The authors would like to thank M. Steczkowski for voice recordings, M. Borczyk for drawings, A. Tr&#261;bczy&#324;ska for virus production, M. Zi&#243;kowska for genotyping and A. Mirgos for assistance with filming. K.R. acknowledges the kind gift of the recombinant adeno-associated virus (rAAV) expressing fluorescent protein mCherry under the control of CaMK promoter from K. Deisseroth. This project was carried out at the core facilities of Laboratory of Animal Models and Laboratory of Tissue Structure and Function, Centre of Neurobiology, Nencki Institute of Experimental Biology, with the use of CePT infrastructure financed by the European Union &#8211; the European Regional development Fund within the Operational Programme &#34;Innovative Economy&#34; for 2007-2013. This work was supported by grants from National Science Centre: Sonata Bis 2012/05/E/NZ4/02996, Harmonia 2013/08/M/NZ3/00861, Symfonia 2013/08/W/NZ24/00691 to K.R. and Sonata Bis 2014/14/E/NZ4/00172 to R.C.

All experimental procedures described below were approved by Local Ethical Committee at the Nencki Institute of Experimental Biology, Polish Academy of Sciences.
Note: Some of the scenes in the associated video are accelerated. Speed factor is indicated in these scenes.
1. Surgery Preparation
<ol>
	<li>Sterilize all tools, glass containers for liquids and cotton swabs in the autoclave. Use dispensable gloves. Clean the surgical table, the stereotaxic frame and all the surrounding area with 70% ethanol. Use a sterile surgical pad to create a sterile space for all the sterilized equipment. Cut gelfoam into small pieces and soak them in sterile saline. 
	Note: According to the Guide for the Care and Use of Laboratory Animals, ethanol is neither a sterilant nor a high-level disinfectant. It should only be used as a cleaning/defatting agent on previously sterilized surfaces.</li>
	<li>Put the animal in the induction chamber and set the isoflurane level to 5% and oxygen flow to 2 L/min. This procedure should take about 3 min.</li>
	<li>Take the animal out of the induction chamber. Use tail or toe pinches in order to ensure that the animal is fully sedated.</li>
	<li>Using a precise trimmer shave the hair from the back of the head (between the ears) up to the eyes.</li>
	<li>Place the animal in the stereotaxic frame and stabilize the head with ear bars.</li>
	<li>Set anesthesia levels to 1.5-2% isoflurane and 0.3 L/min oxygen.</li>
	<li>Apply the eye ointment.</li>
	<li>Inject the animal subcutaneously with Tolfedine (4 mg/kg), Butomidor (2 mg/kg) and Baytril (5 mg/kg) to prevent inflammation, pain and infection, respectively.</li>
	<li>Inject the animal intramuscularly with Dexamethasone (0.2 mg/kg) to prevent brain swelling. 
	Note: It is possible to inject Dexamethasone subcutaneously or intraperitoneally to prevent muscle damage.</li>
	<li>Clean the skin using sterile cotton swabs with Betadine followed by 70% ethanol.</li>
	<li>Change the gloves and spray them with 70% ethanol. 
	Note: While using disposable gloves do not touch the sterile field. Touch the animal only with the tips of sterile surgical instruments and sterile swabs.</li>
</ol>
2. Cranial Window Surgery
<ol>
	<li>Lift the skin with forceps and using micro scissors incise the skin horizontally along the base of the head and then obliquely to the front point between the eyes. Remove the skin flap.</li>
	<li>Apply lidocaine ointment with a sterile swab on the periosteum to prevent excessive bleeding and pain.</li>
	<li>Use sterile cotton swabs or a scalpel to remove the periosteum. Dry the skull with sterile swabs.</li>
	<li>Using a sterile needle apply dense cyanoacrylate glue on the skin edges to immobilize them and to prevent from contact with dental cement. Wait for the glue to dry.</li>
	<li>Lay a sterile 3 mm coverglass over the skull anteriorly to the lambdoid suture. Center the coverslip at RSC coordinates: AP, bregma -2.8; ML, bregma 0. Mark the coverslip edges by scratching the skull surface with a sterile needle. Put the coverglass back into the sterile container with 70% ethanol.</li>
	<li>Use a high-speed dental drill with small diameter burr to outline a 3 mm diameter circle. Clean the drilling site from the bone dust with sterile saline dipped swabs. Use the gelfoam and swabs to stop the occasional bleeding and clean the bone.</li>
	<li>In between drilling check the bone thickness with fine forceps by gently touching the bone circle and checking its mobility. Keep in mind that the bone is thicker on the suture&#39;s area. Stop the drilling when the bone circle is mobile and only an even, thin layer of bone is left on the circumference. Clean the operational field of all the remaining bone dust with saline dipped swabs.</li>
	<li>Drop the sterile saline on the drilling area, covering the drilled circle. Carefully pry the bone circle with fine forceps and then gently but firmly remove the bone by lifting it upwards. Be careful not to skew the bone circle while lifting it to prevent possible damage to the dura.</li>
	<li>Gently apply the gelfoam soaked in sterile saline on the dura to help stop the bleeding. Wait until all bleeding is fully stopped. Carefully remove the gelfoam not to disturb the clotting process. 
	Note: The suture area is highly vascularized, so the bleeding at this point might prove to be profound. It is essential to wait for the sufficient time for the bleeding to stop completely. It is helpful to keep the saline-soaked gelfoam cooled by placing it on ice.</li>
</ol>
3. Virus Injection
<ol>
	<li>Attach the infusion pump to the stereotactic tower and connect the controller.</li>
	<li>Insert the 35G needle into the syringe. Flush the syringe 10 times with ethanol to sterilize it and 10 times with sterile saline to remove traces of ethanol. Remove air bubbles from the syringe. Insert the syringe into the pump. 
	Note: Consider using other disinfectants.</li>
	<li>Thaw a single dose of rAAV2/1mCherry preparation (1012 pfu is recommended) and keep it on ice. Fill the syringe with the virus solution.</li>
	<li>Center the needle on the bregma and then gently insert into the hippocampus using the following coordinates: AP -2, ML +/- 1.0, DV -1. These coordinates will be located near the edge of the craniotomy. Wait for 5 min&#160;for the tissue to stabilize.</li>
	<li>Inject 0.7 &#181;l of the rAAV2/1mCherry solution at the rate of 50 nl/min. Wait 10 min&#160;for the virus to fully adsorb. Gently remove the needle. Blot with gelfoam if bleeding occurs. Repeat with the contralateral site.</li>
</ol>
4. Cranial Window Implantation
<ol>
	<li>Lay the sterile, dried coverglass on the top of the dura in the drilled circle frame. Hold the coverglass with the forceps to gently flatten the dura and bring coverglass&#39; edges closer to the skull surface. 
	Note: It is possible that the coverglass disrupts the clot and the bleeding resumes. If that is the case, lift the coverglass, place it in alcohol, dry and return to step 2.9.</li>
	<li>Using a sterile needle apply the dense cyanoacrylate glue on the coverglass edges to attach them to the skull. Wait for the glue to dry.</li>
	<li>Place a fixation bar (M2 nut or a custom made design) in the front part of the skull. Apply the cyanoacrylate glue over the edges of the bar. Wait for the glue to dry.
	<ol>
		<li>Place the fixation bar in a position that will enable horizontal positioning of the cranial window during imaging session. Place it as distant as possible from the window. If it is placed too close to the window, the bar and the screw connecting it with the custom-made holder might pose as an obstacle for the objective during the imaging process.</li>
	</ol>
	</li>
	<li>Prepare the dental acrylic and apply it on the skull surface around the glass. It is helpful to form a crater-like shape around the window. It will create a cavity for water applied later for imaging with the water objective.</li>
	<li>Create a cap with the dental acrylic, covering the rest of the operational area, skin edges, fixation bar, reinforcing the crater around the cranial window. Wait for the dental cement to harden.</li>
	<li>Remove the animal from the stereotaxic frame and put it into the recovery chamber.</li>
	<li>Wait for the animal to recover from the surgery while observing the physiological functions.</li>
	<li>Apply post-operative analgesia (carprofen, 10 mg/kg) and antibiotics treatment (baytril, 5 mg/kg) for 48 hr.</li>
</ol>
5. Imaging
<ol>
	<li>Start the Ti:Sapphire laser, power up the microscope. The system used in this experiment is equipped with a two-photon laser, OPO system and dual GaAsP PMT.</li>
	<li>Put the animal in the induction chamber and induce anesthesia.</li>
	<li>Remove the animal from the induction chamber and place in the gas anesthesia mask under the microscope. Decrease the oxygen flow to 0.3 L/min and the isoflurane concentration to 1.5-2%.</li>
	<li>Fix the animal to the custom microscope frame with a M2 screw (or another custom system). Level the cranial window. 
	Note: It is possible to use the microscope manufacturer&#39;s head fixation system, although the specific custom frame gives better results (improved head stability, constant positioning in multiple sessions during a chronic experiment).</li>
	<li>Using the widefield microscope settings and a low magnification objective center the view on one of the sides of retrosplenial cortex and focus it on the coverslip surface.</li>
	<li>Apply a droplet of water into the crater-like acrylic well. Switch to a long distance water immersion objective. Move the objective towards the cranial window until the water meniscus connects the specimen and objective.</li>
	<li>Switch to two-photon settings and begin scanning the specimen top to bottom using lowest zoom. The crossing of dura mater will be visible as a glare of high non-specific signal.</li>
	<li>Adjust the microscope acquisition settings in both channels (GFP and mCherry) according to the signal strength from fluorescent cells in order to cover the entire dynamic range.</li>
	<li>After finding a suitable neuron (with the dendritic tree separated from other cells) perform an initial scan using only the GFP filterset with lowest zoom and z-distance of 5 microns.</li>
	<li>Obtain a maximum projection of the scanned stack and print it for annotations (using inverted colors).</li>
	<li>Set zoom to a value that will allow to image the desired morphological details. Image the entire dendritic tree in the GFP and mCherry channels using maximum projection as guide.</li>
</ol>

<ol>
	<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>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>Trachtenberg, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+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>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=Imaging+neocortical+neurons+through+a+chronic+cranial+window.">Imaging neocortical neurons through a chronic cranial window.</a> Cold Spring Harb Protoc. 2012, 694-701 (2012).</li><li>Chow, D. K., 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=Laminar+and+compartmental+regulation+of+dendritic+growth+in+mature+cortex.">Laminar and compartmental regulation of dendritic growth in mature cortex.</a> Nat Neurosci. 12, 116-118 (2009).</li><li>Czajkowski, R., 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=Encoding+and+storage+of+spatial+information+in+the+retrosplenial+cortex.">Encoding and storage of spatial information in the retrosplenial cortex.</a> Proc Natl Acad Sci U S A. 111, 8661-8666 (2014).</li><li>Czajkowski, R., 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=Superficially+projecting+principal+neurons+in+layer+V+of+medial+entorhinal+cortex+in+the+rat+receive+excitatory+retrosplenial+input.">Superficially projecting principal neurons in layer V of medial entorhinal cortex in the rat receive excitatory retrosplenial input.</a> J Neurosci. 33, 15779-15792 (2013).</li><li>Feng, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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>Couey, J. J., 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=Recurrent+inhibitory+circuitry+as+a+mechanism+for+grid+formation.">Recurrent inhibitory circuitry as a mechanism for grid formation.</a> Nat Neurosci. 16, 318-324 (2013).</li><li>Miyashita, T., Rockland, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+projections+from+the+hippocampus+to+the+retrosplenial+cortex+in+the+rat.">GABAergic projections from the hippocampus to the retrosplenial cortex in the rat.</a> European Journal of Neuroscience. 26, 1193-1204 (2007).</li></ol>

The authors (K.L., M.R., K.R.) have rights for patent pending (PL410001) for Holder frame used in this Article.

In the current paper we present a protocol for simultaneous two-photon in vivo imaging of the synaptic inputs and postsynaptic targets in RSC through a cranial window. The implantation procedure consist of several key steps. First, the animal is deeply anesthetized and fixed in the stereotactic frame, then the skull over RSC is thinned with a drill along the marked circular lines and the circular bone is removed. After the bleeding is stopped, the rAAV2/1mCherry is injected into the hippocampus, and t...

Two-photon microscopy revolutionized the observation of brain activity in living and behaving animals. Since its introduction in 1990 it quickly gained popularity and is now implemented as one of the most interesting and innovative approaches towards examination of numerous aspects of brain activity in vivo 1,2. These applications include blood flow measurements, neuronal activation (e.g., using calcium level indicators or immediate early genes expression) and the morphology of neuronal cells. An increasing number of laboratories use two-photon microscopes, implementing the technique throughout the scientific world as a new standard for in vivo brain imaging.
The standard approach involves implantation of the cranial window (a round hole in the cranium covered with a cover glass) over the barrel or visual cortex of the mouse brain 3. Next, depending on the experimental protocol, the mouse undergoes a series of visualization and behavioral training sessions, allowing to monitor the changes in the brain activity and neuronal morphology over time 4,5. In both cases the craniotomy only affects the parietal bone, without crossing the sutures. It is largely believed that the main drawback of the technique is its limited application to easily accessible cortexes such as the barrel or visual cortex. Implantation of the cranial window over other regions poses a lot of difficulties, due to excessive bleeding and/or spatial hindrance.
In this paper we propose the implantation of the cranial window above the retrosplenial cortex (RSC) as another possible region of interest for two-photon in vivo microscopy 6. RSC is an important element of the brain circuit responsible for spatial memory formation. Anatomically, RSC is a part of a neuronal network connecting cortical, hippocampal, and thalamic regions 7. It is heavily involved in a range of behaviors, such as spatial learning and extinction as well as spatial navigation 6.
In order to visualize the morphological changes of the neurons we use a transgenic mouse line expressing green fluorescent protein (GFP) under the thy1 promoter. In these mice, GFP is expressed in approximately 10% of the neurons in the brain allowing for clear visualization of the cortical axons and dendrites using two-photon microscopy 8. Another innovation that we propose is the injection of a recombinant adeno-associated virus serotype 2/1 (rAAV2/1) coding a red fluorescent protein (mCherry) under a neuron-specific camkii promoter 9 into the deeper structures of the brain projecting to RSC, such as the hippocampus. The expression of rAAV2/1mCherry in the hippocampus of Thy1-GFP mouse allows for simultaneous visualization of pre- and postsynaptic elements of the hippocampo-cortical synapses 10. The rAAV-driven expression of mCherry requires two to three weeks for the protein to reach sufficient level in the axonal terminals. This period is consistent with the usual time required for recovery from craniotomy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Drug</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>AErrane 8DG9623</td>
			<td>5-2% pre-operative</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>AErrane 8DG9623</td>
			<td>1.5-2% during surgery</td>
		</tr>
		<tr>
			<td>Dexametasone</td>
			<td>Scan Vet</td>
			<td>Dexasone 2mg/ml</td>
			<td>0.2 mg/kg intramuscular</td>
		</tr>
		<tr>
			<td>Baytril</td>
			<td>Bayer</td>
			<td>2.50%</td>
			<td>5 mg/kg subcutaneously</td>
		</tr>
		<tr>
			<td>Tolfedine</td>
			<td>Vetoquinol</td>
			<td>4%</td>
			<td>4 mg/kg subcutaneously</td>
		</tr>
		<tr>
			<td>Butomidor</td>
			<td>Richter Pharma</td>
			<td>10 mg/ml</td>
			<td>2 mg/kg subcutaneously</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>KRKA-Polska</td>
			<td>Rycarfa 50mg/ml</td>
			<td>10 mg/kg subcutaneously</td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td>Jelfa</td>
			<td>Lignocainum</td>
			<td>topically</td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td>Jelfa</td>
			<td>20 mg/g</td>
			<td>topically</td>
		</tr>
		<tr>
			<td>Surgery</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gelfoam</td>
			<td>Ethicon</td>
			<td>Spongostan dental; REF MS0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Dedra</td>
			<td>Lubrithal</td>
			<td>topically</td>
		</tr>
		<tr>
			<td>CA glue</td>
			<td>Pelikan Daniel</td>
			<td>20G Huste</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental acrylic</td>
			<td>SpofaDental</td>
			<td>Duracryl Plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>Stoelting</td>
			<td>51500D</td>
			<td></td>
		</tr>
		<tr>
			<td>Tool</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coverglass</td>
			<td>Harvard Apparatus</td>
			<td>HSE-64-0720</td>
			<td>3 mm diameter</td>
		</tr>
		<tr>
			<td>Dental drill</td>
			<td>Sigmed</td>
			<td>Keystone KVet</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation bar</td>
			<td>Custom made</td>
			<td>N/A</td>
			<td>M2 or M3 screw nuts could be used</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Renex</td>
			<td>PN-7B-SA</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>Falcon</td>
			<td>BM.183.180</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>KOZO</td>
			<td>XTL6445T</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Holder frame</td>
			<td>Custom made</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-photon microscope</td>
			<td>Zeiss</td>
			<td>Upright Axio Examiner Z1</td>
			<td>Laser unit: Coherent Chameleon 690-1040nm with Optical Parametric Oscillator 1050-1300nm. Objectives: EC-PLAN-NEUFLUAR 10x/0.1 and LD Plan-APOCHROMAT 20x/1.0. Detection: Zeiss bandpass filters BP 500-550 (GFP) and BP 570-610 (mCherry) separated by beam splitter at 560nm and coupled to two GaAsP photodetectors.&#160;</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Virus</td>
			<td>gift from K. Deisseroth</td>
			<td></td>
			<td>Recombinant adeno-associated virus (rAAV) expressing fluorescent protein mCherry under the control of CaMK promoter</td>
		</tr>
	</tbody>
</table>

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.

The expression of GFP in a subset of neurons in the Thy1-GFP reporter mouse allows in vivo imaging of the cortical dendrites and local axonal projections in RSC. Figure&#160;1A shows maximum projection of a stack of images with multiple GFP-positive dendrites visible. The cell body is obscured by an artery. Figure&#160;1B shows a single plane zoomed image (digital zoom 3x) of the dendritic branch indicated in 1A. Details of dendritic morphology (...

Watch this Scientific Journal Video about Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex at JoVE.com

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.

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 ...

simultaneous-two-photon-vivo-imaging-synaptic-inputs-postsynaptic

Research

JoVE Journal

Neuroscience

11.4K Views.  Nencki Institute of Experimental Biology. 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.

Video: Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

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 ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

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 ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

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 ...

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 ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

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 ...