PROJECT SUMMARY Faced with a continuous stream of data, the hippocampus must strike a balance between forming new, discrete memories (pattern separation) and generalizing information across similar experiences (pattern completion). Computational and experimental studies over the past 50 years have begun to delineate the hippocampal circuits involved in these processes, yet a fundamental question is still unanswered: How do the different subregions within the hippocampus integrate time-varying information during experience to form episodic memories? Whether exploring a novel environment, scanning a visual scene, or recognizing a familiar tune, the sensations used to construct and recall memories are not experienced simultaneously but rather as a dynamic stream of information or temporal pattern. Our proposal seeks to understand how the hippocampus, and in particular the dentate gyrus (DG) and CA1 subfields, integrate time-varying information during learning to perform temporal pattern separation and completion. Understanding these mechanisms will provide crucial insights into why our ability to learn and remember declines with age and how hippocampal pathology leads to significant memory impairment in disorders such as Alzheimer’s disease. In order to understand how different subregions process temporal information and to fully capture learning in real time, one must be able to measure activity within two or more networks simultaneously with single cell resolution. In principle, this can be accomplished using two-photon calcium imaging of distinct neuronal populations labeled with green and red fluorescence indicators respectively. However, many circuits in the mammalian brain, including the hippocampus, are laminar where networks are separated by hundreds of microns or more in depth. Microscope objectives are optimized to image only a single focal plane, and optical aberrations severely degrade the laser excitation spot when large axial displacements of ~100 µm or greater from the objective’s ideal focal plane are introduced. To overcome this challenge, we propose to construct a novel two photon imaging system that utilizes both remote focusing and adaptive optics to focus two laser sources with distinct excitation wavelengths at different arbitrary depths. This novel two-photon microscope will allow us to record neuronal activity within both the CA1 and deeper DG subfields simultaneously for the first time, investigating how these subregions process temporal information and modify network activity during learning