Fig. 5: Gas5 knockdown impairs the mobility and clustering of RNA granules and regulates intrinsic excitability.
From: Fear extinction is regulated by the activity of long noncoding RNAs at the synapse
a Schematic representation of the viral CIRTS knockdown construct. b expression of CIRTS along dendrites in primary cortical neurons. Time chased after 10 min of KCl induction is indicated. Arrowheads show CIRTS puncta (red) (n = 8 field of views). Scale bar = 5 µm. green, MAP2. c qRT-PCR performed on primary cortical neurons transduced with either control (n = 7 independent replicates) or CIRTS-Gas5 (n = 6 independent replicates) (two-tailed unpaired Student’s t test, t(9.329) = 3.584, p = 0.0056). **p < 0.01. Error bars represent S.E.M. d Representative primary cortical neurons dendritic region of interest illustrated from i) control and ii) a CIRTS-Gas5 knockdown neuron. Calibration bar shows the log10 diffusion coefficient, yellow colors represent areas of lower diffusion. Scale bar, 2.5 μm. Graph displaying (e) the mean squared displacement (μm2) and (f) the area under the MSD curve (μm2 s) for control (n = 6 independent replicates) and CIRTS-Gas5 knockdown neurons (n = 5 independent replicates) (two-tailed unpaired Student’s t test, p = 0.0303). *p < 0.05. Mean ±S.E.M. are plotted. g 3D representation of a region of interest highlights individual clusters (top), and their appearance in time (bottom). Spatiotemporal clusters were analyzed for their (h) radius (μm), (i) cluster lifetime (s), (j) density in clusters (traj/μm2) and (k) cluster average MSD (μm2) (Control, n = 1244 clusters; CIRTS-Gas5, n = 1811 clusters) (two-tailed unpaired Student’s t test, radius, t(2760) = 3.042, p = 0.0024; cluster lifetime, t(2921) = 5.354, p < 0.0001; density in clusters, t(2950) = 5.596, p < 0.0001; cluster average, t(2724) = 1.967, p = 0.0493). *p < 0.05, **p < 0.01, ****p < 0.0001. Mean ± S.E.M. are plotted. l Traces of mEPSCs recorded in primary cortical neurons in the presence of control or CIRTS-Gas5 knockdown. Plot showing (m) mEPSC amplitude and (n) mEPSC frequency of primary cortical neurons in the presence of control (n = 16 independent replicates) or CIRTS-Gas5 knockdown (n = 10 independent replicates) (two-tailed unpaired Student’s t test, amplitude, t(20.89) = 3.667, p = 0.0014; frequency, t(20.82) = 2.74, p = 0.0123). *p < 0.05, **p < 0.01. Error bars represent S.E.M.
