Name: Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue (Video) | JoVE Journal
Uploaded: 2012-10-29T13:00:00.000+00:00
Duration: 10 min 18 s
Description: Learn fiber-optic implantation for chronic optogenetic stimulation of neural circuits in awake, behaving mice. This method minimizes tissue damage while enabling precise light delivery to study brain function.

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5 minutes

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.

*All materials along with respective manufacturers and/or vendors are listed below the protocol.
1. Assembly of Implant
<ol>
	<li>Prepare a mixture of heat-curable fiber optic epoxy by adding 100 mg of hardener to 1 g of resin.</li>
	<li>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.</li>
	<li>Insert a LC ceramic ferrule with a 127 μm bore into the vice, convex side pointed down.</li>
	<li>Insert the fiber optic into the ferrule. The fiber optic should slide in smoothly and marginally protrude beyond the convex end of the ferrule (Figure 1a).</li>
	<li>Apply one drop of heat-curable fiber optic epoxy to the flat end and heat with heat gun until epoxy turns black. The epoxy should fill the ferrule as it is heated and before curing. The epoxy should cure within ~1 min of constant heat application.</li>
	<li>Clean off any epoxy along the sides of the ferrule, as it will obstruct interfacing with the coupler.</li>
	<li>Polish the convex end of the ferrule using a LC fiber optic polishing disc (FOPD) on aluminum oxide polishing sheets on the polishing pad (Figure 1b). Make circular rotation patterns and polish on four grades in the following order: 5, 3, 1 0.3 μm grit.</li>
	<li>Cut the fiber optic at the flat end to the appropriate length such that it targets the region of interest. The length can be determined using the stereotaxic atlas.</li>
	<li>Test the implant by connecting it to the laser via the coupler cord described below. The polished end of the implant is inserted into sleeve of the coupler and should make direct contact with the opposing ferrule. The implant should be able to maintain 10 mW of light output, measured at the tip of the implant fiber. A bad implant will have a weak focal point near the tip of the fiber optic.</li>
	<li>Store the finished implants (Figure 1c) in foam until use.</li>
</ol>
2. Assembly of Fiber Optic Coupler Cord
<ol>
	<li>Prepare a mixture of heat-curable fiber optic epoxy as above.</li>
	<li>Measure and cut an appropriate length of 220 μm fiber optic with 200 μm core by scoring it with a wedge-tip carbide scribe. The length of the fiber should allow the mouse to move freely around the housing but not allow the mouse to chew through the fiber.</li>
	<li>Insert the fiber optic into a length of furcation tubing slightly longer than the fiber optic length. The tubing should have an inner diameter slightly larger than the fiber optic.</li>
	<li>Strip ~25mm at one end of the fiber optic and insert it into the metal end of a Multimode FC MM Ferrule Assembly with 230 μm bore until it stops. The fiber optic should stick out through the ferrule end (Figure 2a).</li>
	<li>Secure the connection with cyanoacrylate (super glue) at the metal end. Cover the connection with a Connector Boot and polish the ferrule end with a FC FOPD. Make circular rotation patterns and polish on four grades in the following order: 5, 3, 1, 0.3 μm grit (Figure 2b).</li>
	<li>Strip and insert the other end of the fiber optic into a LC ceramic ferrule (230 μm i.d. bore) with the convex end distal. Apply a drop of epoxy to the flat end and heat until cured.</li>
	<li>Polish the convex end of the ferrule using a FC FOPD on aluminum oxide polishing sheets as described above.</li>
	<li>Slide a LC ferrule sleeve over the convex end of the ferrule until the midpoint of the sleeve.</li>
	<li>Place heat-shrink tubing over the furcation tubing and sleeve and heat to secure and protect the connection (Figure 2c).</li>
	<li>Test the coupler by connecting it to the laser source and measuring the light output through the coupler with a spectrophotometer. The light loss between the laser output and the measured coupler output should not exceed 30%.</li>
</ol>
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.
<ol>
	<li>Anesthetize the mouse with an intraperitoneal injection Ketamine/Xylazine mixture 100 and 10 mg/kg respectively using a 30-gauge needle.</li>
	<li>Shave the scalp with clippers. Wipe the scalp with 70% isopropyl alcohol followed by Betasept wipe (4% Chlorhexidine solution) for two min, repeating once.</li>
	<li>Place the mouse in the sterotaxic rig and secure the head, ensuring that the skull is level. Apply ophthalmic ointment to eyes to prevent dryness and postoperative pain. Maintain anesthesia using volatized isoflurane (1-3% diluted with oxygen depending on the physiological state of the mouse, which should be continuously monitored by response to a tail pinch).</li>
	<li>Make an incision through the midline of the scalp, exposing the cranium from the eye orbits to lambda. Push aside connective tissue as necessary.</li>
	<li>Use Serafin clamps to hold back the skin and maintain an access to the cranium (Figure 3a).</li>
	<li>Etch a checkered pattern throughout the surface of the cranium with a dental pick. Wash debris away with sterile saline. Dry thoroughly.</li>
	<li>Apply hydrogen peroxide (3%) to the exposed cranium with a cotton swab for ~2-3 sec to create micropores. Wash multiple times and dry thoroughly. Alternatively, anchors can be screwed into the cranium, as described in Sparta et al. (2012).</li>
	<li>Again, etch a checkered pattern throughout the cranium with a dental pick and wash away debris with saline. Dry thoroughly.</li>
	<li>Using a rotary tool, make a small burr hole craniotomy (&lt;1 mm in diameter) with a sterile drill bit (autoclaved) above the region of interest, determined by the stereotaxic atlas calibrated to bregma and lambda. Be careful not to break the dura or damage any tissue. Wash away debris and dry thoroughly.</li>
	<li>Insert the fiber optic ferrule (implant) into the probe holder and connect to the stereotaxic arm.</li>
	<li>Position the implant in place directly above the region of interest using the stereotaxic arm (Figure 3b). If inserting the optical fiber in the brain tissue, the fiber should be advanced slowly at a rate of ~2 mm/min. The ferrule should rest on the remaining cranium.</li>
	<li>Prepare a mixture of dental cement. The mixture should have a low enough viscosity to easily apply across the cranium. Mixture will be usable for 2-4 min.</li>
	<li>Using a sterile toothpick, apply a thin, even layer of dental cement across the cranium and onto the lower portion of the implant. The base layer of dental cement should cover as much surface area on the cranium as possible. Do not let the dental cement come into contact with the skin of the mouse. This will lead to increased difficulty in suturing as well as irritation to the mouse. If the dental cement does come into contact with the skin, allow it to partially dry such that the entire layer of cement can be peeled off before setting.</li>
	<li>Allow it to dry completely.</li>
	<li>Apply even layers of dental cement to form a small mound over the cranium and around the implant, allowing each layer to dry completely (Figure 3c). Leave ~3-5 mm of the convex end of the ferrule clean of cement to allow for a smooth, unobstructed connection.</li>
	<li>Suture the scalp over the mound of dental cement and around the implant using sterile, single-use silk braided sutures (6-0) with a C22 needle. Remove after 7 days. Optional: Use Vet Bond for additional binding after suturing. Be sure to use minimal Vet Bond. Excess can lead to severe skin damage due to scratching.</li>
	<li>Immediately after surgery, mouse should be subcutaneously injected with Ketprophen (5 mg/kg) to decrease postoperative pain. This should be repeated 24 hr later. Apply topical analgesics (Bupivicaine) and antibiotics (Neosporin) to the sutured skin and around the base of the implant.</li>
	<li>Place the mouse in a cage over a heating blanket for recovery from anesthesia. Place the mouse in a sterile, postoperative recovery cage. The recovery cage should not contain any bedding in order to maintain temperature and avoid asphyxiation. The mouse can be returned to the original cage or a new cage once awake.</li>
</ol>
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, &gt;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.
<img alt="Fiber optic preparation process; includes ferrule cutting, polishing disc use, and epoxy curing." src="/files/ftp_upload/50004/50004fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
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. <a href="https://www.jove.com/files/ftp_upload/50004/50004fig1large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Fiber optic preparation with connector boot, ferrule polishing, LC sleeve, and concentric light." src="/files/ftp_upload/50004/50004fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
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.
<img alt="Mouse cranial window surgery setup for in vivo brain imaging study; step-by-step procedure shown." src="/files/ftp_upload/50004/50004fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
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) &gt;1 month after implantation, the skin has healed around the implant and there are no signs of irritation.
<img alt="Optogenetics setup with solid-state laser and fiber optic implant in rodent for neural stimulation." src="/files/ftp_upload/50004/50004fig4.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
Figure 4. Schematic diagram of the functional system

<ol><li>Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Millisecond-timescale%2C+genetically+targeted+optical+control+of+neuronal+activity%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Millisecond-timescale, genetically targeted optical control of neuronal activity.</a> Nat Neurosci. 8, 1263-1268 (2005).</li>
<li>Arenkiel, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+Vivo+Light-Induced+Activation+of+Neural+Circuitry+in+Trangenic+Mice+Expressing+Channelrhodopsin-2%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">In Vivo Light-Induced Activation of Neural Circuitry in Trangenic Mice Expressing Channelrhodopsin-2.</a> Neuron. 54, 205-218 (2007).</li>
<li>Gradinaru, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+and+cellular+approaches+for+diversifying+and+extending+optogenetics%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Molecular and cellular approaches for diversifying and extending optogenetics.</a> Cell. 141, 165-16 (2010).</li>
<li>Luo, L., Callaway, E. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+dissection+of+neural+circuits%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Genetic dissection of neural circuits.</a> Neuron. 57, 634-660 (2008).</li>
<li>Arenkiel, B. R., Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+genetic+and+imaging+technologies+for+circuit+based+neuroanatomy%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Molecular genetic and imaging technologies for circuit based neuroanatomy.</a> Nature. 461, 900-907 (2009).</li>
<li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optogenetic+interrogation+of+neural+circuits%3A+technology+for+probing+mammalian+brain+structures%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures.</a> Nat. Protoc. 5, 439-456 (2010).</li>
<li>Adamantidis, A. R., Zhang, F., Aravanis, A. M., Deisseroth, K., de Lecea, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+substrates+of+awakening+probed+with+optogenetic+control+of+hypocretin+neurons%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Neural substrates of awakening probed with optogenetic control of hypocretin neurons.</a> Nature. 450, 420-424 (2007).</li>
<li>Sparta, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Construction+of+implantable+optical+fibers+for+long-term+optogenetic+manipulation+of+neural+circuits%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Construction of implantable optical fibers for long-term optogenetic manipulation of neural circuits.</a> Nature Protocols. 7, 12-23 (2012).</li>
<li>Stuber, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Excitatory+transmission+from+the+amygdala+to+nucleus+accumbens+facilitates+reward+seeking%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Excitatory transmission from the amygdala to nucleus accumbens facilitates reward seeking.</a> Nature. 475, 377-380 (2011).</li>
<li>Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optogenetic+stimulation+of+a+hippocampal+engram+activates+fear+memory+recall%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Optogenetic stimulation of a hippocampal engram activates fear memory recall.</a> Nature. 484, 381-385 (2012).</li>
</ol>

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 ...

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

fiber-optic implantation for chronic optogenetic stimulation of brain tissue

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

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.

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.

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

The aim of the following experiment is to activate and study neuronal circuits long term in awake, behaving mice. This method is adapted from the concept originally described in Sparta etal. This is achieved by first assembling a fiber optic couple cord, which connects the optical rotary joint to the fiber optic implant.Next, the fiber optic is implanted over the brain tissue of interest to create a passage for the light to travel directly to the channel redin expressing neurons. Finally, the light source is connected to the optical rotary joint in order to create a serially connected path from the light source to the brain tissue. Ultimately, light induced firing of channel redsin expressing neurons can be evaluated based on in vivo, electrophysiology, and behavioral studies.The main advantage of this technique over existing methods like guide cannulas is that it minimizes the damage to the brain tissue caused by repeated insertion and removal of an optical fiber. Though this method can provide insight into neural circuit function, it can also be applied to other systems to better understand the synapse and circuit dependent mechanisms that underlies certain types of neurological disorders. Visual demonstration of this method is critical.As the implantation steps are difficult to master, each seemingly minor step contributes greatly to the overall stability and reliability of the method. Begin by cutting 35 millimeters of a 125 micron diameter fiber optic with a 100 micron core by positioning a wedge tip carbide scribe perpendicular to the fiber optic and scoring the fiber in a single unidirectional motion followed by pulling straight out. Now, insert an lc ceramic feral with a 127 micron in a diameter bore convex side pointed down into a vice and then insert the fiber optic piece into the feral.The fiber optic should slide in smoothly and marginally protrude beyond the convex end of the feral. Next apply one drop of freshly prepared heat curable fiber optic epoxy to the flat end of the feral, and then apply a heat gun on the epoxy. The epoxy should fill the feral as it is heated and is cured when it turns black.After curing, clean any epoxy off the sides of the feral, then using an lc fiber optic polishing disc makes circular rotation patterns to polish the convex end of the feral on aluminum oxide Polishing sheets on a polishing pad on four decreasing grades. Then cut the fiber optic at the flat end to the appropriate length for targeting the region of interest. To test the polished fiber optic, insert the polished end of the implant into the sleeve of the coupler.Until direct contact with the opposing feral is felt, a bad implant will have a weak focal point near the tip of the fiber optic, whereas a good implant will transmit a smooth concentric circle of light without output. Powers of up to 10 milliwatts store the finished implants in foam until use. Begin this step by measuring and then scoring an off 220 micron diameter fiber optic with a 200 micron core to allow a mouse to move freely but not allow the mouse to chew the fiber.Then insert the fiber optic into a piece of furation tubing with an inner diameter slightly larger and a length slightly shorter than those of the fiber optic. Now, strip around 25 millimeters from one end of the fiber optic and insert it into the metal end of a multimode FC MM feral assembly. With a 230 micron in a diameter bore until it stops, the fiber optic should stick out through the feral end.Next, secure the connection with cyanoacrylate at the metal end and cover the connection with a connector Boot polish the feral end with an FC polishing disc as just demonstrated. After stripping the other end of the fiber optic, insert it into an lc ceramic feral with just the convex end distal. Then apply a drop of epoxy to the flat end and heat until cured as just demonstrated.After polishing the convex end of the feral as just demonstrated, slide an lc feral sleeve over the convex end of the feral until the feral reaches the midpoint of the sleeve. Then place a heat shrink tubing over the ation tubing and sleeve and heat the heat shrink tube into secure and protect the connection. Finally, connect the feral to the laser source and then test it by measuring the light output through the coupler with a spectrophotometer, the light loss between the laser output and measured coupler output should not exceed 30%before beginning the surgery.Disinfect the surgical area with isop profile alcohol followed by chlorhexidine solution. Next, place the mouse in the stereotaxic rig and secure the head ensuring that the skull is level. Apply ophthalmic ointment to the animal's eyes to prevent dryness and postoperative pain.Now make an incision through the midline of the scalp to expose the cranium from the eye orbits to the lambda suture. Use serafin clamps to hold back the skin and maintain access to the cranium. Then use a dental pick to etch a checkered pattern throughout the surface of the cranium.Wash away the resulting debris with sterile saline. After drying thoroughly use a cotton swab to apply 3%hydrogen peroxide to the exposed cranium from around two to three seconds to create micropores. Then wash the skull multiple times with more sterile saline and dry it thoroughly.After etching, washing and drying the checkerboard pattern into the cranium, again, insert the fiber optic feral implant into the probe holder and then connect it to the stereotaxic arm. Determine the bur hole location with a stereotactic atlas calibrated to bgma and lambda. Then use a rotary dental drill to make a small bur hole craniotomy less than one millimeter in diameter above the region of interest, being careful not to break the dura or damage any tissue.Using the stereotaxic arm position the implant in place directly above the region of interest, the feral should rest on the surrounding cranial tissue. Now use a sterile toothpick to apply a thin even layer of freshly prepared dental cement across the cranium and onto the lower portion of the implant. Being careful not to come into contact with the skin of the animal, the base layer of dental cement should cover as much surface area on the cranium as possible.The most difficult aspect of this procedure is applying the dental cement without it touching the mass skin. To ensure success, apply small amounts of cement at a time in thin layers and wait for each layer to completely dry before adding the next one. Apply even layers of the dental cement to form a small mound on top of the cranium and around the implant.Leave the three to five millimeters of the convex end of the feral clean of cement to allow for a smooth unobstructed connection. Then suture the scalp over the mound of dental cement and around the implant vet bond adhesive can be used for additional binding after suturing. Finally, apply topical analgesics and antibiotics to the suture skin and around the base of the implant, and then place the mouse in a cage for postoperative recovery.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. While polished fiber optics should transmit light in a uniform concentric circle similar to the circle shown here as can be seen in this image with careful implantation and suturing, the implant causes no visible irritation to the mouse and can remain in place for long-term studies without any significant degradation of the fiber optic or to the amount of light transmitted Functional behavioral assays in an awake mouse show a direct response from stimulation through a fiber optic implanted over the motor cortex as evidenced by this in vivo electrophysiology video data showing the activation of targeted neurons through the implant and the behavioral response of the animal during brain stimulation. Following this procedure, other methods like photo stimulation of targeted brain regions can be performed in order to answer additional questions such as what are the patterns of circuit connectivity associated with the target cells?After watching this video, you should have a good understanding of how to assemble fiber optic implants and coupler cords, and how to implant them over brain tissue expressing optogenetic porters.

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JoVE Journal

Neuroscience

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

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.

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

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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.

Optogenetic Functional MRI

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

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Here, we present the fabrication method of an optrode system with optical fibers for light delivery and an electrode array for neural recording. In vivo experiments with transgenic mice expressing channelrhodopsin-2 show the feasibility of the system for simultaneous optogenetic stimulation and electrophysiological recording.

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

This protocol details a surgical procedure for performing spinal cord surgery and for implanting and securing an optical shank over the spinal cord in rodents.

Implantation of Optoelectronic Devices in the Rodent Spinal Cord

We have developed a simplified and cost-effective approach for electrode fabrication and conducted recordings of signals across multiple regions in freely moving mice. Utilizing optogenetics, alongside multi-region electrophysiology and calcium signal recording, enabled the revelation of neuronal activities across regions in the seizure kindling model.

An Integrated Method for Crafting Flexible and Convenient Electrophysiological Optrodes for Multi-Region In Vivo Recording

This protocol provides an integrated method that includes viral delivery, optrode implantation, photothrombotic induction, simultaneous recording of astrocyte and neuronal activity, and optogenetic stimulation of astrocytes. This integrated method aims to provide concurrent investigation of the astrocyte-neuron relationship across the acute-to-chronic stages of ischemic stroke.

An Integrated Method for Photothrombotic Stroke Modeling and In Vivo Optrode Recording of Neuronal and Astrocytic Activity in Behaving Mice

Learn fiber-optic implantation for chronic optogenetic stimulation of neural circuits in awake, behaving mice. This method minimizes tissue damage while enabling precise light delivery to study brain function.

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

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