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.

<ol>
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Neuroscience. 20 (6), 330-345 (2019).</li><li>Chung, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-density,+long-lasting,+and+multi-region+electrophysiological+recordings+using+polymer+electrode+arrays.">High-density, long-lasting, and multi-region electrophysiological recordings using polymer electrode arrays.</a> Neuron. 101 (1), 21-31 (2019).</li><li>Jun, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+integrated+silicon+probes+for+high-density+recording+of+neural+activity.">Fully integrated silicon probes for high-density recording of neural activity.</a> Nature. 551 (7679), 232-236 (2017).</li><li>Kozai, T. D., Jaquins-Gerstl, A. S., Vazquez, A. L., Michael, A. C., Cui, X. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasmall+implantable+composite+microelectrodes+with+bioactive+surfaces+for+chronic+neural+interfaces.">Ultrasmall implantable composite microelectrodes with bioactive surfaces for chronic neural interfaces.</a> Nature Materials. 11 (12), 1065-1073 (2012).</li><li>Chen, R., Canales, A., Anikeeva, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+recording+and+modulation+technologies.">Neural recording and modulation technologies.</a> Nature Reviews Materials. 2 (2), 16093 (2017).</li><li>Szostak, K. M., Grand, L., Constandinou, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+interfaces+for+intracortical+recording:+requirements,+fabrication+methods,+and+characteristics.">Neural interfaces for intracortical recording: requirements, fabrication methods, and characteristics.</a> Frontiers in Neuroscience. 11, 665 (2017).</li><li>Subbaroyan, J., Martin, D. C., Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+finite-element+model+of+the+mechanical+effects+of+implantable+microelectrodes+in+the+cerebral+cortex.">A finite-element model of the mechanical effects of implantable microelectrodes in the cerebral cortex.</a> Journal of Neural Engineering. 2 (4), 103-113 (2005).</li><li>Park, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+optogenetics+with+multifunctional+flexible+polymer+fibers.">One-step optogenetics with multifunctional flexible polymer fibers.</a> Nature Neuroscience. 20 (4), 612-619 (2017).</li><li>Guo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+composite+with+carbon+nanofibers+aligned+during+thermal+drawing+as+a+microelectrode+for+chronic+neural+interfaces.">Polymer composite with carbon nanofibers aligned during thermal drawing as a microelectrode for chronic neural interfaces.</a> ACS Nano. 11 (7), 6574-6585 (2017).</li><li>Armstrong-James, M., Millar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+fibre+microelectrodes.">Carbon fibre microelectrodes.</a> Journal of Neuroscience Methods. 1 (3), 279-287 (1979).</li><li>Garris, P. A., Ciolkowski, E. L., Pastore, P., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efflux+of+dopamine+from+the+synaptic+cleft+in+the+nucleus+accumbens+of+the+rat+brain.">Efflux of dopamine from the synaptic cleft in the nucleus accumbens of the rat brain.</a> Journal of Neuroscience: The Official Journal of the Society of Neuroscience. 14 (10), 6084-6093 (1994).</li><li>Guitchounts, G., Markowitz, J. E., Liberti, W. A., Gardner, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+carbon-fiber+electrode+array+for+long-term+neural+recording.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=64-channel+carbon+fiber+electrode+arrays+for+chronic+electrophysiology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+circuit+dynamics+are+homeostatically+tuned+to+criticality+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+cross-orientation+suppression+and+size+tuning+and+the+role+of+experience.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+in+vivo+stability+assessment+of+carbon+fiber+microelectrode+arrays.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-small+carbon+fiber+electrode+recording+site+optimization+and+improved+in+vivo+chronic+recording+yield.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+linear+8.4+&#181;m+diameter+16+channel+carbon+fiber+electrode+arrays+for+single+unit+recordings.">Insertion of linear 8.4 &#181;m 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+low-impedance+tetrodes+by+electroplating+with+additives.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold-plated+electrode+with+high+scratch+strength+for+electrophysiological+recordings.">Gold-plated electrode with high scratch strength for electrophysiological recordings.</a> Scientific Reports. 9 (1), 2985 (2019).</li><li>Lebedev, M. A., Nicolelis, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-machine+interfaces:+From+basic+science+to+neuroprostheses+and+neurorehabilitation.">Brain-machine interfaces: From basic science to neuroprostheses and neurorehabilitation.</a> Physiological Reviews. 97 (2), 767-837 (2017).</li></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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#956;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&#8211;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 &#39;C&#39; 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/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/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/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/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/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/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/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. ...

The bench top construction described here is one of the few currently available electrode technologies that allow recordings from dense clusters of neurons and follow many of those neurons over time. Carbon fiber microelectrode arrays are inexpensive to build. They're small, biocompatible, and move with the brain, which allows healthy and stable recordings.Carbon fibers are extremely thin and initially, they're very difficult to see. However, after a few days of working with them, they become easier to manipulate. To begin with, lay a piece of double-sided tape on two long sides of the cassette, aligning the edge of the tape with the inner edge of the cassette.To prepare a carbon fiber manipulation tool, wrap a small piece of adhesive film around a 30 gauge needle to form a sharp but flexible point with the film. Next, separate individual fibers from the baked bundle and lay them parallel with the short side of the cassette keeping a two to three millimeter distance. After ensuring the fitting of 20 to 30 fibers on each cassette, seal the fibers in place by laying clear tape over the double stick tape, and place the filled cassettes in the cassette holder.To assemble the carbon fiber microelectrode array, start feeding each grounding wire through the appropriate channel on the connector end of the jig and continue until two ends are equal in length. Then twist them together to secure them on the jig. Apply UV-cured dental cement to secure the wire while ensuring not to get any dental cement within the open channel from where the wire is fed through.Use the UV curing wand to cure the dental cement for 20 seconds and align the jig and vice such that the connector end, the basin, and the funnel tip are visible. Next, orient the jig so the funnel points away from the user and the connector ends facing the user. Then slide the 25 gauge needle tip against the cassette at the potential fiber removal site and cut a single carbon fiber out of the cassette.Hold the freshly cut free end of the fiber using a carbon fiber tool and using the needle, cut the other end of the fiber away from the cassette. Then again use the carbon fiber tool to pick up the carbon fiber such that one end is about one centimeter in length from the tool. Use the fiber-attached carbon fiber tool to feed the shorter end of the fiber through the funnel piece from the middle basin of the jig.Continue to feed the fiber through the jig funnel until most of the length of the fiber is through. Feed the back portion of the fiber through an available channel using the carbon fiber tool. Continue to feed through the back until about five millimeters of fiber is sticking out or cut to size if necessary.To remove the insulation at the connector end, use a standard spark wheel lighter to pass the flame over the exposed fibers, then feed the flamed fiber through the jig so that the flamed fiber portion is now within the channel while ensuring that no fibers are sticking out of the back of the jig. Apply UV-cured dental cement to the basin fibers of the jig to fill the entire basin, covering the openings of both channels and funnel. After removing the jig from the vice and orienting the jig so that the funnel points down, secure the jig with the connector end pointing up.Fill the needle with silver print and carefully insert the needle into one channel until stopped by the dental cement. To fill the channel with paint, slowly depress the syringe while removing the needle from the channel, then dip a cotton tip applicator in the paint thinner and clean the base of the jig surface of any paint. Now insert the head stage connector in the proper orientation by aligning the pins with the channels.Once the head stage connector sits straight, upright, and is as flushed to the jig as possible, secure the head stage connector to the jig by applying dental cement along the edge where the head stage connector meets the jig, then UV cure for 20 seconds. In order to cut the electrode tip to the desired length, lower the electrode into a beaker of deionized or distilled water until the funnel tip is fully submerged and held normal to the surface. Now bring the individual carbon fibers together by removing the electrode from the water.Lay the electrode so that the fibers lay flush on the guide surface and cut the fibers to the desired length with a scalpel in a rolling motion, then attach the electrode to the multielectrode impedance tester using the appropriate adapter and test the impedance. Now lower the electrode tip about two millimeters into a microcentrifuge tube of 0.1 molar PBS, and insert grounding wire into the microcentrifuge tube. To reduce the impedance of the electrode tip, inject a positive current with the chosen amplitude and duration, and then rinse the fibers in deionized or distilled water to clean.Next, to perform electroplating in the gold-plating solution, lower the electrode bundle tip about two millimeters into the microcentrifuge tube in the plating mixture and insert the grounding wire into the microcentrifuge tube. Then set appropriate parameters for electroplating. Rinse the fibers thoroughly with deionized or distilled water after electroplating.The representative SEM image of prepared carbon fiber showed a gold-plating solution concentrated at the tip. The fiber electroplating in gold resulted in decreased impedances suitable for the recording. Impedance values from 300 channels after initial cutting, positive current injection, and electroplating showed decreased impedance values after each processing step.Moderate gold electroplating durations produced the small and rounded deposits on the carbon fiber bundle tips. In addition, stable 64-channel electrophysiological activity in the retrosplenial cortex of a freely behaving adult male mouse was analyzed. The results demonstrated the consistency of recording quality, robust single unit detection, consistency over time as shown by non-normalized spike waveforms, and stable noise floor.Also, raw voltage traced 11 months after implantation showed robust local field potential. A representative example of an acute 16-channel carbon fiber microelectrode array recording acquired from the primary visual cortex of an adult female ferret is also shown here. Carbon fiber microelectrode arrays allow the short-term or chronic recording of nearby neurons in the brain over long periods of time, which is essential for understanding neural circuits.

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