Journal: EMBO Reports

Article Title: miR-7 controls glutamatergic transmission and neuronal connectivity in a Cdr1as-dependent manner

doi: 10.1038/s44319-024-00168-9

Figure Lengend Snippet: Glutamatergic phenotype is reverted by sustained miR-7 overexpression. ( A ) Quantification of spontaneous glutamate release calculated as spontaneous frequency. Violin plots: integration of the synapses from all animals used (WT: n = 4 animals and 662 synapses; Cdr1as-KO: n = 3 animals and 421 synapses; “Methods”). Synapses with fluorescence value = 0 were removed. Kernel-Density Estimate plotted (“Methods”). Red line: median. P value: Mann–Whitney U test. ( B ) Quantification of AP-evoked glutamate release calculated as evoked probability. Violin plots: integration of the synapses from all animals used (WT: n = 4 animals and 335 synapses; Cdr1as-KO: n = 3 animals and 270 synapses; “Methods”). Synapses with fluorescence value = 0 were removed. Kernel-Density Estimate plotted (“Methods”). Red line: median. P value: Mann–Whitney U test; ***<0.001. ( C ) Left panel: Burst Frequency—total number of single-electrode bursts divided by the duration of the analysis (600 s), in Hz. Middle panel: IBI—time in seconds between bursts averaged over the duration of the analysis. Right panel: Number of spikes of single-electrode bursts averaged over the duration of the analysis. Each dot represents a single-electrode recording from two independent primary cultures (WT Control = 57; WT bicuculline = 64; Cdr1as-KO Control = 92; Cdr1as-KO Bicuculline = 31 electrodes). Horizontal line: median. P value: one-way ANOVA; ***<0.001. Multiple comparison correction test: Bonferroni. All not statistically significant comparisons are not shown. ( D ) Schematic representation of the miR-7a overexpression and the mCherry control constructs. Viral transduction performed at DIV7 (AAV, “Methods”). mCherry became visible at DIV14 and experiments were performed at DIV21. ( E ) Quantification of miR-7a upregulation based on reads from overexpression construct found in mRNA-Seq of WT and Cdr1as-KO neurons at DIV21, 14 days post miR-7a overexpression (“Methods”). Bar plots represent the mean value of four independent biological replicates per genotype. P value: U Mann–Whitney; n.s = 0.12. Error Bars: standard deviation. ( F ) Glutamate secretion assay performed on neuronal media collected from DIV18-21 WT and Cdr1as-KO neurons infected with control or miR-7a overexpression construct. Violin plots represent the integration of the secreted glutamate concentration quantified in independent primary cultures (WT = 12; WT + miR-7 overexpression = 12; Cdr1as-KO = 12; Cdr1as-KO + mirR-7 overexpression = 24 independent replicates). Glutamate concentrations are calculated based on a glutamate titration curve (“Methods”, Fig. ). The baseline concentration of glutamate in control media without cells was 0.28 µM (4612 RLU). Red line: median. P value: one-way ANOVA; *0.033, ***<0.001. Multiple comparison correction test: Bonferroni. All not statistically significant comparisons are not shown. .

Article Snippet: The sequence of pri-miR-7a was amplified from mouse brain tissue and cloned into an AAV vector (Addgene Plasmid #99126), driven by a neuronal promoter (hSyn1) and expressed together with mCherry fluorescent reporter.

Techniques: Over Expression, Fluorescence, MANN-WHITNEY, Control, Comparison, Construct, Transduction, Standard Deviation, Infection, Concentration Assay, Titration

Journal: EMBO Reports

Article Title: miR-7 controls glutamatergic transmission and neuronal connectivity in a Cdr1as-dependent manner

doi: 10.1038/s44319-024-00168-9

Figure Lengend Snippet: Sustained miR-7 expression reverted these effects. ( A ) Mean Firing Rate: Total number of spikes per single-electrode divided by the duration of the analysis (600 s), in Hz. Median value across timepoints with correspondant SD is plotted. Each timepoint represents a single-electrode recording from four independent primary cultures (WT = 187; WT + miR-7 overexpression = 194; Cdr1as-KO = 189; Cdr1as-KO + miR-7 overexpression = 200 electrodes); transduction time (AAV, DIV7) and reporter first visualization (mCherry, DIV14) are indicated. P value: two-way ANOVA; *0.033, **0.002, ***<0.001. Multiple comparison correction test: Bonferroni, all other comparisons were not statistically significant (not shown). ( B ) Burst Frequency: Total number of single-electrode bursts divided by the duration of the analysis, in Hz. Panel plotted as in ( A ). ( C ) Burst Duration: Average time (sec) from the first spike to last spike in a single-electrode burst. Panel plotted as in ( A ). ( D ) Asynchrony: The ability of neurons to generate APs simultaneously was calculated as area under the well-wide pooled inter-electrode cross-correlation. Higher areas indicate lower synchrony (Halliday et al, 2006, “Methods”). Median value across timepoints with correspondant SD is plotted. Each timepoint represents a network recording (“Methods”) from four independent primary cultures (WT = 11; WT + miR-7 overexpression = 26; Cdr1as-KO = 12; Cdr1as-KO + miR-7 overexpression = 28 electrodes); transduction time (AAV, DIV7) and reporter first visualization (mCherry, DIV14) are indicated. All metrics apply to network bursts across single wells within 20 ms. P value: two-way ANOVA; *0.033, **0.002, ***<0.001. Multiple comparison correction test: Bonferroni, all other comparisons were not statistically significant (not shown). ( E ) Oscillation: Average across network bursts of the interspike interval coefficient of variation (ISI CoV) (standard deviation/mean of the interspike interval) within network bursts. Oscillation is a measure of how the spikes from all of the neurons are organized in time. WT = 11–26; Cdr1as-KO: 12–28 independent network recordings. Panel plotted as in ( D ). ( F ) Burst Peak: Maximum number of spikes per second in the average network burst. The peak of the average network burst histogram divided by the histogram bin size to yield spikes per sec (Hz). WT = 11–26; Cdr1as-KO: 12–28 independent network recordings. Panel plotted as in ( D ). .

Article Snippet: The sequence of pri-miR-7a was amplified from mouse brain tissue and cloned into an AAV vector (Addgene Plasmid #99126), driven by a neuronal promoter (hSyn1) and expressed together with mCherry fluorescent reporter.

Techniques: Expressing, Over Expression, Transduction, Comparison, Standard Deviation