作者希望感谢肯塔基大学微电极技术中心 (CenMeT) 和俄亥俄州立大学实验动物资源中心 (乌拉尔) 的贡献。

仔猪 (Sus 鲷) 是通过一个当地农场接收的, 由俄亥俄州立大学机构动物保育和使用委员会 (IACUC) 预先批准。经过该议定书的批准, 动物实验是按照 IACUC 政策进行的。
1. 仔猪和仔猪的搬运
<ol><li>以系统和随机的方式利用雄性和雌性仔猪, 以消除任何潜在的基于性别的混杂根据到达指南19。 注: 由于最大脑发育期在仔猪出生后3-5 天内, 实验仅以仔猪3-5 天的年龄进行。</li>
	<li>确保仔猪在试验前至少24小时到达 vivarium, 以便对环境进行驯化。 注: 训练有素的兽医人员提供日常动物护理。仔猪被保存在单独的温度维持, 不断监测的网箱, 并获得营养完整的牛奶乳粉ad 随意, 以防止低血糖。仔猪还保持不加奶 (零每 os), 至少3小时麻醉前, 并提供毛毯和玩具, 以确保正常水平的刺激。如果可能的话, 把多只小猪放在同一个笼子里, 允许社会化。</li>
</ol>2. 仔猪模型研究中的环境协定的开发和定制
注: 该技术采用酶促生物协定, 预涂酶和电镀的 m 苯二胺盐酸盐 (mPD)。电极是定制的设计与40毫米刚性轴和生产用于仔猪 (图 1)。
<ol><li>为避免冗余, 请参阅前面描述的15 (图 2) 的详细说明和校准。</li>
</ol>3. 用于仔猪的自定义立体定向装置的麻醉和使用
<ol><li>麻醉动物使用一个麻醉工作站与适当的呼吸机和监测设备, 并监测生理参数, 如脉搏氧, 无创血压, 心电图和温度贯穿整个以前描述的19的整个实验。对仔猪进行插管和通气, 并对七氟醚进行1最小肺泡浓度 (MAC) (约 2.5-3%) 的麻醉, 以达到3.5 小时的水平. 确保这些实验的训练有素的实验室工作人员在场。在准备皮肤之前, 用电动快刀将手术部位上的皮毛去掉。 注: 使用麻醉的浓度和持续时间允许实验模拟手术过程中实际麻醉暴露的时间长度。此外, 七氟醚是儿科人群中最常用的全麻, 使调查对其安全性具有极其重要的意义。</li>
	<li>在放置到立体定向的框架, 开始一个罗库溴铵负荷剂量2.5 毫克/千克和输液1.5 毫克/公斤/小时, 以防止动物运动, 而在安全的框架。<ol>
<li>一旦适当的麻醉深度被脚趾捏确认, 把小猪放在小猪特定的立体定位框架内。</li>
		<li>在立体定向的框架内提供足够的填料, 将小猪放在强制空气变暖垫上, 附加填料 (例如, 流化变位机) 以防止压力溃疡。</li>
		<li>将上颌骨的牙齿放在齿条上 (图 3)。 注: 牙棒应在足够的水平, 以保持头骨非常牢固到位。</li>
		<li>固定和收紧耳朵内的穿透耳棒, 注意确保小猪处于中线位置。将侧面的尖尖尖端放置在耳道内, 并牢固地插入耳棒, 以听到与鼓膜穿透有关的 "pop" 声音。将耳条牢固地附着在头骨上, 并在每一侧插入到相等的深度, 以防止头骨在实验期间的移动 (图 4, 面板 A)。 注意: 保持仔猪的体温 (37.8-38.6 °c) 并持续监测整个过程中的温度对常温的维护至关重要。这可以通过通过毯子和/或热灯完成。一定要在适当的距离放置散热灯, 以避免烧伤动物的皮肤。</li>
	</ol>
</li>
	<li>用小心的方法在头骨上创建一个4-6 厘米的中线切口, 以避免用手术刀把头骨打进。一旦切口, 使用温和的缩回和钝的解剖, 以提高头皮从头骨。用纱布垫轻轻擦洗头骨, 清除任何结缔组织并暴露缝合线 (图 4, 面板 B)。 注意: 没有必要在非生存实验中保持不孕。然而, 在生存实验中必须保持不孕。</li>
	<li>如果需要, 进一步反映头皮, 以暴露感兴趣的区域并确定开颅手术的预定位置 (图 5, 面板 A)。使用外科演练创建大约0.25 厘米2的开颅手术窗口 (根据实验目标可能会更大或更小) 覆盖感兴趣的结构, 使用谨慎不伤害硬脑膜或底层大脑 (图5 , 面板 B)。使用精细的外科剪刀来切除脑组织覆盖的硬脑膜 (图 5, 面板 C)。</li>
	<li>通过确保 headstage 的金属臂与小猪 stereotax 的机器人, 将电极尽可能垂直地放置在 bregma 上。尽量降低电极, 不接触头骨表面。记录 bregma 的坐标 (图 6, 面板 A)。<ol>
<li>确定前、后外侧坐标以及与 bregma 相关的兴趣结构的深度。使用一个物种和年龄合适的立体定向图谱确定立体定向坐标。在这种情况下, 使用专门为小猪20开发的立体定向图谱。</li>
		<li>重新定位电极, 使其具有适当的前后和内侧侧位置, 确保微电极和仪器都垂直于表面。将伪参考电极置于头皮下, 确保与动物接触 (图 6, 面板 B)。</li>
		<li>慢慢地, 轻轻地, 用立体定向的手臂将电极降低到大脑的适当深度。在最后2毫米, 使用液压 microdrive 进一步驱动电极到确切的位置 (图 6, 面板 C)。 注: 电极位置应位于开颅窗。电极深度会因感兴趣的大脑结构而异。在非生存实验数据收集完成后, 没有必要关闭切口。</li>
	</ol>
</li>
</ol>4. 七氟醚麻醉下胞外谷氨酸的测定
<ol><li>确保仔猪在整个过程中持续进行生理监测。仔猪麻醉 inhalationally (通过面锥) 为程序准备。</li>
	<li>在该多边环境协定的植入后, 等待30分钟, 允许电极达到基线, 以确保为 3 h (图 7) 获得正确的测量结果。</li>
	<li>0.25% 布比卡因 (1 毫升/千克) 在术后疼痛管理的手术现场被管理。此外, 持续释放丁丙诺啡是根据需要皮下 q72h。</li>
</ol>5. 灌注和牺牲
<ol><li>执行以前描述的21的灌注和脑组织收集过程。对于非生存手术, 动物在试验后立即被安乐死, 但仍在全身麻醉下。</li>
	<li>采取固定的小猪大脑的大体横断面, 并使用光显微镜可视化电极的轨道, 如前所述的22 , 以允许验证多边环境协定的放置, 确保适当的位置在感兴趣的领域。</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+hippocampal+glutamate+and+spontaneous+seizure+in+the+conscious+human+brain.">Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain.</a> Lancet. 341 (8861), 1607-1610 (1993).</li><li>Reinstrup, 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=Intracerebral+microdialysis+in+clinical+practice:+baseline+values+for+chemical+markers+during+wakefulness,+anesthesia,+and+neurosurgery.">Intracerebral microdialysis in clinical practice: baseline values for chemical markers during wakefulness, anesthesia, and neurosurgery.</a> Neurosurgery. 47 (3), 701-710 (2000).</li><li>Cavus, I., 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=Extracellular+metabolites+in+the+cortex+and+hippocampus+of+epileptic+patients.">Extracellular metabolites in the cortex and hippocampus of epileptic patients.</a> Ann Neurol. 57 (2), 226-235 (2005).</li></ol>

格雷格葛哈德是 Quanteon 有限责任公司的主要所有者。乔治. 金特罗·库维德斯和杰森 Burmeister 曾担任 Quanteon 有限责任公司的顾问。

从实验开始, 小猪的生理动态平衡必须按照本实验室先前发布的21中的描述进行维护。最小的监测应包括脉冲氧, 心电图, capnography, 无创血压和温度。需要训练有素的调查人员, 以便能够适当纠正生理扰动 (例如, 如、低体温/热疗、缺氧、低血压、心律失常)。
在归纳之前,体外在已知条件下进行校准, 以建立多边环境协定的功能和选择性。多边环境协定的校准和电镀是有效利用该技术的关键。在校准过程中可能会出现许多可能的错误。校准可以识别这些问题以及不正确的电镀, 导致错误的干扰响应。编写了一个更详细的表格, 说明了在多边环境协定响应中可能发生的错误, 以及值得注意的原因和建议的解决方案, 这些都应该成为可能的疑难解答的有用工具 (表 1)。必须注意的是, 在校准和电镀之前, 应检查玻璃参考电极是否存在气泡或白色变色, 因为两者都将消极地影响多边环境协定的功能和记录的准确性。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>症状</td>
			<td>原因</td>
			<td>纠正措施</td>
		</tr>
<tr>
<td rowspan="2">无信号</td>
			<td>电极未连接</td>
			<td>正确地将电极连接到 headstage 和 headstage, 以快速探头系统。</td>
		</tr>
<tr>
<td>对快速探头系统没有动力</td>
			<td>打开快速系统背面的电源开关</td>
		</tr>
<tr>
<td rowspan="5">信号噪声</td>
			<td rowspan="2">被血液污染的电极</td>
			<td>在电极插入过程中持续冲洗脑表面</td>
		</tr>
<tr>
<td>立即在 dH2O 中漂洗电极</td>
		</tr>
<tr>
<td>酶膜松动</td>
			<td>清洁和 recoat 电极</td>
		</tr>
<tr>
<td>未插入或涂覆参考电极</td>
			<td>外套和放置参考电极更远的头皮下</td>
		</tr>
<tr>
<td>电极检测脑表面运动</td>
			<td>通常发生在浅层结构中。如果可能, 将电极深度插入 (一次1毫米)</td>
		</tr>
<tr>
<td rowspan="2">动物运动</td>
			<td>动物没有得到充分的保护</td>
			<td>移动后方向的动物, 以更好地安全 earbars 的头骨。如有必要, 提升躯干, 使身体更好地对准。</td>
		</tr>
<tr>
<td>动物麻醉不充分</td>
			<td>验证麻醉设备的完整性。滴定麻醉的有效剂量和管理肌内的罗库溴铵剂量 (5 毫克/千克)</td>
		</tr>
<tr>
<td rowspan="3">电极放置不准确</td>
			<td>电极未正确对齐</td>
			<td>调整电极, 同时保持与 headstage 的正确连接。</td>
		</tr>
<tr>
<td rowspan="2">立体定向坐标不准确</td>
			<td>确保所引用的小猪图谱不使用另一个参照点或对齐平面。</td>
		</tr>
<tr>
<td>小心不要通过评分头骨来掩盖缝合痕迹。</td>
		</tr>
</tbody></table>表 1: 在仔猪中使用的疑难解答说明.可能的原因和纠正措施, 以协助优化和故障排除。
一个用于小猪的立体定向图谱用于确定感兴趣区域的立体定位坐标, 例如 bregma18。应妥善保护耳棒, 以确保头骨水平和完全固定。应注意在头皮中线切口, 以避免评分的头骨, 因为这可能会影响可视化缝合线。开颅窗应大到足以容纳多边环境协定。
该议定书提出了一些技术挑战, 需要有一个库存充足的操作套件和一个熟练掌握该议定书的外科和麻醉方面的调查员/小组。该模型还提出了财政限制, 因为小猪模型比啮齿动物模型更昂贵;然而, 它的成本大大低于使用非人类灵长类动物, 这可以花费上千美元。使用多边环境协定技术提出了自己的挑战, 因为手工涂敷和电镀电极的过程需要熟练的调查员或助理, 以确保足够的选择性和可靠的功能。电极本身是脆弱的, 因为它们是由陶瓷制成的, 因此如果没有观察到适当的谨慎, 就容易损坏。电极受到来自其他电气设备的干扰, 这些装置可以在录音中制造噪音, 并可在操作现场的血液中产生噪声, 咬合记录地点。需要专用设备带来额外的负担, 因为立体定向手术框架必须是定制的, 以固定的小猪头骨在植入过程中。立体定向的框架, 谷氨酸氧化酶, 和电极本身都是昂贵的。此外, 在过去十年中缺乏一只小猪立体定位图谱, 造成技术上的限制, 需要特定的专门知识来确定小猪大脑深层结构的具体位置。开发一个新的立体定向图谱, 也许使用磁共振成像, 将大大提高在仔猪中使用这种技术的能力。
仔猪是一种临床上相关的模型, 主要是由于这一物种和人类新生儿之间存在的相似性, 因为两者都具有相似的大脑结构和发展。与更常用的模型, 如老鼠或老鼠, 小猪有一个更大的中枢神经系统的相似性与人类, 这借给了模型的结果可译性。小猪模型是另外便宜和涉及较少复杂处理比非人类灵长类动物模型。小猪模型旨在研究麻醉可能诱发发育性神经毒性的过程, 测量其对神经损伤的贡献, 并与混淆变量引起的损害问题作斗争。例如, 缺氧可能被误解为麻醉剂造成的损害, 因为它对大脑有全球性的影响。仔猪的使用与人类医学中使用的外科和麻醉条件相同, 以确保结果的逼真度。
用陶瓷为基础的多边环境技术消除了与现代微透析技术相关的一些缺点。微透析与诸如多边环境协定等安培方法相比, 具有有限的时间和空间分辨率, 可以在多达10赫兹23的多个微观区域连续记录谷氨酸事件。这种快速采样率消除了局部神经递质扩散的混杂因子, 这是慢采样方法 (如微透析24) 所固有的。此外, 多边环境协定是一种比微透析探针更少的侵入性方法, 在插入的过程中可能导致显著的胶质, 并且可以改变插入点22的神经递质活动。
以前的研究使用了一系列哺乳动物模型, 测量技术和大脑区域, 表明了基本的谷氨酸水平, 可与使用这种技术发现。这表明, 在适应仔猪模型时, 多边环境协定技术提供了对体内谷氨酸浓度 (表 2) 的有效记录。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>作者 (年)</td>
			<td>录音技术</td>
			<td>动物模型</td>
			<td>年龄</td>
			<td>脑区 (s)</td>
			<td>平均基底谷氨酸浓度 (µM)</td>
		</tr>
<tr>
<td>Hascup 等 (2008)23
</td>
			<td>(以酶为基础)</td>
			<td>啮齿动物</td>
			<td>20-24 周</td>
			<td>前额叶皮质, 纹状体</td>
			<td>3.3 ~ 1.0;5.0 @ 1。2</td>
		</tr>
<tr>
<td>Hascup 等 (2010)25
</td>
			<td>(以酶为基础)</td>
			<td>啮齿动物</td>
			<td>3-6 月</td>
			<td>海马</td>
			<td>4.7-10。4</td>
		</tr>
<tr>
<td>卢瑟福等 (2007)9
</td>
			<td>(以酶为基础)</td>
			<td>啮齿动物</td>
			<td>3-6 月</td>
			<td>前额叶皮质, 纹状体</td>
			<td>44.9 ~ 4.7;7.3 @ 0。9</td>
		</tr>
<tr>
<td>Miele 等 (1996)26
</td>
			<td>微透析 (酶基)</td>
			<td>啮齿动物</td>
			<td>-</td>
			<td>纹 状 体</td>
			<td>3.6 @ 0。5</td>
		</tr>
<tr>
<td>日 et 等 (2006)27
</td>
			<td>(以酶为基础)</td>
			<td>啮齿动物</td>
			<td>3-6 月</td>
			<td>额叶皮层, 纹状体</td>
			<td>1.6 @ 0.3; 1.4 @ 0。2</td>
		</tr>
<tr>
<td>金特罗·库维德斯等 (2007)28
</td>
			<td>(以酶为基础)</td>
			<td>非人类灵长类动物</td>
			<td>5.3-5.5 年</td>
			<td>前运动神经皮质, 运动皮层</td>
			<td>3.8 ~ 1.7;3.7 @ 0。9</td>
		</tr>
<tr>
<td>斯蒂芬斯等。 (2010)29
</td>
			<td>协定 [Spencer-Gerhardt-2 (SG-2)]</td>
			<td>非人类灵长类动物</td>
			<td>11-21 年</td>
			<td>壳</td>
			<td>8.53</td>
		</tr>
<tr>
<td>Kodama 等 (2002)30
</td>
			<td>微透析 (酶基)</td>
			<td>非人类灵长类动物</td>
			<td>-</td>
			<td>前额叶皮质</td>
			<td>1.29-2.21</td>
		</tr>
<tr>
<td>盖文等 (2003)31
</td>
			<td>微透析 (酶基)</td>
			<td>非人类灵长类动物</td>
			<td>青少年</td>
			<td>纹 状 体</td>
			<td>28.74 @ 2.73</td>
		</tr>
<tr>
<td>期间和斯宾塞 (1993)32
</td>
			<td>微透析 (酶基)</td>
			<td>人类</td>
			<td>18-35 年</td>
			<td>海马</td>
			<td>20.3 @ 6。6</td>
		</tr>
<tr>
<td>Reinstrup 等 (2000)33
</td>
			<td>微透析 (酶基)</td>
			<td>人类</td>
			<td>-</td>
			<td>额叶皮质</td>
			<td>16 @ 16</td>
		</tr>
<tr>
<td>Cavus 等 (2005)34
</td>
			<td>微透析 (酶基)</td>
			<td>人类</td>
			<td>15-52 年</td>
			<td>大脑皮层</td>
			<td>2.6 @ 0。3</td>
		</tr>
</tbody></table>表2。不同动物模型基底胞外谷氨酸 levelsacross 的比较。对在健康清醒和麻醉动物中使用微透析或电极建立正常胞外谷氨酸水平的研究进行的选择性审查。
使用多边环境协定技术来监测仔猪模型中的体内谷氨酸浓度, 可以使今后对仔猪神经功能的评估成为麻醉后的结果。已经计划了生存实验, 这将进一步了解麻醉对人类新生儿认知幸福的长期影响。生存实验将允许在麻醉暴露后长时间进行行为测试和谷氨酸变化监测。儿童在有可能以手术干预的形式经历生理压力的情况下接受麻醉也很常见。未来的研究, 以解决的影响, 手术的神经损伤和增加神经毒性将允许更准确的模拟一个共同的临床设置为儿童。使用替代动物模型也是可行的, 这也是通过慢性植入对这些模型进行研究, 使我们能够追踪与神经毒性相关的行为变化。技术本身是多才多艺的, 所以未来的研究不需要局限于谷氨酸水平的分析 (例如, GABA, 胆碱, 赖氨酸,等).可以分析)。

每年, 数以百万计的儿童在美国的1个侵入性和非侵入性程序中都接受麻醉。多年来, 麻醉提供者已经向父母们保证了麻醉药的安全性, 即使是在小孩和新生儿中也是如此。然而, 在1999年发现, 在早期的生命中, n-甲基-d-天门冬氨酸受体亚型的瞬态阻断可能会导致大鼠的神经细胞凋亡2。最近, FDA 发布了一项药物安全通讯, 要求麻醉药品的标签包括对一般麻醉药的警告, 以及它们对3岁以下儿童发育大脑的潜在负面影响 (美国食品和药物管理局, 2017)。然而, 仍然需要阐明可能的机制和潜在的保护措施。
谷氨酸和伽玛氨基丁酸 (GABA) 等神经递质的正常活动对正常神经发育的发生至关重要。虽然大部分涉及的路径仍然是难以捉摸的, 神经递质系统很可能会参与, 因为麻醉剂是已知的调节这些途径, 以产生无意识。特别是, 兴奋性神经递质谷氨酸导致兴奋时, 其活动失调。这种神经递质通常涉及神经发生、神经系统可塑性、突触和神经生长, 以及其他一些重要的脑功能。然而, 长期活化谷氨酸受体可能导致兴奋和神经细胞凋亡, 特别是在压力条件下, 如手术, 缺氧, 减少能源可用性3。谷氨酸与 NMDA 受体的结合已被证明导致 Na+和 Cl−流入。随后的退极化被认为导致 ca2 +通道打开和释放胞内 Ca2 +存储4。这种功能障碍可能导致一连串的代谢紊乱, 最终减少神经细胞的增殖, 增加炎症, 并导致神经元死亡。尽管有这些假设, 但实际机制仍不清楚5。谷氨酸失调由于其在细胞凋亡中的作用, 它代表了一种新的途径, 它可能有助于以前记录的神经元凋亡机制, 这是一种重要的特征。
神经过程研究的障碍之一是它们的高度复杂性, 特别是在神经元发育的设置中。最初几个月的生命是最容易受伤的时期, 在这期间, 神经发育的重要步骤, 如生理凋亡 (神经修剪), synaptogenesis, gliogenesis 和髓鞘形成发生6.鉴于神经元通信的复杂性以及在不破坏正常中枢神经功能的情况下研究这些过程的困难, 已经开发出了针对体内检测和量化重要元素的新技术。神经元通讯。
本研究采用酶联的多边环境技术, 作为研究仔猪模型中的作用机理的一种新方法。该技术可用于研究复杂的体内电化学脑过程, 包括谷氨酸失调。纳入多边环境协定的4通道白金记录站点 (2 个谷氨酸敏感的站点和2个定点站点) 允许自我引用, 这有助于检测的准确性。此外, 将排除层应用于每个电极, 通过防止其他干扰分子检测到7来赋予选择性。此外, 与早期技术相比, 该多边环境协定的低调设计使组织创伤最小。这一特性赋予了更高的空间分辨率, 有利于研究大脑中的微观区域。例如, 海马体 (齿状回、CA1、CA2) 的离散区域可以专门研究8。关于多边环境协定的功能的具体细节以前已被描述为9。
与电化学技术相比, 微透析结合了在兴趣溶液和类似成分溶液之间放置的膜, 允许检测胞外流体变化10。虽然微透析是神经系统的重要支柱, 长期以来一直用于神经递质的检测, 但它具有低时间分辨、谷氨酸变化延迟检测和重大组织损伤的缺点11。
多边环境协定可以间接检测神经递质, 如谷氨酸, 乙酰胆碱, 胆碱, 通过使用适当的氧化酶, 产生活性记者分子, 如 H2O2或 O212,13.
多边环境协定技术已被广泛应用于大鼠和非人类灵长类动物, 用于研究病理生理学过程中的神经毒性, 而不是7,14。在其中一些病理生理学过程中, 多边环境协定技术已被用于研究阿尔茨海默病、癫痫、外伤性脑损伤, 以及药理作用对突触通讯的影响8,15,16,17. 尽管多边环境协定已被用于研究大鼠和非人类灵长类动物的这些病态, 但人类与仔猪大脑的高度发展相似性使得对仔猪的气候变化技术的适应成为一种非常适宜的研究方法。(s) 机制18。

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adaptation of microelectrode array technology for the study of anesthesia-induced neurotoxicity in the intact piglet brain

每年都有数以百万计的儿童接受麻醉以进行多种手术。然而, 在动物和人类的研究中, 对儿童麻醉的安全性提出质疑, 将麻醉药作为发展中大脑的潜在毒性。迄今为止, 没有研究成功地阐明了麻醉可能是神经毒性的机制。动物研究允许对这种机制进行调查, 而新生仔猪则是研究这些效应的优良模型, 因为它们与人脑的发育相似性显著。
该协议采用酶基微电极阵列技术作为研究麻醉诱发神经毒性机制的新方法。多边环境协定允许对体内神经递质活动进行实时监测, 并提供异常的时间和空间分辨率。假设麻醉神经毒性是由谷氨酸失调部分引起的, 而多边环境协定则提供一种测定谷氨酸的方法。该技术在仔猪模型中的新实现, 为研究这一问题提供了一个独特的机会。

这一概念验证的研究与酶为基础的陶瓷多边环境技术在小猪模型可以提供特殊的洞察力的谷氨酸动力学基础。这项研究进一步表明, 酶为基础的多边环境协定技术可以成功地适应于仔猪模型, 以测量生理和麻醉相关的变化, 神经递质活动, 高灵敏度, 高的空间和时间分辨率。在我们的实验中, 通过临床相关的方法和标准维持生理动态平衡, 没有小猪表现出生理扰动的迹象。
所获得的数据表明, 多边环境协定能够精确和空间地解决神经递质测量的皮质和亚皮层脑结构。使用立体定向设备可以明确识别参考表面结构 (bregma), 以便始终如一地定位感兴趣的区域, 而不管小猪的大小和解剖的个体差异。清晰的缝线可视化有助于在微米范围内一致地在区域内放置多边环境协定的准确性 (图 4)。获得对大脑皮质表面的接触是微小的创伤, 微小的出血, 确保任何体内谷氨酸动力学不是由于无意的系统性或局部侮辱 (图 5)。自定义, 刚性的多边环境, 然后与小猪的额平面垂直对齐 (图 6)。在插入之前不能正确地对齐多边环境协定, 可能会阻止目标区域的精确空间记录, 尤其是皮层下区域。
实时体内谷氨酸测量是在3-4 天的老仔猪 (n = 4) 下, 在 2.5-3% 七氟醚麻醉 (大约 1 MAC) 的海马中采取的。探头测量被记录在4赫兹和转换成浓度使用基于校准参数的线性回归 (图 8, 面板 a)。对于每一个时间点, 两个谷氨酸敏感点的信号平均之前, 减去平均哨兵信号, 以产生一个纠正谷氨酸信号。通过应用移动平均来更好地可视化总趋势 (图 8, 面板 B), 这些连续测量得到平滑。平均基底谷氨酸浓度为 4.61, 0.02 µM, 在麻醉暴露过程中保持相对稳定。通过分析信号中与哨兵信号不相关的峰值 (R2 < 0.5), 并超过了信噪比为 3 (图 9, 面板 a), 在一个动物中发现了瞬态谷氨酸活动。在 3.5 h (图 10, 面板 a) 期间检测到总共116个瞬态峰值。在1µM 范围内 (图 10, 面板 B) 中通常观察到产生的瞬态峰值的振幅。为了量化每个瞬态的持续时间, 获得了每个最大峰值值到衰变80% 所需的时间 (t80) (图 9, 面板 B)。3.5 小时记录期间所有谷氨酸瞬态的平均 t80为 4.68 @ 0.82 s (图 10, 面板 C)。这些数据表明, 有可能准确地测量长期和瞬态神经递质的活动, 在皮层下区域的麻醉仔猪大脑。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57391/57391fig1.jpg"> 图 1: SG-2 微电极阵列类型的可视比较.SG-2 阵列包含两个谷氨酸敏感的站点和两个谷氨酸不敏感的定点站点 (每个站点150µm x 20 µm)。(A) 左侧显示挠性轴微电极阵列。刚性轴微电极阵列是为仔猪使用而设计的, 允许在大型动物中进行更深的植入。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57391/57391fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57391/57391fig2.jpg"> 图 2: 微电极阵列的制备和校准过程概述.总的多边环境协定的准备和校准持续约一周。涂层酶、排斥层和校准分析物是特定于感兴趣的神经递质的。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57391/57391fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57391/57391fig3.jpg"> 图 3: 在立体定向设备中放置小猪.小猪的嘴巴直接放在嘴上, 在犬齿后面。穿透耳棒插入耳道, 以确保头骨的后端。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57391/57391fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57391/57391fig4.jpg"> 图 4: 将小猪放置在用于开颅手术的立体定向装置中.(A) 小猪的头部在自定义立体定向框架内紧紧地固定在一起, 确保了多边环境协定的一致放置。穿透耳棒的等距位置是可见的。(B) 沿头皮中线前后切口。避免颅骨的评分, 以可视化冠状和矢状缝线和优化 bregma 的可视性。显示的刻度条表明切口的相对大小和开颅窗的位置。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57391/57391fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57391/57391fig5.jpg"> 图 5: 用于访问海马体的开颅手术.(A) 头皮进一步反映出根据立体定向坐标来暴露多边环境协定插入的近似位置。带圆圈区域被标记 (黑点) 以指导开颅手术。(B) 将颅骨瓣移除的开颅窗 (0.25 厘米2), 以暴露底层硬脑膜。(C) 将脑膜仔细移除, 以暴露未组织外伤的表面皮层。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57391/57391fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57391/57391fig6.jpg"> 图 6: 将定位和插入到海马中.(A) 在 bregma 上放置多边环境协定, 以确定海马体的相对立体定位位置。(B) 在脑表面立体定向放置多边环境协定, 以确定海马插入深度。银伪参考电极安全地放置在头皮下 (用箭头表示)。(C) 在适当深度插入的多边环境协定, 以获得实时的体内胞外谷氨酸测量海马。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57391/57391fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57391/57391fig7.jpg"> 图 7: 在30分钟基线期间的协定行为.最初的快速增加对应于使用机器人的环境协定的下降到海马体。一旦多边环境协定达到适当的深度 (虚线), 基线周期就开始了。胞外谷氨酸的测量将减少30分钟的时间, 不应该被解释为生理学读数。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57391/57391fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57391/57391fig8.jpg"> 图 8: 在七氟醚麻醉下的一只新生猪海马体内的实时胞外谷氨酸测量.(A) 在七氟醚麻醉下, 一只新生猪海马谷氨酸浓度的移动平均值 (使用10数据点)。测量是在4Hz 的3小时后, 一个简短的30分钟基线期。(B) 用移动平均每15分钟平滑谷氨酸测量, 以更好地可视化趋势。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57391/57391fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/57391/57391fig9.jpg"> 图 9: 七氟醚麻醉下新生儿猪海马中瞬态谷氨酸活性的鉴定.(A) 在实时谷氨酸跟踪中指示瞬时谷氨酸峰值 (以红色表示)。当信噪比超过 3, 其信号与哨兵信号 (R2 < 0.5) 不相关时, 峰值被认为是重要的。(B) 在图 9(面板 A) 中标识的具有代表性的瞬态峰值。虚线表示峰值衰减80% 所需的总工期。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57391/57391fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/57391/57391fig10.jpg"> 图 10: 七氟醚麻醉下新生猪海马谷氨酸瞬态的特征.(A) 在15分钟箱中有许多瞬时谷氨酸峰值。当信噪比超过 3, 其信号与哨兵信号 (R2 < 0.5) 不相关时, 峰值被认为是重要的。(B) 瞬时谷氨酸峰值的振幅。误差线表示平均值的标准错误。(C) 表示瞬时谷氨酸峰值的 T80 。误差线表示平均值的标准错误。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57391/57391fig10large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain at JoVE.com

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

微电极阵列技术在完整仔猪脑麻醉诱导神经毒性研究中的应用

本研究探讨了用酶基微电极阵列技术监测仔猪体内的活体神经递质活性的新方法.假设谷氨酸失调有助于麻醉神经毒性的机制。在此, 我们提出了一种适应多边环境技术的协议, 研究麻醉诱发神经毒性的机制。

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question ...

adaptation-microelectrode-array-technology-for-study-anesthesia

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.4K 次观看.  Ohio State University College of Medicine. 本研究探讨了用酶基微电极阵列技术监测仔猪体内的活体神经递质活性的新方法.假设谷氨酸失调有助于麻醉神经毒性的机制。在此, 我们提出了一种适应多边环境技术的协议, 研究麻醉诱发神经毒性的机制。

视频: Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

可伸缩流控喷油器阵列病毒完整的3 - D的大脑回路为目标

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

科研

神经科学

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

高密度的自由移动，使用基于聚酰亚胺的微电极的小鼠的脑电图记录

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

特定脑区的兴奋毒性损伤的成年大鼠立体定向手术治疗

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

设计和超轻重量的制作，可调节的多电极电生理记录小鼠探头

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most neuroimaging or neurophysiological investigations in humans have been confined to the study of &#947;-Amino-Butyric-Acid-(GABA)-receptor-agonist-based anesthetics, while the effects of dissociative N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthetics ketamine, nitrous oxide (N2O) and xenon (Xe) are largely unknown. This paper describes the methods underlying the simultaneous recording of magnetoencephalography (MEG) and electroencephalography (EEG) from healthy males during inhalation of the gaseous anesthetic agents N2O and Xe. Combining MEG and EEG data enables the assessment of electromagnetic brain activity during anesthesia at high temporal, and moderate spatial, resolution. Here we describe a detailed protocol, refined over multiple recording sessions, that includes subject recruitment, anesthesia equipment setup in the MEG scanner room, data collection and basic data analysis. In this protocol each participant is exposed to varying levels of Xe and N2O in a repeated measures cross-over design. Following relevant baseline recordings participants are exposed to step-wise increasing inspired concentrations of Xe and N2O of 8, 16, 24 and 42%, and 16, 32 and 47% respectively, during which their level of responsiveness is tracked with an auditory continuous performance task (aCPT). Results are presented for a number of recordings to highlight the sensor-level properties of the raw data, the spectral topography, the minimization of head movements, and the unequivocal level dependent effects on the auditory evoked responses. This paradigm describes a general approach to the recording of electromagnetic signals associated with the action of different kinds of gaseous anesthetics, which can be readily adapted to be used with volatile and intravenous anesthetic agents. It is expected that the method outlined can contribute to the understanding of the macro-scale mechanisms of anesthesia by enabling methodological extensions involving source space imaging and functional network analysis.

Simultaneous magnetoencephalography and electroencephalography provides a useful tool to search for common and distinct macro-scale mechanisms of reductions in consciousness induced by different anesthetics. This paper illustrates the empirical methods underlying the recording of such data from healthy humans during N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthesia during inhalation of nitrous oxide and xenon.

在健康志愿者的气体麻醉剂氙和一氧化二氮的管理中记录大脑电磁活动

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most ...

微电极阵列技术在完整仔猪脑麻醉诱导神经毒性研究中的应用

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