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 glucose, 10 mM HEPES, pH 7.4, 373 mOsm).</li>
	<li>Prepare saline buffer (Ca2+ and Mg2+ free solution - CMF): 76.55 g/L NaCl, 3.05 g/L KCl, 1.65 g/L Na2HPO4, 0.610 g/L KH2PO4, 21.95 g/L glucose, and 7.90 g/L NaHCO3.</li>
	<li>Prepare incubating solution (with Fura-2 in Krebs solution:) 5 &#181;M Fura-2-acetoxymethyl ester (Fura-2), 0.1% fatty acid-free bovine serum albumin (BSA), and 0.02% Poloxamer 407.</li>
	<li>For mixed population or glia culture, prepare DMEM/F12 with 10% fetal calf serum (FCS) and 40 mg/L gentamicin as the medium.</li>
	<li>For neuron enriched culture, prepare DMEM/F12 with 1% FCS, neuronal cell culture supplement, 40 mg/L and gentamicin as the medium.</li>
</ol>
2. Retina dissection and cell culture preparation
<ol>
	<li>Open the 8-day old chick embryo (E8) egg where the air cell is located.</li>
	<li>Remove the egg contents to a Petri dish and proceed with the embryo euthanasia by decapitation with a pair of tweezers.</li>
	<li>Bring the head to a clean Petri dish and pour some calcium and magnesium free solution (CMF) to wash it.</li>
	<li>Remove the eyes, taking care not to damage it in the process. 
	NOTE: Try to avoid cutting or shredding eye&#39;s layers before removing it from the eye cavity.</li>
	<li>Bring the eyes to a clean Petri dish containing CMF.</li>
	<li>Begin the eye dissection by removing the lens.</li>
	<li>Make 3 or 4 longitudinal cuts in the eye, starting by the hole left by the lens. Make cuts by pulling the tweezers, holding the eye sclera in opposite directions.</li>
	<li>Remove the transparent vitreous body with caution, making sure that the retina is not bound to it.</li>
	<li>Detach the retina from the pigmented epithelium and remove any remaining tissue.</li>
	<li>Cut the clear retina in small pieces.</li>
	<li>Transfer the retina to another recipient and briefly centrifuge (~1,800 x g for 1 min) it to remove the CMF.</li>
	<li>Enzymatically dissociate the retina with 1 mL of 0.25% trypsin, by incubating it at 37 &#176;C for 10 min.</li>
	<li>Stop the reaction by adding 1 mL of medium containing 10% FCS.</li>
	<li>Wash the retina 2 or 3 times with medium. The wash consists of cycles of adding medium and removing it by centrifugation (~1,800 x g for 1 min).</li>
	<li>Add 2 mL of complete medium per retina and gently mechanically dissociate the retina by pipetting it up and down. 
	NOTE: Here, investigators can choose which type of culture will be prepared. For an enriched neuronal culture, use DMEM-F12 + neuronal supplement + 1% FCS + antibiotics. For mixed population or purified glia, use DMEM-F12 + 10% FCS + antibiotics.</li>
	<li>Count the cells and dilute them to the desired density. 
	NOTE: Ideally, this should be a low-density culture with ~ 2 x 106 cells or less.</li>
	<li>Add 50 &#181;L of the cell suspension to each coverslip.</li>
	<li>Coverslip treatment
	<ol>
		<li>Incubate coverslips for at least 1 h (ideally overnight) in 1 mL of 10-50 &#181;g/mL poly-L-lysine in sterilized purified water at 37 &#176;C.</li>
		<li>Remove the poly-L-lysine solution and wash coverslips with sterilized purified water 2-3 times.</li>
		<li>Let coverslips dry in a safety cabinet with UV lights turned on. At this point, store them in a sealed container at 4 &#176;C for up to 1 month.</li>
		<li>Optional step: For neuron enriched cultures, add 50 &#181;L of 10-20 ng/mL laminin in PBS or medium to each coverslip for 2 h at 37 &#176;C. After incubation, remove laminin solution excess and the coverslip is ready to use.</li>
	</ol>
	</li>
	<li>Incubate cells at 37 &#176;C at a 5% CO2 atmosphere until they attach to the glass. This should take about 1-2 h.</li>
	<li>Add 1 mL of complete medium to each well and return the plate to the incubator until the day of the experiment. If needed, change the medium every 2-3 days.</li>
</ol>
3. Loading cells with Fura-2 AM
<ol>
	<li>Reconstitute a 50 &#181;g vial of Fura-2 AM with 50 &#181;L of DMSO.</li>
	<li>To prepare working Fura-2 AM solution, add 3 &#181;L of 10% Poloxamer 407, 7.5 &#181;L of Fura-2 AM in DMSO and Krebs solution q.s.p. to 1.5 mL.</li>
	<li>Sonicate the mixture for 7 min in a water bath.</li>
	<li>Wash the coverslip with the cell culture 3x with Krebs solution before incubating it in Fura-2.</li>
	<li>Incubate at 37 &#176;C and 5% CO2 for 30 min.</li>
	<li>After incubation, wash it 3x again and transfer it to another recipient containing Krebs and protected from the light. 
	&#8203;NOTE: Working Fura-2 AM solution remains stable for 24 hours if protected from the light.</li>
</ol>
4. Calcium imaging of cells
<ol>
	<li>Before each run, add silicon to the coverslip support and chamber to avoid leakage during the experiment.</li>
	<li>Wash the bottom of the coverslip with distilled water to avoid salt crystallization on the microscope lens.</li>
	<li>Put the coverslip on the support, gently pressing the borders.</li>
	<li>Attach the support and chamber to the microscope and start perfusing cells with Krebs solution. 
	NOTE: Cell perfusion during single cell calcium imaging experiments is calibrated to a flow rate of 0.5 mL/min, and it takes 8-10 s for the platform solution to be fully replaced.</li>
	<li>Have a look at the cells and select an appropriate field of view.</li>
	<li>Manually select the cell bodies based on their distinct morphology.</li>
	<li>Before applying any stimulus, wait for baseline stabilization. Each stimulus should take about 30 seconds. 
	&#8203;NOTE: Prepare all the solutions immediately before each experiment.</li>
	<li>Evaluate the variations in [Ca2+]i by quantifying the ratio of the fluorescence emitted at 510 nm following alternate excitation (750 milliseconds) at 340 and 380 nm.</li>
	<li>Process acquired values using a fluorescence analysis software.</li>
	<li>Express experimental results in a table where each row represents an individual cell, and each line a time point.</li>
</ol>
5. Data processing
<ol>
	<li>Using a spreadsheet software, plot the variation of Fura-2 fluorescence ratio values of singular cells separately or of all of them at the same time.</li>
	<li>To quantify the number of reactive cells to determined stimulus, set a cutoff of 30% increase in calcium baseline levels</li>
</ol>

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The authors have nothing to disclose.

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><tbody><tr><td>15 mm coverslip</td><td>Paul Marienfeld GmbH &amp;amp; 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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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 Ca&lt;sup&gt;2+&lt;/sup&gt; 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>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M4880</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</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>NaHCO&lt;sub&gt;3&lt;/sub&gt;</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 ...

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

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JoVE Journal

Neuroscience

4.4K Views.  Universidade Federal do Rio de Janeiro. 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.

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

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 ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Developmental Biology

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions 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+ ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

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

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

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

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

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

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

Calcium Imaging in Neurons

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and neurotransmitter release from synaptic vesicles. Within the cell, calcium concentration is highly dynamic due to the presence of pumps that selectively transport these ions in response to a variety of signals. Calcium imaging takes advantage of intracellular calcium flux to directly visualize calcium signaling in living neurons.This video begins with an overview of the key reagents used for this technique, known as calcium indicator dyes. The discussion includes an introduction to the commonly used dye Fura-2 and some basic principles behind how both ratiometric and non-ratiometric calcium indicators work. Next, a typical calcium imaging experiment is presented, from preparing the cells and dye to capturing and analyzing the fluorescent images. Finally, several experimental applications of calcium imaging are provided, such as the study of neuronal network activity and sensory processing.

Calcium Imaging in Neurons Using Fura-2

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and ...

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Advanced Biology

Establishing a Whole-Cell Configuration for Two-Photon Calcium Imaging of Brain Slices

Source: Camir&#233;, O., et al. <a href="https://app.jove.com/v/56776">Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices.</a> J. Vis. Exp. (2018).
This video demonstrates a protocol for whole-cell patch-clamp recording and two-photon calcium imaging in brain hippocampal slices. This method allows to study the dynamics of local Ca2+ transients (CaTs) in dendrites of different neuronal types in acute brain slices.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review.
1. Preliminary Preparation (Optional: ...

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

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Chapters in this video

Functional Calcium Imaging in Developing Cortical Networks

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

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

Calcium Imaging in Neurons

Establishing a Whole-Cell Configuration for Two-Photon Calcium Imaging of Brain Slices