这个工作是由自然科学和加拿大金吉尔伯特工程研究理事会资助项目拥有全宗德追忆魁北克一个助学金-桑特。

动物被遵守处理与当地动物护理委员会规定，按照加拿大委员会关于动物保护的准则。大鼠收容单独在恒定条件下（21-22ºC温度和湿度的40-50％），其中包括一个12小时黑暗的光周期，开始于上午08:00。周小球和自来水在整个研究可随意。的输送由供后3天的环境适应期，实验前是允许的。雄性SD大鼠间300-800克体重已经经常使用该协议。 1. MI手术<ol><li>诱导麻醉的氯胺酮60 mg / kg和赛拉嗪10mg / kg的腹膜内注射。通过无反射确认适当麻醉时，爪子被挤压。 </li><li>剃手术部位。插管大鼠气管内管:导管（16G1.77英寸），然后将动物侧卧位。放置在加热垫上动物保持温度37℃左右。连接气管内管异氟醚2％。 </li><li>准备手术部位与葡萄糖酸洗必泰和异丙醇。在手术过程中，使用无菌手套和消毒器械。应用眼药膏双眼防止干燥，而在麻醉下。把无菌区对动物和旁边的动物另一个无菌区的仪器。 </li><li>切开皮肤，手术刀片＃10。剥离肌肉组织用止血钳夹。发生在腹卧位动物。打开胸壁用止血钳和定位胸部拉钩。打开心包用止血钳夹。 </li><li>以产生冠状动脉阻塞，循环中的下行冠状动脉周围360毫米长的4-0丝线缝合，通过连续的心肌组织。插入丝线的两端成14G，1.25厘米长的塑料管。 </li><li>拉动丝线缝合及脓液的两端小时管向下靠着动脉以阻塞它，进行40分钟。安全闭塞夹紧塑料管与止血钳夹。 </li><li> 40分钟后，通过释放止血钳，接着除去软管和丝线释放闭塞。 注意:深水对照大鼠经历相同的手术过程减去冠状动脉的闭塞。 </li><li>前闭合胸壁，放置一个柔性导管（18G，4.5厘米）至胸腔将空气抽出胸部用5-ml注射器，以防止气胸。 </li><li>关闭胸腔腔2-0缝合，缝合肌肉用4-0丝线缝合，用3-0丝线缝合皮肤。 </li><li>前关闭最后一个皮肤点，再通过导管在胸部空腔先前放置抽离空气，用5-ml注射器的胸部。 </li><li>最后缝合皮肤点。停止异氟醚通气。 </li><li>监测大鼠的觉醒。不要让动物无人看管，直到它有倍健ð足够的意识，以保持胸骨斜卧。 </li><li>治疗的大鼠，每8小时24小时用镇痛药（丁丙诺啡，0.05毫克/千克IP）中，用抗生素（duplocillin的单次剂量，0.2毫克/千克IP）。 </li><li>手术后，房子只单独直到时间的牺牲， 即后3天心肌梗死。 </li><li>梗塞大小，主要坏死，表示为梗塞组织的该区域在左心室的风险比例。这可以通过使用三苯基着色11测量。 </li></ol> 2.杏仁核隔离<ol><li>首先将动物头锥形袋，用它的鼻子伸出的窄端光圈。安全的大鼠，以避免任何运动。上断头台正确地定位它的头，剪刀的刀片之间。斩首的动物。 </li><li>在培养皿中的老鼠的贴装头保持在碎冰。上执行碎冰（4℃）中组织分离的所有过程。 </li><li>剖开SKULL用剪刀，小心地拉起，避免下毁损的组织分离骨鞘骨咬骨钳。 </li><li>放置头骨底部和脑的后腹侧表面之间的刮刀的平头刀刃，并仔细分离大脑从颅骨轻轻沿头骨底部向前推动刀片直到大脑可以慢慢举起出的头骨。丢弃的头骨。 </li><li>将大脑在它的背部表面。确定下丘脑（结构小脑前），减少大脑冠在其前端的和后端的前后;丢弃大脑的前部和后部部分，如果没有必要用于其他用途。 </li><li>可视化的杏仁核小球颞叶下方，两侧，毗邻下丘脑。 </li><li>翻转大脑平在其正面年底，背水面离实验者。 </li><li>开始与脑的左侧或右侧。分开半球，然后从相邻的杏仁核皮层。切方式约8mm这种松散皮质以允许扁桃体的隔离。切掉扁桃体与手术刀。 </li><li>通过沿暗缝线横跨它运行时，几乎各半切割隔离从其centromedial部扁桃体的基底部分。这种结构并不总是容易辨认。 </li><li>将杏仁核子部分单独确定的小瓶中，并保持在碎冰。 </li><li>重复解剖过程与其他半球。 </li><li>浸入液氮小瓶1分钟。保持小瓶在-80℃的冰箱中待用。 </li></ol> 3，caspase-3活性<ol><li>制备裂解缓冲液与蔗糖的0.32M，1％的Triton-X-100的Tris-HCl，pH值8，5mM的乙二胺四乙酸，二硫苏糖醇的2毫米，苯甲基磺酰氟1mM的，10微克/微升亮肽素的10毫和10微克/微升胃蛋白酶抑制素A的10微克/μL抑肽酶。 </li><li>添加150微升裂解缓冲液至每个样品（5-10毫克）。置于冰上。超声处理在冰上每个样品进行5秒，最大强度（40瓦）。孵育组织在裂解缓冲液在冰上30分钟。涡旋每个样品5分钟。 </li><li>通过将样本交替地在液氮中，并在恒温控制的加热板设定为37℃，进行3次冷冻/解冻循环。在13000克离心机组织（4 ℃）10分钟。小心取出上清液，保持管冰。 </li><li>制备在0和10微克/毫升牛血清白蛋白标准品的蛋白浓度。准备空白，只有缓冲区。 </li><li>加入200μlBradford试剂到每个标准。阅读在595nm处的标准。对于蛋白水平，加入5微升样品上清液至795微升蒸馏水和200μl的Bradford试剂的。读每个样品在595nm处并量化蛋白标准曲线。 </li><li>准备反应缓冲液，用50mM的Tris-HCl，pH值7，5mM的MgCl 2的的&lt;/次&gt;，乙二醇四乙酸为1mM，3-胆酰胺丙基0.1％）二甲基铵基] -1-丙磺酸盐和二硫苏糖醇1mM的。 </li><li>补充蛋白质的25微克至反应缓冲液和caspase-3底物，以获得正和负反应性样品。对于阴性样品中，添加反应缓冲液，25微克蛋白质，0.4微升AC-DEVD-CHO 800μM和0.8微升AC-DEVD-AMC的10mM的。对于阳性样品，加25微克蛋白质反应缓冲液和0​​.8微升AC-DEVD-AMC的10mM的。处理所有阴性和阳性样品一式三份。通过添加反应缓冲液完成每个样品的最终体积达到200微升。 </li><li>通过加入188.7微升反应缓冲液0.5微升AC-DEVD-CHO 800微米的，0.8微升AC-DEVD-AMC的10mM和10μl的裂解缓冲液产生阴性对照。对于阳性对照，加入189.2微升反应缓冲液，0.8微升AC-DEVD-AMC的10mM和10μl的裂解缓冲液。 </li><li>孵育样品和在37℃。在黑暗中3小时对照</li><li>停止与600微升甘氨酸0.4 M和氢氧化钠0.4 M（pH为10），每个样品和控制反应。 </li><li>荧光定量荧光分光光度法在365 nm，发射465纳米波长的激发波长。加入2毫升蒸馏水，以在玻璃试管反应。阅读对照和样品10秒，1分每一秒。 </li><li>量化具体活动每个样品中:（（阴性样品减去阳性样品的荧光）2.8毫升×容积荧光）由（的蛋白质25毫克孵化3小时×数量时）划分。 </li></ol>

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作者什么都没有透露。

我们与此协议在过去10年的经验导致了关键的方法问题的鉴定。首先，在碎冰上培养皿快速大脑解剖重要的是要保持制剂的质量。第二，必须认识到，组织超声处理较短的脑杏仁核相对于其他类型的组织，如心脏（10秒）或肾脏（20秒）。第三，大应注意在添加到样品之前仔细混合基板。我们遇到变量信号当混合顺序是不充分的。第四，读取样品时无气泡应该存在于石英池中。否则，该信号可能被改变。 我们提出在过去的几年中的变化，以增加可重复性的结果:增加衬底体积，以避免体积小于1微升。仍然，小变化可以连续测定之间发生，和至少3个对照样品应当用于控制的变化。这些控件的荧光措施被平均，平均为100％。实验样品在荧光措施转化在对照样品的百分比。 该技术的另一个限制是，组织只有一部分使用，因此，caspase-3的活性可能被低估。事实上，细胞凋亡的时间窗口短于任何给定的细胞，即使它出现在相邻区域处的采样1,4,15的时间可能被错过。反过来也是可能的:细胞凋亡可能是在组织中的某些部分特别强烈，导致胱天蛋白酶-3活性的高估。我们建议使用剩余的组织（杏仁核）的互补性技术，如TUNEL法或Bax蛋白/ Bcl2的比例（参见导言）的。 荧光分析法具有至少两个优点Western印迹。首先，它直接生成的量化数据。第二，它可以测量酶促一个ctivity本身而不是蛋白质或RNA的表达，这可能是不相关的，并且已知的是蛋白表达可以在没有中酶活性的任何变化增加。此外，荧光分光光度法可以在其它组织或用不同的动物物种中进行，并且可以为其它蛋白酶亚型进行调节，以不同的底物，从而使安全可以用比较的。 总之，本文件说明了如何使用荧光分光光度法来测量胱天蛋白酶-3活性在杏仁核，提供凋亡发生在边缘系统的这个区域的MI后3天的证据。

胱天蛋白酶或半胱氨酸依赖性天冬氨酸定向蛋白酶是一个家族参与各种稳态processess 1中的酶。胱天蛋白酶可以根据其在细胞凋亡或炎症中的作用被广义地分类。胱天蛋白酶-1，-4，-5和-12参与炎症而那些从事凋亡可以分划为引发剂胱天蛋白酶（胱天蛋白酶-8，-9）和胱天蛋白酶刽子手（胱天蛋白酶-3，-6和-7 ）。 胱天蛋白酶在许多病理发挥显著的作用，主要表现在疾病，其中细胞凋亡能量过大，心肌梗死（MI）或脑缺血后观察到。 在我们的实验中使用的MI后综合征的大鼠模型中，经确定的caspase-3不仅在心肌2而且在其中牵涉情绪和情感3-6的控制边缘系统激活。它还可以看出，caspase-3的不同区域被激活边缘系统，例如杏仁核和海马，以及局部缺血损伤后4约3天内，活化的峰。有趣的是，caspase-3的活性与心肌梗死后的行为障碍，并通过药理和营养干预其衰减减少这些伤害，提示caspase-3和心梗后抑郁7-12之间可能存在的联系。 Caspase-3的活化可通过各种技术来测量。 Western印迹台确定胱天蛋白酶的酶活性，可检测活性酶以及它们的酶原形式。 Western印迹，但是，是半定量，和小的变化可以通过微弱的信号-噪声比13丢失。更好的方法是基于在基板（DEVD-AMC）的由胱天蛋白酶-3，释放一个荧光化合物，并且可以通过荧光分光光度法测量的裂解。 Caspase-3的活化是凋亡的可靠指标。细胞凋亡钙的测定n为不稳定的，因为发生的时间窗口短，和邻近细胞可以吞噬凋亡小体或细胞14。凋亡可以通过其它技术，如末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）测定法来进一步证实，其检测从凋亡信号传导级联6，或亲/抗凋亡蛋白的比率，如Bax的/得到的DNA片段BCL2 6。

<table>
	<tbody>
		<tr>
			<td>Spectroflurometer</td>
			<td>Photon technology Instrument (now Horiba)</td>
			<td>Ratiomaster M-40</td>
			<td>with DeltaRAM&#160; Random Access Monochromator&#160;</td>
		</tr>
		<tr>
			<td>Respirator</td>
			<td>Harvard apparatus</td>
			<td>683</td>
			<td>small animal ventilation</td>
		</tr>
		<tr>
			<td>Glass cuvette</td>
			<td>NSG precision cells</td>
			<td>3G10</td>
			<td>Optical Glass (340-2,500nm) 10 mm path</td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Cole Parmer</td>
			<td>4710&#160; series</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td>Varian</td>
			<td>Cary 50 Bio</td>
			<td></td>
		</tr>
	</tbody>
</table>

caspase-3 activity in the rat amygdala measured by spectrofluorometry after myocardial infarction

心肌梗死（MI）具有在生理和行为水平显着中期和长期的后果，但所涉及的机制仍不清楚。我们的实验室已开发出心肌梗死后综合征，显示受损心脏的功能，在边缘系统的神经元丧失，认知障碍和抑郁的行为迹象的大鼠模型。在神经元水平，胱天蛋白酶-3的活化介导的MI后凋亡不同边缘区域，如杏仁核 - 3天达到峰值后心肌梗塞。认知和行为障碍出现2-3周后MI和这些与caspase-3的活性的措施相关统计学。这里描述的协议被用来诱发心肌梗死，收集杏仁核组织和使用荧光分光光度法测定caspase-3活性。为了诱导心肌梗死，所述下行冠状动脉被遮挡40分钟。该协议为caspase-3的活性评价心梗开始后第3天:被处死老鼠和杏仁核中分离RAPI从大脑DLY。样品迅速在液氮中冷冻并保持在-80℃直至实际分析。以评估的caspase-3的激活的技术是基于一个衬底（DEVD-AMC）由胱天蛋白酶-3，释放可通过荧光分光光度法来测量一个荧光化合物的裂解。该方法是定量的和可再现的，但所需要的设备是昂贵的，用于量化样品的过程是耗时的。这一技术可以应用到其他组织中，如心脏和肾脏。 DEVD-AMC可以通过其它基片代替，以测量其他胱天蛋白酶的活性。

扁桃体分离出8假（对照）和8 MI大鼠缺血/再灌注协议诱导后三天。使用可以由活性的caspase-3被裂解并通过荧光分光光度法检测出的荧光caspase-3的活性测定在这个组织上。正和负的测量（见步3.11，3.12和3.16），一式三份在10秒进行测量和，用1点在每个秒。阳性和阴性对照之间的差异，计算和的差异为这个实验期间进行的所有假组织的平均设定在100％。结果从MI大鼠根据本参考文献被缩放的， 图1示出了最后的结果:它表明相比假（P &lt;0.05）中的MI大鼠显著更高的活性。 <img alt="图1" src="/files/ftp_upload/53207/53207fig1.jpg" /> 图1. Caspase-3的在心肌梗死大鼠杏仁核活性，表示为平均活性的假对照百分比，设定为100％（8只大鼠每组）。 *表示显著的组间差异（P &lt;0.05）。数据表示为平均值±SEM表示。

Watch this Scientific Journal Video about Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction at JoVE.com

Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

Myocardial infarction (MI) is not only followed by impaired cardiac function but also by apoptosis in the amygdala, a brain region involved in the behavioral consequences of MI. This protocol describes how to induce MI, collect amygdala tissue and measure caspase-3 activity, a marker of apoptosis, therein.

Caspase-3活性在鼠杏仁核测量用荧光分析法心肌梗死后

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still ...

caspase-3-activity-rat-amygdala-measured-spectrofluorometry-after

Research

JoVE Journal

Neuroscience

11.6K 次观看.  Hospital du Sacre-Coeur de Montreal. Myocardial infarction (MI) is not only followed by impaired cardiac function but also by apoptosis in the amygdala, a brain region involved in the behavioral consequences of MI. This protocol describes how to induce MI, collect amygdala tissue and measure caspase-3 activity, a marker of apoptosis, therein.

视频: Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

焦距脑缺血模型大鼠大脑中动脉的血管内缝合闭塞

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

科研

神经科学

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

量化的活动顺</em在小鼠视网膜>调控元件外植体电穿孔

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

监测劈开caspase - 3活性和钾诱导膜去极化使用活细胞成像和Cleaveable荧光染料基板的永生的少突胶质细胞凋亡

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

基因表达的变化大鼠海马后，前丘脑核脑深部刺激分析

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

海马神经元活动的可视化免疫组化空间后学习神经发育障碍的小鼠模型

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

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

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when compared to warm-blooded vertebrates. Because of this, in vitro experiments in turtles can be performed for extended periods of time to investigate the neural signals and control of their target actions. Using an isolated head preparation, we measured the kinematics of eye movements in turtles, and their modulation by electrical signals carried by cranial nerves. After the brain was removed from the skull, leaving the cranial nerves intact, the dissected head was placed in a gimbal to calibrate eye movements. Glass electrodes were attached to cranial nerves (oculomotor, trochlear, and abducens) and stimulated with currents to evoke eye movements. We monitored eye movements with an infrared video tracking system and quantified rotations of the eyes. Current pulses with a range of amplitudes, frequencies, and train durations were used to observe effects on responses. Because the preparation is separated from the brain, the efferent pathway going to muscle targets can be examined in isolation to investigate neural signaling in the absence of centrally processed sensory information.

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

This protocol describes how to use an in vitro isolated turtle head preparation to measure the kinematics of their eye movements. After removal of the brain from the cranium, cranial nerves can be stimulated with currents to quantify rotations of the eye and changes in pupil sizes.

体外刺激对龟脑神经的运动测量

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

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also demonstrate that traumatic brain injuries cause changes to the gut microbiota. However, mechanisms underlying the bidirectional regulation of the brain-gut axis remain unknown. Currently, few models exist for studying the changes in gut microbiota after traumatic brain injury. Therefore, the presented study combines protocols for inducing traumatic brain injury using a lateral fluid percussion device and analysis of caecum samples following injury for investigating alterations in the gut microbiome. Alterations of the gut microbiota composition after traumatic brain injury are determined using 16S-rDNA sequencing. This protocol provides an effective method for studying the relationships between enteric microorganisms and traumatic brain injury.

Presented here is a protocol to induce diffuse traumatic brain injury using a lateral fluid percussion device followed by the collection of the caecum content for gut microbiome analysis.

研究小鼠创伤性脑损伤后凯库姆微生物群的改动

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also ...

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.

Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

电刺激在大鼠中建立急性庞廷梗死

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

This protocol describes the construction of a low-cost microinjection system, its stereotaxic implantation into deep-brain structures and the procedure for timed microinjections of tetrodotoxin in awake and unrestrained rats. The goal is to reveal the participation of hypothalamic structures in the regulation of ovulation by inhibiting their neural activity.