Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Summary

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

Abstract

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro¹ and in vivo^2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits^4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula^6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.⁸ for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage⁹ using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice¹⁰ with minimal tissue damage.

Protocol

*All materials along with respective manufacturers and/or vendors are listed below the protocol.

1. Assembly of Implant

1. Prepare a mixture of heat-curable fiber optic epoxy by adding 100 mg of hardener to 1 g of resin.
2. Measure and cut approximately 35 mm of 125 μm fiber optic with 100 μm core by scoring it with a wedge-tip carbide scribe. Position the scribe perpendicular to the fiber optic and score in a single, unidirectional motion. Cutting the fiber completely will damage the fiber core.
3. Insert a LC ceramic ferrule with a 127 μm bore into the vice, convex side pointed down.
4. Insert the fiber optic....

Discussion

Optogenetics is a powerful new technique that allows unprecedented control over specific neuronal subtypes. This can be exploited to modulate neural circuits with anatomic and temporal precision, while avoiding the cell-type indiscriminate and invasive effects of electrical stimulation through an electrode. Implantation of fiber optics allows for consistent, chronic stimulation of neural circuits over multiple sessions in awake, behaving mice with minimal damage to tissue. This system, originally pioneered by Sparta .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LC Ferrule Sleeve	Precision Fiber Products (PFP)	SM-CS125S	1.25 mm ID
FC MM Pre-Assembled Connector	PFP	MM-CON2004-2300	230 μm Ferrule
FC MM Pre-Assembled Connector	PFP	MM-CON2004-2300	230 μm Ferrule
Miller FOPD-LC Disc	PFP	M1-80754	For LC ferrules
Furcation tubing	PFP	FF9-250	900 μm o.d., 250 μm i.d.
MM LC Stick Ferrule 1.25 mm	PFP	MM-FER2007C-1270	127 μm ID Bore
MM LC Stick Ferrule 1.25 mm	PFP	MM-FER2007C-2300	230 μm ID Bore
Heat-curable epoxy, hardener and resin	PFP	ET-353ND-16OZ
FC/PC and SC/PC Connector Polishing Disk	ThorLabs	D50-FC	For FC ferrules
Digital optical power and Energy Meter	ThorLabs	PM100D	Spectrophotometer
Polishing Pad	ThorLabs	NRS913	9" x 13" 50 Durometer
Aluminum oxide Lapping (Polishing) Sheets: 0.3, 1, 3, 5 μm grits	ThorLabs	LFG03P, LFG1P, LFG3P, LFG5P
Standard Hard Cladding Multimode Fiber	ThorLabs	BFL37-200	Low OH, 200 μm Core, 0.37 NA
Fiber Stripping Tool	ThorLabs	T10S13	Clad/Coat: 200 μm / 300 μm
SILICA/SILICA Optical Fiber	Polymicro Technologies	FVP100110125	High -OH, UV Enhanced, 0.22 NA
1x1 Fiberoptic Rotary Joint	doric lenses	FRJ_FC-FC
Mono Fiberoptic Patchcord	doric lenses	MFP_200/230/900-0.37_2m_FC-FC
Heat shrink tubing, 1/8 inch	Allied Electronics	689-0267
Heat gun	Allied Electronics	972-6966	250 W; 750-800 °F
Cotton tipped applicators	Puritan Medical Products Company	806-WC
VetBond tissue adhesive	Fischer Scientific	19-027136
Flash denture base acrylic	Yates Motloid	ColdPourPowder+Liq
BONN Miniature Iris Scissors	Integra Miltex	18-1392	3-1/2"(8.9cm), straight, 15 mm blades
Johns Hopkins Bulldog Clamp	Integra Miltex	7-290	1-1/2"(3.8 cm), curved
MEGA-Torque Electric Lab Motor	Vector	EL-S
Panther Burs-Ball #1	Clarkson Laboratory	77.1006
Violet Blue Laser System	CrystaLaser	CK473-050-O	Wavelength: 473 nm
Laser Power Supply	CrystaLaser	CL-2005
Dumont #2 Laminectomy Forceps	Fine Science Tools	11223-20
Probe	Fine Science Tools	10140-02
5"Straight Hemostat	Excelta	35-PH
Vise with weighted base	Altex Electronics	PAN381