Two-photon imaging of calcium signaling in neurons and astrocytes

In this thesis I describe my research on the modulation of calcium (Ca 2+) signaling in both neurons and astrocytes in the acute brain slice preparation. The high degree of light scattering in brain tissue presents significant challenges for studying the spatio-temporal dynamics of intracellular Ca2+. In the first two chapters I review the technical aspects of using two-photon microscopy and fluorescent Ca2+ indicators to image Ca2+ changes in subcellular neural compartments in the brain slice. In chapter three I introduce the two areas of investigation of this thesis work that may have important implications for our understanding of both physiological and pathophysiological brain functioning; (1) modulation of dendritic Ca2+ signaling in the prefrontal cortex (PFC) and (2) Ca2+ signaling at the astrocyte-vascular interface. Dopamine (DA) plays an essential role in the physiological functions mediated by the PFC although the cellular mechanisms underlying the actions of DA are largely unknown. I examined DA modulation of Ca2+ influx into the apical dendrites of layer V/VI pyramidal neurons of the PFC during action potential back-propagation. In chapter four I show that the apical dendrites of PFC pyramidal neurons express DA D1 receptors that inhibit N-type Ca2+ channels and reduce dendritic Ca2+ entry. Astrocytes communicate via intracellular Ca2+ signaling mechanisms. In chapter five I examine the role that Ca2+ signaling in astrocyte endfeet plays in the regulation of cerebral vascular dynamics. Two-photon Ca2+ uncaging was used to determine that increases in intracellular Ca2+ concentrations in astrocyte endfeet induce vascular constrictions. Constrictions induced in the cerebrovasculature by increased Ca2+ in astrocyte endfeet represents a previously unknown mechanism for the regulation of cerebral blood flow.