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.

visualizing-shifts-on-neuron-glia-circuit-with-calcium-imaging

Research

JoVE Journal

Neuroscience

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

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

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 ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

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 ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

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.

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 ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...