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1Department of Molecular & Human Genetics, Baylor College of Medicine (BCM), 2Department of Neuroscience, Baylor College of Medicine (BCM), 3Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital
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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.
Ung, K., Arenkiel, B. R. Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue. J. Vis. Exp. (68), e50004, doi:10.3791/50004 (2012).
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 vivo2,3. 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 circuits4-6. 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 cannula6,7; 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.
*All materials along with respective manufacturers and/or vendors are listed below the protocol.
1. Assembly of Implant
2. Assembly of Fiber Optic Coupler Cord
3. Surgical Implantation
*This is a tips-only procedure. Instruments are sterile but gloves do not need to be due to constant manipulation between the instruments and equipment.
Proper assembly of the fiber optic implant and coupler results in minimal photon loss between the light source and the end of the fiber optic in the region of interest. Well-polished fiber optics should transmit light in a uniform, concentric circle (Figure 2d). With careful implantation and suturing, the implant causes no visible irritation to the mouse and can remain in place for long-term studies (Figure 3d, >1 month, unpublished observations) without any significant degradation of the fiber optic or the amount of light transmitted. Improper implantation or suturing can cause irritation and can result in the mouse scratching through its scalp, exposing the dental cement, or breakage of the ferrule from the dental cement due to persistent manipulation. A schematic diagram of the assembled system can be seen in Figure 4.
Figure 1. Assembly of implantable fiber optics. (a) The fiber optic is inserted into the ferrule, marginally protruding beyond the convex end indicated by the arrowhead. (b) The convex end of the ferrule is polished using a FOPD on progressively finer grades of polishing sheets. (c) The finished implantable fiber optic. Click here to view larger figure.
Figure 2. Assembly of fiber optic coupler used to tether the fiber optic rotary joint to the implant. (a) Fiber optic sticking through the ferrule assembly. (b) The ferrule side of the assembly is inserted into the FOPD and polished using progressively finer grades of polishing paper. (c) The ferrule sleeve is fitted over the ferrule and secured with heat shrink tubing. (d) The finished fiber optic coupler should produce a concentric light with minimal photon loss.
Figure 3. Surgical implantation the fiber optics. (a) The entire surface of the cranium is exposed and connective tissue is cleared. (b) The fiber optic implant is held in position with the stereotaxic arm. (c) Dental cement is applied fixing the fiber optic implant to the cranium. (d) >1 month after implantation, the skin has healed around the implant and there are no signs of irritation.
Figure 4. Schematic diagram of the functional system
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 et al.8 and modified to fit our purposes, goes one step beyond the implanted cannula and fixes the fiber optic in place in the region of interest to ensure consistent targeting between sessions in long-term studies. The implants can be adapted to stimulate different regions of the brain.
Various steps within this method require precision and attention to detail. Each junction of fiber optic coupling is necessarily polished to ensure minimal light loss. After polishing, the ends should be examined under a microscope to verify that there is no damage to the fiber core. If light loss between the source and the measured output exceeds 30%, each part should be repolished to allow maximum photon flux or the part should be discarded and remade. If the ferrule does not slide into the sleeve, there is likely debris inside the sleeve obstructing the ferrule. When attaching and removing the coupler cord to the implant, force should be applied directly parallel to the axis of the implant. Due to the fact that mammalian tissue scatters light heavily and the relatively low energy of blue light, the implant should be positioned such that the tip of the fiber is within 500 μm of the region of interest, where >10% of initial light power density persists6. During implantation, the base layer of dental cement is the critical step, as it is this layer that fixes the implant to the cranium. The subsequent layers secure the implant to the base layer and provide protection. The base layer will not adhere well if the cranium is not completely dry; if any section is not adhered well, it is likely that manipulation from the mouse will dislodge the entire implant. Alternatively, anchors for the dental cement can be screwed into the cranium for a more secure fixture.
In behavioral studies, external light leak may provide an unintended cue to the mouse. External light leak is most likely to occur at the connection between the implant and coupler cord directly over the mouse. In order to minimize light leakage, the heat-shrink tubing can be further extended such that it completely covers the ferrule sleeve to provide extra shielding against leakage. If this option is pursued, the heat-shrink tubing will cover the window in the sleeve that provides visual feedback for direct contact between ferrules and contact should be determined with tactile feedback.
Towards further development of this technique, it is possible to implant multiple fiber optics onto a single mouse using additional stereotaxic arms, as described in Sparta et al8. This would enable more complex studies through differential wavelength stimulation in the same region in a temporally specific manner or simultaneous stimulation of different regions. Additionally, fiber optics can be coupled with electrodes (optrode) for in vivo electrophysiology for local stimulation and recording.
We would like to acknowledge that this technique was originally described by Sparta et al., 2012 and has been easily adapted for use in our lab.
|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|
|Vise with weighted base||Altex Electronics||PAN381|
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