Name: large volume, behaviorally-relevant illumination for optogenetics in non-human primates
Uploaded: 2017-10-03T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Large Volume, Behaviorally-relevant Illumination for Optogenetics in Non-human Primates at JoVE.com

LCA 承认来自 NDSEG 奖学金, NSF GRFP, 和麦戈文研究所的朋友的资助。EP 承认资助哈利和尤尼斯 Nohara UROP 基金, 麻省理工学院的 1995年 UROP 基金, 和麻省理工学院 UROP 基金。ESB 承认来自 NIH 2R44NS070453-03A1、"让哈维奖" 和 "纽约干细胞基金会-罗伯逊奖" 的资助。RD 承认来自 NIH EY017292 的资金。迈克尔. 威廉姆斯在拍摄前帮助团队组织和收集补给品。

注意: 所有动物程序都符合 NIH 的指导方针, 并获得了麻省理工学院动物保育委员会的批准. 
 1. 照明灯制作 <ol> <li> 使用一对锋利的剪刀切割250和 #181 的部分; m 直径塑料光纤, 至少10厘米长于所需总照明长度. </li>
	<li> 从塑料光纤的一端取下 15-20 厘米的聚乙烯护套, 使用22口径的钢丝剥皮器. </li>
	<li> 在表虎钳钳夹中保护纤维的最远端 3-5 厘米的末端. </li>
	<li> 握住纤维的一端紧扣在一只手固定的稳定拉。纤维应平行于地板, 垂直于副。在整个加热和冷却过程中保持纤维的恒定张力 (下面的步骤1.5 和 1.6), 使纤维保持平直和绷紧. </li>
	<li> 使用双温热枪的最低设置 (570/1000 和 #176; F), 将纤维的剥离部分加热到直径为 60-100 和 #956; m, 或关于人的头发的直径. </li>
	<li> 保持纤维上的恒定张力, 同时让纤维冷却。如果不保持张力, 可能会导致纤维卷曲. </li>
	<li> 在解剖显微镜下确认变薄尖端的直径。另外, 你可以使用卡尺代替。
	<ol> <li> 如果光纤不够薄, 请根据需要重复步骤1.4 到1.6 以达到所需的笔尖直径. </li>
		<li> 可选如果 re-pulling, 则在纤维的最窄部分放置一个小标记, 如果它再次加热, 使热枪的中心目标是纤维的最窄部分. </li>
		<li> 如果纤程太薄, 请重新开始. </li>
	</ol> </li> <li> 一旦光纤已完全冷却并且达到了所需的直径, 则从两侧的最窄点将光纤捏3厘米。用一个快速, 尖锐的拉沿轴的纤维, 分离两侧创建一个锥形尖端. </li>
	<li> 在低功耗下检查锥形尖端 ( 例如, 在解剖显微镜下的 , 4X)。如果笔尖是分叉的或卷曲的 ( 图 1f ), 则丢弃光纤. </li>
	<li> 准备蚀刻锥形尖端, 把一条实验室胶带放在尖端的末端, 留下最后5毫米暴露。该胶带保护纤维免受蚀刻超过所需的光发射长度。根据靶区的灵长类皮层厚度, 选择该针尖长度。一个较长或较短的长度可以暴露取决于所需的照明长度。如果需要不同的蚀刻尖端长度, 则可以将实验室磁带的边缘从锥形尖端移到更近或更远的地方. </li>
	<li> 折叠小 (〜1英寸 x 2 英寸) 正方形的5和 #956; m 碳化硅研磨片在拇指和食指之间。将纤维的尖端放在研磨片的两面之间, 用小的圆周运动轻轻地蚀刻纤维, 就像在拇指和食指之间滚动一个 BB。频繁地转动纤维, 以便所有边均匀地被蚀刻。纤维顶端会出现和 #8220; 粗糙和 #8221; 在蚀刻过的区域, un-etched 区域通常看起来平滑. </li>
	<li> 重复步骤1.11 与3和 #956; m 氧化铝研磨板. </li>
	<li> 在与蚀刻尖端相对的照明灯的末端贴上一个连接器。这使得照明器可以连接到光缆或激光器. 
	 注意: 这种方法不同于套圈固定玻璃/二氧化硅光纤的首选方法。由于金属套圈比塑料光纤硬, 因此在胶合后抛光纤维和套圈作为一个单元, 会损坏塑料光纤, 因为在抛光过程中产生的细小金属碎片可以嵌入到平坦的纤维中。
	<ol> <li> 从照明灯的另一端取下大约5毫米的聚乙烯护套, 使用22口径的钢丝剥皮器. </li>
		<li> 用热刀将另一端平切, 以平滑表面. </li>
		<li> 抛光与连续地更细的研磨板的相反末端单位: 5 和 #181; m, 3 和 #956; m, 1 和 #956; m 和0.3 和 #181; m. </li>
		<li> 将平面相对端插入260和 #181; m 内径不锈钢套圈, 直到它与套圈的一端齐平. </li>
		<li> 直观地证实, 相反的一端是平滑和均匀抛光的纤维显微镜. </li>
		<li> 将光纤从套圈中取出, 并将套圈垂直贴在表的边缘, 并将光纤指向下方. </li>
		<li> 用塑料环氧树脂填充1毫升注射器, 并在注射器上附上一个钝18口径的针头. </li>
		<li> 使用针将环氧树脂放入套圈中。完全用环氧树脂填充套圈. </li>
		<li> 将光纤插入插芯. </li>
		<li> 擦除任何多余的环氧树脂从抛光表面的纤维与湿不起毛的擦拭之前, 如果需要干燥。否则, 过量的环氧树脂可以在 12-24 小时后被去除. </li>
		<li> 将纤维轻轻地储存直到校准, 并在套圈上放置一个防尘帽. </li>
	</ol> </li> </ol> 2。照明灯校准和质量控制 注意: 这些校准方法用于评估光纤尖端不同距离的光输出非线性。不均匀的光分布通常起因于 #8220; 颠簸和 #8221; 或 #8220; 波浪 #8221; 锥度. 
<ol> <li> 使用光吸收箔 ( 图 2 ) 创建集成球体顶部的光屏蔽膜片。
	 注意: 在处理轻吸箔时戴上手套, 防止皮肤油脂产生反光的污迹。
	<ol> <li> 折叠2和 #8221; 4 #8221; 一块光吸收箔在本身形成一个2和 #8221; x 2 和 #8221; 正方形和 #160; 另一种方法是切割2和 #8221; x 2 和 #8221; 正方形与一个轻的吸收的边和一个光反射的边 ( 即 , 一面是标准铝箔);然而, 折叠方法是更安全的, 因为它提供了一个沉闷的边缘处理膜片在下一步. 和 #160; </li> <li> 将实验室磁带或磁带沿正方形的边缘折叠以绑定横杆边缘。这两者都保持双方在一起, 防止从尖锐的边缘削减. </li>
		<li> 使用26口径针在广场中心穿刺一个孔. </li>
		<li> 从积分球体上卸下大螺钉盖, 并盖上膜片的开口。把洞放在中间。如果膜片被创造了以一个轻的吸收和一个反射的边, 安置轻的吸收的边和光反射的边, 面对集成球形的里面. 
		 注意: 为了防止灰尘和碎片进入积分球体, 并保持孔的中心, 确保膜片的外部的集成球体与实验室磁带. </li>
	</ol> </li> <li> 在500和 #181 中测量沿尖端的光输出; m 增量使用微或立体定向臂和集成球体. 
	 注意: 激光照射时, 应佩戴合适波长的安全护目镜。
	<ol> <li> 将光纤的另一端连接到激光或光源, 但尚未触发光输出. </li>
		<li> 将照明灯固定在立体定向支架上 (preferably 与实验室磁带) 的尖端 7-10 毫米以下的持有人的底部. </li>
		<li> 将立体定向支架固定在立体定向臂中. </li>
		<li> 将照明灯的尖端与膜片中心的孔对齐。当尖端与横膈膜完全相同的水平时, 微为零. </li>
		<li> 关闭房间指示灯并将积分球零除. </li>
		<li> 触发激光 (或其他光源), 并使用集成球体测量总的光功率输出. 
		 注意: 使用尽可能低的光输出进行校准测试. </li>
		<li> 降低光纤500和 #181; m 进入集成球体并重复步骤 2.2.6. </li>
		<li> 继续将光纤降低到集成球体中, 并在500和 #181 中测量总光输出; m 增量, 直到总的光功率水平关闭. 
		 警告: 当整个尖端在球体中时, 总的光功率应该是水平的。不要继续降低超过 1-2 毫米超过蚀刻尖端长度的纤维。如果尖端接触到积分球体的底部, 它可能熔化和/或破坏积分球体. </li>
	</ol> </li> <li> 通过绘制总的光功率输出来确认一个均匀蚀刻的光纤, 将其作为光纤被降低到集成球体以确认线性度的函数. </li> </ol>
 3. 在体内 光照 
 注意: 在这里, 这些方法使用塑料模型显示, 而不是非人类灵长类. 
<ol> <li> 植入在实验之前, 在感兴趣的大脑区域植入了25毫米直径的记录室. </li>
	<li> 在实验前进行解剖磁共振成像 (MRI)。在室内放置一个自定义的记录网格, 并用无菌的外科润滑剂填满它, 以允许网格可视化和确定硬脑膜和靶脑结构的水平. </li>
	<li> 在每个测试会话之前准备一个塔式微驱动器。
	<ol> <li> 在驱动器上的中间和下部夹具上贴一条带有斜角尖的25口径导向管。使用两个夹具确保导管保持平直。如果需要, 可以手动移动这些夹具. 
		 注意: 导管可环氧树脂或焊接到夹具上. </li>
		<li> 在同一驱动器上的上钳上固定大音量照明器。此夹具连接到驱动马达. </li>
		<li> 将大容量的照明灯通过导管完全穿线, 并确认照明灯的尖端延伸过导管的尖端. 
		 注意: 超过导管尖端的照明灯长度等于从硬脑膜到靶脑区的下边缘的距离, 如确定的 通过 MRI. </li>
		<li> 在防腐溶液中浸泡导管和照明灯 ( 如 、chlorohexidine) 进行灭菌. </li>
	</ol> </li> <li> 在干净的室内放置一个自定义的灭菌网格, 并将其固定在预先方向上, 并将螺钉放在会议厅一侧。此处显示的网格有 1 mm 间距;但是, 可以根据需要自定义间距. 
	 注意: 这应该是相同的网格和方向使用的解剖 MRI. </li>
	<li> 在记录室中以预先确定的方向保护立体定向微驱动器支架. </li>
	<li> 用无菌的钝钳将照明器拉起, 从导管中缩回灭菌的照明灯。照明灯尖端应为 5-10 毫米以上的引导管尖端. 
	 注意: 照明灯将弓向外的引导管和钳。这不会损坏灵活的照明灯. </li>
	<li> 将微驱动器贴在支架上, 并将导管置于目标网孔中. </li>
	<li> 降低微驱动器的阶段, 直到引导管刚刚通过硬脑膜的水平. 
	 注意: 如果需要, 第二个微驱动器与电极可以放在舞台上, 并降低到一个不同的网孔。该电极上的局部场电位将显示一个距离相关的光伪迹, 可用于确定光源位置. </li>
	<li> 尝试用无菌镊子手动降低照明灯。
	<ol> <li> 如果有任何阻力, 请收回照明灯 5-10 毫米, 等待10分钟, 让组织和解, 再试一次. </li>
		<li> 如果在第二次尝试时仍有阻力, 请将照明灯尖端缩回引导管中, 向下引导管 0.25 mm, 然后重试. </li>
		<li> 重复, 直到照明灯通过导水管滑动而无任何阻力. </li>
		<li> 降低照明灯, 直到它绷紧. 
		 警告: 不要用力地推挤照明灯反对抵抗。在这些情况下, 导管通常没有穿透硬脑膜充分。强力推动纤维会使它自身弯曲, 防止纤维进入大脑并可能破坏尖端 ( 图 1g ). </li>
	</ol> </li> <li> 将光源的另一端上的套圈连接到较大的光学设置或激光器上. </li>
	<li> 确保非人类灵长类动物没有可见光。
	<ol> <li> 使用黑屏蔽或光吸收箔来完全包含记录塔. </li>
		<li> 将所有光学连接放置在由光吸收箔制成的屏蔽信封中. </li>
		<li> 用吸光箔或黑色电子胶带屏蔽所有跳线. </li>
		<li> 作为一种额外的预防措施, 在非人类灵长类动物后面的实验中放置一个与之相同的浅色发光二极管 (led) 库, 并以2.5 赫兹的频率连续闪烁 led。这可以防止眼睛完全适应黑暗。如果有疏忽的光漏, 这种闪光将有助于防止泄漏作为行为触发. </li>
	</ol>
	</li>
</ol>

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虽然 optogenetic 工具被广泛用于研究啮齿类动物的疾病和生理学, 但对大脑大容量的照明技术的挑战限制了非人类灵长类动物的遗传学的使用。在猴子的开创性研究中使用了大的光功率密度 (~ 100 兆瓦/毫米2到20瓦特/毫米2) 来照亮小容量, 也许和 #60; 1 mm3, 并报告了轻微的行为影响与兴奋 opsins 在皮层4, 9、10...

Optogenetic 工具, 允许毫秒精确, light-driven 神经元控制被广泛用于研究啮齿动物和无脊椎动物的功能生理学和行为。然而, 技术上的挑战限制了非人类灵长类大脑中遗传学的使用, 它的体积 ~ 100 x 大于啮齿动物的大脑1。
为了促进非人类灵长类动物的遗传学研究, 一个照明灯被设计来解决两个相互竞争的目标: 大体积光照和最小穿透损伤。以前试图解决其中一个问题的做法是代价高昂的。束纤维照亮更大的容量, 但以增加的直径, 并且, 因而, 损坏2,3。锥形玻璃纤维可减少穿透损伤, 但将光线聚焦到发光表面区域和 #60; 100 µm2 4,5。外部的大脑照明通过一个窗口的硬脑膜绕过的挑战, 穿透伤害和可能允许大体积照明, 但它只能用于一些肤浅的大脑区域6。
为了创建一个大体积, 小直径的照明灯 (图 1a), 塑料光纤的尖端是热锥和核心和熔覆蚀刻 (图 1b, c)。不同于将光聚焦到一个窄点的锥形光纤, 蚀刻允许光线在尖端的两侧均匀地逸出, 从而在大面积上分布光源 (图 1d, e)。由于穿透损伤与穿透直径成正比, 因此该照明灯没有比传统纤维更大的穿透损伤, 但它有和 #62; 100x. 光发射表面积和光功率的 1/第100更广泛地提供光密度在脑幻影 (1.75% 琼脂糖) (图 1e)。蒙特卡罗模型 (图 1f) 说明了传统光纤和大体积照明灯在光功率密度相等时与发光表面之间的差异。每个照明灯使用集成球体 (图 2a, b) 进行单独校准, 以确保沿尖端均匀分布 (图 2c)。
这种大体积的照明灯已经验证了 optogenetic 操作的行为和神经元射击在非人灵长类动物。纤维针尖长度可以定制的任何大脑区域和每一个动物的个人接受领域的地图。该照明灯可以配对与穿透电极的神经录音, 跨越的长度照明。此外, 由于纤维可以携带任何颜色的可见光, 它可以与任何可用的 optogenetic 分子配对。

<table><tbody><tr><td>Plastic optical fiber</td><td>Industrial fiber optics</td><td>SK-10</td><td>250 micron diameter, Super Eska line</td></tr><tr><td>Wire stripper</td><td>Klein Tools</td><td>11047</td><td>22 gauge</td></tr><tr><td>Vise Clamp</td><td>Wilton</td><td>11104</td><td>Generic table mount vice clamp</td></tr><tr><td>Dual temperature heat gun</td><td>Milwaukee</td><td>8975-6</td><td>570 / 1,000 &amp;deg;F</td></tr><tr><td>Lab marker</td><td>VWR</td><td>52877</td><td></td></tr><tr><td>Dissection microscope</td><td>VistaVision</td><td>82027-156</td><td>Stereo microscope w/ dual incandescent light, 2X/4X magnification, available from VWR</td></tr><tr><td>Lab tape</td><td>VWR</td><td>89097-972</td><td>4 pack of violet color; however, tape color does not matter</td></tr><tr><td>Silicon carbide lapping sheet</td><td>ThorLabs</td><td>LF5P</td><td>5 micron grit, 10 pack</td></tr><tr><td>Aluminum oxide lapping sheet</td><td>ThorLabs</td><td>LF3P</td><td>3 micron grit, 10 pack</td></tr><tr><td>Aluminum oxide lapping&amp;nbsp; sheet</td><td>ThorLabs</td><td>LF1P</td><td>1 micron grit, 10 pack</td></tr><tr><td>Calcined alumina lapping sheet</td><td>ThorLabs</td><td>LF03P</td><td>0.3 micron grit, 10 pack</td></tr><tr><td>Hot knife</td><td>Industrial fiber optics</td><td>IF370012</td><td>60 Watt, heavy duty</td></tr><tr><td>Fiber inspection scope</td><td>ThorLabs</td><td>FS201</td><td>optional</td></tr><tr><td>Stainless Steel Ferrule</td><td>Precision fiber optics</td><td>MM-FER2003SS-265</td><td>265 micron inner diameter</td></tr><tr><td>1 mL syringe</td><td>BD</td><td>14-823-30</td><td>Luer-lok tip is preferable to reduce risk of leakage, but not strictly needed</td></tr><tr><td>Plastic epoxy</td><td>Industrial fiber optics</td><td>40 0005</td><td></td></tr><tr><td>18 gauge blunt needle</td><td>BD</td><td>305180</td><td>1.5 inch length</td></tr><tr><td>Lint-free wipe (KimWipe)</td><td>ThorLabs</td><td>KW32</td><td>available from many vendors</td></tr><tr><td>Light absorbing foil</td><td>ThorLabs</td><td>BKF12</td><td></td></tr><tr><td>Electrical tape</td><td>3M</td><td>Temflex 1700</td><td>Optional, may substitute other brands / models</td></tr><tr><td>26 gauge sharp needle&amp;nbsp;</td><td>BD</td><td>305111</td><td>0.5 inch length</td></tr><tr><td>Micromanipulator</td><td>Siskiyou</td><td>70750000E</td><td>may substitute other brands/models</td></tr><tr><td>Steretactic arm</td><td>Kopf</td><td>1460</td><td>may substitute other brands/models</td></tr><tr><td>Laser safety goggles</td><td>KenTeK</td><td>KCM-6012</td><td>must be selected based on the color of laser used, example given here</td></tr><tr><td>Laser or other light source</td><td>vortran</td><td>Stradus 473-50</td><td>example of blue laser</td></tr><tr><td>Integrating sphere</td><td>ThorLabs</td><td>S142C</td><td>Attached power meter, also available from ThorLabs, item #PM100D</td></tr><tr><td>Ultem recording chamber</td><td>Crist instrument company</td><td>6-ICO-J0</td><td>Customized with alignment notch</td></tr><tr><td>Tower microdrive with clamps</td><td>NAN</td><td>DRTBL-CMS</td><td></td></tr><tr><td>Guide tube</td><td>Custom</td><td>N/A</td><td>Made from 25 gauge spinal needle (BD) or blunt tubing</td></tr><tr><td>NAN driver system</td><td>NAN</td><td>NANDrive</td><td></td></tr><tr><td>Custom grid design</td><td>custom</td><td>custom</td><td>plans available upon request</td></tr><tr><td>Blunt forceps</td><td>FischerScientific</td><td>08-875-8A</td><td>generic stainless steel blunt forceps</td></tr><tr><td>Digital calipers</td><td>Neiko</td><td>01407A</td><td>available on amazon.com. May select a finer resolution caliper for more precise measurements.</td></tr><tr><td>Patch cable</td><td>ThorLabs</td><td>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><td>Clear plastic dust caps</td><td>ThorLabs</td><td>CAPF</td><td>Package of 25</td></tr><tr><td>ceramic split mating sleeve</td><td>Precision Fiber Products, Inc.</td><td>SM-CS1140S</td><td></td></tr></tbody></table>

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

该协议描述了一个大体积的照明灯, 这是为非人类灵长类大脑中的 optogenetic 操纵而开发的。照明灯是一种经蚀刻后的改性塑料光纤, 其发光表面积 #62; 100x。除了描述大容量照明灯的构造外, 本协议还详细介绍了用于确保均匀光分布的质量控制校准。此外, 该协议还描述了插入和移除大容量照明灯的技术。表面和深层结构都可以被照亮。这个大体积的照明灯不需要物理耦合到一个电极, 因为照明灯是由塑料, 而不是玻璃, 它会简单地弯曲的情况下, 传统的光纤将粉碎。因为这个照明灯提供光超过行为相关的组织体积 (≈ 10 mm3) 没有比传统的光纤更大的穿透损伤, 它促进行为研究使用遗传学在非人灵长类动物。

在非人灵长类动物的大脑容量的照明允许行为相关的 optogenetic 操作。Acker et al.(2016) 使用这个大容量的照明灯与红色被转移的 Halorhodopsin, 下颌7研究前额眼睛领域的世俗贡献 (FEF) 到记忆被引导的扫视在二只恒河猴。具体来说, FEF 神经元注射了一个含有颌骨的病毒载体, 然后用红光照亮, 在目标表示、延迟周期或记忆引导的扫视任务的运动准备期间, 使用大体积的照明灯。<sup ...

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

建立了一个组织穿透照明灯的协议, 以提供光超过大体积的最小直径。

非人类灵长类动物遗传学的大体积、行为相关光照

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

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

Universidad San Sebastian

Research

JoVE Journal

Behavior

8.0K 次观看.  Massachusetts Institute of Technology. 建立了一个组织穿透照明灯的协议, 以提供光超过大体积的最小直径。

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

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

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

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

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

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

科研

神经科学

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

单个锥体神经元的高保真光遗传学控制方法<em&gt;在体内</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

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.

在小鼠的文室内使用光遗传学的两种不同的实时位置偏好范式

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

行为学

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

神经活性的光遗传学操作，调节自由移动小鼠的行为

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of ...

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities.
Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small-molecule substrate, a luciferin-can be harnessed to activate photosensory proteins, thereby adding another dimension to light stimulation and enabling the manipulation of a multitude of light-mediated functions in cells across temporal and spatial scales.

生物发光光遗传学2.0:利用生物发光在体外和体内激活光敏蛋白

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate ...

生物化学

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

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

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

指南<em&gt;在体内</em&gt;单单元从Optogenetically鉴定皮质抑制性录像

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

A MRI-Based Toolbox for Neurosurgical Planning in Nonhuman Primates

In this paper, we outline a method for surgical preparation that allows for the practical planning of a variety of neurosurgeries in NHPs solely using data extracted from magnetic resonance imaging (MRI). This protocol allows for the generation of 3D printed anatomically accurate physical models of the brain and skull, as well as an agarose gel model of the brain modeling some of the mechanical properties of the brain. These models can be extracted from MRI using brain extraction software for the model of the brain, and custom code for the model of the skull. The preparation protocol takes advantage of state-of-the-art 3D printing technology to make interfacing brains, skulls, and molds for gel brain models. The skull and brain models can be used to visualize brain tissue inside the skull with the addition of a craniotomy in the custom code, allowing for better preparation for surgeries directly involving the brain. The applications of these methods are designed for surgeries involved in neurological stimulation and recording as well as injection, but the versatility of the system allows for future expansion of the protocol, extraction techniques, and models to a wider scope of surgeries.

The method outlined below aims to provide a comprehensive protocol for the preparation of nonhuman primate (NHP) neurosurgery using a novel combination of three-dimensional (3D) printing methods and MRI data extraction.

非人类灵长类动物神经外科规划基于MRI的工具箱

In this paper, we outline a method for surgical preparation that allows for the practical planning of a variety of neurosurgeries in NHPs solely using ...

生物工程

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we demonstrate the use of magnetic resonance (MR)-guided convection enhanced delivery (CED) of optogenetic viral vectors into primary somatosensory (S1) and motor (M1) cortices of macaques to obtain efficient, widespread cortical expression of light-sensitive ion channels. Adeno-associated viral (AAV) vectors encoding the red-shifted opsin C1V1 fused to yellow fluorescent protein (EYFP) were injected into the cortex of rhesus macaques under MR-guided CED. Three months post-infusion, epifluorescent imaging confirmed large regions of optogenetic expression (&#62;130 mm2) in M1 and S1 in two macaques. Furthermore, we were able to record reliable light-evoked electrophysiology responses from the expressing areas using micro-electrocorticographic arrays. Later histological analysis and immunostaining against the reporter revealed widespread and dense optogenetic expression in M1 and S1 corresponding to the distribution indicated by epifluorescent imaging. This technique enables us to obtain expression across large areas of the cortex within a shorter period of time with minimal damage compared to the traditional techniques and can be an optimal approach for optogenetic viral delivery in large animals such as NHPs. This approach demonstrates great potential for network-level manipulation of neural circuits with cell-type specificity in animal models evolutionarily close to humans.&#160;

Here, we demonstrate magnetic resonance (MR)-guided convection enhanced delivery (CED) of viral vectors into the cortex as an efficient and simplified approach for achieving optogenetic expression across large cortical areas in the macaque brain.

在在线 MRI 图像的指导下,对流增强将视遗传阿德诺相关病毒载体输送到 Rhesus Macaque 的皮质

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we ...

用于 NHP 神经外科计划的 MRI 自动 3D 大脑和颅骨建模

A Neural Implant Design Toolbox for Nonhuman Primates

This paper describes an in-house method of 3D brain and skull modeling from magnetic resonance imaging (MRI) tailored for nonhuman primate (NHP) neurosurgical planning. This automated, computational software-based technique provides an efficient way of extracting brain and skull features from MRI files as opposed to traditional manual extraction techniques using imaging software. Furthermore, the procedure provides a method for visualizing the brain and craniotomized skull together for intuitive, virtual surgical planning. This generates a drastic reduction in time and resources from those required by past work, which relied on iterative 3D printing. The skull modeling process creates a footprint that is exported into modeling software to design custom-fit cranial chambers and headposts for surgical implantation. Custom-fit surgical implants minimize gaps between the implant and the skull that could introduce complications, including infection or decreased stability. By implementing these pre-surgical steps, surgical and experimental complications are reduced. These techniques can be adapted for other surgical processes, facilitating more efficient and effective experimental planning for researchers and, potentially, neurosurgeons.

作者聚焦：简化的大脑和颅骨建模以增强 NHP 研究中的神经外科计划

This paper outlines automated processes for nonhuman primate neurosurgical planning based on magnetic resonance imaging (MRI) scans. These techniques use procedural steps in programming and design platforms to support customized implant design for NHPs. The validity of each component can then be confirmed using three-dimensional (3D) printed life-size anatomical models.

非人类灵长类动物遗传学的大体积、行为相关光照

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