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Small Research Grant 2011

Small Research Grant 2011

Localising action potential initiation under conditions of neuronal plasticity

Thomas Watkins

Dravet Syndrome UK were thrilled to award a small grant of £1,000 to neuroscience student Thomas Watkins. Tom, a medical student at King’s College Hospital in London, outlined a study to us entitled ‘Localising action potential initiation under conditions of neuronal plasticity’ and Dravet Syndrome UK were delighted to be in a position to support Tom’s work.

The study is now complete and Tom reported that “this eight week summer studentship has produced interesting results which will enable further work to be done to understand the structure, function and plasticity of the AIS.

The AIS is where the genetic mutations which result in Dravet syndrome and other sodium channel epilepsies exert their effect and a greater understanding of it will hopefully contribute to the development of treatments for these conditions in the future. I graduated from my intercalated BSc in Neuroscience this year with first class honours and have started my clinical training.

I hope to go on to do a PhD related to the study of epilepsy and would like to thank DSUK for providing the funding for this project which has given me invaluable experience and skills related to my future career”.

Tom’s full report is below:

The AIS is a highly specialised subcompartment of neurons which contains unique molecular constituents including a high density of voltage-gated sodium channels and the scaffolding protein Ankyrin G.

This high density is believed to make the AIS the site of action potential (AP) initiation. A large body of convergent evidence from diverse functional approaches in different types of neuron shows that AP initiation does occur in the proximal axon.

However, no-one had yet managed to link the site of AP initiation to the molecularly specialised AIS region, or to subcompartments within that AIS region (e.g. the distal high densities of Nav1.6). This is especially important given recent evidence from Dr Grubb’s lab that there is a huge and unexpected degree of plasticity at AIS – a neuron’s recent past electrical activity can determine location of the AIS structure within its axon, and this is thought to be a mechanism for controlling neuronal excitability (Grubb & Burrone 2010).

These two experiments have contributed to the understanding of AIS structure and movement and will assist the research group in planning further work

This project combined structural measures of AIS location using live immunohistochemical label for a scaffolding protein localised to the AIS called Neurofascin with electrophysiological recording of action potentials.

During my time in the lab I learnt to patch clamp neurons and successfully recorded action potentials at the AIS in live cells as marked by the antibody for Neurofascin. The preliminary data gathered appears to indicate that, in the cases recorded, the action potential was generated at the AIS before the cell body.

The cell body is the area of the neuron where the whole cell patch was made and the current which caused the cell to fire an AP was injected. This provides exciting new evidence directly linking the structural proteins of the AIS and by extension the sodium channels attached to them to the location of initial action potential generation.

Unfortunately we were unable to record from cells under conditions of neuronal plasticity in which the AIS had moved due to the time taken obtaining recordings and Dr Grubb’s team will continue this area of the project.

As well as electrophysiological recordings Dr Grubb’s team work on elucidating the mechanism of AIS movement which is linked to controlling neuronal excitability. Whilst waiting for cells to mature to the appropriate ages for electrophysiological recordings I assisted in confocal image acquisition and the analysis of an experiment carried out by Mark Evans, a PhD student in Dr Grubb’s laboratory.

This involved treating dissociated hippocampal cultures with a drug called Taxol. I helped to modify a computer program to analyse the effect of the Taxol and found that this microtubule stabilising drug causes the cells in culture to form multiple secondary processes which express structural proteins usually only found at the AIS.

The drug also prevented the movement of the AIS in the neurons’ original axons under conditions simulating periods of heightened excitability which would normally induce it.

We then performed an experiment to evaluate the effect of a drug called Latrunculin which disrupts actin filaments. This was found to cause the AIS of the cells in culture to break apart into multiple small sections and preliminary analysis seems to indicate that it also prevents AIS movement under conditions of high levels of neuronal excitability.

However, I hope to assist in the further analysis which will be carried out on the data to characterise the distributions of the small sections of AIS which resulted from the application of the drug.

This eight week summer studentship has produced interesting results which will enable further work to be done to understand the structure, function and plasticity of the AIS. The AIS is where the genetic mutations which result in Dravet syndrome and other sodium channel epilepsies exert their effect and a greater understanding of it will hopefully contribute to the development of treatments for these conditions in the future.

I graduated from my intercalated BSc in Neuroscience this year with first class honours and have started my clinical training. I hope to go on to do a PhD related to the study of epilepsy and would like to thank DSUK for providing the funding for this project which has given me invaluable experience and skills related to my future career.