The overall goal of this procedure is to introduce a way to study the functional integration of the adult born dentate granule cells. This is accomplished firstly by stereotaxic injection of retrovirus into the dentate gyrus of adult mice to label the newborn dentate granule cells. The second step of the procedure is to perfuse the mice at certain time stages with cold cutting solution and remove the brain.
The third step of the procedure is to prepare acute brain slices with a vibrato. The fourth step of the procedure is to perform whole cell patch clamp recordings on newborn dentate granule cells, and stimulate the molecular layer of acute brain slices. Ultimately, results can be obtained that show functional synaptic integration of newborn neurons by recording and analyzing evoked postsynaptic currents on newborn dentate granule neurons.
The main advantages of this technique are retroviral leveling of newborn neurons in the adult brain and acute brain slash preparation from the adult animals. This method can help answer key questions in the field of adult neurogenesis, such as the functional loss of newborn neurons in the adult brain. Prepare for the virus injection by removing the retrovirus for green fluorescent protein GFP or red fluorescent protein D tomato from the freezer and thawing it on ice Thaw.
Two microliters of retrovirus per animal anesthetize a four to six week old female mouse. Test for anesthesia by pinching the pore, mount the mouse on a stereotaxic frame, shave the hair on the head and wipe the skin with 70%ethanol. Use a scalpel to cut the skin and expose the skull.
Adjust the nose position of the mouse so that bgma and lambda earn the same horizontal plane using a dental drill with a 0.6 millimeter drill bit. Drill four holes through the skull at the coordinates shown here. Quickly fill a one microliter Hamilton flat tip syringe with the defrosted retrovirus.
Position it above one of the holes created at minus two millimeters bgma to inject the retrovirus into the dentate gyrus. Lower the syringe to a vertical depth of 2.5 millimeters. Inject 0.5 microliters of retrovirus at a rate of 0.25 microliters per minute to prevent fluid backflow.
Wait two minutes after each injection before slowly withdrawing the syringe. Briefly rinse the external surface of the syringe tip with sterile PBS and an applicator to remove traces of blood. Use the same procedure to inject 0.5 microliters of retrovirus into the other three sites at ventral depths of either 2.5 or 3.2 millimeters.
Close the wound with sterile surgical suture material and return the animal to its housing until the newborn neurons have reached the desired maturity date. After 21 days have elapsed, prepare for slicing the mouse brain by making the necessary solutions. Chill the cutting solution on ice at zero to four degrees Celsius and bubble it with 95%oxygen, 5%carbon dioxide for at least 30 minutes.
Place the artificial cerebral spinal fluid or A CSF at room temperature and bubble it with 95%oxygen, 5%carbon dioxide for at least 30 minutes. After 30 minutes on ice, set up the ice called oxygen saturated cutting solution for cardiac perfusion. After anesthetizing the mouse perform cardiac perfusion.
Then quickly remove the brain and place it in a Petri dish containing cutting solution and a piece of filter paper in the Petri dish. Cut off the cerebellum and then make a coronal cut one to two millimeters anterior to the injection sites to make a flat surface. For mounting super.
Glue the brain onto a vibram platform. Mount the platform into the vibrams cutting chamber filled with oxygenated cutting solution. Lower the blade and set the cutting window on the Vibram.
Begin slicing the tissue. Cut each slice into two halves along the midline. Use a large bore pipet to transfer these slices to an incubating chamber containing room.
Temperature A CSF bubbled with 95%oxygen 5%carbon dioxide. Continue slicing until the anterior end of the hippocampus is visible. Incubate the slices bubbling continuously in the 32 degrees Celsius water bath for 30 to 60 minutes.
Return the incubating chamber to rim temperature for the duration of the recording. The slicers are now ready for recording. Begin by perfusing the recording chamber with 32 to 34 degrees Celsius oxygen saturated A CSF at about two milliliters per minute.
Use a large boar perpet to transfer a slice into the recording chamber. Place a U-shaped platinum wire with nylon strings on top of the slice to hold it down under low magnification. Identify the molecular layer of the dentate gyrus.
Then lower the stimulation electrode into the molecular layer of the dentate gyrus. Under high. Identify the newborn dentate granule cells in the sub granular zone or granular layer by the expression of the green or red fluorescent protein labeling.
Locate newborn neurons expressing GFP or D tomato by shifting between DIC and fluorescent filters. Select a cell with relatively bright fluorescence and a healthy cell body for whole cell patch clamp recording. Insert a glass micropipet with a three to six mega ohm tip resistance filled with the appropriate internal solution into a micropipet holder on the amplifier head stage under the high magnification objective of the microscope.
Lower the micropipet by a micro manipulator to the selected dentate granule cell and form a whole cell patch. Inject a series of currents under current clamp mode and record the firing properties of the newborn neuron. Apply single pulse or paired pulse electrical tii via the stimulation electrode and record the evoked post-synaptic currents.
If there is no post-synaptic response, move the stimulation electrode to another position in the molecular layer and try to record the post-synaptic currents evoked from the new stimulus location. After finishing the experiment, use clamp fit to measure the amplitude of the evoked postsynaptic responses from the newborn neurons. This figure shows GFP expression in newborn neurons in the dentate gyrus of an adult mouse 21 days post-injection of GFP Retrovirus.
Note, the newborn neurons are easily visualized and that both the dendrites and axons are robustly labeled. The dendrites are the processes that extend from the soma into the molecular layer of the dentate gyrus, and the axons are also visible in the hilos and ca three area. This figure shows whole cell current clamp recordings from a newborn dentate granule cell at 21 days after injection of P-U-X-G-F-P retrovirus in an adult mouse.
The top trace shows action potentials elicited by current injection. This figure shows post-synaptic currents elicited in a newborn dentate granule cell 21 days after virus injection. By stimulating in the molecular layer.
The existence of the post-synaptic response shows that the newborn neuron has already integrated into the existing neural circuits and is receiving synaptic inputs from ENT of various origins After its development. This technique piped the way for the researchers in the field of adult neurogenesis to explore the functional integration of newborn neurons in the adult brain.
