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.
