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 reconoce la financiación de una beca de la NDSEG, la GRFP NSF y los amigos del Instituto McGovern. EP reconoce financiación el Harry y Eunice Nohara UROP fondo, la clase del MIT de 1995 UROP fondo y el fondo de la UROP de MIT. ESB reconoce fondos del NIH 03A1 2R44NS070453 el Premio de Harvey del IET y el Premio de Fundación Robertson de células madre de Nueva York. RD reconoce la financiación de los NIH EY017292. Michael Williams ayudó el equipo para organizar y reunir suministros antes de la filmación.

Nota: todos los procedimientos animales estaban de acuerdo con las directrices de los NIH y fueron aprobados por el Instituto de Massachusetts de tecnología Comité de cuidado Animal.
 1. iluminador fabricación <ol><li>Use un par de tijeras afiladas para cortar un tramo de 250 μm de diámetro fibra óptica de plástico que por lo menos 10 cm de largo mayor que el deseado iluminador total.</li>
	<li>Sacar 15-20 cm de chaqueta de polietileno de un extremo de la fibra óptica de plástico c...

<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>

Mientras que optogenetic herramientas son ampliamente utilizados para el estudio de la enfermedad y la fisiología en roedores, el desafío técnico de iluminar los volúmenes grandes del cerebro limitó el uso de optogenetics en primates no humanos. Pionera en estudios en monos utiliza densidades de energía de luz grande (~ 100 mW/m2 a 20 W/mm2) para iluminar pequeños volúmenes, tal vez &#60; 1 mm3y efectos conductuales modestos reportados con opsins excitatorias de la corteza<sup cla...

Optogenetic herramientas, que permiten un control neuronal exacto milisegundo, impulsado por la luz son ampliamente utilizadas para estudiar la fisiología funcional y comportamiento en roedores e invertebrados. Sin embargo, desafíos técnicos limitó el uso de optogenetics en el cerebro de primates no humanos, que tiene un volumen ~ 100 x más grande que el cerebro de roedores 1.
Para facilitar la optogenetics estudios en primates no humanos, un iluminador fue diseñado para abordar dos objetivos concurrentes: iluminación de gran volumen y penetración mínimo daño. Los intentos anteriores para abordar uno de estos problemas vienen en la cara del otro. Paquetes de fibras se iluminan grandes volúmenes pero con mayor diámetro y, por lo tanto, daños2,3. Fibras de vidrio afilado reducen el daño de la penetración, pero estrecho foco luz a superficies de emisión de luz &#60; 100 μm2 4,5. Iluminación externa del cerebro a través de una ventana en la dura elude el reto de daños de penetración y puede permitir la iluminación de gran volumen, pero sólo puede ser utilizado para algunos superficiales cerebro áreas6.
Para crear un iluminador de gran volumen, de pequeño diámetro (Figura 1a), la punta de un plástico óptico de fibra es cónico y el núcleo y el revestimiento son grabadas (Figura 1bc). A diferencia de otras fibras afiladas que enfocar luz en un punto estrecho, el grabado permite que la luz escape uniformemente hacia fuera los lados de la punta, por lo tanto, distribución de luz ampliamente sobre un área grande (figura 1 de). Debido a daños de penetración son proporcional al diámetro de la penetración, este iluminador no tiene más daño de penetración que una fibra convencional, pero tiene &#62; 100 x la luz emitiendo luz superficie de área y ofrece más ampliamente con 1/100 la potencia de luz densidad en un cerebro fantasma (1.75% agarosa) (Figura 1e). Un modelo Monte Carlo (figura 1f) ilustra la diferencia de propagación de luz entre una fibra convencional y el iluminador de gran volumen cuando tienen densidades de energía de luz igual como su superficies de emisión de luz. Cada iluminador es calibrada individualmente usando una esfera integradora (Figura 2a, b) para asegurar la distribución lumínica a lo largo de la punta (figura 2C).
Este iluminador de gran volumen ha sido validado con la manipulación de optogenetic de comportamiento y de disparo neuronal en primates no humanos. La longitud de la punta de la fibra puede ser adaptada a cualquier área del cerebro y al mapa de campo receptivo individual de cada animal. El iluminador puede ser asociado con un electrodo de penetración para grabaciones neuronales que abarcan la longitud de la iluminación. Además, porque la fibra puede llevar cualquier color de la luz visible, puede ser asociado con cualquiera de las moléculas de optogenetic disponible disponibles.

<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

Este protocolo describe un iluminador de gran volumen, que fue desarrollado para la manipulación de optogenetic en el cerebro de primates no humanos. El iluminador es una modificado fibra óptica de plástico con punta de grabado al agua fuerte, tal que la luz que emite la superficie es &#62; 100 x de una fibra convencional. Además de describir la construcción del iluminador de gran volumen, este protocolo detalla la calibración de control de calidad para asegurar la mejor distribución lumínica. Además, este protocolo describe técnicas para insertar y retirar el iluminador de gran volumen. Estructuras superficiales y profundas pueden ser iluminadas. Este iluminador de gran volumen no necesita estar físicamente junto a un electrodo, y porque la luz está hecha de plástico, no de vidrio, simplemente se dobla en circunstancias cuando destruiría fibras ópticas tradicionales. Porque este iluminador ofrece luz sobre volúmenes de tejido conductualmente relevantes (≈ 10 mm3) sin mayor daño de penetración de una fibra óptica convencional, que facilita estudios de comportamiento usando optogenetics en primates no humanos.

La iluminación de los volúmenes grandes del cerebro en primates no humanos permite la manipulación del comportamiento relevante optogenetic. Acker et al. (2016) utiliza este iluminador de gran volumen con la halorodopsina rojo-cambiado de puesto, las mordazas 7 para estudiar la contribución temporal del campo frontal del ojo (FEF) para Movimientos sacádicos memoria-dirigida en dos monos rhesus. Específicamente, las neuronas de la FEF fueron inyectadas con un vector viral que contien...

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

Se presenta un protocolo para construir un iluminador penetrante de tejido para ofrecer luz sobre grandes volúmenes con diámetro mínimo.

Gran volumen, iluminación conductualmente relevantes para Optogenetics en Primates no humanos

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.

Research

JoVE Journal

Behavior

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

Un procedimiento para estudiar el efecto de la prolongada restricción de alimentos en heroína Buscando en ratas abstinentes

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

Investigación

Comportamiento

Barnes Maze Testing Strategies with Small and Large Rodent Models

Barnes Maze estrategias de ensayo con pequeñas y grandes modelos de roedores

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

Los métodos para explorar la influencia de arriba hacia abajo Procesos Visuales en Comportamiento Motor

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

El descubrimiento de golpe Sordera: Trastornos Detección de ritmo con los dedos sincronizada Tapping y tareas de sincronización de percepción

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

Un procedimiento para observar inducida Contexto-Renovación-pavloviano acondicionado Alcohol comportamiento de búsqueda en ratas

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

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

Evaluación de la cognición social en primates no humanos mediante una red de Computarizado Dispositivo Automatizado Learning Systems (ALDM) prueba

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

La dimensión social del estrés: Manipulaciones experimentales de Apoyo Social e Identidad Social en el Trier Social Estrés Prueba

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

Using Facial Electromyography to Assess Facial Muscle Reactions to Experienced and Observed Affective Touch in Humans

Usando la electromiografía Facial para evaluar las reacciones del músculo Facial tacto afectivo experimentado y observado en los seres humanos

"Affective" touch is believed to be processed in a manner distinct from discriminatory touch and to involve activation of C-tactile (CT) afferent fibers. ...

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

En Vivo protocolo de impactos controlados cabeza sub-concusión para la validación de datos del estudio de campo

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

Evaluar la coherencia de las narrativas cortas de los padres con respecto a su hijo utilizando el procedimiento de muestra de habla de cinco minutos

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