changes from discussion with Lukas

Frontiers_manuscript/For_Submission/Koch_frontiers_diff.tex

@@ -236,10 +236,13 @@ 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.
 
-The effects of altered ion channel properties on firing is generally influenced by the other ionic currents present in the neuron \DIFaddbegin \DIFadd{as summarized in Figure \ref{fig:summary}}\DIFaddend . 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. 
+The effects of altered ion channel properties on firing is generally influenced by the other ionic currents present in the neuron \DIFaddbegin \DIFadd{as illustrated in Figure \ref{fig:summary}}\DIFaddend . 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. \DIFaddbegin \DIFadd{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.}\DIFaddend
+%provide more specificity and thus 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.
 
+%understanding of the neuron-type level firing effects of ion channel mutations gained through 
 
 \bibliographystyle{Frontiers-Harvard} 
 \bibliography{Koch_ref}
@@ -265,7 +268,7 @@ With this study we suggest that neuron-type specific effects are vital to a full
 	\includegraphics[width=0.5\linewidth]{firing_characterization_arrows.jpg}
         \linespread{1.}\selectfont
         \caption[]{Characterization of firing with AUC and rheobase. \textbf{(A)} The area under the curve (AUC) of the repetitive firing frequency-current (fI) curve.  \textbf{(B)}
-Changes in firing as characterized by \(\Delta\)AUC and \(\Delta\)rheobase occupy four quadrants separated by no changes in AUC and rheobase. Representative schematic fI curves in red with respect to a reference (or wild type) fI curve (blue) depict the general changes associated with each quadrant.}
+Changes in firing as characterized by \(\Delta\)AUC and \(\Delta\)rheobase occupy four quadrants separated by no changes in AUC and rheobase. Representative schematic fI curves in red with respect to a reference (or wild type) fI curve (blue) depict the general changes associated with each quadrant. \DIFaddbeginFL \DIFaddFL{Square root functions are used as fI curves for illustration purposes.} }
         \label{fig:firing_characterization}
 \end{figure}
 

Frontiers_manuscript/For_Submission/Koch_reviewer_comments.tex

@@ -70,15 +70,16 @@ We thank Reviewer 1 for their comments. Although previous studies (Hedrich et al
 
 \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. \textcolor{red}{Change anything in manuscript?}
+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. \textcolor{red}{This has altered in the manuscript to be more explicit.}
+
 
 \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 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 or not relevant 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. \textcolor{red}{Change anything in manuscript?}
+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 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. \textcolor{red}{Change anything in manuscript?}
 
 \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 theoretical framework 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 such as that presented in this study enables the reduction of animal experimentation. Although we recognize the importance and value in collection 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. \textcolor{red}{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 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. 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. \textcolor{red}{This has been added to the Modelling limitations section of the discussion.}
 
 
 \section{Reviewer 2}
@@ -88,6 +89,7 @@ Although we agree that data obtained from animal experiments is invaluable in th
 
 \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}
 
+Figure 1 has been updated to reflect the general biological characteristics of each model.
 
 \begin{figure}[!h]
         \centering
@@ -106,10 +108,12 @@ 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 2-5, rheobase is calculated by a 2 step process as outlined in the methods. Briefly described this methods 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. The manuscript has been updated to explicitly state this.
+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 2-5, rheobase is calculated by a 2 step process as outlined in the methods. Briefly described this methods 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.
 
 \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 6.
+
 \begin{figure}[!h]
         \centering
         \includegraphics[width=\linewidth]{summary_fig.jpg}