This work was supported by the National Multiple Sclerosis Society grant RG4595A1/T to DD and the NIH/NINDS grants NS051470, NS052189 and NS066361 to K.A. Figures and movies adapted and/or reprinted from Davalos et al., J Neurosci Methods. 2008 Mar 30;169(1):1-7 Copyright 2008, with permission from Elsevier.

1. Building the spinal stabilization device
<ol>
	<li>Order the Narishige STS-A Compact Spinal Cord Clamps and the Narishige MA-6N head holding adaptor.</li>
	<li>Custom design and make a stainless steel base plate to hold the two Narishige parts in alignment so that the animal's head is supported while its spinal column and tail are clamped. Keep in mind that the entire device should fit under the microscope lens usually on a lowered microscope stage.</li>
</ol>
2. Animal surgery
<ol>
	<li>Pre-heat the microscope chamber with an air heating device and maintain it at 37 &deg;C.</li>
	<li>Weigh the animal and make the anesthetic mix using 100 mg ketamine, 15 mg xylazine and 2.5 mg acepromazine per kg of body weight, diluted in 0.9% NaCl solution under sterile conditions.</li>
	<li>Anesthetize the animal by injecting the anesthetic mix (KXA) intraperitoneally. Regular toe pinching should be performed to ensure deep anesthesia throughout the experiment. Supplement with half the original dose hourly or as needed.</li>
	<li>Use a small animal heating pad to provide thermal support while performing the surgical procedure.</li>
	<li>Apply artificial tears ointment over the eyes to prevent corneal dehydration and damage.</li>
	<li>Shave the back of the animal first with a trimming device and then with a sharp blade to completely remove hair from the area around the incision.</li>
	<li>Remove shaved hairs and disinfect the shaved skin surface with an alcohol pad.</li>
	<li>Perform a small (~1.5 cm) longitudinal incision of the skin over the desired spinal level (thoracic vertebrae shown here).</li>
	<li>Expose the back musculature by carefully retracting the subcutaneous connective tissue.</li>
	<li>Carefully separate the paravertebral muscles from the spinal column at the desired level. Perform the fewest possible incisions to detach the muscles from both sides of each spinous process and then scrape the detached muscles away from the laminar surface.</li>
	<li>Choose the lamina that will be removed and prepare the sites of entry for the spinal clamps on each side of the spinal column, immediately rostral and caudal to the laminectomy. Carefully displace muscular tissue to make four pockets where the clamps will be inserted.</li>
	<li>Lift the spinal column with the straight toothed-tip forceps to allow safe insertion of the curved blunt-tip scissors under the lamina without touching the spinal cord. Perform the laminectomy by slowly cutting the bone on one side and then on the other side.</li>
	<li>Use a cotton swab or insert precut small gelfoam absorbable gelatin sponge pieces next to the incisions to limit minor bleeding. Use a small vessel cauterizer to control further bleeding if necessary. Use preheated sterile artificial cerebrospinal fluid (ACSF) to wash tissue.</li>
</ol>
3. Stabilization of the spinal column and preparation for in vivo imaging
<ol>
	<li>Place the animal on an elevation pad on the base plate of the spinal stabilization device and secure the head in the head holding adaptor.</li>
	<li>Place the spinal clamps of the STS-A device along the anterior-posterior axis of the animal in the pre-prepared pockets flanking the laminectomy (Figure 1). The two clamps should be placed at an angle of ~45&deg; relative to the animal's rostro-caudal axis to allow enough space for lowering a water immersion lens over the exposed spinal cord (Figure 1).</li>
	<li>Place the third clamp of the STS-A device at the base of the tail so the animal's body can be suspended in the air after removal of the elevation pad for the duration of the imaging experiments (Figure 1).</li>
	<li>Build a small well of Gelseal (Amersham Biosciences Corp.) around the exposed spinal cord to facilitate the maintenance of the spinal cord in ACSF and the immersion of the microscope lens in this solution for in vivo imaging.</li>
</ol>
4. In vivo imaging of the mouse spinal cord with two-photon microscopy
<ol>
	<li>Transfer the animal on the spinal stabilization device inside the preheated chamber covering the microscope and secure it on a lowered microscope stage placing the laminectomy straight under the lens (Figure 1).</li>
	<li>Lower a water-immersion lens carefully into the ACSF solution making sure that it does not touch the spinal clamps or the Gelseal.</li>
	<li>Use epifluorescence to identify the area of interest and focus on it. Switch to laser scanning mode and perform in vivo imaging using the appropriate two-photon laser excitation wavelength, dichroics and bandpass filters for the fluorophores present in the imaged tissue.</li>
</ol>
5. Repetitive imaging and post-operative care
<ol>
	<li>At the end of imaging experiments, remove the mouse from the spinal stabilization device and carefully wipe the Gelseal from the area around the laminectomy. Clean the area well.</li>
	<li>Restore and suture the back muscles over the laminectomy.</li>
	<li>Restore and suture the skin over the laminectomy, swab it with betadine.</li>
	<li>Provide 1 ml Lactated Ringers solution (Baxter Healthcare) as a nutrient and hydration supplement, as well as analgesic treatment subcutaneously (0.1 mg/kg buprenorphine).</li>
	<li>Administer antiseptic treatment intraperitoneally (0.03 ml per mouse, 2.27% enrofloxacin injectable antibacterial solution).</li>
	<li>Place animal on a heating pad until full recovery from anesthesia and subsequently house it individually.</li>
	<li>Repeat antiseptic administration daily for the first 3-5 days after surgery and analgesic treatment every 8-12 hours for 2-3 days post-operatively. Monitor animal daily to ensure normal behavior and full recovery.</li>
	<li>For re-imaging through the same laminectomy, reopen the sutured skin and muscles and repeat the steps described in sections 2 - 4.</li>
	<li>Re-locate the previously imaged area by using the blood vasculature as a map as previously described 10.</li>
</ol>
6. Representative results
All animal procedures were performed under the guidelines set by institutional Animal Care and Use Committees at the University of California, San Francisco and are in accordance with Federal regulations. A picture of the spinal stabilization device and a schematic showing the positioning of a mouse on the device under a microscope lens is shown in Figure 1. Allowing adequate room for breathing movements underneath the animal's body ensures stable in vivo imaging in the spinal cord. Figure 2 shows the close relationship between microglia and the vasculature as it was imaged in vivo in the spinal cord of Cx3cr1GFP/+ transgenic mice18, in which microglia are endogenously labeled with GFP. Figure 3 shows examples of repetitive in vivo imaging as it was performed in the same spinal cord areas in mice expressing a fluorescent protein in spinal axons (YFP-H line 3) and microglia (in the Cx3cr1GFP/+ mice).
 
 
<img alt="Figure 1" src="/files/ftp_upload/2760/2760fig1.jpg" /> 
Figure 1. Stabilization of the mouse spinal column for in vivo imaging using two-photon microscopy. (A) A custom-made steel base plate is used to support and align the STS-A Narishige compact spinal cord clamps and the MA-6N Narishige head holding adaptor as shown here. (B) Proper positioning of adult transgenic mice anesthetized with a KXA anesthetic on the spinal stabilization device. The insert shows the placement of the spinal clamps immediately rostrally and caudally to the laminectomy and the exposed spinal cord tissue.
<img alt="Figure 2" src="/files/ftp_upload/2760/2760fig2.jpg" /> 
Figure 2. In vivo imaging of high density microglial cells and blood vessels in the spinal cord of anesthetized mice. Projected but non-aligned z-stacks of (A) highly dense GFP-positive microglia (green) in the spinal cord of a CX3CR1GFP/+ mouse in close proximity with blood vessels (red, labeled with i.v. injection of rhodamine dextran). (B) High magnification image labeled as in (A) of a single microglial cell attached to the wall of a blood vessel with processes extended around the vessel and towards the spinal cord parenchyma. Scale bars, 10&mu;m.
<img alt="Figure 3" src="/files/ftp_upload/2760/2760fig3.jpg" /> 
Figure 3. Repetitive in vivo imaging of the same axonal segments and microglia as they were relocated and reimaged in the spinal cord of anesthetized mice on different days. (A) YFP-labeled axons on days 0 and 5. (B). The same vascular structures and microglial cells around them imaged in vivo in the spinal cord of Cx3cr1GFP/+ mice on days 0 and 1. Scale bars, 10&mu;m.
Movie 1. Representative timelapse sequence acquired in vivo from the mouse spinal cord. This sequence shows in detail fine microglial process dynamics (green) and their interactions with the vasculature (red, labeled with i.v. injection of rhodamine dextran) over time within a tissue densely populated with fluorescent structures. The raw images were only corrected for background noise, brightness and contrast and the timelapse movie was constructed by z-projecting sequentially acquired image stacks, without image alignment, averaging or z-selection of individual planes. Blood vessels go through a similar range of z planes as the images of microglia. Z plane depth: 38 &mu;m. <a alt="Movie 1" href="https://www.jove.com/files/ftp_upload/2760/Davalos_Movie_1.mov">Click here to watch the movie.</a>
Movie 2. Timelapse sequence demonstrating the raw stability of the imaging method at the level of a single z-plane. Fast acquisition of the same single z-plane in the spinal cord of CX3CR1GFP/+ mice placed on the spinal stabilization device under KXA anesthesia, demonstrates minimal image displacement between consecutive frames at a scanning rate of 1 frame/s that is faster than the breathing rate of the mouse. The minor residual displacement is probably due to heartbeat. <a alt="Movie 2" href="https://www.jove.com/files/ftp_upload/2760/Davalos_Movie_2.mov">Click here to watch the movie.</a>

<ol>
	<li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248, 73-76 (1990).</li><li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu. Rev. Biochem. 67, 509-544 (1998).</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, 41-51 (2000).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nat. Methods. 2, 932-940 (2005).</li><li>Germain, R. N., Miller, M. J., Dustin, M. L., Nussenzweig, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+the+immune+system:+progress,+pitfalls+and+promise.">Dynamic imaging of the immune system: progress, pitfalls and promise.</a> Nat. Rev. Immunol. 6, 497-507 (2006).</li><li>Misgeld, T., Kerschensteiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+the+diseased+nervous+system.">In vivo imaging of the diseased nervous system.</a> Nat. Rev. Neurosci. 7, 449-463 (2006).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50, 823-839 (2006).</li><li>Davalos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+mediates+rapid+microglial+response+to+local+brain+injury+in+vivo.">ATP mediates rapid microglial response to local brain injury in vivo.</a> Nat. Neurosci. 8, 752-758 (2005).</li><li>Nimmerjahn, A., Kirchhoff, F., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+microglial+cells+are+highly+dynamic+surveillants+of+brain+parenchyma+in+vivo.">Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.</a> Science. 308, 1314-1318 (2005).</li><li>Grutzendler, J., Kasthuri, N., Gan, W. B. <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+dendritic+spine+stability+in+the+adult+cortex.">Long-term dendritic spine stability in the adult cortex.</a> Nature. 420, 812-816 (2002).</li><li>Svoboda, K., Denk, W., Kleinfeld, D., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dendritic+calcium+dynamics+in+neocortical+pyramidal+neurons.">In vivo dendritic calcium dynamics in neocortical pyramidal neurons.</a> Nature. 385, 161-165 (1997).</li><li>Trachtenberg, J. T. <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+imaging+of+experience-dependent+synaptic+plasticity+in+adult+cortex.">Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex.</a> Nature. 420, 788-794 (2002).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytic+Ca2++signaling+evoked+by+sensory+stimulation+in+vivo.">Astrocytic Ca2+ signaling evoked by sensory stimulation in vivo.</a> Nat. Neurosci. 9, 816-823 (2006).</li><li>Christie, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+arrest+of+individual+senile+plaques+in+a+model+of+Alzheimer's+disease+observed+by+in+vivo+multiphoton+microscopy.">Growth arrest of individual senile plaques in a model of Alzheimer's disease observed by in vivo multiphoton microscopy.</a> J. Neurosci. 21, 858-864 (2001).</li><li>Tsai, J., Grutzendler, J., Duff, K., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrillar+amyloid+deposition+leads+to+local+synaptic+abnormalities+and+breakage+of+neuronal+branches.">Fibrillar amyloid deposition leads to local synaptic abnormalities and breakage of neuronal branches.</a> Nat. Neurosci. 7, 1181-1183 (2004).</li><li>Grutzendler, J., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+synaptic+plasticity+and+pathology+in+the+living+mouse+brain.">Two-photon imaging of synaptic plasticity and pathology in the living mouse brain.</a> NeuroRx. 3, 489-496 (2006).</li><li>Takano, T., Han, X., Deane, R., Zlokovic, B., Nedergaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+astrocytic+Ca2++signaling+and+the+microvasculature+in+experimental+mice+models+of+Alzheimer's+disease.">Two-photon imaging of astrocytic Ca2+ signaling and the microvasculature in experimental mice models of Alzheimer's disease.</a> Ann. N. Y. Acad. Sci. 1097, 40-50 (2007).</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+CX3CR1+Function+by+Targeted+Deletion+and+Green+Fluorescent+Protein+Reporter+Gene+Insertion.">Analysis of Fractalkine Receptor CX3CR1 Function by Targeted Deletion and Green Fluorescent Protein Reporter Gene Insertion.</a> Mol. Cell. Biol. 20, 4106-4114 (2000).</li><li>Kerschensteiner, M., Schwab, M. E., Lichtman, J. W., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+axonal+degeneration+and+regeneration+in+the+injured+spinal+cord.">In vivo imaging of axonal degeneration and regeneration in the injured spinal cord.</a> Nat. Med. 11, 572-577 (2005).</li><li>Kim, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+microscopy+imaging+of+intact+spinal+cord+and+cerebral+cortex+reveals+requirement+for+CXCR6+and+neuroinflammation+in+immune+cell+infiltration+of+cortical+injury+sites.">Two-photon laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury sites.</a> J. Immunol. Methods. 352, 89-100 (2010).</li><li>Shakhar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+T+cell-dendritic+cell+interactions+precede+the+development+of+both+tolerance+and+immunity+in+vivo.">Stable T cell-dendritic cell interactions precede the development of both tolerance and immunity in vivo.</a> Nat. Immunol. 6, 707-714 (2005).</li><li>Tadokoro, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+T+cells+inhibit+stable+contacts+between+CD4++T+cells+and+dendritic+cells+in+vivo.">Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo.</a> J Exp Med. 203, 505-511 (2006).</li><li>Lindquist, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+dendritic+cell+networks+in+vivo.">Visualizing dendritic cell networks in vivo.</a> Nat. Immunol. 5, 1243-1250 (2004).</li><li>Schwickert, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=vivo+imaging+of+germinal+centres+reveals+a+dynamic+open+structure.">vivo imaging of germinal centres reveals a dynamic open structure.</a> Nature. 446, 83-87 (2007).</li></ol>

We have nothing to disclose.

The method described here allows for stable and repetitive in vivo imaging of densely populated fluorescent cellular structures in the spinal cord of anesthetized mice using two-photon microscopy. The achieved stability is a result of a custom-made spinal stabilization device and an anesthetic regimen that reduces respiratory-induced movement artifact. The spinal stabilization device allows breathing space underneath the mouse body and can be built using commercially available spinal clamps and head mounting pie...

<table border="1">
	<tbody>
		<tr>
			<td>	 Name of the reagent </td>
			<td>	 Company </td>
			<td>	 Catalogue number </td>
			<td>	 Comments </td>
		</tr>
		<tr>
			<td>Rhodamine B dextran</td>
			<td>Invitrogen</td>
			<td>D1841</td>
			<td>70 kDa, diluted in 
			ACSF (3% w/v)</td>
		</tr>
		<tr>
			<td>Ketamine HCl</td>
			<td>Bionichepharma</td>
			<td>NDC No: 67457-001-10</td>
			<td>Injectable, 50mg/ml</td>
		</tr>
		<tr>
			<td>Anased</td>
			<td>Lloyd Labs</td>
			<td>NADA No: 139-236</td>
			<td>Xylazine injectable, 
			20mg/ml</td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Vedco</td>
			<td>NADA No: 117-531</td>
			<td>Injectable,10mg/ml</td>
		</tr>
		<tr>
			<td>Artificial tears 
			ointment</td>
			<td>Phoenix 
			pharmaceutical</td>
			<td>NDC No: 57319-760- 
			25</td>
			<td>Lubricant</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Fisher</td>
			<td>19-061617</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>McPherson-Westcott 
			Scissors</td>
			<td>World Precision 
			Instruments</td>
			<td>555500S</td>
			<td>Curved, blunt-tip 
			scissors</td>
		</tr>
		<tr>
			<td>Straight Forceps</td>
			<td>World Precision 
			Instruments</td>
			<td>555047FT</td>
			<td>Toothed tip forceps</td>
		</tr>
		<tr>
			<td>Small vessel cauterize</td>
			<td>Fine Science Tools</td>
			<td>18000-00</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gelfoam</td>
			<td>Pharmacia,Pfizer Inc.</td>
			<td>Mixer Mill MM400</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compact spinal cord 
			clamps</td>
			<td>Narishige</td>
			<td>STS-A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Head holding adaptor</td>
			<td>Narishige</td>
			<td>MA-6N</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gelseal</td>
			<td>Amersham 
			Biosciences Corp.</td>
			<td>80-6421-43</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lactated Ringers</td>
			<td>Baxter Healthcare</td>
			<td>2B8609</td>
			<td>&nbsp;</td>
		</tr>

		<tr>
			<td>Buprenex</td>
			<td>Reckit Benckiser 
			Pharmaceuticals Inc.</td>
			<td>NDC No: 12496- 
			6757-1</td>
			<td>Buprenorphine, 
			injectable</td>
		</tr>
		<tr>
			<td>Baytril</td>
			<td>Bayer</td>
			<td>NADA 140-913</td>
			<td>Enrofloxacin, 
			antibacterial injectable 
			2.27% (20ml)</td>
		</tr>
		<tr>
			<td>Heating pad - Large</td>
			<td>Fine Science Tools</td>
			<td>21060-10</td>
			<td>&nbsp;</td>
		</tr>

	</tbody>
</table>

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.

Watch this Scientific Journal Video about In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy at JoVE.com

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 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 ...

in-vivo-imaging-of-the-mouse-spinal-cord-using-two-photon-microscopy

Research

JoVE Journal

Neuroscience

23.7K Views.  University of California, San Francisco. 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.

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

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 ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

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 ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

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 ...

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...