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.remotexs.ntu.edu.sg/files/ftp_upload/59957/59957fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>&lt;strong&gt;3D Printed Stereotax Adapter Parts (3) and Base Piece (1)&lt;/strong&gt;</td><td>N/A</td><td>N/A</td><td>3d print parts, suggest &amp;lt;30 &amp;mu;m resolution for minimal hand finishing of parts. Files available at:</td></tr><tr><td>https://github.com/jasonechung/PolymerProbe3dParts</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Dental Acrylic (Hygenic Repair Resin, Coltene type II quick set)&lt;/strong&gt;</td><td>Colten/Whaledent</td><td>8886784, 8881627</td><td>Dental acrylic for use during implant construction</td></tr><tr><td>&lt;strong&gt;Hydraulic Micromanipulator (x2)&lt;/strong&gt;</td><td>Narishige Group</td><td>MO-10</td><td>1-axis micromanipulator</td></tr><tr><td>&lt;strong&gt;Kapton Polyimide Tape&lt;/strong&gt;</td><td>Bertech</td><td>PPTDE-1/2</td><td>Double-sided tape</td></tr><tr><td>&lt;strong&gt;Kopf Stereotax Arm&lt;/strong&gt;</td><td>&amp;nbsp;Kopf Instruments</td><td>103088R, 103088L</td><td>Standard rodent stereotax</td></tr><tr><td>&lt;strong&gt;Light Curable Dental Acrylic, Vivid Flow&lt;/strong&gt;</td><td>Coltene/Whaledent</td><td>D33-01-00</td><td>Light curable dental acrylic for use during implant construction</td></tr><tr><td>&lt;strong&gt;Loctite Gel Control&lt;/strong&gt;</td><td>&amp;nbsp;Henkel Corp.</td><td>&amp;nbsp;234790 1364076 1735574 1752699</td><td>Cyanoacrylate for adhering silicon shuttle to corresponding 3d printed part</td></tr><tr><td>&lt;strong&gt;Metabond Quick Cement&lt;/strong&gt;</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>&lt;strong&gt;Polymer Electrode Arrays and Silicon Insertion Shuttles&lt;/strong&gt;</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>&lt;strong&gt;Silicone Gel Kit, Low Viscosity&lt;/strong&gt;</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>&lt;strong&gt;Silicone, Medium-Viscosity Kit&lt;/strong&gt;</td><td>World Precision Instruments</td><td>&amp;nbsp;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.remotexs.ntu.edu.sg/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 ...

chronic-implantation-of-multiple-flexible-polymer-electrode-arrays

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.6K Views.  University of California San Francisco. De methode maakt het mogelijk monitoring van grote, gedistribueerde neuronale populaties over dagen, weken of maanden, waardoor vragen over hoe deze populaties veranderen en ondersteunen cognitieve functies binnen handbereik. De implantatie van meerdere polymeerapparaten en de bijbehorende implantaatconstructie maken samen een opnameplatform dat in staat is om high-channel count, multi-region, long term, continuous en stable recordings te maken. Het aantonen van de procedure zal Clay Smyth zi...

Video: Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Bioengineering

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

Microelectrode arrays for neural interface devices that are made of biocompatible thin-film polymer are expected to have extended functional lifetime because the flexible material may minimize adverse tissue response caused by micromotion. However, their flexibility prevents them from being accurately inserted into neural tissue. This article demonstrates a method to temporarily attach a flexible microelectrode probe to a rigid stiffener using biodissolvable polyethylene glycol (PEG) to facilitate precise, surgical insertion of the probe. A unique stiffener design allows for uniform distribution of the PEG adhesive along the length of the probe. Flip-chip bonding, a common tool used in microelectronics packaging, enables accurate and repeatable alignment and attachment of the probe to the stiffener. The probe and stiffener are surgically implanted together, then the PEG is allowed to dissolve so that the stiffener can be extracted leaving the probe in place. Finally, an in vitro test method is used to evaluate stiffener extraction in an agarose gel model of brain tissue. This approach to implantation has proven particularly advantageous for longer flexible probes (&gt;3 mm). It also provides a feasible method to implant dual-sided flexible probes. To date, the technique has been used to obtain various in vivo recording data from the rat cortex.

Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.

Microelectrode  arrays for neural interface devices that are made of biocompatible thin-film  polymer are expected to have extended functional lifetime ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics.&#160;This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters.&#160;This article develops a protocol for in vitro and in vivo testing of neural recording electrodes.&#160;Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed.&#160;This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models.&#160;The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio.&#160;This protocol can easily be adapted to test other electrode materials and designs.&#160;The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators.&#160;The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. Recent technological progress has enabled the fabrication of microelectrode arrays that allow recording the activity of multiple individual neurons in the neocortex of the human brain. These arrays have the potential to bring unique insight onto the neuronal correlates of cerebral function in health and disease. Furthermore, the identification and decoding of volitional neuronal activity opens the possibility to establish brain-computer interfaces, and thus might help restore lost neurological functions. The implantation of neocortical microelectrode arrays is an invasive procedure requiring a supra-centimetric craniotomy and the exposure of the cortical surface; thus, the procedure must be performed by an adequately trained neurosurgeon. In order to provide an opportunity for surgical training, we designed a procedure based on a human cadaver model. The use of a formaldehyde-fixed human cadaver bypasses the practical, ethical and financial difficulties of surgical practice on animals (especially non-human primates) while preserving the macroscopic structure of the head, skull, meninges and cerebral surface and allowing realistic, operating room-like positioning and instrumentation. Furthermore, the use of a human cadaver is closer to clinical daily practice than any non-human model. The major drawbacks of the cadaveric simulation are the absence of cerebral pulsation and of blood and cerebrospinal fluid circulation. We suggest that a formaldehyde-fixed human cadaver model is an adequate, practical and cost-effective approach to ensure proper surgical training before implanting microelectrode arrays in the living human neocortex.

We designed a procedure in which a formaldehyde-fixed human cadaver is used to assist neurosurgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain.

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. ...

Medicine

Open-source Toolkit: Benchtop Carbon Fiber Microelectrode Array for Nerve Recording

Conventional peripheral nerve probes are primarily fabricated in a cleanroom, requiring the use of multiple expensive and highly specialized tools. This paper presents a cleanroom "light" fabrication process of carbon fiber neural electrode arrays that can be learned quickly by an inexperienced cleanroom user. This carbon fiber electrode array fabrication process requires just one cleanroom tool, a Parylene C deposition machine, that can be learned quickly or outsourced to a commercial processing facility at marginal cost. This fabrication process also includes hand-populating printed circuit boards, insulation, and tip optimization. 
The three different tip optimizations explored here (Nd:YAG laser, blowtorch, and UV laser) result in a range of tip geometries and 1 kHz impedances, with blowtorched fibers resulting in the lowest impedance. While previous experiments have proven laser and blowtorch electrode efficacy, this paper also shows that UV laser-cut fibers can record neural signals in vivo. Existing carbon fiber arrays either do not have individuated electrodes in favor of bundles or require cleanroom fabricated guides for population and insulation. The proposed arrays use only tools that can be used at a benchtop for fiber population. This carbon fiber electrode array fabrication process allows for quick customization of bulk array fabrication at a reduced price compared to commercially available probes.

Here, we describe fabrication methodology for customizable carbon fiber electrode arrays for recording in vivo in nerve and brain.

Conventional peripheral nerve probes are primarily fabricated in a cleanroom, requiring the use of multiple expensive and highly specialized tools. This ...

Subdural Soft Electrocorticography (ECoG) Array Implantation and Long-Term Cortical Recording in Minipigs

Neurological impairments and diseases can be diagnosed or treated using electrocorticography (ECoG) arrays. In drug-resistant epilepsy, these help delineate the epileptic region to resect. In long-term applications such as brain-computer interfaces, these epicortical electrodes are used to record the movement intention of the brain, to control the robotic limbs of paralyzed patients. However, current stiff electrode grids do not answer the need for high-resolution brain recordings and long-term biointegration. Recently, conformable electrode arrays have been proposed to achieve long-term implant stability with high performance. However, preclinical studies for these new implant technologies are needed to validate their long-term functionality and safety profile for their translation to human patients. In this context, porcine models are routinely employed in developing medical devices due to their large organ sizes and easy animal handling. However, only a few brain applications are described in the literature, mostly due to surgery limitations and integration of the implant system on a living animal.
Here, we report the method for long-term implantation (6 months) and evaluation of soft ECoG arrays in the minipig model. The study first presents the implant system, consisting of a soft microfabricated electrode array integrated with a magnetic resonance imaging (MRI)-compatible polymeric transdermal port that houses instrumentation connectors for electrophysiology recordings. Then, the study describes the surgical procedure, from subdural implantation to animal recovery. We focus on the auditory cortex as an example target area where evoked potentials are induced by acoustic stimulation. We finally describe a data acquisition sequence that includes MRI of the whole brain, implant electrochemical characterization, intraoperative and freely moving electrophysiology, and immunohistochemistry staining of the extracted brains.
This model can be used to investigate the safety and function of novel design of cortical prostheses; mandatory preclinical study to envision translation to human patients.

Subdural Soft Electrocorticography ECoG Array Implantation and Long-Term Cortical Recording in Minipigs

Here, we present a method for the long-term performance and safety assessment of soft subdural electrode arrays in a minipig model, describing surgical method and tools, postoperative magnetic resonance imaging, electrophysiology of the auditory cortex, electrochemical properties of the implant, and postmortem immunochemistry.

Neurological impairments and diseases can be diagnosed or treated using electrocorticography (ECoG) arrays. In drug-resistant epilepsy, these help ...

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. Development of these tools is crucial for understanding complex behaviors and cognition and for advancing clinical applications. However, it remains a challenge to densely record from cell populations stably and continuously over long time periods. Many popular electrodes, such as tetrodes and silicon arrays, feature large cross-diameters that produce damage upon insertion and elicit chronic reactive tissue responses associated with neuronal death, hindering the recording of stable, continuous neural activity. In addition, most wire bundles exhibit broad spacing between channels, precluding simultaneous recording from a large number of cells clustered in a small area. The carbon fiber microelectrode arrays described in this protocol offer an accessible solution to these concerns. The study provides a detailed method for fabricating carbon fiber microelectrode arrays that can be used for both acute and chronic recordings in vivo. The physical properties of these electrodes make them ideal for stable and continuous long-term recordings at high cell densities, enabling the researcher to make robust, unambiguous recordings from single units across months.

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

This protocol describes a procedure for constructing carbon fiber microelectrode arrays for chronic and acute in vivo electrophysiological recordings in mouse (Mus musculus) and ferret (Mustela putorius furo) from multiple brain regions. Each step, following the purchase of raw carbon fibers to microelectrode array implantation, is described in detail, with emphasis on microelectrode array construction.

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. ...

Stimulating Mouse Brain with a Low-Cost EEG and Millimeter-Sized Coil

Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, utilizing a millimeter-sized coil. Using conventional screw electrodes combined with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain. In addition, we explain how a millimeter-sized coil is produced using low-cost equipment usually found in laboratories. Practical procedures for fabricating the flexible multielectrode array substrate and the surgical implantation technique for screw electrodes are also presented, which are necessary to produce low-noise EEG signals. Although the methodology is useful for recording from the brain of any small animal, the present report focuses on electrode implementation in an anesthetized mouse skull. Furthermore, this method can be easily extended to an awake small animal that is connected with tethered cables via a common adapter and fixed with a TMS device to the head during recording.The present version of the EEG-TMS system, which can include a maximum of 32 EEG channels (a device with 16 channels is presented as an example with fewer channels) and one TMS channel device, is described. Additionally, typical results obtained by the application of the EEG-TMS system to anesthetized mice are briefly reported.

Author Spotlight: Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo  

A low-cost electroencephalographic recording system combined with a millimeter-sized coil is proposed to drive transcranial magnetic stimulation of the mouse brain in vivo. Using conventional screw electrodes with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain in response to transcranial magnetic stimulation.

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, ...

A Cost-Effective, Versatile Probe Implant System for Electrophysiology in Mice

The DREAM Implant: A Lightweight, Modular, and Cost-Effective Implant System for Chronic Electrophysiology in Head-Fixed and Freely Behaving Mice

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. However, current methods for chronically implanting probes present steep trade-offs between cost, ease of use, size, adaptability, and long-term stability.
This protocol introduces a novel chronic probe implant system for mice called the DREAM (Dynamic, Recoverable, Economical, Adaptable, and Modular), designed to overcome the trade-offs associated with currently available options. The system provides a lightweight, modular and cost-effective solution with standardized hardware elements that can be combined and implanted in straightforward steps and explanted safely for recovery and multiple reuse of probes, significantly reducing experimental costs.
The DREAM implant system integrates three hardware modules: (1) a microdrive that can carry all standard silicon probes, allowing experimenters to adjust recording depth across a travel distance of up to 7 mm; (2) a three-dimensional (3D)-printable, open-source design for a wearable Faraday cage covered in copper mesh for electrical shielding, impact protection, and connector placement, and (3) a miniaturized head-fixation system for improved animal welfare and ease of use. The corresponding surgery protocol was optimized for speed (total duration: 2 h), probe safety, and animal welfare.
The implants had minimal impact on animals&#39; behavioral repertoire, were easily applicable in freely moving and head-fixed contexts, and delivered clearly identifiable spike waveforms and healthy neuronal responses for weeks of post-implant data collection. Infections and other surgery complications were extremely rare.
As such, the DREAM implant system is a versatile, cost-effective solution for chronic electrophysiology in mice, enhancing animal well-being, and enabling more ethologically sound experiments. Its design simplifies experimental procedures across various research needs, increasing accessibility of chronic electrophysiology in rodents to a wide range of research labs.

Here, we introduce a lightweight, cost-effective probe implant system for chronic electrophysiology in rodents optimized for ease of use, probe recovery, experimental versatility, and compatibility with behavior.

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. ...

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

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