A Flp-dependent G-CaMP9a transgenic mouse for neuronal imaging in vivo

Sakamoto, Masayuki; Inoue, Masatoshi; Takeuchi, Akihito; Kobari, Shigetaka; Yokoyama, Tatsushi; Horigane, Shin-ichiro; Takemoto-Kimura, Sayaka; Abe, Manabu; Sakimura, Kenji; Kano, Masanobu; Kitamura, Kazuhiro; Fujii, H.; Bito, Haruhiko

doi:10.1016/j.crmeth.2022.100168

Cited by 11 publications

(7 citation statements)

References 61 publications

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“…To further investigate the relationship between Ca 2+ and cAMP in vivo , a drifting grating stimulus of 8 directions was applied to induce cell-specific Ca 2+ transients in L2/3 neurons of the V1 ( Figure 5A ). In vivo two-photon imaging revealed that RCaMP3 showed direction-selective Ca 2+ transients in response to 4 seconds of drifting gratings, consistent with previous studies (Chen et al, 2013; Dana et al, 2016; Sakamoto et al, 2022a) (Figures 5B-5D; Figure S7 ). Interestingly, cAMPinG1-ST also showed direction-selective cAMP transients preceded by Ca 2+ transients, which were selective to the same direction (Figures 5B-5D; Figure S7 ).…”

Section: Resultssupporting

confidence: 90%

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A multicolor suite for deciphering population coding in calcium and cAMPin vivo

Yokoyama

Manita

Uwamori

et al. 2023

Preprint

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cAMP is a pivotal second messenger regulated by various upstream pathways including Ca2+ and G protein-coupled receptors (GPCRs). To decipherin vivocAMP dynamics, we rationally designed cAMPinG1, an ultrasensitive genetically encoded green cAMP indicator that outperformed its predecessors in both dynamic range and cAMP affinity. Two-photon cAMPinG1 imaging detected cAMP transients in the somata and dendritic spines of neurons in the mouse visual cortex on the order of tens of seconds. In addition, multicolor imaging with a highly sensitive new red Ca2+ indicator RCaMP3 allowed simultaneous measurement of population patterns in Ca2+ and cAMP in hundreds of neurons. We identified Ca2+-induced cAMP responses that represented specific information, such as direction selectivity in vision and locomotion, as well as GPCR-induced cAMP responses. Overall, our multicolor suite revealed that information encoded in Ca2+ and GPCRs signaling is integrated and stored as cAMP transients for longer periodsin vivo.

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Section: Resultssupporting

confidence: 90%

“…Craniotomy was performed as described previously (Sakamoto et al, 2022a). Mice were anesthetized by the anesthetic mixture described above.…”

Section: Methodsmentioning

confidence: 99%

A multicolor suite for deciphering population coding in calcium and cAMPin vivo

Yokoyama

Manita

Uwamori

et al. 2023

Preprint

Self Cite

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“…Optimizing the performance of GECIs and other protein sensors is a critical step for transforming a promising concept into a widely used tool. For example, the optimization of the first-generation GCaMP GECI has continued for more than 20 years, with gradual increases in the number of screened constructs for the newer generations of the sensor, and with the recent jGCaMP8, X-CaMPG, and G-CaMP9a descendants showing superior performance compared to earlier generations ( Nakai et al, 2001 ; Inoue et al, 2019 ; Zhang et al, 2021 ; Sakamoto et al, 2022 ). Switching to large-scale, in-vitro -based optimization pipelines is not limited to GECIs, but was also demonstrated for improving the performance of genetically encoded voltage sensors ( Kannan et al, 2018 , 2021 ) and glutamate sensors ( Aggarwal et al, 2022 ).…”

Section: Discussionmentioning

confidence: 99%

Enhanced detection sensitivity of neuronal activity patterns using CaMPARI1 vs. CaMPARI2

Das

Margevicius²,

Borovicka³

et al. 2023

Front. Neurosci.

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Calcium-modulated photoactivatable ratiometric integrator (CaMPARI) is a calcium ion (Ca2+)- and light-dependent genetically encoded fluorescent activity integrator that can capture snapshots of neuronal activity through an irreversible process known as photoconversion. This unique property was previously used to label neurons based upon their tuning properties in order to map synaptic connectivity and to record large-scale neuronal activity in freely moving mice without attaching any mechanical device to them. The latest version of CaMPARI (CaMPARI2) was engineered to enhance the contrast generated by photoconverting the green protein to the activity-dependent red form and to reduce the Ca2+-independent photoconversion rate compared to the first generation of CaMPARI (CaMPARI1). However, here we show that this optimization process also resulted in reduced photoconversion efficiency of active neurons in the mouse cortex and hippocampus. Through side-by-side comparison of the two CaMPARI sensors under several experimental conditions, we show that CaMPARI1 exhibits a substantially higher red-to-green ratio in active cells than CaMPARI2. In addition, we show that CaMPARI1 also functions as a more sensitive traditional Ca2+ sensor than CaMPARI2 by producing larger activity-driven dynamic fluorescence changes in the observed neurons. Therefore, we conclude that during the optimization process of CaMPARI2, some of the sensor’s characteristics were not predicted properly by in vitro screening assays, and therefore in vivo screening and validation steps should be included in future optimization attempts to increase the predictability of screening pipelines.

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“…In several other later designs, CaM and M13 were fused to the N- and C-terminals of the (cp-)FP 230,240,244 so that the Ca 2+ -binding action will trigger the conformation change around the original Tyr145 site. Later versions of calcium indicators have fine-tuned the binding constant for Ca 2+ through random sequence mutation, so that the sensor can work with different concentrations of Ca 2+ , 245–248 such as the fast-GCaMPs/GCaMP6f series with faster kinetics, 246,249,250 and are suitable to track large population of neurons using two photon 251 /wide-field imaging. 252 As designing red FP-based calcium indicators has been proven to be challenging, the red/infra-red calcium indicators are bacterial phytochrome-based, 177,253,254 which need either a single GAF domain with the mutated cysteine site (for BV-binding) or include both PAS and GAF domains with a sensor module inserted between the two domains.…”

Section: Genetically Encoded Fluorescent Probesmentioning

confidence: 99%