Chloride regulation in neuronal development and epilepsy

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.

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.