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. 2010 Aug;120(8):2661-71.
doi: 10.1172/JCI42219. Epub 2010 Jul 12.

Axon initial segment dysfunction in a mouse model of genetic epilepsy with febrile seizures plus

Verena C Wimmer  1 , Christopher A Reid, Suzanne Mitchell, Kay L Richards, Byron B Scaf, Bryan T Leaw, Elisa L Hill, Michel Royeck, Marie-Therese Horstmann, Brett A Cromer, Philip J Davies, Ruwei Xu, Holger Lerche, Samuel F Berkovic, Heinz Beck, Steven Petrou
Affiliations

Axon initial segment dysfunction in a mouse model of genetic epilepsy with febrile seizures plus

Verena C Wimmer et al. J Clin Invest. 2010 Aug.

Abstract

Febrile seizures are a common childhood seizure disorder and a defining feature of genetic epilepsy with febrile seizures plus (GEFS+), a syndrome frequently associated with Na+ channel mutations. Here, we describe the creation of a knockin mouse heterozygous for the C121W mutation of the beta1 Na+ channel accessory subunit seen in patients with GEFS+. Heterozygous mice with increased core temperature displayed behavioral arrest and were more susceptible to thermal challenge than wild-type mice. Wild-type beta1 was most concentrated in the membrane of axon initial segments (AIS) of pyramidal neurons, while the beta1(C121W) mutant subunit was excluded from AIS membranes. In addition, AIS function, an indicator of neuronal excitability, was substantially enhanced in hippocampal pyramidal neurons of the heterozygous mouse specifically at higher temperatures. Computational modeling predicted that this enhanced excitability was caused by hyperpolarized voltage activation of AIS Na+ channels. This heat-sensitive increased neuronal excitability presumably contributed to the heightened thermal seizure susceptibility and epileptiform discharges seen in patients and mice with beta1(C121W) subunits. We therefore conclude that Na+ channel beta1 subunits modulate AIS excitability and that epilepsy can arise if this modulation is impaired.

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Figures

Figure 1
Figure 1. Construction of the CW mouse model.
(A) Targeting strategy for creating of the β1(C121W) knockin mouse model. Black triangles, Scn1b exons; white and red triangles, loxP sites; gray arrows, PKG/neo cassettes. Recombination between the 2 loxP sites highlighted in red led to generation of CW knockin mice. (B) Top panel: Southern blot analysis showing correct genomic targeting of the floxed cassette (KIfl) using 5′ (left), 3′ (middle), and Neo (right) probes. Bottom panel: Southern blot analysis of PGKneo excision and retention of exon3 carrying the C121W mutation. The blot shown in the left image was stripped and reprobed for Neo (expected band size: approximately 16 kb, middle) and Cre (expected band size: approximately 10 kb, right). (C) Western blots. Top panel: untransfected control HEK cells and HEK cells transfected with cDNA coding for mouse β1-EGFP. Bottom panel: whole-brain extracts of P16 WW, CC, and CW mutant mice. Identical protein levels in all 3 genotypes. (D) Normal ECoGs in somatosensory cortex of P37 CC (black) and CW mice (red).
Figure 2
Figure 2. CW mice show FS phenotype.
(A) ECoGs from somatosensory (S1) and motor cortex (M1) with simultaneous depth recording of hippocampal field potentials (HC). Core temperature increase was nonlinear; selected temperatures are indicated at the bottom. Arrowhead indicates start of heating; dashed vertical line represents start of behavioral arrest phenotype, which occurred at 38.5°C in this mouse. The first signs of epileptiform activity were seen in the hippocampus. (B) Thermal seizure threshold is reduced in C121W knockin mice. CC (n = 46), CW (n = 58), WW (n = 25); ANOVA with Bonferroni’s post-hoc test; CC versus CW, *P < 0.05; CC versus WW, **P < 0.001; CW versus WW, **P < 0.001.
Figure 3
Figure 3. The AIS localization of β1 is disrupted by the C121W mutation.
Green indicates virally expressed β1(C121)-EGFP (A–E) or mutant β1(W121)-EGFP (F–J). Gray shows AIS visualized by immunostaining against Ank. Red shows virally expressed tdTomato (A–D and F–I) or Panα staining (E and J). Merge of the 3 channels with Ank is depicted in blue. (A) Pyramidal neurons in CA3 region of the hippocampus. (B) Pyramidal neurons in hippocampal subiculum (Subi). (C) Layer 2/3 pyramidal neuron in primary somatosensory cortex (S1). (D) Purkinje cell (PC) in cerebellum. Arrows indicate AIS containing β1(C121)-EGFP. (E) High magnification of plasma membrane delineating the AIS in a CA3 pyramidal neuron; membrane colocalization of β1(C121)-EGFP with Na+ channel α subunits and Ank. (F–I) As in A–D for β1(W121)-EGFP. Arrows show that β1(W121) does not localize to AIS, identified by anti-Ank staining. (J) Proximal β1(W121)-EGFP clusters do not colocalize with Na+ channel α subunits or Ank, indicating intracellular retention. Scale bars: 20 μm (A–D, F–I); 1 μm (E and J).
Figure 4
Figure 4. The β1(C121W) mutation does not affect AIS targeting of α subunits.
Green shows staining against Na+ channel α subunit; red shows staining against Ank. Left columns, wild-type (CC) tissue; right columns, homozygous mutant (WW) tissue. (A and D) NaV1.1 in proximal axon of inhibitory neuron (IN, molecular layer in hippocampus). (B and E) NaV1.2 is evenly distributed in pyramidal cell AIS (CA3). (C and F) NaV1.6 gradient with maximum in distal AIS (the “appendage” visible in F is an AIS crossing in close proximity, CA3). Scale bar: 5 μm
Figure 5
Figure 5. CW neurons are more excitable.
Black bars/squares: CC, n = 13 neurons from 8 mice; red bars/ triangles: CW, n = 13 neurons from 6 mice. (A) Representative example traces for selected drive currents of a CC subicular neuron. Asterisk indicates burst at beginning of current step. Complete current step protocol used shown at top. (B) As A but for CW cell. (C) Duration of burst events (*P = 0.038 or less for multiple Student’s t tests; population average P = 0.0019). (D) Number of spikes per burst (*P = 0.025 or less for multiple Student’s t tests; population average P = 0.0065). (E) Frequency of tonic firing increased in CW cells (*P = 0.019 or less for multiple Student’s t tests). (F) AP threshold determined for first AP in each sweep (averaged across driving currents; CC, –48.74 ± 0.32 mV; CW, –51.43 ± 0.23 mV; CC, n = 121 APs; CW, n = 140; Mann-Whitney U test, *P < 0.0001).
Figure 6
Figure 6. Altered AIS AP initiation in CW mice.
Black line/bars: CC, n = 13 neurons from 8 mice; red line/bars: CW, n = 13 neurons from 6 mice. (A) Averaged AP waveforms showing increased amplitude in CW cells (CC, 85.28 ± 0.2914 mV; CW, 97.52 ± 0.1081 mV; amplitude only analyzed for drive currents > 120 pA; CC, n = 97 APs; CW, n = 118; Mann-Whitney U test, P < 0.0001). (B) Time-aligned second derivative of AP waveform (d2V/dt2). (C) Magnification of boxed area in B; second derivative peaks illustrating significantly increased acceleration and increased peak-to-peak time in CW neurons (error bars represent SEM). (D) Comparison of phase plot of AP waveforms (dVdt/ dt) from CC and CW neurons. (E) Increased voltage acceleration in CW AIS (CC, n = 118 APs; CW, n = 139; Mann-Whitney U test, *P < 0.0001). (F) Increased delay between AIS and somatic peaks (CC, n = 103 APs; CW, n = 124; Mann-Whitney U test; *P < 0.0001). (G) Comparison of temperature sensitivity of AIS kinetics. Red solid (CW) and black (CC) lines are recordings made at 34°C as shown in D. Blue traces are recordings from the same genotypes but made at 22°C recording temperature (CW, light blue; CC, dark blue; CC, n = 71 APs from 8 cells; CW, n = 57 APs from 6 cells; P = 0.91, Student’s t test). Time bar: 0.5 ms or 0.1 ms (C); Vm bar: 20 mV; d2/V/dt2 bar: 1000 mV/s–2.
Figure 7
Figure 7. Modeling suggests that wild-type β1 subunits reduce the voltage-dependent opening of AIS Na+ channels.
(A) Comparison of first APs elicited by current injection into a neuron model an AIS/soma Na+ conductance ratio of 15. APs are aligned at threshold as defined for the physiological data. Shifts in AIS Na+ channel V1/2 from 0 to –15 mV are color-coded green to black. V1/2 of the soma was held constant. Time bar: 1 ms; Vm bar: 50 mV. (B) Second derivative of the voltage traces shown in A, illustrating AIS-specific changes in AP initiation (cf. Figure 6, D and E). Traces are aligned to the second peak in the second derivative to more clearly demonstrate changes in peak acceleration and axo-somatic delay. (C–E) Influence of changes in V1/2 of AIS relative Na+ current density (AIS/soma Na+ conductance ratios between 2 and 15) on Vm and acceleration reflecting AIS AP initiation (C), somatic AP generation (D), and axo-somatic delay, calculated as the temporal separation of the 2 peaks in the second derivative (E).
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References

    1. Brackenbury WJ, Djamgoz MB, Isom LL. An emerging role for voltage-gated Na+ channels in cellular migration: regulation of central nervous system development and potentiation of invasive cancers. Neuroscientist. 2008;14(6):571–583. doi: 10.1177/1073858408320293. - DOI - PMC - PubMed
    1. Brackenbury WJ, Isom LL. Voltage-gated Na+ channels: potential for beta subunits as therapeutic targets. Expert Opin Ther Targets. 2008;12(9):1191–1203. doi: 10.1517/14728222.12.9.1191. - DOI - PMC - PubMed
    1. Wallace RH, et al. Febrile seizures and generalized epilepsy associated with a mutation in the Na+-channel beta1 subunit gene SCN1B. Nat Genet. 1998;19(4):366–370. - PubMed
    1. Scheffer IE, et al. Temporal lobe epilepsy and GEFS+ phenotypes associated with SCN1B mutations. Brain. 2007;130(pt 1):100–109. doi: 10.1093/brain/awl272. - DOI - PubMed
    1. Patino GA, et al. A functional null mutation of SCN1B in a patient with Dravet syndrome. J Neurosci. 2009;29(34):10764–10778. doi: 10.1523/JNEUROSCI.2475-09.2009. - DOI - PMC - PubMed

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