We thank Dr. Masashi Kondo and Dr. Masanori Matsuzaki for providing virus vectors. This work was supported by Grants-in-Aid for Scientific Research (20H00481, 20A301, 20H05894, 20H05895 to S.O.), the Japan Agency for Medical Research and Development (JP19gm1310003 and JP17gm5010003 to S.O. and JP19dm0207082 to H.M.), the UTokyo Center for Integrative Science of Human Behavior (CiSHuB), the Japan Science and Technology Agency Moonshot R&#38;D (JPMJMS2024 to H.M.), Brain Science Foundation (to H. M.).

All experiments were approved by the Animal Ethics Committee and the Genetic Recombinant Experiment Safety Committee of Graduate School of Medicine and Faculty of Medicine, the University of Tokyo, and performed according to their guidelines.
1. Preparation
<ol>
	<li>Sterilize all the instruments and apparatuses necessary for the surgery using autoclave or 70% ethanol.</li>
	<li>Glass pipette preparation.
	<ol>
		<li>Firmly fix a 75 &#181;L calibrated capillary to the holder of the micropipette puller. Set the puller to the program with the recommended parameters as P (pressure) = 500, HEAT = 680, PULL = 30, VEL = 60, TIME = 180.</li>
		<li>After finishing the program (usually 4.5 to 5 s), the capillary is divided into two glass pipettes with tapering tails. Then, break the distal part of the tails with tweezers to keep the tip diameter within 30-50 &#181;m. For this, break the tails 1 cm distal to the end of the beveled part.</li>
	</ol>
	</li>
	<li>For craniotomy, prepare a cranial window by gluing a larger outer glass disc, 4 mm in diameter, 0.15 &#177; 0.02 mm in thickness, to a smaller inner glass disc, 2 mm in diameter, 0.525 &#177; 0.075 mm in thickness, using a UV light curing reagent (Figure 2A).</li>
</ol>
2. AAV injection
<ol>
	<li>Preparation of injection apparatus
	<ol>
		<li>Place a glass pipette connected to a 26 G Hamilton syringe through a tube onto a pipette holder of a stereotaxic instrument, and then tilt the pipette holder 60&#176; anteriorly from the vertical axis (Figure 1A,B).</li>
		<li>Fill the glass pipette, the syringe, and the connecting tube with liquid paraffin. Place the syringe on a microinjector.</li>
	</ol>
	</li>
	<li>Surgical procedure 
	NOTE: The following surgery was performed in a sterilized booth. A stereomicroscope was used for the surgery if necessary.
	<ol>
		<li>Anesthetize a male CX3CR1-EGFP mouse (detailed information in the Table of Materials) at 7 to 8 weeks old (bodyweight 22-26 g) with 3% isoflurane in a gas-tight chamber. After 3 min, take out the mouse from the chamber when it loses voluntary movement except breathing, and then switch to continuous anesthesia by a nasal tube with 2% isoflurane. 
		NOTE: Isoflurane is known to activate microglia. However, because imaging is performed 4 weeks after the surgery, there is no effect of isoflurane on the mice during imaging. Although isoflurane anesthesia is used for a few minutes when mice are placed in the stereotaxic instrument before imaging (step 4.2.1), we found that the effect of this short anesthesia is negligible.</li>
		<li>During surgery, keep the mouse warm with a heating pad at 37 &#176;C to prevent hypothermia. Apply eye ointment on both eyes to prevent corneal dryness and administer a meloxicam injection, subcutaneously (5 mg/kg). Attach the mouse to an auxiliary ear bar, and then fix the mouse on the stereotaxic instrument with its dorsal side up.</li>
		<li>Adjust the isoflurane concentration to an appropriate percentage (1%-2%) during the surgery while monitoring the breath and heart rate. Remove the hair using depilatory cream and disinfect the head several times in a circular motion with both an iodine-based or chlorhexidine-based scrub and alcohol.&#160;Then, inject 0.1 mL of 1% lidocaine subcutaneously and apply sterile drapes to secure the surgical site.</li>
		<li>Incise the scalp open along the midline with scissors and make the incision about 2 cm long, to ensure a good exposure of the skull above the right primary visual cortex. After the incision of the scalp, irrigate the head with saline throughout the whole procedure to prevent the exposed skull and brain from drying.</li>
		<li>Remove the periosteum on the exposed skull using forceps. Drill the skull on the stereotaxic coordinates: 3 mm lateral to the midline, 0.5 mm anterior to the lambda line, to create a small hole with approximately 0.5 mm diameter. Rinse off blood and tissue debris from the drill bit, if necessary.</li>
		<li>Fill the glass pipette with 1 &#181;L of rAAV2/1-hSyn-R-CaMP1.07 using the following procedure at a titer of 8.3 x 1012 vector genomes/mL22.
		<ol>
			<li>Advance the syringe to eject 1 &#181;L of liquid paraffin from the glass pipette. Place a piece of transparent film, approximately 2 cm x 2cm, on the exposed skull of the mouse and expel a droplet of 1 &#181;L of AAV solution on the film using a pipettor.</li>
			<li>Place the glass pipette tip into the droplet of AAV solution on the film and gently pull the syringe to aspirate the AAV solution. After the ejection, turn the T-shape stopcock to release the pressure inside the tube and the glass pipette.</li>
		</ol>
		</li>
		<li>Insert the glass pipette to a depth of 500 &#181;m from the brain surface through the hole created in step 2.2.5, and then inject 0.5 &#181;L of AAV solution using the microinjector (Figure 1C). Use the injection volume flow rate of 2.0 &#181;L/h; an injection of 0.5 &#181;L takes 15 min, and then subsequently wait for 5 min.</li>
		<li>After the injection, gently withdraw the glass pipette and rinse the brain surface with saline.</li>
	</ol>
	</li>
</ol>
3. Cranial window implantation
NOTE: Cranial window implantation follows the AAV injection on the same day.
<ol>
	<li>Mark a 2.5 mm diameter circle on the skull, centered 3 mm laterally from the lambda. Create a circular groove along the mark on the skull by drilling. Clean the debris as it may adversely affect the visibility of the skull and apply saline during the drilling process to prevent heating up.</li>
	<li>To check whether the drilling depth in the circular groove is sufficient to remove the central skull fragment, gently press the central skull with forceps. If it moves vertically with little resistance, the drilling depth is sufficient.</li>
	<li>When the groove reaches enough depth, insert the tip of the forceps into the bottom of the central skull fragment and gently lift and remove it to expose the brain surface (Figure 1D).</li>
	<li>Using a 27 G needle, prick and tear the dura at the edge of the exposed brain surface. Insert the tip of the forceps through the hole made at the edge of the dura. Hold the dura and peel it off to expose the brain surface. 
	NOTE: If bleeding occurs, rinse the brain surface with saline until the bleeding stops.</li>
	<li>Using forceps, disperse hemostatic fibers one by one in saline in a 3.5 mm dish, and then place the hemostatic fibers along the margins of the hole in which the transected dura is present.</li>
	<li>Place a cranial window prepared in step 1.3 on the exposed brain surface, and then adhere it to the skull with instant glue while gently pressing the window so that the window is in close contact with the skull.</li>
	<li>Afterward, apply instant glue to the whole exposed skull, and then carefully attach a head-plate onto the skull so that the window locates at the center of the square hole of the head-plate (Figure 2C). Ensure that the surface of the head plate is parallel to the surface of the glass window.</li>
	<li>After the glue has sufficiently hardened, apply dental cement to the exposed skull to reinforce the attachment between the head and the head-plate (Figure 2). 
	NOTE: If the experiments include the presentation of visual stimuli to mice, stray light to the imaging area should be avoided. It is advisable to blacken the cement by mixing it with the activated charcoal for the reduction of light reflection.</li>
	<li>Withdraw the mouse from the anesthesia after the cement has hardened sufficiently (about 20 min). Then, put the mouse into a new cage to recover alone.</li>
	<li>After confirming its consciousness and voluntary movement, indicating full recovery, put the mouse back in the home cage. During the recovery, administer a second, or more if necessary, meloxicam dose (once every 24 hours for 1-3 days) if obvious pain-indicating behaviors occur.</li>
</ol>
4. In vivo two-photon imaging of microglia and neuronal calcium dynamics
<ol>
	<li>Habituation of mice to the microscope apparatus
	<ol>
		<li>Three weeks after the surgery, induce anesthesia in the mouse with 3% isoflurane, and set the mouse under the 25x objective lens of a two-photon microscope using a custom-made stereotaxic instrument (Figure 3C). For the setup here, the mouse is set on the top of a treadmill and allowed to run freely.</li>
		<li>Approximately 10 min later, remove the mouse from the microscope stage and return it to the home cage. Repeat the habituation at least 3 times every alternate day before two-photon image acquisition.</li>
	</ol>
	</li>
	<li>Two-photon image acquisition 
	NOTE: We recommend performing the image acquisition more than 4 weeks after surgery, as microglial activation gradually returns to the basal level.
	<ol>
		<li>As in step 4.1, induce anesthesia in the mouse with 3% isoflurane and set the mouse under the objective lens of the two-photon microscope using a custom-made stereotaxic instrument.</li>
		<li>Install the custom-made shading device and an LCD monitor for visual stimulation as described below (Figure 3D).
		<ol>
			<li>Position the lens to focus on the brain surface, and then set this lens position as the original Z-position. Keep the x-y coordinates constant and elevate the objective lens; remove the mouse and stereotaxic instrument from the objective lens and treadmill.</li>
			<li>Attach a shading device on the top of the head plate using silicone, which is blackened by mixing with charcoal powder, and ensure the space between the head plate and the shading device is well-sealed.</li>
			<li>Fill the shading device with distilled water, and then fix the mouse and the stereotaxic frame again under the objective and on the treadmill. Carefully reset the focal plane at the brain surface, checking the depth of the objective lens. 
			NOTE: To avoid the risk of damaging the objective lens, set the xyz coordinates first, and then apply the shading device of the objective lens. It is also reasonable to install the cover first, and then adjust the coordinates, but cautious handling is necessary for this option.</li>
			<li>Cover the objective lens with black aluminum foil to avoid light contamination from the LCD monitor used for visual stimulation through the objective lens (Figure 3D). Set a 10-inch LCD monitor at 12.5 cm in front of the mouse&#39;s eyes to present visual stimuli (Figure 3D).</li>
		</ol>
		</li>
		<li>Configure the fluorescence emission collection filters to EGFP fluorescence (525/50 nm emission filter) and R-CaMP fluorescence (593/46 nm emission filter) and the excitation wavelength to 1,000 nm. Acquire images with the spatial resolution of 0.25 &#181;m/pixel using a 25x 1.1 NA objective and GaAsP PMTs.</li>
		<li>Find the imaging region in which R-CaMP(+) neurons and EGFP(+) microglia can be simultaneously imaged in layer 2/3. For this setup, data obtained in Figure 4 and Video 1 were at a depth of 100 &#181;m from the pial surface, using a live image preview. Keep the power of the excitation laser as low as possible to avoid photo-bleaching and damage caused by increased local temperature in the imaging region.</li>
		<li>Acquire images at the frame rate of 30 Hz. Simultaneously with the image acquisition, present a drifting-grating visual stimuli at 100% contrast, 1.5 Hz, 0.04 cycles per degree in 12 directions at 6 orientations from 0&#176; to 150&#176; in 30&#176; steps to the mouse17.</li>
		<li>After the image acquisition, remove the mouse from the microscope stage, detach the shading device and the stereotaxic instrument from the mouse, and return the mouse to its home cage.</li>
	</ol>
	</li>
	<li>Data registration
	<ol>
		<li>Convert and save the acquired image data (nd2 format in this case) as a series of tiff image files for each color channel, using Fiji or the software supplied with the microscope.</li>
		<li>Apply Turboreg, a plugin of Fiji, to perform registration with mode = translation. Use averaged image as the reference image with the tiff image files of EGFP channel.</li>
		<li>Perform the following processing for each frame using MATLAB. 
		NOTE: Although MATLAB is used in the following processing, the description mainly focuses on the intention of each step so that data can be analyzed by other programming software such as Python.
		<ol>
			<li>Use the imread function to read two EGFP tiff images before and after registration of the same frame, respectively. Use the imread function to read the R-CaMP tiff image of the same frame.</li>
			<li>Use the normcorr2 function to calculate the correlation function between the vectorized variables of the two GFP images read in step 4.3.3.1.</li>
			<li>Use the max function to detect the linear index with the highest cross-correlation of the correlation function, and then use the ind2sub function to calculate the movement position of the registered image from its original image.</li>
			<li>Use the imwarp function to translate the R-CaMP image to the position calculated in step 4.3.3.3, and then use the imwrite function to save the registered image of R-CaMP.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Generation of calcium traces
	<ol>
		<li>Perform the following processing using MATLAB. Use the imread function to read all the registered R-CaMP tiff images generated in step 4.3.3.4. If the registered images have been already loaded as a variable in workspace, omit this step.</li>
		<li>Use the mean function to generate a time-projection image. Use the imshow function to show the averaged image as a figure; use the roipoly function to generate a binary ROI mask of the target structure (dendritic spine in this data).</li>
		<li>Using the mean function with the images obtained by multiplying the binary mask with images of each frame as input variables, calculate the averaged fluorescence intensity within the ROI for each frame.</li>
		<li>As described previously17, to the variable obtained in step 4.4.3, apply the butter worth frequency filter at cutoff = 1.6 s, for cutting high-frequency noise, and then apply the sliding median filter at the time window = 200 s, for cutting low-frequency noise.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

We describe the protocol of AAV injection and craniotomy for simultaneous imaging of microglial dynamics and neuronal activity in awake mice as well as data processing. This technique can uncover the coordination between microglial dynamics and neuronal activity on time scales ranging from sub-second to tens of seconds.
The surgery protocol involves several technically demanding steps. AAV injection is one of the critical steps. Unsuccessful AAV injection may cause a significant reduction in the expression of R-CaMP. There are two main reasons: clogged glass pipettes and tissue damage. The clogging of glass pipettes reduces or completely blocks the ejection of AAV solution. This situation can be avoided by coloring the AAV solution with a dye such as fast green and visually confirming the success of the injection. Tissue damage caused by AAV injection prevents exogenous gene expression near the center of the injection site. The damage induced by AAV injection is likely to be caused by a sudden increase in the efflux from the tip of the glass pipette after the accumulation of the pressure inside the pipette. Therefore, the outflow of AAV solution from the glass pipette should be constant during the injection. Selecting glass pipettes with a slightly wider diameter at their tips would solve this problem. In cranial window implantation, the speed of surgery is critical. If the surgery takes too long, the brain tissue can be severely damaged. Accordingly, repeated practice is necessary to ensure the speed and smoothness of surgery. In addition, during the 4 weeks from surgery to imaging, the quality of imaging may be reduced by tissue regeneration between the cranial window and the brain tissue by the conventional method17. The method here overcomes this issue by applying thick glasses for the inner glass disc of the cranial windows to inhibit tissue regeneration and keep the window clear. In the system described here, this effect was evident by using an inner glass disc with a thickness of 0.525 &plusmn; 0.075 &#181;m.
The method can be applied successfully to mice older than 4 weeks, but the application to younger mice may be problematic. In juvenile mice, the skull shows rapid and prominent growth, which induces a mismatch between the cranial bone and the glass window.
In some cutting-edge studies, in vivo two-photon imaging was used to study microglia-neuron interactions12,21,23. Especially in the pioneering work by Merlini et al., they performed simultaneous in vivo imaging of microglial dynamics and neural activity within the cell bodies21. In this method, by using thicker inner glasses for cranial windows, we could suppress motion artifacts in the depth orientation and achieve the stable measurement of neuronal activity in microstructures, such as dendritic spines. This method would help investigate synapse-microglial process interactions in awake mice.
Recently, in vitro study demonstrated that thin filopodia-like microglial processes have faster motility than thick processes, suggesting the importance of their faster motility in surveillance19. The system here can track the motility of thin microglial processes with a temporal resolution from a sub-second to a few tens of seconds. This property helps elucidate the functional significance of their motility for surveillance in vivo.
In the future, the combination of this imaging technique with interventions, such as optogenetics24 or chemogenetics25, applied to local neural circuits or interregional neural connections would shed light on novel microglial functions in synaptic development and plasticity which sculpt characteristics of neural circuits. Also, further integration of imaging, intervention, and behavioral tasks would contribute to revealing the coordination of microglia and neurons underlying specific behaviors.

There is growing evidence that the internal state of the body constantly influences the brain functions in animals1,2,3,4,5. Accordingly, to gain a deeper understanding of brain functions, it is crucial to elucidate how glial cells in the brain monitor biological conditions in the periphery and relay the information to neurons.
Microglia, immune cells in the brain, are involved in synaptic development and plasticity, which sculpt characteristics of neural circuits in the brain6,7,8,9. For instance, the pioneering work by Wake et al. demonstrated that microglial processes make contact with synapses in a neuronal activity-dependent manner in the mouse neocortex and that artificial ischemia induces synapse loss following prolonged contact with microglia10. Tremblay et al. found that alteration of visual experience changes the modality of microglial interaction with synapses. During the critical period when dendritic spine turnover in the primary visual cortex (V1) is increased by binocular deprivation, dark adaptation reduces the motility of microglial processes and increases both their contact frequency with synaptic clefts and the number of cellular inclusions in microglia11. These results suggest that microglial processes sense neural activity and their surrounding environment to remodel neural circuits. Furthermore, a recent study reported that microglial surveillance differs between awake and anesthetized conditions, suggesting the importance of experiments using awake mice for investigating neuron-microglia communication under physiological conditions12.
In vivo two-photon calcium imaging is a powerful tool for the recording of calcium dynamics, reflecting ongoing neuronal firing, in hundreds of neurons simultaneously in a living animal13,14,15. Calcium imaging in neurons generally requires a temporal resolution of more than a few Hz to track rapid neuronal responses16,17. In contrast, previous studies tracking microglial dynamics have sampled microglial structures at a relatively low temporal resolution of less than 0.1 Hz18,19,20. One recent study applied simultaneous two-photon imaging to understand neuron-microglia communication21. However, it is still unclear how dynamic microglial processes respond to the surrounding neural activity at a temporal resolution of more than a few Hz in awake mice. In order to address this issue, we describe a simultaneous in vivo two-photon imaging method of neural activity and microglial dynamics with a temporal resolution higher than 1 Hz in awake mice. This method allows us to achieve stable imaging in awake mice at a higher frame rate (maximum, 30 Hz with the pixel frame size of 512 x 512 pixels) and provides a more favorable way to investigate the surveillance behavior of microglia or their interaction with neuronal activity in awake mice.

<table><tbody><tr><td>10-inch LCD monitor</td><td>EIZO</td><td>DuraVision FDX1003</td><td>For presenting grating visual stimuli</td></tr><tr><td>26G Hamilton syringe</td><td>Hamilton</td><td>701N</td><td></td></tr><tr><td>27G needle</td><td>Terumo</td><td>NN-2719S</td><td></td></tr><tr><td>AAV-hSyn-R-CAMP1.07</td><td>N/A</td><td>N/A</td><td>Kindly gifted from Prof. Matsuzaki&#039;s laboratory&amp;nbsp;</td></tr><tr><td>Activated charcoal powder</td><td>Nacalai tesque</td><td>07909-65</td><td></td></tr><tr><td>Black aluminum foil</td><td>THORLABS</td><td>BKF12</td><td></td></tr><tr><td>CX3CR1-EGFP mouse</td><td>Jackson laboratory</td><td>IMSR_JAX: 005582</td><td>&lt;em&gt;CX3CR-1EGFP/+&lt;/em&gt; knock-in/knock-out mice expressing EGFP in microglia in the brain under the control of the endogenous &lt;em&gt;Cx3cr1 &lt;/em&gt;locus.</td></tr><tr><td>DENT SILICONE-V</td><td>Shofu Inc</td><td>N/A</td><td>For the attachment of a shading device to a head-plate</td></tr><tr><td>Dental cement (quick resin, liquid)</td><td>Shofu Inc</td><td>N/A</td><td>AB</td></tr><tr><td>Dental cement(quick resin, powder)</td><td>Shofu Inc</td><td>N/A</td><td>B Color 3</td></tr><tr><td>Drill</td><td>Toyo Associates</td><td>HP-200</td><td></td></tr><tr><td>Glass capillary pipette</td><td>Drummond Scientific Company</td><td>2-000-075</td><td></td></tr><tr><td>Glass disc (large)</td><td>Matsunami</td><td>N/A</td><td>4mm in diameter, 0.15&amp;plusmn;0.02mm in thickness</td></tr><tr><td>Glass disc (small)</td><td>Matsunami</td><td>N/A</td><td>2mm in diameter, 0.525&amp;plusmn;0.075mm in thickness</td></tr><tr><td>Head-plate</td><td>Customized</td><td>N/A</td><td>Material: SUS304, thickness: 0.5mm, see Figure2B for the shape</td></tr><tr><td>Hemostatic fiber</td><td>Davol Inc</td><td>1010090</td><td></td></tr><tr><td>ImageJ Fiji software</td><td></td><td>Free software</td><td>For data registration</td></tr><tr><td>Instant glue (Aron alpha)</td><td>Daiichi Sankyo</td><td>N/A</td><td></td></tr><tr><td>Isoflurane</td><td>Pfizer</td><td>N/A</td><td></td></tr><tr><td>Lidocaine</td><td>Astrazeneca</td><td>N/A</td><td></td></tr><tr><td>MATLAB 2017b</td><td>MathWorks</td><td>N/A</td><td>For data registration and processing</td></tr><tr><td>Meloxicam</td><td>Tokyo Chemical Industry</td><td>M1959</td><td></td></tr><tr><td>Microinjector</td><td>KD Scientific</td><td>KDS-100</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument Company</td><td>P-97</td><td></td></tr><tr><td>Multi-photon excitation microscope</td><td>NIKON</td><td>N/A</td><td>The commercial name is &quot;A1MP+&quot;.</td></tr><tr><td>Objective lens</td><td>NIKON</td><td>N/A</td><td>The commercial name is &quot;CFI75 apochromat 25xC W&quot;.</td></tr><tr><td>Paraffin Liquid</td><td>Nacalai tesque</td><td>26132-35</td><td></td></tr><tr><td>Psychtoolbox</td><td></td><td>Free software</td><td>For presenting grating visual stimuli</td></tr><tr><td>Shading device</td><td>Customized</td><td>N/A</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>M165 FC</td><td></td></tr><tr><td>Stereotaxic instrument</td><td>Narishige Scientific Instrument</td><td>SR-611</td><td>For the surgery</td></tr><tr><td>Stereotaxic instrument</td><td>Customized</td><td>N/A</td><td>For fixing mice under the two-photon microscope</td></tr><tr><td>Stopcock</td><td>ISIS</td><td>VXB1079</td><td></td></tr><tr><td>Surgical silk</td><td>Ethicon</td><td>K881H</td><td></td></tr><tr><td>Treadmill</td><td>Customized</td><td>N/A</td><td></td></tr><tr><td>UV light curing agent</td><td>Norland Products Inc</td><td>NOA 65</td><td></td></tr><tr><td>Vaporizer</td><td>Penlon</td><td>Sigma Delta</td><td>Anesthetic machine</td></tr></tbody></table>

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.

We performed the AAV injection and cranial window implantation in V1 of an 8-week-old CX3XR1-EGFP transgenic mouse as described in this protocol. Four weeks after the surgery, we performed simultaneous in vivo two-photon imaging of R-CaMP-based neural activity and microglial dynamics in layer 2/3 of V1 (Figure 4 and Video 1). During the imaging, the mouse was placed on a treadmill and allowed to run freely. Images were acquired at the frame rate of 30 Hz with the spatial resolution of 0.25 &#181;m/pixel using a 25x, 1.1 NA objective and GaAsP PMTs. Both EGFP and R-CaMP were excited at a wavelength of 1,000 nm, and EGFP fluorescence (525/50 nm emission filter) and R-CaMP fluorescence (593/46 nm emission filter) were collected simultaneously. After the registration of acquired data to minimize motion artifacts, every four frames were averaged into one frame to reduce background noise. Grating visual stimuli were presented to the mouse, and the visual responses of neurons and individual dendritic spines were analyzed (Figure 4D). Microglial processes showed fast dynamics and changed their morphology within 10 s (Figure 4E&#160;and Video 1). As detailed in the Discussion section, when the AAV injection failed, the expression of R-CaMP could not be detected. If the surgery was not successful, the signals of EGFP and R-CaMP could not be observed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64111/64111fig01.jpg" /> 
Figure 1: AAV injection. (A) A glass pipette was connected to a 26 G Hamilton syringe using a silicon tube. (B) The set-up for AAV injection. (C) AAV solution was injected via the glass pipette tilted 60&#176; anteriorly from the vertical axis. (D)&#160;Schematic diagram of the procedure of open skull (described in step 3.3). <a href="https://www.jove.com/files/ftp_upload/64111/64111fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64111/64111fig02v2.jpg" /> 
Figure 2: Cranial window and head-plate. (A) Schematic diagram of cranial window implantation. (B) Custom-made head-plates were used. For imaging in the right hemisphere, the shorter arm should be used on the right side. (C) Dorsal view of a mouse with a cranial window and an implanted head plate. (D) High magnification view of the cranial window shown in Figure 2C. (E) Dorsal view of a mouse fixed with the custom-made stereotaxic instrument. <a href="https://www.jove.com/files/ftp_upload/64111/64111fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64111/64111fig03.jpg" /> 
Figure 3: The set-up for in vivo two-photon imaging. (A) Dorsal view of shading device. (B) Side view of shading device. (C)&#160;View of a mouse with the shading device on the head plate under the objective lens. The mouse is placed on the treadmill. (D) View under the two-photon excitation microscope. A monitor presenting visual stimuli is placed on the right side of the figure. The objective lens is covered with the shading device and black aluminum foil to avoid light from the monitor to the objective lens. <a href="https://www.jove.com/files/ftp_upload/64111/64111fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64111/64111fig04.jpg" /> 
Figure 4: Microglial dynamics and visual responses in a dendritic spine. (A-C) Time-averaged images of EGFP(+) microglia and R-CaMP(+) neurons in layer 2/3 of V1 in a 12-week-old CX3XR1-EGFP transgenic mouse. Signal intensity in each channel was normalized independently. (D) Visual responses in a dendritic spine. Stripe diagrams with black arrows indicate the direction of the grating stimuli presented in the timing indicated by gray columns. In calcium traces, gray lines indicate individual responses, and a black line indicates their average. In the right inset, a yellow arrowhead indicates the dendritic spine in which the region of interest (ROI) was generated to acquire the calcium traces. (E) Microglial process (cyan arrowhead) showed rapid retraction in 10 s. <a href="https://www.jove.com/files/ftp_upload/64111/64111fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Two-photon imaging of neuronal activity and microglial dynamics. Imaging data of EGFP(+) microglia and R-CaMP(+) neurons in layer 2/3 of V1 in a 12-week-old CX3XR1-EGFP transgenic mouse. On the left side of the movie, magenta indicates R-CaMP in neurons, and green indicates EGFP in microglia. The center of the movie corresponds to the EGFP signal, and the right side corresponds to the R-CaMP signal. Signal intensity in each channel was normalized independently. The frame rate of the movie is 40 times faster than the actual rate. Scale bar = 10 &#181;m <a href="https://www.jove.com/files/ftp_upload/64111/220330CatMovieForJoVE_IntNormalized-2.mp4" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice at JoVE.com

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

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 ...

simultaneous-imaging-microglial-dynamics-neuronal-activity-awake

Research

JoVE Journal

Neuroscience

2.2K Views.  The University of Tokyo. Ons protocol maakt de gelijktijdige beeldvorming van microgliadynamiek en neuronale activiteit bij wakkere muizen mogelijk. Het kan op grote schaal worden toegepast om het surveillancegedrag van microglia of de interactie met neuronen te onderzoeken. Het voordeel van deze methode is dat bewegingsartefacten beeldgegevens niet gemakkelijk vervuilen vanwege robuuste hoofdfixatie.Ook stelt herstel na beeldvorming met de hoge framesnelheid ons in staat om bewegingsartefacten uit de gegevens...

Video: Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

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.

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 ...

Behavior

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

Medicine

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 ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Immunology and Infection

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

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 ...

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal ...

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. Under homeostatic conditions, microglia possess a highly ramified morphology. This facilitates their monitoring of the surrounding environment through the continuous extending and retracting of their processes. During brain injury and inflammation, however, microglia become activated and undergo dramatic morphological changes, retracting their ramified processes and swelling their cell body. This facilitates activities such as migration and phagocytosis, which microglia undertake to navigate the brain environment to a less pathological state.
This close relationship between microglial morphology and changes in their activity have enabled considerable insights into various microglial functions. However, such morphological and activity changes are themselves phenomena that can result from any number of intracellular signaling pathways. Moreover, the time-lag between stimulus and response, as well as the highly compartmentalized morphology of microglia, make it difficult to isolate the causative mechanisms that underpin function. To solve this problem, we developed a genetically modified mouse line in which a highly sensitive fluorescent Ca2+-indicator protein is specifically expressed in microglia.
After describing methods for in vivo microglial Ca2+ imaging, this paper presents a structured analysis approach that classifies this Ca2+ activity to rationally defined subcellular regions, thus ensuring that the spatial and temporal dimensions of the encoded information are meaningfully extracted. We believe that this approach will provide a detailed understanding of the intracellular signaling rules that govern the diverse array of microglial activities associated with both higher brain functions and pathological conditions.

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

In this paper, we describe a protocol for in vivo imaging of microglial Ca2+ activity and subsequent analysis of its spatiotemporal dynamics. This method enables thorough characterization of how microglia respond to changes in the brain environment, appropriately capturing the fine spatiotemporal scales at which such events occur.

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. ...

Multilayer Imaging with Microprism in Head-Fixed and Freely Moving Animals

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for studying the structure, function, and plasticity of living brain tissues. In comparison to conventional electrophysiology, fluorescence microscopy can capture the neural activity as well as the morphology of the cells, enabling long-term recordings of the identified neuron populations at single-cell or subcellular resolution. However, high-resolution imaging typically requires a stable, head-fixed setup that restricts the movement of the animal, and the preparation of a flat surface of transparent glass allows visualization of neurons at one or more horizontal planes but is limited in studying the vertical processes running across different depths. Here, we describe a procedure to combine a head plate fixation and a microprism that gives multilayer and multimodal imaging. This surgical preparation not only gives access to the entire column of the mouse visual cortex but allows two-photon imaging in a head-fixed position and one-photon imaging in a freely moving paradigm. Using this approach, one can sample identified cell populations across different cortical layers, register their responses under head-fixed and freely moving states, and track the long-term changes over months. Thus, this method provides a comprehensive assay of the microcircuits, enabling direct comparison of neural activities evoked by well-controlled stimuli and under a natural behavioral paradigm.

Author Spotlight: Comparative Imaging of Neural Activity in Awake and Freely Moving States

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

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