Tagging active neurons by soma-targeted Cal-Light

Abstract

Verifying causal effects of neural circuits is essential for proving a direct circuit-behavior relationship. However, techniques for tagging only active neurons with high spatiotemporal precision remain at the beginning stages. Here we develop the soma-targeted Cal-Light (ST-Cal-Light) which selectively converts somatic calcium rise triggered by action potentials into gene expression. Such modification simultaneously increases the signal-to-noise ratio of reporter gene expression and reduces the light requirement for successful labeling. Because of the enhanced efficacy, the ST-Cal-Light enables the tagging of functionally engaged neurons in various forms of behaviors, including context-dependent fear conditioning, lever-pressing choice behavior, and social interaction behaviors. We also target kainic acid-sensitive neuronal populations in the hippocampus which subsequently suppress seizure symptoms, suggesting ST-Cal-Light’s applicability in controlling disease-related neurons. Furthermore, the generation of a conditional ST-Cal-Light knock-in mouse provides an opportunity to tag active neurons in a region- or cell-type specific manner via crossing with other Cre-driver lines. Thus, the versatile ST-Cal-Light system links somatic action potentials to behaviors with high temporal precision, and ultimately allows functional circuit dissection at a single cell resolution.

CA1 Engram Cell Dynamics Before and After Learning

“…Future experiments may more systematically characterize differences between tagging techniques 48,494/16/24 12:36:00 PM and their consequential impacts on functional readouts of engram population activity. In addition to the TRAP2 system, newly developed methods that utilize faster timescale light-dependent systems include Cal-Light 50 , scFLARE 51,52 and FLiCRE 53 which permit genetic tagging of cells on a seconds-timescale. However, care should be taken when selecting methods as these more temporally restricted tools rely on light activation for tagging that may preclude simultaneous photoexcitation for functional imaging.…”

“…Pairwise correlations were grouped by cell identity and the mean correlation coefficient for all engram/engram, engram/non-engram or non-engram/engram was calculated for each animal. For Supplementary Figure 5C-D, the correlations were calculated for bin sizes of 8,10,16,20,25,32,40,50,64,80, 100, and 125 frames. To evaluate if Spearman coefficients were statistically greater than zero, we performed a shuffle of our traces.…”

Molecular tools for recording and intervention of neuronal activity

“…However, Cal-Light system is improved in terms of its targeting subcellular compartments. Using the soma-targeting version of Cal-Light ( Hyun et al, 2022 ), its signal-noise ratio and dependency on action potential (AP) could be improved.…”

“…Another improved version of a system for light-gated gene expression focuses on expression in specific subcellular compartments. Soma-Targeted (ST)-KA2 ( Hyun et al, 2022 ) is expressed mainly in the somata to facilitate the detection of more AP-dependent neural ensembles. Specific expression in certain compartments, as well as the incorporation of required domains in a single polypeptide chain, has been studied.…”

“…Unlike conventional tagging proteins that simply reflect neuronal activity, Cal-Light can initiate gene expression according to the combined action of light and calcium. Neuronal ensembles can be controlled with the use of Cal-Light using a reporter gene in a vector for expression of an inhibitory opsin such as halorhodopsin (NpHR), as described in Figure 2 ( Hyun et al, 2022 , Lee et al, 2017b ). A previous study reported that suppression of NpHR-positive neurons tagged during epileptic seizures resulted in amelioration of seizure symptoms, suggesting the usefulness of this tool for examining causalities between neural activity and behavior ( Hyun et al, 2022 ).…”