To monitor translation at SN-MN synapses during neuronal plasticity, we generated translational reporters of sensorin, a SN-specific peptide neurotransmitter whose mRNA localizes to distal neuronal processes and concentrates at synapses in SNs paired with MNs (15
). Sensorin translation is required for synapse stabilization between SNs and MNs (27
) and both sensorin translation and secretion are required for 5HT-induced LTF of Aplysia
SN-MN synapses (31
). To generate sensorin translational reporters, we fused the 5’ and 3’ UTRs of sensorin to the coding region of the photoconvertible fluorescent protein dendra2 (32
). Dendra2 switches fluorescence irreversibly from green to red following UV illumination, allowing newly synthesized proteins (green) to be differentiated from proteins synthesized prior to photoconversion (red). Addition of the 5’ and 3’UTRs of sensorin to the dendra2 coding sequence generated a reporter whose mRNA localization was indistinguishable from endogenous sensorin mRNA (; S1, S2
). Specifically, the reporter mRNA localized to neurites of isolated SNs, and concentrated at SN-MN synapses as indicated by ectopic labeling of presynaptic terminals by expression of VAMP-mCherry and labeling of MNs with Alexa647 (). Reporters containing either the 3’UTR or the 5’UTR alone revealed that the 3’UTR was sufficient for localization to neurites, while addition of the 5’UTR was required for targeting to synapses (Fig. S2
). Thus distinct cis-acting elements mediate neuritic and synaptic mRNA localization.
Translation reporter mRNA colocalizes with endogenous sensorin mRNA at synapses
To visualize local translation of the reporter during LTF induced by bath application of 5X5HT, we expressed the reporter in SNs paired with MNs, removed the SN soma, and photoconverted dendra2 (Fig. S1, S3
, SOM). Newly synthesized protein (green) had to result from local translation in the neurite because the soma was no longer present. While 5x5HT induces transcription-dependent LTF, 24 hr (but not 48 hr) facilitation occurs in a translation-dependent manner in the absence of a SN soma, indicating that the initial events involved in persistent LTF can be monitored in SNs lacking cell bodies (33
). We imaged red and green channels before the first application and immediately after the fifth application of 5HT. Control cultures were stimulated with five applications of vehicle (5X artificial seawater, ASW), or were untreated. Very little green signal but robust red signal was detected following UV illumination, indicating efficient photoconversion (). After the fifth pulse of 5HT, green dendra2 signal increased at multiple sites within the neurite, and this was completely blocked by the translational inhibitor anisomycin (10 µM, , S4
). Modest increases in green dendra2 fluorescence were observed in control cultures following application of 5XASW, which were also blocked by anisomycin (10 µM, , S4
). This modest increase in green dendra2 fluorescence represents basal translation because it was also observed in untreated cultures (, S4
). Immediately following imaging, we fixed the cells and performed FISH for the reporter mRNA. 143 of 147 (97%) of sites with new translation contained concentrated reporter mRNA. Thus the subcellular localization of new translation correlated with the subcellular localization of reporter mRNA with remarkable accuracy. Because the reporter mRNA specifically concentrated in VAMP-positive synapses (, S2
)), we conclude that the reporter was translated at synapses.
Bath application of 5X5HT stimulates translation of reporter mRNA at synapses
We quantified new translation as ΔF/F (x100 = % change), normalized to the red dendra2 signal (which serves as a volume control, SOM, ). When stimuli were applied in the presence of anisomycin, there was no significant change in green signal, and thus no translation (2.3±3.4% for Ani+5XASW; −8.9±4.9% for Ani+5X5HT). In untreated or vehicle-treated cultures, we observed a 41.2±3.7% and 43.5±3.6% increase in translation. Following stimulation with 5X5HT, we observed a 155.9±18.7% increase in translation. Thus a basal level of sensorin translation occurs in neurites, which is significantly increased following stimuli that induce LTF (***p<0.001, ANOVA with Bonferroni’s multiple comparison test).