The overall goal of this experimental protocol is to test the effects of endocrine disrupting compounds, like bisphenols, on the network spiking activity of vertebrate neuronal networks established on multi-electrode arrays. This method can help answer key questions in developmental neurobiology and environmental toxicology such as the mechanism of action of environmental toxins on the development of network activity in the embryonic brain. The main advantage of this technique is that the effects on neuronal network parameters, like the mean firing rate, the total number of spikes and the spike amplitude as well as synchrony, can now be studied.

Our previous study showed that EDCs affect word and behavior which is a direct output of neuronal networks, and our protocol now aims to understand the effect of EDCs on these networks. On the day of neuron plating, remove a vial of extracellular matrix from the negative 20 degrees Celsius freezer, spray with 70%ethanol and place on ice. Be sure to thaw out the extracellular matrix on ice.

Do not thaw out at room temperature as the ECM polymerizes above zero degrees Celsius. Work in the BSL2 hood to dilute the extracellular matrix to 25%by adding 300 microliters of cold neurobasal medium to the 100 microliter aliquot. Then, use a P200 pipette to add 100 microliters of 25%extracellular matrix solution to the center of the multi-electrode array taking care not to touch the electrodes.

Immediately remove the ECM leaving a thin film on the surface. Cover the multi-electrode array and place in the tissue culture incubator until ready to plate the neurons. Begin chick neuron isolation by sterilizing the outer shell of an E7 egg with 70%ethanol.

It's important to maintain sterile conditions from this point onward. Ensure all instruments are sterilized with 70%ethanol and solutions are sterile. It is helpful to use a dissection hood, but it is not absolutely necessary for dissections if it is performed quickly.

Then after retrieving and decapitating the embryo, cut around the eyes and remove the eyeballs. Next, use Dumont number five fine forceps and spring scissors to 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 peel membrane carefully.

Transfer the forebrain into another Petri dish containing HBSS and cut it into small pieces of about two millimeters with spring scissors. Use a sterile transfer pipette to transfer the pieces of forebrain into a 15-milliliter centrifuge tube. After the pieces of forebrain sink to the bottom of the centrifuge tube, remove as much HBSS as possible.

Next, add one milliliter of pre-warmed, 0.5%trypsin EDTA and incubate at 37 degrees Celsius for 15 minutes. Use a Pasteur pipette to carefully remove the trypsin without disturbing the pieces of tissue. Add one milliliter of neurobasal medium and let the pieces of tissue sink to the bottom.

After removing the medium and repeating the wash, add two milliliters of neurobasal medium and triturate gently until no more pieces of tissue are seen. Dilute the resuspended cells one to 10 with neurobasal medium. Count viable cells using trypan blue dye and a hemocytometer.

After counting, plate the dissociated cells on ECM-coated, multi-electrode arrays at a density of 2, 200 cells per square millimeter. The next day, add 10 micromolar BPA in neurobasal medium to the treated, multi-electrode arrays. On the day of acquisition, replace the culture medium with fresh neurobasal medium and return the arrays to the CO2 incubator for a minimum of two hours before recording.

Start the recording software and set the temperature of the multi-electrode array to 37 degrees Celsius by clicking on the temperature icon. Set the acquisition parameters by selecting streams in the left window panel and right-clicking on the muse icon. Then, select add processing and spike detector and click OK in the pop-up window.

Spike detector six by STD will appear below the file name. 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.

Then, 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. Click on the clock icon at the bottom of the screen and change the information in the settings section to record every 5.1 minutes and record for five minutes.

This will be started immediately and will be executed once. After retrieving the multi-electrode array from the incubator, place it on the recording unit and lock it into place. Start recording the network activity by clicking on start record in the scheduled recording section.

Typically, a recording time of five minutes is sufficient, although, longer recordings of up to 10 minutes can be obtained. After recording, return the multi-electrode array to the incubator. The average number of spikes is significantly lower in BPA-treated E7 chick forebrain cultures when compared to control cultures.

The average synchrony index is significantly lower in BPA-treated E7 chick forebrain cultures when compared to control cultures. Once mastered, this technique can be done in less than three hours if done properly. While attempting this procedure, it is important to remember to maintain sterile conditions at all time even while recording the spike activity.

Following this procedure, the effect of potential drugs, like antiepileptics and antidepressants, on network activity can be assessed as a step towards understanding their mechanism of action. Many EDCs have subtle effects on the brain during development which manifest themselves as changes in behavior. Behavior is a direct output of the underlying network activity.

Our protocol aims to dissect the effects of these compounds on the network activity.

Summary

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