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 &mu;m fiber optic with 100 &mu;m 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 wi...

<ol>
	<li>Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale,+genetically+targeted+optical+control+of+neuronal+activity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Light-Induced+Activation+of+Neural+Circuitry+in+Trangenic+Mice+Expressing+Channelrhodopsin-2.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+approaches+for+diversifying+and+extending+optogenetics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+neural+circuits.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetic+and+imaging+technologies+for+circuit+based+neuroanatomy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+interrogation+of+neural+circuits:+technology+for+probing+mammalian+brain+structures.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+substrates+of+awakening+probed+with+optogenetic+control+of+hypocretin+neurons.">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/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, 12-23 (2012).</li><li>Stuber, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitatory+transmission+from+the+amygdala+to+nucleus+accumbens+facilitates+reward+seeking.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+stimulation+of+a+hippocampal+engram+activates+fear+memory+recall.">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 border="1">
	<tbody>
		<tr>
			<td>Name of the Reagent or Equipment</td>
			<td>Company</td>
			<td>Catalogue #</td>
			<td>Comments</td>
		</tr>
		<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 &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 &mu;m o.d., 250 &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 &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 &mu;m ID Bore</td>
		</tr>
		<tr>
			<td>Heat-curable epoxy, hardener and resin</td>
			<td>PFP</td>
			<td>ET-353ND-16OZ</td>
			<td>&nbsp;</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" x 13" 50 Durometer</td>
		</tr>
		<tr>
			<td>Aluminum oxide Lapping (Polishing) Sheets: 0.3, 1, 3, 5 &mu;m grits</td>
			<td>ThorLabs</td>
			<td>LFG03P, LFG1P, LFG3P, LFG5P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Standard Hard Cladding Multimode Fiber</td>
			<td>ThorLabs</td>
			<td>BFL37-200</td>
			<td>Low OH, 200 &mu;m Core, 0.37 NA</td>
		</tr>
		<tr>
			<td>Fiber Stripping Tool</td>
			<td>ThorLabs</td>
			<td>T10S13</td>
			<td>Clad/Coat: 200 &mu;m / 300 &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>&nbsp;</td>
		</tr>
		<tr>
			<td>Mono Fiberoptic Patchcord</td>
			<td>doric lenses</td>
			<td>MFP_200/230/900-0.37_2m_FC-FC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heat shrink tubing, 1/8 inch</td>
			<td>Allied Electronics</td>
			<td>689-0267</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heat gun</td>
			<td>Allied Electronics</td>
			<td>972-6966</td>
			<td>250 W; 750-800 &deg;F</td>
		</tr>
		<tr>
			<td>Cotton tipped applicators</td>
			<td>Puritan Medical Products Company</td>
			<td>806-WC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VetBond tissue adhesive</td>
			<td>Fischer Scientific</td>
			<td>19-027136</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Flash denture base acrylic</td>
			<td>Yates Motloid</td>
			<td>ColdPourPowder+Liq</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BONN Miniature Iris Scissors</td>
			<td>Integra Miltex</td>
			<td>18-1392</td>
			<td>3-1/2"(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"(3.8 cm), curved</td>
		</tr>
		<tr>
			<td>MEGA-Torque Electric Lab Motor</td>
			<td>Vector</td>
			<td>EL-S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Panther Burs-Ball #1</td>
			<td>Clarkson Laboratory</td>
			<td>77.1006</td>
			<td>&nbsp;</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>&nbsp;</td>
		</tr>
		<tr>
			<td>Dumont #2 Laminectomy Forceps</td>
			<td>Fine Science Tools</td>
			<td>11223-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Probe</td>
			<td>Fine Science Tools</td>
			<td>10140-02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5"Straight Hemostat</td>
			<td>Excelta</td>
			<td>35-PH</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vise with weighted base</td>
			<td>Altex Electronics</td>
			<td>PAN381</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue at JoVE.com

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

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 ...

fiber-optic-implantation-for-chronic-optogenetic-stimulation-brain

Research

JoVE Journal

Neuroscience

44.3K visualizações.  Baylor College of Medicine (BCM). O desenvolvimento de Optogenetics agora fornece os meios para estimular precisamente neurônios geneticamente definidas e circuitos, tanto In vitro E In vivo. Aqui, descrevemos a montagem e implantação de uma fibra óptica para fotoestimulação crônica do tecido cerebral.