This work was supported in part by Japan Society for the Promotion of Science Overseas Research Fellowships (HO), Endowed Scholar Program (TK), Human Frontier Science Program (TK), Brain Research Foundation (TK), Faculty Science and Technology Acquisition and Retention Program (TK), Brain &#38; Behavior Research Foundation (TK), and by The Sumitomo Foundation Research Grant (JY), NARSAD Young Investigator Research Grant (JY). We thank W. Marks for valuable comments and suggestions during the preparation of the manuscript.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas Southwestern Medical Center.
1. Preparations of microdrive array parts
<ol>
	<li>Print the microdrive array parts using a 3D printer using dental model resin (Figure 1A,B). Ensure that the thickness of individual 3D printed layers is less than 50 &#181;m to keep the small holes on the printed parts clear and viable. 
	NOTE: The microdrive array consists of five parts (Figure 1C): (1) the main body of the microdrive array, which includes nine microdrive-screws for tetrodes and one screw for a silicon-probe (Figure 1Ca-d). The coordination of the tetrode bundle and hole for the opto-silicon probe at the bottom depends on the target brain area&#8217;s coordinates (Figure 1Cd); (2) a shuttle to attach a silicon-probe or optrode (Figure 1Ce); (3) a probe electrical connecter mount to hold the silicon probe connecter (Figure 1Cf); (4) a fiber ferrule holder that clamps to the center part of the body to prevent undesired movements of the implanted opto-silicon probe when plugging/unplugging an optical fiber connector (Figure 1Cg); and (5) a shielding cone that provides physical and electrical shielding to the microdrive array for stable recording (Figure 1Ch). The total weight of the microdrive array is 5.9 g, including the shielding cone (Table 1). If holes are clogged in the printed parts, drill out the holes using drill bits: #76 for the inner holes and #68 for the outer holes for tetrode-microdrive screws, #71 for tetrode microdrive-screw supporter hole, and #77 for the holes for the guide-posts at the bottom of the body.</li>
	<li>Insertion of guide posts into the microdrive array body.
	<ol>
		<li>Cut two 16 mm lengths of 26-Ga stainless steel wire. Gently sharpen the wire tips using a rotary grinder.</li>
		<li>Insert the wires into the bottom holes of the body (Figure 2A). Apply a small amount of cyanoacrylate glue at the bottom of the body to secure the guide posts.</li>
	</ol>
	</li>
</ol>
2. Opto-silicon probe preparation
<ol>
	<li>Prepare the microdrive screw for a silicon-probe. 
	NOTE: The microdrive screw for the silicon probe consists of a custom screw (300 &#181;m pitch), supporting a support tube, and an L-shape tube (Figure 2B).
	<ol>
		<li>Prepare the mold for the microdrive head (Figure 2C). To construct the mold, prepare the 3D-printed plastic pattern of the microdrive (Figure 2Ca). Then, pour liquid silicone gel after making a temporal wall by putting tapes around the pattern. Remove air bubbles by shaking gently, wait until it is cured, then remove the silicone-gel mold from the pattern (Figure 2Cb).</li>
		<li>Cut 18 mm and 9.5 mm lengths of 23 G stainless wire using a rotary grinder. Roughen the top 2&#8211;3 mm of the wires with a rotary grinder to enhance adhesion of the dental acrylic.</li>
		<li>Take one custom screw and apply small amount of silicon oil to reduce the friction with the dental acrylic. Set the wires and a custom-screw to the mold.</li>
		<li>Pour dental acrylic into the mold using a syringe to eliminate air bubbles around the wires and the screws. Air bubble contamination will make the microdrive fragile. Wait until the dental acrylic is fully cured, then take off the microdrive screws from the mold. Bend 6 mm of the longer wire tip to a 60&#176; angle using pliers.</li>
		<li>Check the quality of the microdrive screws (e.g., cracks, air-bubbles, and friction) to rotate the screw. If there is high friction, rotate the screw until they become smooth using an electric screw driver with a customized driver tip, which couples with the microdrive screw.</li>
		<li>Install the microdrive screw into the microdrive array body to check whether it moves up and down smoothly by turning the screw. Threads for the screw are automatically created when inserting the screw into the hole of the body.</li>
	</ol>
	</li>
	<li>Prepare the shuttle (Figure 3Aa).
	<ol>
		<li>Cut two 5 mm lengths of polyetheretherketone (PEEK) tubing using sharp scissors. Align the tubes at both sides of the shuttle. Glue the tubes and shuttle using epoxy.</li>
		<li>Apply small amount of silicon oil on the guide posts. Check the quality of the shuttle by inserting onto the guide posts of the microdrive array body. Make sure the shuttle moves smoothly without excessive friction.</li>
	</ol>
	</li>
	<li>Prepare an optorode (Figure 3Ab). This step can be skipped if an optogenetic experiment is not required.
	<ol>
		<li>Cleave the optical fiber to 21 mm in length using a ruby cutter. Grind the fiber tip to make the tip flat and shiny.</li>
		<li>Gently place the optical fiber on the front side of the silicon-probe. The fiber tip is positioned 200&#8211;300 &#181;m above the top of the electrode sites. Hold the fiber temporarily with transparent tape.</li>
		<li>Glue the optical fiber to the base of the silicon-probe using small amount of epoxy. Wait for at least 5 h until the epoxy is fully cured. 
		NOTE: It is recommended to attach the optical fiber on the same side as the electrode sites. Attaching the fiber at the backside may prevent light from properly illuminating the recording sites.</li>
	</ol>
	</li>
	<li>Attach the shuttle to the silicon probe (Figure 3Ac): apply a small amount of epoxy at the back of the silicon-probe&#8217;s base. Attach the bottom part of the shuttle to the silicon-probe&#8217;s base, and gently hold in position for 2&#8211;3 min to avoid formation of a gap between the shuttle and silicon-probe base during initial cure of the epoxy. Wait for at least 5 h until the epoxy is completely cured.</li>
	<li>Carefully insert the shuttle tubes onto the guide posts of the main body under the microscope (Figure 3B). During this procedure, hold the groove of the shuttle with fine tweezers.</li>
	<li>Insert the microdrive-screw into the screw hole by turning the screw. Engage the silicon probe and microdrive-screw by inserting the tip of the L-shape wire into the groove of the shuttle head (Figure 3C).</li>
	<li>Attach the probe electrical connecter holder to the microdrive array body (Figure 3D).
	<ol>
		<li>Cut two #0 screws to 3.5 mm thread length. Grind the tips to remove burrs.</li>
		<li>Place the probe connecter holder on the body. Place the silicon probe electrical connector into the holder.</li>
		<li>Secure the silicon probe connector in the holder using epoxy, and be sure to not glue it to the microdrive array body to allow for the recovery procedure of the silicon probe. Insert the screws to hold the probe connecter holder.</li>
	</ol>
	</li>
	<li>Attach the ferrule-holder to the opto-silicon probe and microdrive array body (Figure 3D).
	<ol>
		<li>Cut two #0 screws to 6 mm thread length. Grind the tips to remove burrs.</li>
		<li>Grind the outside of two #0 machine screw nuts to make small hex nuts with 2.5&#8211;3.0 mm outer diameter to reduce the weight and space.</li>
		<li>Insert the screws into component A of the holder. Glue the screw heads using epoxy.</li>
		<li>Apply small amount of silicon grease to component A and B to reduce friction with the body. Insert component A into the body, then temporally hold using inverse tweezers.</li>
		<li>Place component B onto component A&#8217;s screws. Thread the customized nuts into the screws. Use pliers to tighten the nuts to secure the ferrule holder on the body.</li>
		<li>Insert the fiber ferrule into the groove of the fiber ferrule holder (component B). Ensure that the fiber ferrule is sticking 4&#8211;5 mm out from the holder.</li>
		<li>Apply small amount of epoxy between the ferrule and the holder groove. Wait until the epoxy is fully cured and check that the ferrule does not move. Check the shuttle and ferrule holder for smooth motion by loosening the nuts before turning the microdrive-screw.</li>
		<li>Check the working distance of the probe. Ensure that the probe tip completely retracts into the body when the ferrule-holder is at the top position while the shuttle tubes are still associated with the guide-posts. The maximum working distance is determined by the length of the silicone probe and the target brain region.</li>
		<li>If the microdrive-screw is loose, apply small amount of dental acrylic around the screw to add more threads for support. When it is cured, rotate the screw to check tightness and stability.</li>
	</ol>
	</li>
</ol>
3. Tetrode preparation
NOTE: This procedure is similar to previously published articles8,19,20,39.
<ol>
	<li>Prepare the microdrive screws for the tetrode. The microdrive for a tetrode consists of a custom-machined screw and a 23 G tubing (Figure 2B). This procedure is similar to section 2.1.</li>
	<li>Make a bundle of 30 G stainless steel tubing that has a 5.5 mil wire inside. In this case, a total of nine 30 G tubing (eight recording tetrodes and one reference electrode) were used.</li>
	<li>Thread the 30 G bundle from the bottom of the drive body, and secure them with 20 G thin-walled tubing to the main body. Trim the bottom of the bundle with a rotary grinder to make the tip even and flush. Trim top part of the 30 G tubes with a rotary grinder so that the 30 G tube sticks out about 0.5 mm from the main body.</li>
	<li>Load 5.5 mil polyimide insulating tubes into the 30 G tubing. Prepare tetrode wires and load them into a 32-channel electric interface board (EIB). Check electrical connection with the impedance tester before final precision cut.</li>
	<li>Lower electrode tip impedance to 250&#8211;350 k&#937; with gold plating solution. Fix all tetrodes with superglue.</li>
	<li>Fill excessive gap between polyimide tube and tetrode with mineral oil for sealing and lubrication. Route the ground wire to the EIB. 
	NOTE: If necessary, the optical fiber can be integrated along tetrode wires12.</li>
</ol>
4. Attaching the shielding cone
<ol>
	<li>Paint silver conductive shielding paint on the inside of the printed cone. Place the microdrive array inside of the cone (Figure 3E).</li>
	<li>Cut two #0 screws to 3.5 mm thread length. Fasten the screws from the outside of the cone to hold the microdrive array in place.</li>
	<li>Apply silver paint around the screw head to electrically connect the shielding cone with electrical ground. Check the electrical connectivity between the ground wire and cone. Apply a small amount of epoxy between the microdrive array body and shielding cone to securely attach the body. 
	NOTE: Another way to prepare the shielding cone is to use aluminum tape40 (Figure 3F). First, prepare the pattern paper for the shielding cone after sticking aluminum foil to the paper (Figure 3Fa). Then, roll the paper and attach it to the microdrive body using a small amount of cyanoacrylate glue (Figure 3Fb). The weight of this cone is 0.72 g and total weight of the microdrive array is reduced to 4.7 g (Table 1).</li>
</ol>
5. Implant surgery
NOTE: This procedure is modified from previously published articles18,39,41 for dual-site implantation. Ensure that the weight of the animal is over 25 g for the microdrive implant for faster recovery after the surgery.
<ol>
	<li>Preparation
	<ol>
		<li>To prepare a ground screw, attach the silver wire to a skull screw and apply silver paint. Then, attach a gold pin to the opposite side of the wire using silver paint.</li>
		<li>Prepare the drive holding adapter to hold the microdrive array to a stereotactic device. Attach a male connecter to a stainless handle using epoxy. Make sure that alignment of the connecter and stainless handle is straight.</li>
		<li>In the case that histological confirmation is needed after recording, apply Di-I to the tetrodes or backside of the silicon-probe38.</li>
		<li>Lower the silicon-probe down to be desired depth. Loosen the nuts of the ferrule holder using pliers, lower the silicon-probe (opto-silicon probe) by turning the silicon-probe&#8217;s microdrive-screw, then fasten the nuts to secure the ferrule holder. When implanting tetrodes in hippocampal area CA1 and a silicon-probe in MEC, the distance between the tetrode cannula and silicon-probe&#8217;s tip is 3&#8211;4 mm.</li>
	</ol>
	</li>
	<li>Set the anesthetized mouse (0.8%&#8211;1.5% isoflurane) in a stereotaxic device. The anesthetic condition of the mouse is confirmed by absence of the toe-pinch reflex. Apply clear ointment to the eyes to prevent drying. Cover the eyes with a piece of foil to protect from strong surgical light exposure.</li>
	<li>Disinfect the mouse&#8217;s scalp with iodine and isopropanol after shaving the fur. Make a 1.5&#8211;2.0 cm incision at the scalp using standard surgical scissors, and remove the tissue over the skull using cotton swabs after subcutaneously applying lidocaine.</li>
	<li>Align the mouse head with the stereotaxic tool. Ensure that the height difference between bregma and lambda is less than 100 &#956;m. Determine the craniotomy location using an atlas and mark these locations with a sterilized pencil.</li>
	<li>Anchor the skull screws (0.8 mm diameter, 0.200 mm thread pitch) by rotating them 1.5 turns (0.3 mm) on the skull, using surgical tweezers and a screwdriver after drilling 8&#8211;11 holes in the skull using 0.5 mm drill bit. 
	NOTE: 2&#8211;4 holes in the frontal skull, 2&#8211;3 holes in each side of the parietal skull, and 1&#8211;2 holes in the interparietal skull are suggested.</li>
	<li>Attach the ground screw to the hole by rotating it one turn (0.2 mm) after drilling a hole in the interparietal bone. Ensure that this hole does not penetrate through the bone into the brain case; otherwise, cerebellar signals will contaminate the recording. Check that the impedance is less than 20 k&#8486; at 1 kHz between the ground screw and skull screws using an impedance meter. 
	NOTE: Larger impedance will cause the introduction of motion artifacts during recording.</li>
	<li>Perform the craniotomy at the marked locations. The dura can be left intact in mice.</li>
	<li>Connect the male pin of the ground screw and the microdrive array&#8217;s ground connector. Check connectivity using the impedance meter by measuring between the ground screw and shielding.</li>
	<li>Set the microdrive array to the adapter, set it to the stereotaxic device, and slowly lower the silicon probe until the desired depth. Ensure that the tetrode bundles are placed above the brain surface but still inside of the microdrive array when the silicon probe is inserted into the brain (Figure 4A).</li>
	<li>Carefully apply the silicon grease to seal the area of the silicon probe and the tetrode bundle (Figure 4B). Put a small amount of the silicon grease at the tip of a 20 G needle and apply the grease around the probes using the needle. Repeat until silicon grease completely covers the area around the probe so that dental acrylic does not flow onto or underneath the electrodes/probes. Be careful not to let the grease touch the electrode sites, otherwise it will dramatically increase the impedance of the recording sites.</li>
	<li>Apply dental acrylic to fix the microdrive array to the anchoring screws in the skull. 
	NOTE: It is recommended to apply dental acrylic in three layers to avoid the excessive heat produced during curing of the acrylic.</li>
	<li>Remove the adapter from the microdrive array carefully. Inject 1 mL of PBS subcutaneously to prevent dehydration. Inject 5 mg/kg meloxicam subcutaneously as an analgesic treatment.</li>
	<li>Cover the silicon-probe connector by a piece of tape to prevent any dirt from getting inside of the electrical connections. Cover the microdrive array using a plastic paraffin film and tape it in place.</li>
	<li>Administer appropriate analgesic treatment for 3 days (e.g., subcutaneous injections of 2 mg/kg meloxicam once per day). Allow 3&#8211;5 days for recovery before starting the tetrode adjustment. The implanted mouse after the recovery period is shown in Figure 4C.</li>
</ol>
6. Recovering the silicon-probe (Figure 4D)
<ol>
	<li>Inject ketamine (75 mg/kg) and dexmedetomidine (1 mg/kg) anesthetics intraperitoneally and confirmed absence of the toe-pinch reflex. Fix the anesthetized mouse by directly perfusing 4% paraformaldehyde through the heart using a hood. Surgical methods for rodents are described previously42.</li>
	<li>Loosen the nuts of the ferrule holder using a plier. Then, carefully move it to the top of the body by turning the adjusting screw to fully retract the silicon-probe towards inside of the microdrive array body. Fasten the nuts to hold the probe at the top position.</li>
	<li>Take the mouse brain out from the bottom by cracking the skull from the side. The microdrive array is now separated from the animal.</li>
	<li>Completely remove the L-shaped microdrive-screw that drives the silicon-probe. Loosen and take out the nuts of the ferrule holder using pliers. Take out the component A of the ferrule holder.</li>
	<li>Unscrew the probe connector mount and detach from the drive body. Check that the probe connector mount can come off from the microdrive array body.</li>
	<li>Hold the top part of the shuttle with tweezers, then carefully slide the silicon-probe assembly out from the microdrive array.</li>
	<li>Clean the probe tip with contact lens cleaner (first with enzyme, then 3% hydrogen peroxide) for at least 1 day. Carefully wipe the electrode tip using isopropanol pads under the microscope. Keep the probe in a static-free storage box. 
	NOTE: The shuttle and probe connector mount remain attached to the silicon probe and can be reused in the next implantation. 
	NOTE: Some silicon probes are not tolerable with hydrogen peroxide. In this case, use the contact lens solution containing proteolytic enzyme only.</li>
	<li>To reuse the microdrive array body for the next surgery, remove the dental acrylic using a combination of fine-tip drills and nippers. Then, recover the skull-screws by immersing the removed dental acrylic into acetone. Note that the acetone will dissolve plastic parts of the microdrive array.</li>
	<li>Remove the epoxy between the microdrive body and shielding cone using a scalpel. 
	NOTE: No additional parts need to be printed again for the next surgery if the microdrive is not broken.</li>
</ol>

The authors have nothing to disclose.

The protocol demonstrates how to construct and implant a hybrid microdrive array that allows recording of neural activities from two brain areas using independent adjustable tetrodes and a silicon-probe in freely behaving mice. It also demonstrates optogenetic experiments and the recovery of the silicon probe after experiments. While adjustable silicon probe33 or opto-silicon probe36 implantation are previously demonstrated in mice, this protocol has clear advantages in the simultaneous tetrode array and opto-silicon probe implantation to provide flexible choice of implanted probe types. The type of implanted probe can be switched depending on the aim of the experiment, such as multi-shank probes27,44 or ultra-density Neuropixels21,45. The coordination and angle of implantation7 can be easily modified at the 3D object design stage as needed. For example, dual-site or even triple-site recording is possible during learning tasks across memory-related brain structures, such as the hippocampus46, entorhinal cortex47, prefrontal cortex48, amygdala49, and cingulate cortex50.
There are several critical procedures for successful implant and recording. Due to the fragility of silicon-based probes, any mechanical vibrations or impacts to the microdrive array should be minimized during assembly. For example, opening the clogged holes using a drill should be finished before loading the silicon probe into the microdrive array. Also, it should be emphasized to carefully check the ground connection in each step during microdrive array construction and implant surgery to ensure stability of the recorded data. Unstable or high-impedance connections to the ground cause heavy noise and motion related artifacts during the recording session. For stable recordings, it is recommended to wait 1-2 weeks after the surgery to avoid electrode drift because the brain tissue is negatively affected by the implant surgery. However, signal quality on the silicon probe recovers after 1-2 weeks from the surgical trauma based on previous experience. It is recommended to use single-housing to prevent damage to the implanted microdrive array by other mice. For the optogenetic experiment, it is important to note that most silicon-probes induce photo-artifacts in response to light-stimulations51, while others are designed to minimize photo-artifacts52 (there are photo-artifact reduced silicon-probes that are commercially available).
The weight of the microdrive array (5.9 g) is heavier than the typical microdrives described in previous articles12,53, mainly due to the microdrive array body (~21% of the total weight), shielding cone (~31%), and metal parts (screws and nuts: ~22%). It is recommended to use mice with weights of over 25 g (~2-3 months old for C57BL/6 mice54,55) for implant surgery, because mice with adequate body weights tend to recover earlier. For this reason, this microdrive array may be not the best solution for juvenile mice. While devices that are 5%-10% of the mouse&#8217;s bodyweight are often guided to be tolerated for implants12,56 (although there is no supporting published data for this57), this microdrive array weighs ~24% of the bodyweight of 25 g mice (~19% when using the alternative cone described below).
However, the implanted adult mice were able to freely move around and jump around in the home cages. Mice implanted with a similar microdrive array weight (~4.5 g) have previously been shown to perform the behavioral task (linear maze task) even under food restriction13,17. The disadvantage of weight is not a problem during recording, as a counterweight balancing system18,34,58 or headpost system59 will support the microdrive array. In addition, the total weight of the microdrive array can be reduced by lowering the height or reducing the thickness of the shielding cone and modifying the design to utilize smaller screws.
Using the current 3D printing material, the thickness of the shielding cone can be reduced up to ~0.3 mm (from the current thickness of ~0.6 mm). The cone height can be reduced ~5 mm as long as the tetrode wires can still be covered. Exposure of the tetrode wires will result in breakage of the wires and failure of the long-term recording. Alternatively, preparation of the shielding cone using paper and aluminum tape can reduce the cone weight to ~0.7 g (~15% of total weight; reduced 20% from the total weight of the original microdrive array); although, these are a tradeoff with the physical strength. In addition, the size of the microdrive (current shielding cone: 4.2 x 4.0 x 2.6 cm = major axis x minor axis x height) can be an obstacle to food and water access if they are provided from the top of the animal cage. As long as they are provided on the cage floor or from the sidewall, the microdrive does not disturb natural behaviors of mice, such as eating, drinking, grooming, rearing, or nesting60.
In conclusion, this microdrive protocol provides researchers with flexible choices for recording from multiple brain areas in freely moving mice for understanding the dynamics and functions of long-ranging neural circuits.

It is crucial to understand how neuronal activity supports cognitive process, such as learning and memory, by investigating how different brain regions dynamically interact with each other. To elucidate dynamics of the neural activity underlying cognitive tasks, large-scale extracellular electrophysiology has been conducted in freely moving animals with the aid of microdrive arrays1,2,3,4. In the past two decades, several types of microdrive array have been developed to implant electrodes into multiple brain regions for rats5,6,7,8 and mice9,10,11,12. Nonetheless, current microdrive designs generally do not allow for the use of multiple probe types, forcing researchers to choose a single electrode type with specific benefits and limitations. For example, tetrode arrays work well for densely populated brain regions such as the dorsal hippocampus CA11,13, while silicon probes give a better geometrical profile for studying anatomical connections14,15.
Tetrodes and silicon probes are often used for in vivo chronic recording, and each has its own advantages and disadvantages. Tetrodes have been proven to have significant advantages in better single unit isolation than single electrodes16,17, in addition to cost effectiveness and mechanical rigidity. They also provide higher yields of single unit activities when combined with microdrives8,18,19,20. It is essential to increase the number of simultaneously recorded neurons for understanding the function of neural circuits21. For example, large numbers of cells are needed to investigate small populations of functionally heterogeneous cell types such as time-related22 or reward coding23 cells. Much higher cell numbers are required to improve the decoding quality of spike sequences13,24,25.
Tetrodes, however, have a disadvantage in recording spatially distributed cells, such as in the cortex or thalamus. In contrast to tetrodes, silicon probes can provide spatial distribution and interaction of local field potentials (LFPs) and spiking activities within a local structure14,26. Multi-shank silicon probes further increase the number of recording sites and allow recording across single or neighboring structures27. However, such arrays are less flexible in the positioning of electrode sites compared to tetrodes. In addition, complex spike sorting algorithms are required in high-density probes to extract information about action potentials of neighboring channels to mirror the data acquired by tetrodes28,29,30. Hence, the overall yield of single units is often less than tetrodes. Moreover, silicon probes are disadvantageous due to their fragility and high cost. Thus, the choice of tetrodes vs. silicon probes depends on the aim of the recording, which is a question of whether obtaining a high yield of single-units or spatial profiling at the recording sites is prioritized.
In addition to recording neural activity, optogenetic manipulation has become one of the more powerful tools in neuroscience to examine how specific cell types and/or pathways contribute to neural circuit functions13,31,32,33. However, optogenetic experiments require additional consideration in microdrive array design to attach the fiber connector to stimulation light sources34,35,36. Often, connecting fiber-optics requires a relatively large force, which may lead to a mechanical shift of the probe in the brain. Therefore, it is not a trivial task to combine an implantable optical fiber to conventional microdrive arrays.
For the above reasons, researchers are required to optimize the selection of the type of electrode or to implant an optical fiber depending on the aim of the recording. For example tetrodes are used to achieve higher unit yield in hippocampus1,13, while silicon probes are used to investigate the laminar depth profile of cortical areas, such as the medial entorhinal cortex (MEC)37. Currently, microdrives for simultaneous implantation of tetrodes and silicon probes had been reported for rats5,11. However, it is extremely challenging to implant multiple tetrodes and silicon probes in mice because of the weight of the microdrives, limited space on the mouse head, and spatial requirements for designing the microdrive to employ different probes. Although it is possible to implant silicon probes without a microdrive, this procedure does not allow for adjustment of the probe and lowers the success rate of silicon-probe recovery12,38. Furthermore, optogenetic experiments require additional considerations in microdrive array design. This protocol demonstrates how to construct and implant a microdrive array for chronic recording in freely moving mice, which allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe. This microdrive array also facilitates optogenetic experiments and recovery of the silicon probe.

<table>
	<tbody>
		<tr>
			<td>#00-90 screw</td>
			<td>J.I. Morris</td>
			<td>#00-90-1/8</td>
			<td>EIB screws</td>
		</tr>
		<tr>
			<td>#0-80 nut</td>
			<td>Small Parts</td>
			<td>B00DGB7CT2</td>
			<td>brass nut for holding fiber ferrule holder</td>
		</tr>
		<tr>
			<td>#0-80 screw</td>
			<td>Small Parts</td>
			<td>B000FMZ57G</td>
			<td>brass machine screw for probe connector mount, fiber ferrule holder, and shielding cone</td>
		</tr>
		<tr>
			<td>22 Ga polyetheretherketone tubes</td>
			<td>Small Parts</td>
			<td>SLPT-22-24</td>
			<td>for attaching to the shuttle, 0.025 inches inner diameter</td>
		</tr>
		<tr>
			<td>23 Ga stainless tubing</td>
			<td>Small Parts</td>
			<td>HTX-23R</td>
			<td>for tetrode</td>
		</tr>
		<tr>
			<td>23 Ga stainless wire</td>
			<td>Small Parts</td>
			<td>HTX-23R-24-10</td>
			<td>for L-shape/support wire</td>
		</tr>
		<tr>
			<td>26 Ga stainless wire</td>
			<td>Small Parts</td>
			<td>GWX-0200</td>
			<td>for guide-posts</td>
		</tr>
		<tr>
			<td>30 Ga stainless wire</td>
			<td>Small Parts</td>
			<td>HTX-30R</td>
			<td>for tetrode</td>
		</tr>
		<tr>
			<td>3-D CAD software package</td>
			<td>Dassault Syst&#232;mes</td>
			<td>SolidWorks 2003</td>
			<td></td>
		</tr>
		<tr>
			<td>3D printer</td>
			<td>FormLab</td>
			<td>Form2</td>
			<td></td>
		</tr>
		<tr>
			<td>5.5mil polyimide insulating tubes</td>
			<td>HPC Medical</td>
			<td>72113900001-012</td>
			<td></td>
		</tr>
		<tr>
			<td>aluminum foil tape</td>
			<td>Tyco</td>
			<td>Tyco Adhesives 617022 Aluminum Foil Tape</td>
			<td>for the alternative shielding cone</td>
		</tr>
		<tr>
			<td>conductive paste</td>
			<td>YSHIELD</td>
			<td>HSF54</td>
			<td>for shielding cone</td>
		</tr>
		<tr>
			<td>customized screws for silicon-probe microdrive</td>
			<td>AMT</td>
			<td>UNM1.25-HalfMoon</td>
			<td>half-moon stainless screw, 1.5 mm diameter, 300 &#181;m thread pitch</td>
		</tr>
		<tr>
			<td>customized screws for tetrode microdrive</td>
			<td>AMT</td>
			<td>Yamamoto_0000-160_9mm</td>
			<td>slotted stainless screw, 0.5 mm diameter, 160 &#181;m thread pitch, custom-made to order for our design</td>
		</tr>
		<tr>
			<td>dental acrylic</td>
			<td>Stoelting</td>
			<td>51459</td>
			<td></td>
		</tr>
		<tr>
			<td>dental model resin</td>
			<td>FormLab</td>
			<td>RS-F2-DMBE-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Dremel rotary tool</td>
			<td>Dremel</td>
			<td>model 800</td>
			<td>a grinder</td>
		</tr>
		<tr>
			<td>drill bit</td>
			<td>Fine Science Tool</td>
			<td>19007-05</td>
			<td></td>
		</tr>
		<tr>
			<td>electric interface board</td>
			<td>Neuralynx</td>
			<td>EIB-36-Narrow</td>
			<td></td>
		</tr>
		<tr>
			<td>epoxy</td>
			<td>Devcon</td>
			<td>GLU-735.90</td>
			<td>5 minutes epoxy</td>
		</tr>
		<tr>
			<td>eye ointment</td>
			<td>Dechra</td>
			<td>Puralube Ophthalmic Ointment</td>
			<td>to prevent mice eyes from drying during surgery</td>
		</tr>
		<tr>
			<td>fiber polishing sheet</td>
			<td>Thorlabs</td>
			<td>LFG5P</td>
			<td>for polishing the optical fiber</td>
		</tr>
		<tr>
			<td>fine tweezers</td>
			<td>Protech International</td>
			<td>15-368</td>
			<td>for loading/recovering the silicon probe</td>
		</tr>
		<tr>
			<td>gold pins</td>
			<td>Neuralynx</td>
			<td>EIB Pins Small</td>
			<td></td>
		</tr>
		<tr>
			<td>ground wire</td>
			<td>A-M Systems</td>
			<td>781500</td>
			<td>0.010 inch bare silver wire</td>
		</tr>
		<tr>
			<td>headstage preamp</td>
			<td>Neuralynx</td>
			<td>HS-36</td>
			<td></td>
		</tr>
		<tr>
			<td>impedance meter</td>
			<td>BAK electronics</td>
			<td>Model IMP-2</td>
			<td>1 kHz testing frequency</td>
		</tr>
		<tr>
			<td>mineral oil</td>
			<td>ZONA</td>
			<td>36-105</td>
			<td>for lubricating screws and wires</td>
		</tr>
		<tr>
			<td>optical fiber</td>
			<td>Doric</td>
			<td>MFC_200/260-0.22_50mm_ZF1.25(G)_FLT</td>
			<td></td>
		</tr>
		<tr>
			<td>Recording system</td>
			<td>Neuralynx</td>
			<td>Digital Lynx 4SX</td>
			<td></td>
		</tr>
		<tr>
			<td>ruby fiber scribe</td>
			<td>Thorlabs</td>
			<td>S90R</td>
			<td>for cleaving the optical fiber</td>
		</tr>
		<tr>
			<td>silicon grease</td>
			<td>Fine Science Tool</td>
			<td>29051-45</td>
			<td></td>
		</tr>
		<tr>
			<td>silicon probe</td>
			<td>Neuronexus</td>
			<td>A1x32-Edge-5mm-20-177</td>
			<td>Fig. 3, 4A, 4B, 5</td>
		</tr>
		<tr>
			<td>silicon probe</td>
			<td>Neuronexus</td>
			<td>A1x32-6mm-50-177</td>
			<td>Fig. 4C</td>
		</tr>
		<tr>
			<td>silicon probe washing solution</td>
			<td>Alcon</td>
			<td>AL10078844</td>
			<td>contact lens cleaner</td>
		</tr>
		<tr>
			<td>silicone lubber</td>
			<td>Smooth-On</td>
			<td>Dragon Skin 10 FAST</td>
			<td>for preparation of microdrive mold</td>
		</tr>
		<tr>
			<td>silver paint</td>
			<td>GC electronic</td>
			<td>22-023</td>
			<td>silver print II coating, used for ground wires</td>
		</tr>
		<tr>
			<td>skull screw</td>
			<td>Otto Frei</td>
			<td>2647-10AC</td>
			<td>0.8 mm diameter, 0.200 mm thread pitch</td>
		</tr>
		<tr>
			<td>standard surgical scissors</td>
			<td>ROBOZ</td>
			<td>RS-5880</td>
			<td></td>
		</tr>
		<tr>
			<td>stereotaxic apparatus</td>
			<td>Kopf</td>
			<td>Model 942</td>
			<td></td>
		</tr>
		<tr>
			<td>super glue</td>
			<td>Loctite</td>
			<td>LOC230992</td>
			<td>for applying to guide-posts</td>
		</tr>
		<tr>
			<td>surgical tweezers</td>
			<td>ROBOZ</td>
			<td>RS-5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrode Twister</td>
			<td>Jun Yamamoto</td>
			<td>TT-01</td>
			<td></td>
		</tr>
		<tr>
			<td>tetrode wires</td>
			<td>Sandvik</td>
			<td>PX000004</td>
			<td></td>
		</tr>
	</tbody>
</table>

hybrid microdrive system with recoverable opto-silicon probe and tetrode for dual-site high density recording in freely moving mice

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, conventional microdrive designs often only allow use of one type of electrode to record from single or multiple regions, limiting the yield of single-unit or depth profile recordings. It also often limits the ability to combine electrode recordings with optogenetic tools to target pathway and/or cell type specific activity. Presented here is a hybrid microdrive array for freely moving mice to optimize yield and a description of its fabrication and reuse of the microdrive array. The current design employs nine tetrodes and one opto-silicon probe implanted in two different brain areas simultaneously in freely moving mice. The tetrodes and the opto-silicon probe are independently adjustable along the dorsoventral axis in the brain to maximize the yield of unit and oscillatory activities. This microdrive array also incorporates a set-up for light, mediating optogenetic manipulation to investigate the regional- or cell type-specific responses and functions of long-range neural circuits. In addition, the opto-silicon probe can be safely recovered and reused after each experiment. Because the microdrive array consists of 3D-printed parts, the design of microdrives can be easily modified to accommodate various settings. First described is the design of the microdrive array and how to attach the optical fiber to a silicon probe for optogenetics experiments, followed by fabrication of the tetrode bundle and implantation of the array into a mouse brain. The recording of local field potentials and unit spiking combined with optogenetic stimulation also demonstrate feasibility of the microdrive array system in freely moving mice.

The microdrive array was constructed within 5 days. The timeline of microdrive preparation is described in Table 2. Using this microdrive, nine tetrodes and one silicon probe were implanted into the hippocampal CA1 and MEC of the mouse [21 week old/29 g body weight male pOxr1-Cre (C57BL/6 background)], respectively. This transgenic mouse expresses Cre in MEC layer III pyramidal neurons. The mouse was injected with 200 nL of AAV5-DIO-ChR2-YFP (titer: 7.7 x 1012 gc/mL) into the MEC 10 weeks before the electrode implant. LFPs were recorded using a low-pass filter (1-500 Hz), and spiking units were detected using a high-pass filter (0.8-5 kHz). Light stimulation (&#955; = 450 nm) was performed using a 1 ms pulse width at 10.6 mW intensity measured at the end of the fiber connector. The reference electrode for the tetrode recording was placed in the white matter using a dedicated tetrode wire. The reference for the silicon probe recording was set as the top channel of the probe.
After the tetrode adjustment, behavioral performance was tested on a linear track (Figure 5A) and in an open field (Figure 5B). In both experiments, the mouse explored freely for ~30 min (Figure 5Aa,b,c; Figure 5Ba,b,c). The electrophysiological signals were successfully recorded without severe motion-related noise throughout the recording session (Figure 5Ad,e; Figure 5Bd,e). Next, light stimulation was performed at the MEC to stimulate MEC layer III neurons that project to the CA143 (Figure 6A). Spontaneous spiking activities (Figure 6B,C) and LFPs (Figure 6D) were recorded from the tetrodes and silicon probe when the mouse was sleeping. LFPs recorded in the tetrodes showed large ripple activities, suggesting that all tetrodes were positioned in the vicinity of the CA1 pyramidal cell layer. Light-induced responsive activities were first observed in MEC, followed by in CA1 with 13-18 ms latency (Figure 6E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60028/60028fig1.jpg" /> 
Figure 1: Microdrive array overview. (A) A skeleton view of the microdrive array, from the tetrode side (a) and silicon-probe side (b). (B) A real image of the loaded microdrive array, viewed from the tetrode side (a) and from the silicon-probe side (b). The microdrive array is placed on the jig stage in panel (b). (C) Individual 3D-printed microdrive array parts. (a-d) The microdrive array body, viewed from four different angles (a: tetrode side view; b: silicon-probe side view; c: top view; d: bottom view). A magnified view of the dashed line in panel (c) is shown in Figure 2A. (e) The shuttle, which holds and allows adjusting of the silicon-probe. A silicon probe is attached at the dashed line in panel (e). (f) The probe-connecter holder, which holds a 32-channel silicon-probe connecter. (g) The fiber ferrule holder, which holds an optical fiber ferrule to prevent from the movement of the probe when plugging/unplugging the fiber connector with the light-source. This part consists of two components: [panel (g) and components A and B]. (h) The printed shielding cone, which provides physical and electrical shielding when painted with conductive material. The cone window allows the ability to see inside of the structure during microdrive array preparation, which is eventually covered by a piece of tape or 3D-printed material. <a href="https://www.jove.com/files/ftp_upload/60028/60028fig1large.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/60028/60028fig2.jpg" /> 
Figure 2: Preparation of guide-posts and microdrive screws on the main body. (A) Guide post-preparation. (a) Magnified view of the microdrive array body shown in Figure 1Cc. (b) Guide post-insertion into the holes of the body. (B) The microdrive-screw designs. (a) The microdrive-screw for a silicon-probe, which consists of a 300 &#181;m pitch custom screw, supporting tube, and L-shape tube. (b) The microdrive-screw for a tetrode, which consists of a 160 &#181;m pitch custom screw and 30 G stainless guide tube. (C) Fabrication of the top piece of the microdrive-screws: (a) Preparation of 3D-printed patterns of the anti-mold for the microdrive-screw. The picture shows a pattern for the silicon-probe microdrive-screw. (b) The mold made using the anti-mold pattern (a) and silicon-rubber material. Assembled microdrive-screws are produced by inserting custom-screws and wires/tube, and pouring dental acrylic in each well. Inset: magnified view of the wells of the mold. <a href="https://www.jove.com/files/ftp_upload/60028/60028fig2large.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/60028/60028fig3.jpg" /> 
Figure 3: Microdrive array assembly. (A) Preparation of an opto-silicon probe. (a) Attaching two plastic guide tubes to the shuttle. (b) Gluing the optical fiber to the silicon-probe. (c) Attaching the shuttle to the opto-silicon probe. In this picture, the bottom part of the shuttle (dashed line) is attached to the silicon-probe&#8217;s base [backside of (b)]. The shuttle and the silicon probe shank should be in parallel. (B) Loading the opto-silicon probe shuttle assembly into the guide-posts of the microdrive array body. (C) Relative position of the silicon-probe microdrive when the probe is completely retracted into the body (a) and when positioned at the lowest in the drive body (b). The L-shape wire is inserted into the groove on the shuttle. (D) An exploded view of the fiber ferrule holder and the probe connector mount. (E) Shielding cone attached. The conductive material is painted inside of the cone. (F) Alternative shielding cone using a paper and aluminum tape. (a) A pattern paper. (b) An attached alternative shielding cone, which reduces 1.1 g of weight compared to the 3D-printed version. <a href="https://www.jove.com/files/ftp_upload/60028/60028fig3large.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/60028/60028fig4.jpg" /> 
Figure 4: Sealing the probes during surgery and recovery of the silicon probe. (A) The microdrive array and the mouse skull after craniotomy, before applying silicon-grease. The silicon-probe is inserted about 2mm into the brain at this time. (B) Applying silicon-grease around the silicon-probe and tetrode bundles to protect the probes from dental acrylic. (C) The chronically implanted mouse after the recovery period, when the mouse is walking (a), grooming (b), and when connected to the recording cable with the counter-balancing pulley system (c). (D) The recovered silicon probe, before (a) and after (b) immersion into the cleaning solution. The biological tissues in (a) are removed after the cleaning process (b). <a href="https://www.jove.com/files/ftp_upload/60028/60028fig4large.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/60028/60028fig5.jpg" /> 
Figure 5: Examples of simultaneous tetrode/silicon-probe recording in the hippocampal CA1 and medial entorhinal cortex (MEC) from the behaving mouse. (A) Recording on the linear track. (a) The linear track used for the recoding. (b) Trajectories of the mouse exploration for ~30 min on the track. (c) Behavioral performance on the linear track. (d-e) Representative LFP recordings from the tetrode (d) and the silicon-probe (e). (B) Recording in the open field. (a) The open field chamber used for the recoding. (b) Trajectories of the mouse exploration for ~30 min in the chamber. (c) Behavioral performance in the open field. (d,e) Representative LFP recordings from the tetrode (d) and the silicon-probe (e). LED is attached to the head amplifier to record the positions of the mouse. The linear track and the open-field chamber are connected with the electrical ground to reduce electrostatic noise. <a href="https://www.jove.com/files/ftp_upload/60028/60028fig5large.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/60028/60028fig6.jpg" /> 
Figure 6: Representative results of simultaneous recordings in the CA1 and MEC and optogenetic stimulation. (A) Expression of AAV5-DIO-ChR2-YFP after 4 weeks of injection. MEC layer III pyramidal neurons that project their axons from dorsal MEC to dorsal CA1. Dashed lines: ori, stratum oriens; pry, stratum pyramidale; rad, stratum radiatum; mol, stratum lacunosum moleculare. (B) Representative spike recording from one of the tetrodes. (a) 2D cluster projections of spikes recorded from the tetrode. (b) Examples of the average spike waveform of three clusters, which are indicated by dashed lines in (a). (C) Representative spike recording from one of the silicon-probe electrode sites. (a) 2D cluster projections of spike principal components. (b) Examples of the average spike waveform of three clusters. Spike clusters (pink and green) are separated from the noise clusters (blue). The clusters in (B,C) are calculated using KlustaKwik software. (D) Traces of spontaneous LFPs simultaneously recorded from the tetrodes in CA1 (a) and the silicon probe in MEC (b). Black arrows indicate the tetrode shown in (B) and silicon-probe electrode site shown in (C). (E) LFP responses to pulsed optical stimulation (10.6 mW, 1 ms; filled red arrowhead) from the tetrodes in CA1 (a) and silicon probe in MEC (b). <a href="https://www.jove.com/files/ftp_upload/60028/60028fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>grams/one</td>
			<td>number</td>
			<td colspan="2">sum [gram]</td>
		</tr>
		<tr>
			<td>main body</td>
			<td>1.25</td>
			<td>1</td>
			<td>1.25</td>
			<td></td>
		</tr>
		<tr>
			<td>shuttle</td>
			<td>0.04</td>
			<td>1</td>
			<td>0.04</td>
			<td></td>
		</tr>
		<tr>
			<td>probe connecter mount</td>
			<td>0.19</td>
			<td>1</td>
			<td>0.19</td>
			<td></td>
		</tr>
		<tr>
			<td>fiber ferrule holder</td>
			<td>0.1</td>
			<td>1</td>
			<td>0.1</td>
			<td></td>
		</tr>
		<tr>
			<td>shielding cone</td>
			<td>1.82</td>
			<td>1</td>
			<td>1.82</td>
			<td>(0.72)*</td>
		</tr>
		<tr>
			<td>conductive paste</td>
			<td>0.2</td>
			<td>1</td>
			<td>0.2</td>
			<td></td>
		</tr>
		<tr>
			<td>machine screw (#00, 2 mm), to hold EIB</td>
			<td>0.05</td>
			<td>2</td>
			<td>0.1</td>
			<td></td>
		</tr>
		<tr>
			<td>machine screw (#0-80, 3.5 mm)</td>
			<td>0.06</td>
			<td>4</td>
			<td>0.24</td>
			<td></td>
		</tr>
		<tr>
			<td>machine screw (#0-80, 6mm)</td>
			<td>0.09</td>
			<td>2</td>
			<td>0.18</td>
			<td></td>
		</tr>
		<tr>
			<td>nut</td>
			<td>0.03</td>
			<td>2</td>
			<td>0.06</td>
			<td></td>
		</tr>
		<tr>
			<td>microdrive (tetrode)</td>
			<td>0.05</td>
			<td>9</td>
			<td>0.45</td>
			<td></td>
		</tr>
		<tr>
			<td>microdrive (silicon probe)</td>
			<td>0.29</td>
			<td>1</td>
			<td>0.29</td>
			<td></td>
		</tr>
		<tr>
			<td>silicon probe</td>
			<td>0.28</td>
			<td>1</td>
			<td>0.28</td>
			<td></td>
		</tr>
		<tr>
			<td>electric interface board</td>
			<td>0.6</td>
			<td>1</td>
			<td>0.6</td>
			<td></td>
		</tr>
		<tr>
			<td>total</td>
			<td></td>
			<td></td>
			<td>5.8</td>
			<td>(4.7)*</td>
		</tr>
	</tbody>
</table>
Table 1: Individual weight of each microdrive array part. The total weight of the microdrive array was 5.9 g after fixing the protective cone with epoxy (*in the case of using an alternative shielding cone using a paper and aluminum tape).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>procedures</td>
			<td>time</td>
		</tr>
		<tr>
			<td>microdrive preparation</td>
			<td></td>
		</tr>
		<tr>
			<td>3D parts printing</td>
			<td>1 day</td>
		</tr>
		<tr>
			<td>optrode preparation</td>
			<td></td>
		</tr>
		<tr>
			<td>Prepare the mold for the microdrive head</td>
			<td>1 day*</td>
		</tr>
		<tr>
			<td>Microdrive head preparation</td>
			<td>3 h</td>
		</tr>
		<tr>
			<td>Attaching an optical fiber</td>
			<td>3 h</td>
		</tr>
		<tr>
			<td>Attaching a shuttle</td>
			<td>3 h</td>
		</tr>
		<tr>
			<td>tetrode preparation</td>
			<td></td>
		</tr>
		<tr>
			<td>Prepare the mold for the microdrive head</td>
			<td>1 day*</td>
		</tr>
		<tr>
			<td>Microdrive heads preparation</td>
			<td>3 h</td>
		</tr>
		<tr>
			<td>Loading tetrode wires</td>
			<td>1 day</td>
		</tr>
		<tr>
			<td>Attaching the shielding cone</td>
			<td></td>
		</tr>
		<tr>
			<td>Painting shielding paint</td>
			<td>overnight*</td>
		</tr>
		<tr>
			<td>Attaching to the microdrive body</td>
			<td>3 h</td>
		</tr>
		<tr>
			<td colspan="2">* these procedure can be conducted in parallel</td>
		</tr>
	</tbody>
</table>
Table 2: The timeline of the microdrive preparation. The 3D-parts printing, waiting for curing the silicone rubber/dental acrylic/epoxy, and loading the tetrode wires take the majority of the time of the microdrive array preparation, in total 4-5 days.
Supplementary Files: The supplementary files include 3D model data of five microdrive parts in both .sldprt and .stl format. The original 3D model files were created with the software Solidworks2003. <a href="https://www.jove.com/files/ftp_upload/60028/Supplementary_Files.zip">Please click here to download this file.</a>

Watch this Scientific Journal Video about Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice at JoVE.com

Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice

This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple purposes.

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, ...

hybrid-microdrive-system-with-recoverable-opto-silicon-probe-tetrode

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.9K Views.  University of Texas Southwestern Medical Center. This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple purposes.

Video: Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform natural behavior. We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allowed us to record activity from multiple neurons simultaneously while a tethered cockroach turned or altered walking speed. While a major advance, tethered preparations provide access to limited behaviors and often lack feedback processes that occur in freely moving animals. We now present a modified version of that technique that allows us to record from the central complex of freely moving cockroaches as they walk in an arena and deal with barriers by turning, climbing or tunneling. Coupled with high speed video and cluster cutting, we can now relate brain activity to various parameters of the movement of freely behaving insects.

We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allows us to monitor activity in individual units of tethered cockroaches. Here we present a modified version of that technique that allows us to also record brain activity in freely moving insects.

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform ...

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle (Mercynorrhina torquata)

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight insect behavior. This technology enables the development of an insect-machine hybrid system using a living insect platform described in this protocol. Moreover, this protocol presents the system configuration and free flight experimental procedures for evaluating the function of the flight muscles in an untethered insect. For demonstration, we targeted the third axillary sclerite (3Ax) muscle to control and achieve left or right turning of a flying beetle. A thin silver wire electrode was implanted on the 3Ax muscle on each side of the beetle. These were connected to the outputs of a wireless backpack (i.e., a neuromuscular electrical stimulator) mounted on the pronotum of the beetle. The muscle was stimulated in free flight by alternating the stimulation side (left or right) or varying the stimulation frequency. The beetle turned to the ipsilateral side when the muscle was stimulated and exhibited a graded response to an increasing frequency. The implantation process and volume calibration of the 3 dimensional motion capture camera system need to be carried out with care to avoid damaging the muscle and losing track of the marker, respectively. This method is highly beneficial to study insect flight, as it helps to reveal the functions of the flight muscle of interest in free flight.

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata

This protocol describes the process of constructing an insect-machine hybrid system and carrying out wireless electrical stimulation of the flight muscles required to control the turning motion of a flying insect.

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...

Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have revealed that gut microbiota-derived metabolites modulate brain-mediated physiological functions through intricate gut-brain pathways in the host. Short-chain fatty acids (SCFAs) are the major bacteria-derived metabolites produced during dietary fiber fermentation by the gut microbiome. Secreted SCFAs from the gut can act at multiple sites in the periphery, affecting the immune, endocrine, and neural responses due to the vast distribution of SCFAs receptors. Therefore, it is challenging to differentiate the central and peripheral effects of SCFAs through oral and intraperitoneal administration of SCFAs. This paper presents a video-based method to interrogate the functional role of SCFAs in the brain via a guide cannula in freely moving mice. The amount and type of SCFAs in the brain can be adjusted by controlling the infusion volume and rate. This method can provide scientists with a way to appreciate the role of gut-derived metabolites in the brain.

Gut-derived microbial metabolites have multifaceted effects leading to complex behavior in animals. We aim to provide a step-by-step method to delineate the effects of gut-derived microbial metabolites in the brain via intracerebroventricular delivery via a guide cannula.

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have ...