Two-photon Calcium Imaging in Mice Navigating a Virtual Reality Environment

Summary

Here we describe the experimental procedures involved in two-photon imaging of mouse cortex during behavior in a virtual reality environment.

Abstract

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically identified cells during behavior^1-6. Here we describe methods to perform two-photon imaging in mouse cortex while the animal navigates a virtual reality environment. We focus on the aspects of the experimental procedures that are key to imaging in a behaving animal in a brightly lit virtual environment. The key problems that arise in this experimental setup that we here address are: minimizing brain motion related artifacts, minimizing light leak from the virtual reality projection system, and minimizing laser induced tissue damage. We also provide sample software to control the virtual reality environment and to do pupil tracking. With these procedures and resources it should be possible to convert a conventional two-photon microscope for use in behaving mice.

Introduction

Two-photon imaging of calcium indicators (genetically encoded like GCaMP5⁷ or R-GECO⁸, or synthetic dyes like OGB or Fluo4) has emerged as a powerful method of measuring neuronal activity in behaving mice^1-6. It enables the simultaneous measurement of the activity of hundreds of cells at near-single action potential resolution, up to approximately 800 µm below the brain surface^9,10. Moreover, using genetically encoded calcium indicators (GECIs) neuronal activity can be measured chronically^5,11,12, and in genetically defined cell types¹³. Together, these methods provide a degree of temporal and spat....

Protocol

All animal procedures were approved and carried out in accordance with guidelines of the Veterinary Department of the Canton Basel-Stadt.

1. Hardware and Software Setup

1. Two-photon scanning microscope setup:
   1. Use a pulsed infrared laser as an illumination source (pulse width <120 fsec).
   2. Use a scan head composed of an 8 or 12 kHz resonant scanner and a standard galvanometer. Note: This enables frame rates of 40 or 60 Hz at 750 x 400 pixels. A high frame rate is critical for minimizing brain motion induced image distortion. Moreover, fast resonant scanning results in a higher signal yield ....

Results

The image quality in two-photon calcium imaging of cell populations labeled with a GECI largely depends on the quality of the cranial window implant. Two weeks following virus injection the cranial window should be inspected for clarity. There should be no granulation tissue or bone regrowth visible (Figure 1A). Moreover, the pattern of superficial blood vessels should remain unchanged and boundaries of the vasculature should be sharply defined. At the same time, GECI expression can also be checked. Boli.......

Discussion

The key to the success of behavioral two-photon imaging is the stability of the preparation in two ways:

1. Over the course of days post window implantation, inflammatory responses of the tissue can lead to enhancement of the formation of granulation tissue and cartilage that will hinder or even prevent imaging.
2. During the experiment the brain has to be stable enough to prevent motion artifacts from corrupting neural activity related fluorescence signal.

To keep the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cover slips (diameter = 3-5 mm)	Menzel		window implant
InSight DeepSee laser	Spectra-Physics		microscope
12 kHz Resonance scanner	Cambridge Technology	G1-003-30026	microscope
Galvometer	Cambridge Technology	G6215H	microscope
Digitizer	National Instruments	NI 5772	microscope
FPGA	National Instruments	PXIe 7965R	microscope
Acquisition card	National Instruments	PCIe 6363	microscope
Emission filter 525/50	Semrock	FF03-525/50-25	microscope
Piezo-electric z-drive	Physikinstrumente	P-726.1CD	microscope
Controller for Piezo-electric drive	Physikinstrumente	E665 LVPZT	microscope
Objective 16X, 0.8NA	Nikon	CFI75	microscope
Current amplifier	Femto	DHPCA-100	microscope
Photomultiplier tube	Hamamatsu		microscope
USB Camera without IR filter	ImagingSource	DMK22BUC03	pupil tracking
Objective 50 mm	ImagingSource	M5018-MP	pupil tracking
Macro adapter rings	ImagingSource	LAexSet	pupil tracking
Optical computer mouse	Logitech	G500	motion tracking
Styrofoam ball 20 cm	e.g. idee-shop.de	08797.00.15	virtual environment
LED projector	Samsung	SP-F10M	virtual environment
Acquisition card	National Instruments	NI 6009	virtual environment
Panda3D game engine		www.panda3d.org	virtual environment
Numpy library for Python		www.scipy.org	virtual environment
Scipy library for Python		www.scipy.org	virtual environment
NI-DAQmx driver	National Instruments	www.ni.com	virtual environment
Ultrasound gel	Dahlhausen	5701.0342.10	imaging