Ion homeostasis in optic nerve astrocytes

The brain is a complex organ that possesses the characteristics of learning, memory and personality. This is achieved by complex interplay between the best known cell type in the brain, the neuron. However, there is a second kind of cell type in the brain, the glial cell. The most common type of glial cell is the astrocyte, named after its star-like appearance. Astrocytes out-number neurons and play important roles in regulating the environment within which neurons operate. Neuronal activity results in inbalances in the extracellular space of the brain, and astrocytes act to counter these unwanted effects. Recently, we have learned that astrocytes are also involved in the processing of information in the brain, a role previously though to be restricted to neurons. In order to perform this signal processing function, astrocytes must maintain tight control of their intracellular environment. This is particularly true for pH, any large variation of which will severely compromise the astrocytes ability to communicate with neighbouring cells. Previous studies have examined astrocyte ionic handling in reduced preparation, such as cultured cells removed from the brain and maintained in glass dishes. This grant will apply cell imaging and ion-sensitive electrode techniques to reveal how astrocytes within the normal brain manage to regulate their internal pH and ion concentrations while at the same time controlling the extracellular environment as required by neurons. Understanding how astrocytes achieve this balancing act will tell us a lot about how the brain functions.

The objective is to generate a description of pH and ion regulation in brain astrocytes under physiological conditions, and how this is related to extracellular ionic regulation. The post-natal day 0 rat optic nerve will be used as a model since it contains an effectively isolated population of GFAP (+) astrocytes of mature phenotype. These is also an unrivalled database of cellular and molecular information about this preparation. We will measure directly pHi and ionic concentration changes using fluorescent dyes and pHe and extracellular ionic changes using ion-sensitive microelectrodes. Selective groups of transporters will be activated and/or inhibited using specific ionic conditions and drugs. We will also determine how the regulation of intracellular pH, Na+ and Ca2+ are linked in these cells. For example, previous studies indicate that Na-dependent pH regulation is important in astrocytes, but the extent to which this couples regulation of pHi and [Na+]i is not clear. Various astrocyte functions are dependent upon [Na+]i, for example glutamate re-uptake. The link between pH and Na+ will be investigated by duel loading of ion-sensitive dyes into the cells, displacing pHi, and seeing how pHi and [Na+]i are correlated. A similar approach will be used to test the dependence of [Ca2+]i upon pHi, focusing upon the role of Na-Ca exchange. We will also see how activation and/or block of specific transporters affects extracellular homeostasis and how these transporters regulate activity-dependent ionic changes.

Impact Summary

Academic impact:

Enhancing the knowledge economy: The grant addressing an important black hole in our understanding of how the brain works and will represent a significant advance in neuroscience. Since astrocytes play so many roles in the CNS, a proper understanding of how they function to regulate ionic changes in the extracellular space will be of interest to a very wide academic audience.

Ionic regulation of the extracellular space is of significance not only for the physiology of the brain, but also for its pathophysiology and its pharmacology. For example, the actions of many drugs are pH dependent and a better understanding of how brain pH is regulated and how drugs may affect pH will be of interest to clinicians and drug companies. On a global scale, the relevance of the study to developing white matter injury is key, since cerebral palsy is a far bigger clinical problem in second and third world countries than here in the West.

Economic and societal Impact:

The project is a piece of basic science that will have significance for a number of wider societal interests. For example, by gaining a better understand of the physiology of developing white matter our understanding of some very important disease states will be improved. Developing white matter is the target of the disorder cerebral palsy and our understanding of this disability is poor. Cerebral palsy is the most common of all human birth disorders and involves either necrotic damage or disruption of developing white mater. The results of this project will therefore be of interest not only to the clinical and basic scientists involved in this area, but also to a large cohort of patients and their families.

Application and engagement: Findings will be disbursed through the normal academic channels which include publication of articles and giving lectures at national and international meetings.