Добірка наукової літератури з теми "JRCaMP1a"

Mitoflashes are spontaneous transients of the biosensor mt-cpYFP. In cardiomyocytes, mitoflashes are associated with the cyclophilin D (CypD) mediated opening of mitochondrial permeability transition pore (mPTP), while in skeletal muscle they are considered hallmarks of mitochondrial respiration burst under physiological conditions. Here, we evaluated the potential association between mitoflashes and the mPTP opening at different CypD levels and phosphorylation status by generating three CypD derived fusion constructs with a red shifted, pH stable Ca2+ sensor jRCaMP1b. We observed perinuclear mitochondrial Ca2+ efflux accompanying mitoflashes in CypD and CypDS42A (a phosphor-resistant mutation at Serine 42) overexpressed myofibers but not the control myofibers expressing the mitochondria-targeting sequence of CypD (CypDN30). Assisted by a newly developed analysis program, we identified shorter, more frequent mitoflash activities occurring over larger areas in CypD and CypDS42A overexpressed myofibers than the control CypDN30 myofibers. These observations provide an association between the elevated CypD expression and increased mitoflash activities in hindlimb muscles in an amyotrophic lateral sclerosis (ALS) mouse model previously observed. More importantly, feeding the mice with sodium butyrate reversed the CypD-associated mitoflash phenotypes and protected against ectopic upregulation of CypD, unveiling a novel molecular mechanism underlying butyrate mediated alleviation of ALS progression in the mouse model.

Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging.

All-optical methods for imaging and manipulating brain networks with high spatial resolution are fundamental to study how neuronal ensembles drive behavior. Stimulation of neuronal ensembles using two-photon holographic techniques requires high-sensitivity actuators to avoid photodamage and heating. Moreover, two-photon-excitable opsins should be insensitive to light at wavelengths used for imaging. To achieve this goal, we developed a novel soma-targeted variant of the large-conductance blue-light-sensitive opsin CoChR (stCoChR). In the mouse cortex in vivo, we combined holographic two-photon stimulation of stCoChR with an amplified laser tuned at the opsin absorption peak and two-photon imaging of the red-shifted indicator jRCaMP1a. Compared to previously characterized blue-light-sensitive soma-targeted opsins in vivo, stCoChR allowed neuronal stimulation with more than 10-fold lower average power and no spectral crosstalk. The combination of stCoChR, tuned amplified laser stimulation, and red-shifted functional indicators promises to be a powerful tool for large-scale interrogation of neural networks in the intact brain.

Abstract Electrical activity and intracellular Ca 2+ transients are key features of cardiomyocytes. They can be measured using organic voltage- and Ca 2+-sensitive dyes but their photostability and phototoxicity means they are unsuitable for long-term measurements. Here, we investigated whether genetically-encoded voltage and Ca 2+ indicators (GEVIs and GECIs) delivered as modified mRNA (modRNA) into human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) would be accurate alternatives allowing measurements over long periods. These indicators were detected in hiPSC-CMs for up to 7 days after transfection and did not affect responses to proarrhythmic compounds. Furthermore, using the GEVI ASAP2f we observed action potential prolongation in long QT syndrome models, while the GECI jRCaMP1b facilitated the repeated evaluation of Ca 2+ handling responses for various tyrosine kinase inhibitors. This study demonstrated that modRNAs encoding optogenetic constructs report cardiac physiology in hiPSC-CMs without toxicity or the need for stable integration, illustrating their value as alternatives to organic dyes or other gene delivery methods for expressing transgenes.

The anatomical and functional organization of the motor cortex and its role in forelimb movement control were deeply investigated over the past years. Extensive research showed two spatially segregated functional areas related to forelimb control: the caudal forelimb area (CFA) and the rostral forelimb area (RFA). Many studies suggest that these two areas are a part of a highly integrated computational unit with distinct motor functions. Recently, optogenetic motor mapping revealed that distinct complex movements are related to segregated cortical functional modules. Although studying optogenetically-evoked behaviors already provides a powerful way to investigate the neuronal pathways related to motor output, decoding the functional engagement and interdependence of cortical motor circuits are still two largely unexplored field. The “all-optical” interrogation of neuronal circuits constitute a successful strategy to causally dissect the functional organization of the motor cortex, since it combines optogenetics and optical indicators to simultaneously record and manipulate the activity of selected neuronal populations using light. During my Ph.D., I contributed to develop a one-photon all-optical strategy to causally investigate the neuronal activity patterns in RFA and CFA driving optogenetically-evoked complex movements in awake head-fixed mice. To this aim, we first examined four different red-shifted Genetically Encoded Calcium Indicator (GECI) , identifying jRCaMP1a as the best indicator for detecting in vivo neuronal activity on multiple cortical areas simultaneously. Then, we combined the widely used blue-sensitive opsin, ChannelRhodopsin2 (ChR2) with jRCaMP1a for detecting in vivo large-scale stimulated cortical dynamics. Once we demonstrated that our one-photon all-optical approach was cross-activation free, we exploited it to causally investigate RFA and CFA cortical activity patterns associated with two optogenetically-evoked complex movements, the grasp-like and the locomotion-like movement. We showed stereotyped and reproducible spatiotemporal propagation patterns of calcium dynamics per movement category highlighting a direct contribution of defined patterns to complex movement execution. Furthermore, we demonstrated that movement-specific cortical activity maps were bounded on discrete function modules centred on the related light-based motor maps, providing clear evidence of their independent functional organization. Importantly, the visualization of the cortical activity elicited by optogenetic stimulation allowed us to identify a third cortical functional module evoking grasp, which is characterized by segregated large-scale cortical dynamics. We named it Lateral Forelimb Area (LFA). In conclusion, our one-photon large-scale all-optical system led to a robust classification of the connectivity, independence, and hierarchy of three functional cortical regions involved in performing evoked complex movements. The results obtained during my Ph.D. provide important insights on the physiological interplay of brain activity and motor control which could be further applied to the investigation of the altered cortical activity patterns in pathological conditions.