这项工作得到了皇家墨尔本医院神经科学基金会（A2087）的支持。

本研究由弗洛里动物伦理委员会（墨尔本大学，编号 22-025UM）根据澳大利亚科学目的动物护理和使用规范批准。从动物资源中心（澳大利亚）获得的 C57BL/6 雄性小鼠（8 周）用于本研究。
1. Headstage设计与制造
注意: 头部舞台 PCB 板是一个紧凑的 14 毫米 x 12 毫米四层板，设计用于直接放置在动物的头部。它使用商用放大器芯片（参见 材料表），所有设计和 Gerber 文件均可在线获取（GitHub 链接:https://github.com/dechuansun/Intan-headstage/tree/main/pcbway）。
<ol><li>向制造商提供以下规格:板厚:0.6 mm;最小跟踪/间距:4 mils;最小孔径:0.2 mm。</li>
	<li>在PCB组装过程中， 遵循以下顺序:<ol><li>使用设置为 350 °C 的热风枪将放大器芯片焊接到板上。</li>
		<li>焊接无源元件。</li>
		<li>焊接 SPI 连接器和电极连接器（参见 材料表）。</li>
	</ol></li>
	<li>在显微镜下检查焊接以保证质量。使用环氧树脂将 SPI 连接器固定到位，以增加稳定性。</li>
	<li>使用第三方记录软件和控制板（见 材料表）进行信号采集。有关详细说明，请参阅软件用户指南。</li>
	<li>设计的头台支持 8 个通道。在软件中，启用通道 8、9、12、13、20、21、22 和 23 进行录制。</li>
</ol>2. 电极制造
<ol><li>将 PFA 涂层的钨丝（参见 材料表）切割成不同电极类型的特定长度:前额叶皮层电极 （12 mm）、海马电极 （10 mm） 和接地电极 （6 mm）。</li>
	<li>将黄铜管（参见 材料表）切成 3 毫米的段。</li>
	<li>使用打火机去除每根焊丝末端 2 毫米的涂层，然后将电极丝牢固地焊接到黄铜管上。黄铜管的内径为 0.45 mm，外径为 0.60 mm。</li>
	<li>对于接地电极，将 M1.2 不锈钢螺钉（参见 材料表）焊接到电极上。将基于磷酸的助焊剂涂在螺钉上以增强焊接。焊接后，用酒精清洁螺丝。 注意: 在焊接过程中戴上手套进行保护。</li>
</ol>3. 外科手术
<ol><li>在用3%异氟烷和1L / min氧气流量的麻醉室中麻醉小鼠。</li>
	<li>将麻醉的小鼠放在加热垫上，并将其固定在立体定位框架中（参见 材料表）。</li>
	<li>将异氟烷的维持率调整为2.5-3%，并将氧气流量降低至500 mL/min。使用脚趾捏，确认动物是否仍处于深度麻醉状态。</li>
	<li>皮下注射0.5mg/kg的卡洛芬，并涂抹眼膏以保护眼睛。</li>
	<li>用聚维酮碘和80%乙醇剃须和消毒小鼠的头部。</li>
	<li>沿着头皮的中线做一个 8 毫米的切口，去除切口区域的结缔组织。</li>
	<li>使用过氧化氢清洁颅骨表面，注意不要接触周围的皮肤。</li>
	<li>将 bregma 和 lambda 标志对齐到同一水平，以便准确放置电极（bregma 和 lambda 是矢状缝合线与冠状缝线和 lambdoid 缝合线相交的地方）。</li>
	<li>在指定坐标处为参比电极/接地电极、锚螺钉（0.9 mm 钻头毛刺）和有源电极（0.3 mm 钻头毛刺）钻孔。</li>
	<li>将定制的电极（第 2 步）连接到立体定位框架臂上，并确保其垂直于大脑。</li>
	<li>将电极植入海马CA1区域（AP - 1.8 mm，ML - 1.3 mm，DV - 1.4 mm）。 注:AP，前后位;ML， 内侧;DV，背腹侧。</li>
	<li>在前额叶皮层（AP - 2.0 mm，ML - 0.3 mm，DV - 1.7 mm）中重复电极植入。</li>
	<li>用市售的强力粘合剂和牙科粘结剂固定电极（参见 材料表）。</li>
	<li>植入两个 1.2 毫米地脚螺钉（AP - 1.8 毫米，ML -1.6 毫米）以防止移动。</li>
	<li>将参比/接地电极直接与硬脑膜直接接触，位于 lambda 标志后方 2 毫米和单侧 2 毫米处。</li>
	<li>将电极的黄铜管侧连接到多通道插座连接器（参见 材料表），接地电极位于中间。</li>
	<li>在中间引脚的外部使用 0.8 mm 的热缩管进行隔离。</li>
	<li>用粘合剂和牙科粘合剂固定电极、锚固件螺钉和连接器。</li>
</ol>4.术后护理
<ol><li>为了减轻术后疼痛，根据疼痛评估，每 12-24 小时皮下注射 5-10 mg/kg 卡洛芬，持续三天。</li>
	<li>在开始任何记录或实验程序之前，为动物提供一周的恢复期。</li>
</ol>5. 录音程序
<ol><li>处理动物15分钟，每天两次，连续三天。</li>
	<li>轻轻地将鼠标周围的手合拢，不要施加过大的压力，从而捡起鼠标。</li>
	<li>将头盔板放在动物的头上30分钟，每天一次，连续三天。</li>
	<li>在录音当天，让动物适应录音室 30 分钟。</li>
	<li>将动物放在法拉第笼内的小型记录室中，以减少外部电气干扰。连接自定义头台进行录制。</li>
	<li>打开录音软件并选择 2.00 kHz 采样率。通过选择每个频道并按 空格键来禁用除 13 和 20 之外的所有频道。</li>
	<li>在 硬件带宽窗口中，将下限带宽设置为 2 Hz ，将上限带宽设置为 100 Hz。</li>
	<li>在 软件滤波窗口中，将低通滤波器调整为 100 Hz ，将高通滤波器调整为 2 Hz。</li>
	<li>通过单击" 选择文件名"来选择存储路径，然后单击" 录制"。</li>
	<li>以 10 分钟的适应期开始每个记录会话，然后进行 15 分钟的基线脑电图记录。</li>
	<li>基线记录后， 通过 腹膜内注射给药，并继续记录另外30分钟，不得拖延。 注意:有关所用药物的详细信息，请参阅"结果"部分。</li>
</ol>

小鼠大脑中的 LFP 记录用于认知研究和药物分析

<ol>
	<li>Einevoll, G. T., Kayser, C., Logothetis, N. K., Panzeri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+and+analysis+of+local+field+potentials+for+studying+the+function+of+cortical+circuits.">Modelling and analysis of local field potentials for studying the function of cortical circuits.</a> Nat Rev Neurosci. 14 (11), 770-785 (2013).</li><li>Buzsaki, G., Anastassiou, C. A., 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=The+origin+of+extracellular+fields+and+currents-EEG,+ECOG,+LFP+and+spikes.">The origin of extracellular fields and currents-EEG, ECOG, LFP and spikes.</a> Nat Rev Neurosci. 13 (6), 407-420 (2012).</li><li>Sigurdsson, T., Stark, K. L., Karayiorgou, M., Gogos, J. A., Gordon, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+hippocampal-prefrontal+synchrony+in+a+genetic+mouse+model+of+schizophrenia.">Impaired hippocampal-prefrontal synchrony in a genetic mouse model of schizophrenia.</a> Nature. 464 (7289), 763-767 (2010).</li><li>Witton, 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=Disrupted+hippocampal+sharp-wave+ripple-associated+spike+dynamics+in+a+transgenic+mouse+model+of+dementia.">Disrupted hippocampal sharp-wave ripple-associated spike dynamics in a transgenic mouse model of dementia.</a> J Physiol. 594 (16), 4615-4630 (2016).</li><li>Englot, D. J., Konrad, P. E., Morgan, V. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+network+disruption+in+the+degenerative+dementias.">Functional network disruption in the degenerative dementias.</a> Lancet Neurol. 10 (9), 829-843 (2011).</li><li>Sigurdsson, T., Duvarci, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal-prefrontal+interactions+in+cognition,+behavior+and+psychiatric+disease.">Hippocampal-prefrontal interactions in cognition, behavior and psychiatric disease.</a> Front Syst Neurosci. 9, 190 (2015).</li><li>Sun, D., 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=Effects+of+antipsychotic+drugs+and+potassium+channel+modulators+on+spectral+properties+of+local+field+potentials+in+mouse+hippocampus+and+pre-frontal+cortex.">Effects of antipsychotic drugs and potassium channel modulators on spectral properties of local field potentials in mouse hippocampus and pre-frontal cortex.</a> Neuropharmacology. 191, 108572 (2021).</li><li>Bokil, H., Andrews, P., Kulkarni, J. E., Mehta, S., Mitra, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronux:+A+platform+for+analyzing+neural+signals.">Chronux: A platform for analyzing neural signals.</a> J Neurosci Methods. 192 (1), 146-151 (2010).</li><li>Bozkurt, A., Lal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+flexible+printed+circuit+technology+based+microelectrode+array+for+extracellular+stimulation+of+the+invertebrate+locomotory+system.">Low-cost flexible printed circuit technology based microelectrode array for extracellular stimulation of the invertebrate locomotory system.</a> Sens Actuator A Phys. 169 (1), 89-97 (2011).</li><li>Du, 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=High-resolution+mapping+of+in+vivo+gastrointestinal+slow+wave+activity+using+flexible+printed+circuit+board+electrodes:+Methodology+and+validation.">High-resolution mapping of in vivo gastrointestinal slow wave activity using flexible printed circuit board electrodes: Methodology and validation.</a> Ann Biomed Eng. 37, 839-846 (2009).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroscience.+Histological+Staining+of+Neural+Tissue.">Neuroscience. Histological Staining of Neural Tissue.</a> JoVE. , (2023).</li></ol>

作者没有什么可透露的。

这里介绍的协议概述了构建定制头台的过程，该头台专门设计用于同时记录海马体（HIP）和前额叶皮层（PFC）中的双重局部场电位（LFP）。该协议中提供的详细步骤为研究人员提供了足够的信息，以彻底检查每个区域内以及HIP和PFC之间的信号通信。
定制设计的头级采用商用放大器芯片。虽然目前的配置支持 8 个通道的记录，但头级可以很容易地适应整个 32 个通道的容量，从?...

局部场电位 （LFP） 是指从细胞外空间记录的电活动，反映了一组局部神经元的集体活动。它们表现出多种频率范围，从 1 Hz 的慢波到 100 Hz 或 200 Hz 的快速振荡。 特定频段与学习、记忆和决策等认知功能有关 1,2。LFP 特性的变化已被用作各种神经系统疾病的生物标志物，包括痴呆和精神分裂症 3,4。分析 LFP 记录可以为与这些疾病相关的潜在病理机制和潜在的治疗策略提供有价值的见解。
双 LFP 记录是一种用于测量两个特定大脑区域内部和之间的局部电活动的技术。这种技术提供了一个宝贵的机会来研究不同大脑区域内部和之间发生的错综复杂的神经动力学和信号通信。先前的研究表明，检测单个大脑区域神经元特性的改变可能很复杂，但可以观察到区域间皮层通信的变化 5,6。因此，利用双LFP记录为解决这个问题提供了一种有效的方法。
海马-前额叶连接在调节认知功能方面起着至关重要的作用，功能障碍与各种神经系统疾病有关 7,8。这些区域的双电极记录可以提供有关这些相互作用的信息。不幸的是，关于在这些区域之间执行双电极LFP记录的方法的信息有限。此外，市售的记录设备通常价格昂贵，并且缺乏对特定实验设计的适应性。记录LFP的传统方法涉及使用屏蔽电缆将记录设备连接到植入动物大脑的电极。然而，这种方法容易受到运动伪影和环境噪声的影响，从而影响录制信号的质量和可靠性。
该协议描述了一种在小鼠海马体和前额叶皮层中执行双电极LFP记录的综合程序，使用可以放置在动物头部的低成本定制设计的头部。这些方法使研究人员能够研究两个离散大脑区域内的特定区域振荡模式，并探索这些区域之间的区域间信息交换和连通性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Brass tube&#160;</td>
			<td>Albion Alloys, USA</td>
			<td>Inside diameter of 0.45 mm</td>
		</tr>
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			<td>Carprofen&#160;</td>
			<td>Rimadyl, Pfizer Animal Health&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial amplifier chip</td>
			<td>Intantech</td>
			<td>RHD 2132</td>
		</tr>
		<tr>
			<td>Control board</td>
			<td>Intantech</td>
			<td>RHD recording system</td>
		</tr>
		<tr>
			<td>Dental cement&#160;</td>
			<td>Paladur</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat shrinks</td>
			<td>Panduit</td>
			<td>0.8 mm diameter</td>
		</tr>
		<tr>
			<td>M1.2 stainless steel screw</td>
			<td>Watch tools</td>
			<td>Clock and watch screw</td>
		</tr>
		<tr>
			<td>Multichannel socket connector&#160;</td>
			<td>Harwin, AU</td>
			<td>1.27 mm pitch, PCB socket</td>
		</tr>
		<tr>
			<td>PFA-coated tungsten wires&#160;</td>
			<td>A-M SYSTEMS, USA</td>
			<td>Inside diameter of 150 &#181;m&#160;</td>
		</tr>
		<tr>
			<td>Phosphoric acid-based flux</td>
			<td>Chip Quik</td>
			<td>CQ4LF-0.5</td>
		</tr>
		<tr>
			<td>Recording software</td>
			<td>Intantech</td>
			<td>RHX recording software</td>
		</tr>
		<tr>
			<td>Stereotactic Frame</td>
			<td>World Precision Instruments</td>
			<td>Mouse stereotactic instrument</td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>UHU</td>
			<td>Ultra fast</td>
		</tr>
	</tbody>
</table>

dual extracellular recordings in the mouse hippocampus and prefrontal cortex

记录局部场电位 （LFP） 的技术是一种电生理学方法，用于测量局部神经元群的电活动。它是认知研究的重要工具，特别是在海马体和前额叶皮层等大脑区域。这些区域之间的双LFP记录特别令人感兴趣，因为它们允许探索区域间信号通信。然而，执行这些记录的方法很少被描述，大多数商业记录设备要么价格昂贵，要么缺乏适应特定实验设计的适应性。本研究提出了一种在小鼠海马体和前额叶皮层中执行双电极 LFP 记录的综合方案，以研究抗精神病药物和钾通道调节剂对这些区域的 LFP 特性的影响。该技术可以测量LFP的特性，包括每个大脑区域内的功率谱以及两者之间的相干性。此外，还为这些实验开发了一种低成本、定制设计的记录设备。总之，该协议提供了一种在不同大脑区域记录高信噪比信号的方法，有助于研究大脑内的区域间信息通信。

此处显示的结果表明，在四组C57BL / 6雄性小鼠（每组n = 8;年龄:8周;体重:24.0±0.42g）中测试的几种药物对局部场电位（LFPs）特性的影响。测试的药物包括抗精神病药物氯氮平、钾通道调节剂 4-氨基吡啶 （4-AP） 和瑞替加滨，以及对照载体生理盐水。
如图 1 所示，将小鼠放置在一个小记录室中，并使用定制设计的头部舞台从海马体 （HIP） 和前额叶皮层 （P...

Watch this Scientific Journal Video about Dual Extracellular Recordings in the Mouse Hippocampus and Prefrontal Cortex at JoVE.com

作者聚焦:使用双 LFP 记录方案研究药物对小鼠大脑信号的影响

该协议概述了使用定制设计的记录设备和电极来记录局部场电位并研究小鼠海马体和前额叶皮层中的信息流。

小鼠海马体和前额叶皮层中的双重细胞外记录

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal ...

本研究提出了一种在小鼠海马体和前额叶皮层中执行双电极 LFP 记录的综合方案，以研究药物对这些区域的 LFP 特性的影响。该协议提供了一种低成本和超轻的脑信号记录设备，该设备针对小鼠的认知研究进行了全面优化。该研究提供了一种在不同脑区记录高信噪比信号的方法，有助于研究大脑内部的区域间信息通信。

dual-extracellular-recordings-mouse-hippocampus-prefrontal

Research

JoVE Journal

Neuroscience

Dual Extracellular Recordings in the Mouse Hippocampus and Prefrontal Cortex

1.2K 次观看.  The University of Melbourne. 本研究提出了一种在小鼠海马体和前额叶皮层中执行双电极 LFP 记录的综合方案，以研究药物对这些区域的 LFP 特性的影响。该协议提供了一种低成本和超轻的脑信号记录设备，该设备针对小鼠的认知研究进行了全面优化。该研究提供了一种在不同脑区记录高信噪比信号的方法，有助于研究大脑内部的区域间信息通信。

视频: 作者聚焦:使用双 LFP 记录方案研究药物对小鼠大脑信号的影响

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar tasks. Participants' behavior during task performance in an fMRI scanner can then be correlated to the brain activity using the blood-oxygen-level-dependent signal. We measure behavior to be able to sort correct trials, where the subject performed the task correctly and then be able to examine the brain signals related to correct performance. Conversely, if subjects do not perform the task correctly, and these trials are included in the same analysis with the correct trials we would introduce trials that were not only for correct performance. Thus, in many cases these errors can be used themselves to then correlate brain activity to them. We describe two complementary tasks that are used in our lab to examine the brain during suppression of an automatic responses: the stroop1 and anti-saccade tasks. The emotional stroop paradigm instructs participants to either report the superimposed emotional 'word' across the affective faces or the facial 'expressions' of the face stimuli1,2. When the word and the facial expression refer to different emotions, a conflict between what must be said and what is automatically read occurs. The participant has to resolve the conflict between two simultaneously competing processes of word reading and facial expression. Our urge to read out a word leads to strong 'stimulus-response (SR)' associations; hence inhibiting these strong SR's is difficult and participants are prone to making errors. Overcoming this conflict and directing attention away from the face or the word requires the subject to inhibit bottom up processes which typically directs attention to the more salient stimulus. Similarly, in the anti-saccade task3,4,5,6, where an instruction cue is used to direct only attention to a peripheral stimulus location but then the eye movement is made to the mirror opposite position. Yet again we measure behavior by recording the eye movements of participants which allows for the sorting of the behavioral responses into correct and error trials7 which then can be correlated to brain activity. Neuroimaging now allows researchers to measure different behaviors of correct and error trials that are indicative of different cognitive processes and pinpoint the different neural networks involved.

The goal of our research is to correlate behavior to brain activity. Accurate behavioral measures and imaging techniques allow us to elucidate brain-behavior relationships.

关联从人类前额叶皮层的fMRI信号的行为反应:检查的认知过程，使用任务分析

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar ...

科研

神经科学

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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 ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

低聚淀粉样蛋白-β的立体灌注到小鼠海马

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用<em&gt;在子宫内</em&gt;电穿孔的C57BL / 6小鼠

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic field pulses. The initiation of cortical activation or its modulation depends on the background activation of the neurons of the cortical region activated, the characteristics of the coil, its position and its orientation with respect to the head. TMS combined with simultaneous electrocephalography (EEG) and neuronavigation (nTMS-EEG) allows for the assessment of cortico-cortical excitability and connectivity in almost all cortical areas in a reproducible manner. This advance makes nTMS-EEG a powerful tool that can accurately assess brain dynamics and neurophysiology in test-retest paradigms that are required for clinical trials. Limitations of this method include artifacts that cover the initial brain reactivity to stimulation. Thus, the process of removing artifacts may also extract valuable information. Moreover, the optimal parameters for dorsolateral prefrontal (DLPFC) stimulation are not fully known and current protocols utilize variations from the motor cortex (M1) stimulation paradigms. However, evolving nTMS-EEG designs hope to address these issues. The protocol presented here introduces some standard practices for assessing neurophysiological functioning from stimulation to the DLPFC that can be applied in patients with treatment resistant psychiatric disorders that receive treatment such as transcranial direct current stimulation (tDCS), repetitive transcranial magnetic stimulation (rTMS), magnetic seizure therapy (MST) or electroconvulsive therapy (ECT).

The protocol presented here is for TMS-EEG studies utilizing intracortical excitability test-retest design paradigms. The intent of the protocol is to produce reliable and reproducible cortical excitability measures for assessing neurophysiological functioning related to therapeutic interventions in the treatment of neuropsychiatric diseases such as major depression.

侧前额叶皮质联合经颅磁刺激与脑电图的研究

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

莱纳尔皮质-希波坎普斯有机切片文化中的癫痫发生模型

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. Traditional in vitro electrophysiological techniques use electrical stimulation to evoke synaptic transmission. A major drawback of this method is its nonspecific nature; all axons in the region of the stimulating electrode will be activated, making it difficult to attribute an effect to a particular afferent connection. This issue can be overcome by replacing electrical stimulation with optogenetic-based stimulation. We describe a method for combining optogenetics with in vitro patch-clamp recordings. This is a powerful tool for the study of both basal synaptic transmission and synaptic plasticity of precise anatomically defined synaptic connections and is applicable to almost any pathway in the brain. Here, we describe the preparation and handling of a viral vector encoding channelrhodopsin protein for surgical injection into a pre-synaptic region of interest (medial prefrontal cortex) in the rodent brain and making of acute slices of downstream target regions (lateral entorhinal cortex). A detailed procedure for combining patch-clamp recordings with synaptic activation by light stimulation to study short- and long-term synaptic plasticity is also presented. We discuss examples of experiments that achieve pathway- and cell-specificity by combining optogenetics and Cre-dependent cell labeling. Finally, histological confirmation of the pre-synaptic region of interest is described along with biocytin labeling of the post-synaptic cell, to allow further identification of the precise location and cell type.

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

离体 从内侧前额叶皮层到外嗅皮层的远距离突触传递和可塑性的光遗传学询问

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. ...

Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex

Golgi impregnation, using the Golgi staining kit with minor adaptations, is used to impregnate dendritic spines in the rat hippocampus and medial prefrontal cortex. This technique is a marked improvement over previous methods of Golgi impregnation because the premixed chemicals are safer to use, neurons are consistently well impregnated, there is far less background debris, and for a given region, there are extremely small deviations in spine density between experiments. Moreover, brains can be accumulated after a certain point and kept frozen until further processing. Using this method any brain region of interest can be studied. Once stained and cover slipped, dendritic spine density is determined by counting the number of spines for a length of dendrite and expressed as spine density per 10 &#181;m dendrite.

The protocol describes a modification of the rapid Golgi method, which can be adapted to any part of the nervous system, for staining neurons in the hippocampus and medial prefrontal cortex of the rat.