The overall goal of this procedure is to virally transduce neurons throughout the brain. This is accomplished by cryo anesthetization of newborn pups, followed by transfer of the neonate to the cooled stage for injection. Next, identify the injection site for targeting the lateral ventricles between the Lambda suture and each eye or lateral from the sagittal suture halfway between Lambda and Bgma.
The final step is to insert the needle to the correct depth and slowly infuse up to two microliters of virus solution into the lateral ventricle of each hemisphere. Ultimately, a fluorescent tag or immunofluorescence for the gene of interest is used to show the transgene expression pattern resulting from intraventricular injection. Generally, individuals new to this technique will struggle with accurately targeting the lateral ventricles.
This technique is demonstrated through both freehand intracranial injection as well as through stereotactic injection, demonstrating the freehand technique will be jone another postdoc in my laboratory. To begin, prepare a 10 microliter injection syringe with a 32 gauge needle for general postnatal day zero neonates. Then place a small aluminum plate on ice to cool and place a dry task wipe on top of the plate to protect the pup skin from the cold metal.
This serves as a flat cold surface for anesthetizing and injecting the pups. Next, prepare a warming pad suitable for keeping neonatal mice warm before and after injection. Once the newborn pups have begun to nurse, remove approximately half of them from the cage and leave the other half with their mother.
Place the collected pups on the warming pad while awaiting injection. Transfer one pup from the warming pad onto the cold metal plate to induce hypothermia.Anesthesia. Wait two to three minutes for the pup to become fully anesthetized.
Confirm anesthesia by very gently squeezing a paw and monitor for lack of movement or respiration for free. Hand intracranial injection load five microliters of diluted adeno associated virus, or a A V with 0.05%Trian blue into the injection syringe. Gently wipe the head of the anesthetized pup with a cotton swab soaked in 70%Ethanol.
Identify the injection sites at two fifths of the distance from the lambda suture to each eye and alternative injection site is located at approximately 0.8 to one millimeter lateral from the sagittal suture halfway between Lambda and Bgma. These landmarks are visible through the skin at postnatal day zero. Mark the injection site with a non-toxic laboratory pen.
Hold the syringe with a scale visible for monitoring the volume of solution dispensed. Ensure that the thumb can reach the top of the plunger. Then remove the thumb from the plunger while positioning the needle.
To avoid accidentally dispensing virus, lay the pup on its side with its head directly under the syringe. Turn the P'S head so that the marked injection site is facing up and gently but firmly. Hold this position with an open hand.
Hold the syringe perpendicular to the surface of the skull and insert the needle at the marked injection site to a depth of approximately three millimeters. Inject the needle until the resistance decreases slightly indicating that the needle has penetrated into the lateral ventricle. Begin slowly injecting virus while monitoring the volume dispensed from the syringe.
Administer a maximum volume of two microliters into the ventricle. Slowly withdraw the needle. Allow the first injection site to close before injecting the other hemisphere.
If the needle is in the correct position, the dye will spread to fill the ventricle. Cool the neonatal stage to between four and eight degrees Celsius by adding 100%ethanol and dry ice to the reservoir. At the front end of the block lock.
Maintain the temperature above one degree Celsius to avoid frostbite of the pups. Load five microliters of diluted A a V with 0.05%trian blue into the injection syringe held by the stereotaxic manipulator. Later, gently place the pup's head between the ear bars of the neonatal frame.
Make sure the head is level in the Y axis by checking that the line between lambda and Bgma is parallel to the stage. Make sure the head is level in the x-axis by checking that an imagined line between the ears or a line between the eyes is parallel to the stage. Gently wipe the anesthetized pup's head with a cotton swab soaked in 70%ethanol.
Then use the stereotaxic manipulator to position the syringe above Lambda and zero. The x and Y coordinates. Move the stereotaxic arms to a position 0.8 millimeters lateral and 1.5 millimeters anterior to the lambda suture or standard postnatal day zero pups slowly lower the needle into the injection site.
The surface of the skull will indent and then release. Once the needle has penetrated the skin, retract the needle until the skull recovers its normal concave shape, but keep the bevel of the needle under the skin zero. The Z coordinate at this point.
Insert the needle until Z equals negative 1.7 millimeters and then retract to negative 1.5 millimeters. Slowly inject the virus. Each hemisphere can accept up to two microliters of solution.
Keep the syringe in place for 30 to 60 seconds after completing the injection and then slowly retract the needle over one to two minutes. Repeat for the contralateral hemisphere using negative coordinates in the x axis for the injection site. After completing injections into both hemispheres, place the pup back on the warming pad until its body temperature and skin color returned to normal and the pup begins to move.
Next, remove the remaining unin injected pups to the warming pad and return the injected pups to the cage after they recover normal movement. Repeat the procedure until all mice have been injected. Successful intraventricular viral injection yields widespread and robust.
Neuronal expression, YFP or TD tomato fluorescent gene constructs were packaged into a a V eight and injected into the lateral ventricles of neonatal mice. High viral titers resulted in dense labeling of the olfactory bulb, striatum, cerebral cortex, hippocampus, and cerebellum. Labeling was also apparent in cerebellar purkinje neurons, but notably absent from cerebellar granule neurons which are not yet present in large numbers at postnatal day zero injection of fewer viral particles resulted in sparse labeling and lower intensity expression.
Using AA V eight within a concentration range between 10 to the seventh and 10 to the 10th particles per is an easy and reliable way to control the degree of transgene mosaicism produced by injection. Multiple transgenes can be expressed by co injecting two or more viruses. Co injection of two viruses packaged into the same serotype, biases the resulting transduction to overlapping neuronal populations so that many cells express both transgenes at lower concentrations.
The expression patterns become more independent and the percentage of co labeled cells is decreased. Non-overlapping expression can also be achieved by-in injecting different a a v serotypes co injection of AAV one and AAV eight. Encoding distinct fluorescent reporters produces a largely independent pattern of dual mosaicism Once mastered.
This technique can be completed in five to 10 minutes per neonate if done properly.
