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>

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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 inflammatory immune response to a minimum the cranial window implantation surgery should be carried out as quickly as possible and direct damage to the brain should be avoided at all costs. Of paramount importance is to ensure that the cranial window implant remains airtight for the entire duration of the experiment to prevent inflammation.
To minimize brain motion related imaging artifacts take care not to damage or remove the dura mater during the surgery. Remaining locomotion induced brain motion can be corrected for when motion is confined to the plane parallel to the imaging plane (the x/y plane). The maximum tolerable standard deviation of motion in x/y is roughly 5 &#181;m. For motion along the z-axis this value is roughly 1 &#181;m, such that the extent of the movement is smaller than the resolution limit of the microscope.
The low speed of the resonant scanner at the scan path turnaround points can lead to laser induced tissue damage. To prevent this, the laser can be blocked at the turnarounds using a blanker in the scan telescope. If this blanker is set correctly and average laser power under the objective is kept below 50 mW, imaging is possible for extended periods of time over multiple days without causing any visible laser damage. An alternative method to a mechanical blanking of the laser is to use a fast Pockels cell to reduce laser power at the turnarounds. This method however has the disadvantage that Pockels cells typically introduce undesirable beam distortions.
The aptitude of head fixed rodents to navigate virtual environments has made it possible to chronically image populations of cells in cortex of mice performing a wide range of behavioral tasks3,4,6,22. However, it is worthwhile to note that head-fixation inherently constrains behavior and locomotion on a spherical treadmill is in many ways not equivalent to unrestrained locomotion or normal behavior.
In the future, the strength of the virtual reality imaging approach described here will likely be the combination of calcium imaging with targeted optogenetic stimulation during behavior. With this it would be possible to optically manipulate presynaptic cells and image their post-synaptic targets while monitoring the resultant behavioral changes. In addition, as calcium indicators freely diffuse within the cell, it will also be possible to measure activity selectively in subcellular compartments during behavior. Due to movement induced brain motion, such recordings have been limited by the difficulty to obtain stable recordings.

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

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 of labeled cells should be clearly visible under an epifluorescence microscope (Figure 1B).
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 (Figure 2). The scan path of the resonant scanner is sinusoidal resulting in nonlinear image distortion. During the turnaround points data are not acquired due to image stretching in this part of the scan path. If the light source of the visual stimulation system, here the LEDs of the projector, is correctly timed to the turnaround points, light leak should only be apparent during this time (Figure 2). The flickering frequency of the light source has to be twice the scan frequency. Key to making this work are the fast rise and fall times of the LEDs and controlling electronics (Figure 3).
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50885/50885fig1highres.jpg" src="/files/ftp_upload/50885/50885fig1.jpg" /> 
Figure 1. Top view of a cranial window in a head fixed mouse two weeks after surgery. (A) The head bar is optimized in weight and shape to increase stability for behavior during imaging experiments, while reducing burden on the animal. The head bar is made from aluminum and has a thickness of 1 mm. Scale bar is 5 mm. (B) Epifluorescence image of GCaMP5 expression after four injections (AAV2/1-EF1&#945;-GCaMP5) of approximately 80 nl. The injection depth is approximately 400 &#181;m. Scale bar is 1 mm. (C) Schematics of the head bar. All measurements in mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50885/50885fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50885/50885fig2highres.jpg" src="/files/ftp_upload/50885/50885fig2.jpg" /> 
Figure 2. Improving signal to noise by reducing light leak. The LED light source in the projector of the virtual reality environment is synchronized to the resonant scanner to turn on only during the scan turnaround periods when data are not recorded (orange boxes). The laser blanker was removed from the microscope for this example to illustrate the effect of light leak resulting from visual stimulation. The image (average of 8 sec of data) was taken in mouse visual cortex transfected with AAV2/1-EF1&#945;-GCaMP5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50885/50885fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50885/50885fig3highres.jpg" src="/files/ftp_upload/50885/50885fig3.jpg" /> 
Figure 3. Circuit diagram of the electronics used to flicker a conventional LED projector. The gating circuit is driven by a TTL input to synchronize fast flickering with the resonant scanner. C1: capacitor 1nF; D1, D2: diodes 1N4148; INV1: Inverter HEF40106 (1 of 6 used); LED1 &#8211; LED of the beamer; R1: resistor 47 &#937;; R2: resistor 2.2 k&#937;; R3: resistor 100 &#937;; T1, T5, T6, T7: transistors IF9321; T2: transistor BC846; T3: transistor BC170; T4: transistor BC856. Note that this circuit has to be repeated for all three color channels (red, green, blue) of the projector. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50885/50885fig3highres.jpg" target="_blank">Click here to view larger image.</a>

Watch this Scientific Journal Video about Two-photon Calcium Imaging in Mice Navigating a Virtual Reality Environment at JoVE.com

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

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Behavior

30.9K Views.  Friedrich Miescher Institute for Biomedical Research. Here we describe the experimental procedures involved in two-photon imaging of mouse cortex during behavior in a virtual reality environment.

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

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

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 . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

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 .

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Biology

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Neuroscience

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

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows 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

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.

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

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.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

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.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

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

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

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

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.

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

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

Head-restrained behavioral experiments in mice allow neuroscientists to observe neural circuit activity with high-resolution electrophysiological and optical imaging tools while delivering precise sensory stimuli to a behaving animal. Recently, human and rodent studies using virtual reality (VR) environments have shown VR to be an important tool for uncovering the neural mechanisms underlying spatial learning in the hippocampus and cortex, due to the extremely precise control over parameters such as spatial and contextual cues. Setting up virtual environments for rodent spatial behaviors can, however, be costly and require an extensive background in engineering and computer programming. Here, we present a simple yet powerful system based upon inexpensive, modular, open-source hardware and software that enables researchers to study spatial learning in head-restrained mice using a VR environment. This system uses coupled microcontrollers to measure locomotion and deliver behavioral stimuli while head-restrained mice run on a wheel in concert with a virtual linear track environment rendered by a graphical software package running on a single-board computer. The emphasis on distributed processing allows researchers to design flexible, modular systems to elicit and measure complex spatial behaviors in mice in order to determine the connection between neural circuit activity and spatial learning in the mammalian brain.

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.

Head-restrained behavioral experiments in mice allow neuroscientists to observe neural circuit activity with high-resolution electrophysiological and ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Author Spotlight: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

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.

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

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

Summary

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Chapters in this video

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

Multi-layer Cortical Ca²⁺ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

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

In vivo Calcium Imaging in Mouse Inferior Olive

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

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

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

Calcium Imaging in Mouse Superior Colliculus