我们感谢 Puttemans、马克 de Paep、萨拉 de Pril、Wouter Depuydt、阿斯特丽德何曼思、Piet Kayenbergh、Gerrit Meulemans、克利斯朵夫 Ulens 和 Stijn Verstraeten 的技术和行政援助。

所有技术程序都是按照国家卫生研究院关于实验室动物的护理和使用指南和欧盟指令 2010/63/欧盟的规定进行的, 并得到了鲁汶的伦理委员会的批准。
1. 清醒行为猴细胞外记录的一般方法
<ol><li>训练动物执行你的具体研究问题所需的视觉和马达任务。确保动物能够在同一记录会话期间灵活地切换任务, 以便广泛地测试神经元, 并更好地了解驱动神经响应的特征 (图 2-3)。<ol>
<li>训练动物在视觉上被引导的抓住 (VGG; 抓住 ' 在光 ') 评估反应的 visuomotor 组分。注意: 独立于所选任务, 在训练阶段开始前至少三天内逐步限制液体摄入量。<ol>
<li>在实验课的整个时间内限制猴子的头部。</li>
			<li>在第一次疗程中, 将手对侧放在休息位置的录音室, 帮助动物到达并抓住物体, 每次尝试后给予人工奖励。</li>
			<li>在每次试验结束时, 把猴子的手放在休息的位置上。</li>
			<li>每几个试验, 松开猴子的手, 等待几秒钟观察, 如果动物自发地发起运动。</li>
			<li>当猴子到达目标时, 应用人工奖励。</li>
			<li>当获得正确的到达阶段, 帮助动物举起 (或拉扯) 对象和奖励手工。</li>
			<li>就像在1.1.1.4 和 1.1.1.5, 松开猴子的手, 等待几秒钟观察动物是否自发地启动运动。当运动正确执行时给予奖励。</li>
			<li>在手术过程中, 尽可能多地纠正到达、手部位置和手腕方向。</li>
			<li>重复以上步骤, 直到动物自动执行序列。</li>
			<li>加载自动任务。当动物在预定的时间执行到达和掌握动作时, 它会自动得到奖赏。</li>
			<li>逐渐增加对象的持有时间。</li>
			<li>介绍在物体底部投射固定点的激光。然后添加眼睛跟踪器, 以监视对象要掌握的眼睛位置。</li>
		</ol>
</li>
		<li>训练动物在记忆引导下抓取 (MGG) 来调查运动成分的反应, 不受刺激的视觉成分的影响。<ol>
<li>克制猴子的头。</li>
			<li>按照 VGG 描述的相同步骤, 确保动物在电子定义的窗口中的任务期间保持激光固定。对于这个版本的任务, 灯在固定期结束时熄灭。</li>
		</ol>
</li>
		<li>训练猴子被动固定, 以解决视觉反应和形状的选择性。<ol>
<li>克制猴子的头。</li>
			<li>使用 CRT (3D 刺激的被动固定) 或 LCD 显示器 (被动固定2D 刺激) 向猴子呈现视觉刺激。</li>
			<li>在屏幕中心显示固定点, 叠加在视觉刺激上。</li>
			<li>奖励动物在每次表现刺激之后, 逐渐增加固定期直到达到任务的标准。</li>
		</ol>
</li>
	</ol>
</li>
	<li>使用无菌工具, 窗帘和长袍进行手术。<ol>
<li>麻醉动物与氯胺酮 (15 毫克/千克, 肌肉) 和 medetomidine 盐酸 (0.01-0 04 毫升/千克肌肉), 并定期确认麻醉通过检查动物对刺激, 心率, 呼吸率和血液的反应压力。</li>
		<li>保持全身麻醉 (丙泊酚10毫克/千克/小时静脉注射), 并使用气管管来管理氧气。在麻醉下使用 lanolim 软膏防止眼部干燥。</li>
		<li>使用0.5 毫升丁丙诺啡 (静脉注射 0.3mg) 提供镇痛。在手术中心率增加的情况下, 可以进行额外的剂量治疗。</li>
		<li>用陶瓷螺钉和牙科丙烯酸植入 MRI 兼容头柱。在严格的无菌条件下执行所有的生存手术。为充分维护无菌领域, 使用一次性无菌手套, 口罩和无菌器械。</li>
		<li>以解剖学磁共振成像 (MRI) 为指导;霍斯利-克拉克坐标), 在感兴趣的地区做一个开颅手术, 并植入猴子头骨上的录音室。使用标准记录室进行单单元细胞外记录或多电极 microdrive, 用于同时记录多个神经元。</li>
		<li>手术后, 停止静脉注射丙泊酚, 直到自发呼吸恢复。不要让动物无人看管, 直到它恢复知觉并在社会群体中引入动物才完全康复。</li>
		<li>根据机构兽医的建议, 提供术后镇痛;例如使用 Meloxicam (5 毫克/毫升肌肉)。</li>
		<li>手术6周后再开始实验。这样可以更好地将头部贴在头骨上, 保证动物完全从干预中恢复。</li>
	</ol>
</li>
	<li>使用 MRI (用于单单元细胞外记录) 和计算机断层扫描 (CT; 用于多电极录音) 对记录区域进行本地化。<ol>
<li>用2% 硫酸铜溶液填充玻璃毛细血管, 并将其插入记录网格中。</li>
		<li>进行结构 MRI (切片厚度: 0.6 毫米)。</li>
	</ol>
</li>
	<li>神经活动的监测。<ol>
<li>使用阻抗为 0.8-1 MΩ的钨电极。</li>
		<li>使用23G 不锈钢导水管和液压 microdrive 将电极通过硬脑膜插入。</li>
		<li>对于穗识别, 放大和过滤300和5000赫兹之间的神经活动。</li>
		<li>对于局部场电位 (LFP) 记录, 放大并过滤1至170赫兹之间的信号。</li>
	</ol>
</li>
	<li>监视眼睛信号<ol>
<li>在动物的眼睛前调整红外线摄像头, 以获得足够的瞳孔图像和角膜反射。</li>
		<li>使用红外线摄像头在500赫兹的瞳孔位置取样。</li>
	</ol>
</li>
</ol>2. 研究背部区域的对象选择性
<ol><li>执行视觉引导抓取 (VGG)。<ol>
<li>根据研究的目标选择正确的抓取设置: 旋转木马安装或机器人安装 (图 3)。</li>
		<li>对于旋转木马安装程序, 运行 VGG 任务:<ol>
<li>让猴子把手对在完全黑暗中的静止位置的记录半球, 以启动序列。</li>
			<li>在可变时间之后 (intertrial 间隔: 2,000-3, 000 毫秒), 应用红色激光 (固定点) 在对象的基地 (距离:28 cm 从猴子的眼睛)。如果动物保持它的凝视在电子定义的固定窗口 (+/-2.5°) 为 500 ms, 照亮对象从上面与光源。</li>
			<li>在可变延迟 (300-1500 毫秒) 后, 程序的激光调光 (视觉去提示) 指示猴子从静止位置抬起手, 并达到, 抓住和保持对象的可变时间间隔 (持有时间: 300-900 毫秒)。</li>
			<li>当动物正确地执行整个序列时, 用一滴果汁奖励它。</li>
		</ol>
</li>
		<li>对机器人设置使用类似的任务序列。<ol>
<li>至于旋转木马的设置, 让猴子把手对到记录半球在完全黑暗的静止位置, 以启动序列。</li>
			<li>在可变时间之后 (intertrial 间隔: 2,000-3, 000 毫秒), 照亮对象上的 LED (固定点) (从内; 距离:28 厘米从猴子的眼睛)。同样, 如果动物保持它的凝视在电子定义的固定窗口 (+/-2.5°) 为 500 ms, 照亮对象从内部与白色光源。</li>
			<li>在可变延迟 (300-1500 毫秒) 后, 关掉 LED (视觉去提示), 指示猴子从静止位置抬起手, 到达, 抓住并按住对象的可变间隔 (保持时间: 300-900 毫秒)。</li>
			<li>当动物正确地执行整个序列时, 用一滴果汁奖励它。</li>
		</ol>
</li>
		<li>在任务期间, 量化猴子的表现, 特别注意时间。测量在走信号和手运动开始 (反应时间) 之间的时间, 以及运动和物体升力之间的距离 (抓住时间)。</li>
	</ol></li>
	<li>执行记忆引导抓取 (MGG;"抓住黑暗")。使用 MGG 任务确定神经元是 visuomotor 的还是以运动为主的。 注意: 该序列类似于 VGG 所描述的顺序, 但对象在完全黑暗中被抓住。<ol>
<li>与 VGG 任务相同的是, 让猴子把手对在完全黑暗的静止位置上的记录半球开始序列。</li>
		<li>在可变时间之后 (intertrial 间隔: 2,000-3、000毫秒), 应用红色激光/LED (固定点) 表示固定点 (在对象的底部为旋转木马设置, 在对象的中心为机器人设置; 距离: 从猴子的眼睛28厘米).如果动物保持它的凝视在电子定义的固定窗口 (+/-2.5°) 为500毫秒, 照亮对象。</li>
		<li>经过固定的时间 (400 毫秒), 关掉灯。</li>
		<li>在一个可变延迟期 (300-1500 毫秒) 后, 光偏移, 昏暗/关闭固定点 (去提示), 以指示猴子举起手和到达, 抓住, 并持有的对象 (持有时间: 300-900 毫秒)。</li>
		<li>每当动物正确地执行整个序列, 给一滴果汁作为奖励。</li>
	</ol></li>
	<li>进行被动固定。至于 VGG 任务, 根据研究的目标选择最合适的设置 (旋转木马或机器人设置)。 注意: 可以执行两种不同的被动固定任务: 现实世界物体的被动固定 (使用在旋转木马和机器人设置中的对象) 和被动固定3维/2 维图像的物体。<ol>
<li>对现实世界中的物体进行被动固定。<ol>
<li>目前的固定点 (红色激光为旋转木马设置投射在对象的底部和红色 LED 在机器人设置)。</li>
			<li>如果动物保持它的凝视在电子定义的固定窗口 (+/-2.5°) 为 500 ms, 照亮对象为 2000 ms。</li>
			<li>如果动物保持它的凝视在窗口内为1000毫秒, 奖励它与一滴果汁。</li>
		</ol>
</li>
		<li>对物体进行3维/2 维图像的被动固定。<ol>
<li>在黑色背景上呈现所有视觉刺激 (亮度为 8 cd/米2), 使用显示器 (分辨率为 1280 x 1024 像素), 配有快速衰减 P46-phosphor, 并在120赫兹 (查看距离:86 厘米) 操作。</li>
			<li>在3D 测试中, 通过在显示器 (CRT 显示器) 上交替左、右眼图像, 结合两个铁电液晶百叶窗, 呈现刺激立体感。找到这些百叶窗在猴子的眼睛前面, 操作在60赫兹和同步到显示器的垂直折回。</li>
			<li>通过在屏幕中央显示一个小正方形 (固定点; 0.2° x 0.2°) 开始试用。如果眼睛位置保持在电子定义的1°正方形窗口 (比实际对象小得多) 至少500毫秒, 则在屏幕上呈现视觉刺激, 总时间为500毫秒。</li>
			<li>当猴子保持稳定的固定, 直到刺激抵消, 奖励它一滴果汁。</li>
			<li>为了对形状选择性进行充分的研究, 在被动固定任务中, 在以下序列中运行一组2D 图像的综合测试电池。</li>
			<li>运行搜索测试。使用一组广泛的图像 (表面图像) 测试单元格的视觉选择性;图 4A), 包括在 VGG 中掌握的对象的图片。对于这个和所有后续的视觉任务, 将最强响应 (称为 "首选图像") 的图像与神经元响应较弱的第二个图像 (称为 "nonpreferred 图像") 进行比较。如果研究中的神经元也对物体的图像作出反应, 则寻找驱动细胞反应性的特定刺激成分 (轮廓测试、接受田间试验和还原试验)。</li>
			<li>运行轮廓测试。从真实物体的原始表面图像 (包含纹理、阴影和透视的2D 或3D 图像), 获得相同刺激形状 (剪影和轮廓) 的渐进简化版本;图 4B)。每个条件收集至少10个试验, 以确定神经元是否喜欢原始的表面, 剪影或轮廓从原始的形状。</li>
			<li>运行一个接受域 (RF) 测试。为了映射神经元的 RF, 在显示器上显示不同位置的物体的图像 (在此实验中, 35 位置; 3°的刺激大小), 覆盖中央视野19,20。在合理的时间内在所有可能的位置收集足够的刺激重复, 减少刺激持续时间 (闪光刺激; 刺激持续时间: 300 毫秒, intertrial 间隔: 300 毫秒)。</li>
			<li>运行还原测试。使用在 RF 中心显示的轮廓碎片进行还原测试, 以确定最小有效形状特征 (MESF)。通过裁剪主坐标轴上每个原始轮廓形状的轮廓 (图 3B), 在 Photoshop 中生成一组刺激。将 MESF 设计为最小的形状片段, 它唤起的响应至少是完整的轮廓响应的 70%, 而不是显着小于该响应8。</li>
			<li>为了更好地估计位置依赖性 (刺激位置对片段选择性的影响), 运行两个不同的测试。使用位于原始大纲形状中所占位置的碎片运行缩减测试。在形状的质心处运行还原测试。</li>
			<li>在此阶段, 使用 MESF 运行一个新的 RF 映射。</li>
		</ol>
</li>
	</ol></li>
</ol>

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作者没有什么可透露的。

对背流进行研究的综合方法需要仔细选择行为任务和视觉测试: 视觉和抓取范式可以根据区域的特定属性组合或单独使用。
在这篇文章中, 我们提供的例子, 神经活动记录在 F5p 和在响应一个子集的视觉和运动任务, 但非常相似的反应可以观察到其他前沿领域, 如45B 区和 F5a。
我们提出了两个实验装置来研究物体在抓取过程中的神经表现。用垂直旋转传送带 (图 3A) 包含多达六个对象, 我们可以向猴子呈现不同的对象。旋转传送带允许不同的目标对象 (形状、大小、体积等) 的呈现, 要求不同的抓取策略 (动力手柄与精密手柄)。
第二个更复杂的方式来呈现对象在单细胞录音是通过商业机器人手臂和爪 (图 3B)。在这种情况下, 机器人通过抓取物体 (图 3B) 来启动试验, 并将其移动到空间中的特定位置, 在整个黑暗中, 而猴子的手停留在静止的位置。此外, 事件序列在两个设置中是相同的。然而, 机器人的使用允许对实验参数进行广泛的操作 (对象的距离, 在 frontoparallel 平面中的位置, 或物体的方向)。最后, 如图 3B的右面板所示, 机器人也可以被编程来掌握不同的物体 (在我们的情况下, 板和球体)。
这一实验方法允许确定驱动 visuomotor 神经元在抓取过程中对物体观察反应的目标特征。但是, 这种方法也有局限性。每次测试, 一些神经元将被排除在进一步的测试中 (例如,没有对对象的图像的响应, 没有轮廓选择性), 因此实验的结论只能与显示任务相关的所有神经元的子集有关。在抓取过程中的活动。然而, 在我们以前的研究8, 大多数 (83%) 的神经元显示视觉反应的对象观察在抓取期间也有选择地响应对象的图像, 和大部分的后神经元 (90%)对这些图像的轮廓版本也有选择性。因此, 我们的测试协议可能是适当的, 很大一部分的所有视觉应答神经元的顶叶和额叶皮质。
一些 visuomotor 神经元, 最有可能在与运动相关的脑部皮层, 如区域 F5p, 可能只响应对象在抓取任务的背景下, 从来没有回应的图像的对象 (即使与双目视差) 呈现在显示。尽管如此, 我们还是可以利用机器人来研究这些神经元的特性。通过这个实验装置, 我们可以在 frontoparallel 平面的被动固定 (类似于射频测试), 在不同的3D 方向和不同距离的动物的不同位置呈现对象, 我们可以结合扫视眼睛运动朝向对象与对象抓住21。
我们的目的不是为研究顶额神经元提供单一或刚性的实验性协议, 而是强调必须采取全面和动态的方法, 并为研究中的神经元专门设计的任务和测试。例如, 关于视觉选择性, 我们的协议可以很容易地适应于研究神经元对物体的其他视觉特性。例如, 我们遵循了一个非常相似的方法, 当调查的3D 选择性12和 F5a 神经元13在抓取期间。我们还结合抓取执行和详细的视觉测试与行动视频, 在调查的行动观察反应在22。同样, 许多其他实验任务, 不包括在这里, 也可以添加到我们的协议, 取决于要解决的科学问题。这些任务包括研究刺激的纯粹物理特征 (例如,刺激大小) 和认知方面, 如刺激熟悉23或生物相关性 (偏爱的形状, 是生物相关的如面孔24)。
在这些领域的进一步研究将使人们更好地了解网络, 并将使我们能够改进所使用的协议类型。

人类和非人类灵长类动物拥有执行复杂运动动作的能力, 包括物体抓取。为了成功地完成这些任务, 我们的大脑需要完成将内部对象属性转换为马达命令。这种转变依赖于位于顶叶和腹侧前运动神经皮层1,2,3 (图 1) 的背皮层区域的复杂网络。
从猴子和人类的病变研究4,5, 我们知道, 背部视觉流-起源于初级视觉皮层和定向到后顶叶皮层-参与了空间视觉和运动规划行动。然而, 大多数的背流区域并不专门用于处理独特的类型。例如, 前 intraparietal 区 (本) 是背视流的终末期区域之一, 它包含了各种神经元, 不仅在抓取6、7、8期间, 而且在视觉检查对象7,8,9,10。
类似于 F5, 位于腹前运动神经皮层 (PMv) 的神经元, 在视觉固定和对象抓取过程中也有反应, 这对于将视觉信息转化为运动动作11是很重要的。这个区域的前部 (界别分组 F5a) 包含有选择性地响应三维 (3D, 视差定义) 图像12,13, 而位于凸性 (F5c) 中的界别分组包含神经元特征为镜子属性1,3, 射击时, 动物执行或观察行动。最后, 后 F5 区 (F5p) 是一个与手相关的领域, 有很大比例的 visuomotor 神经元响应观察和掌握3D 对象14,15。F5, 面积 45B, 位于弧形沟的下支, 也可能涉及两个形状处理16,17和抓取18。
在顶叶和额叶皮层检测对象选择性是很有挑战性的, 因为很难确定这些神经元对哪些功能做出反应, 以及这些神经元的接受领域是什么。例如, 如果一个神经元响应一个板块, 而不是一个锥, 这些物体的特征是驱动这种选择性: 2D 轮廓, 3D 结构, 深度方向, 或多种不同特征的组合？为了确定在对象固定和抓取过程中响应的神经元的关键对象特征, 需要使用对象的图像和相同图像的缩小版本来进行各种视觉测试。
在 F5 和小鼠的神经元中, 有相当一部分不仅对物体的视觉呈现做出反应, 而且当动物在黑暗中抓住这个物体时 (即在没有视觉信息的情况下)。这种神经元可能不会对无法掌握的物体的图像作出反应。因此, 视觉和运动成分的反应是紧密相连的, 这使得它很难研究神经元对象表示在这些地区。由于 visuomotor 神经元只能用真实世界的物体来测试, 所以我们需要一个灵活的系统, 在不同的位置在视觉领域和不同的方向呈现不同的物体, 如果我们想确定哪些特征对这些都是重要的。神经元。后者只能通过能够在视觉空间的不同位置呈现不同物体的机器人来实现。
本文旨在为研究顶额神经元的研究者提供一个实验指南。在以下各节中, 我们将提供实验室中用于分析醒猴猴 (猕猴猕猴) 抓取和视觉对象反应的一般协议。

<table><tbody><tr><td>Grasping robot</td><td>GIBAS Universal Robots</td><td>UR-6-85-5-A</td><td>Robot arm equipped with a gripper</td></tr><tr><td>Carousel motor</td><td>Siboni</td><td>RD066/&amp;dagger;20 MV6, 35x23 F02</td><td>Motor to be implemented in a custom-made vertical carousel. It allows the rotation of the carousel.</td></tr><tr><td>Eye tracker</td><td>SR Research</td><td>EyeLink II</td><td>Infrared camera system sampling at 500 Hz</td></tr><tr><td>Filter</td><td>Wavetek Rockland</td><td>852</td><td>Electronic filters perform a variety of signal-processing functions with the purpose of removing a signal&#039;s unwanted frequency components.</td></tr><tr><td>Preamplifier</td><td>BAK ELECTRONICS, INC.</td><td>A-1</td><td>The Model A-1 allows to reduce input capacity and noise pickup and allows to test impedance for metal micro-electrodes</td></tr><tr><td>Electrodes</td><td>FHC</td><td>UEWLEESE*N4G</td><td>Metal microelectrodes (* = Impedance, to be chosen by the researcher)</td></tr><tr><td>CRT monitor</td><td>Vision Research Graphics</td><td>M21L-67S01</td><td>The CRT monitor is equipped with a fast-decay P46-phosphor operating at 120 Hz</td></tr><tr><td>Ferroelectric liquid crystal shutters</td><td>Display Tech</td><td>FLC Shutter Panel; LV2500P-OEM</td><td>The shutters operate at 60 Hz in front of the monkeys and are synchronized to the vertical retrace of the monitor</td></tr></tbody></table>

investigating object representations in the macaque dorsal visual stream using single-unit recordings

以前的研究表明, 猕猴大脑顶额区的神经元可以高度选择性地进行现实世界中的物体、视差定义的曲面以及真实世界物体 (无差异) 的图像, 类似于在腹视流中描述。此外, 顶锋区域被认为可以将视觉对象信息转换成适当的马达输出, 如抓取时手的预整形。为了更好地描述 visuomotor 变换所涉及的皮层网络中的对象选择性, 我们提供了一个测试电池, 目的是分析顶额区域神经元的视觉对象选择性。

图 5绘制了一个示例神经元的响应, 从区域 F5p 测试了四个对象: 两个不同的形状-一个球体和一个板块-显示两个不同的大小 (6 和3厘米)。这个特殊的神经元不仅响应大球体 (最佳刺激; 左上板), 而且还对大板 (左下板)。相比之下, 对较小对象的响应较弱 (右上角和下面板)。
图 6显示了在 VGG 和被动固定中测试的一个神经元的例子。这个神经元不仅在抓取 (VGG 任务, 面板 A), 而且对屏幕上显示的物体的2D 图像的视觉呈现 (被动固定包括在抓取任务中使用的对象的图片) 的响应;图 6B)。请注意, 被动固定任务中的首选刺激方案不一定是要掌握的对象, 而是另一种2D 图片, 动物没有以前的抓取经验 (橙色)。图 6C显示了使用首选和 nonpreferred 图像进行测试时该单元格的 RF。在还原测试中获得的响应示例如图 6D所示。这个例子神经元响应测试中的最小片段 (1 1.5°)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57745/57745fig1v2.jpg"> 图1。顶前端网络涉及视觉对象的处理和电机的规划和执行.后顶叶区的项目到区域 PFG, 45B 和 F5a, 然后 F5p, M1, 最后, 到脊髓。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57745/57745fig2v2.jpg"> 图2。用于测试对象选择性的决策树: 用于测试神经元群中 visuomotor 反应的实验性协议.VGG 任务可以跟随 MGG 或视觉任务 (被动固定)。两种不同的被动固定任务可以根据感兴趣的区域来考虑: 现实世界的被动固定和2D 物体图像的被动固定。灵长类 visuomotor 系统进化来支持对真实物体的操控, 而不是对象6、13的图像, 因此, 预测那些具有马达主导成分的区域将会显著地对真实的, graspable 的物体的视觉反应。但是, 只有使用减少方法来详细探讨形状选择性, 这样可以更容易地实现与对象的图像。在2D 被动固定的任务中, 一个积极的答案 (表明对物体图像的视觉选择性) 意味着可以进一步改善神经元的反应。这导致我们运行一个新的实验任务, 探索较低水平的特点, 在刺激。相反, 负面反应表明实验的结束。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57745/57745fig3v2.jpg"> 图3。Visuomotor 设置.(A).旋转木马设置。左面板: 旋转木马设计 (猴子看不见)。右面板: 旋转板的细节显示对象被抓住和猴子手接近它。有一个垂直旋转传送带包含多达六个对象, 我们可以向猴子呈现不同的对象。(B)机器人安装。左面板: 机器人安装的前视图。右面板: 机器人提供的四种不同物体的细节 (小/大板; 小/大球体)。第二个更复杂的方式来呈现的对象在单细胞录音是通过一个商业机器人手臂配备了爪。对于 A 和 B, 在视觉固定过程中, 事件序列是相同的, 但在旋转木马设置中, 对象从上面和机器人设置中被照亮, 对象从内部被照亮。在把握阶段, 任务略有不同。而在旋转木马设置中, 去提示是由激光调光指示;在机器人安装中, 固定指示灯完全关闭。另一个区别是指两个设置的特定功能。虽然旋转木马的设置可以主要用于测试对象的选择性在一个独特的位置在视觉空间, 与机器人设置, 我们可以编程的距离, 对象被抓住, 在 frontoparallel 平面上的位置, 甚至诱发抓取过程中物体方向的扰动 (例如,物体在到达阶段的快速45°旋转)。这两个系统允许不同的目标对象的表现, 不同的抓取性质 (大小, 体积等), 要求不同的抓取策略 (功率握与精确握)。(C). VGG 任务 (旋转木马设置) 的示例。1. 固定: 在我们的旋转木马 VGG 任务中, 猴子将其对侧的手放在静止位置装置上以启动序列。接下来, 将激光投射到要被抓住的物体上, 这仍然是完全黑暗的。2. 光照: 如果动物在围绕物体的电子定义窗口周围保持稳定的固定时间, 则该物体由外部光源 (任务的视觉相位) 照亮。最后, 在变量延迟后, 激光变暗, 作为视觉去线索, 并指示猴子启动抓取动作。该动物得到奖励, 以达到, 抓住和举起的对象 (由光纤电缆检测)。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57745/57745fig4v2.jpg"> 图4。视觉刺激。(A).用于评估视觉形状选择性的刺激集的示例。(B).从最初的表面图像, 我们产生的视觉刺激 (3D 表面, 2D 表面, 剪影, 轮廓和碎片) 逐步简化版本。通过在较小的段中划分轮廓, 我们搜索最小有效形状特征 (MESF) 唤起视觉选择性。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57745/57745fig5v2.jpg"> 图5。VGG 任务测试与机器人安装 (机器人安装在图 3B).我们提出了四不同的对象在同一位置的深度: 大球体 (左上), 大板 (左下), 小板 (右下), 和小球体 (右上角)。神经元响应与对象中的光起始对齐 (纸盒大小为20毫秒)。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57745/57745fig6v2.jpg"> 图6。用 VGG (抓取旋转木马) 和被动固定任务记录的脑功能神经元。(A)在抓取过程中的活动。Peristimulus-时间直方图, 显示了一个脑电图神经元的反应 (神经元响应对对象的光开始)。(B)同一神经元的视觉反应, 当测试与一组广泛的2D 图像的真实世界的对象, 包括一个图像的对象要掌握 (在两个不同的方向: 水平与垂直)。(C).接受字段映射。2D 插值映射, 表示对 A 和 B 中神经元的首选 (左) 和 nonpreferred (右) 刺激的平均响应, 当测试时, 3°图像的对象。为了构建地图, 我们量化了网络神经元响应 (通过减去基线活动) 获得的屏幕上的35个不同位置 (由虚线网格线的交叉点表示;[00]: 中央位置;+ 6°方位: 对侧), 间隔2°分开, 覆盖信息学和对侧的视觉 hemifields。颜色表示神经反应的强度 (变化在0和细胞的最大响应之间)。(D).颜色树图, 表示同一神经元的归一化净响应 (发射率减去基线活动), 如图 6A-C中的首选和 nonpreferred 刺激 (首选和 nonpreferred 图像的轮廓)在标准的减少试验 (减少测试与片断位于被占领的位置在原始的外形形状; 4 片断刺激, 第一排; 8 片段刺激, 第二排; 16 片段刺激, 第三排)。每个圆圈中的颜色表示响应大小 (1 = 28 峰值/秒)。

Watch this Scientific Journal Video about Investigating Object Representations in the Macaque Dorsal Visual Stream Using Single-unit Recordings at JoVE.com

Investigating Object Representations in the Macaque Dorsal Visual Stream Using Single-unit Recordings

本文给出了 visuomotor 变换中顶-额神经元的对象选择性分析的详细协议。

用单单元记录法研究猕猴背视流中的对象表示

Previous studies have shown that neurons in parieto-frontal areas of the macaque brain can be highly selective for real-world objects, disparity-defined ...

investigating-object-representations-macaque-dorsal-visual-stream

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

8.2K 次观看.  Katholieke Universiteit Leuven. 本文给出了 visuomotor 变换中顶-额神经元的对象选择性分析的详细协议。

视频: Investigating Object Representations in the Macaque Dorsal Visual Stream Using Single-unit Recordings

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

单机在体内</em从大鼠视交叉>录音

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

科研

神经科学

A Pressure Injection System for Investigating the Neuropharmacology of Information Processing in Awake Behaving Macaque Monkey Cortex

The top-down modulation of feed-forward cortical information processing is functionally important for many cognitive processes, including the modulation of sensory information processing by attention. However, little is known about which neurotransmitter systems are involved in such modulations. A practical way to address this question is to combine single-cell recording with local and temporary neuropharmacological manipulation in a suitable animal model. Here we demonstrate a technique combining acute single-cell recordings with the injection of neuropharmacological agents in the direct vicinity of the recording electrode. The video shows the preparation of the pressure injection/recording system, including preparation of the substance to be injected. We show a rhesus monkey performing a visual attention task and the procedure of single-unit recording with block-wise pharmacological manipulations.

Here, we show the pressure injection of neuropharmacological substances during single-cell recording in an awake, behaving macaque monkey. This procedure allows pharmacological manipulation in the direct vicinity of a cortical recording site.

对调查信息处理的神经药理学清醒行为猕猴皮质压力喷射系统

The top-down modulation of feed-forward cortical information processing is functionally important for many cognitive processes, including the modulation ...

行为学

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

多层皮质Ca<sup&gt; 2+</sup&gt;使用棱镜探针和小型荧光显微镜成像自由移动的小鼠

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

移动硅探针的神经行为鼠害的大型记录

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Simultaneous Eye Tracking and Single-Neuron Recordings in Human Epilepsy Patients

Intracranial recordings from patients with intractable epilepsy provide a unique opportunity to study the activity of individual human neurons during active behavior. An important tool for quantifying behavior is eye tracking, which is an indispensable tool for studying visual attention. However, eye tracking is challenging to use concurrently with invasive electrophysiology and this approach has consequently been little used. Here, we present a proven experimental protocol to conduct single-neuron recordings with simultaneous eye tracking in humans. We describe how the systems are connected and the optimal settings to record neurons and eye movements. To illustrate the utility of this method, we summarize results that were made possible by this setup. This data shows how using eye tracking in a memory-guided visual search task allowed us to describe a new class of neurons called target neurons, whose response was reflective of top-down attention to the current search target. Lastly, we discuss the significance and solutions to potential problems of this setup. Together, our protocol and results suggest that single-neuron recordings with simultaneous eye tracking in humans are an effective method to study human brain function. It provides a key missing link between animal neurophysiology and human cognitive neuroscience.

We describe a method to conduct single-neuron recordings with simultaneous eye tracking in humans. We demonstrate the utility of this method and illustrate how we used this approach to obtain neurons in the human medial temporal lobe that encode targets of a visual search.

人类癫痫患者同步眼动追踪和单神经记录

Intracranial recordings from patients with intractable epilepsy provide a unique opportunity to study the activity of individual human neurons during ...

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

自由移动鸽子穿越三个阶段学习记录单个神经元&#39;动作电位

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal&#8217;s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.

This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

警报，头抑制大鼠子皮层的大脑结构中Juxtacellular监测和本地化单神经元

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures ...

Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a comprehensive survey of the morphological characteristics observed among a selective neuronal population. Combination of these techniques constitutes a novel approach for the collection and analysis of neuroanatomical data. Together, these techniques enable large-scale, and therefore more comprehensive, sampling of selective neuronal populations and establish unbiased quantitative methods for describing morphologically unique neuronal classes within a population.
The protocol outlines the use of modified rabies virus to selectively label neurons. G-deleted rabies virus acts like a retrograde tracer following stereotaxic injection into a target brain structure of interest and serves as a vehicle for the delivery and expression of EGFP in neurons. Large numbers of neurons are infected using this technique and express GFP throughout their dendrites, producing &#34;Golgi-like&#34; complete fills of individual neurons. Accordingly, the virus-mediated retrograde tracing method improves upon traditional dye-based retrograde tracing techniques by producing complete intracellular fills.
Individual well-isolated neurons spanning all regions of the brain area under study are selected for reconstruction in order to obtain a representative sample of neurons. The protocol outlines procedures to reconstruct cell bodies and complete dendritic arborization patterns of labeled neurons spanning multiple tissue sections. Morphological data, including positions of each neuron within the brain structure, are extracted for further analysis. Standard programming functions were utilized to perform independent cluster analyses and cluster evaluations based on morphological metrics. To verify the utility of these analyses, statistical evaluation of a cluster analysis performed on 160 neurons reconstructed in the thalamic reticular nucleus of the thalamus (TRN) of the macaque monkey was made. Both the original cluster analysis and the statistical evaluations performed here indicate that TRN neurons are separated into three subpopulations, each with unique morphological characteristics.

This protocol describes large-scale reconstructions of selective neuronal populations, labeled following retrograde infection with a modified rabies virus expressing fluorescent markers, and independent, unbiased cluster analyses that enable comprehensive characterization of morphological metrics among distinct neuronal subclasses.

基于形态学度量大规模重建和独立，公正聚类分类神经元选择性人群

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a ...

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 ...

用于小鼠电生理学的经济高效、多功能探针植入系统

The DREAM Implant: A Lightweight, Modular, and Cost-Effective Implant System for Chronic Electrophysiology in Head-Fixed and Freely Behaving Mice

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. However, current methods for chronically implanting probes present steep trade-offs between cost, ease of use, size, adaptability, and long-term stability.
This protocol introduces a novel chronic probe implant system for mice called the DREAM (Dynamic, Recoverable, Economical, Adaptable, and Modular), designed to overcome the trade-offs associated with currently available options. The system provides a lightweight, modular and cost-effective solution with standardized hardware elements that can be combined and implanted in straightforward steps and explanted safely for recovery and multiple reuse of probes, significantly reducing experimental costs.
The DREAM implant system integrates three hardware modules: (1) a microdrive that can carry all standard silicon probes, allowing experimenters to adjust recording depth across a travel distance of up to 7 mm; (2) a three-dimensional (3D)-printable, open-source design for a wearable Faraday cage covered in copper mesh for electrical shielding, impact protection, and connector placement, and (3) a miniaturized head-fixation system for improved animal welfare and ease of use. The corresponding surgery protocol was optimized for speed (total duration: 2 h), probe safety, and animal welfare.
The implants had minimal impact on animals&#39; behavioral repertoire, were easily applicable in freely moving and head-fixed contexts, and delivered clearly identifiable spike waveforms and healthy neuronal responses for weeks of post-implant data collection. Infections and other surgery complications were extremely rare.
As such, the DREAM implant system is a versatile, cost-effective solution for chronic electrophysiology in mice, enhancing animal well-being, and enabling more ethologically sound experiments. Its design simplifies experimental procedures across various research needs, increasing accessibility of chronic electrophysiology in rodents to a wide range of research labs.

作者聚焦:通过虚拟现实和动物电生理学了解自然神经元计算

Here, we introduce a lightweight, cost-effective probe implant system for chronic electrophysiology in rodents optimized for ease of use, probe recovery, experimental versatility, and compatibility with behavior.

DREAM 植入物:一种轻便、模块化且经济高效的植入系统，用于头部固定和行为自由的小鼠的慢性电生理学

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. ...

用单单元记录法研究猕猴背视流中的对象表示

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