Paradoxical hyperexcitability from NaV1.2 sodium channel loss in neocortical pyramidal cells

Figures

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  Figure 1.. Scn2a knockout increases AP excitability, with intact axonal propagation (A) APs (spikes) per 300-ms stimulation epoch for each current amplitude in Scn2a+/+ (black), Scn2a+/− (cyan), and Scn2a−/− (red) cells. Brown and gray areas highlight first 50 ms and last 100 ms of epoch, respectively. (B) APs versus current for 3 Scn2a conditions, color coded as in (A). Bars are mean ± SEM; n = 77 WT, 40 Scn2a+/−, 60 Scn2a−/− cells. Top: entire 300-ms epoch. Middle: first 50 ms. Bottom: last 100 ms. Asterisk indicates F/I slope between 100 and 300 pA. p < 0.01, Kruskal-Wallis test. Scale bar: 20 μm. (C) Scanning differential contrast and two-photon fluorescent image of simultaneous recordings in a Scn2a−/− pyramidal neuron of APs evoked from soma and recorded at axon bleb 212 μm from axon hillock. (D) Conduction delay of AP threshold between soma and bleb in each recording versus axon length and AP amplitude in bleb in Scn2a+/+ (black, n = 9) and Scn2a−/− (red, n = 6) cells. (E) Scanning differential contrast and two-photon fluorescent image detailing bouton scan sites for AP-evoked calcium imaging of a Scn2a−/− neuron (insets). Responses to burst of five APs at boutons 1–3 are shown as…

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  Figure 2.. Scn2a knockout impairs somatodendritic excitability (A) Rheobase AP waveform from all neurons. Note reduction in AP height with loss of NaV1.2. (B) AP plotted as voltage versus time (top) and dV/dt versus voltage (phase-plane, bottom). Different phases of the AP are color-coded across panels to indicate different phases of the AP corresponding to initiation of AP in AIS, the soma, and peak repolarization. (C) Phase plane of data shown in (A). (D) Amplitude and threshold of APs at rheobase. Circles are single cells. Boxplots are median, quartiles, and 90% tails. Data color-coded as in (A). *p < 0.001, Kruskal-Wallis test. (E) Peak of AIS and somatic components of the rising phase of the AP and minimum of the falling phase of the AP. *p < 0.001, Kruskal-Wallis test. (F) Left: morphology of imaged neuron. Middle: 2-photon calcium imaging of AP-evoked calcium transients throughout apical tuft dendrites in Scn2a+/+ (black, n = 10), Scn2a+/− (cyan, n = 7), and Scn2a−/− (red, n = 6) cells. Right: transient amplitude shown for first of five bursts (top) and area under the curve from stimulus onset to stimulus offset +100 ms (bottom). Circles and bars are means ± SEM. *p < 0.01,…

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  Figure 3.. Scn2a loss in dendrites increases AP excitability in compartmental models (A) Compartmental model of layer 5 pyramidal cell with NaV1.2 and NaV1.6 distributed in the AIS soma and dendrites as shown. Purple and green for NaV1.2 and NaV1.6 show relative densities in dendrite, soma, and AIS in model. Note different scale bars for each compartment. (B) Firing patterns of models in which NaV1.2 is reduced to 50% or 0% in all compartments. Data are color-coded as in Figures 1 and 2. (C) Phase planes and F/I curves for each model. Note that increase in F/I is appreciable only in Scn2a−/− conditions. (D) APs elicited by 350-pA current and underlying whole-cell Na and K currents with progressive loss of NaV1.2. (E) Charge transfer (Q) ratio (Na/K) for each AP as a function of NaV1.2 density. 100% is WT levels, and 0% is Scn2a−/−. (F) Timing of first APs in conditions noted in (D) and (E) (identical color coding). Note depolarization of AHP and advanced AP2 timing with NaV1.2 loss. (G) Reducing K conductance in WT model recapitulates hyperexcitability observed in Scn2a−/− cells due to depolarization of AHP (inset). (H) Increasing K conductance in Scn2a−/− model reduces excitability, in…

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  Figure 4.. Dynamic-clamp injection of NaV1.2 or Ktransient conductance rescues excitability (A) AP firing response to 300-pA, 300-ms current before (red) and after (gray) injection of 1.25 μS NaV1.2 conductance via somatic recording electrode. Dashed line is aligned to last AHP in baseline conditions. (B) As in (A), but for 200 nS of Ktransient. Baseline is in red, and Ktransient injection is in purple. (C) Top: phase planes of first AP for each condition in (A) and (B). Bottom: summary data for interleaved WT cells (n = 5) in identical recording conditions and for all Scn2a−/− cells with NaV1.2 (n = 9) or KV1.2 (n = 18) conductance injection. Circles are single cells with lines connecting paired data. n applies to all panels. (D) Highlight of timing of first three APs in WT and Scn2a−/− before and after NaV1.2 conductance injection. Inter-spike intervals (ISIs) for APs 1–2 and 2–3 are plotted in (E). (E) Top left: ISI1 versus ISI2 at 300-pA current injection for all WT (black) and Scn2a−/− (red) cells in population in Figure 1. Other panels show change in ISIs from Scn2a−/− baseline (red) after conductance injection (other colors). Lines connect values from the same cell. (F) Minimum…