Studying Brain Function Using Non-human Primate Models
Tian-Le Xu, Long-Jun Wu in Nonclassical Ion Channels in the Nervous System, 2021
Besides the construction of the disease model, the transgenic engineering technique can also serve as an exploitation tool for neurobiology. GCaMP, a genetically encoded calcium indicator fused with GFP, can monitor the neuronal activity in the brain of a living animal via the optical technique. Park’s group utilized a high tier lentiviral vector to generate the GCaMP transgenic marmosets under the control of the CMV promotor and Synapsin promotor (124). In additions, Tomioka’s group created a tetracyclin-inducible transgene expression (tet-on) system and achieved target exogenous gene conditional control (125). The ever-expanding development of optogenetics and chemogenetics techniques enbales more precise and deeper understanding of the human nerve system by transgenic NHP model.
TRPML Subfamily of Endolysosomal Channels
Bruno Gasnier, Michael X. Zhu in Ion and Molecule Transport in Lysosomes, 2020
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
Yu Chen, Babak Kateb in Neurophotonics and Brain Mapping, 2017
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.
Preclinical target validation for non-addictive therapeutics development for pain
Published in Expert Opinion on Therapeutic Targets, 2022
Richard Hargreaves, Karen Akinsanya, Seena K. Ajit, Neel T. Dhruv, Jamie Driscoll, Peter Farina, Narender Gavva, Marie Gill, Andrea Houghton, Smriti Iyengar, Carrie Jones, Annemieke Kavelaars, Ajamete Kaykas, Walter J. Koroshetz, Pascal Laeng, Jennifer M. Laird, Donald C. Lo, Johan Luthman, Gordon Munro, Michael L. Oshinsky, G. Sitta Sittampalam, Sarah A. Woller, Amir P. Tamiz
When defining pain phenotypes in in vitro models, researchers should carefully consider the choice of phenotype in terms of scalability and reliability as it relates to the human condition. The most reliable measures of in vitro phenotypes that relate to clinical pain phenotypes have yet to be determined. Artificial intelligence (AI)-based approaches can be leveraged to facilitate large-scale and complex screens that detect emerging patterns among in vitro models. When technologies are less scalable, high-throughput methods should be employed earlier in the process to screen multiple replicates for promising targets, and more limited throughput technologies can be used to tease out mechanisms for secondary validation. For example, multielectrode arrays (MEAs) are a limited throughput technology that can be used to measure action potentials in cultured samples, whereas GCaMP reporters can be used to screen in more high-throughput formats (e.g. 96- or 384-well plates) to measure bulk calcium signaling.
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.
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