High-performance calcium sensors for imaging activity in neuronal populations and microcompartments

Dana, Hod; Sun, Yi; Mohar, Boaz; Hulse, Brad K.; Kerlin, Aaron; Hasseman, J.; Tsegaye, Getahun; Tsang, Arthur; Wong, Allan; Patel, Ronak; Macklin, John J.; Yang, Chen; Konnerth, Arthur; Jayaraman, Vivek; Looger, Loren L.; Schreiter, Eric R.; Svoboda, Karel; Kim, Douglas S.

doi:10.1038/s41592-019-0435-6

Cited by 956 publications

(933 citation statements)

References 49 publications

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“…Additionally, to test whether a reduced shuttling of lactate from glia to neurons in chk CRISPR mutants impairs the ability of neurons to maintain an adequate energy level under increased activity, we determined possible alterations in calcium handling in chk CRISPR mutants, a parameter expected to be affected by low energy levels. For this, we expressed the recently described genetically‐encoded calcium sensor JGCaMP7s (Dana et al, ) in motor neurons ( C380 > JGCaMP7s ) in control animals or chk mutants. Figure a shows that in control neurons PTX exposure induces an increase in JGCaMP7s fluorescence that returned near to basal levels after 20 minutes (Figure b) similar to the signal observed using the sensor GCaMP6f (Figure a).…”

Section: Resultsmentioning

confidence: 99%

Neuronal lactate levels depend on glia‐derived lactate during high brain activity in Drosophila

González-Gutiérrez

Ibacache

Esparza

et al. 2019

Glia

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Lactate/pyruvate transport between glial cells and neurons is thought to play an important role in how brain cells sustain the high‐energy demand that neuronal activity requires. However, the in vivo mechanisms and characteristics that underlie the transport of monocarboxylates are poorly described. Here, we use Drosophila expressing genetically encoded FRET sensors to provide an ex vivo characterization of the transport of monocarboxylates in motor neurons and glial cells from the larval ventral nerve cord. We show that lactate/pyruvate transport in glial cells is coupled to protons and is more efficient than in neurons. Glial cells maintain higher levels of intracellular lactate generating a positive gradient toward neurons. Interestingly, during high neuronal activity, raised lactate in motor neurons is dependent on transfer from glial cells mediated in part by the previously described monocarboxylate transporter Chaski, providing support for in vivo glia‐to‐neuron lactate shuttling during neuronal activity.

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Section: Resultsmentioning

confidence: 99%

Neuronal lactate levels depend on glia‐derived lactate during high brain activity in Drosophila

González-Gutiérrez

Ibacache

Esparza

et al. 2019

Glia

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“…For this we measured cytosolic Ca 2+ responses of intracellular tachyzoites and exposed them to fluctuations of host cytosolic Ca 2+ by stimulating them with a variety of agonists that act specifically on the host cell. We used Genetically Encoded Ca 2+ Indicators (GECIs) (14,15) expressed in the cytosol of HeLa cells infected with T. gondii tachyzoites expressing either cytosolic GCaMP6f or luminal PV targeted jGCaMP7f (16) (See Material and Methods and Table S1). We grew HeLa cells on coverslips, transfected them with red GECIs and infected these cells with green GECI-expressing tachyzoites.…”

Section: Calcium Influx In Intracellular Parasitesmentioning

confidence: 99%

“…We therefore investigated how host cytosolic Ca 2+ signaling interacted or affected the cytosolic Ca 2+ levels of intracellular tachyzoites residing within a PV. With this aim, we expressed Ca 2+ genetic indicators (GECIs) like RGECO or jRGECO1a in the cytoplasm of HeLa cells [15,16] and infected them with T. gondii tachyzoites expressing either cytosolic GCaMP6f or jGCaMP7f [17] targeted to the lumen of the PV (See Material and Methods and Table S1). We grew Hela cells on coverslips, transfected them with the a GECI expressing plasmid and infected these cells with GECI-expressing tachyzoites.…”

Section: Parasites Are Liable To Host Ca 2+ Fluxesmentioning

confidence: 99%

The Role of Potassium and Host Calcium Signaling inToxoplasma gondiiegress

Vella

Moore

et al. 2020

Preprint

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Toxoplasma gondii, an obligate intracellular parasite, is capable of invading virtually any nucleated cell. Active invasion by the parasite is a precise process and intracellular parasites reside and replicate inside a parasitophorous vacuole (PV). The membrane of the PV functions as a molecular sieve allowing for its ionic milieu to be in equilibrium with the host cytosol. The parasite would thus be exposed to the ionic fluctuations of the host cytoplasm, such as increases in host cytosolic Ca 2+ during Ca 2+ signaling events. Ca 2+ is a universal signaling molecule and both the host cell and T. gondii will utilize Ca 2+ signaling to stimulate diverse cellular functions. Using T. gondii tachyzoites and host cells expressing genetically encoded Ca 2+ indicators, we demonstrate that host Ca 2+ signaling impacts intracellular Ca 2+ levels of the parasite. We propose that Ca 2+ influx derived from host Ca 2+ signaling into the parasite is utilized to maintain the intracellular Ca 2+ stores replenished as the parasite replicates within the low Ca 2+ environment of the host cell.Intracellular Ca 2+ store(s) release is needed to reach the initiation spike in Ca 2+ that meets the threshold of Ca 2+ needed to initiate the tightly coordinated process of egress. Post activation of the signal and subsequent microneme secretion, would cause breakdown of the host cell and allow for extracellular Ca 2+ influx, causing a second, larger spike in Ca 2+ and activation of the glideosome, the main motility machinery. To directly deliver precise concentrations of ions needed for egress, we used patch-clamped infected host cells and monitored parasite egress. In doing so, we discovered an alternative role for K + in timing parasite egress. This is the first study using wholecell patch clamp to study the role of ions such as K + and Ca 2+ in T. gondii egress. Author SummaryToxoplasma gondii is an intracellular parasite that causes disease by engaging in multiple rounds of a lytic cycle which consists of invasion, replication inside a parasitophorous vacuole (PV) and egress, resulting in lysis of the host cell. In intracellular parasites, the release of Ca 2+ from intracellular stores forms part of a series of signaling events that culminate in stimulation of motility and egress. This means that intracellular parasites must be able to uptake Ca 2+ while replicating to keep their intracellular stores filled to be used during egress. This is not an insignificant task considering that they are inside a PV surrounded by the host cytosol which contains Ca 2+ below 100 nM. We present data showing that the host Ca 2+ increases are able to 3 reach the parasite cytosol through a Ca 2+ influx mechanism sensitive to nifedipine. This influx causes oscillations that are the result of Ca 2+ increase and re-uptake by intracellular stores. A Ca 2+ threshold was needed for egress, extracellular Ca 2+ influx was relevant for egress and potassium had a modulatory effect on egress. We used infected patched host cells, a novel approach that leaves the host ...

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“…For example, an action potential can be triggered in a GCAMP-expressing neuron using electrophysiology or optogenetics while measuring fluorescence (e.g. (Dana et al, 2019)). This will give the kernel needed to convolve the action potential train to model the fluorescence transient or, equivalently, to deconvolve the fluorescence transient to obtain the underlying action potential train (I am setting aside here potential nonlinearities of the fluorescence response).…”

Section: Work Aroundmentioning

confidence: 99%

The impact of reporter kinetics on the interpretation of data gathered with fluorescent reporters

Sabatini

2019

Preprint

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Fluorescent reporters of biological functions are used to monitor biochemical events and signals in cells and tissue. For neurobiology, these have been particularly useful for monitoring signals in the brains of behaving animals. In order to enhance signal-to-noise, fluorescent reporters typically have kinetics that are slower than that of the underlying biological process. This low-pass filtering by the reporter renders the fluorescence transient a leaking integrated version of the biological signal. Here I discuss the effects that low-pass filtering, or more precisely of integrating by convolving with an exponentially decaying kernel, has on the interpretation of the relationship between the reporter fluorescence transient and the events that underlie it. Unfortunately, when the biological events being monitored are impulse-like, such as the firing of an action potential or the release of neurotransmitter, filtering greatly reduces the maximum correlation coefficient that can be found between the events and the fluorescence signal. This can erroneously support the conclusion that the fluorescence transient and the biological signal that it reports are only weakly related. Furthermore, when examining the encoding of behavioral state variables by nervous system, filtering by the reporter kinetics will favor the interpretation that fluorescence transients encode integrals of measured variables as opposed to the variables themselves. For these reasons, it is necessary to take into account the filtering effects of the indicator by deconvolving with the convolution kernel and recovering the underlying biological events before making conclusions about what is encoded in the signals emitted by fluorescent reporters.

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High-performance calcium sensors for imaging activity in neuronal populations and microcompartments

Cited by 956 publications

References 49 publications

Neuronal lactate levels depend on glia‐derived lactate during high brain activity in Drosophila

Neuronal lactate levels depend on glia‐derived lactate during high brain activity in Drosophila

The Role of Potassium and Host Calcium Signaling inToxoplasma gondiiegress

The impact of reporter kinetics on the interpretation of data gathered with fluorescent reporters

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