作者感谢波莱特和丽莎 Pezzino 在这项研究中为秘书提供支持, 菲尔奥尔巴赫先生为技术支持。作者还感谢 Drs 和迈克尔. 琼斯 (圣路易斯大学医学院) 介绍我们到体外孤立的头部准备。支持这一合作的资金是由生物学系 (罗伯特-s 大通基金)、学术研究委员会和拉斐特大学的神经科学项目提供的。最后, 这项工作献给2016年9月28日去世的菲尔奥尔巴赫先生;他退役了扫描电子显微镜, 并确认其5轴阶段的用处, 以便在本议定书中使用。他的友谊和足智多谋将被大大地怀念。

注: 红耳滑块龟, 男性和女性, 从供应商购买。海龟被安置在一个温暖的动物套房里, 里面有两个60加仑的浴缸, 装有砖群岛, 在 250 W 红外线灯下晒黑。环境保持在一个14/10 小时的光/暗循环, 水温在22摄氏度。灯在凌晨6:00 打开, 在下午8:00 关闭。装有过滤系统的坦克每周都被清理干净, 海龟每隔一天就被喂食ad 随意。对红耳滑块龟的护理和以下所描述的所有实验过程32,33是批准的机构动物护理和使用委员会 (IACUC) 在拉斐特学院。
1. 设备设置
<ol><li>准备龟铃的解决方案。按此顺序将以下内容添加到蒸馏水中: 氯化钠96.5 毫米 (58.44 克/摩尔), 氯化钾2.6 毫米 (74.56 克/摩尔), 氯化镁2.0 毫米 (203.31 克/摩尔), 碳酸氢钠31.5 毫米 (84.01 克/摩尔), 葡萄糖20.0 毫米 (180.16 克/摩尔), 浓缩盐酸调整 pH 值为 7.51, 氯化钙4.0 毫米 (110.98 克/摩尔) (见材料表)。在添加每个盐时混合溶液。 注意: 浓缩盐酸是危险的 (皮肤, 眼睛, 吸入和摄取危险)。</li>
	<li>从5厘米长的毛细管玻璃 (参见材料表) 中, 通过对不同尺寸的内径进行火焰抛光, 以适应不同厚度的颅神经, 给出吸入电极的提示。<ol>
<li>用一个小文件在一条毛细管玻璃上蚀刻一条线。放置在纸巾和打破一半。</li>
		<li>慢慢地将毛细管玻璃的一端滚入本生燃烧器的火焰中。使用解剖范围和光纤光源定期检查刀尖的大小、平滑度和对称性 (请参阅材料表)。 注: 对于头部宽度介于20和30毫米之间的龟, 最佳拟合内径大小通常范围从0.4 到0.8 毫米为动眼神经神经 (nIII), 0.3 至0.6 毫米为滑车神经 (), 0.2 至0.4 毫米为展神经 (nVI)。</li>
	</ol>
</li>
	<li>清洁和组织 Rongeurs, 钝解剖探针, microscissors, 细钳, 弯曲钳, 和刀柄与刀片 (见材料表) 的解剖。 注: 器械灭菌是可选的。</li>
</ol>2. 麻醉和安乐死
<ol><li>把海龟放在冰桶里60分钟 cryoanesthetize。</li>
	<li>弄死龟通过斩首使用一个小动物断头台 (见材料表)。<ol>
<li>用小的称量铲轻轻地撬开动物的颚, 这样钩子就可以插入, 并在下颚尖下转动。</li>
		<li>用稳定的压力拉动动物的头部通过断头台。迅速斩首的动物。</li>
	</ol>
</li>
	<li>把放在解剖盘里。有足够的海龟手的解决方案, 以灌溉组织。Oxygenize 95/5% O2/CO2的解决方案 (请参见材料表)。</li>
	<li>在盘子外面放置冰块, 保持4摄氏度的组织。</li>
</ol>3. 解剖
<ol><li>利用解剖范围和光纤光源进行解剖。</li>
	<li>取下下颌。在口腔中放置一个钝的解剖探针, 以提供更容易的头部处理。用手术刀把齿骨骨连接到颅骨上。用 rongeurs 把下颚从颅骨上拉出来。使用 rongeurs 的皮肤和肌肉从他们的附件在颅骨的背部和侧面区域。</li>
	<li>取下脊椎柱。<ol>
<li>识别颅骨尾部的椎柱。弯曲椎柱腹部以暴露脊髓。用 microscissors 剪脊髓。使用 rongeurs 从颅骨中取出椎柱和其他组织, caudally。</li>
	</ol>
</li>
	<li>切断颅神经后从颅骨中取出大脑。<ol>
<li>从孔大口径开始, 用 rongeurs 在背颅骨上切开两个切口。使伤口表面避免损害大脑下方。</li>
		<li>用 rongeurs 小心拉下背颅骨。使用 microscissors 移除脑膜, 以暴露其余的大脑。删除足够的脑膜, 直到嗅球, 在前颅腔, 可以识别 (见图 1A)。如有必要, 继续用龟铃的解决方案来灌溉大脑。</li>
		<li>使用弯曲钳轻轻拉大脑 caudally 和产生轻微的紧张的脑神经。小心地切断和删除的嗅觉灯泡和大脑与弯曲钳。</li>
		<li>用 microscissors 轻轻地将中脑推向中线, 露出颅神经;nIII, 大约0.6 毫米, 可以看到在前的《圣经》, 和 nIII 的直径将略小于。在它们附加到中脑的地方剪切 nIII 和. 在另一侧重复此操作。</li>
		<li>用 microscissors 切开左、右视神经 (nII)。然后将脑干倾斜到一侧。观察 nVI 从桥脑和髓质交界附近的腹面出现 (见图 1C);nVI 直径小, 约0.3 毫米左右 nVI。</li>
		<li>用细钳和 microscissors 从龟中取出脑干的其余部分。一旦颅骨空了, 检查颅腔地板。确定 nIII、nVI 和。</li>
	</ol>
</li>
	<li>用细钳和 microscissors 去除上、下眼盖。</li>
</ol>4. 眼部运动的校准
<ol><li>使用刚性表 (请参阅材料表) 以支持框架和其他工具的放置。将放到框架卡盘中 , 使头部的背表面平行于地平线 , 用一个小气泡级在头骨上休息。大致定位在框架的水平和垂直旋转中心的眼睛之一。</li>
	<li>将红外线摄像机安装在红外发光二极管 (LED) 上, 它是基于视频的眼球跟踪系统的一部分 (参见材料表), 距离龟眼大约12厘米。<ol>
<li>把相机的角度看45度以上的眼睛视线。LED 应在11点位置时, 看相机镜头。将 LED 沿眼睛的光轴居中。相机将略微偏离轴 (从眼睛上方看)。</li>
		<li>调整相机的距离从眼睛, 以便相机的看法是最大的填充眼球。确保眼睛的角 (canthi) 位于水平视图的边缘。</li>
	</ol>
</li>
	<li>将摄像机连接到基于视频的眼睛跟踪系统来处理数据。将信号分割成 DVD 录音机以捕获原始视频。聚焦相机, 以获得清晰的眼睛图像。注意使用三度的线性调整 (x, y, z) 与框架提供的眼睛在相机视图的中心位置。</li>
	<li>使用视频跟踪系统所提供的程序, 通过设置阈值和对比度来检测暗瞳孔。<ol>
<li>使用鼠标, 点击 "视频" 菜单和 "模式" 选择 "高精度", 以捕获图像的采样率为30赫兹 (分辨率为640像素 x 480 线)。此外, 在 "视频" 下, 为 "瞳孔类型" 和 "椭圆 (旋转椭圆)" 选择 "暗瞳孔" 作为 "瞳孔分割法"。</li>
		<li>在 "EyeCamera" 窗口中, 单击 "瞳孔搜索区域调整" 图标 (在中心有一个点的小垂直矩形)。使用鼠标拖动一个矩形来限制瞳孔周围的区域。避免可能与瞳孔混淆的暗区域。</li>
		<li>在 "控件" 窗口中, 确认选中 "自动图像" 和 "正锁阈值跟踪" 框。单击 "自动阈值" 以优化扫描的密度, 这将显示为暗瞳孔上的绿色点。</li>
	</ol>
</li>
	<li>校准视频显示的眼睛跟踪程序, 以旋转的框架12.5 ° (+/-) 围绕其水平轴和10° (+/-) 围绕其垂直轴。<ol>
<li>在 "控件" 窗口中, 单击 "显示"。检查框中的 "凝视" 和 "传感" 的 "凝视点", "校验地区", 和 "几何网格"。在检查 "几何网格" 下的方框后, 会弹出一个窗口, 并说 "刺激显示几何必须在几何网格显示之前进行测量。你想现在这样做吗？为 "是" 选择 "Y"。</li>
		<li>使用鼠标, 点击 "窗口" 菜单, 并选择 "刺激"。"刺激" 窗口将打开显示一个垂直和水平线交叉在中心的显示屏。将线条的长度测量到最近的 mm. 按键盘上的 "Esc" 退出 "刺激" 窗口。</li>
		<li>使用鼠标, 点击 "刺激" 菜单, 并选择 "几何设置"。输入刚刚测量过的线条的长度。调整查看距离, 使水平线的度数/线等于25°, 垂直线的20°。单击 "存储" 按钮并关闭窗口。</li>
		<li>在 "EyeSpace" 窗口中, 选择 "数据点" 的校准次数为 "9"。当海龟的眼睛定位在中心, 点击中心数据点, 并点击 "重新出现" 按钮。 注: "刺激" 窗口将打开, "准备就绪" 将出现在屏幕的中心。一个框将出现在中心位置, 然后消失。在它消失后, "刺激" 窗口将关闭。中心位置现在应该校准。</li>
		<li>通过旋转框架右/左, +12.5 °/-12.5 °, 向上/向下 +10 °/-10 °来校准剩余的数据点, 重复该过程。</li>
	</ol>
</li>
	<li>为了校准扭转旋转, 请使用基于视频的眼睛跟踪程序所提供的模板拟合算法。该算法根据虹膜的标记设置零位置, 并计算当标记从瞳孔质心偏移时的旋转角度。<ol>
<li>使用鼠标指针, 点击 "窗口" 菜单, 并选择 "扭转"。单击 "扭" 窗口中的 "开始" 按钮。在 "EyeCamera" 窗口中, 将在眼睛的图像上显示一个弧线。</li>
		<li>在虹膜中存在不规则标记的位置, 使用滑块调整圆弧的半径、角度和长度。选中 "实时图形" 和 "调整后自动设置" 框。如有必要, 调整 "控件" 窗口中的亮度和对比度, 并重新阈值暗瞳孔 (请参见步骤 4)。单击 "设置模板" 按钮。</li>
	</ol>
</li>
	<li>将标尺放在与瞳孔相同的焦距平面上, 并记录完整相机视图的宽度。该值稍后将用于确定瞳孔的实际宽度。</li>
</ol>5. 吸入电极在颅神经上的定位以唤起眼球运动
<ol><li>小心地放置一个针参考电极到连接或肌肉组织留在头部。</li>
	<li>将吸入电极 (参见材料表) 置于颅神经上, 使用机器人和解剖范围安装在臂上。使用光纤光源来查看和引导放置。<ol>
<li>将神经的大小与毛细管玻璃尖相匹配。尝试和错误是必要的, 以获得一个最好的配合周围的神经直径 (见步骤1.2 的大小建议)。将玻璃尖放到吸入电极上。填充吸入电极与响铃的解决方案, 并调整在注射器内的体积约一半的能力。</li>
		<li>小心地移动电极的玻璃尖端使用机器人到一个位置在选定的神经的切口之上。确保铃声的解决方案填满头骨, 尖端在其表面下方。如有必要, 使用建模粘土来筑坝, 使铃声的溶液从颅骨漏出。</li>
		<li>拉回注射器的柱塞。 注意: 真空会将神经吸收到毛细管尖端的末端。一个良好的配合是指的神经留在尖端, 很少或没有额外的真空应用。</li>
	</ol>
</li>
</ol>6. 刺激颅神经及眼球运动分析
<ol><li>使用具有当前隔离装置的一般用途神经/肌肉刺激器 (请参阅材料表) 通过吸入电极刺激颅神经。<ol>
<li>使用电缆将吸入电极连接到当前隔离装置。将引线从 pin 参考电极连接到隔离装置的接地连接。</li>
		<li>使用刺激器和隔离装置上的刻度盘和开关选择电流的参数。使用的电流范围从1到100µA, 频率为10到400赫兹. 使用1或2毫秒的脉冲在列车持续 100, 500, 或 1000 ms。</li>
	</ol>
</li>
	<li>记录刺激的时间。 注: 晶体管-晶体管-逻辑 (TTL) 脉冲与从刺激器的电流传递同步, 并通过电缆实时通信, 以视频为基础的眼球跟踪系统的输入通道。一个软件模块提供了与视频为基础的眼睛跟踪程序控制通信。<ol>
<li>要可视化当前应用程序的时间及其对眼球运动的影响, 请单击 "PenPlots" 菜单。选择 "x 凝视位置"、"Y 凝视位置"、"扭转" 和 "瞳孔宽度", 显示用于 X 和 Y 眼睛位置、扭转和瞳孔宽度的实时原始数据地块。还可以从 "PenPlots" 菜单中选择 "秒 & 标记", 以显示带有刻度线的计时图, 该图形以1秒的间隔显示。 注: 大写字母 "T" 将出现, 标记 TTL 脉冲的开始, 同时与当前应用程序一起发生。</li>
		<li>要存储由电流诱发的眼球运动数据, 请单击 "文件" 菜单, 然后在 "数据" 下选择 "新数据文件"。输入一个文件名, 然后按 "Enter"。保存数据可以暂停, 然后使用键命令 ("Ctrl" + "p") 组合重新启动。实验会话完成后, 在 "数据" 下选择 "关闭数据文件" 菜单。</li>
		<li>要帮助跟踪应用的电流类型, 请单击 "窗口" 菜单并选择 "数据垫"。将出现 "键盘/DataMarker" 窗口。点击一个字母或一个数字, 以确定当前刺激被传递到神经的参数。 注: 例如, "x" 可以代表10µA. 单击 "x" 可将其输入实时存储到数据文件中, 用于即席分析。它也出现在 "PenPlot" 为持续观察的 "秒 & 标记"。</li>
	</ol>
</li>
	<li>分析眼部追踪系统的数据。<ol>
<li>以文本分隔格式打开保存的数据文件, 并将其设置为一个可供选择的跨页程序, 以便组织数据并进行统计分析。<ol>
<li>将文件中存储的瞳孔宽度值转换为实际大小 (mm)。</li>
			<li>将 X 和 Y 眼睛位置和 torsions 的值转换为度数单位, 并使用约定来描述眼球运动;因此, 旋转的正方向: intorsion、仰角和内收;和负面的旋转方向: extorsion, 抑郁症和诱拐。</li>
		</ol>
</li>
		<li>将页眉信息复制到新工作表中。这将包括屏幕大小 (宽度和高度) 和查看距离的值。以30赫兹速率收集的八列数据将跟随标题信息。</li>
		<li>返回包含原始数据的工作表。在标题为 "标记" 的最后一栏中, 为 "X" 做一个 "查找", 以找到刺激应用于10µA 的位置. 发现 "T" 的发病率, 标志着当前刺激的开始。复制15行 (0.5 s) 的数据发生在 "t" 和90帧之后的 "t" (3.0 s);即, 3.5 的总计。将新工作表中的数据粘贴到页眉信息下。</li>
		<li>在 "PupilWidth" 之后插入一个空列。在空白列中, 转换为 mm 的校准尺寸: 水平瞳孔直径 = "PupilWidth" x 相机视图宽度的尺寸</li>
		<li>在 "X_Gaze" 和 "Y_Gaze" 之后插入2个空列。将位置正常化为查看屏幕的尺寸: 坐标 (0, 0), 在屏幕左上角延伸到 (1, 1) 在右下角。在第一个空白列中, 将位置转换为在屏幕中心有 (0、0) 的坐标系。包括转换到屏幕尺寸的 mm: X = (0.5 x 宽度) – ("X_Gaze" x 宽度);Y = (0.5 x 高度) – ("Y_Gaze" x 高度) 注意: 手术顺序为左眼。该序列将需要逆转的右眼, 以便遵循的反绑架和积极的内收的公约。</li>
		<li>在第二列中, 使用三角函数转换成角度 (°) 旋转: 水平旋转 = arctan (X/查看距离); 垂直旋转 = arctan (Y/查看距离)</li>
		<li>扭转已经显示单位的程度, 但要符合 intorsion 的积极性, 如果测量是在左眼, 乘以-1。对于右眼, 不需要乘法。程序代码顺时针旋转为正值。</li>
		<li>绘制瞳孔直径和旋转作为时间的函数。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Kikillus, K. H., Hare, K. M., Hartley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimizing+false-negatives+when+predicting+the+potential+distribution+of+an+invasive+species:+A+bioclimatic+envelope+for+the+red-eared+slider+at+global+and+regional+scales.">Minimizing false-negatives when predicting the potential distribution of an invasive species: A bioclimatic envelope for the red-eared slider at global and regional scales.</a> Anim Conserv. 13, 5-15 (2010).</li><li>Lutz, P. L., Rosenthal, M., Sick, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+without+oxygen:+turtle+brain+as+a+model+of+anaerobic+metabolism.">Living without oxygen: turtle brain as a model of anaerobic metabolism.</a> Mol Physiol. 8, 411-425 (1985).</li><li>Lutz, P. L., Milton, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negotiating+brain+anoxia+survival+in+the+turtle.">Negotiating brain anoxia survival in the turtle.</a> J Exp Biol. 207, 3141-3147 (2004).</li><li>Storey, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoxia+tolerance+in+turtles:+Metabolic+regulation+and+gene+expression.">Anoxia tolerance in turtles: Metabolic regulation and gene expression.</a> Comp Biochem Physiol A-Mol Integr Physiol. 147 (2), 263-276 (2007).</li><li>Granda, A. M., Dearworth, J. R., Subramaniam, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balanced+interactions+in+ganglion-cell+receptive+fields.">Balanced interactions in ganglion-cell receptive fields.</a> Vis Neurosci. 16, 319-332 (1999).</li><li>Dearworth, J. R., Granda, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplied+functions+unify+shapes+of+ganglion-cell+receptive+fields+in+retina+of+turtle.">Multiplied functions unify shapes of ganglion-cell receptive fields in retina of turtle.</a> J Vis. 2 (3), 204-217 (2002).</li><li>Nesbit, S. C., Van Hoof, A. G., Le, C. C., Dearworth Jr, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+recording+of+light+responses+from+optic+nerve+fibers+and+the+caudal+photoreceptor+in+the+crayfish.">Extracellular recording of light responses from optic nerve fibers and the caudal photoreceptor in the crayfish.</a> J Undergrad Neurosci Educ. 14 (1), A29-A38 (2015).</li><li>McMahon, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+and+circulatory+compensation+to+hypoxia+in+crustaceans.">Respiratory and circulatory compensation to hypoxia in crustaceans.</a> Resp Phsiol. 128 (3), 349-364 (2001).</li><li>Leigh, R. J., Zee, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The neurology of eye movements. , (1999).</li><li>Robinson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+measuring+eye+movement+using+a+scleral+search+coil+in+a+magnetic+field.">A method of measuring eye movement using a scleral search coil in a magnetic field.</a> IEEE Trans Biomed Eng. 10, 137-145 (1963).</li><li>Judge, S. J., Richmond, B. J., Chu, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+magnetic+search+coils+for+measurement+of+eye+position:+an+improved+method.">Implantation of magnetic search coils for measurement of eye position: an improved method.</a> Vis Res. 20, 535-538 (1980).</li><li>Ong, J. K. Y., Halswanter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+torsional+eye+movements+by+tracking+stable+iris+features.">Measuring torsional eye movements by tracking stable iris features.</a> J Neurosci Meth. 192, 261-267 (2010).</li><li>Kimmel, D. L., Mammo, D., Newsome, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+eye+non-invasively:+simultaneous+comparison+of+the+scleral+search+coil+and+optical+tracking+techniques+in+the+macaque+monkey.">Tracking the eye non-invasively: simultaneous comparison of the scleral search coil and optical tracking techniques in the macaque monkey.</a> Front Behav Neurosci. 6 (49), 1-17 (2012).</li><li>Otero-Millan, J., Roberts, D. C., Lasker, A., Zee, D. S., Kheradmand, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knowing+what+the+brain+is+seeing+in+three+dimensions:+A+novel,+noninvasive,+sensitive,+accurate,+and+low-noise+technique+for+measuring+ocular+torsion.">Knowing what the brain is seeing in three dimensions: A novel, noninvasive, sensitive, accurate, and low-noise technique for measuring ocular torsion.</a> J Vis. 15 (14), 1-15 (2015).</li><li>Demski, L. S., Bauer, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+movements+evoked+by+electrical+stimulation+of+the+brain+in+anesthetized+fishes.">Eye movements evoked by electrical stimulation of the brain in anesthetized fishes.</a> Brain Behav Evol. 11, 109-129 (1975).</li><li>Gioanni, H., Bennis, M., Sansonetti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+and+vestibular+reflexes+that+stabilize+gaze+in+the+chameleon.">Visual and vestibular reflexes that stabilize gaze in the chameleon.</a> Vis Neurosci. 10, 947-956 (1993).</li><li>Straka, H., Dieringer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+organization+principles+of+the+VOR:+lessons+from+frogs.">Basic organization principles of the VOR: lessons from frogs.</a> Prog Neurobio. 73 (4), 259-309 (2004).</li><li>Voss, J., Bischof, H. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+movements+of+laterally+eyed+birds+are+not+independent.">Eye movements of laterally eyed birds are not independent.</a> J Exp Biol. 212 (10), 1568-1575 (2009).</li><li>Ariel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+eye+movements+in+the+turtle.">Independent eye movements in the turtle.</a> Vis Neurosci. 5, 29-41 (1990).</li><li>Ariel, M., Rosenberg, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+synaptic+drugs+on+turtle+optokinetic+nystagmus+and+the+spike+responses+of+the+basal+optic+nucleus.">Effects of synaptic drugs on turtle optokinetic nystagmus and the spike responses of the basal optic nucleus.</a> Vis Neurosci. 7, 431-440 (1991).</li><li>Balaban, C. D., Ariel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+"beat-to-beat"+interval+generator+for+optokinetic+nystagmus.">A "beat-to-beat" interval generator for optokinetic nystagmus.</a> Biol Cybern. 66, 203-216 (1992).</li><li>Keifer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+eye-blink+reflex+model:+Role+of+excitatory+amino+acid+receptors+and+labeling+of+network+activity+with+sulforhodamine.">In vitro eye-blink reflex model: Role of excitatory amino acid receptors and labeling of network activity with sulforhodamine.</a> Exp Brain Res. 97, 239-253 (1993).</li><li>Keifer, J., Armstrong, K. E., Houk, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+classical+conditioning+of+abducens+nerve+discharge+in+turtles.">In vitro classical conditioning of abducens nerve discharge in turtles.</a> J Neurosci. 15, 5036-5048 (1995).</li><li>Rosenberg, A. F., Ariel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+optokinetic+eye+movements+in+turtles+that+incorporates+properties+of+retinal+slip+neurons.">A model for optokinetic eye movements in turtles that incorporates properties of retinal slip neurons.</a> Vis Neurosci. 13, 375-383 (1996).</li><li>Ariel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-loop+optokinetic+responses+of+the+turtle.">Open-loop optokinetic responses of the turtle.</a> Vis Res. 37, 925-933 (1997).</li><li>Anderson, C. W., Keifer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+conditioned+abducens+nerve+responses+in+a+highly+reduced+in+vitro+brainstem+preparation+from+the+turtle.">Properties of conditioned abducens nerve responses in a highly reduced in vitro brainstem preparation from the turtle.</a> J Neurophysiol. 81, 1242-1250 (1999).</li><li>Keifer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+classical+conditioning+of+the+turtle+eyeblink+reflex:+Approaching+cellular+mechanisms+of+acquisition.">In vitro classical conditioning of the turtle eyeblink reflex: Approaching cellular mechanisms of acquisition.</a> Cerebell. 2, 55-61 (2003).</li><li>Zhu, D., Keifer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+controlling+trigeminal+and+auditory+nerve-evoked+abducens+eyeblink+reflexes+in+pond+turtles.">Pathways controlling trigeminal and auditory nerve-evoked abducens eyeblink reflexes in pond turtles.</a> Brain Behav Evol. 64, 207-222 (2004).</li><li>Jones, M. S., Ariel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+unilateral+eighth+nerve+block+on+fictive+VOR+in+the+turtle.">The effects of unilateral eighth nerve block on fictive VOR in the turtle.</a> Br Res. 1094, 149-162 (2006).</li><li>Jones, M. S., Ariel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology,+intrinsic+membrane+properties,+and+rotation-evoked+responses+of+trochlear+motoneurons+in+the+turtle.">Morphology, intrinsic membrane properties, and rotation-evoked responses of trochlear motoneurons in the turtle.</a> J Neurophysiol. 99 (3), 1187-1200 (2008).</li><li>Krenz, J. G., Naylor, G. J. P., Shaffer, H. B., Janzen, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+phylogenetics+and+evolution+of+turtles.">Molecular phylogenetics and evolution of turtles.</a> Mol Phylogenet Evol. 37 (1), 178-191 (2005).</li><li>Dearworth, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+trochlear+nerve+in+eye+abduction+and+frontal+vision+of+the+red-eared+slider+turtle+(Trachemys+scripta+elegans).">Role of the trochlear nerve in eye abduction and frontal vision of the red-eared slider turtle (Trachemys scripta elegans).</a> J Comp Neur. 52, 3464-3477 (2013).</li><li>Dearworth, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pupil+constriction+evoked+in+vitro+by+stimulation+of+the+oculomotor+nerve+in+the+turtle+(Trachemys+scripta+elegans).">Pupil constriction evoked in vitro by stimulation of the oculomotor nerve in the turtle (Trachemys scripta elegans).</a> Vis Neurosci. 26, 309-318 (2009).</li><li>Mead, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFEL+TOUR:+a+description+of+the+introduction+to+FUN+electrophysiology+labs+workshop+at+Bowdoin+College,+July+27-30,+and+the+resultant+faculty+learning+community.">IFEL TOUR: a description of the introduction to FUN electrophysiology labs workshop at Bowdoin College, July 27-30, and the resultant faculty learning community.</a> J Undergrad Neurosci Educ. 5, A42-A48 (2007).</li><li>Jackson, D. C., Ultsch, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+hibernation+under+the+ice+by+turtles+and+frogs.">Physiology of hibernation under the ice by turtles and frogs.</a> J Exp Zool A Ecol Genet Physiol. 313 (6), 311-327 (2010).</li><li>Romano, J. M., Dearworth, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pupil+constriction+evoked+by+stimulation+of+the+ciliary+nerve+in+the+red-eared+slider+turtle+(Trachemys+scripta+elegans).">Pupil constriction evoked by stimulation of the ciliary nerve in the red-eared slider turtle (Trachemys scripta elegans).</a> J Penns Acad Sci. 85, 4-8 (2011).</li><li>Miller, J. M., Robins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraocular-muscle+forces+in+alert+monkey.">Extraocular-muscle forces in alert monkey.</a> Vis Res. 32, 1099-1113 (1992).</li><li>Gamlin, P. D., Miller, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraocular+muscle+motor+units+characterized+by+spike-triggered+averaging+in+alert+monkey.">Extraocular muscle motor units characterized by spike-triggered averaging in alert monkey.</a> J Neurosci Meth. 204, 159-167 (2011).</li><li>Quaia, C., Ying, H. S., Optican, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Viscoelastic+properties+of+passive+eye+muscle+in+primates.+III:+Force+elicited+by+natural+elongations.">The Viscoelastic properties of passive eye muscle in primates. III: Force elicited by natural elongations.</a> PLOS ONE. 5, A236-A254 (2010).</li><li>Anderson, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+primate+oculomotor+plant+revealed+by+effects+of+abducens+microstimulation.">Dynamics of primate oculomotor plant revealed by effects of abducens microstimulation.</a> J Neurophys. 101, 2907-2923 (2009).</li><li>Maxwell, J. H., Harless, M., Morlock, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+surgery.">Anesthesia and surgery.</a> Turtles: Perspective and Research. , 127-152 (1979).</li><li>AVMA Panel on Euthanasia. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+Veterinary+Medical+Association.">American Veterinary Medical Association.</a> J Am Vet Med Assoc. 218 (5), 669-696 (2001).</li><li>Clarke, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+the+pupil's+response+to+light+in+the+hooded+rat.">Shaping the pupil's response to light in the hooded rat.</a> Exp Br Res. 176, 641-651 (2007).</li><li>Bennett, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+anesthesia+and+chemical+restraint+in+reptiles.">A review of anesthesia and chemical restraint in reptiles.</a> J Zoo Wild Med. 22 (3), 282-303 (1991).</li><li>Bickler, P. E., Buck, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+Tolerance+in+Reptiles,+Amphibians,+and+Fishes:+Life+with+Variable+Oxygen+Availability.">Hypoxia Tolerance in Reptiles, Amphibians, and Fishes: Life with Variable Oxygen Availability.</a> Ann Rev Physiol. 69, 145-170 (2007).</li></ol>

作者没有什么可透露的。

关键步骤:
本议定书内的关键步骤如下: 1) 解剖和护理, 以保持横断神经的生存能力;2) 将吸入电极大小与颅神经匹配, 提供一致的反应;3) 将头部放置在框架内, 为眼睛的旋转提供适当的校准。
故障 排除:
解剖可能具有挑战性, 但在完成了几次之后, 这些步骤就应该变得相对笔直。如果神经出现不反应, 最可能的原因是解剖失败。在大脑的切除...

在电生理实验中使用红耳滑块龟的基本原理:
红耳滑块龟 (Trachemys 龟线虫) 被认为是世界上最严重的入侵物种之一1 , 可以表明生态系统有问题。红耳滑块龟之所以如此成功的原因是很不清楚, 但它可能部分是由于他们的耐受生理和拥有的神经组织, 可以生存在缺氧条件下2,3,4.使用它们进行实验并不会威胁到他们的数量, 并以最小的努力, 电生理准备可以保持在延长的持续时间内可行, 只要18小时5,6。这种好处类似于使用无脊椎动物 (如小龙虾7) 的优势, 它也有能力抵御低氧水平的8。
眼球运动测量技术:
用非人类灵长类测量额眼动物眼球运动的方法得到了很好的发展9。眼睛在轨道旋转大约三个轴: 水平, 垂直和扭转。磁搜索线圈方法通常被认为是最可靠的测量旋转, 但侵入, 要求小线圈插入到动物的 scleras10,11。基于视频的系统也可以测量旋转, 具有非侵入性的优点。随着新的图像处理技术的发展, 增强了其功能, 使基于视频的系统成为考虑12、13、14的一种有吸引力的替代方案。
为测量 nonmammals 的眼球运动而开发的技术已经没有那么重要了。度量值要么是低分辨率, 要么仅描述部分旋转15、16、17、18。缺乏发展可以部分地归咎于训练 nonmammals 遵循视觉目标的困难。虽然眼球运动已在红耳滑块龟19,20,21,22,23,24,25 ,26,27,28,29,30, 由于训练动物跟踪目标的挑战, 他们的眼球运动的精确运动学是不好的理解。
红耳滑块龟一般被认为是侧眼脊椎动物, 但因为他们可以完全收回他们的头到他们的外壳31, 由甲壳的侧面视觉场显著闭塞发生32。结果是他们的视觉视线被逼向前方, 使它们的行为更像额眼哺乳动物。因此, 它们作为开发测量眼球运动方法的模型也提供了一个独特的进化视角。
本工作中描述的协议使用体外分离头准备, 以确定红耳滑块龟眼运动的运动学。大脑从头骨中解剖, 使脑神经完好无损。头部被放置在一个框架内, 以校准眼球运动, 并通过电刺激颅神经支配眼睛肌肉唤起反应。眼睛旋转的措施是由一个基于视频的系统, 使用软件算法, 跟踪黑暗的瞳孔和虹膜的标记。该制剂为测量眼外 (即、水平、垂直和扭转旋转) 的运动提供了一个机会:32和人工 (即, 瞳孔改变)33运动。
传出神经通路分析模型系统:
更普遍地说, 该方法为调查人员提供了一个机会, 研究传出神经信号如何产生眼球运动时, 肌肉从他们的放松状态开始, 并在缺乏综合感官信息处理的大脑32, 33。因此, 眼睛的运动学可以被检查在一个模型系统中, 他们是单独处理的传出神经通路离开大脑和 synapsing 到肌肉。

<table><tbody><tr><td>Red-eared slider turtles</td><td>Kons Scientific</td><td>&lt;em&gt;Trachemys scripta elegans&lt;/em&gt;</td><td>Large size (carapace length 15-20 cm)</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich Co. LLC.</td><td>S5886</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich Co. LLC.</td><td>P5405</td><td></td></tr><tr><td>Magnesium choride</td><td>Sigma-Aldrich Co. LLC.</td><td>M7304</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich Co. LLC.</td><td>S5761</td><td></td></tr><tr><td>Dextrose</td><td>Sigma-Aldrich Co. LLC.</td><td>C5767</td><td></td></tr><tr><td>Concentrated hydrochloric acid</td><td>Sigma-Aldrich Co. LLC.</td><td>H7020</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich Co. LLC.</td><td>C7902</td><td></td></tr><tr><td>pH meter</td><td>Oakton</td><td>pH 6+</td><td></td></tr><tr><td>Suction stimulation electrode</td><td>A-M Systems</td><td>573000</td><td>Bipolar suction electrode. Note that 573000 has been replaced with 573050.</td></tr><tr><td>Capillary glass</td><td>A-M systems</td><td>626000</td><td>Single-barrel borosilicate capillary glass without microfilament, length 10 cm, outside diameter 1.0 mm, inner diameter 0.50 mm</td></tr><tr><td>Alternative suction stimulation electrode</td><td>A-M Systems</td><td>573050</td><td>Bipolar suction electrode. Requires larger diameter capillary glass: 627000, outside diameter 1.2 mm, inner diameter 0.68 mm</td></tr><tr><td>Stereoscope</td><td>Lieca</td><td>GZ7</td><td>Magnification range, 10x &amp;ndash; 70x</td></tr><tr><td>Fiber optic light source</td><td>Amscope</td><td>HL250-A</td><td>150W Fiber optical microscope illuminator light box</td></tr><tr><td>Rongeurs</td><td>Carolina Biological Supply Company</td><td>625654</td><td>stainless steel, straight spring, 5.25&quot;</td></tr><tr><td>Blunt dissection probe</td><td>Carolina Biological Supply Company</td><td>627405</td><td>Huber mall probe, double-ended probe and seeker, 6&quot;</td></tr><tr><td>Microscissors</td><td>Carolina Biological Supply Company</td><td>623555</td><td>Iris microdissecting scissors, stainless steel, 0.5&quot; blades, 4.75&quot; long</td></tr><tr><td>Fine forceps</td><td>Sigma-Aldrich Co. LLC.</td><td>F6521</td><td>Jewelers forceps, dumont No. 5, inox alloy, 4.25&quot;</td></tr><tr><td>Curved forceps</td><td>Sigma-Aldrich Co. LLC.</td><td>Z168696</td><td>Medium tip, curved forceps, stainless steel, 4&quot;</td></tr><tr><td>Scalpel handle</td><td>Sigma-Aldrich Co. LLC.</td><td>S2896</td><td>Scalpel handles, No. 3, stainless steel</td></tr><tr><td>Scalpel blade</td><td>Sigma-Aldrich Co. LLC.</td><td>S2771</td><td>Scalpel blades, No. 11, steel</td></tr><tr><td>Guillotine</td><td>Harvard Apparatus</td><td>73-1918</td><td>Kleine guillotine type 7575</td></tr><tr><td>Spatula</td><td>Sigma</td><td>Z648299</td><td>Micro spoon and spatula weighing set. Use small spatula: 5.9&amp;rdquo; long x 0.07&amp;rdquo; diameter handle with square end: 0.17&amp;rdquo; x 1.3&amp;rdquo; long, other end round: 0.17&amp;rdquo; x 1.27&amp;rdquo; long</td></tr><tr><td>Hook</td><td>Autozone</td><td>98069</td><td>SureBilt hook and pick set. Use grinder to dull sharp points of hook to prevent injury to animals mouth.</td></tr><tr><td>95/5% O&lt;sub&gt;2&lt;/sub&gt;/CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Airgas, Inc.</td><td>X02OX95C2003102</td><td>5% Carbon dioxide balance oxygen certified standard gas mixture, size 200 Cylinder, CGA-296</td></tr><tr><td>Regulator</td><td>Airgas, Inc.</td><td>Y11244D296-AG</td><td>Single stage brass 0-100 psi analytical cylinder regulator CGA-296 with needle outlet. Use brass adjustable airline pipe valve to go from 3/8&quot;, inner diameter, vinyl airline tubing connected to regulator to a 3/16&quot;, inner diameter, airline connection going to airstone or glass pasteur pipette.</td></tr><tr><td>Adjustable airline pipe valve</td><td>Doctors Foster and Smith</td><td>CD-12061</td><td>Brass valve</td></tr><tr><td>Rigid table</td><td>Unknown</td><td>Unknown</td><td>Auto-clave door laid on top of a sturdy table. Nine 5&quot; diameter tennis balls isolate vibrations from the top surface of the table.</td></tr><tr><td>5&quot; tennis ball</td><td>Petco Animal Supplies, Inc.</td><td>712868</td><td>Petco Jumbo Pet Tennis Ball: balls are unsliced and held within an integrated frame on the underside part of the autoclave door.</td></tr><tr><td>Alternative vibration isolation table</td><td>Newport Corporation</td><td>INT1-36-6-N</td><td>Rigid vibration control system, integrity 1: Surface dimensions, 3&#039; x 6&#039;</td></tr><tr><td>Gimbal</td><td>ISI, International Scientific Instruments, Inc.</td><td>Stage from SUPER III-A Scanning EM</td><td>5-axis eucentric stage: X, Y, and Z linear movements, &amp;plusmn;20 mm, 0.1 mm precision; Rotations, vertical, &amp;plusmn;10&amp;deg;, and horizontal, &amp;plusmn;12.5&amp;deg;, with 1.25&amp;deg; precision. Note: from decommission instrument.</td></tr><tr><td>Chuck for gimbal</td><td>Unknown</td><td>Unknown</td><td>Chuck from an old microtome of unknown manufacture was machined to fit the shaft of the specimen holder of the Scanning EM stage</td></tr><tr><td>Alternative gimbal</td><td>ThorLabs, Inc.</td><td>GN2/M with MBT602/M</td><td>Dual-axis goniometer (GN2/M) mounted on 3-axis microblock stage with thumbscrew adjusters (MBT602/M): design a chuck to hold turtle head with eye at 12.7 mm above top surface of goniometer (distance to point of rotation)</td></tr><tr><td>Video-based eye tracking system</td><td>Arrington Research, Inc.</td><td>ViewPoint EyeTracker, PC-60</td><td>Tracking method: Infrared video by dark pupil; Black and white camera (Item BC02): 30 Hz, 640 x 480; System requirements: Windows 2000, XP, 7, 8, 8.1, 10; Visual range: Horizontal +/- 44&amp;deg;; vertical +/- 20&amp;deg;; Accuracy ~0.5&amp;deg;; Spatial resolution ~0.15&amp;deg;; Pupil size resolution ~0.03 mm; Eye data: X, Y position of gaze, pupil height and width, torsion, delta time, total time, and regions of interest (ROI); Real-time communication (Item 0022): 4-Channel AnalogOut with eight TTL input channels to mark codes into the data file</td></tr><tr><td>Multi-position magnetic base</td><td>Harbor Freight Tools</td><td>Pittsburg, item #5645</td><td>Magnetic holder reaches up to 12&quot; and produces 45 lbs. of magnetic pull. Use to position camera. Machine thread holes onto the end of the rod to mount cameras.</td></tr><tr><td>Micromanipulator</td><td>Kopf</td><td>900</td><td>5 axis manipulation for mount of suction electrode: X, Y, Z linear travel, 2 axis of rotation</td></tr><tr><td>Dissection scope on boom</td><td>Lieca</td><td>GZ6</td><td>Magnification range, 6.7x &amp;ndash; 40x</td></tr><tr><td>Nerve/muscle stimulator</td><td>Astro-Med Grass Telefactor</td><td>Grass S88</td><td>Dual pulse voltage stimulator: two output channels that can be operated independently or synchronized to generate non-isolated constant voltage pulses (10 mv to 150 V). Pulses can be single (10 &amp;mu;sec to 10 sec), repetitive (0.01 Hz to 1 KHz), and trains (1 ms to 10 s) and synchronized with TTL inputs and output. Send TTL outputs via the output channels of a DB25 connector to the TTL input channels of the ViewPoint EyeTracker. Note: Astro-Med Grass Telefactor is no longer in business.</td></tr><tr><td>Current isolation device</td><td>Astro-Med Grass Telefactor</td><td>PSIU6</td><td>Current stimulus isolation unit: enables safe delivery of constant currents by the S88 to the preparation. The PSIU6 connects by a BNC cable to one of the output channels of the S88. Multiplier switches on the PSIU6 allow the S88 to generate a wide array of current amplitudes ranging from 0.1 &amp;micro;A to 15 mA.</td></tr><tr><td>Alternative nerve/muscle stimulator with isolation</td><td>A-M Systems</td><td>2100</td><td>Isolated Pulse Stimulator: Unit has built-in isolator to produce constant currents.</td></tr></tbody></table>

ocular kinematics measured by in vitro stimulation of the cranial nerves in the turtle

在动物被安乐死后, 他们的组织开始死亡。海龟提供了一个优势, 因为他们的组织的生存时间较长, 特别是与热血脊椎动物相比。正因为如此, 在海龟体内进行的体外实验可以在较长时间内进行, 以调查神经信号和控制其目标动作。采用分离头制备方法, 测量了龟眼运动的运动学, 并对其进行了神经电信号调制。大脑从头骨中取出后, 将颅骨神经完整, 解剖头被放置在一个框架内以校准眼球运动。玻璃电极附着于颅神经 (动眼神经、滑车和展), 并以电流刺激以唤起眼球运动。我们用红外线视频跟踪系统和眼睛的量化旋转来监测眼球运动。电流脉冲的幅度, 频率, 和列车持续时间被用来观察对反应的影响。由于制剂与大脑分离, 进入肌肉靶的传出通路可以单独检查, 以便在没有中央处理的感官信息的情况下研究神经信号。

图 1显示从描述解剖的视频中拍摄的图像的剧照。图像提供的神经的典型位置之前, 从大脑切割。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56864/56864fig1.jpg"> 图 1: 从解剖视频中捕捉到的图像的剧照, 以显示视神经 (nII)、动眼神经 (nIII)、滑车神经 (展) 的位置, 以及神经 (nVI).(A) ...

Watch this Scientific Journal Video about Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle at JoVE.com

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

本协议描述如何使用体外分离制剂来测量其眼球运动的运动学。大脑从颅骨切除后, 可以用电流刺激颅神经, 以量化眼睛的旋转和瞳孔大小的变化。

体外刺激对龟脑神经的运动测量

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

ocular-kinematics-measured-vitro-stimulation-cranial-nerves

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

8.4K 次观看.  Lafayette College. 本协议描述如何使用体外分离制剂来测量其眼球运动的运动学。大脑从颅骨切除后, 可以用电流刺激颅神经, 以量化眼睛的旋转和瞳孔大小的变化。

视频: Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.

The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.

VisioTracker，创新的自动化到动眼神经分析方法

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

科研

神经科学

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

视频oculography小鼠

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

Three Dimensional Vestibular Ocular Reflex Testing Using a Six Degrees of Freedom Motion Platform

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the vestibular organ results in mild to severe equilibrium problems, such as vertigo, dizziness, oscillopsia, gait unsteadiness nausea and/or vomiting. A good and frequently used measure to quantify gaze stabilization is the gain, which is defined as the magnitude of compensatory eye movements with respect to imposed head movements. To test vestibular function more fully one has to realize that 3D VOR ideally generates compensatory ocular rotations not only with a magnitude (gain) equal and opposite to the head rotation but also about an axis that is co-linear with the head rotation axis (alignment). Abnormal vestibular function thus results in changes in gain and changes in alignment of the 3D VOR response.
Here we describe a method to measure 3D VOR using whole body rotation on a 6DF motion platform. Although the method also allows testing translation VOR responses 1, we limit ourselves to a discussion of the method to measure 3D angular VOR. In addition, we restrict ourselves here to description of data collected in healthy subjects in response to angular sinusoidal and impulse stimulation.
Subjects are sitting upright and receive whole-body small amplitude sinusoidal and constant acceleration impulses. Sinusoidal stimuli (f = 1 Hz, A = 4&deg;) were delivered about the vertical axis and about axes in the horizontal plane varying between roll and pitch at increments of 22.5&deg; in azimuth. Impulses were delivered in yaw, roll and pitch and in the vertical canal planes. Eye movements were measured using the scleral search coil technique 2. Search coil signals were sampled at a frequency of 1 kHz.
The input-output ratio (gain) and misalignment (co-linearity) of the 3D VOR were calculated from the eye coil signals 3.
Gain and co-linearity of 3D VOR depended on the orientation of the stimulus axis. Systematic deviations were found in particular during horizontal axis stimulation. In the light the eye rotation axis was properly aligned with the stimulus axis at orientations 0&deg; and 90&deg; azimuth, but gradually deviated more and more towards 45&deg; azimuth.
The systematic deviations in misalignment for intermediate axes can be explained by a low gain for torsion (X-axis or roll-axis rotation) and a high gain for vertical eye movements (Y-axis or pitch-axis rotation (see Figure 2). Because intermediate axis stimulation leads a compensatory response based on vector summation of the individual eye rotation components, the net response axis will deviate because the gain for X- and Y-axis are different.
In darkness the gain of all eye rotation components had lower values. The result was that the misalignment in darkness and for impulses had different peaks and troughs than in the light: its minimum value was reached for pitch axis stimulation and its maximum for roll axis stimulation.
Case Presentation
Nine subjects participated in the experiment. All subjects gave their informed consent. The experimental procedure was approved by the Medical Ethics Committee of Erasmus University Medical Center and adhered to the Declaration of Helsinki for research involving human subjects.
Six subjects served as controls. Three subjects had a unilateral vestibular impairment due to a vestibular schwannoma. The age of control subjects (six males and three females) ranged from 22 to 55 years. None of the controls had visual or vestibular complaints due to neurological, cardio vascular and ophthalmic disorders.
The age of the patients with schwannoma varied between 44 and 64 years (two males and one female). All schwannoma subjects were under medical surveillance and/or had received treatment by a multidisciplinary team consisting of an othorhinolaryngologist and a neurosurgeon of the Erasmus University Medical Center. Tested patients all had a right side vestibular schwannoma and underwent a wait and watch policy (Table 1; subjects N1-N3) after being diagnosed with vestibular schwannoma. Their tumors had been stabile for over 8-10 years on magnetic resonance imaging.

A method is described to measure three-dimensional vestibulo ocular reflexes (3D VOR) in humans using a six degrees of freedom (6DF) motion simulator. The gain and misalignment of the 3D angular VOR provide a direct measure of the quality of vestibular function. Representative data on healthy subjects are provided

三维前庭眼反射测试使用六度自由度运动平台

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the ...

The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish

Zebrafish are a proven model for vision research, however many of the earlier methods generally focused on larval fish or demonstrated a simple response. More recently adult visual behavior in zebrafish has become of interest, but methods to measure specific responses are new coming. To address this gap, we set out to develop a methodology to repeatedly and accurately utilize the optokinetic response (OKR) to measure visual acuity in adult zebrafish. Here we show that the adult zebrafish's visual acuity can be measured, including both binocular and monocular acuities. Because the fish is not harmed during the procedure, the visual acuity can be measured and compared over short or long periods of time. The visual acuity measurements described here can also be done quickly allowing for high throughput and for additional visual procedures if desired. This type of analysis is conducive to drug intervention studies or investigations of disease progression.

Zebrafish have been a valuable tool for many research areas. Here we demonstrate a method to elicit a visual response and calculate a functional visual acuity in the adult zebrafish.

在视动反应在斑马鱼视力的定量测量

Zebrafish are a proven model for vision research, however many of the  earlier methods generally focused on larval fish or demonstrated a simple  ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

光学相干层析成像和光运动响应在小鼠和大鼠中的结构和功能视觉系统读数

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury are essential for translating novel therapeutic strategies from rodent models to clinical application due to their closer similarities to humans in size and anatomy. Here we describe some in vivo methods to evaluate the function and structure of the retinal ganglion cells (RGCs) and optic nerve (ON) in large animals, including visual evoked potential (VEP), pattern electroretinogram (PERG) and optical coherence tomography (OCT). Both goat and non-human primate were employed in this study. By presenting these in vivo methods step by step, we hope to increase experimental reproducibility among different labs and facilitate the usage of large animal models of optic neuropathies.

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

Here we demostrate several in vivo tests (flash visual evoked potential, pattern electroretinogram and optic coherence tomography) in goat and rhesus macaque to understand the structure and function of the optic nerve and its neurons.

体内 大型动物视网膜神经节细胞及视神经功能及结构的评估方法

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury ...

A New Optokinetic Testing Method to Measure Rat Vision

Optokinetic nystagmus (OKN) is a reflexive eye movement initiated by the motion of visual stimuli in the field of vision. The head-tracking movement associated with OKN is commonly used as a measure of visual function in rodents. To record OKN responses in normal and experimental rats, a simple and inexpensive apparatus has been developed. This setup uses two tablet screens to display the OKN visual stimulus consisting of high contrast black and white stripes generated using the OKN Stripes Visualization Web Application, a freely available software. The rat is placed inside a clear Plexiglass holder that limits&#160;movement so that the&#160;rat&#39;s head continuously faces the OKN display screen. The position of the rat holder can be changed to adjust the distance between the rat and the display screen. A micro-camera positioned above the rat holder is used to record the rat&#39;s visual activities. These recordings can be used for quantitative assessments. Based on the presence or absence of clear head-tracking, the OKN responses at different spatial frequencies can be determined. The collected data demonstrates a novel technique for reliable measurement of visual acuity in normal and retinal degenerate rats.

<meta charset="utf8"></head><body>一种测量大鼠视力的新光动测试方法

The Optokinetic Nystagmus (OKN) behavioral testing method is used for the assessment of visual acuity in rodents. Here a simple method is demonstrated that can be easily set up in research laboratories for a reliable assessment of visual function in both normal and experimental rats.

一种测量大鼠视力的新光动测试方法

Optokinetic nystagmus (OKN) is a reflexive eye movement initiated by the motion of visual stimuli in the field of vision. The head-tracking movement ...

评估小鼠的视动反射反应：一种新的高精度方案

Quantification of Visual Feature Selectivity of the Optokinetic Reflex in Mice

The optokinetic reflex (OKR) is an essential innate eye movement that is triggered by the global motion of the visual environment and serves to stabilize retinal images. Due to its importance and robustness, the OKR has been used to study visual-motor learning and to evaluate the visual functions of mice with different genetic backgrounds, ages, and drug treatments. Here, we introduce a procedure for evaluating OKR responses of head-fixed mice with high accuracy. Head fixation can rule out the contribution of vestibular stimulation on eye movements, making it possible to measure eye movements triggered only by visual motion. The OKR is elicited by a virtual drum system, in which a vertical grating presented on three computer monitors drifts horizontally in an oscillatory manner or unidirectionally at a constant velocity. With this virtual reality system, we can systematically change visual parameters like spatial frequency, temporal/oscillation frequency, contrast, luminance, and the direction of gratings, and quantify tuning curves of visual feature selectivity. High-speed infrared video-oculography ensures accurate measurement of the trajectory of eye movements. The eyes of individual mice are calibrated to provide opportunities to compare the OKRs between animals of different ages, genders, and genetic backgrounds. The quantitative power of this technique allows it to detect changes in the OKR when this behavior plastically adapts due to aging, sensory experience, or motor learning; thus, it makes this technique a valuable addition to the repertoire of tools used to investigate the plasticity of ocular behaviors.

作者聚焦：一种研究小鼠视动反射视觉特征选择性的准确定量方法 

Here, we describe a standard protocol for quantifying the optokinetic reflex. It combines virtual drum stimulation and video-oculography, and thus allows precise evaluation of the feature selectivity of the behavior and its adaptive plasticity.

小鼠视动反射视觉特征选择性的量化

The optokinetic reflex (OKR) is an essential innate eye movement that is triggered by the global motion of the visual environment and serves to stabilize ...

体外刺激对龟脑神经的运动测量

摘要

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此视频中的章节

VisioTracker，创新的自动化到动眼神经分析方法

视频oculography小鼠

三维前庭眼反射测试使用六度自由度运动平台

在视动反应在斑马鱼视力的定量测量

光学相干层析成像和光运动响应在小鼠和大鼠中的结构和功能视觉系统读数

体内大型动物视网膜神经节细胞及视神经功能及结构的评估方法

一种测量大鼠视力的新光动测试方法

一种测量大鼠视力的新光动测试方法

小鼠视动反射视觉特征选择性的量化

小鼠视动反射视觉特征选择性的量化

体外刺激对龟脑神经的运动测量

摘要

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此视频中的章节

VisioTracker，创新的自动化到动眼神经分析方法

视频oculography小鼠

三维前庭眼反射测试使用六度自由度运动平台

在视动反应在斑马鱼视力的定量测量

光学相干层析成像和光运动响应在小鼠和大鼠中的结构和功能视觉系统读数

体内 大型动物视网膜神经节细胞及视神经功能及结构的评估方法

一种测量大鼠视力的新光动测试方法

一种测量大鼠视力的新光动测试方法

小鼠视动反射视觉特征选择性的量化

小鼠视动反射视觉特征选择性的量化

体内大型动物视网膜神经节细胞及视神经功能及结构的评估方法