Improved methods for marking active neuron populations

Abstract

Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetics and reduced fluorescence after chemical fixation. In this work, we develop CaMPARI2, an improved sensor with brighter green and red fluorescence, faster calcium unbinding kinetics and decreased photoconversion in low calcium conditions. We demonstrate the improved performance of CaMPARI2 in mammalian neurons and in vivo in larval zebrafish brain and mouse visual cortex. Additionally, we herein develop an immunohistochemical detection method for specific labeling of the photoconverted red form of CaMPARI. The anti-CaMPARI-red antibody provides strong labeling that is selective for photoconverted CaMPARI in activated neurons in rodent brain tissue.

rsCaMPARI: an erasable marker of neuronal activity

“…1f). The calcium affinity of rsCaMPARI is therefore similar to previously described CaMPARIs 15,16 and GCaMPs [10][11][12] , suggesting that the dynamic range of rsCaMPARI calcium sensitivity falls within the physiological range of neuronal calcium transients.…”

“…To engineer an erasable calcium marker, we took inspiration from CaMPARI and CaMPARI2 since they were previously shown to function well in several in vivo preparations 15,16 . We introduced CaMPARI2 mutations onto a mEos3.1 25 scaffold and converted the protein from a green-to-red photoconvertible FP to a reversibly switchable FP that cycles between bright green and dim states via an H62L substitution within the chromophore, as was previously demonstrated by the production of the mGeos FPs from EosFP 20 .…”

“…Recently we introduced CaMPARI 15 (calcium-modulated photoactivatable ratiometric integrator) and its improved variant CaMPARI2 16 , a genetically encoded fluorescent sensor that enables spatiotemporally precise labelling of active neuron ensembles in large tissue volumes under widefield illumination. CaMPARI is a photoconvertible FP where efficient green-to-red photoconversion only occurs at elevated intracellular calcium concentration and is gated by userdefined violet light illumination.…”

Freeze-frame imaging of synaptic activity using SynTagMA

“…A strategy to overcome this limit is to rapidly 'freeze' activity in a defined time window and read it out at high resolution later. The Ca 2+ -modulated photoactivatable ratiometric integrator CaMPARI undergoes an irreversible chromophore change from green to red when the Ca 2+ bound form is irradiated with violet (390-405 nm) light (Fosque et al, 2015;Moeyaert et al, 2018). CaMPARI has been successfully applied to map the activity of thousands of neurons in zebrafish, Drosophila, and in mouse (Bohra et al, 2018;Fosque et al, 2015;Patel and Cox, 2017;Zolnik et al, 2017).…”

“…By anchoring CaMPARI to either pre-or postsynaptic compartments, we developed tools that mark active synapses in short time windows defined by violet illumination. Three steps were necessary to create a Synaptic Tag for Mapping Activity (SynTagMA): 1) We improved the brightness and conversion efficiency of CaMPARI (Moeyaert et al, 2018); 2) We targeted CaMPARI2 (F391W_L398V) to either presynaptic boutons by fusing it to synaptophysin (preSynTagMA) or to the postsynaptic density by fusing it to an intrabody against PSD95 (Gross et al, 2013) (postSynTagMA); 3) We developed an analysis workflow that corrects for chromatic aberration, tissue displacement (warping) and automatically finds regions of interest (i.e. postsynapses or boutons) to quantify green and red fluorescence.…”