A Cost-Effective and Minimally Invasive Protocol for Chronic Multi-Site Electroencephalography Recording in Freely Moving Mice

Summary

This protocol was developed to create a reliable and easily accessible system for recording freely moving rodent EEG.

Abstract

Electroencephalography (EEG) serves as a fundamental tool for tracking electrical activity in the brain and has become increasingly important in both clinical diagnostics and preclinical animal research. While rodent EEG offers a powerful platform for modeling human neurological disorders, long-term recordings are often limited by the high cost, invasiveness, or technical complexity of existing systems. Here, we present a reliable, accessible, economically viable, and minimally intrusive protocol for chronic, multi-site cortical EEG acquisition in freely moving mice. The technique involves fabricating platinum-iridium wires with screw electrodes connected to a universal serial bus (USB)-type interface, which are anchored epidurally using bone screws and secured with adhesive and dental cement. The system allows for stable signal capture over several months, as demonstrated in wild-type C57BL/6J mice with an 85% post-operative survival rate (n = 20). EEG was sampled at 1,000 Hz and filtered between 1 Hz and 500 Hz using a differential amplifier setup. To validate the system, pentylenetetrazol (20 mg/kg) was administered to induce seizure-like activity, leading to a marked increase in interictal discharges-from 2-5 at baseline to 20-51 events post-injection-accompanied by increased signal amplitude. Discharges were identified based on waveform morphology and signal characteristics matching recognized electrophysiological criteria. The setup is compatible with behavioral video tracking and open-source analysis pipelines, and supports integration with pharmacological, genetic, or neuromodulation studies. This method provides a stable and adaptable platform for investigating brain network dynamics in rodent models, with applications in epilepsy research and beyond.

Introduction

Electroencephalography (EEG) provides a direct measure of brain electrical activity and is widely used in diagnosing epilepsy¹. In recent years, EEG techniques have been increasingly adapted for use in small animal models to study neurological disorders, sleep physiology, and brain network dynamics^2,3,4. With the advent of genetically modified mouse models and significant advancements in recording technologies, rodent EEG has become an essential tool for preclinical research, offering valuable insights into translational mechanisms relevant to human brain health^5,6.

Capturing and manipulating neural activity simultaneously is critical for studying brain dynamics at both macro- and micro-circuit levels. This dual approach enables real-time monitoring of the neural networks involved in seizure initiation and progression. Epilepsy, characterized by recurrent and often unpredictable seizures, arises from disruptions in complex brain circuits⁴. Despite extensive research, the mechanisms governing seizure spread and onset remain incompletely understood, in part due to limited use of technologies that allow for simultaneous recordings across multiple brain areas⁷.

EEG recording from animal models that recapitulate human neurological disorders has revealed interictal spikes, spike-and-wave discharges, and seizure onset patterns that closely mirror those seen in human patients^8,9, thereby advancing our understanding of electrophysiological changes taking place in the human brain. Similarly, EEG abnormalities detected in models of Alzheimer's disease, autism spectrum disorder, and schizophrenia provide critical insights into disrupted neural oscillatory activity and highlight potential therapeutic targets¹⁰.

Despite differences in brain anatomy and scale, mouse and human EEGs exhibit striking similarities^2,11. Both exhibit characteristic oscillatory frequency bands -- delta, theta, alpha, beta, and gamma -- with power spectra generally following a 1/f distribution^2,11. Transitions between brain states, such as the emergence of delta activity during non-rapid eye movement (NREM) sleep or increased gamma synchronization during attention and memory tasks, are conserved across species. Moreover, epileptiform activity, including high-frequency oscillations (HFOs) and ictal patterns, show comparable temporal and spectral profiles in both mice and humans⁷.

The conserved features of rodent and human EEG make mouse models highly translational for studying brain disorders. Mouse EEG allows invasive, high-resolution recordings enabling detailed circuit-level analysis¹². It also supports testing of neuromodulatory strategies, including pharmacological agents, optogenetic control, and closed-loop stimulation^13,14, which can be coupled with behavioral and molecular tools to precisely interrogate neural dysfunction.

Nevertheless, caution must be taken when extrapolating findings. The smaller size and simpler cortical structure of the mouse brain can affect signal propagation, and cognitive event-related potentials (ERPs) may be less prominent compared to humans¹⁵. Ensuring consistency in electrode placement, signal referencing, and analytical pipelines is critical to improving reproducibility and cross-laboratory validity¹⁶.

Recording EEG in small animals presents both opportunities and challenges. Mouse EEG is typically acquired using chronically implanted electrodes positioned either epidurally on the skull surface or as depth electrodes inserted into subcortical structures^17,18. Surface electrodes, such as screw or wire electrodes placed on the dura mater, are minimally invasive and suitable for long-term, longitudinal recordings. Such electrodes are below the bone, which provides a high impedance barrier to recording. Accordingly, dural electrodes in rodents can be thought of as more comparable to electrocorticography (eCoG) than to EEG. Depth electrodes, in contrast, offer higher spatial resolution and enable access to deep brain regions such as the hippocampus, thalamus, or amygdala, facilitating studies on seizure networks and oscillatory coupling across regions distribution^2,11.

Modern EEG systems for rodents support multi-channel recording with high temporal resolution and are often coupled with video monitoring for behavioral correlation¹⁸. Additionally, wireless EEG systems allow for untethered, real-time acquisition in freely behaving animals, minimizing movement constraints and stress¹⁹. Though with time these modernizations were made commercially available, they are either very expensive or are too invasive to study the rodent model over a long period of time.

To address this gap, we present a versatile and cost-effective protocol for multi-site EEG acquisition in rodent models, suitable for long-term studies with minimal invasiveness. This EEG recording approach is particularly suitable for studies requiring high-resolution assessment of cortical activity in mice under controlled experimental conditions. It is best applied when (i) the animal can tolerate brief anesthesia during electrode implantation and fully recover, (ii) stable head positioning or minimal movement is ensured during recordings, (iii) long-term or repeated monitoring of neural activity is desired, and (iv) the experimental design can accommodate single- or limited multi-animal recording depending on amplifier capacity. Investigators should consider these constraints to determine whether this method aligns with their experimental objectives.

Protocol

This protocol received approval from the Augusta University Animal Care and Use Committee. The National Institutes of Health Guide for Care and Use of Laboratory Animals was followed at each step of the experiment. All possible measures were implemented to minimize the number of animals utilized in this study. The mice used for this study are 2-month-old wild-type C57Bl6J. The time required to perform each step is included in the respective steps.

1. Preparing plug pins with electrodes (5 min)

1. Use USB type B female plug pins with 5 pins to solder with 0.003" platinum-iridium wire. Remove the coat from the wire before soldering it with the pin. The fourth pin out of the five pins is the ground pin; do not use it to connect to the electrodes (Figure 1A). Use the other 4 pins to connect the electrodes.
2. Bend the pins by about 45° and wrap the wires around each pin twice to make sure the wires remain connected properly and do not come off (Figure 1A).
3. Add a bit of solder rosin flux paste to each pin to clean the surface of the pin so it can be soldered effectively.
4. Add a small piece of solder wire to each pin, use a soldering iron to melt the solder wire, and permanently attach the pin to the platinum-iridium wire (Figure 1B-D).
   NOTE: Use a 45° forceps to hold the wire, and use another one to wrap around the pin. Do the whole procedure under a bright-field microscope. Cut the remaining wire after wrapping and soldering, and keep the other end attached to the electrodes. Sterilize the connector with platinum-rhodium wire and the electrodes before the surgery.

2. Implanting electrodes (30 min)

1. Anesthetize the mouse in an anesthesia chamber and mount the mouse on the stereotaxic stage. Make sure that the mouse is not under-dosed before mounting it into the stage by pinching the hind leg and waiting till it stops responding to the pinch. Quickly start the isoflurane/oxygen vaporizer on the nose at a rate of 1.5% v/v.
2. Inject the mouse with carprofen (5 mg/kg) to reduce inflammatory responses and pain, and dexamethasone (0.1 mg/kg IM) to reduce inflammation and brain swelling. Use eye ointment to keep the eyes moist, as long exposure to anesthetics might dry the eyes and damage them.
3. Use a hair remover or shave the top of the skull to expose the skin. Follow aseptic technique to clean the area of incision using 70% ethanol and iodine pads.
4. Make a clear single stroke incision in the middle of the head with a scalpel. Cut around the incision to expose the site of electrode implantation. Make sure muscles are not exposed (Figure 2A).
5. Wash with 0.9% NaCl solution and dry the surface. Scrub the surface with cotton swabs soaked or dipped in hydrogen peroxide. Connective tissue on the bone that is persistent may be removed with a scalpel. Wait at least 1 min after scrubbing.
6. Wash with 0.9% NaCl solution and dry the surface before starting to drill.
7. Use a dental drill to make the surface of the skull rough to promote acrylic adhesion.
8. Measure and point the locations of the electrode implantation (AP: +2, -4, ML: ±1.5)²⁰ (Figure 2B).
9. Drill the surface using 1.4 mm drills and put 4 bone screws (1.5 mm) in the hole using precision screwdrivers. Make sure the drilling does not make holes through the bone. The screws should not make a hole in the brain, but rather touch the surface (Figure 2C).
10. Wash with a 0.9% NaCl solution if there is any blood and wait for it to dry up. Use a vacuum if necessary.
11. Wrap the wires attached to the USB connector around each of the screws 2-4 times and cut the leftover wires (Figure 2D).
12. Put a small drop of super glue (Loctite) on the top of each screw and allow it to dry to make the connection strong. Make sure the connector is placed in the middle of the skull and not touching the skull or any of the electrodes (Figure 2E).
13. Use cyanoacrylate to slowly build the headcap a little bit at a time. Avoid applying a layer that is too thick, as it can heat the bone. Attach a big screw on the flat surface of the connector, which does not contact the bone or animal, using dental cement to aid USB cable plugging in (Figure 2F).
14. Let the head cap dry for at least 2 min before unhooking the mouse from the stereotaxic stage. Observe the mouse for the next 24 h on a heat pad.
15. Treat the mouse with carprofen for the next 7 days at a dosage of 5 mg/kg every 12 h. The mouse will be ready for EEG recording after 2 weeks of waiting to promote bone growth at the acrylic interface.
    NOTE: Try to keep the connector as close to the skull as possible. Make sure it does not touch the skull or the bone screws. Make sure the wires are not connected to each other at either end. Be careful about Cyanoacrylate getting into the USB connector and eyes.

3. Recording EEG (65 min)

1. Record EEG of each mouse for 1 h, 30 min before Pentylenetetrazol (PTZ) treatment and 30 min after 20 mg/Kg PTZ treatment.
2. Plug the USB cable into the connector. Connect the USB cable to a differential alternating current (AC) amplifier using alligator clips with the Gain set to 10k and filtered between 0.1 Hz and 250 Hz²¹. Sample EEG signal at 1000 Hz (Figure 3).
3. Record the outputs from the amplifier simultaneously on the computer and display them on an oscilloscope for real-time monitoring. Connect the amplifier outputs to a data acquisition and signal conditioning (e.g., NIDAQ) USB device attached to the computer (Figure 3).
4. Use the Python code to record the EEG signal (available at https://github.com/schrodi159/blakelabeeg). The code digitally filters the signal at 0.1 Hz and 250 Hz.
   NOTE: Recording EEG in a room without electrical interference improves the quality of the data and recording.

4. Data interpretation

1. Use the Python code to visualize data (available at https://github.com/schrodi159/blakelabeeg). Identify interictal discharges from each recording (Figure 4A).
   NOTE: Interictal discharge was considered if the waveform is twice the baseline and has a duration less than 200 ms²² and follows at least 4 out of the following 5 criteria- di- or triphasic wave with a pointed peak, different duration than background, asymmetry of wave form, background activity is disrupted by the presence, and followed by an after going slow wave^8,20,22.

Results

Electrodes were implanted in 20 mice on a C57Bl6J background, including 5 wild-type C57Bl6J mice, with a post-operative survival rate of 85% maintained up to 6 months after the surgery (Figure 4B). After 6 months, the headcaps began to detach, and all mice were euthanized at 8 months of age. The average weight of the headcaps assembly, including the connector and screws, was 1.13 g (n = 20). The mice regained consciousness within 30 min post-surgery and resumed normal movement and gazing wit...

Discussion

In this study, we developed and validated a robust, cost-effective, and minimally invasive protocol for chronic, multi-site EEG recording in freely moving mice. The successful implantation of screw electrodes and custom-fabricated platinum-iridium electrodes soldered to a USB connector, along with the observed survival rate and recording quality, demonstrates the feasibility and reliability of the technique in chronic recordings. Notably, this protocol allows for precise electrophysiological data collection without exten...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.003" platinum iridium wire	AM systems	777000
1.5 mm Bone screws	Antrin Miniature specialities		000-120X1/16 SL BIND MS SST
AC amplifier	AM Systems
Carprofen	Covetrus	11695-6935-1
Coldpac Denture acrylic base	Yates Motloid Company	https://www.yates-motloid.com/collections/coldpac/denture-base
Dental drill 1.4 mm	Volvere GX	N/A
Dexamethasone	VEDCO	50989-437-12
Eye lube	Optix Care	https://theoptixcare.com/
Hydrogen peroxide	Sigma Aldrich	7722-84-1
NIDAQ USB	National Instruments	https://www.ni.com/en/shop/data-acquisition/entry-level-usb-daq.html	Data acquisition and signal conditioning USB
Pentylenetetrazol	Caymen Chemicals	18682
Solder rosin flux paste	Weller	https://www.weller-tools.com/us/en/consumer/products/soldering-accessories/
Solder wire 0.32"	Radio Shack	N/A
Super glue	Loctite	N/A
USB type B female plugs	QM Seller	X00292ABU9