Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

The protocol given below has been standardized to test the effects of BPA exposure during (early) embryogenesis, and may be modified for use with BPS, BPF, or other EDC in chick embryonic neurons.

This protocol follows the institutional policies of Delaware State University and the NIH policy for avian embryos. The protocol also is in confirmation with DSU's material and chemical safety guidelines.

1. Material Setup

1. Dissolve BPA (m.w. 228.29) in 10% ethanol (v/v) in water to make a 1 mM stock solution (0.23 mg per mL of 10% ethanol). Make subsequent dilutions (0.1 µM, 0.5 µM, 1 µM, 5 µM, and 10 µM) in neurobasal medium.
   Caution: BPA is an environmental toxin and care should be exercised when handling the powder.
2. Incubate chick eggs in a commercially available tabletop egg incubator at 37 °C. Incubate eggs for 7 days for E7 embryos.
3. Prepare MEAs. Sterilize the required number of MEAs by soaking in 70% ethanol for 3 to 4 h and then rinse three times in approximately 10 mL of sterile distilled water in the BSLII hood.
   NOTE: The MEAs contain 64 nanoporous platinum electrodes arranged in an 8 x 8 grid. The electrode diameter is 30 µm and the spacing between the electrodes is 200 µm. Do not use denatured ethanol for sterilizing the MEAs as this will lead to corrosion of the MEA.
4. Place the MEAs inside a sterile container with lid (to maintain sterile conditions) and move to a table top incubator. Bake for at least 6 hours at 55 °C. This step is critical for proper sterilization and the temperature should not exceed 60 °C. Maintaining sterility, move the dish containing MEAs to the BSLII hood for storage until further use.
   NOTE: We use an oven safe glass baking dish with plastic lid and surface sterilize it with 70% ethanol and place in the BSLII hood for drying.
5. Aliquot the extra cellular matrix ECM solution. Thaw the vial at 4 °C overnight and freeze 100 µL aliquots at -20 °C.
   NOTE: ECM is shipped frozen. ECM should not be brought to room temperature until ready to coat as it polymerizes at room temperature and, once polymerized, it is impossible to coat. ECM without growth factors is preferred to prevent additional effects due to unknown factors.
6. Sterilize all dissection instruments (Dumont #5 forceps, curved forceps, small scissors, and spring scissors) and self-sealing material coated dissection dishes with 70% ethanol and let them dry in the dissection hood.

2. Chick Embryonic Neuron Culture and Network Activity

1. On the day of plating, remove a vial of ECM from the -20 °C freezer, spray with 70% ethanol, and place on ice. Dilute to 25% by adding 300 µL cold neurobasal medium inside BSLII hood.
2. Using a P200 pipetteman add 100 µL of 25% ECM to the center of the MEA taking care not to touch the electrodes and remove immediately leaving a thin film on the surface. Cover the MEA and place in the CO₂ incubator (37 ° C and 5% CO₂) until ready to plate the neurons.
3. Sterilize the outer shell of an E7 egg (embryonic day 7, incubated at 37 °C for 7 days) with 70% ethanol. Decapitate the embryo into a Sylgard bottom dissection dish containing cold sterile Hank's Balanced Salts Solution (HBSS) without calcium. Cut around the eyes and remove the eyeballs. Using DuMont #5 fine forceps and spring scissors make an incision on the ventral side and remove the outer layers of skin to expose the forebrain and optic tectum. Peel and remove the pial membrane carefully. Transfer the forebrain into another petri dish and cut it into small pieces of about 2 mm with spring scissors.
   NOTE: There should not be any blood vessels attached to the forebrain after removal of the pial membrane. If available, it is preferable to perform the dissection in a sterile dissection hood, although we have obtained fairly good results when dissection is performed outside a hood as long as the instruments and dissection area are thoroughly sterilized with 70% ethanol.
4. Using a sterile transfer pipette, collect the pieces of forebrain into a 15 mL centrifuge tube and remove as much of the HBSS as possible after letting the pieces of forebrain sink to the bottom of the centrifuge tube.
5. Add 1 mL of 0.05% Trypsin/EDTA (pre-warmed to 37 °C) and incubate at 37 °C for 15 minutes.
6. Using a Pasteur pipette, carefully remove trypsin without disturbing the pieces of tissue and add 1 mL neurobasal medium. Let the pieces of tissue sink to the bottom and remove the medium. Repeat this wash once more. This step is to wash off the trypsin/EDTA.
7. Add 2 mL of neurobasal medium and start triturating. Trituration involves taking a sterile fire-polished Pasteur pipette and passing the tissue through it several times gently until no more pieces of tissue are seen. If any pieces remain after repeated passes, just leave them to settle to the bottom.
   NOTE: Avoid frothing while triturating – if froth does form, stop and remove by aspiration.
8. Dilute the re-suspended cells 1:10 with neurobasal medium and count viable cells using Trypan Blue dye and a hemocytometer. Mix 50 µL of the diluted cells with 50 µL of Trypan Blue solution in a 0.5 mL centrifuge tube. Add 10 µL to the hemacytometer after placing on the coverslip and count the bright clear cells (blue cells are dead and should not be counted).
   NOTE: Typical cell numbers from a single E7 optic tectum range between 1 and 5 x 10⁷ cells/mL.
9. Plate the dissociated cells on ECM coated MEAs at a density of 2,200 cells/square mm. For our MEA system, this translates to approximately 130,000 cells per MEA. Add neurobasal medium to bring the volume in the MEA to 1 mL and place MEAs in the CO₂ incubator overnight for cell attachment and neurite extension (Figure 1).
   NOTE: For MEAs of different areas and electrode numbers, different cell densities could be tried – in general, higher cell density results in greater network activity, but also leads to shorter lived cultures.
10. The next day, add BPA to a final concentration of 10 µM in neurobasal medium from the 1 mM stock made in step 1.1 to the treated MEA. To the control MEA, add the same volume of the 10% ethanol in water (v/v) solution.
11. Replace spent medium with neurobasal medium containing 10 µM BPA every other day until 7 days in vitro (7DIV) and every day thereafter, as the network activity increases.
    NOTE: As the network activity increases, metabolic activity increases and media changes need to be more frequent. Do not let the medium turn orange.

3. Recording Neuronal Network Activity

1. On the day of acquisition, exchange the culture medium with fresh neurobasal medium and return the MEAs to the CO₂ incubator for a minimum of 2 h before recording. Start the recording software and set the temperature of the MEA to 37 °C by clicking on the temperature icon (Supplemental Figure 3.1a-b).
   NOTE: Recording can begin as early as day 3 of plating and can continue as long as the cultures are alive.
2. Set up the acquisition parameters by right-clicking on the "Muse" icon (under Streams in the left window panel) and select "Add Processing" and "Spike Detector" and click OK in the pop up window. "Spike Detector (6 x STD)" will appear below the file name (Supplemental Figure 3.2a-b).
3. Next right click on the Spike Detector, select "Add Processing" and "Burst Detector" and click OK in the pop up window. "Burst Detector" (ISI) will appear below the Muse icon (Supplemental Figure 3.3a-b).
4. Next right click on Burst Detector, select "Add Processing" and "Neural Statistics Compiler." In the pop up window, ensure that File Header, Aggregated Well Statistics and Synchrony are selected. Click OK. "Statistics Compiler" will appear below (Supplemental Figure 3.4a-b).
   NOTE: This will set the adaptive threshold crossing (6x STD filter), the inter-spike interval at 100 ms with a minimum number of spikes at 5, the mean firing rate detection at 10 s with a synchrony parameter at 20 ms, and the minimum spike rate at 0.083333 spikes per min.
5. Set up the scheduled recording for the recording time of 5 minutes (300,000 ms) by clicking on the clock icon at the bottom. This is achieved by changing the information in the setting section to record every 5.1 minutes and record for 5 minutes. This will be started immediately and will be executed once (Supplemental Figure 3.5).
6. Ensure that the temperature of the MEA has reached 37 °C before moving the MEA. Remove the MEA from the incubator, place it on the recording unit, and lock down the MEA. Start recording the network activity by clicking on the start record in the scheduled recording section or the record icon in the "File Play" window. Typically, a recording time of 5 minutes (300,000 ms) is sufficient, although longer recordings of up to 10 minutes can be obtained.
7. After recording, return the MEA to the incubator.
   NOTE: Care must be taken to maintain sterility and to minimize the time the MEA is outside the incubator.

4. Data Analysis

1. Analysis of the raw data can be done offline using the recording software. Click on the File pull down menu and open recording. Select the raw data file to be analyzed. Replay the raw recordings to capture the required spiking parameters.
2. To obtain the Average Number of Spikes, use the Spike Detector module and for obtaining Synchrony Index, use the Neural Statistics Compiler module.
3. Load the Spike Detector, Burst Detector and Statistics Compiler Modules as before (Section 3).
4. From the pull-down menus within the Spike Detector, Burst Detector, and Statistics Compiler module windows, select Spike List, Network Burst List, and Advanced Metrics, respectively.
5. Click the record button to start recording the data file which will create a .csv file in the directed folder. The spike parameters – the total number of spikes and the Synchrony Index – will be recorded in the .csv file.
   NOTE: Real time analysis while recording raw data is not recommended as this could consume processor time and computational resources.