Engineering a calcium reporter gene for magnetic resonance imaging of neural activity

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PROJECT SUMMARY To encode sensory experience and generate specific behaviors, the mammalian brain relies on functional coupling between networks of neurons distributed over multiple regions of the brain. Imaging techniques, which can observe molecular events at a brain-wide scale, are therefore needed to understand how these interconnected networks function as a whole. Fluorescent genetically encoded calcium indicators (GECIs) enable optical recording of calcium dynamics (a reliable measure of spiking activity) from well-defined cell types; but operate over small volumes (~ 1 mm3), limiting access to neural signals in localized neural circuits. Broader areas (~ 10 x 10 mm2) may be accessed with widefield optical methods; but light scattering limits imaging to sub- cortical depths in mice. On the other hand, techniques like functional MRI (fMRI) permit activation mapping in arbitrarily large brain volumes; but the ensuing signal gives only an indirect indication of neural activity based on localized changes in hemodynamic parameters. Histological techniques like c-fos immunostaining can also reveal whole-brain activation patterns; but only at a single time point per subject, thus failing to adequately capture dynamically evolving network properties. These collective limitations create a critical technological gap for large-scale monitoring of neural activity inside the intact mammalian brain. In this project, we will pilot steps to address the above challenge by developing genetic tools for imaging calcium with MRI; and we will examine their ability to faithfully monitor calcium dynamics in cultured neurons. The proposed sensors will be based on a manganese-binding protein class known as calprotectin, which we introduced in 2021 as the first protein-based MRI sensor for calcium. These first generation sensors are responsive to calcium concentrations exceeding 5 μM, which is much larger than the typical dynamic range (0.1-1 μM) experienced in neurons during activity. Starting with the parental sensor protein and building on a strong set of preliminary data, our goals here will be to optimize sensitivity for calcium sensing down to the neuronal range (Specific Aim 1), establish methods to genetically express the sensors in mammalian neurons, examine potential sensor-associated toxicity and take remedial steps if needed, and finally show that we can use these sensors to dynamically image calcium signals in neurons stimulated with agonists and optogenetics (Specific Aim 2). The primary outcome of this work will be the development of new neuroscience tools for directly observing calcium dynamics, while combining brain-wide access with all the advantages of genetic encoding, including cell-specific targeting, long-term expression, and access to a wide array of neuroscience methods ranging from axon-tracing and BBB-crossing viruses to transgenic animals. The work here will thus set the stage for previously impossible in vivo studies on identifying functionally coupled networks involved in everything from learning to executive control and decision making, as well as how these connections are modified in disease states.