This work was supported by the Dutch Research Council (NWO; Crossover Program 17619 &#34;INTENSE&#34;, TS) and has received funding from the European Union&#39;s Seventh Framework Program (FP7/2007-2013) under grant agreement No. 600925 (Neuroseeker, TS, FB, PT), as&#160;well&#160;as from the Max Planck Society.

A Cost-Effective, Versatile Probe Implant System for Electrophysiology in Mice

<ol>
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IMEC Neuropixels 1.0 User Manual V1.0.8 Available from: <a target="_blank" href="https://www.neuropixels.org/_files/ugd/328966_ca209d53ffb346b3bf98be39b903efa9.pdf">https://www.neuropixels.org/_files/ugd/328966_ca209d53ffb346b3bf98be39b903efa9.pdf</a> (2023)</li><li>Baranauskas, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+nanotube+composite+coating+of+neural+microelectrodes+preferentially+improves+the+multiunit+signal-to-noise+ratio.">Carbon nanotube composite coating of neural microelectrodes preferentially improves the multiunit signal-to-noise ratio.</a> J Neural Eng. 8 (6), 066013 (2011).</li><li>Niell, C. M., Stryker, M. 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TS, AN, and MNH are co-founders of 3Dneuro B.V., which manufactures the open-source microdrives and Faraday crowns used in this protocol. FB and PT are part of the scientific advisory board of 33Dneuro B.V.. FB and PT do not receive any financial compensation for this position.

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 V&#246;r&#246;slakos 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 &#62;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. <a href="https://www.jove.com/files/ftp_upload/66867/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
To create a system that reconciles these different requirements more seamlessly, the DREAM implant was designed on the basis of the V&#246;r&#246;slakos 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&#39; 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 V&#246;r&#246;slakos et al.21. <a href="https://www.jove.com/files/ftp_upload/66867/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
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&#39;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&#160;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 &#181;m 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.

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,&#160;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&#39;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&#39;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 &#39;crown&#39;, 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&#160;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.&#160;

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05&#34; Solder Tail Socket</td>
			<td>Mill-Max</td>
			<td>853-93-100-10-001000</td>
			<td></td>
		</tr>
		<tr>
			<td>1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;- Reagent tetramethylindocarbocyanine perchlorate (&#39;DiI&#39;; DiIC18(3))</td>
			<td>ThermoFisher</td>
			<td>D282</td>
			<td>Lipophilic dye used for easier histological verification of the probe location</td>
		</tr>
		<tr>
			<td>Adhesive Putty (Blu-Tack)</td>
			<td>Bostik</td>
			<td>308590110</td>
			<td>Variations (e.g. by Pritt) should be available in your stationary store</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma Aldrich</td>
			<td>A1296</td>
			<td>Make with saline for conductivity.</td>
		</tr>
		<tr>
			<td>Amplifier (Miniamp-64)</td>
			<td>Cambridge Neurotech</td>
			<td></td>
			<td>Miniature and implantable amplifier and digitiser. Alternative Implantable digitiser, or implantable Omnetics connector use possible.</td>
		</tr>
		<tr>
			<td>Analgesic Cream (EMLA Cream)</td>
			<td>Aspen</td>
			<td>39699/0088</td>
			<td>Analgesic cream used for operative pain containing prilocaine, lidocaine.</td>
		</tr>
		<tr>
			<td>Angled Spacer</td>
			<td>3DNeuro</td>
			<td></td>
			<td>Angled spacer for non-perpendicular drive mounting.. Open souce, also available at https://github.com/zero-noise-lab/dream-implant/</td>
		</tr>
		<tr>
			<td>Blue light curing LED</td>
			<td>B.A. International</td>
			<td>818223</td>
			<td>Curing light for primer polymerisation. 420-480 nm wavelength</td>
		</tr>
		<tr>
			<td>Bone wax</td>
			<td>SMI</td>
			<td>Z046</td>
			<td>Wax to protect craniotomy and probe post surgery.</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Elanco Europe LTD</td>
			<td>401513</td>
			<td>Injectable Buprenorphine solution (0.3 mg/mL)</td>
		</tr>
		<tr>
			<td>Copper mesh</td>
			<td>Dexmet</td>
			<td>3CU6-050FA</td>
			<td>Copper mesh used to electrically and physically shield probe and craniotomy.</td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue (Loctite)</td>
			<td>Loctite</td>
			<td>1363589</td>
			<td>Cyanoacrylate gel glue</td>
		</tr>
		<tr>
			<td>Dental Cement (SuperBond C&#38;B)</td>
			<td>Sun Medical</td>
			<td>K058E</td>
			<td>Dental cement (SuperBond)</td>
		</tr>
		<tr>
			<td>Depilation Cream (Veet)</td>
			<td>Veet</td>
			<td>310000091434</td>
			<td>Hair removal cream for removal of hair around surgical site.</td>
		</tr>
		<tr>
			<td>Enrofloxacin (Baytril)</td>
			<td>Elanco Europe LTD</td>
			<td>00879/4117</td>
			<td>Injectable enrofloxacin solution (25 mg/mL)</td>
		</tr>
		<tr>
			<td>Faraday crown</td>
			<td>3DNeuro</td>
			<td></td>
			<td>3D printed implantable protective cage. Open souce, also available at https://github.com/zero-noise-lab/dream-implant/</td>
		</tr>
		<tr>
			<td>Faraday ring</td>
			<td>3DNeuro</td>
			<td></td>
			<td>3D printed implantable protective ring for faraday cage. Open souce, also available at https://github.com/zero-noise-lab/dream-implant/</td>
		</tr>
		<tr>
			<td>Haemostatic Sponge</td>
			<td>SMI</td>
			<td>ZHG101010</td>
			<td>Absorbable gelatin haemostatic sponge</td>
		</tr>
		<tr>
			<td>Heat Shrink Tubing</td>
			<td>HellermannTyton</td>
			<td>TA32-9/3 BK</td>
			<td>Heat Shrink tubing for making soft tipped forceps</td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>Braunol</td>
			<td>9322507</td>
			<td>Aqueous povidone-iodine solution.</td>
		</tr>
		<tr>
			<td>Metamizole (Novalgin)</td>
			<td>Sanofi-Aventis Gmbh</td>
			<td>4527098</td>
			<td>Injectable Metamizole (500mg/mL)</td>
		</tr>
		<tr>
			<td>Metamizole (Novalgin)</td>
			<td>Sanofi-Aventis Gmbh</td>
			<td>1553758</td>
			<td>Metamizole solution</td>
		</tr>
		<tr>
			<td>Microdrive (R2Drive)</td>
			<td>3DNeuro</td>
			<td></td>
			<td>Recoverable Metal micro drive with moveable shuttle. Open souce, also available at https://buzsakilab.github.io/ 3d_print_designs/</td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma-Aldrich</td>
			<td>M5310-100ML</td>
			<td>Oil used as solvent to create craniotomy protection gel.</td>
		</tr>
		<tr>
			<td>Non-Shedding Wipes (Kimtech)</td>
			<td>Kimtech</td>
			<td>7552</td>
			<td>Non-shedding wipes</td>
		</tr>
		<tr>
			<td>Primer</td>
			<td>Bisco</td>
			<td>B-7202P</td>
			<td>Universal skull adhesive preventing moisture from deteriorating the cement and providing a solid base to build up cement onto.</td>
		</tr>
		<tr>
			<td>R2Drive holder</td>
			<td>3DNeuro</td>
			<td></td>
			<td>Stereotactic attachment for mounting R2Drive. Open souce, also available at https://buzsakilab.github.io/ 3d_print_designs/</td>
		</tr>
		<tr>
			<td>Self-adherent wrap</td>
			<td>3M</td>
			<td>VB050</td>
			<td>Protective wrap for implant post surgery</td>
		</tr>
		<tr>
			<td>Silicon probe (H2)</td>
			<td>Cambridge Neurotech</td>
			<td></td>
			<td>Chronically implantable linear silicon probe with 32 channels. Alternative Probe use possible.</td>
		</tr>
		<tr>
			<td>Silicone Elastomer (Duragel)</td>
			<td>Cambridge Neurotech</td>
			<td></td>
			<td>Silicone Elastomer</td>
		</tr>
		<tr>
			<td>Silicone Plaster (Kwikcast)</td>
			<td>WPI</td>
			<td>KWIK-CAST</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver conductive epoxy</td>
			<td>MG Chemicals</td>
			<td>8331D-14G</td>
			<td>Silver epoxy</td>
		</tr>
		<tr>
			<td>Size 5 Dumont forceps</td>
			<td>FSTools</td>
			<td>11251-10</td>
			<td>Small forceps for lifting bone flap.</td>
		</tr>
		<tr>
			<td>Stainless steel wire, Teflon coated</td>
			<td>Science Products GmBH</td>
			<td>SS-3T</td>
			<td>Ground wire</td>
		</tr>
		<tr>
			<td>Stereotax (RWD)</td>
			<td>RWD</td>
			<td>68803</td>
			<td>Stereotax for surgical procedures on mice.</td>
		</tr>
		<tr>
			<td>Tergazyme</td>
			<td>Alconox</td>
			<td>1304</td>
			<td>A possible enzymatic cleaner to clean probe</td>
		</tr>
		<tr>
			<td>Two Part Fast setting Epoxy Resin</td>
			<td>Gorilla</td>
			<td>EP3</td>
			<td>Epoxy for permanent bonding of DREAM implant parts.</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors Round Handle</td>
			<td>FSTools</td>
			<td>15403-08</td>
			<td>0.075mm straight tipped spring rebound veterinary scissors.</td>
		</tr>
		<tr>
			<td>Veterinary Cyanoacrylate glue (Vetbond)</td>
			<td>3M</td>
			<td>70-0068-5256-3</td>
			<td>Veterinary cyanoacrylate glue</td>
		</tr>
	</tbody>
</table>

the dream implant: a lightweight, modular, and cost-effective implant system for chronic electrophysiology in head-fixed and freely behaving mice

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&#39; 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.

This protocol presents a chronic implantation system that enables researchers to implement lightweight, cost-effective and safe chronic electrophysiology recordings in behaving mice (Figure 1). The main factors that determine successful application of this approach include: complete cement coverage of the skull, a minimally invasive and properly protected craniotomy, secure attachment of the microdrive and wiring to the skull and complete continuity of protective Faraday material. When these points are accounted for, high-quality recordings can be reached consistently. Here representative results pertaining to the following main aspects of surgery success are shown:
1) Is the implant interfering with animal behavior or well-being? 
2) Is signal quality high, and can signals be maintained over prolonged periods of time? 
3) Can recordings be combined easily with task performance?
To assess the impact of the implant on animal behaviour, we analysed tracked locomotion patterns in five implanted animals. Figure 2A shows an example of an animal freely moving inside of a play cage for 10 min before and 1 week after implant. One can see that movement patterns are unchanged. This observation is confirmed by Figure 2B,&#160;C showing the distributions of movement speeds and head directions across animals. Both running speed and head directions were largely unchanged before and after implantation, and if anything, running speeds seemed to be slightly elevated after surgery. Supplementary Video 1 shows a short video recording of an animal 6 days after implantation surgery. Typical home cage behaviors like locomotion, grooming, rearing and foraging in the home environment are all visible and indicate successful recovery from surgery, as well as general health. The low behavioral impact of the implant is most likely due to its low weight and manageable height.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66867/66867fig02.jpg" /> 
Figure 2: Locomotion before and after surgery. (A) Example locomotion of an animal before (left panel) and after (right panel) implantation. x/y coordinates are in centimeters, points show position of the animal at each timepoint over a period of 10 min. (B) Distribution of movement speeds in cm/s for 5 sessions before and 3 sessions after implantation in 5 animals. (C) Kernel density for probability of movement in different directions, for the same sessions analyzed in(B). <a href="https://www.jove.com/files/ftp_upload/66867/66867fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Next, the signal quality in Local Field Potential (LFP) and spiking activity across recording sites is assessed. Here, we show representative data from cortical recordings in the primary visual cortex (V1). For validation, putative single-unit activity was extracted from broadband neuronal signals recorded in V1 of an awake mouse using Kilosort 3 (see Figure 3). Figure 3A shows the location of extracted single units on the probe shank, Figure 3B shows the corresponding spike waveforms, and Figure 3C shows the spiking responses of the same neurons to a current source density (CSD) protocol. In this paradigm, widefield flashes were presented with a duration of 300 ms at a frequency of 1 Hz (i.e., 300 ms on, 700 ms off) over 200 trials. Finally, Figure 3D shows the same units&#39; responses to a visual receptive field mapping protocol, consisting of 2000 frames of randomly selected black and white squares on a grey background, and each presented for 16.6 ms. Squares covered 12 degrees of visual angle each and were selected from a field of 15 x 5 possible locations so that the mapping paradigm covered a visual space of -90 to +90 degrees azimuth and -30 to +40 degrees elevation in total. Firing rate responses to each stimulus frame were extracted by analyzing the maximum firing rate across a 16.6 ms window, subject to a delay of between 40-140 ms, identified as optimal per channel based on the maximum activity in each window. This type of recording can be used to guide adjustment of the insertion depth of each electrode and to assess signal quality after the implant surgery.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66867/66867fig03.jpg" /> 
Figure 3: Recorded neuronal signals. (A)&#160;Inferred location of single units sorted by Kilosort 3 spike sorting package along the probe&#39;s electrode contacts. (B)&#160;Spike waveforms for the same units shown in A across 5 ms of time. Thin lines: Individual spike waveforms. Thick lines: Average spike waveform. (C) Raster plot of spikes in response to a current source density (CSD) paradigm presenting 300 ms widefield flashes followed by a 700 ms black screen. Responses are shown for the same units as in A and B. Superimposed colored lines represent peri-stimulus time histograms (PSTHs) of the same responses. Firing rates for the PSTHs were calculated in 10 ms bins and then normalized by the maximum firing rate across the entire PSTH. Time 0 is centered around the widefield flash stimulus. (D) Estimated receptive fields of the same units as in A-C, measured by a Sparse Noise Receptive Field Mapping paradigm. Each plot shows average firing rate activity over a 16.6ms analysis window in response to the onset (left panel) or offset (right panel) of white and black square stimuli. Stimuli were presented for the duration of 16.6 ms, located randomly across a 5 x 15 square grid spanning 180 degrees of visual angle horizontally and 70 degrees of visual angle vertically. Firing rate activity was z-scored across the entire receptive field grid (see color bar). <a href="https://www.jove.com/files/ftp_upload/66867/66867fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Recording quality remained high across repeated recordings for weeks to months. Figure 4A shows longitudinal LFP recordings from one animal over 15 weeks. LFPs were recorded in response to the CSD paradigm described above (see Figure 3A-C). Figure 4A shows averaged LFP responses 500 ms following flash onset. In this example, we used a linear probe with 32 channels, with an interelectrode distance of 25 &#181;m. Note that on day 18, the probe depth was adjusted, shifting the probe downwards by 600 &#181;m. Both before and after this adjustment, LFP signals remained stable across recording days.
Consistent with this, spike waveforms of putative single units were discernible over many recordings. Figure 4B shows representative example spike waveforms from three recording sessions across a month of recordings, demonstrating that single unit activity can be identified successfully over time. Figure 4C shows the overall number of putative single units extracted from chronic recordings in six animals, spanning a window of up to 100 days. Single units were defined according to the default criteria of kilosort 3.0 (see Supplementary Table 1). As one can see, the number of clearly defined single units typically amounted to ~40 in the first-week post-implantation, and then dropped off gradually, moving towards an apparently stable asymptote of ~20 units. Given that these recordings were conducted using linear 32-channel probes, this equates to an expected yield of about 1.25 single units per electrode directly after implantation, declining to approx. 0.65 single units per electrode in long-term recordings. Repeated connection to the implant&#39;s amplifier/connector over sessions did not appear to impact either recording quality or implant stability since the Faraday crown that holds the amplifier/connector can withstand repeated forces of over 10 Newton, an order of magnitude larger than even the maximal mating forces required by standard connectors (see Supplementary Video 2).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66867/66867fig04.jpg" /> 
Figure 4: Stability of neuronal recordings over time. (A)&#160;Average LFP activity in response to a widefield flash CSD stimulus, shown across all 32 channels of a chronically implanted probe from 3-110 days post-implant. The red vertical line denotes the probe being lowered to a new location due to channels 0-8 recording from outside the brain by Day 18 post-surgery. (B) Spike waveforms of three example units from the same chronic implant recorded repeatedly across four weeks. Thin lines: Individual spike waveforms. Thick superimposed line: Average spike waveform. (C) The number of putative single units detected by Kilosort 3 across recording days for 6 animals (see inset legend). The red square denotes the days when the probe was moved. The dotted line denotes the number of electrodes per implant used in these recordings (32). <a href="https://www.jove.com/files/ftp_upload/66867/66867fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Finally, by providing a modular system including a microdrive as well as a wearable Faraday cage and a headplate that doubles as an implant base and a device for head-fixation, this protocol enables the integration of chronic electrophysiology with head-fixed behavior. Here, example data from mice traversing a virtual environment on a spherical treadmill are shown. Figure 5A shows running-related spiking activity of 20 units in an example trial. Figure 5B shows the diverse but robust relationships between running speed and spiking activity of individual spike-sorted units, as well as a population average for the same effect in Figure 5C, confirming the well-established effect of locomotor activity on neuronal activity in rodent V124.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66867/66867fig05.jpg" /> 
Figure 5: Neuronal responses during head-fixed behavior. (A) Raster plot of single unit responses across an example trial, with running speed (purple line) and average firing rates across all single units (light blue line) superimposed. (B)&#160;Single unit activity during different running speed categories, shown for six example units. (C) Average spiking activity across all single units in one example session, plotted across the five quinitiles of the running speed distribution. Running speeds in this session ranged from 0 to 0.88 meters/second. <a href="https://www.jove.com/files/ftp_upload/66867/66867fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table 1: Table showing default parameters used by Kilosort 3 when identifying single units in the recordings shown in Figure 3, Figure 4, and Figure 5. <a href="https://www.jove.com/files/ftp_upload/66867/Supplementary Table 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Video 1: Video showing animal locomotor activity post implant. Video taken after 5 day recovery phase is complete, showing normal locomotor behavior, as well as adaption to the size and weight of the implant. The animal can be seen normally exploring a play cage containing environmental enrichment. <a href="https://www.jove.com/files/ftp_upload/66867/Supplementary video 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Video 2: Video showing force being applied onto the assembled Faraday crown. The forces withstood by the Faraday crown are approximately one order of magnitude larger than the connection force needed for standard connectors such as 4-pin polarized nano connectors. <a href="https://www.jove.com/files/ftp_upload/66867/Supplementary Video 2.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 1: Figure showing images of the drive holder. Printable design files can be found in the corresponding Github repository (https://github.com/zero-noise-lab/dream-implant/). <a href="https://www.jove.com/files/ftp_upload/66867/Supplementary Figure 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Template for copper mesh. Print the template with the original scaling and use the stencil for cutting out the copper mesh (step 2.12). Use the scale bar for verifying and, if necessary, adjusting the scaling of the print. <a href="https://www.jove.com/files/ftp_upload/66867/Supplementary Figure 2.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Photo series showing the assembly steps of the implant during surgery. Two microdrives, as well as two amplifiers, are installed in this case. <a href="https://www.jove.com/files/ftp_upload/66867/Supplementary Figure 3.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 4: Drawing of mouse skull featuring example placement of drives, craniotomies (in green), and GND/REF pin (in red). Pin location is suggested due to placement in the cerebellum, which is unlikely to interfere with cortical recordings. <a href="https://www.jove.com/files/ftp_upload/66867/Supplementary Figure 4.zip" target="_blank">Please click here to download this File.</a>

The DREAM Implant: A Lightweight, Modular, and Cost-Effective Implant System for Chronic Electrophysiology in Head-Fixed and Freely Behaving Mice

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. ...

the-dream-implant-lightweight-modular-cost-effective-implant-system

Research

JoVE Journal

Neuroscience

801 Views.  Radboud University. 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.

Video: The DREAM Implant: A Lightweight, Modular, and Cost-Effective Implant System for Chronic Electrophysiology in Head-Fixed and Freely Behaving Mice

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

Long-term Sensory Conflict in Freely Behaving Mice

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

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

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

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...

Microinjectrode System for Combined Drug Infusion and Electrophysiology

This microinjectrode system is designed for drug infusion, electrophysiology, and delivery and retrieval of experimental probes, such as microelectrodes and nanosensors, optimized for repeated use in awake, behaving animals. The microinjectrode system can be configured for multiple purposes: (1) simple arrangement of the cannula for placement of an experimental probe that would otherwise be too fragile to penetrate the dura mater, (2) microfluidic infusion of a drug, either independently or coupled to a cannula containing an experimental probe (i.e., microelectrode, nanosensor). In this protocol we explain the step by step construction of the microinjectrode, its coupling to microfluidic components, and the protocol for use of the system in vivo. The microfluidic components of this system allow for delivery of volumes on the nanoliter scale, with minimal penetration damage. Drug infusion can be performed independently or simultaneously with experimental probes such as microelectrodes or nanosensors in an awake, behaving animal. Applications of this system range from measuring the effects of a drug on cortical electrical activity and behavior, to understanding the function of a specific region of cortex in the context of behavioral performance based on probe or nanosensor measurements. To demonstrate some of the capabilities of this system, we present an example of muscimol infusion for reversible inactivation of the frontal eye field (FEF) in rhesus macaque during a working memory task.

We present a microinjectrode system designed for electrophysiology and assisted delivery of experimental probes (i.e., nanosensors, microelectrodes), with optional drug infusion. Widely available microfluidic components are coupled to a cannula containing the probe. A step-by-step protocol for microinjectrode construction is included, with results during muscimol infusion in macaque cortex.

This microinjectrode system is designed for drug infusion, electrophysiology, and delivery and retrieval of experimental probes, such as microelectrodes ...

Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this purpose, an in vivo imaging technique that detects calcium transients in neurons using genetically encoded calcium indicators (GECIs), a miniaturized fluorescence microscope, and a gradient refractive index (GRIN) lens has been developed and successfully applied to many brain structures1,2,3,4,5,6. This imaging technique is particularly powerful because it enables chronic simultaneous imaging of genetically defined cell populations for a long-term period up to several weeks. Although useful, this imaging technique has not been easily applied to brain structures that locate deep within the brain such as amygdala, an essential brain structure for emotional processing and associative fear memory7. There are several factors that make it difficult to apply the imaging technique to the amygdala. For instance, motion artifacts usually occur more frequently during the imaging conducted in the deeper brain regions because a head-mount microscope implanted deep in the brain is relatively unstable. Another problem is that the lateral ventricle is positioned close to the implanted GRIN lens and its movement during respiration may cause highly irregular motion artifacts that cannot be easily corrected, which makes it difficult to form a stable imaging view. Furthermore, because cells in the amygdala are usually quiet at a resting or anesthetized state, it is hard to find and focus the target cells expressing GECI in the amygdala during baseplating procedure for later imaging. This protocol provides a helpful guideline for how to efficiently target cells expressing GECI in the amygdala with head-mount miniaturized microscope for successful in vivo calcium imaging in such a deeper brain region. It is noted that this protocol is based on a particular system (e.g., Inscopix) but not restricted to it.

In vivo microendoscopic calcium imaging is an invaluable tool that enables real-time monitoring of neuronal activities in freely behaving animals. However, applying this technique to the amygdala has been difficult. This protocol aims to provide a useful guideline for successfully targeting amygdala cells with a miniaturized microscope in mice.

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this ...

Head Implants for the Neuroimaging of Awake, Head-Fixed Rats

Anesthetics, commonly used in preclinical and fundamental scientific research, have a depressive influence on the metabolic, neuronal, and vascular functions of the brain and can adversely influence neurophysiological results. The use of awake animals for research studies is advantageous but poses the major challenge of keeping the animals calm and stationary to minimize motion artifacts throughout data acquisition. Awake imaging in smaller-sized rodents (e.g., mice) is very common but remains scant in rats as rats are bigger, stronger, and have a greater tendency to oppose movement restraints and head fixation over the long durations required for imaging. A new model of neuroimaging of awake, head-fixed rats using customized hand-sewn slings, 3D-printed head implants, head caps, and a headframe is described. The results acquired following a single trial of single-whisker stimulation suggest an increase in the intensity of the evoked functional response. The acquisition of the evoked functional response from awake, head-fixed rats is faster than that from anesthetized rats, reliable, reproducible, and can be used for repeated longitudinal studies.

A detailed new procedure for functional imaging of awake, head-fixed rats is described.

Anesthetics, commonly used in preclinical and fundamental scientific research, have a depressive influence on the metabolic, neuronal, and vascular ...

Micro-Drive Implant for Recording Daily Neural Activity in Juvenile Mice

A Lightweight Drive Implant for Chronic Tetrode Recordings in Juvenile Mice

In vivo electrophysiology provides unparalleled insight into the sub-second-level circuit dynamics of the intact brain and represents a method of particular importance for studying mouse models of human neuropsychiatric disorders. However, such methods often require large cranial implants, which cannot be used in mice at early developmental time points. As such, virtually no studies of in vivo physiology have been performed in freely behaving infant or juvenile mice, despite the fact that a better understanding of neurological development in this critical window would likely provide unique insights into age-dependent developmental disorders such as autism or schizophrenia. Here, a micro-drive design, surgical implantation procedure, and post-surgery recovery strategy are described that allow for chronic field and single-unit recordings from multiple brain regions simultaneously in mice as they age from postnatal day 20 (p20) to postnatal day 60 (p60) and beyond, a time window roughly corresponding to the human ages of 2 years old through to adulthood. The number of recording electrodes and final recording sites can be easily modified and expanded, thus allowing flexible experimental control of the in vivo monitoring of behavior- or disease-relevant brain regions across development.

Author Spotlight: A Lightweight Drive Implant for Chronic Tetrode Recordings in Juvenile Mice  

Here, we describe a micro-drive design, surgical implantation procedure, and post-surgery recovery strategy that allow for chronic field and single-unit recordings from multiple brain regions simultaneously in juvenile and adolescent mice across a critical developmental window from postnatal day 20 (p20) to postnatal day 60 (p60) and beyond.

In vivo electrophysiology provides unparalleled insight into the sub-second-level circuit dynamics of the intact brain and represents a method of ...

Multilayer Imaging with Microprism in Head-Fixed and Freely Moving Animals

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for studying the structure, function, and plasticity of living brain tissues. In comparison to conventional electrophysiology, fluorescence microscopy can capture the neural activity as well as the morphology of the cells, enabling long-term recordings of the identified neuron populations at single-cell or subcellular resolution. However, high-resolution imaging typically requires a stable, head-fixed setup that restricts the movement of the animal, and the preparation of a flat surface of transparent glass allows visualization of neurons at one or more horizontal planes but is limited in studying the vertical processes running across different depths. Here, we describe a procedure to combine a head plate fixation and a microprism that gives multilayer and multimodal imaging. This surgical preparation not only gives access to the entire column of the mouse visual cortex but allows two-photon imaging in a head-fixed position and one-photon imaging in a freely moving paradigm. Using this approach, one can sample identified cell populations across different cortical layers, register their responses under head-fixed and freely moving states, and track the long-term changes over months. Thus, this method provides a comprehensive assay of the microcircuits, enabling direct comparison of neural activities evoked by well-controlled stimuli and under a natural behavioral paradigm.

Author Spotlight: Comparative Imaging of Neural Activity in Awake and Freely Moving States

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for ...