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 will damage the fiber core.</li>
	<li>Insert a LC ceramic ferrule with a 127 &mu;m 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 &mu;m 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 &mu;m fiber optic with 200 &mu;m 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 &mu;m 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 &mu;m grit (Figure 2b).</li>
	<li>Strip and insert the other end of the fiber optic into a LC ceramic ferrule (230 &mu;m 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="Figure 1" src="/files/ftp_upload/50004/50004fig1.jpg" /> 
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="Figure 2" src="/files/ftp_upload/50004/50004fig2.jpg" /> 
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="Figure 3" src="/files/ftp_upload/50004/50004fig3.jpg" /> 
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="Figure 4" src="/files/ftp_upload/50004/50004fig4.jpg" /> 
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/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

44.4K Views.  Baylor College of Medicine (BCM). 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.

Video: Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

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

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

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

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.

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...