Figures and data in The Na+ leak channel NALCN controls spontaneous activity and mediates synaptic modulation by α2-adrenergic receptors in auditory neurons

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8 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements

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Spontaneous firing is absent in NALCN conditional knockout (cKO) mice.

(A) Representative cell-attached recording from a spontaneously spiking cartwheel cell (CWC) in control. Noradrenaline (NA; 10 µM) applied to the bath in the middle trace, and washout on the right-most trace. (B) Spontaneous spike rate in control, NA, and washout, showing that NA eliminated spontaneous spiking in CWCs (n = 6 cells). (C) Representative trace showing absence of spontaneous firing in a CWC from a NALCN cKO mouse. NA had no effect on firing. (D) Summary of spontaneous firing and lack of NA effect in CWC from NALCN cKO mice (n = 5 cells). See Figure 1—source data 1.

Figure 1—source data 1 Source data for Figure 1B, D.: https://cdn.elifesciences.org/articles/89520/elife-89520-fig1-data1-v1.xlsx
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Figure 1—figure supplement 1

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NALCN conditional knockout (cKO) mice show no difference in auditory brainstem response (ABR) from wildtype (WT).

(A) Example traces of WT (left) and NALCN cKO (right: red) ABR responses to 8 kHz pure tones presented at different sound intensities. Arrows indicate ABR wave I threshold. (B) ABR thresholds for WT and NALCN cKO mice were similar for various pure tones tested for mouse hearing (p>0.95 for all frequencies, ANOVA/Sidak’s multiple-comparison test). (C) Example ABR trace from WT (black) and NALCN cKO (red) in response to 8 kHz pure tone at 80 dB SPL. (**D–G**) Maximum wave I and II amplitude (**D, F**) and latencies (**E, G**) to 8 kHz tone stimulation presented at 80 dB SPL were not different between WT (N = 4) and NALCN cKO animals (N = 4). See Figure 1—figure supplement 1—source data 1.

Figure 1—figure supplement 1—source data 1 Source data for Figure 1—figure supplement 1B.: https://cdn.elifesciences.org/articles/89520/elife-89520-fig1-figsupp1-data1-v1.xlsx
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Figure 1—figure supplement 2

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Biocytin-filled cartwheel cells (CWCs) from NALCN conditional knockout (cKO) mice.

(**A, B**) Two examples of CWC that were labeled with biocytin imaged using confocal microscopy. Characteristic of CWC, spiny dendrites extend up to the ependymal layer while a fine axon ramifies within the molecular and cell body layers of the dorsal cochlear nucleus (DCN).

Figure 2 with 1 supplement

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NALCN is required for noradrenaline (NA)-mediated shift in rheobase.

(A1) Profile of voltage responses to current steps. Simple and complex spikes evoked by +200 pA. (A2) Raster plot of spike timings during different level of current injection. (**B1, B2**) Same cell as in (A) but in the presence of 10 μM NA (indicated by red traces and markers). (**C, D**) As in (**A, B**) but for recordings from a cartwheel cell (CWC) from a NALCN conditional knockout (cKO) mouse. (E) Spike rate calculated from data in (A2) and (B2) plotted as a function of current level. (F) Data from (E) normalized to peak firing rate. (G) Spike rate calculated from data in (C2) and (D2) plotted as a function of current level. (H) Data from (G) normalized to peak firing rate. (I), (J), raw and normalized frequency-intensity plots (respectively) for current responses from one WT cell in control solutions and in presence of 10 mM NA + 1 mM idazoxan. (K) Summary data (mean ± SEM) for ratio of change in rheobase of CWC from WT and KO mice with 10 μM NA, NA + idazoxan, and hyperpolarization conditions. Significance: *<0.05; **<0.01; ***<0.001. Dashed line in (**A1, C1, D1**) indicates 0 mV. See Figure 2—source data 1.

Figure 2—source data 1 Source data for Figure 2K.: https://cdn.elifesciences.org/articles/89520/elife-89520-fig2-data1-v1.xlsx
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Figure 2—figure supplement 1

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Noradrenaline (NA) effect on rheobase and spiking is voltage dependent.

(A) Profile of voltage responses to current steps. Simple and complex spikes evoked in response to depolarizing current steps. (B) Raster plot of spike timings during different level of current injection. (**C, D**) Same cell as in (B) but membrane hyperpolarized by 2 mV (see dashed line). (E) Normalized spike rate plotted as a function of current level of cells A and C. (F) Average data for spike rate from Ctrl and bias current injected conditions. Hyperpolarizing current injection show increase in rheobase and suppression of evoked firing (rheobase Ctrl = 70.88 ± 7.68 pA, hyperpolarized = 125 ± 12.90 pA, N = 6, p=0.0009).

Figure 3

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Noradrenaline (NA) evoked a Ba²⁺-resistant outward current that required NALCN.

(A) Outward current in response to NA puff (100 µM, 50 ms) in whole-cell voltage-clamp mode (– 65 mV) before (black) and after (red) block of GIRK channels by bath application of 100 μM Ba²⁺. (B) As in (A), but from a cartwheel cell (CWC) from a NALCN conditional knockout (cKO) mouse. (C) Average data showing responses to NA puff in control conditions and after the block of GIRK channels from control (n = 5) and NALCN cKO mice (n = 6). In the KO, the NA response was markedly reduced and was Ba²⁺ sensitive. Dashed line indicates initial current level. See Figure 3—source data 1.

Figure 3—source data 1 Source data for Figure 3C.: https://cdn.elifesciences.org/articles/89520/elife-89520-fig3-data1-v1.xlsx
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Figure 4

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GABA_B receptors activate outward current mediated by NALCN and GIRK channels.

(A) Traces from one neuron showing baclofen puff (100 µM, 50 ms) evoked outward current in control solution (black) and with 100 μM Ba²⁺ in bath (red). (B) As in (A) but for a cartwheel cell (CWC) from a NALCN conditional knockout (cKO) mouse. (C) Averaged data showing Ba²⁺ sensitivity of the noradrenaline (NA) response in wildtype (WT) and knockout (KO) tissue. Ba²⁺ produced a significant block in all cases, while KO CWC showed significantly smaller baclofen responses. (D) The degree of block by Ba²⁺ was significantly greater in KO mice, indicating that more of the baclofen response is mediated by GIRK channels after KO of NALCN. Dashed line indicates initial current level. See Figure 4—source data 1.

Figure 4—source data 1 Source data for Figure 4C, D.: https://cdn.elifesciences.org/articles/89520/elife-89520-fig4-data1-v1.xlsx
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Figure 5

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NALCN current evoked by Ca²⁺ reduction is inhibited by α2 receptors.

(A) Shifting bath Ca²⁺ from 2 mM to 0.1 mM evokes a slow inward current that is then rapidly reduced by subsequent wash-in of 10 μM noradrenaline (NA). (B) Group data showing the magnitude of inward current shift in 0.1 mM Ca²⁺ (black) and the significantly smaller shift in 0.1 mM Ca²⁺ plus NA (blue). N = 18 cells. (C) Experiment as in (A) but for a cartwheel cell (CWC) from a NALCN conditional knockout (cKO) mouse. (D) As in (B), but for CWC from knockout (KO) tissue. N = 6 cells. (E) Experiment as in (A) but in continuous presence of 1 µM idazoxan. NA failed to block the low-Ca²⁺-evoked current. (F) The magnitude of inward current blocked by NA was significantly greater in wildtype (WT) compared to KO cells. All neurons voltage-clamped to –70 mV. Statistical significance: *p<0.05; **p<0.01; ***p<0.001. Extracellular solution contained TTX, NBQX, MK-801, strychnine, SR95331, and apamin. Dashed line indicates initial current level. See Figure 5—source data 1.

Figure 5—source data 1 Source data for Figure 5B, D, F.: https://cdn.elifesciences.org/articles/89520/elife-89520-fig5-data1-v1.xlsx
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Figure 6

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NALCN current evoked by Ca²⁺ reduction is inhibited by GABA_B receptors.

(A) Shifting bath Ca²⁺ from 2 mM to 0.1 mM evokes a slow inward current that is then rapidly reduced by subsequent wash-in of 10 μM baclofen. (B) Experiment as in (A) but for a cartwheel cell (CWC) from a NALCN conditional knockout (cKO) mouse. (C) Group data showing the magnitude of inward current shift in 0.1 mM Ca²⁺ (black) and the significantly smaller shift in 0.1 mM Ca²⁺ plus baclofen (red). N = 18 cells. (D) As in (C), but for CWC from knockout (KO) tissue. N = 6 cells. (E) The magnitude of inward current blocked by baclofen was significantly greater in wildtype (WT) compared to KO cells. All neurons voltage-clamped to –70 mV. Statistical significance: *p<0.05; **p<0.01; ***p<0.001. Extracellular solution contains TTX, NBQX, MK-801, strychnine, SR95331, and apamin. Dashed line indicates initial current level. See Figure 6—source data 1.

Figure 6—source data 1 Source data for Figure 6C, D, E.: https://cdn.elifesciences.org/articles/89520/elife-89520-fig6-data1-v1.xlsx
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Figure 7

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Baclofen and noradrenaline (NA) act on the same population of NALCN channels.

(A) Representative example of NALCN current evoked by shift from 2 mM Ca²⁺ to 0.1 mM Ca²⁺, followed by bath application of baclofen (20 µM) and subsequent application of NA (10 µM) with baclofen still present. In the presence of baclofen, the rapid decline in inward current normally evoked by NA was absent. (B) Summary plot of NALCN current amplitude evoked by 0.1 mM Ca²⁺ and after baclofen and subsequent NA application (wildtype [WT], N = 9). NA failed to produce a significant change after baclofen application. (C) Percentage block of 0.1 Ca²⁺ current in baclofen, or NA alone, compared to the block of current by NA in a background of baclofen. Baclofen completely occluded response to subsequent application of NA. Statistical significance, ***p<0.001. Dashed line indicates initial current level. See Figure 7—source data 1.

Figure 7—source data 1 Source data for Figure 7B, C.: https://cdn.elifesciences.org/articles/89520/elife-89520-fig7-data1-v1.xlsx
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Figure 8

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Noradrenaline (NA) enhances cartwheel cell (CWC)-mediated IPSCs in wildtype (WT) but not in NALCN cKO cells.

(A) Example average trace of evoked IPSCs recorded from a CWC with a CsCl pipette fill, before (black) and after (blue) bath application of 10 μM NA. (B) Diary plot of effect of NA application (applied during black bar) of the IPSCs amplitude in cell (A). (C) Example average trace of evoked IPSCs recorded from a CWC as in (A), but from a NALCN cKO mouse. Data shown before (black) and after (blue) bath application of 10 μM NA. (D) Diary plot of effect of NA application in (C). (E) Summary plot of percent change of IPSCs amplitude after NA application in WT and knockout (KO) mice (WT, N = 8; KO, N = 10). See Figure 8—source data 1.

Figure 8—source data 1 Source data for Figure 8E.: https://cdn.elifesciences.org/articles/89520/elife-89520-fig8-data1-v1.xlsx
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Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers
Strain, strain background (Mus musculus)	C57BL/6J	Jackson Laboratory	RRID:JAX:000664
Strain, strain background (M. musculus)	GlyT2-EGFP, Tg(Slc6a5-EGFP)1Uze	MGI, Zeilhofer et al., 2005	RRID:MGI:J:145521
Strain, strain background (M. musculus)	B6(Cg)- Nalcn^{tm1c(KOMP)Wtsi}/ DrenJ	MGI and Jackson Laboratory	RRID:JAX_030718
Strain, strain background (M. musculus)	Tg(Slc6a5-cre)KF109Gsat	MGI	RRID:MGI:4367229
Chemical compound, drug	Strychnine hydrochloride	Sigma	Cat# S8753
Chemical compound, drug	SR-95531 hydrobromide	Tocris Bioscience	Cat# 1262
Chemical compound, drug	NBQX disodium salt	Tocris Bioscience	Cat# 1044
Chemical compound, drug	(+)-MK-801 hydrogen maleate	Sigma	Cat# M107
Chemical compound, drug	L-(-)-Norepinephrine (+)-bitartrate salt monohydrate	Sigma	Cat# A9512
Chemical compound, drug	(±)-Norepinephrine (+)-bitartrate salt	Sigma	Cat# A0937
Chemical compound, drug	Idazoxan hydrochloride	Sigma	Cat# I6138-100MG
Chemical compound, drug	Apamin	Alomone Labs	Cat# STA-200
Chemical compound, drug	D-AP5	Tocris Bioscience	Cat# 0106
Chemical compound, drug	Barium chloride	Sigma	Cat# 202738
Software, algorithm	pClamp 10	Molecular Devices	RRID:SCR_011323
Software, algorithm	Igor Pro 8	WaveMetrics	RRID:SCR_000325
Software, algorithm	NeuroMatic	Rothman and Silver, 2018; DOI:10.3389/fninf.2018.00014	RRID:SCR_004186
Software, algorithm	Axograph	Axograph	RRID:SCR_014284
Software, algorithm	Prism 9	GraphPad	RRID:SCR_002798
Software, algorithm	Excel	Microsoft	RRID:SCR_016137
Software, algorithm	Affinity Designer	Serif	RRID:SCR_016952