Loss of Navβ4-Mediated Regulation of Sodium Currents in Adult Purkinje Neurons Disrupts Firing and Impairs Motor Coordination and Balance

Abstract

The resurgent component of voltage-gated Na⁺ (Nav) currents, I_NaR, has been suggested to provide the depolarizing drive for high-frequency firing and to be generated by voltage-dependent Nav channel block (at depolarized potentials) and unblock (at hyperpolarized potentials) by the accessory Navβ4 subunit. To test these hypotheses, we examined the effects of the targeted deletion of Scn4b (Navβ4) on I_NaR and on repetitive firing in cerebellar Purkinje neurons. We show here that Scn4b^-/- animals have deficits in motor coordination and balance and that firing rates in Scn4b^-/- Purkinje neurons are markedly attenuated. Acute, in vivo short hairpin RNA (shRNA)-mediated "knockdown" of Navβ4 in adult Purkinje neurons also reduced spontaneous and evoked firing rates. Dynamic clamp-mediated addition of I_NaR partially rescued firing in Scn4b^-/- Purkinje neurons. Voltage-clamp experiments revealed that I_NaR was reduced (by ∼50%), but not eliminated, in Scn4b^-/- Purkinje neurons, revealing that additional mechanisms contribute to generation of I_NaR.

Keywords: Scn4b(−/−); Scn4b-targeted shRNA; cerebellum; dynamic clamp; resurgent sodium current.

Figure 1. Targeted disruption of Scn4b results in impaired motor performance

A, Schematic of the approach used to disrupt the Scn4b locus B, Nav channel α and β subunit transcript expression levels in cerebellar lysates from Scn4b^−/− and WT animals. C, Western blots of lysates prepared from the cerebella of WT and Scn4b^−/− animals probed with a polyclonal anti-Navβ4 (AbCam) antibody. D, Balance and motor coordination were evaluated by quantifying performance on the elevated balance beam (see Experimental Procedures). E–F, The mean ± SEM times to cross the 11 mm (E) and the 5 mm (F) beams, however, were significantly (**** P < .0001; ** P < .01; two-way ANOVA) longer for the Scn4b^−/− (N = 11), than the WT (N = 12), animals. G–H, Analysis of the numbers of hindlimb footslips revealed very few placement errors for both Scn4b^−/− and WT animals on the 11 mm beam, whereas the Scn4b^−/− animals had significantly (P < .01; two-way ANOVA) more footslips than WT animals on the 5 mm beam.

Figure 2. High frequency firing is attenuated in adult Scn4b^−/− Purkinje neurons

A, Representative whole-cell recordings obtained from adult WT (upper, black) and Scn4b^−/− (lower, red) Purkinje neurons in acute cerebellar slices. B. The mean ± SEM spontaneous firing frequency measured in adult Scn4b^−/− (red; N = 5, n = 23) Purkinje neurons was significantly (*** P < .001; Student’s t-test) lower than in WT (black; N = 5, n = 23) cells. C–F, Repetitive firing rates in response to depolarizing current injections from the baseline (C, D) and following a (−0.5 nA) hyperpolarizing current injection (E, F) were also measured. Representative recordings from WT Purkinje neurons are shown in C and E; the protocols are displayed below the records. Mean ± SEM evoked firing rates were significantly (** P < .01; two-way ANOVA) lower in Scn4b^−/− (N = 4, n = 10–14), than in WT (N = 3, n = 12–16), Purkinje neurons both at baseline (D) and following the −0.5 nA prepulse (F). See also Figure S3.

Figure 3. Acute in-vivo Navβ4 knockdown in mature Purkinje neurons also disrupts high frequency firing

A, Representative fluorescence images of an acute cerebellar slice prepared from a WT adult mouse 3 weeks following injection of Scn4b-targeted shRNA-expressing AAV1. The low magnification image (A1) shows widespread tdTomato expression throughout an entire lobule of the cerebellum (scale bar = 100 μm) and the higher magnification image (A2) shows a single AAV1-transduced Purkinje neuron (scale bar = 50 μm). B, Representative current-clamp recordings from td-Tomato-expressing Purkinje neurons in slices prepared two weeks following injections of the non-targeted shRNA- (upper; black) or the Scn4b-targeted shRNA- (lower; red) expressing AAV1. C, Purkinje neurons expressing the Scn4b-targeted shRNA (red; N = 2; n = 17) fired at significantly (** P < .01; Student’s t-test) lower rates than cells expressing the non-targeted shRNA (black; N = 2; n = 11). D, Repetitive firing rates in response to varying amplitude depolarizing current injections following −0.5 nA prepulses were measured in experiments similar to those in Figure 2F. E, Mean ± SEM evoked firing rates are significantly (* P < .05, two-way ANOVA) lower in Purkinje neurons expressing the Scn4b-targeted shRNA (N = 2; n = 13, red dashed line) than in Purkinje neurons expressing the non-targeted shRNA (N = 2; n = 11, black dashed line). The data in Figure 2F were replotted here to facilitate direct comparison between the effects of the targeted deletion and the acute knockdown of Scn4b.

Figure 4. Effects of the targeted deletion of Scn4b on Nav currents in adult Purkinje neurons

Representative whole-cell Nav currents recorded from adult WT (A) Purkinje neurons in acute cerebellar slices; the voltage-clamp paradigm is illustrated above the records and the currents are shown in the color of the corresponding voltage step. The raw, unsubtracted Nav current records are shown in the boxed inset. The peak amplitudes of the transient Nav currents evoked at each test potential were measured, and peak (transient) Nav conductances were determined and normalized (in the same cell) to the maximal Nav conductance. B, Mean ± SEM normalized peak transient Nav conductances in WT (black; N = 5; n = 12) and Scn4b^−/− (red; N = 5; n = 13) adult Purkinje neurons plotted as a function of the test potential. C, Representative recordings of Nav currents evoked at 0 mV from various conditioning voltages in a WT Purkinje neuron are shown; the voltage-clamp protocol is illustrated above the current records and the currents are shown in the color of the corresponding voltage step. The persistent component of the Nav currents has been digitally subtracted. D, The peak transient Nav current amplitudes evoked at 0 mV from each conditioning voltage were measured and normalized to the peak transient Nav current measured after the −120 mV conditioning voltage step (in the same cell). The mean ± SEM normalized peak Nav current amplitudes in WT (black; N = 5; n = 15) and Scn4b^−/− (red; N = 3; n = 14) Purkinje neurons were plotted as a function of the conditioning voltage and fitted with first order Boltzmann functions. E, Nav currents evoked at −40 mV following a prepulse to +30 mV were also recorded in ACSF with 300 μM CdCl₂ and 5 mM TEA before and during perfusion of ACSF containing 1 μM TTX (E1). The currents recorded at various times (indicated by different colors) during the perfusion of the TTX-containing bath are shown and the TTX-sensitive currents were obtained by subtraction of the currents recorded before and after the start of TTX perfusion (E2).

Figure 5. Peak transient Nav current density is higher in acutely isolated neonatal Scn4b^−/−, than in WT, Purkinje neurons

A, Differential interference contrast (DIC) photomicrographs of acutely isolated neonatal cerebellar Purkinje neurons; scale bars = 50 μm. B, Representative recordings of whole-cell transient Nav currents evoked at various test potentials; the voltage steps are shown below the current records in the corresponding colors. C, The mean ± SEM transient Nav current densities measured in isolated P11–P18 WT (N = 7; n = 12) and Scn4b^−/− (N = 6; n = 12) Purkinje neurons are plotted as a function of the test potential. See also Figure S2. D, The mean ± SEM inactivation tau at 0 mV and −20 mV are similar in WT (N = 7; n = 11) and Scn4b^−/− (N = 6; n = 12) Purkinje neurons. E, Mean ± SEM normalized transient Nav conductances are plotted as a function of test potential. The Boltzmann fits to the data reveal that the voltage-dependences of activation of the transient Nav currents in P11–P18 WT (N = 7; n = 12) and Scn4b^−/− (N = 6; n = 13) Purkinje neurons are similar. F, The voltage-dependences of inactivation of the transient Nav currents were measured using a protocol as described in Supplemental Experimental Procedures. The Boltzmann fits to the mean ± SEM normalized data reveal that the voltage-dependences of steady-state inactivation in WT (N = 7; n = 10) and Scn4b^−/− (N = 6; n = 13) Purkinje neurons are similar. G, I_NaP was measured 10 ms after the onset of the test potential. Boltzmann fits revealed that the voltage-dependences of activation of I_NaP are similar in WT (N = 7; n = 12) and Scn4b^−/− (N = 6; n = 13) Purkinje neurons. H, To compare the relative amplitudes of I_NaP and I_NaT in WT and Scn4b^−/− Purkinje neurons, the magnitude of the peak Nav tail current, measured at −90 mV following the 0 mV voltage step, was divided by the peak I_NaT amplitude in the same cell. I, Recovery of Nav currents in WT and Scn4b^−/− Purkinje neurons from inactivation was measured at 0 mV using the three pulse protocol illustrated; representative Nav currents following each recovery period are shown below. The peak Nav current elicited during the second depolarizing step to 0mV, following each recovery period, was normalized to the peak Nav current measured during the first depolarizing step. Mean ± SEM values were plotted as a function of the time between steps. J, Representative action-potential voltage-clamp command waveform (from a holding voltage of −80mV) (upper) and TTX-sensitive currents (below) recorded in WT (black) and Scn4b^−/− (red) Purkinje neurons are shown.

Figure 6. I_NaR is attenuated, but not eliminated, in acutely isolated P11–P18 Scn4b^−/− Purkinje neurons

A, Representative TTX-sensitive Nav currents evoked in low (50 mM) Na⁺ bath solution in response to a voltage ramp from +20 to −100 mV (displayed below the current records) in isolated WT (black) and Scn4b^−/− (red) Purkinje neurons. B, The normalized current values reveal that the TTX-sensitive inward currents were significantly (** P < .01, Student’s t-test) smaller in Scn4b^−/−, than in WT, cells. C, Representative TTX-sensitive I_NaR recorded at various potentials following depolarizations to +30 mV in a WT Purkinje neuron with 154 mM Na⁺ in the bath; the voltage command is displayed below the records and the arrow indicates the peak I_NaR. D, Representative peak I_NaR evoked at −45 mV in isolated WT (black) and Scn4b^−/− Purkinje neurons. E, Mean ± SEM peak I_NaR in neonatal Scn4b^−/− (red; N = 5; n = 10) Purkinje neurons are significantly (* P < .05, two-way ANOVA) attenuated compared with WT (black; N = 5; n = 10) Purkinje neurons. I_NaP, measured 80 ms into the sweep, was digitally subtracted prior to the analysis. F, The time constants (tau) of I_NaR decay are similar in neonatal WT (n = 10) and Scn4b^−/− (n = 10) Purkinje neurons.

Figure 7. Dynamic clamp-mediated addition of I_NaR rescues high frequency firing in Scn4b^−/− Purkinje neurons

A, Schematic of a thirteen state Markov model describing the transient, persistent and resurgent components of the Nav currents in cerebellar Purkinje neurons (see: Supplemental Experimental Procedures B, Brief depolarizing current injections (bottom traces) were delivered to a model cell and the resulting change in voltage (upper trace), as well as the RTXI current injected (middle trace), were measured. The red dashed line reveals the voltage change of the model cell without the addition of I_Na. C, Left- To isolate and apply the resurgent portion of the I_Na model in dynamic clamp, if the proportion of channels in the OB state was increasing, the RTXI current was set to 0. Right- With the fast transient portion of the Nav current excluded, the resurgent Nav current was isolated. See also Figure S4. D, Change in the instantaneous firing frequency of a Purkinje neuron in response to 0.0–2.5 nA (x-axis) of depolarizing current with different amounts of modeled I_NaR added to the cell via dynamic clamp. E, Representative action potentials (black) and RTXI currents (red) are illustrated. Upper traces show I_NaR in a Purkinje neuron firing at 48 Hz, and the lower traces show I_NaR in a Purkinje neuron firing at 288 Hz. Boxes (on the right) provide increased temporal resolution to display I_NaR activation during a single action potential; the red arrow indicates I_NaR during the inter-spike interval and the blue arrow indicates I_NaR during the upswing of the action potential. F, Plots of the changes in the O (blue), OB (green) and C5 (yellow) states as a function of time during action potentials; I_NaR is also illustrated (red). The proportion of channels in the OB state (green arrow) is decreasing during the inter-spike interval. G, Purkinje neuron action potential (black) waveforms generated in Matlab with the I_NaR model applied. Hyperpolarization of the membrane voltage in 5 mV increments alters the time course of I_NaR activation during the inter-spike interval.