![]() Following repeated sparse activation of Schaffer collateral axons, postSynTagMA marks a small subset of synapses on CA1 pyramidal cells as highly active. We use spine-localized postSynTagMA to visualize the extent of action potential back-propagation into the large apical dendritic tree of hippocampal pyramidal cells. Bouton-localized preSynTagMA allows us to distinguish active and inactive axons. Three steps were necessary to create a Synaptic Tag for Mapping Activity (SynTagMA): (1) We introduced point mutations in CaMPARI to generate a new probe, CaMPARI2, with improved brightness and conversion efficiency 11 (2) We target CaMPARI2 (F391W_元98V) to either presynaptic boutons by fusing it to synaptophysin (preSynTagMA) or to the postsynaptic density by fusing it to an intrabody against PSD95 15 (postSynTagMA) (3) We present 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. By anchoring CaMPARI to either pre- or postsynaptic compartments, we are able to mark active synapses a in short time window defined by violet light illumination. As CaMPARI was designed to diffuse freely within the cytoplasm, it does not preserve subcellular details of Ca 2+ signaling. CaMPARI has been successfully applied to map the activity of thousands of neurons in zebrafish, Drosophila, and in mouse 10, 12, 13, 14. 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 10, 11. In general, the need to choose between high temporal or high spatial resolution limits the information we can extract from the brain with optical methods.Ī strategy to overcome this limit is to rapidly ‘freeze’ activity in a defined time window and read it out at high resolution later. Projection microscopy can be a very efficient approach 9, but only in situations where the fluorescent label is restricted to one or very few neurons. Multi-beam scanning designs have been proposed, but due to scattering of emitted photons, they do not produce sharp images at depth 7, 8. Most functional imaging experiments are therefore limited to cell bodies, i.e., low spatial resolution 5, or monitor the activity of a few synapses within a single focal plane 6. However, the tradeoff between spatial and temporal resolution makes it impossible with this technology to simultaneously measure fluorescence in the thousands of synapses of even a single pyramidal neuron. Excellent genetically-encoded sensors for calcium, glutamate and voltage have been developed 1, 2, 3, which when combined with two-photon laser-scanning microscopy can monitor the activity of neurons even down to the synaptic level in highly light-scattering brain tissue 4. On the network level, neuroscience faces an extreme ‘needle in the haystack’ problem: it is thought to be impossible, in practice, to create a map of all synapses that are active during a specific sensory input or behavior. The physical changes underlying learning and memory likely involve alterations in the strength and/or number of synaptic connections. Together, these tools provide an efficient method for repeatedly mapping active neurons and synapses in cell culture, slice preparations, and in vivo during behavior. To analyze large datasets, we show how to identify and track the fluorescence of thousands of individual synapses in an automated fashion. Targeted to excitatory postsynapses, postSynTagMA creates a snapshot of synapses active just before photoconversion. ![]() Targeted to presynaptic terminals, preSynTagMA allows discrimination between active and silent axons. 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. Here we introduce SynTagMA to tag active synapses in a user-defined time window. ![]() ![]() 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. Information within the brain travels from neuron to neuron across billions of synapses. ![]()
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