terminals. However, GECIs’ ability to bind Ca2+ at physiologically relevant concentration makes them strong candidates to affect calcium homeostasis and alter synaptic transmission by exogenously increasing Ca2+ buffering. In the present study, we show that genetically expressed GCaMP6m modulates SV release probability at the mouse calyx of Held synapse. GCaMP6m expression for approximately three weeks decreased initial SV release for both low-frequency stimulation and high-frequency stimulation trains, and slowed presynaptic short-term depression. However, GCaMP6m does not affect quantal events during spontaneous activity at this synapse. This study emphasizes the careful use of GECIs as monitors of neuronal activity and inspects the role of these transgenic indicators which may alter calcium-dependent physiological responses.
“…Additionally, under physiological conditions, mitochondrial Ca 2+ uniporters (MCUs) are mainly responsible for mediating Ca 2+ uptake, while Na + -Ca 2+ -Li + exchangers (NCLXs) are mainly responsible for mediating Ca 2+ exportation within mitochondria. Moreover, mitochondrial Ca 2+ signalling is tightly coupled to IP 3 R-mediated Ca 2+ release from the astrocyte endoplasmic reticulum (ER) [ 37 ]. Fig.…”
Section: Astrocyte Mitochondria Are Involved In the Regulation Of Neu...mentioning
“…Ca 2+ events are terminated by Ca 2+ removal through the plasma membrane by Ca 2+ ATPase (PCMA) or by uptake to Ca 2+ stores by ER calcium ATPase (SERCA) (Bazargani and Attwell, 2016). Elongated mitochondria in astrocytic branchlets can also actively uptake intracellular Ca 2+ by mitochondria Ca 2+ uniporters (Zhang and Ding, 2018).…”
Glia are as numerous in the brain as neurons and widely known to serve supportive roles such as structural scaffolding, extracellular ionic and neurotransmitter homeostasis, and metabolic support. However, over the past two decades, several lines of evidence indicate that astrocytes, which are a type of glia, play active roles in neural information processing. Astrocytes, although not electrically active, can exhibit a form of excitability by dynamic changes in intracellular calcium levels. They sense synaptic activity and release neuroactive substances, named gliotransmitters, that modulate neuronal activity and synaptic transmission in several brain areas, thus impacting animal behavior. This “dialogue” between astrocytes and neurons is embodied in the concept of the tripartite synapse that includes astrocytes as integral elements of synaptic function. Here, we review the recent work and discuss how astrocytes via calcium-mediated excitability modulate synaptic information processing at various spatial and time scales.
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