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

Резюме

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.

Аннотация

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.

Введение

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 w....

протокол

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

1. Prepare or purchase aqueous silk fibroin (5-7.5% w/v).

2. Mix Aqueous Silk with AAV Expression Vectors

1. Choose an AAV expression vector to drive the optogenetic protein or fluorescent indicator of choice.
   Note: To minimize the v....

Результаты

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 µm diameter) can readily accommodate 200 nL of silk/AAV. With practice, experimenters can achieve highly reliable expression arou.......

Обсуждение

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.......

Материалы

Name	Company	Catalog Number	Comments
Aqueous silk fibroin	Sigma	5154-20ML	Aqueous Silk Fibroin (5% w/v) for making films
Microinjector to deposit silk/AAV	Drummond	3-000-207	Nanoject III nanoliter injector
Manipulator to hold implants	Narashige	MM-33	Micromanipulator
Stereoscope to visualize silk deposits	AmScope	SM-6TX-FRL	3.5X-45X Trinocular articulating zoom microscope with ring light
Vacuum chamber to store implants	Ablaze	N/A	3.5 Quart Vacuum Vac Degassing Chamber
Optional, implant holder for storage	N/A	N/A	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.
Optical fiber	Thorlabs	FT200EMT	Ø200 µm Core Multimode Optical Fiber for fiber implants
Ferrules	Kientec	FZI-LC-230	LC Zirconia Ferrule for fiber implants
Various materials for manufacturing chronic fiber implants	Various	N/A	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).
Tapered fiber implants	Optogenix	Lambda-B	Tapered fiber implants
GRIN lenses	GoFoton	CLH-100-WD002-002-SSI-GF3	GRIN lenses
Small glass cranial windows	Warner	64-0726 (CS-3R-0)	Small round cover glass, #0 thickness
Large glass cranial windows	Warner	64-0731 (CS-5R-0)	Small round cover glass, #0 thickness
Various materials for manufacturing cranial windows	Various	N/A	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.