Fig. 1: Mechanisms of release and uptake by ATLAS.
From: ATLAS: a rationally designed anterograde transsynaptic tracer
a, Western blot of lysate from cortical cultures. IB, immunoblot. b, DNA constructs for testing of ATLAS. Arrows denote promoters. c, Live cultured neuron expressing ss-AF-YFP (green) and PSD-95.FingR-tagRFP (magenta). Scale bar, 5 μm. d, Cultured neurons expressing ss-AF-YFP fixed and colabeled for endogenous clathrin. Scale bar, 5 μm. e, Neurons exposed to AF-HA protein (green) for 1 h and colabeled for endogenous MAP2 (magenta). Scale bar 25 μm. f, Same as e, but with the addition of 10 mg ml−1 chlorpromazine to the culture medium. Scale bar, 25 μm. g, Release of SEP from presynaptic sites in cultured cortical neurons expressing VAMP2-At-BACEcs-SEP. PSD-95.FingR-tagRFP (magenta), SEP (green). Scale bar, 1 μm. h, Same as g, but with VAMP2-At-BACEcs-AF-SEP. Scale bar, 1 μm. i, τ of VAMP2-At-BACEcs-AF-SEP is significantly increased versus VAMP2-At-BACEcs-SEP (n = 5, unpaired two-tailed t-test with Welch’s correction). j, Adding 100 mM KCl to the culture medium causes a significant increase in the frequency of release events of VAMP2-At-BACEcs-SEP (n = 5, paired two-tailed t-test). k,l, Graphs of the differential (DF) of SEP fluorescence for VAMP2-At-BACEcs-AF-SEP with control medium (k) versus with medium containing 100 mM KCl (l). Each spike denotes a single release event and does not reflect the duration of visible SEP fluorescence. m, Paired patch clamp recording of CA1 neurons in hippocampal slices (n–p). EC, entorhinal cortex. n, Biolistic transfection of CA1 pyramidal neurons with ss-AF-YFP did not significantly affect AMPAR-eEPSC amplitude (n = 10 pairs, P = 0.31, paired, two-tailed t-test). Scatterplots show eEPSC amplitudes for pairs of neighboring untransfected and transfected cells (open circles) with corresponding mean ± s.e.m. (filled circles). Insets show representative current traces from control and transfected neurons with stimulation artifacts subtracted. o, Same as n for NMDAR-eEPSC amplitude (n = 8 pairs, P = 0.70, paired, two-tailed t-test). p, Right, bar graph shows mean ± s.e.m. amplitude ratio of second AMPAR-eEPSC versus first (n = 6, P = 0.19, paired, two-tailed t-test). Left, representative current traces from control (gray) and transfected (orange) without stimulation artifacts. Scale bar, 50 ms. NS, not significant.
