| Dynamic imaging of neuronal function is one of the main techniques capable of resolving the emergent function of brain circuits, but no single technique is sufficient to capture all levels of information necessary to understand these networks. Specifically, calcium indicators can measure action potentials from individual neurons but are unable to detect membrane potential dynamics of the cell body and dendrites. In contrast, voltage sensitive dye imaging can measure neuronal transmembrane voltage changes occurring during synaptic activation, but is often unable to detect action potential firing in individual neurons within heterogeneous networks. I combined calcium and voltage imaging to create a novel imaging technique that merges the strengths of both methods to more completely evaluate the overall behavior of a network, allowing me to resolve both synaptic potentials and action potential firing in hippocampus microcircuits.;I built a system that combined two-photon excitation microscopy and epifluorescence microscopy in order to image voltage and calcium signals from the same tissue using the same objective lens. Integration of these imaging modalities required optimization of compatible dyes, lenses, filters, and custom development of analytical software. Implementation of this integrated microscope allowed me to image both calcium and voltage signals from multi-stained hippocampal slices with minimal interference between the two modalities.;I next investigated the postnatal development of circuit function of the hippocampal dentate gyrus using the combined calcium and voltage imaging system. I found that the gating function of the dentate exhibited a prolonged maturation, which could contribute to altered behavioral characteristics evident in mouse pups relative to adult animals. I also found that granule cells exhibit precise, deterministic firing characteristics at all ages. In addition, I found strong asymmetry in cellular activation within the infra- and suprapyramidal blades of the dentate gyrus.;This new microscope is capable of imaging dynamic circuit activity in virtually any tissue in the brain or spinal cord, revealing both network and individual neural properties. This is critical in elucidating mechanisms underlying both healthy function and disease states in these various microcircuits. This knowledge can help guide efforts directed towards development of effective preventative methods as well as improve our current treatment strategies in various neurologic and psychiatric disorders. |
