Freeze-frame imaging of synaptic activity using SynTagMA

Abstract

Information within the brain travels from neuron to neuron across billions of synapses. At any given moment, only a small subset of neurons and synapses are active, but finding the active synapses in brain tissue has been a technical challenge. Here we introduce SynTagMA to tag active synapses in a user-defined time window. Upon 395–405 nm illumination, this genetically encoded marker of activity converts from green to red fluorescence if, and only if, it is bound to calcium. Targeted to presynaptic terminals, preSynTagMA allows discrimination between active and silent axons. Targeted to excitatory postsynapses, postSynTagMA creates a snapshot of synapses active just before photoconversion. To analyze large datasets, we show how to identify and track the fluorescence of thousands of individual synapses in an automated fashion. Together, these tools provide an efficient method for repeatedly mapping active neurons and synapses in cell culture, slice preparations, and in vivo during behavior.

Freeze-Frame Imaging of Dendritic Calcium Signals With TubuTag

“…For the previously published synaptically localized calcium integrator SynTagMA ( Perez-Alvarez et al, 2020 ), we acquired separate image stacks to collect green and red fluorescence, exciting at 980 and 1040 nm, respectively. Green images had very little autofluorescence and were used to mask out non-synaptic fluorescence in the red channel.…”

“…A topic of considerable interest is the possibility that synapses with synchronous activity are able to stabilize each other when situated close together, leading over time to a clustered input organization ( Kleindienst et al, 2011 ; Iacaruso et al, 2017 ). In principle, an activity tag exclusively located at excitatory synapses like postSynTagMA ( Perez-Alvarez et al, 2020 ) could be used to investigate the input organization of individual neurons, but photoconversion of a group of spines could also result from a local dendritic calcium event. Combining SynTagMA and TubuTag might be a way to resolve this ambivalence: red spines on a green section of dendrite would be incontrovertible evidence for synchronous synaptic activity.…”

“…TubuTag is CaMPARI2 ( Moeyaert et al, 2018 ) fused to human α-tubulin, high affinity TubuTag contains 2 point mutations in the CaMPARI2 domain, F391W and L398V. To generate high affinity TubuTag, we replaced mCherry from pcDNA3.1 mCherry-human-alpha-tubulin (a gift from Marina Mikhaylova) with the CaMPARI moiety from preSynTagMA ( Perez-Alvarez et al, 2020 ; Addgene ID 119738) using Hin dIII and Bsp 1407I restriction sites. To insert the CaMPARI-tubulin fusion construct into a pAAV backbone, we first replaced in the C-terminus the restriction site Xho I by Hin dIII using PCR.…”

“…To generate a lasting record of synaptic activity that can be read-out later, we previously developed SynTagMA ( Perez-Alvarez et al, 2020 ), a synaptically targeted variant of CaMPARI-2 ( Moeyaert et al, 2018 ). This calcium integrator photoconverts irreversibly from green to red if it is bound to calcium and simultaneously illuminated by violet light ( Fosque et al, 2015 ; Moeyaert et al, 2018 ).…”

Freeze-frame imaging of dendritic calcium signals with TubuTag

“…Optical recording in vivo requires a priori knowledge of input sites since imaging is typically limited to a single plane containing a few dendrites (Helmchen et al, 1999;Takahashi et al, 2020). To generate a lasting record of synaptic activity that can be readout later, we previously developed SynTagMA (Perez-Alvarez et al, 2020), a synaptically-targeted variant of CaMPARI-2 (Moeyaert et al, 2018). This calcium integrator photoconverts irreversibly from green to red if it is bound to calcium and simultaneously illuminated by violet light (Fosque et al, 2015;Moeyaert et al, 2018).…”