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. 2022 Jun 15;5:589. doi: 10.1038/s42003-022-03446-1

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a, b Basal synaptic transmission in acute hippocampal slices. a Input–output (I/O) curves obtained by plotting the field excitatory postsynaptic potentiation (fEPSP) slope against stimulus intensity were comparable between Mkln1^–/– and Mkln1^+/+ slices. b The paired-pulse ratio was recorded across different interstimulus intervals (ISI). Note the significantly larger paired-pulse facilitation (PPF) in Mkln1^–/– slices relative to Mkln1^+/+ slices (genotype: F(1,31) = 12.31, P < 0.01). PPF was maximal at ISI = 40 ms in Mkln1^–/– slices (^**P < 0.01) and decreased with larger intervals. Mkln1^+/+ (n = 17 slices); Mkln1^–/– slices (n = 16 slices). c, d Mkln1^–/– mice showed impaired late-LTP induced by high-frequency stimulation (HFS) trains in the Schaffer collateral-CA1 region (arrows: 3× 1 s stimulations of 100 Hz at 0, 6, and 12 min). c The averaged fEPSP-slope values normalized to 30-min baseline. Despite comparable initial induction of LTP in Mkln1^–/– and Mkln1^+/+ slices, the potentiation effect decayed more rapidly in Mkln1^–/– slices (time × genotype: F(90,1800) = 1.48, P < 0.01), leading an overall significant reduction in LTP (genotype: F(1,20) = 6.33, P < 0.05) by the end of a 4 h recording. Diamond points illustrate baseline recording in the CA1 region. Inset: Representative fEPSP traces (Dashed lines correspond to baseline while solid lines represent LTP). d Averaged fEPSP-slope values from 120–240 min (Late-LTP), which are significantly decreased in Mkln1^–/– slices (Independent t-test: t(20)= −3.18, P < 0.01). Data are represented as mean ± SEM. Mkln1^+/+ (N = 7 mice); Mkln1^–/– (N = 8 mice). Baseline: Mkln1^+/+ (n = 12 slices), Mkln1^–/– (n = 10 slices); HFS: Mkln1^+/+ (n = 12 slices), Mkln1^–/– (n = 10 slices). e Immunoblots of fractionated extracts from hippocampal tissue showing separation into soluble (supernatant) and insoluble (pellet) fractions. Enrichment of synaptotagmin (Syt) and PSD-95 is shown in the soluble and insoluble fractions, respectively. f, g Analysis of actin levels in Mkln1^+/+ and Mkln1^–/– hippocampal tissue lysates. f The upper panel shows representative blots of actin levels in the soluble and insoluble fractions. Protein levels were normalized to the housekeeping gene, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). g Compared with Mkln1^+/+ tissue, actin protein levels were significantly increased in insoluble fractions of Mkln1^–/– hippocampal tissue (independent t-test with Welch’s correction: t(8.13) = 2.34, P < 0.05). Mkln1^+/+ (N = 8); Mkln1^–/– (N = 8) from 3 independent preparations. Data are expressed as a percentage of control ± SEM. h Representative images of Mkln1^+/+ and Mkln1^–/– dissociated hippocampal neurons stained with phalloidin. Scale bar, 5 µm. i Frequency distribution showing F-actin immunoreactivity within phalloidin-stained regions of interest (ROI). The intensity of F-actin labeling in actin-rich compartments is significantly higher in Mkln1^–/– neurons than Mkln1^+/+ controls (two-sample KS test: D = 0.24, P < 0.0001, Mkln1^+/+: 1054; Mkln1^–/–: 1067 dendritic protrusions). j Normalized phalloidin intensity measures were significantly increased in Mkln1^–/– actin-positive ROIs compared with Mkln1^+/+ controls (independent t-test: t (38) = 2.88, P < 0.01). Mkln1^+/+ (n = 20); Mkln1^–/– (n = 20) neurons from two to three independent preparations. Data are expressed as a percentage of control ± SEM. k–n Elimination of muskelin leads to an increase in the spine F-actin stable pool. k FRAP analysis of GFP-actin 1 day after transfection of cultured hippocampal neurons (DIV 13); representative spine heads before and at different points after the laser bleaching impulse. Imaging and photobleaching conditions were similar for both conditions. Scale bar = 2 µm. l Analysis of GFP-actin fluorescence recovery shows that the plateau of the recovery curve in Mkln1^–/– spines does not approach the same level as Mkln1^+/+ spines. m The stable actin fraction measured from FRAP curves of individual spines is significantly increased in Mkln1^–/– spines (genotype: β = 16.98, t = 5.49, P < 0.0001). n The GFP-actin recovery half-time of the dynamic F-actin pool is comparable for Mkln1^–/– and Mkln1^+/+ spines. Scatter plots depict results per spine, and the line within bar graphs corresponds to the group mean value. Mkln1^+/+ (n = 17 neurons, 47 spines), Mkln1^–/– (n = 25 neurons, 94 spines; 2 independent replications from four to six embryos per genotype.