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. 2009 Jun 19;324(5934):1536-40.
doi: 10.1126/science.1173205. Epub 2009 May 14.

Synapse- And Stimulus-Specific Local Translation During Long-Term Neuronal Plasticity

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Free PMC article

Synapse- And Stimulus-Specific Local Translation During Long-Term Neuronal Plasticity

Dan Ohtan Wang et al. Science. .
Free PMC article

Abstract

Long-term memory and synaptic plasticity require changes in gene expression and yet can occur in a synapse-specific manner. Messenger RNA localization and regulated translation at synapses are thus critical for establishing synapse specificity. Using live-cell microscopy of photoconvertible fluorescent protein translational reporters, we directly visualized local translation at synapses during long-term facilitation of Aplysia sensory-motor synapses. Translation of the reporter required multiple applications of serotonin, was spatially restricted to stimulated synapses, was transcript- and stimulus-specific, and occurred during long-term facilitation but not during long-term depression of sensory-motor synapses. Translational regulation only occurred in the presence of a chemical synapse and required calcium signaling in the postsynaptic motor neuron. Thus, highly regulated local translation occurs at synapses during long-term plasticity and requires trans-synaptic signals.

Figures

Figure 1
Figure 1. Translation reporter mRNA colocalizes with endogenous sensorin mRNA at synapses
(A) Colocalization of 5’3’UTR sensorin reporter mRNA and endogenous sensorin mRNA. Expression vectors encoding the control (CtrlUTR) or sensorin (5’3’UTR) translational reporter were microinjected into DIV2 Aplysia SNs (isolated or paired with MNs). Cultures were fixed 48 hrs later and processed for double-label FISH using DIG-labeled dendra2 riboprobes and biotin-labeled sensorin riboprobes. Representative confocal images of DIC/merged (A1, 2, 7 and 8), dendra reporter mRNA in red (A3, 5, 9 and 11) and endogenous sensorin mRNA in green (A4, 6, 10 and 12). FISH signals are shown in isolated SNs (A1, 3, 4, 7, 9 and 10) and in SNs paired with MNs (A2, 5, 6, 8, 11 and 12). Scale bar: 50µm. Colocalization was quantified as colocalization coefficient (0, no colocalization; 1, perfect colocalization); (B) VAMP-mCherry and reporter were co-expressed in SN; MN was labeled with Alexa647. Synapses are detected as VAMP-mCherry-positive varicosities contacting the MN. B1: Low magnification images of reporter protein (green), VAMP-mCherry (red) and MN (blue) with boxes of higher magnification images in a1 and b1. Cells were fixed after imaging and processed for reporter mRNA FISH in B2, a2 and b2. Scale bar: 50µm.
Figure 2
Figure 2. Bath application of 5X5HT stimulates translation of reporter mRNA at synapses
Sensorin translational reporter was expressed in Aplysia SNs co-cultured with MNs, the SN soma was removed and dendra2 was UV photoconverted from green to red throughout the neuronal arbor. (A1) Low magnification image of dendra-reporter SN-MN co-culture. The dashed circle outlines the removed soma. (A2 and A3) High magnification DIC/merged image of area in the red box in A1, before (A2) and after (A3) UV photoconversion. (A4 and A5) Green dendra2 signal following photoconversion, before (A4) and after (A5) serotonin stimulation (5X5HT). Lack of green dendra2 indicates efficient photoconversion; new green dendra2 reveals significant new translation post 5x5HT. (A6 and A7) Photoconverted red dendra2 signal before (A6) and after (A7) 5x5HT shows SN volume. A8: Cells were fixed at the end of the experiment and processed for FISH with antisense dendra2 riboprobes. Arrowheads in A5 and A8 point to the sites of new (green) dendra2 protein synthesis colocalizing with reporter mRNA clusters. Scale bar: 50µm. See fig. S4 for Basal, 5XASW, Ani+5X5HT, Ani+5XASW results. (B) Quantification of new translation as ΔF/F, ***p<0.001, ANOVA and Bonferroni multiple comparison test.
Figure 3
Figure 3. Synapse-, stimulus- and transcript-specific translation
Sensorin translational reporter was expressed in SNs paired with MNs, SN soma was removed and dendra2 was UV photoconverted from green to red. Stimuli were locally applied to subsets of SN-MN synapses using perfusion electrodes (see fig S3). (A, B, C) Low magnification image of dendra2-reporter expressing coculture. Dashed circle indicates removed SN soma; blue arrowheads indicate direction of perfusion; black squares denote regions imaged before and after local perfusion; white squares denote imaged non-perfused regions. (A-C panels 1–4) Pseudocolored images of green dendra2 signal after photoconversion and before (pre) and after (post) local perfusion. (A-C panels 5 and 6) reporter mRNA FISH images. Arrowheads in A2 and A5 point to new translation colocalizing with reporter mRNA. A: 5’3’UTR reporter, local perfusion of 5x5HT. B: 5’3’UTR reporter, local perfusion of 5xFMRFa. C: 3’UTR, local perfusion of 5x5HT. See fig S6–9 for images of pre-UV green and post-UV red photoconverted dendra2 signal, 5XASW controls, and 1X5HT stimulation. Scale bar: 50µm; (D) Quantification of translation as ratio of ΔF/F at perfused compared to non-perfused sites reveals translation occurs only with 5x5HT. ***p<0.0001, Wilcoxon-Mann Whitney test (also see fig S10 for group data).
Figure 4
Figure 4. Local stimulation with 5X5HT does not stimulate translation of sensorin reporter in isolated SNs or in SNs paired with non-target MNs
Sensorin translational reporter was expressed in (A) isolated Aplysia SNs,or (B) SNs cocultured with nontarget L11 MNs. Local perfusion of 5X5HT was as described in Fig 3. (A, B) Low magnification image of dendra2-reporter expressing SNs. Dashed circle indicates removed soma; blue arrowheads indicate direction of perfusion; black squares denote regions imaged before and after local perfusion; white squares denote imaged non-perfused regions. (A and B panels 1–4) Pseudocolored images of green dendra2 signal after photoconversion and before (pre) and after (post) local perfusion. (A and B panels 5 and 6) reporter mRNA FISH. See fig S9 for images of red photoconverted dendra2 signal. Scale bar: 50µm; (C) Quantification of translation as ratio of ΔF/F at perfused compared to non-perfused sites reveals no increase in translation with either 5X5HT or 5XASW (Wilcoxon-Mann-Whitney test; also see group data and RNA intensity quantification in S13).
Figure 5
Figure 5. Calcium signaling in motor neuron is required for 5HT-induced translation of reporter in sensory neuron
Sensorin translational reporter was expressed in Aplysia SNs co-cultured with target LFS MNs. The SN soma was removed, either vehicle (A) or BAPTA (50mM, B) was microinjected into the MN, dendra2 was photoconverted, and 5X5HT was locally perfused. (A, B) Low magnification image of dendra2-reporter expressing coculture. Dashed circle indicates removed soma; blue arrowheads indicate direction of perfusion; black squares denote regions imaged before and after local perfusion; white squares denote imaged non-perfused regions. (A and B panels 1–4) Pseudocolored images of green dendra2 signal after photoconversion and before (pre) and after (post) local perfusion. (A and B panels 5 and 6) Reporter mRNA FISH images. See fig S13 for images of pre-UV and photoconverted dendra2 signal. Scale bar: 50µm; (D) Quantification of translation as ratio of ΔF/F at perfused compared to non-perfused sites reveals that BAPTA injection into the MN blocks 5HT-induced translation in the SN (***p<0.0005, t-test).

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