Developing next generation multiphoton systems to reveal cortico-thalamic interactions underlying short-term memory in behaving mice - One of the goals of systems neuroscience is to understand how sensory information is transformed into goal-
directed behavior via diverse brain regions and circuits. To achieve this aim, it is critical to elucidate computations
performed within specific layers of the cortex by specific cell classes and the communication dynamics between
multiple brain regions. Two-photon microscopy has been used successfully to perform functional brain imaging
at the single-cell level mice, but its penetration is limited by tissue scattering to the top layers of the cortex. I have
developed a 3-photon microscope to overcome this challenge. Today, the main drawback of 3-photon
microscope is its relatively modest speed, limiting its use for multi-site imaging. Optimizing instrument design
and imaging protocol to overcome this limitation is required for broad end-user acceptance. In this proposal, I
will construct and optimize a combined 2-photon and 3-photon microscope for multi-site, superficial and deep
brain imaging at single-cell resolution. Specifically, I have first developed a custom-made 3-photon microscope
with optimized laser and microscope parameters (Aim 1a). Optimizing these parameters can improve imaging
speed and imaging depth while lowering the average laser power to avoid damage in the live mouse brain. The
microscope performance improvement has been validated by performing functional imaging in the primary visual
cortex of GCaMP6 mice to characterize visual responses of each cortical layer and subplate. In addition, I will
characterize the effective attenuation lengths (EAL) of higher visual areas in awake mice with label-free imaging
and laser-ablation methods. Then, I will demonstrate the microscope’s performance by examining cell-specific
differences within a layer 6 (L6) of V1. Since neuronal responses to visual stimuli are modulated by the cortical
state such as arousal, or reward expectation, I will image adjacent sets of neurons with distinct projections to the
lateral geniculate nucleus (LGN) and lateral posterior (LP) regions (e.g., cortico-cortical [CC] and cortico-thalamic
[CT] neurons in L6) in primary and higher visual areas to reveal circuit-based response types within a single
cortical layer using retrobead-based tracing methods (Aim 1b). Next, I have developed custom-made 2-photon
wide-field microscope to perform neuronal recordings and manipulations in the primary visual cortex and higher
visual areas (Aim 2a). I have improved imaging speed and field of view by implementing multifocal multiphoton
microscopy (MMM). Multiple foci two-photon excitation efficiency will be optimized by coupling a diffractive
element (DOE) with customized intermediate optics. High sensitivity single-photon counting detection will be
achieved using a novel avalanche photodiode array detector. To demonstrate microscope performance and
which brain regions are necessary for a well-established goal-directed behavioral paradigm, I will perform SLM-
based two-photon optogenetics while imaging expert animals (Aim 2b). In addition to imaging and stimulating
neuronal activity across superficial depths at single regions and at multiple regions, it is necessary to image and
optogenetically manipulate neuronal activity at multiple depths, at targeted locations, and for identified neurons,
in order to determine the causality of neuronal subpopulations in behavior. Here, I will design and implement
two- and three-photon MMM systems to extend the depth performance of MMM for multi-site neuronal recording
across multiple regions and multiple layers and integrate this system with the 2-photon optogenetics system
implemented in Aim 2a (Aim 3a). I will use this technology for modulating specific components of the cortico-
cortical and cortico-thalamo-cortical projections of V1-V2-PPC-MC circuit (Aim 3b).
