Here, we introduce a lightweight, cost-effective probe implant system for chronic electrophysiology in rodents optimized for ease of use, probe recovery, experimental versatility, and compatibility with behavior.
Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. However, current methods for chronically implanting probes present steep trade-offs between cost, ease of use, size, adaptability, and long-term stability.
This protocol introduces a novel chronic probe implant system for mice called the DREAM (Dynamic, Recoverable, Economical, Adaptable, and Modular), designed to overcome the trade-offs associated with currently available options. The system provides a lightweight, modular and cost-effective solution with standardized hardware elements that can be combined and implanted in straightforward steps and explanted safely for recovery and multiple reuse of probes, significantly reducing experimental costs.
The DREAM implant system integrates three hardware modules: (1) a microdrive that can carry all standard silicon probes, allowing experimenters to adjust recording depth across a travel distance of up to 7 mm; (2) a three-dimensional (3D)-printable, open-source design for a wearable Faraday cage covered in copper mesh for electrical shielding, impact protection, and connector placement, and (3) a miniaturized head-fixation system for improved animal welfare and ease of use. The corresponding surgery protocol was optimized for speed (total duration: 2 h), probe safety, and animal welfare.
The implants had minimal impact on animals' behavioral repertoire, were easily applicable in freely moving and head-fixed contexts, and delivered clearly identifiable spike waveforms and healthy neuronal responses for weeks of post-implant data collection. Infections and other surgery complications were extremely rare.
As such, the DREAM implant system is a versatile, cost-effective solution for chronic electrophysiology in mice, enhancing animal well-being, and enabling more ethologically sound experiments. Its design simplifies experimental procedures across various research needs, increasing accessibility of chronic electrophysiology in rodents to a wide range of research labs.
Electrophysiology with chronically implanted silicon probes has emerged as a powerful technique for investigating neural activity and connectivity in behaving animals, particularly in mice, due to their genetic and experimental tractability1. Laminar silicon probes, in particular, have proven to be an invaluable tool for identifying functional relationships within cortical columns2 and for relating the dynamics of large neuronal populations to behavior in a way that was impossible previously3.
Two complementary approaches are the current gold standards for recording neural activity in vivo: two-photon microscopy4,5 and extracellular electrophysiology6. The choice of recording methodology constrains the nature of the readouts that can be obtained: two-photon microscopy is particularly well-suited to longitudinal studies of individually identifiable neurons in large populations across time but suffers from high equipment costs and is limited to superficial layers of the cortex in intact brains. In addition, the typical temporal resolution of ~30 Hz limits its ability to capture ongoing neuronal dynamics7,8.
In contrast, electrophysiological recordings offer high temporal resolution (up to 40 kHz) to track neuronal activity moment by moment, can be applied widely across species as well as across cortical depths, and have relatively low-cost setups compared to two-photon microscopy. However, the identification of individual neurons, as well as longitudinal tracking of neuronal populations, are difficult to achieve. This especially applies to wire electrodes, e.g., tetrodes, and to acute electrode insertions. Besides lacking the ability to track neurons across recording sessions9, repeated acute insertions cause local trauma10 that mounts an immune response11, increasing the chance of infection and gliosis. This ultimately reduces the stability of recorded neuronal activity and life expectancy of experimental animals, limiting the scope of longitudinal studies featuring acute electrophysiological recordings to just a few days12.
Chronic high-density silicon probe recordings aim to combine some of the best attributes of acute electrophysiology and two-photon imaging. They can track neural population dynamics across sessions with only a somewhat lowered ability to identify individual neurons compared to two-photon imaging13. These recordings provide high flexibility in the spatial placement and precise temporal resolution of the recorded signals, as well as improved longevity and well-being of experimental animals compared to acute recordings14. Furthermore, in contrast to acute recordings, chronic electrophysiology necessitates only a single implantation event, effectively reducing the risk of infection and tissue damage and minimizing stress on the animals15. Collectively, these advantages make chronic electrophysiology a powerful tool for investigating the organization and function of the nervous system.
However, commonly used chronic implantation techniques for mice constrain researchers to make significant trade-offs between compatibility with behavioral recordings, implant weight, replicability of implants, financial costs, and overall ease of use. Many implant protocols are not designed to facilitate the reuse of probes16, steeply raising the effective cost of individual experiments and thus making it financially difficult for some labs to use chronic electrophysiology. They also often require extensive in-house prototyping and design work, for which the expertise and resources may not be present.
On the other hand, integrated implant systems17 offer a more widely accessible solution for chronic electrophysiology in rodents. These systems are designed to integrate a microdrive holding the probe with the remainder of the implant to simplify implant handling and surgical procedures. However, once implanted, such systems can be top-heavy and limit the experimenter's ability to flexibly adapt an experiment to different target coordinates. Often, their weight precludes implants in smaller animals, potentially impairs animal movement and induces stress18. This can disproportionately affect research on juvenile and female cohorts, as weight limitations are more likely to affect these groups.
Additionally, not all integrated systems allow for adjustment of electrode positions post-implantation. This is relevant, as gliosis or scarring due to probe insertion19, especially in the initial 48 h after implantation20, can reduce the quality of the recorded neuronal activity. Micro-adjustments to the probe insertion depth can limit these negative effects on signal integrity. Therefore, micropositioning mechanisms, commonly called microdrives, can be beneficial even in probes with a large number of electrodes distributed across their length.
To overcome such trade-offs, we introduce a novel chronic electrophysiology implant system for mice that addresses the limitations of previous designs by offering a lightweight, cost-effective, and modular solution. The DREAM implant system is designed to weigh less than 10% (~2.1 g) of a mouse's typical body weight, ensuring animal welfare and minimal impact on behavior. Validation of the DREAM implant design shows minimal impact on behavioral key metrics such as locomotion – which can be significantly impacted in rodents when loads are placed on the cranium. This can benefit experimental paradigms that utilize freely moving as well as head-fixed animals by boosting animal well-being and allowing more ethologically sound experiments.
The system includes a microdrive for flexible adjustment of recording depth up to 7 mm and can be adapted to different types of probes and recording devices, providing researchers with a cost-effective and versatile tool for various experimental applications. The system is routinely combined with a metal microdrive21, which offers consistent probe recovery compared to other systems (expected average recovery rate: approx. three reliable reuses per probe) and drastically reduces the cost of individual experiments.
The design features a 3D-printed protective Faraday cage, allowing for cheap yet robust protection from electrophysiological noise, mechanical impacts, and infectious materials, enabling stable and noise-free recordings that suffer from minimal infection rates. This implantable cage consists of the so-called 'crown', designed for impact protection and to provide structure for the conductive metal mesh coating of the Faraday cage, and the crown ring, which serves as a mount for an implantable amplifier and/or probe connector (see Figure 1).
Finally, the headplates included in the modular implant system are designed to be compatible with a novel, efficient head-fixation system without adding extra bulk to the implant. In contrast to other existing systems, it does not require tightening small screws close to the implant, speeding up the fixation of mice in the experimental setup, and improving the experimenter-animal relationship, as well as behavioral adherence. At the same time, the headplate is used as a base on which to build the other modules of the DREAM chronic electrophysiology system.
Design files for the DREAM implant are published as open-source hardware at https://github.com/zero-noise-lab/dream-implant/. In the following sections, the design and fabrication of the DREAM implant system will be described, its successful implementation in a mouse model will be demonstrated, and its potential applications and advantages compared to existing systems will be discussed.
This manuscript presents a protocol for the fast, safe, and standardized implantation of probes, which also allows probe recovery and reuse at the end of the experiment. The approach makes use of a modular system of implant components, specifically a microdrive, which is compatible with all common silicon probes and recording systems, a headplate that can be used for head-fixed behavioral experiments, and a wearable Faraday cage to protect the implant. This constellation allows users to flexibly adapt their implant to different experimental paradigms, such as head-fixed versus freely moving behavior or implant miniaturization (without Faraday cage) versus increased long-term signal robustness (with Faraday cage) – without having to sacrifice the standardization of the implant in the process.
This approach makes chronic electrophysiological recordings more standardized (through prefabricated elements that do not require assembly by hand), less costly (through probe recovery), less time-consuming (by simplifying surgery steps), and more easily compatible with animal welfare and behavior (through decreased implant size and stress-free head fixation). As such, this protocol aims to make electrophysiological implants in behaving rodents attainable for a broader range of researchers beyond the pioneering labs at the cutting edge of the field.
To achieve this aim, the protocol presented here minimizes the trade-off between several often equally crucial aspects of microdrive implants, namely flexibility, modularity, ease of implantation, stability, overall cost, compatibility with behavior, and probe reusability. Currently, available approaches often excel at some of these aspects but at a steep cost to other features. For instance, for use cases that demand absolute implant stability over long time periods, the best implant approach may be to directly cement the probe onto the skull25. However, this also prevents probe reuse, as well as repositioning of recording sites in case of bad recording quality, and it is incompatible with standardized implant placement. Similarly, while the AMIE drive provides a lightweight, low-cost solution for recoverable implantation of probes, it is limited to single probes and restricted in the placement of the target coordinates17. At the opposite end of the spectrum, some commercially available nano-drives (see Table 116,17,21,26,27,28,29,30) are extremely small, can be placed freely on the skull, and maximize the number of probes that can be implanted in a single animal16. However, they are expensive compared to other solutions, require experimenters to be highly skilled for successful implant surgeries, and prohibit probe reuse. The microdrive developed by Vöröslakos et al.21, a lightweight version of which is also part of this protocol, sacrifices small implant size for better ease-of-use, lower price, and probe reusability
Table 1: Comparison of popular strategies for chronic probe implants in rodents. Availability: whether the microdrive is open source (for researchers to build themselves), commercially available, or both. Modularity: Integrated systems consist of one or few components that are in a fixed relation to each other, while modular systems allow free placement of the probe /microdrive relative to the protection (head gear/Faraday cage) after production of the implant (e.g., at time of surgery). Modularity was determined from published information or implantation protocols of the listed implants. Headfix: Yes: The implant has mechanisms for head-fixation integrated into its design, X: The implant leaves the space to add an extra headplate for fixation without big issues, No: The design of the implant likely creates space issues or requires substantial design modifications for use with head fixation. Probe placement: Restricted: Probe location is limited at the implant design stage. Flexible: Probe location can be adjusted even during surgery. Number of probes: the number of probes that could be implanted. Note that implanting >2 probes on a mouse does pose a significant challenge independent of the chosen implant system. Probe reusability: yes, if the probes can, in theory, be reused. Weight/size: weight and bulkiness of the implant. Please click here to download this Table.
To create a system that reconciles these different requirements more seamlessly, the DREAM implant was designed on the basis of the Vöröslakos implant21, but with several fundamental modifications. First, to reduce overall implant weight, the microdrive used here is produced in machined aluminum rather than 3D-printed stainless steel, and the Faraday crown is miniaturized, achieving an overall weight reduction of 1.2-1.4 g depending on the choice of headplate material (see Table 2). Second, the headplate surrounding the microdrive was designed to allow for an integrated head fixation mechanism that enables fast and stress-free head fixation while doubling as a base for the Faraday cage, giving access to most potential target areas for neuronal recordings and adding only minimal weight to the implant. The flat shape of the fixation mechanism and lack of protrusions also ensure minimal impairment of animals' visual field or locomotion (see Figure 2A–C), a clear improvement over previous systems31,32. The Faraday crown and ring that are fixed onto the headplate were also substantially altered compared to previous designs. They now do not require any ad-hoc adaptation (e.g., in terms of connector placement) or soldering throughout the surgery, removing potential causes of implant damage and unpredictable variance in implant quality. Instead, the DREAM implant provides multiple standardized crown ring variations that allow placing each connector at one of four pre-defined positions, minimizing variability and effort during surgery. Finally, by optimizing the implant system for probe recovery, the DREAM implant allows experimenters to drastically cut the cost as well as preparation time per implant since the microdrive and probe can typically be recovered, cleaned, and reused together.
For a more exhaustive overview of the trade-offs posed by different implant systems, see Table 1. While the approach presented here does generally not provide maximal performance compared to all other strategies, e.g., in terms of size, stability, or cost, it operates in the upper range across all these parameters, making it more easily applicable to a wide range of experiments.
Three aspects of the protocol are particularly crucial to adapt to each specific use case: The constellation of ground and reference, the technique for cementing the microdrive, and implant validation via neuronal recording. First, when implanting the ground and reference pins, the goal was to identify the sweet spot between mechanical/electrical stability and invasiveness. While, e.g., floating silver wires embedded in agar are less invasive than bone screws33, they are likely more prone to becoming dislodged over time. The use of pins, coupled with agar, ensures a stable electrical connection whilst also having the advantage of being easier to control during insertion, avoiding tissue trauma. Ground pins cemented to the skull are unlikely to become dislodged, and in the event of the wire becoming separated from the pin, reattachment is usually simple due to the larger surface area and stability of the implanted pin.
Table 2: Comparison of component weights between the DREAM implant and the implant described by Vöröslakos et al.21. Please click here to download this Table.
Second, cementing of the microdrive should generally occur prior to the insertion of the probe in the brain. This prevents lateral movement of the probe inside the brain if the microdrive is not perfectly fixed in the stereotactic holder during insertion. To check the placement of the probe before cementing the microdrive in place, one can briefly lower the tip of the probe shank to ascertain where it will contact the brain since extrapolating the touchdown position can be difficult given the microscope's parallax shift. Once the microdrive position is established, one optionally can protect the craniotomy with silicone elastomer prior to cementing the microdrive to ensure that the cement does not accidentally make contact with the craniotomy; however, lowering the probe through the silicone elastomer is not recommended, as silicone elastomer residue can be pulled into the brain and cause inflammation and gliosis.
Third, depending on the experimental protocol used, a test recording directly after surgery may or may not be useful. Largely, neuronal activity recorded right after probe insertion will not be directly representative of activity recorded chronically, due to factors such as transient brain swelling and tissue movement around the probe, meaning that both insertion depth as well as spike waveforms are unlikely to stabilize directly. As such, immediate recordings can mainly serve to ascertain general signal quality and implant integrity. It is recommended that the moveable microdrive sled be utilized in subsequent days post-surgery once the brain has stabilized to fine-tune the position. This also helps to avoid moving the probe by more than 1000 µm per day, minimizing damage to the recording site and thus improving recording site longevity.
Finally, users may wish to adapt the system to record from more than one target location. As this system is modular, the user has a lot of leeway on how to assemble and place components in relation to each other (see above and Supplementary Figure 3 and Supplementary Figure 4). This includes modifications that would allow a horizontally extended shuttle to be mounted on the microdrive, allowing for multiple probes or large multi-shank probes to be implanted, as well as the implantation of multiple individual microdrives (see Supplementary Figure 3 and Supplementary Figure 4). Such modifications only require the use of an adapted crown ring, with an increased number of mounting zones for connectors/interface boards/headstages. However, the space limitations of this design are dictated by the animal model, in this case, the mouse, which makes stacking multiple probes onto one microdrive more attractive in terms of footprint than implanting several microdrives independently of each other. The microdrives used here can support stacked probes, and thus, the only real limitation is the number of headstages or connectors that can fit the space and weight constraints defined by the animal model. Spacers can also be used to further increase non-vertical mounting and insertion paths.
In conclusion, this protocol allows for inexpensive, lightweight, and importantly adjustable implantation of a probe, with the added benefit of a microdrive design that prioritizes probe recovery. This tackles the problems of the prohibitive cost of single-use probes, the high barrier of surgical and implantation skills, as well as the fact that commercial solutions for chronic implantation are often difficult to adapt to unique use cases. These issues pose a pain point to labs already using acute electrophysiology and a deterrent to those that do not yet undertake electrophysiology experiments. This system aims to facilitate the wider uptake of chronic electrophysiology research beyond these limitations.
The authors have nothing to disclose.
This work was supported by the Dutch Research Council (NWO; Crossover Program 17619 "INTENSE", TS) and has received funding from the European Union's Seventh Framework Program (FP7/2007-2013) under grant agreement No. 600925 (Neuroseeker, TS, FB, PT), as well as from the Max Planck Society.
0.05" Solder Tail Socket | Mill-Max | 853-93-100-10-001000 | |
1,1’-dioctadecyl-3,3,3’,3’- Reagent tetramethylindocarbocyanine perchlorate (’DiI’; DiIC18(3)) | ThermoFisher | D282 | Lipophilic dye used for easier histological verification of the probe location |
Adhesive Putty (Blu-Tack) | Bostik | 308590110 | Variations (e.g. by Pritt) should be available in your stationary store |
Agar | Sigma Aldrich | A1296 | Make with saline for conductivity. |
Amplifier (Miniamp-64) | Cambridge Neurotech | Miniature and implantable amplifier and digitiser. Alternative Implantable digitiser, or implantable Omnetics connector use possible. | |
Analgesic Cream (EMLA Cream) | Aspen | 39699/0088 | Analgesic cream used for operative pain containing prilocaine, lidocaine. |
Angled Spacer | 3DNeuro | Angled spacer for non-perpendicular drive mounting.. Open souce, also available at https://github.com/zero-noise-lab/dream-implant/ | |
Blue light curing LED | B.A. International | 818223 | Curing light for primer polymerisation. 420-480 nm wavelength |
Bone wax | SMI | Z046 | Wax to protect craniotomy and probe post surgery. |
Copper mesh | Dexmet | 3CU6-050FA | Copper mesh used to electrically and physically shield probe and craniotomy. |
Cyanoacrylate glue (Loctite) | Loctite | 1363589 | Cyanoacrylate gel glue |
Dental Cement (SuperBond C&B) | Sun Medical | K058E | Dental cement (SuperBond) |
Depilation Cream (Veet) | Veet | 310000091434 | Hair removal cream for removal of hair around surgical site. |
Faraday crown | 3DNeuro | 3D printed implantable protective cage. Open souce, also available at https://github.com/zero-noise-lab/dream-implant/ | |
Faraday ring | 3DNeuro | 3D printed implantable protective ring for faraday cage. Open souce, also available at https://github.com/zero-noise-lab/dream-implant/ | |
Haemostatic Sponge | SMI | ZHG101010 | Absorbable gelatin haemostatic sponge |
Heat Shrink Tubing | HellermannTyton | TA32-9/3 BK | Heat Shrink tubing for making soft tipped forceps |
Iodine | Braunol | 9322507 | Aqueous povidone-iodine solution. |
Microdrive (R2Drive) | 3DNeuro | Recoverable Metal micro drive with moveable shuttle. Open souce, also available at https://buzsakilab.github.io/ 3d_print_designs/ |
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Mineral Oil | Sigma-Aldrich | M5310-100ML | Oil used as solvent to create craniotomy protection gel. |
Non-Shedding Wipes (Kimtech) | Kimtech | 7552 | Non-shedding wipes |
Primer | Bisco | B-7202P | Universal skull adhesive preventing moisture from deteriorating the cement and providing a solid base to build up cement onto. |
R2Drive holder | 3DNeuro | Stereotactic attachment for mounting R2Drive. Open souce, also available at https://buzsakilab.github.io/ 3d_print_designs/ |
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Self-adherent wrap | 3M | VB050 | Protective wrap for implant post surgery |
Silicon probe (H2) | Cambridge Neurotech | Chronically implantable linear silicon probe with 32 channels. Alternative Probe use possible. | |
Silicone Elastomer (Duragel) | Cambridge Neurotech | Silicone Elastomer | |
Silicone Plaster (Kwikcast) | WPI | KWIK-CAST | |
Silver conductive epoxy | MG Chemicals | 8331D-14G | Silver epoxy |
Size 5 Dumont forceps | FSTools | 11251-10 | Small forceps for lifting bone flap. |
Stainless steel wire, Teflon coated | Science Products GmBH | SS-3T | Ground wire |
Stereotax (RWD) | RWD | 68803 | Stereotax for surgical procedures on mice. |
Tergazyme | Alconox | 1304 | A possible enzymatic cleaner to clean probe |
Two Part Fast setting Epoxy Resin | Gorilla | EP3 | Epoxy for permanent bonding of DREAM implant parts. |
Vannas Spring Scissors Round Handle | FSTools | 15403-08 | 0.075mm straight tipped spring rebound veterinary scissors. |
Veterinary Cyanoacrylate glue (Vetbond) | 3M | 70-0068-5256-3 | Veterinary cyanoacrylate glue |
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