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. 2013 Jul 22;591(Pt 19):4807–4825. doi: 10.1113/jphysiol.2013.257253

Figure 1. Biolistic transfection and voltage clamp data.

Figure 1

Biolistic (gene gun) transfection with cDNA for wild-type (Kv2.1 WT) or pore mutant Kv2.1 (Kv2.1 DN) altered whole cell outward currents in neocortical pyramidal neurons. A, confocal projection from Z-stack illustrating GFP expression in pyramidal and non-pyramidal neurons in layer 2/3 of rat somatosensory cortex in organotypic slice culture. Cells were transfected with cDNA for GFP and a non-conducting Kv2.1 pore mutant that acts as a dominant negative (Kv2.1 DN). Scale bar = 25 μm. B, left: IR/DIC image of transfected layer 2/3 pyramidal cell. Note gold ‘bullet’ (black circle) in cell. Scale bar = 25 μm. Right: FITC fluorescent image of the same cell, showing GFP expression. C, whole cell voltage clamp records from a non-transfected control cell. A family of voltage steps were made in 10 mV increments, nominally between –68 and +32 mV (see inset in D). Traces are shown for steps to +32 mV (black) and –2 mV (red). D, similar traces from a cell transfected with GFP and the Kv2.1 DN construct. Note smaller current amplitude and more obvious A-type current at –10 mV. E, traces for a cell transfected with GFP plus the Kv2.1 WT cDNA (Kv2.1 overexpression). Note the greatly enhanced current amplitude. F, bar chart showing mean amplitude (at 200 ms after step initiation) for non-transfected control (Ctl) vs. dominant negative (Kv2.1 DN) groups for pyramidal cells in layer 2/3 (L2/3), layer 5 (L5) and combined layers 2/3 and 5 (all). There were no significant differences between the various non-transfected (Ctl) groups. For all layers, the Kv2.1 DN cells had lower current amplitudes than matched non-transfected (Ctl) cells. G, for all layers combined, there was a significant reduction in current amplitude (at –2 mV) in the Kv2.1 DN group vs. the non-transfected group. There was no difference between non-transfected cells and cells transfected with GFP alone. Current amplitude was significantly increased in cells overexpressing the Kv2.1 WT cDNA (GFP non-transfected control group not shown).