visualization of calcium influx in ventral midbrain neurons derived from mouse embryos

Visualization of Calcium Influx in Ventral Midbrain Neurons Derived from Mouse Embryos

Prepare 1 liter of the HEPES recording buffer, 200 milliliters of 20 micromolar glutamate recording buffer, and 200 milliliters of 10 micromolar NBQX recording buffer according to manuscript directions. Fill a sterile millimetre Petri dish with 3 milliliters of the recording buffer.Take the Petri dish with the infected cultures from the incubator, then carefully grab the edge of one coverslip with fine tip forceps, and transfer it into the dish with the recording buffer. Place the remaining coverslip in medium back into the 37 degrees Celsius incubator, and transport the dish with recording buffer to the confocal microscope.Start the imaging software. While it is initializing, start the peristaltic pump and place the line into the recording buffer. Then calibrate the flow to 2 milliliters per minute. Transfer the infected coverslip from the Petri dish to the recording bath with fine forceps. Using the 10x water immersion objective and brightfield light, find the plane of focus, and look for a region with a high density of neuron cell bodies. Then switch to the 40X objective and refocus the sample.Select and apply Alexa Flour 488 in the dyes list window. In order to prevent overexposure and photobleaching of the fluorophores, start with low HV and laser power settings. For the Alexa Fluor 488 channel, set the HV to 500, the gain to 1x, and the offset to zero. Set the power to 5% for the 488 laser line, increase the pinhole size to 300 micrometers, and use the focus x2 scanning option to optimally adjust emission signals to sub saturation levels.Settings can then be adjusted for optimal visibility of each channel. Once the microscope settings are optimized, move the stage to locate a region with multiple cells displaying spontaneous changes in GCaMP6f fluorescence, and focus on the desired plane for imaging. Use the clip rect tool to clip the imaging frame to a size that can achieve a frame interval of just under one second. Set the interval window to a value of 1.0 and the Num window to 600.To capture a t-series movie, select the time option, then use the XYt scanning option to begin imaging. Watch the imaging progress bar, and move the line from the HEPES recording buffer into the 20 micromolar glutamate recording buffer at the appropriate time point. When imaging is complete, select the series done button, and save the finished t-series movie. Continue to perfuse the glutamate for an additional five minutes, so that the cultured neurons have been exposed to glutamate for a total of 10 minutes.Repeat this process for each coverslip to be imaged. It is possible to view calcium traces in neuronal cell bodies immediately following the experiment. Use the ellipse tool to draw the desired number of ROIs around neuronal soma, and use the series analysis button to visualize the traces. After the additional five minute exposure to glutamate, remove the coverslip from the bath with fine forceps, and place it back into the Petri dish with the recording buffer until all imaging is completed.

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&#160;1. Plating the cellsNOTE: Based on experience, about 100,000 viable cells per embryo are collected. 2&#8211;3-month-old timed pregnant mice typically have litter sizes of 8-10 embryos; therefore, a rough estimate for total yield of cells per timed pregnant mouse is approximately 1 million cells.<ol><li>Using a hemocytometer, perform a cell count and then dilute the suspension to 2,000 cells/&#181;L using cell culture medium. Triturate briefly to mix.</li><li>Remove coverslips with laminin solution from the incubator and aspirate the remaining laminin solution from the coated coverslips using a vacuum. Plate quickly to avoid the coverslips from drying completely. Pipette 100 &#181;L (2.0 x 105&#160;cells/coverslip) onto each coverslip and place Petri dishes into a 37 &#176;C incubator for 1 h.</li><li>Carefully add 3 mL of cell culture medium to each dish and place back into the 37 &#176;C incubator. Preform half medium changes 2 times per week for 2 weeks.</li></ol>2. Infection of cell culture at 14 (days&#160;in vitro) DIV with adeno-associated viral (AAV) vectors<ol><li>For each dish prepare 1 mL of serum free DMEM medium with 1 &#181;L of hSyn-GCaMP6f AAV (1.0 x 1013&#160;titer)</li><li>Aspirate the cell culture medium from each dish and replace with 1 mL of serum free Dulbecco's Modified Eagle Medium (DMEM) containing hSyn-GCaMP6f. Place dishes back into the 37 &#176;C incubator for 1 h.</li><li>Aspirate the serum free medium containing AAVs and replace with 3 mL of cell culture medium. Place dishes back into the 37 &#176;C incubator. We have found that 5-7 days of AAV infection allows for ideal levels of GCaMP expression. Continue to change medium every 2-3 days throughout this period of viral infection.</li></ol>3. Live confocal Ca2+&#160;imaging between 19-21 days in vitro (DIV)NOTE: Imaging can be done between 5-7 days following viral infection. This is the ideal window to achieve visible expression of the fluorophore at levels which allow for detection of spontaneous Ca2+&#160;activity.<ol><li>Preparation of recording buffers<ol><li>To make 1 L of HEPES recording buffer, add: 9.009 g of NaCl, 0.3728 g of KCl, 0.901 g of D-glucose, 2.381 g of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2 mL of 1 M CaCl2&#160;stock solution, and 500 &#181;L of 1 M MgCl2&#160;stock solution to 800 mL of sterile distilled H2O. Bring the pH to 7.4 with NaOH. Bring to a final volume of 1 L.</li><li>To make 200 mL of 20 &#181;M glutamate recording buffer, dilute 40 &#181;L of 100 mM glutamate stock solution into 200 mL of HEPES recording buffer described above.</li></ol></li><li>Confocal imaging<ol><li>Fill a sterile 35 mm Petri dish with 3 mL of recording buffer.</li><li>Remove a 35 mm Petri dish with infected cultures from the 37 &#176;C incubator. Using fine tip forceps, carefully grab the edge of one coverslip and transfer it quickly into the Petri dish filled with recording buffer. Place the remaining coverslip in medium back into the 37 &#176;C incubator. Transport the dish with the recording buffer to the confocal microscope.</li><li>Start the imaging software. Proceed to the next step while it initializes.</li><li>Start the peristaltic pump and place the line into the recording buffer. Calibrate the speed of flow to be 2 mL/min.</li><li>Transfer the infected coverslip from the 35 mm Petri dish into the recording bath.</li><li>Using the 10x water immersion objective and bright-field (BF) light, find the plane of focus and look for a region with a high density of neuron cell bodies. Switch to the 40x water immersion objective and use BF light to refocus the sample.</li><li>In the &#8220;Dyes list&#8221; window within FluoView select AlexaFluor 488 and apply it.</li><li>AAV expressions can be variable; therefore, in order to prevent overexposure and photobleaching of the fluorophores, start with low HV and laser power settings. For the AlexaFluor 488 channel, set the high voltage (HV) to 500, the gain to 1x, and offset to 0. For the 488 laser line set the power to 5%. To increase the effective volume imaged in the z-plane, increase the pinhole size to 300 &#181;m. Use the &#8220;focus x2&#8221; scanning option to optimally adjust emission signals to sub-saturation levels. From here, settings can be adjusted until ideal visibility of each channel is achieved. NOTE: To accurately capture the full range of Ca2+&#160;fluxes with GCaMP, adjust the baseline HV and laser power settings to allow for an increase in fluorescent intensity without oversaturating the detector.</li><li>Once microscope settings are optimized, move the stage to locate a region with multiple cells displaying spontaneous changes in GCaMP6f fluorescence and focus to the desired plane for imaging.</li><li>Use the &#8220;Clip rect&#8221; tool to clip the imaging frame to a size that can achieve a frame interval of just under 1 second. This is necessary to set the imaging interval at 1 frame per second.</li><li>Set the &#8220;Interval&#8221; window to a value of 1.0 and the &#8220;Num&#8221; window to 600. NOTE: In order to deliver different recording buffers at the desired time point (300 s), it is important to calibrate the latency of the pump to deliver the new solution to the bath. This will be dependent on the solution perfusion rate (2 mL/min) and the length of the line used to pump solution.</li><li>To capture a t-series movie select the &#8220;Time&#8221; option and then use the &#8220;XYt&#8221; scanning option to begin imaging.</li><li>Watch the imaging progress bar and move the line from the HEPES recording buffer into the 20 &#181;M glutamate recording buffer at the appropriate time point (e.g., if the latency of the pump is calibrated to deliver solution at 60 s, move the line into the glutamate buffer at 240 frames in order to deliver glutamate at 300 s).</li><li>When imaging is complete, select the&#160;Series Done&#160;button and save the finished t-series movie. Continue to perfuse 20 &#181;M Glutamate for an additional 5 min, so that the cultured neurons have been exposed to glutamate for a total of 10 min. Repeat this process for each coverslip to be imaged.</li><li>Following the additional 5 min exposure to 20 &#181;M Glutamate, remove the coverslip from the bath and place back into the 35mm Petri dish containing recording buffer until the day of imaging is completed.</li></ol></li></ol>

<figure class='table'><table class='ck-table-resized'><colgroup><col style='width:25%;'><col style='width:25%;'><col style='width:25%;'><col style='width:25%;'></colgroup><tbody><tr><td style='padding:0px 3px;vertical-align:bottom;'>35 mm uncoated plastic cell culture dishes</td><td style='padding:0px 3px;vertical-align:bottom;'>VWR</td><td style='padding:0px 3px;vertical-align:bottom;'>25382-348</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>Fiber Optic Illuminator, 100V</td><td style='padding:0px 3px;vertical-align:bottom;'>Kent Scientific</td><td style='padding:0px 3px;vertical-align:bottom;'>KSC5410</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>Filter System, PES 22UM 250ML</td><td style='padding:0px 3px;vertical-align:bottom;'>VWR</td><td style='padding:0px 3px;vertical-align:bottom;'>28199-764</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>Fluoview 1000 confocal microscope</td><td style='padding:0px 3px;vertical-align:bottom;'>Olympus</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>Fluoview 1200 confocal microscope</td><td style='padding:0px 3px;vertical-align:bottom;'>Olympus</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>Hanks-balanced Salt Solution (HBSS) 1x</td><td style='padding:0px 3px;vertical-align:bottom;'>ThermoFisher</td><td style='padding:0px 3px;vertical-align:bottom;'>14175095</td><td style='padding:0px 3px;vertical-align:bottom;'>500 mL</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>HEPES</td><td style='padding:0px 3px;vertical-align:bottom;'>VWR</td><td style='padding:0px 3px;vertical-align:bottom;'>101170-478</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>HeraCell 150 CO&#8322;&#160;incubator</td><td style='padding:0px 3px;vertical-align:bottom;'>Heraeus (ThermoFisher)</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>Laminin</td><td style='padding:0px 3px;vertical-align:bottom;'>Sigma-Aldrich</td><td style='padding:0px 3px;vertical-align:bottom;'>L2020</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>L-glutamic acid</td><td style='padding:0px 3px;vertical-align:bottom;'>VWR</td><td style='padding:0px 3px;vertical-align:bottom;'>97061-634</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>Magnesium Chloride (MgCl&#8322;), andydrous</td><td style='padding:0px 3px;vertical-align:bottom;'>Sigma-Aldrich</td><td style='padding:0px 3px;vertical-align:bottom;'>M8266</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>MPII Mini-Peristaltic Pump, 115/230 VAC, 50/60 Hz</td><td style='padding:0px 3px;vertical-align:bottom;'>Harvard Apparatus</td><td style='padding:0px 3px;vertical-align:bottom;'>70-2027</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>pAAV.Syn.GCaMP6f.WPRE.SV40</td><td style='padding:0px 3px;vertical-align:bottom;'>Addgene</td><td style='padding:0px 3px;vertical-align:bottom;'>100837-AAV1</td><td style='padding:0px 3px;vertical-align:bottom;'>Titer: 1.00E+13 gc/ml</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>Potassium Chloride (KCl), anhydrous</td><td style='padding:0px 3px;vertical-align:bottom;'>Sigma-Aldrich</td><td style='padding:0px 3px;vertical-align:bottom;'>746436</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>Pump Head Tubing Pieces For MPII</td><td style='padding:0px 3px;vertical-align:bottom;'>Harvard Apparatus</td><td style='padding:0px 3px;vertical-align:bottom;'>55-4148</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>Sodium Chloride (NaCl), anhydrous</td><td style='padding:0px 3px;vertical-align:bottom;'>Sigma-Aldrich</td><td style='padding:0px 3px;vertical-align:bottom;'>746398</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr><td style='padding:0px 3px;vertical-align:bottom;'>Time-pregnant female C57BL/6 mice</td><td style='padding:0px 3px;vertical-align:bottom;'>Texas A&amp;M Institue for Genomic Medicine</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td><td style='padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr></tbody></table></figure>

Source:&#160;<a style='text-decoration:none;' href='https://appjove.unicartagenaproxy.elogim.com/v/61481/quantifying-spontaneous-ca2-fluxes-and-their-downstream-effects-in-primary-mouse-midbrain-neurons'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Bancroft, E. A.,&#160;et al<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>. Quantifying Spontaneous Ca<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:0.6em;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>2+<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2020)</a> &#160;This video demonstrates the real-time visualization of calcium influx in ventral midbrain neurons derived from mouse embryos. Using a viral vector to express a calcium indicator, the neurons are imaged under a confocal microscope to track changes in fluorescence intensity during spontaneous and neurotransmitter-induced calcium activity.

Source:&#160;Bancroft, E. A.,&#160;et al. Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons.&#160;J. Vis. ...

Take a coverslip with a culture of ventral midbrain neurons derived from mouse embryos.The cells are infected with a viral vector to express a calcium indicator.Transfer the coverslip to the recording chamber of a confocal microscope perfused with a recording buffer.Using visible light, identify a region containing multiple neurons, then switch to fluorescence imaging.At low intracellular calcium levels, the indicator remains unbound, exhibiting low fluorescence intensity.Intrinsic properties of the neurons generate spontaneous action potentials, triggering voltage-gated calcium channels to open and allow calcium influx.The excess calcium binds to the indicator, inducing a conformational change that increases fluorescence intensity.Record the fluorescence intensity over time to track the spontaneous calcium activity.Introduce a neurotransmitter at a high concentration, which binds to receptors on the neurons, generating continuous action potentials. &#160;The sustained voltage-gated calcium channel activation prolongs calcium influx, causing a sustained increase in fluorescence intensity.

7 Görüntüleme. Visualization of Calcium Influx in Ventral Midbrain Neurons Derived from Mouse Embryos

Video: Visualization of Calcium Influx in Ventral Midbrain Neurons Derived from Mouse Embryos - Deney

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.

Xenopus laevis'te Nöronal Öncül Karakterizasyonu için Floresan Kalsiyum Görüntüleme ve Daha Sonra Yerinde Hibridizasyon

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 ...

JoVE Journal

Gelişim Biyolojisi

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

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

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

Kalsiyum Görüntüleme Tekniği ile Nöron-Glia Devresindeki Değişimlerin Görselleştirilmesi

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

Nörobilim

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

Kendisinin geçici eşleme ve parçacık analiz teknikleri intrasellüler Ca sinyal2 + in Situ ölçmek için uygulamaları

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

Visualization of Calcium Influx in Ventral Midbrain Neurons Derived from Mouse Embryos

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Visualization of Calcium Influx in Ventral Midbrain Neurons Derived from Mouse Embryos