We would like to thank Greg Guitchounts for guidance with electrode design and construction and Tim Gardner for opening up his lab and facilities to us. We would like to thank Christos Michas for his assistance with PDS use at the Bio-Interface and Technology core facility and Neil Ritter, Jon Spyreas, and David Landesman for their help in designing early versions of the 16-channel jig. We would like to thank Tim Cavanaugh for his assistance with SEM imaging at the Center for Harvard Nanoscale Systems at Harvard.

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<li>Szostak, K. M., Grand, L., Constandinou, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+interfaces+for+intracortical+recording%3A+requirements%2C+fabrication+methods%2C+and+characteristics%5BTitle%5D)+AND+%22Frontiers+in+Neuroscience%22%5BJournal%5D)">Neural interfaces for intracortical recording: requirements, fabrication methods, and characteristics.</a> Frontiers in Neuroscience. 11, 665(2017).</li>
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<li>Guitchounts, G., Markowitz, J. E., Liberti, W. A., Gardner, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+carbon-fiber+electrode+array+for+long-term+neural+recording%5BTitle%5D)+AND+%22Journal+of+Neural+Engineering%22%5BJournal%5D)">A carbon-fiber electrode array for long-term neural recording.</a> Journal of Neural Engineering. 10 (4), 046016(2013).</li>
<li>Guitchounts, G., Cox, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((64-channel+carbon+fiber+electrode+arrays+for+chronic+electrophysiology%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">64-channel carbon fiber electrode arrays for chronic electrophysiology.</a> Scientific Reports. 10 (1), 3830(2020).</li>
<li>Ma, Z., Turrigiano, G. G., Wessel, R., Hengen, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortical+circuit+dynamics+are+homeostatically+tuned+to+criticality+in+vivo%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Cortical circuit dynamics are homeostatically tuned to criticality in vivo.</a> Neuron. 104 (4), 655-664 (2019).</li>
<li>Popovic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+cross-orientation+suppression+and+size+tuning+and+the+role+of+experience%5BTitle%5D)+AND+%22Journal+of+Neuroscience%3A+The+Official+Journal+of+the+Society+of+Neuroscience%22%5BJournal%5D)">Development of cross-orientation suppression and size tuning and the role of experience.</a> Journal of Neuroscience: The Official Journal of the Society of Neuroscience. 38 (11), 2656-2670 (2018).</li>
<li>Patel, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+in+vivo+stability+assessment+of+carbon+fiber+microelectrode+arrays%5BTitle%5D)+AND+%22Journal+of+Neural+Engineering%22%5BJournal%5D)">Chronic in vivo stability assessment of carbon fiber microelectrode arrays.</a> Journal of Neural Engineering. 13 (6), 066002(2016).</li>
<li>Welle, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultra-small+carbon+fiber+electrode+recording+site+optimization+and+improved+in+vivo+chronic+recording+yield%5BTitle%5D)+AND+%22Journal+of+Neural+Engineering%22%5BJournal%5D)">Ultra-small carbon fiber electrode recording site optimization and improved in vivo chronic recording yield.</a> Journal of Neural Engineering. 17 (2), 026037(2020).</li>
<li>Patel, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insertion+of+linear+8.4+%C2%B5m+diameter+16+channel+carbon+fiber+electrode+arrays+for+single+unit+recordings%5BTitle%5D)+AND+%22Journal+of+Neural+Engineering%22%5BJournal%5D)">Insertion of linear 8.4 µm diameter 16 channel carbon fiber electrode arrays for single unit recordings.</a> Journal of Neural Engineering. 12 (4), 046009(2015).</li>
<li>Ferguson, J. E., Boldt, C., Redish, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Creating+low-impedance+tetrodes+by+electroplating+with+additives%5BTitle%5D)+AND+%22Sensors+and+Actuators.+A%2C+Physical%22%5BJournal%5D)">Creating low-impedance tetrodes by electroplating with additives.</a> Sensors and Actuators. A, Physical. 156 (2), 388-393 (2009).</li>
<li>Vafaiee, M., Vossoughi, M., Mohammadpour, R., Sasanpour, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gold-plated+electrode+with+high+scratch+strength+for+electrophysiological+recordings%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Gold-plated electrode with high scratch strength for electrophysiological recordings.</a> Scientific Reports. 9 (1), 2985(2019).</li>
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</ol>

The authors declare no financial conflicts of interest.

This protocol describes each step necessary for constructing a functional CFEA for both acute and chronic use. The process described is customizable to the researcher&#39;s needs, making it an accessible and inexpensive option for monitoring single neurons over months. The protocol demonstrates the feasibility of recording both robust single-unit activity within minutes of implantation in an anesthetized animal, and across four months in an awake, behaving animal, illustrating the potential of these CFEAs to study short-term and long-term changes in neural responses.
The steps of the protocol described have been thoroughly tested and improved upon over time to yield an efficient procedure that can be completed quickly, at a low marginal cost (&#60;$100.00), with the capability of recording unambiguous single units, densely and stably over months. The construction steps can be completed in less than one day and will produce electrophysiological signals that are comparable to any leading commercial array. The CFEAs also have a much smaller footprint (16-channel bundle of fibers has a diameter of ~26 &#181;m) than similar commercial arrays, and their biocompatibility makes them suitable for long-term use13. Importantly, there are several critical steps and instructions that must be followed in order to produce a functioning CFEA with comparable performance.
Due to the fragility of the carbon fibers, they must be handled with utmost care. Handling them with sharp forceps or other tools may result in breakage of the fibers. Additionally, it is important to construct the CFEAs in a space with limited air movement so that the fibers do not blow away. When flaming the back portion of the fibers, the lighter only needs to be moved in a back-and-forth motion very briefly, for approximately 1 s. The steps following this removal of insulation are crucial for constructing an electrode with working channels. The flamed tips should be fed into the jig without any additional contact. Then, when filling the basin with dental cement, it is important that the cement is carefully applied and completely fills the channels and funnel basin, closing off the openings without filling them. The dental cement should then be completely cured with UV light before proceeding. Once this is complete, silver paint should be injected into each channel until completely filled but not spilling out. This is the most variable step in the process. Any over-filling can produce crosstalk between channels, and insufficient filling can result in a connection failure. If unable to inject silver paint using a 25 G needle, it is likely that the solution is too viscous and, in this case, a small amount of paint thinner can be added to create a more fluid solution. Once all the channels are filled, and the headstage connector is inserted, it is important to allow the array to cure for 24 h prior to securing the connector with dental cement. We found that failure to do so lowered the number of connected channels. Applying a generous amount of dental cement is also important so that the connector does not disconnect when interfacing with the signal acquisition system. If they become detached, it is possible to attempt reconnection with the repeated filling of channels with silver paint, but the user should test the impedance values of the CFEA to assess the number of connected channels. Allowing the dental cement to cure overnight also serves to prevent potential detachment.
Measuring the impedance of the electrode will provide an accurate estimation of connected channels. This can be done after submerging the ground and reference wires and the carbon fiber tips in PBS. We have observed that a high impedance (&#62;15 M&#937;) is indicative of an open, unconnected channel. Prior to injecting current and electroplating, a connected channel can have a range of impedance values that should significantly decrease with this process. The average number of connected channels (impedance &#60; 4 M&#937; after current injection) per 16-channel electrode was 12.96 &#177; 2.74 (mean &#177; SD; N = 48 electrodes). A number of electroplating times were tested, and 30 s produced superior signal isolation among the recording sites (Figure 5). While it has been well established that PEDOT-pTS12,24,25,26&#160;and PEDOT-TFB21 provide reliable options for preparing carbon fiber recording sites, we found that plating with gold, a proven and dependable method for electroplating electrodes for chronic implantation27,28, increased the ease of implantation and prevented the electrode tips from clumping together. In producing final impedance values of less than 0.2 M&#937; on average, this method proves comparable to values achieved using PEDOT-TFB21 and PEDOT-pTS26.
When implanting the microelectrode array, it is important to visually follow the insertion of the carbon fiber tips under the microscope. Successful insertion should be apparent, with no bending of the fibers. If the fibers appear to be buckling, it is unlikely that they will successfully enter the brain. In this case, the angle of the probe should be adjusted for a second attempt. This process can continue until the insertion of the probe is successful. Once the electrode is at the desired depth, we have found that waiting at least 30 min will allow the probe to settle for optimal signal acquisition (acute recordings).
The CFEAs described, in addition to their small footprint and biocompatibility, offer a robust, customizable alternative to commercial arrays due to their ease of construction and low cost. The greatest limitation to the CFEAs detailed in this protocol is their scalability. Due to the manual nature of their construction, scaling up to designs with hundreds of recording sites may not be practical. Additionally, advances in microelectrode array fabrication using nanotechnology will enable larger-scale population recordings than the methods described here. However, this protocol delivers CFEA accessibility to labs interested in benchtop fabrication of carbon fiber electrodes. We observed no loss of stability or decreased robustness in spike amplitude over the duration of the 120-day chronic experiments, as indicated by a representative single channel typical of our observations on that time scale (Figure 6A-E). Additionally, the CFEAs show the capacity for persistent single-unit activity, as four single units remained discernible 11 months after implantation in mouse (Figure 6G,H). It is also possible to obtain stable, single-unit recordings acutely (Figure 7), which offers an advantage over many other commercial electrodes for the study of single neurons over short time periods. In the future, the development of such flexible, biocompatible probes with minimal diameters will enable the study of complex processes. These tools will provide substantial utility in the advancement of neural technology, including applications in brain-machine interfaces (BMIs), which require continuous, long-term stability29.

Electrodes and electrode arrays are valuable tools for understanding how the brain processes information at the neuronal level. While electrophysiological recordings have been achievable for over two centuries1, it is still not possible to simultaneously measure the activity of entire neural circuits at the spatial and temporal resolution required to capture the spiking of individual neurons. Although non-invasive methods, such as electroencephalography2, positron emission topography3, and functional magnetic resonance imaging4 allow for whole-brain measurements, they cannot achieve the spatial and temporal resolution necessary for resolving the activity of neural circuits2,5. In contrast, imaging methods such as optical imaging using voltage-sensitive dyes or genetically encoded calcium indicators can achieve single-unit spatial resolution, but they pose issues such as low temporal resolution and poor selectivity3,4,5,6. Electrical recordings are a powerful alternative to these methods. Recording electrodes provide unparalleled temporal resolution and allow the user to make measurements with spike-time precision in any region of the brain7. Additionally, chronically implanted multielectrode arrays (MEAs) enable large-scale (tens to hundreds of cells), single-cell recordings in behaving animals over a period of days to months8,9. However, silicon probes that record at higher densities have a large footprint and are highly invasive, and chronically implanted arrays often generate an inflammation response, tissue encapsulation, and neuronal death10,11,12,13.
The limitations of existing electrodes have resulted in recent innovations that allow for stable, high-resolution, long-term recordings. Typical electrodes consist of a metallic conductor, such as tungsten or platinum-iridium, or are silicon- or polymer-based. While metal-based microwire arrays can maintain long-term, stable recordings, they have a much larger footprint, with a single wire&#39;s diameter ranging from 10-200 &#181;m14. In contrast, silicon-based electrode arrays yield recordings with high spatial resolution, but due to their relatively rigid design, they are typically unable to maintain the signal and record from the same neurons over many months15. Recent developments in silicon-based arrays have resulted in electrodes that can reliably perform chronic recordings, but these arrays cannot be used to record from deep brain regions in larger animals and are intended for linear recordings9. Advances in polymer arrays have resulted in increased flexibility and recording stability of single units and offer the potential for high-density recordings in the near future but with limited availability at present8,16,17. Carbon fibers allow for high-density recordings with off-the-shelf materials that are described here.
Carbon fiber recording microelectrodes have been used for decades, with the first carbon fiber electrodes consisting of a single carbon-fiber inserted into a glass micropipette. These microelectrodes were used for single-unit extracellular recordings, and although the signal-to-noise ratio was comparable to the best tungsten-in-glass microelectrodes, they were advantageous due to their flexibility, lower impedance values, and simplicity to manufacture18,19. Efforts to develop carbon fiber electrode arrays have recently accelerated due to the biosensing capabilities of carbon fibers. In addition to their increased biocompatibility and exceptional electrical conductivity, they feature a unique set of properties, including high-temperature resistance, low relative density, high tensile strength, low bending stiffness, high detection sensitivity, and a small cross-sectional area10,12. All of these properties have motivated the development of carbon fiber microelectrode arrays (CFEAs) that facilitate chronic, stable, high-yield recordings of single neurons. Such CFEAs can now be crafted by hand20,21&#160;(Figure 1), yielding microelectrode arrays that can hold single neurons over months. Described here is an accessible construction process for CFEAs that has been adapted in two ways for acute and chronic recordings of individual neurons in two species.

<table><tbody><tr><td>#10 scalpel blade</td><td>Fisher Scientific</td><td>14-840-15</td><td>Building tool</td></tr><tr><td>16-channel CFEA Jig</td><td>Realize Inc.</td><td></td><td>CFMA component</td></tr><tr><td>16-channel Omnetics connector</td><td>Omnetics</td><td>A79014-001</td><td>CFMA component</td></tr><tr><td>25 G needle</td><td>Fisher Scientific</td><td>14-840-84</td><td>Building tool - sharp-tipped</td></tr><tr><td>30 G needle</td><td>Fisher Scientific</td><td>14-841-03</td><td>Building tool</td></tr><tr><td>31 G stainless steel 304 hypodermic round tubing</td><td>Small Parts Inc</td><td>B000FMYN38</td><td>For guide tube</td></tr><tr><td>32-channel CFEA jig</td><td>Realize Inc.</td><td></td><td>CFMA component</td></tr><tr><td>32-channel Omnetics connector</td><td>Omnetics</td><td>A79022-001</td><td>CFMA component</td></tr><tr><td>6 in cotton tip applicators</td><td>Fisher Scientific</td><td>22-363-156</td><td>Building tool</td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A16P4</td><td>Building tool</td></tr><tr><td>AutoCad 3D printing software</td><td>Autodesk</td><td></td><td>Computer-aided design tool/ 3D modeling software</td></tr><tr><td>Autodesk Fusion 360</td><td>Autodesk</td><td></td><td>Computer-aided design tool/ 3D modeling software</td></tr><tr><td>BD disposable syringes</td><td>Fisher Scientific</td><td>14-823-30</td><td>1 mL</td></tr><tr><td>Carbon fibers</td><td>Good Fellow USA</td><td>C 005725</td><td>7 &amp;mu;m epoxy sized</td></tr><tr><td>Cassettes and cassette holder</td><td></td><td></td><td>For coating fibers</td></tr><tr><td>Clear tape</td><td>Scotch</td><td></td><td>For coating raw fibers</td></tr><tr><td>Deionized water</td><td></td><td></td><td>Electroplating component</td></tr><tr><td>Double-sided tape</td><td>Scotch</td><td></td><td>For coating raw fibers</td></tr><tr><td>Flowable Dental Composite</td><td>Pentron</td><td>Flow-It ALC</td><td>CFMA component/ UV cured dental cement</td></tr><tr><td>Gold plating solution</td><td>Sifco ASC</td><td>5355</td><td>10.0-20.0% glycerol, 1.0-5.0% ethylenediamine, 1.0-5.0% acetic acid (ethylenedinitrilo)tetra-, dipotassium salt, 5.0-10.0% butanoic acid, mercapto-monogold(1+) sodium salt, 1.0&amp;ndash;5.0% potassium metabisulfite, 55.0-82.0% water</td></tr><tr><td>Jewelry clamp</td><td>Amazon</td><td>B00GRABH9K</td><td>Building tool</td></tr><tr><td>JRClust</td><td></td><td></td><td>Ferret spike sorting software</td></tr><tr><td>Lighter</td><td>BIC</td><td>LCP62DC</td><td>Building tool</td></tr><tr><td>Micromanipulator</td><td>Scientifica</td><td>PS-7000C</td><td>For guide tube</td></tr><tr><td>Microscissors</td><td>Fisher Scientific</td><td>08-953-1B</td><td>Building tool</td></tr><tr><td>MountainSort</td><td></td><td></td><td>Mouse spike sorting software</td></tr><tr><td>NanoZ 16-channel adapter</td><td>Multi-channel systems</td><td>ADPT-nanoZ-NN-16</td><td>Electroplating component</td></tr><tr><td>NanoZ 32-channel adapter</td><td>White Matter</td><td>NZA-OMN-32 rev A</td><td>Electroplating component</td></tr><tr><td>NanoZ multi-electrode impedance tester</td><td>White Matter</td><td></td><td>Electroplating component</td></tr><tr><td>Parafilm</td><td>Fisher Stockroom</td><td>13-374-10</td><td>Semi-transparent, flexible film with adhesive properties</td></tr><tr><td>Parylene &#039;C&#039; Dimer</td><td>Specialty Coating Systems</td><td>980130-C-01LBE</td><td>For coating raw fibers</td></tr><tr><td>PEG 8000</td><td>Fisher Scientific</td><td>25322-68-3</td><td>Electroplating component</td></tr><tr><td>Phosphate-buffered saline</td><td></td><td></td><td>Electroplating component</td></tr><tr><td>Polyimide tubing</td><td>MicroLumen</td><td>BRAUNI001</td><td>For guide tube</td></tr><tr><td>Rotary tool</td><td>Dremel</td><td>300124</td><td>For guide tube</td></tr><tr><td>Scalpel handle</td><td>Fine Science Tools</td><td>10003-12</td><td>Building tool</td></tr><tr><td>Silver conductive coating</td><td>MG Chemicals</td><td>842AR Super Shield</td><td>CFMA component</td></tr><tr><td>Stereo microscope with range 6.7:1</td><td>Motic</td><td>SMZ-168</td><td>Building tool</td></tr><tr><td>Sticky notes</td><td>Post-it</td><td></td><td>Building tool</td></tr><tr><td>Tissue wipes</td><td>Kimtech Science</td><td>34155</td><td>Building tool</td></tr><tr><td>Tungsten wire</td><td>A-M Systems</td><td>797550</td><td>CFMA component</td></tr><tr><td>UV curing wand</td><td>Woodpecker</td><td></td><td>Building tool</td></tr><tr><td>Vacuum deposition chamber</td><td>Specialty Coating Systems</td><td></td><td>Labcoter 2 (PDS 2010)</td></tr></tbody></table>

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.

With the completion of this protocol, stable recordings of single-unit spiking activity will be possible. These microelectrode arrays are customizable in material, channel count, and headstage adapter based on the researcher&#39;s needs. Electroplating fibers in gold results in decreased impedances suitable for recording (Figure 4 and Figure&#160;5). If the user intends to record chronically, measurements can be made after the animal has recovered from the surgical procedure. Chronic procedures have resulted in stable, single-unit recordings for at least 120 days. A representative recording is shown in Figure 6, illustrating stable 64-channel electrophysiological activity in the retrosplenial cortex of a freely behaving, adult male mouse. If an acute preparation is intended, recordings can begin shortly after implantation (~30 min). This will allow time for the electrode to settle in the brain. Figure 7 provides a representative example of an acute 16-channel CFEA recording acquired from the primary visual cortex of an adult female ferret. Spike sorting in mouse and ferret was performed with spike sorting software (see Table of Materials).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62760/62760fig01.jpg" /> 
Figure 1: Anatomy of 16- and 32-channel carbon fiber microelectrode arrays (CFEAs). (A) Schematics of 32-channel (top) and 16-channel (bottom) CFEA from three different views. The 16-channel CFEA features an extended design for handling purposes. The 32-channel design features a flat face that allows for two jigs to be combined for a 64-channel CFEA. Both the diagrams have identifying structures labeled with dimensions. The connector end indicates the location of the connector insertion, and GND/REF channels indicate where the grounding wire is inserted. The funnel basin refers to the location that the fibers pass through to be overlaid with UV light-cured dental cement, and the funnel tip signifies the site from where the fibers exit the jig. The funnel tip is divided into quadrants to minimize fibers clinging together and creating damage. The fibers are later pulled into a single bundle with the use of the dental cement. Jigs are 3D printed using SLA resin printers. Diagrams are enlarged to show details. (B) Constructed CFEA. Diagram has identifying structures labeled. The blue bundle tip represents the segment of the carbon fibers that acquire recording measurements. The gray within the funnel basin and surrounding the connector is indicative of UV light-cured dental cement that holds carbon fibers in place in the funnel basin and secures the connector to the jig. The purple wire represents the grounding wire. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62760/62760fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62760/62760fig02.jpg" /> 
Figure 2: Loading of raw carbon fibers into cassettes for parylene C coating. (A) Carbon fibers are loaded onto cartridges overlaid with two strips of double-sided tape (blue). Each cassette is loaded with ~25 fibers. (B) Cassettes are loaded into a laser-cut holder (gray) in preparation for parylene C coating. Each holds ten cassettes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62760/62760fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62760/62760fig03.jpg" /> 
Figure 3: Carbon fiber microelectrode array (CFEA) bundle construction schematic. (A) 16 individual, coated carbon fibers (black) are threaded through the 32-channel 3D-printed jig (gray). (B) Carbon fiber tips are cut with micro-scissors, leaving excess fiber equal to the height of the jig base, extending out of the jig base. (C) A standard plastic spark wheel lighter is quickly passed over the excess fiber to remove parylene C insulation. The top right schematic shows the removal of parylene from 9 of the 12 fibers. (D) Fibers are reinserted into the jig until the fiber end is flush with the base. The top-right schematic shows the reinsertion of 9 fibers with uninsulated (gray) fiber tips housed inside the jig base. The jig is then flipped over and steps A-D are repeated to thread the opposite 16 channels. (E) The jig is filled with dental cement to secure the fibers. Silver print is injected into each well of the jig base. (F) The male connector is inserted into the jig base. (G) CFEA and scalpel are frozen in a -20 &#176;C freezer. The array tip is cut to the desired length, leaving 32 even fibers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62760/62760fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62760/62760fig04.jpg" /> 
Figure 4: Tip treatment and electroplating. (A) Electrode tips are first placed into 0.1 M PBS, where current is passed through each electrode. The tips are then rinsed and transferred to a gold plating solution, where they are electroplated with the current. (B) SEM images of prepared carbon fiber show gold plating solution concentrated at the tip. Scale bar represents 4 &#181;m. (C) Impedance values from 168 channels after initial cutting (purple; 3.11 M&#937; &#177; 0.42 M&#937;, median &#177; SE, n = 168 fibers), positive current injection (pink; 1.23 M&#937; &#177; 0.36 M&#937;, median &#177; SE, n = 168 fibers), and electroplating (orange; 0.19 M&#937; &#177; 0.15 M&#937;, median &#177; SE, n = 168 fibers) show decreased impedance values after each processing step. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62760/62760fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62760/62760fig05.jpg" /> 
Figure 5: Moderate gold electroplating durations produce small, rounded deposits on carbon fiber bundle tips. The carbon fiber tips pictured are all from different microelectrode arrays, reflecting different durations of injected current for impedance reduction or gold-plating. Images additionally depict the parylene C coating, which insulates the carbon fibers and prevents any acquisition of signal from a location other than the tips of the fibers. (A) Scanning electron microscopy image of carbon fiber tips after freezing and making a single cut with a razor blade. Scale bars represent 10 &#181;m. (B) Same as A but then followed with injection of positive current for 10 s. (C) Same as B but then electroplated with gold for 5 s. (D) Same as B but then electroplated with gold for 15 s. (E) Same as B but then electroplated with gold for 30 s. (F) Same as B but then electroplated with gold for 120 s. We found that electroplating for 30 s at a current of -0.05 &#181;A was optimal for electrophysiological recordings. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62760/62760fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62760/62760fig06.jpg" /> 
Figure 6: Chronic extracellular recordings in freely behaving mouse retrosplenial cortex with carbon fiber microelectrode arrays show persistent, stable neural activity. (A) Eleven&#160;bandpassed voltage traces were recorded simultaneously. Subsequent traces recorded from the first channel (top row) are plotted in B to show durability across time. The remaining ten rows demonstrate the consistency of recording quality and show robust activity across the array. Scale bar to the left of each trace represents a 200 &#181;V potential. (B) Bandpassed data from the same fiber as in the top trace in A, expanded to show robust activity across a 120-day continuous recording. (C) Clustering reveals robust single unit detection over months. Traces represent the average waveform of a continuously observable representative single unit across 120 days, extracted from the fiber plotted in B at each timepoint. (D) Mean, non-normalized spike waveforms from C stacked to demonstrate consistency over time. (E) Carbon fiber recordings demonstrate a stable noise floor over many months. Standard deviation of the noise floor (trace minus spiking activity) in B shows no progressive change in noise. Bars represent mean contamination. Error bars represent standard deviation. (F) Scale drawing of a mouse with a chronically implanted CFEA and headstage.&#160;(G) Raw voltage trace (top) 11 months after implantation shows robust LFP. Bandpassed voltage trace (bottom) shows steady neural activity. (H) Mean spike waveform of the neuron recorded on the fiber from C, underlaid by the first 1,000 incidences of spiking activity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62760/62760fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62760/62760fig07.jpg" /> 
Figure 7: Carbon fiber microelectrode array (CFEA) recordings from the ferret primary visual cortex. (A) Waveforms of spike sorted single units recorded from a 16-channel CFEA. Action potentials from single neurons were often evident on multiple channels at slightly differing amplitudes. (B) Direction tuning curves from selected neurons. Colors correspond to recorded units in A. Arrows indicate the direction of stimulus movement. Scale bars indicate the response rate. Error bars indicate the mean response with standard error. The horizontal dashed line represents the same cell&#39;s spontaneous firing rate during exposure to a blank screen. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62760/62760fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings at JoVE.com

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.

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. ...

construction-implementation-carbon-fiber-microelectrode-arrays-for

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.7K Views.  Washington University in St. Louis. De hier beschreven bench top-constructie is een van de weinige momenteel beschikbare elektrodetechnologieën die opnames van dichte clusters van neuronen mogelijk maken en veel van die neuronen in de loop van de tijd volgen. Koolstofvezel micro-elektrode arrays zijn goedkoop om te bouwen. Ze zijn klein, biocompatibel en bewegen met de hersenen mee, wat gezonde en stabiele opnames mogelijk maakt.Koolstofvezels zijn extreem dun en in eerste instantie zijn ze erg moeilijk te zien. Na een ...

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

Construction of Microdrive Arrays for Chronic Neural Recordings in Awake Behaving Mice

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field potentials (LFPs) from populations of neurons and action potentials from individual cells, as the animal engages in experimentally relevant tasks. Chronically implanted microdrives allow for brain recordings to last over periods of several weeks. Miniaturized drives and lightweight components allow for these long-term recordings to occur in small mammals, such as mice. By using tetrodes, which consist of tightly braided bundles of four electrodes in which each wire has a diameter of 12.5 &mu;m, it is possible to isolate physiologically active neurons in superficial brain regions such as the cerebral cortex, dorsal hippocampus, and subiculum, as well as deeper regions such as the striatum and the amygdala. Moreover, this technique insures stable, high-fidelity neural recordings as the animal is challenged with a variety of behavioral tasks. This manuscript describes several techniques that have been optimized to record from the mouse brain. First, we show how to fabricate tetrodes, load them into driveable tubes, and gold-plate their tips in order to reduce their impedance from M&Omega; to K&Omega; range. Second, we show how to construct a custom microdrive assembly for carrying and moving the tetrodes vertically, with the use of inexpensive materials. Third, we show the steps for assembling a commercially available microdrive (Neuralynx VersaDrive) that is designed to carry independently movable tetrodes. Finally, we present representative results of local field potentials and single-unit signals obtained in the dorsal subiculum of mice. These techniques can be easily modified to accommodate different types of electrode arrays and recording schemes in the mouse brain.

The design and assembly of microdrives for in vivo electrophysiological recordings of brain signals from the mouse is described. By attaching microelectrode bundles to sturdy driveable carriers, these techniques allow for long-term and stable neural recordings. The lightweight design allows for unrestricted behavioral performance by the animal following drive implantation.

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field ...

Behavior

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

Biology

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential amplitude of action potential signatures over the channels of a tetrode allows for the isolation of single-unit activity from multi-unit signals.  The ability to precisely control the stereotaxic location and depth of the tetrode is critical for studying coordinated neural activity across brain regions.  In combination with a micro-drive array, it is possible to achieve precise placement and stable control of many tetrodes over the course of days to weeks.  In this protocol, we demonstrate how to fabricate and condition tetrodes using basic tools and materials, install the tetrodes into a multi-drive tetrode array for chronic in-vivo recording in the rat, make ground wire connections to the micro-drive array, and attach a protective cone onto the micro-drive array in order to protect the tetrodes from physical contact with the environment.



Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

In this protocol we demonstrate how to fabricate and condition tetrodes for use with a micro-drive array, which was designed for chronic electrophysiological recordings in rats. In addition, we illustrate the final stages of micro-drive array construction, which includes installing ground wires and a protective cone.

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential ...

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 ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

Bioengineering

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural underpinnings of brain disease and injury. As electrodes are miniaturized to the scale of individual cells, a corresponding rise in the interface impedance limits the quality of recorded signals. Additionally, conventional electrode materials are stiff, resulting in a significant mechanical mismatch between the electrode and the surrounding brain tissue, which elicits an inflammatory response that eventually leads to a degradation of the device performance. To address these challenges, we have developed a process to fabricate flexible microelectrodes based on Ti3C2 MXene, a recently discovered nanomaterial that possesses remarkably high volumetric capacitance, electrical conductivity, surface functionality, and processability in aqueous dispersions. Flexible arrays of Ti3C2 MXene microelectrodes have remarkably low impedance due to the high conductivity and high specific surface area of the Ti3C2 MXene films, and they have proven to be exquisitely sensitive for recording neuronal activity. In this protocol, we describe a novel method for micropatterning Ti3C2 MXene into microelectrode arrays on flexible polymeric substrates and outline their use for in vivo micro-electrocorticography recording. This method can easily be extended to create MXene electrode arrays of arbitrary size or geometry for a range of other applications in bioelectronics and it can also be adapted for use with other conductive inks besides Ti3C2 MXene. This protocol enables simple and scalable fabrication of microelectrodes from solution-based conductive inks, and specifically allows harnessing the unique properties of hydrophilic Ti3C2 MXene to overcome many of the barriers that have long hindered the widespread adoption of carbon-based nanomaterials for high-fidelity neural microelectrodes.

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural ...

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 ...

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.

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 ...

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.

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

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

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