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...

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 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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

14.7K 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

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

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

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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 ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

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 ...

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral (dorsal root ganglia) and central tissues (cortex, subventricular zone, organoid) that are dynamically studied in terms of calcium shifts. The model chosen to illustrate the results is the retina, a simple tissue with complex cellular interactions. Calcium is a universal messenger involved in most of the important cellular roles. We explain in a step-by-step protocol how retinal neuron-glial cells in culture can be prepared and evaluated, envisioning calcium shifts. In this model, we differentiate neurons from glia based on their selective response to KCl and ATP. Calcium permeable receptors and channels are selectively expressed in different compartments. To analyze calcium responses, we use ratiometric fluorescent dies such as Fura-2. This probe quantifies free Ca2+ concentration based on Ca2+-free and Ca2+-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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

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Functional Calcium Imaging in Developing Cortical Networks

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

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

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

Dissection of Local Ca²⁺ Signals in Cultured Cells by Membrane-targeted Ca²⁺ Indicators

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

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Imaging Local Ca²⁺ Signals in Cultured Mammalian Cells