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@ -111,13 +111,19 @@ Ion channels determine neuronal excitability and mutations that alter ion channe
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}. 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. \textcolor{red}{what effect does the mutation have on firing behaviour? - first motivation, about where this should lead}
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.
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. 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.
One of the most realistic methods, but also the most time extensive and expensive, is the generation of a mouse line with the prefered mutation.
%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}. %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. %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.
The generation of mice lines is costly and behavioural characterization of new mice lines is required to assess similarities to patient symptoms. Although the generation of mouse lines is desirable for a clinical disorder characterized by a specific ion channel mutation, this approach becomes impractical for disorders associated with a collection of distinct mutations in a single ion channel. Because of the lack of adequate experimental approaches, a great need is present for the ability to assess the impacts of ion channel mutations on neuronal firing. A more general understanding of the effects of changes in current properties on neuronal firing may help to understand the impacts of ion channel mutations. Specifically, modelling approaches can be used to assess the impacts of current property changes on firing behaviour, bridging the gap between changes in the biophysical properties induced by mutations and clinical symptoms. Conductance-based neuronal models enable insight into the effects of ion channel mutations with specific effects of the resulting ionic current as well as enabling \textit{in silico} assessment of the relative effects of changes in biophysical properties of ionic currents on neuronal firing . The effects of altered voltage-gated potassium channel \Kv function is of particular interest in this study as it gives rise to the \IKv current and is associated with episodic ataxia type 1. Furthermore, modelling approaches enable predictions of the effects of specific mutation and drug induced biophysical property changes. The generation of mice lines is costly and behavioural characterization of new mice lines is required to assess similarities to patient symptoms. Although the generation of mouse lines is desirable for a clinical disorder characterized by a specific ion channel mutation, this approach becomes impractical for disorders associated with a collection of distinct mutations in a single ion channel. Because of the lack of adequate experimental approaches, a great need is present for the ability to assess the impacts of ion channel mutations on neuronal firing. A more general understanding of the effects of changes in current properties on neuronal firing may help to understand the impacts of ion channel mutations. Specifically, modelling approaches can be used to assess the impacts of current property changes on firing behaviour, bridging the gap between changes in the biophysical properties induced by mutations and clinical symptoms. Conductance-based neuronal models enable insight into the effects of ion channel mutations with specific effects of the resulting ionic current as well as enabling \textit{in silico} assessment of the relative effects of changes in biophysical properties of ionic currents on neuronal firing . The effects of altered voltage-gated potassium channel \Kv function is of particular interest in this study as it gives rise to the \IKv current and is associated with episodic ataxia type 1. Furthermore, modelling approaches enable predictions of the effects of specific mutation and drug induced biophysical property changes.