changed some stuff in intro

manuscript.tex

@@ -109,7 +109,13 @@ Ion channels determine neuronal excitability and mutations that alter ion channe
 \section*{Introduction (750 Words Maximum - Currently 673)}
 %\textit{The Introduction should briefly indicate the objectives of the study and provide enough background information to clarify why the study was undertaken and what hypotheses were tested.}
 
-Neuronal ion channels are vital in determining neuronal excitability, action potential generation and firing patterns \citep{bernard_channelopathies_2008, carbone_ion_2020}. In particular, the properties and combinations of ion channels and their resulting currents determine the firing properties of the neuron \citep{rutecki_neuronal_1992, pospischil_minimal_2008}. However, ion channel function can be disturbed, resulting in altered ionic current properties and altered neuronal firing behaviour \citep{carbone_ion_2020}. Ion channel mutations are a common cause of such channelopathies and are often associated with hereditary clinical disorders \citep{bernard_channelopathies_2008, carbone_ion_2020}. The effects of these mutations are frequently determined at a biophysical level, however assessment of the impact of mutations on neuronal firing and excitability is more difficult. Experimentally, transfection of cell cultures or the generation of mutant mice lines are common approaches. Cell culture transfection does not replicate the exact interplay of endogenous currents nor does it take into account the complexity of the nervous system including factors such as expression patterns, intracellular regulation and modulation of ion channels as well as network effects. Transfected currents are characterized in isolation and the role of these isolated currents in the context of other currents in a neuron cannot be definitively inferred. The effects of individual currents \textit{in vivo} also depend on the neuron type they are expressed in and which roles these neurons have in specific circuits. Complex interactions between different cell types \textit{in vivo} are neglected in transfected cell culture.  Additionally, transfected currents are not present with the neuron-type specific cellular machinery present \textit{in vivo} and are even transfected in cells of different species. Furthermore, culture conditions can shape ion channel expression \citep{ponce_expression_2018}.
+Neuronal ion channels are vital in determining neuronal excitability, action potential generation and firing patterns \citep{bernard_channelopathies_2008, carbone_ion_2020}. In particular, the properties and combinations of ion channels and their resulting currents determine the firing properties of the neuron \citep{rutecki_neuronal_1992, pospischil_minimal_2008}. However, ion channel function can be disturbed, resulting in altered ionic current properties and altered neuronal firing behaviour \citep{carbone_ion_2020}. Ion channel mutations are a common cause of such channelopathies and are often associated with hereditary clinical disorders \citep{bernard_channelopathies_2008, carbone_ion_2020}. 
+
+The effects of these mutations are frequently determined at a biophysical level in heterologous expression systems that contain no endogenous ionic currents. \textcolor{red}{why?} These experiments can determine how the voltage dependent kinetics have changed, but only allow for rough predictions about how the firing rate of a neuron is affected. 
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+Transfection of primary neuron cultures can overcome some of these limitations and changes in firing behaviour can more readily be assessed \cite{Scalmani2006, Liu2019}. However, the relative expression and conductance of the transfected ion channel in relation to endogenous currents can be variable. As the firing behaviour and dynamics of neuronal models can be dramatically altered by altering relative current amplitudes \citep{rutecki_neuronal_1992, pospischil_minimal_2008,Kispersky2012, golowasch_failure_2002, barreiro_-current_2012}, primary neuronal do probably not provide definitive insight into the effects of a channelopathy on \textit{in vivo} firing. 
+ 
+%The effects of these mutations are frequently determined at a biophysical level, however assessment of the impact of mutations on neuronal firing and excitability is more difficult. Experimentally, transfection of cell cultures or the generation of mutant mice lines are common approaches. Cell culture transfection does not replicate the exact interplay of endogenous currents nor does it take into account the complexity of the nervous system including factors such as expression patterns, intracellular regulation and modulation of ion channels as well as network effects. Transfected currents are characterized in isolation and the role of these isolated currents in the context of other currents in a neuron cannot be definitively inferred. The effects of individual currents \textit{in vivo} also depend on the neuron type they are expressed in and which roles these neurons have in specific circuits. Complex interactions between different cell types \textit{in vivo} are neglected in transfected cell culture.  Additionally, transfected currents are not present with the neuron-type specific cellular machinery present \textit{in vivo} and are even transfected in cells of different species. Furthermore, culture conditions can shape ion channel expression \citep{ponce_expression_2018}.
 
 Ion channel transfection of primary neuronal cultures can overcome some of the limitations of cell culture expression. In transfected neuronal cell cultures firing can more readily be assessed as endogenous currents are present, however the expressed and endogenous versions of the same ion channel are present in the cell \cite{Scalmani2006, Smith2018}. To avoid the confound of both expressed and endogenous current contributing to firing, a drug resistance can be introduced into the ion channel that is transfected and the drug is used to silence the endogenous version of this current \cite{Liu2019}.  Although addition of TTX-resistance to \(\textrm{Na}_{\textrm{V}}\) does not alter the gating properties of these channels \cite{Leffler2005}, the relative expression and conductance of the transfected ion channel in relation to endogenous currents can be variable and non-specific blocking of ion channels not affected by the channelopathy may occur. As the firing behaviour and dynamics of neuronal models can be dramatically altered by altering relative current amplitudes \citep{rutecki_neuronal_1992, pospischil_minimal_2008,Kispersky2012, golowasch_failure_2002, barreiro_-current_2012}, primary neuronal cultures provide a useful general indication as to the effects of ion channel mutations but do not provide definitive insight into the effects of a channelopathy on \textit{in vivo} firing.