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

Summary

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.

Abstract

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 (rAAV^mCherry) 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.

Introduction

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 rAAV2/1^mCherry 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.

Protocol

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

1. 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.
2. 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.
3. Take the animal out of the induction chamber. Use tail or toe pinches in order to ensure that the animal is fully sedated.
4. Using a precise trimmer shave the hair from the back of the head (between the ears) up to the eyes.
5. Place the animal in the stereotaxic frame and stabilize the head with ear bars.
6. Set anesthesia levels to 1.5-2% isoflurane and 0.3 L/min oxygen.
7. Apply the eye ointment.
8. 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.
9. 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.
10. Clean the skin using sterile cotton swabs with Betadine followed by 70% ethanol.
11. 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.

2. Cranial Window Surgery

1. 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.
2. Apply lidocaine ointment with a sterile swab on the periosteum to prevent excessive bleeding and pain.
3. Use sterile cotton swabs or a scalpel to remove the periosteum. Dry the skull with sterile swabs.
4. 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.
5. 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.
6. 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.
7. 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'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.
8. 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.
9. 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.

3. Virus Injection

1. Attach the infusion pump to the stereotactic tower and connect the controller.
2. 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.
3. Thaw a single dose of rAAV2/1^mCherry preparation (10¹² pfu is recommended) and keep it on ice. Fill the syringe with the virus solution.
4. 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 for the tissue to stabilize.
5. Inject 0.7 µl of the rAAV2/1^mCherry solution at the rate of 50 nl/min. Wait 10 min for the virus to fully adsorb. Gently remove the needle. Blot with gelfoam if bleeding occurs. Repeat with the contralateral site.

4. Cranial Window Implantation

1. 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' 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.
2. 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.
3. 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.
   1. 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.
4. 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.
5. 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.
6. Remove the animal from the stereotaxic frame and put it into the recovery chamber.
7. Wait for the animal to recover from the surgery while observing the physiological functions.
8. Apply post-operative analgesia (carprofen, 10 mg/kg) and antibiotics treatment (baytril, 5 mg/kg) for 48 hr.

5. Imaging

1. 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.
2. Put the animal in the induction chamber and induce anesthesia.
3. 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%.
4. 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's head fixation system, although the specific custom frame gives better results (improved head stability, constant positioning in multiple sessions during a chronic experiment).
5. 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.
6. 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.
7. 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.
8. 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.
9. 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.
10. Obtain a maximum projection of the scanned stack and print it for annotations (using inverted colors).
11. 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.

Results

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 1A shows maximum projection of a stack of images with multiple GFP-positive dendrites visible. The cell body is obscured by an artery. Figure 1B shows a single plane zoomed image (digital zoom 3x) of the dendritic branch indicated in 1A. Details of dendritic morphology (spines, fi...

Discussion

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 rAAV2/1^mCherry is injected into the hippocampus, and t...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Drug
Isoflurane	Baxter	AErrane 8DG9623	5-2% pre-operative
Isoflurane	Baxter	AErrane 8DG9623	1.5-2% during surgery
Dexametasone	Scan Vet	Dexasone 2mg/ml	0.2 mg/kg intramuscular
Baytril	Bayer	2.50%	5 mg/kg subcutaneously
Tolfedine	Vetoquinol	4%	4 mg/kg subcutaneously
Butomidor	Richter Pharma	10 mg/ml	2 mg/kg subcutaneously
Carprofen	KRKA-Polska	Rycarfa 50mg/ml	10 mg/kg subcutaneously
Lidocaine	Jelfa	Lignocainum	topically
Lidocaine	Jelfa	20 mg/g	topically
Surgery
Gelfoam	Ethicon	Spongostan dental; REF MS0005
Eye ointment	Dedra	Lubrithal	topically
CA glue	Pelikan Daniel	20G Huste
Dental acrylic	SpofaDental	Duracryl Plus
Stereotaxic frame	Stoelting	51500D
Tool
Coverglass	Harvard Apparatus	HSE-64-0720	3 mm diameter
Dental drill	Sigmed	Keystone KVet
Fixation bar	Custom made	N/A	M2 or M3 screw nuts could be used
Forceps	Renex	PN-7B-SA
Micro scissors	Falcon	BM.183.180
Dissection microscope	KOZO	XTL6445T
Imaging
Holder frame	Custom made	N/A
Two-photon microscope	Zeiss	Upright Axio Examiner Z1	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.
Reagent
Virus	gift from K. Deisseroth		Recombinant adeno-associated virus (rAAV) expressing fluorescent protein mCherry under the control of CaMK promoter