Name: in vivo targeted expression of optogenetic proteins using silk/aav films
Uploaded: 2019-02-26T14:00:00.000+00:00
Duration: 6 min 11 s
Description: Watch this Scientific Journal Video about In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films at JoVE.com

The authors wish to thank J. Vazquez for illustrations, D. Kaplan and C. Preda for reagents and helpful guidance, and the labs of B. Sabatini and C. Harvey for in vivo imaging. Microscopy was made possible by M. Ocana and the Neurobiology Imaging Center, supported in part by the Neural Imaging Center as part of a National Institute of Neurological Disorders and Stroke (NINDS) P30 Core Center grant (NS072030). This work was supported by the GVR Khodadad Family foundation, the Nancy Lurie Marks foundation, and by NIH grants, NINDS R21NS093498, U01NS108177 and NINDS R35NS097284 to W.G.R, and by an NIH postdoctoral fellowship F32NS101889 to C.H.C.

All experiments involving animals were performed in accordance with protocols approved by the Harvard Standing Committee on Animal Care following guidelines described in the US NIH Guide for the Care and Use of Laboratory Animals. Adult C57BL/6 mice of either sex (6-15 weeks of age) were used for all experiments.
1. Obtain Aqueous Silk Fibroin
<ol>
	<li>Prepare or purchase aqueous silk fibroin (5-7.5% w/v).</li>
</ol>
2. Mix Aqueous Silk with AAV Expression Vectors
<ol>
	<li>Choose an AAV expression vector to drive the optogenetic protein or fluorescent indicator of choice. 
	Note: To minimize the volume of silk/AAV that must be applied to implants while still driving robust expression, stock-titer AAV (stock titers typically obtained from vector cores are around ~1013 gc/mL) is recommended.</li>
	<li>Immediately prior to coating implants, thaw an aliquot of AAV and combine with 5-7.5% aqueous silk fibroin (this mixture will be referred to as silk/AAV). In a 200 &#181;L PCR tube, mix aqueous fibroin and AAV in a 1:1 ratio (for cranial windows use 1:4) immediately prior to the application. Gently pipette the solution in and out several times to thoroughly mix the fibroin and AAV.</li>
	<li>Keep silk/AAV mixture on ice prior to use.</li>
</ol>
3. Prepare Equipment for Fabrication and Storage of Silk/AAV-Coated Devices
<ol>
	<li>Procure equipment for coating optical fibers and Gradient-Index (GRIN) lenses (Figures 1, 2).
	<ol>
		<li>Construct a stable ferrule holder. To hold ceramic ferrules, drill 1.25 mm holes in a block of &#188;&#8221; sheet acrylic. Tap holes to insert set screws from the side to hold ferrules in place. 
		&#8203;Note: Any clamp can be used for this purpose.</li>
		<li>Position a manipulator with sub-millimeter precision to move the optical fibers (stereotaxic apparatus or other precision micromanipulator).</li>
		<li>Assemble a stable holder to position the microinjector.</li>
		<li>Use a stereoscope to visualize optical fibers and silk droplet.</li>
		<li>Position a light source to illuminate the optical fibers.</li>
	</ol>
	</li>
	<li>Prepare equipment for coating cranial windows (Figure 3).
	<ol>
		<li>Choose any P10 pipettor.</li>
		<li>Obtain a container with lid. 
		Note: Any container with a silicone bottom is suggested&#8212;the soft bottom facilitates lifting up cranial windows.</li>
	</ol>
	</li>
	<li>Prepare equipment to store finished implants (Figure 4).
	<ol>
		<li>Obtain a small (1-5 L) vacuum chamber.</li>
		<li>Make sure that there is space to store implants in a 4 &#176;C refrigerator.</li>
	</ol>
	</li>
</ol>
4. Apply Silk/AAV Film to Devices
<ol>
	<li>Coating optical fibers to drive focal expression at the fiber tip
	<ol>
		<li>Prepare chronic fiber implants as previously described4.</li>
		<li>Prior to use, rinse implants with ethanol, then with ultrapure water to ensure that the optical fibers are clean. 
		Note: Silk films adhere more reliably to clean glass surfaces.</li>
		<li>Prepare a device to hold fiber ferrules. For typical 1.25 mm diameter ferrules, use a block &#188; inch clear acrylic, with ~1.3 mm holes, and tapped set screws entering from the side to hole implants firmly in place (Figure 1A).</li>
		<li>Mount the ferrule holder into a stereotaxic apparatus (or any manipulation solution with submillimeter precision) equipped with a microinjector. Place the ferrule holder above the microinjector and apply the silk/AAV mixture from below. 
		Note: This is because applications of large volumes from above resulted in silk/AAV that was not restricted to the tip. However, the application of many small sequential volumes from above or below can produce AAV/silk deposits that are confined to the tip (although we prefer to apply from below).</li>
		<li>Pull a standard intracranial injection pipette from borosilicate glass capillary.
		<ol>
			<li>To make it easier to</li>
			<li>To produce an injection tip with a clean flat tip of the desired diameter, hold one pipette in each hand and use the thicker part of the taper on one pipette to score the other pipette at the desired break location.</li>
			<li>Gently rub back and forth in a sawing motion (the glass-on-glass scoring method).</li>
			<li>After scoring the pipette, apply gentle pressure to the tip of the scored pipette with the body of the other pipette to achieve a clean break.</li>
		</ol>
		</li>
		<li>Position a stereoscope to give a clear view of the optical fiber faces. 
		Note: Magnification should be sufficient to accurately position the injection pipette above the face of optical fibers.</li>
		<li>Insert fiber implants into holder with the brain-side of the optical fiber facing downward.</li>
		<li>Load the injection pipette with silk/AAV solution, as for any standard intracranial injection5. Load the amount required for the number of implants being made, plus ~30% extra to accommodate losses due to pipettes clogging. For example, if 10 implants are being made, then load with 100 nL deposits and withdraw ~1.3 &#181;L. 
		Note: Silk/AAV may dry at the pipette tip in between ejections, which can clog the pipette. Large diameter pipettes (50-100 &#181;m) are less likely to clog. Clogs can be dislodged by gentle brushing down the tip of the pipette with a wet paper wipe or alcohol swab.</li>
		<li>Maneuver the injection pipette until it is touching or nearly touching the center of the optical fiber surface. Eject 10-20 nL of silk/AAV solution. Withdraw the pipette. 
		Note: The rate of delivery is not critical, but typical rates are 5-20 nL/s.</li>
		<li>Observe the bolus of silk/AAV on the flat surface which appears as a liquid dome that dries to a flat film within ~1 min (Figure 1B).</li>
		<li>Repeat steps 4.1.9-4.1.10 until the desired amount of silk/AAV is deposited (a total of 20-200 nL for most applications). When preparing multiple implants, apply silk/AAV to one implant and then move on to coat other implants before returning to the first.</li>
		<li>Allow 1 h for drying before moving implants.</li>
		<li>Vacuum desiccate overnight at ~125 Torr (-25 in. Hg), 4 &#176;C. Do this by placing the entire ferrule holder into a vacuum chamber.</li>
		<li>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, be relatively thin (&#62;100 &#181;m), and symmetrical (Figure 1C). 
		Note: Large or asymmetrical silk/AAV films may dislodge from the fiber during implantation (Figure 1D). The most common cause of problems arises from the application of single large volumes rather than the sequential application of many small volumes.</li>
	</ol>
	</li>
	<li>Coating tapered optical fibers to drive expression along the fiber axis
	<ol>
		<li>Obtain tapered optical fiber implants and perform steps 4.1.2-4.1.8, except that the tapered fiber is positioned laterally such that it is perpendicular to the injector (Figure 2A). Position the injector above the tapered fiber. 
		Note: Loading liquid droplets onto tapered fibers poses added challenges, because surface tension tends to cause droplets to jump back onto the injection pipette or migrate up the tapered fiber. Smaller injection pipettes (30-50 &#181;m diameter) help to overcome this problem but increase the risk that the injection pipette will clog. Due to surface tension, droplets tend to adhere to the area of largest surface area, so the optimal injection pipette size is dependent on the size of the tapered fiber and one&#8217;s tolerance for the occasional clog.</li>
		<li>Position the silk/AAV injection pipette against the side of the optical fiber at the beginning of the taper. Make sure the injection pipette is touching the optical fiber.</li>
		<li>Eject 20 nL of silk/AAV to start the coating process. Ensure that the droplet adheres to the optical fiber and remains at the interface of the fiber/pipette. Gently wick the droplet towards the end of the fiber tip as the silk/AAV dries (~45 s). Keep the injecting pipette in contact with the drying droplet to avoid clogging the pipette tip. 
		Note: Each deposit should coat approximately 400 &#181;m of the tapered fiber (Figure 2B).</li>
		<li>When the first bolus has dried almost completely, eject another 20 nL and continue wicking the droplet along the taper. 
		Note: The liquid silk will adhere to the dried silk, anchoring one end of the droplet as the pipette moves along the taper.</li>
		<li>Repeat step 4.2.4 by ejecting small amounts of silk/AAV, and gradually drawing the solution up the side of the taper. 5-6 ejections are sufficient to traverse the surface of a 2.5 mm taper.</li>
		<li>To drive more uniform expression around all sides of the fiber, rotate the fiber and repeat steps 4.2.2-4.2.5 until the desired amount of silk/AAV has been deposited.</li>
		<li>If a hanging strand of dried silk/AAV extends beyond the fiber tip, carefully cut the strand with scissors, or use the ejection pipette to bend the strand back and adhere it to the taper of the fiber.</li>
		<li>Allow 1 h for drying before moving implants.</li>
		<li>Vacuum desiccate overnight in 4 &#176;C. The entire ferrule holder can be placed into a vacuum chamber.</li>
		<li>Evaluate the shape and position of the resulting silk film under a high-power microscope. 
		Note: Films need not be entirely uniform but should not have bumps that extend more than 100 &#181;m beyond the surface of the fiber to minimize damage to surrounding tissue during implantation (Figure 3C). To minimize film size, it is critical that each droplet is completely dry before subsequent deposits are made.</li>
	</ol>
	</li>
	<li>Coating GRIN lens implants
	<ol>
		<li>Obtain GRIN lenses6,7 and repeat steps 4.1.2-4.1.8. The injector can be mounted above.</li>
		<li>Deposit silk/AAV in a single ejection (1 &#181;L for a 1.0 mm diameter lens). 
		Note: This will yield a dome of liquid that adheres to the face of the lens, and dries to produce a uniform film (100-200 &#181;m thick). However, in the event that a single large ejection dries unevenly and produces a film that is thicker near the edges of the GRIN lens, try depositing multiple smaller droplets (100-200 nL) in the center of the lens surface (allowing each droplet to dry before depositing the next) to ensure that the film will drive expression at the center of the field of view.</li>
		<li>Allow 1 h for drying before moving implants.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Coating glass cranial windows
	<ol>
		<li>Prepare glass cranial windows by adhering two 3 mm diameter round coverslips (no. 1 thickness) to one 5 mm diameter window with optical adhesive (for details, see Goldey et al. 20148).</li>
		<li>Mix silk:virus in a ratio of 1:4 to reduce the total amount of silk in the film. Excessive quantities of silk do not dissolve beneath cranial windows after implantation. Titration experiments may be required to determine the ratio and volume that gives the desired expression profile.</li>
		<li>Hand pipette a 5 &#181;L droplet onto the surface of the 3 mm (brain facing) coverslip. The droplet should spread out to cover the entire glass surface (Figure 3).</li>
		<li>Allow 2-3 h for drying before moving windows.</li>
	</ol>
	</li>
</ol>
5. Storing silk/AAV-coated implants
<ol>
	<li>Store silk/AAV-coated optical fibers in a cooled vacuum desiccator (~125 Torr, 4 &#176;C) prior to use (Figure 4A).</li>
	<li>Do not store cranial windows and GRIN lenses under vacuum, as large silk films stored under vacuum fail to fully dissolve after implantation. Implant cranial windows and GRIN lenses immediately after drying, or within a day of manufacture if stored at atmospheric pressure and 4 &#176;C.</li>
</ol>
6. Implanting the Devices
<ol>
	<li>Prepare animals for implant surgery as previously described4.
	<ol>
		<li>Briefly, anesthetize mice with an intraperitoneal injection of ketamine/xylazine (100/10 mg/kg) and check the depth of anesthesia using a gentle toe-pinch. Shave the skull in the area of the implant and clean the scalp with iodine and alcohol.</li>
		<li>Mount animals in a stereotaxic device and supplement anesthesia using a mixture of oxygen and isoflurane (1-2%). Make an incision in the scalp over the area of interest, and perform a craniotomy large enough to accommodate the implant.</li>
	</ol>
	</li>
	<li>Implant optical fibers9 and microendoscope lenses10 according to previously published procedures. Handle implants with care, as the silk/AAV deposit can be knocked off by an imperfect craniotomy, or by the implant catching on the edge of the skull. Lower the implant into the brain slowly (~2 mm/min).</li>
	<li>Implant cranial windows as describe previously8. Do not touch the coated side of the window and avoid rinsing the window with fluid if performing gavage, as this may wash away the virus. To achieve maximal expression, perform a durotomy.</li>
</ol>
7. Evaluating the Expression and Troubleshooting
<ol>
	<li>To evaluate expression of virally-expressed proteins, allow ~2-3 weeks for the virus to drive expression, then perform intracardial perfusion with 4% paraformaldehyde in phosphate buffered saline solution11 and process brain tissue for fluorescent microscopy12.</li>
	<li>Evaluate the expression by using fluorescent microscopy to image the expression pattern of fluorophore-tagged optogenetic proteins.</li>
	<li>If the level of expression is insufficient, increase the amount of virus in coatings by either increasing the total volume of the silk/AAV coating, or preferably by using a higher titer virus.</li>
</ol>

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The authors have nothing to disclose.

The use of silk/AAV to target the expression of optogentic proteins overcomes limitations of approaches that are currently in use. Although many studies successfully use AAV injections to express optogenetic proteins, it is challenging to align expression to the tip of optical fibers, to regions around the length of tapered fibers, and to the viewing region of a GRIN lens. Because of misalignment between optical components and optogenetic expression, stereotaxic injections can be unreliable, and many experiments fail. The silk/AAV labelling method we describe here solves this problem. It also simplifies the procedure by eliminating a second surgical step and in some cases eliminating the need for a second surgery. It can also be difficult to use viruses to obtain widespread expression beneath cranial windows, and experimenter typically perform long surgeries to inject virus in multiple locations. The ability to obtain widespread expression over large cortical regions by simply coating cranial windows with silk/AAV is a simplification that eliminates the need for the many invasive injections.
Another potential advantage of the silk/AAV method is that it might induce less inflammation in the neural tissues compared to viral injections. Injecting high-titer AAV into the brain can cause inflammatory responses such as reactive astrocytosis that have the potential to alter cellular and circuit properties14,15 (although such potential complications are usually ignored). Silk films induce little immunogenic response on their own13 and silk/AAV films are expected to release virus over the course of many hours or days16, which may lower the viral load in the surrounding tissue and reduce immunogenic responses. With conventional approaches in which implantation of a device is preceded by an AAV injection, inflammatory responses can arise from both the implantation and the injection. In the future it will be desirable to systematically compare conventional approaches and the silk/AAV method to determine whether silk/AAV films reduces overall inflammatory responses.
Several steps are critical to the successful use of silk/AAV films. Most importantly, the coating of optical fibers must be done carefully as described in the Methods and the location of the dried films should be assessed carefully by visual inspection under a microscope to ensure that the films are compact, in the correct location, and adhere to the face of the optical fiber. Any silk/AAV on the sides of the optical fiber will lead to expression outside the region of interest, and misshaped films that protrude beyond the face of the fiber may break off during implantation and lead to unreliable or no expression. The techniques we describe for applying silk/AAV to implantable devices can be adapted to use of any materials are that are readily available and allow the precise deposition of small volumes of silk/AAV.
A bit of practice is required to achieve accurate, reproducible results. If expression is observed along the track of the fiber, it is likely that the silk film dried on the side of the fiber rather than the fiber face. Repeat the manufacturing process and closely inspect dried implants for signs that films are drying on the side of the fiber. Because silk/AAV films are optically transparent, it may help to practice applying silk mixed with dye (Fast Green or a similar dye) to better visualize the shape of resulting films (Figure 2C). If there is no expression, it is probable that the silk film dislodged from the fiber tip during implantation. We suggest the use of stock-titer virus when making implants. For optical fibers, this reduces the total volume that must be applied to small diameter fibers. If the size of the coating is a concern consider waiting longer between each 10 nL application to allow complete drying of the deposited droplet. Silk/AAV droplets dry faster under a warm lamp. For cranial windows, high-titer virus may be necessary to supply adequate viral load across the pia or dura. Certain types of implants may dissolve silk and release AAV more readily than others. We have found that cranial windows implanted over the surface of the brain require a lower ratio of silk/virus to achieve reliable expression, perhaps owing to different cerebral spinal fluid dynamics or protease activity. If expression cannot be increased by increasing effective AAV concentrations, decreasing the volume of aqueous silk is a plausible alternative.
Finally, it is important to store the optical components properly and implant them quite soon after they are prepared. We have shown that coated fibers that are refrigerated under vacuum can be stored for many days prior to use. Vacuum storage removes residual moisture17 which may reduce the solubility of silk films, and also help maintain viral efficacy. Ideally, optical fibers should be implanted within 24 hours of fabrication. However, we find that silk/AAV-coated fibers stored under vacuum drive similar levels of expression when implanted 7 days after fabrication (Figure 4B). In contrast, coated cranial windows and GRIN lenses drove the most reliable expression when they were dried at room temperature and used within hours of preparation. The reason for this disparity remains unclear. Further studies may be needed to refine preparation and storage conditions to further extend the storage time.
Silk/AAV-coated cranial windows have considerable potential because they drastically shorten surgical times and are extremely simple to manufacture, but at present this method has limitations. Coated cranial windows uniformly label large areas of cortex and drive sufficient expression in layer 2/3 for GCaMP imaging, with somewhat less expression in deeper layers. However, stereotaxic injections drive more robust expression and provide more control over the layers targeted for expression. Reliable expression was only achieved when the dura was removed. Although the dura is often removed for many 2-photon imaging experiments to improve image quality8, for many experiments it is desirable to obtain labelling in a less invasive manner. We have therefore explored our ability to use silk/AAV to label cortical regions without removing the dura. We obtained some labelling, but it is possible that this was a consequence of damaging the dura in the process of preparing the craniotomy. Further study is needed for coated cranial windows to be used to reliably label the cortex without removing the dura.
The preparation of aqueous silk fibroin from cocoons of Bombyx Mori is described in detail in Rockwood et al. (2011)18. Aqueous silk fibroin is now commercially available (5% w/v). Although most of our experiments were performed using aqueous silk fibroin stocks prepared in our lab (5-7.5% w/v), we have obtained similar results using commercial aqueous fibroin. Aqueous fibroin is stable at 4 &#176;C for up to 3 months, after which it spontaneously transitions from liquid to hydrogel18. We recommend that fibroin stocks be divided into ~1 mL aliquot and stored at -80 &#176;C. A 1 mL working aliquot (sufficient for coating hundreds of implants) can be stored at 4 &#176;C and used until it begins to gel. Be careful not shake, vortex, agitate or aggressively pipette aqueous fibroin, as shear forces can lead to gelation19,20.
Silk/AAV films permit a wide range of expression patterns, from widespread cortical expression under cranial windows, to precise subcortical expression at the tip of a small-diameter optical fibers. These techniques were developed to take advantage of common AAV expression vectors but could likely be used to disperse other expression vectors like Lentiviruses or rabies viruses into the brain. Silk films could also be manufactured into three-dimensional shapes to improve viral release into tissue. For example, in order to drive strong expression beneath cortical windows without the use of a durotomy, cranial windows could be coated with arrays of silk microneedles that would pierce the dura and release virus in deeper cortical layers21. Further refinement will likely lead to improved properties of virus release, and new applications for silk/AAV films.

The past decade has produced an explosion of engineered light-sensitive proteins for monitoring and manipulating neural activity1. Viruses offer unparalleled flexibility for expressing these optogenetic tools in the brain. Compared to transgenic animals, viruses are far easier to produce, transport, and store, allowing fast implementation of the newest optogenetic tools. Expression can be targeted genetically to distinct neuronal populations, and viruses designed for the retrograde transport can even be used to target expression based on neuronal connectivity2.
Viruses are usually introduced with stereotaxic injections, which can be time-consuming and challenging. Precisely targeting small regions can be difficult, while driving expression over broad areas often requires many injections. Moreover, when an optical device is subsequently implanted into the brain to deliver light in vivo, the implant must be properly aligned with the viral injection. Here, we describe an easily-implemented method for delivering viral vectors to the tissue around an implanted device using silk fibroin films3. Silk fibroin is commercially available, well-tolerated by neural tissues, and can be used to produce materials with varied properties. Silk films can be applied to implants using common laboratory equipment like microinjection pipettes or hand pipettes. Silk/AAV films eliminate the requirement for two surgical procedures and ensure that virus-mediated expression is properly aligned to the optical implant. The resulting expression is constrained to the tip of fibers, and results in less unwanted expression along the fiber track than stereotaxic injections.
In addition to producing targeted expression at the tip of small fibers, silk/AAV films can be used to drive widespread (&#62;3 mm diameter) cortical expression beneath cranial windows. In vivo 2-photon imaging of fluorescent activity sensors has become an indispensable tool for evaluating the role of neuronal activity in driving sensory and cognitive processing. However, to drive uniform expression over the broad cortical areas, experimenters often perform multiple injections. These injections can be extremely time-consuming and can lead to inconsistent expression across the field of view. In contrast, silk/AAV-coated cranial windows are extremely easy to manufacture, greatly reduce the time required for surgeries, and most remarkably drive expression hundreds of microns below the cortical surface.

<table><tbody><tr><td>Aqueous silk fibroin</td><td>Sigma</td><td>5154-20ML</td><td>Aqueous Silk Fibroin (5% w/v) for making films</td></tr><tr><td>Microinjector to deposit silk/AAV</td><td>Drummond</td><td>3-000-207</td><td>Nanoject III nanoliter injector</td></tr><tr><td>Manipulator to hold implants</td><td>Narashige</td><td>MM-33</td><td>Micromanipulator</td></tr><tr><td>Stereoscope to visualize silk deposits</td><td>AmScope</td><td>SM-6TX-FRL</td><td>3.5X-45X Trinocular articulating zoom microscope with ring light</td></tr><tr><td>Vacuum chamber to store implants</td><td>Ablaze</td><td>N/A</td><td>3.5 Quart Vacuum Vac Degassing Chamber</td></tr><tr><td>Optional, implant holder for storage</td><td>N/A</td><td>N/A</td><td>To store premade optical fibers, drill a grid of ~4 mm-deep holes with a diameter just larger than the ferrule diameter into a plastic block.</td></tr><tr><td>Optical fiber</td><td>Thorlabs</td><td>FT200EMT</td><td>&amp;Oslash;200 &amp;micro;m Core Multimode Optical Fiber for fiber implants</td></tr><tr><td>Ferrules</td><td>Kientec</td><td>FZI-LC-230</td><td>LC Zirconia Ferrule for fiber implants</td></tr><tr><td>Various materials for manufacturing chronic fiber implants</td><td>Various</td><td>N/A</td><td>For detailed procedure, see Ung K, Arenkiel BR. Fiber-optic implantation for chronic optogenetic stimulation of brain tissue. Journal of visualized experiments: JoVE. 2012(68).</td></tr><tr><td>Tapered fiber implants</td><td>Optogenix</td><td>Lambda-B</td><td>Tapered fiber implants</td></tr><tr><td>GRIN lenses</td><td>GoFoton</td><td>CLH-100-WD002-002-SSI-GF3</td><td>GRIN lenses</td></tr><tr><td>Small glass cranial windows</td><td>Warner</td><td>64-0726 (CS-3R-0)</td><td>Small round cover glass, #0 thickness</td></tr><tr><td>Large glass cranial windows</td><td>Warner</td><td>64-0731 (CS-5R-0)</td><td>Small round cover glass, #0 thickness</td></tr><tr><td>Various materials for manufacturing cranial windows</td><td>Various</td><td>N/A</td><td>For detailed procedure, see Goldey GJ et al. Removable cranial windows for long-term imaging in awake mice. Nature protocols. 2014 Nov;9(11):2515.</td></tr></tbody></table>

in vivo targeted expression of optogenetic proteins using silk/aav films

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical methods for manipulating and monitoring the activity of neurons in vivo. These types of experiments rely on two main components: 1) implantable devices that provide optical access to the brain, and 2) light-sensitive proteins that change neuronal excitability or provide a readout of neuronal activity. There are a number of ways to express light-sensitive proteins, but stereotaxic injection of viral vectors is currently the most flexible approach because expression can be controlled with genetic, anatomical, and temporal precision. Despite the great utility of viral vectors, delivering the virus to the site of optical implants poses numerous challenges. Stereotaxic virus injections are demanding surgeries that increase surgical time, increase the cost of studies, and pose a risk to the animal's health. The surrounding tissue can be physically damaged by the injection syringe, and by immunogenic inflammation caused by the abrupt delivery of a bolus of high-titer virus. Aligning injections with optical implants is especially difficult when targeting small regions deep in the brain. To overcome these challenges, we describe a method for coating multiple types of optical implants with films composed of silk fibroin and Adeno-associated viral (AAV) vectors. Fibroin, a polymer derived from the cocoon of Bombyx mori, can encapsulate and protect biomolecules and can be processed into forms ranging from soluble films to ceramics. When implanted into the brain, silk/AAV coatings release virus at the interface between optical elements and the surrounding brain, driving expression precisely where it is needed. This method is easily implemented and promises to greatly facilitate in vivo studies of neural circuit function.

To assess the success of silk/AAV films in driving expression, we perfused animals 2-3 weeks after implantation and prepared brain slices from the region of interest. Fluorescence images of fluorophore-tagged optogenetic proteins (ChR2-YFP) provided a measure of the extent of expression (Figure 1D). Typical optical fibers (230 &#181;m diameter) can readily accommodate 200 nL of silk/AAV. With practice, experimenters can achieve highly reliable expression around the tip of implanted fibers (Figure 5).
To assess expression driven by silk/AAV-coated cranial windows, begin imaging beginning 7-10 days after implantation. We have used two-photon imaging for visualization, but other methods such as fluorescence imaging with a CCD could also be used. 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, we suggest performing a durectomy prior to implanting the window, and/or increasing the amount of virus in the film. We achieved the best expression using a 1:4 mixture of silk and stock-titer AAV, respectively. While this represents a substantially larger number of viral particles than are usually used in stereotaxic injections, the decreased surgical time counters the marginal additional cost of virus. Meanwhile, if silk films fail to dissolve beneath the window, further reduce the amount of silk used to coat the window. The total amount of silk in coated windows is 10-100 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 than can dissolve silk films13. However, the presence of some silk is essential to achieving expression beneath windows3, likely because a film made of virus alone is washed away by interstitial fluid during surgery.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58728/58728fig1.jpg" /> 
Figure 1: Applying silk/AAV films to optical fibers. (A) Chronic fiber implants are placed fiber side-down into a holder (inset) mounted on a XYZ translator. A fixed microinjector below the fibers dispenses silk/AAV onto the fiber tips. A stereoscope allows visualization of the process. (B) Apply silk/AAV to fiber tips in small volumes (10-20 nL). After ejecting a bolus, retract the pipette and allow ~60 s for the droplet to dry to a flat film. Repeat the process until the required volume has been applied to the fiber tip. (C) Inspect silk coatings. Optimal coatings should be centered on the fiber tip (left), while improper coatings extend outward from the fiber face making them more prone to dislodge from the fiber (right). (D) Representative fibers coated with 200 nL of silk/AAV, and the resulting AAV-driven ChR2-YFP expression 2 weeks after implantation. The compact silk/AAV coating on the left resulted in robust expression, while the coating on the right protruded past the face of the fiber and resulted almost no expression, likely because the silk/AAV did not adhere to the optical fiber during implantation. Scale bars 0.2 mm (fibers) and 1.0 mm (brain slices). <a href="https://www.jove.com/files/ftp_upload/58728/58728fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58728/58728fig2.jpg" /> 
Figure 2: Setup for coating tapered fiber implants. (A) The microinjector is mounted above the tapered fiber holder, and tapered fibers are positioned orthogonally to the ejection syringe. (B) Begin at the widest point (inset) and eject small volumes while moving the ejection syringe towards the point of the taper. This results in a continuous coating along the length of the taper. (C) Representative tapered fiber coated with silk mixed with Fast Green to aid in visualization. <a href="https://www.jove.com/files/ftp_upload/58728/58728fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58728/58728fig3.jpg" /> 
Figure 3: Coating cranial windows. Silk/AAV can be applied cranial windows using a hand pipette. A standard 3 mm diameter window can be coated with a 5 &#181;L droplet, which will slowly dry to a flat film. Inset: GCaMP6f expression resulting from silk/AAV-coated cranial windows implanted with and without durectomies. This figure has been adapted from Jackman et al. (2018)3. <a href="https://www.jove.com/files/ftp_upload/58728/58728fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58728/58728fig4.jpg" /> 
Figure 4: Storing silk/AAV coated implants. (A) To remove residual moisture and preserve viral efficacy, implants should be stored under vacuum at 4 &#176;C until used. Implants stored this way remain viable for at least 7 days. (B) Expression resulting from 4 silk/AAV coated fibers implanted after 7 days of storage. <a href="https://www.jove.com/files/ftp_upload/58728/58728fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58728/58728fig5.jpg" /> 
Figure 5: Silk/AAV-GFP coated optical fibers reliably drive expression. Fluorescent images of slices from 24 consecutive striatal implants. Each implant was coated with 100-400 nL of 1:1 silk/AAV-GFP. This cohort of implants indicates the ability of silk to restrict expression to the implant site (in this case the dorsal striatum). GFP fluorescence is indicated in green; DAPI staining is shown in blue. <a href="https://www.jove.com/files/ftp_upload/58728/58728fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films at JoVE.com

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

Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

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

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical ...

in-vivo-targeted-expression-optogenetic-proteins-using-silkaav

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.4K Views.  Oregon Health and Science University. Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

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

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Genetics

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

Optogenetic Functional MRI

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional magnetic resonance imaging (ofMRI), which is a novel technique that combines the relatively high spatial resolution of high-field fMRI with the precision of optogenetic stimulation. Fiber optics that enable delivery of specific wavelengths of light deep into the brain in vivo are implanted into regions of interest in order to specifically stimulate targeted cell types that have been genetically induced to express light-sensitive trans-membrane conductance channels, called opsins. fMRI is used to provide a non-invasive method of determining the brain's global dynamic response to optogenetic stimulation of specific neural circuits through measurement of the blood-oxygen-level-dependent (BOLD) signal, which provides an indirect measurement of neuronal activity. This protocol describes the construction of fiber optic implants, the implantation surgeries, the imaging with photostimulation and the data analysis required to successfully perform ofMRI. In summary, the precise stimulation and whole-brain monitoring ability of ofMRI are crucial factors in making ofMRI a powerful tool for the study of the connectomics of the brain in both healthy and diseased states.

This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional ...

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we demonstrate the use of magnetic resonance (MR)-guided convection enhanced delivery (CED) of optogenetic viral vectors into primary somatosensory (S1) and motor (M1) cortices of macaques to obtain efficient, widespread cortical expression of light-sensitive ion channels. Adeno-associated viral (AAV) vectors encoding the red-shifted opsin C1V1 fused to yellow fluorescent protein (EYFP) were injected into the cortex of rhesus macaques under MR-guided CED. Three months post-infusion, epifluorescent imaging confirmed large regions of optogenetic expression (&#62;130 mm2) in M1 and S1 in two macaques. Furthermore, we were able to record reliable light-evoked electrophysiology responses from the expressing areas using micro-electrocorticographic arrays. Later histological analysis and immunostaining against the reporter revealed widespread and dense optogenetic expression in M1 and S1 corresponding to the distribution indicated by epifluorescent imaging. This technique enables us to obtain expression across large areas of the cortex within a shorter period of time with minimal damage compared to the traditional techniques and can be an optimal approach for optogenetic viral delivery in large animals such as NHPs. This approach demonstrates great potential for network-level manipulation of neural circuits with cell-type specificity in animal models evolutionarily close to humans.&#160;

Here, we demonstrate magnetic resonance (MR)-guided convection enhanced delivery (CED) of viral vectors into the cortex as an efficient and simplified approach for achieving optogenetic expression across large cortical areas in the macaque brain.

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we ...

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

Injections of AAV Vectors for Optogenetics in Anesthetized and Awake Behaving Non-Human Primate Brain

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of optogenetics, however, requires that safety and efficacy be demonstrated in animal models, ideally in non-human primates (NHPs), because of their neurological similarity to humans. The number of candidate vectors that are potentially useful for neuroscience and medicine is vast, and no high-throughput means to test these vectors yet exists. Thus, there is a need for techniques to make multiple spatially and volumetrically accurate injections of viral vectors into NHP brain that can be identified unambiguously through postmortem histology. Described herein is such a method. Injection cannulas are constructed from coupled polytetrafluoroethylene and stainless-steel tubes. These cannulas are autoclavable, disposable, and have low minimal-loading volumes, making them ideal for the injection of expensive, highly concentrated viral vector solutions. An inert, red-dyed mineral oil fills the dead space and forms a visible meniscus with the vector solution, allowing instantaneous and accurate measurement of injection rates and volumes. The oil is loaded into the rear of the cannula, reducing the risk of co-injection with the vector. Cannulas can be loaded in 10 min, and injections can be made in 20 min. This procedure is well suited for injections into awake or anesthetized animals. When used to deliver high-quality viral vectors, this procedure can produce robust expression of optogenetic proteins, allowing optical control of neural activity and behavior in NHPs.

As currently implemented, optogenetics in non-human primates requires injection of viral vectors into the brain. An optimal injection method should be reliable and, for many applications, capable of targeting individual sites of arbitrary depth that are readily and unambiguously identified in postmortem histology. An injection method with these properties is presented.

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of ...

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific genes. Multiple means of assessing non-coding DNA and various chromatin states have proven useful in predicting the presence of enhancer sequences in the genome, but validating the activity of these sequences and finding the organs and developmental stages they are active in is a labor-intensive process. Recent advances in adeno-associated virus (AAV) vectors have enabled the widespread delivery of transgenes to mouse tissues, enabling in vivo enhancer testing without necessitating a transgenic animal. This protocol shows how a reporter construct that expresses EGFP under the control of a minimal promoter, which does not drive significant expression on its own, can be used to study the activity patterns of candidate enhancer sequences in the mouse brain. An AAV-packaged reporter construct is delivered to the mouse brain and incubated for 1-4 weeks, after which the animal is sacrificed, and brain sections are observed under a microscope. EGFP appears in cells in which the tested enhancer is sufficient to initiate gene expression, pinpointing the location and developmental stage in which the enhancer is active in the brain. Standard cloning methods, low-cost AAV packaging, and expanding AAV serotypes and methods for in vivo delivery and standard imaging readout make this an accessible approach for the study of how gene expression is regulated in the brain.

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

This protocol describes a novel rAAV-based transient enhancer-reporter assay. This assay can be used to induce enhancer-driven expression in vivo in the mouse brain.

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific ...

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. Traditional in vitro electrophysiological techniques use electrical stimulation to evoke synaptic transmission. A major drawback of this method is its nonspecific nature; all axons in the region of the stimulating electrode will be activated, making it difficult to attribute an effect to a particular afferent connection. This issue can be overcome by replacing electrical stimulation with optogenetic-based stimulation. We describe a method for combining optogenetics with in vitro patch-clamp recordings. This is a powerful tool for the study of both basal synaptic transmission and synaptic plasticity of precise anatomically defined synaptic connections and is applicable to almost any pathway in the brain. Here, we describe the preparation and handling of a viral vector encoding channelrhodopsin protein for surgical injection into a pre-synaptic region of interest (medial prefrontal cortex) in the rodent brain and making of acute slices of downstream target regions (lateral entorhinal cortex). A detailed procedure for combining patch-clamp recordings with synaptic activation by light stimulation to study short- and long-term synaptic plasticity is also presented. We discuss examples of experiments that achieve pathway- and cell-specificity by combining optogenetics and Cre-dependent cell labeling. Finally, histological confirmation of the pre-synaptic region of interest is described along with biocytin labeling of the post-synaptic cell, to allow further identification of the precise location and cell type.

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. ...

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

Summary

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Chapters in this video

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

Optogenetic Functional MRI

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Injections of AAV Vectors for Optogenetics in Anesthetized and Awake Behaving Non-Human Primate Brain

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex