Differential roles of Na_V1.2 and Na_V1.6 in neocortical pyramidal cell excitability

Reviewer #1 (Public review):

Summary:

Prior research indicates that NaV1.2 and NaV1.6 have different compartmental distributions, expression timelines in development, and roles in neuron function. The lack of subtype-specific tools to control Nav1.2 and Nav1.6 activity however has hampered efforts to define the role of each channel in neuronal behavior. The authors attempt to address the problem of subtype specificity here by using aryl sulfonamides (ASCs) to stabilize channels in the inactivated state in combination with mice carrying a mutation that renders NaV1.2 and/or NaV1.6 genetically resistant to the drug. Using this innovative approach, the authors find that action potential initiation is controlled by NaV1.6 while both NaV1.2 and NaV1.6 are involved in backpropagation of the action potential to the soma, corroborating previous findings. Additionally, NaV1.2 inhibition paradoxically increases the firing rate, as has also been observed in genetic knockout models. Finally, the potential anticonvulsant properties of ASCs were tested. NaV1.6 inhibition but not NaV1.2 inhibition was found to decrease action potential firing in prefrontal cortex layer 5b pyramidal neurons in response to current injections designed to mimic inputs during seizure. This result is consistent with studies of loss-of-function Nav1.6 models and knockdown studies showing that these animals are resistant to certain seizure types. These results lend further support for the therapeutic promise of activity-dependent, NaV1.6-selective, inhibitors for epilepsy.

Strengths:

(1) The chemogenetic approaches used to achieve selective inhibition of NaV1.2 and NaV1.6 are innovative and help resolve long-standing questions regarding the role of Nav1.2 and Nav1.6 in neuronal electrogenesis.

(2) The experimental design is overall rigorous, with appropriate controls included.

(3) The assays to elucidate the effects of channel inactivation on typical and seizure-like activity were well selected.

Weaknesses:

(1) The potential impact of the YW->SR mutation in the voltage sensor does not appear to have been sufficiently assessed. The activation/inactivation curves in Figure 1E show differences in both activation and inactivation at physiologically relevant membrane voltages, which may be significant even though the V1/2 and slope factors are roughly similar.

(2) Additional discussion of the fact that channels are only partially blocked by the ASC and that ASCs act in a use-dependent manner would improve the manuscript and help readers interpret these results.

(3) NaV1.6 was described as being exclusively responsible for the change in action potential threshold, but when NaV1.6 alone was inactivated, the effect was significantly reduced from the condition in which both channels were inactivated (Figure 4E). Similarly, Figure 6C shows that blockade of both channels causes threshold depolarization prior to the seizure-like event, but selective inactivation of NaV1.6 does not. As NaV1.2 does not appear to be involved in action potential initiation and threshold change, what is the mechanism of this dissimilarity between the NaV1.6 inactivation and combined NaV1.6/ NaV1.2 inactivation?

(4) The idea that use-dependent VGSC-acting drugs may be effective antiseizure medications is well established. Additional discussion or at least acknowledgement of the existing, widely used, use-dependent VGSC drugs should be included (e.g. Carbamazepine, Lamotrigine, Phenytoin). Also, the idea that targeting NaV1.6 may be effective for seizures is established by studies using genetic models, knockdown, and partially selective pharmacology (e.g. NBI-921352). Additional discussion of how the results reported here are consistent with or differ from studies using these alternative approaches would improve the discussion

Reviewer #2 (Public review):

The authors used a clever and powerful approach to explore how Nav1.2 and Nav1.6 channels, which are both present in neocortical pyramidal neurons, differentially control firing properties of the neurons. Overall, the approach worked very well, and the results show very interesting differences when one or the other channel is partially inhibited. The experimental data is solid and the experimental data is very nicely complemented by a computational model incorporating the different localization of the two types of sodium channels.

In my opinion the presentation and interpretation of the results could be improved by a more thorough discussion of the fact that only incomplete inhibition of the channels can be achieved by the inhibitor under physiological recording conditions and I thought the paper could be easier to digest if the figures were re-organized. However, the key results are well-documented.

Reviewer #3 (Public review):

Summary:

The authors used powerful and novel reagents to carefully assess the roles of the voltage gated sodium channel (NaV) isoforms in regulating the neural excitability of principal neurons of the cerebral cortex. Using this approach, they were able to confirm that two different isoforms, NaV1.2 and NaV1.6 have distinct roles in electrogenesis of neocortical pyramidal neurons.

Strengths:

Development of very powerful transgenic mice in which NaV1.2 and/or NaV1.6 were modified to be insensitive to ASCs, a particular class of NaV blocker. This allowed them to test for roles of the two isoforms in an acute setting, without concerns of genetic or functional compensation that might result from a NaV channel knockout.

Careful biophysical analysis of ASC effects on different NaV isoforms.

Extensive and rigorous analysis of electrogenesis - action potential production - under conditions of blockade of either NaV1.2 or NaV1 or both.

Weaknesses:

Some results are overstated in that the representative example records provided do not directly support the conclusions.

Results from a computational model are provided to make predictions of outcomes, but the computational approach is highly underdeveloped.

Author response:

We thank the reviewers and editors for these careful and constructive comments. Based on these comments, we plan to perform new experiments and revised analysis, summarized as follows:

(1) A more thorough analysis and experimental test of the effects of YW->SR variants on baseline AP excitability in neurons in the absence of any pharmacology.

(2) More details on modeling of selective block of Na_V1.2 and Na_V1.6.

(3) Revisions to text, figure contents, and figure order to better convey key points and better frame these findings in the context of current clinically available anti-seizure medications that interact with sodium channels.