Instructions for the low-cost construction and surgical implantation of a chronic transcranial high-density electroencephalographic montage into mice are provided. Signal recording, extraction, and processing techniques are also described.
Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network connectivity, are applicable to an expanding variety of questions in neuroscience. Performing these kinds of analyses in a rodent model requires higher electrode density than traditional screw electrodes can accomplish. While higher-density electroencephalographic montages for rodents exist, they are of limited availability to most researchers, are not robust enough for repeated experiments over an extended period of time, or are limited to use in anesthetized rodents.1-3 A proposed low-cost method for constructing a durable, high-count, transcranial electrode array, consisting of bilaterally implantable headpieces is investigated as a means to perform advanced electroencephalogram analyses in mice or rats.
Procedures for headpiece fabrication and surgical implantation necessary to produce high signal to noise, low-impedance electroencephalographic and electromyographic signals are presented. While the methodology is useful in both rats and mice, this manuscript focuses on the more challenging implementation for the smaller mouse skull. Freely moving mice are only tethered to cables via a common adapter during recording. One version of this electrode system that includes 26 electroencephalographic channels and 4 electromyographic channels is described below.
Neuronal activity can be recorded extracellularly with various levels of granularity from microscopic (individual action potentials) to mesoscopic (local field potentials) to macroscopic (electroencephalogram). These brainwave traces are classically analyzed in the frequency domain to characterize behavioral, neurophysiological, or electrophysiological states. This can be done with a single biopotential,4 but sparse density EEG recordings cannot resolve the spatial component of neuronal activity. Modern electroencephalogram analysis relies on multiple electrodes to produce detailed maps of spatiotemporal distribution of cortical activity in order to correlate that activity with specific psychological conditions and physiologic processes.5-7 Two of the more commonly used categories of analysis requiring high-density EEG montages are electrical source imaging and neural network connectivity measures.8-11
Electrical source imaging involves the localization of functionally active brain regions. Topographical mapping of the electrode array can visualize the current source density of the electrical activity within the brain during event related potentials (ERPs) and evoked potentials (EPs). Electrical source localization is commonly used in both seizure studies as well as in power distribution analyses.12-15 Since EEG has high temporal resolution, EEG studies permit real time evaluation of ERPs and EPs as well as temporally precise post hoc analysis.3,11,12
Associating cognitive states and functions with the interplay of oscillations seen on the electroencephalogram is the ultimate goal of the various measures of neural network connectivity. Numerous studies have shown synchronization and phase locking of oscillations among different brain regions are associated with specific states of arousal, attention, and action.6,13,14,16-19 Demonstrating such signal associations among brain regions requires high-density arrays that permit assessments of network connectivity.
Source localization and network analyses of EEG signals originated with studies in humans, but investigations into the neuronal basis for these signals necessarily involve animal models, as they require invasive techniques that are otherwise impossible in humans. In order to replicate these analyses in rodent models, a method for capturing high-density EEG signals in a rodent's brain is needed. While other groups have constructed high-density microelectrode arrays for use in mice, such approaches are of limited availability to researchers without access to nanofabrication facilities, are not robust enough for repeated experiments over an extended period of time, or are limited to use in anesthetized mice.1-3,7 A low cost alternative protocol for constructing chronic high-density, transcranial electrode array is demonstrated here.
The signal acquisition approach described here is not limited to EEG, but includes electromyographic (EMG) signals. Acquisition of EMG signals can be a complementary approach for defining behavior state and is particularly useful for sleep studies. This approach provides an intermediate between expensive, ultra-high-density intracranial grids, and the limited lead numbers possible with traditional screw electrodes that are insufficient for advanced analysis approaches. The headpiece design is easily constructed and affordable for high-throughput studies. Use of this acquisition system in conjunction with assorted genetic or pharmacologic manipulative techniques within rodent models can help uncover the mechanisms of cortical oscillation generation, behavioral divergences from true genotypic differences, source localization of ERPs and EPs, and large-scale network communication.
The low-cost construction and surgical steps necessary in order to properly attain a 26 channel, high-density EEG montage in a mouse is described. Proper epidural electrode contact is critical in acquiring quality EEG signals in this system. Two steps within the protocol address this issue: pin trimming to match brain contour, and headpiece implantation prior to acrylic reinforcement. It is important not to cut a pin too short during the construction phase. When implanting the headpieces, it is imperative to check pin placement before the final acrylic reinforcement. One way to confirm proper electrode contact is through impedance testing. Ostensibly, impedances of 5-10 kΩ suggest proper epidural placement.26 Impedance measurements demonstrate the headpieces' durability, as electrode impedance values are stable within this 5-10 kΩ range for at least 4 months after implantation. The other essential step involves aligning the EMG pins with the two posterior-most rows of the 2 x 7 EEG brick. This is critical for adapter connection, as misaligned EMG and EEG pins will result in an inability to connect the adapter or bent adapter pins.
A major advantage of this acquisition system is the ease of modifying the shape of the electrode array in order to optimize varied experimental needs. Customized electrode arrangements that are optimally suited for specific experiments can be readily created. Customization for specific experiments could potentially combine EEG with cannula for directed drug delivery for combined pharmacological, electroencephalographic, and behavioral studies.27 Headpieces, adapters, and surgical procedures are easily tailored to a wide number of studies when following the methods described in the protocol above. A second major advantage of this acquisition system is its low cost. At present, this acquisition system can record 128 input channels on up to 4 separate cables, permitting simultaneous recordings from 4 mice or if desired, rats with higher density grids. Such expansion would only require extra cables and adapters.
This approach to high-density EEG acquisition addresses drawbacks of other high-density EEG acquisition methods in mice. The system described in this work is handily constructed with simple materials and uses open source hardware and software that is inexpensive and stable, allows for repeated measurements in the same animal over months, permits free movement during an experiment, and does not require mice to be anesthetized for recording. Limitations of this system are that it has only been validated to date in mice that weigh 20 g or more, and are older than 12 weeks. Smaller or younger mice may have difficulty with the headpiece implantation. A secondary limitation of this methodology is the inability to precisely control electrode depth after headpiece fabrication. However, this same limitation applies to traditional screw EEG electrodes since there is no way to precisely know the pre-mortem screw depth relative to the cortical surface. Troubleshooting for this method typically involves properly shielding interfering signal from the mouse when tethered in order to obtain noise-free signal.
High-density EEG arrays are essential for the complex spatiotemporal analyses of EEG data that are the new normal in modern EEG interpretation. While spatial distribution of a visual evoked potential is illustrated, data acquired using this system can be analyzed using electrical source imaging techniques and neuronal connectivity measures. A 60% to 70% reduction in contact area between these electrode pins compared to traditional screw contacts permits more precise signal localization, as shown in Figure 4. Employing high-density analytic techniques in genetically modified mice, following pharmacological intervention, or in animals with intrinsic pathology such as seizure disorders can help discern the mechanisms generating specific cortical oscillations, localize sources of ERPs and EPs, and reveal large-scale network properties. By better paralleling human systems, this approach will improve small animal models of human neurophysiology and neuropathology, providing easier translation of discoveries made in rodent models to scientific and clinical relevance in humans.
The authors have nothing to disclose.
This work was supported by the Foundation for Anesthesia Education and Research Mentored Research Training Grant (ARM), by the National Institutes of Health grants GM107117 (MBK) and GM088156 (MBK), and by the Department of Anesthesiology and Critical Care at the University of Pennsylvania, Perelman School of Medicine.
32 Channel RHD2132 amplifier headstage | Intan Technologies | C3314 | |
Aquistion Board | Open Ephys | v2.2 | |
100 Position Receptable Connector | Digi-Key | ED85100-ND | Headpiece |
Acetone (1L) | Sigma Aldrich | 179973-1L | |
Razor Blade (100pack) | McMaster Carr | 3962A4 | |
Wire-Cutting Pliers | MSC Industrial | 321786 | |
2-Part Epoxy | McMaster Carr | 7605A18 | |
PFA Coated Silver Wire (25ft) | A-M Systems | 787000 | EMG Wire |
CircuitWriter Pen | MCM Electronics | 200-175 | Silver Applicator for Electrode Tips |
36 Position Dual Row Male Nano-Miniature Connector | Omnetics Connector Corporation | A79028-001 | Headpiece to Amplifier Adapter |
Conn Strip Header 2 x 50 | Digi-Key | ED83100-ND | Headpiece to Amplifier Adapter |
Clidox Base and Acitvator | Pharmacal | 95120F & 96120F | Sterilant |
Isoflurane | Priamal Enterprises Ltd | 66794-019-10 | |
Oxygen | Airgas | OX USP300 | |
Closed Loop Temperature Controller | CWE Inc. | 08-130000 | |
Curved Scissors | FST | 14085-09 | |
0.25% Bupivicaine Hydrochloride | Hospira | 0409-1159-02 | Local Anesthetic |
Meloxicam 5mg/mL | Henry Schein | 6451602845 | Pain/Inflammation Relief |
0.9% Sodium Chloride | Hospira | 0409-4888-20 | Fluids |
Cefazolin | Hospira | 0409-0806-01 | Antibacterial |
No.11 Disposable Scapel (20 pk) | Feather | 2975#11 | |
Micro Serrefines | FST | 18052-3 | |
Cotton Swabs (1000 pk) | MSC Industrial | 8749574 | |
0.5mm Micro Drill Bit | FST | 19007-05 | |
Stereotaxic Drill | Kopf | Model 1471 | |
Curved Forceps | Roboz | RS-5136 | |
Methyl Methacrylate | A-M Systems | 525000 | Cement for headpiece |
Methyl Methacrylate Crosslinking Compound | A-M Systems | 526000 | |
Curved Hemostats | FST | 13003-10 | Aide in Adapter Connection |
RHD2000 standard SPI interface cable (3ft) | Intan Technologies | C3203 | |
Cantilever Arm | Instech | MCLA | |
Micro Spatula (12 pk) | Fischer Scientific | S50822 | |
Digital Soldering Station | MCM Electronics | 21-10115 | |
Rosin Core Solder 60/40 Tin/Lead | MCM Electronics | 21-1045 | |
Color Craze Nail Polish with Hardeners (Nitrocellulose based) | L.A. Colors | CNP508 | |
Small Animal Stereotaxic Instrument with Digital Display Console | Kopf | Model 940 |