Name: Two-photon Calcium Imaging in Mice Navigating a Virtual Reality Environment (Video) | JoVE Journal
Uploaded: 2014-02-20T22:00:00.000+00:00
Duration: 8 min 12 s
Description: Learn two-photon calcium imaging in mice navigating virtual reality using an air-supported treadmill and synchronized LED projector. This method reduces movement artifacts and enables stable neuroactivity recordings during behavioral studies.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by the Friedrich Miescher Institute for Biomedical Research, the Max Planck Society, and the Human Frontiers Science Program.

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
<ol>
	<li>Two-photon scanning microscope setup:
	<ol>
		<li>Use a pulsed infrared laser as an illumination source (pulse width &#60;120 fsec).</li>
		<li>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 and reduces photobleaching16 and phototoxicity.</li>
		<li>Use a Piezo-electric high speed z-stepper to sequentially acquire time interleaved images at multiple depths. Note: This is optional and used to increase data yield at the expense of frame rate.</li>
		<li>Use a mechanical blanker to reduce laser exposure to the tissue. Adjust the width of the blanker depending on the amplitude of the resonant scanner. Note: Due to the sinusoidal nature of the scan path of a resonant scanner, the laser beam moves comparably slow at the scan turnaround points. To prevent damage of the tissue at these turnaround points where laser exposure is greatest, a mechanical blanker is introduced into the focal plane between the scan and tube lenses that stops the laser beam.&#160;</li>
		<li>For data acquisition use a high speed digitizer (800 MHz) in combination with a field-programmable gate array (FPGA). Note: The FPGA high-pass filters and bins the PMT signal into pixels. The combination of the high-pass filter on the FPGA and the effective low-pass filter on the PMT amplifier result in a band-pass filter centered roughly around the repetition rate of the laser (80 MHz). Sample FPGA code is provided as a supplement.</li>
	</ol>
	</li>
	<li>Air-supported treadmill setup based on the design of Dombeck and colleagues2
	<ol>
		<li>Connect the ball holder to an air supply line using an air supply tube of at least 10 mm inner diameter. Maximize the cross section of the ball holder air supply inlet and place ample amount of tubing between the main air supply and the ball holder. Note: Increased cross section and increased length of the air supply tubing reduces noise generation.</li>
		<li>Place a polystyrene foam ball (20 cm diameter) in a custom-designed and 3D-printed ball holder.</li>
		<li>If the behavioral paradigm only requires forward (and backward) locomotion, restrict movement of the ball to the horizontal (pitch) axis by inserting a pin (e.g. a 19 G hypodermic needle) on the side of the ball. Note: Typically animals adapt to the setup much quicker in this reduced rotation paradigm.</li>
		<li>Track movement of the ball using one or two optical computer mice to detect motion around two or all three axes, respectively. Note: The computer mice should ideally operate with an infrared diode for least interference with the imaging.</li>
	</ol>
	</li>
	<li>Virtual reality environment setup
	<ol>
		<li>Prepare the virtual reality environment. Note: A sample environment based on Panda3D, a freely available game engine, is provided as a supplement. The objects of the virtual environment are generated using Wings3D, an open source 3D modeler. The sample program reads position information on a data acquisition card and translates this into movement in the virtual environment. The code also performs the nonlinear transformation necessary for the environment to be projected onto a toroidal screen. The detailed construction of the toroidal screen has been described elsewhere14,17.&#160;</li>
		<li>Use LED projectors or LED backlit computer screens for displaying the virtual environment. Integrate a gating circuit for fast flickering of the LEDs during imaging. Note: An inherent problem in two-photon imaging during visual stimulation is light leak originating from the visual stimulation system, which can be reduced by shielding the objective. However, light leak from the visual stimulation system can be fully abolished by synchronizing the light output of the projector to the turnaround points of the resonant scanner (when data are not acquired), as is done with LED arenas used for visual stimulation during imaging in Drosophila18. This results in an effective high speed alternation of visual stimulation and data acquisition (see Figure 2). The flickering frequency (24 kHz) is orders of magnitude above the flicker fusion threshold, above which flickering is no longer perceived by the animal. The electronic circuit diagram of this modification to a commercial LED projector is shown in Figure 3.</li>
	</ol>
	</li>
	<li>Pupil tracking
	<ol>
		<li>Use a CMOS based video camera (30 fps) for pupil position tracking. Make sure that the camera does not contain an infrared blocking filter. Note: The pupil is visible at high contrast during recordings due to stray light from the two-photon excitation laser exiting through the eye. Pupil position and diameter is extracted in real-time using custom-programmed software based on the design of Sakatani and Isa19.&#160;</li>
	</ol>
	</li>
</ol>
2. Injection of Genetic Calcium Indicator and Window Implant
The injection of a calcium indicator20 and the implantation of the cranial window21 are performed as previously described with the following additions:
<ol>
	<li>Inject the virus into the region of interest using a glass injection pipette backfilled with virus solution and connected to a pressure injection system. Adjust the pressure depending on the pipette inner-diameter, typically approx. 70-140 mbar, to inject approximately 100 nl of virus solution over the course of 1-2 min. Low volume injection pulses delivered at low frequency (0.05 sec pulses at 2 Hz) should be used in order to prevent overflow of the virus along the pipette during injection. Note: In order to monitor the injection volume, the pipette can be marked at small, evenly spaced lengths (e.g. 0.5 mm) under a stereo microscope. Displacement of the meniscus between the markings can then be used to estimate the injected volume.</li>
	<li>Several weeks post injection this should result in a bolus of densely labeled cells with a diameter of approximately 500 &#181;m. &#160;Efficacy of GECI expression in the region of interest depends on the choice of GECI and promoter combination, as well as the infectious titer and serotype of the recombinant AAV used. Note: As a representative example, injection of AAV2/1-EF1&#945;-GCaMP5 with a viral titer of 8 x 1012 GC/ml into primary visual cortex of a mouse results in satisfactory expression at approximately two weeks post injection (Figure 1).</li>
	<li>The head bar should not obstruct the visual field of the mouse, but should be rigid enough to allow for stable imaging during behavior. This typically requires two points of attachment on either side of the head bar (see Figure 1). To firmly attach the head bar to the skull, make sure to maximize and dry the area of exposed skull where the head bar will be attached. Additionally, using a scalpel or a syringe, scratch the skull to create superficial grooves thereby increasing the surface area for gluing. Cover the bone with cyanoacrylate tissue adhesive before attaching the head bar with dental cement.</li>
</ol>
3. Experimental Procedure
<ol>
	<li>Two to three weeks following virus injection, check the cranial window implant for clarity and expression under epifluorescence illumination. Clearly visible and sharply defined boundaries of superficial blood vessels are a good indication that the window implant is suitable for imaging (see Figure 1).</li>
	<li>Measure and adjust laser power to a value below 50 mW at the sample.</li>
	<li>Turn on the airflow to the spherical treadmill.</li>
	<li>For head fixation under the microscope, briefly anesthetize the animal with isoflurane to reduce stress. Place the animal into an isoflurane anesthesia container and wait until the animal stops moving (approximately 10 sec). Remove the animal and immediately head fix it under the microscope.</li>
	<li>Position the visual display system as close as possible to the animal to still allow for access to the animal. The less visual contact the animal has with the experimenter, the less stressed the animal will be during the initial phase of the experiment.</li>
	<li>Make sure the cranial window implant is free of dust and dirt. Clean with water if necessary.</li>
	<li>Apply clear ultrasound gel as immersion medium between the cranial window and water-immersion objective. This solves problems of slow image degradation due to water evaporation or leakage and alleviates the need to mount a cone on the head bar to hold water. Note: Centrifuge the gel before usage to remove all air bubbles, and take care not to create any air bubbles during application of the gel. Air bubbles can severely degrade image quality.</li>
	<li>Wrap a piece of black tape around the objective and head bar for light shielding.</li>
	<li>Move the virtual environment arena to its final position and adjust the video camera for pupil tracking.</li>
	<li>Set the laser blanker to match the scan amplitude used during the experiment.</li>
	<li>Start recording data.</li>
</ol>

<ol><li>Helmchen, F., Fee, M. S., Tank, D. W., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Miniature+Head-Mounted+Two-Photon+MicroscopeHigh-Resolution+Brain+Imaging+in+Freely+Moving+Animals%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">A Miniature Head-Mounted Two-Photon MicroscopeHigh-Resolution Brain Imaging in Freely Moving Animals.</a> Neuron. 31 (6), 903-912 (2001).</li>
<li>Dombeck, D. A., Khabbaz, A. N., Collman, F., Adelman, T. L., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+large-scale+neural+activity+with+cellular+resolution+in+awake%2C+mobile+mice%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Imaging large-scale neural activity with cellular resolution in awake, mobile mice.</a> Neuron. 56, 43-57 (2007).</li>
<li>Dombeck, D. A., Harvey, C. D., Tian, L., Looger, L. L., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+imaging+of+hippocampal+place+cells+at+cellular+resolution+during+virtual+navigation%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">Functional imaging of hippocampal place cells at cellular resolution during virtual navigation.</a> Nat. Neurosci. 13 (11), 1433-1440 (2010).</li>
<li>Harvey, C. D., Coen, P., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Choice-specific+sequences+in+parietal+cortex+during+a+virtual-navigation+decision+task%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Choice-specific sequences in parietal cortex during a virtual-navigation decision task.</a> Nature. 484 (7395), 62-68 (2012).</li>
<li>Huber, D., Gutnisky, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiple+dynamic+representations+in+the+motor+cortex+during+sensorimotor+learning%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Multiple dynamic representations in the motor cortex during sensorimotor learning.</a> Nature. 484 (7395), 473-478 (2012).</li>
<li>Keller, G. B., Bonhoeffer, T., Hübener, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensorimotor+mismatch+signals+in+primary+visual+cortex+of+the+behaving+mouse%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Sensorimotor mismatch signals in primary visual cortex of the behaving mouse.</a> Neuron. 74 (5), 809-815 (2012).</li>
<li>Akerboom, J., Chen, T. -W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+a+GCaMP+Calcium+Indicator+for+Neural+Activity+Imaging%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging.</a> J. Neurosci. 32 (40), 13819-13840 (2012).</li>
<li>Zhao, Y., Araki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+expanded+palette+of+genetically+encoded+Ca2%2B+indicators%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">An expanded palette of genetically encoded Ca2+ indicators.</a> Science. 333 (6051), 1888-1891 (2011).</li>
<li>Mittmann, W., Wallace, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-photon+calcium+imaging+of+evoked+activity+from+L5+somatosensory+neurons+in+vivo%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo.</a> Nat. Neurosci. 14 (8), 1089-1893 (2011).</li>
<li>Katona, G., Szalay, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fast+two-photon+in+vivo+imaging+with+three-dimensional+random-access+scanning+in+large+tissue+volumes%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes.</a> Nat. Methods. 9 (2), 201-208 (2012).</li>
<li>Mank, M., Santos, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genetically+encoded+calcium+indicator+for+chronic+in+vivo+two-photon+imaging%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">A genetically encoded calcium indicator for chronic in vivo two-photon imaging.</a> Nat. Methods. 5 (9), 805-811 (2008).</li>
<li>Margolis, D. J., Lütcke, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reorganization+of+cortical+population+activity+imaged+throughout+long-term+sensory+deprivation%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">Reorganization of cortical population activity imaged throughout long-term sensory deprivation.</a> Nat. Neurosci. 15 (11), 1539-1546 (2012).</li>
<li>Zariwala, H. A., Borghuis, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Cre-dependent+GCaMP3+reporter+mouse+for+neuronal+imaging+in+vivo%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo.</a> J. Neurosci. 32 (9), 3131-3141 (2012).</li>
<li>Harvey, C. D., Collman, F., Dombeck, D. A., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+dynamics+of+hippocampal+place+cells+during+virtual+navigation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Intracellular dynamics of hippocampal place cells during virtual navigation.</a> Nature. 461 (7266), 941-946 (2009).</li>
<li>Hölscher, C., Schnee, A., Dahmen, H., Setia, L., Mallot, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rats+are+able+to+navigate+in+virtual+environments%5BTitle%5D)+AND+%22J.+Exp.+Biol%22%5BJournal%5D)">Rats are able to navigate in virtual environments.</a> J. Exp. Biol. 208, 561-5519 (2005).</li>
<li>Borlinghaus, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MRT+letter%3A+high+speed+scanning+has+the+potential+to+increase+fluorescence+yield+and+to+reduce+photobleaching%29%5BTitle%5D)+AND+%22Microsc.+Res.+Tech%22%5BJournal%5D)">MRT letter: high speed scanning has the potential to increase fluorescence yield and to reduce photobleaching).</a> Microsc. Res. Tech. 69 (9), 689-692 (2006).</li>
<li>Reiff, D. F., Plett, J., Mank, M., Griesbeck, O., Borst, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Virtual+Reality+for+Mice%2C+mousevr.blogspot.com%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">Virtual Reality for Mice, mousevr.blogspot.com.</a> Nat. Neurosci. 13, 973-978 (2010).</li>
<li>Sakatani, T., Isa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+analysis+of+spontaneous+saccade-like+rapid+eye+movements+in+C57BL%2F6+mice%5BTitle%5D)+AND+%22Neurosci.+Res%22%5BJournal%5D)">Quantitative analysis of spontaneous saccade-like rapid eye movements in C57BL/6 mice.</a> Neurosci. Res. 58, 324-331 (2007).</li>
<li>Golshani, P., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+2-photon+calcium+imaging+in+layer+2%2F3+of+mice%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">In vivo 2-photon calcium imaging in layer 2/3 of mice.</a> J. Vis. Exp. (13), (2008).</li>
<li>Holtmaat, A., Bonhoeffer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term%2C+high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nat. Protoc. 4 (8), 1128-1144 (2009).</li>
<li>Schmidt-Hieber, C., Häusser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+mechanisms+of+spatial+navigation+in+the+medial+entorhinal+cortex%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">Cellular mechanisms of spatial navigation in the medial entorhinal cortex.</a> Nat. Neurosci. 16 (3), 325-331 (2013).</li>
</ol>

The authors declare that they have no competing financial interests.

The key to the success of behavioral two-photon imaging is the stability of the preparation in two ways:
<ol>
	<li>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.</li>
	<li>During the experiment the brain has to be stable enough to prevent motion artifacts from corrupting neural activity related fluorescence signal.</li>
</ol>
To keep the...

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 mice1-6. It enables the simultaneous measurement of the activity of hundreds of cells at near-single action potential resolution, up to approximately 800 &#181;m below the brain surface9,10. Moreover, using genetically encoded calcium indicators (GECIs) neuronal activity can be measured chronically5,11,12, and in genetically defined cell types13. Together, these methods provide a degree of temporal and spatial resolution that open up a multitude of new possibilities in the study of neuronal computation in vivo.
Surgical intervention is necessary to expose and label the mouse brain for imaging. Cells are typically transfected using a recombinant adeno-associated virus (AAV) system for GECI delivery and a cranial window is implanted over the injection site to gain optical access to the brain. A head bar is then attached to the skull for head fixation under the two-photon microscope. The design and implementation of these steps is critical as most of the problems with awake imaging arise from instabilities in the preparation. Ideally the procedure described here should allow for chronic imaging of up to several months following the surgery.
To enable visually guided behavior during two-photon imaging, the head fixed mouse sits on an air supported spherical treadmill, which it can use to navigate a virtual reality environment. Locomotion of the mouse on the treadmill is coupled to movement in the virtual environment that is displayed on a toroidal screen surrounding the mouse14,15. Behavioral variables such as locomotion, visual stimulus, and pupil position can be recorded6.
We describe the procedures involved in chronic two-photon imaging in mice exploring a virtual reality environment. The key points addressed are: reduction of movement artifacts, reduction of light leak, maximization of the number of simultaneously recorded cells, and minimization of photo damage. We also provide details on setting up the air-supported treadmill, pupil tracking, and the virtual reality environment. The procedures described here can be used for imaging fluorescently labeled cell populations in head fixed mice&#160;in a potentially wide variety of behavioral paradigms.

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

two-photon calcium imaging in mice navigating a virtual reality environment

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

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 behavior1-6. 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.

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...

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

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically ...

The overall goal of the following experiment is to perform the key aspects of chronic two photon imaging in mice, exploring a virtual reality environment. This is achieved by modifying a two photon microscope for fast and stable imaging in behaving mice. Next, an air supported treadmill capable of locomotion tracking is set up.Then a virtual reality environment is prepared that uses an LED projector synchronized to the microscope in order to minimize light leak. As a result, good and stable image recordings are obtained due to the reduction of movement artifacts, the reduction of light leak, and the minimization of photo damage edge. Although this method can be used to measure neuroactivity during navigation in a virtual reality environment, it can also be easily adapted to a wide variety of different behavioral paradigms that require visual stimulation.All animal procedures were approved and carried out in accordance with the guidelines of the veterinary department of the Canton Basil sta. The two photon microscope used in these experiments consists of a scan head composed of an eight or 12 kilohertz resonant scanner and a standard galvanometer. This enables frame rates of 40 or 60 hertz at 750 times 400 pixels and reduces brain motion induced image distortion, photobleaching and phototoxicity.A piezoelectric high speed Z stepper is used to sequentially acquire time interleaved images at multiple depths. A mechanical blanker is used to reduce laser exposure to the tissue at the turnaround points of the scan path, the blanker has to be adjustable to set its width. Depending on the amplitude of the resonant scanner, connect an air supply tube with at least a 10 millimeter inner diameter to the ball holder of the treadmill and place an BLE amount of tubing between the main air supply and the ball holder to reduce noise.Place a polystyrene foam ball, 20 centimeters in diameter in a custom designed and 3D printed ball holder. If the behavioral paradigm only requires forward and backward locomotion, insert a pin such as a 19 gauge hypodermic needle on the side of the ball. To restrict movement of the ball in the horizontal axis, use one or two optical computer mice to track movement of the ball around two or all three axes respectively.To prepare the virtual reality environment, use LED projectors or LED backlit computer screens for the display. Integrate a gating circuit for fast flickering of the LEDs during imaging. The 24 kilohertz flickering frequency is orders of magnitude above the flicker fusion threshold, above which flickering is no longer perceived by the animal.Adjust the exact flickering frequency and duty cycle to synchronize the light output of the projector to the turnaround points of the resonant scanner for pupil tracking, use a CMOs based video camera. Make sure that the camera does not contain an infrared blocking filter. Approximately two to three weeks after injecting the genetic calcium indicator and implanting a cranial window, use epi fluorescence illumination to check the cranial window implant for clarity and expression.Clearly visible and sharply defined. Boundaries of superficial blood vessels are a good indication that the window implant is suitable for imaging one to two days later. To carry out the two photon experiment, measure and adjust the laser power to a value below 50 milliwatts at the sample.Then turn on the airflow to the spherical treadmill. Next head, fix the animal on the spherical treadmill. The first few times the mouse is head fixed.This process can be eased by briefly sedating the animal with iso fluorine. Once the animal has become accustomed to the procedure, mounting can normally be performed without sedation. Note that it is critical to minimize stress to the animal during this procedure.Then position the visual display system as close as possible to the animal to still allow for access to the animal. Make sure the cranial window implant is free of dirt and clean it with water if necessary, apply clear ultrasound gel as immersion medium between the cranial window and water immersion objective for light shielding. Wrap a piece of black tape around the objective and head bar.Move the virtual environment arena to its final position and adjust the video camera for pupil tracking. Finally, set the laser blanker to match the scan amplitude used during the experiment and start recording data when experiments are complete. Euthanize the animal according to your institution's guidelines.The image quality in two photon calcium imaging of cell populations labeled with a genetic calcium indicator largely depends on the quality of the cranial window implant as demonstrated here when checking the cranial window for clarity after virus injection, there should be no granulation tissue or bone regrowth visible. Moreover, the pattern of superficial blood vessels should remain unchanged and the boundaries of the vasculature should be sharply defined. At the same time, BOI of labeled cells should be clearly visible under an epi fluorescence microscope.Ideally, the preparation is stable enough such that distortions of the two photon images induced by locomotion of the mouse are not visible in the image after frame registration. Light leak from the visual stimulation system can be fully abolished by correctly timing the light output of the projector to the turnaround points of the resonance scanner. When data are not acquired, the flickering frequency of the light source should be twice the scan frequency.In this example, the blanker was removed to illustrate the effect of light leak shown in the orange boxes during visual stimulation. After watching this video, you should have a good understanding of how to perform twofold in imaging, behaving mice, navigating a virtual reality environment. In addition to setting up the imaging and virtual reality systems as described here, the key to success in these experiments is mastering the cranial window implant technique.A clean implant is a prerequisite for high quality images and stable recordings.

two-photon-calcium-imaging-mice-navigating-virtual-reality

Research

JoVE Journal

Behavior

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

Source: Maruoka, H., et. al. Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice. J. Vis. Exp. (2022)     This video shows a two-photon microscopy procedure for imaging neuronal activity and microglial dynamics in an awake mouse during visual stimulation, highlighting calcium transients and cellular interactions.

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in an Awake Mouse

Source: Lee, H. et. al., Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse. J. Vis. Exp. (2020)This video demonstrates in vivo calcium imaging in a mouse using a gradient refractive index, or GRIN lens, and a miniaturized microscope. The setup combines head fixation within a frictionless mobile cage system, stimulating neurons and triggering calcium ion influx. The GRIN lens captures calcium signaling in the brain, enabling...

Real-Time Microendoscopic Calcium Imaging in a Freely Moving Mouse

Source: Thapa, R., et. al., Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging. J. Vis. Exp. (2021)The video demonstrates real-time calcium imaging using a miniscope in a transduced mouse, correlating neuronal activity with behavior.

Real-Time Calcium Imaging of a Mouse Medial Prefrontal Cortex Using a Miniscope

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Calcium Imaging in Mouse Inferior Olive

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Here, we present a simplified open-source hardware and software setup for investigating mouse spatial learning using virtual reality (VR). This system displays a virtual linear track to a head-restrained mouse running on a wheel by utilizing a network of microcontrollers and a single-board computer running an easy-to-use Python graphical software package.

An Open-Source Virtual Reality System for the Measurement of Spatial Learning in Head-Restrained Mice

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

Calcium Imaging in Mouse Superior Colliculus

Learn two-photon calcium imaging in mice navigating virtual reality using an air-supported treadmill and synchronized LED projector. This method reduces movement artifacts and enables stable neuroactivity recordings during behavioral studies.

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

Explore More Videos