作者得到了日本促进科学 KAKENHI 赠款 JP16K11200 和17H02223 协会的支持, 以及金泽医科大学 S2016-8 和 C2017-3 的研究补助金。我们感谢 Yuhichi 大海在拍照时的支持。

所有的程序都是按照日本生理学学会的指导原则, 并经金泽医科大学动物保育委员会批准。
1. 玻璃光纤化学传感架的施工
注: 要建立玻璃光纤化学传感支架, 使用商用电极支架 (图 1A)。
<ol><li>轻轻拉出钢管的压力控制从桶的持有人。</li>
	<li>通过钻孔, 扩大了固定架上的销座一侧的孔。使孔直径为3毫米, 深度为12毫米。</li>
	<li>将不锈钢管 (直径为3毫米, 厚度为0.5 毫米, 长度为8毫米;图 1A2) 在洞里。</li>
	<li>将⌀2.5 mm 套圈 (图 1A1) 上的陶瓷剥离联接套筒插入孔中。</li>
	<li>将电极架的枪管固定在 L 型榫头上, 用环氧树脂胶粘剂。</li>
	<li>使榫头中的孔 (直径为3毫米), 并将其附加到带有螺钉的机械手上。</li>
</ol>2. 头后安装
<ol><li>准备一个由电路板垫片制成的定制的头柱。将柱状电路板间隔成一个立方的形状与铣刀, 并将其切割成2头柱与锯 (图 1B)。用铣刀将头柱底部压扁。</li>
	<li>为研究中的特定神经元类型, 制备一种光敏视蛋白的转基因动物。在这项工作中, 转基因老鼠 (VGAT-ChR2 小鼠), 其中抑制神经元表达 channelrhodopsin-2 (ChR2)11使用。</li>
	<li>麻醉的动物与0.3 毫克/千克的 medetomidine, 4.0 毫克/千克的咪达唑仑和5.0 毫克/公斤的布托啡诺由腹腔管理。为了证实动物完全麻醉, 测试其对尾部捏的反应。</li>
	<li>将鼠标放在加热垫上的立体定位框架中。提前70% 酒精清洗立体定向框架和加热垫。将牙齿放在咬杆的孔中, 轻轻拧紧鼻钳。用耳棒固定头部两侧。当头部正确固定的耳棒, 收紧鼻钳。把眼膏涂抹在眼睛上。</li>
	<li>用小剪刀刮头皮, 用葡萄糖酸氯己定的棉签清洁头皮。手术前, 用高压釜 (121 摄氏度, 15 分钟) 消毒所有手术器械和头部。</li>
	<li>用剪刀把头皮沿中线切开, 把它推到一边。使切口从头部后到眼睛 (大约20毫米)。用棉签蘸上70% 的酒精并干燥头骨, 去掉骨膜。</li>
	<li>将头部贴在机械手上。使用机械手将头部贴在 bregma 周围的头骨上。</li>
	<li>将单体 (4 滴)、催化剂 (1 滴) 和聚合物 (1 小勺) 混合在冰箱里冷藏的小盘子里。将牙科水泥 (低于1克) 放在颅骨的额部和顶叶上, 将头部贴在头骨上。</li>
	<li>牙科水泥变硬后, 从立体定向的框架中取出鼠标。在暴露的皮肤上涂上抗生素软膏。注射 medetomidine 拮抗剂 (0.3 毫克/千克) 和抗生素 (土霉素; 20 毫克/千克)。在笼子的热垫上放置并监视鼠标, 直到它苏醒, 然后将它返回到一个外壳笼中。</li>
	<li>把动物放在一个单独的住房笼子里, 用一些浓缩装置。每天更换动物纸床上用品, 保持笼子清洁, 防止感染。仔细观察动物是否显示出任何不适的迹象。如果怀疑有任何疼痛, 应将利多卡因用于伤口。在头后安装和开颅手术后, 每24小时管理 meloxicam 或卡洛芬48小时。如果 NSAID 不充分, 可以给地方利多卡因.
</li>
</ol>3. 驯化
<ol><li>从麻醉恢复后两天 (最小), 使动物适应记录室的头部固定状态。至少进行5天的驯化训练。</li>
	<li>在训练过程中, 将动物放在录音室中, 将头部固定在一个定制的夹具上。在会议期间, 为了限制移动, 用一台3D 打印机制作的自定义树脂半管 (图 1C) 覆盖车身。 注: 在1天, 驯化疗程应为5分钟长。然后每天增加到10、20、40和60分钟, 分别为2、3、4和5天。</li>
	<li>在每届会议结束时, 给动物一个食物奖励。如果动物在驯化过程中不断挣扎, 停止会话。</li>
</ol>4. 开颅手术
注意: 在驯化后, 开颅手术是在大脑区域进行记录的。开颅术是在麻醉下的立体定向框架下进行的, 如头部安装后。术后程序与头后安装相同。
<ol><li>在开颅手术前, 用棉签用棉签清洁头骨和70% 酒精。</li>
	<li>小心地用牙钻把头骨放在录音现场。当骨头变得足够薄时, 用手术刀的尖端切开它, 然后将它取出。</li>
	<li>将牙科水泥放在暴露的区域周围, 以加强头骨。</li>
	<li>在牙科水泥变得坚硬之后, 用硅胶粘合剂覆盖裸露的大脑区域。术后手术过程如步骤2.8 所述。</li>
</ol>5. 电生理记录
<ol><li>允许动物恢复至少1天后, 开颅术前进行电生理记录。把动物放在录音室里, 就像录音开始时的训练过程中那样。</li>
	<li>使玻璃吸管从硼硅酸盐玻璃毛细血管 (OD = 1.5 毫米, ID = 0.9 毫米, 90.0 毫米长) 拉在电极牵引器。他们的尖端直径是2–3µm, 他们的阻力是第4-6 MΩ时, 他们填充10毫米 PBS。电极的长度为40–50毫米。在一些录音中, 2% neurobiotin 被添加到10毫米 PBS 中。</li>
	<li>将填充的玻璃吸管附着在特制的光纤化学传感支架上。</li>
	<li>将银 Cl 涂层银线 (直径0.2 毫米) 放入玻璃吸管中。通过薄涂层电线将银线连接到 pin。将 pin 连接到录音放大器的 headstage。</li>
	<li>将光纤插补线 (核心 = 960 µm, 包覆 = 1000 µm, NA = 0.63) 与氧化锆箍 (外径 = 2.5 毫米) 连接到光纤化学传感支架上的拆分联接套筒上。将贴片线推入套筒, 直到插针的尖端与玻璃吸管的背面接触。补丁线提供光 (波长 = 465 毫微米) 从 LED。led 指示灯由单通道 led 驱动程序控制。</li>
	<li>在开颅手术上取下硅胶粘合剂。定期将温热生理盐水 (38 °c) 放在脑表面, 以防止在录音过程中大脑表面干燥。如有必要, 用锋利的镊子取出硬脑膜。</li>
	<li>用机械手将玻璃电极插入脑组织中。在插入电极电阻时监视它。当电阻低于预期时, 更换电极, 因为电极的尖端可能损坏。在感兴趣的深度, 通过调整2到5µm 步骤中的深度来搜索和隔离单个单元峰值。</li>
	<li>单单元隔离后, 传送 LED 灯并观察对光刺激的响应。</li>
	<li>在识别细胞类型后记录神经活动。执行 2–3 h 的录制。如果录音是连续的天 (最多2天), 用硅胶粘合剂覆盖开颅手术。当录音结束, 动物被牺牲, 检索头部后, 并把它在丙酮清洗和重用它。</li>
</ol>

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作者没有什么可透露的。

光遗传学已经成为神经科学的有力工具。它用于识别体内特定的神经元类型, 以及操纵特定神经元通路的活动。对不同神经元类型的神经活动的澄清促进了对神经回路机制的理解。在这里, 我们演示了一种方法, 通过一个玻璃电极在清醒的 VGAT-ChR2 小鼠 IC 的芯片上传送光到录音现场。
在描述的方法中有几个关键步骤。首先, 它是一个要求使用的动物有充分和特定的表达 opsins 在目标神经元。为了识别对光的反应与细胞外记录, 有必要的激活视蛋白唤起的峰值在目标神经元。在某些转基因菌株中, 在年轻动物中, 神经元的基因表达水平可能低于14, 而实验的最低年龄也可能是如此。此外, 可能需要进行一些额外的实验, 以确认对光刺激的反应是否特定于目标神经元类型 (如免疫组化)。当目标神经元兴奋时, 有必要区分直接诱发反应与 synaptically 诱发反应, 这可以通过阈值的反应延迟。第二, 确保脑组织尽可能干净是至关重要的。为了防止开颅手术和录音中的出血, 特别需要保持清洁。此外, 在开颅术后, 应防止大脑表面与硅胶粘合剂紧密覆盖表面。
为了有效地从电极尖端传送光, 减少光路中的任何损耗是至关重要的。在我们的光纤化学传感的配置中, 光功率通过电极保持器: 光功率是45兆瓦在光纤补丁线的末端, 而它是3–5兆瓦在玻璃吸管的尖端。如结果所示, 笔尖大小和全长不影响笔尖的光功率 (图 2)。结果表明, 无论玻璃吸管的结构如何, 光衰减都是相当稳定的。这种光衰减在 VGAT-ChR2 小鼠的 ic 神经元的记录中并不重要, 因为我们已经确认在 ic 的腹边边缘 (2 毫米深度) 可以记录光激活的峰值, 尽管它在其他记录配置中可能很重要。这将很难大幅减少光衰减, 但可能有改善的可能性。首先, 使用石英代替硼硅酸盐玻璃作为电极吸管可能是有效的。众所周知, 石英具有更好的光电导率, 因此可以通过电极15减少光损耗。此外, 据报道, 吸管末端的抛光剂减少了吸管和15条线之间接触面的光损耗。如果光衰减的改善是不够的, 它将是有效的使用激光代替 led, 虽然激光成本超过 led。
玻璃吸管电极的优点之一是可以通过向填充溶液添加示踪剂或载体来执行组织学程序。组织学程序将增加大量有用的信息 (记录的位置, 记录的神经元的形态学等) 到电生理数据。此外, 据报道, 在填充液15中使用玻璃电极与阻滞剂进行药理学调查是可能的。与这些优点相比, 玻璃吸管电极的局限性在于它不能从多个孤立单元中进行记录。为了获得多项录音, 可以将电子四极线放入玻璃吸管中, 而不是用单一的银 Cl 涂层导线。在这种情况下, 有必要使用一个更大的尖端大小的吸管 (30–60µm, 电子四极的典型尖端大小), 并让电子四极的末端从吸管的尖端。
虽然以前报告 optrodes 经常需要复杂制造16,17, 玛吉和他的同事开发了光纤化学传感使用标准玻璃吸管和光纤化学传感我们设计了18。在他们的方法, 他们插入一根薄薄的光纤到吸管提供光。为了使纤维足够薄, 以插入吸管的小腿, 他们蚀刻125µm 包覆纤维与9µm 核心到直径8–10µm 在尖端。这种方法的优点是它甚至适用于通常用于离子导入的弯曲吸管。然而, 使用 LED 作为光源可能很难, 因为标准的商用 led 无法通过9 µm 核心光纤获得足够的光功率。

中枢神经系统由不同类型的神经元组成, 具有不同的功能。这些不同类型的神经元在神经回路中的工作方式是神经科学的主要关注之一。然而, 在许多脑区, 由于电穗信号本身没有明显的差异, 有些例外, 因此无法区分电子活动体内记录中的神经元类型。最近在光遗传学的进展取得了突破1,2。利用具有光敏视蛋白 (如channelrhodopsin-2) 的转基因动物在特定的神经元类型中表达, 在体内记录3 中有效地区分神经元类型是可能的,4,5,6。在这些动物中, 光敏感视蛋白的神经元通过在电记录中给予光刺激而兴奋, 但其他神经元却没有。因此, 视蛋白阳性神经元很容易与其他神经元类型区别, 因为它们对光的反应。
在光遗传学实验中, 将光传送到记录站点是至关重要的。作为一种非侵入性的方法, 光通常是从大脑的表面定向的。然而, 由于光的强度降低, 因为它通过脑组织, 这是很难刺激大脑的深层区域从脑的表面。特别是, 当大脑表面的光学透明度较低时, 刺激光很难到达深部的脑区, 而醒着的动物的录音通常也是如此。电生理实验常常是在麻醉动物身上进行的, 因为身体运动会导致录音中的噪音。然而, 众所周知, 麻醉是改变神经反应7,8,9,10。因此, 有必要使用清醒的动物, 以研究神经反应没有人工作用的麻醉。与麻醉动物的实验不同, 电生理记录是在实验中与醒着的动物的手术恢复后进行的。在手术与录音之间的时间间隔内, 组织渗出物经常堆积在脑表面, 使脑表面的光学透明度降低。
在这里, 我们描述了一个方法, 记录从一个清醒的小鼠使用定制玻璃光纤化学传感的单单位录音。在这种方法中, 光通过记录玻璃电极传递, 以便在深部脑区能可靠地刺激被记录的神经元。这种定制的光纤化学传感系统由可访问和廉价的材料组成, 易于组装。

<table><tbody><tr><td>Electrode holder</td><td>Molecular Device</td><td>1-HL-U</td><td>pipette holder for microelectrode amplifier</td></tr><tr><td>Ceramic split mating sleeve</td><td>Thorlabs</td><td>ADAF1</td><td>f2.5 mm ferrule</td></tr><tr><td>Circuit board spacer</td><td>Teishin Denki</td><td>SPA-320</td><td>f8.0 mm, 20.0 mm long</td></tr><tr><td>Stereotaxic frame for mice</td><td>Narishige</td><td>SR-6M-HT</td><td>Stereotaxic instruments for mice</td></tr><tr><td>Manipulator</td><td>Narishige</td><td>NA</td><td>Manual manipulator</td></tr><tr><td>Superbond</td><td>Sun Medical</td><td>M: 204610557</td><td>Dental adhesive resin cement</td></tr><tr><td>Form2</td><td>Formlabs</td><td>NA</td><td>3D printer</td></tr><tr><td>Kwik-Sil</td><td>WPI</td><td>KWIK-SIL</td><td>Low toxicity silicone adhesive</td></tr><tr><td>Borosilicate glass capillaries</td><td>Narishige</td><td>GD-1.5</td><td>OD 1.5 mm, ID 0.9 mm, 90.0 mm long</td></tr><tr><td>Fiber-optic patch cord</td><td>Doric Lenses</td><td>MFP_960/1000/2200-0.63_1m_FCM-ZF2.5</td><td>Monofiberoptic patchcord, OD, 2.5 mm, core = 960 mm, cladding = 1000 mm, NA = 0.63</td></tr><tr><td>Connectrized LED</td><td>Doric Lenses</td><td>LEDC-1B_FC</td><td>Central wave length = 465 nm, output power = 45 mW (Core 960 mm 0.63 NA )</td></tr><tr><td>LED driver</td><td>Doric Lenses</td><td>LEDRV_1CH_1000</td><td>1 ch LED driver, maximum output = 1000 mA</td></tr><tr><td>Electrode puller</td><td>Narishige</td><td>PB-7</td><td>Dual-stage glass micropipette puller</td></tr><tr><td>Borosilicate glass capillary</td><td>Narishige</td><td>GD-1.5</td><td>Bolosilicate glass capillary, OD, 1.5mm, ID, 0.9 mm, 90.0 mm long</td></tr><tr><td>GENTACIN</td><td>MSD CO., Ltd</td><td>185711173</td><td>Antibiotic ointment</td></tr><tr><td>Terramycin&amp;reg;-LA</td><td>Zoetis</td><td>G 333</td><td>Oxytetracycline</td></tr><tr><td>Tg(Slc32a1- COP4*H134R/EYFP)8Gfng/J</td><td>Jackson Labs</td><td>#14548</td><td>VGAT-ChR2 mice</td></tr><tr><td>Multiclamp 700B</td><td>Molecular Devices</td><td>2500-0157</td><td>Microelectrode amplifier</td></tr></tbody></table>

optogenetics identification of a neuronal type with a glass optrode in awake mice

神经回路中不同类型的神经元是如何工作的, 这是神经科学的主要关注。最近在光遗传学的研究进展, 使得在大脑神经区的体内电生理实验中神经元类型的识别成为了一种方法。在光遗传学实验中, 将光传送到记录站点是至关重要的。然而, 通常很难将刺激的光线从大脑的表面传送到大脑的深层区域。特别是, 当大脑表面的光学透明度较低时, 刺激光很难到达深部的脑区, 而醒着的动物的录音通常也是如此。在这里, 我们描述了一个方法, 记录的峰值响应的光从清醒的鼠标使用定制玻璃光纤化学传感。在这种方法中, 光通过记录玻璃电极传递, 这样就可以可靠地在脑深部区域内刺激记录的神经元。这种定制的光纤化学传感系统由可访问和廉价的材料组成, 易于组装。

在图 2中, 我们检查了笔尖大小和玻璃吸管长度对吸管尖端的光功率的影响 (图 2A-B)。光功率测量的光功率计放置1毫米远离尖端。当刀尖尺寸变化时, 全长被设置为 50 2 毫米, 当全长变化时, 刀尖尺寸设置为2.5 µm。玻璃吸管的小腿被设置了到8毫米。尖端的光功率范围从3–5兆瓦 (平均 * SD) 不等;图 2A: 3.77 @ 0.29 兆瓦, n = 20;图 2B: 4.24 @ 0.31 兆瓦, n = 21)。吸管的尖端尺寸 (图 2A) 与光功率几乎不相关 (R = 0.1555)。吸管的长度只与轻的力量轻微地相关以消极方式 (R =-0.2054)。这些结果表明, 吸管的尖端和长度很少影响尖端的光功率。我们还检查了在玻璃吸管中, 溶液 (10 毫米 PBS) 的体积负载量是否影响光功率 (图 2C)。溶液的体积由吸管中溶液的长度来评估。数据记录从4吸管 (尖端大小: 0.25 µm, 全长:50 @ 2 毫米, 小腿的长度: 8 毫米)。我们发现, 改变溶液在吸管中的体积并不影响光功率 (图 2C)。
在图 3中, 我们显示了在 VGAT-ChR2 小鼠 (2–4月大) 的丘 (IC) 下的穗反应记录。IC 是一个听觉中心在中脑和由谷氨酸和 GABAergic 神经元12,13, 这两个响应的声音3,4。在 VGAT-ChR2 小鼠的 IC 中, GABAergic 神经元明确表达 ChR24。在先前的工作中, 发现 GABAergic 和谷氨酸神经元分别被激发或抑制, 当它们被给予光刺激3,4。在醒鼠中, 我们发现两种类型的神经元 (图 3A-B)。当光刺激通过玻璃电极传递时, 它会诱发某些神经元的穗反应 (图 3A)。相比之下, 在其他神经元中, 光刺激抑制声音刺激诱发的穗反应 (图 3B)。结果表明, 玻璃光纤化学传感能记录分离出的单单位活动, 并能可靠地刺激记录神经元的光。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57781/57781fig1.jpg"> 图 1:光纤化学传感持有人和录音配件.面板 (A) 和 (B) 显示光纤化学传感持有人。面板 (A) 显示光纤化学传感持有者的部件结构。(1) 这是一种陶瓷剥离交配套筒。(2) 这是不锈钢管。(3) 这是一个桶的持有人。(4) 这是一个锥形垫圈。(5) 这是聚碳酸酯帽。面板 (B) 显示组装的光纤化学传感持有人。面板 (C) 和 (D) 显示了定制的头柱。面板 (C) 显示头部柱子的侧面视图。面板 (D) 显示头部柱的斜视图。两个头柱 (右) 是由电路板间隔 (左)。面板 (E), (F) 和 (G) 显示定制的树脂半管。面板 (E) 和 (F) 显示树脂半管的尺寸图。面板 (E) 显示树脂半管的前视图。面板 (F) 显示树脂半管的侧面视图。面板 (G) 显示了半管的照片图像。面板中的数字 (E) 和 (F) 表示绘图尺寸 (mm).<a href="/files/ftp_upload/57781/57781fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57781/57781fig2.jpg"> 图 2:玻璃吸管的配置对尖端光功率的影响.(A) 尖端的尺寸是针对轻型电源绘制的。该吸管的全长被设置为 50, 2 毫米。小腿的长度被设置了到8毫米. (B) 将吸管的长度绘制在光功率上。笔尖大小设置为0.25 µm。小腿的长度被设置了到8毫米. (C) 此面板显示解决方案 (10 毫米 PBS) 的体积负载对光功率的影响。4吸管的数据由不同的符号和线条表示。<a href="/files/ftp_upload/57781/57781fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57781/57781fig3.jpg"> 图 3: 光诱发的反应在 IC 的醒 VGAT-ChR2 小鼠.(A) 光刺激 (蓝盒, 0.03 s) 诱发穗反应。(B) 光刺激 (蓝盒, 0.03 s) 抑制声音诱发的穗反应 (灰色框)。上面的3跟踪是对声音的响应, 而在发出声音和光线时, 较低的3迹是响应。<a href="/files/ftp_upload/57781/57781fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice at JoVE.com

Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

光遗传学玻璃光纤化学传感在醒鼠中的神经型鉴定

这项工作介绍了一个方法, 以可靠地执行 optogenetic 单单元记录从清醒的鼠标使用定制玻璃光纤化学传感。

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the ...

optogenetics-identification-neuronal-type-with-glass-optrode-awake

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.7K 次观看.  Kanazawa Medical University. 这项工作介绍了一个方法, 以可靠地执行 optogenetic 单单元记录从清醒的鼠标使用定制玻璃光纤化学传感。

视频: Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

在完整的视网膜电生理特性表达GFP的细胞群

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

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 ...

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

Optogenetically有针对性的前脑电路的激光扫描光刺激

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

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 ...

行为学

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 ...

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser.&#160;Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible.&#160;The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

在鼠背耳蜗核的听觉通路的光遗传学刺激直接可视化

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

监测小鼠睡眠/觉醒状态期间神经回路的光遗传学操作

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective neural modulation or monitoring. As specific types of neuronal cells can be genetically modified to express opsin proteins, optogenetics enables optical stimulation or inhibition of the selected neurons. There have been several technological advances in the optical system for optogenetics. Recently, it was proposed to combine the optical waveguide for light delivery with electrophysiological recording to simultaneously monitor the neural responses to optogenetic stimulation or inhibition. In this study, an implantable optrode array (2x2 optical fibers) was developed with embedded multichannel electrodes. 
A light-emitting diode (LED) was employed as a light source, and a microfabricated microlens array was integrated to provide sufficient light power at the tip of the optical fibers. The optrode array system comprises the disposable part and the reusable part. The disposable part has optical fibers and electrodes, while the reusable part has the LED and electronic circuitry for light control and neural signal processing. The novel design of the implantable optrode array system is introduced in the accompanying video in addition to the procedure of the optrode implantation surgery, optogenetic light stimulation, and the electrophysiological neural recording. The results of in vivo experiments successfully showed time-locked neural spikes evoked by the light stimuli from hippocampal excitatory neurons of mice.

Here, we present the fabrication method of an optrode system with optical fibers for light delivery and an electrode array for neural recording. In vivo experiments with transgenic mice expressing channelrhodopsin-2 show the feasibility of the system for simultaneous optogenetic stimulation and electrophysiological recording.

用于同步光遗传学调制和电神经记录的光驱阵列

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective ...

Installation of Optical Windows in the Brain of Transgenic Mice to Image Neural Activities

Source: Lin, X., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56297">Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice.</a> J. Vis. Exp. (2019).
This video demonstrates the brain&#39;s neural activity recording through an optical window in transgenic mice. The mice express a fluorescent protein that enables the detection of neural activities. A small portion of the skull is carefully removed to install an optical window, and the skull is then covered with black cement to block light before imaging.

在转基因小鼠的大脑中安装光学窗口以对神经活动进行成像

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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