Construction of an Improved Multi-Tetrode Hyperdrive for Large-Scale Neural Recording in Behaving Rats

Podsumowanie

We present the construction of a 3D-printable hyperdrive with eighteen independently adjustable tetrodes. The hyperdrive is designed to record brain activity in freely behaving rats over a period of several weeks.

Streszczenie

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems neuroscience. One key component of this technique consists of the precise placement of multiple electrodes into desired brain regions and the maintenance of their stability. Here, we describe a protocol for the construction of a 3D-printable hyperdrive, which includes eighteen independently adjustable tetrodes, and is specifically designed for in vivo extracellular neural recording in freely behaving rats. The tetrodes attached to the microdrives can either be individually advanced into multiple brain regions along the track, or can be used to place an array of electrodes into a smaller area. The multiple tetrodes allow for simultaneous examination of action potentials from dozens of individual neurons, as well as local field potentials from populations of neurons in the brain during active behavior. In addition, the design provides for simpler 3D drafting software that can easily be modified for differing experimental needs.

Wprowadzenie

In the field of systems neuroscience, scientists study the neural correlates underlying cognitive processes such as spatial navigation, memory, and decision-making. For these types of studies, it is critical to monitor the activity of many individual neurons during animal behavior. Over the past decades, two important advances have been made to meet the experimental needs for extracellular neural recording in small animals^1,2,3. First was the development of the tetrode, a bundle of four microwires used to record neural activity of neurons simultaneously^1,2,4. The differential signal amplitudes of activity across the four channels of a tetrode allows for the isolation of individual neuron activity from many simultaneously recorded cells⁵. In addition, the flexible nature of the microwires allows greater stability of the tetrode minimizing the relative displacement between the tetrode and the target cell population. Tetrodes are now widely used instead of a single electrode for many brain studies in various species, including rodents^1,2,6, primates⁷, and insects⁸. Second was the development of a hyperdrive carrying multiple independently movable tetrodes, which allows for the simultaneous monitoring of neural activity from larger populations of neurons from multiple recording locations^3,9,10,11,12.

The availability of a reliable and affordable multi-tetrode recording device for small animals is limited. The classic hyperdrive, initially developed by Bruce McNaughton¹³, has been successfully used for neural recordings in freely behaving rats in many labs in the past two decades^9,10,14,15. However, for technical reasons, the original components needed to build the McNaughton drive are now very difficult to obtain and are not compatible with recently improved data acquisition interfaces. The other well accepted design of hyperdrive requires the microdrives to be individually handcrafted, which could yield inconsistent results and consume substantial time¹². In order to record neural activity from various brain regions in behaving rats, we developed a new hyperdrive using stereolithographic technology. We sought to satisfy the following requirements: (1) the new hyperdrive must allow precise displacement of tetrodes in the brain and provide stable recording from multiple target regions; (2) the new hyperdrive must be compatible with the magnetic quickclip system recently developed to allow easy connection; and (3) the new hyperdrive can be accurately reproduced with materials easily available. Here, we provide a technique for building the 3D-printable hyperdrive containing eighteen independently movable tetrodes, based upon the McNaughton design. In the protocol, we describe the details of the fabrication process of the new hyperdrive, which we have used successfully to record single-neuron action potentials and local field potentials from the postrhinal and medial entorhinal cortices over weeks in a freely behaving rat during natural foraging tasks.

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Protokół

1. Stereolithography of 3D Models

1. Use stereolithographic techniques to print the hyperdrive parts and accessories. Each hyperdrive is comprised of eighteen shuttles, eighteen shuttle bolts, and one each of all other plastic pieces (Figure 1).
   NOTE: The accessories are not part of the hyperdrive but are necessary for hyperdrive construction.

2. Preparation of Accessories (Figure 2).

1. Preparation of the microdrive rack (Figure 2C).
   1. Clean and expand the smaller through-holes and the larger blind-holes in the rack with a ø 0.71 mm (0.028") drill bit and a ø 0.84 mm (0.033") drill bit, respectively.
   2. Cut a ø 0.89 mm (0.035") welding rod into 17 mm long segments, round both ends, and insert each guide rod into the ø 0.84 mm (0.033") holes on the rack, leaving 11.5 mm outside (flush with the threaded rods).
   3. Fully insert six 0-80 threaded, 15.88 mm (5/8") long flat head screws down into the slots in the rack. Ensure that the guide rods and threaded rods are straight and parallel to one another. Fill the remaining space in the slots with dilute dental cement. Air dry on a benchtop for 15 min.
   4. Glue the welding rods and screws into the rack with thin super glue and allow to air dry for 15 min.
2. Preparation of the core station (Figure 2E) .
   1. Thread the four holes with a 2-56 tap, and use 2-56, 4.76 mm (3/16") long nylon screws to secure the core in the station, if necessary.
3. Preparation of the turning tool (Figure 2F) .
   1. Thread the hole on the handle with a 4-40 tap. Insert the machined tip into the slot in the handle and secure with a 4-40, 4.76 mm (3/16") long cup screw.
4. Preparation of the hyperdrive holder (Figure 2G) .
   1. Thread the screw hole with an 8-32 tap. Use an 8-32, 9.52 mm (3/8") long nylon thumb screw to secure the hyperdrive when in use.
5. Preparation of the rod positioning complex (Figure 2H).
   1. Thread the stem from the side with the larger hole (top) with an 8-32 tap to a depth of about 7 mm. Thread the smaller holes (six in the top, eighteen in the bottom) with a 0-80 tap. Expand the central hole in the top with a ø 4.76 mm (3/16") drill bit, if necessary.
   2. Assemble the stem to the top, using an 8-32, ø 4.76 mm (3/16"), 6.35 mm (1/4") long shoulder screw. Secure the bottom to the top with 0-80, 6.35 mm (1/4") long screws when in use.

3. Preparation of the Hyperdrive Components (Figure 3).

1. Preparation of the hyperdrive nut (Figure 3A).
   1. Using the nut holder (Figure 2D), thread the nut with a 3/8-24 bottoming tap until smooth.
2. Assembly of the hyperdrive core (Figure 3B).
   1. Clean and expand the holes in the core using different sized drill bits (twelve ground wire through-holes (inner ring):ø 0.61 mm (0.024"); the eighteen tetrode through-holes (middle ring): ø 0.66 mm (0.026") first, and then ø 0.71 mm (0.028"); the eighteen guide rod blind-holes (outer ring): ø 0.84 mm (0.033")).
   2. Thread the two through-holes on top of the core and the remaining eight blind-holes (four on the side, four near the bottom) with a 0-80 tap. Use a bottoming tap for the blind-holes.
   3. Create external threads at the base of the core using a 3/8-24 die. Adjust the die properly so the hyperdrive nut will fit over the new threads.
   4. Depending on the number of ground wires desired, insert multiple 6 mm long segments of 23-gauge metal tubing (cannulas) into the ground wire holes in the core, gluing them if necessary. File the ends of the ground wire cannulas until flush with the outside of the core, and clean the cannulas with a ø 0.30 mm (0.012") steel wire.
   5. Fully insert eighteen 0-80, 15.88 mm (5/8") long flat head screws head down into the slots in the core. Do not bend the screws or damage the threads during this process.
   6. Using the rod positioning complex and the core station, position eighteen 17 mm segments of ø 0.89 mm (0.035") welding rod over the guide rod holes in the core and hammer them down to be flush with the screws (about 5 mm).
   7. Correct the positions of the welding rods and screws if necessary, then tighten the central shoulder screw and the surrounding six screws in the rod positioning complex to secure the outward directions of the rods in the core. Screw the nut onto the core (with the rod positioning complex) and fit the core into the hyperdrive holder to allow easier positioning under a stereoscope.
   8. Fill the slots with dilute dental cement to secure the screws to the core and allow air drying for 15 min. Fill 2-3 slots at a time before the dental cement gets too thick. Scrape away any excess dental cement on the core to maintain a proper fit with the shield.
   9. Glue the screws and rods into the core with thin super glue, allow air drying for 15 min.
3. Assembly of the microdrive (Figure 3C) .
   1. Clean and expand the two outer holes in the shuttle with drill bits (smaller hole: ø 0.61 mm (0.024") drill bit; larger hole: ø 0.89 mm (0.035") drill bit).
   2. Insert the shuttle bolt into the bolt holder base. Pay attention to the orientation. Close the bolt holder lid, hold tightly, and thread slowly through the hole in the lid with a 0-80 tap. Tap 2-3 times until smooth.
   3. Insert the shuttle bolt into the shuttle from the side with the smaller opening. Place the shuttle-shuttle bolt complex upside-down in the microdrive assembly station base.
   4. Cut a 15 mm segment of 23 gauge metal tubing and smooth both ends, then position the tubing over the ø 0.61 mm (0.024") hole, guided by the slot on the station Lid. Hammer the cannula into the hole until the upper end is flush with the station Lid.
   5. Remove the outer half of the upper tip of the cannula with a sanding wheel. Clean the cannula with a ø 0.30 mm (0.012") metal wire. Glue the cannula onto the shuttle using thin super glue, making sure not to glue the shuttle bolt to the shuttle, and air dry for 15 min.
   6. Prepare at least eighteen microdrives, test the microdrive on the microdrive rack. Make sure that the shuttle bolt can rotate smoothly in the shuttle and that the entire microdrive moves freely along the length of the threaded rod.
4. Preparation of the central column (Figure 3D).
   1. Sand the top and bottom of the central column until flat, if needed. Thread the two holes in the central column with a 0-80 tap. Insert a 0-80 hex nut (3.18 mm (1/8") wide, 1.19 mm (3/64") high) into each slot.
5. Preparation of the hyperdrive cap (Figure 3E).
   1. Using non-magnetic forceps, glue four magnets (3 mm in diameter, 1 mm thick) into the four wells, matching them to the N and S poles on the electrode interface board.
6. Assembly of the guide cannulas into a bundle (Figure 3F).
   1. Place eighteen 30 gauge, thin wall cannulas (ID 0.19 mm,0.0075") into ø 2.29 mm (0.09") heat-shrink tubes (3-5 mm long, spaced apart along the bundle by 5-10 mm). Make all cannulas flush with one another on one end of the bundle.
   2. Shrink the heat-shrink tubes using a heat gun until the bundle is tight. Squeeze the bundle gently to shape it as desired (round or oval). Confirm that all cannulas are in the correct positions with no twisting, crossing, or bending.
   3. Mark the area(s) for soldering on the cannulas. The unsoldered portion should be 26 mm in length, while the soldered portion should be 5-10 mm. Move the shrinking tubes to the soldering marks to prevent spreading.
   4. Apply flux to one soldering area and solder while rotating the bundle. Cool at room temperature for at least 1 min. Repeat this step to solder the same area two more times. Smooth out the soldered portion by soldering without applying flux and filler material. Cool at room temperature for at least 1 min.
   5. Cut the bundle to the proper length with a diamond wheel at the highest speed, polish both ends to adjust the length (unsoldered part: 26 mm, soldered part: 5-10 mm as desired). Clean the guide cannulas with a ø 0.18 mm (0.007") metal wire under a stereoscope.
7. Preparing the tetrodes. Similar procedures have been described^8,16,17 .
   1. Adjust the height of the horizontal T bar and the position of the magnetic stirrer, so that the horizontal arm at the cross of the T bar is directly above the center of the magnetic stirrer. Hook one end of an S-hook to the center of a small magnetic stir bar, then glue them together. Clean the tetrode making space with compressed air and ethanol wipes.
   2. Circle the two ends of a piece of single tetrode wire around 40 cm in length together, then secure with a piece of copper tape.
   3. Lift the wire circle by holding the copper tape. Place the end opposite to the copper tape onto the horizontal arm of the T bar. Lower the copper tape gently (while the other end is still on the T bar), twist once, and place the copper tape onto the T bar. The tetrode circle is now in a figure eight ("∞") configuration with the copper tape sitting on top of the cross of the horizontal bar.
   4. Hold the copper tape on the T bar with one hand gently. With your other hand, hook the free end of the S-hook (with a magnetic stir attached to the other end) through the bottom of the tetrode wire circle, release the S-hook gently and let it straighten the four wires by the weight of the S hook.
   5. Adjust the height of the horizontal bar until the bottom of the S-hook is about 1 cm above the center of the magnetic stirrer plate.
   6. Bend the edge of the copper tape down to secure it to the horizontal bar. Examine the four straight tetrode wires by eye, then remove any debris.
   7. Turn on the stirrer twisting the four wires at a speed around 60 rpm, until the angle between the two opposite untwisted wires is about 60°.
   8. Set the heat gun to 210 °C, and heat the twisted wires by sweeping the gun along the straight length of the wires from different angles for 2 min to fuse them together by melting the VG bond coat.
   9. Lift the S-hook with stir gently and cut the lower end of the tetrode with fine scissors.
   10. Hold the copper tape on the horizontal bar with a finger, cut the wires from both edges of the copper tape with scissors, and remove the copper tape. Cut the remaining wire on the horizontal bar to release the tetrode.
   11. Place the completed tetrode in a dust-free box for storage. Prepare at least twenty-five tetrodes.

4. Assembly of the hyperdrive (Figure 4).

1. Inserting the guide cannulas into the hyperdrive core (Figure 4A).
   1. Remove the heat-shrink tubes and slide a 4 mm segment of silicon tubing (ID 1.02 mm (0.04"), OD 2.16 mm (0.085")) along the bundle to the soldered/unsoldered border. Wedge the slit in the hyperdrive spacer to widen the central hole, allowing the spacer to slip around the silicon tube. Remove the wedge when the spacer sits at the center of the silicon tube.
   2. Organize the positions of the guide cannulas in the bundle by placing long segments (10 cm) of the ø 0.18mm (0.007") metal wire through each cannula into a specific tetrode hole in the hyperdrive core, preventing any crossover of the wires or cannulas in the process. Bend the ends of the wires to hold them in place.
   3. Push the cannulas through their respective holes in the core, being careful to avoid bending or crossing between them, until the free end of each cannula is at least 2 mm outside the upper end of the tetrode hole. Secure the spacer by screwing the nut onto the core, being careful to prevent the spacer from rotating. Apply a drop of very dilute dental cement from the top of the core onto the junction of the cannulas to secure their relative positions.
   4. Cut the guide wires from the soldered end of the bundle, and remove them from the cannulas by retracting from the free end.
2. Assembly of the microdrives onto the hyperdrive Core (Figure 4B). A detailed spatial arrangement of the microdrives in the hyperdrive has been previously described^11,13.
   1. Load the microdrives slowly and carefully onto each threaded rod of the core. Confirm that (1) the 23 gauge microdrive cannula goes into the tetrode hole smoothly, (2) the 30 gauge guide cannula goes into the 23 gauge microdrive cannula smoothly, and (3) the shuttle bolt turns smoothly along the threaded rod. Screw the microdrives down to 1.0-1.5 mm above the lower end of the threaded rods.
   2. Cut eighteen pieces of polyimide tubing (ID 0.11 mm (0.0045"), OD 0.14 mm (0.0055")) into 38-43 mm segments (length of the guide cannula bundle plus 7 mm). Clean each tube with a ø 0.08 mm (0.003") steel wire.
   3. Invert the core, insert the polyimide tubes carefully into the guide cannulas from the soldered end, and push them all the way in under a stereoscope. Flip the core upright and glue the upper end of the polyimide tube onto the microdrive cannula with thick super glue. Place the core upside-down and let the glue dry for 15 min.
   4. Cut the extra polyimide tubing at the upper end, leaving 0.5-1.0 mm outside of the microdrive cannula.
3. Assembly of the ground wires (Figure 4C).
   1. Cut the number of ground wires necessary to lengths of 25-30 mm from coated steel wire (coated ø 0.20 mm (0.008"), bare ø 0.13 mm (0.005")). Strip 2 mm of the plastic insulation from both tips of the wires and insert one end of each into the ends of 6-8 mm long 30 gauge cannulas. Flatten the ends of the cannulas to secure the connection to their respective wires.
   2. Use a Dremel tool to cut the cannulas in half to create two complete ground wires from each.
   3. Insert the round end of the 30 gauge cannula into the upper end of the ground wire cannula in the core and press to make the insertion tight.
4. Assembly of the electrode interface board (Figure 4D).
   1. Insert the central column into the core and secure with two 0-80, 7.94 mm (5/16") long socket head screws. Glue if necessary to make the central column steady in the core.
   2. Expand the portions of the slots in the EIB-72-QC-Large board that correspond to the two tapped holes in the central column with a ø 1.2 mm tap. Attach the electrode interface board to the central column with two 0-80, 3.97 mm (5/32") long pan head screws. Make sure the board is situated in the center and is secure.
5. Connecting the ground wires (Figure 4E).
   1. Route each ground wire around the central column and connect the exposed free end to the electrode interface board with a gold pin at the designated ground hole.
6. Loading the tetrodes into the hyperdrive, as previously described^16,17 .
   1. Load each tetrode carefully into the polyimide tubes of the microdrives, being careful not to bend them during the process.
   2. Gently feed the free end wires into their designated holes in the electrode interface board and electrically connect them using gold pins.
   3. Cut the tetrodes individually to a proper length. Confirm that the portion of tetrodes protruding from the lower ends of the polyimide tubes after cutting is straight, otherwise replace the entire tetrode and recut.
7. Attaching the shield.
   1. Attach the shield to the core using four 0-80, 3.97 mm (5/32") pan head screws. The numbers on the shield should match with the numbers on the electrode interface board.
8. Plating the tetrode tips.
   1. Plate the tips of the tetrodes using the NanoZ plating device equipped with an ADPT-NZ-EIB-36 connector and an ADPT-EIB-72-QC-HS-36 adaptor¹⁷. Alternatively, plate them manually one by one as described elsewhere¹⁶. Plate the tetrode tips prior to use (e.g., one day before implantation), as impedance will gradually increase over time after plating. Replace the tetrodes that are shorted or obstructed during the process of plating, cut them to a proper length, and re-plate.
9. Finalizing the hyperdrive (Figure 4F).
   1. Glue the tetrodes to their polyimide tubes as previously described¹⁶. Retract all of them back into their guide cannulas so the plated tips are not exposed.
   2. Screw four 0-80, 6.35 mm (1/4") long socket head screws into the four holes near the bottom of the hyperdrive core.
   3. Using a stereoscope, lower each tetrode slowly until the tip of the tetrode is just above the edge of the guide cannula. Meanwhile, locate the position of each tetrode in the guide cannula bundle. The map of the tetrode's position is critical for reconstruction of recording sites.
   4. Attach the cap to the drive and store the hyperdrive properly for implantation.

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Wyniki

We used a newly built hyperdrive to obtain trial results. The drive was equipped with tetrodes constructed from ø 17 µm (0.0007"), polyimide-coated platinum-iridium (90%-10%) wire. The tips of the tetrodes were plated in platinum black solution to reduce electrode impedances to between 100 and 200 kΩ at 1 kHz. The hyperdrive was implanted 4.6 mm left of the midline and 0.5 mm anterior to the transverse sinus on the skull of a 550 g, male Long-Evans rat. Additional groun...

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Dyskusje

Here, we describe the process of constructing a newly developed hyperdrive comprised of eighteen independently movable tetrodes. The drive can be constructed from affordable parts purchased at many available hardware stores, combined with components created by stereolithographic printing. The hyperdrive can be chronically implanted onto a rat's skull using standard surgical procedures and is capable of recording extracellular neural activity while the animal performs various behavioral tasks.

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Materiały

Name	Company	Catalog Number	Comments
Welding rod	Blue Demon	ER308L-035-01T	Stainless steel, 0.035" in diameter
Screw	McMaster	91771A060	Stainless steel, flat head, 0-80 thread, 5/8" in length
Screw	McMaster	91772A051	Stainless steel, pan head, 0-80 thread, 5/32" in length
Screw	McMaster	92196A056	Stainless steel, socket head, 0-80 thread, 5/16" in length
Screw	McMaster	92196A055	Stainless steel, socket head, 0-80 thread, 1/4" in length
Screw	McMaster	95868A131	Nylon,  socket head, 2-56 thread, 3/16" in length, black
Screw nut	McMaster	90730A001	Stainless steel, narrow hex,  0-80 thread
Shoulder screw	McMaster	90298A213	Stainless steel, 8-32 thread, 3/16" in diameter, 1/4" in length
Cup screw	McMaster	92313A105	Stainless steel, 4-40 thread, 3/16" in length
Thumb screw	McMaster	94323A592	Nylon, 8-32 thread, 3/8" in length, black
Magnet	Apex	M3X1MMDI	Neodymium, 3 mm X 1 mm disc
Metal tubing	Small Parts	B00137QHNS	Stainless steel, 23 gauge, 0.0253" OD, 0.013" ID, 0.006" wall
Metal tubing	New England Small Tube	Custom-made	Stainless steel, 30 gauge, 0.012/0.0125" OD, 0.007/0.008" ID, full hard
Heat-shrink tubing	McMaster	7856K72	0.09" ID before shrinking, blue
Silicone tubing	A-M Systems	807300	0.040" ID, 0.085" OD
Polyimide tubing	A-M Systems	823400	0.0045" ID, 0.0005" wall
Ground wire	A-M Systems	791500	0.005" bare, 0.008" coated, half hard
Tetrode wire	California Fine Wire	Custom-made	0.0007" in diameter, platinum-iridium (90%-10%), HML and VG coating
EIB	Neuralynx		EIB-72-QC-Large
Gold pins	Neuralynx		large EIB pins
Tap	Balax	01302-000	M1.2 thread size
Tap	McMaster	2522A811	0-80 thread size, bottoming
Tap	McMaster	2522A771	0-80 thread size, plug
Tap	McMaster	26955A94	3/8"-24 thread size, bottoming
Tap	McMaster	2522A713	2-56 thread size
Tap	McMaster	2522A715	4-40 thread size
Tap	McMaster	2522A718	8-32 thread size
Die	McMaster	2576A457	3/8"-24 thread size, 1" OD
Drill bit	McMaster	30585A82	Wire gauge 65, 0.035" in diameter
Drill bit	McMaster	30585A83	Wire gauge 66, 0.033" in diameter
Drill bit	McMaster	30585A87	Wire gauge 70, 0.028" in diameter
Drill bit	McMaster	30585A88	Wire gauge 71, 0.026" in diameter
Drill bit	McMaster	30585A91	Wire gauge 73, 0.024" in diameter
Drill bit	McMaster	8870A23	3/16" in diameter
Dremel disc	Wagner	31M	Diamond coated, 22 mm in diameter, 0.17 mm in thickness
Steel wire	Precision Brand	21212	0.012" in diameter, full hard
Steel wire	Precision Brand	21007	0.007" in diameter, full hard
Steel wire	A-M Systems	792700	0.003" in diameter, half hard
Super glue	Loctite	LT-40640	# 406
Super glue	Loctite	LT-41550	# 415
Dental acrylic powder	Teets	223-3773	Coral
Dental acrylic liquid	Teets	223-4003