some more fixes

Frontiers_manuscript/Koch_frontiers.tex

@@ -65,7 +65,7 @@ $^{3}$Department of Neurology and Epileptology, Hertie Institute for Clinical Br
 
 \begin{abstract}
 \section{}
-Clinically relevant mutations to voltage-gated  ion channels, called channelopathies, alter ion channel function, properties of ionic currents and neuronal firing.  The effects of ion channel mutations are routinely assessed and characterized as loss of function (LOF) or gain of function (GOF) at the level of ionic currents. However, emerging personalized medicine approaches based on LOF/GOF characterization have limited therapeutic success. Potential reasons are among others that the translation from this binary characterization to neuronal firing is currently not well understood ---  especially when considering different neuronal cell types. Here we investigate the impact of neuronal cell type on the firing outcome of ion channel mutations with simulations of a diverse collection of conductance-based neuron models. We systematically analyzed the effects of changes in ion current properties on firing in different neuronal types.  Additionally, we simulated the effects of known mutations in the \textit{KCNA1} gene encoding the \Kv potassium channel subtype associated with episodic ataxia type~1 (EA1). These simulations revealed that the outcome of a given change in ion channel properties on neuronal excitability is depends on neuron type, i.e. the properties and expression levels of the unaffected ionic currents. Consequently, neuron-type specific effects are vital to a full understanding of the effects of channelopathies on neuronal excitability and are an important step towards improving the efficacy and precision of personalized medicine approaches.
+Clinically relevant mutations to voltage-gated  ion channels, called channelopathies, alter ion channel function, properties of ionic currents and neuronal firing.  The effects of ion channel mutations are routinely assessed and characterized as loss of function (LOF) or gain of function (GOF) at the level of ionic currents. However, emerging personalized medicine approaches based on LOF/GOF characterization have limited therapeutic success. Potential reasons are among others that the translation from this binary characterization to neuronal firing is currently not well understood ---  especially when considering different neuronal cell types. Here we investigate the impact of neuronal cell type on the firing outcome of ion channel mutations with simulations of a diverse collection of conductance-based neuron models. We systematically analyzed the effects of changes in ion current properties on firing in different neuronal types.  Additionally, we simulated the effects of known mutations in the \textit{KCNA1} gene encoding the \Kv potassium channel subtype associated with episodic ataxia type~1 (EA1). These simulations revealed that the outcome of a given change in ion channel properties on neuronal excitability depends on neuron type, i.e. the properties and expression levels of the unaffected ionic currents. Consequently, neuron-type specific effects are vital to a full understanding of the effects of channelopathies on neuronal excitability and are an important step towards improving the efficacy and precision of personalized medicine approaches.
 
 
 
@@ -75,7 +75,7 @@ Clinically relevant mutations to voltage-gated  ion channels, called channelopat
 \end{abstract}
 \section{Introduction}
 The properties and combinations of voltage-gated ion channels are vital in determining neuronal excitability \citep{bernard_channelopathies_2008, carbone_ion_2020, rutecki_neuronal_1992, pospischil_minimal_2008}. However, ion channel function can be disturbed, for instance through genetic alterations, resulting in altered neuronal firing behavior \citep{carbone_ion_2020}. In recent years, next generation sequencing has led to an increase in the discovery of clinically relevant ion channel mutations and has provided the basis for pathophysiological studies of genetic epilepsies, pain disorders, dyskinesias, intellectual disabilities, myotonias, and periodic paralyses \citep{bernard_channelopathies_2008, carbone_ion_2020}.
-Ongoing efforts of many research groups have contributed to the current understanding of underlying disease mechanism in channelopathies, however a complex pathophysiological landscape has emerged for many channelopathies and is likely a reason for limited therapeutic success with standard care.              
+Ongoing efforts of many research groups have contributed to the current understanding of underlying disease mechanism in channelopathies. However, a complex pathophysiological landscape has emerged for many channelopathies and is likely a reason for limited therapeutic success with standard care.              
 
 Ion channel variants are frequently classified in heterologous expression systems as either a loss of function (LOF) or a gain of function (GOF)  in the respective ionic current \citep{Musto2020, Kullmann2002, Waxman2011, Kim2021}. This LOF/GOF classification is often directly used to predict the effects on neuronal firing \citep{Niday2018, Wei2017, Wolff2017,Masnada2017}, which in turn is important for understanding the pathophysiology of these disorders and for identification of potential therapeutic targets \citep{Orsini2018, Yang2018, Colasante2020, Yu2006}. Experimentally, the effects of channelopathies on neuronal firing are assessed using primary neuronal cultures  \citep{Scalmani2006, Smith2018, Liu2019}  or \textit{in vitro} recordings from slices of transgenic mouse lines \citep{Mantegazza2019, Xie2010,Lory2020, Habib2015, Hedrich14874}, but are restricted to limited number of different neuron types. Neuron types differ in many aspects. They may differ in their composition of ionic currents \citep{yao2021taxonomy, Cadwell2016, BICCN2021, Scala2021} and therefore likely respond differently to changes in the properties of a single ionic current. Expression level of an affected gene \citep{Layer2021} and relative amplitudes of ionic currents \citep{rutecki_neuronal_1992, pospischil_minimal_2008,Kispersky2012, golowasch_failure_2002, barreiro_-current_2012} indeed dramatically influence the firing behavior and dynamics of neurons. Mutations in different sodium channel genes have been experimentally shown to affect firing in a neuron-type specific manner based on differences in expression levels of the affected gene \citep{Layer2021}, but also on other neuron-type specific mechanisms \citep{Hedrich14874, makinson_scn1a_2016}.
 
@@ -162,13 +162,13 @@ Mutations in \textit{KCNA1} are associated with episodic ataxia type~1 (EA1) and
 
 \section{Discussion}
 
-To compare the effects of ion channel mutations on neuronal firing of different neuron types, a diverse set of conductance-based models was used and the effect of changes in individual channel properties across conductance-based neuronal models. Additionally, the effects of episodic ataxia type~1 associated (EA1) \textit{KCNA1} mutations were simulated. Changes to single ionic current properties, as well as known EA1 associated \textit{KCNA1} mutations showed consistent effects on the rheobase across neuron types, whereas the effects on AUC of the steady-state fI-curve depended on the neuron type. Our results demonstrate that loss of function (LOF) and gain of function (GOF) on the biophysical level cannot be uniquely transferred to the level of neuronal firing. Thus, the effects caused by different mutations depend on the properties of the other ion channels expressed in a neuron and are therefore depend on the channel ensemble of a specific neuron type. 
+To compare the effects of ion channel mutations on neuronal firing of different neuron types, we used a diverse set of conductance-based models to systematically characterize the effects of changes in individual channel properties. Additionally, we simulated the effects of specific episodic ataxia type~1 associated (EA1) \textit{KCNA1} mutations. Changes to single ionic current properties, as well as known EA1 associated \textit{KCNA1} mutations showed consistent effects on the rheobase across neuron types, whereas the effects on the slope of the steady-state fI-curve depended on the neuron type. Our results demonstrate that loss of function (LOF) and gain of function (GOF) on the biophysical level cannot be uniquely transferred to the level of neuronal firing. Thus, the effects caused by different mutations depend on the properties of the other ion channels expressed in a neuron and are therefore depend on the channel ensemble of a specific neuron type. 
 
 \subsection{Firing Frequency Analysis}
-Although, firing differences can be characterized by an area under the curve of the fI curve for fixed current steps this approach characterizes firing as a mixture of key features: rheobase and the initial slope of the fI curve. By probing rheobase directly and using an AUC relative to rheobase, we disambiguate these features and enable insights into the effects on rheobase and initial fI curve steepness. This increases the specificity of our understanding of how ion channel mutations alter firing across neuron types and enable classification as described in Figure \ref{fig:firing_characterization}. Importantly, in cases when ion channel mutations alter rheobase and initial fI curve steepness in ways that oppose effects on firing (upper left and bottom right quadrants of Figure \ref{fig:firing_characterization}~B) this disamgibuation is important for understanding the outcome of the mutation. In these cases, the regime the neuron is operating in is vital in determining the neuron's firing outcome. If it is in its excitable regime and only occasionally generates an action potential, then the effect on the rheobase is more important. If it is firing periodically with high rates, then the change in AUC might be more relevant. 
+Although differences in neuronal firing can be characterized by an area under the curve of the fI curve for a fixed current range, this approach characterizes firing as a mixture of key features: rheobase and the initial slope of the fI curve. By probing rheobase directly and using an AUC relative to rheobase, we disambiguate these features and enable insights into the effects on rheobase and initial fI curve steepness. This increases the specificity of our understanding of how ion channel mutations alter firing across neuron types and enable classification as described in Figure \ref{fig:firing_characterization}. Importantly, in cases when ion channel mutations alter rheobase and initial fI curve steepness in ways that oppose effects on firing (upper left and bottom right quadrants of Figure \ref{fig:firing_characterization}~B), this disamgibuation is important for understanding the outcome of the mutation. In these cases, the regime the neuron is operating in is vital in determining the neuron's firing outcome. If it is in its excitable regime and only occasionally generates an action potential, then the effect on the rheobase is more important. If it is firing periodically with high rates, then the change in AUC might be more relevant. 
 
 \subsection{Modelling Limitations}
-The models used here are simple and they all capture key aspects of the firing dynamics for their respective neuron. The simple models fall short of capturing the complex physiology, biophysics and heterogeneity of real neurons, nor do they take into account subunit stoichiometry, auxillary subunits, membrane composition which influence the biophysics of ionic currents \citep{Al-Sabi_2013, Oliver_2004, Pongs_2009, Rettig1994}. However, for the purpose of understanding how different neuron-types, or current environments, contribute to diversity in firing outcomes of ion channel mutations, the fidelity of the models to the physiological neurons they represent is of a minor concern. For exploring possible neuron-type specific effects, variety in currents and dynamics across models is of utmost importance. With this context in mind, the collection of models used here are labelled as models A-L to highlight that the physiological neurons they represent is not of chief concern, but rather that the collection of models with different attributes respond heterogeneously to the same perturbation. Additionally, the development of more realistic models is a high priority and will enable neuron-type specific predictions that may aid precision medicine approaches. Thus, weight should not be put on any single predicted firing outcome here in a specific model, but rather on the differences in outcomes that occur across the neuron-type spectrum the models used here represent. %  Menchaca_2012,
+The models used here are simple and they all capture key aspects of the firing dynamics for their respective neuron. The simple models fall short of capturing the complex physiology, biophysics and heterogeneity of real neurons, nor do they take into account subunit stoichiometry, auxillary subunits, membrane composition which influence the biophysics of ionic currents \citep{Al-Sabi_2013, Oliver_2004, Pongs_2009, Rettig1994}. However, for the purpose of understanding how different neuron-types, or current environments, contribute to the diversity in firing outcomes of ion channel mutations, the fidelity of the models to the physiological neurons they represent is of a minor concern. For exploring possible neuron-type specific effects, variety in currents and dynamics across models is of utmost importance. With this context in mind, the collection of models used here are labelled as models A-L to highlight that the physiological neurons they represent is not of chief concern, but rather that the collection of models with different attributes respond heterogeneously to the same perturbation. Additionally, the development of more realistic models is a high priority and will enable neuron-type specific predictions that may aid precision medicine approaches. Thus, weight should not be put on any single predicted firing outcome here in a specific model, but rather on the differences in outcomes that occur across the neuron-type spectrum the models used here represent. %  Menchaca_2012,
 
 \subsection{Neuronal Diversity}
 The nervous system consists of a vastly diverse and heterogenous collection of neurons with variable properties and characteristics including diverse combinations and expression levels of ion channels which are vital for neuronal firing dynamics.
@@ -216,7 +216,7 @@ With this study we suggest that neuron-type specific effects are vital to a full
 \end{figure}
 
 
-\begin{figure}[h!]
+\begin{figure}[!ht]
         \centering
 	\includegraphics[width=0.5\linewidth]{Figures/firing_characterization_arrows.pdf}
         \linespread{1.}\selectfont
@@ -225,7 +225,7 @@ Changes in firing as characterized by \(\Delta\)AUC and \(\Delta\)rheobase occup
         \label{fig:firing_characterization}
 \end{figure}
 
-\begin{figure}[h!]
+\begin{figure}[!ht]
         \centering
         \includegraphics[width=\linewidth]{Figures/AUC_correlation.pdf}
         \linespread{1.}\selectfont
@@ -233,7 +233,7 @@ Changes in firing as characterized by \(\Delta\)AUC and \(\Delta\)rheobase occup
         \label{fig:AUC_correlation}
 \end{figure}
 
-\begin{figure}[h!]
+\begin{figure}[!ht]
         \centering
         \includegraphics[width=\linewidth]{Figures/rheobase_correlation.pdf}
         \linespread{1.}\selectfont
@@ -241,7 +241,7 @@ Changes in firing as characterized by \(\Delta\)AUC and \(\Delta\)rheobase occup
         \label{fig:rheobase_correlation}
 \end{figure}
 
-\begin{figure}[h!]
+\begin{figure}[!ht]
         \centering
         \includegraphics[width=\linewidth]{Figures/simulation_model_comparison.pdf}
         \linespread{1.}\selectfont