All animal experiments were performed in accordance with guidelines approved by the IACUC at the University of Nebraska Medical Center.
1. Before surgery
<ol>
	<li>Prepare pipettes for viral injections. Pull borosilicate glass capillaries using a pipette puller and bevel the pipette at a 20&#176; angle. Sterilize the pipettes overnight.</li>
	<li>Prepare fresh pieces of sterile gel foam by cutting them into small squares. Submerge the gel foam in a sterile microfuge tube containing 0.5 mL of saline. Soak the gel foam in saline for at least 30 min before use.</li>
	<li>Prepare fresh pieces of sponge strips by cutting them into thin strips.</li>
	<li>Twenty minutes before surgery, inject 4-6-week-old GLAST-CreER/GCaMP6f mice intraperitoneally with 0.2 mg/kg dexamethasone to prevent brain swelling and 5 mg/kg carprofen to reduce inflammation. 
	&#8203;NOTE: To induce the recombination and expression of GCaMP6f in approximately 40% of the astrocytes, GLAST-CreER/GCaMP6f mice were injected with 100 mg/kg tamoxifen at 3 weeks for 5 consecutive days.</li>
</ol>
2. Start of surgery
<ol>
	<li>After 20 min, anesthetize the mice with 3% isoflurane with oxygen at a flow rate of 1 L/min for approximately 1.5 min.</li>
	<li>Once anesthetized, place the mouse on a stereotaxic frame that sits on a water re-circulating blanket to maintain a body temperature of 37 &#176;C throughout the surgery. Secure the mouse to the frame using the snout clamp and ear bars. Ensure that the snout clamp and ear bars are stable enough that the head will not move during the procedure, but not so tight that the skull is damaged.</li>
	<li>Maintain anesthesia with 1-1.5% isoflurane with oxygen at a flow rate of 1-2 L/min. Note that some animals may need more or less isoflurane to maintain sedation. Monitor the breathing of the animal as well as its reflexes to toe and tail pinches, and adjust the isoflurane appropriately.</li>
	<li>Apply eye ointment to each eye using a cotton tip applicator to prevent the eyes from drying out during the procedure.</li>
	<li>Using rodent trimmers, shave the hair from the neck to just past the eyes. Use caution to ensure that the whiskers of the animal are not accidentally trimmed.</li>
	<li>Clean the shaved area with one iodine prep pad, followed by one alcohol prep pad.</li>
	<li>Using sterile tissue forceps and surgical scissors, cut and remove the skin from the frontal sutures anterior to the bregma all the way posteriorly to lambda.</li>
	<li>Once the skin is removed, add approximately 0.1 mL of 1% xylocaine (lidocaine with epinephrine 1:200,000) to the skull to minimize bleeding of the skull.</li>
	<li>Using a sterile size 11 carbon steel surgical blade attached to a handle, gently scrape the connective tissue from the skull. Take extra care when removing the connective tissue near the edge of the skull, as any remaining tissue could prevent strong adhesion of the glass or head plate later on.</li>
	<li>Use sterile tissue forceps to remove loose connective tissue from the skull.</li>
	<li>Using sterile cotton tip applicators, clean off any remaining xylocaine from the skull.</li>
	<li>Once completed, thoroughly degrease the skull using a sterile cotton tip applicator dipped in acetone. Use compressed air to immediately blow-dry the skull.</li>
</ol>
3. Craniotomy
<ol>
	<li>Using a fine-point marker and a ruler, measure the appropriate size for the opening at the desired location. Confirm the size of the opening by comparing it to the size of the cover glass (3 or 5 mm in diameter); ensure that the opening is slightly smaller than the size of the cover glass.</li>
	<li>Using a dental drill, gradually trace the outline of the opening, thinning the bone. Drill slowly to prevent drilling through the bone, and drill in concentric circles to facilitate even thinning of the bone.</li>
	<li>After each concentric pass, add a drop or two of sterile saline to the drilled area. Allow the saline to sit for at least 10 s to prevent the bone from overheating and damaging the underlying dura.</li>
	<li>Use a sterile cotton tip applicator to absorb any remaining saline. Blow compressed air over the area to ensure that all remaining saline has evaporated before resuming drilling. Do this to blow away bone debris that remains.</li>
	<li>Repeat steps 3.2-3.4 until the bone is adequately thinned for removal.</li>
	<li>Ensure that the bone is adequately thinned for removal by gently pushing on the central area of the bone with fine forceps to check that the thinned skull moves. The underlying vasculature should be visible where the drilling occurred. Observe that the bone may appear as if it will crack where thinning occurred. 
	NOTE: Training at this stage is crucial to identify when the bone has been thinned appropriately.</li>
	<li>Before attempting to remove the bone, check the mouse to make sure it is completely sedated. If not, gradually increase the isoflurane maintenance to prevent brain swelling when removing the bone.</li>
	<li>Add a small piece of gel foam saturated in saline to the thinned skull. Add one or two additional drops of saline to the thinned skull. 
	NOTE: Having plenty of saline over the thinned skull helps to reduce bleeding and protect the dura when removing the bone flap.</li>
	<li>Using a miniature 15&#176;&#160;pointed blade, carefully insert the blade into the thinned bone and cut along the thinned bone. Keep the gel foam soaked in saline, ready to stop any bleeds that may occur.</li>
	<li>Using forceps, carefully lift and remove the bone. Be as gentle as possible to avoid damage to the underlying tissue and vasculature. Take extreme care not to damage the dura mater.</li>
	<li>Once the bone flap is removed, add a fresh piece of gel foam saturated in saline to the cortex. Add a few drops of saline to prevent the gel foam from drying out, as this will cause damage to the underlying tissue.</li>
</ol>
4. Viral injections
<ol>
	<li>Perform viral injections using a stereotaxic apparatus.
	<ol>
		<li>Prepare AAV1.CaMKII.0.4.Cre (titer of 3 &#215; 1013 genome copies (GC)/mL) at 1:5,000 by serial dilutions in saline.</li>
		<li>Prepare the mixture of AAV1.CaMKII.0.4.Cre (1:5,000) and AAV1.CAG.FLEX.tdTomato (titer of 5 &#215; 1012 GC/mL) on a small piece of parafilm.</li>
		<li>Fill a sterile beveled glass pipette with a tip size of 20 &#181;m with the virus mixture by gently placing the pipette on the solution.</li>
		<li>Lower the pipette so that it just touches the surface of the brain and continue to lower for an additional 200-300 &#181;m for L2/3 injections. Using an intracellular microinjection dispense system (see the Table of Materials), pressure-inject 12-15 times over 2 min (20 psi, 9 ms pulse duration). Observe the meniscus in the pipette drop with each injection to ensure that the pipette is not blocked.</li>
		<li>Once injected, leave the pipette in the brain for 4-5 min to prevent backflow. Slowly retract the pipette.</li>
		<li>Repeat the injections at 2-3 sites (approximately 500 &#181;m apart).</li>
		<li>Discard the used glass pipettes, parafilm, pipette tips, and microfuge tubes used for serial dilutions of the Cre virus into 10% bleach.</li>
	</ol>
	</li>
</ol>
5. Implantation of cranial window
<ol>
	<li>Remove any gel foam, and use a small strip of sponge strips to soak up any remaining saline.</li>
	<li>Add a few drops of the antibiotic enrofloxacin (22.7 &#181;g/mL) to the opening and allow it to remain there for 1 min. After 1 min, use a fresh piece of sponge strip to absorb any remaining enrofloxacin. Repeat this process two more times.</li>
	<li>After the third wash, add a few drops of saline to the opening and allow it&#160;to remain there for 1 min. After 1 min, use a fresh piece of sponge strip to absorb any remaining saline. Repeat this wash process two more times and after the third wash, add a small drop of saline to the opening.</li>
	<li>Place the cover glass over the opening. Use forceps to ensure that the glass is flush over the opening to obtain images of good quality.</li>
	<li>While gently holding the glass in place, apply cyanoacrylate adhesive gel (Table of Materials) around the perimeter of the glass to seal the edges of the window to the skull. Make sure not to let the glue seep under the cover glass, and keep as much glue off the surface of the cover glass as possible.</li>
	<li>Apply a layer of super glue over the adhesive gel. Add a layer of dental cement liquid over the glue to allow it to harden.</li>
	<li>Take an appropriately sized helicopter-type head plate and apply a thin layer of glue around the central opening. Place the head plate over the cover glass, and allow the glue to dry briefly.</li>
	<li>In a 1.5 mL microfuge tube, add dental cement powder to the 0.1 mL mark on the tube. Add 7-8 drops of fast-curing, instant adhesive, and mix. Draw into a 1 mL syringe with a 19 G needle that has been cut to create a larger opening.</li>
	<li>Inject the mixture through the lateral holes of the helicopter bar until it seeps from either side. Apply the dental cement/adhesive mixture to the rest of the exposed skull to fasten the headplate to the skull, which will reduce movement artifacts during imaging.</li>
	<li>Allow the mixture to air-dry for at least 15 min.</li>
	<li>Inject the mouse with 0.5 mL of saline subcutaneously to aid in recovery.</li>
</ol>
6. Post operation
<ol>
	<li>Remove the mouse from the stereotaxic frame and return it to its cage.</li>
	<li>Place a portion of the cage on a water re-circulating blanket, space gel heating pads or isothermal pads to assist with recovery.</li>
	<li>Provide the animal with a small helping of food pellets and wet food, in addition to their regular diet, to further aid in recovery. Add new food pellet and wet food each day after surgery for the duration of recovery.</li>
	<li>The day after surgery, inject mice once with 5 mg/kg carprofen and 5 mg/kg enrofloxacin.</li>
	<li>On days 2-6, inject the mice once with 5 mg/kg carprofen and twice with 5 mg/kg enrofloxacin, pending approval by the local IACUC. Separate the enrofloxacin injections by at least 8 h.</li>
	<li>On days 7-20, inject the mice daily with 5 mg/kg carprofen.</li>
</ol>
7. Animal habituation for imaging
<ol>
	<li>On day 14 after surgery, examine the cranial window for optical clarity. Do not use mice with unclear or otherwise damaged windows (i.e., excessive angiogenesis).</li>
	<li>Check for tdTomato expression under a fluorescence microscope. If labeled cells can be identified, proceed with animal habituation.</li>
	<li>First day of habituation (handling)
	<ol>
		<li>Hold the mouse for a few minutes and return it to its cage after handling. Repeat the handling three times with a 15 min interval between the sessions.</li>
		<li>After the third trial, habituate the mouse by wrapping the animal in a small piece of cloth.</li>
		<li>Hold the mouse wrapped in cloth for approximately 1 min.</li>
		<li>After the trial, return the mouse to its cage. Repeat this process two more times, allowing a 15 min interval between each trial.</li>
	</ol>
	</li>
	<li>Second day of habituation
	<ol>
		<li>Weigh and record the mouse weight.</li>
		<li>Wrap the mouse in cloth, and secure it via its head plate to the head fixation arm of the air-lifted home cage.</li>
		<li>Leave the home cage exposed to the light.</li>
		<li>Allow the mouse to remain secured in the mobile home cage for 15 min.</li>
		<li>After habituation, remove the mouse from the home cage and turn off the airflow.</li>
		<li>Weigh the mouse and return it to its cage. Take care to only include mice that do not lose more than 10% of their body weight. Exclude mice that lose more than 10% of their weight during any day of habituation from imaging experiments.</li>
	</ol>
	</li>
	<li>Day three of habituation and beyond
	<ol>
		<li>Weigh and record the mouse weight before securing it to the air-lifted home cage.</li>
		<li>Secure the mouse to the air-lifted home cage.</li>
		<li>Cover the home cage with something that will provide a dark interior, such as a box, and allow the mouse to remain secured for 30 min.</li>
		<li>After 30 min, uncover the home cage, remove the mouse, and turn off the air flow.</li>
		<li>Weigh and record the mouse weight after habituation.</li>
		<li>Repeat this process every day, increasing the duration the mouse is secured to the home cage by 15 min. Continue this process until the mouse can be secured to the home cage for 1.5 h as this is the approximate duration of a two-photon imaging session.</li>
		<li>To habituate the mouse to noise, perform some habituation sessions on the microscope to acclimate the mouse to the sounds of the laser scanning mirrors. Exclude mice that fail to habituate sufficiently (i.e., vocalizations and stress-induced defecation).</li>
	</ol>
	</li>
</ol>
8. Multiphoton imaging
<ol>
	<li>Commence imaging 3 weeks following surgery to allow for the window to clear.</li>
	<li>Perform imaging on a custom-made or commercially available multiphoton microscope equipped with a Ti:Sapphire laser. Acquire images using a high numerical aperture (NA) water immersion objective. 
	NOTE: Two-channel imaging is achieved by using a 565 nm dichroic mirror and two external photomultiplier tubes. A 535/50 bandpass filter is used to detect GCaMP6f emission, and a 610/75 bandpass filter is used to detect tdTomato. A 25x water immersion objective (1.05 NA) and resonant scanners were used to acquire images shown in Figure 1 and Figure 2. Each region of interest consisted of a stack of images separated axially by 1 &#181;m. Each optical section was collected at 512 x 512 pixels, 0.18&#160;&#181;m/pixel. Images were acquired from the forelimb region of the primary motor cortex as determined by stereotaxic coordinates.</li>
	<li>Set the wavelength of the laser to 920 nm (990 nm if a red-shifted GECI is used).</li>
	<li>Place the mouse on the mobile home cage, and clamp the headplate to the head fixation arm.</li>
	<li>Add a few drops of water to the center of the window.</li>
	<li>Using the wide-field mode of the microscope, select 2-3 positions of easily identifiable vasculature. Save the images of these blood vessels and record the X- and Y-coordinates that appear on the motor controller to relocate the tdTomato-labeled dendrites for subsequent imaging sessions. Adjust the X- and Y-coordinates each time to realign with the saved images of the blood vessels.</li>
	<li>Once the vasculature has been imaged and positions recorded, locate the regions with neurons expressing tdTomato (Figure 1). Image the synaptic structures (dendrites and axons) of neurons and GCaMP6f activity in astrocytes. 
	NOTE: The dendrites will serve as landmarks to reliably return to the same regions and image the same dendrites and astrocytes over repeated days. No detectable shift in the imaging plane was observed with stable head fixation and after habituation to head fixation. Displacement that occurs in the X and Y directions is motion-corrected.</li>
	<li>After imaging, return the mouse to its cage. 
	NOTE: Mice are typically kept on the scope for a maximum of 2 h. When the imaging lasts for a long time, the water under the objective may dry. Water should be added during the experiments. Alternatively, an ultrasonic gel can be used instead of water.</li>
</ol>

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(88), e51869 (2014).</li><li>Holtmaat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nature Protocols. 4 (8), 1128-1144 (2009).</li><li>Hauglund, N. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meningeal+lymphangiogenesis+and+enhanced+glymphatic+activity+in+mice+with+chronically+implanted+EEG+electrodes.">Meningeal lymphangiogenesis and enhanced glymphatic activity in mice with chronically implanted EEG electrodes.</a> Journal of Neuroscience. 40 (11), 2371-2380 (2020).</li><li>De Paola, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+structural+plasticity+of+axonal+branches+and+boutons+in+the+adult+neocortex.">Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex.</a> Neuron. 49 (6), 861-875 (2006).</li><li>Cheng, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+two-photon+calcium+imaging+at+different+depths+with+spatiotemporal+multiplexing.">Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing.</a> Nature Methods. 8 (2), 139-142 (2011).</li><li>Yang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinned-skull+cranial+window+technique+for+long-term+imaging+of+the+cortex+in+live+mice.">Thinned-skull cranial window technique for long-term imaging of the cortex in live mice.</a> Nature Protocols. 5 (2), 201-208 (2010).</li><li>Shih, A. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polished+and+reinforced+thinned-skull+window+for+long-term+imaging+of+the+mouse+brain.">A polished and reinforced thinned-skull window for long-term imaging of the mouse brain.</a> Journal of Visualized Experiments: JoVE. (61), e3742 (2012).</li><li>Helm, P. J., Ottersen, O. P., Nase, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+optical+properties+of+the+mouse+cranium--implications+for+in+vivo+multi+photon+laser+scanning+microscopy.">Analysis of optical properties of the mouse cranium--implications for in vivo multi photon laser scanning microscopy.</a> Journal of Neuroscience Methods. 178 (2), 316-322 (2009).</li><li>Stobart, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+calcium+imaging+of+astrocytes+reveals+distinct+cellular+compartment+responses+to+sensory+stimulation.">Long-term in vivo calcium imaging of astrocytes reveals distinct cellular compartment responses to sensory stimulation.</a> Cerebral Cortex. 28 (1), 184-198 (2018).</li><li>Pryazhnikov, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+two-photon+imaging+in+somatosensory+cortex+of+behaving+mice+reveals+dendritic+spine+formation+enhancement+by+subchronic+administration+of+low-dose+ketamine.">Longitudinal two-photon imaging in somatosensory cortex of behaving mice reveals dendritic spine formation enhancement by subchronic administration of low-dose ketamine.</a> Scientific Reports. 8 (1), 6464 (2018).</li><li>Thrane, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+anesthesia+selectively+disrupts+astrocyte+calcium+signaling+in+the+awake+mouse+cortex.">General anesthesia selectively disrupts astrocyte calcium signaling in the awake mouse cortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (46), 18974-18979 (2012).</li><li>Paukert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norepinephrine+controls+astroglial+responsiveness+to+local+circuit+activity.">Norepinephrine controls astroglial responsiveness to local circuit activity.</a> Neuron. 82 (6), 1263-1270 (2014).</li><li>Delekate, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabotropic+P2Y1+receptor+signalling+mediates+astrocytic+hyperactivity+in+vivo+in+an+Alzheimer's+disease+mouse+model.">Metabotropic P2Y1 receptor signalling mediates astrocytic hyperactivity in vivo in an Alzheimer's disease mouse model.</a> Nature Communications. 5, 5422 (2014).</li></ol>

Here, we have presented a protocol for the implantation of chronic cranial windows for in vivo imaging of cortical astrocytes and neurons in awake, head-restrained mice on an air-lifted home cage. Specific examples have been provided of the cranial window application for imaging astrocytes that express GECIs and neuronal synaptic structures. With the use of multiphoton microscopy, astrocytic calcium signaling dynamics and structural synaptic dynamics can be recorded repeatedly over days.
A chronic cranial window provides good optical imaging quality and allows for multiple imaging sessions to be performed of the same neuronal and glial structures. The approach also makes it possible to perform intracranial viral injections before covering the craniotomy with a cover glass. 
Users should be aware of a number of limitations incurred through the implantation of chronic cranial windows. Experience and much practice are needed to achieve a high-quality window and to ensure animal survival. Moreover, the surgery is invasive and can result in an inflammatory response. Pharmacological treatment during surgery and post-surgical recovery includes anti-inflammatory drugs and antibiotics31. The use of anti-inflammatory drugs may not be appropriate in studies investigating models of neuroinflammation as these drugs may also affect the phenomenon under study.
More recently, meningeal lymphangiogenesis has been shown to occur in response to cranial window implantation32. Thus, the chronic cranial window might not be therefore acceptable for studies investigating meningeal lymphatic vessels. Importantly, a 2-3-week waiting period post-surgery is necessary before imaging experiments31,33. Additionally, the age of the mice, as well as post-operation recovery time, limits the ability to image very early developmental events. While cranial windows can be implanted in younger mice (P15-21)34, possible side effects, such as inflammation, need to be considered.
As an alternative to the chronic cranial window, thinned skull preparations may also be made. This method results in the thinning of the skull over the region of interest before imaging. A thinned skull window is less invasive and overcomes the need for anti-inflammatory drug administration, making it a choice in studies investigating models of neuroinflammation and neuroimmune interactions. However, should repeated imaging be desired, the skull needs to be re-thinned before imaging, and bone can only be re-thinned a limited number of times before image quality degrades35.
Although a modified version of a reinforced thin-skull method that does not require rethinning has been described36, a non-uniform skull thickness may cause spherical aberrations, resulting in the distortion of fluorescent structures37. Thus, when quantitative measurements are being recorded, such as calcium signaling dynamics38 or levels of synaptic proteins27, as opposed to the identification of structures such as dendritic spines, the chronic cranial window provides a more optically stable and reliable preparation. Thus, for longitudinal, in vivo imaging, the insertion of cranial windows is the superior method for examining changes as a result of drug treatment39, training in a behavioral paradigm4,25, and synaptic remodeling in neurological disorders11,27.
The described procedure is technically challenging and provides a barrier for quality image acquisition. Extreme care must be taken at each step to ensure that the window stays free of infection, and damage to the underlying tissue is avoided. This includes gentle scraping of connective tissue on the skull, taking adequate breaks for cooling the bone when drilling, ensuring uniform thinning to facilitate easy bone removal, preventing brain swelling, and extreme care to not damage the dura mater during surgery. Only the best window preparations remain optically transparent for longer periods of imaging.
While changes in synaptic structures can be imaged in anesthetized mice over days, this is not preferred when examining astrocyte calcium signaling as anesthesia has been shown to disrupt astrocyte calcium signaling in vivo40. To perform in vivo imaging in awake, head-restrained mice, it is essential to habituate the animal to the imaging conditions in the mobile home cage, free-floating treadmill, air-lifted Styrofoam ball, or other apparatus. Proper habituation will not only reduce stress during imaging, but also act to minimize movement artifacts during the imaging sessions.
In summary, in vivo multiphoton microscopy through a chronic cranial window is a useful tool for studying structural and functional changes of cells in the cortex. Using cell-specific fluorescent dyes and reporters, it is possible to study cellular morphology, interactions, and activity. With the longitudinal imaging allowed by chronic cranial windows, it is possible to examine how synaptic structures change and develop over time13,14. Using the protocol presented here, it is possible to examine calcium signaling dynamics in astrocytes due to sensory stimulation23,38, locomotion or startle6,41, disease15,42, or other parameters of interest.

The ability to perform in vivo multiphoton fluorescence microscopy in the cortex of mice is paramount to the study of cellular signaling and structure1,2,3,4,5,6,7,8,9, disease pathology10,11, and cellular development12,13. With the implantation of chronic cranial windows, longitudinal imaging is possible, allowing for repeated imaging of cortical areas for days, weeks, or months13,14 in live animals. Multiphoton microscopy is ideal for in vivo, repeated imaging because of improved depth probing and reduced photodamage associated with the infrared laser used. This allows for the study of molecular and cellular dynamics of specific cells in various cortical regions.
Multiphoton microscopy has been used for in vivo imaging of neuronal and glial cells in mice15,16,17,18,19,20. Various strategies can be implemented to label particular cell types and areas of interest. One common approach is to drive the expression of genetically encoded fluorescent proteins in a cell-specific manner using the Cre-Lox recombination system. This can be performed with genetically modified mice, e.g., crossing tdTomato &#34;floxed&#34; mouse (Ai14) with a mouse expressing Cre-recombinase under a promoter of interest21. Alternatively, cell- and site-specific labeling can be achieved with viral injections. Here, a virus encoding Cre recombinase under a cell-specific promotor and a virus encoding a floxed gene of interest are injected into a defined region. Appropriate cell types receiving both viral vectors will then express the desired gene(s). These genes can be structural markers, such as tdTomato, to view changes in cellular morphology22 or genetically encoded calcium indicators (GECIs), such as GCaMP and/or RCaMP, to examine calcium dynamics23. Methods of genetic recombination can be applied individually or in combination to label one or more cell types. A third approach, not requiring transgenic mice or viral constructs (which have limited packaging capacity), is in utero electroporation of DNA constructs24. Depending on the timing of the electroporation, different cell types can be targeted25,26,27.
When performing multiphoton imaging, mice can be imaged while awake or anesthetized. Imaging of awake mice can be performed by securing the mouse via an attached head plate28. This approach is made less stressful by allowing relatively free movement of the animal using methods, such as free-floating, air-supported Styrofoam balls29, free-floating treadmills1, or an air-lifted home cage system where mice are fastened by an attached head plate and allowed to move in an open chamber30. For each of these imaging conditions, it will first be necessary to habituate the mice to the imaging setup. This paper describes the habituation and imaging procedure using an air-lifted home cage system.
This protocol describes the implantation of a chronic cranial window for longitudinal in vivo imaging in the cortex. Here, we will use mice that conditionally express GCaMP6f in astrocytes to monitor calcium signaling dynamics. Further, this paper describes the procedure for viral injections using tdTomato as a label for neurons. This allows the determination of changes in neuronal synaptic structure and/or the availability as a structural marker that enables repeated imaging of the same astrocyte. Throughout the protocol, crucial steps will be highlighted to ensure the best possible quality of images obtained from multiphoton microscopy.

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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.

The quality of the cranial window can be assessed by how crisp the neuronal structures appear. In a good window, dendritic spines are clearly visible (Figure 1). With the structural and positional data stored, the same animal can be imaged repeatedly for days, weeks, or months to examine the same cells (Figure 1). The images in Figure 1 were obtained from the forelimb region of the primary motor cortex (in a 5 mm window). A variety of parameters can be measured, including density and dynamics of dendritic spines and axonal boutons to study structural synaptic plasticity. Astrocytic activity can be studied by analyzing the spatiotemporal dynamics of calcium signaling (Figure 2). Depending on the microscope capabilities (i.e., resonant scanners, piezo motor controlling the objective) and the experimental question, time-lapse imaging can be performed in a single focal plane or in a volume or multiregion to monitor calcium activity at the desired acquisition frequency.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62633/62633fig1v2.jpg" /> 
Figure 1:&#160;Repeated imaging of dendrites and astrocytes over days. A dendrite expressing tdTomato (magenta) allows the identification and repeated imaging of GCaMP6f-expressing astrocytes (white) in close proximity. Ca2+ activity within astrocytes is shown at different time points on different days. Synaptic structural plasticity can be concurrently imaged. Two new spines that appeared on Day 2 are marked with arrows. While one spine persisted and was visible on Day 5 (blue arrow), the other spine was eliminated (green arrow). Scale bar = 10 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62633/62633fig1v2large.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/62633/62633fig02.jpg" /> 
Figure 2: Imaging of astrocytic calcium signaling. Images showing Ca2+ activity from an astrocyte expressing GCaMP6f. Panels show Ca2+ activity recorded from a 4 &#181;m volume at different time points during the acquisition. Calcium signaling in processes and microdomains can be observed during the resting periods. A global event encompassing the entire cell can be observed during a locomotion bout at 188 s. Scale bar = 10 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62633/62633fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice at JoVE.com

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.

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 ...

implantation-cranial-window-for-repeated-vivo-imaging-awake

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.0K Views.  University of Nebraska Medical Center. 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.

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

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 ...

Behavior

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers patients only modest benefits, as most patients do not survive more than five years following diagnosis due to glioma relapse 1,2. The discovery of cancer stem cells in human brain tumors holds promise for having an enormous impact on the development of novel therapeutic strategies for glioma 3. Cancer stem cells are defined by their ability both to self-renew and to differentiate, and are thought to be the only cells in a tumor that have the capacity to initiate new tumors 4. Glioma relapse following radiation therapy is thought to arise from resistance of glioma stem cells (GSCs) to therapy 5-10. In vivo, GSCs are shown to reside in a perivascular niche that is important for maintaining their stem cell-like characteristics 11-14. Central to the organization of the GSC niche are vascular endothelial cells 12. Existing evidence suggests that GSCs and their interaction with the vascular endothelial cells are important for tumor development, and identify GSCs and their interaction with endothelial cells as important therapeutic targets for glioma. The presence of GSCs is determined experimentally by their capability to initiate new tumors upon orthotopic transplantation 15. This is typically achieved by injecting a specific number of GBM cells isolated from human tumors into the brains of severely immuno-deficient mice, or of mouse GBM cells into the brains of congenic host mice. Assays for tumor growth are then performed following sufficient time to allow GSCs among the injected GBM cells to give rise to new tumors-typically several weeks or months. Hence, existing assays do not allow examination of the important pathological process of tumor initiation from single GSCs in vivo. Consequently, essential insights into the specific roles of GSCs and their interaction with the vascular endothelial cells in the early stages of tumor initiation are lacking. Such insights are critical for developing novel therapeutic strategies for glioma, and will have great implications for preventing glioma relapse in patients. Here we have adapted the PoRTS cranial window procedure 16and in vivo two-photon microscopy to allow visualization of tumor initiation from injected GBM cells in the brain of a live mouse. Our technique will pave the way for future efforts to elucidate the key signaling mechanisms between GSCs and vascular endothelial cells during glioma initiation.

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

Medicine

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

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 ...

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 ...

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell types of an animal's brain, upon exposure to exogenous stimuli. Here, the administration of a closed-skull traumatic brain injury (TBI) and simultaneous implantation of a cranial window for subsequent longitudinal intravital imaging in mice is shown. Mice are intracranially injected with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) under a neuronal specific promoter. After 2 to 4 weeks, the mice are subjected to a repetitive TBI using a weight drop device over the AAV injection location. Within the same surgical session, the mice are implanted with a metal headpost and then a glass cranial window over the TBI impacting site. The expression and cellular localization of EGFP is examined using a two-photon microscope in the same brain region exposed to trauma over the course of months.

This study demonstrates delivery of a repetitive traumatic brain injury to mice and simultaneous implantation of a cranial window for subsequent intravital imaging of a neuron-expressed EGFP using two-photon microscopy.

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell ...

Implantation of Cranial Imaging Window in Mouse Model: A Surgical Procedure to Implant Glass-based Coverslip for Stable Optical Access to Regions of Murine Brain

Source: Alieva, M. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/59278/longitudinal-intravital-imaging-brain-tumor-cell-behavior-response-to">Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy.</a>&#160;J. Vis. Exp.&#160; (2019)
This video presents a surgical procedure to implant cranial window in a mouse model for stable optical access to different regions of the brain.

Source: Alieva, M. et al. Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy.&#160;J. Vis. Exp.&#160; ...

JoVE Encyclopedia of Experiments

Cancer Research

Cancers of the Nervous System

In vivo studies

Installation of Optical Windows in the Brain of Transgenic Mice to Image Neural Activities

Source: Lin, X., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56297">Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice.</a> J. Vis. Exp. (2019).
This video demonstrates the brain&#39;s neural activity recording through an optical window in transgenic mice. The mice express a fluorescent protein that enables the detection of neural activities. A small portion of the skull is carefully removed to install an optical window, and the skull is then covered with black cement to block light before imaging.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Summary

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Chapters in this video

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

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

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

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving 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

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

Implantation of Cranial Imaging Window in Mouse Model: A Surgical Procedure to Implant Glass-based Coverslip for Stable Optical Access to Regions of Murine Brain

Installation of Optical Windows in the Brain of Transgenic Mice to Image Neural Activities