这项工作得到了国家普通医学科学研究所 (GM102691, GM1115384)、国家神经疾病和中风研究所 (NS100065) (美国马里兰州贝塞斯达) 和纽约大学系麻醉研究基金的支持。麻醉学 (纽约州, 美国纽约)。

这项研究中的所有程序都得到了纽约大学医学院机构动物护理和使用委员会 (IACUC) 的批准, 认为这符合国家卫生研究所 (NIH) 《实验动物护理和使用指南》。
1. 立体定向插管植入术
<ol><li>使用10-12 大雄性 Sprague-Dawley 大鼠。</li>
	<li>如前所述, 用1.5-2% 异氟醚1,3,5麻醉动物。一旦动物变得没有反应, 以尖锐的钳子强烈捏, 执行手术。一定要高压灭菌所有仪器, 使用无菌手术手套, 并添加眼膏到动物的眼睛, 以防止损坏。</li>
	<li>立体定向植入双侧26规格的引导插管进入 PFC 在12.5 度角与坐标 AP: + 2.9 毫米;ML: +/-1.6 毫米;DV:-2.1 毫米。为了插入插管, 在颅骨上的所需坐标上钻孔, 并根据所使用的插管大小进行孔直径。 注: 在这里, pfc 被研究作为潜在的目标颅内植入物, 因为它在疼痛处理中的重要作用, 这已经在以前的一些研究1,2,7已经证明。为了研究其他特定大脑区域的功能, 研究人员可以根据大脑地图集使用不同的坐标。关于进行颅内手术的更多细节, 见 Goffer等人 (2013年)3、lee 等人 (2015年)1和 sun等人 (2017年)10。</li>
	<li>让老鼠从手术中恢复至少1周。手术后, 在恢复前注射皮下液体, 以帮助支持代谢需求, 并在新闭合的切口中应用局部布比卡因。将动物放在温暖的垫子上, 直到它们醒来, 并监测动物手术 3天, 以确保良好的健康和适当的恢复。</li>
	<li>一旦动物从手术中完全恢复, 开始注射 (下一步)。</li>
</ol>2. 颅内和腹腔内注射
<ol><li>对于颅内注射, 使用 PE-50 管连接在一端10μl 汉密尔顿注射器与33规格的注射器插管, 延伸1.0 毫米超出植入的指南。</li>
	<li>在这些大鼠的 PFC 中注入 0.5Μl (或更少 (如果需要) 研究药物或盐水。因为 PFC 是大鼠中较大的区域, 所以这个量不会扩散到其他区域。然而, 对于较小的大脑区域或小鼠, 使用较小的体积。注射量将取决于大脑的区域和动物的物种。 注: 请注意, 盐水应用作对照, 而不是 DMSO, 因为 DMSO 是神经毒性的。然而, 极少量的 dmso 可能是安全的输液, 因为研究表明, 小于50% 的 dmso (即小于0.3μl 的总体积, 在这种情况下) 可能不会干扰行为研究 8,9。</li>
	<li>在100秒内双边注入体积, 并在去除前保持喷射器插管的额外 60秒, 以允许这种溶液缓慢扩散。</li>
	<li>对于协同药理作用的研究, 通过系统方法共同用药另一种药物。在这种情况下, 注射所需的药物或控制内部, 并在此后立即在腹腔内使用额外的药物。作为一个例子, 我们研究了吗啡和安巴金的协同镇痛作用, 以测试添加剂效应。我们在体内注入了 AMPAkine, 并与腹腔内注射 1 mg/kg 吗啡 (安全的全身剂量)结合使用. 注: 建议先进行颅内注射, 因为它们比腹腔注射更难进行。</li>
</ol>3. 镇痛检测和评估
<ol><li>为了研究颅内注射对大鼠急性疼痛行为的影响, 采用足底试验 (哈格里夫斯试验) 计算出对热刺激的戒断潜伏期。哈格里夫斯的装置通过玻璃平面将红外光束聚焦到老鼠的脚上;老鼠站在玻璃平面上方自由移动。在进行哈格里夫斯测试时, 将红外光束聚焦在大鼠足底区域下。<ol>
<li>首先在注射前进行基线哈格里夫斯的测试, 以建立比较的基线值。</li>
	</ol>
</li>
	<li>在确定基线值之前, 不要注射任何种类的药物。进行5次良好的试验, 间隔5分钟。一个很好的试验是通过明确的戒断来表示的,即当老鼠弯曲膝盖, 抬起脚向上进入身体时。 注: 确保试验间隔 5分钟, 以防止对大鼠过敏。一旦红外光束被破坏, Hargreaves 的装置会自动记录撤离时间。因此, 一定要从运动、换重等方面打折试验, 如果进行打折试验, 等 5分钟, 重复试验。</li>
	<li>计算退出阈值的平均值的5试验。</li>
	<li>在获得基线平均值后, 开始实验, 获得药物输注后的戒断时间。Hargreaves 的测试可以在颅内注射后20-30 进行, 尽管确切的时间可能取决于特定药物的药代动力学。这是为了确保老鼠已经吸收了这种药物, 并正在经历其影响。以与步骤3.1 相同的方式进行此实验。</li>
	<li>计算步骤3.2 中提到的取款阈值。</li>
</ol>

<ol>
	<li>Lee, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+corticostriatal+circuitry+relieves+chronic+neuropathic+pain.">Activation of corticostriatal circuitry relieves chronic neuropathic pain.</a> The Journal of Neuroscience. 35 (13), 5247-5259 (2015).</li><li>Martinez, E., Lin, H. H., Zhou, H., Dale, J., Liu, K., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticostriatal+Regulation+of+Acute+Pain.">Corticostriatal Regulation of Acute Pain.</a> Frontiers in Cellular Neuroscience. 11, 146 (2017).</li><li>Goffer, Y., 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=Calcium-permeable+AMPA+receptors+in+the+nucleus+accumbens+regulate+depression-like+behaviors+in+the+chronic+neuropathic+pain+state.">Calcium-permeable AMPA receptors in the nucleus accumbens regulate depression-like behaviors in the chronic neuropathic pain state.</a> The Journal of Neuroscience. 33 (48), 19034-19044 (2013).</li><li>Carr, K. 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=AMPA+receptor+subunit+GluR1+downstream+of+D-1+dopamine+receptor+stimulation+in+nucleus+accumbens+shell+mediates+increased+drug+reward+magnitude+in+food-restricted+rats.">AMPA receptor subunit GluR1 downstream of D-1 dopamine receptor stimulation in nucleus accumbens shell mediates increased drug reward magnitude in food-restricted rats.</a> Neuroscience. 165, 1074-1086 (2010).</li><li>Su, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPAkines+target+the+nucleus+accumbens+to+relieve+postoperative+pain.">AMPAkines target the nucleus accumbens to relieve postoperative pain.</a> Anesthesiology. 125 (5), 1030-1043 (2016).</li><li>Le, A. M., Lee, M., Su, C., Zou, A., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPAkines+have+novel+analgesic+properties+in+rat+models+of+persistent+neuropathic+and+inflammatory+pain.">AMPAkines have novel analgesic properties in rat models of persistent neuropathic and inflammatory pain.</a> Anesthesiology. 121 (5), 1080-1090 (2014).</li><li>Cooper, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthetisation+of+prefrontal+cortex+and+response+to+noxious+stimulation.">Anaesthetisation of prefrontal cortex and response to noxious stimulation.</a> Nature. 254 (5499), 439-440 (1975).</li><li>Blevins, J. E., Stanley, B. G., Reidelberger, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DMSO+as+a+vehicle+for+central+injections:+tests+with+feeding+elicited+by+norepinephrine+injected+into+the+paraventricular+nucleus.">DMSO as a vehicle for central injections: tests with feeding elicited by norepinephrine injected into the paraventricular nucleus.</a> Pharmacology Biochemistry and Behavior. 71, 277-282 (2002).</li><li>Schmeichel, B. E., Herman, M. A., Roberto, M., Koob, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypocretin+Neurotransmission+Within+the+Central+Amygdala+Mediates+Escalated+Cocaine+Self-administration+and+Stress-Induced+Reinstatement+in+Rats.">Hypocretin Neurotransmission Within the Central Amygdala Mediates Escalated Cocaine Self-administration and Stress-Induced Reinstatement in Rats.</a> Biological Psychiatry. 81, 606-615 (2017).</li><li>Sun, Y., Liu, K., Martinez, E., Dale, J., Huang, D., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPAkines+and+morphine+provide+complementary+analgesia.">AMPAkines and morphine provide complementary analgesia.</a> Behavioural Brain Research. 334 (2017), 1-5 (2017).</li></ol>

作者没有什么可透露的。

在这项研究中, 我们已经证明, 颅内药理学是一个强大的工具, 研究疼痛机制, 并具有作为治疗传递系统的潜力。在我们的协议中, 我们将 AMPAkines 直接送入 PFC, 发现通过增强 PFC 中的谷氨酸信号, AMPAkines 提供了止痛。我们能够通过使用颅内注射结合腹腔注射, 并随后进行疼痛检测来证明这一点。根据止痛效果的证据, 当 Ampakine 被送到 PFC 时, 目前的研究表明, PFC 可能是 Ampakine 的目标。这是颅内药理入?...

据悉, 中枢神经系统在疼痛调节中发挥着关键作用。例如, 大脑中的谷氨酸信号在疼痛1,2的背景下具有调节作用。因此, 有必要研究大脑中有关疼痛的细胞和分子信号通路。此外, 还需要了解特定大脑区域的分子目标是否可以修改以治疗疼痛。目前对大脑疼痛的研究依赖于电生理的体外研究与全身 (腹腔) 药理药物的传递相结合。体外研究在揭示体内疼痛机制方面存在明显缺陷。同时, 系统药物输送并不能描述确切的细胞目标。体内化学和生物制剂的体内注射已成为研究大脑中神经和分子通路的有力工具。近年来, 其他领域已在体内颅内注射成功地研究成瘾和奖励行为和电路路径在啮齿类动物3,4。然而, 在疼痛的情况下,缺乏使用体内颅内药理学。
颅内注射可以精确地将药物注射到大脑的特定区域。此外, 特定的途径和受体可以使用高选择性药物的目标。颅内运载系统与精确药物的结合使我们能够瞄准分子和细胞的疼痛目标。这些药物在颅内分娩后, 研究人员可以观察到啮齿类动物行为的直接影响。从很好的实验中, 啮齿类动物的行为可以与药理学联系起来。
在这个协议中, 我们使用了在前额叶皮层 (PFC) 的 ampakine 输注的例子, 以证明皮质谷氨酸信号在疼痛调节的机制。Ampakine 是已知的合金调节剂的合成化合物。他们已经显示出在动物模型 5,6的能力来缓解急性和慢性疼痛。此前的研究表明, ampakine 的可能作用部位在大脑5,6。PFC 是大脑中的一个区域, 它表现出自上而下的控制, 以调节情绪和行为。其中一些产出预测已被证明是疼痛调节1、2、7的关键。更具体地说, 在 PFC 中谷氨酸信号已被证明可以调节疼痛。因此, PFC 被选定为目标脑区, 用于研究疼痛状态下的 Ampakine。

<table>
	<tbody>
		<tr>
			<td>Sterotaxic Cannula</td>
			<td>PlasticsOne</td>
			<td>8I26GA8MMKIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Syringe</td>
			<td>Hamilton</td>
			<td>8440</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPAkine</td>
			<td>Sigma Aldrich</td>
			<td>C-271</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>Hargreaves Apparatus</td>
			<td>Ugo Basile</td>
			<td>37370</td>
			<td></td>
		</tr>
		<tr>
			<td>Male Sprague-Dawley rats</td>
			<td>Taconic Farms</td>
			<td>NTac:SD</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Surgical gloves</td>
			<td>Dynarex</td>
			<td>6535</td>
			<td></td>
		</tr>
	</tbody>
</table>

intracranial pharmacotherapy and pain assays in rodents

疼痛是一种突出的感官体验, 具有情感和认知维度。然而, 对疼痛的核心机制仍然了解甚少, 阻碍了有效疗法的发展。颅内药理学是了解大脑疼痛的分子和细胞机制以及新治疗方法的重要工具。在这里, 我们提出了一个协议, 结合颅内药理学疼痛行为测试。具体而言, 我们展示了如何将镇痛药物注入到一个特定的大脑区域, 这可能是造成疼痛调节的原因。此外, 为了确定候选药物在中枢神经系统中的作用, 颅内治疗后进行疼痛检测。我们的研究结果表明, 颅内给药的镇痛药在目标区域可以提供缓解啮齿类动物的疼痛。因此, 我们的协议成功地证明, 颅内药理学, 结合疼痛行为测试, 可以是一个强大的工具, 研究大脑中的疼痛机制。

例如, 我们通过插管将 AMPAkine 注入 PFC (图 1)。我们还系统地注入吗啡, 以评估 Ampakine 和吗啡之间的协同镇痛作用。这些结果表明, Ampakine 和吗啡具有添加镇痛作用。这也表明, 颅内注射有能力发现, 至少在一定程度上, 在疼痛的情况下药物激活的机制。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58473/58473fi...

Watch this Scientific Journal Video about Intracranial Pharmacotherapy and Pain Assays in Rodents at JoVE.com

Intracranial Pharmacotherapy and Pain Assays in Rodents

在这里, 我们提出了一个方案, 以执行颅内药理实验, 其次是在啮齿类动物的疼痛行为检测。该协议允许研究人员在大脑中提供分子和细胞靶标, 用于治疗疼痛的药理剂。

颅内药物治疗和啮齿动物疼痛检测

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

这项技术的主要优点是，它可以用来进一步探索大脑中的疼痛机制和药物作用。为了研究颅内注射对大鼠急性疼痛行为的影响，手术一周后，将大鼠放在哈格里夫斯仪器的玻璃板上，并使用该仪器将红外光束聚焦在大鼠脚的植物区域下的玻璃平面上。要研究两种不同研究药物之间的协同药理作用，请使用一块PE-50管连接到一个10微升汉密尔顿注射器，该注射器配有一个33量量的注射器管，该管比植入导轨外延伸一毫米。在100秒内将0.5微升或更少的研究药物注射到一个颅内管中。喷油器管在喷射后再放置 60 秒，以方便溶液缓慢扩散。然后，在内注射感兴趣的第二种药物。注射后20至30分钟，进行植物反射测试五次，如证明。在这个具有代表性的实验中，AMPAkine的颅内分娩显示出一种附加镇痛作用，当与内向内注射吗啡共同管理时，表明颅内注射至少部分地具有在疼痛情况下发现药物激活机制的力量。在尝试此过程时，必须记住密切遵循该过程的时间。按照这个程序，颅内植入物可以与疼痛分析一起使用，以回答有关大脑区域负责给定疼痛表型，药物作用的可能位点，或可能的疼痛药物机制的其他问题。

intracranial-pharmacotherapy-and-pain-assays-in-rodents

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Neuroscience

Unit 1

Drugs for Pain Management: Opioid Analgesics and General Anesthetics

SCIENTISTS IN ACTION

5.1K 次观看.  New York University School of Medicine. 这项技术的主要优点是，它可以用来进一步探索大脑中的疼痛机制和药物作用。为了研究颅内注射对大鼠急性疼痛行为的影响，手术一周后，将大鼠放在哈格里夫斯仪器的玻璃板上，并使用该仪器将红外光束聚焦在大鼠脚的植物区域下的玻璃平面上。要研究两种不同研究药物之间的协同药理作用，请使用一块PE-50管连接到一个10微升汉密尔顿注射器，该注射器配有一个33量量?...

视频: Intracranial Pharmacotherapy and Pain Assays in Rodents

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

颅内注射腺相关病毒载体

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

科研

神经科学

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron precursors (CGNPs) as the cell-of-origin for medulloblastoma1-4. However, the diverse clinical presentations of medulloblastoma subtypes in human patients (nodular, desmoplastic, classical and large cell/anaplastic), and the fact that medulloblastoma is found in a subset of human patients with no ectopic expression of CGNP marker5, suggest that the cellular and molecular origins of medulloblastoma are more complex and far from being completely deciphered. Therefore, it is essential to determine whether there is an alternative medulloblastoma tumor cell-of-origin based on which cell-type specific therapeutic modality can be developed. To this end, intracranial orthotopic allografting of genetically marked tumor cell types followed by subsequent analyses of secondary tumor development in recipients will allow determination of the cellular origin of tumor-initiating cells. Here we describe the experimental protocol for intracranial orthotopic allografting of medulloblastoma cells derived from primary tumor tissue, and this procedure can also be used for transplanting cells from established cell lines.

This protocol describes the isolation and dissociation of mouse medulloblastoma tissue, and subsequent allografting of the tumor cells into immunocompromised recipient mice in order to initiate secondary medulloblastoma.

在免疫功能低下小鼠颅内髓母细胞瘤细胞原位Allografting

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron ...

Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the sensorimotor cortex and the underneath white matter (undercut) so that a specific region of the cerebral cortex is largely isolated from the neighboring cortex and subcortical regions1-3. After a latency of two or more weeks following the surgery, epileptiform discharges can be recorded in brain slices from rodents1; and electrical or behavior seizures can be observed in vivo from other species such as cat and monkey4-6. This well established animal model is efficient to generate and mimics several important characteristics of traumatic brain injury. However, it is technically challenging attempting to make precise cortical lesions in the small rodent brain with a free hand. Based on the procedure initially established in Dr. David Prince's lab at the Stanford University1, here we present an improved technique to perform a surgery for the preparation of this model in mice and rats. We demonstrate how to make a simple surgical device and use it to gain a better control of cutting depth and angle to generate more precise and consistent results. The device is easy to make, and the procedure is quick to learn. The generation of this animal model provides an efficient system for study on the mechanisms of posttraumatic epileptogenesis.

Partially isolated cortex (&#x201C;undercut&#x201D;) is an efficient animal model of posttraumatic epileptogenesis. Here we demonstrate how to make a novel surgical device and use it to make more precise and consistent lesions to generate this model.

准备在鼠害外伤性癫痫的底切模型

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the ...

Habituation and Prepulse Inhibition of Acoustic Startle in Rodents

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3.
We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used.
Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.

Habituation and prepulse inhibition of startle are operational measures of sensory gating. Sensory gating is disrupted in schizophrenia, and some other mental disorders and neurodegenerative diseases. We here describe a standard protocol to assess short-term and long-term habituation as well as prepulse inhibition of acoustic startle responses in rats and mice.

在鼠害声不惊人死不休的习惯和前脉冲抑制

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

运行轮活动在啮齿类动物的昼夜节律的记录和分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

深部脑刺激与同步功能磁共振成像在鼠害

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Acrylic Resin Molding Based Head Fixation Technique in Rodents

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires surgical attachment of metallic frames on the skull. The attached frames are then clamped to a stationary platform resulting in immobilization of the head. However, metallic frames for head fixation have been technically difficult to design and implement in general laboratory environment. In this study, we provide a novel head fixation method. Using a custom-made head fixation bar, head mounter is constructed during implantation surgery. After the application of acrylic resin for affixing implants such as electrodes and cannula on the skull, additional resins applied on top of that to build a mold matching to the port of the fixation bar. The molded head mounter serves as a guide rails, investigators conveniently fixate the animal&#8217;s head by inserting the head mounter into the port of the fixation bar. This method could be easily applicable if implantation surgery using dental acrylics is necessary and might be useful for laboratories that cannot easily fabricate CNC machined metal head-posts.

Traditional head fixation techniques have used metallic frames attached on the skull as an interface for head fixation apparatus. This protocol demonstrates a frameless, acrylic resin molding based head fixation technique for behavioral tasks in rodents.

在鼠害丙烯酸树脂模塑基头固定技术

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

间刺激间隔对啮齿类动物时间处理评估的力量

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

绘制脑血流和电场图：小鼠的电极放置

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p &lt; 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths &lt;5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

作者聚焦：基于刺激的方法改善阿尔茨海默病模型中的脑血流 

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and ...