Automatic Detection of Highly Organized Theta Oscillations in the Murine EEG

The overall goal of this procedure is to analyze pharmacologically induced highly organized theta oscillations in mice using implantable EEG radiotelemetry. This message can help answer key questions in the systemic neuroscience field, such as how to induce, detect, and quantify highly organized theta oscillations in the mouse EEG using long term hippocampal EEG radio-telemetric recordings. The main advantage of this technique is that it helps to analyze highly organized theta oscillations in large scale murine hippocampal EEG recordings.

The implications of this technique extend to our therapy of neuropsychiatric and neurodegenerative diseases such as Alzheimer's disease, because frequency alterations such as theta dysrhythmia are often associated with it. Though this method can provide insight into murine hippocampal theta dysrhythmia, it can also be applied to EEG recordings from other species, including human following adaptation. Generally, if the ritual is new, this method will struggle, because the detection analysis of theta oscillation requires proper EEG electrode implantation and theta induction.

Prior to insertion of the electrodes, scratch the parylene coating from the tip. Next, mechanically clip the extracranial part of an electrode to the stainless steel helix of the transmitter. And shorten them to the required length.

To implant the radio frequency transmitter, place an anesthetized mouse on the surgical table, and continue to deliver isoflurane to the animal through a face mask. Ensure the depth of anesthesia by checking the tail pinch reflex, foot pinch reflex, and respiration rate. Next, remove the body hair from the scalp and disinfect the shaved scalp with 70%ethanol and an iodine based scrub.

Afterward, make a midline incision on the scalp from the forehead to the nuchial region using a scalpel. Then, prepare a subcutaneous pouch on one side of the back of the animal by performing a blood dissection using surgical scissors. Subsequently, insert the transmitter into the subcutaneous pouch and deposit the excess length of the flexible transmitter leads into the pouch.

Following this, fix the skull using a nose clamp. Then place the experimental animal on the stereotaxic frame. And set the ear bars.

Apply 0.3 percent hydrogen peroxide to the skull to remove any tissue and to help visualize the cranial sutures and the craniometric landmarks such as bregma and lambda. Next, drill the holes at the coordinates of choice using a high speed neurosurgical drill in a pressure free mode at maximum velocity. Select the appropriate electrodes and sterilize the electrodes using 70%ethanol prior to implantation.

The electrode type should be carefully chosen according to experimental requirements. Usually, parylene coated tungsten or stainless steel electrodes are the most common. Afterward, fix the electrodes using the glass ionomer cement and wait until the cement has fully cured.

Next, close the scalp using over and over sutures with non-absorbable suture material. For post-operative pain management, administer carprofen subcutaneously once a day for four days post-implantation. Allow the animal to recover for 10 to 14 days before the recordings or injection experiments.

In this procedure, activate the radiofrequency transmitter using the magnetic switch. Perform long term hippocampal EEG recordings for at least 24 to 48 hours. The analysis of EEG amplitude and EEG frequency characteristics of long term recordings provides detailed insight into the circadian dependency of theta oscillations and their association with specific behavior and cognitive conditions or tasks.

For the pharmacological induction of theta oscillations, pretreat the animal with methscopolamine to avoid peripheral muscarinic reactions. Subsequently, administer a single dose of urethane or a muscarinic receptor agonist to the mouse for theta initiation. After about 60 minutes, inject atropine to differentiate atropine sensitive type two from atropine insensitive type one theta oscillations.

And continue recording. To validate the EEG electrode placement, cut the stainless steel electrodes and remove the radio frequency transmitter. Then, sacrifice the mouse and remove the brain from the neurocranium.

To validate the electrode placement, mount the extracted brain on the tissue holder of a Vibroslicer using an adhesive. And cut the brain into 100 micrometer coronal slices. Mount the slices onto glass slides and stain them with nissl blue using standard histological procedure.

Incorporate the animal for analysis only when it meets the correct EEG electrode placement criteria. For the CA1 region, the tip of the deep electrode should be localized inside the CA1 pyramidal layer. Upload the EEG data.

Here is an exemplary 30 minutes EEG segment. Use the complex Morlet wavelet function, displayed here, for time frequency analysis. For time frequency analysis, the frequency range is set between 0.2 to 12 Hz and a step size is set to 0.1 Hz.This color-coded time frequency plot of the EEG segment illustrates the theta and the upper delta frequency ranges.

Here, an extracted 2.5 seconds EEG epoch is analyzed for theta segment. Since the maximum amplitude in the theta range divided by that in the upper delta range is larger than 1.5, this segment is classified as a theta oscillatory epoch. This guarantees that the maximum theta amplitude is at least 50%higher than the amplitude in the upper delta band in the calculated EEG epoch.

This figure shows the correlation of the highly organized theta oscillations and the theta detection measure in EEG recording, in which the theta oscillatory segments can be quantified and analyzed statistically. Here are two 2.5 seconds EEG epochs, visually classified as non-theta and theta segments respectively. And here are the time frequency analysis of the CA1 EEG segments, displayed in the corresponding graphs above in the range of 0.2 to 12 Hz.With the amplitude being color-coded, the time frequency analysis of the non-theta EEG segment exhibits a regular fluctuating theta architecture regarding frequencies in time.

Whereas the segment with highly synchronized theta oscillations is characterized by a regular non-fluctuating high amplitude theta of a nearly constant frequency of six Hz.The ratio of maximum theta to maximum delta amplitude is 1.25 in this map and 4.67 in this map. Clearly classifying this EEG epoch as a theta oscillation EEG segment. Once mastered, this technique can be done in one hour if it is performed properly.

Following this procedure, other methods like time frequency analysis of other frequency bands or automated seizure detection can be performed in order to answer questions like if other EEG bands are affected, how they are coupled and are related to neural hyperexcitability. After its development, this technique paved the way for researchers in the field of systemic neuroscience to explore alterations in hippocampal theta architecture in transgenic mouse models, such as Alzheimer's disease mouse models. After watching this video, you should have a good understanding of how to induce, detect, and analyze highly organized theta oscillations.