The overall goal of this procedure, is to describe how neural and cardiac activities are coordinated during mouse non REM sleep. Through monitoring and analyzing electroencephalographic, electromyographic, and electrocardiographic signals. This method can help answer key questions in the sleep field.
Such as how sleep reconciles two essential and evolutionarily preserved needs. Which are to be both continuous and fragile. The main advantage of this technique is that it uses standard polysomnography to score non REM sleep in a novel way that is based on the variable behavioral responses to external stimuli.
We first had the idea for this method when we observed that sleeping mice showed variable responses to noise such as waking up and sleeping through. Generally individuals new to this method will struggle because it combines surgical skill, bare minimal experimentation, and advancing analysis. Visual demonstration of this method is critical as several essential steps are new to the field.
In particular concerning the behavioral design and data analysis. The implications of this technique extend towards diagnosis of sleep disorders in humans. Because it identifies the moments of sleep that are highly fragile.
To begin the procedure, prepare to EEG ecogeolectrodes using 0.5 centimeter long pieces of gold wire, each soldered on top of a gold plated steel screw. Then clean them in 70%ethanol. Next prepare two EMG-ECG electrodes with three to four centimeter long gold wires.
Bend the wires at a 90 degree angle one centimeter from one end and prepare a coil at the other end. Between the two ends bend the wire to create a small curvature that corresponds to the surface profile of the bone between the cerebellum and lambda. After that prepare a six channel female to male head connector by covering the connector of both female and male pins at the base with tape.
Then add to the pins at the corners some tin to facilitate the soldering process. In this step, place an anesthetized animal on the stereotaxic apparatus by positioning the mouth. This will fix the mouse head.
Then fix the head by positioning the ear bars at the skull with their blunt reverse ends. Subsequently lift the skin at the center of the skull and gently cut the lifted portion of the skin along the mid line from the top of the neck to the level of the eyes. Then remove the scalp.
Ensure that the window is large enough to clearly see the bregma and the lambda fissures of the skull. Fix the skin on both sides with bulldog serafines to ensure access to the bone. Next carefully remove the conjunctive tissue with a scalpel.
Clean the area with iodine based disinfectant and dry the skull with an antiseptic swab. Then using a sharp scalpel blade, scratch the skull to obtain a cleaned and mattified bone surface. Using only the scalpel tip, scratch a grid like meshwork of groves with a distance of one to two millimeters between the grooves.
Afterward, perform four craniotomies in the skull at specific locations with a microdrill. While drilling, blow away the bone dust using a pasteur pipette and clean any bleeding with antiseptic swabs. If bleeding occurs, ensure that it is stopped completely before resuming the process.
Use a hemostatic sponge to accelerate hemostasis. Fix the screw in a hemostatic clamp. On the left hemisphere, screw two gold plated screws through the craniotomies.
Hold the clamp vertically above the craniotomy and rotate the screw while not deviating from the vertical position. Gently open the hemostatic clamp to release the screw. On the right hemisphere screw the previously prepared electrodes through the craniotomies.
Add a drop of glue on the middle part of the skull to ensure tight adhesion of the left EMG-ECG. Carefully lift the border of the skin from the neck muscles with forceps and insert the EMG-ECG wires with the coiled ends in the muscles. To detect the heart ECG signals during sleep, ensure that the EMG-ECG wire is inserted into the muscle to a depth of about 0.8 to one centimeter.
Position the left EMG-ECG next to the posterior left anchoring screw. Repeat the procedure to insert the right EMG-ECG. Position the right EMG-ECG next to the anterior left anchoring screw.
Ensure that the loop ends are placed as far as possible from each other. It is important that the EMG-ECG wires are inserted deeply enough into the muscle in order for the heart rate to be detected reliably. Using a spatula apply the two component epoxy glue to the skull between and around the screws.
Let it dry in the light but protect the eyes of the animal from excessive lighting. Try to place the connector pins as closely as possible to minimize the height of the implant using a small crocodile lamp. Solder the four pins at the corner of the connector to the four wires.
Minimize the time in contact with the soldering tip as this rapidly heats the screws. Next, fill the space between the glue and the connector with dental cement and cover the soldered parts. Create smooth surface and avoid sharp edges that could hurt the animal.
In addition, avoid touching the skin as this leads to itching. Now remove the bulldog serafines. Disinfect the wound with iodine based disinfectant.
If necessary, close the wound with suture points in front and behind the connector. Afterwards, monitor the animal every day for a week and look for weight loss, reduced or abnormal activity, and signs of infection. Five to six days after surgery, connect the recording cable to the head connector on the animal, leaving it in its home cage.
Wait an additional four to five days before the start of recording so that the animal is habituated to the condition and sleeps naturally. With a commercial polysomnographic software, record EEG and EMG-ECG data over 48 hours using typical settings such as 2000 x gain, a 2000 hertz sampling rate at acquisition, down sampled to 200 hertz after acquisition. Apply a 0.7 hertz high pass filter for all the EEG traces and a 10 hertz high pass filter for all the EMG-ECG traces.
In this demonstration, the mouse has been sleeping for more than 40 seconds. Here are the characteristic low frequency, high amplitude signals in the EEG and the low muscle tone in the EMG traces. It is important that the mouse sleep stably for at least 40 seconds before the noise is played to ensure a long base line for the analysis of sleep, prior to wake up or sleep through.
Now an acoustic stimulus, here a white noise, will be delivered. In this case, the mouse wakes up within 10 seconds. Note that the EEG desynchronizes when the mouse wakes up.
In another case, the mouse does not wake up, even though the noise has been delivered for more than 20 seconds. Note that the EEG does not desynchronize. Shown here are the raw traces of the EEG in black and the corresponding EMG-ECG signals in gray, for 40 seconds before the noise onset and during the 20 seconds of noise.
This is a sleep through event. And this is an example of the wake up event. The corresponding analyzed data for sigma power dynamics in the 40 seconds window before noise onset are shown here for sleep through and wake up.
The key result of this analysis is that the sigma power is at its trough before noise onset for a sleep through event and at its peak for a wake up event. In contrast to the wake up and sleep through events presented before, we discarded the events with an early transition into REM sleep. And we also discarded the events with an early wake up before noise onset.
While attempting this procedure, it's important to ensure that EMG signal detects the heart rate throughout non REM sleep. Moreover remember that the analysis procedure needs to be understood and implemented in a software script to extract the infra-slow oscillation. Following this procedure, other methods, such as local electrophysiological recordings, or imaging approaches can be performed to follow activity patterns in defined brain areas.
After redevelopment, this technique paves the way for researchers in the field of human sleep to explore how neural and cardiac activities are organized in the different sleep stages in healthy sleepers and in sleep disorder patients. After watching this video, you should have a good understanding of how to score and analyze non REM sleep retrospectively based on the behavioral reactivity of the sleeping mouse to an acoustic stimulus.
