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 ...

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