In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

This method can help answer key questions in the field of systems neuroscience. Such as how the activity of neuronal ensembles supports sensory and cognitive processing in the brain. The main advantage of this technique is that it facilitates the accurate targeting of optogenetic proteins like Chan rhodopsin and GCaMP to brain regions of interest, without requiring a separate virus injection procedure.

Visual demonstration of this method is critical, as the deposition of silk and virus to small optical implants can take some practice in order to achieve reproducible results. First, mix thawed AAV and five to seven point five percent aqueous silk fibroin in a one to one ratio in a 200 microliter PCR tube. Gently pipette the solution in and out several times to thoroughly mix the AAV and fibroin.

To deposit the silk/AAV, load an injection pipette with the amount of solution required for the number of fiber implants being made, plus approximately 30 percent extra to accommodate losses due to pipette clogging. Next, mount a ferrule holder into a stereotaxic apparatus equipped with a microinjector. Place the ferrule holder above the microinjector and apply the silk/AAV mixture from below.

Maneuver the injection pipette until it is touching or nearly touching the center of the optical fiber surface, and eject 10 to 20 nanoliters of silk/AAV solution. Then, withdraw the pipette. Next, observe the bolus of silk/AAV on the flat surface which appears as a liquid dome that dries to a flat film within approximately one minute.

Repeat the previous steps until the desired amount of silk/AAV is deposited. When preparing multiple implants, apply the silk/AAV solution to one implant and then move on to coat other implants, before returning to the first. After one hour of drying, place the entire ferrule holder into a vacuum chamber and vacuum desiccate overnight at approximately 125 torr and four degrees Celsius.

On the following day, evaluate the shape and position of the resulting silk film under a high-power microscope. Ensure that films are confined to the tip of the optical fiber surface, relatively thin, and symmetrical. For coating tapered optical fibers, position the silk/AAV injection pipette against the side of the optical fiber at the beginning of the taper, making sure that the pipette is touching the optical fiber.

Eject 20 nanoliters of silk/AAV to start the coating process, ensuring that the droplet adheres to the optical fiber and remains at the interface of the fiber and pipette. Gently wick the droplet towards the end of the fiber tip as the silk/AAV dries, and keep the injection pipette in contact with the drying droplet to avoid clogging the pipette tip. When the first bolus has almost completely dried, eject another 20 nanoliters and continue wicking the droplet along the taper.

Repeat the previous step by ejecting small amounts of silk/AAV, and gradually drying the solution up the side of the taper. After drying, evaluate the shape and position of the resulting silk film under a high-power microscope. For coating GRIN lens implants, deposit silk/AAV in a single injection on a GRIN lens implant.

After drying, evaluate the shape and position of the resulting silk film under a high-power microscope to ensure that the film covers the surface of the lens. For coating glass cranial windows, hand-pipette a five-microliter droplet of silk/AAV onto the surface of a three millimeter round cover slip so that the droplet spreads out to cover the entire glass surface. After drying, store the silk/AAV-coated optical fibers in a cooled vacuum desiccator at 125 torr and four degrees Celsius prior to implanting the devices.

Fluorescence images of fluorophore-tagged optogenetic proteins provided a measure of the extent of expression of the silk/AAV films, and typical optical fibers can readily accommodate 200 nanoliters of silk/AAV. With practice, experimenters can achieve highly reliable expression around the tip of implanted fibers. Two possible issues with coated cranial windows are insufficient expression, and silk films that fail to dissolve, and obscure the field of view.

To increase expression, a durectomy can be performed prior to implanting the window, and/or increasing the amount of virus in the film. The best expression was achieved using a one to four mixture of silk and stock-titer AAV, respectively. The amount of silk in coated windows is 10 to 100 times more than on fiber implants, and the film is less embedded in tissue, and thus may not be exposed to the same levels of proteolytic activity that can dissolve silk films.

However, the presence of some silk is essential to achieving your expression beneath windows, likely because a film made of virus alone is washed away by interstitial fluid during surgery. While attempting this procedure, it's important to prepare and store silk-coated implants in the manner we describe. Improper storage may change the properties of silk films, or decrease the efficacy of viral expression vectors.

This technique can be of great utility for researchers in the field of systems neuroscience to explore how neural activity drives sensory and cognitive processing in the brains of mammals such as mice, rats, or even nonhuman primates. Don't forget that working with viral expression vectors can be hazardous and personal protective equipment, such as gloves, goggles, and lab coats, should always be worn while working with Adeno-associated virus.