作者感谢Bryan Krause为这个项目提供的技术支持和指导。
这项工作得到了国际麻醉研究学会（IMRA至AR），美国国立卫生研究院（R01 GM109086至MIB）和美国威斯康星大学麦迪逊分校医学与公共卫生学院麻醉学系的支持。

本协议中描述的涉及动物的所有程序均已获得威斯康星大学麦迪逊分校医学院和公共卫生动物护理和使用委员会的批准。
1.育种小鼠在中间神经元亚群中表达荧光报告蛋白
<ol><li>将纯合子，Cre依赖性tdTomato雄性小鼠与纯合SOM-Cre雌性或纯合PV-Cre雌性小鼠配对。 注意：其他特定的神经元群体可以通过使用适当的Cre线来靶向。</li>
	<li>让杂合子成熟到至少 3 周大后再进行。对于这里描述的实验，基因分型不是必需的，因为纯合亲本产生的后代对于细胞类型特异性Cre重组酶和Cre依赖性报告等位基因都是杂合的。</li>
</ol>2.进行病毒构建体的单侧立体定向注射
<ol><li>按照仪器用户手册中的指示调整进样移液器微量移液器拉拔器的设置（推荐设置见 表1 ）。拉动玻璃微量移液器。</li>
	<li>折断移液器尖端的尖端，使吸头直径约为 30 μm，在几毫米内最小锥度。</li>
	<li>使用先前记录的程序，确定要注射的病毒的适当滴度和体积。在这里描述的实验中，单侧进样 1.0 μL（滴度：3.1-5.7 TU/mL）产生了良好的结果。</li>
	<li>用矿物油回注移液器的全部体积。将移液器装入微量注射器上，并将少量矿物油流过吸头，以确保吸头不堵塞。</li>
	<li>预先填充至少 1.0 μL 的病毒构建体。这些实验中使用的重组腺相关病毒载体是AAV2-hSyn-hChR2（H134R）-EYFP。</li>
	<li>在手术区域安排无菌布。对立体定位手术的工具进行消毒，并将其放在窗帘上。</li>
	<li>使用异氟醚（3%用于诱导，1.5-2%用于维持）和氧气混合物麻醉SOM-tdTomato或PV-tdTomato杂合动物。在整个手术过程中定期确认手术麻醉水平，捏住脚趾。通过每10-15分钟监测一次呼吸，确保动物不会超出麻醉的手术计划。</li>
	<li>剃掉动物的头顶。将70%异丙醇和碘基溶液大量涂抹在手术区域，将眼膏涂抹在眼窝上，以防止膜干燥。将布比卡因/利多卡因（1：1 比例，1.0 mg/kg）皮下注射至手术部位进行局部麻醉。</li>
	<li>将动物放入立体定位框架中。</li>
	<li>使用手术刀沿着覆盖颅骨背表面的皮肤矢状轴切开。使用镊子回缩皮肤。必要时用0.9%盐水补水颅骨。</li>
	<li>在头骨表面，用铅笔轻轻标记前坐标和侧面坐标的交点。在横向平面的适当坐标处钻一个孔（相对于布雷格玛的毫米为单位，扣带皮层（Cg）注射：前0.2，外侧0.3;丘脑后（Po）注射：后2.25，外侧3.4）。 注意：标记应超出毛刺孔的边界，以便为准确放置移液器提供指导。</li>
	<li>打开并平衡空气台。</li>
	<li>立体定位框架的电极操纵器在0°处重新定位以注射到Cg中，或在冠状平面中45°处重新定位以注射到Po中。</li>
	<li>将注射泵连接到电极机械手。将微型注射泵控制器连接到注射泵。</li>
	<li>在步骤2.11中创建的标记的交叉处，在大脑表面附近（但不接触）导航移液器吸头。将移液器沿其纵轴以大约 1 mm/s 的速度推进到大脑中 0.9 mm（以 Cg 注射）或 3.1 mm（以 Po 注射）。等待 10 分钟，然后再继续。</li>
	<li>在 10 分钟（100 nL/min）的时间内注射 1.0 μL 病毒构建体。如果观察到病毒从移液器插入部位涌出，请将注射速率减慢至 50 nL/min。</li>
	<li>注射后，等待10分钟，然后缓慢缩回注射移液器。</li>
	<li>缝合以闭合头皮切口并皮下给予2-5mg / kg美洛昔康。</li>
	<li>停用异氟醚并在麻醉期间监测动物。允许根据机构动物护理和使用委员会描述的程序恢复，包括进一步服用镇痛药。</li>
</ol>3.急性脑切片的制备
<ol><li>在收获组织之前，允许至少3周的病毒构建体表达。</li>
	<li>准备1L人工脑脊液用于切片程序（切片人工脑脊液，sACSF）。成分见 表2 。</li>
	<li>在整个切片过程中，通过气体分散管向sACSF提供溶解的95%O2/5%CO2 混合物。</li>
	<li>为振动刀片切片机准备冰冷浴。将冰冷的标本载物台安装到切片机上，并将蓝宝石刀片固定到位以进行组织切片。</li>
	<li>用3%异氟醚和氧气麻醉小鼠，直到右反射丧失。</li>
	<li>使用断头台将小鼠斩首，并立即将头部浸入 4°C sACSF 中。为了保持组织的健康，请尽快完成以下步骤。</li>
	<li>通过在颅底做一个小切口并轻轻移除每个颅骨板来打开颅腔。轻轻去除下面的硬脑膜。</li>
	<li>当大脑仍在颅腔中时，使用剃须刀片去除小脑。沿着左半球的矢状面进行第二次垂直切口，就在中线的侧面。</li>
	<li>准备组织块进行切片。<ol><li>轻轻地将大脑从颅腔中取出。将大脑放在滤纸上，矢状平面向下。将滤纸引导到阻塞模板上，并将大脑与底层模板轮廓对齐（补充图1）。</li>
		<li>在冕平面上进行两个平行切口，如模板上的线条所示。如有必要，加入一小滴sACSF以保持滤纸湿润。</li>
		<li>在进行步骤3.8.4时，将组织块短暂地置于4°C sACSF中。</li>
		<li>在冰冷的试样阶段涂上少量强力胶。</li>
		<li>从冷 sACSF 中提起组织块。使用吸水毛巾的一角吸走多余的 sACSF。将组织块的后冠平面粘在标本阶段，大脑的背表面面向蓝宝石刀片。</li>
	</ol></li>
	<li>收集500μm厚的冠状脑切片。将感兴趣的切片放在34°C sACSF中的尼龙网（补充图2）上，并使容器达到室温。 注意：对于此处描述的实验，从距前膛后约2.25毫米为中心的冠状部分收集电生理记录，以研究非主要感觉区域，即内侧次级视觉皮层（V2MM）。</li>
</ol>4.含有溶解性挥发性麻醉剂异氟醚的实验性人工脑脊液（eACSF）袋的制备
<ol><li>准备 300 mL 3.0% 异氟醚的储备混合物。<ol><li>在密封的聚四氟乙烯气袋中，将~100mL的95%O 2/5%CO2气体混合物加入20-30mL的液体异氟醚和少量的0.9%盐水中。等待至少30分钟，以使异氟醚在液相和气相之间平衡。</li>
		<li>使用以下公式确定要添加到库存袋中的饱和异氟醚气体 Vsat 的量： <img alt="Equation 1" src="/files/ftp_upload/61333/61333equ01.jpg"> 其中P%库存是库存气体的目标组成（在这种情况下为3%），V库存是库存气袋的最终体积，P异氟醚是异氟醚在室温下的分压（~240mmHg），P总值是大气压（~760mmHg）。</li>
		<li>将计算出的饱和气体量加入空气袋中，并在袋中填充 95% O2/5% CO2 气体混合物，使储液袋的总体积达到 300 mL。</li>
	</ol></li>
	<li>准备2L人工脑脊液用于在实验期间灌注切片（实验ACSF，eACSF）。成分见表2。将 95% O 2/5% CO2 气体混合物溶解到溶液中。</li>
	<li>准备两袋单独的对照和异氟醚溶液。<ol><li>在空的聚四氟乙烯气袋中，加入 600 mL eACSF 和 600 mL 95% O 2/5% CO2 气体混合物。将此袋子标记为对照组。</li>
		<li>在另一个空的聚四氟乙烯气袋中，加入 300 mL 的 eACSF。将此袋子标记为异氟醚。</li>
		<li>选择异氟醚的生理相关平衡气相浓度。使用相当于1.3%异氟醚的气体浓度进行实验。小鼠在吸入异氟醚时失去矫正反射和意识。</li>
		<li>使用以下公式计算室温下的等效气相浓度，P%（T室）31： <img alt="Equation 2" src="/files/ftp_upload/61333/61333equ02.jpg"> 其中P%（T体）为步骤4.3.3中选择的生理相关气相浓度，T室为25°C，T本体为37°C。</li>
		<li>使用以下公式确定从库存气袋V库存中加入异氟醚溶液的气体体积。 <img alt="Equation 3" src="/files/ftp_upload/61333/61333equ03.jpg"> 其中V溶液是异氟醚袋（300mL）中eACSF的体积，P%（T室）由公式（2）输入，λ是异氟烷的盐水/气体奥斯特瓦尔德分配系数（λ = 1.232），P%库存是库存气袋的气相浓度（P%库存= 3.0%）。</li>
		<li>向异氟醚溶液袋中，加入来自气体袋的气体体积，V库存，按步骤4.3.5计算。</li>
		<li>在异氟醚溶液袋中，加入 95% O2/5% CO2 气体混合物，使异氟烷溶液袋中的气体总体积达到 300 mL。</li>
	</ol></li>
	<li>在摇床上摇动对照袋和异氟醚袋至少1小时，以使异氟醚相平衡。</li>
	<li>收集完所有数据后，可以使用麻醉气体监测仪测量异氟醚在袋中剩余溶液上方的平衡气体浓度，以验证正确的浓度。</li>
	<li>报告水性单位中挥发性气体的实验浓度，因为毫摩尔浓度对温度变化更可靠。使用以下公式将室温气相浓度P%（T室）转换为等效水浓度（C水溶液，单位为mM）31： <img alt="Equation 4" src="/files/ftp_upload/61333/61333equ04.jpg"> 其中α是25°C下异氟醚的盐水/气体本生分配系数32。</li>
</ol>5. 多声道录音的硬件和软件准备
<ol><li>根据制造商说明设置16通道数据采集系统。 注意：可以使用几种市售放大器和数据采集系统来收集多通道记录。在这里描述的实验中，模拟信号通过电极参考面板传递到两个放大器，在那里它们被放大（2000x）和滤波（0.1-10kHz）。数据采集系统的模拟输入以40kHz的频率数字化。</li>
	<li>将适当的 16 通道头台适配器固定到显微镜显微操纵器上。调整适配器的方向，使母连接器端口朝下。</li>
	<li>调整该显微操纵器的操作角度，使其向下朝向记录室，相对于水平角度约为70°。</li>
	<li>通过固定在显微操纵器的上台适配器将上台输入连接到 16 x 1 探头进行体外电生理学。</li>
	<li>将头级输出连接器连接到数据采集系统。</li>
	<li>安装适当的数据采集软件。配置 15 个输入通道以对应于来自前 15 个多通道探头触点的输入信号。配置其余通道以接收来自细胞内电极的输入。 注意： 在收集和分析数据时，请注意电极和适配器图，以确保适当的信号与收集它的电极触点相对应。</li>
</ol>6. 光刺激方案的配置
<ol><li>设置光传输系统并安装随附的软件。</li>
	<li>打开软件。选择硬件接线配置，其中触发源（数字/TTL 输出）向光传输系统提供触发输入信号，光传输系统向 470 nm LED 提供触发输出信号。</li>
	<li>安装高倍物镜。使用数码相机校准大倍光物镜，以便与光传输系统配合使用。</li>
	<li>创建光刺激配置文件的新配置文件序列。<ol><li>创建选择模式。在这里描述的实验中，使用直径为150μm的圆来允许轴突末端的层特异性激活。</li>
		<li>要构建配置文件序列，请为任意数量的试验中的每一个复制并粘贴此配置文件。</li>
		<li>创建包含任何光强度、脉冲持续时间或脉冲数的波形的波形列表。</li>
		<li>将波形随机分配给每个配置文件。每个配置文件及其分配的波形对应于来自数字TTL输入或一个试验的一个触发脉冲。</li>
		<li>保存配置文件序列。</li>
	</ol></li>
	<li>在数据采集软件中，创建一个新协议。<ol><li>将试验数设置为等于刚刚创建的配置文件序列中的配置文件数。</li>
		<li>选择信号输入以匹配步骤 5.6 中配置的输入。配置提供单个数字TTL输出的协议，在数字触发之前和之后从这16个输入通道记录适当的时间。</li>
	</ol></li>
</ol>7.将多通道探针置于离体脑组织切片中
<ol><li>以 3-6 mL/min 的速度灌注气泡 eACSF（不在密封袋中）。</li>
	<li>将包含感兴趣区域的脑切片转移到显微镜灌注室的网状网格上。用铂金竖琴锚（见 补充图3）。</li>
	<li>旋转网格，使多通道探头远端的电极触点线近似垂直于皮亚表面。</li>
	<li>在宽场照明和显微操纵器的精细控制下，将多通道探头朝向切片表面。</li>
	<li>旋转滤光片立方转盘以接合适当的滤光片立方体，以可视化在皮质传入轴突末端表达的荧光报告蛋白。如有必要，旋转切片以更精确地将探头与皮亚表面对齐。</li>
	<li>将探针放置在切片平面的正上方，沿x轴与最终目标位置相差~200μm，在被记录的组织区域的边界之外留下至少一个通道</li>
	<li>通过沿其纵轴移动机械手，将探头缓慢插入切片中。为了尽量减少对组织的损害，仅将探头推进到尖锐尖端刚好在组织表面下方可见的程度。这将最大限度地减少对组织的损害，同时仍然确保电极触点与组织接触。</li>
</ol>8. 膜片钳靶神经元并获得全细胞构型
<ol><li>将 eACSF 源切换到袋装控制溶液。</li>
	<li>鉴定荧光标记的细胞，以进行靶向膜片钳记录。<ol><li>将光圈光圈限制为最小直径。使用低倍物镜，使组织聚焦。</li>
		<li>将光线集中在与多通道探头相邻（但不重叠）的组织区域上。</li>
		<li>接合高倍（40x或60x）水浸物镜，小心避免多通道探头和物镜接触。</li>
		<li>旋转滤光片立方体转盘以接合适当的滤光片组，以允许对表达Cre依赖性荧光标记物的细胞进行成像。</li>
		<li>鉴定荧光标记的细胞作为膜片钳记录的靶标。抬起物镜以腾出足够的空间来降低贴片移液器。</li>
	</ol></li>
	<li>用内部溶液（表2）加载贴片移液器（见表1），并将移液器安装到电极支架中。使用 1 mL 注射器，施加相当于 ~0.1mL 空气的正压。</li>
	<li>将贴片移液器降低到溶液中。在目视引导下聚焦移液器吸头。</li>
	<li>使用先前演示的步骤从目标细胞获得全细胞记录33。</li>
	<li>如果计划评估电池内在特性的变化（例如，输入电阻，响应当前步骤的动作电位放电率），请进行这些记录。否则，请转到下面的轴突刺激协议。</li>
</ol>9. 轴突末端的层特异性光遗传学激活
<ol><li>操纵 x-y 平面中的视场，使光刺激配置文件与切片上的所需位置对齐。</li>
	<li>加载光激励协议并准备光传输系统以接收数字TTL脉冲。</li>
	<li>光遗传学激活轴突末端，同时记录细胞外场电位和细胞内膜波动。</li>
	<li>将eACSF源切换到异氟醚溶液并洗涤药物15分钟。如有必要，在冲洗期间收集自发录音。</li>
	<li>重复步骤 9.2-9.3。</li>
	<li>将eACSF源切换到对照溶液并洗出药物20分钟。如有必要，在冲洗期间收集自发记录。</li>
	<li>重复步骤 9.2-9.3。</li>
</ol>

<ol>
	<li>Baumgart, J. P., 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=Isoflurane+inhibits+synaptic+vesicle+exocytosis+through+reduced+Ca2++influx,+not+Ca2+-exocytosis+coupling.">Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx, not Ca2+-exocytosis coupling.</a> Proceedings of the National Academy of Sciences U.S.A. 112 (38), 11959-11964 (2015).</li><li>Herring, B. E., Xie, Z., Marks, J., Fox, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+inhibits+the+neurotransmitter+release+machinery.">Isoflurane inhibits the neurotransmitter release machinery.</a> Journal of Neurophysiology. 102 (2), 1265-1273 (2009).</li><li>Xie, Z., 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=Interaction+of+anesthetics+with+neurotransmitter+release+machinery+proteins.">Interaction of anesthetics with neurotransmitter release machinery proteins.</a> Journal of Neurophysiology. 109 (3), 758-767 (2013).</li><li>Crick, F., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+framework+for+consciousness.">A framework for consciousness.</a> Nature Neurosciences. 6 (2), 119-126 (2003).</li><li>Koch, C., Massimini, M., Boly, M., Tononi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+correlates+of+consciousness:+progress+and+problems.">Neural correlates of consciousness: progress and problems.</a> Nature Reviews Neurosciences. 17 (5), 307-321 (2016).</li><li>Dehaene, S., Changeux, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+theoretical+approaches+to+conscious+processing.">Experimental and theoretical approaches to conscious processing.</a> Neuron. 70 (2), 200-227 (2011).</li><li>Friston, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+theory+of+cortical+responses.">A theory of cortical responses.</a> Philosophical Transactions of the Royal Society of London B Biological Sciences. 360 (1456), 815-836 (2005).</li><li>Mashour, G. A., Hudetz, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bottom-Up+and+Top-Down+Mechanisms+of+General+Anesthetics+Modulate+Different+Dimensions+of+Consciousness.">Bottom-Up and Top-Down Mechanisms of General Anesthetics Modulate Different Dimensions of Consciousness.</a> Frontiers in Neural Circuits. 11, 44 (2017).</li><li>Voss, L. J., Garcia, P. S., Hentschke, H., Banks, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Effects+of+General+Anesthetics+on+Cortical+Network+Activity+Using+Ex+Vivo+Preparations.">Understanding the Effects of General Anesthetics on Cortical Network Activity Using Ex Vivo Preparations.</a> Anesthesiology. 130 (6), 1049-1063 (2019).</li><li>Redinbaugh, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thalamus+Modulates+Consciousness+Via+Layer-Specific+Control+of+Cortex.">Thalamus Modulates Consciousness Via Layer-Specific Control of Cortex.</a> Neuron. 105 (4), 0896 (2020).</li><li>Carhart-Harris, R. L., Friston, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REBUS+and+the+Anarchic+Brain:+Toward+a+Unified+Model+of+the+Brain+Action+of+Psychedelics.">REBUS and the Anarchic Brain: Toward a Unified Model of the Brain Action of Psychedelics.</a> Pharmacological Reviews. 71 (3), 316-344 (2019).</li><li>Mak-McCully, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+cortical+and+thalamic+activity+during+non-REM+sleep+in+humans.">Coordination of cortical and thalamic activity during non-REM sleep in humans.</a> Nature communications. 8 (1), 15499 (2017).</li><li>Alkire, M. T., Hudetz, A. G., Tononi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consciousness+and+anesthesia.">Consciousness and anesthesia.</a> Science. 322 (5903), 876-880 (2008).</li><li>Sanders, R. D., Maze, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noradrenergic+trespass+in+anesthetic+and+sedative+states.">Noradrenergic trespass in anesthetic and sedative states.</a> Anesthesiology. 117 (5), 945-947 (2012).</li><li>Hemmings, H. C., 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=Towards+a+Comprehensive+Understanding+of+Anesthetic+Mechanisms+of+Action:+A+Decade+of+Discovery.">Towards a Comprehensive Understanding of Anesthetic Mechanisms of Action: A Decade of Discovery.</a> Trends in Pharmacological Sciences. 40 (7), 464-481 (2019).</li><li>Nourski, K. V., 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=Auditory+Predictive+Coding+across+Awareness+States+under+Anesthesia:+An+Intracranial+Electrophysiology+Study.">Auditory Predictive Coding across Awareness States under Anesthesia: An Intracranial Electrophysiology Study.</a> Journal of Neurosciences. 38 (39), 8441-8452 (2018).</li><li>Liu, X., 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=Propofol+disrupts+functional+interactions+between+sensory+and+high-order+processing+of+auditory+verbal+memory.">Propofol disrupts functional interactions between sensory and high-order processing of auditory verbal memory.</a> Human Brain Mapping. 33 (10), 2487-2498 (2012).</li><li>Hentschke, H., Raz, A., Krause, B. M., Murphy, C. A., Banks, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+cortical+network+activity+by+the+general+anesthetic+isoflurane.">Disruption of cortical network activity by the general anesthetic isoflurane.</a> British Journal of Anaesthesiology. 119 (4), 685-696 (2017).</li><li>Lee, U., 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=Disruption+of+frontal-parietal+communication+by+ketamine,+propofol,+and+sevoflurane.">Disruption of frontal-parietal communication by ketamine, propofol, and sevoflurane.</a> Anesthesiology. 118 (6), 1264-1275 (2013).</li><li>Ferrarelli, F., 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=Breakdown+in+cortical+effective+connectivity+during+midazolam-induced+loss+of+consciousness.">Breakdown in cortical effective connectivity during midazolam-induced loss of consciousness.</a> Proceedings of the National Academy of Science U.S.A. 107 (6), 2681-2686 (2010).</li><li>Ku, S. W., Lee, U., Noh, G. J., Jun, I. G., Mashour, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+inhibition+of+frontal-to-parietal+feedback+connectivity+is+a+neurophysiologic+correlate+of+general+anesthesia+in+surgical+patients.">Preferential inhibition of frontal-to-parietal feedback connectivity is a neurophysiologic correlate of general anesthesia in surgical patients.</a> PLoS.One. 6 (10), 25155 (2011).</li><li>Lee, M., 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=Network+Properties+in+Transitions+of+Consciousness+during+Propofol-induced+Sedation.">Network Properties in Transitions of Consciousness during Propofol-induced Sedation.</a> Scientific Reports. 7 (1), 16791 (2017).</li><li>Murphy, M., 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=Propofol+anesthesia+and+sleep:+a+high-density+EEG+study.">Propofol anesthesia and sleep: a high-density EEG study.</a> Sleep. 34 (3), 283-291 (2011).</li><li>Murphy, C., Krause, B., Banks, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+effects+of+isoflurane+on+cortico-cortical+feedback+afferent+responses+in+murine+non-primary+neocortex.">Selective effects of isoflurane on cortico-cortical feedback afferent responses in murine non-primary neocortex.</a> British Journal of Anaesthesiology. 123 (4), 488-496 (2019).</li><li>Raz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+effect+of+isoflurane+on+top-down+versus+bottom-up+pathways+in+sensory+cortex.">Preferential effect of isoflurane on top-down versus bottom-up pathways in sensory cortex.</a> Frontiers in System Neurosciences. 8, 191 (2014).</li><li>Schrouff, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+functional+integration+decreases+during+propofol-induced+loss+of+consciousness.">Brain functional integration decreases during propofol-induced loss of consciousness.</a> Neuroimage. 57 (1), 198-205 (2011).</li><li>Petreanu, L., Mao, T., Sternson, S. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+subcellular+organization+of+neocortical+excitatory+connections.">The subcellular organization of neocortical excitatory connections.</a> Nature. 457 (7233), 1142-1145 (2009).</li><li>Cruikshank, S. J., Urabe, H., Nurmikko, A. V., Connors, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathway-Specific+Feedforward+Circuits+between+Thalamus+and+Neocortex+Revealed+by+Selective+Optical+Stimulation+of+Axons.">Pathway-Specific Feedforward Circuits between Thalamus and Neocortex Revealed by Selective Optical Stimulation of Axons.</a> Neuron. 65 (2), 230-245 (2010).</li><li>Gredell, J. A., Turnquist, P. A., MacIver, M. B., Pearce, 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=Determination+of+diffusion+and+partition+coefficients+of+propofol+in+rat+brain+tissue:+implications+for+studies+of+drug+action+in+vitro.">Determination of diffusion and partition coefficients of propofol in rat brain tissue: implications for studies of drug action in vitro.</a> BJA: British Journal of Anaesthesia. 93 (6), 810-817 (2004).</li><li>Benkwitz, C., 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=Determination+of+the+EC50+amnesic+concentration+of+etomidate+and+its+diffusion+profile+in+brain+tissue:+implications+for+in+vitro+studies.">Determination of the EC50 amnesic concentration of etomidate and its diffusion profile in brain tissue: implications for in vitro studies.</a> Anesthesiology. 106 (1), 114-123 (2007).</li><li>Franks, N. P., Lieb, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+actions+of+volatile+general+anaesthetics+at+molecular+and+cellular+levels.">Selective actions of volatile general anaesthetics at molecular and cellular levels.</a> British Journal of Anaesthesia. 71 (1), 65-76 (1993).</li><li>Honemann, C. W., Washington, J., Honemann, M. C., Nietgen, G. W., Durieux, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partition+coefficients+of+volatile+anesthetics+in+aqueous+electrolyte+solutions+at+various+temperatures.">Partition coefficients of volatile anesthetics in aqueous electrolyte solutions at various temperatures.</a> Anesthesiology. 89 (4), 1032-1035 (1998).</li><li>Au - Segev, A., Au - Garcia-Oscos, F., Au - Kourrich, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+Patch-clamp+Recordings+in+Brain+Slices.">Whole-cell Patch-clamp Recordings in Brain Slices.</a> Journal of Visualized Experiments. (112), e54024 (2016).</li><li>Lin, J. Y., Lin, M. Z., Steinbach, P., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+engineered+channelrhodopsin+variants+with+improved+properties+and+kinetics.">Characterization of engineered channelrhodopsin variants with improved properties and kinetics.</a> Biophysical Journal. 96 (5), 1803-1814 (2009).</li><li>Lin, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+user's+guide+to+channelrhodopsin+variants:+features,+limitations+and+future+developments.">A user's guide to channelrhodopsin variants: features, limitations and future developments.</a> Experimental Physiology. 96 (1), 19-25 (2011).</li><li>Banks, M. I., Pearce, 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=Dual+actions+of+volatile+anesthetics+on+GABAA+IPSCs:+dissociation+of+blocking+and+prolonging+effects.">Dual actions of volatile anesthetics on GABAA IPSCs: dissociation of blocking and prolonging effects.</a> Anesthesiology. 90 (1), 120-134 (1999).</li><li>Hagan, C. E., Pearce, R. A., Trudell, J. R., MacIver, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+measures+of+volatile+anesthetics+in+the+aqueous+phase+using+calcium+sensitive+electrodes.">Concentration measures of volatile anesthetics in the aqueous phase using calcium sensitive electrodes.</a> Journal of Neuroscience Methods. 81, 177-184 (1998).</li></ol>

作者没有什么可透露的。

在这篇手稿中，描述了一种用于评估离体脑切片中选择性激活的传入途径的细胞内和细胞外反应的方案。
光遗传学工具和并行记录方案的使用使研究人员能够探测局部人群对来自远处大脑区域的传入输入的反应，同时同时记录来自中间神经元的目标人群。光遗传学技术的使用允许保留和激活传入投影的轴突末端，即使它们的细胞体不再附着。这减轻了以前对 离体 切片...

一个多世纪以来，挥发性麻醉剂在各种临床和学术环境中无处不在。不同类别的麻醉剂具有独特的，通常不重叠的分子靶点1，2，3，但几乎所有麻醉剂都产生无意识。虽然它们的行为影响是完全可预测的，但麻醉剂诱导意识丧失的机制在很大程度上是未知的。麻醉剂可能最终通过对皮质丘脑回路的作用影响意识的水平和内容，破坏整个皮质层次结构的信息整合4，5，6，7，8，9。更广泛地说，皮质丘脑回路的调节可能在实验10或药理学11的意识状态改变中发挥作用，也可能与睡眠12和意识的病理生理障碍13，14有关。
麻醉期间意识丧失和恢复机制的难以捉摸可能部分归因于麻醉剂在细胞、网络和系统水平上的非线性协同作用15。例如，异氟醚抑制所选大脑区域16，17，18内的活动，损害远处大脑区域19，20，21，22，23之间的连接，并以通路特异性方式减少突触反应24，25.麻醉剂的哪些效果，从分子到系统水平，是必要或足以影响意识丧失的尚不清楚。除了使用非侵入性技术对意识进行实质性临床研究19，20，26之外，重要的是实验学家试图解开服务于意识体验的独特细胞和网络相互作用。
通过简化完整大脑中发现的复杂相互作用，离体脑切片允许研究大脑动态系统的孤立组件9。减少切片制备结合了局部神经回路相对完整的解剖结构的优点和体外操作的多功能性。然而，直到最近，方法学上的限制已经排除了对脑切片中长程输入的突触和回路特性的研究27，28;皮质丘脑纤维束的曲折路径使得电刺激几乎不可能激活独立的传入途径。
研究麻醉剂对脑切片制剂的影响提出了额外的挑战。如果没有完整的呼吸和循环系统，必须使用麻醉剂洗澡，并且浓度与估计的效果部位浓度仔细匹配。对于许多静脉麻醉剂，组织中平衡速度的缓慢使得传统的药理学研究费力29，30。研究挥发性气体麻醉剂在离体制剂中的影响更容易处理，但也带来了挑战。这些包括将吸入分压剂量转换为水性浓度，以及需要通过人工脑脊液将药物的改良输送系统输送到组织31。
在这里，描述了研究人员可以利用挥发性麻醉异氟醚的有据可查的物理化学特性将药物输送到离体脑切片的方法，以高时空分辨率激活对感兴趣的皮质区域的通路和层特异性输入，并同时进行层流记录和来自选定神经元群的靶向膜片钳记录。综合起来，这些程序使研究人员能够测量挥发性麻醉剂诱导的几种可观察到的电生理反应特性的变化，从突触到局部网络水平。

<table>
	<tbody>
		<tr>
			<td>2.5x broadfield objective lens</td>
			<td>Olympus</td>
			<td>MPLFLN2.5X</td>
			<td></td>
		</tr>
		<tr>
			<td>40x water immersion objective lens</td>
			<td>Olympus</td>
			<td>LUMPLFLN40XW</td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2/5% CO2 mixture</td>
			<td>Airgas</td>
			<td>Z02OX95R2003045</td>
			<td></td>
		</tr>
		<tr>
			<td>A16 probe</td>
			<td>NeuroNexus</td>
			<td>A16x1-2mm-100-177-A16</td>
			<td>16-channel probe</td>
		</tr>
		<tr>
			<td>AAV2-hSyn-hChR2(H134R)-EYFP</td>
			<td>Karl Deisseroth Lab, UNC Vector Core</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic gas monitor (POET II)</td>
			<td>Criticare</td>
			<td>602-3A</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP, Magnesium Salt</td>
			<td>Sigma Aldrich</td>
			<td>A9187</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J</td>
			<td>The Jackson Laboratory</td>
			<td>007914</td>
			<td>Cre-dependent tdTomato mouse</td>
		</tr>
		<tr>
			<td>B6;129P2-Pvalbtm1(cre)Arbr/J</td>
			<td>The Jackson Laboratory</td>
			<td>008069</td>
			<td>PV-Cre mouse</td>
		</tr>
		<tr>
			<td>Belly Dancer Shaker</td>
			<td>Thomas Scientific</td>
			<td>1210H86-TS</td>
			<td>for equilibration of sealed gas bags</td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>Generic brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Generic brand</td>
			<td></td>
			<td>for silver chloriding patch clamp electrode</td>
		</tr>
		<tr>
			<td>Bupivicaine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Dot Scientific</td>
			<td>DSC20010</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Capillary glass (patch clamp recordings)</td>
			<td>King Precision Glass, Inc.</td>
			<td>KG-33</td>
			<td>Borosilicate, ID: 1.1mm, OD: 1.7mm, Length: 90.0mm</td>
		</tr>
		<tr>
			<td>Capillary glass (viral injections)</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-203-G/X</td>
			<td>3.5&#34;</td>
		</tr>
		<tr>
			<td>Control of junior micromanipulator</td>
			<td>Luigs and Neumann</td>
			<td>SM8</td>
			<td>for control of junior micromanipulator</td>
		</tr>
		<tr>
			<td>Control of manipulators and shifting table</td>
			<td>Luigs and Neumann</td>
			<td>SM7</td>
			<td>for control of multichannel electrode and shifting table</td>
		</tr>
		<tr>
			<td>Digidata 1440A + Clampex 10</td>
			<td>Molecular Devices</td>
			<td>1440A</td>
			<td>Digitizer and software</td>
		</tr>
		<tr>
			<td>E-3603 tubing</td>
			<td>Fisher Scientific</td>
			<td>14171208</td>
			<td>for delivery of 95% O2/5% CO2 gas mixture to incubation chamber + application of pressure during patch clamping</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Dot Scientific</td>
			<td>DSE57060</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>ERP-27 EEG Reference/Patch Panel</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Filling needle</td>
			<td>World Precision Instruments</td>
			<td>50821912</td>
			<td>for filling patch clamp pipettes</td>
		</tr>
		<tr>
			<td>Filter cube for imaging EYFP</td>
			<td>Olympus</td>
			<td>U-MRFPHQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Fisher Scientific</td>
			<td>09801E</td>
			<td>lay over slice template during preparation of tissue block</td>
		</tr>
		<tr>
			<td>Flaming/Brown micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td>2.5x2.5 Box filament</td>
		</tr>
		<tr>
			<td>Gas dispersion tube</td>
			<td>Sigma Aldrich</td>
			<td>CLS3953312C</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass syringe (100 mL)</td>
			<td>Sigma Aldrich</td>
			<td>Z314390</td>
			<td>for filling gas-sealed bags</td>
		</tr>
		<tr>
			<td>Gluconic Acid, Potassium Salt (K-gluconate)</td>
			<td>Dot Scientific</td>
			<td>DSG37020</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Dot Scientific</td>
			<td>DSG32040</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>GTP, Sodium Salt</td>
			<td>Sigma Aldrich</td>
			<td>G8877</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Headstage-probe adaptor</td>
			<td>NeuroNexus</td>
			<td>A16-OM16</td>
			<td>adaptor to connect 16-channel probe to headstage input</td>
		</tr>
		<tr>
			<td>Hemostatic Forceps</td>
			<td>VWR International</td>
			<td>76192-096</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Dot Scientific</td>
			<td>DSH75030</td>
			<td>ACSF,intracellular solution</td>
		</tr>
		<tr>
			<td>HS-16 Headstage</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol (70%)</td>
			<td>VWR International</td>
			<td>101223-746</td>
			<td></td>
		</tr>
		<tr>
			<td>Junior micromanipulator</td>
			<td>Luigs and Neumann</td>
			<td>210-100 000 0090-R</td>
			<td>for manipulation of patch clamp electrode</td>
		</tr>
		<tr>
			<td>LED Light Source Control Module</td>
			<td>Mightex</td>
			<td>BLS-PL02_US</td>
			<td>optogenetic light source control</td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lynx-8 Amplifier</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Lynx-8 Power Supply</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (MgSO4)</td>
			<td>Dot Scientific</td>
			<td>DSM24300</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>mCherry, Texas Red filter cube</td>
			<td>Chroma</td>
			<td>49008</td>
			<td>for imaging tdTomato fluorescent reporter</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette holder</td>
			<td>Fisher Scientific</td>
			<td>NC9044962</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsyringe pump</td>
			<td>World Precision Instruments</td>
			<td>UMP3-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Generic brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MultiClamp 700A</td>
			<td>Molecular Devices/Axon Instruments</td>
			<td>700A</td>
			<td>Amplifier</td>
		</tr>
		<tr>
			<td>Nitrogen (for air table)</td>
			<td>Airgas</td>
			<td>NI200</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh</td>
			<td>Fisher Scientific</td>
			<td>501460083</td>
			<td>stretched over horseshoe of flattened platinum wire, slice rest on top of this during recordings</td>
		</tr>
		<tr>
			<td>Nylon, cut from pantyhose</td>
			<td>Generic brand</td>
			<td></td>
			<td>small piece to create slice platform in incubation chamber, single fibers to create platinum harp</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Fisher Scientific</td>
			<td>NC1697520</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Dot Scientific</td>
			<td>307</td>
			<td>For transferring tissue to rig</td>
		</tr>
		<tr>
			<td>Platinum wire</td>
			<td>VWR International</td>
			<td>BT124000</td>
			<td>2 cm, flattened, to make platinum harp</td>
		</tr>
		<tr>
			<td>Polygon400</td>
			<td>Mightex</td>
			<td>DSI-E-0470-0617-000</td>
			<td>optogenetic light delivery system, comes with PolyScan2 software</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Dot Scientific</td>
			<td>DSP41000</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Potassium Phosphate (KH2PO4)</td>
			<td>Dot Scientific</td>
			<td>DSP41200</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>Sapphire blade (for vibratome)</td>
			<td>VWR International</td>
			<td>100492-502</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Santa Cruz Biotechnology, Inc.</td>
			<td>sc-361445</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealed gas bag</td>
			<td>Fisher Scientific</td>
			<td>109236</td>
			<td></td>
		</tr>
		<tr>
			<td>Shifting table for microscope</td>
			<td>Luigs and Neumann</td>
			<td>380FMU</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (HCO3-)</td>
			<td>Dot Scientific</td>
			<td>DSS22060</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Dot Scientific</td>
			<td>DSS23020</td>
			<td>ACSF, intracellular solution</td>
		</tr>
		<tr>
			<td>Ssttm2.1(cre)Zjh/J (SOM-IRES-Cre)</td>
			<td>The Jackson Laboratory</td>
			<td>013044</td>
			<td>SOM-Cre mouse</td>
		</tr>
		<tr>
			<td>Stereotaxic instrument</td>
			<td>Kopf</td>
			<td>Model 902</td>
			<td>Dual Small Animal</td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>Staples</td>
			<td>886833</td>
			<td>to fix tissue block to specimen stage during slice preparation</td>
		</tr>
		<tr>
			<td>Surgical drill</td>
			<td>RAM Products Inc.</td>
			<td>DIGITALMICROTORQUE</td>
			<td>Microtorque II</td>
		</tr>
		<tr>
			<td>Syringe (1 mL) with LuerLock tip</td>
			<td>Fisher Scientific</td>
			<td>309628</td>
			<td>for application of pressure during patch clamping</td>
		</tr>
		<tr>
			<td>Syringe (1 mL) with slip tip</td>
			<td>WW Grainger, Inc.</td>
			<td>19G384</td>
			<td>for filling patch clamp pipettes</td>
		</tr>
		<tr>
			<td>Syringe Filters</td>
			<td>VWR International</td>
			<td>66064-414</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrating microtome</td>
			<td>Leica Biosystems</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Wypall towels</td>
			<td>Fisher Scientific</td>
			<td>19-042-427</td>
			<td></td>
		</tr>
	</tbody>
</table>

optogenetic activation of afferent pathways in brain slices and modulation of responses by volatile anesthetics

麻醉剂部分通过它们对丘脑皮质回路的作用来影响意识。然而，挥发性麻醉剂对这些回路的不同细胞和网络组件的影响程度尚不清楚。离体脑切片提供了一种方法，研究人员可以通过这种方法探测复杂网络的离散成分，并解开挥发性麻醉剂对诱发反应影响的潜在机制。为了分离脑切片中潜在的细胞类型和途径特异性药物效应，研究人员必须能够独立激活传入纤维途径，识别非重叠的细胞群，并将挥发性麻醉剂应用于水溶液中的组织。在该协议中，描述了测量光遗传学诱发对离体脑切片中新皮层的两个独立传入途径的反应的方法。记录细胞外反应以测定网络活性，并在生长抑素和细小白蛋白阳性中间神经元中进行靶向全细胞膜片钳记录。描述了通过人工脑脊液输送生理相关浓度的异氟醚以调节细胞和网络反应。

协议中描述的步骤时间表如图 1 所示。来自高阶皮质区域或非初级丘脑核的皮质输入在非初级视觉皮层1 层 24 中具有部分重叠的末端场。为了分离独立的丘脑皮质或皮质传入途径，将含有ChR2和eYFP荧光报告基因的病毒载体注射到Po或Cg中。注射半径内的细胞吸收病毒载体，并在2-4周后在体细胞和突出轴突中表达非特异性阳离子通道ChR2和报告基因（<strong class="...

Watch this Scientific Journal Video about Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics at JoVE.com

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

离体脑切片可用于研究挥发性麻醉剂对传入输入的诱发反应的影响。光遗传学用于独立激活丘脑皮质和皮质皮层传入非原发性新皮层，并且用异氟醚调节突触和网络反应。

脑切片中传入通路的光遗传学激活和挥发性麻醉剂对反应的调节

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

optogenetic-activation-afferent-pathways-brain-slices-modulation

Research

JoVE Journal

Neuroscience

2.1K 次观看.  University of Wisconsin. 离体脑切片可用于研究挥发性麻醉剂对传入输入的诱发反应的影响。光遗传学用于独立激活丘脑皮质和皮质皮层传入非原发性新皮层，并且用异氟醚调节突触和网络反应。

视频: Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

科研

神经科学

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Optogenetic激活斑马鱼的体感神经元使用厨师tdTomato的

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

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 ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

在脑片恐惧电路体外光遗传学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海马鼠脑切片的突触密度评价

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

粉扑诱导的全细胞电流在脑切片中的应用

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. Through such combinatorial coding, organisms can detect and discriminate between a myriad of volatile odor molecules. Thus, an odor at a given concentration can be described by an activation pattern of ORs, which is specific to each odor. In that sense, cracking the mechanisms that the brain uses to perceive odor requires the understanding odorant-OR interactions. This is why the olfaction community is committed to &#34;de-orphanize&#34;&#160;these receptors. Conventional in vitro systems used to identify odorant-OR interactions have utilized incubating cell media with odorant, which is distinct from the natural detection of odors via vapor odorants dissolution into nasal mucosa before interacting with ORs. Here, we describe a new method that allows for real-time monitoring of OR activation via vapor-phase odorants. Our method relies on measuring cAMP release by luminescence using the Glosensor assay. It bridges current gaps between in vivo and in vitro approaches and provides a basis for a biomimetic volatile chemical sensor.

Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase.

气相臭受体活化的实时体外监测

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.