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Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.
Virally-mediated gene delivery holds great potential for the study of neurological processes and treatment of brain disorders1,2,3. The great versatility of this technique can also be exploited to fluorescently label cells for imaging both in vitro and in vivo4. Here we demonstrate a detailed procedure for the transduction of neurons and glia in mouse visual cortex using a double-stranded adeno-association virus expressing enhanced green fluorescent protein.
While this technique is relatively straight forward, there are a number of technical details that need to be considered. One important factor is injection-induced tissue damage – therefore it is crucial to use great caution during the surgery and insertion/removal of the micropipette. A failed surgery could result in the alteration of the structures to be imaged. Additionally, after loading the virus into the micropipette, great care must be taken to clear any air bubbles at the pipette tip and ensure the proper volume is injected. Lowering the micropipette slightly beyond the target Z-coordinate then withdrawing it to the proper position can prevent virus from overflowing out of the injection site. When choosing an incubation time, allowing adequate time for viral gene expression should be considered. This will vary based on the virus used. A limited immune response may be elicited by the injection. In our experience, this inflammation is generally restricted to the needle track and can be avoided by imaging away from the injection sight (approximately 50 µms or more). Lastly, if brain sections are to be mounted on slides, use of an antifade mounting medium is suggested to maintain fluorescence.
Intracranial injection of viral vectors has several technical advantages over other labeling techniques. Through the use of stereotaxic coordinates and modulating the volume injected, fluorescent label can be precisely localized to an area of interest. The amount of transduction can be altered by adjusting the volume injected, the virus titer or the survival time, allowing for visualization of either groups of cells or individual cells. Additionally, the use of different viruses (e.g. lentivirus, herpes virus, adenovirus) can also modulate the timing and amount of fluorescent protein expressed as well as the cell types targeted. Overall, this technique can provide for very versatile labeling of different brain elements for imaging.
The animal and experimental protocols were approved by the University of Rochester University Committee on Animal Resources (UCAR) in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals.
This work was made possible by grants from the NIH (EY012977), a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund, the Whitehall Foundation, and the Sloan Foundation (A.K.M.).
Disclosures: The authors have nothing to disclose.
Rebecca L. Lowery, Department of Neurobiology and Anatomy, University of Rochester Medical Center, Rochester, NY, USA, Email: Rebecca_Lowery/at/urmc.rochester.edu.
Ania K. Majewska, Department of Neurobiology and Anatomy, University of Rochester Medical Center, Rochester, NY, USA, Email: Ania_Majewska/at/urmc.rochester.edu.