The overall goal of this protocol for the electric and magnetic stimulation of one-dimensional and two-dimensional cultures is to use neuronal cultures efficiently as a reduced model for brain stimulation from which we can extract insight and important parameters. Using our electric stimulation protocol with one dimensional cultures allows us to measure specific parameters related to neural excitation, for example, chronaxie and rheobase in different compartments of the neuron. The magnetic stimulation technique allows us to compare the effect of magnetic versus electric fields on neuronal cultures.
This model also allows us to better diagnose and treat brain disorders. We propose an effective and easy method for culturing pattern neuronal networks in any design pattern even one-dimensional networks. We can then orient electric and time-varying magnetic fields with respect to these networks to separate and observe contributions from different compartments of the neuron.
The main advantage of this technique is that it does not require human subjects or animal models enabling extensive and exhaustive search of the right parameters for neuronal stimulation. Demonstrating the procedure will be Yuri Burnishev, a technician from my laboratory and Ami Eisen, a PhD student in the laboratory. To begin, prepare patterned neuronal cultures by seeding neural cells on glass coverslips partially pre-coated with a biorejection layer.
To do this, clean 25 millimeter coverslips. And using a Sputtering Machine, coat the coverslip with a biorejecting layer. Adjust the paramaters for the deposition process to create a layer consisting of a thin chrome film of six to 10 angstroms, that ensures coating stability and a 30 angstrom thick gold layer providing the coating with thiol reactivity.
Once the process is completed, remove the coverslip from the Sputtering Machine and examine the obtained coating. Next, assemble a modified pen plotter system by replacing an ink tip of a plotter pen with a sharp metal etching needle. Place a rubber sheet glued to a stiff plate into the plotter so that a sturdy and high-friction surface is ensured during etching.
Then, adjust the sheet's position by pressing the buttons provided on the plotter. Once the rubber sheet is in place, pipette a small drop of water onto its surface and place the pre-coated coverslip over the drop. Next, using the plotter, etch through the metal layers by repetitive scratching generating 200 micrometer-thick lines to create the desired patterns.
Prior to an electric stimulation, place the seeded coverslip into an imaging chamber. To stimulate the cells with an electric field of a constant direction, use two parallel electrodes separated by 11 millimeters and located at opposite sides of the culture plated on the 13 millimeter coverslips. Then, assemble the culture imaging chamber and matching electrode holder such that the wire electrodes are immersed in the extracellular media and positioned one millimeter above the culture.
Then, plug the single electrode pair to the operational amplifier that amplifies the signal from the generator. Apply a single pulse of bipolar square-wave with a 50%duty cycle and amplitude of 22 volts. Use a bipolar wave with no DC component to avoid electrolysis that may occur at the electrodes.
Apply the pulse lasting from 10 microseconds to four milliseconds to ensure effective stimulation but prevent cell injury. To stimulate the cells with electric fields of a changing direction, use two perpendicular pairs of electrodes. Make sure that the two pairs of electrodes as well as their power and monitoring terminals are completely electrically isolated from each other and are floating from earth ground.
To change the electric field orientation, vary the amplitude of the voltage applied to the two electrode pairs. Then, determine the intensity and the direction of the resulting field by vector addition. To generate a fixed amplitude rotating field, apply a sine wave to one electrode pair while feeding the other pair with a cosine wave.
To begin magnetic stimulation, position a neuronal culture, grown in a circular-shaped pattern, under the microscope. Stimulate the culture using a standard circular magnetic stimulation coil and a supporting stimulator which are commercially available. To stimulate the cultures with non-standard magnetic field intensities, connect a custom-made magnetic stimulation coil to the custom-built stimulator.
Then, with an aid of a laser pointer, align the center of the culture to the center of the coil to ensure successful stimulation. Adjust the coil positions so that it remains approximately five millimeters above the center of the culture. Once the stimulation coil is properly positioned, load the stimulator to a potential of up to five kilovolts.
Then, using a high current, high voltage switch, discharge the high voltaging current through the stimulation coil to generate a magnetic pulse that stimulates the activity of the cultured neurons. Finally, mount a pickup coil at the same position as the neuronal culture and measure the intensity of the generated magnetic field. This schematic shows that by applying different amplitudes of voltage pulses to the two pairs of electrodes, the electric fields can be oriented to every angle without the need to manually turn the culture.
Here are the example recordings of calcium transients with electric stimulation at different-applied voltages. When applying a constant duration of electric field, the number of neurons that fire and responds to the electric field shows a cumulative Gaussian distribution as a function of the amplitude of the voltage. And this is an example of a strength-duration curve obtained while employing this protocol.
For the ring cultures responding to the magnetic field, the success rate of stimulation grows with the culture radius. This graph depicts the relation between the ring size and the magnetic field strength. Most successful excitations of the cultures lie in the overlap between the space of the experimentally accessible phase and the high probability region.
Once mastered, the preparation of one-dimensional cultures can be performed within three to four hours. When performing electric and magnetic stimulation, it's important to remember to allow full recovery of the culture from the stimulation step and to make sure that all equipment is floating with respect to the electric ground. By following this procedure, other questions can be addressed such as culturing neurons from different areas of the brain, measuring statistics, for example, of cortical neurons.
Another possibility is to measure parameters of any disease model neuron. After its development, this technique can pave the way for the optimization of the strength and durations needed for effective and safe brain stimulation. After watching this video, you should have a good understanding of how to create any desired pattern of neuronal cultures and to be able to stimulate them electrically or magnetically for the study of interactions of neurons with electromagnetic fields.
