Fig. 1: J∆NI8-1 transduction induces electrophysiological alterations in rat primary hippocampal neurons. | Gene Therapy

Fig. 1: J∆NI8-1 transduction induces electrophysiological alterations in rat primary hippocampal neurons.

From: Genetic mutations in HSV-1 replication-defective vectors: Implications for their safety in gene therapy applications

Fig. 1

A Schematic representation of the J∆NI8-1 recombinant vector genome, in which the Ubiquitin (Ub) promoter driving tdTomato (tdT) expression has been inserted in the ICP4 locus. B Schematic representation of the experimental set-up used for primary neuron transduction with HSV-1-based vectors. C Representative immunofluorescence images showing rat primary hippocampal neurons transduced with J∆NI8-1 at a multiplicity of infection of 1. Representative immunofluorescence images for β-III-Tubulin (C, green); DAPI (C’, blue), tdTomato (C”, red). D Histogram summarizing the average percentage of tdT-positive cells over DAPI in rat primary hippocampal neurons transduced with J∆NI8-1 at a multiplicity of infection of 1, E, F Voltage response of control and J∆NI8-1 transduced neurons to suprathreshold depolarizing ramp currents. Sequential sweeps are represented in grey. The one with greater amplitude current injection is shown in black. Note that J∆NI8-1 transduced neurons require injection of greater amplitude currents to elicit action potentials. G, H Voltage-clamp recordings of control and J∆NI8-1 transfected neurons (Vhold = −60 mV). I–L Histograms summarizing average Resting Membrane Potential, Input Resistance, spontaneous event Frequency, and Instantaneous Frequency of control and J∆NI8-1 transfected neurons. Data are shown as Mean ± SEM. Every dot represents a single cell. I CTRL = −55.7 ± 1.6 mV, J∆NI8-1 = -66 ± 1.4 mV. J CTRL = 296 ± 30 MΩ, J∆NI8-1 = 63 ± 7 MΩ. K CTRL = 0.28 ± 0.21 Hz, J∆NI8-1 = 2.47 ± 0.44 Hz. L CTRL = 0.78 ± 0.47 Hz, J∆NI8-1 = 14.42 ± 2.07 Hz. Statistical significance in (I–L) was calculated using an unpaired Student’s t-test. ***p < 0.001. M Examples of spontaneous activity in control (upper trace) and J∆NI8-1 transduced neurons (lower trace). Note the bursting activity of transduced cells which arises from a highly hyperpolarized resting membrane potential compared to controls. N, O Histograms summarizing average AHP amplitude and AP amplitude of control and J∆NI8-1 transduced neurons. Data are shown as Mean ± SEM. Every dot represents a single cell. N CTRL = −5,1 ± 1,3 mV; J∆NI8-1 = +9,6 ± 1.0 mV. O CTRL = +73 ± 4 mV; J∆NI8-1 = +68 ± 2 mV. Statistical significance in (N, O) was calculated using an unpaired Student’s t-test. ***p < 0.001. P Average voltage traces (V), and their first derivative (V’), of the spontaneous action potential of control (black) and transduced (red) neurons. Below, phase plot highlighting the slope trajectory during the entire AP cycle. Data are the mean of three cells per condition.

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