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Application of In Vivo Ca2+ Imaging in the Pathological Study of Autism Spectrum Disorders
Published in Tian-Le Xu, Long-Jun Wu, Nonclassical Ion Channels in the Nervous System, 2021
In 2001, a genetically encoded calcium indicator (GECI) GCaMP, was first developed by Junichi Nakai, based on a green fluorescent protein (Nakai et al., 2001). It is composed of green fluorescent protain (GFP,) calmodulin (CaM) and M13 domains. Calcium binding with GCaMP results in a structural shift and brighter fluorescenceMiyawaki 1997. Now, many different versions of GECIs have been developed. Among them, the green and red Ca2+ indicators (GCaMP and RCaMP, respectively) are most commonly used. Without a calcium ion, GCaMP cannot be excited to emit light. However, when the calcium ion appears, the structure of the GCaMP can be changed because of the combining of Ca2+. Then, it can be excited to emit green light (Figure 11.2). The repertoire and properties of GECI are constantly improving to further expand the palette of GECIs and to more precisely reflect complex neural dynamics. For example, a quadripolar GECI suite (XCaMP) and a near-infrared Ca2+ indicator were developed recently (Inoue et al., 2019; Qian et al., 2019).
TRPML Subfamily of Endolysosomal Channels
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Nicholas E. Karagas, Morgan A. Rousseau, Kartik Venkatachalam
The use of GECIs provides an alternative and potentially more sophisticated way to measure intracellular [Ca2+], for instance, by targeting to subcellular compartments or genetically defined cell types. The popular GECIs, GCaMPs, are comprised of circularly permutated GFP and calmodulin, with the latter binding to free Ca2+ and thereupon imposing an increase in the fluorescence of the former (Nakai et al., 2001). The power of GECIs is realized upon targeting to specific organelles, which allows for unprecedented spatiotemporal resolution of Ca2+ measurements (Mao et al., 2008; Pologruto et al., 2004). Indeed, by tagging the termini of TRPMLs with derivatives of GCaMP, Ca2+ in the vicinity of the channel can be detected in a cell type or tissue of interest in any genetically tractable organism. This approach allows measurement of Ca2+ changes in the vicinity of endolysosomes when the GCaMP is tagged to TRPML proteins (Samie et al., 2013; Wong et al., 2017). It is, however, important to bear in mind that TRPML-GCaMP can detect Ca2+ in the vicinity of the channel even if the actual source of the cations is not TRPML. As long as adequate free Ca2+ can diffuse to the GCaMP moiety, this signal will be reported by an increase in fluorescence.
Advanced Optical Imaging in the Study of Acute and Chronic Response to Implanted Neural Interfaces
Published in Yu Chen, Babak Kateb, Neurophotonics and Brain Mapping, 2017
Cristin G. Welle, Daniel X. Hammer
Multichannel TPLSM is especially well suited for the investigation of neurovascular coupling because, unlike OCT that has difficulty resolving individual neurons with intrinsic contrast, all the key structures of the neurovascular unit (neurons, support cells, capillaries) can be resolved and simultaneously examined. Kleinfeld et al. used a two-channel approach and custom line scanning to simultaneously image neurons and astrocytes (with the Ca2+ reporter Oregon green bapta-1-AM) and record RBC velocity in capillaries (labeled with fluorescein-dextran) during electrical stimulation of the rat hindlimb (Kleinfeld et al. 2011). They found rapid neuronal Ca2+ transients and flow changes synchronous with stimulation. Spontaneous neuronal activity also occurred separately. Calcium transients in astrocytes occurred on much slower timescales, apparently without any temporal correspondence to stimulation. Kleinfeld et al. used the same dual-channel approach to simultaneously image blood flow and arterial smooth muscle activation using GCaMP (Kleinfeld et al. 2011). The multichannel approach allows moderate examination of the spatiotemporal environment involved in neurovascular coupling and the functional and metabolic response to stimulation or degradation from disease and age.
Cell death assays for neurodegenerative disease drug discovery
Published in Expert Opinion on Drug Discovery, 2019
Jeremy W. Linsley, Terry Reisine, Steven Finkbeiner
Genetically encoded calcium indicators (GECIs) provide a targetable, less toxic alternative to monitor calcium transients. Recently developed GCaMP variants are more sensitive at detecting calcium than dyes and can be used for longitudinal Ca2+ imaging, whereas dyes need replenishment over time and can lead to toxicity [17]. Because they are genetically encoded, researchers can restrict the expression of GECIs to cell types of interest using cell type-specific genetic enhancers and promoters and can be subcellularly targeted to monitor organelles or microdomains within the neuron such as the cell membrane or mitochondria [88]. Recently, Shi et al. used GCaMP to show that human iPSC derived-motor neurons from ALS patients with C9 mutations have increased Ca2+ transients in response to glutamate challenge compared to controls [84]. These authors also found that the enhanced Ca2+ response was associated with an increased frequency of action potentials and shorter survival times. Retigabine reduced the hyperactivity of the ALS iPSC derived-motor neurons and increased their survival, supporting the hypothesis that the abnormal activity was related to degeneration of the ALS motor neurons and drugs targeting this hyperactivity may be neuroprotective.
Inter-relationships among physical dimensions, distal–proximal rank orders, and basal GCaMP fluorescence levels in Ca2+ imaging of functionally distinct synaptic boutons at Drosophila neuromuscular junctions
Published in Journal of Neurogenetics, 2018
It should be stressed that GCaMP signals reflect cytosolic residual Ca2+ accumulation, which takes place in a time scale of hundreds of milliseconds to a few seconds, and involves both Ca2+ influx and clearance mechanisms. In contrast, Ca2+ entry and subsequent vesicle fusion and transmitter release occur in milliseconds. Therefore, depending on the stimulus protocols and local physiological conditions, the two measurements may yield rather different readouts that reflect the states of two steps in the chain of cellular Ca2+ dynamics, from influx, local actions, cytoplasmic accumulation and clearance (cf. Xing & Wu, 2018). Thus, in type Ib synaptic boutons at low Ca2+ condition, the distal–proximal gradient of GCaMP signal gradient does not necessarily imply a similar gradient in transmission strength.
System level analysis of motor-related neural activities in larval Drosophila
Published in Journal of Neurogenetics, 2019
Youngteak Yoon, Jeonghyuk Park, Atsushi Taniguchi, Hiroshi Kohsaka, Ken Nakae, Shigenori Nonaka, Shin Ishii, Akinao Nose
While this work was in progress, a similar study on Drosophila larval functional imaging was published by Lemon et al. (2015). The authors used state-of-the-art multi-view light-sheet microscopy with one- or two-photon excitation to achieve superior temporal and spatial resolution in the 1st and 3rd instar CNS. In the study by Lemon et al. (2015), GCaMP alone was expressed in neurons, and thus the functional unit of the statistical analyses of neuronal activity was voxels but not cells. Instead, in this study, a nuclear marker was co-expressed to allow activity profiling at the cell level. Our study also identified BW-biased neurons that are not described by Lemon et al. (2015). A difficulty common to our study and that of Lemon et al. (2015) was that since all neurons are visualized, it is difficult to know the identity of the neurons that show characteristic activity patterns. Our previous study showed that when GCaMP is expressed in fewer than ∼10 cells in each neuromere, correlation analyses may be used to reveal the outline of the neuron showing specific activity (Park et al., 2018). Thus, applying a functional imaging technique, such as the one described in this study to a large number of relatively sparse Gal4 lines may enable system-level analyses of motor activity while retaining the ability to identify the neurons of interest. Once candidate neurons are identified, their roles may be studied by the use of optogenetics. Furthermore, by combining functional imaging with optogenetical perturbations, system-level analyses of the dynamics of the motor circuits would be possible in the future.