Genetically Targeted Optical Electrophysiology in Intact Neural Circuits

Cao, Guojie; Platisa, Jelena; Pieribone, Vincent A.; Raccuglia, Davide; Kunst, Michael; Nitabach, Michael N.

doi:10.1016/j.cell.2013.07.027

Cited by 248 publications

(228 citation statements)

References 49 publications

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“…A few studies exist for assessing the in vivo performance of genetically encoded calcium sensors in Drosophila neurons 24,25 , so far using older versions of GCaMP that are less reliable for reporting the underlying rapid calcium dynamics. An alternative approach toward resolving action potential frequency and shape will be the use of voltage sensors such as ArcLight 26,27 .…”

Section: Applicationsmentioning

confidence: 99%

A microfluidics-based method for measuring neuronal activity in Drosophila chemosensory neurons

et al. 2016

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Animals possess highly specialized sensory neurons that are capable of perceiving information about a continuously changing environment. Measuring neuronal activity in these cells is an important step toward understanding the basic coding principles, as well as the spatial and temporal dynamics, of sensory systems. The fruit fly Drosophila melanogaster is widely used as a genetic model organism to uncover basic mechanisms of taste coding and odor perception [1][2][3][4][5][6][7] . Recent insights into the molecular and functional mechanisms of sensory perception in Drosophila have been achieved by the use of single-sensillum recordings of adult taste or olfactory sensilla 1,4,[8][9][10][11][12][13] . This method enables sensitive measurements of action potential patterns and shows whether responses are excitatory or inhibitory, but drawing final conclusions about the individual firing neuron is difficult, as each sensillum contains two to four neurons 14 . In the larva, multicellular electrophysiological measurements of the taste system have been performed, but this method is technically challenging and it is not clear how many taste neurons are housed in which sensillum 15 .Microfluidic devices have been used previously to image command interneurons (AVA) and sensory neurons (ASH) in the nematode Caenorhabditis elegans and for studies of axotomy in peripheral sensory neurons of Drosophila larvae [16][17][18] . To implement a similar strategy for Drosophila larval chemosensation, we developed a simple and widely applicable microfluidic chip. We used this custom-made microfluidic device to deliver precise and temporally restricted tastant stimulation in the first published physiological characterization of individual larval gustatory receptor neurons (GRNs) expressing the calcium sensor GCaMP5 (ref. 19). Development of the protocolThe design of the chip is simple. It contains a single channel that is connected to a chamber in which the sample can be mounted to bring it into contact with fluids in the channel. The chamber was originally designed to exactly fit the head of a third-instar Drosophila larva; however, it is also suitable for the proboscis or the leg of an adult fly. The channel for delivering stimulants is relatively broad, designed at a width of 1 mm to reduce flow pressure on the sample. We use a single-channel system in which changes of liquids are performed by the tubing system outside the chip, allowing a relatively slow flow rate inside the chip. Here we provide information required for the design and production of this microfluidic device ( Fig. 1; Supplementary Data), as well as a detailed protocol for live imaging to acquire GCaMP-based calcium imaging (Figs. 2 and 3). By introducing the larval head, or the adult proboscis or leg, into the chamber, neuronal activity can be recorded while chemosensory neurons are in contact with the solution pumped through the channel (Figs. 2 and 3).Overview of the procedure An overview of the workflow involved in implementing the procedure is provided in Figu...

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Section: Applicationsmentioning

confidence: 99%

A microfluidics-based method for measuring neuronal activity in Drosophila chemosensory neurons

et al. 2016

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“…Indeed, GAL4-directed expression of an aequorin transgene in Drosophila was the first example of a calcium reporter animal (Rosay et al, 1997). More recent studies have expressed genetically encoded fluorescent reporters in Drosophila LNv clock neurons (Cao et al, 2013). Other studies in Drosophila, which have also exploited the power of genetically encoded sensors of GPCR activity, elucidated the role of dopamine and octopamine receptors in olfactory memory formation by monitoring calcium and cAMP signals in the mushroom body (MB), the part of the fly brain where olfactory memory is processed (Tomchik and Davis, 2009;Gervasi et al, 2010).…”

Section: Tools For In Vivo Gpcr Researchmentioning

confidence: 99%

Model Organisms in G Protein–Coupled Receptor Research

et al. 2015

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The study of G protein-coupled receptors (GPCRs) has benefited greatly from experimental approaches that interrogate their functions in controlled, artificial environments. Working in vitro, GPCR receptorologists discovered the basic biologic mechanisms by which GPCRs operate, including their eponymous capacity to couple to G proteins; their molecular makeup, including the famed serpentine transmembrane unit; and ultimately, their three-dimensional structure. Although the insights gained from working outside the native environments of GPCRs have allowed for the collection of low-noise data, such approaches cannot directly address a receptor's native (in vivo) functions. An in vivo approach can complement the rigor of in vitro approaches: as studied in model organisms, it imposes physiologic constraints on receptor action and thus allows investigators to deduce the most salient features of receptor function. Here, we briefly discuss specific examples in which model organisms have successfully contributed to the elucidation of signals controlled through GPCRs and other surface receptor systems. We list recent examples that have served either in the initial discovery of GPCR signaling concepts or in their fuller definition. Furthermore, we selectively highlight experimental advantages, shortcomings, and tools of each model organism.

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“…These limitations can be overcome by innovations in optogenetics, namely by the introduction of genetically encoded voltage indicators (GEVIs). Different GEVIs have been used to date mainly for imaging of electric activity in mouse 10,11 and drosophila 12 brain, as well as in fish heart. 13 Recently, the ArcLight GEVI has been transduced in human induced pluripotent stem cell (iPSC)-derived cardiac myocytes for imaging of action potentials (APs) in drug screening applications.…”

mentioning

confidence: 99%

Sensing Cardiac Electrical Activity With a Cardiac Myocyte–Targeted Optogenetic Voltage Indicator

Liao

Boer

Mutoh

et al. 2015

Circulation Research

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Rationale: Monitoring and controlling cardiac myocyte activity with optogenetic tools offer exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac excitation from the cellular to the whole heart level. Objective: To test the hypothesis that cardiac myocyte–targeted voltage-sensitive fluorescence protein 2.3 (VSFP2.3) can be exploited as optogenetic tool for the monitoring of electric activity in isolated cardiac myocytes and the whole heart as well as function and maturity in induced pluripotent stem cell–derived cardiac myocytes. Methods and Results: We first generated mice with cardiac myocyte–restricted expression of VSFP2.3 and demonstrated distinct localization of VSFP2.3 at the t-tubulus/junctional sarcoplasmic reticulum microdomain without any signs for associated pathologies (assessed by echocardiography, RNA-sequencing, and patch clamping). Optically recorded VSFP2.3 signals correlated well with membrane voltage measured simultaneously by patch clamping. The use of VSFP2.3 for human action potential recordings was confirmed by simulation of immature and mature action potentials in murine VSFP2.3 cardiac myocytes. Optical cardiograms could be monitored in whole hearts ex vivo and minimally invasively in vivo via fiber optics at physiological heart rate (10 Hz) and under pacing-induced arrhythmia. Finally, we reprogrammed tail-tip fibroblasts from transgenic mice and used the VSFP2.3 sensor for benchmarking functional and structural maturation in induced pluripotent stem cell–derived cardiac myocytes. Conclusions: We introduce a novel transgenic voltage-sensor model as a new method in cardiovascular research and provide proof of concept for its use in optogenetic sensing of physiological and pathological excitation in mature and immature cardiac myocytes in vitro and in vivo.

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Genetically Targeted Optical Electrophysiology in Intact Neural Circuits

Cited by 248 publications

References 49 publications

A microfluidics-based method for measuring neuronal activity in Drosophila chemosensory neurons

A microfluidics-based method for measuring neuronal activity in Drosophila chemosensory neurons

Model Organisms in G Protein–Coupled Receptor Research

Sensing Cardiac Electrical Activity With a Cardiac Myocyte–Targeted Optogenetic Voltage Indicator

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