The authors thank Ofer Feinerman, Fred Wolf, Menahem Segal, Andreas Neef and Eitan Reuveny for very helpful discussions. The authors thank Ilan Breskin and Jordi Soriano for developing early versions of the technology. The authors thank Tsvi Tlusty and Jean-Pierre Eckmann for help with the theoretical concepts. This research was supported by the Minerva Foundation, the Ministry of Science and Technology, Israel, and by Israel Science Foundation grant 1320/09 and the Bi-National Science Foundation grant 2008331.

Ethics Statement: Procedures involving animal handling were done in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Weizmann Institute of Science, and the appropriate Israeli law. The Weizmann Institute is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The Weizmann Institutional Animal Care and Use Committee approved this study, conducted with hippocampal neurons.
1. Preparation of ...

<ol>
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The authors declare that they have no competing financial interests.

1D patterning is an important tool that can be used for a variety of applications. For example, we have used 1D patterning for creating logic gates from neuronal cultures 29 and more recently to measure the Chronaxie and Rheobase of rat hippocampal neurons 5, and the slowing down of signal propagation velocity of firing activity in Down syndrome hippocampal neurons compared to the wild type (WT) hippocampal neurons 27. The suggested protocol for 1D p...

The interaction between neurons and external electric fields has fundamental implications as well as practical ones. While it is known since the times of Volta that an externally applied electric field can excite tissue, the mechanisms responsible for the production of a resultant action potential in neurons are only recently starting to be unraveled 1,2,3,4. This includes finding answers to questions regarding the mechanism that causes depolarization of membrane potential, the role of membrane properties and of ion channels, and even the region in the neuron that responds to the electric field 2,5. Therapeutic neurostimulation 6,7,8,9,10 methodologies are particularly dependent on this information, which can be crucial for targeting the afflicted areas and for understanding the outcome of the therapy. Such understanding can also help in developing treatment protocols and new approaches for stimulation of different areas in the brain.
Measuring the interaction within the in vivo brain adds an important component to this understanding, but is hampered by the imprecision and low controllability of measurements within the skull. In contrast, measurements in cultures can easily be performed in high volume with high precision, excellent signal to noise performance and a high degree of reproducibility and of control. Using cultures a large variety of neuronal properties of collective network behavior can be elucidated 11,12,13,14,15,16. Similarly, this well controlled system is expected be highly efficient in elucidating the mechanism by which other stimulation methods work, for example how channel opening during optical stimulation in optogenetically active neurons 17,18,19 is responsible for creating action potential.
Here the focus is on describing the development and understanding of tools that can efficiently excite the neuron via an external electric field. In this paper we describe the preparation of two-dimensional and one-dimensional patterned hippocampal cultures, stimulation using different configurations and orientation of a directly applied electric field by bath electrodes, and finally stimulation of two-dimensional and patterned one-dimensional cultures by a time-varying magnetic field, which induces an electric field 5,20,21.

<table>
	<tbody>
		<tr>
			<td>APV</td>
			<td>Sigma-Aldrich</td>
			<td>A8054</td>
			<td>Disconnect the network. Mentioned in Section 2.4.2</td>
		</tr>
		<tr>
			<td>B27 supp</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Plating medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>bicuculline</td>
			<td>Sigma-Aldrich</td>
			<td>14343</td>
			<td>Disconnect the network&#160;. Mentioned in Section 2.4.2</td>
		</tr>
		<tr>
			<td>Borax (sodium tetraborate decahydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>S9640</td>
			<td>Borate buffer. Mentioned in Section 1.1.2</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Frutarom LTD</td>
			<td>5550710</td>
			<td>Borate buffer. Mentioned in Section 1.1.2</td>
		</tr>
		<tr>
			<td>CaCl2 , 1M</td>
			<td>Fluka&#160;</td>
			<td>21098</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Sigma-Aldrich</td>
			<td>C239</td>
			<td>Disconnect the network&#160;. Mentioned in Section 2.4.2</td>
		</tr>
		<tr>
			<td>COMSOL</td>
			<td>COMSOL Inc</td>
			<td>Multiphysics 3.5</td>
			<td>Numerical simulation. Mentioned in Section 3.5.2</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>65146</td>
			<td>Plating medium, Extracellular recording solution&#160;. Mentioned in Section 1.1.1&#160;&#160;&#160; 1.5.2</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Cell Cultures. Mentioned in Section 1.2.4&#160;&#160;&#160; 1.2.6</td>
		</tr>
		<tr>
			<td>FCS(FBS)</td>
			<td>Gibco</td>
			<td>12657-029</td>
			<td>Plating medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F1141</td>
			<td>Bio Coating. Mentioned in Section 1.2.6</td>
		</tr>
		<tr>
			<td>Fluo4, AM</td>
			<td>Life technologies</td>
			<td>F14201</td>
			<td>Imaging of spontaneous or evoked activity&#160;. Mentioned in Section 1.5.1&#160;&#160;&#160; 1.5.3&#160;&#160;&#160; 1.5.5</td>
		</tr>
		<tr>
			<td>FUDR</td>
			<td>Sigma-Aldrich</td>
			<td>F0503</td>
			<td>Changing medium. Mentioned in Section 1.4.1</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Sigma-Aldrich</td>
			<td>G1272</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>GlutaMAX 100X</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>Hepes, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>HI HS&#160;</td>
			<td>BI</td>
			<td>04-124-1A</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1&#160;&#160;&#160; 1.4.1&#160;&#160;&#160; 1.4.2</td>
		</tr>
		<tr>
			<td>KCl,&#160; 3M</td>
			<td>Merck</td>
			<td>1049361000</td>
			<td>Extracellular recording solution. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>Laminin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>Bio Coating. Mentioned in Section 1.2.6</td>
		</tr>
		<tr>
			<td>MEM x 1</td>
			<td>Gibco</td>
			<td>21090-022</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.4.1&#160;&#160;&#160; 1.4.2</td>
		</tr>
		<tr>
			<td>MgCl2 , 1M</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td>Extracellular recording solution. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>NaCl, 4M</td>
			<td>Bio-Lab</td>
			<td>19030591</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>Octadecanethiol</td>
			<td>Sigma-Aldrich</td>
			<td>01858</td>
			<td>Cleaning Cr-Au coated coverslips (1D cultures). Mentioned in Section 1.2.3</td>
		</tr>
		<tr>
			<td>Pluracare F108 NF Prill</td>
			<td>BASF Corparation&#160;</td>
			<td>50475278</td>
			<td>Bio-Rejection Coating, Bio Coating. Mentioned in Section 1.2.4&#160;&#160;&#160; 1.2.6</td>
		</tr>
		<tr>
			<td>Poly-L-lysine 0.01% solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;P47075</td>
			<td>Promote cell division. Mentioned in Section 1.1.4</td>
		</tr>
		<tr>
			<td>Sucrose, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>S1888</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>Thiol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>1858</td>
			<td>Bio-Rejection Coating. Mentioned in Section 1.2.3</td>
		</tr>
		<tr>
			<td>URIDINE</td>
			<td>Sigma-Aldrich</td>
			<td>U3750</td>
			<td>Changing medium. Mentioned in Section 1.4.1</td>
		</tr>
		<tr>
			<td>Sputtering machine</td>
			<td>AJA International, Inc</td>
			<td>ATC Orion-5Series&#160;</td>
			<td>coating glass with thin layers of metal. Mentioned in Section 1.2.2</td>
		</tr>
		<tr>
			<td>Pen plotter&#160;</td>
			<td>Hewlett Packard&#160;</td>
			<td>HP 7475A</td>
			<td>Etching of pattern to the coated coverslip. Mentioned in Section 1.2.5</td>
		</tr>
		<tr>
			<td>Electrodes wires&#160;</td>
			<td>A-M Systems, Carlsborg WA</td>
			<td>767000</td>
			<td>Electric stimulation of neuronal cultures. Mentioned in Section 2.1&#160;&#160;&#160; 2.2&#160;&#160;&#160; 2.3&#160;&#160; 2.4.5</td>
		</tr>
		<tr>
			<td>Signal generator</td>
			<td>BKPrecision</td>
			<td>4079</td>
			<td>Shaping of the electric signal. Mentioned in Section 2.3</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Homemade</td>
			<td></td>
			<td>Voltage amplification of the signal from the signal generator to the electrodes. Mentioned in Section 2.3</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Matrix&#160;</td>
			<td>MPS-3005 LK-3&#160;</td>
			<td>Power supply to the sputtering machine. Mentioned in Section 1.2.2.3</td>
		</tr>
		<tr>
			<td>Transcranial magnetic stimulation</td>
			<td>Magstim, Spring Gardens, UK</td>
			<td>Rapid 2</td>
			<td>Magnetic stimulation of neuronal culture. Mentioned in Section 3.1&#160;&#160; 3.3&#160;&#160; 3.4</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Cognis</td>
			<td>Versamid 140</td>
			<td>Casting of homemade coils. Mentioned in Section 3.4</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Shell</td>
			<td>EPON 815&#160;</td>
			<td>Casting of homemade coils. Mentioned in Section 3.4</td>
		</tr>
		<tr>
			<td>Platinum wires 0.005&#39;&#39; thick; A-M Systems,&#160;</td>
			<td>&#160;Carlsborg WA</td>
			<td>&#160;767000</td>
			<td>Electric stimulation of neuronal cultures. Mentioned in Section 2.1</td>
		</tr>
		<tr>
			<td>Circular magnetic coil</td>
			<td>Homemade</td>
			<td></td>
			<td>Magnetic stimulation of neuronal culture. Mentioned in Section 3.3</td>
		</tr>
		<tr>
			<td>WaveXpress SW</td>
			<td>B&#38;K Precision&#160;</td>
			<td></td>
			<td>Waveform editing software. Mentioned in Section 2.1.32</td>
		</tr>
		<tr>
			<td>Xion Ultra 897</td>
			<td>Andor</td>
			<td></td>
			<td>Sensitive EMCCD camera. Mentioned in Section 2.4.4</td>
		</tr>
	</tbody>
</table>

external excitation of neurons using electric and magnetic fields in one- and two-dimensional cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

The protocol presented allows for easy patterning of neuronal cultures. When it is combined with several methods we developed for stimulation, it enables to make measurements of some intrinsic neuron properties such as Chronaxie and Rheobase5, to compare properties of healthy and diseased neurons27, to find optimal ways to stimulate cultures as a function of their structure and many more novel approaches. Some examples are presented in the n...

Watch this Scientific Journal Video about External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures at JoVE.com

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

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.

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13.1K Views.  The Salk Institute for Biological Studies. 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 s...