Supported by grants from the American Parkinson Disease Association (APDA) and NIH R01NS115809-01 to RS. We thank the Texas A&#38;M Institute for Genomic Medicine (TIGM) for providing timed pregnant mice to generate primary dopaminergic cultures.

<ol>
	<li>Marras, 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=Prevalence+of+Parkinson's+disease+across+North+America.">Prevalence of Parkinson's disease across North America.</a> NPJ Parkinson's Disease. 4, 21 (2018).</li><li>Poewe, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson+disease.">Parkinson disease.</a> Nature Reviews Disease Primers. 3, 17013 (2017).</li><li>Mehta, A., Prabhakar, M., Kumar, P., Deshmukh, R., Sharma, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitotoxicity:+bridge+to+various+triggers+in+neurodegenerative+disorders.">Excitotoxicity: bridge to various triggers in neurodegenerative disorders.</a> European Journal of Pharmacology. 698 (1-3), 6-18 (2013).</li><li>Ambrosi, G., Cerri, S., Blandini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+further+update+on+the+role+of+excitotoxicity+in+the+pathogenesis+of+Parkinson's+disease.">A further update on the role of excitotoxicity in the pathogenesis of Parkinson's disease.</a> Journal of Neural Transmission (Vienna). 121 (8), 849-859 (2014).</li><li>Dong, X. X., Wang, Y., Qin, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+excitotoxicity+and+their+relevance+to+pathogenesis+of+neurodegenerative+diseases.">Molecular mechanisms of excitotoxicity and their relevance to pathogenesis of neurodegenerative diseases.</a> Acta Pharmaceutica Sinica B. 30 (4), 379-387 (2009).</li><li>Vieira, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitotoxicity+through+Ca2+-permeable+AMPA+receptors+requires+Ca2+-dependent+JNK+activation.">Excitotoxicity through Ca2+-permeable AMPA receptors requires Ca2+-dependent JNK activation.</a> Neurobiology of Disease. 40 (3), 645-655 (2010).</li><li>Sebe, J. Y., 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=Ca(2+)-Permeable+AMPARs+Mediate+Glutamatergic+Transmission+and+Excitotoxic+Damage+at+the+Hair+Cell+Ribbon+Synapse.">Ca(2+)-Permeable AMPARs Mediate Glutamatergic Transmission and Excitotoxic Damage at the Hair Cell Ribbon Synapse.</a> Journal of Neuroscience. 37 (25), 6162-6175 (2017).</li><li>Brickley, S. G., Farrant, M., Swanson, G. T., Cull-Candy, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNQX+increases+GABA-mediated+synaptic+transmission+in+the+cerebellum+by+an+AMPA/kainate+receptor-independent+mechanism.">CNQX increases GABA-mediated synaptic transmission in the cerebellum by an AMPA/kainate receptor-independent mechanism.</a> Neuropharmacology. 41 (6), 730-736 (2001).</li><li>Srinivasan, 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=Smoking-Relevant+Nicotine+Concentration+Attenuates+the+Unfolded+Protein+Response+in+Dopaminergic+Neurons.">Smoking-Relevant Nicotine Concentration Attenuates the Unfolded Protein Response in Dopaminergic Neurons.</a> Journal of Neuroscience. 36 (1), 65-79 (2016).</li><li>Henley, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+Identification+of+Living+Dopaminergic+Neurons+in+Midbrain+Cultures+Using+RNA+Sequencing+and+TH-promoter-driven+eGFP+Expression.">Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression.</a> Journal of Visualized Experiments. (120), e54981 (2017).</li><li>Douhou, A., Troadec, J. D., Ruberg, M., Raisman-Vozari, R., Michel, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+promotion+of+mesencephalic+dopaminergic+neurons+by+depolarizing+concentrations+of+K++requires+concurrent+inactivation+of+NMDA+or+AMPA/kainate+receptors.">Survival promotion of mesencephalic dopaminergic neurons by depolarizing concentrations of K+ requires concurrent inactivation of NMDA or AMPA/kainate receptors.</a> Journal of Neurochemistry. 78 (1), 163-174 (2001).</li><li>Lavaur, J., 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=The+noble+gas+xenon+provides+protection+and+trophic+stimulation+to+midbrain+dopamine+neurons.">The noble gas xenon provides protection and trophic stimulation to midbrain dopamine neurons.</a> Journal of Neurochemistry. 142 (1), 14-28 (2017).</li><li>Kritis, A. A., Stamoula, E. G., Paniskaki, K. A., Vavilis, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Researching+glutamate+-+induced+cytotoxicity+in+different+cell+lines:+a+comparative/collective+analysis/study.">Researching glutamate - induced cytotoxicity in different cell lines: a comparative/collective analysis/study.</a> Frontiers in Cellular Neuroscience. 9, 91 (2015).</li><li>Gupta, K., Hardingham, G. E., Chandran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptor-dependent+glutamate+excitotoxicity+in+human+embryonic+stem+cell-derived+neurons.">NMDA receptor-dependent glutamate excitotoxicity in human embryonic stem cell-derived neurons.</a> Neuroscience Letters. 543, 95-100 (2013).</li><li>Surmeier, D. J., Obeso, J. A., Halliday, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+neuronal+vulnerability+in+Parkinson+disease.">Selective neuronal vulnerability in Parkinson disease.</a> Nature Reviews Neuroscience. 18 (2), 101-113 (2017).</li><li>Ramirez, J. M., Tryba, A. K., Pena, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pacemaker+neurons+and+neuronal+networks:+an+integrative+view.">Pacemaker neurons and neuronal networks: an integrative view.</a> Current Opinion in Neurobiology. 14 (6), 665-674 (2004).</li><li>Guzman, J. N., Sanchez-Padilla, J., Chan, C. S., Surmeier, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+pacemaking+in+substantia+nigra+dopaminergic+neurons.">Robust pacemaking in substantia nigra dopaminergic neurons.</a> Journal of Neuroscience. 29 (35), 11011-11019 (2009).</li></ol>

The authors have nothing to disclose.

We describe a long-term primary ventral mesencephalic (VM) cell culture system for high-content analysis of glutamate-mediated apoptosis in neurons. Studies have employed primary midbrain dopaminergic cultures to elucidate excitotoxic mechanisms in the context of PD models11,12. In this study, we employ a combinatorial approach using Genetically Encoded Calcium Indicators (GECIs) to measure Ca2+ activity and further associate this activity with downstream molecular changes, such as initiation of apoptotic signaling cascades4. The method has multiple advantages to other similar cell culture systems. As we have particular interest in excitotoxicity within the context of Parkinson&#8217;s disease, using primary VM cell cultures is ideal. By using different field relocation techniques, such as gridded coverslips or a motorized XY microscope stage combined with TH immunostaining, we can directly study the cell type specific effects of glutamate-mediated apoptosis in ventral midbrain neurons. Additionally, the 3-week cell culture model allows for neurons to develop their full, mature molecular profile, reflecting adult DA neurons9. Previous methods have mainly focused on molecular changes following glutamate-mediated excitotoxicity13,14. The model is unique in its ability to correlate acute changes in neuronal physiology with downstream molecular events in identified cell types. One limitation of the primary culture model is that the dissection technique captures the entire ventral midbrain, including DA and GABAergic neurons as well as neurons from the SNc and VTA. Evidence now suggests that DA neurons of the SNc have selective vulnerability to calcium and eventual cell death compared to DA neurons of the neighboring VTA15. Unfortunately, differentiating SNc from VTA neurons in embryonic cultures has proven difficult with few anatomical landmarks to define these structures in the embryonic brain.
We demonstrate that the primary culture technique allows for quantification of heterogenous spontaneous Ca2+ activity (Figure 1). Therefore, this is an ideal cell culture system model to study tonically active cells, such as pacemaking dopaminergic neurons of the midbrain, neocortical neurons, and GABAergic neurons of the suprachiasmatic nucleus (SCN)16,17. In most applications, Ca2+ imaging does not achieve the same temporal resolution as electrophysiology. Therefore, it is likely that a single Ca2+ event is analogous to a burst of neuronal action potentials. This can be interpreted to mean that Ca2+ imaging allows for relatively accurate measures of abnormal bursting activity in pacemaking cells and is therefore appropriate for a high content screen of Ca2+-mediated excitotoxic cell death.
To achieve and maintain spontaneous Ca2+ activity, it is important to address two key points in the protocol. First is the plating density of the cells following dissection. For primary VM neurons, previous studies have used around 100,000 cells/cm2 9,10. We have adapted the protocol to plate a density of 200,000 cells/cm2, which creates a heterogenous range of spontaneous activity and increases the number of dopaminergic VM neurons present on each coverslip. Since different pacemaking neurons have distinct firing properties16, the plating density needs to be customized to the cell type being studied and optimized in order to achieve ideal levels of spontaneous activity. Second is the incubation time following viral infection of AAVs. Like plating density, this will be dependent on the specific context of the research question and type of AAV being used. For the specific AAV used here, 5 days of incubation following viral infection is ideal to achieve the desired protein expression levels, which allows for dynamic changes in GCaMP fluorescence in order to record Ca2+ activity. Many factors determine how quickly and efficiently an AAV will express its cargo, much of which is outside the scope of this method, but briefly, it is important to consider promoter activity and the rate at which the cargo protein matures and folds.
Another advantage of the method is that it allows for considerable flexibility in format, expression vectors, use of imaging equipment, and the range of scientific questions that can be addressed. In addition, the method enables inquiry into a wide range of specific questions that surround glutamate-mediated excitotoxicity in PD, and other models of nervous system dysfunction. For example, glutamate-mediated excitotoxicity involves multiple receptors and signaling cascades5. By using the method, and as demonstrated with the AMPAR blocker, NBQX in Figure 1, it is possible to dissect out specific components of the excitotoxic glutamate response at a physiological and molecular level. Conceivably, a similar approach using inhibitors of second messenger systems could be used to determine their contribution to excitotoxicity. Additionally, the AAVs used here could be adapted to express GECIs&#160;with cell-specific promoters or AAV-expressed optogenetic sensors that could be used to measure other parameters such as neurotransmitter release.
Apart from primary embryonic dissections and confocal imaging, much of the protocol uses basic laboratory skills that do not require specialized training. Therefore, the limitations to the model include the difficulty of the embryonic dissection technique, the length of time the cells must be cultured to reach maturity, and access to a confocal microscope, or similar imaging apparatus. The many benefits and flexibility of the method outweighs these limitations, making this an ideal model to study the role glutamate-mediated excitotoxicity in nervous system disorders. Finally, this model could be an effective tool to screen novel compounds for anti-apoptotic effects and their ability to preserve DA neuron health.

Parkinson&#8217;s Disease (PD) is the second most common neurodegenerative disorder worldwide, with no known cure. Estimates suggest that PD prevalence will continue to increase and is projected to surpass 1 million diagnoses by the year 2030 in the United States alone1. With few effective treatments currently available to combat PD, there is a pressing need to develop more effective therapies. PD is characterized by a rapid and progressive loss of midbrain dopamine (DA) neurons2. The mechanisms that underlie neurodegeneration in PD are poorly understood. Evidence suggests a likely convergence of multiple mechanisms, such as oxidative stress and&#160;mitochondrial dysfunction, etc. that contribute to the initiation of apoptotic signaling cascades and eventual cell death3.
One such convergent mechanism, glutamate-mediated excitotoxicity has been implicated in multiple neurodegenerative diseases, including PD4. While glutamate-mediated excitotoxicity is thought to work mainly through stimulation of NMDA receptors via an excessive increase in intracellular Ca2+ concentration and eventual initiation of apoptosis, Ca2+-permeable AMPA receptors have also been implicated in the excitotoxic response5,6,7. Therefore, it is of interest to determine the contribution of AMPA receptors to glutamate-mediated apoptosis within a PD model. This can be achieved using NBQX, an AMPA and kainate blocker, which at micromolar concentrations is selective for AMPA&#160;receptors8. Glutamate-mediated excitotoxicity and apoptotic signaling cascades are an ideal downstream target to measure the extent of cell death, and a potential target for therapeutic intervention. Therefore, developing a high-content method for assessing glutamate-mediated modulation of calcium activity and associated downstream signaling in primary ventral mesencephalic (VM) neurons would be valuable for screening novel treatment methods on their ability to preserve neuronal health.
Here, we have developed a protocol in which we express the genetically encoded calcium indicator (GECI), GCaMP6f, using AAV2/5 with the human synapsin (hSyn) promotor to measure the Ca2+ activity of mouse VM primary neurons in response to glutamate application which can be measured at the physiological and molecular level. This high-content screening can be adapted for discovering pharmaceuticals or treatments that modulate Ca2+ activity to preserve the health of VM neurons. We propose that this primary culture model is an effective way to screen for novel PD interventions, based on their ability to preserve the health of VM neurons and mitigate the progression of PD.

<table><tbody><tr><td>10% Formalin/PBS</td><td>VWR</td><td>100496-506</td><td></td></tr><tr><td>10X NA 0.3 water-immersion objective</td><td>Olympus</td><td>UMPLFLN10XW</td><td></td></tr><tr><td>12 mm circular cover glass No. 1</td><td>Phenix Research Products</td><td>MS20-121</td><td></td></tr><tr><td>20X NA 0.85 oil-immersion objective</td><td>Olympus</td><td>UPLSAPO20XO</td><td></td></tr><tr><td>35 mm uncoated plastic cell culture dishes</td><td>VWR</td><td>25382-348</td><td></td></tr><tr><td>40X NA 0.3 water-immersion objective</td><td>Olympus</td><td>LUMPLFLN40XW</td><td></td></tr><tr><td>60X NA 1.35 oil-immersion objective</td><td>Olympus</td><td>UPLSAPO60XO</td><td></td></tr><tr><td>Ampicillin (sodium)</td><td>Gold Bio</td><td>A-301-25</td><td></td></tr><tr><td>B-27 supplement</td><td>ThermoFisher</td><td>17504044</td><td>50x stock</td></tr><tr><td>Binolcular Microscope</td><td>Kent Scientific</td><td>KSCXTS-1121</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A7030</td><td></td></tr><tr><td>Calcium Chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;), anhydrous</td><td>Sigma-Aldrich</td><td>746495</td><td></td></tr><tr><td>Chicken polyclonal anti-Tyrosine Hydroxylase</td><td>Abcam</td><td>ab76442</td><td></td></tr><tr><td>Deoxyribonuclease I (DNase)</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>D-glucose, andydrous</td><td>Sigma-Aldrich</td><td>RDD016</td><td></td></tr><tr><td>DMEM + GlutaMAX medium</td><td>ThermoFisher</td><td>10569010</td><td>500 mL</td></tr><tr><td>Equine serum</td><td>ThermoFisher</td><td>26050088</td><td>heat-inactivated</td></tr><tr><td>Fiber Optic Illuminator, 100V</td><td>Kent Scientific</td><td>KSC5410</td><td></td></tr><tr><td>Filter System, PES 22UM 250ML</td><td>VWR</td><td>28199-764</td><td></td></tr><tr><td>Fluoview 1000 confocal microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Fluoview 1200 confocal microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>GlutaMAX supplement</td><td>ThermoFisher</td><td>35050061</td><td></td></tr><tr><td>Goat polyclonal anti-chicken Alexa Fluor 594</td><td>Abcam</td><td>ab150176</td><td></td></tr><tr><td>Goat polyclonal anti-rabbit Alexa Fluor 594</td><td>Abcam</td><td>ab150077</td><td></td></tr><tr><td>Hanks-balanced Salt Solution (HBSS) 1x</td><td>ThermoFisher</td><td>14175095</td><td>500 mL</td></tr><tr><td>HEPES</td><td>VWR</td><td>101170-478</td><td></td></tr><tr><td>HeraCell 150 CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Heraeus (ThermoFisher)</td><td></td><td></td></tr><tr><td>ImageJ v1.52e</td><td>NIH</td><td></td><td></td></tr><tr><td>IRIS-Fine Scissors (Round Type)-S/S Str/31*8mm/13cm</td><td>RWD</td><td>S12014-13</td><td></td></tr><tr><td>Kanamycin monosulfate</td><td>Gold Bio</td><td>K-120-25</td><td></td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td></td></tr><tr><td>L-Ascorbic acid</td><td>Sigma-Aldrich</td><td>A7506</td><td></td></tr><tr><td>L-glutamic acid</td><td>VWR</td><td>97061-634</td><td></td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;), andydrous</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>MPII Mini-Peristaltic Pump, 115/230 VAC, 50/60 Hz</td><td>Harvard Apparatus</td><td>70-2027</td><td></td></tr><tr><td>MULLER Micro Forceps-Str, 0.15mm Tips, 11cm</td><td>RWD</td><td>F11014-11</td><td></td></tr><tr><td>NBQX</td><td>Hello Bio</td><td>HB0443</td><td></td></tr><tr><td>Neurobasal medium</td><td>ThermoFisher</td><td>21103049</td><td>500 mL</td></tr><tr><td>Normal goat serum (NGS)</td><td>Abcam</td><td>ab7481</td><td></td></tr><tr><td>Origin 2020</td><td>OriginLab</td><td></td><td></td></tr><tr><td>pAAV.Syn.GCaMP6f.WPRE.SV40</td><td>Addgene</td><td>100837-AAV1</td><td>Titer: 1.00E+13 gc/ml</td></tr><tr><td>Papain</td><td>Worthington Biomedical Corporation</td><td>LS003126</td><td></td></tr><tr><td>Penicillin streptomycin</td><td>ThermoFisher</td><td>15140122</td><td>10,000 U/mL</td></tr><tr><td>Phosphate-buffered saline (PBS) 1x</td><td>ThermoFisher</td><td>10010049</td><td>500 mL</td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>P4832</td><td></td></tr><tr><td>Poly-L-ornithine</td><td>Sigma-Aldrich</td><td>P4957</td><td></td></tr><tr><td>Potassium Chloride (KCl), anhydrous</td><td>Sigma-Aldrich</td><td>746436</td><td></td></tr><tr><td>Pump Head Tubing Pieces For MPII</td><td>Harvard Apparatus</td><td>55-4148</td><td></td></tr><tr><td>Rabbit monoclonal anti-caspase-3</td><td>Abcam</td><td>ab32351</td><td></td></tr><tr><td>Sodium Chloride (NaCl), anhydrous</td><td>Sigma-Aldrich</td><td>746398</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S7903</td><td>BioXtra, &amp;ge;99.5% (GC)</td></tr><tr><td>Time-pregnant female C57BL/6 mice</td><td>Texas A&amp;amp;M Institue for Genomic Medicine</td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td>500 mL</td></tr><tr><td>Wide-bore blue pipette tips P1000</td><td>VWR</td><td>83007-380</td><td></td></tr></tbody></table>

quantifying spontaneous ca2+ fluxes and their downstream effects in primary mouse midbrain neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Following initial culturing of cells, we treated VM culture dishes at 14 DIV with 1 &#181;L of AAV hSyn-GCaMP6f and allowed for 5 days of viral expression. On the day of imaging HEPES recording buffer was prepared fresh. We used two conditions; in one condition 20 &#181;M glutamate was applied for 10 min, while in the other condition 5 min of 10 &#181;M NBQX application preceded a 10 min co-application of 10 &#181;M NBQX + 20 &#181;M glutamate. In both conditions, we observed heterogenous and spontaneous changes in GCaMP6f fluorescence, which indicate spontaneous Ca2+ fluxes, as shown in the representative traces (Figure 1A,B, Supplemental Movie 1-2). Application of 20 &#181;M glutamate generated a robust and sustained Ca2+ response in both spontaneously active and quiescent neurons (Figure 1A, Supplemental Movie 1). Application of 10 &#181;M NBQX reduced spontaneous activity, and partially blocked the glutamate response (Figure 1B, Supplemental Movie 2). The extent to which glutamate application stimulated a Ca2+ response in each condition was quantified using area under the curve, peak amplitude, and latency to respond. Both area under the curve and peak amplitude were similar for both the glutamate and NBQX + glutamate treated conditions (Figure 1C), while latency to response was significantly increased in the NBQX + glutamate condition (Figure 2A,B). In addition to quantifying the Ca2+ response to glutamate treatment, we fixed and stained samples with an anti-caspase-3 antibody as a measure of glutamate-mediated apoptosis. We observed a range of caspase-3 activation across the conditions (Figure 3A,B). Caspase-3 activation was quantified by measuring area and mean caspase-3 intensity. When compared to untreated control cells, the average area of cells with caspase-3 activation under glutamate and NBQX + glutamate conditions trended towards significance (Figure 3B). Mean caspase-3 intensity was significantly higher in the glutamate and NBQX + glutamate conditions as compared to untreated controls (Figure 3B). Together, these results demonstrate a high-content framework in which apoptosis of neurons can be measured by quantifying Ca2+ responses to excitotoxic agents and followed up with an analysis of downstream apoptotic events such as caspase-3 activation in the same set of cultures.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61481/61481fig01.jpg" /> 
Figure 1: Cultured ventral mesencephalic neurons display spontaneous Ca2+ activity and are robustly stimulated by glutamate application.&#160;(A) Representative traces of spontaneous Ca2+ activity in VM neurons and their response to 20 &#181;M Glutamate application. (B) Representative traces of spontaneous Ca2+ activity in VM neurons and their response to 10 &#181;M NBQX + 20 &#181;M Glutamate application. (C) Population data showing area under the curve and peak amplitude of Ca2+ traces. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61481/61481fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61481/61481fig02v3.jpg" /> 
Figure 2: AMPAR blockade with NBQX delays response to glutamate application in cultured ventral mesencephalic neurons.&#160;(A) Representative Ca2+ traces of glutamate (gray) and NBQX + glutamate (blue) evoked responses. Average Ca2+ traces of glutamate (black) and NBQX + glutamate (red) are shown overlaid. (B) Population data showing latency to response for glutamate and NBQX + glutamate evoked responses. Percent change between glutamate and NBQX + glutamate conditions is displayed in the right panel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61481/61481fig02v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61481/61481fig03v3.jpg" /> 
Figure 3: Glutamate application increases caspase-3 expression in tyrosine hydroxylase (TH) positive ventral mesencephalic neurons.&#160;(A) Representative confocal images of VM cultures immunostained for caspase-3 (green) and TH (red), scale bar = 10 &#181;m. (B) Population data showing DA neuron area and mean gray value of caspase-3 expression in each condition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61481/61481fig03v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Movie 1: Spontaneous Ca2+ activity and response to glutamate application. 
Spontaneous Ca2+ fluxes in the presence of HEPES recording buffer (0-300 s) followed by application of 20 &#181;M glutamate (301-600 s). Scale bar = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61481/Supplementary_Movie 1_glutamate_ver_2.avi" target="_blank">Please click here to download this video.</a>
Supplementary Movie 2: Spontaneous Ca2+ activity and response to NBQX + glutamate application. 
Spontaneous Ca2+ fluxes in the presence of HEPES recording buffer (0-300 s) followed by application of 10 &#181;M NBQX (301-600 s), and 10 &#181;M NBQX + 20 &#181;M glutamate (601-900 s). Scale bar = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61481/Supplementary_Movie_2_NBQX_glut.avi" target="_blank">Please click here to download this video.</a>

Watch this Scientific Journal Video about Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons at JoVE.com

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.2K Views.  Texas A&M University Health Science Center, College of Medicine. Dit protocol kwantificeert calciumstromen in dopaminerge neuronen, die verloren gaan bij de ziekte van Parkinson. Dit is handig om te begrijpen hoe abnormaal calcium dopaminerge neuron verlies veroorzaakt bij de ziekte van Parkinson. Voor het eerst tonen we spontane calciumstromen in gekweekte primaire midbrainneuronen.Deze methode kan worden gebruikt om specifieke receptorbijdragen die betrokken zijn bij calcium-gemedieerde apoptose te ontleden. Onze methode biedt een laan voor hoge i...

Video: Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

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

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

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 ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

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 ...

Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a cascade of events leading to neurotransmitter release. Moreover, changes in presynaptic calcium levels mediate the activity of many intracellular proteins and play an important role in synaptic plasticity. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases. The neuromuscular junction is a suitable model for studying synaptic plasticity, as it has only one type of neurotransmitter. This article describes the method for loading a calcium-sensitive dye through the cut nerve bundle into the mice's motor nerve endings. This method allows the estimation of all parameters related to intracellular calcium changes, such as basal calcium level and calcium transient. Since the influx of calcium from the cell exterior into the nerve terminals and its binding/unbinding to the calcium-sensitive dye occur within the range of a few milliseconds, a speedy imaging system is required to record these events. Indeed, high-speed cameras are commonly used for the registration of fast calcium changes, but they have low image resolution parameters. The protocol presented here for recording calcium transient allows extremely good spatial-temporal resolution provided by confocal microscopy.

The protocol describes the method of loading a fluorescent calcium dye through the cut nerve into mouse motor nerve terminals. In addition, a unique method for recording fast calcium transients in the peripheral nerve endings using confocal microscopy is presented.

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

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

Subcellular Imaging of Neuronal Calcium Handling In Vivo

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

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

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

Source:&#160;<a style='text-decoration:none;' href='https://app-jove-com.remotexs.ntu.edu.sg/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. ...

JoVE Encyclopedia of Experiments

Neuroimaging

Structural & Functional Brain Imaging

Simultaneous Measurement of Intracellular Calcium and Membrane Potential in a Mouse Cerebral Endothelium

Source: Hakim, M. A. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58832">Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium.</a> J. Vis. Exp. (2019)
The video demonstrates the simultaneous measurement of intracellular calcium levels and changes in membrane potential in endothelial cells of mouse cerebral artery segments. The isolated endothelial tube is loaded with a fluorescent calcium indicator and treated with a drug that induces intracellular calcium release, resulting in a change in membrane potential. The indicator&#39;s fluorescence enables the measurement of calcium levels, and a recording electrode is inserted into a cell to measure changes in membrane potential.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Dissection and Isolation of ...

Quantifying Spontaneous Ca²⁺ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

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