作者想感谢 Mahla 的卡梅隆. 布拉德利, 雷切尔. 安妮和乔安娜. 斯蒂芬。另外还要感谢 Dr. 史蒂文 Mcguigan 作为第二个麻醉师的支持。佩帕帕斯提供了宝贵的麻醉护士监督。马库斯. 斯通欣然地将他的时间和专长用于编辑和拍摄该协议。Dr. Muthukumaraswamy 给出了关于数据分析和结果解释的具体建议。最后, 杰拉德戈特了许多令人振奋的讨论, 帮助执行了一些试点实验, 并在泡沫头支撑设计的中心。
这项研究得到了詹姆斯. 麦克唐纳合作赠款 #220020419 "重建意识", 授予乔治 Mashour, 迈克尔阿维丹, 马克斯 Kelz 和大卫 Liley。

作者没有什么可透露的。

本文概述了一个全面的协议, 同时记录的 MEG 和脑电图在麻醉气体交付与 N2O 和氙。这样的一个协议将是有价值的研究的电磁神经相关的麻醉诱导减少意识。该议定书还预计将推广到其他麻醉气体, 如七氟醚或异氟醚的交付。这将有助于更好地理解的共同的, 具体的和独特的宏观机制, 基础麻醉诱导减少意识的一系列麻醉剂有相当不同的分子模式和行动目标。理解麻醉剂的功能是神经科学的?...

临床前神经证据表明, 人的意识现象依赖于显式神经回路的完整性。这种电路被系统的下降到无意识的影响的观察证实了需要在麻醉过程中使用的神经影像技术, 并使 ' 导航 ' 搜索的神经系统相关性意识.除了睡眠, 麻醉是唯一的方法, 通过它可以控制, 可逆和重现的时尚, 扰乱, 从而解剖, sub-serve 意识的机制, 特别是在宏观尺度全球脑动力学。临床上, 全身麻醉可以定义为催眠/无意识, 静止和镇痛的状态, 仍然是最广泛使用和最安全的医疗干预之一。尽管最终结果的清晰度和效率, 仍然有很大的不确定性有关的作用机制的各种类型的药物引起麻醉性昏迷1。
麻醉剂可以分为静脉注射剂, 特别是异丙酚和巴比妥酸盐, 或挥发性/气态剂, 如七氟醚, 氟烷, 一氧化二氮 (N2O) 和氙 (氙气)。麻醉药理学已经很好地建立了多个细胞靶点确定与麻醉作用。大多数研究对象的行为主要通过 agonism γ-氨基丁酸 (GABA) 受体介导的活动。相反, 氯胺酮, 氙和 n2O 被认为是发挥其作用的主要目标是 n-甲基-d-天门冬氨酸 (NMDA) 谷氨酸受体2,3。其他重要的药理靶点包括钾通道、乙酰胆碱受体和残留的谷氨酸受体、AMPA 和酸, 但是它们对麻醉作用的贡献程度仍然难以捉摸 (全面审查见4)。
在作用机制的变化程度和观察到的生理和神经效应的不同类型的代理人呈现得出的一般结论对其影响有意识加工困难。gaba 剂引起的意识丧失通常以大脑活动的全球变化为特征。这是明显的在高, 低频三角洲 (δ, 0.5-4 Hz) 波浪和减少在高频率, 伽玛 (γ, 35-45Hz) 活动在脑电图 (EEG), 类似慢波睡眠5,6以及脑血流和葡萄糖代谢的广泛减少5,67891011.Boveroux et al。13通过功能性磁共振成像 (fMRI) 显示异丙酚麻醉下的静止状态功能连通性显著下降, 从而增加了这种观察。相比之下, 游离麻醉药对大脑活动的影响却不那么清晰。在某些情况下, 它们与脑血流量和葡萄糖代谢的增加有关14,15,16,1718,19, 20,21当研究由 Rex 和同事22和 Laitio 和同事23,24查看氙气的效果提供了增加和减少大脑的证据活动.类似的不规则性在 EEG 信号的影响上可以看到25,26,27,28。约翰逊et al。29显示了低频带三角洲和θ的总功率的增加, 以及在高密度 EEG 中进行氙麻醉的高频段伽玛的研究, 而在三角洲、θ和alpha 频带30,31和更高频率的氙气32。氙对电头皮活动的影响的这种可变性在α和 beta 频率范围可以被观察, 同时增加33和减少34被报告。
尽管存在上述的差异, 当人们试图查看大脑区域之间功能连通性的变化时, 这幅图开始变得更加一致。然而, 这种措施主要限于在空间或时间分辨率方面必须作出让步的方式。虽然使用 EEG 的研究显示出清晰, 并在一定程度上一致, 在麻醉/镇静期间功能网络的拓扑结构的变化与异丙酚35, 七氟醚,36和 N2O37,广泛间隔的传感器电平 EEG 数据没有足够的空间分辨率来有意义地定义和描绘相应的功能网络的顶点。相反, 研究利用功能磁共振成像和正电子发射断层扫描 (PET) 的优越空间分辨率, 发现类似的拓扑改变在 large-scale 函数连接到 EEG13,38,39 、40、41, 但没有足够的时间分辨率来表征α (8-13 Hz) EEG 带中的相振幅耦合和其他正在出现的重要特征的动态现象麻醉动作12,42。此外, 这些措施并没有直接评估电磁神经活动43。
因此, 为了有意义地推进对与麻醉药作用相关的宏观过程的理解, 必须解决以前提到的调查的局限性;麻醉剂的限制性覆盖范围和非侵入性测量的时空分辨率不足。在此基础上, 作者概述了同时记录脑 (MEG) 和脑电图活动的健康志愿者的方法, 已开发为管理气体游离麻醉剂, 氙和 N2O。
MEG 被利用, 因为它是唯一的非侵入性的神经技术, 除了 EEG, 在毫秒范围内有一个时间分辨率。EEG 的问题是模糊的电磁场的头骨, 这是一个低通过滤器的外皮生成的活动, 而 MEG 是不太敏感的这个问题和问题的音量传导44。可以认为, MEG 具有更高的空间和源定位精度比脑电图45,46。EEG 不允许真正的无参考记录37,47, 但 MEG 确实如此。MEG 系统也通常记录的皮层活动比脑电图更广泛的频率范围, 包括高伽玛48(通常是 70-90 Hz), 这已经被建议参与麻醉剂的催眠作用, 包括氙29和 N2O28。meg 提供神经的活动, 由 eeg 转达的恭维, 因为 eeg 活动涉及细胞外电流, 而 meg 主要反映的磁场产生的细胞内流46,49. 此外, MEG 是特别敏感的电生理活动切向皮层, 而 EEG 主要记录细胞外活动径向到皮层49。因此, 结合 MEG 和 EEG 数据具有 super-additive 优势50。
气体分离剂氙和 N2o 已选择的原因如下: 它们是无嗅的 (氙) 或基本上无嗅 (N2o), 因此可以很容易地利用在控制条件的存在, 当受聘于亚浓度。此外, 它们很适合在实验室环境中进行远程管理和监测, 因为它们的心肺-呼吸抑制剂效果不佳61。氙和较小程度的 N2O, 保留相对较低的最小肺泡浓度-(MAC)-清醒, 其中50% 的病人变得没有反应的口头命令的值为32.6 ± 6.1%51和 63.3 +-7.1%52分别。尽管氙和 N2O 都是 NMDA 受体拮抗剂, 他们不同地调制 EEG-氙似乎表现得更像一个典型的 gaba 剂时, 使用双频谱指数33,53,54(用于 electroencephalographically 监测麻醉深度的几种方法之一)。相比之下, N2O 产生的脑电图效果要低得多, 因为如果使用双频索引26, 则它的监视很差。由于氙有不同的报告脑电图性质的其他游离剂, 但具有相似的特点, 以更普遍研究的 gaba 剂, 其电生理研究有潜力揭示重要与意识的神经相关和相应的功能网络变化有关的特点。由于 nmda 受体介导的活动在学习和记忆中所起的关键作用, 以及它在一系列领域中的牵连作用, 在 nmda 受体上作用的药物可能会更多地揭示依附正常和改变意识的脑网络。精神疾病, 包括精神分裂症和抑郁症80。
本文主要研究了在医院环境下, 同时记录 MEG 和脑电图的气体麻醉剂的要求和复杂的数据收集过程。基本数据分析在传感器水平概述和示例数据提供说明, 高保真录音可以获得最低的头部移动。对于随后的源成像和/或功能连接分析, 通常使用此类数据执行的许多潜在方法都没有描述, 因为这些方法在文献中得到了很好的描述, 并演示了各种选项分析55,56。

<table border="0" cellpadding="0" cellspacing="0" width="681">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Neuromag TRIUX 306-channel MEG system</td>
			<td style="width:115px;">Elekta Oy, Stockholm, SWEDEN</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polhemus Fastrak 3D system</td>
			<td style="width:115px;">Polhemus, VT, USA</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">MEG compatible ER-1 insert headphones</td>
			<td style="width:115px;">Etymotic Research Inc., IL, USA</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Low Density foam head cap, MEG compatible</td>
			<td style="width:115px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">Custom made by research team</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Harness, MEG compatible</td>
			<td style="width:115px;">N/A</td>
			<td style="width:119px;"></td>
			<td style="width:231px;">~3 m long, ~ 5 cm wide, cloth/jute strip to secure participant position on MEG chair</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ambu Neuroline 720 Single Patient Surface Electrodes</td>
			<td style="width:115px;">Ambu, Copenhagen, Denmark</td>
			<td style="width:119px;">72015-K10</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">3.0T TIM Trio MRI system</td>
			<td style="width:115px;">Siemens AB, Erlangen, GERMANY</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Asalab amplifier system</td>
			<td style="width:115px;">ANT Neuro, Enschede, NETHERLANDS</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">this system is no longer manufactured and has been deprecated to 64 channel eego EEG amplifier</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">64-channel Waveguard EEG cap, MEG compatible</td>
			<td style="width:115px;">ANT Neuro, Enschede, NETHERLANDS</td>
			<td style="width:119px;">CA-138</td>
			<td style="width:231px;">size Medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Magnetically shielded cordless battery box</td>
			<td style="width:115px;">ANT Neuro, Enschede, NETHERLANDS</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">Magnetic shielding not provided by manufacturer &#8211; Modified by research team</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">OneStep ClearGel Electrode gel</td>
			<td style="width:115px;">H+H Medizinprodukte GbR, Munster, GERMANY</td>
			<td style="width:119px;">154547</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Akzent Xe Color Anesthesia Machine</td>
			<td style="width:115px;">Stephan GmbH, Gackenbach, GERMANY</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Omron M6-Comfort Blood Pressure Monitor</td>
			<td style="width:115px;">Omron Healthcare, Kyoto, JAPAN</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Xenon gas (99.999% purity)</td>
			<td style="width:115px;">Coregas, Thomastown, VIC, AUSTRALIA</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">we estimate that we use approx 40 L (SATP) per participant</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Medical Nitrous Oxide</td>
			<td style="width:115px;">Coregas, Thomastown, VIC, AUSTRALIA</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">x2 G size cylinders</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Medical Oxygen</td>
			<td style="width:115px;">Coregas, Thomastown, VIC, AUSTRALIA</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">x2 G size cylinders</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Medical Air</td>
			<td style="width:115px;">Coregas, Thomastown, VIC, AUSTRALIA</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">x2 G size cylinders</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Filter Respiratory &#38; HMES with Capno Port Hypnobag</td>
			<td style="width:115px;">Medtronic, MN, USA</td>
			<td style="width:119px;">352/5805</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Yankauer High Adult</td>
			<td style="width:115px;">Medtronic, MN, USA</td>
			<td style="width:119px;">8888-502005</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Quadralite EcoMask anaesthetic masks</td>
			<td style="width:115px;">Intersurgical Australia Pty Ltd</td>
			<td style="width:119px;">7093000/7094000</td>
			<td style="width:231px;">size 3 and size 4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Suction Canister Disp 1200 mL Medival Guardian</td>
			<td style="width:115px;">Cardinal Health, OH, USA</td>
			<td style="width:119px;">65651-212</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Catheter Mount Ext 4-13 cm with&#160; 90A elbow</td>
			<td style="width:115px;">Medtronic, MN, USA</td>
			<td style="width:119px;">330/5667</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Catheter IV Optiva 24g x 19 mm Yellow St Su</td>
			<td style="width:115px;">Smiths Medical, MN, USA</td>
			<td style="width:119px;">5063-INT</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dexamethasone Mylan Injection Vials (4 mg/1 mL)</td>
			<td style="width:115px;">Alphapharm Pty Ltd, Sydney, AUSTRALIA</td>
			<td style="width:119px;">400528517</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ondasetron (4 mg/2 mL)</td>
			<td style="width:115px;">Alphapharm Pty Ltd, Sydney, AUSTRALIA</td>
			<td style="width:119px;">400008857</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="546">
			<td height="546" style="height:546px;width:216px;">Medical resuscitation cart</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td style="width:231px;">The medical resuscitation cart is configured according to the suggested minimal requirements for Adult resuscitation recommended in the document &#34;Standards for Resuscitation: Clinical Practice and Education; June 2014) by the Australian and New Zealand Resuscitation councils and specifically endorsed by multiple professional health care organizations including the Australian and New Zealand College of Anaesthetists.&#160; It includes all the necessary airway and circulatory equipment, as well as the associated pharmacuetical agents to enable full cardio-respiratory resuscitation and support in a non-clinical environment.&#160; Full details can be found at https://resus.org.au/standards-for-resuscitation-clinical-practice-and-education/</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Maxfilter Version 2.2</td>
			<td style="width:115px;">Elekta Oy, Stockholm, SWEDEN</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">Data analysis software provided with Elekta&#39;s Neuromag TRIUX MEG system</td>
		</tr>
	</tbody>
</table>

recording brain electromagnetic activity during the administration of the gaseous anesthetic agents xenon and nitrous oxide in healthy volunteers

麻醉可以说是研究全球意识/无意识的神经系统相关性的唯一系统方法之一。然而到目前为止, 大多数神经影像或神经的研究都局限在人类的γ-氨基丁酸 (GABA)-受体-agonist-based 麻醉剂, 而游离 n-甲基-d-天门冬氨酸 (NMDA) 的影响,受体 antagonist-based 麻醉剂氯胺酮, 一氧化二氮 (N2O) 和氙 (氙气) 大多是未知的。本文介绍了从健康男性吸入气体麻醉剂 N2O 和氙的同时记录图 (MEG) 和脑电图 (eeg) 的方法。结合 MEG 和脑电图数据, 可以评估在高时、中度的麻醉过程中的电磁脑活动。在这里, 我们描述了一个详细的协议, 在多个录音会议, 包括学科招聘, 麻醉设备设置在 MEG 扫描仪室, 数据收集和基本数据分析。在本协议中, 每个参与者在重复的测量交叉设计中暴露于不同级别的氙和 N2O。在相关的基线记录之后, 参与者将会接触到逐步增加的氙和 N2O 的8、16、24和 42%, 以及16、32和47% 分别, 在这期间, 它们的响应程度是通过听觉连续性能任务 (aCPT)。给出了许多记录的结果, 以强调原始数据的传感器级特性、频谱形貌、头部运动的最小化以及对听觉诱发反应的明确水平依赖性效应。这一范式描述了一个一般的方法来记录的电磁信号与行动的不同类型的气体麻醉剂, 这可以很容易地适应用于挥发性和静脉麻醉剂。预计该方法可以帮助理解的宏观规模的机制, 麻醉通过启用方法扩展涉及源空间成像和功能网络分析。

本节利用从一个主题获得的数据, 以证明同时录制的典型特征和这些信息的潜力有助于更好地了解麻醉引起的意识改变状态。为了简化博览会, 结果显示为 i) post-anti-催吐剂管理基线 (基线 3), ii) 0.75 等 MAC 清醒峰值气体浓度 (3 级), N2O (47%) 和氙 (24%) 和 iii) 氙峰值气体浓度 42% (4级)。选择级别3和 4, 因为它们分别为 N2O 和氙的最高稳态级别。此外, 4 级氙涉及明?...

Watch this Scientific Journal Video about Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers at JoVE.com

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

同时图和脑电图提供了一个有用的工具, 以寻找共同和独特的宏观规模的机制, 减少意识的不同麻醉剂诱导。本文阐述了在吸入一氧化二氮和氙时, 从健康人的 n-甲基-d-天门冬氨酸 (NMDA) 受体 antagonist-based 麻醉中记录这些数据的经验方法。

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 ...

recording-brain-electromagnetic-activity-during-administration

Education

Research

JoVE Core

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Statistics

Neuroscience

Unit 1

Measures of Variation

Measures of Relative Standing

Probability Distributions

Estimates

Distributions

Hypothesis Testing

Analysis of Variance

Correlation and Regression

Statistics in Practice

SCIENTISTS IN ACTION

10.9K 次观看.  Swinburne University of Technology. 同时图和脑电图提供了一个有用的工具, 以寻找共同和独特的宏观规模的机制, 减少意识的不同麻醉剂诱导。本文阐述了在吸入一氧化二氮和氙时, 从健康人的 n-甲基-d-天门冬氨酸 (NMDA) 受体 antagonist-based 麻醉中记录这些数据的经验方法。

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

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

在大鼠的药物自我管理的评估超声发声

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

科研

神经科学

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

视网膜节的形态分析

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

微透析的乙醇操作性乙醇的自我管理和乙醇的测定气相色谱

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

鉴定转基因鱼的完整的大脑中的神经元的电活动记录

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

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 ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

同时电生理记录和抑制剂在啮齿动物大脑微注射

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

通过的嗅球钙监控录像的变化温度引起的神经元电活动<em&gt;非洲爪蟾</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

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 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) ...