The gray box indicates the time window for efficient photoconversion of sypCaMPARI. c Activity-dependent photoconversion (ΔR/G 0) versus delay from start of stimulation to violet light from the same experiments in b. Trial 1 shows the effect of illumination without stimulation. b Ratio of red to green fluorescence (R/G 0) from sypCaMPARI boutons illuminated at different times relative to the electrical stimulation. ‘Exact’ & ‘200 ms’ timing share the same color code (cyan) as we used ‘exact’ timing in sypCaMPARI experiments and a 200 ms delay in preSynTagMA experiments. After each trial, new images were acquired. Cultured neurons were 14–22 days old, expressing sypCaMPARI for 8–16 days.Ī A spatial light modulator was used to illuminate parts of the axonal arbor (405 nm, 50 mW cm −2, 100 ms) at different times relative to a brief tetanic stimulation (5 APs). Data are presented as mean ± SEM in d, e, and g. AP fold increase was statistically different from all other stimulation conditions using a one-way ANOVA with Tukey’s post-hoc comparison (* p = 0.032). g The amount of photoconversion (R/G 0) in a similar experiment as e but varying the number of APs in a 50 Hz train (20 light pulses of 1 s duration at 0.1 Hz, 405 nm, 54.1 mW cm −2 Stim: 20 bursts at 50 Hz). f Representative red (magenta, trial 0, trial 1, trial 3) and green (green, trial 0) images of boutons from the experiment in e. Note that after washing out TTX, the R/G 0 ratio (trial 5) increased to the same amplitude as the first instance in control neurons (trial 2). The experiment was performed in the absence or presence of 3 µM tetrodotoxin to block action potentials (control: n = 8 neurons TTX: n = 7 neurons). e Plot of initial red to green ratio of boutons expressing sypCaMPARI at baseline, after photoconverting violet light alone (20 light pulses of 1 s duration at 0.1 Hz, 405 nm, 10.8 mW cm −2) and after simultaneous stimulation with trains of 50 APs at 50 Hz (trials 1–4). d Plot of the maximum ΔF/F versus number of APs from the experiments in b and c. Black line is the average response of n = 3 neurons. c Trial-averaged responses to 30 single APs (green, n = 57 synapses). b Average fluorescence response of sypCaMPARI boutons (green channel emission) to varying numbers of action potentials (APs) evoked at 50 Hz ( n = 6 neurons, 317 synapses). Note the clear punctate labeling of axonal boutons. Together, these tools provide an efficient method for repeatedly mapping active neurons and synapses in cell culture, slice preparations, and in vivo during behavior.Ī Representative image of cultured rat hippocampal neurons expressing sypCaMPARI. 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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