Optically Induced Calcium-Dependent Gene Activation and Labeling of Active Neurons Using CaMPARI and Cal-Light

Abstract

The advent of optogenetic methods has made it possible to use endogeneously produced molecules to image and manipulate cellular, subcellular, and synaptic activity. It has also led to the development of photoactivatable calcium-dependent indicators that mark active synapses, neurons, and circuits. Furthermore, calcium-dependent photoactivation can be used to trigger gene expression in active neurons. Here we describe two sets of protocols, one using CaMPARI and a second one using Cal-Light. CaMPARI, a calcium-modulated photoactivatable ratiometric integrator, enables rapid network-wide, tunable, all-optical functional circuit mapping. Cal-Light, a photoactivatable calcium sensor, while slower to respond than CaMPARI, has the capacity to trigger the expression of genes, including effectors, activators, indicators, or other constructs. Here we describe the rationale and provide procedures for using these two calcium-dependent constructs (1) in vitro in dissociated primary neuronal cell cultures (CaMPARI & Cal-Light); (2) in vitro in acute brain slices for circuit mapping (CaMPARI); (3) in vivo for triggering photoconversion or gene expression (CaMPARI & Cal-Light); and finally, (4) for recovering photoconverted neurons post-fixation with immunocytochemistry (CaMPARI). The approaches and protocols we describe are examples of the potential uses of both CaMPARI & Cal-Light. The ability to mark and manipulate neurons that are active during specific epochs of behavior has a vast unexplored experimental potential.

Red Photoactivatable Genetic Optical-Indicators

“…This should therefore allow to detect the probes in vivo by use of two-photon imaging (Drobizhev et al, 2014). In addition, photoactivation by 405 light can be obtained via widefield epifluoresence, as previously shown for similar probes at similar laser powers (e.g., CaMPARI;Ebner et al, 2019) or via light-guides (Sileo et al, 2015). However, as short wavelengths poorly penetrate tissues and scatter (Carmi et al, 2019), it would seem more attractive should these probes be compatible with two-photon illumination.…”

DeepLabStream enables closed-loop behavioral experiments using deep learning-based markerless, real-time posture detection

“…Direct optogenetic activation and inhibition 46 – 49 of neuronal population based on posture detection might also be possible with DLStream, although our stimulation setup had delays of ~150 ms between detection and manipulation, which may be too slow for certain applications. In our hands, delays were still short enough to allow for targeting activity triggered calcium dynamics by the Cal-Light system 1 , 50 . Using a solution like DLStream the range of detectable behaviors would increase substantially and applications for action- and posture-dependent labeling and subsequent manipulation of different freely moving species are wide-ranging.…”

Coordination between Eye Movement and Whisking in Head-Fixed Mice Navigating a Plus Maze

“…Adult C57BL/6 male mice ( n = 4), weighing 25–32 g were anesthetized with ketamine/xylazine (90 mg/kg/10 mg/kg). Lightweight aluminum head posts were affixed to the skull using Rely X and Jet Acrylic (Ortho-Jet) black cement ( Dominiak et al, 2019 ; Ebner et al, 2019 ). In the 2 d after surgery, analgesia was provided by buprenorphine and carprofen injections.…”