ultrasound-induced neuromodulation using a microelectrode array system

Ultrasound-Induced Neuromodulation Using a Microelectrode Array System

To begin, aspirate the culture medium to fill a single well in a 24-well neuron culture plate having embedded microelectrode arrays or MEA. After preparing three hundred milliliters of degassed and deionized water, carefully fill it in the focussed ultrasound or FUS transducer cone. Using a customized threaded rod, secure the 3D-printed holder to a frame. Position the frame such that the head of the FUS transducer is over the well that will be stimulated. Use a rubber band to secure parafilm over the well on the 24-well MEA plate containing the medium and the human induced pluripotent stem cells or hIPSCs.
Set the FUS parameters on the transducer power output or TPO control panel. The values of the various parameters are mentioned on the screen. Next, apply coupling gel on top of the parafilm over the well, and lower the FUS transducer into the coupling gel, ensuring contact with the gel with minimal air bubbles. Start the FUS sonication by pressing the bottom right button on the TPO, and wait at least five minutes between each round of sonication to allow the neurons to return to a baseline state.
If the connection is appropriate, the trigger pulse generated by the FUS system will automatically align the FUS stimulation sequence with the MEA recording. Analyze the signal by reading the FUS sonication time with the transfer data based on the change in the firing rate associated with the FUS.

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1. Preparation of materials
<ol>
	<li>Aspirate the culture medium and use it to fill a single well in a 24-well neuron culture plate with an embedded microelectrode array (MEA) (Figure 1A).</li>
	<li>Sterilize the parafilm interface, the rubber band, and the focused ultrasound (FUS) cone with its rubber membrane using 70% ethanol for 10 min, and place them in the fume hood for later assembly.</li>
	<li>Degas 300 mL of deionized water and 50 mL of coupling gel. Centrifuge the water and gel at 160 x g for 5 min to avoid inducing cavitation within the coupling medium. 
	NOTE: The original source of the human induced pluripotent stem cells (HiPSCs) is from GM01582 and CIPS cell lines. On average, a density of 5 x 104 motor neurons and 2.5 x 104 astrocytes per well can be achieved.</li>
</ol>
2. Connection and setup of the peripherals
<ol>
	<li>Secure the FUS cone to the transducer using screws, and seal the cone with a flexible rubber membrane in a ventilated sterile hood. Fill the cone with the degassed and deionized (DG-DI) water from step 1.3, and ensure the absence of bubbles in the cone to avoid cavitation.</li>
	<li>Use a customized threaded rod to secure the 3D-printed holder to a frame (Figure 1B). Position the frame such that the head of the FUS transducer is over the well that will be stimulated.</li>
	<li>Use a rubber band to secure parafilm over the well on the 24-well MEA plate containing the medium and the HiPSCs.</li>
	<li>Prepare the FUS system by connecting the ultrasound transducer&#39;s back-end driver electronics, in this case, the transducer power output (TPO), to a 100-240 V power outlet and connecting the matching network to the TPO and the FUS transducer. The matching network ensures efficient electrical coupling between the transducer and the TPO.</li>
	<li>Connect the MEA system to a power outlet (100-240 V) (Figure 2B, Connection 5). Connect the MEA system synchronization port to the TPO (Figure 2B, Connection 3). This connection will synchronize the data acquisition by the MEA system with the FUS stimulation.</li>
	<li>Place the 24-well MEA plate in the MEA system, and remove the lid to enable direct contact between the transducer and the well. Place the transducer 5-10 mm above the well plate to allow room for the degassed coupling gel, as described in step 3.2 (Figure 2A and Figure 1B).</li>
</ol>
3. Stimulation and neuronal signal acquisition
<ol>
	<li>Set the FUS parameters on the TPO control panel&#160; (Table 1).</li>
	<li>Apply the coupling gel on top of the parafilm, and lower the FUS transducer into the coupling gel, ensuring contact with the gel with minimal air bubbles (Figure 1A).</li>
	<li>Start the MEA system recording by clicking on the Start button on the user interface.</li>
	<li>Start the FUS sonication by pressing the bottom right button on the TPO (Figure 2A, Label 7), and wait at least 5 min between each round of sonication to allow the neurons to return to a baseline state.</li>
	<li>Use the trigger pulse generated by the FUS system to align the FUS stimulation sequence to the MEA recording (Figure 1B, Connection 3).</li>
</ol>
Table 1: Focused ultrasound (FUS) parameters set on the TPO
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td>Value</td>
		</tr>
		<tr>
			<td>Max Power/Ch</td>
			<td>1.200 W</td>
		</tr>
		<tr>
			<td>Pactual</td>
			<td>0.749 W/channel</td>
		</tr>
		<tr>
			<td>ISPPA</td>
			<td>10.79 W/cm2</td>
		</tr>
		<tr>
			<td>ISPTA</td>
			<td>0.05 W/cm2</td>
		</tr>
		<tr>
			<td>Burst Length</td>
			<td>0.100 ms</td>
		</tr>
		<tr>
			<td>Frequency</td>
			<td>250.00 kHz</td>
		</tr>
		<tr>
			<td>Focus</td>
			<td>39.800 mm</td>
		</tr>
		<tr>
			<td>Period</td>
			<td>20.000 ms</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>60.000 s</td>
		</tr>
	</tbody>
</table>

Source: Liang, R. et al., <a href="https://app.jove.com/v/65115">Focused Ultrasound Neuromodulation of Human In Vitro Neural Cultures in Multi-Well Microelectrode Arrays.</a> J. Vis. Exp. (2024)
This video demonstrates the method for inducing the modulation of neural activity in cultured human iPSC-derived neurons in a multi-well microelectrode array, or MEA system using a focused ultrasound transducer.

<img alt="Figure 1" src="/files/ftp_upload/22882/22882_Figure1.jpg" /> 
Figure 1: FUS neuromodulation with a multi-well microelectrode array (MEA). (A) Schematic of the setup for FUS neuromodulation with a multi-well MEA. The acoustic waves generated by the FUS transducer propagate through an FUS cone filled with degassed water and are coupled using ultrasound gel. The parafilm is secured to the well using a rubber band to prevent contamination. The ...

1. Preparation of materials

	Aspirate the culture medium and use it to fill a single well in a 24-well neuron culture plate with an embedded ...

Begin with a microelectrode array or MEA system containing a parafilm-covered multi-well MEA plate and a focused ultrasound transducer above it.
One of the MEA wells contains media with human iPSC-derived neurons over embedded microelectrodes, where the neurons communicate with each other via electrical signals.
Set up the transducer for the emission of ultrasound waves.
Apply degassed coupling gel on the parafilm and lower the transducer to establish contact with the gel without air bubbles for efficient ultrasound transmission.
Next, the MEA system records the electrical signals from the neurons via the microelectrodes to obtain a baseline neuronal activity.
Using the transducer, apply continuous high-frequency ultrasound waves, which pass through the coupling gel and parafilm and reach the neurons.
The ultrasound waves excite the neurons and modulate their electrical activity. Re-record this electrical signal.
An increase in neuronal activity after ultrasound treatment, compared to pre-treatment, confirms the successful neuromodulation.

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Ultrasound-Induced Neuromodulation Using a Microelectrode Array System

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