Name: culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies
Uploaded: 2020-05-27T15:00:00
Description: Watch this Scientific Journal Video about Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies at JoVE.com

The authors wish to acknowledge funding from the Lundbeck Foundation Research initiative on Brain Barriers and Drug Delivery (RIBBDD) and Simon Hougners Family Foundation.

1. Preparation of buffers and solutions for cell culturing
<ol>
	<li>Prepare collagen stock solution by dissolving 5 mg of collagen IV from human placenta in 50 mL of PBS overnight at 4 &#176;C. Aliquot the stock solution into 5 mL portions and store at -20 &#176;C.</li>
	<li>Prepare fibronectin stock solution by dissolving 5 mg of fibronectin in 5 mL of sterile water overnight. Store the fibronectin stocks in aliquots of 500 &#181;L at -20 &#176;C. When thawing, add PBS to a final volume of 50 mL to prepare the work s...

<ol>
	<li>Armulik, A., Genove, G., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+Developmental,+Physiological,+and+Pathological+Perspectives,+Problems,+and+Promises.">Pericytes: Developmental, Physiological, and Pathological Perspectives, Problems, and Promises.</a> Developmental Cell. 21 (2), 193-215 (2011).</li><li>Abbott, N. J., Ronnback, L., Hansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-endothelial+interactions+at+the+blood-brain+barrier.">Astrocyte-endothelial interactions at the blood-brain barrier.</a> Nature Reviews Neuroscience. 7 (1), 41-53 (2006).</li><li>Mathiisen, T. M., Lehre, K. P., Danbolt, N. C., Ottersen, O. 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C., 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=In+vitro+models+of+the+blood-brain+barrier:+An+overview+of+commonly+used+brain+endothelial+cell+culture+models+and+guidelines+for+their+use.">In vitro models of the blood-brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use.</a> Journal of Cerebral Blood Flow and Metabolism. 36 (5), 862-890 (2016).</li><li>Zhou, L., Sohet, F., Daneman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Pericytes+from+Rodent+Optic+Nerve+by+Immunopanning.">Purification of Pericytes from Rodent Optic Nerve by Immunopanning.</a> Cold Spring Harbor Protocols. , 608-617 (2014).</li><li>Liu, G. H., 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=Isolation,+Purification,+and+Cultivation+of+Primary+Retinal+Microvascular+Pericytes:+A+Novel+Model+Using+Rats.">Isolation, Purification, and Cultivation of Primary Retinal Microvascular Pericytes: A Novel Model Using Rats.</a> Microcirculation. 21 (6), 478-489 (2014).</li><li>Nayak, R. C., Herman, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+retinal+microvascular+pericytes:+isolation,+propagation,+and+identification.">Bovine retinal microvascular pericytes: isolation, propagation, and identification.</a> Methods In Molecular Medicine. 46, 247-263 (2001).</li><li>Nees, S., 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=Isolation,+bulk+cultivation,+and+characterization+of+coronary+microvascular+pericytes:+the+second+most+frequent+myocardial+cell+type+in+vitro.">Isolation, bulk cultivation, and characterization of coronary microvascular pericytes: the second most frequent myocardial cell type in vitro.</a> American Journal of Physiology-Heart and Circulatory Physiology. 302 (1), H69-H84 (2012).</li><li>Armulik, A., Abramsson, A., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial/pericyte+interactions.">Endothelial/pericyte interactions.</a> Circulation Research. 97 (6), 512-523 (2005).</li><li>Helms, H. C., Brodin, B., Milner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+Molecular+Biology.">Methods in Molecular Biology.</a> Cerebral Angiogenesis: Methods and Protocols. 1135, 365-382 (2014).</li><li>Abbracchio, M. P., Burnstock, G., Verkhratsky, A., Zimmermann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+signalling+in+the+nervous+system:+an+overview.">Purinergic signalling in the nervous system: an overview.</a> Trends in Neurosciences. 32 (1), 19-29 (2009).</li><li>Cai, C., 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=Stimulation-induced+rises+in+cerebral+blood+flow+and+local+capillary+vasoconstriction+depend+on+conducted+vascular+responses+in+brain+capillaries.">Stimulation-induced rises in cerebral blood flow and local capillary vasoconstriction depend on conducted vascular responses in brain capillaries.</a> PNAS. , (2018).</li><li>Tigges, U., Welser-Alves, J. V., Boroujerdi, A., Milner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+and+simple+method+for+culturing+pericytes+from+mouse+brain.">A novel and simple method for culturing pericytes from mouse brain.</a> Microvascular Research. 84 (1), 74-80 (2012).</li><li>Maier, C. L., Shepherd, B. R., Yi, T., Pober, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explant+Outgrowth,+Propagation+and+Characterization+of+Human+Pericytes.">Explant Outgrowth, Propagation and Characterization of Human Pericytes.</a> Microcirculation. 17 (5), 367-380 (2010).</li><li>Orlidge, A., Damore, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+specific+effects+of+glycosaminoglycans+on+the+attachment+and+proliferation+of+vascular+wall+components.">Cell specific effects of glycosaminoglycans on the attachment and proliferation of vascular wall components.</a> Microvascular Research. 31 (1), 41-53 (1986).</li><li>Rhinehart, K., Zhang, Z., Pallone, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++signaling+and+membrane+potential+in+descending+vasa+recta+pericytes+and+endothelia.">Ca2+ signaling and membrane potential in descending vasa recta pericytes and endothelia.</a> American Journal of Physiology-Renal Physiology. 283, F852-F860 (2002).</li><li>Bintig, W., 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=Purine+receptors+and+Ca2++signalling+in+the+human+blood-brain+barrier+endothelial+cell+line+hCMEC/D3.">Purine receptors and Ca2+ signalling in the human blood-brain barrier endothelial cell line hCMEC/D3.</a> Purinergic Signalling. 8 (1), 71-80 (2012).</li><li>Neuhaus, A. A., Couch, Y., Sutherland, B. A., Buchan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+method+to+study+pericyte+contractility+and+responses+to+ischaemia+in+vitro+using+electrical+impedance.">Novel method to study pericyte contractility and responses to ischaemia in vitro using electrical impedance.</a> Journal of Cerebral Blood Flow and Metabolism. 37 (6), 2013-2024 (2017).</li><li>Kamouchi, M., 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=Calcium+influx+pathways+in+rat+CNS+pericytes.">Calcium influx pathways in rat CNS pericytes.</a> Molecular Brain Research. 126 (2), 114-120 (2004).</li><li>Das, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+Causes+Retinal+Pericytes+to+Contract+In+vitro.">ATP Causes Retinal Pericytes to Contract In vitro.</a> Experimental Eye Research. 46, 349-362 (1988).</li></ol>

In this study, we have presented a method to isolate primary pericytes from bovine brains. The described protocol allows culture of this otherwise rather inaccessible cell type. The subsequently obtained cell culture was a nearly homogenous population of pericytes, with little or no contamination with endothelial cells&#160;and glial cells based on cell morphology and protein expression12. Furthermore, we demonstrated a simple and straightforward method to load the pericytes with calcium dyes for ...

Brain capillary pericytes, together with endothelial cells and astrocytes, constitute the NVU1,2,3. The endothelial cells, which form the structural basis of the capillaries, form long cylindrical tubes with a diameter of 5-8 &#181;m. The endothelial cells are sporadically covered with pericytes and surrounded by protrusions from astrocytes; the astrocyte endfeet.
The blood-brain barrier (BBB), situated at the brain capillaries, is the main site for exchange of nutrients, gases and waste products between the brain and the blood. The BBB also protects the brain from endogenous and exogenous neurotoxins and serves as a barrier for the delivery of a large number of drug compounds. The barrier function is a focus area, as well as an obstacle, for drug companies developing central nervous system (CNS) medicines. This has spurred a large interest in investigating the cells of the NVU in culture4. Brain astrocytes and endothelial cells have been cultured and characterized in a number of studies, whereas the studies and protocols for pericyte culture are sparse.
Previously published protocols have described generation of brain capillary pericyte cultures to some degree, using a range of different approaches such as immunopanning5, high- and low-glucose media6, fluorescent-activated cell sorting7, density gradient centrifugation8, etc. Although these methods seem sufficient to obtain cultures of pericytes, some are time consuming, cost expensive and the pericytes obtained might not be ideal due to the number of culture passages that can de-differentiate the pericytes9. Furthermore, the potential of cultured pericytes in in vitro signaling studies has been fairly unexplored until now.
The present work focuses on the generation of pericyte cultures from isolated bovine brain capillaries and the subsequent setup for measurements and imaging studies of changes in intracellular calcium, an important intracellular second messenger. We briefly describe the isolation of capillaries from cortical gray matter (for details see Helms et al.10) and the isolation and culture of pericytes in pure monoculture without contamination with endothelial or glial cells. We then provide a protocol for seeding of pericytes in 96-well plates and loading protocols for the calcium probe Fura-2 AM. Finally, we show how pericytes can be used in real-time confocal imaging in microscope culture chambers and describe the protocols for this.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ATP</td>
			<td>Tocris</td>
			<td>3245</td>
			<td></td>
		</tr>
		<tr>
			<td>Cal-520 AM</td>
			<td>AAT Bioquest</td>
			<td>21130</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell incubator</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Fisher</td>
			<td>Heraeus Multifuge 3SR+</td>
			<td>Standard large volume centrifuge for spinning down cells</td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma Aldrich</td>
			<td>C5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td>Zeiss LSM 510</td>
			<td>Inverted microscope</td>
		</tr>
		<tr>
			<td>Counting chamber</td>
			<td>FastRead</td>
			<td>102</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip cell chamber</td>
			<td>Airekacells</td>
			<td>SC15022</td>
			<td></td>
		</tr>
		<tr>
			<td>Cremophor EL</td>
			<td>Sigma Aldrich</td>
			<td>C5135</td>
			<td>Formerly known as Kolliphor EL</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>471267</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagles Medium</td>
			<td>Sigma Aldrich</td>
			<td>D0819</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>PAA/GE Healthcare</td>
			<td>A15-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma Aldrich</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2 AM</td>
			<td>Thermo Fisher</td>
			<td>F1201</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips 22x22 mm</td>
			<td>VWR International</td>
			<td>631-0123</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14065-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma Aldrich</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>AppliChem Panreac</td>
			<td>A1069</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Olympus</td>
			<td>Olympus CK2</td>
			<td>Upright light microscope with phase contrast</td>
		</tr>
		<tr>
			<td>MEM nonessential amino acids</td>
			<td>Sigma Aldrich</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader</td>
			<td>BMG LabTech</td>
			<td>NOVOstar</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td>Phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>penicillin G sodium/streptomycin sulfate</td>
			<td>Sigma Aldrich</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F127</td>
			<td>Sigma Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma Aldrich</td>
			<td>T4299</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 flask</td>
			<td>Sigma Aldrich</td>
			<td>CLS3972</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning incorporated</td>
			<td>3603</td>
			<td></td>
		</tr>
	</tbody>
</table>

culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Bovine brain capillaries were isolated from fresh brain tissue and Figure 1 presents the capillary seeding and cellular outgrowth over days and subsequent purification&#160;of pericytes. The capillaries are fully attached to the flask at day 1 and on day 2 endothelial sprouting has become visible (Figure 1, day 2). After 4 days, the cellular outgrowth is highly distinctive (Figure 1, day 4a) and the ...

Watch this Scientific Journal Video about Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies at JoVE.com

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

This is a simple and effective method for isolating and culturing primary brain pericytes for study of intercellular calcium signaling. The obtained cell culture is a nearly homogenous population of pericytes, and it's straightforward to load the pericytes for calcium imaging. The methods described here should provide other researchers in the field with strong tools for studying pericyte biology, and intracellular signaling in pericytes in vitro.Begin by thawing one vial of capillaries in a 37 degree Celsius water bath. Once the capillaries have thawed, transfer them to a centrifuge tube with 30 milliliters of DMEM comp, and centrifuge them for five minutes at 500 times G.Then, remove the medium from the tube, and re-suspend the palette in 10 milliliters of fresh DMEM comp. Transfer the suspension to a coated T-75 flask, and allow the capillaries to adhere to the bottom for four to six hours in a 37 degrees Celsius incubator supplied with 10%carbon dioxide.After the incubation, inspect the flask under a light microscope. Fractions of capillaries should now be attached to the bottom of the flask. On day four after seeding the capillaries, inspect them under the microscope to ensure that they are approximately 60 to 70%confluent.Aspirate the medium, and gently wash the cells with PBS. Add two milliliters of thawed trypsin-EDTA, and leave the flask in the incubator for one to three minutes, taking the flask out frequently, and observing cell detachment with the microscope. When the endothelial cells start to round up, gently tap the flask to detach them.When the cells have detached, stop trypsinization by adding 10 milliliters of DMEM comp to the flask. Flush the flask a few times with the medium, and aspirate the endothelial cell suspension, which can now be used for other purposes. Add 10 milliliters of DMEM comp to the flask, and check it under the light microscope to assure that the pericytes are still present and attached to the bottom.Then, put the flask back into the incubator to allow the pericyte-enriched culture to grow. To seed pericytes into coated 96-well plates, detach them as described in the text manuscript. Aspirate the medium, taking care not to disturb the cell pellet, And re-suspend the pellet in one milliliter of fresh DMEM comp.Count the cells using a counting chamber, then calculate the volume of suspension that should be added to each well to seed 10, 000 cells per well. Add the cell suspension, then add enough DMEM comp to the cells to achieve a total volume of 200 microliters per well. When ready to load the pericytes with Fura-2 AM calcium indicator dye, take the 96-well plate with the cells out of the incubator, and aspirate the medium from the wells.Wash the cells twice with assay buffer, and add 100 microliters of loading solution to each well. Wrap the plate with tinfoil to avoid photo bleaching, and incubate it for 45 minutes with 30 RPM shaking at room temperature. After the incubation, aspirate the loading buffer, and wash the cells with the assay buffer twice.Add 100 microliters of fresh to assay buffer, and leave the cells to incubate for 30 minutes. After the incubation, set the temperature of the plate reader to 37 degrees Celsius, and transfer the 96-well plate with cells to the sample plate position, then place the reagent plate with agonist at the reagent plate position. Start by measuring loading of the cells to ensure equal loading of Fura-2 AM in all wells, then perform the measurements with excitation fluorescence wavelength at 340 to 380 nanometers, and the emission wavelength at 510 nanometers.Add 50 microliters of agonist, at a speed of 150 microliters per second, from the reagent plate to each well with cells. Mount a cover slip into the cell chamber, and tighten it to avoid leaks. Add DMEM comp and the calculated volume of cell suspension into each chamber, for a final volume of 500 microliters.Incubate the cell chambers at 37 degrees Celsius and 10%carbon dioxide for six days, or until confluent. Once the cells are confluent, take the cell chambers out of the incubator, and aspirate the medium. Wash the cells twice with assay buffer, and add 500 microliters of loading buffer to each chamber, then incubate them at room temperature for 45 minutes.After the incubation, aspirate the loading buffer, and wash the cells twice with assay buffer, then add 500 microliters of fresh assay buffer to each chamber and incubate them for another 30 minutes at room temperature, to allow cleavage of the AM ester. Replace the buffer with 500 microliters of fresh assay buffer, and proceed with live imaging at a confocal microscope. Mount the cell chamber on the stage of the microscope as gently as possible to avoid disturbance of the cells.Set the excitation wavelength at 488 nanometers, emission at 515 nanometers, two minute sequential image acquisition with five second intervals, and an x, y image size of 512 by 512 pixels. Add 300 microliters of 100 millimolar ATP to the cell chamber, and initiate image acquisition. This protocol was used to purify pericytes from bovine brain capillaries.Cellular outgrowth from the seeded capillaries was imaged over the course of nine days. The capillaries were fully attached to the flask at day one, and by day two, endothelial sprouting was visible. After four days, the cellular outgrowth was distinctive, and the endothelial cells were removed via trypsinization.Remnants of the capillaries were present after the trypsinization, but disappeared from the flask in the following days. After removal of the endothelial layer, the pericytes were allowed to grow until confluency. On day nine, the pericytes reached approximately 80%confluency, and formed islands.Addition of ATP to the Fura-2 loaded pericytes resulted in an increase in cytosolic calcium levels. The response occurred immediately after addition of ATP to the pericytes, and declined slowly over the measured time period. The calcium response was also measured with real-time confocal imaging.Baseline fluorescence was measured, then ATP was added at 64 seconds, and a strong intracellular calcium response was evident. Soon after, the cytosolic calcium compartmentalized in the cells, and a reduction in cell area was visible. By 300 seconds, the cell area heavily reduced, and the fluorescence declined almost to baseline levels.This method has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP, and are able to contract in vitro.

culture-brain-capillary-pericytes-for-cytosolic-calcium-measurements

Research

JoVE Journal

Neuroscience

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 ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

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 ...

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages over conventional multi-photon calcium imaging systems: (1) compact; (2) light-weighted; (3) affordable; and (4) allows recording from freely behaving animals. This protocol describes brain surgeries for deep brain in vivo calcium imaging using a custom-developed miniscope recording system. The preparation procedure consists of three steps, including (1) stereotaxically injecting the virus at the desired brain region of a mouse brain to label a specific subgroup of neurons with genetically encoded calcium sensor; (2) implantation of gradient-index (GRIN) lens that can relay calcium image from deep brain region to the miniscope system; and (3) affixing the miniscope holder over the mouse skull where miniscope can be attached later. To perform in vivo calcium imaging, the miniscope is fastened onto the holder, and neuronal calcium images are collected along with simultaneous behavior recordings. The present surgery protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for deep brain in vivo calcium imaging.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Author Spotlight: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...