The present protocol describes a simple procedure to acquire and analyze the topography of epicranial visual evoked potentials with 32-multichannel thin-film electrodes in the mouse.
Visual evoked potentials (VEP) allow the characterization of visual function in preclinical mouse models. Various methods exist to measure VEPs in mice, from non-invasive EEG, subcutaneous single-electrodes, and ECoG to fully invasive intracortical multichannel visual cortex recordings. It can be useful to acquire a global, topographical EEG-level characterization of visual responses previous to local intracortical microelectrode measurements in acute experimental settings. For example, one use case is to assess global cross-modal changes in VEP topography in deafness models before studying its effects on a local intracortical level. Multichannel epicranial EEG is a robust method to acquire such an overview measure of cortical visual activity. Multichannel epicranial EEG provides comparable results through a standardized, consistent approach to, for example, identify cross-modal, pathological, or age-related changes in cortical visual function. The current study presents a method to obtain the topographical distribution of flash-evoked VEPs with a 32-channel thin-film EEG electrode array in anesthetized mice. Combined with analysis in the time and frequency domain, this approach allows fast characterization and screening of the topography and basic visual properties of mouse cortical visual function, which can be combined with various acute experimental settings.
Mice are a preclinical model of degenerative processes of vision and ophthalmological diseases1,2,3,4. Visual evoked potentials (VEPs) are commonly used to measure cortical visual function and, for example, to assess visual degeneration in pathological models5,6. The VEP latency, conduction time, amplitude, multifocal characteristics, or spatial acuity of cortical visual evoked potentials provide diagnostic information on the functional integrity of the visual system7,8,9.
In mice, cortical visual evoked potentials can be measured across various spatial scales with methods of different complexity from non-invasive EEG, subdermal needle electrodes, and skull-implanted screws, to fully invasive intracranial approaches with epicortical ECoG, to intracortical electrode recordings10,11,12,13,14,15,16,17. These methods have different strengths and weaknesses. For example, a low number of electrodes only provides limited information on cortical VEP distribution, whereas subcutaneous needle electrodes often fail to ensure consistent recording locations. Moreover, implanted screws or fully invasive methods require damaging, penetrating, or removing of the skull and often provide only local information.
In acute experiments, a first global overview of cortical visual function is often desired, which is eventually followed by further experimental steps and compared to local intracortical recordings. For example, one potential use case is utilized first to investigate the EEG-level effects of visual cross-modal reorganization of deafness or hearing loss on VEP topography and cortical visual activity18,19 before studying the impacts on a local intracortical level.
Multichannel EEG recordings with thin-film multi-electrode arrays can provide a systematized VEP topography from the mouse skull20,21,22,23,24. Such epicranial recordings can have advantages over ECoG recordings by leaving the integrity of the skull intact and avoiding direct manipulation of the cortical surface. In addition, thin-film multi-electrodes provide a standardized electrode configuration, allowing the comparison of visual evoked spatio-temporal brain activity between experiments similar to a standardized EEG system in humans25. A standardized framework also facilitates using common EEG analysis toolboxes (e.g., Fieldtrip, Chronux, EEGLAB, and ERPLAB) to analyze mouse EEG in the time and frequency domain or in terms of connectivity26,27,28,29,30,31.
The present protocol describes a procedure for topographical VEP recordings in mice using a 32-channel thin-film electrode. This can be used as part of acute experiments followed by additional experimental steps, such as intracortical microelectrode recordings from specific brain areas. It is demonstrated here how to reliably record epicranial flash evoked VEPs with 32-channel thin-film electrodes from the mouse. In addition, exemplary analysis of topographical VEP recordings in the time and frequency domain is presented.
All animals were handled and housed according to German (TierSchG, BGBl. I S. 1206, 1313) and European Union (ETS 123; Directive 2010/63/EU) guidelines for animal research. The animal experiments were approved by German state authorities (Lower Saxony State Office for Consumer Protection and Food Safety, LAVES) and were monitored by the university animal welfare officer. A 3-month-old male C57BL/6J mouse was used for the present study.
1. Animal details
2. Induction of general anesthesia
3. Physiological monitoring and maintenance of general anesthesia
4. Electrode placement and recording setup
5. EEG recording and VEP measurement
6. Completion of the experiment, electrode removal, and electrode cleaning
7. Basic VEP signal processing: Time and frequency domain
Recording visual evoked potentials with multichannel EEG allows the assessment of the topography of VEP amplitudes, latencies, or frequency components in mice. Figure 2A shows an example of a flash evoked VEP topography recorded with an epicranial 32-channel EEG from a 3-month-old male C57BL/6J mouse. The strongest visual evoked activity occurs in the occipital region above the visual cortex.
Figure 2B shows the voltage distribution over the skull at different time points pre- and post flash onset. The strongest N1 amplitude of the VEP occurs at 50 ms poststimulus above the central occipital regions. This is followed by a potential reversal and a positive peak across the occipital and frontal areas at 300 ms poststimulus. The butterfly plot in Figure 1B shows the peak latency of the VEP N1 component at 46 ms and a positive peak at around 300 ms post onset.
Figure 2C shows the global field power calculated from all the EEG channels37,38. This can be used to analyze the multichannel EEG data and determine the peak latency (46 ms), though it does not show changes at 300 ms poststimulus in the shown example. Moreover, it can evaluate time points of interest where topographical signals spatially change from a common modification.
Figure 2D shows a time-frequency analysis of the response and gamma power in the 30-45 Hz power distribution at different time points. The topographies for gamma power are also centered above the central occipital regions and shift toward more frontal regions at later time points. Below, the time-frequency plot for an occipital single EEG channel with a multi-taper spectrogram shows fast activity during the response.
VEP characteristics are comparable between C57Bl/6J mice of 2-3 months old (Figure 3A). The topography shows consistent spatiotemporal patterns above the occipital and frontal areas at different time points in three example mice (Figure 3B). The spatiotemporal negative potentials occur above the occipital regions at 50 ms post stimulus onset, followed by positivity around 300 ms shifted toward the frontal regions in all mice.
The latencies are also consistent between mice (Figure 3B), with N1 component latencies (46 ms, 44 ms, 57 ms) and amplitudes (0.14 mV, 0.12 mV, 0.13 mV). However, amplitudes differ more frequently because they can be affected by variables, electrode impedance, electrode contacts, and visual status. Similarly, global field power (i.e., the analysis of multichannel EEG data) reflects consistency between animals, with peak latencies (46 ms, 44 ms, 57 ms) reflecting intracortical visual response latencies39.
Figure 1: Schematic of the experimental setup. The mouse is preferably placed inside a soundproof chamber. A temperature-controlled heating pad is used to maintain adequate body temperature throughout the experiment. ECG recordings are assessed with silver wire ECG electrodes, which allow for heart rate monitoring. EEG signals are recorded with a multichannel EEG electrode during visual stimulation elicited with a LED stroboscope. Please click here to view a larger version of this figure.
Figure 2: VEP topography and global field power in the mouse EEG. (A) 32-channel EEG electrode (left) and the topography of VEPs (right) from a 500 ms interval post stimulus onset (right). (B) VEP topographies at different time points after flash stimulation (150 repetitions) to both eyes (top). The distribution of visual evoked potentials shows the concentration of the largest responses in the occipital regions. Below: individual 32 EEG traces for an interval of 500 ms pre- and 500 ms post stimulus onset with negative peak N1. A deflection generated by the stimulus artifact is visible at stimulus onset. (C) Global field power of the EEG signals. (D) Time-frequency representation of visually evoked activity for one occipital channel (bottom). Topographical power distribution in the gamma range (30-40 Hz) at different time points. Please click here to view a larger version of this figure.
Figure 3: Comparison of spatiotemporal profiles of visual evoked potentials between three different mice. (A) VEP topographies of three mice (M1, M2, M3) after flash stimulation to both eyes (average of 150 repetitions). Traces are shown for a duration of 500 ms post stimulus onset. The distribution of visual evoked potentials shows the concentration of the largest responses in the occipital regions. (B) Spatiotemporal profiles are shown at different time points for the three example mice at post 50 ms and post 300 ms relative to stroboscope flash; below are butterfly plots of the responses above for the three mice. VEPs show a prominent negative peak N1. The green line shows the global field power of responses and reflects the latency of EEG activity. Please click here to view a larger version of this figure.
This article describes a method for recording epi-cranial multichannel EEG with thin-film electrodes and how to acquire a consistent topographical representation of visual evoked potentials in the mouse. Here, we exemplarily showed binocular flash stimulation, but this approach can also be applied with other types of visual stimuli (i.e., monocular, spatial gratings, focal visual field) using, for example, a larger display.
A critical step in the protocol is the positioning of the electrode. Precise and consistent electrode positioning must be ensured to allow comparability between experiments. Importantly, the electrode position needs to be documented for later comparison (i.e., operating microscope images). Although different types of thin-film electrodes and configurations (from other distributors) can be used, it is advised to decide on one defined electrode configuration and stick to it for back-comparability in experiments. A second critical aspect is good electrode contact with the skull. The quality of contact influences the electrode impedance and signal quality40. Thus, similar contact to the skull of all the electrode sites is important, especially for analyzing spatial response distributions. If an electrode channel shows a noisy signal (i.e., high-frequency components), it must be ensured that sufficient contact with the skull is provided, and impedances must be tested during the experiments. However, the present VEP recording is quite robust for overall signal quality.
If the researcher wants to reuse the same electrode multiple times, removing the electrode is another important step. It is important to carefully and slowly separate the electrode from the skull by using liquid (e.g., saline) to avoid damaging the contacts during removal. Monitoring animal health and physiological status is an important aspect of all animal experiments. Physiological status can influence comparability. It is advised to proceed fast (i.e., similar timing between experiments) with the protocol. Ensuring physiological body temperature is especially important (i.e., it helps to increase the surrounding temperature and to work in a warm room). One should take special care of the eyes of the mouse, as they reflect the physiological state, but leaving them untouched is often the best way. In case the eyes of the mouse become opaque, this is a sign that the physiological condition of the mouse is degrading. This state is reversible41; however, the animal's physiological condition requires special attention (it must be ensured that the core body temperature is in the physiological range, the heart rate and ECG waves should be checked, and excessively deep anesthesia levels should be avoided).
Monitoring adequate anesthesia depth is essential. It is important to avoid too light and too deep anesthesia, characterized by the presence of burst-suppression in the EEG signal. This can lead to unwanted effects in terms of sensory responsiveness42,43,44. Also, visual response properties change depending on anesthesia depth and the type of anesthetic used45.
An unresolved limitation of this method is that off-the-shelf thin-film electrodes currently do not cover the lateral, temporal regions of the mouse brain. This limits the assessment of EEG signals from temporal regions (e.g., auditory cortex), sensory interactions, and source localization. Specifically, customized EEG arrays can provide a solution here. Another limit is that, depending on the recording system, it may be difficult to assess electrode impedances during the experiment to allow for comparable electrode properties. A third limitation of the method is that it is not fully non-invasive and requires an incision of the skin and general anesthesia. To resolve the latter, one can implant the grid chronically21,22 or apply true non-invasive scalp EEG13.
Regarding the usability in acute experiments, the described epicranial EEG measures represent a good trade-off between different methods. The procedure leaves the cortex and skull intact and allows less manipulation, avoiding potential functional damage to the cortical surface. This potentially provides better consistency in the screening of cortical visual activity.
In contrast to simpler recording methods, using multichannel thin-film EEG recordings significantly improves the information content of experimental data due to the comparability of electrode positions. It provides a repeatable, topographic, and more complete overview compared to single-electrode approaches. Additionally, this approach allows the use of EEG toolboxes (EEGLAB, ERPLAB, Fieldtrip, Chronux), utilizing electrode configuration, and ultimately facilitating data analysis26,27,28,29,30.
Potential applications of multichannel thin-film EEG are visual phenotyping of mice strains and assessment of visual function previous to intracortical recordings. Also, and most importantly, the multichannel EEG approach can easily be extended to assess auditory or somatosensory potentials, respectively.
The authors have nothing to disclose.
This work was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, Cluster of Excellence 2177 "Hearing4all", Project number 390895286).
Bepanthen 5% Dexpantheol | Bayer | Ophtamic gel | |
Cheetah software 5.11 | Neuralnyx | Version 5.11 | Recording software for neurophysiologcal signals |
Digital Lynx SX | Neuralynx | Digital Lynx 16SX | Recording system |
ECG differential amplifier | Otoconsult | WDA2 V1.0 | |
Electric shaver | Aesculap | GT420 | |
Electrode Holder | TSE Systems | 430005-HE | |
Examination light | Heine | HL 5000 | Cold light source lamp |
Heating Pad + Temperature Control system | CWE | TC-1000 Mouse | |
Histoacryl 0.5 mL | B.Braun | Tissue adhesive | |
Infrared heat lamp | Sanitas | SIL 06 | |
Ketamine 10% | WDT | Ketaminhydrochlorid | |
LED stroboscope | Monarch | Nova Strobe PBL | Visual stimulation |
Matlab 2021a | The Mathworks | 2021a | Stimulus control and analysis |
Moria Vessel Clamp | Fine Science Tools | 18320-11 | |
Mouse EEG electrode | NeuroNexus | H32 (Reticular) | 32-channel EEG electrode. Thickness: 20 μm; length: 8.6 mm; width 6.8 mm. Platinum sites: 500 μm diameter |
Mouse Frame | custom made | Information available on request | |
Multifunction I/O device | National Instruments | PCIe-6353 with BNC 2090A | Analog stimulus generation, output, and trigger |
NaCl 0.9% | B.Braun | Isotonic, sterile, nonpyrogenic | |
Neuralynx HS36 | Neuralynx | HS-36 | Headstage |
Neuronexus probe connector | Neuralynx | ADPT-HS36-N2T-32A | Electrode connector |
Oscilloscope | Tektronix | TDS 2014B | |
Progent Intensive Cleaner | Menicon | Protein remover and disinfecting solution for rigid gas permeable lenses | |
Recording PC | HP | HP Z800 | Recording PC |
Rimadyl (Carprofen) | Zoetis | Carprofen | |
Silicon Oil M 1000 | Carl Roth | 4045.1 | |
Silver wire | Science Products | AG-8W | Diameter 203 µm; ECG and reference electrode |
Sound proof chamber | IAC acoustics | ||
Stereotactic Micromanipulator | TSE Systems | 430005-M/P | For EEG electrode placement |
Stimulation PC | Dell | Dell Precision T5810 | Stimulation PC |
Surgical microscope | Zeiss | Op-Mi Focus | |
Surgical tape | 3M | 1527-0 | 1.25 cm x 9.1 m |
Thilo-Tears 3 mg/g | Alcon Pharma GmbH | Ophtamic gel | |
Vaselin Lichtenstein | Winthrop | White vaselin ointment | |
Xylazin 2% | Bernburg | Xylazinehydrochlorid | |
Xylocaine Spray (10 mg/puff) | Aspen | Lidocaine |
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