injectable mesh electronics for stable single-neuron recordings in a mouse brain

Injectable Mesh Electronics for Stable Single-Neuron Recordings in a Mouse Brain

To inject mesh electronics into a mouse brain, use a dental drill and stereotaxic frame to open a craniotomy at the desired coordinates on the skull. Open a second craniotomy away from the injection site for the insertion of a stainless steel grounding screw, or wire.
Then, fix a clamping substrate to the skull with dental cement. An approximately 1-millimeter wide cut in the substrate improves the reliability of the folding step later in the procedure. Afterward, mount the pipette holder with the needle containing mesh electronics into the stereotaxic frame using a right-angle end clamp. Attach the side outlet of the pipette holder to a 5-milliliter syringe fastened in a syringe pump with a 0.5 to 1-meter-long capillary tubing.
Then, use the stereotactic frame to position the tip of the needle at the desired starting location within the brain. Position the camera to display the top of the mesh electronics probe within the glass needle. Initiate the flow by setting the syringe pump to a low speed and pressing Start.
Slowly increase the flow rate if the mesh electronics probe does not move within the needle. As the mesh electronics probe starts to move within the needle, use the stereotactic frame to retract the needle at the same rate as which the mesh electronics probe is being injected, using the marked original position of mesh electronics as a guide.
Continue flowing saline, and retracting the needle until the needle has exited the skull. Then, stop the flow from the syringe pump. In this procedure, use the stereotactic frame to carefully guide the needle to the flat, flexible cable clamping substrate and across the gap, flowing the solution with the syringe pump to generate a slack in the mesh electronics interconnects.
Once the needle is above the clamping substrate and across the gap, resume the flow at a faster rate to eject the mesh electronics input/output pads onto the clamping substrate. Using tweezers in a pipette of deionized water, bend the input/output pads to 90-degree angle as close to the first input/output pad as possible.
Once the input/output pads are aligned, unfolded, and at a 90-degree angle to the mesh stem, dry them in place with gently flowing compressed air. Cut the clamping substrate at a straight edge approximately 0.5 to 1 millimeter from the edge of the input/output pads.
Also, cut off extraneous parts of the clamping substrate that will hinder the insertion into the PCB-mounted 32-channel ZIF connector. Then, insert the input/output pads into the ZIF connector on the PCB and close the latch. Use measurement electronics to measure the impedance between the channels and the ground screw to confirm successful interfacing.
If the impedance values are too high, unlatch the ZIF connector, adjust the insertion, and retest until successful connection is confirmed. Subsequently, cover the ZIF connector and exposed mesh electronics interconnects with dental cement for protection. Flip the PCB at the gap in the substrate and fix the PCB with cement onto the mouse skull.
For freely moving recordings, release the mouse from the restrainer after inserting the preamplifier PCB and grounding the reference screw. Record for the desired length of time using the data acquisition system while the mouse behaves freely.

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1. Stereotaxic Injection of Mesh Electronics into Live Mouse Brain
NOTE: Mice were anesthetized by intraperitoneal injection with a mixture of 75 mg/kg ketamine and 1 mg/kg dexdomitor. The degree of anesthesia was verified with the toe pinch method prior to beginning surgery. The body temperature was maintained by placing the mouse on a 37 &#176;C homeothermic blanket while under anesthesia. Proper sterile technique was implemented for the surgery, including but not limited to autoclaving all metal surgical instruments for 1 h prior to use, using sterilized gloves, using a hot bead sterilizer throughout the surgery, the maintenance of a sterile field around the surgical site, the disinfection of plastic instruments with 70% ethanol, and the&#160;depilated scalp skin was prepped with iodophor prior to incision. For survival surgeries, after the conclusion of the surgery, antibiotic ointment was applied around the wound, and the mouse was returned to a cage equipped with a 37 &#176;C heating pad. Mice were not left unattended until they had regained sufficient consciousness to maintain sternal recumbency. Mice were given buprenorphine&#160;analgesia via intraperitoneal injection at a dose of 0.05 mg/kg body weight every 12 h for up to 72 h following the surgery. Mice were isolated from other animals following surgery. Mice were euthanized via either intraperitoneal injection of pentobarbital at a dose of 270 mg/kg body weight or via transcardial perfusion.
<ol>
	<li>Anesthetize the mouse and fix it in a stereotaxic frame.</li>
	<li>Apply ocular lubricant to the mouse&#39;s eyes to prevent dryness while under anesthesia.</li>
	<li>Use a dental drill and stereotaxic frame to open a craniotomy at the desired coordinates on the skull. Open a second craniotomy away from the injection site for the insertion of a stainless-steel grounding screw or wire.</li>
	<li>Fix a clamping substrate to the skull with dental cement. Cut an approximately 1-mm wide gap in the substrate to improve the reliability of the folding step later in the procedure. 
	NOTE: A flat flexible cable (FFC) cut to a &#34;L&#34; shape works well (Figure 4A), although many materials would work as long as they are of the correct thickness for the 32-channel zero insertion force (ZIF) connector (designed for&#160;0.18 &#177; 0.05 mm thick cables).</li>
	<li>Mount the pipette holder with the needle containing mesh electronics onto the stereotaxic frame using a right-angle end clamp (Figure 2C&#160;and&#160;Figure 3A).</li>
	<li>Attach the side outlet of the pipette holder to a 5 mL syringe fastened in a syringe pump (Figure 3D) using an approximately 0.5&#8211;1 m length of capillary tubing. 
	NOTE: Ensure there are no bubbles in the capillary tubing before connecting it to the pipette holder. Bubbles can interrupt the flow during injection and prevent a smooth, controlled delivery of mesh electronics.</li>
	<li>Use the stereotaxic frame to position the tip of the needle at the desired starting location within the brain. 
	NOTE: The mesh electronics probes used here are designed with recording electrodes spread over a length of ca. 2 mm and with the first electrode located ca. 0.5 mm from the starting edge of the mesh electronics (left-most edge in&#160;Figure 1A). For this reason, the stereotaxic coordinates should be selected such that the starting location is 0.5 mm deeper than the brain region of interest. The spread and location of the recording electrodes within mesh electronics can be selected freely during the mask design process and should be selected so the recording electrodes span the brain region(s) of interest as they are injected along their stereotaxic trajectory.</li>
	<li>Position the camera (Figure 3B) to display the top of the mesh electronics probe within the glass needle. Some software allows the user to draw a line on the screen to mark the original position of the mesh electronics.</li>
	<li>Initiate the flow by setting the syringe pump to a low speed and pressing Start. 10 mL/h is a typical starting flow rate for a 400 &#181;m inner diameter capillary needle. Slowly increase the flow rate if the mesh electronics probe does not move within the needle. 
	NOTE: It is important to minimize the volume of fluid injected into the brain as this can damage the tissue surrounding the injection site. Best results are achieved with injection volumes less than 25 &#181;L per 1 mm of injected mesh length. Ideal values are less than half of this volume; in our laboratory, we typically inject 10&#8211;50 &#181;L per a 4 mm injected mesh length.</li>
	<li>As the mesh electronics probe starts to move within the needle, use the stereotaxic frame to retract the needle at the same rate with which the mesh electronics probe is being injected, using the marked original position of mesh electronics as a guide. 
	NOTE: This procedure, termed the field of view (FoV) injection method, allows for precise delivery of mesh electronics to a targeted brain region without crumpling or dislocation. Often the flow rate can be reduced once the mesh electronics probe begins moving within the needle. In our laboratory, flow rates of 20&#8211;30 mL/h are often required to overcome the static friction between the mesh and capillary needle walls, but the rate can then be reduced to 10 mL/h once the injection process has been initiated. Flow rates and injection volumes are usually smaller for smaller diameter capillary needles.</li>
	<li>Continue flowing saline and retracting the needle until the needle has exited the skull. Stop the flow from the syringe pump.</li>
</ol>
2. Input/output Interfacing
NOTE: At this point, the mesh electronics probe has been injected from the desired starting point within the brain along the chosen trajectory. The needle has been retracted and is just above the craniotomy with the mesh electronics interconnects spanning from the brain to the needle and the input/output (I/O) pads still inside the needle (Figure 4B). This section uses a printed circuit board (PCB;&#160;Figure 4,&#160;Figure 5) to interface to the mesh electronics probe. The PCB connects a ZIF connector to a 32-channel standard amplifier connector through an insulating substrate that becomes the head-stage for neural recording experiments. The PCB is customizable to accommodate various head-stage configurations. Our design files are available by request or from the resource website, meshelectronics.org, and can be used to purchase PCBs inexpensively from vendors of PCB manufacture and assembly services.
<ol>
	<li>Use the stereotaxic frame to carefully guide the needle to the FFC clamping substrate and across the gap, flowing the solution with the syringe pump to generate slack in the mesh electronics interconnects (Figure 4C).</li>
	<li>Once the needle is above the clamping substrate and across the gap, resume the flow at a fast rate to eject the mesh electronics I/O pads onto the clamping substrate (Figure 4D).</li>
	<li>Using tweezers and a pipette of deionized water (DI), bend the I/O pads to ca. 90&#176; angle as close to the first I/O pad as possible. 
	NOTE: The bending is necessary to allow the pads to be inserted into the ZIF connector on a PCB in a subsequent step. The ZIF connector is exactly the same width&#160;as the 32 I/O pads of the mesh electronics probe, so an imperfect 90&#176; bend, or a bend not occurring right before the first I/O pad, will result in having to cut off I/O pads (the left-most pads in&#160;Figure 4E).</li>
	<li>Once the I/O pads are aligned, unfolded, and at a 90&#176; angle to the mesh stem, dry them in place with gently flowing compressed air. 
	NOTE: Mesh electronics probes with fewer than 32 channels can be interfaced to with the same 32-channel interface board. For example, our lab commonly uses 16-channel mesh electronics probes with 32-channel PCBs. This provides extra space within the ZIF connector, making interfacing easier, and the additional uncontacted channels are easily identified as open circuits by means of impedance testing during recording sessions.</li>
	<li>Cut the clamping substrate at a straight edge approximately 0.5&#8211;1 mm from the edge of the I/O pads. Also cut off extraneous parts of the clamping substrate that will hinder the insertion into the PCB-mounted 32-channel ZIF connector (Figure 4F).</li>
	<li>Insert the I/O pads into the ZIF connector on the PCB and close the latch (Figure 4G). Use measurement electronics to measure the impedance between the channels and the ground screw to confirm successful interfacing. If the impedance values are too high, unlatch the ZIF connector, adjust the insertion, and retest until successful connection is confirmed.</li>
	<li>Cover the ZIF connector and exposed mesh electronics interconnects with dental cement for protection. Flip the PCB at the gap in the substrate, and fix the PCB with cement onto the mouse skull (Figure 4H). 
	NOTE: Bending the FFC at the gap reduces the mechanical strain that can sometimes break the mesh electronics interconnects.</li>
	<li>Allow the cement to harden, turning the PCB into a robust, compact head-stage for interfacing during subsequent recording sessions (Figure 4I).</li>
</ol>
3. Neural Recording Experiments
<ol>
	<li>Place the mouse in a tailveiner or another restrainer. Insert the preamplifier PCB into the standard amplifier connector on the head-stage PCB. Use a separate cable to ground the reference screw.</li>
	<li>For restrained recordings, leave the mouse in the restrainer. Record the data using the data acquisition system for the desired period (Figure 5A).</li>
	<li>For freely moving recordings, release the mouse from the restrainer after inserting the preamplifier PCB and grounding the reference screw. Record for the desired length of time using the data acquisition system while the mouse behaves freely (Figure 5B)</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Motorized stereotaxic frame</td>
			<td>World Precision Instruments</td>
			<td>MTM-3</td>
			<td>For mouse stereotaxic surgery</td>
		</tr>
		<tr>
			<td>512-channel recording controller</td>
			<td>Intan Technologies</td>
			<td>C3004</td>
			<td>A component of the neural recording system</td>
		</tr>
		<tr>
			<td>RHD2132 amplifier board</td>
			<td>Intan Technologies</td>
			<td>C3314</td>
			<td>A component of the neural recording system</td>
		</tr>
		<tr>
			<td>RHD2000 3 feet ultra thin SPI interface cable</td>
			<td>Intan Technologies</td>
			<td>C3213</td>
			<td>A component of the neural recording system</td>
		</tr>
		<tr>
			<td>Mouse restrainer</td>
			<td>Braintree Scientific</td>
			<td>TV-150&#160;STD</td>
			<td>Standard 1.25 inch inner diameter; used to restrain the mouse during restrained recording sessions.</td>
		</tr>
		<tr>
			<td>Glass capillary needles</td>
			<td>Drummond Scientific Co.</td>
			<td></td>
			<td>Inner diameter 0.40 mm, outer diameter 0.65 mm. Other diameters are available.</td>
		</tr>
		<tr>
			<td>Micropipette holder U-type</td>
			<td>Molecular Devices, LLC</td>
			<td>1-HL-U</td>
			<td>Used to hold the glass capillary needles during stereotaxic injection</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>NORM-JECT&#174;, Henke Sass Wolf</td>
			<td></td>
			<td>Used for manual loading of mesh electronics into capillary needles</td>
		</tr>
		<tr>
			<td>Polyethylene intrademic catheter tubing</td>
			<td>Becton Dickinson and Company</td>
			<td></td>
			<td>Inner diameter 1.19 mm, outer diameter 1.70 mm</td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Becton Dickinson and Company</td>
			<td></td>
			<td>Used in the syringe pump for injection of mesh electronics in vivo</td>
		</tr>
		<tr>
			<td>Eyepiece camera</td>
			<td>Thorlabs Inc.</td>
			<td>DCC1240C</td>
			<td>Used to view mesh electronics within capillary needles during injection</td>
		</tr>
		<tr>
			<td>ThorCam uc480 image acquisition software for USB cameras</td>
			<td>Thorlabs Inc.</td>
			<td></td>
			<td>Used to view mesh electronics within capillary needles during injection</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>PHD 2000</td>
			<td>Used to flow precise volumes of solution through capillary needles during injection of mesh electronics</td>
		</tr>
		<tr>
			<td>EXL-M40 dental drill</td>
			<td>Osada</td>
			<td>3144-830</td>
			<td>For drilling the craniotomy</td>
		</tr>
		<tr>
			<td>0.9 mm drill burr</td>
			<td>Fine Science Tools</td>
			<td>19007-09</td>
			<td>For drilling the craniotomy</td>
		</tr>
		<tr>
			<td>Hot bead sterilizer 14 cm</td>
			<td>Fine Science Tools</td>
			<td>18000-50</td>
			<td>Used to sterlize surgical instruments</td>
		</tr>
		<tr>
			<td>CM1950 cryosectioning instrument</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Used to slice frozen tissue into sections. Many universities have</td>
		</tr>
		<tr>
			<td>Right-angle end clamp</td>
			<td>Thorlabs Inc.</td>
			<td>RA180/M</td>
			<td>Used to attach the pipette holder to the stereotaxic frame</td>
		</tr>
		<tr>
			<td>Printed circuit board (PCB)</td>
			<td>Advanced Circuits</td>
			<td></td>
			<td>Used to interface between&#160; mesh electronics and peripheral measurement electronics such as the Intan recording system. Advanced Circuits and other vendors manufacture and 
			assemble PCBs based on provided design files. Our PCB design files are available by request or at the resource site meshelectronics.org</td>
		</tr>
		<tr>
			<td>32-channel standard amplifier connector</td>
			<td>Omnetics Connector Corp.</td>
			<td>A79024-001</td>
			<td>Component assembled onto the PCB</td>
		</tr>
		<tr>
			<td>32-channel flat flexible cable (FFC)</td>
			<td>Molex, LLC</td>
			<td>152660339</td>
			<td>Used as a clamping substrate when interfacing to mesh electronics I/O pads with the PCB- mounted ZIF connector</td>
		</tr>
		<tr>
			<td>32-channel zero insertion force (ZIF) connector</td>
			<td>Hirose Electric Co., LTD</td>
			<td>FH12A-32S-0.5SH(55)</td>
			<td>Component assembled onto the PCB</td>
		</tr>
	</tbody>
</table>

Source: Schuhmann Jr., T. G., et al. <a href="https://app.jove.com/v/58003">Syringe-injectable Mesh Electronics for Stable Chronic Rodent Electrophysiology.</a> J. Vis. Exp. (2018).
The video demonstrates the injection of mesh electronics probes into a mouse brain for stable long-term recordings at a single-neuron level. An anesthetized mouse with an exposed skull is secured on a stereotaxic frame, and the probe&#39;s mesh device region is injected into the brain through drilled holes. The probe&#39;s input-output pads are connected to a circuit, and recordings are obtained using a data acquisition system.

<img alt="Figure 1" src="/files/ftp_upload/22726/22726_Figure1.jpg" /> 
Figure 1: Photographs and optical microscope images of plug-and-play mesh electronics.&#160;(A) Tiled optical microscope images of a syringe-injectable mesh electronics probe with plug-and-play I/O. The probe was imaged after the completion of the fabrication steps in&#160;Figure 2&#160;but prior to the release from the Ni-coated substrate. Dashed boxes correspon...

1. Stereotaxic Injection of Mesh Electronics into Live Mouse Brain
NOTE: Mice were anesthetized by intraperitoneal injection with a mixture of 75 mg/kg ...

Begin with an anesthetized mouse with an exposed skull on a stereotaxic frame and cover with a surgical drape.
Drill holes in the skull to access the brain and attach a clamping substrate.
Mount a capillary needle connected to a syringe pump onto the frame.
The needle contains a buffer with a mesh electronics probe, a flexible structure comprising a mesh device region connected to input/output pads.
Position the needle inside the brain. Inject to place the mesh device region within the tissue while retracting the needle until it exits the skull.
Position the needle over the clamping substrate and eject the input/output pads.
Bend the pads to attach them to an electronic circuit board.
Verify the completed circuit and insulate the connections. Allow neurons to connect through the porous mesh.
Connect the circuit to a data acquisition system. Allow the mouse to behave freely to collect stable single-neuron recordings.

17 조회수. 마우스 뇌에서 안정적인 단일 뉴런 기록을 위한 주입 가능한 메시 전자 장치

비디오: 마우스 뇌에서 안정적인 단일 뉴런 기록을 위한 주입 가능한 메시 전자 장치 - 실험

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

경고 설치류에 Transcranial 전기 두뇌 자극

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

연구

JoVE Journal

신경과학

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

만성 Transcranial 전기 자극 및 쥐 Intracortical 녹음

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Construction of Local Field Potential Microelectrodes for in vivo Recordings from Multiple Brain Structures Simultaneously

Researchers often need to record local field potentials (LFPs) simultaneously from several brain structures. Recording from multiple desired brain regions requires different microelectrode designs, but commercially available microelectrode arrays often do not offer such flexibility. Here, the present protocol outlines the straightforward design of custom-made microelectrode arrays to record LFPs from multiple brain structures simultaneously at different depths. This work describes the construction of the bilateral cortical, striatal, ventrolateral thalamic, and nigral microelectrodes as an example. The outlined design principle offers flexibility, and the microelectrodes can be modified and customized to record LFPs from any structure by calculating stereotaxic coordinates and quickly changing the construction accordingly to target different brain regions in either freely moving or anesthetized mice. The microelectrode assembly requires standard tools and supplies. These custom microelectrode arrays allow investigators to easily design microelectrode arrays in any configuration to track neuronal activity, providing LFP recordings with millisecond resolution.

Construction of Local Field Potential Microelectrodes for in vivo Recordings from Multiple Brain Structures Simultaneously

The present protocol describes the construction of custom-made microelectrode arrays to record local field potentials in vivo from multiple brain structures simultaneously.

여러 뇌 구조에서 동시에 생체 내 녹음을위한 로컬 필드 잠재적 인 미세 전극 구축

Researchers often need to record local field potentials (LFPs) simultaneously from several brain structures. Recording from multiple desired brain regions ...

Injectable Mesh Electronics for Stable Single-Neuron Recordings in a Mouse Brain

내레이션 대본

Injectable Mesh Electronics for Stable Single-Neuron Recordings in a Mouse Brain