| Historically, experimental methods to investigate astrocyte function in the intact brain have been limited by the fact that glia are electrically silent. This has led to a heavy reliance on calcium imaging techniques in the study of astrocyte function. In the past decade, the widespread use of this technique, particularly in vivo, has pushed the boundaries of what can be accomplished with fluorescent imaging. This series of studies is dedicated to taking that boundary a step further, utilizing the technology currently available to ask fundamental questions regarding the neurovascular unit (NVU), and concluding with some perspectives on the advances still needed to fully elucidate the functional role of astrocytes within the NVU. This dissertation is focused on astrocytic function in relation to the vascular compartment, astrocytic function in relation to the neuronal compartment, and finally, astrocytic function in relation to each other (Fig. A.1). In considering the vascular aspect of the NVU, we compare the in vivo temporal characteristics of the cerebral blood vessel dilation and the astrocytic calcium response to increased neuronal activity. We provide evidence of a significant temporal mismatch that challenges the popular idea of a calcium-dependent astrocytic role in triggering neurovascular coupling. In considering the metabolic aspect of the NVU, we report here the first application of 2-photon laser scanning microscopy (TPLSM) to functional imaging of intrinsically fluorescent beta-nicotinamide adenine dinucleotide (NADH) as a means to visualize single cell metabolism in vivo. We demonstrate, through this new imaging modality, that astrocytes respond readily and rapidly to evoked neuronal activity. We suggest that, contrary to what is indicated by their calcium activity, astrocytes are actually very sensitive to neuronal activity, and do respond on a relevant time scale, but do not necessarily act as mediators in translating neuronal activity to the vasculature. Given their prominent yet mysterious place within the NVU, there is a great need to identify suitable methodologies to describe the large repertoire of astrocytic calcium dynamics, ranging from subcellular compartments to network-level waves. Toward this end, we report on the application of optical flow image analysis to calcium signaling dynamics in spontaneously forming networks of astrocytes and neurons. By using information gained from a reduced preparation in conjunction with the whole animal approach, we hope to contribute to the set of tools that will be necessary to accurately assess the role(s) of astrocytes within the NVU. |
