Name: Author Spotlight: Deciphering Memory and Learning Through Neural Implants for Multi&amp;#45;Region Brain Studies
Uploaded: 2024-04-26T13:30:00
Description: Read this Scientific Article Protocol about The TD Drive: A Parametric, Open-Source Implant for Multi-Area Electrophysiological Recordings in Behaving and Sleeping Rats at JoVE.com

The authors would like to thank Angela Gomez Fonseca for the inspiration to develop the drive and all the students who ran pilot experiments with the animals, Milan Bogers, Floor van Ravenswoud, and Eva Severijnen. This work was supported by the Dutch Research Council (NWO; Crossover Program 17619 &#34;INTENSE&#34;).

The present study was approved by the Dutch Central Commissie Dierproeven (CCD) and conducted according to the Experiments on Animals Act (protocol codes: 2020-0020-006 &#38; 2020-0020-010). Male Lister Hooded rats of 9-12 weeks on arrival were used. The reagents and the equipment used in the protocol are listed in the Table of Materials. See Supplementary Figure 1 and Supplementary Figure 2 for the steps of the drive-building process.
1...

Open&amp;#45;Source Implant for Multi&amp;#45;Region Electrophysiological Monitoring in Rats

<ol>
	<li>Deffieux, T., Demen&#233;, C., Tanter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Ultrasound+Imaging:+A+New+Imaging+Modality+for+Neuroscience.">Functional Ultrasound Imaging: A New Imaging Modality for Neuroscience.</a> Neuroscience. 474, 110-121 (2021).</li><li>Finn, E. S., Poldrack, R. A., Shine, J. 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D., Contero, M., Company, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parametric+CAD+modeling:+An+analysis+of+strategies+for+design+reusability.">Parametric CAD modeling: An analysis of strategies for design reusability.</a> Comput Aided Des. 74, 18-31 (2016).</li><li>Kloosterman, F., 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=Micro-drive+array+for+chronic+in+vivo+recording:+Drive+fabrication.">Micro-drive array for chronic in vivo recording: Drive fabrication.</a> J Vis Exp. (26), e1094 (2009).</li><li>Voigts, J., Siegle, J. H., Pritchett, D. L., Moore, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+flexDrive:+An+ultra-light+implant+for+optical+control+and+highly+parallel+chronic+recording+of+neuronal+ensembles+in+freely+moving+mice.">The flexDrive: An ultra-light implant for optical control and highly parallel chronic recording of neuronal ensembles in freely moving mice.</a> Front Syst Neurosci. 7, (2013).</li><li>Voigts, J., Newman, J. P., Wilson, M. A., Harnett, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+easy-to-assemble,+robust,+and+lightweight+drive+implant+for+chronic+tetrode+recordings+in+freely+moving+animals.">An easy-to-assemble, robust, and lightweight drive implant for chronic tetrode recordings in freely moving animals.</a> J Neural Eng. 17 (2), 026044 (2020).</li><li>Sheng, T., 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=A+novel+3D-printed+multi-driven+system+for+large-scale+neurophysiological+recordings+in+multiple+brain+regions.">A novel 3D-printed multi-driven system for large-scale neurophysiological recordings in multiple brain regions.</a> J Neurosci Methods. 361, 109286 (2021).</li><li>V&#246;r&#246;slakos, M., Petersen, P. C., V&#246;r&#246;slakos, B., Buzs&#225;ki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+microdrive+and+head+cap+system+for+silicon+probe+recovery+in+freely+moving+rodent.">Metal microdrive and head cap system for silicon probe recovery in freely moving rodent.</a> eLife. 10, e65859 (2021).</li><li>Mishra, A., Marzban, N., Cohen, M. X., Englitz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+neural+microstates+in+the+VTA-striatal-prefrontal+loop+during+novelty+exploration+in+the+rat.">Dynamics of neural microstates in the VTA-striatal-prefrontal loop during novelty exploration in the rat.</a> bioRxiv. , (2020).</li><li>Allen, L. 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=RatHat:+A+self-targeting+printable+brain+implant+system.">RatHat: A self-targeting printable brain implant system.</a> eNeuro. 7 (2), (2020).</li><li>. Soldering Safety Available from: <a target="_blank" href="https://safety.eng.cam.ac.uk/safe-working/copy_of_soldering-safety">https://safety.eng.cam.ac.uk/safe-working/copy_of_soldering-safety</a> (2018)</li><li>. Harvard Soldering Safety Guidelines Available from: <a target="_blank" href="https://www.ehs.harvard.edu/sites/default/files/soldering_safety_guidelines.pdf">https://www.ehs.harvard.edu/sites/default/files/soldering_safety_guidelines.pdf</a> (2019)</li><li>Samanta, A., 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=CBD+lengthens+sleep+but+shortens+ripples+and+leads+to+intact+simple+but+worse+cumulative+memory.">CBD lengthens sleep but shortens ripples and leads to intact simple but worse cumulative memory.</a> iScience. 26 (11), 108327 (2023).</li><li>Machado, F., Malpica, N., Borromeo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parametric+CAD+modeling+for+open+source+scientific+hardware:+Comparing+OpenSCAD+and+FreeCAD+Python+scripts.">Parametric CAD modeling for open source scientific hardware: Comparing OpenSCAD and FreeCAD Python scripts.</a> PLOS One. 14 (12), e0225795 (2019).</li><li>Schwarz, C., 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=The+head-fixed+behaving+rat:+Procedures+and+pitfalls.">The head-fixed behaving rat: Procedures and pitfalls.</a> Somatosens Mot Res. 27 (4), 131-148 (2010).</li><li>Gardiner, T. 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Presented in this article is an adaptable implant for bilateral, symmetric multi-area wire electrode recordings for freely-moving rats.
The ability to easily adjust the implant by changing predefined parameters was one of the motivations for the creation of the TD Drive. While aiming to maximize the flexibility for changing parameters, inherent constraints in the relations between them necessarily impose limits to this adaptability. No limits are set by default for the anteroposterior paramete...

The multi-area nature of brain interactions during wake and sleep makes it difficult to exhaustively study the ongoing physiological processes. While approaches such as functional MRI (fMRI) and functional ultrasound (fUS) allow sampling of brain activity from whole brains1,2, they exploit neurovascular coupling to infer brain activity from hemodynamic activity, limiting their temporal resolution2. In addition, fMRI requires the placement of the research subject in an MRI scanner, prohibiting experiments with freely moving animals. Optical imaging of calcium dynamics with single or multiphoton imaging enables cell type-specific recordings of hundreds of neurons simultaneously3. However, head-mounted microscopes such as the Miniscope3, which do allow freely moving behavior, are usually limited to imaging superficial cortical areas in intact brains4. While the diameter of their field of view on the cortex can be in the order of 1 mm, the space requirements of these head-mounted microscopes can make it difficult to target several, especially adjacent, areas. Therefore, to capture multi-area brain dynamics in wake and sleep accurately, extracellular electrophysiology, recorded with electrodes implanted in the brain areas of interest, is one of the methods of choice due to its high temporal resolution and spatial precision5. In addition, it allows the characterization of sleep dynamics in animals compatible with analyses obtained from human EEG, increasing the translational value of this method6.
Classically, studies recording brain activity with extracellular electrodes have employed individual wire electrodes or electrode bundles, such as tetrodes7. State-of-the-art probes such as the Neuropixels probe8 allow targeting several areas simultaneously, given that they are aligned on an axis that allows implanting the probe along that axis without impairing the animal. However, accurate simultaneous recordings of multiple, spatially separated areas still remain challenging, with existing methods being either costly or time-intensive.
In recent years, additive manufacturing methods such as stereolithography have become broadly available. This allowed researchers to develop novel electrode implants that were adaptable to their experimental requirements9, for example, simplified repeatable targeting of multiple brain areas. Frequently, these implant designs are also shared with the academic community as open-source hardware, allowing other researchers to adapt them to their own purposes. The degree of adaptability of specific implants varies both as a result of how the implant is designed and how it is shared. Parametric modeling10 is a popular approach in computer-aided design, in which different components of the design are linked by interdependent parameters and a defined design history. Implementing a parametric approach for designing implants increases their reusability and adaptability10, as changing individual parameters automatically updates the complete designs without the need for complex re-modeling of the design. A consequential necessity is that the design itself is shared in an editable format that preserves the parametric relationships and design history. File formats that only represent geometric primitives, such as STL or STEP, make subsequent parametric modifications of published models unfeasible.
While tetrode hyperdrives11,12,13 enable recordings from dozens of tetrodes, their assembly and implantation are time-intensive, and their quality is largely dependent on the skill and experience of the individual researcher. In addition, they usually combine the guide tubes that direct the recording electrodes to their target location in one or two larger bundles, therefore limiting the number and spread of areas that can be targeted efficiently.
Other implants14,15 expose the complete skull and allow for the free placement of multiple individual microdrives that carry the recording electrodes. While the placement of independent microdrives16 during surgery time maximizes flexibility, it increases surgery time and can make it difficult to target multiple adjacent areas due to the space requirements of the individual microdrives. In addition, while the implants are open source, they are only published as STL files, making modification difficult.
An example of a drive with a more inherent parametric philosophy is the RatHat17. By providing a surgical stencil that covers the whole dorsal surface of the skull, it allows precise targeting of multiple brain targets without the use of a stereotactic frame during surgery. Multiple implant variations for cannulas, optrodes, or tetrodes are available. However, while the drive is free to use for academic purposes, it is not published open-source, creating a hurdle for researchers to evaluate and use the implant.
Presented in this article is the TD Drive (see Figure 1), a novel 3D-printable implant for extracellular electrode recordings in rats. The TD Drive aims to overcome some of the drawbacks of existing solutions: it allows to target multiple brain areas, mirrored across both hemispheres, with independent wire electrodes simultaneously. Due to its simple design, it can be assembled in a few hours at a relatively low cost by less experienced researchers. The TD Drive is published open-source, in easily modifiable file formats to allow researchers to adjust it to their specific needs. Incorporating a parametric 3D modeling approach from the beginning of the TD Drive&#39;s design process allows the parameters necessary to be changed to be abstracted: to change target locations, researchers can simply edit the parameters representing their dorsoventral and anteroposterior coordinates, without the need for re-designing the drive themselves. The files to modify and manufacture the TD Drive can be found at <a href="https://github.com/3Dneuro/TD_Drive">https://github.com/3Dneuro/TD_Drive</a>.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66457/66457fig01.jpg" /> 
Figure 1: Overview of the TD Drive. (A) Rendering of a TD Drive with a protective cap. (B) Rendering with inner parts shown. The TD Drive features (a)&#160;multiple, parametrically adjustable recording locations for fixed and moveable electrode wires, an EIB with (b) a high-density Omnetics connector compatible with common tethered and wireless data acquisition systems, and (c) an intuitive channel mapping optimized for recordings with Intan/Open Ephys systems (see Supplementary Figure 1) and (d) a cap to protect the implant during tethered recordings and when no headstage is connected. (C) A guide stencil on the bottom of the TD Drive facilitates the placement of guide cannulas and serves as a redundant verification of implant locations during surgery. <a href="https://www.jove.com/files/ftp_upload/66457/66457fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The implant design was piloted in n = 4, validated in n = 8, and confirmed in n= 8 Lister Hooded rats that performed different tasks. The first 4 animals were used to develop the drive and adjust parameters. Then, a full pilot was run with 8 animals (shown in results). A second cohort of 8 animals was run and included in the implant survival analysis. The implant was compatible with tethered sleep recordings and open field recordings (Object Exploration) as well as wireless recording in a large maze (HexMaze 9 m x 5 m) using two different commercial recording systems and headstages. The two cohorts of 8 were recorded with two different acquisition systems - tethered for longer sleep recordings and wireless for large maze exploration recordings. We can conclude that this simple wire drive allows for long-running experiments with larger cohorts by less experienced researchers to enable sleep stage analysis as well as oscillation analysis in multiple brain areas. This is in contrast to most electrophysiology implants to date, which, due to difficulty and time intensity, allow for smaller animal cohorts and usually need very experienced experimenters. However, with this drive, no individual neuron activity can be recorded; thus, the use is limited to investigations of local field potential (LFP) and summation activity.

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	<tbody>
		<tr>
			<td>0.5 mm drill bit&#160;</td>
			<td>McMaster</td>
			<td>2951A38</td>
			<td></td>
		</tr>
		<tr>
			<td>1.27 mm pitch interconnected SIP/DIP socket (Mill-Max)</td>
			<td>Mouser Electronic</td>
			<td>575-003101</td>
			<td>For essembling and connection of EEG &#38; GND screws</td>
		</tr>
		<tr>
			<td>5 minute epoxy&#160;</td>
			<td>Bison</td>
			<td>Commercially available</td>
			<td>regular off-the-shelf epoxy</td>
		</tr>
		<tr>
			<td>cyanoacrylate glue</td>
			<td>Loctite</td>
			<td></td>
			<td>Super Glue-3&#160;</td>
		</tr>
		<tr>
			<td>EEG wire</td>
			<td>Science Products GmbH</td>
			<td>7SS-2T</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode wire</td>
			<td>Science Products GmbH</td>
			<td>NC7620F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>LC</td>
			<td></td>
			<td>For standard pre-operative sterilization procedure of drive</td>
		</tr>
		<tr>
			<td>Fine forceps (5)</td>
			<td>FST</td>
			<td>91150-20</td>
			<td>For wire bundle preperation and handling</td>
		</tr>
		<tr>
			<td>Form 3B</td>
			<td>Formlabs</td>
			<td></td>
			<td>3D printer used to 3D print the self-printed parts of the TD drive</td>
		</tr>
		<tr>
			<td>Gold pins (small)</td>
			<td>Neuralynx, Inc.</td>
			<td>9885</td>
			<td>Attachment of electorde wires to EIB board</td>
		</tr>
		<tr>
			<td>Ground wire</td>
			<td>Science Products GmbH</td>
			<td>SS-3T/A</td>
			<td></td>
		</tr>
		<tr>
			<td>High-density connector&#160;</td>
			<td>LabMaker GmbH/Omnetics</td>
			<td>A79026-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Lister Hodded rats</td>
			<td>Charles River Laboratories</td>
			<td>Crl:LIS</td>
			<td>we used male rats, 9-12 weeks of age at arrival</td>
		</tr>
		<tr>
			<td>M1 brass insert</td>
			<td>AliExpress</td>
			<td>Commercially available</td>
			<td>https://aliexpress.com/item/33047616164.html</td>
		</tr>
		<tr>
			<td>M1 tap</td>
			<td>McMaster</td>
			<td>2504A33</td>
			<td></td>
		</tr>
		<tr>
			<td>M1x16 screw</td>
			<td>Bossard</td>
			<td>1096613</td>
			<td></td>
		</tr>
		<tr>
			<td>M1x3 stainless steel screws&#160;</td>
			<td>Screws and More</td>
			<td>84213_14985</td>
			<td></td>
		</tr>
		<tr>
			<td>M2.5x5 polyimide screws</td>
			<td>Screws and more</td>
			<td>7985PA25S_50</td>
			<td></td>
		</tr>
		<tr>
			<td>mineral oil</td>
			<td>McMaster</td>
			<td>1244K14</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>Etos</td>
			<td>Commercially available</td>
			<td>For color coding EEG and GND wires</td>
		</tr>
		<tr>
			<td>painter&#39;s tape</td>
			<td>Gamma</td>
			<td>Commercially available</td>
			<td>For wire bundle preperation</td>
		</tr>
		<tr>
			<td>Pin vise</td>
			<td>McMaster</td>
			<td>8455A16</td>
			<td></td>
		</tr>
		<tr>
			<td>plotting paper</td>
			<td>Canson</td>
			<td>Commercially available</td>
			<td>For wire bundle preperation</td>
		</tr>
		<tr>
			<td>polyimide tubes</td>
			<td>Amazon / Small Parts</td>
			<td>TWPT-0159-30-50</td>
			<td>AWG, 0.0159&#34; ID, 0.0219&#34; OD, 0.0030&#34; Wall, 30&#34; Length</td>
		</tr>
		<tr>
			<td>RHD 32-channel headstage with accelerometer</td>
			<td>Intan Technologies, LLC</td>
			<td>C3324</td>
			<td>For tethered recordings in the sleepbox</td>
		</tr>
		<tr>
			<td>RHD 3-ft (0.9 m) standard SPI cables</td>
			<td>Intan Technologies, LLC</td>
			<td>C3203</td>
			<td>From commutator to headstage</td>
		</tr>
		<tr>
			<td>RHD 6-ft (1.8 m) standard SPI cables</td>
			<td>Intan Technologies, LLC</td>
			<td>C3206</td>
			<td>From OpenEphys box to commutator</td>
		</tr>
		<tr>
			<td>Slip Ring with Flange</td>
			<td>Adafruit</td>
			<td>1196</td>
			<td>Commutator: 22 mm diameter, 12 wires</td>
		</tr>
		<tr>
			<td>Solder flux&#160;</td>
			<td>Griffon S-39 50 ml</td>
			<td>Commercially available</td>
			<td>For soldering EEG &#38; GND screws</td>
		</tr>
		<tr>
			<td>soldering paste</td>
			<td>Amazon</td>
			<td>B08CBZ5HC5</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel M2 nut&#160;</td>
			<td>McMaster</td>
			<td>93935A305</td>
			<td></td>
		</tr>
		<tr>
			<td>Tethered recording setup&#160;</td>
			<td>OpenEphys</td>
			<td>Acquasition Board</td>
			<td></td>
		</tr>
		<tr>
			<td>Wireless recording logger</td>
			<td>SpikeGadgets</td>
			<td>miniLogger 32</td>
			<td>For wireless recordings in the task</td>
		</tr>
		<tr>
			<td>Wireless recording setup</td>
			<td>SpikeGadgets</td>
			<td>Main Control Unit (MCU) incl. breakout board and RF transceiver</td>
			<td>For wireless recordings in the task</td>
		</tr>
	</tbody>
</table>

the td drive: a parametric, open-source implant for multi&#45;area electrophysiological recordings in behaving and sleeping rats

Intricate interactions between multiple brain areas underlie most functions attributed to the brain. The process of learning, as well as the formation and consolidation of memories, are two examples that rely heavily on functional connectivity across the brain. In addition, investigating hemispheric similarities and/or differences goes hand in hand with these multi-area interactions. Electrophysiological studies trying to further elucidate these complex processes thus depend on recording brain activity at multiple locations simultaneously and often in a bilateral fashion. Presented here is a 3D-printable implant for rats, named TD Drive, capable of symmetric, bilateral wire electrode recordings, currently in up to ten distributed brain areas simultaneously. The open-source design was created employing parametric design principles, allowing prospective users to easily adapt the drive design to their needs by simply adjusting high-level parameters, such as anterior-posterior and mediolateral coordinates of the recording electrode locations. The implant design was validated in n = 20 Lister Hooded rats that performed different tasks. The implant was compatible with tethered sleep recordings and open field recordings (Object Exploration) as well as wireless recording in a large maze using two different commercial recording systems and headstages. Thus, presented here is the adaptable design and assembly of a new electrophysiological implant, facilitating fast preparation and implantation.

Author Spotlight: Deciphering Memory and Learning Through Neural Implants for Multi&amp;#45;Region Brain Studies

The TD Drive: A Parametric, Open-Source Implant for Multi&#45;Area Electrophysiological Recordings in Behaving and Sleeping Rats

Our research goal is to better understand the neural mechanisms underlying memory processing, especially to examine the interactions between specific brain regions in real time during different learning time points, and also during sleep. In this regard, here we developed a novel drive implant that enables us to look at neural activity simultaneously across multiple brain regions in freely moving rats during task and rest periods. To record brain activity in behaving animals, researchers commonly use self-made complex implants with dozens of individually movable wire electrodes.However, using and implanting these devices successfully can take years of training. Our drive is easy to build and does not need expert hands, making this kind of research more accessible. The TD drive was designed based on three ideas, contrasting it to other complex designs.It should allow for recording of multiple brain areas bilaterally, be relatively easy and quick to build, and researchers should be able to adapt the design themselves without having to have advanced knowledge of 3D design. We aim to understand, how do different brain regions interact with each other when rodents perform simple versus complex behavior tasks? How does learning and different drugs affect these interactions between brain regions during sleep, and how are these interactions during sleep related to memory performance?

Using the instructions provided in the protocol, the TD Drive could be built easily by multiple experimenters. After drive development (n = 4), a full pilot was run with eight animals. An additional batch of eight animals was implanted, and experimental data collection was performed. As data analysis has not been completed on these animals, they have been included in the survival analysis, but not in other analyses (e.g., targeting or histology). Implant surgery was performed 2 weeks after arrival (see <strong class="xfi...

Read this Scientific Article Protocol about The TD Drive: A Parametric, Open-Source Implant for Multi-Area Electrophysiological Recordings in Behaving and Sleeping Rats at JoVE.com

Here, we present a unique, 3D-printable implant for rats, named TD Drive, capable of symmetric, bilateral wire electrode recordings, currently in up to ten distributed brain areas simultaneously.

Intricate interactions between multiple brain areas underlie most functions attributed to the brain. The process of learning, as well as the formation and ...

the-td-drive-parametric-open-source-implant-for-multiarea

Research

JoVE Journal

Neuroscience

Fabrication of TD Drive Implants for Electrophysiological Recordings of Sleeping and Live Rats 

To begin, create the 3D models. Click on upload to open the drive body design in the Autodesk Fusion software. Under the Modify tab, click on Change parameters.Adjust the coordinates for the first recording location by entering the anterior posterior coordinate and anterior posterior site one and the medial-lateral coordinate in medial-lateral site one and press OK.On the 3D printed model, use an M1 tap to tap the guide holes for the shuttle screws, which are the extension of the counter sink holes. Then slide a 3D-printed shuttle onto an M1 by 16 screw and use an M1 brass insert to hold the 3D printed shuttle in place. Using a small amount of soldering paste, solder the brass insert to the screw.After the shuttle assembly is cooled down, gently rotate the 3D-printed shuttle multiple times around the screw. Next, to assemble the drive, cut commercially available polyimide tubes to approximately 25 millimeters in length. Insert the polyimide guide tubes into the drive body and finalize the insertion using tweezers.Using a thin needle or toothpick, apply a small amount of liquid cyanoacrylate glue to the holes at the top of the drive body to fix the guide tubes in place. Also apply cyanoacrylate glue to the interface between the guide tubes and the guide stencil at the bottom of the drive body. Cut the polyimide guide tubes on the bottom such that they extend approximately one millimeter beyond the center pedestals of the drive body.Insert two shuttle assemblies in the drive body. Screw the electrode interface board to the drive body with M-2.5 by five polyamide screws. Then insert a stainless steel M2 nut into the extrusion on the left cap half and fix it with cyanoacrylate glue.Tap the hole in the front of the right cap half with an M1 tap. Next, prepare two metal plates as the surface for creating the electrode wire bundles. Attach plotting paper to the first plate and not-too-sticky painters tape onto the second plate with the sticky surface pointing up.Draw a clear line with an angle of 60 degrees on the plotting paper. For each electrode bundle, cut the electrode wire into the required number of pieces with the specified length. Gently pick up the four wires by touching them with a fingertip and place them as close as possible next to each other on the painters tape.Under a microscope, use fingers or forceps to place the wires together as close as possible. Apply a thin layer of liquid cyanoacrylate glue to the first two centimeters of the top of the bundle. After the setup dries fully, remove the wire bundle from the tape and transfer it to the plate with the plotting paper.For the retrosplenial cortex bundles, make a straight cut at the bottom of the array. For the hippocampus electrode bundles, place the array on the plotting paper such that it intersects the 60 degree line. Then use the line as a guide to make a cut at an angle of 60 degrees to the direction of the wires.Then using a razor or scalpel blade, carefully split the shortest of the four wires, cutting perpendicular to the wire direction. For the prefrontal cortex bundles, split the bottom of the array into two two-wire bundles. Shorten one of the two-wire bundles by one millimeter by cutting it perpendicular to the wire direction.Then cut two pieces of six centimeter length for the ground wire. Also cut eight pieces of six centimeters length for the electroencephalogram wire. Next, place an M1 by three stainless steel screw in a third hand.Wrap the de-insulated side of the ground or electroencephalogram wire around the shank of the screw. Apply a small amount of solder flux and solder the wire to the screw. Place a SIP DIP pin in the third hand so that the female side is accessible.Insert the de-insulated part of the opposite side of the wire into the SIP DIP pin and solder the wire to the pin. Next, place another SIP DIP pin in the holder with the male side accessible. Solder the de-insulated side of the other wire to the male side of the pin.Use a multimeter to verify that there is a continuous connection between the screw and the de-insulated wire end of the wire pin assembly when both assemblies are connected. To load the wire bundles into the drive, attach the drive to a holder. Once the drive body is stable, carefully slide one of the wire bundles into the respective polyimide tube.Use thin forceps to grab one of the wires and carefully bend it towards the desired hole and insert it. Once inserted, use a gold pin to pin it into the electrocorticography hole. Next, when the fixed wire arrays are aligned, apply a small amount of strong epoxy glue to the top of the guide tubes to fix the bundles in place.To fix the movable hippocampus wire arrays, first move the shuttle to the highest required position. Then push the wire bundles into the U-shaped opening of the shuttle and glue them in place with a small amount of a strong epoxy glue. Carefully insert the open end of the wire pin assembly of a ground wire through one of the through-holes marked ground and fix it using a gold pin.Remove the drive from the holder. Take care not to bend any the wire assemblies. To recover the electrode interface board after experimentation, gently push soft tweezers between the board and the drive body, or carefully lift the EIB by hand to release the remaining cyanoacrylate bond.The TD drive was implanted into eight rats for a pilot run, and implant surgeries were carried out two weeks post-arrival. Post-surgery, animals showed adaptability to the implants with fixed electrodes in frontal and retrosplenial regions and adjustable hippocampal bundles to enhance signal coverage. Sleep recordings were performed on tethered rats in a recording box, while wake data was recorded as wireless recordings in a larger maze.In seven out of eight animals, all target sites were reached on at least one hemisphere. While some animals began to lose their implants after two months, most retained them for up to 100 days. During this time, the local field potential remains stable, as observed by Delta oscillations in the hippocampal channel.