improved rebuttal

Frontiers_manuscript/For_Submission/Koch_frontiers_revised.tex

@@ -188,7 +188,7 @@ The effects of changes in channel properties depend in part on the neuronal mode
 
 Therefore, the transfer of LOF or GOF from the current to the firing level should be used with caution; the neuron type in which the mutant ion channel is expressed may provide valuable insight into the functional consequences of an ion channel mutation. Experimental assessment of the effects of a patient's specific ion channel mutation \textit{in vivo} is not generally feasible at a large scale. Therefore, modelling approaches investigating the effects of patient specific channelopathies provide a viable method bridging between characterization of changes in biophysical properties of ionic currents and the firing consequences of these effects. In both experimental and modelling studies on the effects of ion channel mutations on neuronal firing the specific dependency on neuron type should be considered.
 
-Our simulations demonstrate that the effects of altered ion channel properties on firing is generally influenced by the other ionic currents present in the neuron as illustrated in Figure \ref{fig:summary}. In channelopathies the effect of a given ion channel mutation on neuronal firing therefore depends on the neuron type in which those changes occur \citep{Hedrich14874, makinson_scn1a_2016, Waxman2007, Rush2006}. Although certain complexities of neurons such as differences in neuron-type sensitivities to current property changes, interactions between ionic currents, cell morphology and subcellular ion channel distribution are neglected here, it is likely that this increased complexity \textit{in vivo} would contribute to the neuron-type dependent effects on neuronal firing. The complexity and nuances of the nervous system, including neuron-type dependent firing effects of channelopathies explored here, likely underlie shortcomings in treatment approaches in patients with channelopathies. Accounting for neuron-type dependent firing effects provides an opportunity to improve the efficacy and precision in personalized medicine approaches. Although this is not experimentally feasible, improved modelling and simulations methods to predict neuron-type dependent effects may provide an opportunity to inform therapeutic strategies that are more specific and thus have greater efficacy.
+Our simulations demonstrate that the effects of altered ion channel properties on firing is generally influenced by the other ionic currents present in the neuron as illustrated in Figure \ref{fig:summary}. In channelopathies the effect of a given ion channel mutation on neuronal firing therefore depends on the neuron type in which those changes occur \citep{Hedrich14874, makinson_scn1a_2016, Waxman2007, Rush2006}. Although certain complexities of neurons such as differences in neuron-type sensitivities to current property changes, interactions between ionic currents, cell morphology and subcellular ion channel distribution are neglected here, it is likely that this increased complexity \textit{in vivo} would contribute to the neuron-type dependent effects on neuronal firing. The complexity and nuances of the nervous system, including neuron-type dependent firing effects of channelopathies explored here, likely underlie shortcomings in treatment approaches in patients with channelopathies. Accounting for neuron-type dependent firing effects provides an opportunity to improve the efficacy and precision in personalized medicine approaches. Although this is not experimentally feasible, improved modelling and simulation methods to predict neuron-type dependent effects may provide an opportunity to inform therapeutic strategies that are more specific and thus have greater efficacy.
 
 With this study we suggest that neuron-type specific effects are vital to a full understanding of the effects of channelopathies at the level of neuronal firing. Furthermore, we highlight the use of modelling approaches to enable relatively fast and efficient insight into channelopathies.
 

Frontiers_manuscript/For_Submission/Koch_reviewer_comments.tex

@@ -61,58 +61,36 @@
 \maketitle
 
 
+We thank the three reviewers for their comments that help to improve our manuscript.
+
 \section{Reviewer 1}
 
 \textit{Although the proposed study for publication presents an up-to-date approach, it does not bring any innovation beyond the previous studies (Hedrich et al., 2014; Makinson et al., 2016; Waxman, 2007; Rush et al., 2006).}
 
-We thank the reviewer for their comments. Although previous studies (Hedrich et al., 2014; Makinson et al., 2016; Waxman, 2007; Rush et al., 2006) referred to by the reviewer and discussed in our manuscript investigate differences in outcomes of ion channel mutations in different neuronal types, these studies have addressed specific instances in which this is the case. For instance, Waxman, 2007 and Rush et al., 2006 demonstrate the dependence of Nav1.7 mutations on the presence/absence of Nav1.8. However whether the outcome of the mutation is graded with different levels of Nav1.8 or whether this effect is modulated by other channels is not investigated nor known. In this context, our work furthers the current knowledge that certain ion channel mutations can have different effects on firing in specific contexts (i.e. cell types) by generalizing this viewpoint to demonstrate that the effects of any ion channel mutation will depend in the neuron-type and its ion channel composition. This suggests that holistic understanding of the effects of ion channel mutations likely requires a more specific neuron-type dependent level of understanding than is generally used currently.  Although many differences can exist between neuron types, this study demonstrates that different ion channel composition alone is sufficient to cause neuron type specific ion channel mutation firing effects. The ``Ionic Current Environments Determine the Effect of Ion Channel Mutations'' section of the discussion has been updated to clarify this.
+Although previous studies (Hedrich et al., 2014; Makinson et al., 2016; Waxman, 2007; Rush et al., 2006) referred to by the reviewer and discussed in our manuscript investigate differences in outcomes of ion channel mutations in different neuronal types, these studies have addressed specific instances in which this is the case. For instance, Waxman, 2007 and Rush et al., 2006 demonstrate the dependence of Nav1.7 mutations on the presence/absence of Nav1.8. However whether the outcome of the mutation is graded with different levels of Nav1.8 or whether this effect is modulated by other channels is not investigated nor known. In this context, our work furthers the current knowledge that certain ion channel mutations can have different effects on firing in specific contexts (i.e. cell types) by generalizing this viewpoint to demonstrate that the effects of any ion channel mutation will depend on the ion channel composition of different neuron types. This suggests that holistic understanding of the effects of ion channel mutations likely requires a more specific neuron-type dependent level of understanding than is generally used currently.  Although many differences can exist between neuron types, this study demonstrates that different ion channel composition alone is sufficient to cause neuron type specific ion channel mutation firing effects. The ``Ionic Current Environments Determine the Effect of Ion Channel Mutations'' section of the discussion has been updated to clarify this.
 
 
 % reduce down to changes in currents, in vivo lots of other changes, but show that it happens already on the level of currents - experiments don't show this
 
 \textit{The authors say that taking into account neuron type-dependent firing effects offers an opportunity to increase efficacy and precision in personalized medicine approaches, but they do not elaborate on how this applies to the clinic.}
 
-Currently personalized medicine approaches, although promising, have limited efficacy. Increased efficacy in these approaches likely will result with more therapeutic specificity, however different avenues exist to increase specificity. Although we do not offer a directly actionable clinical outcome, we propose that understanding of the neuron-type level firing effects of ion channel mutations may provide an opportunity for therapeutic strategies that provide more specificity and thus greater efficacy. Although this is not experimentally feasible, improving modelling and simulations methods may fill this gap. This has altered in the manuscript to be more explicit.
-
+Although we do not offer a directly actionable clinical outcome, our results suggest that neuron-type specific firing effects of ion channel mutations occur and need to be taken into account for therapeutic strategies. Although this is experimentally difficult, improved modelling and simulation methods may fill this gap. We added a similar sentence right before the last paragraph of the discussion.
 
 \textit{Considering that even a single mutation in the organism will affect many neuron types (cells) and the signaling pathways to which it is associated, how can all other possible effects be eliminated?}
 
-We thank the reviewer for their question. The complexity noted in the reviewers question is, in our opinion, vital to the outcome of any ion channel mutation. With this study we demonstrate this to be the case at a neuron-type ion channel composition level and suggest that distillation of this complexity into a LOF or GOF characterization in terms of firing is not meaningful unless tied to a neuron type. Although we make no claim as to at which point certain aspects of the complexity become less relevant or even irrelevant to the outcome of an ion channel mutation, we use the simulation presented within this study to encourage investigation into neuron type specific effects and to use LOF/GOF firing characterizations with caution.
+We fully agree that the effects of a single mutation are potentially much more complex than the ones we described in our manuscript. It is hard to imagine how all of this complexity should be captured or controlled for. Nevertheless clinicians around the globe base their therapeutic strategies on LOF and GOF effects measured in the biophysical properties of ion channel variants, ignoring all this complexity --- what else should they do? With our manuscript we want to emphasize this complexity on the still relatively simple example of differences in ion channel composition between different neuron types. We use the simulations presented within our study to encourage investigation into neuron type specific effects and to use LOF/GOF firing characterizations with caution. And we suggest simulations to be used for large scale screenings across neuron types.
 
 \textit{Failure to analyze the effects of the obtained data on living organisms casts a shadow over the reliability of the results. The study would be much more valuable if the authors could at least support their results with animal experiments. Otherwise, it will not go beyond being an evaluation study based on already known mutations. This limits the originality of the study.}
 
-Although we agree that data obtained from animal experiments is invaluable in the investigation of channelopathies and in neuroscience and neurology generally, the modelling and simulation in this study serves to provide a theoretical framework (Figures 3 and 4) to explain why neuron-type level investigation is important in understanding of ion channel mutations. Furthermore, efforts to reduce animal experimentation are ongoing and modelling approaches such as that presented in this study enables the reduction of animal experimentation. Previous experimental studies (Hedrich et al., 2014; Makinson et al., 2016; Waxman, 2007; Rush et al., 2006) have demonstrated neuron-type specific effects of ion channel mutations. Therefore, experimental efforts should be focused on validation and investigation of specific cases rather than accumulation of evidence for the general neuron-type dependence. Furthermore, the development of neuron type specific models enables informed hypothesis generation for such electrophysiological experimentation and enables testing of hypotheses that are difficult to test experimentally. Although we recognize the importance and value in the discovery of new mutations, further investigation and analysis of known mutations and their effects is also valuable and essential for understanding the effects of ion channel mutations at multiple levels of scale. This has been added to the Modelling limitations section of the discussion.
+Although we agree that data obtained from animal experiments is invaluable in the investigation of channelopathies and in neuroscience and neurology generally, the modelling and simulation in this study serves to provide a theoretical framework (Figures 3 and 4) to explain why neuron-type level investigation is important in understanding of ion channel mutations. Furthermore, efforts to reduce animal experimentation are ongoing and modelling approaches such as that presented in this study enables the reduction of animal experimentation. Previous experimental studies (Hedrich et al., 2014; Makinson et al., 2016; Waxman, 2007; Rush et al., 2006) have demonstrated neuron-type specific effects of ion channel mutations. Therefore, experimental efforts should be focused on validation and investigation of specific cases rather than accumulation of evidence for the general neuron-type dependence. Furthermore, the development of neuron type specific models enables informed hypothesis generation for such electrophysiological experimentation and enables testing of hypotheses that are difficult to test experimentally. Although we recognize the importance and value in the discovery of new mutations, further investigation and analysis of known mutations and their effects is also valuable and essential for understanding the effects of ion channel mutations at multiple levels of scale. This has been added to the ``Modelling limitations'' section of the discussion.
 
 
 \section{Reviewer 2}
 \textit{The work presented by the authors "Loss or Gain of Function? Effects of Ion Channel Mutations on Neuronal Firing Depend on the Neuron Type" is an approach to elucidate how the loss or gain of function mutation in ionic currents can alter the firing rate of action potentials in different neuronal types. They analyze changes in the rheobase and the area under the curve of the fI curves. Although the approach is adequate to distinguish LOF or GOF, intermediate cases are not easy to analyze. I emphasize that the authors ponder a large part of the limitations of the method used in a broad discussion. However, I believe that the authors should make some clarifications that allow the reader to better understand the information from the results.}
 
-
-
 \textit{Authors should include a schematic representing the general biological characteristics of each model tested. Even this schematic could be appended to the model description section}
 
-We thank the reviewer for their comments. Figure 1 has been updated to reflect the general anatomical characteristics of each model and Figure 2 has been added to reflect the ion current composition of each model. The manuscript has been updated to reflect these changes.
-
-%\begin{figure}[!h]
-%        \centering
-%        \includegraphics[width=\linewidth]{diversity_in_firing_diagram.jpg}
-%        \linespread{1.}\selectfont
-%	\caption[]{Diversity in Neuronal Model Firing. Spike trains (left), frequency-current (fI) curves (right) for Cb stellate  \textbf{(A)}, RS inhibitory  \textbf{(B)}, FS \textbf{(C)}, RS pyramidal  \textbf{(D)}, RS inhibitory +\Kv  \textbf{(E)}, Cb stellate +\Kv  \textbf{(F)}, FS +\Kv  \textbf{(G)}, RS pyramidal +\Kv  \textbf{(H)}, STN +\Kv  \textbf{(I)}, Cb stellate \(\Delta\)\Kv  \textbf{(J)}, STN \(\Delta\)\Kv  \textbf{(K)}, and STN  \textbf{(L)} neuron models. Models are sorted qualitatively based on their fI curves. Black markers on the fI curves indicate the current step at which the spike train occurs. The green marker indicates the current at which firing begins in response to an ascending current ramp, whereas the red marker indicates the current at which firing ceases in response to a descending current ramp (see Supplementary Figure S1). A schematic illustrating the anatomical locations of the models is included \textbf{(M)}, however single compartment models are used for each cell type.} 
-%        \label{fig:diversity_in_firing}
-%\end{figure}
-%
-%\begin{figure}[!h]
-%        \centering
-%        \includegraphics[width=\linewidth]{model_g.jpg}
-%        \linespread{1.}\selectfont
-%	\caption[]{Diversity in Neuronal Model Current Composition. Distributions of maximal current conductances (\(\mathrm{g}_{\mathrm{max}}\)) for Cb stellate  \textbf{(A)}, RS inhibitory  \textbf{(B)}, FS \textbf{(C)}, RS pyramidal  \textbf{(D)}, RS inhibitory +\Kv  \textbf{(E)}, Cb stellate +\Kv  \textbf{(F)}, FS +\Kv  \textbf{(G)}, RS pyramidal +\Kv  \textbf{(H)}, STN +\Kv  \textbf{(I)}, Cb stellate \(\Delta\)\Kv  \textbf{(J)}, STN \(\Delta\)\Kv  \textbf{(K)}, and STN  \textbf{(L)} neuron models. Models are sorted as in Figure \ref{fig:diversity_in_firing}.}
-%\end{figure}
-
-
-
-\FloatBarrier
-
-%We thank Reviewer 2 for their comments.
+Thank you for your suggestion. We updated Figure 1 to reflect the general anatomical origin of each model and we added a new Figure 2 to reflect the ion current composition of each model. The manuscript has been updated to reflect these changes.
 
 \textit{In Figure 1 the authors should comment in more detail on the behavior of the fI curve (B)}
 
@@ -120,20 +98,11 @@ This model, the RS inhibitory model ceases to fire with large current steps as e
 
 \textit{Bearing in mind that the authors mention that each model may have hysteresis depending on the stimulation ramp that was applied in figure 1, under what conditions was the rheobase determined in figure 2-5? It is also not clear which model they use to build figure 2A.}
 
-Although hysteresis is important both from a dynamical systems perspective and in a physiological context, we exclude further analysis of hysteresis in our simulations past Figure 1 to be as applicable as possible to commonly used protocols in the assessment of ion channel mutations. For Figures 3-6 (formerly 2-5), rheobase is calculated by a 2 step process as outlined in the methods. Briefly described this method uses a sequence of coarse current step amplitudes to identify the step interval in which firing begins. This interval is then explored at high resolution with a second sequence of current step inputs. Rheobases from step current  protocols were used for subsequent analysis in order to maintain relatability to commonly used experimental measures. For Figure 2, square root functions were used for illustration purposes. The manuscript has been updated to explicitly state these clarifications.
+Although hysteresis is important both from a dynamical systems perspective and in a physiological context, we exclude further analysis of hysteresis in our simulations past Figure 1 to be as applicable as possible to commonly used protocols in the assessment of ion channel mutations. For Figures 3-6 (formerly 2-5), rheobase is calculated by a 2 step process as outlined in the methods. Briefly described this method uses a sequence of coarse current step amplitudes to identify the step interval in which firing begins. This interval is then explored at high resolution with a second sequence of current step inputs. Rheobases from step current  protocols were used for subsequent analysis in order to maintain relatability to commonly used experimental measures. For Figure 2, square root functions were used for illustration purposes. The methods part of the manuscript has been updated to explicitly state these clarifications.
 
 \textit{A final outline summarizing the main results of the main models and the general limitations of this approach could be included.}
 
-A summary figure has been added as Figure 7 and the discussion updated accordingly.
-
-%\begin{figure}[!h]
-%        \centering
-%        \includegraphics[width=\linewidth]{summary_fig.jpg}
-%        \linespread{1.}\selectfont
-%	\caption[]{Summary of neuron type dependence of channelopathies. The wildtype channel (blue) is mutated (red) the effects of it in different neuron types (green, orange and grey) each with a unique set of ion channels (see inset axes) determines the effect of firing seen on the right by the shift from the blue wildtype fI curve to the red fI curve for the mutated ion channel.}
-%\end{figure}
-%
-%\FloatBarrier
+Thank you for this good suggestion. We added a summary figure as Figure 7 and updated the next-to-last paragraph of the discussion accordingly.
 
 \section{Reviewer 3}
 \textit{With this study, authors aimed to investigate the impact of neuronal cell type on the firing outcome of ion channel mutations. Study were conducted with simulations on conductance-based neuron models.
@@ -142,6 +111,6 @@ Results are evaluated well. Presentation of findings is fine.
 Finding are clearly showing that the neuronal type and the respective ionic current composition is of vital importance and must be considered when assessing the effects of mutations on neuronal firing.
 I can feel that the text overhauled many times (may be past review processes). So the manuscript can be published as it is in my opinion.}
 
-We thank the reviewer for their comments and review.
+Thank you very much for your positive evaluation!
 
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