Exploring Computational Networks Across an Entire Brain at Single Cell Resolution

1 2 1,2 1,2Hayli Spence , Anne Boulet , Mario Capecchi , Dimitri Tränkner1 2Department of Biology and Human Genetics

AbstractLarge scale monitoring of neuronal activity in mouse models currently depends on live-imaging using expensive and training-intensive multi-photon technology or activity-snapshots based on immediate early-gene expression with high background and poorly definable test intervals. To overcome these limitations, we generated a mouse line for the expression of the neuronal activity reporter CaMPARI. CaMPARI changes from green to red fluorescence with high intracellular calcium and photoconversion (PC) light. High intracellular calcium is naturally present in active neurons, PC light is provided by the experimenter when and where a snapshot of neuronal activity is required and can be delivered through single or multiple light guides deep into the brain.

Large Scale Recordings of Neuronal Activity. A) Live Imaging Techniques (e.g. 2-photon microscopy) rely on transient fluorescence. The time to scan a brain volume cannot exceed signal length, thereby limiting spatial resolution. In contrast, activity snapshots (e.g., using cFOS immunohistochemistry) sacrifice temporal for maximal spatial resolution. B) Spatio-temporal resolution of various techniques to measure neuronal activity. IEG: immediate early genes; fMRI: functional magnetic resonance imaging; EEG: electroencephalography; MEG: magnetoencephalography.

Activity Snapshots using CaMPARI. A-C: modified from Fosque et al., 2015. A) CaMPARI is derived from the stony corral protein EOS. By fusing calcium binding calmodulin and a 2+calmodulin/Ca binding peptide (M13) to either end, CaMPARI photoconversion (PC) becomes calcium dependent (B). C) CaMPARI PC in cultured neurons without (left) and with (right)

electrical stimulation. D) Short PC light exposures expand the temporal resolution of CaMPARI based activity snapshots to brief time intervals.

lslThe CaMPARI reporter mouse. A) We have generated a mouse line for cre-recombinase dependent expression of CaMPARI in select cell types (CaMPARI ). The engineered gene has been targeted to the Rosa26 locus, an euchromatine segment on mouse chromosome 6. Crossing this reporter line with, e.g., the driver line EMX1-cre, results in CaMPARI expressing excitatory

lslneocortical neurons. B) CaMPARI-expressing cells of an EMX1-cre/CaMPARI mouse in fixed tissue imaged with a confocal microscope (left) and living tissue imaged with a 2-photon microscope (right). Scale bars are 50 mm.

lsl 2CaMPARI photoconversion in the reporter mouse. A) An EMX1-cre/CaMPARI mouse received PC light (150 mW/mm ) for 30 min (pulsed at 4 sec on/1 sec off) transcranially supplied to the right hemisphere. Green (left) and red (right) fluorescence of CaMPARI-expressing cells without (B) and with (C) PC light. Fold backround (fb) red fluorescence versus fb green fluorescence of cells found in B (D) and C (E). Red CaMPARI fluorescence is detected only after PC light exposure, with stronger CaMPARI expression also leading to stronger red CaMPARI fluorescence; this bias can be removed by forming the ratio of red and green CaMPARI fluorescence. F) PC is little dependent on distance from the PC light source. G) Average PC of cells separated by 0.75 to 0.95 mm from the PC light source is not significantly different (n.s.) from that of cells separated by 0.95 to 1.15 mm; p>0.1 (Mann-Whitney rank sum). Cells without (green circles) and with ( ) detectable PC; : linear regression. Scale bars are 50 mm.red circles black lines

ConclusionOur newly developed CaMPARI-reporter mouse can be used to monitor neuronal activity during defined time periods using a ratiometric approach. Preliminary data suggest that experiments relying on robust CaMPARI expression and optimized activity integration are more likely to be informative.

THANK YOU!

Loren Looger and Eric Schreiter (Janelia Farm). The Capecchi lab, in particular Kerry Prettyman and Kay Higgins. Chris Rodesch (University of Utah Cell Imaging Facility). NIH grant R01MH093595

x EMX1-creA B

lslThe CaMPARI reporter mouse

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CaMPARI fluorescence measured as

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background fluorescencefold background (fb) =

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tissue processingand imaging

activity mappingexperiment

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fMRI

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IEG Immunohistochemistry

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Fosque et al., 2015

CaMPARI

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Interested in this mouse?

contact: Dr. Tränkner (dtrankner@genetics.utah.edu)

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