Enhanced K_Na1.1 Channel Underlies Cortical Hyperexcitability and Seizure Susceptibility after Traumatic Brain Injury

Reviewer #1 (Public Review):

Summary
General Comments
The authors present an interesting study that aims to resolve the contribution of the sodium-activated potassium channel (KNa1.1) to acquired, trauma-induced epilepsy. To this end, the authors first aim to develop a mouse model that consistently generates seizures. Using controlled cortical impact (CCI) methods to induce traumatic brain injuries (TBIs) that range from mild to severe, the authors demonstrate that behavioral deficits correlate with the extent of brain damage. Interestingly, despite the differences in behavioral scores, the spontaneous seizure phenotype was similar across mice with a range of TBI-associated tissue loss. However, when challenged with the chemoconvulsant pentylenetetrazol (PTZ), mice with more severe TBI exhibited more severe seizures.

After establishing a model of moderate TBI, the authors then show that moderate brain injury transiently upregulates the perilesional expression of KNa1.1. Moreover, the authors provide some evidence that the expression of inhibitory neuron markers is downregulated in the perilesional region following moderate TBI, whereas the expression of excitatory neuron markers is unchanged. Consistent with this finding is the functional observation that neurons receive less inhibitory signaling following TBI, whereas excitatory signaling is unchanged. Inhibitory neurons also fire less robustly following moderate TBI.

The authors then show that deletion of KNa1.1 in mice provides a moderate level of protection against pharmacologically induced seizures following TBI. In aggregate, the authors propose a model wherein inhibitory neuron-specific upregulation of KNa1.1 following TBI selectively reduces the excitability of inhibitory neurons. In turn, this reduced excitability of inhibitory neurons promotes perilesional tissue hyperexcitability and, ultimately, seizures. Although this model is compelling, readers should be aware that the authors only utilized PTZ-induced seizures following TBI to resolve differences between WT and KO animals. It remains unclear whether WT mice have a robust spontaneous seizure phenotype 14 days after moderate TBI, and whether deleting KNa1.1 reduces this spontaneous seizure phenotype. In general, the combined use of TBI and a chemoconvulsant to evaluate epileptic phenotypes diminishes this reviewer's enthusiasm for the clinical impact of the author's conclusions; although, I appreciate that "capturing [spontaneous] seizures is challenging in terms of animal numbers and long-term recording, which hinders high-throughput studies" (line 302).

Finally, the authors seemed to have missed an opportunity to determine if the electrophysiological changes observed in WT mice following TBI (i.e., Figure 5) are eliminated in the KNa1.1 KO mouse. That is, the authors show that KNa1.1 contributes to the intrinsic firing properties of uninjured tissue (Figure 6). But does deleting KNa1.1 also restore inhibitory neuron excitability associated with TBI? The inclusion of such data would strengthen the conclusion that the reduced inhibitory neuron excitability following TBI is indeed the result of changes in KNa1.1 expression.

Strengths:
(1) The development of a TBI model with a range of behavioral phenotypes.

(2) The inclusion of knockout mouse.

(3) The inclusion of functional data regarding the intrinsic and synaptic properties of neurons following TBI.

Weaknesses:
(1) A missed opportunity to better utilize the knockout animal to test the hypothesis that KNa1.1 drives changes in intrinsic excitability following TBI.

(2) The combined use of TBI and chemoconvulsants to suss out differences in seizure phenotypes.

Reviewer #2 (Public Review):

Summary:
Authors hypothesized that modulation of KNa1.1 channel specifically in inhibitory interneurons contributes to the hyperactivity of neurons in the peripheral cortex at the lesion site, enhances seizure susceptibility to PTZ-induced seizures, and promotes the occurrence of PTE. They test this hypothesis in a mouse model of TBI induced by controlled cortical impact in wild-type and kcnt1 knock-out (KO) mice. The authors performed a series of experiments including behavioral assessment, electrographic recordings in vivo and in vitro, western blotting, and immunofluorescence imaging with the goal of investigating the contributory role of KNa1.1 channel to post-traumatic epileptogenesis.

Strengths:
The hypothesis is innovative, focusing on the specific role of the KNa1.1 channel in the development of PTE post-TBI. The use of a comprehensive set of techniques, including EEG, whole cell patch clamp, western blot, immunofluorescence imaging, and behavioral assessments, provides a diverse data set. The study makes initial steps in correlating specific molecular changes with functional outcomes in TBI models, offering potential pathways for therapeutic intervention.

Weaknesses:

1. The study presents interesting findings on early changes in protein expression and electrophysiological properties following TBI. However, I would like to draw attention to the timeline of EEG and cellular assessments that require further clarification or consideration. The patch clamp recordings and other assays were conducted within 14 days post-TBI, while EEG recordings with or without PTZ testing were performed at 3 months post-injury. This temporal gap leaves a period where changes in electrophysiological properties and PTE status are not accounted for. Since epileptogenesis post-TBI involves a dynamic process spanning from hyperacute and acute phases to chronic development, capturing these changes continuously or at least at more frequent intervals (on and off bi-weekly) could provide a more comprehensive understanding of this progression. In the current study design, the one-week duration of EEG recordings at the 3-month timepoint raises the possibility that some seizures might have occurred undetected between the early post-injury phase and the EEG recording period. This gap could potentially affect the interpretation of results, especially when correlating early post-injury cellular changes with later seizure activity and thresholds and hence is a significant limitation to data interpretation. Experiments using western blots, immunofluorescence, and patch clamp were done at an early timepoint hence the relevance of these datasets to PTE status outcome is not established.

2. While referencing Nichols et al., 2015, to justify the 14-day timeline for characterizing seizures is understandable, it is important to consider differences in animal models (juvenile rats in Nichols vs. adult mice in the present study) which might influence the generalizability of the findings.

3. Behavior: Authors performed behavioral assays using the rotarod technique evaluating the hanging time of mice with different severity of TBI (mild, moderate, severe). The purpose of the testing is explained as 'to sort out an appropriate TBI model'. The authors also measured mortality rates 'to attain a stable model'. It is not clear what is assumed by the terms 'appropriate' or 'stable' model. Furthermore, the relevance of this to post-traumatic epileptogenesis is unclear. Additionally, the mortality in the Sham group within 2 weeks of craniectomy is not explained.

4. Seizure assessment: authors report seizure severity in PTZ-induced seizures but no mention about the severity of spontaneous seizures between different TBI severity modalities. When characterizing the PTZ-induced seizures, the mild TBI group does not have generalized seizures. Does this mean that al all 6 tested animals in the mild TBI group had exclusively focal seizures? What about the spontaneous seizure occurrence: were all seizures generalized or were any focal too? Did that differ between mild, moderate, and severe TBI?

5. Experiments with KCNT1 KO mice: in all experiments with a mutant mouse line, authors only used them in TBI group. Without the Sham group, it is difficult to discern whether any observed changes in seizure susceptibility in the KCNT1 KO TBI group are due to gene deletion, the TBI, or a combination of both. This group would provide a crucial comparison point to isolate the effects of the KCNT1 knockout from those of TBI. This limits the ability to make comprehensive conclusions about the role of the KCNT1 gene in seizure susceptibility following TBI.

6. While the current study showed interesting data about the KNa1.1 changes early after TBI, the study design and disconnect between early and late electrophysiology experiments timeline, does not establish a correlative or causative link between KNa1.1 and post-traumatic epileptogenesis since it remained unresolved whether KCNT1 KO mice developed no PTE (or less severe/ less frequent seizures at 3 months) compared with WT mice and what are the seizure properties of KCNT1 KO Sham mice compared to WT TBI and Sham groups. The hypothesis was that modulation of KNa1.1 channel specifically in inhibitory interneurons contributes to the hyperactivity of neurons in the peripheral cortex at the lesion site, enhances seizure susceptibility to PTZ-induced seizures, and promotes the occurrence of PTE. The part about 'promotes the occurrence of PTE' was not established.

7. NeuN is not the best marker of neurons in the context of TBI since TBI affects its expression patterns which will influence the interpretation of co-localization results. Unlike NeuN, Nissl staining is less likely to be affected by factors that alter protein expression, such as TBI. Therefore, it can be a more stable marker for identifying neurons in injured brain tissue.

8. Statistics: Authors report only SEM, which shows the precision of the mean and it will decrease as the sample size increases and does not reflect the data variability.