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 pipette that is ideal for use will have a tip diameter of 20 - 40 &#956;m and a shank length of 6 - 7 mm. Autoclave the pipette and surgical instruments. The drill is sterilized by wiping with 70% ethanol.</li>
	<li>Store the virus AAV2/5-GfaABC1D.cyto-GCaMP3.SV40 (1.5 x 1013 gc/ml), at -80 &#176;C in 10 &#956;l aliquots. Just before surgery, take out the aliquots out of the freezer and store on ice until used for filling the micropipette.</li>
	<li>Fill the micropipette with virus using a syringe pump. Tightly connect one end of a piece of tubing, through an appropriately sized tubing compression fitting, to a glass syringe with a reinforced plunger. Attach the autoclaved micropipette to the other end of the piece of tubing through an appropriately sized removable needle compression fitting.</li>
	<li>Before loading the viral vector, remove air bubbles from the pumping system including tubing, syringe and micropipette by filling the system with mineral oil colored with Sudan red IV (~1 mg/50 ml) in a 1 ml syringe with a needle (27 G).</li>
	<li>Place a clean piece of parafilm under the glass micropipette, and dispense 5 - 10 &#956;l of the viral vector onto the parafilm.</li>
	<li>Suck the viral vector by moving the plunger of the syringe backward at 0.5 &#956;l/min, and mark the boundary between the vector and the oil on the glass pipette.</li>
</ol>
1.2) Anesthesia, Hair Cutting, Head Fixation and Preparation for Craniotomy
<ol>
	<li>Put a mouse (P49-63, C57BL/6) into the chamber filled with N2O and O2, and 5% isoflurane. Assess the depth of anesthesia by loss of purposeful movement and a slowed respiratory rate (~60 - 90/min). Cut hair on the head when the mouse is anesthetized.</li>
	<li>Fit the mouse in the stereotaxic frame, with its head secured by blunt ear bars and its nose placed into an anesthesia and ventilation system. Maintain continuous isoflurane at 2 - 3%. Apply artificial tears ointment to both eyes since mice lose their blink reflex under anesthesia.</li>
	<li>Administer 0.05 ml of Buprenorphine (0.1 mg/ml) by subcutaneous injection for pain relief.</li>
	<li>Clean surgical area with 10% povidone iodine and 70% alcohol three times, alternating between the two solutions and starting with povidone iodine. Start from the front and wipe toward the back to remove any already cut hairs.</li>
	<li>Make a skin incision on top of the head which starts between the eyes and ends between the ears.</li>
</ol>
1.3) Craniotomy and Microinjections
<ol>
	<li>Remove the periosteum by swiping with a cotton swab and then dry the surface of the skull with dental cotton balls. Make a mark around the injection site, and drill around the edge of the mark using a small steel burr powered by a high speed drill.</li>
	<li>Remove the bone and clean the surface with saline.</li>
	<li>Place the pipette into the left dorsal lateral striatum with the following coordinates (mm): anterior-posterior +0.8, medial-lateral: +2.0, dorsal-ventral from the pial surface: -2.4 13. Note that these coordinates mean that the tip of the microinjection needle sits just above the dorsolateral striatum (Figure 1A, B), meaning that it only slightly penetrates within this nucleus. Avoid damaging blood vessels. If the pipette happens to be right on a blood vessel, slightly adjust the coordinates by ~0.1 - 0.2 mm.</li>
	<li>Start injection with injection rate set at 0.2 &#956;l/min, and typically inject 1 to 1.5&#160;&#956;l of virus.</li>
	<li>Leave the pipette in place after the injection for 10 min, and then withdraw the pipette slowly.</li>
	<li>Finish by closing the surgical wound with continuous suture external nylon suture.</li>
</ol>
1.4) Recovery
<ol>
	<li>Put the mouse in a clean cage on a heating pad for 24 hr. Do not return a mouse that has undergone surgery to the company of other animals until fully recovered. Do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Add Trimethoprim Sulfamethoxiazole (5 ml per 500 ml water, containing 40 mg trimethoprim and 200 mg sulfamethoxiazole) in the water for a week. Administer Buprenorphine two times per day for up to 3 days after surgery.</li>
	<li>Maintain the mouse on a 12 hr light-dark cycle, fed and watered daily, and is weighted once per day up to 3 days after surgery. In these 3 days, any mouse with a loss of more than 10% body weight is considered as compromised, and will be euthanized instead of being subjected to experimentation.</li>
	<li>Separately, change the mouse cage twice per week, and monitor for general health at least once per day. Typically, the mouse is used within 2 - 3 weeks after the stereotaxic microinjections.</li>
</ol>
2. Acute Brain Slice Preparation for Confocal Ca2+ Imaging
2.1) Preparation of Cutting and Recording Solutions.
<ol>
	<li>Prepare cutting solution comprising (mM): 194 sucrose, 30 NaCl, 4.5 KCl, 10 D-glucose, 1 MgCl2, 1.2 NaH2PO4, and 26 NaHCO3, saturated with 95% O2 and 5% CO2.</li>
	<li>Prepare recording solution comprising (mM): 124 NaCl, 4.5 KCl, 1 MgCl2, 10 D-glucose, 2 CaCl2, 1.2 NaH2PO4, and 26 NaHCO3; pH 7.3 - 7.4, 290 - 295 mOsm, saturated with 95% O2 and 5% CO2. Fill the brain slice holding beaker with recording solution, and keep it at 34 &#176;C.</li>
</ol>
2.2) Slicing
<ol>
	<li>Two to three weeks after stereotaxic microinjections, deeply and terminally anesthetize the mouse, and then decapitate it.</li>
	<li>Extract the brain, and use a blade to remove the uninjected right hemisphere, and mount the left one onto the vibratome tray using super glue. Fill the vibratome tray with ice cold cutting buffer, and keep saturating it with 95% O2 and 5% CO2.</li>
	<li>Cut coronal or sagittal striatal brain slices at 300&#160;&#956;m thickness. Usually, 4 - 5 coronal, or 6 - 7 sagittal striatal slices can be collected.</li>
	<li>Transfer the slices to the slice holding beaker warmed at 34 &#176;C, and keep them there for 30 min before storing them at room temperature for subsequent recording.</li>
	<li>Incubate the brain slices in oxygenated recording buffer at room temperature for at least 30 min before [Ca2+]i imaging.</li>
</ol>
3. Immunohistochemistry (IHC)
<ol>
	<li>Two weeks after virus injection, perfuse the mouse transcardially with PBS followed by 10% formalin in PBS.</li>
	<li>Remove, post-fix overnight, and cryoprotect the brain in buffered 30% sucrose for at least 2 days.</li>
	<li>Prepare 40 &#956;m sections using a cryostat microtome.</li>
	<li>Rinse sections 3 times with PBS at 10 min interval.</li>
	<li>Incubate sections for 1 hr in PBS with 10% normal goat serum and with 0.5% Triton X-100.</li>
	<li>Incubate sections overnight at 4 &#176;C in primary antibody prepared in PBS with 0.5% Triton X-100. The primary antibodies used were as follows: chicken anti-GFP 1:1,000, mouse anti-S100&#946; 1:400, mouse anti-NeuN 1:600, mouse anti-glutamine synthetase 1:300.</li>
	<li>Rinse sections 3 times with PBS at 10 min intervals.</li>
	<li>Incubate sections with the secondary antibodies prepared in PBS with 10% normal goat serum for 2 hr at room temperature. The second antibodies were as follows: goat anti-mouse Alexa546 1:500, goat anti-chicken Alexa488 1:500.</li>
	<li>Mount the sections on glass slides, dry and apply several drops of antifade mounting medium, then slowly cover the sections with a glass coverslip. Store the slides at 4 &#176;C.</li>
	<li>Take images on a confocal microscope with a 40X oil immersion lens with a numerical aperture of 1.3.</li>
</ol>
4. Confocal [Ca2+]i Imaging
NOTE: [Ca2+]i imaging was done using a confocal microscope with a 40X water immersion objective lens with a numerical aperture of 0.8.
<ol>
	<li>Between 1 and 5 hr after slicing, place slice in the recording chamber and superfuse with oxygenated recording buffer at room temperature with flow rate of 1 - 2 ml per min. Then place a platinum harp with nylon strings on top of the slice to minimize movement during the experiment.</li>
	<li>Visualize and locate the dorsal striatum with bright field luminescence under a 10X water immersion objective.</li>
	<li>Switch to the 40X water immersion objective lens, and turn on the 488&#160;nm line of the Argon laser. By changing the focal plane, find cells located about 20 &#956;m below the slice surface, displaying basal fluorescence in soma, branches and branchlets. Of note, compromised cells usually display fluorescence level above average, which should be avoided.</li>
	<li>Use &#8216;frame scan&#8217; mode to start scanning. Usually, the laser intensity adjusted to 0.5 &#8211; 5% of the maximum output is sufficient to image [Ca2+]i with cyto-GCaMP3. For monitoring [Ca2+]i dynamics in the whole territory of astrocytes, the &#8216;frame scan&#8217; with a scan rate of 1&#160;sec per frame or faster is recommended, but this needs to be decided depending on the experiment in question.</li>
	<li>If necessary, to image [Ca2+]i in processes, use digital magnification of 2 - 3 fold to monitor 1 - 2 astrocytes in the whole field of view.</li>
	<li>To avoid saturated pixels during recording, use &#8216;HiLo&#8217; lookup mode to pseudocolor the cells. Always start with a &#8220;pilot recording&#8221; for about 5 min, to test if red color appears as indication of saturation. If so, lower the laser output power, or lower the PMT/Gain/offset values and perform another pilot recording to test the pixel values are in the non saturated range. Typically, the combined parameters used for imaging astrocyte [Ca2+]i in the striatum are as follows: laser output power 0.5 - 5% (of 10 mW), PMT 550 - 650 Volts, Gain 2.0 - 3.5X, and offset 0 - 5%.</li>
</ol>

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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 serotype 5 is critical to achieve the specificity. One limitation of the technique described here is that virus infection and surgery procedure may cause astrocyte reactivity, especially with high-titer viruses19. Importantly, in the current study, AAV2/5 microinjections with a relative low titer did not cause decreases in GS that have been associated with astrocyte reactivity19. Similar controls have been reported for hippocampal astrocytes8. However, it is important to emphasize the necessity of these controls for each brain area to be studied, not least because astrocytes are heterogeneous20, and because they likely perform different functions in distinct regions of the brain.
One of the important factors that may affect the robustness of the targeted gene expression using virus microinjection is the coordinates in use, which varies among different mouse strains and changes over the development of the animals. Only C57BL/6 mice of ~8 weeks old were used throughout this study. It has been also shown that viral expression of GFP in striatal neurons and astrocytes in rats could be detected 4 days post injection, reached a plateau by 2 - 4 weeks post injection, and then remained stable for 9 months post injection 21. The time course of cyto-GCaMP3 expression has not been precisely determined here, but at least two weeks for virus expression are recommended.
The approach of using AAV2/5 mediated expression of GECIs in striatal astrocytes can now be used to better understand astrocyte [Ca2+]i signaling in the striatum. Specifically, cyto-GCaMP3 expression level is sufficiently high to permit imaging of [Ca2+]i signals in small populations of astrocytes (~10 cells) and within the entire territories of single astrocytes. The detailed analysis of [Ca2+]i signals is still ongoing, but to date [Ca2+]i signals have been detected from distal processes as far as ~50 &#181;m away from the soma (Figure 4). Detailed experiments on astrocyte [Ca2+]i signals and their correlative and causative functions within the striatum are feasible. In the future, refined approaches (i.e., promoters) are needed to deliver GECIs to genetically defined populations of astrocytes so that one can explore calcium signaling within diverse populations of astrocytes20.

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><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 GfaABC&lt;sub&gt;1&lt;/sub&gt;D.cyto-GCaMP3</td><td>Addgene</td><td>44331</td><td>a plasmid sent to UPenn Vector Core for virus packaging</td></tr><tr><td>1 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&amp;reg;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&amp;beta;</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 striatum (Figure 1B). We detected cyto-GCaMP3 using GFP antibodies and also stained the slices with a known astrocyte marker, S100&#946;. Cyto-GCaMP3 expression was only found in S100&#946; positive cells. No expression was found in neurons visualized with NeuN (Figure 2A and 2B), suggesting astrocyte specific expression. Importantly, cyto-GCaMP3 expression was detected in ~60% of S100&#946; positive cells (Figure 2C).
Astrocytes respond to brain insults such as injury, ischemia and infection by displaying changes that have become known as astrocyte reactivity, which represents a spectrum of potential changes from mild to severe15. We sought to evaluate if virus microinjection caused overt astrocyte reactivity. It was previously shown that reduced expression levels of Glutamine Synthetase (GS) were associated with the reactive astrocytes16,17. Therefore, GS expression in the striatum of WT and virus injected mice was compared. We found no significant changes in GS expression in striatal astrocytes following virus injection (Figure 3A - C), indicating no overt astrocyte reactivity using GS levels as a metric. This general approach could be expanded in future work to evaluate astrocyte reactivity using other markers of reactivity. However, note that GFAP levels may not be a suitable way to measure reactivity in striatal astrocytes, because GFAP is not expressed at detectable levels in most striatal astrocytes under basal conditions18. In summary, by using IHC we conclude cyto-GCaMP3 expression was robust and specific to astrocytes in the striatum, and that virus injection did not cause astrocyte reactivity as assessed by GS expression level changes.
We next prepared acute brain slices from virus injected mice to image [Ca2+]i in striatal astrocytes. Acute 300 &#956;m thick sagittal or coronal slices were prepared, and after a period of recovery the slices were placed in a recording chamber on a confocal microscope for [Ca2+]i imaging using the 488 nm line of an Argon laser. Striatal astrocytes expressing cyto-GCaMP3 could easily be identified in most (usually all) of the striatal slices (~6 - 7 slices per mouse), as they showed visible green fluorescence in cells with clearly bushy morphology (Figure 4). A typical 5&#160;min recording at 1 Hz from a representative field is shown in Figure 4A; at least 10 astrocytes displaying cyto-GCaMP3 could be imaged and are plotted in Figure 4A as Regions of Interest (ROI). Higher magnification imaging with an additional digital zoom of 2.0 - 3.0 was necessary to identify single astrocytes (Figure 4B). Under these circumstances, entire astrocyte territories were revealed by cyto-GCaMP3 expression, as shown in Figure 4B. Spontaneous Ca2+ signals were easily measured in the somata as well as in the branches and branchlets of astrocytes6.
<img alt="Figure 1" src="/files/ftp_upload/51972/51972fig1highres.jpg" /> 
Figure 1: Viral delivery of GECIs (e.g. cyto-GCaMP3) by AAV2/5 into the adult mouse striatum. A. Schematic illustrates the protocol for AAV2/5 microinjections into the dorsolateral striatum. B. Shows the approximate position of the microinjection needle in relation to the striatum and the coordinates used for stereotaxic injections. The image of a Nissl-stained slice in panel B was downloaded from ALLEN BRAIN ATLAS.
<img alt="Figure 2" src="/files/ftp_upload/51972/51972fig2highres.jpg" /> 
Figure 2: Cyto-GCaMP3 expression was specific to striatal astrocytes. A - B. Representative images showing that cyto-GCaMP3 expression colocalizes with a marker for astrocytes (S100&#946;) (B), but not with a marker for neurons (NeuN) (A). C. Summary bar graph for colocalization experiments like those shown in A and B.
<img alt="Figure 3" src="/files/ftp_upload/51972/51972fig3highres.jpg" /> 
Figure 3: Cyto-GCaMP3 expression within striatal astrocytes did not cause changes in the expression of GS. A - B. Representative images of GCaMP3 and GS IHC from mice that had received AAV2/5 for GCaMP3. Images for control non injected mice are also shown. C. Bar graph summarizes results from experiments such as those in A and B (n.s. indicates not significant using an unpaired Student&#8217;s t test, p &#60;0.05).
<img alt="Figure 4" src="/files/ftp_upload/51972/51972fig4highres.jpg" /> 
Figure 4: Representative examples of [Ca2+]i signals recorded from striatal astrocytes with cyto-GCaMP3. A. A flattened z-stack image showing 10 astrocytes (numbered 1 - 10).The traces below show [Ca2+]i signals recorded over 5 min from the 10 cells shown in A. B. A flattened and zoomed-in z-stack image for a single astrocyte.The traces below show [Ca2+]i signals recorded over 5 min for the single cell shown in B.

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

14.8K Views.  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. 

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

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Biology

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Real-Time Imaging for Neuron Activity in the Dentate Gyrus Cells of Mice 

In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal activity in awake animals during behavior tasks. Since the hippocampus is closely associated with episodic and spatial memory, it has become an essential brain region in&#160;this field&#39;s research. In recent research, engram cells and place cells were studied by recording the neural activities in the hippocampal CA1 region using the miniature microscope in mice while performing behavioral tasks including open-field and linear track. Although the dentate gyrus is another important region in the hippocampus, it has rarely been studied with in vivo imaging due to its greater depth and difficulty for imaging. In this protocol, we present in detail a calcium imaging process, including how to inject the virus, implant a GRIN (Gradient-index) lens, and attach a base plate for imaging the dentate gyrus of the hippocampus. We further describe how to preprocess the calcium imaging data using MATLAB. Additionally, studies of other deep brain regions that require imaging may benefit from this method.

Author Spotlight: Investigating Neural Activity of Dentate Gyrus Granule Cells with Miniature Microscope

The dentate gyrus of the hippocampus carries out essential and distinct functions in learning and memory. This protocol describes a set of robust and efficient procedures for in vivo calcium imaging of granule cells in the dentate gyrus in freely moving mice.

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal ...

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

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