The overall aim of this procedure is to measure EEG defined sleep and lactate concentration during optogenetic stimulation at precise stereotaxic coordinates. Surgically implant the necessary electroencephalographic leads and cannulas for a lactate sensor and fiber optic cable. Then insert the fiber optic cable and pre calibrated lactate sensor into their guide cannulas in the animal's skull.
Next, adjust the blue light stimulus intensity to achieve the desired electrophysiological response. Then proceed to collect the electroencephalographic, electromyographic and lactate concentration data while the animal is allowed to spontaneously behave and or sleep. Compared to other methods like pharmacological studies of the EEG, this experimental approach has the advantage of monitoring brain biochemistry and electrophysiology while the activity of neurons is manipulated in real time on a sub-second time scale.
So potential to yield novel insights for the sleep research field is high. For instance, one can identify metabolic and electrophysiological concomitants of slow wave sleep. This type of experiment can also be applied in other areas of neuroscience, such as learning and memory, synaptic plasticity, or seizure disorders.
The surgical technique is technically challenging. It requires a good deal of dexterity, but that can be achieved through practice. In this visual demonstration, I will iterate the critical steps of some complicated surgical procedures that will compliment the written description.
Once an animal has recovered from surgery, the challenge is to properly insert the lactate sensor and fiber optic cable. Jonathan and I will demonstrate how to use a firm hand when restraining the animal to insert these two delicate pieces of equipment. Finally, we'll show how to optimize the EEG response by adjusting the intensity of the stimulus in real time.
For this experiment, use transgenic mice that express the blue light sensitive cation channel. Channel adopts in two in cerebral cortical neurons. First anesthetize the mouse using 5%isof fluorine, 95%oxygen for induction while maintaining anesthesia with 3%isof fluorine, 97%oxygen.
Make a medial incision on the top of the skull from between the eyes to the back of the skull. Clean the skull with hydrogen peroxide and sterile saline. Locate and mark B and Lambda for determining stereotaxic coordinates for electrode optogenetic stimulus configurations prime screw holes with a high speed dental drill with a 0.5 millimeter ball bur bit followed by a 0.7 millimeter ball bur bit.
Insert the electrode kelo gram screws into the holes with a slotted hand screwdriver. Drive them in approximately four to five revolutions to obtain the desired depth. Hold the guide cannulas in place with a stereotaxic cannula, and then affix them to the skull and anchors with acrylic cement To maintain patency, each guide cannula should contain a dummy cannula or stylet from the time of surgery to the time of experimentation.
Once EEGs and guide cannulas are positioned in the skull, bond them together with a thin layer of dental acrylic cement. After the cement sets position the plastic connector above the dried cement mound sold at the ends of the wires emanating from the EEG leads to the contacts on the plastic connector in case the wire leads in cement. Then draw the electromyogram wires through the nucle muscles by sliding them into the barrel of a 21 gauge needle.
Pierced through the muscle tie a double surgeon's knot a five zero nylon suture around these wires just distal to where they exit the muscle suture back together the skin that was retracted to access the muscle tissue with single interrupted surgeon knots using a reverse cutting P three needle and five zero nylon suture. Once the mc stimulus unit is programmed, run it as a standalone signal generator as a five volt binary on-off signal. Connect the mc stimulus unit to the TTL enabled power unit of the laser with BNC connectors.
Connect the laser power unit to the laser via a ribbon cable. Also connect the laser to the male FC connector on the originating fiber optic patch cable. Connect the originating fiber optic cable to the rotary joint.
Commutator Fiber optic rotary joint serves as a commutator. As the animal moves about its cage, the rotary joint rotates to prevent breakage of the fiber optic cables due to rotational torque. With plastic cable ties affix the commutator to a metal stand placed above the cylindrical cage in which the animal is housed.
Restrain the mouse by using one hand to pin the mouse under a cupped palm. Orient the head between the experimenter's middle and index finger. Now clear the guide cannula of debris, debris using a sterile 25 gauge needle.
Then insert the fiber optic cable by hand and fasten it to the fiber optic guide Cannula with a threaded screw cap. Control the depth of insertion of the fiber optic cable in the brain by a suture knot tied on the fiber optic cable at a fixed distance from the flat cleaved end. The sensor is e equated in phosphate buffered saline and exposed to three concentrations of L lactate in stepwise fashion.
Per manufacturer protocols, Insert the pre calibrated sensor into the skull mounted lactate guide cannula in a manner identical to the fiber optic cable insertion procedure. Connect the lactate sensor to the pre amplifier of the pinnacle 8, 400 biosensor with bipolar plug connectors. Then attach this pre amplifier to the eight pin connector on the surgically implanted head mount.
Before collecting data, use the laser intensity control knob to adjust the intensity of the optogenetic stimulus. The amplitude of the EEG response will vary across animals due to factors that have not been studied systematically. It's therefore necessary to adjust the intensity of the optogenetic stimulus and verify that the EEG response is adequate When the desired response is achieved.
Collect data using the Pinnacle 8, 400 system with the neuro score. A SEIA interface classify the sleep states by visual inspection of the EEG and EMG data process. The data in ten second epochs as wake non rapid eye movement, sleep, or rapid eye movement sleep based on the EEG and EMG.
This biosensor optogenetic stimulus configuration shows the surgically implanted EEG electrodes, lactate sensor, and cannula for optogenetic stimulation. In the absence of optogenetic stimulation, this mouse underwent spontaneous sleep. Wake state transitions while E-E-G-E-M-G and cerebral lactate concentration were monitored continuously wake and the two subtypes of sleep.
Rapid eye movement and non rapid eye movement are defined based on the EEG and the EMG. Interestingly, the lactate biosensor current rises as a function of low amplitude EEG, and falls as a function of high amplitude EEG. Both channels of the EEG are responsive to optogenetic stimuli delivered in the frontal cortex.
After watching this video, you should have a good understanding of how to surgically implant a mouse for simultaneous optogenetic manipulation and measurement of the electroencephalogram, electromyogram, and lactate concentration in the brain. Once mastered, this surgical technique can be properly performed in 90 to 120 minutes. Remember to constantly monitor the animal's respiration as a gauge of anesthesia depth.
If the animal breathes less frequently than once every four to five seconds, anesthesia may be too deep. If the animal breathes more frequently than once every two seconds, the anesthesia may be too light. Remember to take the appropriate safety precautions when using the gas anesthetic agent isof fluorine.
Ensure adequate ventilation in the surgical suite to protect your retina from the laser used for optogenetic stimulation. Either wear protective goggles or enclose the animal in a light proof cabinet. You'll wanna practice implanting the lactate biosensor and the fiber optic cable in a fully awake mouse.
Mice are not compliant with these procedures and must be immobilized. However, the restraint cannot be so forceful as to injure the animal or prevent respiration.
