Spontaneous firing is absent in NALCN KO mice. A, Representative cell-attached recording from a spontaneously spiking CWC in control. 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 KO mouse. NA had no effect on firing. D, Summary of spontaneous firing and lack of NA effect in CWC from NALCN KO mice (n = 5 cells).

NALCN is required for 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 presence of 10 μM NA (indicated by red traces and markers). C, D as in A, B but for recordings from a CWC from a NALCN KO mouse. E, Spike rate calculated from data in A2 and B2 plotted as 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 function of current level. H, Data from G normalized to peak firing rate. I, 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 zero mV.

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 (−70 mV) before (black) and after (red) block of GIRK channels by bath application of 100 μM Ba2+. B, As in A, but from a CWC from a NALCN KO 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 KO mice (n= 6). In the KO, the NA response was markedly reduced, and was Ba2+ sensitive. Dashed line indicates initial current level.

GABAB 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 CWC from a NALCN KO mouse. C, Averaged data showing Ba2+ sensitivity of the NA response in WT and 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.

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 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 CWC from a NALCN KO mouse. D, As in B, but for CWC from 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 WT as 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, apamin. Dashed line indicates initial current level.

NALCN current evoked by Ca2+ reduction is inhibited by GABAB 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 CWC from a NALCN KO 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 KO tissue. N=6 cells. E, The magnitude of inward current blocked by baclofen was significantly greater in WT as 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, apamin. Dashed line indicates initial current level.

Baclofen and 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 (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.

NA enhances CWC-mediated IPSCs in WT but not NALCN CWC KO. 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 KO 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 KO mice. (WT, N = 8; KO, N = 10)

NALCN KO mice show no difference in auditory brainstem response (ABR) from wildtype (WT). A. Example traces of WT (left) and NALCN KO (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 KO 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 KO (red) in response to 8 KHz pure tone at 80 dB SPL. D-H, Maximum wave I and II amplitude (D, F) and latencies (E, H) to 8 KHz tone stimulation presented at 80 dB SPL were not different between WT (N = 4) and NALCN KO animals (N = 4).

Biocytin-filled CWCs from NALCN KO 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 DCN.

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 function of current level of cell 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).