Glia play many critical yet poorly understood roles in normal brain physiology, as well as in a wide range of neuropathologies. Far from being neutral glue of the nervous system, glia are heterogeneous, active players in axonal pathfinding, synapse formation and signal transmission efficacy. Serious conditions arise when these functions become defective. Astrocytes, for example, have been implicated in neurodevelopmental diseases such as Rett syndrome, fragile X mental retardation and in epileptogenesis. Microglia are involved in the initiation and progression of neurological disorders including Alzheimer's disease, Parkinson's disease, multiple sclerosis, as well as obsessive compulsive disorder and schizophrenia. Progress in understanding the causes of these diseases would be much faster if better tools were available for monitoring and manipulating glial cell activity. Intracellular calcium transients represent such convenient, measurable signals that strongly correlate with cellular activity. Here we propose to implement the latest generation of genetically encoded calcium indicators (GECI) into mouse reporters, using specific gene knock-in techniques. We will also explore the potential of using an alternative site-specific recombination system in the mouse, based on Dre, a tyrosine recombinase similar to Cre but possessing a distinct DNA specificity. Availability of this alternative genetic labeling technology will enable experiments investigating the relationship between two distinct cell populations, such as specific neurons and glia, without any confounding issues of dye loading efficiency, viral injections, etc., because all the required tools can be brought together by breeding. This technology will make it possible to stimulate, for example, a specific population of neurons with channel rhodopsin and monitor surrounding glia with calcium indicators, and vice versa. We will evaluate these experiments in acute brain slices, with ultrafast two-photon imaging technology in a raster scanning mode or using a powerful new approach termed targeted path scanning for rapid investigation of neural networks. The proposed tools will allow us to study reactive astrocytes in mouse models of temporal lobe epilepsy and to investigate Hoxb8 mutant microglia, which have been implicated in obsessive compulsive-like behavior in mice. Temporal lobe epilepsy is the most common type of epilepsy in humans. Only recently, astrocytes have been shown to play an important role in the disease progress. Dramatic changes occur in the expression of neurotransmitter receptors, voltage gated ion channels, inflammatory cytokines, and a variety of other proteins in response to seizure activity. Astrocytic networks will be studie in normal and epileptic brain slices, establishing an experimental framework for drug development and testing. Microglia arise from several developmental origins, including one in the Hoxb8 expression domain. Microglia are sensors of neuronal activity. We will be able to determine if Hoxb8 mutant microglia respond to neural stimuli correctly. Taken together, the proposed work will advance the set of tools available to address the underlying causes of glial and other disorders.
Glia strongly affect nervous system performance and health, contributing to a wide range of illnesses including Rett syndrome, Alzheimer's disease, Parkinson's disease, epilepsy and OCD. Genetic tools generated in this project will allow investigators studying any of these and other disorders to manipulate and monitor glial activity. This will provide a framework for the development and testing of new therapeutic approaches to disorders involving astrocytes and microglia.
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