1. Neuroscience

Enhanced excitability of cortical neurons in low-divalent solutions is primarily mediated by altered voltage-dependence of voltage-gated sodium channels

  1. Briana J Martiszus
  2. Timur Tsintsadze
  3. Wenhan Chang
  4. Stephen M Smith  Is a corresponding author
  1. Section of Pulmonary & Critical Care Medicine, VA Portland Health Care System, United States
  2. Department of Medicine, Division of Pulmonary & Critical Care Medicine, Oregon Health & Science University, United States
  3. Endocrine Research Unit, Veterans Affairs Medical Center and University of California, San Francisco, United States
https://doi.org/10.7554/eLife.67914
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Cite this article
  1. Briana J Martiszus
  2. Timur Tsintsadze
  3. Wenhan Chang
  4. Stephen M Smith
(2021)
Enhanced excitability of cortical neurons in low-divalent solutions is primarily mediated by altered voltage-dependence of voltage-gated sodium channels
eLife 10:e67914.
https://doi.org/10.7554/eLife.67914
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6 figures, 17 tables and 1 additional file

Figures

Figure 1 with 2 supplements
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CaSR deletion reduces divalent-dependent excitability.

(A) Spontaneous voltage traces at RMP following the application of solutions with different divalent concentrations (T1.1 (upper traces), T0.2 (middle), and T1.1 recovery (lower)) recorded in three …

Figure 1—source data 1

Action potential frequency and resting membrane potential in conventional WT, NesCre and Casr-/- neurons in T1.1 or T0.2 with no current injection.

RMP units are mV and each sub-column represents measurements from a single neuron.

https://cdn.elifesciences.org/articles/67914/elife-67914-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
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Casr expression levels reduced in Casr-/- neurons.

Casr expression levels shown as delta delta with actin used as normalizing gene. Average values of 1.0, 0.02, and 0.65 for NesCre, Casr-/-, and conWT respectively with each genotype reflecting the …

Figure 1—figure supplement 2
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CaSR deletion reduces divalent-dependent excitability following the generation of action potentials elicited by current injections.

(A) The neurons were held at RMP in T1.1 (zero basal current injection) or at the same potential in T0.2 (hyperpolarizing current injection as described in Figure 1H). The current injection is shown …

Figure 2
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CaSR deletion does not affect divalent-dependent excitability at equivalent membrane potential.

(A) Exemplary traces showing the divalent-dependent increase in neuronal excitability following the switch from T1.1 to T0.2 (change indicated by upper trace) in NesCre (blue) and Casr-/- (red) …

Figure 2—source data 1

Action potential frequency in NesCre and Casr-/- neurons in T1.1 or T0.2 with standing currents Ia and Ib.

The action potential frequency is in log base 10 and each sub-column represents measurements from a single neuron.

https://cdn.elifesciences.org/articles/67914/elife-67914-fig2-data1-v2.xlsx
Figure 3
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CaSR deletion and external divalent concentration affect VGSC current gating.

(A) Exemplary traces showing VGSC currents activated by voltage steps from −80 in 10 mV increments (left), in nucleated patches isolated from NesCre (blue) and Casr-/- (red) neurons in solutions T1.1…

Figure 4
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CaSR deletion and external divalent concentration do not significantly affect VGPC current gating.

(A) Exemplary traces showing VGPC currents activated by voltage steps from −80 in 10 mV increments (left), in a NesCre neuron in solutions T1.1 and T0.2. The outward currents elicited by the 50 ms …

Figure 5 with 1 supplement
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VGSC current activation by decreased external divalent concentration.

(A,B) Plots illustrating the responses of the basal currents in two WT neurons during application of T1.1 and T0.2 before and during TTX or TTX and Gd3+. Average basal currents were measured over 50 …

Figure 5—figure supplement 1
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VGSC current activation by decreased external divalent concentrationin Casr-/- neurons.

(A) Plot of average basal current measurements (filled circles) and individual neurons (open circles) in each solution condition in Casr-/- (n = 9) neurons. Each basal current represents the average …

Figure 6
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Divalent-dependent depolarization is almost entirely mediated via VGSCs.

(A) The response of the membrane potential in two WT neurons during application of T1.1 and T0.2 before and during TTX or TTX and Gd3+. T1.1 and T0.2 application is indicated by vertical shading …

Figure 6—source data 1

Depolarization elicited by switch from T1.1 to T0.2 that was sensitive to TTX, Gd3+, or resistant to both blockers in conventional WT and Casr-/- neurons.

Depolarization units are volts and each sub-column represents measurements from a single neuron.

https://cdn.elifesciences.org/articles/67914/elife-67914-fig6-data1-v2.xlsx

Tables

Table 1
Action potential frequency.
ANOVA tableSSDFMSF (DFn, DFd)P value
Interaction130.0264.99F (2, 54)=3.193p=0.0489
[Ca2+]o on AP count594.41594.4F (1, 54)=29.21p<0.0001
Genotype136.1268.04F (2, 54)=3.368p=0.0418
Subjects (matching)10915420.20F (54, 54)=0.9925p=0.5110
Residual10995420.35
Table 2
RMP.
ANOVA tableSSDFMSF (DFn, DFd)P value
Interaction14.36114.36F (1, 37)=1.035p=0.3155
[Ca2+]o on RMP438.91438.9F (1, 37)=31.65p<0.0001
Genotype151311513F (1, 37)=19.10p<0.0001
Subjects (matching)29303779.19F (37, 37)=5.710p<0.0001
Residual513.23713.87
Table 3
Action potential frequency.
ANOVA tableSSDFMSF (DFn, DFd)p Value
Interaction0.340710.3407F (1, 35)=0.4758p=0.4949
[Ca2+]o at hyperpolarizing injection8.26218.262F (1, 35)=11.54p=0.0017
Genotype0.538010.5380F (1, 35)=0.7309p=0.3984
Subjects (matching)25.76350.7360F (35, 35)=1.028p=0.4679
Residual25.06350.7161
Table 4
Action potential frequency.
ANOVA tableSSDFMSF (DFn, DFd)p Value
Interaction0.609010.6090F (1, 35)=0.2048p=0.6536
[Ca2+]o at depolarizing injection45.09145.09F (1, 35)=15.17p=0.0004
Genotype5.98215.982F (1, 35)=0.9959p=0.3252
Subjects (matching)210.2356.006F (35, 35)=2.020p=0.0204
Residual104.1352.973
Table 5
Action potential threshold.
ANOVA tableSSDFMSF (DFn, DFd)p Value
Interaction0.522510.5225F (1, 27)=0.07478p=0.7866
[Ca2+]o on AP threshold394.61394.6F (1, 27)=56.48p<0.0001
Genotype54.34154.34F (1, 27)=2.284p=0.1424
Subjects (matching)642.52723.80F (27, 27)=3.406p=0.0011
Residual188.6276.987
Table 6
Action potential frequency.
ANOVA tableSSDFMSF (DFn, DFd)p Value
Interaction0.430530.1435F (3, 87)=0.3481p=0.7906
[Ca2+]o and I22.2337.410F (3, 87)=17.97p<0.0001
Genotype0.134110.1341F (1, 29)=0.2005p=0.6577
Subjects (matching)19.41290.6692F (29, 87)=1.623p=0.0445
Residual35.87870.4123
Table 7
Action potential frequency.
Sidak's multiple comparisons testMean diff.95% CI of diff.Significant?SummaryAdjusted p value
T1.1 Ia vs. T0.2 Ia−1.059−1.498 to −0.6200Yes****<0.0001
T1.1 Ia vs. T0.2 Ib−0.6203−1.059 to −0.1813Yes**0.0016
T1.1 Ia vs. T1.1 Ib−0.1163−0.5554 to 0.3227Nons0.9797
T0.2 Ia vs. T0.2 Ib0.4387−0.0003709 to 0.8778Nons0.0503
T0.2 Ia vs. T1.1 Ib0.94270.5037 to 1.382Yes****<0.0001
T0.2 Ib vs. T1.1 Ib0.50400.06496 to 0.9431Yes*0.0160
Table 8
Membrane potential with Ia.
ANOVA tableSSDFMSF (DFn, DFd)p Value
Interaction131.71131.7F (1, 29)=4.055p=0.0534
[Ca2+]o949.21949.2F (1, 29)=29.22p<0.0001
Genotype162.71162.7F (1, 29)=4.874p=0.0353
Subjects (matching)968.02933.38F (29, 29)=1.028p=0.4711
Residual942.02932.48
Table 9
Action potential threshold recorded at −70 mV.
ANOVA tableSSDFMSF (DFn, DFd)p Value
Interaction0.0665810.06658F (1, 25)=0.004070p=0.9496
[Ca2+]o845.11845.1F (1, 25)=51.66p<0.0001
Genotype391.01391.0F (1, 25)=10.52p=0.0033
Subjects (matching)929.52537.18F (25, 25)=2.273p=0.0225
Residual408.92516.36
(B) Action potential threshold recorded at −70 mV
Interaction2.008e-0712.008e-07F (1, 28)=2.800p=0.1054
[Ca2+]o1.415e-0611.415e-06F (1, 28)=19.73p=0.0001
Genotype3.050e-0713.050e-07F (1, 28)=0.4545p=0.5057
Subjects (matching)1.879e-05286.710e-07F (28, 28)=9.358p<0.0001
Residual2.008e-06287.170e-08
(C) Action potential threshold recorded at −70 mV
Interaction0.000160210.0001602F (1, 28)=6.758p=0.0147
[Ca2+]o0.000482110.0004821F (1, 28)=20.34p=0.0001
Genotype0.00119310.001193F (1, 28)=5.891p=0.0219
Subjects (matching)0.005669280.0002025F (28, 28)=8.541p<0.0001
Residual0.0006637282.370e-005
Table 10
Voltage-gated sodium channel current V0.5 for inactivation.
ANOVA tableSSDFMSF (DFn, DFd)p Value
Interaction12.00112.00F (1, 18)=0.7743p=0.3905
[Ca2+]o397313973F (1, 18)=256.2p<0.0001
Genotype27.49127.49F (1, 18)=0.5632p=0.4627
Subjects (matching)878.51848.81F (18, 18)=3.148p=0.0097
Residual279.11815.50
Table 11
Voltage-gated sodium channel current V0.5 for activation.
ANOVA tableSSDFMSF (DFn, DFd)p Value
Interaction4.81414.814F (1, 17)=0.5668p=0.4618
[Ca2+]o834.51834.5F (1, 17)=98.24p<0.0001
Genotype157.61157.6F (1, 17)=4.813p=0.0424
Subjects (matching)556.71732.75F (17, 17)=3.855p=0.0040
Residual144.4178.494
Table 12
Voltage-gated potassium channel currents at 60 mV.
ANOVA tableSSDFMSF (DFn, DFd)p Value
Interaction1.226e-01834.086e-019F (3, 57)=0.2271p=0.8772
[Ca2+]o and time7.270e-01832.423e-018F (3, 57)=1.347p=0.2683
Genotype6.054e-01716.054e-017F (1, 19)=1.231p=0.2811
Subjects (matching)9.345e-016194.919e-017F (19, 57)=27.33p<0.0001
Residual1.026e-016571.800e-018
Table 13
divalent-dependent basal currents at −70 mV.
ANOVA tableSSDFMSF (DFn, DFd)p Value
Treatment6.871e-02123.435e-021F (1.495, 17.94)=13.30p=0.0007
Individual (between rows)5.669e-022124.725e-023F (12, 24)=0.1828p=0.9981
Residual (random)6.201e-021242.584e-022
Total1.364e-02038
Table 14
Post hoc testing of divalent-dependent basal currents at −70 mV.
Sidak's multiple comparisons testMean diff.95% CI of diff.Significant?SummaryAdjusted p value
TTX sens vs. Gd3+ sens−2.247e-011−4.391e-011 to −1.033e-012Yes*0.0392
TTX sens vs. Rem−3.158e-011−4.899e-011 to −1.418e-011Yes***0.0009
Gd3+ sens vs. Rem−9.111e-012−2.146e-011 to 3.240e-012Nons0.1789
Table 15
divalent-dependent depolarization.
ANOVA tableSSDFMSF (DFn, DFd)p Value
Treatment0.000394420.0001972F (1.219, 13.41)=12.83p=0.0022
Individual (between rows)0.0001037119.423e-006F (11, 22)=0.6132p=0.7982
Residual (random)0.0003381221.537e-005
Total0.000836135
Table 16
Post hoc testing of blocker sensitive fractions of the divalent-dependent depolarization.
Sidak's multiple comparisons testMean diff.95% CI of diff.Significant?SummaryAdjusted p value
TTX sens vs. Gd3+ sens0.0065280.001005 to 0.01205Yes*0.0215
TTX sens vs. Rem0.0074280.002879 to 0.01198Yes**0.0028
Gd3+ sens vs. Rem0.0009−0.001301 to 0.003101Nons0.5311
Key resources table
Reagent type
(species) or
resource		DesignationSource or
reference		IdentifiersAdditional
information
Gene (M. musculus)CasrGenBankCasr
Strain, strain background (M. musculus)Mouse wild-type strain C57BL/6J × 129×1The Jackson LaboratoryRRID:MGI:5652742
Genetic reagent, strain background (M. musculus)Mouse expressing Nestin-cre mutationThe Jackson Laboratory as used in Sun et al., 2018Stock No. 003771C57/BL6J and 129S4 background strain
Genetic reagent, strain background (M. musculus)Mouse with Lox mutation to delete exon 7 of CasrLaboratory of Dr. Wenhan Chang, UCSF
(Chang et al., 2008)		Casrfl/flC57/BL6J and 129S4 background strain
Sequence-based reagentCasrApplied BiosystemsMm00443377_m1Quantitative PCR Mouse probe set
Sequence-based reagentActbApplied BiosystemsMm04394036_g1Quantitative PCR Mouse probe set
Sequence-based reagentNes-Cre1 primerIDTGCAAAACAGGCTCTAGCGTTCG
Sequence-based reagentNes-Cre2 primerIDTCTGTTTCACTATCCAGGTTACGG
Sequence-based reagentP3U primerIDTTGTGACGGAAAACATACTGC
Sequence-based reagentLox R primerIDTGCGTTTTTAGAGGGAAGCAG

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