Name: visualizing shifts on neuron-glia circuit with the calcium imaging technique
Uploaded: 2022-04-08T15:00:00
Description: Watch this Scientific Journal Video about Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique at JoVE.com

Grants, sponsors, and funding sources: MH is recipient of a PhD CNPq fellowship. HRF is recipient of a postdoc fellowship supported by CNPq (HRF grant number 152071/2020-2). RAMR is supported by CNPq and FAPERJ (grant numbers E-26/202.668/2018, E-26/010.002215/2019, 426342/2018-6 and 312157/2016-9 and INCT-INNT (National Institute for Translational Neuroscience).

All experiments involving animals were approved by and carried out following the guidelines of the Institutional Animal Care and Use Committee of the Federal University of Rio de Janeiro, following the &#34;Principles of Laboratory Animal Care&#34; (NIH, Bethesda, USA); permit number IBCCF-035 for fertilized White Leghorn chicken eggs.
1. Preparation of solutions
<ol>
	<li>Prepare Krebs solution: (132 mM NaCl, 4 mM KCl, 1.4 mM MgCl2, 2.5 mM CaCl2, 6 mM ...

<ol>
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T., Poo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+itself+promotes+the+developmental+switch+of+neuronal+GABAergic+responses+from+excitation+to+inhibition.">GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition.</a> Cell. 105 (4), 521-532 (2001).</li><li>Schitine, 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=Ampakine+CX546+increases+proliferation+and+neuronal+differentiation+in+subventricular+zone+stem/progenitor+cell+cultures.">Ampakine CX546 increases proliferation and neuronal differentiation in subventricular zone stem/progenitor cell cultures.</a> European Journal of Neuroscience. 35 (11), 1672-1683 (2012).</li><li>Xapelli, 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=Modulation+of+subventricular+zone+oligodendrogenesis:+a+role+for+hemopressin.">Modulation of subventricular zone oligodendrogenesis: a role for hemopressin.</a> Frontiers in Cellular Neuroscience. 8, 59 (2014).</li><li>Ribeiro-Resende, V. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+lacking+GD3+synthase+display+morphological+abnormalities+in+the+sciatic+nerve+and+neuronal+disturbances+during+peripheral+nerve+regeneration.">Mice lacking GD3 synthase display morphological abnormalities in the sciatic nerve and neuronal disturbances during peripheral nerve regeneration.</a> PLoS One. 9 (10), 108919 (2014).</li><li>Xapelli, 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=Activation+of+type+1+cannabinoid+receptor+(CB1R)+promotes+neurogenesis+in+murine+subventricular+zone+cell+cultures.">Activation of type 1 cannabinoid receptor (CB1R) promotes neurogenesis in murine subventricular zone cell cultures.</a> PLoS One. 8 (5), 63529 (2013).</li><li>Kubrusly, R. C. 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=Neuro-glial+cannabinoid+receptors+modulate+signaling+in+the+embryonic+avian+retina.">Neuro-glial cannabinoid receptors modulate signaling in the embryonic avian retina.</a> Neurochemistry International. 112, 27-37 (2018).</li><li>Egan, T. M., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+calcium+ions+to+P2X+channel+responses.">Contribution of calcium ions to P2X channel responses.</a> Journal of Neuroscience. 24 (13), 3413-3420 (2004).</li><li>Illes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P2X7+Receptors+Amplify+CNS+Damage+in+Neurodegenerative+Diseases.">P2X7 Receptors Amplify CNS Damage in Neurodegenerative Diseases.</a> International Journal of Molecular Sciences. 21 (17), (2020).</li><li>Faroni, 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=Purinergic+signaling+mediated+by+P2X7+receptors+controls+myelination+in+sciatic+nerves.">Purinergic signaling mediated by P2X7 receptors controls myelination in sciatic nerves.</a> Journal of Neuroscience Research. 92 (10), 1259-1269 (2014).</li><li>Freitas, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+Induce+Cell+Death+and+Promote+P2X7+Receptor+Signaling+in+Retinal+Glial+Progenitors+in+Culture.">Cannabinoids Induce Cell Death and Promote P2X7 Receptor Signaling in Retinal Glial Progenitors in Culture.</a> Molecular Neurobiology. 56 (9), 6472-6486 (2019).</li><li>Freitas, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione-Induced+Calcium+Shifts+in+Chick+Retinal+Glial+Cells.">Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells.</a> PLoS One. 11 (4), 0153677 (2016).</li><li>Yang, X. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+receptors+for+glutamate+and+GABA+in+retinal+neurons.">Characterization of receptors for glutamate and GABA in retinal neurons.</a> Progress in Neurobiology. 73 (2), 127-150 (2004).</li><li>Reis, R. A., Kubrusly, R. C., de Mello, M. C., de Mello, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+coupling+of+NMDA+receptor+with+ip3+production+in+cultured+cells+of+the+avian+retina.">Transient coupling of NMDA receptor with ip3 production in cultured cells of the avian retina.</a> Neurochemistry International. 26 (4), 375-380 (1995).</li><li>L&#243;pez-Colom&#233;, A. M., Ortega, A., Romo-de-Vivar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitatory+amino+acid-induced+phosphoinositide+hydrolysis+in+M&#252;ller+glia.">Excitatory amino acid-induced phosphoinositide hydrolysis in M&#252;ller glia.</a> Glia. 9 (2), 127-135 (1993).</li><li>Ventura, A. L., de Mello, F. G., de Melo Reis, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+dopamine+research+in+retina+cells.">Methods of dopamine research in retina cells.</a> Methods in Molecular Biology. 964, 25-42 (2013).</li><li>Miras-Portugal, M. T., Sebasti&#225;n-Serrano, &. #. 1. 9. 3. ;., de Diego Garc&#237;a, L., D&#237;az-Hern&#225;ndez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+P2X7+Receptor:+Involvement+in+Neuronal+Physiology+and+Pathology.">Neuronal P2X7 Receptor: Involvement in Neuronal Physiology and Pathology.</a> Journal of Neuroscience. 37 (30), 7063-7072 (2017).</li><li>Illes, P., Khan, T. M., Rubini, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+P2X7+Receptors+Revisited:+Do+They+Really+Exist.">Neuronal P2X7 Receptors Revisited: Do They Really Exist.</a> Journal of Neuroscience. 37 (30), 7049-7062 (2017).</li><li>Schitine, C. 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=Functional+plasticity+of+GAT-3+in+avian+M&#252;ller+cells+is+regulated+by+neurons+via+a+glutamatergic+input.">Functional plasticity of GAT-3 in avian M&#252;ller cells is regulated by neurons via a glutamatergic input.</a> Neurochemistry International. 82, 42-51 (2015).</li><li>Ferreira, D. D., Stutz, B., de Mello, F. G., Reis, R. A., Kubrusly, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caffeine+potentiates+the+release+of+GABA+mediated+by+NMDA+receptor+activation:+Involvement+of+A1+adenosine+receptors.">Caffeine potentiates the release of GABA mediated by NMDA receptor activation: Involvement of A1 adenosine receptors.</a> Neuroscience. 281, 208-215 (2014).</li><li>Faria, R. X., Freitas, H. R., Reis, R. 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=P2X7+receptor+large+pore+signaling+in+avian+M&#252;ller+glial+cells.">P2X7 receptor large pore signaling in avian M&#252;ller glial cells.</a> Journal of Bioenergetics and Biomembranes. 49 (3), 215-229 (2017).</li><li>Passos, 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=Regulation+of+the+Serotonergic+System+by+Kainate+in+the+Avian+Retina.">Regulation of the Serotonergic System by Kainate in the Avian Retina.</a> Cellular and Molecular Neurobiology. 39 (7), 1039-1049 (2019).</li><li>de Melo Reis, R. A., Ventura, A. L., Schitine, C. S., de Mello, M. C., de Mello, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+glia+as+an+active+compartment+modulating+nervous+activity+in+the+vertebrate+retina:+neurotransmitters+and+trophic+factors.">M&#252;ller glia as an active compartment modulating nervous activity in the vertebrate retina: neurotransmitters and trophic factors.</a> Neurochemical Research. 33 (8), 1466-1474 (2008).</li><li>Harada, K., Kamiya, T., Tsuboi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gliotransmitter+Release+from+Astrocytes:+Functional+Developmental,+and+Pathological+Implications+in+the+Brain.">Gliotransmitter Release from Astrocytes: Functional Developmental, and Pathological Implications in the Brain.</a> Frontiers in Neuroscience. 9, 499. 9, 499 (2015).</li></ol>

We have used the retina tissue to show that calcium responses mediated by KCl or ATP are clearly compartmentalized into neuronal and glial responses, respectively (Figure 1). Although some data in the literature imply that P2X7 receptors are expressed in neurons, which regulate neuronal activity and synaptic neurotransmitter release20, other authors question the existence of neuronal P2X7 receptors. Indeed, current results sustain the idea that primary glial P2X7 rece...

Due to the universal properties of calcium as a second messenger, this ion is involved in a vast number of signaling activities: gene transcription, birth and death, proliferation, migration and differentiation, synaptic transmission, and plasticity. Hence, a method capable of tracking calcium activation dynamics with fidelity and agility would provide a way to observe unique spatial-temporal responses. Such a method is the cellular calcium imaging technique, which correlates calcium shifts functional data with specific cell phenotypes based on their distinct responses.
Ca2+ probes were first developed in the 1980&#39;s, with later improvements allowing these molecules to be used in live cell assays1. As a chemical indicator, Fura-2 is considered to be the standard for quantitative [Ca2+]i measurements. The acetoxymethyl (AM) ester of this indicator (i.e., Fura-2 AM) easily permeates the cell membrane and can reach intracellular concentrations 20-fold greater than the incubation dilution (e.g., [5 &#181;M]o/[100 &#181;M]i). Another advantage of Fura-2 is that it has good photobleaching resistance; thus, imaging this indicator for longer periods of time will not greatly affect its fluorescence capabilities. Finally, Fura-2 is sensitive to a wide range of calcium levels, from ~100 nM to ~100 &#181;M, and has a Kd of ~145 nM, which is comparable to the resting [Ca2+]i2. Later, cell calcium imaging was developed with better fluorescent microscopes and computation methods, together with ratiometric probes that are not affected by dye loading.
Every cell expresses different calcium devices (pumps, transporters, receptors, and channels) that contribute to the final response as a particular signature. The important tip is to find selective responses of different types of cells correlated with their phenotypic expression. Accordingly, there are at least two different receptors that operate through calcium shifts: ionotropic receptors that permeate Ca2+ in a fast mode and slow metabotropic receptors coupled to signaling pathways and intracellular stocks that release Ca2+ activated by second messengers, such as inositol triphosphate and cyclic ADP-ribose3.
For example, progenitor cells express nestin in the immature retina and show GABAA receptors depolarized by GABA (or muscimol)4. This happens due to the Cl- electrochemical gradient with high intracellular Cl&#8722; levels; as the tissue develops, KCC2 transporters switch from excitation on progenitors to inhibition on mature GABAergic neurons5. On the other hand, stem cells that express sox-2 at the immature subventricular zone (SVZ) of postnatal rodents also present metabotropic H1 receptors activated by histamine increasing Ca2+ in a slow manner6. A second metabotropic receptor from the protease-activated receptor-1 (PAR-1) family, activated by thrombin and downstream to G(q/11) and phospholipase C (PLC), gives slow Ca2+ shifts in oligodendrocytes (that express O4 and PLP) generated from multipotent SVZ neural stem cells7.
In general, neurons express voltage-dependent calcium channels as well as major neurotransmitter receptors permeable to Ca2+, as glutamatergic (AMPA, NMDA, kainate) and peripheral and central nicotinic receptors. Potassium chloride is usually used as a depolarizing agent to activate peripheral neurons, as the dorsal root ganglion neurons8 or central neurons, as from subventricular zone9 or retina10. On the other hand, ATP is acknowledged as the major gliotransmitter (in addition to D-serine), which activates selective Ca2+ permeable P2X members, as P2X7 and P2X4. Both receptors present equivalent Ca2+ currents, similar to the ones shown by NMDA receptors acknowledged as the largest Ca2+ currents activated by transmitters11. P2X7 receptors are highly expressed on microglia, but at a lower density on astrocytes and oligodendrocytes, having a role in the release of proinflammatory cytokines12. P2X7 receptors are also expressed on Schwann cells13 and M&#252;ller glia in the retina14,15.
The retina is known to show almost all transmitters seen in the brain. For instance, the vertical axis (photoreceptors, bipolar and retinal ganglion cells) is mainly glutamatergic, with calcium-permeable AMPA or kainate receptors expressed in OFF-bipolar cells and mgluR6 expressed in ON-bipolar cells16. Curiously, all three receptors are also found in M&#252;ller glia, which are coupled to calcium and inositol triphosphate pathways17,18. The horizontal inhibitory axis, made by horizontal and amacrine cells, secrete not only GABA, but also dopamine, acetylcholine, and other classical neurotransmitters. Amacrine cells are the main types of cells found in the avian retinal cultures, showing several types of calcium operated channels, as glutamatergic, purinergic, nicotinic and voltage dependent calcium channels. For this reason, this is an excellent model to evaluate different properties of calcium shifts among neurons and glia.
Therefore, the combination of different receptors and channels summed to selective phenotypic markers during development with distinct agonist response patterns allows unique signatures in stem, progenitor, neuron, astrocyte, oligodendrocyte, and microglia that operate through selective signaling devices.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mm coverslip</td>
			<td>Paul Marienfeld GmbH &#38; Co. KG</td>
			<td>111550</td>
			<td>Cell suport</td>
		</tr>
		<tr>
			<td>510 nm long-pass filter</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A1852</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>Suplement</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolSNAP digital camera</td>
			<td>Roper Scientific, Trenton, NJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Neon</td>
			<td>1466</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/ F-12</td>
			<td>Gibco</td>
			<td>12400-24</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>Excel Software</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F9665</td>
			<td>Suplement</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Axiovert 200; Carl Zeiss</td>
			<td>B 40-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2 AM</td>
			<td>Molecular Probes</td>
			<td>F1221</td>
			<td>Ratiometric Ca2+ indicator</td>
		</tr>
		<tr>
			<td>Gentamicin Sulfate</td>
			<td>Calbiochem</td>
			<td>1405-41-0</td>
			<td>antibiotics</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Lambda DG-4 apparatus</td>
			<td>Sutter Instrument, Novato, CA</td>
			<td>DG-4PLUS/OF30</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Gibco</td>
			<td>23017-015</td>
			<td>Help cell adhesion</td>
		</tr>
		<tr>
			<td>Metafluor software</td>
			<td>Universal Imaging Corp. West Chester, PA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M4880</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Vetec</td>
			<td>129</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Isofar</td>
			<td>310</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Vetec</td>
			<td>306</td>
			<td></td>
		</tr>
		<tr>
			<td>PH3 platform</td>
			<td>Warner Intruments, Hamden, CT</td>
			<td>64-0286</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Molecular Probes</td>
			<td>P6866</td>
			<td>nonionic, surfactant</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td>Help cell adhesion</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25%</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td>Dissociation enzyme</td>
		</tr>
	</tbody>
</table>

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.

Here, we used retina cells in culture from embryonic day 8 chicks to investigate how neurons and glia signal in terms of calcium shifts. Cultures were prepared essentially as described15,19 as mixed neuron-glial cells (at a density of &#8805; 1 x 106 cell/dish) at a stage of 7 days in vitro (Figure 1A). Alternatively, enriched neuronal cells prepared in low density (5 x 105 cell/dish), seeded on treated poly-L-l...

Watch this Scientific Journal Video about Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique at JoVE.com

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

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 ...

The overall goal of this procedure is to monitor changes in cytosolic calcium concentrations in live cells to differentiate neuronal or glia responses based on potassium chloride, or ATP stimuli. Calcium is an important second messenger involved in several cellular processes, including neurotransmission, plasticity, and apoptosis. The advent of high selective fluorescent calcium dye, such as Fura-2 AM, associated with the development of better fluorescent microscopes and computation methods, yielded high resolution optical data with a high degree of spatial and temporal resolution to image calcium signaling on living cells and organisms.This protocol is adaptable to enriched neuron, purified glia, or mixed population cultures. It tracks which cell types responded to determinate stimuli based on the differential response to potassium chloride and ATP. Potassium chloride change cell's membrane potential.And this opens voltage gated calcium channels, expressed essentially by neurons. On the other hand, ATP activates P2X7 large part channel, present mainly in glial cells. By coupling potassium chloride and ATP stimuli with other drugs, it is possible to see which compartment of the nervous system is responding to them, based on the increase of intracellular calcium concentration.For this procedure, you'll need some surgical instruments or tweezers. Use a biosafety cabinet and perform best practice to avoid contamination. To start the chicken retina culture, carefully open a fertilized egg by the bottom, where the air cell is located.Remove the contents to a Petri dish and proceed with the embryo donation by decapitation with a pair of tweezers. Once the head is removed, bring it to a clean Petri dish and add some calcium and magnesium free solution to it. Next, remove the eyes, taking care not to damage them in the process.Bring the eye to another Petri dish containing CMF solution. With a pair of tweezers, begin to dissect the eye by removing the lens. Then, starting by the hole left by the lens, do three or four longitudinal cuts in the eye.Remove the transparent vitreous body with caution. Make sure the retina is not bind to it. Next, gently detach the retina from the pigmented epithelium.Remove any remaining epithelial tissue. When the retina is totally clear, cut it in small pieces. Take all the retina tissue with the help of an automated pipette.Briefly centrifuge the retina to remove all the CMF. Add 1 mL of 0.25%trypsin. And incubate the tissue at 37 C for 10 minutes.Stop trypsin reaction by adding 1 mL of medium containing 10%fetal calf serum. Centrifuge the retina to remove all the trypsin. Wash the cells two or three times with medium containing 10%FCS.Next, add 2 mL of medium per retina and gently mechanically dissociate it. Dilute the cells so you can properly count them with an hemocytometer. Use 15 mm coverslips protected with poly-L-lysine and laminin to help cell adhesion.Pipette 50 L of your diluted cells on each coverslip. Incubate the cells at 37 C in 5%CO2 atmosphere. And chill them to attach the glass.This should take about one to two hours. Add 1 mL of medium and return it to the incubator until the day of the experiment. If needed change half of the medium every two to three days.Reconstitute a 50 g vial of Fura-2 AM with 50 L of DMSO. Krebs solution is a good option to transfer cells during the experiment, because it doesn't interfere with fluorescence measurement. Preheat the solution to 37 before beginning.Add Poloxamer 407. Add Fura-2 AM and DMSO. Sonicate it for seven minutes in a water bath.Prepare a 6-well plate adding Krebs solution to three wells and the working Fura-2 solution on another. Wash the coverslip contain your cells three times before adding to the Fura-2 well. Incubate the cells at 37 C and 5%CO2 atmosphere for 30 minutes in a dark incubator.After incubation, wash it three times again. Transfer it to another recipient containing Krebs and protect it from the light. It is possible to reuse the prepared Fura-2 to incubate more coverslips with cells.Add silicon to the coverslip support and chamber before each run to avoid solution leakage. Take out the coverslip from Krebs solution and carefully wash the bottom with distilled water to avoid salt crystallization on the microscope lens. Put the coverslip on the support, pressing the borders with caution.Attach the coverslip support and chamber to the microscope, and start perfusing the cells with Krebs solution. Select appropriate cells and choose a good field. Manually determine the regions of interest by selecting cell bodies based on their distinct morphology.The variations of calcium concentration were evaluated by quantifying the ratio of the fluorescence emitted at 510 nm following alternate excitation at 340 and 380 nm. After adding potassium chloride it's possible to see many cells glowing and the fluorescence response raising on the graph. Potassium chloride opens voltage gated calcium channels, presents mainly in neurons.Each individual line on the graph represents the unique region of interest. Therefore, it is possible to track individual cell responses during the experiment. ATP stimuli opens P2X7 receptor, essentially expressed by glial cells.This is an enriched neuron culture. Therefore, there are few glial cells here. This is the reason why so few cells respond to the ATP stimuli.Acquired values were processed using the Metafluor software. Experimental results are expressed in an Excel table, where each row represent an individual cell, and each line, a time point. Using Excel software, it is possible to plot calcium variations of individual cells separately, or of all of them at the same graph.To quantify the amount of reactive cells to any stimulus, set a cutoff of 30%increasing calcium baseline levels. Here we use retina cells in culture from embryonic day 8 chicks to investigate how neurons and glia signal in terms of calcium shifts, after receiving 50 mm potassium chloride and 1 mm ATP stimuli. In each experiment, about 100 cells were readily analyzed.Figure A represents a mixed culture containing both neurons and glia, that was maintained for one week. When we quantify the responses, it is possible to see that about half of the analyzed cells answer potassium chloride, and the other half responded to ATP. Figure B refers to a neuron enriched culture.It was incubated for only three days, therefore, most glial cells haven't differentiated yet. In this case, 89%of cells answer potassium chloride, reinforcing the prevalence of neurons. Figure C shows a purified glia culture.These cells were maintained for 10 days with medium changes every three days. After that time most neuron dies, leaving only glia. Indeed, this culture was solely activated by ATP.This methodology has been used broadly due to the universal properties of calcium as a second messenger. The phenotypical analysis can be enhanced by complimenting this methodology with immunochemistry of fixed cells after calcium imaging. This protocol could also be adapted to other mixed cell systems expressing other calcium channels.The important tip is to find selective responses of different types of cells correlated with their phenotypic expression.

Research

JoVE Journal

Neuroscience

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

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