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

Reviewer #1 (Public Review):

This study uses electrophysiological techniques in vitro to address the role of the Na+ leak channel NALCN in various physiological functions in cartwheel interneurons of the dorsal cochlear nucleus. Comparing wild type and glycinergic neuron-specific knockout mice for NALCN, the authors show that these channels 1) are required for spontaneous firing, 2) are modulated by noradrenaline (NA, via alpha2 receptors) and GABA (through GABAB receptors), 3) how the modulation by NA enhances IPSCs in these neurons.

This work builds on previous results from the Trussell's lab in terms of the physiology of cartwheel cells, and from other labs in terms of the role of NALCN channels, that have been characterized in more and more brain areas somewhat recently; for this reason, this study could be of interest for researchers that work in other preparations as well. The general conclusions are strongly supported by results that are clearly and elegantly presented.

I have a few comments that, in my opinion, might help clarify some aspects of the manuscript.

1. It is mentioned throughout the manuscript, including the abstract, that the results suggest a closed apposition of NALCN channels and alpha2 and GABAB receptors. From what I understand, this conclusion comes from the fact that GABAB receptors activate GIRK channels through a membrane-delimited mechanism. Is it possible that these receptors converge on other effectors, for example adenylate cyclase (see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374141/).

2. In Figure 2G, the neurons from NALCN KO mice appear to reach a significantly higher frequency than those from WT (figure 2E, 110 vs. 70 spikes/s). Was this higher frequency a feature of all experiments? The results mention a rundown of peak firing rate due to whole-cell dialysis, but, from what I understand, the control conditions should be similar for all experiments.

3. Also in Figure 2, the firing patterns for neurons from WT and NALCN KO mice appear to be quite different, with spikes appearing to be generated during the hyperpolarization of the bursts in the second half of the current step for WT neurons but always during the depolarization in KO neurons. Was this always the case? If so, could NALCN channels be involved in this type of firing? Along these lines, it would be interesting to show an example of a firing pattern of neurons from WT mice in the presence of NA, which inhibits NALCN channels.

4. It might be interesting to discuss how the hyperpolarization induced by the activation of GIRK channels and inhibition of NALCN channels could have different consequences due to their opposite effect on the input resistance.

Reviewer #2 (Public Review):

This is a very interesting paper with several important findings related to the working mechanism of the cartwheel cells (CWC) in the dorsal cochlear nucleus (DCN). These cells generate spontaneous firing that is inhibited by the activation of α2-adrenergic receptors, which also enhances the synaptic strength in the cells, but the mechanisms underlying the spontaneous firing and the dual regulation by α2-adrenergic receptor activation have remained elusive. By recording these cells with the NALCN sodium-leak channel conditionally knocked, the authors discovered that both the spontaneous firing and the regulation by noradrenaline (NA) require NALCN. Mechanistically, the authors found that activation of the adrenergic receptor or GABAB receptor inhibits NALCN. Interestingly, these receptor activations also suppress the low [Ca2+] "activation" of NALCN currents, suggesting crosstalk between the pathways. The finding of such dominant contribution of the NALCN conductance to the regulation of firing by NA is somewhat surprising considering that NA is known to regulate K+ conductances in many other neurons.

The studies reveal the molecular mechanisms underlying well known regulations of the neuronal processes in the auditory pathway. The results will be important to the understanding of auditory information processing in particular, and, more generally, to the understanding of the regulation of inhibitory neurons and ion channels. The results are convincing and are clearly presented.

Reviewer #3 (Public Review):

The study by Ngodup and colleagues describes the contribution of sodium leak NALCN conductance on the effects of noradrenaline on cartwheel interneurons of the DCN. The manuscript is very well-written and the experiments are well-controlled. The scope of the study is of high biological relevance and recapitulates a primary finding of the Khaliq lab (Philippart et al., eLife, 2018) in ventral midbrain dopamine neurons, that Gi/o-coupled receptors inhibit NALCN current to reduce neuronal excitability. Together these studies provide unequivocable evidence for NALCN as a downstream target of these receptors. There are no major concerns. I have only minor suggestions:

Minor
1. As introduced in the introduction, NALCN is inhibited by extracellular calcium which has led to some discourse of the relevance of NALCN when recorded in 0.1 mM calcium. A strength of this study is the effect of NA on NALCN is recorded in physiological levels of calcium (1.2 mM). I suggest including the concentration of extracellular calcium in the aCSF in the Results section instead of relying on the reader to look to the Methods.

2. It would be interesting to include the basal membrane properties of the KO compared to wildtype, including membrane resistance and resting membrane potential. From the example recording in Figure 2, one might think that the KOs have lower membrane resistance, so it is interesting that the 2 mV hyperpolarization produced similar effects on rheobase. In addition, from the example in Figure 2G, it appears that NA has an effect on firing frequency with large current injection in the KO. Is this true in grouped data and if so, is there any speculation into how this occurs?

3. Please expand on the rationale for why GABAB and alpha2 must be physically close to NALCN. To my knowledge, the mechanism by which these receptors inhibit NALCN is not known. Must it be membrane-delimited?

Author Response

Reviewer #1 (Public Review):

This study uses electrophysiological techniques in vitro to address the role of the Na+ leak channel NALCN in various physiological functions in cartwheel interneurons of the dorsal cochlear nucleus. Comparing wild type and glycinergic neuron-specific knockout mice for NALCN, the authors show that these channels 1) are required for spontaneous firing, 2) are modulated by noradrenaline (NA, via alpha2 receptors) and GABA (through GABAB receptors), 3) how the modulation by NA enhances IPSCs in these neurons.

This work builds on previous results from the Trussell's lab in terms of the physiology of cartwheel cells, and from other labs in terms of the role of NALCN channels, that have been characterized in more and more brain areas somewhat recently; for this reason, this study could be of interest for researchers that work in other preparations as well. The general conclusions are strongly supported by results that are clearly and elegantly presented.

I have a few comments that, in my opinion, might help clarify some aspects of the manuscript.

1. It is mentioned throughout the manuscript, including the abstract, that the results suggest a closed apposition of NALCN channels and alpha2 and GABAB receptors. From what I understand, this conclusion comes from the fact that GABAB receptors activate GIRK channels through a membrane-delimited mechanism. Is it possible that these receptors converge on other effectors, for example adenylate cyclase (see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374141/).

It will be of interest to test the roles of adenylyl cyclase modulation in the control of NALCN, as a complement to the studies we have presented here.

1. In Figure 2G, the neurons from NALCN KO mice appear to reach a significantly higher frequency than those from WT (figure 2E, 110 vs. 70 spikes/s). Was this higher frequency a feature of all experiments? The results mention a rundown of peak firing rate due to whole-cell dialysis, but, from what I understand, the control conditions should be similar for all experiments.

The peak firing rates in control solutions for WT and KO CWC are not statistically different.

1. Also in Figure 2, the firing patterns for neurons from WT and NALCN KO mice appear to be quite different, with spikes appearing to be generated during the hyperpolarization of the bursts in the second half of the current step for WT neurons but always during the depolarization in KO neurons. Was this always the case? If so, could NALCN channels be involved in this type of firing? Along these lines, it would be interesting to show an example of a firing pattern of neurons from WT mice in the presence of NA, which inhibits NALCN channels.

The specific pattern of spikes in CWC is quite variable from trial-to-trial or cell-to-cell, as it is dependent on multiple CaV and calcium dependent K channels subtypes, and is not dependent on the genotypes used here. The primary effects observed in the KO are in background firing and sensitivity to NA, both reflected alterations in rheobase. The firing pattern example requested was shown in the raster plot of fig 2B2.

1. It might be interesting to discuss how the hyperpolarization induced by the activation of GIRK channels and inhibition of NALCN channels could have different consequences due to their opposite effect on the input resistance.

We considered this as a point of discussion, but decided that making sense of it would depend on assumptions about the location of the channels (dendritic vs somatic, distance to AIS) that we do not have data for. For example, a dendritic increase in resistance through NALCN block, leading to a hyperpolarization of the soma, might have actions similar to a somatic hyperpolarizing conductance increase by GIRK, as far as the voltage at the AIS is concerned.

Reviewer #3 (Public Review):

The study by Ngodup and colleagues describes the contribution of sodium leak NALCN conductance on the effects of noradrenaline on cartwheel interneurons of the DCN. The manuscript is very well-written and the experiments are well-controlled. The scope of the study is of high biological relevance and recapitulates a primary finding of the Khaliq lab (Philippart et al., eLife, 2018) in ventral midbrain dopamine neurons, that Gi/o-coupled receptors inhibit NALCN current to reduce neuronal excitability. Together these studies provide unequivocable evidence for NALCN as a downstream target of these receptors. There are no major concerns. I have only minor suggestions:

Minor

1. As introduced in the introduction, NALCN is inhibited by extracellular calcium which has led to some discourse of the relevance of NALCN when recorded in 0.1 mM calcium. A strength of this study is the effect of NA on NALCN is recorded in physiological levels of calcium (1.2 mM). I suggest including the concentration of extracellular calcium in the aCSF in the Results section instead of relying on the reader to look to the Methods.

Will do.

1. It would be interesting to include the basal membrane properties of the KO compared to wildtype, including membrane resistance and resting membrane potential. From the example recording in Figure 2, one might think that the KOs have lower membrane resistance, so it is interesting that the 2 mV hyperpolarization produced similar effects on rheobase. In addition, from the example in Figure 2G, it appears that NA has an effect on firing frequency with large current injection in the KO. Is this true in grouped data and if so, is there any speculation into how this occurs?

Will do.

1. Please expand on the rationale for why GABAB and alpha2 must be physically close to NALCN. To my knowledge, the mechanism by which these receptors inhibit NALCN is not known. Must it be membrane-delimited?

Given the known membrane delimited modulation of GIRK by GABAB, and that alpha2 and GABAB receptors appear to share the same population of NALCN channels, and that alpha2 receptors do not appear to target GIRK channels, we felt the simplest explanation would be coupling through G-proteins, with spatial segregation of different receptor/channel pools providing the means for separating GIRK and NALCN effects. However, the involvement of an additional second messenger is testable.