2015 — 2017 |
Campbell, Robert E. (co-PI) [⬀] Clack, Nathan G (co-PI) [⬀] Drobijev, Mikhail Hughes, Thomas E (co-PI) [⬀] |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Northern Lights Collaboration For Better 2-Photon Probes @ Montana State University - Bozeman
? DESCRIPTION (provided by applicant): The Northern Lights collaboration for better 2-photon probes will combine the expertise of four very different investigators and laboratories from Montana and Canada with complementary interests and skills to tackle a fundamental problem in imaging of the activity of the living brain. It is now well agreed that genetically encoded, fluorescent biosensors are uniquely suited to provide the specificity and versatility necessary to image the activity of defined cells in the brain. It is also clear that to image cells in thick slies or whole brains it will be necessary to turn to 2- photon (nonlinear) imaging, which penetrates deeper into living tissues and causes less damage. Furthermore, 2-photon signals provide additional molecular-level information that 1- photon signals lack. Many years have gone into developing better fluorescent proteins and biosensors for linear imaging. Unfortunately, directly optimizing probes for the 2-photon nonlinear properties has been beyond the reach of biosensor engineers because of the overwhelming complexity of the current state-of-the-art 2-photon characterization. This is a critical impediment for the BRAIN initiative, because robust 2-photon probes with high brightness and stability are needed. Here we describe a three year program to develop a new process and new instruments using available ultrafast lasers and advanced experimental designs that will empower biosensor engineers involved in the BRAIN project to evolve the next generation of multi photon probes.
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0.948 |
2021 |
Campbell, Robert E. Dana, Hod Michael Paquet, Marie-Eve |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Optimization of Campari For Large-Scale, Cellular-Resolution Activity Recording in Freely-Moving Mice @ Cleveland Clinic Lerner Com-Cwru
Project Summary The goal of this proposed research is to optimize a dual-use calcium ion sensor for recording single-cell activity from the entire dorsal cortex or hippocampus of freely-moving mice. It is widely accepted that optical imaging with genetically-encoded fluorescent calcium sensors is currently the only method to obtain measurements of genetically-identified neuronal populations with dense sampling. Over the past several years, the recording capabilities of two-photon microscopes have been improved to record from millimeter-scale tissue, and new miniaturized microscopes have been developed to record from moving mice. However, most behaviors arise from collective interactions between neurons from multiple brain areas, which cannot be simultaneously monitored with these systems. Therefore, there is a clear need to develop a new approach to directly monitor the synchronized activity of distributed neural circuits. CaMPARI is a unique calcium sensor that can detect activity in two calcium-dependent ways: 1) permanent color change (green to red) upon illumination with violet light, a process known as photoconversion, and 2) dynamic changes in fluorescence intensity. Our data show that CaMPARI allows recording of brain activity from freely-behaving mice, without using microscope objectives or implanted devices. Moreover, natural degradation of the red CaMPARI protein enables multiple longitudinal measurements. However, previous attempts to improve CaMPARI using in vitro assays reduced some of its in vivo properties, which resulted in low dynamic recording sensitivity and a low photoconversion rate that requires long illumination times to accumulate a sufficient amount of red protein. Therefore, the project goal is to optimize CaMPARI to allow sensitive recording of cellular-resolution, cortex-wide activity snapshots in freely-moving mice, followed by subsequent dynamic recording from the same mouse using two- and three-photon microscopy. To optimize CaMPARI?s performance, we will combine in vitro testing in purified protein, HEK cells, and neurons, and the most predictive assay: large-scale in vivo screening of ~30-fold more constructs than previous studies. Aim 1 will focus on enhancing CaMPARI?s photoconversion efficiency to facilitate large-scale recordings in freely-moving mice. Aim 2 will focus on improving CaMPARI?s dynamic recording properties and sensitivity. In Aim 3, we will combine beneficial mutations from Aims 1-2 to generate a new CaMPARI with optimized photoconversion and dynamic recording capabilities. Our proof-of-concept experiments will demonstrate multi- regional cortical mapping during a battery of behavioral and cognitive tests to detect cellular-resolution changes in cortex-wide activity patterns. This optimized CAMPARI is expected to facilitate new hypothesis-driven studies by providing volumetric, multi-regional brain activity data of genetically-targeted neurons during cognitive and behavioral testing of freely-moving mice, enabling studies that involve both head-fixation and free movement in the same mice, and to utilize complementary techniques like optogenetic stimulation and single-cell sequencing methods to enable studying the properties of active (red-labeled) cells during behavioral studies.
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0.907 |