Written by Eric Casey, 2022

Construction of chronically implantable arrays for multi-site in-vivo electrophysiological recordings

Recording electrical activity, either local field potentials (LFP) or spiking, at multiple areas of the brain allows studying the brain at the level of circuits. This brings the potential of investigating properties that emerge from the interaction of multiple populations of neurons, such as measures of functional connectivity between brain areas and the activity of functional circuits (also called electomes). This protocol describes a method that combines the use of tetrodes, printable circuit boards (PCB) and 3D printing to build arrays of 16 channels split in 4 tetrodes, capable of recording from up to 4 brain areas (one tetrode in each brain area) (Fig 1A). These arrays can record both LFP and spikes for months, and the design of the 3D printed frame can be changed to target fewer brain areas and different coordinates. This protocol focuses on arrays that are built with Molex connectors and PCB adapted to them (Fig 1B). Molex connectors are approximately 20-fold cheaper than Omnetics, allowing for larger scale experiments. However, since the headstages of Plexon rigs and the nanoZ are only compatible with Omnetics connectors, an Omnetics-Molex adapter is needed. The method to build the Omnetics-Molex adapter is described at the Appendix: Omnetics-Molex adapter, at the end of this protocol. The designs of the PCBs and 3D printed frame, as well as the PCB for the adapter, are included in the folder of this protocol. Finally, almost identical procedures can be followed to build arrays with Omnetics connectors, and the design for the PCB for Omnetics connectors (Fig 1C) is also included in the folder of this protocol.

Fig 1. Chronically implantable arrays for multi-site in-vivo electrophysiological recordings. A) Finished array. B) PCB with Molex connector. C) PCB with Omnetics connector.

The construction of the arrays consist in the following steps:

1. Soldering PCB and connector
2. Soldering ground wire
3. Testing PCB for short-circuits and misconnections
4. Making tetrodes
5. Attaching tetrodes to PCB
6. Printing 3D frame
7. Attaching tetrodes and PCB to 3D printed frame
8. Cutting tetrodes to their final length
9. Straightening bent tetrodes
10. Testing impedances and gold plating
11. Final test for short-circuits and misconnections
12. Cover the gold pins with epoxy

1) Soldering PCB to connector

We have different designs of PCBs, here I’ll only focus on the 16 channels for Molex connectors, but the procedure is mostly the same for other PCBs and connectors (Fig 2).

Fig 2. PCB for 16-Channels, 2 grounds and Molex connector.

Since the connectors have 20 channels, there are 20 contacts in the PCB, but those at each end are all for ground wires (Fig 2). There are 16 small holes for connecting electrodes, and 2 big holes for ground wires (Fig 2). The first step is soldering the Molex connector to the PCB. That can be achieved with solder paste and soldering molds. There are 2 molds: one for Omnetics connectors, and one for Molex connectors. Make sure to use the Molex one, easily recognizable for having 2 rows of 10 holes each (Fig 3A), while the Omnetics one has rows with 8 and 10 holes respectively (Fig 3B).

Fig 3. Soldering PCB to connector. A) Mold for soldering 20-channels Molex connector to PCB. B) Mold for soldering 16-channels Omnetics connector. C) Using two L-shaped frames and tape, the PCB can be held to align it with the mold and put the solder paste.

1. Hold the PCB with frames (they are with the molds) and tape (Fig 3C).
2. Align the mold and the PCB such that each of the 20 connections of the PCB are visible through the small holes of the mold, and tape it (taping is not always necessary, but sometimes makes this step easier).
3. Put solder paste near to the holes, and slide a rigid object with a flat surface (like the plastic card that comes with the mold), applying pressure towards the PCB, to spread the solder paste to the holes.
4. Remove the mold pulling from a corner; ideally, the PCB should look like Fig 4A.

Fig 4. PCB with uncured solder paste. A) After putting solder paste using the mold and before adding the connector. B) Whit the connector.

5. Place the connector on the PCB using the white lines as a reference (each pin of the connector should be on a PCB connection, Fig 4B).
6. Put the PBC in the infrared heater (Fig 5A) on program one (Fig 5B). When the program finishes, the solder should be hard and the connector should be attached to the PCB (Fig 5D, compare with Fig 5C which shows the same PCB before going through the infrared heater). Alternatively, a heat gun can be used, making sure of not blowing the PCB and connector.
7. Put epoxy on the connections between PCB and connector and the sides of the connector to attach them strongly, as soldering is not enough to prevent disconnections due to posterior handling. Avoid to block the near electrode holes with epoxy.

2) Soldering ground wire/s:

1. Cut a piece of silver wire of ~5 cm
2. Remove the insulation of one tip (~3 mm)
3. Use solder and a classical soldering machine to solder the stripped end of the silver wire to one of the ground holes.
4. Optional: repeat with the other ground hole if two ground wires are required.

Fig 5. Using an infrared heater to solder the connector to PCB. A) Infrared heater with a PCB inside. B) Screen of the infrared heater screen, set to wave 1 which is the most appropriate program. C) PCB with solder paste and connector, before soldering with the infrared heater. D) PCB soldered to connector, after infrared heater.

3) Testing PCB for short-circuits and misconnections

Use a voltmeter (Fig 6A) in impedance mode to check for short circuits and misconnections:

1. Check both ground holes against each other: they should be connected (Fig 6B).
2. Check a ground hole against each electrode hole: there should not be any connection (Fig 6B).
3. Check every electrode holes pair combination: there should not be any connection (Fig 6B).
4. Check for connection between the PCB holes and the connector: place one voltmeter tip in a hole, and slide the other tip through the connections of the connector (Fig 6B). The voltmeter should beep at some point, otherwise it indicates that there is a misconnection.

Fig 6. Testing PCB for short-circuits and misconnections. A) Soldered PCBs and a voltmeter, used to test PCBs for short-circuits and misconnections. B) Connections to be checked. Numbers are according to the order in which they are explained in the text.

4) Making tetrodes (Eric's method)

I will describe a method to make teetrodes using the Open Ephys tetrodes twister (https://open-ephys.org/twister, Fig 7A) and nichrome wire (Fig 7B).

Fig 7. Materials to make tetrodes. A) Open Ephys tetrodes twister. B) Nichrome wire.

1. Cut a piece of nichrome wire of a length of 50 cm approximately.
2. Bend the wire to make a loop, and cut it to make two wires of 25 cm each, approximately (Fig 8A-B).
3. While holding both wires together from one of their tips, wet your fingers with water and slide them through both wires together (Fig 8C); this will stick the wires together (although this is not a necessary step, it eases considerably the following steps).
4. While holding both wires together from one of their tips, bend the two wires together to form another loop, putting the four tips together, and hold the tips together with tape (Fig 8D).
5. Hold the nichrome wires from the loop using an elevated horizontal rod, separating each wire with tape, and hold the taped tips with the clip of the twister (Fig 8E-F).
6. Set the tetrode twister to 40 spins forward and 5 backward (this generally works well for the indicated length of wires, but it is not critical), and start the twister. If the forward rotations are too many, the wires will get over-coiled and eventually break; in that case, reduce the number of spins forward.
7. Once the twister finishes, push up the holder slightly to release tension and cut the tip of the tetrode just above the tape (Fig 8G).
8. Keep the tetrodes on a soft surface in a safe place (Fig 8H).

Fig 8. Making tetrodes. A-D) Starting from a piece of nichrome wire of ~50cm, bend it to make a loop (A), and cut it in two pieces of equal size (B); stick both pieces together by sliding wet fingers through them (C), make another loop and stick the four tips together with tape (D). E) To twist the loop of wires to make a tetrode, an Open Ephys tetrodes twister and an elevated, horizontal rod are used. F) Magnification of F, showing the wires held from the loop with an elevated and horizontal rod, using tape to separate the wires from each other, and from the tape using the clip of the twister. G) Once twisted, the clip is slightly pushed up to release tension, and then the tip of the tetrode is cut close to the tape. H)Three finished tetrodes.

5) Attaching tetrodes to PCB (Eric's method)

This step consists in threading each wire of each tetrode through the channel holes of the PCB, and fastening them with the gold pins. Fastening with gold pins serves two functions: first, it holds the wire to the PCB; second, it removes the insulation of the wire, allowing for an electrical connection between the wire and the PCB.

1. Cut the loop of a tetrode through the middle (Fig 9A).
2. Thread each wire of the tetrode through consecutive channel holes (Fig 9B-C). It is critical that the channel's holes are consecutive to ensure that each channel records from the expected brain area (Fig 9E). Make sure that the tip of the tetrode will point to the correct side when the PCB is coupled to the 3D frame at Step 7: Attaching tetrodes and PCB to 3D printed frame (Fig 9C).
3. Thread the gold pins through the channel holes to fasten the wires (Fig 9C, and Fig 10).
4. Cut the wires at the side of the gold pins (not the tetrode), as short as possible to prevent them from touching nearby pins and causing connections between channels (Fig 9D).
5. Repeat steps 1 to 4 for every tetrode.

This step relies very much on how much practice the user has, and the way to do it depends on each user. However, the following is a list of suggestions to successfully complete this step (and keep your sanity =] ).

• Use a flexible arm to hold the PCB while attaching the tetrodes (Fig 9B-D).
• Always hold tetrodes with hands or tweezers with plastic tips (Fig 9B-C); avoid using tweezers with metal tips. Avoid bending or holding the tetrode in the part that will be implanted. Most of the length of the tetrode will be cut at the end, so holding the tetrode from the tip is recommended.
• Vary the light level and position to make it easier to see the wires. Using a scope helps sometimes.
• Use a plier with one tip cut to push the gold pins into the channel holes (Fig 10).

Fig 9. Attaching tetrodes to PCB. A) Tetrode indicating where the loop should be cut to attach the wires to the holes of the PCB. B-C) Procedure to attach tetrodes to the PCB: holding the PCB with a flexible arm, the wires of the tetrode are threaded individually through each hole. The side to where the tetrode should point and the side where the gold pins must be put are indicated in C. D) PCB with a tetrode attached, indicating where to cut the wires. E) PCB with a tetrode attached, view under a scope. Each wire is connected to a different hole, and the holes must be consecutive (1 to 4 in this case).

Fig 10. Using gold pins to attach wires to PCB holes. A) A plier with one tip cut. B) Use magnetized tweezers to gently put the gold pin into the hole, after the wire has been threaded. C) Use the plier with one tip cut to press the gold pin for it to get tightly attached to the hole. The longer tip is used to push the gold pin.

4) Making tetrodes & 5) Attaching tetrodes to PCB (Habby's method)

This method is largely the same as Eric's, but you will be attaching the wires to the PCB first then twisting the tetrodes. You will still use the Open Ephys tetrodes twister, nichrome wire, and the gold pins.

1. Cut a piece of nichrome wire of a length of 25 cm approximately.
2. Thread the wire through one of the channel holes, and thread the gold pin through that channel to fasten the wire at about half way.
3. Loop the other half of the wire through, and secure it with the gold pin.
4. You should now see a small loop on the gold pin's side. Cut off the connected wire so that the two channels aren't connected.
5. Repeat the above steps at another nearby consecutive channel holes to make 2 more.
6. Now hold all 4 pieces and secure the end with a small piece of tape. You car wet your fingers with water and slide them through the wires to help with sticking the wires together.
7. Hold the PCB flat with an octopus clamp so that the wires are resting vertically. Clamp the taped tips with the clip of the twister.
8. Set the tetrode twister to 30 spins forward and 5 backward. If it's getting too tight, you can change the settings.
9. Once the twister finishes, push up the holder slightly to release tension and unclip the tetrode twister.
10. Repeat the above steps for every tetrode.

6) Printing 3D frame

Print the 3D frame that will hold the PCB and tetrodes in the resin printer. A design to target the nucleus accumbens, prelimbic cortex, basolateral amygdala and ventral hippocampus (Fig 11A) can be found here: https://www.tinkercad.com/things/eDnOUgjttzG?sharecode=--Gtaq_sZYnWQLwjnWfw5LS_WctWCSpyz0KBggHi7xk . The number and the relative positions of the guiding tubes can be modified to target different brain areas, and modifications can be made to add optical fibers, electrodes for electrical stimulation, etc. Given the thinness of the guiding tubes, it will be necessary to manually remove the non-cured resin from the tubes by submerging a piece of optical fiber in isopropanol and passing it through the guiding tubes. If the rate of blocked tubes is too high, the holes can be opened with a microdrill of 0.4 mm diameter?.

7) Attaching tetrodes and PCB to 3D printed frame

This step consists in passing each tetrode through the guiding tubes of the frame, and then attaching the PCB to the holder (Fig 11A-B). Finally, tetrodes and PCB are glued to the frame. In this step, it is critical to make sure to pass the appropriate tetrode through the correct tube, and to be consistent across arrays; this means, the tetrode expected to target the brain area corresponding to that tube. This will ensure that every group of four channels will record from the same region in all the arrays. While the way to accomplish this step depends on the user, the following instructions may be a useful guide:

1. Hold the PCB with a flexible arm with the tetrodes pointing up (Fig 11C-D).
2. Cut the tetrodes for their length to be ~1cm different from each other, such that the first to be threaded will be the longest and the last one will be the shortest. This will facilitate putting the tetrodes in order without bending them excessively.
3. Holding the 3D printed frame with fingers and a tetrode with plastic tip tweezers, pass the first tetrode through the first guiding tube. Repeat for the successive tetrodes-tubes pairs (Fig 11C-D). Once the four tetrodes are in their tubes, the 3D printed frame can be released as it will be held by the tetrodes, as long as it is in vertical position.
4. Pass the ground wires though the ground wire holes (see Fig 11A).
5. Pass the tetrodes and ground wires through their holes until the 3D printed frame and the PCB are ~1cm away from each other, then place the small border of the PCB on the PCB holder of the 3D printed frame (see Fig 11A-B).
6. Put a fast glue (as Superglue) in the border between the PCB and the PCB holder of the 3D printed frame.
7. If required, put optic fibers (already cut to their appropriate length) in the fiber holder.
8. Use epoxy to glue the tetrodes, ferrule of the fibers and PCB to the 3D printed frame.

Fig 11. Attaching tetrodes and PCB to 3D printed frame. A) Three different views of the 3D printed frame, indicating its parts. B) PCB with Molex connector soldered and glued. The dashed rectangle indicates the edge that must be inserted in the PCB holder of the 3D printed frame. C-D) Threading tetrodes through guiding tubes of the 3D printed frame. A flexible arm is used to hold the pcb with the tetrodes pointing up, and the 3D printed frame is held and slightly moved in order to pass the tetrodes through the guiding tubes, while orienting the tetrodes with plastic tip tweezers.

8) Cutting tetrodes to their final length

Cut each tetrode to its desired length, according to the dorsoventral coordinates of the targeted areas in the brain. While the exact length is not very important (as long as tetrodes are long enough to reach the target brain areas, it is fine if they are a few millimeters larger than necessary), their relative length (how long a tetrode is compared with the others) must be as precise as possible. Using a scope, a caliper or ruler and a thin scissor, cut each tetrode to to a length of ~2 mm longer than the distance between the targeted area and the top of the brain (~2mm will ensure enough distance to prevent the guiding tubes from touching the skull) (Fig 12A). The scissor must be placed at 90° respect to the tetrode and must have a very good edge. Marking the point of the fiber to be cut with a marker first, may facilitate this step (Fig 12B).

9) Straightening bent tetrodes

It is very common for tetrodes to not be straight at this step. If that happens to one or more tetrodes, gently push each of them with plastic tip tweezers to straighten them. Do not grab the tetrodes nor apply pressure at this step, just push them gently.

Fig 12. Cutting tetrodes to its final length using a scope and a ruler. In B, a marker was used to mark the point to cut under the scope, and the this mark was used as reference to cut the tetrode without the scope.

10) Testing impedances and gold plating

Once the array is finished, the impedance of each channel must be measured to verify that it is low enough for good quality recordings. In general, impedances will be around 2-3 MΩ, or even higher (measured at 1 kHz). Then, tetrodes are electroplated to reduce impedances to ~200 kΩ (measured at 1 kHz), which increases the signal-to-noise ratio and allows for the recording of small amplitude signals; this is critical to record single unit spikes. When electric current passes through a solution of a salt of gold, gold cations are reduced to metallic gold at the tip of the electrode (cathode) and deposit, creating a gold coat that increases the conductive surface of the electrode tip, which lowers its impedance. We perform impedance testing and gold plating with NanoZ, whose software and manual can be downloaded here. To use NanoZ with our arrays, the files “prefs.ini” and “electrodes.ini” located at “...\AppData\Local\nanoZ” must be replaced by the files of the same name that can be found with this protocol. These modified versions of prefs.ini and electrodes.ini replace the default connectors and probes by a connector and probe that maps the 16-channels probe of Omnetics. In addition, to use the nanoZ with the arrays of this protocol, which have Molex connectors, a Omnetics-Molex adapter must be used (Fig 13, also see Appendix: Omnetics-Molex adapter for more information about the adapter).

Fig 13. Setup for electroplating using NanoZ. NanoZ must be holded with an appropriate frame, and the Omnetics-Molex adapter is plugged to the probe, and the array is connected to the adapter. Gold solution is also shown. A-C) Side, back and front view, respectively. D) Side view of the tetrodes tips immersed in gold solution.

1. Hold the nanoZ with an appropriate support (a scope’s frame with a 3D printed holder will work well, see Fig 13A), connect it to a computer and start the NanoZ software.
2. Connect the Omnetics-molex adapter to the NanoZ probe. Make sure to connect it in the appropriate orientation (see Fig 13B-C), otherwise the ground (as well as a number of channels) will be disconnected and the circuit will not be closed (see Error messages and troubleshooting using NanoZ).
3. Connect the array to the Omnetics-Molex adapter (Fig 13A-D).
4. Test impedances. To do this, immerse the tips of all tetrodes and the ground wire/s in saline (solution of NaCl 0.09% in water), select the “Test impedances” mode at the NanoZ software and set the parameters to 1004Hz of test frequency, 40 cycles and a pause of 200 msec, and click “Test probe” (Fig 14A-B, top). A report window should appear with the impedances of each channel, which can be saved as a text file (Fig 14A-B, bottom). A good sanity check is to test impedances without immersing the tetrode tips in saline, which will report high impedances (~20 MOhm or more, Fig 14A, bottom), and then test them again with the tips in saline, to confirm a reduction in the impedance (Fig 14B, bottom), which indicates that exist an electrical connection between the tetrodes wires and the connector.
5. Perform electroplating of the tetrodes. To do this, put the gold solution in a 15mL tube lid (or any vessel of similar size), and immerse the tips of all tetrodes and the ground wire/s in the gold solution (Fig 13D). Then, select the electroplating mode at the NanoZ software, set parameters to “Match impedances mode” mode, plating current of 0.2 µA and target of 200 kOhm at 1004 Hz; also set the number of runs, interval duration and pause to a reasonable trade off of result quality and duration (4 runs, 5 seconds interval and 1 second pause may be a good starting point). Finally, click “Autoplate” to start the electroplating (Fig 14C, top). A report window should appear, displaying the initial impedance and the impedance after electroplating, for each channel (Fig 14C, bottom). This report can be saved as a text file. Note that these reports will show open circuits in blue, good impedances in green and short circuits in red; however, this classification is based on thresholds set at the “Condition” field (Fig 14A-C, bottom), and channels marked in red in general will not have short circuits, but just impedances below the condition of the NanoZ. To further verify absence of short circuits, perform step 11: Final test for short-circuits and misconnections.
6. Recover gold solution. Gold solution can be reused several times and still achieve a good reduction of impedance.

Error messages and troubleshooting using NanoZ The two most common error that can be obtained during this procedures are: a. Signal clipped: this means that there is a short circuit in that channel, and the impedance will not be shown in the row of that channel but the row will be highlighted in yellow instead (Fig 14C, bottom, rows 5 and 6). If this happens in all the channels, or many of them, discarding the array will be the best option. If it happens in one or few channels, those channels can be discarded during the data analysis, once the recordings are done. b. Plating voltage out of compliance: this appears during electroplating and means that, given the current impedance, the voltage necessary to achieve the platting current is too high for the NanoZ to achieve it (Fig 14D). This happens when the circuit is open, and there are three possible scenarios: ● If it happens in a single channel, that channel may not have an electrical connection between the tetrode and the PCB, or between the PCB and the connector (the last is less likely if step 3, Testing PCB for short-circuits and misconnections, was performed correctly). In this case, that channel should be discarded during the analysis (and not used for average referencing during the recording). ● If it happens in all channels: ○ The simplest scenario is that the Omnetics-Molex adapter was plugged incorrectly, which causes the ground to not be connected. To fix it, change the orientation of the adapter and try again. Also, make sure that the ground wire is immersed in the gold solution and its tip is stripped. ○ If the prior does not fix it, the ground wire may not be well connected to the PCB. Replace the ground wire and make sure that the end that goes into the gold solution is stripped.

Fig 14. NanoZ software settings and reports. Setting (top) and report windows (bottom) for impedance testing without immersing the tetrodes tips in saline (A), impedance testing with the tetrodes tips immersed in saline (B), electroplating with gold (C), and “Plating voltage out of compliance” error during electroplating (D).

11) Final test for short-circuits and misconnections

This step is almost the same as step 3, but touching the gold pins of each channel instead of the holes. Using a flexible arm to hold the array will facilitate this step. Use a voltmeter in impedance mode to check for short circuits and misconnections:

1. Check both ground holes against each other: they should be connected.
2. Check a ground hole against each electrode gold pin: there should not be any connection
3. Check all electrode gold pins pair combinations: there should not be any connection
4. Check for connection between the PCB holes and the connector: place one voltmeter tip on a ground hole or a channel gold pin, and slide the other tip through the connections of the connector. The voltmeter should beep at some point, otherwise it indicates that there is a misconnection.

12) Cover the gold pins with epoxy

This step is optional, but it will prevent the gold pins from loosening. Cover all the gold pins with a layer of epoxy.

Appendix: Omnetics-Molex adapter

This protocol focuses on building arrays using Molex connectors, which are approximately 20-fold cheaper than Omnetics connectors. However, in order to use these arrays in a Plexon rig that uses headstages for Omnetics connectors, an adapter is needed to connect the arrays to the rig headstage, as well as to the NanoZ. The construction of an Omnetics-Molex adapter requires a PCB (the design can be found in this same folder) capable of connecting a female Molex connector at one side, and a female Omnetics connector at the other side (Fig 15, middle and right). This allows to make an electrical connection between the array with a male Molex connector (Fig 15, left) and the rig headstage or the Nanoz probe. The construction is almost identical to step 2, Soldering PCB to connector, but female Molex must be soldered in this case, and after soldering the Molex connector, an Omnetics connector must be soldered.

Building an Omnetics-Molex adapter:

1. Hold the PCB with frames (they are with the molds) and tape, with the contacts for the Molex connector pointing up.
2. Align the mold for Molex and the PCB such that each of the 20 connections of the PCB are visible through the small holes of the mold, and tape it (taping is not always necessary, but sometimes can make this step easier).
3. Put solder paste near to the holes, and slide a rigid object with a flat surface (like the plastic card that comes with the mold), applying pressure towards the PCB, to spread the solder paste to the holes.
4. Remove the mold pulling from a corner.
5. Place the female Molex connector on the PCB (each pin of the connector should be on a PCB connection).
6. Put the PBC in the infrared heater (Fig 5A) on program one (Fig 5B). When the program finishes, the solder should be hard and the Molex connector should be attached to the PCB.
7. Repeat steps 1 to 6, but using a mold for Omnetics and a female Omnetics.
8. Test adapter for short-circuits and misconnections. To do this, connect an array PCB with a Molex connector (like the one of Fig 5D) and a ground wire soldered (it could also be an array to which the gold pins have not been covered with epoxy) to the adapter, and connect the adapter to the 16-Channels headstage of the Plexon rig. While checking the signal in the Plexon software, touch one by one all the holes (or gold pins) of the array PCB with the tip of the ground wire. The signal of the channel corresponding to that hole should become flat, as it will be electrically connected to the ground.
9. Remove all connections and glue the Molex and Omnetics connectors to the PCB, putting epoxy on the connections between the PCB and the connectors, and the sides of the connectors.

Fig 15. Array PCB with male Molex connector (left) and adapter with female Molex (middle) and Omnetics (right) connectors.