Fig 2: Downregulation of Nup358 expression in cortical neurons. Cortical neurons transfected at 5 DIV, with shRNA constructs co-expressing GFP to downregulate Nup358 (shNup358). As a control, neurons were transfected with a non-targeted shRNA construct (shCTRL). Immunostaining was conducted for Nup358 (red), and the nuclei are labeled with DAPI (blue). Asterisks are used to mark the transfected neuronal cells (green) with a substantial decrease in the Nup358 signal compared to the control untransfected cells. Scale bar: 10 µm.

Fig 3: The effect of Nup358 downregulation on the spontaneous activity of cultured mouse cortical neurons. (A) Tracks of membrane potential changes recorded in gap-free mode in the control scrambled-treated (left) and shNup358-treated (right) neurons. (B) Different properties of the resting membrane potentials are computed from the traces shown in (A). Each dot in the graphs represents an individual cell, and the dashed line in the violin plots is the median; the left and right dashed lines are the upper and lower quartiles, respectively. n = 14. * p < 0.05, Student’s unpaired t-test.

Fig 4: The downregulation of Nup358 increases the firing frequency of cortical neurons. (A) Representative recordings of membrane potentials in scrambled and shNup358-treated neurons in response to 1000 ms long whole-cell current injections. (B) Raster plot showing the temporal locations of action potential firing over current injection trials. Each bar in the raster indicates one action potential, and each row represents an independently recorded neuron. (C) Relative frequency distributions of the mean Inter Spike Interval (ISI) computed from the neurons assessed in (B). n = 6 + 6. µ, Gaussian fit mean; σ, Gaussian fit S.D.; RMSE, root-mean-square deviation.

Fig 5: Nup358-dependent modifications of sodium currents’ biophysical properties. (A) Cs-based isolation of voltage-gated sodium currents (INa) in response to voltage steps between −80 and +40 mV. (B) I-V plot of normalized peak INa currents plotted with current density as a function of test voltage. (C) Normalized mean conductance versus voltage plot of INa activation of the data in (B). The line represents the average Boltzmann fit to data. (D) Representative currents of INa steady-state inactivation (inset, voltage protocol). Currents were normalized to the current elicited from a holding potential of −80 mV and used to construct the steady-state inactivation. Plotted points represent the mean of the normalized currents and are fitted with a Boltzmann function. (E) Representative INa current traces were obtained using the protocol shown in the inset to study the recovery time course from fast inactivation. The plot represents the current recovery as a function of the interstimulus interval (Δt). Recoveries were fitted with one exponential.