Linear and non-linear fluorescence imaging of neuronal activity

Optical imaging of neuronal activity offers new possibilities for understanding brain physiology. The predominant methods in neuroscience for measuring electrical activity require electrodes inserted into the tissue. Such methods, however, provide limited spatial information and are invasive. Optical methods are less physically invasive and offer the possibility for simultaneously imaging the activity of many neurons. In this thesis one- and two-photon fluorescence microscopy techniques were applied to several in vivo and in vitro mammalian preparations. Using one-photon absorption fluorescence microscopy and gradient index (GRIN) lens optics, cortical electrical activity in response to electric stimulation was resolved in three-dimensions at high-speed in the primary somatosensory cortex of the mouse in vivo using voltage-sensitive dyes. Imaging at depths up to 150 μm below the cortex surface, it was possible to resolve depth-dependent patterns of neuronal activity in response to cortical and thalamic electric stimulation. The patterns of activity were consistent with known cortical cellular architecture. In a qualitatively different set of experiments, one-photon fluorescence microscopy via voltage-sensitive dyes was successfully employed to image an in vitro preparation of the perfused rat brainstem during the process of respiratory rhythmogenesis. Imaging results yielded insights into the spatial organization of the central respiratory rhythm generation region in the ventrolateral medulla. A multifocal two-photon scanning microscope was constructed, and design and operation principles are described. Utilizing the novel device, anatomical and functional two-photon imaging via potentiometric dyes and calcium dyes is described, and the results of in vivo versus in vitro imaging are compared. Anatomical imaging results used either functional probe background fluorescence or green fluorescent protein (GFP) expression. Spectroscopic experiments measuring the two-photon absorption (TPA) cross sections, Σ2 of various fluorophores are described as well. Utilizing single-beam two-photon microscopy, action potentials were recorded optically from individual (∼1 μm) nerve terminals of the intact mouse neurohypophysis, in a single sweep. Single-trial recordings of action potentials exhibited signal-to-noise ratios ∼5 and fractional fluorescence changes of up to ∼10%. These results represent the first single-trial optical recording of action potentials from individual nerve terminals in an intact mammalian preparation using 180° detection, and may serve as an alternative to invasive electrode arrays for studying neuronal systems in vivo