Name: quantifying spontaneous ca2+ fluxes and their downstream effects in primary mouse midbrain neurons
Uploaded: 2020-09-09T14:00:00
Duration: 6 min 54 s
Description: Watch this Scientific Journal Video about Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons at JoVE.com

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>
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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 (CaCl2), 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 CO2 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 (MgCl2), 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, &#8805;99.5% (GC)</td>
		</tr>
		<tr>
			<td>Time-pregnant female C57BL/6 mice</td>
			<td>Texas A&#38;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/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/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/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/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/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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