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 Vec...

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk+Fibroin+Films+Facilitate+Single-Step+Targeted+Expression+of+Optogenetic+Proteins.">Silk Fibroin Films Facilitate Single-Step Targeted Expression of Optogenetic Proteins.</a> Cell Reports. 22 (12), 3351-3361 (2018).</li><li>Ung, K., Arenkiel, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber-optic+implantation+for+chronic+optogenetic+stimulation+of+brain+tissue.">Fiber-optic implantation for chronic optogenetic stimulation of brain tissue.</a> Journal of Visualized Experiments. (68), e50004 (2012).</li><li>Lowery, R. L., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+injection+of+adeno-associated+viral+vectors.">Intracranial injection of adeno-associated viral vectors.</a> Journal of Visualized Experiments. (45), (2010).</li><li>Ghosh, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+integration+of+a+fluorescence+microscope.">Miniaturized integration of a fluorescence microscope.</a> Nature Methods. 8 (10), 871-878 (2011).</li><li>Cai, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+shared+neural+ensemble+links+distinct+contextual+memories+encoded+close+in+time.">A shared neural ensemble links distinct contextual memories encoded close in time.</a> Nature. 534 (7605), 115-118 (2016).</li><li>Goldey, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removable+cranial+windows+for+long-term+imaging+in+awake+mice.">Removable cranial windows for long-term imaging in awake mice.</a> Nature Protocols. 9 (11), 2515-2538 (2014).</li><li>Sparta, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+implantable+optical+fibers+for+long-term+optogenetic+manipulation+of+neural+circuits.">Construction of implantable optical fibers for long-term optogenetic manipulation of neural circuits.</a> Nature Protocols. 7 (1), 12-23 (2011).</li><li>Resendez, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+cortical,+subcortical+and+deep+brain+neural+circuit+dynamics+during+naturalistic+mammalian+behavior+with+head-mounted+microscopes+and+chronically+implanted+lenses.">Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses.</a> Nature Protocols. 11 (3), 566-597 (2016).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), (2012).</li><li>Park, J. J., Cunningham, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin+sectioning+of+slice+preparations+for+immunohistochemistry.">Thin sectioning of slice preparations for immunohistochemistry.</a> Journal of Visualized Experiments. (3), 194 (2007).</li><li>Cao, Y., Wang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradation+of+silk+biomaterials.">Biodegradation of silk biomaterials.</a> International Journal of Molecular Sciences. 10 (4), 1514-1524 (2009).</li><li>Jackman, S. L., Beneduce, B. M., Drew, I. R., Regehr, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achieving+high-frequency+optical+control+of+synaptic+transmission.">Achieving high-frequency optical control of synaptic transmission.</a> Journal of Neuroscience. 34 (22), 7704-7714 (2014).</li><li>Ortinski, P. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+induction+of+astrocytic+gliosis+generates+deficits+in+neuronal+inhibition.">Selective induction of astrocytic gliosis generates deficits in neuronal inhibition.</a> Nature Neuroscience. 13 (5), 584-591 (2010).</li><li>Hines, D. J., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+controlled+release+from+silk+fibroin+films.">Mechanisms of controlled release from silk fibroin films.</a> Biomacromolecules. 12 (3), 804-812 (2011).</li><li>Hu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+silk+material+structure+by+temperature-controlled+water+vapor+annealing.">Regulation of silk material structure by temperature-controlled water vapor annealing.</a> Biomacromolecules. 12 (5), 1686-1696 (2011).</li><li>Rockwood, D. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Materials+fabrication+from+Bombyx+mori+silk+fibroin.">Materials fabrication from Bombyx mori silk fibroin.</a> Nature Protocols. 6 (10), 1612-1631 (2011).</li><li>Yucel, T., Cebe, P., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vortex-induced+injectable+silk+fibroin+hydrogels.">Vortex-induced injectable silk fibroin hydrogels.</a> Biophysical Journal. 97 (7), 2044-2050 (2009).</li><li>Wang, X., Kluge, J. A., Leisk, G. G., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonication-induced+gelation+of+silk+fibroin+for+cell+encapsulation.">Sonication-induced gelation of silk fibroin for cell encapsulation.</a> Biomaterials. 29 (8), 1054-1064 (2008).</li><li>Lee, J., Park, S. H., Seo, I. H., Lee, K. J., Ryu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+repeatable+fabrication+of+high+A/R+silk+fibroin+microneedles+using+thermally-drawn+micromolds.">Rapid and repeatable fabrication of high A/R silk fibroin microneedles using thermally-drawn micromolds.</a> European Journal of Biopharmaceutics. 94, 11-19 (2015).</li></ol>

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. Th...

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>&#216;200 &#181;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 arou...

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 ...

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.

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

Research

JoVE Journal

Neuroscience

8.4K visualizações.  Oregon Health and Science University. Este método pode ajudar a responder perguntas-chave no campo da neurociência de sistemas. Como a atividade dos conjuntos neuronais suporta o processamento sensorial e cognitivo no cérebro. A principal vantagem desta técnica é que ela facilita o direcionamento preciso de proteínas optogenéticas como a rodopsina chan e GCaMP para regiões de interesse cerebral, sem exigir um procedimento separado de injeção de vírus.A demonstração visual deste método é fundamental, pois a d...