Kv2 channels regulate firing rate in pyramidal neurons from rat sensorimotor cortex

Abstract

The largest outward potassium current in the soma of neocortical pyramidal neurons is due to channels containing Kv2.1 α subunits. These channels have been implicated in cellular responses to seizures and ischaemia, mechanisms for intrinsic plasticity and cell death, and responsiveness to anaesthetic agents. Despite their abundance, knowledge of the function of these delayed rectifier channels has been limited by the lack of specific pharmacological agents. To test for functional roles of Kv2 channels in pyramidal cells from somatosensory or motor cortex of rats (layers 2/3 or 5), we transfected cortical neurons with DNA for a Kv2.1 pore mutant (Kv2.1W365C/Y380T: Kv2.1 DN) in an organotypic culture model to manipulate channel expression. Slices were obtained from rats at postnatal days (P7-P14) and maintained in organotypic culture. We used biolistic methods to transfect neurons with gold 'bullets' coated with DNA for the Kv2.1 DN and green fluorescent protein (GFP), GFP alone, or wild type (WT) Kv2.1 plus GFP. Cells that fluoresced green, contained a bullet and responded to positive or negative pressure from the recording pipette were considered to be transfected cells. In each slice, we recorded from a transfected cell and a control non-transfected cell from the same layer and area. Whole-cell voltage-clamp recordings obtained after 3-7 days in culture showed that cells transfected with the Kv2.1 DN had a significant reduction in outward current (∼45% decrease in the total current density measured 200 ms after onset of a voltage step from -78 to -2 mV). Transfection with GFP alone did not affect current amplitude and overexpression of the Kv2.1 WT resulted in greatly increased currents. Current-clamp experiments were used to assess the functional consequences of manipulation of Kv2.1 expression. The results suggest roles for Kv2 channels in controlling membrane potential during the interspike interval (ISI), firing rate, spike frequency adaptation (SFA) and the steady-state gain of firing. Specifically, firing rate and gain were reduced in the Kv2.1 DN cells. The most parsimonious explanation for the effects on firing is that in the absence of Kv2 channels, the membrane remains depolarized during the ISIs, preventing recovery of Na(+) channels from inactivation. Depolarization and the number of inactivated Na(+) channels would build with successive spikes, resulting in slower firing and enhanced spike frequency adaptation in the Kv2.1 DN cells.

Figure 1. Biolistic transfection and voltage clamp data

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).

Figure 2. Immunocytochemistry

We stained organotypic slices with the Neuromab Kv2.1 monoclonal antibody (K89/34). Slices contained non-transfected cells and cells with GFP fluorescence, indicating biolistic transfection with GFP plus the Kv2.1 DN construct (A, D and B, E), or GFP plus overexpression of Kv2.1 WT (C, F). Note the differing exposures in D vs. E and F. In E and F we focused on the enhanced, brighter staining of the transfected cells at the expense of viewing non-transfected cells, and thus the background is dimmer. Non-transfected cells (no GFP fluorescence) showed a typical patchy distribution of clusters of channels on the soma and proximal apical dendrites (D). The staining pattern was similar for some cells expressing the Kv2.1 DN (e.g. cell with arrow in D, cell marked with double arrow in E). Other cells transfected with the Kv2.1 DN (cell marked with single arrow in E) and most cells transfected with the Kv2.1 WT appeared more intensely stained with a more continuous pattern. The Kv2.1 WT cells in F (arrow, asterisk) showed intense staining compared to non-transfected cells but retained a clustered distribution. Scale bars = 25 μm.

Figure 3. Passive properties and single AP properties

Transfection with the Kv2.1 DN did not lead to changes in passive properties or properties of single APs. A, superimposed representative APs from a non-transfected (Ctl) layer 2/3 pyramidal cell and Kv2.1 DN cell from the same slice and layer. We found no differences between Kv2.1 DN and non-transfected control groups for any measured parameters of single APs (amplitude, threshold, dV/dt, half-width: APs elicited with suprathreshold 10 ms current injection). B, traces from a different pair of non-transfected cell (left) and Kv2.1 DN cell (right) indicate no changes in input resistance in the Kv2.1 DN cells. C, left: bar chart indicating no significant differences for input resistance between Kv2.1 DN and non-transfected (Ctl) cells. Right: rheobase did not differ in the Kv2.1 DN group vs. non-transfected (Ctl: n= 12 pairs of cells).

Figure 4. Repetitive firing

A, voltage trace in response to 200 pA current injection (500 ms) in a non-transfected layer 5 pyramidal cell. B, response of Kv2.1 DN transfected cell in same slice and layer as A (200 pA step current injection). C, plot of average firing frequency vs. injected current (I) for the cells shown in A and B. Note faster firing and steeper f–I slope in non-transfected (Ctl) cells (black circles) vs. Kv2.1 DN (red circles). D, response of same cell as in A to a larger (300 pA) current injection. Note faster firing vs. A. E, response of same Kv2.1 DN cell as in B to 300 pA current injection. Note faster initial firing vs. B, followed by a decline in spike height and eventual spike failure. F, plot of average firing frequency vs. injected current (I) for 12 pairs of non-transfected (Ctl) and Kv2.1 DN cells, as well as GFP alone (n= 7 cells) and Kv2.1 WT (n= 8 cells). Note lower slope (gain) and slower firing in the Kv2.1 DN cells (red). G, response of same cells as in A and B to a current injection at 1.5× rheobase. The non-transfected trace is black and Kv2.1 DN trace is red (there was little difference between the cells at this current). H, response of same cells in A and B to a larger current injection (3× rheobase). Same colour scheme as in G. Note depolarized V_m during ISIs and spike failure in the Kv2.1 N cell. I, plot of average firing frequency vs. multiple of rheobase. We compared non-transfected vs. Kv2.1 DN cells separately for layers 2/3 and for layer 5. In each case, the Kv2.1 DN cells fire slower and gain is lower. Individual data points are not included for clarity.

Figure 5. Spike frequency adaptation

A, repetitive firing from the same cells as in Fig. 4A and B for the time epochs indicated. Non-transfected (Ctl) trace is black and Kv2.1 DN trace is red. Aa, Firing is similar initially. The Kv2.1 DN trace becomes more depolarized during ISIs and firing slows towards the end of the first 100 ms of firing. Ab, firing in the same cells at 240–340 ms after initial current injection. Note the depolarized ISI and voltage threshold, slowed firing, and reduced spike amplitude in the Kv2.1 DN cell. B, plots for instantaneous firing frequency (1/ISI) normalized to the second ISI and as a function of ISI number. Experimental groups are indicated by colour (inset in E). Non-transfected (Ctl) cells are shown in black, cells transfected with GFP alone in green, Kv2.1 DN cells in red and cells with overexpressed Kv2.1 WT in blue. While GFP alone or Kv2.1 WT caused no significant change in this relationship, the Kv2.1 DN group showed a significantly greater decrease in firing frequency with increasing spike number (spike frequency adaptation: SFA). C, population data for the minimum voltage attained during the ISI for non-transfected and Kv2.1 DN cells as a function of spike number during firing (at 3× rheobase). The ISIs became more depolarized with successive spikes and this change was greater in Kv2.1 DN cells. D, spike voltage threshold became more depolarized with spike number and this effect appeared more pronounced in Kv2.1 DN cells vs. non-transfected control cells. E, the maximum rate of rise for the spike (dV/dt) decreased with spike number during firing and this effect was magnified in the Kv2.1 DN cells (P= 0.04). The difference between the Kv2.1 DN and Kv2.1 WT groups was significantly different (ANOVA and post-hoc Tukey's multiple comparisons test). F, the maximum rate of decay for the spike (dV/dt) slowed during firing and this effect was magnified in the Kv2.1 DN cells (P= 0.04). The difference between the Kv2.1 DN and Kv2.1 WT groups was significantly different (ANOVA and post-hoc Tukey's multiple comparisons test).

Figure 6. Phase relationships during repetitive firing in control and Kv2.1 DN cells

A and B, the trajectories of all APs during a 500 ms current injection at 3× rheobase. C and D, the trajectories of 10 spikes elicited by 10 suprathreshold, 5 ms current injections at 50 Hz. A, phase plot of APs during repetitive firing in a non-transfected cell. Note the relatively stable trajectories after the initial AP. B, phase plot of APs during repetitive firing in a Kv2.1 DN cell. Note the greater changes in trajectories of successive APs compared to those in A. There were greater changes in AP amplitude, voltage during ISIs and dV/dt in the Kv2.1 DN cells. C, non-transfected cell: phase plot of APs elicited by 10 stimuli (10 ms) at 50 Hz. The trajectories were more stable than during repetitive firing (e.g. A). D, Kv2.1 DN cell: phase plot of APs elicited by 10 stimuli (10 ms) at 50 Hz. The trajectories are stable and similar to the non-transfected cell in C.

Figure 7. Trains of APs elicited at 50 Hz with 10 ms suprathreshold current injections

A, a train of APs in a non-transfected (Ctl) layer 5 pyramidal cell elicited by 10 APs at 50 Hz and shown with a slow time base to emphasize the afterhyperpolarization (AHP) following the spikes. Inset, box plot showing no differences in the peak AHP between non-transfected and Kv2.1 DN cells for this protocol (no significant differences). B, a train of APs elicited by 10 APs at 50 Hz in a cell transfected with the Kv2.1 DN in layer 5 of the same slice as cell in A. Inset, box plot showing no differences in the slow AHP (sAHP: measured at 500 ms) between non-transfected (Ctl) and Kv2.1 DN cells for this protocol (no significant differences). C, AP train shown in A except with expanded time scale. Inset, stimulus protocol. D, AP train shown in B except with expanded time scale. E, peak AP amplitude for the first and 10th AP compared between non-transfected (Ctl) and Kv2.1 DN cells. There were no differences between groups for the initial AP, the 10th AP or the changes between APs 1 and 10. F, AP voltage threshold for the first and 10th AP compared between non-transfected (Ctl) and Kv2.1 DN cells. There were no differences between groups for the initial AP, the 10th AP, or the changes between APs 1 and 10. G, AP rate of rise (dV/dt up) for the first and 10th AP compared between non-transfected (Ctl) and Kv2.1 DN cells. There were no differences between groups for the initial AP, the 10th AP, or the changes between APs 1 and 10.