Name: large volume, behaviorally-relevant illumination for optogenetics in non-human primates
Uploaded: 2017-10-03T15:00:00
Description: Watch this Scientific Journal Video about Large Volume, Behaviorally-relevant Illumination for Optogenetics in Non-human Primates at JoVE.com

LCA acknowledges funding from an NDSEG fellowship, the NSF GRFP, and the Friends of the McGovern Institute. EP acknowledges funding the Harry and Eunice Nohara UROP Fund, the MIT Class of 1995 UROP Fund, and the MIT UROP Fund. ESB acknowledges funding from NIH 2R44NS070453-03A1, the IET Harvey Prize, and the New York Stem Cell Foundation-Robertson Award. RD acknowledges funding from NIH EY017292. Michael Williams helped the team to organize and gather supplies prior to filming.

Note: All animal procedures were in accordance with the NIH guidelines and were approved by the Massachusetts Institute of Technology Committee on Animal Care.
1. Illuminator Fabrication
<ol>
	<li>Use a pair of sharp shears to cut a section of 250 &#181;m diameter plastic optical fiber that is at least 10 cm longer than the desired total illuminator length.</li>
	<li>Remove 15 - 20 cm of polyethylene jacket from one end of the plastic optical fiber using a 22 gauge wire stripper.</li>
	<li>S...

<ol>
	<li>Herculano-Houzel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+brain+in+numbers:+a+linearly+scaled-up+primate+brain.">The human brain in numbers: a linearly scaled-up primate brain.</a> Front Hum Neurosci. 3, 31 (2009).</li><li>Tamura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+glass-coated+tungsten+microelectrode+enclosing+optical+fibers+for+optogenetic+exploration+in+primate+deep+brain+structures.">A glass-coated tungsten microelectrode enclosing optical fibers for optogenetic exploration in primate deep brain structures.</a> J Neurosci Meth. 211 (1), 49-57 (2012).</li><li>Diester, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optogenetic+toolbox+designed+for+primates.">An optogenetic toolbox designed for primates.</a> Nat Neurosci. 14 (3), 387-397 (2011).</li><li>Dai, J., Brooks, D. I., Sheinberg, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+and+Electrical+Microstimulation+Systematically+Bias+Visuospatial+Choice+in+Primates.">Optogenetic and Electrical Microstimulation Systematically Bias Visuospatial Choice in Primates.</a> Curr biol. 24 (1), 63-69 (2014).</li><li>Ozden, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+coaxial+optrode+as+multifunction+write-read+probe+for+optogenetic+studies+in+non-human+primates.">A coaxial optrode as multifunction write-read probe for optogenetic studies in non-human primates.</a> J Neurosci Meth. 219 (1), 142-154 (2013).</li><li>Ruiz, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics+through+windows+on+the+brain+in+the+nonhuman+primate.">Optogenetics through windows on the brain in the nonhuman primate.</a> J Neurophysiol. 110 (6), 1455-1467 (2013).</li><li>Chuong, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+optical+inhibition+with+a+red-shifted+microbial+rhodopsin.">Noninvasive optical inhibition with a red-shifted microbial rhodopsin.</a> Nat Neurosci. 17 (8), 1123-1129 (2014).</li><li>Acker, L., Pino, E. N., Boyden, E. S., Desimone, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FEF+inactivation+with+improved+optogenetic+methods.">FEF inactivation with improved optogenetic methods.</a> Proc Natl Acad Sci U S A. 113 (46), 7297-7306 (2016).</li><li>Jazayeri, M., Lindbloom-Brown, Z., Horwitz, 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=Saccadic+eye+movements+evoked+by+optogenetic+activation+of+primate+V1.">Saccadic eye movements evoked by optogenetic activation of primate V1.</a> Nat Neurosci. 15 (10), 1368-1370 (2012).</li><li>Gerits, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetically+Induced+Behavioral+and+Functional+Network+Changes+in+Primates.">Optogenetically Induced Behavioral and Functional Network Changes in Primates.</a> Curr Biol. 22 (18), 1722-1726 (2012).</li><li>Ohayon, S., Grimaldi, P., Schweers, N., Tsao, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccade+modulation+by+optical+and+electrical+stimulation+in+the+macaque+frontal+eye+field.">Saccade modulation by optical and electrical stimulation in the macaque frontal eye field.</a> J Neurosci. 33 (42), 16684-16697 (2013).</li><li>Cavanaugh, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+inactivation+modifies+monkey+visuomotor+behavior.">Optogenetic inactivation modifies monkey visuomotor behavior.</a> Neuron. 76 (5), 901-907 (2012).</li></ol>

While optogenetic tools are widely used to study disease and physiology in rodents, the technical challenge of illuminating large brain volumes has limited the use of optogenetics in non-human primates. Pioneering studies in monkeys used large light power densities (~100 mW/mm2 to 20 W/mm2) to illuminate small volumes, perhaps &#60; 1 mm3, and reported modest behavioral effects with excitatory opsins in the cortex4,9,<s...

Optogenetic tools, which allow for millisecond-precise, light-driven neuronal control are widely used to study functional physiology and behavior in rodents and invertebrates. However, technical challenges have limited the use of optogenetics in the non-human primate brain, which has a volume ~100x larger than the rodent brain 1.
To facilitate optogenetics studies in non-human primates, an illuminator was designed to address two competing goals: large volume illumination and minimal penetration damage. Previous attempts to address one of these concerns have come at the expensive of the other. Bundles of fibers illuminate larger volumes but with increased diameter, and, thus, damage2,3. Tapered glass fibers reduce penetration damage, but narrowly focus light to light emitting surface areas &#60;100 &#181;m2 4,5. External brain illumination through a window in the dura circumvents the challenge of penetration damage and may allow for large volume illumination, but it can only be used for a few superficial brain areas6.
To create a large-volume, small diameter illuminator (Figure 1a), the tip of a plastic optical fiber is heat tapered and the core and cladding are etched (Figure 1b,c). Unlike other tapered fibers that focus light to a narrow point, the etching allows light to escape evenly out the sides of the tip, thus, distributing light broadly over a large area (Figure 1d,e). Because penetration damage is proportional to penetration diameter, this illuminator has no more penetration damage than a conventional fiber, yet it has &#62;100x the light emitting surface area and delivers light more broadly with 1/100th the light power density in a brain phantom (1.75% agarose) (Figure 1e). A Monte Carlo model (Figure 1f) illustrates the difference in light spread between a conventional fiber and the large volume illuminator when they have equal light power densities as their light emitting surfaces. Each illuminator is individually calibrated using an integrating sphere (Figure 2a,b) to ensure even light distribution along the tip (Figure 2c).
This large volume illuminator has been validated with optogenetic manipulation of both behavior and neuronal firing in non-human primates. The fiber tip length may be customized to any brain area and to each animal&#8217;s individual receptive field map. The illuminator may be paired with a penetrating electrode for neuronal recordings that span the length of illumination. Further, because the fiber can carry any color of visible light, it can be paired with any of the available optogenetic molecules available.

<table border="0" cellpadding="0" cellspacing="0" width="853">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:403px;">Plastic optical fiber</td>
			<td style="width:323px;">Industrial fiber optics</td>
			<td style="width:64px;">SK-10</td>
			<td style="width:64px;">250 micron diameter, Super Eska line</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:403px;">Wire stripper</td>
			<td style="width:323px;">Klein Tools</td>
			<td align="right" style="width:64px;">11047</td>
			<td>22 gauge</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:403px;">Vise Clamp</td>
			<td style="width:323px;">Wilton</td>
			<td align="right" style="width:64px;">11104</td>
			<td>Generic table mount vice clamp</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:403px;">Dual temperature heat gun</td>
			<td style="width:323px;">Milwaukee</td>
			<td style="width:64px;">8975-6</td>
			<td>570 / 1000&#176;F</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:403px;">Lab marker</td>
			<td style="width:323px;">VWR</td>
			<td align="right" style="width:64px;">52877</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:403px;">Dissection microscope</td>
			<td style="width:323px;">VistaVision</td>
			<td style="width:64px;">82027-156</td>
			<td>Stereo microscope w/ dual incandescent light, 2x/4x magnification, available from VWR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:403px;">Lab tape</td>
			<td style="width:323px;">VWR</td>
			<td style="width:64px;">89097-972</td>
			<td>4 pack of violet color; however, tape color does not matter</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:403px;">Silicon carbide lapping sheet</td>
			<td style="width:323px;">ThorLabs</td>
			<td style="width:64px;">LF5P</td>
			<td>5 micron grit, 10 pack</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:403px;">Aluminum oxide lapping sheet</td>
			<td style="width:323px;">ThorLabs</td>
			<td style="width:64px;">LF3P</td>
			<td>3 micron grit, 10 pack</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:403px;">Aluminum oxide lapping&#160; sheet</td>
			<td style="width:323px;">ThorLabs</td>
			<td style="width:64px;">LF1P</td>
			<td>1 micron grit, 10 pack</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:403px;">Calcined alumina lapping sheet</td>
			<td style="width:323px;">ThorLabs</td>
			<td style="width:64px;">LF03P</td>
			<td>0.3 micron grit, 10 pack</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:403px;">Hot knife</td>
			<td style="width:323px;">Industrial fiber optics</td>
			<td style="width:64px;">IF370012</td>
			<td>60 Watt, heavy duty</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:403px;">Fiber inspection scope</td>
			<td style="width:323px;">ThorLabs</td>
			<td style="width:64px;">FS201</td>
			<td>optional</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:403px;">Stainless Steel Ferrule</td>
			<td style="width:323px;">Precision fiber optics</td>
			<td style="width:64px;">MM-FER2003SS-265</td>
			<td>265 micron inner diameter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:403px;">1 mL syringe</td>
			<td style="width:323px;">BD</td>
			<td style="width:64px;">14-823-30</td>
			<td>Luer-lok tip is preferable to reduce risk of leakage, but not strictly needed</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:403px;">Plastic epoxy</td>
			<td style="width:323px;">Industrial fiber optics</td>
			<td>40 0005</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:403px;">18 gauge blunt needle</td>
			<td style="width:323px;">BD</td>
			<td align="right" style="width:64px;">305180</td>
			<td>1.5 inch length</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:403px;">Lint-free wipe (KimWipe)</td>
			<td style="width:323px;">ThorLabs</td>
			<td>KW32</td>
			<td>available from many vendors</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:403px;">Light absorbing foil</td>
			<td style="width:323px;">ThorLabs</td>
			<td style="width:64px;">BKF12</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:403px;">Electrical tape</td>
			<td style="width:323px;">3M</td>
			<td style="width:64px;">Temflex 1700</td>
			<td>Optional, may substitute other brands / models</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:403px;">26 gauge sharp needle&#160;</td>
			<td style="width:323px;">BD</td>
			<td align="right" style="width:64px;">305111</td>
			<td>0.5 inch length</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:403px;">Micromanipulator</td>
			<td style="width:323px;">Siskiyou</td>
			<td style="width:64px;">70750000E</td>
			<td>may substitute other brands/models</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:403px;">Steretactic arm</td>
			<td style="width:323px;">Kopf</td>
			<td align="right" style="width:64px;">1460</td>
			<td>may substitute other brands/models</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:403px;">Laser safety goggles</td>
			<td style="width:323px;">KenTeK</td>
			<td>KCM-6012</td>
			<td>must be selected based on the color of laser used, example given here</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:403px;">Laser or other light source</td>
			<td style="width:323px;">vortran</td>
			<td style="width:64px;">Stradus 473-50</td>
			<td>example of blue laser</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:403px;">Integrating sphere</td>
			<td style="width:323px;">ThorLabs</td>
			<td>S142C</td>
			<td>Attached power meter, also available from ThorLabs, item #PM100D</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:403px;">Ultem recording chamber</td>
			<td style="width:323px;">Crist instrument company</td>
			<td style="width:64px;">6-ICO-J0</td>
			<td>Customized with alignment notch</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:403px;">Tower microdrive with clamps</td>
			<td style="width:323px;">NAN</td>
			<td style="width:64px;">DRTBL-CMS</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:403px;">Guide tube</td>
			<td style="width:323px;">Custom</td>
			<td style="width:64px;">N/A</td>
			<td>Made from 25 gauge spinal needle (BD) or blunt tubing</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:403px;">NAN driver system</td>
			<td style="width:323px;">NAN</td>
			<td style="width:64px;">NANDrive</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:403px;">Custom grid design</td>
			<td style="width:323px;">custom</td>
			<td style="width:64px;">custom</td>
			<td>plans available upon request</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:403px;">Blunt forceps</td>
			<td style="width:323px;">FischerScientific</td>
			<td style="width:64px;">08-875-8A</td>
			<td>generic stainless steel blunt forceps</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:403px;">Digital calipers</td>
			<td style="width:323px;">Neiko</td>
			<td style="width:64px;">01407A</td>
			<td>available on amazon.com. May select a finer resolution caliper for more precise measurements.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:403px;">Patch cable</td>
			<td style="width:323px;">ThorLabs</td>
			<td style="width:64px;">FG200LCC-custom</td>
			<td>This is one example of many possible patch cables. As long as the fiber diameter is less than or equal to the fiber diameter of the large volume illuminator and as long as the connectors interface, any patch cable (glass or plastic, vendor purchased or made in the lab) is fine for this application.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:403px;">Clear plastic dust caps</td>
			<td style="width:323px;">ThorLabs</td>
			<td style="width:64px;">CAPF</td>
			<td>Package of 25</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:403px;">ceramic split mating sleeve</td>
			<td style="width:323px;">Precision Fiber Products, Inc.</td>
			<td style="width:64px;">SM-CS1140S</td>
			<td></td>
		</tr>
	</tbody>
</table>

large volume, behaviorally-relevant illumination for optogenetics in non-human primates

This protocol describes a large-volume illuminator, which was developed for optogenetic manipulations in the non-human primate brain. The illuminator is a modified plastic optical fiber with etched tip, such that the light emitting surface area is &#62; 100x that of a conventional fiber. In addition to describing the construction of the large-volume illuminator, this protocol details the quality-control calibration used to ensure even light distribution. Further, this protocol describes techniques for inserting and removing the large volume illuminator. Both superficial and deep structures may be illuminated. This large volume illuminator does not need to be physically coupled to an electrode, and because the illuminator is made of plastic, not glass, it will simply bend in circumstances when traditional optical fibers would shatter. Because this illuminator delivers light over behaviorally-relevant tissue volumes (&#8776; 10 mm3) with no greater penetration damage than a conventional optical fiber, it facilitates behavioral studies using optogenetics in non-human primates.

The illumination of large brain volumes in non-human primates allows for behaviorally-relevant optogenetic manipulation. Acker et al. (2016) used this large volume illuminator with the red-shifted Halorhodopsin, Jaws 7 to study the temporal contribution of the frontal eye field (FEF) to memory-guided saccades in two rhesus monkeys. Specifically, FEF neurons were injected with a viral vector containing Jaws and then illuminated with red-light using the large volume illuminator during eithe...

Watch this Scientific Journal Video about Large Volume, Behaviorally-relevant Illumination for Optogenetics in Non-human Primates at JoVE.com

Large Volume, Behaviorally-relevant Illumination for Optogenetics in Non-human Primates

A protocol to build a tissue penetrating illuminator for delivering light over large volumes with minimal diameter is presented.

This protocol describes a large-volume illuminator, which was developed for optogenetic manipulations in the non-human primate brain. The illuminator is a ...

The overall goal of this protocol is to create a large volume tissue illuminator that is well suited to optogenetic manipulations in non human primates. This method can help answer key questions in the field of neuroscience, such as how do populations of neurons in the different brain regions contribute to behavior, and how can temporally specific neuronal firing patterns in one region impact another region? The main advantage of this technique is that it uses light, and thus allows optogenetic manipulation of large behaviorally relevant brain volumes with minimal tissue damage.The implications of this technique extend toward translational medicine because of the similarities between the human and non-human primate brains. Generally, individuals new to this method will struggle because it takes some practice to apply the proper amount of pressure when pulling these fibers. To begin this procedure, use a pair of sharp shears to cut a section of 250 micrometer diameter plastic optical fiber that is at least 10 centimeters longer than the desired total illuminator length.Next, remove 15 to 20 centimeter of polyethylene jacket from one end of the plastic optical fiber using a 22-gauge wire stripper. Then, secure the distal-most three to five centimeters of the stripped end of the fiber in a table vice clamp. Hold the jacketed end of the fiber taut in one hand, with a constant steady pull.The fiber should be parallel to the floor, pulled away from the vice. Maintain this constant tension on the fiber throughout heating and cooling, so that the fiber remains straight and taut as it thins. Using the lowest setting of a dual temperature heat gun, heat the stripped section of the fiber until it is thinned to a diameter of 60 to 100 micrometers, or about the diameter of a human hair.Maintain the constant tension on the fiber while allowing it to cool. Failure to maintain tension or overheating the fiber may cause the fiber to curl and or break. To help maintain constant tension on the fiber as you heat it, use your non-dominant hand so you don't have to let go when you're using the heat gun.Also, you can gently wrap the fiber around your palm to get an even better grip. Once the fiber has fully cooled, and the desired diameter has been achieved, pinch the fiber three centimeters from the narrowest point on both sides. With a quick sharp pull along the axis of the fiber, separate the sides to create a tapered tip.Then, examine the tapered tip in low power under a dissection microscope. If the tip is forked or curled, discard the fiber. Even with a lot of practice, about a third of fibers will have forked or curled tips.We just discard those fibers rather than trying to repair them, since it's so quick and inexpensive to pull another one. Prepare to etch the tapered tip by placing a piece of lab tape near the end of the tip, leaving the desired tip length exposed. Here the last five millimeters are exposed.The tape protects the fiber from etching above the desired length of light emission. After that, fold a small square of five micrometer silicon carbide lapping sheet over between the thumb and forefinger. Place the tip of the fiber between the two sides of the lapping sheet, and gently etch the fiber using small circular motions.Frequently rotate the fiber so that all sides are etched evenly. Next, remove about five millimeters of the polyethylene jacket from the opposite end of the illuminator using a 22-gauge wire stripper. Then, cut the opposite end flat with a hot knife to smooth the surface.Affix a connector on the end of the illuminator opposite to the etched tip. This allows the illuminator to connect to an optical cable or laser. Afterward, polish the opposite end flat with successively finer lapping sheets.Subsequently, insert the flat opposite end into a 260 micrometer inner diameter stainless steel ferrule until it is flush with the end of the ferrule. Visually confirm that the opposite end is smoothly and evenly polished with a fiber microscope. Tape the ferrule vertically on the edge of the table with the smaller opening of the ferrule pointing down.Then, fill a one milliliter syringe with plastic epoxy and attach a blunt 18-gauge needle to the syringe. Following this, use the needle to apply epoxy down into the ferrule. Fill the ferrule completely with epoxy.Insert fiber into the ferrule so that it is flush with the bottom of the ferrule. If needed, wipe any excess epoxy from the polished surface of the fiber with a wet lint-free wipe prior to drying. In this step, prepare a tower microdrive prior to each testing session.Then affix a 25-gauge guide tube with a beveled tip to the lower clamp on the drive. Secure the large volume illuminator in the upper clamp on the same drive. This clamp is attached to the drive motor.Afterward, thread the large volume illuminator through the guide tube fully, and confirm that the tip of the illuminator extends past the tip of the guide tube. Alternately, lab tape may be applied to the fiber so that the tape, rather than the fiber itself, is clamped, to protect the fiber. Next, soak the guide tube and illuminator in an antiseptic solution for sterilization.Place a custom sterilized grid with one millimeter hole spacing inside the clean chamber, and secure it at a prespecified orientation with the screw on the side of the chamber. Retract the sterilized illuminator from the guide tube by pulling the illuminator up using sterile blunt forceps. The tip of the illuminator should be five to 10 millimeters above the tip of the guide tube.Affix the microdrive to the holder and place the guide tube into the target grid hole. Then, lower the microdrive stage until the guide tube is just through the level of the dura. At the same time, lower the illuminator manually with sterile forceps.If there is any resistance, retract the illuminator five to 10 millimeters, wait 10 minutes, and try again. If there is still resistance on the second attempt, retract the illuminator tip into the guide tube, lower the guide tube 0.25 millimeters, and try again. Connect the ferrule on the other end of the illuminator to the larger optic setup or laser.This figure shows that the error rates increased significantly with illumination. And the raster plots here show the inhibition of neurons spanning the 2.5 millimeter thickness of cortex. And the local field potential showing the light artifact spanning 4.5 millimeters.Once mastered, a large volume illuminator can be constructed using this technique in 10 to 15 minutes, plus epoxy drying time. Calibration adds another half hour or so, so to maximize efficiency, we usually make several illuminators at once. While attending this procedure, it's important to remember that many of the illuminators won't meet quality control standards.That's okay. If they fail tip inspection, we discard them. But if they fail calibration, we usually repolish them and repeat the calibration.After its development, this technique helped pave the way for researchers in the field of neuroscience to use optogenetics to suppress neuronal firing and modulate behavior in non-human primates. After watching this video, you should have a good understanding of how to construct a large volume tissue illuminator.

large-volume-behaviorally-relevant-illumination-for-optogenetics-non

Research

JoVE Journal

Behavior

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Barnes Maze Testing Strategies with Small and Large Rodent Models

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.&#160;bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.&#160;distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.&#160;random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.&#160;shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.

The dry-land Barnes maze is widely used to measure spatial navigation ability in response to mildly aversive stimuli. Over consecutive days, performance (e.g.&#160;latency to locate escape cage) of control subjects improves, indicative of normal learning and memory. Differences between rats and mice necessitate apparatus and methodology changes that are detailed here.

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land ...

Methods to Explore the Influence of Top-down Visual Processes on Motor Behavior

Kinesthetic awareness is important to successfully navigate the environment. When we interact with our daily surroundings, some aspects of movement are deliberately planned, while others spontaneously occur below conscious awareness. The deliberate component of this dichotomy has been studied extensively in several contexts, while the spontaneous component remains largely under-explored. Moreover, how perceptual processes modulate these movement classes is still unclear. In particular, a currently debated issue is whether the visuomotor system is governed by the spatial percept produced by a visual illusion or whether it is not affected by the illusion and is governed instead by the veridical percept. Bistable percepts such as 3D depth inversion illusions (DIIs) provide an excellent context to study such interactions and balance, particularly when used in combination with reach-to-grasp movements. In this study, a methodology is developed that uses a DII to clarify the role of top-down processes on motor action, particularly exploring how reaches toward a target on a DII are affected in both deliberate and spontaneous movement domains.

It is unclear how top-down signals from the ventral visual stream affect movement. We developed a paradigm to test motor behavior towards a target on a 3D depth inversion illusion. Significant differences are reported in both deliberate, goal-directed movements and automatic actions under illusory and veridical viewing conditions.

Kinesthetic awareness is important to successfully navigate the environment. When we interact with our daily surroundings, some aspects of movement are ...

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Assessment of Social Cognition in Non-human Primates Using a Network of Computerized Automated Learning Device (ALDM) Test Systems

Fagot &#38; Paleressompoulle1 and Fagot &#38; Bonte2 have published an automated learning device (ALDM) for the study of cognitive abilities of monkeys maintained in semi-free ranging conditions. Data accumulated during the last five years have consistently demonstrated the efficiency of this protocol to investigate individual/physical cognition in monkeys, and have further shown that this procedure reduces stress level during animal testing3. This paper demonstrates that networks of ALDM can also be used to investigate different facets of social cognition and in-group expressed behaviors in monkeys, and describes three illustrative protocols developed for that purpose. The first study demonstrates how ethological assessments of social behavior and computerized assessments of cognitive performance could be integrated to investigate the effects of socially exhibited moods on the cognitive performance of individuals. The second study shows that batteries of ALDM running in parallel can provide unique information on the influence of the presence of others on task performance. Finally, the last study shows that networks of ALDM test units can also be used to study issues related to social transmission and cultural evolution. Combined together, these three studies demonstrate clearly that ALDM testing is a highly promising experimental tool for bridging the gap in the animal literature between research on individual cognition and research on social cognition.

Assessment of Social Cognition in Non-human Primates Using a Network of Computerized Automated Learning Device ALDM Test Systems

Fagot &#38; Paleressompoulle1 have published an automated learning device (ALDM) aimed at testing individual cognitive abilities in semi-free ranging monkeys. The main goal of our protocol is to use a network of ALDM test units to study social cognition in non-human primates.

Fagot &#38; Paleressompoulle1 and Fagot &#38; Bonte2 have published an automated learning device (ALDM) for the study of cognitive abilities of monkeys ...

The Social Dimension of Stress: Experimental Manipulations of Social Support and Social Identity in the Trier Social Stress Test

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations supportive behavior of other people as well as positive social relationships can act as powerful resources to cope with stress. In order to study the interplay between these variables, this protocol describes two effective experimental manipulations of social relationships and supportive behavior in the laboratory. In the present article, these two manipulations are implemented in the Trier Social Stress Test (TSST)&#8212;a standard stress induction paradigm in which participants are subjected to a simulated job interview. More precisely, we propose (a) a manipulation of the relationship between different protagonists in the TSST by making a shared social identity salient and (b) a manipulation of the behavior of the TSST-selection committee, which acts either supportively or unsupportively. These two experimental manipulations are designed in a modular fashion and can be applied independently of each other but can also be combined. Moreover, these two manipulations can also be integrated into other stress protocols and into other standardized social interactions such as trust games, negotiation tasks, or other group tasks.

Previous research on the social dimension of stress has focused on two important variables: social identity and social support. This protocol introduces an effective experimental manipulation of these two social variables and describes their implementation in a standard stress induction paradigm (Trier Social Stress Test).

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations ...

In Vivo Protocol of Controlled Subconcussive Head Impacts for the Validation of Field Study Data

Subconcussive hits pose a threat to neuronal health as they have shown to induce neuronal structural damage and functional impairment without causing outward symptomology and appear to be a key contributor to an irreversible neurodegenerative disease, chronic traumatic encephalopathy (CTE). In addition, athletes can incur more than 1,000 of these hits per season. The subconcussive soccer heading model (SSHM) is a relevant, reproducible, and leading method of isolating and examining the effects of these subconcussive head impacts. By controlling variables such as ball traveling speed, the frequency of impacts, interval, ball placement to the head, as well as by measuring head impact magnitude, the SSHM provides the scientific community with a superior avenue of investigating the acute subconcussive effects on neuronal health. In this paper, we demonstrate the utility of SSHM in studying a time-course expression of neurofilament-light polypeptide (NF-L) in plasma in a repeated measures fashion. NF-L is an axonal injury marker that has previously been shown to be elevated in boxers and football players following subconcussive head trauma. Thirty-four adult aged soccer players were recruited and randomly assigned to either a soccer heading (n = 18) or kicking (n = 16) group. The heading group executed 10 headers with soccer balls projected at a velocity of 25 mph over 10 min. The kicking group followed the same protocol with 10 kicks. Plasma samples were obtained before and at 0 h, 2 h, and 24 h after heading/kicking and assessed for NF-L expressions. The heading group showed a gradual increase in plasma NF-L expression and peaked at 24 h after the heading protocol, whereas the kicking group remained consistent across the time points. These results confirmed the NF-L data from clinical field studies, encouraging the use of SSHM to validate clinical subconcussion data.

The subconcussive soccer heading model is a safe and concise methodological approach to isolate and measure the effects of subconcussive head impacts.

Subconcussive hits pose a threat to neuronal health as they have shown to induce neuronal structural damage and functional impairment without causing ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

Assessing the Coherence of Parents&#039; Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...

Two Different Real-Time Place Preference Paradigms Using Optogenetics within the Ventral Tegmental Area of the Mouse

Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with behavioral testing in validated paradigms allows the measurement of behavioral consequences upon stimulation of distinct neurons in real-time with high spatial and temporal selectivity, and thus the establishment of causal relationships between neuronal activation and behavior. Here, we describe a step-by-step protocol for a real-time place preference (RT-PP) paradigm, a modified version of the classical conditioned place preference (CPP) test. The RT-PP is performed in a three-compartment apparatus and can be utilized to answer if optogenetic stimulation of a specific neuronal population is rewarding or aversive. We also describe an alternative version of the RT-PP protocol, the so-called neutral compartment preference (NCP) protocol, which can be used to confirm aversion. The two approaches are based on extensions of classical methodology originating from behavioral pharmacology and recent implementation of optogenetics within the neuroscience field. Apart from measuring place preference in real time, these setups can also give information regarding conditioned behavior. We provide easy-to-follow step-by-step protocols alongside examples of our own data and discuss important aspects to consider when applying these types of experiments.

Here we present two easy-to-follow step-by-step protocols for place preference paradigms using optogenetics in mice. Using these two different setups, preference and avoidance behaviors can be solidly assessed within the same apparatus with high spatial and temporal selectivity, and in a straightforward manner.

Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with ...