Name: simultaneous two-photon in vivo imaging of synaptic inputs and postsynaptic targets in the mouse retrosplenial cortex
Uploaded: 2016-03-13T14:00:00
Description: 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

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...

<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>

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 ...

Research

JoVE Journal

Neuroscience

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

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 ...

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. ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

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

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 ...

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 ...

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

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 ...

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 ...

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

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 ...

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+ ...

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

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

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...