New recipes with CaMPARI for ‘snapshots’ of synaptic circuit activity

The most powerful way to study cerebral network function and connectivity is by tracking the activity of large populations of neurons in relation to a stimulus or behaviour. Various techniques have been applied to monitor neuronal activity. For instance, labelling based on immediate early gene (IEG) expression (such as cFos and Arc) reveals a long-lasting trace of plasticity-related neuronal activity. This permits post hoc analysis in relatively large brain volumes, but it most likely excludes neurons exhibiting modest activity levels. On the contrary, calcium indicators such as GCaMPs (Chen et al. 2013) can report neuronal activity in real time with high sensitivity. However, due to the transient nature of calcium events, simultaneous visualization or post hoc analysis of activated neurons in large brain volumes proved to be challenging. Ideally, for functional synaptic circuit mapping, one would like to employ a method that combines the better of the enduring but somewhat enigmatic IEG-based labelling and the exact but fleeting calcium indicators. The recently developed activity reporter CaMPARI (calcium-modulated photoactivatable ratiometric integrator) potentially fulfils these requirements (Fosque et al. 2015). CaMPARI is a new type of fluorescent calcium indicator that efficiently undergoes an irreversible green-to-red conversion upon violet light illumination and binding of calcium (Fosque et al. 2015). The experimenter defines the time window of photoconversion, which can be repeatedly synchronized with a chosen stimulus. As a result, the intensity of red fluorescence scales with the sum of the calcium levels present at the time of all photoconversion epochs. This feature has been employed to generate a lasting ‘snapshot’ of evoked neuronal activity in various animal models such as zebrafish larvae, flies and in head fixed adult mice (Fosque et al. 2015). The study by Zolnik et al. (Zolnik et al. 2017), published in this issue of The Journal of Physiology, further characterizes and expands the possible applications of CaMPARI. They show that CaMPARI photoconversion is effective at low violet light intensities and linearly correlates with the dose of light (Fosque et al. 2015). This feature may extend the use of CaMPARI to experiments in which violet light delivery is challenging, such as in scattering tissue in vivo. The authors also cleverly employed CaMPARI’s sensitivity to reveal, in addition to spiking, sub-threshold synaptic activity in mouse brain slices, thereby offering a method for functional synaptic connectivity mapping. Subthreshold synaptic potentials typically generate localized dendritic calcium events, which are insufficient to be detected at the soma using transient calcium indicators. Nonetheless, Zolnik et al. demonstrate that repeatedly combining the violet light illumination with subthreshold stimulation causes a persistent accumulation of photoconverted CaMPARI at the soma. Thus, post hoc measurements based on CaMPARI can reveal a complete functional map of postsynaptic neurons that receive supraas well as subthreshold inputs (Fig. 1). Taking further advantage of CaMPARI’s potential to reveal subthreshold activity,