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 the cover glass is fixed to the skull over the drilled area. Finally the fixation bar is secured on the head and the animal is placed in the recovery chamber for 48 hr. After approximately 2-3 weeks needed for the virus expression, the RSC can be visualized. The imaging protocol comprises of the following steps. First, the animal is anesthetized and fixed under the microscope. The focus is then set using the widefield microscope, the system is switched into the two-photon mode and the channels of interest (GFP and mCherry) are visualized.
The presented technique offers a major improvement over the previously described protocols. In the standard approach, only one type of a label could be detected. It could be used to image local axonal projections and dendritic trees, but no long range connectivity studies were possible. By combining two fluorescent proteins we enabled simultaneous tracing of pre- and postsynaptic elements. This allows long term in vivo monitoring of putative synaptic connections.
In order to obtain the optimal results in the described procedure, it is important to pay attention to several critical steps. During lifting the bone circle in step 2.8 any damage to the dura may cause inflammation and impair transparency of the cranial window. Insufficient stoppage of bleeding in step 2.9 or at any other step of the procedure results in blood accumulation under the window and significantly reduces the field of view. After injecting the virus it is vital to wait for at least 10 min&#160;before removing the needle, in order for the virus to infuse into the tissue at the injection site only. It limits the possibility of unwanted infection of the cortex with the virus during needle removal. The area of the viral transfection should be examined with a post hoc histological analysis of the brain tissue. Any mCherry expression in cells along the needle trace should be avoided. The standard recovery time is 3 weeks. This period is sufficient for the virus to reach stable expression in the synaptic projections and for the cranial window to fully heal and stabilize. The animals should be single housed in order to prevent removal of the cranial window implant by the cage mates. The use of an enlarged cage might be considered in order to avoid accidental damage of the cranial window by hitting the metal bars. Stable fixation of the mouse under the microscope is essential. Any head movement, including breathing movements, may cause significant reduction in quality of the obtained images. It is also helpful to position the cranial window horizontally to the objective, to limit possible problems with acquiring equal focus for the entire plane of the window. Clear criteria for resolving spines and boutons should be applied. Generally, boutons are defined as axonal swellings having a diameter at least 3 times bigger than the preceding fiber 7. Spines are defined as clearly distinguishable protrusions from the dendrite shaft that contain a bulbous head. Further division into populations of thin, stubby, mushroom and branched spines is possible 3. Due to the relatively poor axial resolution of the two-photon microscopy, analysis of spines that project along the optical axis should be avoided 3. In order to clearly distinguish functional boutons and spines, a post hoc immunolabeling can be performed in order to identify pre- and postsynaptic markers 7.
Although the presented protocol proved to be the most favorable in our experimental designs, it is possible to modify it in order to fit different experimental goals. Sometimes it is more suitable to use injectable anesthesia (such as ketamine-xylasine) instead of isoflurane, but it is important to adequately adjust the dosage, keeping in mind differences in drug susceptibility between mice of different strains, age and sex. It is possible to use a trepan bur instead of a spherical one for the drilling, but it could increase the risk of damage to the dura. Sterile saline may be successfully replaced with artificial cerebrospinal fluid (ACSF), but it is important to keep it sterile at all times during preparing and operation. ACSF is stable for 3-4 weeks after preparation, and if any contamination occurs before this time it should be immediately discarded. Different head fixation devices may be applied, depending on the experimental design, including the possibility of observing the awake mouse under the two-photon microscope.
As any other technique, this one also has its limitations. The two-photon microscope allows for visualization of the brain tissue up to 500 &#181;m deep from the dura surface. For examination of the deeper brain structures additional modifications must be applied. Our protocol allows access to most of RSC, but the part of the structure hidden under the superior sagittal sinus is still not accessible. The resolution of the two-photon microscope is not sufficient for the identification of a specific synapse as well as detailed morphological analysis of dendritic spines. Additional techniques, such as correlative electron microscopy must be applied in order to confirm the existence of a suspected structure. It is also important to mention that this is a relatively difficult surgical technique and it is not recommended for an unexperienced operator.
The presented technique may be applied in a wide range of experiments. It may be modified for investigation of different brain regions accessible with the two-photon microscope. It enables simultaneous monitoring of axonal and dendritic alterations during variety of physiological and pathological states including cognitive processes and aging or progression of neurologic and psychiatric conditions. It allows the use of cell-specific promoters to visualize projections originating at precisely defined neuronal subsets. It may also be adjusted to fit the protocols of experiments on awake and behaving animals. Furthermore, calcium- or pH- sensitive proteins can be expressed in the brain in order to visualize not only the neuronal morphology but also changes in cell activity and function. Another possible modification of the approach is the use of a different rAAV serotype for mCherry expression. The chimeric 2/1 serotype provides robust expression at the injection site with sufficiently rapid onset (2-3 weeks). The mCherry levels remained stable for at least 12 weeks after initial onset and no retrograde labeling was detected in our experiments. In order to obtain retrograde labeling, a different serotype might be used, such as rAAV9. The imaging sessions can be performed at any frequency, however at least 24-hr&#160;interval is recommended in order to allow proper recovery of the animal after anesthesia. If applied properly, this technique allows performing multiple imaging sessions of the same region over the course of several months. For long-term experiments (longer than 6 months), a Cre/LoxP system can be used with the recombinase delivered with the AAV vector into a floxed GFP mouse line.

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><tbody><tr><td>&lt;strong&gt;Drug&lt;/strong&gt;</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>&lt;strong&gt;Surgery&lt;/strong&gt;</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>&lt;strong&gt;Tool&lt;/strong&gt;</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>&lt;strong&gt;Imaging&lt;/strong&gt;</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.&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</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 (spines, filopodia) are clearly visible. The GFP channel is acquired by using the band pass emission filter 500-550 nm.
An injection of the rAAV2/1mCherry into the dorsal hippocampus allows visualization of the hippocampal axons and synaptic boutons terminating in RSC. These terminals can be detected in the mCherry channel (the band pass emission filter 570-610 nm).
<img alt="Figure 1" src="/files/ftp_upload/53528/53528fig1.jpg" /> 
Figure1. Two channel in vivo two-photon imaging of RSC neurons and hippocampal projections to RSC.&#160;(A) An overview of the GFP-expressing cells in a fragment of RSC (image shown in inverted colors). Maximum projection shown of a 100 &#181;m thick stack taken at low magnification (0.7x digital zoom). (B) Single optical plane of the fragment indicated in (A) acquired at high magnification (3x digital zoom) in the GFP channel. (C) Single optical plane fragment indicated in (A) using the mCherry acquisition settings. See Protocol for details of the detection filters. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53528/53528fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

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

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

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 ...

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

Behavior

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 ...

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 Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

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

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

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

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Neuronal Activity Mapping in Developing Mouse Sensory Cortex

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following the discovery of numerous genes contributing to these developmental changes, it is now known that neuronal activity also substantially modulates these processes. In the developing cerebral cortex, neurons exhibit synchronized activity patterns that are specialized to each primary sensory area. These patterns markedly differ from those observed in the mature cortex, emphasizing their role in regulating area-specific developmental processes. Deficiencies in neuronal activity during development can lead to various brain diseases. These findings highlight the need to examine the regulatory mechanisms underlying activity patterns in neuronal development. This paper summarizes a series of protocols to visualize primary sensory areas and neuronal activity in neonatal mice, to image the activity of individual neurons within the cortical subfields using two-photon microscopy in vivo, and to analyze subfield-related activity correlations. We show representative results of patchwork-like synchronous activity within individual barrels in the somatosensory cortex. We also discuss various potential applications and some limitations of this protocol.

Author Spotlight: Deciphering Neural Circuit Formation from Two-Photon Microscopy and Single Neuron Imaging

Primary sensory areas in the neocortex exhibit unique spontaneous activities during development. This article describes how to visualize individual neuron activities and primary sensory areas to analyze area-specific synchronous activities in neonatal mice in vivo.

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following ...

Two-Photon In Vivo Imaging of the Mouse Retina

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/61970/transpupillary-two-photon-in-vivo-imaging-of-the-mouse-retina'>Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).</a>This video demonstrates a technique for in vivo retinal imaging using two-photon microscopy. Anesthetized mice are prepared for imaging by securing their heads, Applying eye lubricant, and placing a coverslip over the eye. Retinal ganglion cells (RGCs) expressing fluorescent proteins are first visualized using epifluorescence for alignment. Finally, two-photon imaging is employed, allowing for high-resolution visualization of RGCs.

Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).This video demonstrates a technique ...

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

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Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

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

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

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

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

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

Two-Photon In Vivo Imaging of the Mouse Retina