Thy1-GCaMP6f (line GP5.1)

Thy1-GCaMP6f (line GP5.5)

www.janelia.org/genie

Context

GCaMP6 transgenic mice for neuronal activity imaging in vivo

Hod Dana, Caiying Guo, Brenda C. Shields, Amy Hu, Tsai-Wen Chen, Rex A. Kerr, Vivek Jayaraman, Loren L. Looger, Karel Svoboda, Douglas S. Kim

Genetically-Encoded Neuronal Indicator and Effector Project, Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

Expression in neocortex Layer 2/3 Layer 5

Thy1-GCaMP6s (line GP4.3)

Thy1-GCaMP6f (line GP5.5)

Thy1-GCaMP6f (line GP5.11)

Thy1-GCaMP3 (NIH line 10)

In vivo functional imaging in V1 Expression in hippocampus

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Methods

Thy1-GCaMP6s (line GP4.3)

Visual responses of 58 responsive cells (out of 498 tested)

ΔF/F

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•  Mice were perfused with paraformaldehyde at 6-8 weeks of age (no additional staining)

•  Coronal sections (50µm thickness) of the entire brain were cut

•  Images were taken with a wide-field microscope

•  Selected regions were imaged using a confocal microscope

•  Expression levels from individual cells were calculated using custom analysis scripts

Conclusions

Estimating expression per neuron

Semi-automated software was used to segment cell bodies and calculate mean signal per cell

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•  Thy1-GCaMP6-WPRE mice have interesting expression patterns in the cortex and hippocampus

•  Expression levels are sufficient for in vivo imaging

•  Additional mouse lines are being screened

•  Transgenic mice will be deposited at The Jackson Laboratory

•  GCaMP6 protein calcium indicators (6s,6m, and 6f) enable neuronal calcium imaging with unprecedented sensitivity (Chen et al., 2013)

•  GCaMP sensors can be highly expressed in neurons using viral gene transduction

•  Viral infection is invasive and variable

•  With AAV-based transduction expression levels increase over time and eventually affect cell health

•  Transgenic mice obviate the need for viral gene transduction and can show stable expression over time

Moving gratings in eight different orientations were presented to anesthetized mice. Responses were recorded in V1 using 2-photon microscopy (scanimage.org)

In vivo functional imaging in V1

Current limitations •  Expression levels in Thy-1 transgenic

mice are low compared to viral injection

•  L2/3 functional imaging experiments in Thy-1 transgenic mice required 50-100 mW average laser power (940 nm) compared with 10-25 mW using viral injection

•  The number of responsive cells was relatively low, likely due to low expression level and poor signal-to-noise ratio

Expression cassettes Thy1-GCaMP3 NIH line 10 (Xu et al., 2012)

Cre-dependent GCaMP3 Ai38 (Zariwala et al., 2012)

References Xu et al. Nonlinear dendritic integration of sensory and motor input during an active sensing task. Nature, 2012 Zariwala et al. A cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo. J. Neurosci., 2012 Chen et al. Ultrasensitive fluorescent proteins for imaging neural activity. Nature, 2013

Thy1-GCaMP6s

Thy1-GCaMP6f

CA1 CA3 Dentate Gyrus

Whole brain widefield imaging [selected sections, Thy1-GCaMP6f (line GP5.5)] Anterior Posterior

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873.28 NNN 39

Averaged image

GCaMP6f expression using viral injection

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Image was acquired with 13% of excitation power used for acquiring images from transgenic mouse brain sections

Thy1 promoter GCaMP6f WPRE pA

Thy1 promoter GCaMP6s WPRE pA

Thy1 promoter GCaMP3 pA

GCaMP6s V1 functional imaging (viral injection)

Motor cortex Somatosensory cortex Visual cortex Visual cortex (AAV infection)