Custom-built micro-drives enable the sub-millimeter targeting of cortical recording sites with linear silicon arrays.
The marmoset monkey provides an ideal model for examining laminar cortical circuits due to its smooth cortical surface, which facilitates recordings with linear arrays. The marmoset has recently grown in popularity due to its similar neural functional organization to other primates and its technical advantages for recording and imaging. However, neurophysiology in this model poses some unique challenges due to the small size and lack of gyri as anatomical landmarks. Using custom-built micro-drives, researchers can manipulate linear array placement to sub-millimeter precision and reliably record at the same retinotopically targeted location across recording days. This protocol describes the step-by-step construction of the micro-drive positioning system and the neurophysiological recording technique with silicon linear electrode arrays. With precise control of electrode placement across recording sessions, researchers can easily traverse the cortex to identify areas of interest based on their retinotopic organization and the tuning properties of the recorded neurons. Further, using this laminar array electrode system, it is possible to apply a current source density analysis (CSD) to determine the recording depth of individual neurons. This protocol also demonstrates examples of laminar recordings, including spike waveforms isolated in Kilosort, which span multiple channels on the arrays.
The common marmoset (Callithrix jacchus) has quickly grown in popularity as a model to study brain function in recent years. This growing popularity is due to the accessibility of the marmoset's smooth cortex, the similarities in neural functional organization with humans and other primates, and the small size and fast breeding rate1. As this model organism has grown in popularity, there has been rapid development in the neurophysiological techniques suited for use in the marmoset brain. Electrophysiology methods are widely used in neuroscience to study the activity of single neurons in the cortex of both rodents and primates, resulting in unparalleled temporal resolution and location access. Due to the relative novelty of the marmoset monkey as a model of visual neuroscience, the optimization of awake-behaving electrophysiology techniques is still evolving. Previous studies have shown the establishment of robust protocols for electrophysiology in anesthetized preparations2, and early awake-behaving neurophysiology studies have shown the reliability of single-channel tungsten electodes3. In recent years, researchers have established the use of silicon-based microelectrode arrays for awake-behaving neurophysiology4. However, the marmoset poses unique targeting challenges due to its small brain size and lack of anatomical landmarks. This protocol outlines how to construct and use a micro-drive recording system suitable for the marmoset that allows for the recording of large populations of neurons with silicon linear arrays while producing minimal tissue damage.
Working with the marmoset poses a challenge due to the smaller scale of the retinotopic maps in the visual cortex as compared to larger primates. A slight shift of the electrodes by just 1 mm can result in significant changes in the maps. Moreover, researchers often need to alter the placement of the electrodes between the recording sessions to obtain a broader range of retinotopic positions in the visual cortex. Current semi-chronic preparations do not allow for the adjustment of the electrode positioning daily or with enough precision to target specific locations at sub-millimeter scales5. With this in mind, the proposed micro-drive system utilizes an X-Y electrode stage that mounts a lightweight micro-drive to a recording chamber and allows for the sub-millimeter targeting of cortical sites. The moveable X-Y stage components allow for vertical and horizontal movement of the linear array in order to traverse the cortical areas systematically, which is required to identify areas of interest (via retinotopy and tuning properties). Across recording sessions, researchers can also manually adjust the X-Y stage to shift the targeted sites within the area. This is a key advantage over alternative techniques using semi-chronic recording preparations, which do not have easy electrode targeting mechanisms.
The micro-drive is a versatile tool that enables the attachment of various silicon arrays to be mounted for lowering into the cortex. In this protocol, a custom probe with two 32-channel linear arrays spaced 200 µm apart was used for the investigation of laminar circuits spanning the cortical depth. Most methods for probing the neural circuitry typically sample the electrical potentials or single units averaged across all the layers of the cerebral cortex. However, recent research has revealed intriguing findings about cortical laminar microcircuits6. By utilizing the micro-drive, researchers can use laminar probes and make fine adjustments to the recording depth to ensure comprehensive sampling across all the layers.
This system can be constructed with commercially available components and is easily modified for different experimental techniques or probes. The key advantages of this preparation are the ability to change the X-Y recording position with sub-millimeter precision and to control the depth of the recording within the cortex. This protocol presents step-by-step instructions for constructing the X-Y stage micro-drive and neurophysiology recording techniques.
The experimental procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocols for the experimental and behavioral procedures were approved by the University of Rochester Institutional Animal Care and Use Committee.
1. Construction of the micro-drive containing the electrode for recording (Figure 1)
NOTE: Custom-built X-Y stages holding multi-channel linear silicon arrays allow for sub-millimeter targeting of the recording sites.
2. Polytrode plating of electrodes to reduce the overall impedance (Figure 2)
NOTE: For the best recording quality, it is useful to electrode-plate the silicon electrode arrays with a poly(3,4-ethylenedioxythiophene) solution (PEDOT). This method has been shown to increase the signal-to-noise ratio7,8.
3. Surgical placement of the head cap, chambers, and craniotomy (Figure 3A-C)
NOTE: In this work, at the termination of the study, the animal was anesthetized under isoflurane and received intramuscular (IM) injections of ketamine, followed by an intraperitoneal (IP) injection of euthasol. The brain was extracted following transcardial perfusion with saline followed by 10% formalin.
4. Neurophysiology recording setup (Figure 3D-F)
NOTE: The animal handling steps will vary depending on the lab and experiment. The following steps are specific to the placement of the micro-drive and the penetration of the dura for the recordings.
5. Performing the neurophysiology recording experiment (Figure 4)
NOTE: Here, the method for lowing the electrode arrays into the cortex is described; this method has been optimized to avoid excessive dimpling of the underlying tissue. An increase in noise in the electrophysiological recordings provides a good indication of dimpling prior to penetrating the silastic and entering the brain. Once in the brain, if there is too much dimpling, the researcher may notice units shifting on the probe even without the manipulation of the drive (units gradually moving across channels in depth), or alternatively, the researcher may notice suppression of the neural activity, particularly at superficial sites on the probe. In those conditions, the drive is retracted to relieve dimpling and facilitate better recordings.
This protocol describes how to build an X-Y electrode stage (Figure 1) that allows for the sub-millimeter targeting of sites and maintains reliable positioning across separate recording sessions. The reliability of the X-Y positioning is illustrated in Figure 6, which demonstrates that two recording sessions conducted a week apart showed a 70.8% overlap in their mean RF locations (Figure 6A). Furthermore, minor adjustments to the micro-drive positioning can support precise movement in the retinotopic space. Small X and Y stage movements up to 0.2 mm can be made by referencing the predrilled holes in each stage, which are 2 mm apart. In a series of recording sessions that transversed the MT/MTC border using minor positioning adjustments (Figure 6B), precise movements in retinotopic space could be seen (Figure 6C). The recording sites for the MT and MTC aligned with electrophysiology maps previously depicted in Solomon and Rosa2. Similarly, with this protocol, the Z-positioning can be tracked by the number of turns to the preferred electrode depth. While the Z-positioning of the drive to place electrodes in cortical depth must be adjusted daily and includes some variation, it is possible to obtain estimates of depth in early visual areas offline by using current source density analysis (CSD), as described previously16.
Using this X-Y stage technology, researchers can perform neurophysiological recording with linear arrays while the subjects perform a variety of foraging tasks for visual receptive field mapping and simple saccade tasks17. Electrode placement techniques are shown in Figure 4, which highlights the importance of retraction to alleviate pressure on the tissue and prevent long-term cortical damage. Using two shank linear arrays allows for sampling a large number of neurons across cortical depths during each recording. In this work, the spike-sorted array data obtained using Kilosort showed that the principle component features of a selection of spikes in a cluster were distinct from background spikes and that the recordings were stable over time (Figure 5).
Figure 1: Construction of the electrode micro-drive. The construction of an electrode micro-drive is possible after obtaining all the necessary components outlaid in section 1, step 1. Each successive step (section 1, steps 2-10) shows a before and an after image to aid in the visualization of the actions required. This construction is explained fully in protocol section 1. Please click here to view a larger version of this figure.
Figure 2: Constructed micro-drive and electroplating setup. Overview image of the completed micro-drive from the (A) top and (B) bottom. (C) The micro-drive is attached to a protective sleeve (same dimensions as the chambers on the animal's implant, with an extended length for protection) and then attached to the animal's chamber by tightening three side screws. (D) Configuration of the micro-drive on the impedance tester for electroplating. Please click here to view a larger version of this figure.
Figure 3: Schematic of the head cap and chamber placement (A) Drawing representation of an animal with a head cap, head post, and MT chamber. (B) Image of an MT chamber 6 months after craniotomy and sealed with a thin layer of silastic. (C) Schematic of the chamber and micro-drive preparation. The illustration shows the chamber preparation and the thin layer of silastic for use in the recordings. The micro-drive is secured to the recording chamber by tension from the side screws. (D) One should move parallel to the chamber to attach the micro-drive to the recording chamber. Coming from an angle will likely result in hitting the side of the chamber or the cement sides of the craniotomy. (E) The micro-drive secured on an example head cap. (F) Depiction of the three screws on the micro-drive to be tightened for secure placement. Note: One screw sits behind the connector and is usually tightened from above. The screws are tightened in a repeatable order to ensure drive location reliability. Please click here to view a larger version of this figure.
Figure 4: Neurophysiology recordings (A) Depiction of the electrode before and after it touches the dura (top). High-pass-filtered recording signal from a single probe in a neural recording before and after the probe touches the dura (bottom). (B) Depiction of an electrode causing dimpling after initially popping through the silastic (top). High-pass-filtered recording signal from one probe in a neural recording after the probe has popped through the silastic (bottom). Channels with neurons are labeled as filled. (C) Depiction of the electrode in the cortex before and after retraction (top). High-pass-filtered recording signal from one probe in a neural recording before and after a 500 µm retraction; the units have shifted one channel (bottom). Please click here to view a larger version of this figure.
Figure 5: Post-processing of neurophysiology data. (A) Depiction of a laminar probe (left) and a selection of spike waveforms viewed across each channel (right). (B) Example Kilosort output. (Left) Example waveform across several channels with a peak waveform on channel 46. (Top right) The principle component features of a selection of spikes in the selected cluster (blue) against a set of background spikes (uniformly spaced in time across the entire recording, grey). Spiking clusters are easily discriminated from background spikes. (Bottom right) The amplitude of a selection of spikes belonging to the selected cluster over time. The recordings show stability over time for this example neuron. Please click here to view a larger version of this figure.
Figure 6: Reliability of the X-Y stage positioning (A) Mean receptive field (RF) contours for two recording sessions 1 week apart with the same micro-drive coordinates, showing 70.8% overlap. (B) An MT/MTC map showing the successive targeting locations across recording sessions. The solid lines and numbers indicate eccentricity from the fovea in degrees. The dashed lines indicate the horizontal meridian (HM). The bolded lines indicate the vertical meridian (VM). (C) The corresponding RF locations for each shank outlined with a circle marking the center location show that minor adjustments to the micro-drive positioning can support precise movement in the retinotopic space. Panel B has been adapted with permission from Solomon and Rosa2. Please click here to view a larger version of this figure.
Several methods (e.g., chronic, semi-chronic, acute) are currently available for performing neurophysiology experiments in non-human primates. The common marmoset poses unique challenges for neurophysiology experiments due to its small size and lack of gyri as anatomical landmarks. This requires researchers to use neurophysiological landmarks such as the retinotopy and tuning properties of areas of interest to identify the recording targets. Therefore, when initially mapping out a cortical area, daily adjustments to the electrode positioning may be needed. Current preparations for marmoset neurophysiology often use semi-chronic probe positioning, which does not allow for easy-access positioning mechanisms5. This protocol demonstrates a lightweight micro-drive for stable linear array recordings that enables flexible positioning with precise sub-millimeter movements across sessions to map out the retinotopy within a chamber.
The current approach, which involves acute daily electrode penetrations for precise electrode targeting and movement across retinotopic maps, differs from chronic electrode array implants in marmosets5. However, acute preparations intrinsically have drawbacks, such as an increased experimental preparation time and an increased risk of infection, as well as an increased risk of cortical tissue damage due to repeated penetrations. While the use of silastic has been optimized in this preparation to limit the risk of infection, applying the silastic does require some troubleshooting to ensure it is done correctly and to obtain a dry seal. This preparation also demands care to avoid electrode damage during the drive mounting and requires care in tightening the drive screws to ensure reliable targeting across sessions.
Long-term cortical damage can be avoided in this preparation when applied correctly with a secure drive, slow penetration of the tissue, and the avoidance of major blood vessels. Additionally, the use of silastic in the craniotomy to prevent infections has been optimized in this technique. The brain is covered with a thin layer of silastic after the craniotomy is performed to prevent infection and keep the interior of the chamber dry14. When the dura is clear from blood and granulation tissue (which is possible with a tight silastic seal), this enables the visualization of the major blood vessels in the underlying tissue and for targeting the silicon probes to avoid hitting the blood vessels during recordings. This technique requires some troubleshooting during learning, since it is critical that the silastic seal is thin (<1 mm) in the center to allow electrode penetration without excessive dimpling while also being thick at the edges to provide mechanical purchase. These methods will be valuable in investigating the circuitry role of the layers across the marmoset cortex and in preventing tissue damage during multiple penetrations.
Using custom-printed plastic components along with commercially available supplies, this micro-drive design is easy to use once fabricated and can be personalized for different recording probes. While this method has been shown to be reliable, the initial fabrication can be difficult and costly to learn. However, the components of the probe are relatively economical to purchase, and, once constructed, the probes have been shown to last up to 1 year with continual use.
One of the key advantages of this preparation is the precise and repeatable targeting of areas of interest due to the X-Y stage. With this technology, researchers are able to map the retinotopic locations within the recording chambers and record from different areas across days using a coordinate system. To achieve this, it is critical to ensure the correct placement of the probe daily and to tighten all the exterior screws in a repeatable manner. Changes to the exterior screw tightness can lead to small shifts in the electrode placement across days.
Acute recordings offer the benefit of repeated independent measurements and can result in a higher yield of single-unit data. However, semi-chronic or chronic recordings may also offer advantages. In small animals such as mice, chronic recordings can be made with high-density silicon probes to obtain stable, long-term population recordings over multiple days18. Eliminating the repeated insertions of electrodes into the brain for each recording session can shorten the overall experimental preparation time and reduce the risk of infection or cortical tissue damage. Therefore, when evaluating the use of chronic or acute recording preparations, it is essential to consider the specific experimental requirements.
The proposed micro-drive design allows the flexibility of different recording techniques and array usage, meaning it could be easily adopted into existing experimental setups and pipelines. Furthermore, the flexibility of the array mounting on the micro-drive may lead to possible future applications, such as controlling laser stimulation for optogenetic purposes. This method may enable future studies to improve on targeting for laser stimulation in the marmoset.
The authors have nothing to disclose.
This work was supported by the National Institutes of Health (NIH) grant R01 EY030998 (J.F.M., A.B., and S.C.). This method is based on methods developed in Coop et al. (under review, 2022; https://www.biorxiv.org/content/10.1101/2022.10.11.511827v2.abstract). We would like to thank Dina Graf and members of the Mitchell lab for help with the marmoset care and handling.
1/4 Hp burr drill bit | McMaster & Carr | Cat# 43035A32 | Carbide Bur with 1/4" Shank Diameter, Rounded Cylinder Head, trade Number SC-1, single Cut(https://www.mcmaster.com/products/bur-bits/burs-7/?s=1%2F4%22+bur+bits) |
1x1mm Crist Grid | Crist Instruments | 1 mm x 1 mm Grid | https://www.cristinstrument.com/products/implant-intro/grids |
91% isopropyl alcohol | Medline | N/A | https://www.medline.com/product/Medline-Isopropyl-Rubbing-Alcohol/Bulk-Alcohol/Z05-PF03807?question=91%25%20isopropyl%20alcohol |
Acquisition Board | Open-Ephys | N/A | https://open-ephys.org/acquisition-system/eux9baf6a5s8tid06hk1mw5aafjdz1 |
Bacitracin Ointment | Medline: Cosette Pharmaceuticals Inc | N/A | https://www.medline.com/product/Bacitracin-Ointment/Antibiotics/Z05-PF86957?question=bacitr |
Blunt straight Forceps | Medline | N/A | https://www.medline.com/category/Central-Sterile/Surgical-Instruments/Forceps/Z05-CA16_02_20/products |
Bone wax | Medline | ETHW31G | https://www.medline.com/product/Ethicon-Bone-Wax/Bone-Wax/Z05-PF61528?question=bonewax |
C&B Metabond Quick Adhesive Cement System | Parkell, Inc. | SKU: S380 | https://www.parkell.com/C-B-Metabond-Quick-Adhesive-Cement-System |
Clavamox | MWI Animal Health | N/A | |
Contact lens solution | Bausch and lomb | Various sources available | |
Custom Printed 3D printed parts | ProtoLab | https://marmolab.bcs.rochester.edu/resources.html | |
DB25-G2 25 Pin Male Plug Port Signal Connector | Various Sources | DB25-G2 25 | DB25-G2 25 Pin Male Plug Port Signal 2 Row Terminal Breakout Board Screw Nut Connector |
diamond saw attachement for dremmel | Dremmel | 545 Diamond Wheel | https://www.dremel.com/us/en/p/545-26150545ab |
Digitizing Head-stages | Intan | RHD 32channel (Part #C3314) | https://intantech.com/RHD_headstages.html?tabSelect=RHD32ch&yPos=120.80 000305175781 |
EDOT | Sigma Aldrich | Product # 483028 | https://www.sigmaaldrich.com/US/en/product/aldrich/483028 |
Helping Hands | Harbor Freight | N/A | https://www.harborfreight.com/helping-hands-60501.html |
Hook Electrical Clips | Various Sources | N/A | Hook test Cable wires |
Interface Cables (RHD 3-ft (0.9 m) ultra thin SPI cable) | Intan | Part #C3213 | https://intantech.com/RHD_SPI_cables.html |
Lab jack | Various Sources | N/A | https://www.amazon.com/Stainless-Steel-Scissor-Stand-Platform/dp/B07T8FM85H/ref=asc_df_B07T8FM85H/?tag=&linkCode=df0&hvadid=366343 827267&hvpos=&hvnetw=g&hvrand =2036619536500717246&hvpone =&hvptwo=&hvqmt=&hvdev=c&hv dvcmdl=&hvlocint=&hvlocphy=900 5674&hvtargid=pla-795933567991& ref=&adgrpid=71496544770&th=1 |
Meloxicam | MWI Animal Health | N/A | |
Micro-drive | Crist Instrument | 3-NRMD | https://www.cristinstrument.com/products/microdrives/miniature-microdrive-3-nrmd |
Multi-channel linear silicon arrays with 64 channel connector | NeuroNexus | A1x32-5mm-25-177 | https://www.neuronexus.com/products/electrode-arrays/up-to-10-mm-depth/ |
NanoZ Omentics Adapter- 32 Channel | NeuraLynx | ADPT-NZ-N2T-32 | https://neuralynx.com/hardware/adpt-nz-n2t-32 |
NanoZ System | Plexon | NanoZ Impedence Tester | https://plexon.com/products/nanoz-impedance-tester/ |
Narishige Micromanipulator | Narishige | Stereotaxic Micromanipulator | https://usa.narishige-group.com/ |
Open-Ephys GUI | Open-Ephys | https://open-ephys.org/ | |
Polyimide Tubing (OD(in): 0.021 / ID(in) 0.018 ) | Various Sources (Chamfr) | Chamfr Cat#HPC01895 | https://chamfr.com/sellers/teleflex-medical-oem-llc/ |
Primate Chair | Custom made by University of Rochester Machine Shop | Designs online | https://marmolab.bcs.rochester.edu/resources.html |
Poly(sodium 4-styrenesulfonate) (PSS) | Sigma Aldrich | Product # 243051 | https://www.sigmaaldrich.com/US/en/product/aldrich/243051 |
RHD USB Interface board | Intan | RHD2000 Evaluation Board Version 1.0 | https://intantech.com/RHD_USB_interface_board.html |
Silastic gel | World Precision Instuments | # KWIK-SIL | Low Toxicity Silicone Adhesive ((https://www.wpiinc.com/kwik-sil-low-toxicity-silicone-adhesive) |
Slow release buprenorphine | Compounding Pharmacy | ||
Stainless steel wire 36 gauge | McMaster & Carr | Cat# 6517K11 | Round Bend-and-Stay Multipurpose 304 Stainless Steel Wire, Matte Finish, 1-Foot Long, 0.008" Diameter |
Stanley 6-Piece Precision Screwdriver Set | Stanley | 1.4mm flathead screwdriver | https://www.amazon.com/Stanley-Tools-6-Piece-Precision-Screwdriver/dp/B076621ZGC/ref=sr_1_3?crid=237VSK5FNFP9N&keywords= stanley+66-052&qid=1672764369&sprefix= stanley+66-052%2Caps%2C90&sr=8-3 |
Steel Screws | McMaster & Carr | type 00 stainless steel hex screws and 1/8” in length | https://www.mcmaster.com/ |
Steel Tube | McMaster & Carr | 28 gauge stainless steel tubing | https://www.mcmaster.com/tubing/multipurpose-304-stainless-steel-6/id~0-055/ |
Superglue | Loctite | SuperGlue Gel Control | https://www.loctiteproducts.com/en/products/fix/super-glue/loctite_super_gluegelcontrol.html |