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><li>Epsztein, J., Brecht, M., Lee, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+determinants+of+hippocampal+CA1+place+and+silent+cell+activity+in+a+novel+environment%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Intracellular determinants of hippocampal CA1 place and silent cell activity in a novel environment.</a> Neuron. 70 (1), 109-120 (2011).</li>
<li>Okun, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diverse+coupling+of+neurons+to+populations+in+sensory+cortex%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Diverse coupling of neurons to populations in sensory cortex.</a> Nature. 521 (7553), 511-515 (2015).</li>
<li>Jun, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fully+integrated+silicon+probes+for+high-density+recording+of+neural+activity%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Fully integrated silicon probes for high-density recording of neural activity.</a> Nature. 551 (7679), 232-236 (2017).</li>
<li>Znamenskiy, P., Kim, M. -H., Muir, D. R., Iacaruso, M. F., Hofer, S. B., Mrsic-Flogel, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+specificity+of+recurrent+inhibition+in+visual+cortex%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Functional specificity of recurrent inhibition in visual cortex.</a> Neuron. 112 (6), 991-1000 (2024).</li>
<li>Rowland, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Propagation+of+activity+through+the+cortical+hierarchy+and+perception+are+determined+by+neural+variability%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Propagation of activity through the cortical hierarchy and perception are determined by neural variability.</a> Nat Neurosci. 26 (9), 1584-1594 (2023).</li>
<li>Roth, M. M., Dahmen, J. C., Muir, D. R., Imhof, F., Martini, F. J., Hofer, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thalamic+nuclei+convey+diverse+contextual+information+to+layer+1+of+visual+cortex%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Thalamic nuclei convey diverse contextual information to layer 1 of visual cortex.</a> Nat Neurosci. 19 (2), 299-307 (2016).</li>
<li>Zong, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+two-photon+calcium+imaging+in+freely+moving+mice%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Large-scale two-photon calcium imaging in freely moving mice.</a> Cell. 185 (7), 1240-1256 (2022).</li>
<li>Demas, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-speed%2C+cortex-wide+volumetric+recording+of+neuroactivity+at+cellular+resolution+using+light+beads+microscopy%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">High-speed, cortex-wide volumetric recording of neuroactivity at cellular resolution using light beads microscopy.</a> Nat Methods. 18 (9), 1103-1111 (2021).</li>
<li>Buzsáki, G., Anastassiou, C. A., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+origin+of+extracellular+fields+and+currents+EEG%2C+ECoG%2C+LFP+and+spikes%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">The origin of extracellular fields and currents EEG, ECoG, LFP and spikes.</a> Nat Rev Neurosci. 13 (6), 407-420 (2012).</li>
<li>Polikov, V. S., Tresco, P. A., Reichert, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Response+of+brain+tissue+to+chronically+implanted+neural+electrodes%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">Response of brain tissue to chronically implanted neural electrodes.</a> J Neurosci Methods. 148 (1), 1-18 (2005).</li>
<li>Savya, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+spatiotemporal+dynamics+of+astrocyte+reactivity+following+neural+electrode+implantation%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">In vivo spatiotemporal dynamics of astrocyte reactivity following neural electrode implantation.</a> Biomaterials. 289, 121784(2022).</li>
<li>Perge, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intra-day+signal+instabilities+affect+decoding+performance+in+an+intracortical+neural+interface+system%5BTitle%5D)+AND+%22J+Neural+Eng%22%5BJournal%5D)">Intra-day signal instabilities affect decoding performance in an intracortical neural interface system.</a> J Neural Eng. 10 (3), 036004(2013).</li>
<li>Pachitariu, M., Steinmetz, N., Kadir, S., Carandini, M., Harris, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kilosort%3A+realtime+spike-sorting+for+extracellular+electrophysiology+with+hundreds+of+channels%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">Kilosort: realtime spike-sorting for extracellular electrophysiology with hundreds of channels.</a> bioRxiv. , 061481(2016).</li>
<li>Buzsáki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+recording+of+neuronal+ensembles%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Large-scale recording of neuronal ensembles.</a> Nat Neurosci. 7 (5), 446-451 (2004).</li>
<li>Kloosterman, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micro-drive+array+for+chronic+in+vivo+recording%3A+Drive+fabrication%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Micro-drive array for chronic in vivo recording: Drive fabrication.</a> J Vis Exp. (26), e1094(2009).</li>
<li>Jacobs, T., Darch, H., Holtzman, T., De Zeeuw, C. I., Romano, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standard+operating+protocol%3A+Implantation+of+Cambrige+NeuroTech+chronic+silicon+probe+and+mini-amp-64+digital+headstage+in+mice%5BTitle%5D)+AND+%22Protocol+Exchange%22%5BJournal%5D)">Standard operating protocol: Implantation of Cambrige NeuroTech chronic silicon probe and mini-amp-64 digital headstage in mice.</a> Protocol Exchange. , (2023).</li>
<li>Juavinett, A. L., Bekheet, G., Churchland, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+adaptable%2C+reusable%2C+and+light+implant+for+chronic+Neuropixels+probes%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">An adaptable, reusable, and light implant for chronic Neuropixels probes.</a> bioRxiv. , (2024).</li>
<li>Kozai, T. D. Y., Jaquins-Gerstl, A. S., Vazquez, A. L., Michael, A. C., Cui, X. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain+tissue+responses+to+neural+implants+impact+signal+sensitivity+and+intervention+strategies%5BTitle%5D)+AND+%22ACS+Chem+Neurosci%22%5BJournal%5D)">Brain tissue responses to neural implants impact signal sensitivity and intervention strategies.</a> ACS Chem Neurosci. 6 (1), 48-67 (2015).</li>
<li>Nguyen, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micro-drive+array+for+chronic+in+vivo+recording%3A+tetrode+assembly%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Micro-drive array for chronic in vivo recording: tetrode assembly.</a> J Vis Exp. (26), e1098(2009).</li>
<li>Biran, R., Martin, D. C., Tresco, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+cell+loss+accompanies+the+brain+tissue+response+to+chronically+implanted+silicon+microelectrode+arrays%5BTitle%5D)+AND+%22Exp+Neurol%22%5BJournal%5D)">Neuronal cell loss accompanies the brain tissue response to chronically implanted silicon microelectrode arrays.</a> Exp Neurol. 195 (1), 115-126 (2005).</li>
<li>Vöröslakos, M., Petersen, P. C., Vöröslakos, B., Buzsáki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metal+microdrive+and+head+cap+system+for+silicon+probe+recovery+in+freely+moving+rodent%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Metal microdrive and head cap system for silicon probe recovery in freely moving rodent.</a> eLife. 10, e65859(2021).</li>
<li>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/pubmed?term=((Carbon+nanotube+composite+coating+of+neural+microelectrodes+preferentially+improves+the+multiunit+signal-to-noise+ratio%5BTitle%5D)+AND+%22J+Neural+Eng%22%5BJournal%5D)">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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+visual+responses+by+behavioral+state+in+mouse+visual+cortex%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Modulation of visual responses by behavioral state in mouse visual cortex.</a> Neuron. 65 (4), 472-479 (2010).</li>
<li>Okun, M., Carandini, M., Harris, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long+term+recordings+with+immobile+silicon+probes+in+the+mouse+cortex%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">Long term recordings with immobile silicon probes in the mouse cortex.</a> bioRxiv. , (2015).</li>
<li>Chung, J., Sharif, F., Jung, D., Kim, S., Royer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micro-drive+and+headgear+for+chronic+implant+and+recovery+of+optoelectronic+probes%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Micro-drive and headgear for chronic implant and recovery of optoelectronic probes.</a> Sci Rep. 7 (1), 2773(2017).</li>
<li>Bimbard, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+adaptable%2C+reusable%2C+and+light+implant+for+chronic+Neuropixels+probes%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">An adaptable, reusable, and light implant for chronic Neuropixels probes.</a> bioRxiv. , (2024).</li>
<li>Jones, E. A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+recoverable+Neuropixels+in+mice%5BTitle%5D)+AND+%22protocols.io%22%5BJournal%5D)">Chronic recoverable Neuropixels in mice.</a> protocols.io. , (2023).</li>
<li>Neuronexus Products - dDrive. , Available from: 
 <a target="_blank" href="https://www.neuronexus.com/products/accessories/microdrives/ddrive/">https://www.neuronexus.com/products/accessories/microdrives/ddrive/</a> (2024).</li>
<li>van Daal, R. J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Implantation+of+Neuropixels+probes+for+chronic+recording+of+neuronal+activity+in+freely+behaving+mice+and+rats%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Implantation of Neuropixels probes for chronic recording of neuronal activity in freely behaving mice and rats.</a> Nat Protoc. 16 (7), 3322-3347 (2021).</li>
<li>Guo, Z. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Procedures+for+behavioral+experiments+in+head-fixed+mice%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Procedures for behavioral experiments in head-fixed mice.</a> PLoS One. 9 (2), e88678(2014).</li>
<li>Groblewski, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+standardized+head-fixation+system+for+performing+large-scale%2C+in+vivo+physiological+recordings+in+mice%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">A standardized head-fixation system for performing large-scale, in vivo physiological recordings in mice.</a> J Neurosci Methods. 346, 108922(2020).</li>
<li>Vasilev, D., Raposo, I., Totah, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brightness+illusions+evoke+pupil+constriction+preceded+by+a+primary+visual+cortex+response+in+rats%5BTitle%5D)+AND+%22Cereb+Cortex%22%5BJournal%5D)">Brightness illusions evoke pupil constriction preceded by a primary visual cortex response in rats.</a> Cereb Cortex. 33 (12), 7952-7959 (2023).</li>
</ol>

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><tbody><tr><td>0.05&quot; Solder Tail Socket</td><td>Mill-Max</td><td>853-93-100-10-001000</td><td></td></tr><tr><td>1,1&#039;-dioctadecyl-3,3,3&#039;,3&#039;- Reagent tetramethylindocarbocyanine perchlorate (&#039;DiI&#039;; 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&amp;amp;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

962 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

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

Genetics

Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test interventions at various stages of the disease process. These animal models are particularly difficult to implement in immature rodents, a classic model organism for laboratory study of these disorders. Recording continuous EEG in young animal models of seizures and other neurological disorders presents a technical challenge due to the small physical size of young rodents and their dependence on the dam prior to weaning. Therefore, there is not only a clear need for improving pre-clinical research that will better identify those therapies suitable for translation to the clinic but also a need for new devices capable of recording continuous EEG in immature rodents. Here, we describe the technology behind and demonstrate the use of a novel miniature telemetry system, specifically engineered for use in immature rats or mice, which is also effective for use in adult animals.

Here we demonstrate the use of a wireless enabling technology for electroencephalogram (EEG) in neonatal rodent models of human disease. With telemetry, there are no encumbering connections, thus allowing natural behaviors.

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test ...

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

Subdural Soft Electrocorticography (ECoG) Array Implantation and Long-Term Cortical Recording in Minipigs

Neurological impairments and diseases can be diagnosed or treated using electrocorticography (ECoG) arrays. In drug-resistant epilepsy, these help delineate the epileptic region to resect. In long-term applications such as brain-computer interfaces, these epicortical electrodes are used to record the movement intention of the brain, to control the robotic limbs of paralyzed patients. However, current stiff electrode grids do not answer the need for high-resolution brain recordings and long-term biointegration. Recently, conformable electrode arrays have been proposed to achieve long-term implant stability with high performance. However, preclinical studies for these new implant technologies are needed to validate their long-term functionality and safety profile for their translation to human patients. In this context, porcine models are routinely employed in developing medical devices due to their large organ sizes and easy animal handling. However, only a few brain applications are described in the literature, mostly due to surgery limitations and integration of the implant system on a living animal.
Here, we report the method for long-term implantation (6 months) and evaluation of soft ECoG arrays in the minipig model. The study first presents the implant system, consisting of a soft microfabricated electrode array integrated with a magnetic resonance imaging (MRI)-compatible polymeric transdermal port that houses instrumentation connectors for electrophysiology recordings. Then, the study describes the surgical procedure, from subdural implantation to animal recovery. We focus on the auditory cortex as an example target area where evoked potentials are induced by acoustic stimulation. We finally describe a data acquisition sequence that includes MRI of the whole brain, implant electrochemical characterization, intraoperative and freely moving electrophysiology, and immunohistochemistry staining of the extracted brains.
This model can be used to investigate the safety and function of novel design of cortical prostheses; mandatory preclinical study to envision translation to human patients.

Subdural Soft Electrocorticography ECoG Array Implantation and Long-Term Cortical Recording in Minipigs

Here, we present a method for the long-term performance and safety assessment of soft subdural electrode arrays in a minipig model, describing surgical method and tools, postoperative magnetic resonance imaging, electrophysiology of the auditory cortex, electrochemical properties of the implant, and postmortem immunochemistry.

Neurological impairments and diseases can be diagnosed or treated using electrocorticography (ECoG) arrays. In drug-resistant epilepsy, these help ...

Bioengineering

An Implantable System For Chronic In Vivo Electromyography

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in assessing neuromuscular function. Chronic recording of EMG activity reflecting a muscle&#8217;s reinnervation status after nerve injury has been limited, due to the invasive nature of traditional EMG recording techniques. In this regard, an implantable system is designed for long-term, in vivo EMG recording and nerve stimulation. It has been applied and tested in a study on reinnervation of laryngeal muscles. This system consists of 1) two bipolar electrode nerve cuffs and leads for stimulating each of two nerves: the recurrent laryngeal nerve (RLN) and internal branch of the superior laryngeal nerve (SLN); 2) two EMG recording electrodes and leads for each of the two laryngeal muscles: posterior cricoarytenoid (PCA) muscle and thyroarytenoid-lateral cricoarytenoid (TA-LCA) muscle complex; and 3) a skin receptacle interfacing all implanted lead terminals to an external recording preamplifier and stimulator using a connection cable. The wire leads are Teflon-coated, multi-filament, type 316 stainless steel. They are coiled and can stretch during body movement of the awake animal to prevent lead breakage and electrode migration. This system is implanted during an aseptic surgery. Afterwards, baseline EMG recordings are performed before the RLN is transected in the second surgery to study muscle reinnervation. Throughout the study, multiple physiological sessions are conducted in the anesthetized animal to obtain evoked and spontaneous EMG activity that reflects the reinnervation status of laryngeal muscles. The system is compact, free of infection over the course of the study, and highly durable. This implantable system can provide a reliable platform for research in which long-term recording or nerve stimulation is required in an anesthetized or freely moving animal.&#160;

Presented here is a protocol for the manufacturing of an implantable system for in vivo chronological recording of evoked and spontaneous electromyographic potentials. The system is applied to the investigation of reinnervation of laryngeal muscles following nerve injury. &#160;&#160;

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in ...

Stimulating Mouse Brain with a Low-Cost EEG and Millimeter-Sized Coil

Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, utilizing a millimeter-sized coil. Using conventional screw electrodes combined with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain. In addition, we explain how a millimeter-sized coil is produced using low-cost equipment usually found in laboratories. Practical procedures for fabricating the flexible multielectrode array substrate and the surgical implantation technique for screw electrodes are also presented, which are necessary to produce low-noise EEG signals. Although the methodology is useful for recording from the brain of any small animal, the present report focuses on electrode implementation in an anesthetized mouse skull. Furthermore, this method can be easily extended to an awake small animal that is connected with tethered cables via a common adapter and fixed with a TMS device to the head during recording.The present version of the EEG-TMS system, which can include a maximum of 32 EEG channels (a device with 16 channels is presented as an example with fewer channels) and one TMS channel device, is described. Additionally, typical results obtained by the application of the EEG-TMS system to anesthetized mice are briefly reported.

Author Spotlight: Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo  

A low-cost electroencephalographic recording system combined with a millimeter-sized coil is proposed to drive transcranial magnetic stimulation of the mouse brain in vivo. Using conventional screw electrodes with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain in response to transcranial magnetic stimulation.

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, ...

Detecting Spreading Depolarization Using AC Amplifiers and Non-Invasive Imaging

Combining Imaging and Electrophysiology to Visualize and Record Spreading Depolarizations in Mice

Spreading Depolarizations (SDs) are massive events in the brain that often go undetected due to their slow propagation through gray matter. Because SD detection can be elusive, it is optimally confirmed using multiple methods. This protocol describes methods for combining imaging and electrophysiology to detect SDs in a manner that most laboratories can reliably and easily adopt. SDs occur following traumatic brain injuries, stroke, subarachnoid hemorrhages, ischemia, and migraine aura. Historically, SDs have been recorded using DC amplifiers, which can resolve the slow extracellular shift and the depression in high-frequency activity. However, DC amplifiers are nearly impossible to use for chronic in vivo recordings. This protocol employs a common AC amplifier for in vivo electrophysiology recordings to confirm high-frequency depression, along with non-invasive imaging necessary to detect the propagating wave of SD. These methods can be reliably adopted and/or modified for most experimental approaches to confirm the presence or absence of SDs following brain injury.

This protocol demonstrates the combination of imaging and electrophysiology to reliably detect spreading depolarizations in adult mice following a mild traumatic brain injury.

Spreading Depolarizations (SDs) are massive events in the brain that often go undetected due to their slow propagation through gray matter. Because SD ...

Injectable Mesh Electronics for Stable Single-Neuron Recordings in a Mouse Brain

Source: Schuhmann Jr., T. G., et al. <a href="https://app.jove.com/v/58003">Syringe-injectable Mesh Electronics for Stable Chronic Rodent Electrophysiology.</a> J. Vis. Exp. (2018).
The video demonstrates the injection of mesh electronics probes into a mouse brain for stable long-term recordings at a single-neuron level. An anesthetized mouse with an exposed skull is secured on a stereotaxic frame, and the probe&#39;s mesh device region is injected into the brain through drilled holes. The probe&#39;s input-output pads are connected to a circuit, and recordings are obtained using a data acquisition system.

1. Stereotaxic Injection of Mesh Electronics into Live Mouse Brain
NOTE: Mice were anesthetized by intraperitoneal injection with a mixture of 75 mg/kg ...

JoVE Encyclopedia of Experiments

Neurophysiology

Stimulation and Modulation Techniques

Implanting a Wireless Bidirectional Microelectrode System in a Rat Brain

Source: Melo-Thomas, L., et al., <a href="https://app.jove.com/v/56299">A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats.</a> J. Vis. Exp. (2017)
The video demonstrates a procedure for implanting a wireless bidirectional multichannel electrode unit into a rat brain to record and stimulate multichannel neural networks.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Electrophysiology Techniques

Implanting an On-Head Electrode System in a Rat Model for Real-Time Seizure Detection

Source: Koz&#225;k, G., et al. <a href="https://app.jove.com/v/56669">Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats.</a> J. Vis. Exp. (2018).
This video demonstrates the method of implanting an on-head electrode system in an anesthetized rat for real-time seizure detection. The procedure includes placing stimulation and recording electrodes, securing them with dental cement, and connecting them to a head-mounted amplifier.