During sleep and quiet wakeful states, the neocortex exhibits large-scale slow-wave (SW) activity. The synchronous generation of the neocortical SWs implies the existence of an as-yet unknown spatiotemporal coordinating mechanism with long-range afferents driving inhibitory interneurons.

The Yoshihiro Yoshihara lab at the RIKEN Center for Brain Science sought to examine the role of the claustrum in this brain-wide cortical activity. In their recent report, The claustrum coordinates cortical slow-wave activity; the authors report that the claustrum is the neural network mechanism that facilitates the synchronous neocortical SWs.

To assess the role of the claustrum in this phenomenon, the authors generated a claustrum-specific Cre-expressing transgenic mouse line. The use of in vitro and in vivo techniques enabled the genetic visualization and manipulation of a subpopulation of claustral glutamatergic neurons.

Here, we address the author’s use of the ELG-2 Datalogger Wireless EEG system- a telemetry system designed to reduce the behavioral restrictions and data noise associated with the traditional telemetry systems.

Tethered EEG:
Traditional in vivo electroencephalography (EEG) experiments use a tethered system, where implanted electrodes are connected to the recording platform through a long cable. These systems, however, are plagued with issues due to the very nature of the tether. The long cables often tangle and restrict the animal, disrupting natural behavior. If the tether gets caught or the animal’s movement pulls too hard, damage to the cable or complete disconnections are possible. Damaged or frayed cables will cause data artifacts while complete disconnections will result in an abrupt end to data collection and injury to the animal.

Furthermore, tethered EEG equipment also suffers from poor signal-to-noise ratios. The tether acts as an antenna which can pick up electrical noise from AC power lines, motors, and other outside sources. These artifacts within the data complicate processing and interpretation. While Faraday cages may limit the noise from outside sources, they do not shield against the motion artifacts that are caused by movement of the tether itself.

Another issue evident in the traditional tethered EEG experiments is the limitation of animal scale-up. Additional tethers pose the threat of tangling and interfere with natural animal behavior as well as social interaction. Excessive environmental and motion artifacts are particularly evident when simultaneously recording EEGs from multiple rodents in close proximity. The use of Faraday cages inhibits the study of social interactions and ultimately takes up more physical space per animal in the laboratory. Limitations in the software and hardware systems of tethered EEG also reduce the possibilities of animal scale-up.

Wireless EEG:
The Amuza ELG-2 Datalogger Wireless EEG system enables researchers to wirelessly record EEG, electromyography (EMG), and local field potential (LFP) activity in freely moving animals. Designed to remove motion artifacts, the wireless lightweight headstage is small enough to sit on the head of an animal without disrupting natural behavior. The simple and flexible electrode design connects directly to a well-shielded headstage. Not only does this system reduce electrical noise from outside sources, but it also reduces implantation surgery time and complexity compared to the traditional PCB-based electrodes. The headstage is also easily removed and attached to the electrode implants, allowing researchers to interchange the device between animals. Since data is directly stored onto a microSD card, there is no signal interference across devices and animal scaleup is unlimited.

The ELG-2 Datalogger Wireless EEG system is also customizable to meet the demands of the researchers and their individual experimental needs. The standard ELG-2 Datalogger Wireless EEG set is designed to record from four channels but can be altered to record from as few as two, to as many as seven. The standard set has two screws for EEG, two silver wires for EMG, and one screw for reference and ground. If you want to record LFP activity, electrode configurations can be custom made to fit your experimental needs. Furthermore, the electrode set uses a universal 1.27 mm pitch pin and socket connector, making it easy for you to create your own electrode sets. The standard sampling frequency is 100 Hz but can be increased to 200 Hz.

In the Narikiyo, K. et al study, the authors opted to use two customized ELG-2 Datalogger Wireless EEG systems to simultaneously record from two mice. The headstage was designed to record from 5-channels with a 200 Hz sampling frequency. They created their own electrode configuration to record LFPs in the neocortex and monitor sleep/wake states with EEG and EMG recordings. Their setup included three perfluoroalkoxy-coated stainless steel wires for LFP recordings, one screw for EEG, one silver wire for EMG, and one screw for ground and reference.

Experimental Design:
To assess the role of the claustrum in the synchronous generation of widespread neocortical SW activity during sleeping and awake rest states, the authors generated a claustrum-specific Cre-expressing transgenic mouse line.

Male and female mice, one to eight months of age, were used for anatomical experiments and slice physiology to determine the neural network connectivity between the claustrum and the neocortex. Male mice, two to six months of age, were used for in vivo EEG and optogenetic experiments to assess the underlying temporal relationship between the claustrum and the neocortex with respect to SW activity.

The authors performed an in vivo loss-of-function experiment with the ELG-2 Datalogger Wireless EEG system in freely moving mice to examine the necessity of the claustrum for synchronized cortical SW generation. The Cre-positive claustral neurons were selectively ablated via AAV-mediated injection of the Cre-dependent diphtheria toxin A subunit (DTA). Control mice were injected with GFP.

The ELG-2 Datalogger Wireless EEG electrodes were surgically implanted into anesthetized mice, 2 weeks after injection. The LFP wire electrodes were implanted into the deep layers of the right prefrontal cortex (coordinates: AP: 2.5–2.8, ML: 0.5–1.5, DV: 1.0–1.5), anterior cingulate cortex (coordinates: AP: 1.0, ML: 0.5, DV: 1.3), and parietal cortex (AP: −2.0, ML: 1.5, DV: 0.4). The EEG electrode and EMG wire electrode were implanted into the left parietal cortex (AP: -2.0; ML: 2.0) and neck muscle, respectively. The electrical ground and reference electrode was implanted above the cerebellum.

Recordings were performed in the home cage under a 12-hour light/12-hour dark cycle, 6 to 12 weeks after AAV injection. Mice were acclimated to the weight and presence of the headstage with a dummy device overnight before experimentation. LFPs were recorded to detect SW activity in the deep layers of the cortex. The EEG and EMG electrodes were used to monitor the behavioral states of the mice.

Results
The generation of a genetic neural circuit map of the claustrum’s input and output connectivity revealed widespread reciprocal connections between the Cre-expressing claustral glutamatergic neurons and cortical areas.

In vitro whole-cell patch clamp recordings during optogenetic stimulation demonstrated that photostimulation of claustral neurons evoked excitatory postsynaptic potentials in all types of neurons but predominantly drove spike responses in inhibitory interneurons.

In vivo EEG in head-fixed mice revealed that the claustral glutamatergic neurons were more active during SW periods as compared to non-SW, suggesting that the claustrum activity is more closely related to SW activity than it is to the sleep state. When combined with optogenetic stimulation, in vivo EEG revealed that claustrum-induced SW activity via photostimulation mirrors spontaneous SW activity and suggests that the claustrum can regulate SW generation in neocortical areas through the coordinated activation of inhibitory interneurons.

The recorded LFPs during the loss of function experiment with the ELG-2 Datalogger Wireless EEG system revealed that the claustral-neuron-ablated mice showed a dramatic attenuation of the SW activity during SW sleep and awake rest as compared to the control. The authors indicate that the results demonstrate the importance of the claustrum in the synchronous coordination of cortical SW activity.

Discussion
Through the genetic visualization and manipulation of the Cre-expressing claustral glutamatergic neurons, the authors were able to identify the claustrum as a brain structure that can serve as the spatiotemporal mechanism that generates the synchronous neocortical SW activity during sleep and awake rest.

The use of the ELG-2 Datalogger Wireless EEG system allowed the authors to wirelessly record SW activity in the neocortex while monitoring the behavioral states of mice. This system promotes the natural behavior of animals while removing the artifacts commonly associated with traditional tethered EEG experiments. The ability to accurately and efficiently record in vivo telemetry data from freely moving animals without behavioral restrictions enables reproducible data acquisition across experiments and allows future research to build upon the established findings.

Narikiyo, K., Mizuguchi, R., Ajima, A., Shiozaki, M., Hamanaka, H., Johansen, J.P., Mori, K., Yoshihara, Y. The claustrum coordinates cortical slow-wave activity. Nature Neuroscience, 2020;23(6): 741-753. doi:10.1038/s41593-020-0625-7

Amuza Wireless Neuroscience
The traditional tethered systems do not only impose barriers in EEG experiments, they also hinder natural animal behavior and produce data artifacts during in-vivo optogenetic and fiber photometry experiments. The Amuza TeleOpto and TeleFipho systems are easy to use wireless systems, designed for reproducibility and animal comfort.