The majority of the work and the personnel involved were supported by NIH grant NS060677 and partly by NIH grants MH099559 and MH104069 (to BSK). Some of the work was also supported by the CHDI Foundation.

All animal protocols were in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at UCLA.
1.1) Prepare Micropipette and AAV2/5 Virus Loading
<ol>
	<li>Use fine borosilicate glass micropipettes for the injection of the virus. Pull the micropipette using a two-step pulling program with a vertical puller. Bevel the pipette at an angle of 40&#176; by using a pipette grinder. The ...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+targeted+optical+sensor+to+monitor+calcium+signals+in+astrocyte+processes.">A genetically targeted optical sensor to monitor calcium signals in astrocyte processes.</a> Nat Neurosci. 13, 759-766 (2010).</li><li>Shigetomi, E., 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+calcium+microdomains+within+entire+astrocyte+territories+and+endfeet+with+GCaMPs+expressed+using+adeno-associated+viruses.">Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses.</a> J Gen Physiol. 141, 633-647 (2013).</li><li>Shigetomi, E., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+near+plasma+membrane+and+global+intracellular+calcium+dynamics+in+astrocytes.">Measuring near plasma membrane and global intracellular calcium dynamics in astrocytes.</a> J Vis Exp. 26, (2009).</li><li>Reeves, A. M., Shigetomi, E., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+loading+of+calcium+indicator+dyes+to+study+astrocyte+physiology:+key+limitations+and+improvements+using+morphological+maps.">Bulk loading of calcium indicator dyes to study astrocyte physiology: key limitations and improvements using morphological maps.</a> J Neurosci. 31, 9353-9358 (2011).</li><li>Li, D. D., Agulhon, C., Schmidt, E., Oheim, M., Ropert, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+for+investigating+astrocyte-to-neuron+communication.">New tools for investigating astrocyte-to-neuron communication.</a> Frontiers in Cellular Neuroscience. 7, (2013).</li><li>Davila, D., Thibault, K., Fiacco, T. A., Agulhon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+molecular+approaches+to+understanding+astrocyte+function+in+vivo.">Recent molecular approaches to understanding astrocyte function in vivo.</a> Front Cell Neurosci. 7, 272 (2013).</li><li>Paxinos, G., Franklin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2012).</li><li>Perea, G., Yang, A., Boyden, E. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes:+biology+and+pathology.">Astrocytes: biology and pathology.</a> Acta Neuropathol. 119, 7-35 (2010).</li><li>Eid, 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=Loss+of+glutamine+synthetase+in+the+human+epileptogenic+hippocampus:+possible+mechanism+for+raised+extracellular+glutamate+in+mesial+temporal+lobe+epilepsy.">Loss of glutamine synthetase in the human epileptogenic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy.</a> Lancet. 363, 28-37 (2004).</li><li>Eid, T., Williamson, A., Lee, T. S., Petroff, O. A., de Lanerolle, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+and+astrocytes--key+players+in+human+mesial+temporal+lobe+epilepsy.">Glutamate and astrocytes--key players in human mesial temporal lobe epilepsy.</a> Epilepsia. 49 Suppl 2, 42-52 (2008).</li><li>Tong, X., 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=Astrocyte+Kir4.1+ion+channel+deficits+contribute+to+neuronal+dysfunction+in+Huntington's+disease+model+mice.">Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice.</a> Nat Neurosci. 17, 694-703 (2014).</li><li>Ortinski, P. I., 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=Selective+induction+of+astrocytic+gliosis+generates+deficits+in+neuronal+inhibition.">Selective induction of astrocytic gliosis generates deficits in neuronal inhibition.</a> Nat Neurosci. 13, 584-591 (2010).</li><li>Zhang, Y., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+heterogeneity:+an+underappreciated+topic+in+neurobiology.">Astrocyte heterogeneity: an underappreciated topic in neurobiology.</a> Curr Opin Neurobiol. 20, 588-594 (2010).</li><li>Reimsnider, S., Manfredsson, F. P., Muzyczka, N., Mandel, R. 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The authors have nothing to disclose.

The methods described herein have allowed us to express cyto-GCaMP3 in striatal astrocytes in vivo for subsequent [Ca2+]i imaging in situ. This method has advantages over using transgenic or knock-in mice, including robust expression of the targeted protein, rapidity and flexibility of experimental implementation and anatomical specificity. The expression of GCaMP3 using AAV2/5 was found to be specific and robust. The combination of GFAP GfaABC1D promoter with AAV of the...

Astrocytes are ubiquitous and abundant glial cells of the brain. It is well established that astrocytes serve vital support and homeostatic roles including buffering of K+ concentration in the extracellular space, uptake of neurotransmitters as well as providing nutrients. However, recent studies show that they also display [Ca2+]i signals, which occur spontaneously and are increased by neuronal activity1. The existence of astrocyte [Ca2+]i signaling has been increasingly thought to trigger their communication with neurons, and as such has been interpreted as a form of &#8220;Ca2+ excitability&#8221; within astrocytes. The available data over the last two decades suggest two settings in which astrocytes and neurons may communicate, perhaps in a bidirectional manner. First, astrocytes often respond with an increase in [Ca2+]i when activated by neurotransmitters and neuromodulators released from neurons2. Second, [Ca2+]i increases within astrocytes cause the release of signaling molecules from astrocytes that in turn may affect neurons and blood vessels. Evidence suggests that molecules released from astrocytes lead to changes in the functions of synapses, circuits and ultimately behavior3-5 via astrocyte-to-neuron signaling. However, this remains a rapidly developing research area, and it has been argued that a better and detailed understanding of astrocyte [Ca2+]i is needed to resolve some of the current uncertainties6.
In past work, it was demonstrated that bulk loading of organic Ca2+ indicator dyes into astrocytes fails to reliably detect [Ca2+]i signals within entire astrocytes in culture and in situ7-10. These findings have been discussed by us and others6,11,12. The emerging picture is that [Ca2+]i signals within astrocyte processes (e.g., branches and branchlets), which are the primary sites for interactions with neurons and blood vessels, have rarely been explored in detail. Recently, the use of genetically encoded calcium indicators (GECIs) such as cytosolic GCaMP3, GCaMP5G and GCaMP6 and plasma membrane tethered versions (e.g., Lck-GCaMP3) has permitted the study of [Ca2+]i signals in small compartments of astrocytes such as thin processes, near the plasma membrane and within entire territories7,8. However, GECIs have one disadvantage over organic Ca2+ indicator dyes and that is the requirement for genetic methods to deliver the encoding genes selectively to astrocytes in vivo for periods of weeks for the GECIs to be appropriately expressed. Expression in vivo is typically achieved using transgenic mice, knock-in mice or with virus based delivery approaches. In the present JoVE article we report methods and procedures employed to deliver GECIs to striatal astrocytes using adeno-associated viruses. We focus on cyto-GCaMP3 as an example, but the same basic procedure works for any other GECI or fluorescent protein based reporter.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Syringe Pump</td>
			<td>Harvard Apparatus</td>
			<td>704506</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Capillaries</td>
			<td >World Precision Instruments</td>
			<td >1B100-4</td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipette puller</td>
			<td >Narishige</td>
			<td >PC-10</td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipette grinder</td>
			<td >Narishige</td>
			<td >EG-40</td>
			<td></td>
		</tr>
		<tr >
			<td >pZac2.1 GfaABC1D.cyto-GCaMP3</td>
			<td >Addgene</td>
			<td>44331</td>
			<td >a plasmid sent to UPenn Vector Core for virus packaging</td>
		</tr>
		<tr >
			<td >I mL syringe</td>
			<td >BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr >
			<td >syringe needle</td>
			<td >BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr >
			<td >AAV2/5 virus</td>
			<td >UPenn vector core</td>
			<td >NA</td>
			<td></td>
		</tr>
		<tr >
			<td >Sudan red IV</td>
			<td >Sigma-Aldrich</td>
			<td >67386</td>
			<td></td>
		</tr>
		<tr >
			<td >Mineral oil</td>
			<td >CVS Pharmacy</td>
			<td>152355</td>
			<td></td>
		</tr>
		<tr >
			<td >Cryostat</td>
			<td >Leica</td>
			<td>CM3050 S</td>
			<td></td>
		</tr>
		<tr >
			<td >Stereotaxic instrument</td>
			<td>David Kopf Instruments</td>
			<td >900LS</td>
			<td></td>
		</tr>
		<tr >
			<td >High Speed Rotary Micromotor Kit</td>
			<td >FOREDOM</td>
			<td >K.1070</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde</td>
			<td >Santa cruz biotechnology</td>
			<td >sc-281692</td>
			<td></td>
		</tr>
		<tr >
			<td >Super Glue</td>
			<td>Krazy&#174;Glue</td>
			<td>KG925</td>
			<td></td>
		</tr>
		<tr >
			<td >Microslicer</td>
			<td >Ted Pella</td>
			<td >DTK-Zero 1</td>
			<td></td>
		</tr>
		<tr >
			<td >Confocal microscopes</td>
			<td >Olympus</td>
			<td >FV300 and FV1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Normal goat serum</td>
			<td >Vector</td>
			<td >S-1000</td>
			<td></td>
		</tr>
		<tr >
			<td >chicken anti-GFP</td>
			<td >Abcam</td>
			<td >ab13970</td>
			<td></td>
		</tr>
		<tr >
			<td >mouse anti-s100&#946;</td>
			<td >Sigma-Aldrich</td>
			<td >S2532</td>
			<td></td>
		</tr>
		<tr >
			<td >mouse anti-NeuN</td>
			<td >Millipore</td>
			<td >MAB377</td>
			<td></td>
		</tr>
		<tr >
			<td >mouse anti-glutamine synthetase</td>
			<td >Millipore</td>
			<td >MAB302</td>
			<td></td>
		</tr>
		<tr >
			<td >goat anti-mouse-Alexa546</td>
			<td >Invitrogen</td>
			<td >A11003</td>
			<td></td>
		</tr>
		<tr >
			<td >goat anti-chicken-Alexa488</td>
			<td >Invitrogen</td>
			<td >A11039</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope Slides</td>
			<td >Fisher Scientific</td>
			<td >12-550-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Cover Glass</td>
			<td >Fisher Scientific</td>
			<td >12-548-5J</td>
			<td></td>
		</tr>
		<tr >
			<td >Mounting Medium</td>
			<td >Vector</td>
			<td >H-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging intracellular ca2+ signals in striatal astrocytes from adult mice using genetically-encoded calcium indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

For astrocyte specific expression of cyto-GCaMP3 in the striatum, we used adeno-associated virus (AAV) of the 5 serotype, and the GFAP GfaABC1D promoter (Figure 1A), which has been previously shown to drive robust GCaMP3 and reporter gene expression in hippocampal and cortical astrocytes8,14. Two weeks after virus microinjection into the mouse striatum, the mouse (~10 weeks old) was perfused and IHC was performed on thin brain sections to evaluate cyto-GCaMP3 expression in the stria...

Watch this Scientific Journal Video about Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators at JoVE.com

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

imaging-intracellular-ca2-signals-striatal-astrocytes-from-adult-mice

Research

JoVE Journal

Neuroscience

14.7K visualizações.  University of California Los Angeles. The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ. 