Roles of specific Kv channel types in repolarization of the action potential in genetically identified subclasses of pyramidal neurons in mouse neocortex

Abstract

The action potential (AP) is a fundamental feature of excitable cells that serves as the basis for long-distance signaling in the nervous system. There is considerable diversity in the appearance of APs and the underlying repolarization mechanisms in different neuronal types (reviewed in Bean BP. Nat Rev Neurosci 8: 451-465, 2007), including among pyramidal cell subtypes. In the present work, we used specific pharmacological blockers to test for contributions of Kv1, Kv2, or Kv4 channels to repolarization of single APs in two genetically defined subpopulations of pyramidal cells in layer 5 of mouse somatosensory cortex (etv1 and glt) as well as pyramidal cells from layer 2/3. These three subtypes differ in AP properties (Groh A, Meyer HS, Schmidt EF, Heintz N, Sakmann B, Krieger P. Cereb Cortex 20: 826-836, 2010; Guan D, Armstrong WE, Foehring RC. J Neurophysiol 113: 2014-2032, 2015) as well as laminar position, morphology, and projection targets. We asked what the roles of Kv1, Kv2, and Kv4 channels are in AP repolarization and whether the underlying mechanisms are pyramidal cell subtype dependent. We found that Kv4 channels are critically involved in repolarizing neocortical pyramidal cells. There are also pyramidal cell subtype-specific differences in the role for Kv1 channels. Only Kv4 channels were involved in repolarizing the narrow APs of glt cells. In contrast, in etv1 cells and layer 2/3 cells, the broader APs are partially repolarized by Kv1 channels in addition to Kv4 channels. Consistent with their activation in the subthreshold range, Kv1 channels also regulate AP voltage threshold in all pyramidal cell subtypes.

Keywords: AmmTx3; dendrotoxin; guangxitoxin; potassium channel; somatosensory cortex.

Fig. 1.

Effects of 100 nM α-dendrotoxin (DTX) on the action potential. Action potentials (APs) were elicited with a 5-ms, just-suprathreshold current injection (Figs. 1–5). All experiments (Figs. 1–5) were performed in the presence of 20 μM DNQX, 50 μM AP-5, and 100 μM picrotoxin to block fast synaptic transmission via AMPA, NMDA, or GABA_A receptors, respectively. A: representative traces for single APs in a layer 2/3 pyramidal neuron before and after application of 100 nM DTX. Inset: same APs at longer time base. B: summary data for AP half-width (AP HW: width of the AP at one-half amplitude relative to the resting potential) in layer 2/3 pyramidal cells. C: summary data for AP voltage threshold (Threshold) in layer 2/3 pyramidal cells. D: representative traces for single APs in an etv1 pyramidal neuron (layer 5) before and after application of 100 nM DTX. Scale bars also apply to A and G. Inset: same APs at longer time base. E: summary data for AP half-width (AP HW) in etv1 pyramidal cells. F: summary data for AP voltage threshold (Threshold) in etv1 pyramidal cells. G: representative traces for single APs in a glt pyramidal neuron (layer 5) before and after application of 100 nM DTX. Inset: same APs at longer time base; scale also applies to insets in A and D. H: summary data for AP half-width (AP HW) in glt pyramidal cells. I: summary data for AP voltage threshold (Threshold) in glt cells. *Significant difference between control and drug (paired t-test, P < 0.05).

Fig. 2.

Effects of 100 nM guangxitoxin-1E (GxTx; blocks K_v2 channels) on the action potential. A: representative traces for single APs in a layer 2/3 pyramidal neuron before and after application of 100 nM GxTx. GxTx had no effect on the AP in these cells. Inset: same APs at longer time base. B: summary data for AP half-width (AP HW) in layer 2/3 pyramidal cells. C: summary data for AP voltage threshold (Threshold) in layer 2/3 pyramidal cells. D: representative traces for single APs in an etv1 pyramidal neuron (layer 5) before and after application of 100 nM GxTx. GxTx had no effect on the AP in these cells. Scale bars also apply to A and G. Inset: same APs at longer time base. E: summary data for AP half-width (AP HW) in etv1 pyramidal cells. F: summary data for AP voltage threshold (Threshold) in etv1 pyramidal cells. G: representative traces for single APs in a glt pyramidal neuron (layer 5) before and after application of 100 nM GxTx. GxTx had no effect on the AP in these cells. Inset: same APs at longer time base; scale also applies to insets in A and D. H: summary data for AP half-width (AP HW) in glt pyramidal cells. I: summary data for AP voltage threshold (Threshold) in glt cells.

Fig. 3.

Effects of 4 mM 4-aminopyridine (4-AP) on the action potential. A: representative traces for single APs in a layer 2/3 pyramidal neuron before and after application of 4 mM 4-AP. Inset: same APs at longer time base. B: summary data for AP half-width (AP HW) in layer 2/3 pyramidal cells. C: summary data for AP voltage threshold (Threshold) in layer 2/3 pyramidal cells. D: representative traces for single APs in an etv1 pyramidal neuron (layer 5) before and after application of 4 mM 4-AP. Scale bars also apply to A and G. Inset: same APs at longer time base. E: summary data for AP half-width (AP HW) in etv1 pyramidal cells. F: summary data for AP voltage threshold (Threshold) in etv1 pyramidal cells. G: representative traces for single APs in a glt pyramidal neuron (layer 5) before and after application of 4 mM 4-AP. Inset: same APs at longer time base; scale also applies to insets in A and D. H: summary data for AP half-width (AP HW) in glt pyramidal cells. I: summary data for AP voltage threshold (Threshold) in glt cells. *Significant difference between control and drug (paired t-test, P < 0.05).

Fig. 4.

Effects of 150 μM BaCl₂ on the action potential. A: representative traces for single APs in a layer 2/3 pyramidal neuron before and after application of 150 μM BaCl₂. Inset: same APs at longer time base. B: summary data for AP half-width (AP HW) in layer 2/3 pyramidal cells. C: summary data for AP voltage threshold (Threshold) in layer 2/3 pyramidal cells. D: representative traces for single APs in an etv1 pyramidal neuron (layer 5) before and after application of 150 μM BaCl₂. Scale bars also apply to A and G. Inset: same APs at longer time base. E: summary data for AP half-width (AP HW) in etv1 pyramidal cells. F: summary data for AP voltage threshold (Threshold) in etv1 pyramidal cells. G: representative traces for single APs in a glt pyramidal neuron (layer 5) before and after application of 150 μM BaCl₂. Inset: same APs at longer time base; scale also applies to insets in A and D. H: summary data for AP half-width (AP HW) in glt pyramidal cells. I: summary data for AP voltage threshold (Threshold) in glt cells. *Significant difference between control and drug (paired t-test, P < 0.05).

Fig. 5.

Effects of 200 nM AmmTx3 on the action potential. A: representative traces for single APs in a layer 2/3 pyramidal neuron before and after application of 200 nM AmmTx3 (blocks K_v4 channels associated with DPP6 or DPP10 subunits; Maffie et al. 2013). Inset: same APs at longer time base. B: summary data for AP half-width (AP HW) in layer 2/3 pyramidal cells. C: summary data for AP voltage threshold (Threshold) in layer 2/3 pyramidal cells. D: representative traces for single APs in an etv1 pyramidal neuron (layer 5) before and after application of 200 nM AmmTx3. Scale bars also apply to A and G. Inset: same APs at longer time base. E: summary data for AP half-width (AP HW) in etv1 pyramidal cells. F: summary data for AP voltage threshold (Threshold) in etv1 pyramidal cells. G: representative traces for single APs in a glt pyramidal neuron (layer 5) before and after application of 200 nM AmmTx3. Inset: same APs at longer time base; scale also applies to insets in A and D. H: summary data for AP half-width (AP HW) in glt pyramidal cells. I: summary data for AP voltage threshold (Threshold) in glt cells. *Significant difference between control and drug (paired t-test, P < 0.05).

Fig. 6.

Specificity of blockers for the A-type current. We used outside-out macropatches from the somas of layer 5 neurons to test whether 4-AP (4 mM), BaCl₂ (150 μM), and AmmTx3 (200 nM) are specific blockers of the transient A-type current or if they also block other components of the whole cell outward current. Similar numbers of etv1 and glt cells were tested; however, all examples shown were from glt cells. The same color scheme was used in A–C for the control trace (Ctl; black), the trace in the presence of blocker (blue trace), and the blocker-sensitive trace (red; obtained by subtracting the blocker trace from the Ctl trace). A: 4-AP. we applied 4 mM 4-AP to 6 cells (3 etv1 and 3 glt). 4-AP blocked most of the transient, A-type current and <1/2 of the current at 500 ms in this exemplar cell. Inset: voltage protocol for all drugs (A–C). B: BaCl₂. We applied BaCl₂ to 12 cells (6 etv1 and 6 glt). BaCl₂ blocked most of the early transient K⁺ current but also blocked most of the persistent current (at 500 ms). Outward holding current was also reduced by BaCl₂. C: AmmTx3. we applied AmmTx3 to 12 cells (8 etv1 and 4 glt). AmmTx3 blocked most of the transient, A-type current and almost none of the persistent current (at 500 ms) in this exemplar cell. D: summary box plots for block of peak and steady-state (ss, current at end of step) currents by 4-AP, BaCl₂, and AmmTx3. *Significant difference between control and drug (paired t-test, P < 0.05).