Chloride regulation in neuronal development and epilepsy

Lead Research Organisation: University of Oxford
Department Name: Pharmacology

Abstract

The nervous system is characterised by highly organised ‘circuits’ in which specific groups of nerve cells, or neurons, interconnect. The way neurons communicate depends upon their three dimensional structure and the type of connections that they form with one another. Neural circuits are laid down during development and errors at these early stages can lead to problems later in life. For instance, epilepsy and schizophrenia are just two disorders where abnormal developmental events are thought to underlie aberrant communication in the adult brain.

This research focuses upon chloride, a key ion in the nervous system. It has been observed that the level of chloride inside neurons normally decreases during the period that neural circuits are developing. In contrast, neurons in the brains of epilepsy patients have abnormally high levels of chloride. Using neural tissue from rodents, the experiments in this project will examine how changes in chloride levels influence the structure and connections of growing neurons. Furthermore, it will address how molecules that control chloride levels are themselves regulated during development.

Findings from this work will advance our understanding of neonatal development, and also our understanding of the cellular changes that occur in common disorders of the nervous system.

Technical Summary

A question of major importance in neuroscience is how neural circuits are formed during normal development and in the diseased brain. The physical shape of a neuron dictates which cells it is able to interact with and neural activity is known to be a key regulator of precise neuronal morphology. Since ionic regulation mechanisms underpin both voltage events and the actions of neurotransmitters, these represent fundamental aspects of any activity-dependent process. Recent work has suggested that chloride regulation may play a particularly important role during neural circuit formation and this is the focus of the current research. As neurons are growing, intracellular chloride levels are high due to the expression patterns of transporter proteins. NKCC1, a transporter protein that moves chloride into the cell is highly expressed during early development while the expression of KCC2, a transporter that moves chloride out of the cell, is low and increases with age. The consequence of high intracellular chloride is that activation of chloride-permeable receptors, such as the GABA-type-A receptor, is likely to generate membrane depolarisations that can trigger local intracellular signalling events. Intriguingly, abnormally high chloride levels have also been observed in a number of pathological conditions in the mature brain. In epilepsy for instance, chronic reduction in KCC2 transporter activity is thought to result in chloride accumulation. The first objective of this research proposal is to examine the significance of elevated intracellular chloride for the normal growth of dendrites. This will be investigated by prematurely reducing intracellular chloride via molecular (expression constructs and RNAi knockdown) and pharmacological approaches. The impact upon dendritic growth will be quantified by performing time-lapse confocal imaging of growth events and local calcium-mediated signaling events. The second objective will again use molecular and pharmacological techniques in order to address whether abnormally elevated chloride levels in mature neurons are necessary and/or sufficient to trigger morphological changes that are characteristic of epileptic tissue, such as a reduction in the density of dendritic ?spines?. Finally, the work will investigate how KCC2 protein is regulated in neurons. Novel molecular tools will be used to visualise the subcellular localisation of KCC2 and to monitor its insertion/removal from the plasma membrane under conditions of altered neural activity. It is anticipated that the work will provide new and important information regarding chloride regulation in development and disease, and results will be disseminated through publication in journals and presentations at scientific and medical conferences.

Publications


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Ellender TJ (2014) Excitatory effects of parvalbumin-expressing interneurons maintain hippocampal epileptiform activity via synchronous afterdischarges. in The Journal of neuroscience : the official journal of the Society for Neuroscience
Ilie A (2012) Adenosine release during seizures attenuates GABAA receptor-mediated depolarization. in The Journal of neuroscience : the official journal of the Society for Neuroscience
Raimondo JV (2012) Genetically encoded proton sensors reveal activity-dependent pH changes in neurons. in Frontiers in molecular neuroscience
Raimondo JV (2016) Tight Coupling of Astrocyte pH Dynamics to Epileptiform Activity Revealed by Genetically Encoded pH Sensors. in The Journal of neuroscience : the official journal of the Society for Neuroscience
Raimondo JV (2015) Ion dynamics during seizures. in Frontiers in cellular neuroscience
Raimondo JV (2012) Short-term ionic plasticity at GABAergic synapses. in Frontiers in synaptic neuroscience
Richards BA (2010) In vivo spike-timing-dependent plasticity in the optic tectum of Xenopus laevis. in Frontiers in synaptic neuroscience
 
Description EP Abraham Cephalosporin Trust Fund, Equipment grant (Live cell fluorescence imaging)
Amount £12,465 (GBP)
Organisation University of Oxford 
Department E P A Cephalosporin Fund
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 01/2007 
End 01/2008
 
Description Oxford University John Fell Fund Equipment grant (Development of synaptic circuits in the brain)
Amount £38,400 (GBP)
Organisation University of Oxford 
Department The John Fell Fund
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 01/2006 
End 01/2008
 
Description PhD project: Chloride Transporter Proteins in Neuronal Plasticity
Amount £36,000 (GBP)
Organisation The Wellcome Trust Ltd 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2007 
End 09/2010
 
Description PhD project: Regulation of fast postsynaptic inhibition in epilepsy
Amount £36,000 (GBP)
Organisation The Wellcome Trust Ltd 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2009 
End 09/2012
 
Title DNA plasmids 
Description DNA plasmids for manipulating chloride transporter proteins, enabling overexpression, knockdown and live cell monitoring of protein trafficking. 
Type Of Material Biological samples 
Year Produced 2009 
Provided To Others? Yes  
Impact The materials have been used as part of a collaboration, which has resulted in a publication in the Journal of Physiology (PMID: 21911617). 
URL http://europepmc.org/abstract/MED/21911617
 
Title Data analysis scripts. 
Description A software suite which we have developed for the analysis of calcium events in cells and morphological properties. 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact Use by other research groups in related fields. 
 
Description Molecular interactions during synaptic development 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution Conducted pilot experiments.
Collaborator Contribution Supported pilot project to examine novel protein interaction during synaptic development.
Impact Presentation of data at one international conference.
Start Year 2006
 
Description Molecular regulation of a chloride transporter protein 
Organisation Medical Research Council (MRC)
Department MRC Anatomical Neuropharmacology Unit
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Significant intellectual input and sharing of resources.
Collaborator Contribution Intellectual input and sharing of resources.
Impact Presentation of data at two international conferences. First manuscript submitted to Neuron.
Start Year 2006
 
Description In vivo experimentation 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Contributed case study for the opening of the Biomedical Sciences Building, Oxford University.

Website was cited by several major news groups.
Year(s) Of Engagement Activity 2008,2009
 
Description School science days 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Presenting scientific data to an audience of 16-18 year old school students. One aim of this scheme is to encourage students from disadvantaged backgrounds to study science at University.

Positive feedback from students and teachers involved.
Year(s) Of Engagement Activity 2006,2007
 
Description Talk at Local Primary School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact A science-based talk given to 60 Primary School children, aged 4-5 years. This included a presentation followed by a series of 'hands-on' experiments that the children performed.

Very positive feedback from the school and request to run similar sessions in future years.
Year(s) Of Engagement Activity 2009