Name: implantation of a cranial window for repeated in vivo imaging in awake mice
Uploaded: 2021-06-22T15:00:00
Description: Watch this Scientific Journal Video about Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice at JoVE.com

All animal experiments were performed in accordance with guidelines approved by the IACUC at the University of Nebraska Medical Center.
1. Before surgery
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	<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...

<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. 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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.
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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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			<td>Mylan Institutional, LLC.</td>
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			<td>Dissecting Microscope</td>
			<td>Nikon</td>
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			<td>Duralay Liquid&#160; (dental cement liquid)</td>
			<td>Patterson Dental</td>
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			<td></td>
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		</tr>
		<tr>
			<td>Eye Ointment</td>
			<td>Dechra</td>
			<td>17033-211-38</td>
			<td>Surgery Supply</td>
		</tr>
		<tr>
			<td>Fiber Lite High Intensity Illuminator</td>
			<td>Dolan-Jenner Industries</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Forceps (Large)</td>
			<td>World Precision Instruments</td>
			<td>14099</td>
			<td>Surgery Tools</td>
		</tr>
		<tr>
			<td>Forceps (Small)</td>
			<td>World Precision Instruments</td>
			<td>501764</td>
			<td>Surgery Tools</td>
		</tr>
		<tr>
			<td>GCaMP6f B6; 129S-Gt(ROSA)26Sortm95.1(CAGGCaMP6f)Hze/J</td>
			<td>The Jackson Laboratory</td>
			<td>Stock No: 024105</td>
			<td>Mouse line</td>
		</tr>
		<tr>
			<td>Germinator</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>GLAST-CreER Tg(Slc1a3-cre/ERT) 1Nat/J</td>
			<td>The Jackson Laboratory</td>
			<td>Stock No: 012586</td>
			<td>Mouse Line</td>
		</tr>
		<tr>
			<td>Headplate</td>
			<td>Neurotar</td>
			<td>Model 1, Model 3</td>
			<td>Surgery Supply</td>
		</tr>
		<tr>
			<td>Hemostatic forceps</td>
			<td>World Precision Instruments</td>
			<td>501705</td>
			<td>Surgery Tools</td>
		</tr>
		<tr>
			<td>Holder for 15o Pointed Blade</td>
			<td>World Precision Instruments</td>
			<td>501247</td>
			<td>Surgery Tools</td>
		</tr>
		<tr>
			<td>Holder for Scalpel Blades</td>
			<td>World Precision Instruments</td>
			<td>500236</td>
			<td>Surgery Tools</td>
		</tr>
		<tr>
			<td>Iodine Prep Pads</td>
			<td>Avantor</td>
			<td>15648-926</td>
			<td>Surgery Supply</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal</td>
			<td></td>
			<td>Surgery Supply</td>
		</tr>
		<tr>
			<td>Isoflurane table top system with Induction Box</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Isoflurane Vaporizer</td>
			<td>SurgiVet</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Krazy Glue</td>
			<td>Office Depot</td>
			<td>KG517</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Loctite 401</td>
			<td>Henkel</td>
			<td>40140</td>
			<td>fast-curing instant adhesive</td>
		</tr>
		<tr>
			<td>Loctite 454</td>
			<td>Fisher Scientific</td>
			<td>NC9194415</td>
			<td>cyanoacrylate adhesive gel</td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Multiphoton Microscope</td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Nitrogen</td>
			<td>Matheson</td>
			<td>NI M200</td>
			<td>Gas</td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>Matheson</td>
			<td>OX M250</td>
			<td>Gas</td>
		</tr>
		<tr>
			<td>Picospritzer</td>
			<td>Parker</td>
			<td></td>
			<td>intracellular microinjection dispense system</td>
		</tr>
		<tr>
			<td>Pipette Tips</td>
			<td>Rainin</td>
			<td>17014340</td>
			<td>Surgery Supply</td>
		</tr>
		<tr>
			<td>Rodent Hair Trimmer</td>
			<td>Wahl</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Saline (0.9% Sodium Chloride)</td>
			<td>Med Vet International</td>
			<td>RX0.9NACL-30BAC</td>
			<td>Surgery Supply</td>
		</tr>
		<tr>
			<td>Scalpel Blades, Size 11</td>
			<td>Integra</td>
			<td>4-111</td>
			<td>Surgery Tools</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>World Precision Instruments</td>
			<td>503667</td>
			<td>Surgery Tools</td>
		</tr>
		<tr>
			<td>Stereotaxic Instrument</td>
			<td>Stoelting</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Sugi Sponge Strips (sponge strips)</td>
			<td>Kettenbach Dental</td>
			<td>31002</td>
			<td>Surgery Supply</td>
		</tr>
		<tr>
			<td>SURGIFOAM (gel foam)</td>
			<td>Ethicon</td>
			<td>1972</td>
			<td>Surgery Supply</td>
		</tr>
		<tr>
			<td>Syringe with 26 G Needle</td>
			<td>BD</td>
			<td>309625</td>
			<td>Surgery Supply</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma Aldrich</td>
			<td>T5648-1G</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Ti:Sapphire Laser</td>
			<td>Coherent</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Transfer Pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-711-9AM</td>
			<td>Surgery Supply</td>
		</tr>
		<tr>
			<td>Water Blanket</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Xylocaine MPF with Epinephrine (1:200,000), 10 mg/mL</td>
			<td>Fresenius Kabi USA</td>
			<td></td>
			<td>Drug</td>
		</tr>
	</tbody>
</table>

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 ...

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 ...

The protocol describes how to perform viral injections and implant a cranial window for imaging of brain cells in awake, head-restrained mice. The advantage of this method is that structure and activity of neurons and astrocytes can be imaged repeatedly over multiple sessions. This approach can be used to determine how neural cells are altered in models of neurodevelopmental, or neurodegenerative disorders, or whether experience-dependent plasticity is impaired.Begin by securing an anesthetized mouse to a stereotaxic frame using the snout clamp and ear bars. Using sterile surgical tools, cut and remove the skin from the frontal sutures anterior to the bregma to posteriorly to lambda. Using a sterile size 11 carbon steel surgical blade, gently scrape all the connective tissue from the skull.With the help of a dental drill, gradually drill the skull bone along the outline of the opening in concentric circles to facilitate even thinning of the bone. After each concentric pass, add a drop, or two of saline to the drilled area, and allow the saline to sit for at least 10 seconds before resuming the drilling. Gently push on the central area of the skull bone with fine forceps to check that the skull is adequately thinned and moves.Ensure that the underlying vasculature where the drilling occurred is intact, and that the bone is not cracked. Carefully insert a miniature 15-degree pointed blade into the thinned bone and cut and use the gel foam soaked in saline to stop any bleeds that may occur. Using forceps, carefully lift and remove the bone, taking care to not damage the dura mater.For the injection, fill a sterile beveled glass pipette with a 20-micrometer tip with the virus mixture. Then lower the pipette to touch the surface of the brain, and continue to lower for an additional 200 to 300 micrometers for layer 2/3 injections. Using an intracellular microinjection dispense system, pressure inject the system 12 to 15 times over two minutes.For the cranial window implantation, place the cover glass over the opening in the skull. Use forceps to ensure that the glass is flush over the opening. To seal the edges of the glass window to the skull, apply cyanoacrylate adhesive gel around the perimeter of the glass surface.Over the adhesive gel, apply a layer of glue. Then add a layer of dental cement liquid. When dental cement hardens, apply a thin layer of glue around the central opening of the helicopter type head plate, and place the helicopter type head plate of the appropriate size over the cover glass.Allow the glue to dry. Add dental cement powder in a 1.5-milliliter microfuge tube up to the 0.1 milliliter mark. Mix seven to eight drops of fast-curing instant adhesive into the powder, and draw the resultant mixture into a one-milliliter syringe with a 19 gauge needle that has been cut to create a larger opening.Inject the powder 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 head plate to the skull. Wrap the mouse in cloth, and secure it via its head plate to the head fixation arm of the airlifted home cage, then leave the home cage exposed to light.After a habituation time of 15 minutes, remove the mouse from the home cage. Using the wide field mode of the microscope, select two to three positions of easily identifiable vasculature. Save the images of the blood vessels, and record the x and y-coordinates that appear on the motor controller.Image the synaptic structures of neurons, and GCaMP6f activity in astrocytes. In the representative analysis, the quality of the cranial window was evaluated with repeated imaging of dendrites and GCaMP6f-expressing astrocytes over days. In a good window, the neuronal structures appeared crisp with clearly visible dendritic spines.Two new spines appeared on day two of imaging. Only one spine persisted and was visible on day five. Calcium activity was studied in astrocytes expressing GCaMP6f at different time points.A global event encompassing the entire cell was witnessed during a locomotion bout. The two critical steps of this protocol are that the bone needs to be adequately thinned prior to removal of the bone, and the proper placement of the cover glass over the opening. Imaging can be combined with behavioral analysis, either during imaging, or post-behavior, such as learning a new motor skill, to determine how neuronal and astrocyte structure and function is modulated by learning.

Research

JoVE Journal

Neuroscience

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

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

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Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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

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 ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

Piezo High Accuracy Surgical Osteal Removal (PHASOR): A Technique for Improved Cranial Window Surgery in Mice

Piezo High Accuracy Surgical Osteal Removal PHASOR: A Technique for Improved Cranial Window Surgery in Mice

Multiphoton microscopy has been widely adapted for imaging neurons in vivo. Repeated imaging requires implantation of a cranial window or repeated ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

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

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 ...