The overall goal of this experiment is to observe the effect of high-frequency stimulation on neuronal activity in neurogenesis. This method can help answer key questions in an aligned cellular and molecular mechanisms of high-frequency deep brain stimulation, such as the induction of neuron activity and the neurogenesis in hippocampus. The advantage of this technique is that it demonstrates the powerful effect of high-frequency TBS on neuron activity and the neurogenesis in the adult brain.
Though this method can provide insight into the effects of high-frequency TBS on the mouse dentate gyrus, it can also be applied to other systems such as the basal ganglia and the subthalamic regions for the brain disorders in the clinic. To begin this procedure, place an anesthetized mouse onto the stereotactic frame. Align the ear bands with its head, and gently tighten them to support the centered head.
Grasp the skin anterior to its ears and remove about one square centimeter of it over the skull. Then remove the muscle and other attachment overlying the skull with a cotton swab to expose the skull surface. Next, apply hydrogen peroxide solution to dry the skull in order to identify the sagittal and lambda suture lines.
Then, identify the bregma and lambda. The bregma point will be used as the original point to set the nucleus position. After that, level the skull on the horizontal plane using these two points.
Make sure that the heights of these two points are almost the same in the dorsal ventral plane. In addition, make sure that the skull is leveled in the medial lateral plane. Next, mount a homemade two channel copper micro wire electrode into the rotating electrode holder on the stereotactic surgical frame.
Place the electrode directly above the bregma and set it as original site. Then, move the electrode gently to the correct position above the skull, without touching it. Use the digital display on the stereotactic surgical frame to indicate the anterior posterior, medial lateral, and dorsoventral positions.
When the desired site is identified for the electrode insertion mark the position with a black marker. Afterward, use the dorsoventral stereotactic adjustments to raise the electrode holder. Then, sterilize the drill bit with 70%ethanol.
Use a handheld micromanipulator assisted drill, make a burr hole precisely at the marked position. Use a sterile cotton swab to stop any bleeding during the operation. Keep drilling until dura is exposed.
Use a bent needle to remove any bone scraps that are generated during the drilling, and make a pinhole on the dura without damaging the underlying soft brain tissue. To confirm that the burr hole is made properly at the desired position, lower the electrode to make sure that it inserts through the burr hole smoothly without touching any obstacle. If no resistance is detected, slowly insert the electrode to the target depth from the surface of the skull.
Then, apply dental cement using a small spatula to hold the electrode in place. As the cement thickens, mold it around the inserted electrode and other bone screws to form a nice, smooth cap. Next, disengage the electrode from the electrode holder.
Remove the mouse from the stereotactic frame, and return it to a warm cage. Let the mouse move freely and allow it to recover for two days in the cage. To deliver stimulation, connect the implanted electrode to a programmable stimulator via leads.
Set up the software interface. Then, set the HFS parameters as six series of six trains of six pulses at 400 Hertz, and the stimulation intensity of 200 milliamps. Deliver HFS for the desired period of time as set up.
For the control group, implant electrodes in the mice without delivering any stimulation. For CFAS staining, stimulate the experimental mice two times for one day. For BrdU labeling, apply appropriate stimulations to both the acute stimulation group and the chronic stimulation group.
During the course of the stimulation, make sure the mouse is awake. Remember to insure the leads are not twisted. When the stimulation is done, carefully disconnect the electrode pin from the electrode holder in the connector of the recording system.
For the CFAS and notch one staining, about three hours after the last HFDBS, anesthetize the mice by injecting pentobarbital to achieve a surgical plane of anesthesia. For BrdU labeling, about 12 hours after the last HFDBS stimulation, give six injects of BrdU to the control and the stimulated mice at two hour intervals. 36 hours after the last injection, deeply anesthetize the mice with pentobarbital and transcardially profuse it with 50 milliliters of cold PBS followed by 50 milliliters of cold 4%paraformaldehyde.
Remove its head and make an incision on the skin to expose the skill. Using forceps, chip off the skull slightly and carefully remove the whole brain. Then, post fix the brain is 4%paraformaldehyde for an additional two days, and transfer it to 30%sucrose solution at four degrees Celsius overnight.
Using a cryostat, slice the brain into 20 micron coronal sections. Transfer and mount the sections to the glass slide with a brush, and store them a minus 20 degrees Celsius. Afterward, transfer the slides into normal PBS containing no fixative.
Wash the sections with PBS three to four times for five minutes each to remove any excess fixative. For the BrdU labeled group, incubate the sections in two molar HCL for 30 minutes at 37 degree Celsius, and rinse them in a 0.1 molar borate buffer. After 10 minutes, wash the sections two times in PBS for five minutes each time.
Subsequently, incubate the sections in blocking solution for one hour at room temperature. Then, incubate the sections with anti-CFAS polyclonal antibody, anti-notch one antibody, or anti-BrdU antibody in a blocking solution at four degrees Celsius overnight. Was the sections three times in PBS for 10 minutes each time, then incubate them with Alexa Fluor 568 conjugated anti-rabid, Alexa Fluor 488 conjugated anti-goat, or Alexa Fluor 488 conjugated anti-rat secondary antibody for one hour at room temperature.
Was the sections three times in PBS for 10 minutes each time. Subsequently, cover the immuno labeled brain sections using an antifade reagent mounting medium containing DAPI. Let the sections air dry, and store them protected from light at four degrees Celsius.
Shown here is an experimental paradigm for the HFS treatment in the generation of BrdU labeled proliferating neurons in the DG.The animals were divided into an acute and a chronic group to receive different patterns of stimulations. Afterward, the animals in both groups were injected with BrdU on day six. On day eight, the animals were anesthetized and profused for the preparation of cryostat sections.
Here are the images of the BrdU immunofluorescent staining post-HFDBS in the DG.A quantitative analysis of the neuronal production indicated that the acute stimulation in the DG could not enhance neurogenesis. While the chronic stimulation in the DG significantly up regulated the neurogenesis. Compared to the control group, the number of BrdU labeling cells increased significantly in the chronic group, both in the ipsilateral and contralateral hemisphere.
These are the images of the BrdU immunofluorescent staining post-HFDBS in the PP sub-region. A quantitative analysis of the neuronal production indicated that either the acute or chronic stimulation in the PP could not enhance the neurogenesis significantly. The number of BrdU labeling cells was relatively stable with or without the HFS treatment.
While attempting this procedure, it's important to remember to locate the electrode properly, and to deliver the stimulation successfully. After it's development, this technique paved the way for researchers in the field neuroscience high-frequency stimulation enrolled in the models. After watching this video, you should have a good understanding of how to deliver high-frequency stimulation to deep brain nucleus.
