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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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 Two-dimensional (2D) and One-dimensional (1D) Hippocampal Cultures

1. Preparation of coverslips for 2D cultures.
   1. Prepare plating medium (PM) composed of: 0.9 mL minimum essential media (MEM)+3G, 0.05 mL fetal calf serum (FCS), 0.05 mL heat inactivated horse serum (HI HS) and 1 µL of B27 supplement. Note: MEM+3G contains for every 500 mL of MEM x 1, 1 mL of gentamycin, 5 mL of stabilized L-glutamine 100x (see Table of Materials/Reagents) and 5 mL of 60% D-(+)-glucose.
   2. Prepare borate buffer composed of: 1.9 g borax (sodium tetraborate decahydrate) in 200 mL double distilled water (DDW) (mix at 60 °C) and 1.24 g boric acid in 200 mL DDW. Titrate final solution to pH 8.5 using 1 M HCl. Note: The final solution is 400 mL 0.1 M borate buffer.
   3. Immerse glass coverslips in 65% nitric acid for 2 h. Rinse three times in DDW followed by three rinses with absolute analytical reagent (ABS AR) ethanol.
   4. Pass each coverslip briefly through the flame of a Bunsen burner two or three times for 1 - 2 s, and then leave to incubate overnight in 24 well plates with 1 mL poly-L-lysine 0.01% solution diluted 1:5 in borate buffer (0.1 M, pH 8.4). Then rinse coverslips in DDW three times and leave with 1 mL PM per well in a standard 37 °C, 5% CO₂ incubator overnight.
2. Preparation of coverslips for 1D cultures.
   1. Clean glass coverslips by immersion in a base piranha solution consisting of 75 mL DDW, 25 mL 25% ammonia solution and 25 mL 30% hydrogen peroxide. Place solution with the glass coverslips on a heating plate at ~ 50 °C for 30 min and then dry the glass coverslips with nitrogen.
   2. Coat coverslips first with a thin chrome film (99.999%) of 6 Å thickness followed by a 30 Å layer of gold (99.999%), using either vapor or sputter deposition.
      1. To achieve a sputtering rate of 0.05 – 1 Å /s use a sputtering machine with 2 e-beams and a vacuum system of 260 l/s with target sizes 2" and 4". Use a rotation stage that can go from 0 – 100 rounds per minute (RPM). Use a direct current (DC) sputter power of 0 – 750 Watts.
      2. Use a rotation of 30 rpm and argon 99.999% to get plasma at 10 mTorr pressure in the chamber.
      3. Operate the power supply of the sputtering guns at 40 W DC sputter power for the chrome, leading to ~ 0.12 Å/s coverage rate, and at 10 W DC sputter power for the gold, leading to ~ 0.28 Å/s.
   3. Dissolve 0.1 g 1-octadecanethiol in 100 mL ABS AR ethanol using ultrasound for 30 min. Place Cr-Au coated coverslips for 2 h in this solution, then wash with ethanol ABS AR and dry with nitrogen.
   4. Prepare a solution of 100 mL Dulbecco's phosphate buffered saline (D-PBS) and 3.5 g of a tri-block co-polymer (see Table of Materials/Reagents) by stirring for 1 - 2 h at 600 - 700 rpm. Place coverslips in the solution for 1 h. Dry coverslips with nitrogen.
   5. Mechanically etch the desired pattern by scratching the bio-rejection layer²². Do this using a pen plotter, where the pen is replaced by an etching needle. Scratch the pattern through the metal layers to reach the underlying glass. Control this process by a computer to achieve a replicated desired pattern. Patterns formed by this process are demonstrated in Figure 2 and Figure 3.
   6. Prepare a bio-compatible layer of 100 mL D-PBS, 3.5 g tri-block co-polymer, 35.7 µL/mL fibronectin and 29 µl/mL laminin.
      1. Sterilize coverslips in ultra-violet light for at least 10 min. Incubate coverslips in the prepared bio-compatible solution overnight.
         NOTE: The bio-compatible layer will form only where the bio-rejection layer has been etched off in the previous step.
      2. On the next day wash coverslips two times with P-DBS. Incubate coverslips in PM overnight. Coverslips are now ready for cell plating.
3. Perform dissection according to standard procedures that have been published extensively previously^23,24.
   1. In brief, extract hippocampus or cortex from rat embryos, typically at day E19, or from mice, typically at day E17^23,24.
   2. Dissociate cells first in papain solution for 20 - 30 min, followed by mechanical trituration²⁴ with glass pipettes whose tips are fire polished.
      NOTE: If the cells come from genetically modified mice then the tissue from each embryo should be maintained in a separate 1.5 mL plastic tube during the entire process.
   3. Count cells with trypan blue before seeding.
      NOTE: For genetically modified animals the counting should be done separately for each embryo.
   4. For 2D cultures, seed mouse neurons at 750,000 and rat neurons at 850,000 cells per well. For 1D, seed at 650,000 cells per well. Shake plate slightly immediately after seeding to ensure homogeneous coverage of the coverslip.
4. Maintenance of the neuronal cultures.
   1. Prepare changing medium (CM) composed of (per mL): 0.9 mL MEM+3G, 0.1 mL HI HS, 10 µL 5-fluoro-2′-deoxyuridine (FUDR) with uridine 100x.
   2. Prepare final medium (FM) composed of (per mL): 0.9 mL MEM+3G and 0.1 mL HI HS.
   3. Replace PM with 1-1.5 mL CM after 4 days in vitro (DIV). At 6 DIV, replace 50% of the CM with fresh CM. At DIV 8, change the medium to 1.5 mL FM, followed by a 50% change of FM every 2 days. After about one week spontaneous synchronous activity emerges.
5. Imaging of spontaneous or evoked activity in neuronal cultures with fluorescent dyes.
   1. Prepare a solution of 50 µg calcium sensitive fluorescent dye (see Table of Materials/Reagents) in 50 µL DMSO (dimethyl sulfoxide).
   2. Prepare extracellular recording solution (EM) containing (in mM) 10 HEPES, 4 KCl, 2 CaCl₂, 1 MgCl₂, 139 NaCl, 10 D-glucose, 45 sucrose (pH 7.4).
   3. Incubate neuronal culture in 2 mL EM with 8 µL of the calcium sensitive fluorescent dye solution for 1 h. Protect from light and gently rotate to ensure homogenous spread of the dye to the cells.
   4. Replace solution with fresh EM prior to imaging. The fluorescent imaging is demonstrated in Figure 4.
   5. Image in a fluorescence microscope with optical filters for calcium fluorescence imaging (excitation peak at 488 nm, emission peak at 520 nm), using a camera and software capable of quantifying the intensity of any region of interest (ROI) within the field of view of the microscope.

2. Electric Stimulation of Cultures

NOTE: The basic setup for electric stimulation is shown in Figure 1. A cover slip on which the neuronal culture has been grown for about 14 days is placed in a Petri dish under a fluorescence microscope. Electrical activity of the neurons is imaged using calcium sensitive dyes while a voltage is applied via two pairs of bath electrodes that are positioned outside the culture. The electrodes are driven by a signal generator whose output is amplified by a dual channel amplifier. Voltage control for stimulation is preferred over the more standard current control^25,26 because the electric field vectors are determined directly, thus enabling straightforward vector addition and combination. This does require a careful check of the uniformity of the electric field, which can be performed over the whole sample for the case of voltage control. When using voltage control care should be taken to avoid any ground loops and the homogeneity of the electric field should be verified (see 2.2 below).

1. For electric stimulation with a homogeneous electric field use a pair of parallel electrode wires.
   1. Use electrodes made of platinum with a thickness on the order of 0.005'' (127 µm). When used with the 13 mm coverslips, ensure that the distance between the two electrodes is around 11 mm, and position the electrodes 1 mm above the culture.
      NOTE: To make the electrode holder (Figures 5A and 6A) use polytetrafluoroethylene (PTFE). Drill narrow holes through the PTFE to insert the electrodes. The device should be higher than the extracellular solution so that the top end, where the electrodes are exposed, will never come in contact with the solution. For insulation, use epoxy glue on any part of the electrode leads that might be exposed.
   2. Use a square pulse shape with a 50% duty cycle, with no DC component to avoid electrolysis. Vary pulse duration between 10 µs and 4 ms to cause effective stimulation without burning the culture. Ensure that the amplitude is in the order of ± 22 V (see Figure 5). The square pulse can be observed on an oscilloscope connected in parallel to the electrodes.
      NOTE: For easy programming of any desired waveform, use a commercial waveform editing software (see Materials list). Enter graphically the desired waveform and send it to the waveform generator.
2. To test for field homogeneity use a probe electrode. Use a grid of at least 1 mm x 1 mm to allow the probe to be moved in the area between electrodes and measure electric potential.
   1. Measure electric potential. Use to calculate the electric field. Use one of the electrodes as a reference electrode. Measure the electric field with varying pulse durations between 100 µs to 4 ms (see Figure 5B for an example of 100 µs pulse duration) to verify that the field is homogeneous within the range of stimulating durations.
      NOTE: See Figure 5D for an example of a measured electric field homogeneity when the pulse duration was 1 ms.
3. Use 2 perpendicular pairs of bath electrodes to produce more complicated electric field shapes, and to be able to orient the field in different directions (see Fig. 6B). The device with 2 pairs of electrodes will be used, and the signal on each pair of the electrodes will be observed on two separate oscilloscopes.
   1. Position the electrodes 1 mm above the culture and at a distance of 10 - 11 mm from each other. Make sure that both electrode pairs are floating (have no ground connection), and do not have any common ground by measuring the resistance between any set of electrodes and verifying absence of short circuits between any of the electrodes. Verify that all equipment used, which is connected to the electrodes (such as the oscilloscopes, the amplifiers, the signal generators, etc.) is floating with respect to ground by checking that all equipment is floating and that none of the reference electrodes is touching any grounded equipment.
   2. To change field orientation, vary the amplitude of the voltage fed to the two electrode pairs with respect to each other (see Figure 7A). For example, for 0° use ± 22 V on the electrode pair which are perpendicular to the pattern and 0 V for the other electrode. For 45°, use ± 15.6 V on both pairs of electrodes with no phase lag, for an amplitude vector sum of 15.6²+15.6²=22².
   3. To apply a rotating field use one single waveform of a sine voltage pulse and one single waveform of a cosine voltage pulse to the two pairs of electrodes to produce a fixed amplitude rotating electric field (see Figure 6B).
      NOTE: As can be viewed in Figure 6B, when using one cycle of a sine wave with ± 22 V in one electrode and one cycle of a cosine wave with ± 22 V in the other electrode, the vector sum is a rotating electric field with the cycle duration same as the sine and cosine waves, and with an amplitude of ± 22 V.
4. To measure and calculate Chronaxie and Rheobase of axons in the neuronal culture vs. dendrites in the same culture perform the following steps.
   1. Use a calcium sensitive fluorescent dye for calcium imaging as described in the Table of Materials/Reagents and in 1.5) with a 1D culture patterned on thin (170 µm), long (10 mm) lines. Calcium sensitive dye will be applied to the culture, and incubated for 45 minutes. The dye will be washed by replacing with a fresh recording solution.
   2. Disconnect the network to achieve population statistics of the direct response to electrical stimulation, without the effect of synaptic transmission. To do so, apply a combination of 40 µM of bicuculline, which blocks the inhibitory action of gamma-Aminobutyric acid-A(GABA_A) receptors, 10 µM of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), to block the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) and kainate receptors and 20 µM of (2R)-amino-5-phosphonovaleric acid (APV), which blocks the N-methyl-D-aspartate (NMDA) receptors. The blockers will be added to the recording solution.
   3. Apply a square pulse as described in 2.1 and 2.3 with varying time durations between 100 µs and 4 ms. The signal can be observed on the oscilloscope.
   4. Use a fluorescence microscope with an image acquisition program (see 1.5) to monitor the intensity of the calcium transients at several ROIs, each containing a few hundred neurons. Acquire images using a sensitive EMCCD camera (see Materials list), capable of an image acquisition rate of at least 20 frames per second. The change in light intensity is proportional to the amount of neurons which were stimulated. Estimate the fraction of stimulated neurons using the relative change in intensity.
   5. For each pulse duration (100 µs to 4 ms), change the amplitude of the voltage applied to the electrodes starting at a voltage where no change in intensity is seen (a few Volts), to the voltage where the changes in intensity due to the applied voltage has saturated (Up to ± 22 V).
      NOTE: The intensity change will be distributed as a cumulative Gaussian⁵ with respect to the applied voltage for stimulation for each time duration of the voltage pulse.
   6. Fit a cumulative Gaussian distribution for the intensity vs. the applied voltage for each duration and extract the Gaussian mean from this fit.
      NOTE: This mean is the representative voltage to which the neurons responded.
   7. At the end of this process obtain for each stimulation duration a mean voltage to which the neurons responded. Use these pairs of durations and strengths to plot the Strength-Duration curves (see Figure 7).

3. Magnetic Stimulation of Cultures

Note: The basic setup for magnetic stimulation is shown in Figure 2. On top right is shown an inverted fluorescence microscope that is used to image calcium sensitive dyes in the neurons. The magnetic coil (blue circles) is positioned about 5 mm concentrically above a neuronal ring culture, (blue outline). A pickup coil (red circle) on the circumference of the Petri dish monitors the voltage induced by the magnetic pulse. On the top left is shown the measured dynamics of the magnetic stimulator (MS) coil with a capacitor voltage load of 5,000 kV, as integrated from the pickup coil. The induced electric field (calculated for a ring radius of 14 mm) is depicted in green while the magnetic field is depicted in blue. On the bottom are shown images of the neuronal culture. At bottom left is a bright field image of a patterned 24-mm coverslip. The white areas are the neurons. The photographed pattern consists of concentric ring cultures with different radii. At the bottom right is a zoom onto a short segment of the rings, showing individual neurons. For a scale, the rings' width is about 200 µm.

1. Grow the neurons in a circular ring pattern (etched as described in 1.2.5) for 1D culture stimulation. Use a calcium sensitive fluorescent dye for calcium imaging (as described in section 1.5) with a 1D culture patterned on thin (170 µm), long (10 mm) lines.
   1. Use a circular magnetic coil and position a Petri dish approximately 5 mm below and concentric with the coil. Use a custom coil of approximately 30-mm (inner diameter, 46-mm outer diameter) coil with an inductance of L = 90 mH driven by a homemade or commercial MS loaded with a maximal voltage of 5 kV.
   2. Discharge a high voltage and current through a conducting coil using a high-current-high-voltage switch to magnetically stimulate neuronal cultures. The magnetic stimulator (MS) can be built as described in²¹ using large capacitors, on the order of 100 mF, to obtain a high voltage of 1 - 5 kV. Alternatively use a commercially available MS (see Table of Materials/Reagents).
      NOTE: Use a 0.254 mm thick and 6.35 mm wide polyester-coated rectangular copper wire to fabricate a homemade coil²¹. Turn wires on custom made frames, insulated with glass fibers and cast in epoxy (see Table of Materials/Reagents). Alternatively use commercially available coils (see Table of Materials/Reagents).
2. Use rotating magnetic fields to stimulate 2D cultures.
3. Now, use the rotating magnetic fields to stimulate 2D cultures. Fire the TMS with no culture in the dish, at different intensities, to show the linear correlation of the coil reading with the intensities.
4. Next, while recording calcium transients with a neuronal culture, start firing the TMS at increasing intensities while recording both calcium transients and the coil. At first, network bursts observed as large calcium transients, should not synchronize with pick up coil TMS spikes. Continuing to increase intensity, at some point, the calcium transients start to become synchronized with the TMS spikes. Alternatively, or in addition, after achieving a synchronized response, start decreasing the intensities until synchronization is abolished, to determine the TMS threshold.
   NOTE: Conditions should be maintained strictly fixed for each of the setups, using the exact volume every time, the same vessels and exact coil positions and orientations.
   1. Mount the pickup coil on the base of the recording dish so that it is in a plane parallel to the neuronal culture and in a fixed position with respect to the culture.
      NOTE: This ensures that the dependence of the positioning of the pickup coil with respect to the magnet is faithful to the position of the culture and that any discrepancies in the positioning will be negligible on the pickup coil readings.
      1. Use two independent coils that are positioned perpendicular to each other (Figure 8B) to generate a rotating magnetic and induced electric field. Connect each coil to its own MS. Ensure that the stimulators discharge similar currents at a phase lag of 90 degrees (Figure 10A), resulting in a rotating magnetic field which scans 270 degrees of the real space at a maximum field of ~ 270 V/m (Figure 10B).
      2. Position the neuronal culture inside a spherical glass container filled with the external recording solution (EM).
      3. Monitor stimulation (change in fluorescence intensity of the neurons) with the camera as described in step 2.4.4.
      4. In the case of the cross coils (Figure 8B), position the pickup coil parallel and at a specific distance below the neuronal culture. Carefully maintain this configuration throughout the experiments.
5. Calculate analytically the electric field for 1D configuration by E_max=k₁Br, where E_max is the maximum amplitude of the induced electric field and is directed along the tangent of the rings with radius r. B is the amplitude of the magnetic pulse and k₁ is a dimensional proportionality constant that can be measured using the pickup coil (Figure 8A).
6. Use a numerical simulation package (see Material list) to numerically simulate the electric field²¹.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
APV	Sigma-Aldrich	A8054	Disconnect the network. Mentioned in Section 2.4.2
B27 supp	Gibco	17504-044	Plating medium. Mentioned in Section 1.1.1
bicuculline	Sigma-Aldrich	14343	Disconnect the network. Mentioned in Section 2.4.2
Borax (sodium tetraborate decahydrate)	Sigma-Aldrich	S9640	Borate buffer. Mentioned in Section 1.1.2
Boric acid	Frutarom LTD	5550710	Borate buffer. Mentioned in Section 1.1.2
CaCl2 , 1 M	Fluka	21098	Extracellular recording solution. Mentioned in Section 1.5.2
CNQX	Sigma-Aldrich	C239	Disconnect the network. Mentioned in Section 2.4.2
COMSOL	COMSOL Inc	Multiphysics 3.5	Numerical simulation. Mentioned in Section 3.5.2
D-(+)-Glucose, 1 M	Sigma-Aldrich	65146	Plating medium, Extracellular recording solution. Mentioned in Sections 1.1.1 and 1.5.2
D-PBS	Sigma-Aldrich	D8537	Cell Cultures. Mentioned in Sections 1.2.4 and 1.2.6
FCS (FBS)	Gibco	12657-029	Plating medium. Mentioned in Section 1.1.1
Fibronectin	Sigma-Aldrich	F1141	Bio Coating. Mentioned in Section 1.2.6
Fluo4AM	Life technologies	F14201	Imaging of spontaneous or evoked activity. Mentioned in Sections 1.5.1, 1.5.3, and 1.5.5
FUDR	Sigma-Aldrich	F0503	Changing medium. Mentioned in Section 1.4.1
Gentamycin	Sigma-Aldrich	G1272	Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1
GlutaMAX 100x	Gibco	35050-038	Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1
Hepes, 1 M	Sigma-Aldrich	H0887	Extracellular recording solution. Mentioned in Section 1.5.2
HI HS	BI	04-124-1A	Plating medium, Changing medium, Final medium. Mentioned in Sections 1.1.1, 1.4.1, and 1.4.2
KCl,  3 M	Merck	1049361000	Extracellular recording solution. Mentioned in Section 1.5.2
Laminin	Sigma-Aldrich	L2020	Bio Coating. Mentioned in Section 1.2.6
MEM x 1	Gibco	21090-022	Plating medium, Changing medium, Final medium. Mentioned in Section 1.4.1    1.4.2
MgCl2 , 1 M	Sigma-Aldrich	M1028	Extracellular recording solution. Mentioned in Section 1.5.2
NaCl, 4 M	Bio-Lab	19030591	Extracellular recording solution. Mentioned in Section 1.5.2
Octadecanethiol	Sigma-Aldrich	01858	Cleaning Cr-Au coated coverslips (1D cultures). Mentioned in Section 1.2.3
Pluracare F108 NF Prill	BASF Corparation	50475278	Bio-Rejection Coating, Bio Coating. Mentioned in Sections 1.2.4 and 1.2.6
Poly-L-lysine 0.01% solution	Sigma-Aldrich	P47075	Promote cell division. Mentioned in Section 1.1.4
Sucrose, 1 M	Sigma-Aldrich	S1888	Extracellular recording solution. Mentioned in Section 1.5.2
Thiol	Sigma-Aldrich	1858	Bio-Rejection Coating. Mentioned in Section 1.2.3
URIDINE	Sigma-Aldrich	U3750	Changing medium. Mentioned in Section 1.4.1
Sputtering machine	AJA International, Inc	ATC Orion-5Series	coating glass with thin layers of metal. Mentioned in Section 1.2.2
Pen plotter	Hewlett Packard	HP 7475A	Etching of pattern to the coated coverslip. Mentioned in Section 1.2.5
Electrodes wires	A-M Systems, Carlsborg WA	767000	Electric stimulation of neuronal cultures. Mentioned in Sections 2.1, 2.2, 2.3, and 2.4.5
Signal generator	BKPrecision	4079	Shaping of the electric signal. Mentioned in Section 2.3
Amplifier	Homemade		Voltage amplification of the signal from the signal generator to the electrodes. Mentioned in Section 2.3
Power supply	Matrix	MPS-3005 LK-3	Power supply to the sputtering machine. Mentioned in Section 1.2.2.3
Transcranial magnetic stimulation	Magstim, Spring Gardens, UK	Rapid 2	Magnetic stimulation of neuronal culture. Mentioned in Sections 3.1, 3.3, and 3.4
Epoxy	Cognis	Versamid 140	Casting of homemade coils. Mentioned in Section 3.4
Epoxy	Shell	EPON 815	Casting of homemade coils. Mentioned in Section 3.4
Platinum wires 0.005'' thick; A-M Systems,	Carlsborg WA	767000	Electric stimulation of neuronal cultures. Mentioned in Section 2.1
Circular magnetic coil	Homemade		Magnetic stimulation of neuronal culture. Mentioned in Section 3.3
WaveXpress SW	B&K Precision		Waveform editing software. Mentioned in Section 2.1.32
Xion Ultra 897	Andor		Sensitive EMCCD camera. Mentioned in Section 2.4.4