FIGURE SUMMARY
Title

Visualization of newly synthesized neuronal RNA in vitro and in vivo using click-chemistry

Authors
Akbalik, G., Langebeck-Jensen, K., Tushev, G., Sambandan, S., Rinne, J., Epstein, I., Cajigas, I., Vlatkovic, I., Schuman, E.
Source
Full text @ RNA Biol.

Visualization of newly synthesized RNA in cultured hippocampal neurons. (A) Illustration of the method. (B) An increase in the somatic and dendritic RNA signal is observed with increasing concentrations of EU. Fire lookup table (LUT) represents fluorescence intensity of labeled RNA (pixel intensities 0–255). Arrowheads (purple) indicate RNA granules. Scale bar for B and C, 50 μm (C) Block of transcription or absence of EU results in a severely diminished nascent RNA signal. The dendrites were detected using an anti-MAP2 antibody (green). (D) Representative straightened dendrites following 5 mM EU for the indicated treatment times. Left, proximal; right, distal. Scale bar 20 μm. LUT, Fire (pixel intensities 0–255) (E) Mean intensity of the nascent RNA fluorescence in MAP2-defined dendritic area. Per time point, 20–42 dendrites from 2 independent experiments were analyzed. ****p < 0.0001, *p = 0.0254, n.s. (non significant) p = 0.6304, *p = 0.0313, respectively. (F) Heatmap shows the RNA signal along the dendrite. LUT, Spring. (G) Electrical and synaptic properties after 5 mM EU treatment for 12 h. Upper-left, representative traces of mEPSCs from the control and EU-treated neurons; upper-right, mEPSCs amplitude in control (n = 7) and EU-treated neurons (n = 7), p = 0.8844; lower-left, mEPSC frequency in control (n = 7) and EU-treated neurons (n = 7), p = 0.6342, lower-right, resting potential in control (n = 8) and EU-treated neurons (n = 8), p = 0.9707.

PABP and Y10B colocalize with newly synthesized RNA. (A, B) Left panels: Representative dendrites showing the colocalization of the fluorescent signals from PABP or Y10B and nascent RNA. Only a defined length (104 μm) of the original straightened dendrite is shown. Antibody leave-out control (no anti-PABP) does not show any signal. Scale bars: 10 μm. LUT for PABP, Cyan Hot; LUT for RNA, Fire (pixel intensities 0–255). The dendritic marker MAP2, magenta. Right panels: Graph of the correlation of the signal from RNA granules and PABP or Y10B along the original length of the representative dendrites shown. corr., correlation coefficient.

Depolarization of hippocampal neurons induces a decrease in detectable nascent RNA in the somata and dendrites. (A, B) Left panels: Representative dendrites showing a decrease in newly synthesized RNA signal after KCl treatment. The dendritic marker MAP2, green. Scale bars, 10 μm. Right panels: Graph showing the average intensity of RNA fluorescence in dendrites (n = 10–16) or somata (n = 9–19) in MAP2-defined dendritic area. Nascent RNA signal decreased significantly in both dendrites (top, **p = 0.0043, n.s. = 0.9999, **p = 0.0093, respectively; bottom, **p = 0.0023) and somata (top, **p = 0.0029, bottom, *p = 0.0129) following KCl treatment.

In vivo labeling of newly synthesized RNA in the larval zebrafish brain. (A) Experimental procedure. (B) Left. Representative images of the larval zebrafish head (dorsal view) showing the time-dependent increase in nascent RNA signal in specific regions (green arrows) of the tectum. LUT, Fire (pixel intensities 0–255). Right. Graph representing the time-dependent increase in the normalized mean fluorescence intensity of RNA signal for the indicated conditions. Scale bar, 25 μm. n = 7–9 larvae from 2 independent experiments. *p = 0.0221, p = 0.0001, n.s., (not significant). LUT, Fire. (C) Left. Representative images of the larval zebrafish head (dorsal view) showing PTZ-induced increase in the newly synthesized RNA signal in the indicated region of the tectum. Scale bar, 25 μm. LUT, Fire (pixel intensities 0–255). Right. Graph representing the PTZ- induced increase in the normalized mean intensity of the newly synthesized RNA signal for the indicated conditions. n = 22–24 larvae from 4 independent experiments. *p = 0.0133, **p = 0.0062, ****p < 0.0001.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ RNA Biol.