Name: a thin-skull window technique for chronic two-photon in vivo imaging of murine microglia in models of neuroinflammation
Uploaded: 2010-09-19T17:00:00
Duration: 12 min 7 s
Description: Watch this Scientific Journal Video about A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation at JoVE.com

We thank Emily A. Kelly for the head plate design.
We are grateful for the support of NIH awards to H.A.G.: PO1 MH64570, RO1 MH56838, RO1 MH078989, R21 MH03851 and the generous support of the Geoffrey Waasdorp Pediatric Neurology Fund without which this work would not have been possible. A.K.M is funded by the NIH (EY019277), Whitehall Foundation, the Alfred P. Sloan Foundation and a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund. M.E.T. is funded by a Fonds de la recherche en sante du Quebec (FRSQ) postdoctoral training award. D.F.M. is a trainee in the Medical Scientist Training Program, funded by NIH T32 GM07356 and T32 AI049815.

1. Preparing the Animal for Imaging
<ol>
<li>In our experiments, we use adult CX3CR1GFP/+ mice that express GFP in mononuclear cell lineages, including microglia.4 Different strains of mice can be used to meet the needs of a specific project.</li>
<li>Anesthetize the mouse with an intraperitoneal injection of a three drug cocktail consisting of 0.05 mg/kg Fentanyl, 5.0 mg/kg Midazolam, and 0.5 mg/kg Medetomidine.5 Surgical preparation for TSCW starts after the animal no longer responds to painful stimuli, such as a tail pinch. Throughout the surgery and imaging, we keep the animal on a heating pad to prevent post-anesthesia hypothermia. If necessary, a booster dose of one-third the original dose of the anesthetic cocktail can be given to restore the original anesthetic plane.</li>
<li>Cover the animal's eyes with a protective ophthalmic ointment to keep the eyes moist during anesthesia, which suppresses the animal's blink reflex. Remove the hair from the scalp of the animal using a razor, shears, scissors, or chemical methods. Disinfect the scalp using a 10% povidone-iodine solution and 70% ethanol. All surgical instruments need to be autoclaved, sterilized with a glass-bead sterilizer, or disinfected with 70% ethanol.</li>
<li>Make a midline incision of the scalp using small scissors, starting 4-5 mm caudal to the skull and advancing forward to the front of the eyes. It is important that this incision be long enough that the skin will not interfere with the gluing of the head plate that is used to stabilize the mouse head during two-photon imaging (Figure 1). Identify the area to be thinned under a dissecting scope. Avoid areas of interest directly located over cranial sutures, as the skull is less stable in these areas and underlying large vessels and meninges will interfere with imaging.</li>
<li>Apply 100% ethanol followed by 10% solution of ferric chloride with a sterile cotton swab to dry the membranes on top of the skull and scrape them away using fine tweezers or a razor blade. Failure to remove the membranes results in unstable head plate attachment.</li>
<li>Place a thin layer of the Permabond 910 glue around the edges of the viewing window underneath the head plate. Place the head plate coated with glue over the area of interest on the animal's skull with light pressure (Figure 1). It is important that the head plate is only glued to the bone of the skull. Any skin or membranes that are caught between the head plate and the skull will decrease the stability of the bond. Apply a small amount of acrylate around the viewing window using a syringe to instantly bond the glue. Finally, apply a small amount of Loctite 454 to the edges of the viewing window to prevent leaks.</li>
<li>Screw the head plate to the animal holder (Figure 1) and check the stability of the head plate under a dissection scope by lightly probing with a pair of forceps. There should be no movement of the skull relative to the head plate. Place a drop of saline on the viewing window to ensure there are no leaks.</li>
</ol>
2. Preparing the Thinned Skull Cortical Window
<ol>
<li>In preparation for thinning the skull, dry the skull using a combination of sterile cotton swabs and compressed air. We initially thin the skull with a sterile IRF 007 drill bit in a Microtorque II drill set at 4000 rpm. Very gently, begin thinning a 2-2.5 mm diameter circular area of the skull. Use only light sweeping motions nearly parallel to the skull; use no direct downward pressure. Stop drilling every 20-30 seconds to remove bone dust using the compressed air. These breaks in drilling allow the skull to cool so there is no heat-induced damage to the underlying brain tissue.
<ol class="lalpha">
<li>	As the drilling progresses, notice the transition to the moister spongy bone layer (i.e. the "diploe"). Once this layer is reached, exercise extra caution with the drill.</li>
</ol></li>
<li>Occasionally check the thinness of the skull by placing saline over the thinned area and viewing under the dissecting scope. Saline will further enable heat dissipation. As the skull is thinned, smaller vasculature becomes visible.</li>
<li>Use a sterile dental microblade to achieve the final thinning under saline, as the microblade provides much more tactile feedback about the stability of the skull. This allows for much thinner preparations than with the drill alone. From our experience, the optimal skull thickness is 10-30 &mu;m.
<ol class="lalpha">
<li>	It is important to check the thinness of the skull under epifluorescence multiple times during the first few attempts at making a thin skull window. The sharpness and depth of visible microglia and vasculature give a good indication as to when the window is ready for imaging.</li>
</ol></li>
<li>	Once the window is ready, glue a custom-made rectangular piece of #0 coverglass with a dimension of less than 2 mm on each side over the window. Using a 1.0 mm diameter glass pipette, place a small drop of cyanoacrylate glue on the thinned skull area and carefully lower down the small piece of coverslip, then press gently against the skull to squeeze out excess amount of the glue. It is important to avoid bubble formation between the glass and the glue since trapped air will cause the area underneath to become optically opaque. The cyanoacrylate glue is used because it remains transparent when dry and also prevents bone and membrane regrowth keeping the skull under the window thin and translucent. If there is any glue on top of the cover glass, use the microblade to carefully remove it once the cover glass is in place and the glue is dry.</li>
<li>An analgesic (for example, Buprenorphine 2 mg/kg sub-cutaneously) is administered immediately after the surgery and re-administered at any signs of pain, including reluctance to move, eat or drink, weight loss, salivation, piloerection, respiratory sounds, etc.</li>
</ol>
3. Two-photon Imaging
<ol>
<li>In preparation for two-photon imaging, cover the TSCW with saline and locate the area of interest under epifluorescence (Figure 2). To enable subsequent imaging of the area, take a picture using a photographic camera.</li>
<li>For two-photon imaging, we use a custom-made two-photon microscope6 with a Ti:Sapphire laser tuned to 920 nm. Fluorescence is detected by using a photomultiplier tube in whole-field detection mode and a 580/180 emission filter. A 20X water-immersion lens (0.95 N.A.) is used throughout the imaging session. The maximum output of the laser power at the objective is set between 50 and 65 mW.</li>
<li>Select an area of interest in cortical layers I or II (up to 150 &mu;m below the pial surface). To facilitate re-imaging, take pictures of the same field of view at 1X, 2X, and 3X digital zoom. Blood vessels can serve as landmarks to easily find the same field of view during subsequent imaging sessions. Under a zoom of 3X, multiple z-stacks with 80-90 Z-steps in each stack and a 0.69 &mu;m step can be acquired every 5 minutes for up to 30 minutes, which enables a good sampling of microglial morphology and behavior over time (Figure 3). Between acquisitions of z-stacks, one should verify the level of saline over the TSCW, as well as the animal's depth of anesthesia.</li>
</ol>
4. Injection of the HIV-1 Neurotoxin, Tat
<ol>
<li>First, silanize the inner lining of a 35 gauge needle and 10 &mu;L Microvolume syringe to prevent Tat deposition. Withdraw the silanizing solution (Sigmacote) until it fills both the needle and syringe. Allow the solution to sit for 5 minutes at room temperature. Empty the solution from the syringe, remove the plunger, and allow the syringe and needle to dry completely. Finally, wash the syringe and needle thoroughly with deionized water.</li>
<li>Perform a small craniotomy 0.5 mm in diameter either 3 mm rostral or lateral to the TSCW where two-photon images have been obtained, using a smaller drill bit and carefully thinning the skull until a pair of forceps with sharp tips can remove the thinned layer of bone away easily. We use a 10 &mu;L Microvolume syringe fitted with 35 gauge needle controlled by an Ultramicropump III syringe pump mounted on a 3-axis micromanipulator and after lining up the needle tip to the craniotomy, it is carefully advanced to a depth of 700 to 900 &mu;m below the pial surface where 3 &mu;L Tat1-72 (or other pro-inflammatory agents) or saline (or control vehicle) is delivered at 80 nL/min.</li>
</ol>
5. Animal Housing
<ol>
<li>If the animal is to be imaged multiple times in a single day, cover the open area of the skull with a mixture of opthalmic ointment and Vaseline to prevent discomfort or damage to the skull in between imaging sessions. Disconnect the support bridge from the small head-plate (see Figure 1B) and place the animal in a warmed cage with easily accessible food and water. All animals are to be housed singly after surgery at all times. Once the imaging sessions for an animal are complete for a given day, carefully remove the entire head-plate and suture the skin back over the skull with #6 or smaller suture. Again, house the animals singly with easily accessible food and water between experimental days. Check the animals daily for any signs of stress, pain, or infection.</li>
</ol>
6. Representative Results
 <img src="/files/ftp_upload/2059/2059fig1.jpg" alt="Figure 1" /> 
Figure 1. Stabilization of animal for surgery and two-photon imaging (A) and head plate design (B).
 <img src="/files/ftp_upload/2059/2059fig2.jpg" alt="Figure 2" /> 
Figure 2. GFP-labeled microglia visualized with epifluorescence.
 <img src="/files/ftp_upload/2059/2059fig3.jpg" alt="Figure 3" /> 
Figure 3. GFP-labeled microglia visualized with two-photon imaging after implantation of the TSCW, approximately 50 &mu;m below the pial surface. The single two-photon sections taken 15-30 minutes after implantation of the TSCW (day 0), as well as 7 and 17 days later, demonstrate that the windows stays clear while microglia remain inactivated in absence of inflammatory insults. The z-projections taken 6 and 24 hours post-injection of the HIV neurotoxin tat exhibit impressive morphologic changes of activated microglia. Scale bar: 10 &mu;m.

<ol>
	<li>Mostany, R., &amp;amp, P. o. r. t. e. r. a. -. C. a. i. l. l. i. a. u., C, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+craniotomy+surgery+procedure+for+chronic+brain+imaging.">A craniotomy surgery procedure for chronic brain imaging.</a> J Vis Exp. 12, (2008).</li><li>Holtmaat, A. <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> Nat Protoc. 4, (2009).</li><li>Yang, G. <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 Protoc. 5, 213-21 (2010).</li><li>Jung, S. <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+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol Cell Biol. 20, 4106-41 (2000).</li><li>Mrsic-Flogel, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostatic+regulation+of+eye-specific+responses+in+visual+cortex+during+ocular+dominance+plasticity.">Homeostatic regulation of eye-specific responses in visual cortex during ocular dominance plasticity.</a> Neuron. 54, 961-96 (2007).</li><li>Majewska, A., Yiu, G., &amp;amp, Y. u. s. t. e., R, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+custom-made+two-photon+microscope+and+deconvolution+system.">A custom-made two-photon microscope and deconvolution system.</a> Pflugers Arch. 441, 2-3 (2000).</li><li>Sharer, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+of+HIV-1+infection+of+the+central+nervous+system.">Pathology of HIV-1 infection of the central nervous system.</a> A review. J Neuropathol Exp Neurol. 51, (1992).</li><li>Everall, I. P., Aliberti, J., &amp;amp, B. a. l. c. e. r. z. y. k., M, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+density+in+the+superior+frontal+and+temporal+gyri+does+not+correlate+with+the+degree+of+human+immunodeficiency+virus-associated+dementia.">Neuronal density in the superior frontal and temporal gyri does not correlate with the degree of human immunodeficiency virus-associated dementia.</a> Acta Neuropathol (Berl. 88, 538-53 (1994).</li><li>Masliah, E., Banati, R. B., &amp;amp, Q. u. i. n. l. a. n., M, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+injury+is+a+pathological+substrate+for+human+immunodeficiency+virus-related+cognitive+disorders.">Dendritic injury is a pathological substrate for human immunodeficiency virus-related cognitive disorders.</a> Ann Neurol. 42, 963-96 (1997).</li><li>Everall, I. P., Banati, R. B., &amp;amp, U. p. e. n. d. e. r., B, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+synaptic+density+is+reduced+in+mild+to+moderate+human+immunodeficiency+virus+neurocognitive+disorder.">Cortical synaptic density is reduced in mild to moderate human immunodeficiency virus neurocognitive disorder.</a> Brain Pathol. 9, (1999).</li><li>Glass, J. D., Fedor, H., Wesselingh, S. L., &amp;amp, M. c. A. r. t. h. u. r., C, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+quantitation+of+human+immunodeficiency+virus+in+the+brain:+correlations+with+dementia.">Immunocytochemical quantitation of human immunodeficiency virus in the brain: correlations with dementia.</a> Ann Neurol. 38, (1995).</li><li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, (2000).</li></ol>

No conflicts of interest declared.

There is growing consensus that the actual substrate for HIV-1 associated neurocognitive deficits (HAND) is destruction of synaptic architecture, presumably caused by pro-inflammatory mediators released from HIV-1 infected mononuclear phagocytes. Microglial activation, multinucleated giant cells, and gliosis due to astrocytic hypertrophy are considered nonspecific hallmarks of HIV-1 infection and inflammation in the CNS 7. In contrast, the severity of premortem neurologic disease has been correlated with loss of synaptic complexity, as well as macrophage burden in the CNS 8,9,10,11. However, dynamic interactions between microglia, peripheral infiltrating macrophages, and neurons in patients with HAND simply cannot be modeled using conventional static processing obtained from conventional immunocytochemical studies. Recent studies from our laboratories have suggested that at least some of these interactions between peripheral and central mononuclear cells and neurons can be modeled in part by stereotactic injection of the HIV-1 protein Tat1-72 into brain parenchyma (Lu, Marker, Tremblay, Qi, and Gelbard, unpublished data). We therefore decided to apply in vivo two photon imaging to examine the interplay between monocyte-derived macrophages, brain resident microglia and neurons, in a tractable small animal model for neuroAIDS. While open-skull or thinned-skull preparations afford a single examination of cortical architecture for a brief period of time (hours), investigators interested in the time course of subacute events cannot use these preparations without artifactually activating microglia/macrophage due to surgical manipulation of the calvarial window. Here we demonstrate a simple way of circumventing these limitation by gluing a tiny piece of glass coverslip over the thinned skull area preventing the calvarium from regenerating in the TSCW as well as maintaining translucency for &ge;3 weeks. We further demonstrate that this technique allows us to monitor the behavior of peripheral monocytes entering cerebral microvasculature as well as brain-resident microglia in response to stereotactic injection of HIV Tat1-72 in the cortex of mice heterozygous for CX3CR1/GFP (to identify cells of mononuclear lineage). This technique can easily be adapted to use on mice with different genetic backgrounds; for example, we routinely use heterozygous CX3CR1/GFP X Thy-1 YFP 12 mice to investigate interactions between monocyte-derived macrophages, brain resident microglia and neurons in in vivo models of neuroinflammation relevant to HAND. Our TSCW preparation allows investigators to study cell-cell interactions between the periphery and CNS that may occur over periods of weeks, in which the animal can be repeatedly imaged before and after experimental treatment. Thus, each animal can serve as its own control. One caveat for this TSCW model is that the target cells under investigation need to be either genetically engineered to express enhanced fluorescent proteins (eXFP) or be labeled with a fluorescent dye without mechanical breach of the calvarium; and that eXFP expression must be robust enough to allow cellular architecture to be visualized after repeated imaging sessions over a period of weeks.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Fentanyl Citrate</td>
<td>&nbsp;</td>
<td>Hospira</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Midazolam hydrochloride</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Medetomidine hydrocholoride (Domitor)</td>
<td>&nbsp;</td>
<td>Pfizer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Heating pads</td>
<td>&nbsp;</td>
<td>Beyond Bodi Heat</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tobramycin and dexamethasone ophthalmic ointment (TobraDex)</td>
<td>&nbsp;</td>
<td>Alcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Povidone-iodine 10% solution</td>
<td>&nbsp;</td>
<td>Betadine</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ferric chloride 10% solution</td>
<td>&nbsp;</td>
<td>Ricca Chemical Company</td>
<td>3120-32</td>
<td>&nbsp;</td>
<tr>
<td>Methyl cyanoacrylate 910</td>
<td>&nbsp;</td>
<td>Permabond</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cyanoacrylate 454</td>
<td>&nbsp;</td>
<td>Loctite</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>TEETS denture material crosslinking methyl methacrylate</td>
<td>&nbsp;</td>
<td>Co-Oral-lte Dental Mfg CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microtorque II handpiece kit</td>
<td>&nbsp;</td>
<td>Pearson</td>
<td>R14-0002</td>
<td>&nbsp;</td>
<tr>
<td>IRF 007 drill bits</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>19008-07</td>
<td>&nbsp;</td>
<tr>
<td>Norland bendable microblade full radius</td>
<td>&nbsp;</td>
<td>Salvin Dental</td>
<td>BLAD-NORLAND-69</td>
<td>&nbsp;</td>
<tr>
<td>Cyanoacrylate</td>
<td>&nbsp;</td>
<td>The Original Superglue</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>#0 glass coverslip</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>63750-01</td>
<td>&nbsp;</td>
<tr>
<td>10 &mu;l Microvolume syringe</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>ILS010LT</td>
<td>&nbsp;</td>
<tr>
<td>UltraMicroPump III</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>UMP3-1</td>
<td>&nbsp;</td>
<tr>
<td>35 gauge NanoFil needle</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>NL35BL-2</td>
<td>&nbsp;</td>
<tr>
<td>Ball Mill, Carbide bits #1/4</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>501860</td>
<td>&nbsp;</td>
<tr>
<td>Sigmacote</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>SL2-100</td>
<td>&nbsp;</td>
<tr>
<td>Photomultiplier tube </td>
<td>&nbsp;</td>
<td>Hamamatsu</td>
<td>HC125-02</td>
<td>&nbsp;</td>
<tr>
<td>Ti:Sapphire laser Mai-Tai</td>
<td>&nbsp;</td>
<td>Spectra-Physics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a thin-skull window technique for chronic two-photon in vivo imaging of murine microglia in models of neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

Watch this Scientific Journal Video about A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation at JoVE.com

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

a-thin-skull-window-technique-for-chronic-two-photon-vivo-imaging

Research

JoVE Journal

Neuroscience

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

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

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

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

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

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

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

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 ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...