Name: chronic implantation of multiple flexible polymer electrode arrays
Uploaded: 2019-10-04T12:30:00
Duration: 8 min 54 s
Description: Watch this Scientific Journal Video about Chronic Implantation of Multiple Flexible Polymer Electrode Arrays at JoVE.com

This work was supported by NINDS grant U01NS090537 to L.M.F and V.M.T., NIMH grant F30MH109292 to J.E.C, and NIMH grant F30MH115582 to H.R.J. J.E.C. and H.R.J. are also supported by NIGMS MSTP grant #T32GM007618. The Flatiron Institute is a division of the Simons Foundation.

J.E.C and L.M.F. are inventors on a pending patent related to the work described here.

This is a method for the implantation of multiple polymer electrode arrays to distributed brain areas for recording of single units over months. This method represents an 8x increase in recording channels and 4x increase in number of insertions from the closest large-scale polymer-array based system2,3. That system utilized a polymer mesh injection-based system in mouse but did not report an absolute number of putative single-units and thus a comparison of single neuron yield is not possible.
The method for insertion of a flexible device is based on an earlier protocol from Felix et al.39, with important modifications: a three-piece insertion apparatus for independent motion of the silicon shuttle during retraction, and tethering of the array at its target depth prior to retraction of the shuttle, which together eliminate the need for the quick withdrawal described in the original protocol. These changes minimize tissue damage and maintain array stability during retraction of the shuttle. Other flexible device implantation strategies, such as temporarily stiffening devices with bio dissolvable materials, are compatible with subsequent steps in this protocol. Securing the devices within the implant necessitated integrating previously validated strategies for covering the brain and protecting the delicate device ribbons.
Due to their fragility, care and attention are required to avoid directly contacting or otherwise transmitting force to the polymer electrode arrays and the silicon insertion shuttles. Particularly when working with multiple devices, insertion should be observed under a microscope to avoid interference of one device with another. In general, it is possible to handle an electrode array gently with plastic tipped forceps, avoiding the traces. Such a strategy is appropriate, for example, if the polymer electrode array begins to retract with the insertion shuttle. This can occur if the PEG is not completely dissolved, or due to surface tension of saline or CSF between the polymer and silicon.
One of the most common recoverable errors is array detachment from the insertion shuttle. This can occur at insertion, as the brain dimples and pressure at the device tip increases, if the array and shuttle are imperfectly aligned or if condensation has partially dissolved the PEG. To re-adhere an array, raise it as high as possible above the brain surface and wait for it to dry (approximately 5 min).
A critical aspect of planning a multi-array implantation surgery is the design of the base piece to accommodate all implant targets and sit without gaps against the contour of the skull. The base piece is a small plastic piece that is fixed to the skull after skull cleaning, screw placement, and partial craniectomies, prior to the insertion of the arrays. It has three functions: 1) to hold saline for dissolving the PEG following array insertion but before silicon shuttle retraction, 2) to provide a location above the skull surface to which the arrays can be attached by polyimide wings, thereby allowing strain relief along the ribbon above its insertion point in the brain, and 3) to hold artificial dural sealant, which stabilizes and protects the arrays and brain. The base piece can be fashioned by hand or 3D-printed. It was observed that draining and drying the base piece of saline are very important preceding device insertion. These steps prevent condensation and separation of the array and insertion shuttle. Drying the base piece is also critical to filling the base piece with artificial dural sealant. It is also important that the base piece not leak, as a film of silicone gel is difficult to remove from the skull and will prevent adhesion of dental acrylic for reliable chronic attachment of the implant to the skull. It is expected that any low-viscosity, biocompatible silicone elastomer could be used to fill the craniectomies and base piece, with a higher viscosity silicone elastomer surrounding it and the exposed polymer array ribbons.
Advances in polymer nanofabrication will translate to polymer-based electrode arrays, reducing feature sizes and increasing the possible number of electrodes in an array closer to those of silicon devices15,16,17,18,19. Similarly, the cross-sectional areas of polymer devices will shrink alongside feature sizes, providing even better biocompatibility8. Again, as is being accomplished with silicon devices, integration with amplifying, digitizing, and multiplexing chips17 will further enable larger-scale neural recording.

An ideal neural implant would record from a very large number of individual neurons in distributed brain areas over weeks to months. Flexible polymer electrode arrays provide electrophysiological recordings with the longevity to record for months and the stability to track individual neurons1,2,3. However, the same mechanical properties that reduce shearing damage4 and confer biocompatibility and recording capability2,3,5,6,7,8 pose a challenge to their insertion into the brain relative to their rigid counterparts. Previous work accomplished a maximum of four 32-channel arrays, but the total yield of sorted putative single neurons is unreported2,3,9. Conversely, silicon-based electrode arrays have been used in high-density, multi-region implants, but these technologies lack either the ability to record spikes from neurons over months (longevity) or to track the same neurons (stability) on that timescale, or the density to record from hundreds of individual neurons across multiple brain regions. The method presented here overcomes the low number of insertions in current polymer electrode array-based methods, thereby providing means for the electrophysiologic recording of large numbers of individual neurons in multiple anatomically distant regions for months, with the stability to record from the same individual neurons across many days.
There is some debate regarding the importance of using a polymer substrate instead of microwire- or silicon- based strategies. As demonstrated by Dhawale et al.10, microwires are indeed capable of months-long stable recordings in rodents, though the implants were limited to 16 tetrodes in a single region. Scaling up the size of the microwire implant reaches a relatively high upper limit, with up to 1792 implanted channels achieved in a non-human primate11. However, construction of the microwire arrays is incompatible with silicon nanofabrication processes and is, therefore, extremely time consuming, requiring manual handling of each channel individually during the construction12,13,14. As such, it is not clear if this technology could support an order of magnitude increase in recording channels.
Current silicon devices can place hundreds or even over a thousand electrodes on a single monolithic device15,16,17,18,19. The latest silicon fabrication processes generate devices with smaller cross-sectional areas, regardless of the material, resulting in less glial activation20,21,22,23,24 and more compliant devices. There is a variability in reports of silicon probe single-unit recording longevity, with some indicating that relatively large silicon probes can provide long-term recording25,26. Notably, the latest commercially-available silicon devices17 have the longevity to record for several months and have cross-sectional areas very similar to the shanks used in the method described here (Jun et al. 201717: 70 &#181;m x 20 &#181;m, devices described here and in Chung et al. 20191: 68 &#181;m &#8211; 80 &#181;m x 14 &#181;m). Due to the difference in stability, this probe has not been demonstrated to be able to record from the same neurons over weeks. This likely is due to some combination of the use of rigid silicon as well as direct tethering to the skull, known to cause micromotion, instability, and gliosis at the array-brain interface27,28. To construct a device that can move with the neural tissue, materials that are soft5,29 and flexible7 are required. Many available polymers (see Geddes and Roeder30, Fattahi et al.31, and Weltman et al.32 for reviews) have the flexibility and stability of microwires and are also compatible with the nanofabrication processes, which allow the dense packing of silicon devices.
Several neural implantation issues are specific to the use of flexible polymer electrode arrays. The first of these is the insertion of the array, as flexible arrays lack the rigidity to be advanced into the brain like silicon- or microwire-based strategies. The majority of insertion strategies for flexible devices depend on a temporary stiffening of the substrate as is done in this method (see Weltman et al.32 for review). There are five notable strategies that do not make use of a rigid shuttle. First, there are methods that make use of materials that transition from rigid to compliant upon implantation33,34. A drawback of this strategy is that it requires a relatively large cross-sectional area to achieve the force required for penetration of brain tissue before buckling as dictated by Euler&#8217;s buckling force calculation35. This increase in cross-sectional area will negatively impact the health of the surrounding tissue20,21,22,23,24. Second is the use of a removable supporting structure above the brain36, though this requires time-consuming removal or dissolution of scaffolding to maintain a minimal unsupported length (and high buckling force). Alternatively, it would require the array to be inserted with a longer unsupported length, thereby requiring a stiffer array substrate or a larger array cross-sectional area. Third is pre-penetration to open a hole for the flexible array to be inserted in afterward35. This requires precise realignment or relatively large pre-penetration diameter, and electrode array rigidity and cross-sectional area to permit unsupported insertion. Fourth is the use of dissolvable coatings to stiffen the flexible device. This significantly increases the cross-sectional area and acute damage caused by insertion, even when special precautions are taken to preserve the sharp tip of a device37. Fifth is the injection of the polymer array. This strategy has had success in achieving implants with up to four 32-ch insertions2, but requires using a far larger cross-sectional area for insertion, a 250 &#181;m &#8211; 1.5 mm outer diameter glass capillary tube9, causing greater acute damage. In contrast, using a removable shuttle, while adding cross-sectional area to the acute insertion, allows for the use of the stiffest possible materials, and can, therefore, be the theoretical minimum size when inserting an arbitrarily flexible device. Thus, insertion using a rigid shuttle is currently the most attractive option for inserting flexible devices.
There are two requirements of any insertion shuttle approach: a suitably stiff substrate and a way to couple the flexible device to the substrate. Insertion shuttle materials are typically silicon38,39,40,41, stainless steel8,42, or tungsten43,44,45, with stiffer materials allowing for smaller cross-sectional areas. These are typically affixed using an adhesive such as polyethylene glycol (PEG)8,38,39,42,43, electrostatic forces40, or direct physical coupling45,46. In all cases, the challenges are the alignment and coupling of the electrode array and insertion shuttle before insertion and decoupling after insertion. Recounted below is a refinement of the method introduced by Felix et al.39 to temporarily brace the electrode array with a silicon insertion shuttle, attached using PEG, that is removed after insertion of the array to its target depth.
A second challenge presented by flexible devices within a chronic implant is that of stabilizing the device within the brain while still allowing for the device to be integrated into an implant attached to the skull. The brain moves relative to the skull due to natural pulsations, post-traumatic edematous changes, impact, and other causes, and the electrode array must therefore be at least somewhat free to move relative to where it is affixed to the skull and recording hardware. This is achieved using a 3D-printed plastic base piece, custom-designed for each set of implant targets, that has multiple functions: a saline reservoir during implantation, location to tether the polymer arrays, and housing for silicone gel. The tethering location above the skull and silicone gel work together to create a larger radius of curvature for the array and thereby allow for larger compressive forces on the array. This in turn allows for movement of brain relative to the anchor points of the array (skull) to be translated into buckling load.
Further challenges include the need to house multiple arrays and provide ample strain relief for the animal to freely behave without transfer of vibrations or impact forces to the electrode arrays, which can cause motion relative to neural tissue. Adaptations to solutions that have been used in similar applications where the brain must be stable relative to a rigid recording window have addressed this challenge. An artificial dural sealant silicone gel (Table of Materials), which has previously been demonstrated to be non-toxic and prevent CSF leakage47, provides counter-pressure to the brain to prevent outward swelling and to stabilize the array at the brain surface. An additional layer of protection is added to the device ribbons by the medium-viscosity, surgical grade silicone elastomer, previously demonstrated for use in sealing chronic neural electrode implants48. Finally, the silicone-buffered implant and headstage is encased with 3D-printed pieces custom designed to maintain a low center of mass for minimal reduction of the animal&#8217;s normal mobility.
This protocol starts with a flexible polymer microelectrode array mounted to a silicon insertion shuttle. It proceeds with mounting of the array-shuttle device to the 3D-printed insertion pieces, describes the surgical technique and implant construction steps required to successfully implant an animal, and is capable of supporting sixteen polymer multi-electrode arrays implanted in eight anatomically distant regions in a single rat1.
This protocol assumes the starting materials of polymer electrode arrays attached by the biodissolvable adhesive polyethylene glycol (PEG) to a silicon insertion shuttle, as shown in Felix et al.39, and at least two independently movable insertion pieces: one to which the silicon shuttle will be glued and one to which the electrode array&#8217;s connector will be adhered. This protocol also uses a third insertion piece to more securely attach the two insertion pieces to a micron-scale micromanipulator. All files for 3D printing can be found at: https://github.com/jasonechung/PolymerProbe3DParts
Each polymer electrode array, used in this method is comprised of two to four recording shanks, a ribbon that conveys the electrical traces, and, at the end of the ribbon, a hardware connector or printed circuit board. The electrode array and ribbon are fixed atop the silicon shuttle with PEG. Each ribbon has a 2 cm long x 1 mm thick polyimide tube attached to the ribbon via UV curable epoxy, extending perpendicular to the length of the ribbon. Each device (electrode array and insertion shuttle) must be loaded onto the 3D-printed insertion pieces that will be used to insert the array into the brain and retract the shuttle (Figure 1). In this design, the hydraulic insertion micromanipulator (green, Table of Materials) moves the entire insertion apparatus (piece 1, piece 2 and the retraction micromanipulator, orange) to its target depth. Once the array has been detached from the insertion apparatus and fixed, the second, retraction micromanipulator (orange) retracts piece 1 and the attached shuttle independently from the rest of the insertion apparatus, removing the shuttle without displacing the array.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59957/59957fig01.jpg" /> 
Figure 1: Inserter components. 
(A) Pieces 1 and 2 are temporarily fixed to each other with a removable screw and will later be docked onto the retraction micromanipulator piston (orange). (B) The array and insertion shuttle are adhered to piece 1 and the array connector is attached to piece 2 with double-sided tape. Piece 3 connects the retraction micromanipulator and pieces 1 and 2 to the insertion micromanipulator (green). The insertion micromanipulator is fixed to a stereotactic adapter for implant positioning. Pieces 1-3 are pictured in their relative sizes. Piece 4 is a stabilizing piece for proper alignment of the insertion shuttle. <a href="https://www.jove.com/files/ftp_upload/59957/59957fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td rowspan="2">3D Printed Stereotax Adapter Parts (3) and Base Piece (1)</td>
			<td rowspan="2">N/A</td>
			<td rowspan="2">N/A</td>
			<td>3d print parts, suggest &#60;30 &#956;m resolution for minimal hand finishing of parts. Files available at:</td>
		</tr>
		<tr>
			<td>https://github.com/jasonechung/PolymerProbe3dParts</td>
		</tr>
		<tr>
			<td>Dental Acrylic (Hygenic Repair Resin, Coltene type II quick set)</td>
			<td>Colten/Whaledent</td>
			<td>8886784, 8881627</td>
			<td>Dental acrylic for use during implant construction</td>
		</tr>
		<tr>
			<td>Hydraulic Micromanipulator (x2)</td>
			<td>Narishige Group</td>
			<td>MO-10</td>
			<td>1-axis micromanipulator</td>
		</tr>
		<tr>
			<td>Kapton Polyimide Tape</td>
			<td>Bertech</td>
			<td>PPTDE-1/2</td>
			<td>Double-sided tape</td>
		</tr>
		<tr>
			<td>Kopf Stereotax Arm</td>
			<td>&#160;Kopf Instruments</td>
			<td>103088R, 103088L</td>
			<td>Standard rodent stereotax</td>
		</tr>
		<tr>
			<td>Light Curable Dental Acrylic, Vivid Flow</td>
			<td>Coltene/Whaledent</td>
			<td>D33-01-00</td>
			<td>Light curable dental acrylic for use during implant construction</td>
		</tr>
		<tr>
			<td>Loctite Gel Control</td>
			<td>&#160;Henkel Corp.</td>
			<td>&#160;234790 1364076 1735574 1752699</td>
			<td>Cyanoacrylate for adhering silicon shuttle to corresponding 3d printed part</td>
		</tr>
		<tr>
			<td>Metabond Quick Cement</td>
			<td>Parkell</td>
			<td>S380</td>
			<td>For direct application to skull to create strong connection between skull and implant</td>
		</tr>
		<tr>
			<td>Polymer Electrode Arrays and Silicon Insertion Shuttles</td>
			<td>Lawrence-Livermore National Laboratory</td>
			<td>N/A</td>
			<td>Fabricated at Lawrence-Livermore National Laboratory, polyimide electrode arrays, silicon insertion shuttle</td>
		</tr>
		<tr>
			<td>Silicone Gel Kit, Low Viscosity</td>
			<td>Dow Corning</td>
			<td>03/80</td>
			<td>Low-viscosity silicone gel for filling of 3d printed base piece</td>
		</tr>
		<tr>
			<td>Silicone, Medium-Viscosity Kit</td>
			<td>World Precision Instruments</td>
			<td>&#160;Kwik-Sil</td>
			<td>Medium-viscosity silicone gel for protection of polymer electrode arrays</td>
		</tr>
	</tbody>
</table>

chronic implantation of multiple flexible polymer electrode arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Following this protocol, a 1,024-channel neural implant recording yielded 375 single units1 (sorted with MountainSort50, noise overlap &#60; 0.03, isolation &#62; 0.96, 512 channels used for single unit recording, Figure 9A). This protocol can be used to implant different numbers of devices, with different channel counts and specifications, to different combinations of recording targets. Using the same protocol, single unit recording longevity has been demonstrated for at least 160 days1 in data from 19 devices (18 32-channel devices in prefrontal cortices, one 64-channel device in orbitofrontal cortex) across three different rats (Figure 9B). One of the three animals had a digital electrical failure resulting in an inability to record from four devices. Of the remaining 15/19 devices, there was a recording yield average of ~1 single unit per channel. Individual devices had yields of only a few single units up to ~2 units per channel. It is typical to see very different yields on devices implanted in the same animal in the same region.
In addition, a different surgical team following the protocol described here implanted six additional animals each with a combination of 4-6 32-channel devices targeted to orbitofrontal cortex and nucleus accumbens, and a tetrode hyperdrive (total implant weight approximately 50 g). One animal had an implant detach within one month of surgery. A second animal died during the post-operative recovery period, likely unrelated to the protocol steps described here. The remaining four animals remained healthy with stable implants that for the length of experiment, which lasted 4-11 months. Single unit counts were similar to those previously reported for 32-channel devices.&#160;
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/59957/59957fig09.jpg" /> 
Figure 9: Single-unit yield and recording longevity. 
(A) Number of putative single-unit clusters from 512 channels (of the 1,024-channel implant), stratified by quality metric thresholds. Automated curation using MountainSort (noise overlap 0.03, isolation 0.96, black box in upper right) resulted in the identification of 375 single units from the 512 channels. Reproduced with permission from Neuron [Figure 2A]1. (B) Single-unit yields for polymer arrays per channel (left y-axis) or per 16-channel shank (right y-axis) over 160 days post-implantation (x-axis) in rats. Solid line is the mean cell yield across 8 shanks, dotted lines &#177; 1 SE. Individual time points per shank are shown as color-coded dots by region. Reproduced with permission from Neuron [Figure 3A]1. <a href="https://www.jove.com/files/ftp_upload/59957/59957fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Chronic Implantation of Multiple Flexible Polymer Electrode Arrays at JoVE.com

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...

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