The design and assembly of microdrives for in vivo electrophysiological recordings of brain signals from the mouse is described. By attaching microelectrode bundles to sturdy driveable carriers, these techniques allow for long-term and stable neural recordings. The lightweight design allows for unrestricted behavioral performance by the animal following drive implantation.
State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field potentials (LFPs) from populations of neurons and action potentials from individual cells, as the animal engages in experimentally relevant tasks. Chronically implanted microdrives allow for brain recordings to last over periods of several weeks. Miniaturized drives and lightweight components allow for these long-term recordings to occur in small mammals, such as mice. By using tetrodes, which consist of tightly braided bundles of four electrodes in which each wire has a diameter of 12.5 μm, it is possible to isolate physiologically active neurons in superficial brain regions such as the cerebral cortex, dorsal hippocampus, and subiculum, as well as deeper regions such as the striatum and the amygdala. Moreover, this technique insures stable, high-fidelity neural recordings as the animal is challenged with a variety of behavioral tasks. This manuscript describes several techniques that have been optimized to record from the mouse brain. First, we show how to fabricate tetrodes, load them into driveable tubes, and gold-plate their tips in order to reduce their impedance from MΩ to KΩ range. Second, we show how to construct a custom microdrive assembly for carrying and moving the tetrodes vertically, with the use of inexpensive materials. Third, we show the steps for assembling a commercially available microdrive (Neuralynx VersaDrive) that is designed to carry independently movable tetrodes. Finally, we present representative results of local field potentials and single-unit signals obtained in the dorsal subiculum of mice. These techniques can be easily modified to accommodate different types of electrode arrays and recording schemes in the mouse brain.
The use of the microelectrode technique for recording extracellular neural signals in vivo has a long and valued tradition in neuroscience 1, 2. The ability to record electrical activity from many brain regions in freely behaving animals is, however, a more recent technology that is becoming increasingly common as the software packages for the acquisition, analysis and discrimination of neural signals becomes more sophisticated and user-friendly 3, 4. The technological advances on the software side have also been accompanied by reductions in the weight and bulk of the implantable devices, which have been scaled down sufficiently for recording in small mammals, such as mice. By using lightweight (mostly plastic) components, researchers are able to construct microdrives that allow for independent positioning of electrodes or tetrodes to target a wide variety of brain regions 5-7. Even deep brain structures, such as the amygdala 6 and the striatum 5, can be routinely targeted with the selection of an appropriately long drive screw. These recording techniques allow researchers to obtain high-fidelity neural signals and are in register with the electrical activity of single neurons recorded intracellularly 8, 9. Using these types of microdrives, we have successfully recorded single-units from mice for up to two months after implantation 10. In addition, the lightweight nature of the devices (approximately 1.5-2.0 g) has resulted in behavioral performance that is comparable to non-implanted mice in many behavioral tasks. In particular, we have demonstrated that implanted mice exhibit normal performance in the novel object recognition task 10 and the object place task (unpublished data).
The use of microdrives coupled to multiple tetrodes allows researchers to monitor and analyze neural activity at the network level while also recording from multiple single-units within the brain. Recording with these tetrodes has several major advantages for unit identification purposes and enables the high accuracy acquisition and discrimination of multiple single-units 11. We describe how to fabricate and gold-plate tetrode bundles and then subsequently load them into driveable electrode carriers. One type of drive carrier we describe is commercially available and the other is a simple, but easily expandable, drive design that can accommodate multiple carriers and tetrode arrangements without a significant investment of resources.
1. Tetrode Fabrication
2. Custom Microdrive Assembly
3. VersaDrive Assembly
4. Gold-plating of Electrode Tips
After implanting the microdrive and lowering the electrodes to the intended brain targets, an amplified data acquisition system, such as a Neuralynx Lynx-8, is needed for recording neural signals. Representative neural recordings of local field potentials (LFPs) and single-unit action potentials (often termed “spikes”) from the mouse dorsal subiculum are shown in Figure 2. LFP signals were sampled at 3 kHz and band-pass filtered between 0.1-500 Hz (Figure 2A and 2B). Figure 2A shows four continuously sampled LFP channels with a poorly grounded signal. These signals appear extremely noisy and easily saturate within the given amplitude range. Figure 2B shows an example of four channels from well-grounded LFP signals, with clearly visible network oscillations in the theta range (4-12 Hz). LFP signals were visualized in the “1D Data Viewer” using NeuroExplorer (version 4.0) data analysis software.
Single-unit spike channels were sampled at 30 kHz and band-pass filtered between 0.5-9 kHz. The main advantage of using tetrodes is that they allow signals from putative individual neurons to be triangulated between several recording points for improved unit discrimination. The left panel of Figure 2C shows an example of a poor tetrode recording because the four electrode wires have registered essentially identical spike waveforms throughout the recording session. Over a thousand individual spike waveforms (n = 1458) were overlaid on top of each other for each channel of the tetrodes, with the averaged waveform shown in yellow. This pattern was likely due to the fusing of the wires together during tetrode fabrication (during the insulation melting step), causing the bundle of wires to effectively act as a single recording electrode. It is also possible that this pattern was due to a referencing artifact or that a source that is equidistant from all four electrodes was responsible for the signal. These possibilities cannot be excluded but they can be minimized. Typically within the amplifier system, a recording channel can be user-assigned to be a reference channel. This helps to troubleshoot problematic ground connections and references and allows flexibility in obtained low-noise recordings. The right panel of Figure 2C shows an example of a good tetrode recording showing overlaid spike waveforms (n = 1939) from a putative unit with different amplitudes across the four tetrode wires. This type of spike recording pattern allows for improved unit discrimination during subsequent offline clustering and separation. Spike waveforms were visualized using Plexon Offline Sorter (version 2.8.8).
Name of Reagent/Material | Company | Catalog Number | Comments |
0.0005″ (12.5 μM) diameter Platinum-Iridium wire | California Fine Wire | CFW#100-167 | HML VG insulated www.calfinewire.com |
0.002″ (50 μM) diameter Stableohm 675 wire | California Fine Wire | CFW# 100-188 | HML insulated Ni-Cr |
polyamide tubing | Polymicro Technologies | 1068150020 | 99 micron I.D., 166 micron O.D. www.polymicro.com |
brass guides | World Plastics Inc | 3.3 x 6.6 mm | |
Delrin blocks | World Plastics Inc | 3.13 x 2.5 mm | |
Fillister head brass screws | J.I. Morris Co. | 00-90 x 1/2 | drive screw www.jimorrisco.com |
hex brass nuts | J.I. Morris Co. | 00-90 | |
Fillister head brass screws | J.I. Morris Co. | 000-120 x 3/32 | EIB mount and ground screw |
plexiglass acrylic | Canal Street Plastics | 5 mm thick, clear, www.cpcnyc.com | |
cyanoacrylate | Krazy Glue | 2 g tube | |
electronic interface board | Neuralynx | EIB-18 | www.neuralynx.com |
non-cyanide gold solution | SIFCO | SIFCO 5355 | www.sifcoasc.com |
VersaDrive 4 | Neuralynx | four tetrode model | |
tetrode assembly station | Neuralynx | ||
motorized tetrode spinner | Neuralynx | tetrode spinner 2.0 | |
VersaDrive jig | Neuralynx | ||
soldering iron | Radio Shack | 64-2802B | www.radioshack.com |
nanoZ | Neuralynx | ||
small bit drill/driver | Ram Products | Rampower 35 | with footpedal controller, www.ramprodinc.com |
drill bits | Small Parts, Inc. | 3/32″ bits, www.smallpartsinc.com | |
dissecting microscope | Olympus | SZ-60 | www.olympusamerica.com |
heat gun | Alphawire | Fit gun 3 | use setting “1” only, www.alphawire.com |
26 AWG copper wire | Arcor Electronics | F26 | for ground wires, www.arcorelectronics.com |
soldering flux | Eagle | 2 oz, #205 | |
0.02″ diameter solder | Kester | 24-6337-0010 | www.kester.com |
benchtop vise | Vacu-Vise | Model 300 | |
fiber optic light | Nikon | MKII | dual light arms, www.nikon.com |
5-min epoxy | Allied Electronics | 25 ml, www.alliedelec.com | |
fine tweezers | Roboz Surgical Instrument Co. | RS-4907, RS-5010 | INOX material, www.roboz.com |
micro dissecting scissors | Roboz Surgical Instrument Co. | RS-5880 |
Table 1. Materials and reagents used for constructing tetrodes and microdrives.
Figure 1. Completed VersaDrive 4 assembly. This picture shows a completed VersaDrive with four tetrodes extending from the bottom and two ground wires protruding from the sides. The green chip on top is an Omnetics adapter that plugs directly into the VersaDrive and is used to connect with a Neuralynx amplifier system.
Figure 2. Representative local field potentials and single-unit spike waveforms. Local field potentials (LFP) and single-unit recordings obtained from electrodes implanted in the dorsal subiculum of a mouse. A. The traces represent 10 sec of LFP signals shown from four continuously sampled channels with a poor electrical ground. B. The traces represent 3 sec of LFP signals from four continuously sampled channels with a proper electrical ground, which show a stable, continuous baseline with visible network oscillations in the theta range (4-12 Hz). Episodes of theta activity are marked by grey bars. C. The left panel shows an example of a poor tetrode spike pattern, with all four channels displaying comparable spike waveforms from a putative individual unit. The right panel shows an example of good tetrode spike waveforms with a range of waveforms across the four channels. The average waveforms for each tetrode channel are shown in yellow; scale bars, 2 msec (x axis), 200 μV (y axis). Click here to view larger figure.
We have described a set of techniques for constructing light and compact microdrives for the recording of extracellular unit and field potential activity in mice. By building custom microdrives with bases fashioned from acrylic glass (methyl methacrylate), the core system can be easily adapted for multiple drives and for the targeting of a wide array of neural regions. We have successfully modified the system for recording from multiple brain targets and with larger arrays for recordings in mice. With further modification, motorized drive elements can be incorporated to allow for remote, and potentially more precise, electrode placement 7.
We would like to stress that these recording devices give the researcher flexibility in utilizing either single microwires or wire bundles, such as tetrodes. Larger diameter single microwires are more robust and better suited for the recording of LFPs within brain tissue. While tetrodes can also be used to record LFPs, they are optimized for the isolation of single-unit action potentials 8, 11. In our laboratory, stable recordings of single-units have been obtained for up to 8 weeks following implantation. However, these recordings are not of the same putative units over that entire time. In our hands, a single-unit can be followed over several recording sessions (30 min each) that span a period of 3 days, reflecting an inter-session stability 10. On the other hand, robust LFPs and network oscillations can be recorded throughout the entire post-implantation period, especially with the use of larger diameter wire such as 50 μm (0.002″) wire. Note that the methods described here apply to unilateral recording of brain structures, but they can be easily modified for bilateral recordings. For example, when building custom microdrives, the appropriate distance between the drives must be determined beforehand in order to properly target brain structures bilaterally.
As microdrive components become more lightweight and the software to analyze neural signals improves, the library of potential brain targets and testable hypotheses within neuroscience continues to expand. It is clear that, since their inception 1, 12, brain recordings from awake behaving animals have greatly advanced our understanding of how neurons and networks of neurons encode behaviorally relevant events 3, 4,13,14. In particular, brain recordings from genetically modified mice have allowed the identification of molecular cascades that are crucially involved in neural encoding 15-17. Importantly, the technique has only recently been applied to clinically oriented issues 17, 18.
Advances in the fabrication of tetrodes and the increased availability of manufactured solutions will further facilitate the movement of this technology into addressing human diseases and ailments 19, 20. And while the penetration of electrodes into brain tissue is invasive in nature, these recordings offer invaluable information from individual neurons that cannot be obtained with technologies such as functional imaging. Thus, in both animal models and humans, awake behaving recordings using moveable microdrives will continue to provide indispensable information about neural ensembles, neural coding, topographic specificity, and network oscillations within the brain.
The authors have nothing to disclose.
We thank Daniel Carpi for his help and early contributions to this project. We also thank Lucrecia Novoa for her assistance with artwork and images. This work was supported by NIH/NIAID program grant 5P01AI073693-03.
Name of Reagent/Material | Company | Catalog Number | Comments |
0.0005″ (12.5 μM) diameter Platinum-Iridium wire | California Fine Wire | CFW#100-167 | HML VG insulated www.calfinewire.com |
0.002″ (50 μM) diameter Stableohm 675 wire | California Fine Wire | CFW# 100-188 | HML insulated Ni-Cr |
polyamide tubing | Polymicro Technologies | 1068150020 | 99 micron I.D., 166 micron O.D. www.polymicro.com |
brass guides | World Plastics Inc | 3.3 x 6.6 mm | |
Delrin blocks | World Plastics Inc | 3.13 x 2.5 mm | |
Fillister head brass screws | J.I. Morris Co. | 00-90 x 1/2 | drive screw www.jimorrisco.com |
hex brass nuts | J.I. Morris Co. | 00-90 | |
Fillister head brass screws | J.I. Morris Co. | 000-120 x 3/32 | EIB mount and ground screw |
plexiglass acrylic | Canal Street Plastics | 5 mm thick, clear, www.cpcnyc.com | |
cyanoacrylate | Krazy Glue | 2 g tube | |
electronic interface board | Neuralynx | EIB-18 | www.neuralynx.com |
non-cyanide gold solution | SIFCO | SIFCO 5355 | www.sifcoasc.com |
VersaDrive 4 | Neuralynx | four tetrode model | |
tetrode assembly station | Neuralynx | ||
motorized tetrode spinner | Neuralynx | tetrode spinner 2.0 | |
VersaDrive jig | Neuralynx | ||
soldering iron | Radio Shack | 64-2802B | www.radioshack.com |
nanoZ | Neuralynx | ||
small bit drill/driver | Ram Products | Rampower 35 | with footpedal controller, www.ramprodinc.com |
drill bits | Small Parts, Inc. | 3/32″ bits, www.smallpartsinc.com | |
dissecting microscope | Olympus | SZ-60 | www.olympusamerica.com |
heat gun | Alphawire | Fit gun 3 | use setting “1” only, www.alphawire.com |
26 AWG copper wire | Arcor Electronics | F26 | for ground wires, www.arcorelectronics.com |
soldering flux | Eagle | 2 oz, #205 | |
0.02″ diameter solder | Kester | 24-6337-0010 | www.kester.com |
benchtop vise | Vacu-Vise | Model 300 | |
fiber optic light | Nikon | MKII | dual light arms, www.nikon.com |
5-min epoxy | Allied Electronics | 25 ml, www.alliedelec.com | |
fine tweezers | Roboz Surgical Instrument Co. | RS-4907, RS-5010 | INOX material, www.roboz.com |
micro dissecting scissors | Roboz Surgical Instrument Co. | RS-5880 |
Table 1. Materials and reagents used for constructing tetrodes and microdrives.