这项研究得到了国立护理研究所 (K99/R00NR014369) 的支持。

所描述的所有方法都是由芝加哥伊利诺伊大学的机构动物护理委员会批准的。
1. 创建双极北美放射学会电极
<ol><li>要制造电极, 切两件不锈钢丝每约18毫米长。切割一条聚乙烯 (PE-50) 油管约15毫米长。将两条导线进油管, 使导线两端突出。</li>
	<li>从导线的两端除去绝缘物;修剪电线留下2-3 毫米裸露的电线。一端, 在暴露的导线上卷曲男性针脚。将针脚安全地焊接到导线上, 确保连接器条内的针脚牢固, 并覆盖与环氧树脂的连接。 注意: 避免焊接的另一种方法是使用快速连接/释放鳄鱼夹子。</li>
	<li>在电极的另一端, 从导线的两端除去绝缘, 留下2-3 毫米暴露的导线。弯曲这部分电线, 以创建小 "V" 形钩在绝缘线。 注: 这是电极的一部分, 将与肾交感神经接触。密封这一端以防止流体进入油管是很重要的。有机硅或环氧树脂有效地可以使用。</li>
</ol>2. 管理麻醉和准备手术场所
<ol><li>对雄性大鼠进行麻醉 (年龄9-11 周, 重150-400 克)。通过腹腔 (IP) 注射, 管理戊巴比妥钠50毫克/千克。为了评估手术中的麻醉稳定面, 每15分钟检查一次脚趾捏反射, 并根据需要重新进行麻醉。 注: 戊巴比妥钠 (戊巴比妥) 用于以前的研究, 以达到持续的麻醉平面, 不干扰北美放射学会4,5,6的调制。该协议是为非生存手术, 因此没有恢复/术后监测期。</li>
	<li>根据机构动物护理指导方针 (i.) 准备手术地点; 剃掉老鼠的腹部、背部和头部; 用10% 聚维酮碘溶液清洗皮肤; 涂抹眼部润滑剂; 把老鼠放在暖气垫上)。在实验期间保持体温在37摄氏度。</li>
</ol>3. Cannulate 股静脉 (供静脉进入)
<ol><li>添加肝素到0.9% 无菌生理盐水 (达到20单位/毫升)。用22G 针将血气盐水填充1毫升注射器。将15厘米的 PE-50 油管连接到针上, 并用溶液填充油管。</li>
	<li>与鼠仰卧卧, 创建一个2厘米水平切口通过腹股沟区。采用棉尖涂抹器, 解剖结缔组织, 露出股静脉和动脉。为保持切口开放, 无论是应用单钩弹性手术留在外科领域与丝绸胶带或使用小止血剂。</li>
	<li>使用止血剂在90°角度弯曲22G 针的尖端, 作为导管介绍人10。</li>
	<li>显微镜下的血管可视化。用弯钳轻轻地将静脉和动脉分开。在静脉下放置两个12厘米长的5-0 丝缝合线 (一个远端和一个近端);用同样的方式把缝线放在动脉下面。<ol>
<li>将远端 (底) 缝线系在静脉咬合;使用丝带或小止血剂将此缝合线的边缘固定在手术领域。轻轻拉紧静脉, 但不要用这么多的力量, 船只会撕裂。将静脉垂直于外科医生的显性手。</li>
	</ol>
</li>
	<li>在近端缝合中使用松动的半结, 简单咬合静脉。用细腻的止血钳, 轻轻夹住这缝合咬合血流。将22G 针与弯曲尖端握在非显性手上;使用主手将油管与镊子扣紧。</li>
	<li>用导管介绍人 (步骤 3.3) 在静脉穿刺一个小孔, 并将 PE-50 导管 (预填充血气生理盐水) 插入容器内;使用弯曲的针保持打开的容器开放, 并协助将导管的尖端定位到容器10。<ol>
<li>释放近端缝合, 轻轻冲洗0.2 毫升的血气生理盐水进入静脉;提前导管。检查血液从静脉回流, 以确保适当的放置。完成近端结, 并与远端缝合领带, 确保静脉内的导管。</li>
	</ol>
</li>
	<li>在实验中使用静脉通路来管理补充麻醉或药物和收集血液。如果需要定期进行静脉输液和血液取样, 请合并3路连接器。每15分钟检查一次有害的脚趾捏反射, 滴定麻醉。</li>
</ol>4. Cannulate 股动脉用于平均动脉压力监测
<ol><li>显微镜下的动脉可视化。类似于静脉插管的方法, 系远端缝合 (步骤 3.4) 咬合动脉;将此缝合线的边缘固定在带丝胶带的外科手术场上, 并将动脉垂直于外科医生的主导手。</li>
	<li>使用压力传感器/输液系统时的动脉通路<ol>
<li>将压力传感器/油管连接至500毫升0.9% 盐水袋。用盐水冲洗油管, 除去所有气泡, 将袋子放在压力诱导袋内加压系统。</li>
		<li>如步骤3.1 所述, 通过22G 针填充1毫升注射器和血气盐水, 并将15厘米的 PE-50 连接到针 (冲洗油管与血气生理盐水)。</li>
		<li>在近端缝合中使用松散的半结来短暂咬合动脉。将导管介绍人 (步骤 3.3) 放在非显性手上;用血管插管钳将 PE-50 的远端放在占主导地位的手上。用弯曲的22G 针在动脉内穿刺一个孔, 并将套管插入血管内。</li>
		<li>释放近端缝合, 轻轻冲洗0.2 毫升的血气生理盐水进入动脉, 尽量提前导管。检查动脉血返回以确保正确放置。完成近端结, 并与远端缝合领带, 确保导管内动脉。将动脉线连接到压力传感器/油管。 注: 导管远端部分可贴在大鼠后肢上, 以保证动脉线的安全。另一种方法的容器插管是描述的 Jespersen et al。11他们的协议不同于使用拉钩传播切口, 胶-而不是缝合-安全油管, 并且方法不包括弯曲的针介绍人。</li>
	</ol>
</li>
	<li>使用遥测的动脉通路<ol>
<li>在动脉插管前, 在高放大倍数下检查压力传感导管。确保导管没有气泡/碎片, 并有一个完整的半月板之间的液体填充 (近端) 和凝胶填充 (远端) 成分。在每次植入之前, 将凝胶在导管的远端端重新填充。用磁铁打开发射机;在手术中监测 BP, 以承受完美的安置。</li>
		<li>在近端缝合中使用松动的半结, 短暂咬合股动脉。将导管介绍人 (步骤 3.3) 放在非显性手上。用导管插管钳握住遥测装置套管的尖端, 避免从尖端中置换凝胶。</li>
		<li>用弯曲的22G 针在动脉内穿刺一个孔, 用导管插管钳将套管插入动脉, 以避免从尖端中置换凝胶。尽量提前插管。使用近端和远端缝合连接, 确保压力导管的安全。</li>
		<li>将遥测植入物的身体塞在切口附近的侧面, 并用4-0 尼龙缝线缝合切割针。在记录期结束时, 通过磁铁将遥测装置关闭, 以节省电池寿命。</li>
	</ol>
</li>
</ol>5. 将大鼠定位在立体定向手术框架中以进入大脑
<ol><li>将大鼠轻轻地移动到立体定向手术框架中的俯卧位。</li>
	<li>将大鼠放置在耳棒之间, 调整切牙条以平衡 lambda 和 bregma 的高度。定位可能取决于大鼠的应变、体重和中枢神经系统目标的位置。</li>
	<li>通过头皮中线做一个2厘米 rostrocaudal 手术刀切口。使用棉尖的喷头, 牢固地去除头骨表面的结缔组织。将过氧化氢应用于颅骨, 以帮助可视化 bregma、lambda 和中线缝合。</li>
	<li>使用大鼠大脑图谱来指导目标12, 钻一个毛刺孔截骨, 大小为电极进入, 通过头骨。</li>
</ol>6. 隔离肾交感神经
<ol><li>将导线北美放射学会电极 (步骤 1.1-1.3) 连接到10X 预放大器和微电极放大器。</li>
	<li>将肾神经通过腹膜后切口隔离, 或后, 将大鼠固定在立体定向的框架内。一旦大鼠在立体定向的框架内, 定位北美放射学会电极。使手术刀切开从4-5 厘米以下的肋骨在尾部方向, 稍侧向脊柱。钝解剖切口, 可视化椎旁肌肉。</li>
	<li>用剪刀做一个非常肤浅的1-2 厘米 rostrocaudal 切口在那里脂肪满足肌肉。使用棉尖的喷头, 将脂肪从肌肉中传播到想象肾脏。重要的是不要进入腹膜的空间。</li>
	<li>使用拉钩轻轻地将肾脏与椎旁肌肉分开, 以可视化肾动脉和腹主动脉。不要过度伸展血管, 以免损害肾脏神经。使用 2 "x 2" 棉纱布垫浸泡在盐水中, 以保护肾脏免受伤害。</li>
	<li>在高放大倍数下, 识别切口口袋中的肾神经。神经束最容易在由主动脉和肾动脉形成的直角上可见。肾神经密切跟随肾动脉从主动脉到肾脏。</li>
	<li>选择将放置在记录电极上的神经束的一段。用显微解剖镊子轻轻地解剖周围组织/血管的神经纤维。</li>
	<li>将导线北美放射学会电极固定在支架上 (例如, 附着在支撑架上的鳄鱼夹子)。将电极降低到神经段的水平。用神经钩轻轻地将肾神经段抬到电极上, 而不伸展神经。 注: 神经应在绝缘线的两个 "V" 形钩内休息, 并与神经平行。电极线不能接触任何其他组织, 血液, 或淋巴液。</li>
	<li>用矿物油填充切口, 防止暴露的肾交感神经变得干燥。在切口的皮肤上使用一个末端的接地夹, 另一端附着在法拉第笼上。</li>
	<li>使用高和低通滤波 (10 赫兹和3赫) 将信号定向到放大器。调整增益高达 10 k 包括一个音频显示器, 以评估爆破模式的北美放射学会。使用采样率, 范围介于 200万赫兹 4、5、6、7、8之间。假设 CNS 操作被假定为快速/简短的交感神经应答, 请使用增加的采样速率。</li>
</ol>7. 记录数据
<ol><li>通过反射注射1毫升生理盐水或10µg/毫升肾上腺素 (0.1 毫升) 静脉注射, 评估北美放射学会记录的质量。如图 1所示, 输液应增加 BP 并抑制北美放射学会。平均动脉压力增加 60-80 mmHg 是足够的肾 sympathoinhibition4,13,14。</li>
	<li>如果需要, 调整电极位置以改善信号。如果神经不接触电极上的两个钩子或任何组织、血液或淋巴液与导线接触, 则需要重新定位。 注意: 重新定位的需要是基于神经放电的听觉特征。<ol>
<li>如果北美放射学会的爆发不是周期性地与心脏循环发生, 如果有任何干扰的录音, 然后小心地重新定位电极。</li>
		<li>呼吸运动也会影响北美放射学会记录的质量, 通过将电极轻轻地移动到肌肉运动不会在呼吸中扰乱电极的位置来改善信号。</li>
	</ol>
</li>
	<li>一旦获得清晰的信号, 通过提取矿物油和应用硅胶来覆盖切口口袋中的神经/电极连接, 确保北美放射学会电极到位。在凝胶完全设置之前不要移动老鼠。</li>
	<li>在连续记录北美放射学会和平均动脉压力的同时执行 CNS 操作协议。如果 microinjector/脉冲发生器用于脑干操作, 则可以将该装置的逻辑信号引入北美放射学会/BP 记录, 以记录中枢神经系统操作的时间。</li>
	<li>实验完成后, 通过粉碎硅胶与脊柱肌肉之间记录电极的神经, 确定噪声水平。记录至少三十年代的这个 "零" 值为北美放射学会4,5,6。作为量化噪声的另一种方法, 管理一个短作用的节阻滞剂, 如阿托品, 六甲铵, chlorisondamine, 或 pentolinium 酒石酸8,15,16,17。</li>
	<li>小心地取出北美放射学会电极, 从电线电极上取出硅胶的任何痕迹。保存电极以供重新使用。关闭遥测发射机并取出, 注意不要损坏导管的尖端。</li>
</ol>8. 安乐死 (Transcardiac 灌注)
<ol><li>通过注射染料或荧光、产生电解损伤或通过检测 c-fos 表达来识别中枢神经系统操作的位置。</li>
	<li>如果大脑分析需要固定, 准备 transcardiac 灌注大鼠。评估脚趾捏反射, 以确保大鼠仍然深麻醉。必要时提供补充麻醉。在通风罩中进行多聚甲醛固定剂的 transcardiac 灌注。 注意: 狂热的皮肤/眼部刺激性。</li>
	<li>将油管插入灌注泵中, 并以0.9% 生理盐水为质数。</li>
	<li>5-6 厘米侧切口通过皮肤和腹壁立即下肋骨笼和打开胸腔。小心地将肝脏与隔膜分开。用弯曲钝剪刀在隔膜上做一个小切口。将0.1 毫升肝素直接注入左心室。</li>
	<li>通过一个灌注针进入左心室 (一个不锈钢饲针工作很好的这一步), 要么刺穿心脏或切割一个小切口使用锋利的剪刀, 通过饲针通过, 所以尖端是可见的通过主动脉壁 (但不应达到主弓)。使用手术或电子夹子固定针到位。</li>
	<li>使用输液泵, 管理0.9% 正常生理盐水 (室温)。立即创建一个2-3 毫米切口在右心房创建一个出口盐水冲洗。不要切断降主动脉。继续盐水冲洗, 直到肝脏改变颜色从红色或褐色到淡黄色, 大约400毫升的输液2-3 分钟。</li>
	<li>停止泵。将灌流液切换至固定器 (e, 10% 福尔马林或4% 多聚甲醛);注入400毫升超过2-3 分钟. 移除大脑并在固定溶液中保存标本在4摄氏度过夜, 然后将组织转移到30% 蔗糖 (30 毫升磷酸酯缓冲盐水中溶解的毫升蔗糖) 至少100天或直到大脑下沉, 用于抗冻前 cryogenetic 切片18。</li>
</ol>9. 分析数据
<ol><li>全波矫正原北美放射学会以获得绝对值。全波矫正十年代段的原始噪声信号。重要的是排除任何受低信噪比影响的研究。在量化北美放射学会的研究中, 调查人员采用了先验标准, 如要求信噪比超过2:1 至 6:117、19、20。</li>
	<li>计算非重叠段 (µV) 的平均整流北美放射学会, 并减去噪声估计 (µV)。根据实验的目的, 调查人员可以选择时间间隔, 如十年代 (图 1) 或1秒. 通过使用生理学软件中的波形分析选项或将数据导出到电子表格中计算方法来计算平均值选定的时间间隔。</li>
	<li>为了在不同的动物之间正常化, 表达价值作为进一步分析的百分比变化从基线。使用参数统计进行分组比较。</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+sympathetic+nerve+activity+chronically+in+rats:+Surgery+techniques,+assessment+of+nerve+activity,+and+quantification.">Recording sympathetic nerve activity chronically in rats: Surgery techniques, assessment of nerve activity, and quantification.</a> American Journal of Physiology-Heart and Circulatory Physiology. 305 (10), H1407-H1416 (2013).</li><li>Miki, K., Kosho, A., Hayashida, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+continuous+measurements+of+renal+sympathetic+nerve+activity+and+cardiovascular+function+during+exercise+in+rats.">Method for continuous measurements of renal sympathetic nerve activity and cardiovascular function during exercise in rats.</a> Experimental Physiology. 87 (1), 33-39 (2002).</li><li>Huetteman, D. A., Bogie, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+blood+pressure+monitoring+in+laboratory+rodents+via+implantable+radio+telemetry.">Direct blood pressure monitoring in laboratory rodents via implantable radio telemetry.</a> Methods in Molecular Biology. 573, 57-73 (2009).</li><li>Jespersen, B., Knupp, L., Northcott, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoral+arterial+and+venous+catheterization+for+blood+sampling,+drug+administration+and+conscious+blood+pressure+and+heart+rate+measurements.">Femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements.</a> Journal of Visualized Experiments. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Afferent+renal+denervation+impairs+baroreflex+control+of+efferent+renal+sympathetic+nerve+activity.">Afferent renal denervation impairs baroreflex control of efferent renal sympathetic nerve activity.</a> American Journal of Physiology, Regulatory, Integrative and Comparative Physiology. 295 (6), R1882-R1890 (2008).</li><li>Guild, S. 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=Quantifying+sympathetic+nerve+activity:+problems,+pitfalls+and+the+need+for+standardization.">Quantifying sympathetic nerve activity: problems, pitfalls and the need for standardization.</a> Experimental Physiology. 95 (1), 41-50 (2010).</li><li>Stocker, S. D., Hunwick, K. J., Toney, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+paraventricular+nucleus+differentially+supports+lumbar+and+renal+sympathetic+outflow+in+water-deprived+rats.">Hypothalamic paraventricular nucleus differentially supports lumbar and renal sympathetic outflow in water-deprived rats.</a> Journal of Physiology. 15 (563 Pt 1), 249-263 (2005).</li><li>Stocker, S. D., Gordon, K. W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+receptors+in+the+hypothalamic+paraventricular+nucleus+contribute+to+insulin-induced+sympathoexcitation.">Glutamate receptors in the hypothalamic paraventricular nucleus contribute to insulin-induced sympathoexcitation.</a> Neurophysiology. 113 (5), 1302-1309 (2015).</li><li>Saponjic, J., Radulovacki, M., Carley, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+glutamate+into+the+pedunculopontine+tegmental+nuclei+of+anesthetized+rat+causes+respiratory+dysrhythmia+and+alters+EEG+and+EMG+power.">Injection of glutamate into the pedunculopontine tegmental nuclei of anesthetized rat causes respiratory dysrhythmia and alters EEG and EMG power.</a> Sleep and Breathing. 9 (2), 82-91 (2005).</li><li>DiBona, G. F., Jones, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+renal+nerve+activity+responses+to+baroreceptor+denervation+in+hypertensive+rats.">Dynamic analysis of renal nerve activity responses to baroreceptor denervation in hypertensive rats.</a> Hypertension. 37 (4), 1153-1163 (2001).</li><li>Kunitake, T., Kannan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discharge+pattern+of+renal+sympathetic+nerve+activity+in+the+conscious+rat:+spectral+analysis+of+integrated+activity.">Discharge pattern of renal sympathetic nerve activity in the conscious rat: spectral analysis of integrated activity.</a> Journal of Neurophysiology. 84 (6), 2859-2867 (2000).</li><li>Machado, B. H., Bonagamba, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+L-glutamate+into+the+nucleus+tractus+solitarii+increases+arterial+pressure+in+conscious+rats.">Microinjection of L-glutamate into the nucleus tractus solitarii increases arterial pressure in conscious rats.</a> Brain Research. 576 (1), 131-138 (1992).</li><li>Murakami, 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=Inhalation+anesthesia+is+preferable+for+recording+rat+cardiac+function+using+an+electrocardiogram.">Inhalation anesthesia is preferable for recording rat cardiac function using an electrocardiogram.</a> Biological and Pharmaceutical Bulletin. 37 (5), 834-839 (2014).</li><li>Nakamura, T., Tanida, M., Niijima, A., Hibino, H., Shen, J., Nagai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+stimulation+affects+renal+sympathetic+nerve+activity+and+blood+pressure+in+rats.">Auditory stimulation affects renal sympathetic nerve activity and blood pressure in rats.</a> Neuroscience Letters. 416 (2), 107-112 (2007).</li><li>Bardgett, M. E., McCarthy, J. J., Stocker, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamatergic+receptor+activation+in+the+rostral+ventrolateral+medulla+mediates+the+sympathoexcitatory+response+to+hyperinsulinemia.">Glutamatergic receptor activation in the rostral ventrolateral medulla mediates the sympathoexcitatory response to hyperinsulinemia.</a> Hypertension. 55 (2), 284-290 (2010).</li><li>Brozoski, D. T., Dean, C., Hopp, F. A., Seagard, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+blockade+of+endocannabinoids+in+the+NTS+modulates+baroreflex-evoked+sympathoinhibition.">Uptake blockade of endocannabinoids in the NTS modulates baroreflex-evoked sympathoinhibition.</a> Brain Research. 1059 (2), 197-202 (2005).</li><li>Barman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+can+we+learn+about+neural+control+of+the+cardiovascular+system+by+studying+rhythms+in+sympathetic+nerve+activity?.">What can we learn about neural control of the cardiovascular system by studying rhythms in sympathetic nerve activity?.</a> International Journal of Psychophysiology. 103, 69-78 (2016).</li><li>Charkoudian, N., Wallin, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sympathetic+neural+activity+to+the+cardiovascular+system:+integrator+of+systemic+physiology+and+interindividual+characteristics.">Sympathetic neural activity to the cardiovascular system: integrator of systemic physiology and interindividual characteristics.</a> Comprehensive Physiology. 4 (2), 825-850 (2014).</li><li>Malpas, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sympathetic+nervous+system+overactivity+and+its+role+in+the+development+of+cardiovascular+disease.">Sympathetic nervous system overactivity and its role in the development of cardiovascular disease.</a> Physiological Reviews. 90 (2), 513-557 (2010).</li><li>Chen, W. W., Xiong, X. Q., Chen, Q., Li, Y. H., Kang, Y. M., Zhu, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+sympathetic+afferent+reflex+and+its+implications+for+sympathetic+activation+in+chronic+heart+failure+and+hypertension.">Cardiac sympathetic afferent reflex and its implications for sympathetic activation in chronic heart failure and hypertension.</a> Acta Physiologica. 213 (4), 778-794 (2015).</li><li>Linz, 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=Modulation+of+renal+sympathetic+innervation:+Recent+insights+beyond+blood+pressure+control.">Modulation of renal sympathetic innervation: Recent insights beyond blood pressure control.</a> Clinical Autonomic Research. , (2018).</li></ol>

安妮. 芬克是国际数据科学客户咨询委员会的成员。

测量北美放射学会的关键步骤包括: (1) 在将肾脏与椎旁肌分离时避免肾动脉和神经的伸展, 以及将神经段放在记录电极上时 (2) 仔细解剖肾神经纤维从周围组织/血管, (3) 确保电极线没有组织, 血液, 或淋巴液, (4) 防止神经干燥, 通过应用矿物油的肾神经和硅胶的神经电极单位。为了进行故障排除, 必须确保记录系统充分接地。为了获得清晰的北美放射学会信号, 在嵌入硅胶之前, 可以在可视化和侦听?...

关于心血管系统自主控制的临床前发现告知管理疾病的策略, 如高血压、心力衰竭和慢性肾脏疾病。交感神经系统的过度活动和减少的迷走神经心脏口气贡献高血压 (BP)1。慢性升高的肾交感神经流出增强儿茶酚胺分泌, 减少肾血流量, 对心血管/肾脏系统2,3有害后果。为了定义导致自主功能障碍的神经生物学途径, 啮齿类动物的研究对于确定中枢神经系统 (CNS) 神经元调节交感神经参数是很重要的。本议定书的目的是提供有关测量肾交感神经活动 ( 北美放射学会 ) 和 BP 的技术信息 , 并概述在麻醉大鼠中枢神经系统操作中对急感神经变化进行量化的方法。
急性 (非生存) 北美放射学会测量 (持久极小到 h) 是有用的, 当科学家将探测中枢神经系统药理, 电, 或 optogenetically 麻醉大鼠, 以确定特定核的功能。利用这些方法, 研究了孤立核、periaqueductal 灰、pedunculopontine tegmentum 和延髓延髓髓质等结构, 确定了调节交感神经参数4的神经生物学通路, 5,6,7。这一方法对于确定在慢性自主功能障碍8、9的慢性病模型中进一步研究中枢神经系统目标是很重要的。为了完成这些实验, 实验室需要一个焊接铁, 外科显微镜, 立体定向框架, 微电极放大器, 和音频显示器。根据实验室中存在的影响电子噪音的因素, 手术/录音区域可能需要法拉第笼/接地带, 以减少北美放射学会记录中的电噪声。如果大脑分析需要组织固定, 需要一个灌注泵和通风罩。数据可以用多种生理学软件/数据采集 (模拟数字转换器) 单元4、5进行数字化和记录, 并采用不同的分析方法和兼容性, 用于合并遥测信号。.

<table>
	<tbody>
		<tr>
			<td>Stainless steel wire</td>
			<td>A-M Systems; Sequim, WA</td>
			<td>791000</td>
			<td>RSNA electrode</td>
		</tr>
		<tr>
			<td>Polyethylene (PE-50) tubing</td>
			<td>VWR; Radnor, PA</td>
			<td>63019-048</td>
			<td>RSNA electrode; vessel cannulation</td>
		</tr>
		<tr>
			<td>Miniature pin connector</td>
			<td>A-M Systems; Sequim, WA</td>
			<td>520200</td>
			<td>RSNA electrode</td>
		</tr>
		<tr>
			<td>Crimping tool</td>
			<td>Daniels Manufacturing Corp.; Orlando, FL</td>
			<td>M22520</td>
			<td>RSNA electrode</td>
		</tr>
		<tr>
			<td>Connector strip</td>
			<td>Amphenol; Clinton Township, MI</td>
			<td>221-2653</td>
			<td>RSNA electrode</td>
		</tr>
		<tr>
			<td>J-B Kwik Epoxy</td>
			<td>J-B Weld, Sulphur Springs, TX</td>
			<td>8270</td>
			<td>RSNA electrode</td>
		</tr>
		<tr>
			<td>Silicone</td>
			<td>Permatex; Hartford, CT</td>
			<td>2222</td>
			<td>RSNA electrode</td>
		</tr>
		<tr>
			<td>Heparin sodium; Injectable (10 mL vial, 1000 U/mL)</td>
			<td>KV Veterinary Supply; David City, NE</td>
			<td>P03466</td>
			<td>Venous line patency</td>
		</tr>
		<tr>
			<td>Phenylephrine HCl; Injectable (1 mL vial; 10 mg/mL)</td>
			<td>ACE Surgical Supply; Brockton, MA</td>
			<td>950-6312</td>
			<td>Testing renal sympathoinhibition</td>
		</tr>
		<tr>
			<td>Single-hook elastic surgical stays</td>
			<td>Harvard Apparatus; Holliston, MA</td>
			<td>72-2595</td>
			<td>Incision</td>
		</tr>
		<tr>
			<td>Silk surgical tape</td>
			<td>3M, Minneapolis, MN</td>
			<td>1538-0</td>
			<td>Secure surgical stays</td>
		</tr>
		<tr>
			<td>Needles, 20 G</td>
			<td>Sigma-Aldrich; St. Louis, MO</td>
			<td>Z192554-100EA</td>
			<td>Vessel cannulation</td>
		</tr>
		<tr>
			<td>Dumont #7 curved forceps</td>
			<td>Fine Science Tools; Foster City, CA</td>
			<td>11274-20</td>
			<td>Vessel cannulation</td>
		</tr>
		<tr>
			<td>5-0 silk suture ties</td>
			<td>Braintree Scientific; Braintree, MA</td>
			<td>SUT-S 106</td>
			<td>Vessel cannulation</td>
		</tr>
		<tr>
			<td>Delicate hemostatic forceps</td>
			<td>Roboz Surgical Instrument Co.; Gaithersburg, MD</td>
			<td>RS-7117</td>
			<td>Vessel cannulation and RSNA surgery</td>
		</tr>
		<tr>
			<td>Crile Hemostatic forceps</td>
			<td>Fine Science Tools; Foster City, CA</td>
			<td>13004-14</td>
			<td>Needle bending</td>
		</tr>
		<tr>
			<td>Telemetry transmitter</td>
			<td>Data Sciences International; Minneapolis, MN</td>
			<td>PA-10</td>
			<td>Mean arterial pressure monitoring (telemetry)</td>
		</tr>
		<tr>
			<td>Re-gel syringe</td>
			<td>Data Sciences International; Minneapolis, MN</td>
			<td>276-0038-001</td>
			<td>Transmitter reuse (telemetry)</td>
		</tr>
		<tr>
			<td>Disposable pressure transducer</td>
			<td>Transpac; San Clemente, CA</td>
			<td>MI-1224</td>
			<td>Mean arterial pressure monitoring</td>
		</tr>
		<tr>
			<td>Clear-Cuff pressure infuser</td>
			<td>MILA International Inc.; Florence, KY</td>
			<td>2281339</td>
			<td>Mean arterial pressure monitoring</td>
		</tr>
		<tr>
			<td>Vessel cannulation forceps</td>
			<td>Fine Science Tools; Foster City, CA</td>
			<td>00574-11</td>
			<td>Catheter insertion</td>
		</tr>
		<tr>
			<td>Black monofilament nylon 4-0 suture on reverse cutting needle</td>
			<td>McKesson Medical-Surgical; San Francisco, CA</td>
			<td>S661GX</td>
			<td>Secure telemetry transmitter</td>
		</tr>
		<tr>
			<td>Telemetry receiver</td>
			<td>Data Sciences International; Minneapolis, MN</td>
			<td>RPC-1</td>
			<td>Mean arterial pressure monitoring (telemetry)</td>
		</tr>
		<tr>
			<td>LabChart Pro (software), PowerLab (acquisition hardware)</td>
			<td>AD Instruments; Colorado Springs, CO</td>
			<td>ML846, MX2 matrix 2.0 (Compatible with Data Science International telemetry)</td>
			<td>3 options for software/acquisition hardware</td>
		</tr>
		<tr>
			<td>SciWorks (software), DataWave (acquisition hardware)</td>
			<td>DataWave Technologies, Loveland, CO</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Spike 2 (software), Micro1401-3</td>
			<td>Cambridge Electronic Design Ltd., London UK</td>
			<td>1401-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-drill</td>
			<td>Roboz Surgical Instrument Co.; Gaithersburg, MD</td>
			<td>RS-6300</td>
			<td>CNS surgery</td>
		</tr>
		<tr>
			<td>Stereotaxic surgery frame</td>
			<td>Stoelting; Wood Dale, IL</td>
			<td>51600</td>
			<td>CNS surgery</td>
		</tr>
		<tr>
			<td>Microelectrode amplifier with 10X pre-amplifier</td>
			<td>A-M Systems; Sequim, WA</td>
			<td>1800-2</td>
			<td>RSNA recording</td>
		</tr>
		<tr>
			<td>Retractors</td>
			<td>Fine Science Tools; Foster City, CA</td>
			<td>17009-07</td>
			<td>RSNA surgery</td>
		</tr>
		<tr>
			<td>Micro-dissecting tweezers</td>
			<td>Fine Science Tools; Foster City, CA</td>
			<td>11251-10</td>
			<td>RSNA surgery</td>
		</tr>
		<tr>
			<td>Micro-hook</td>
			<td>Fine Science Tools; Foster City, CA</td>
			<td>10064-14</td>
			<td>RSNA surgery</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Fisher Scientific; Waltham, MA</td>
			<td>8042-47-5</td>
			<td>RSNA surgery</td>
		</tr>
		<tr>
			<td>Audio monitor</td>
			<td>A-M Systems; Sequim, WA</td>
			<td>3300</td>
			<td>RSNA surgery</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Wacker, Munchen; Germany</td>
			<td>RT601A-B</td>
			<td>RSNA surgery</td>
		</tr>
		<tr>
			<td>Electrical clips</td>
			<td>Tyco Electronics; Schaffhausen, Switzerland</td>
			<td>EB0283-000</td>
			<td>Grounding or securing perfusion needle</td>
		</tr>
		<tr>
			<td>Bonn scissors, straight/sharp points</td>
			<td>Roboz Surgical Instrument Co; Gaithersburg, MD</td>
			<td>RS-5840</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>Gavage needle</td>
			<td>Harvard Apparatus; Holliston, MA</td>
			<td>75-0286</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>Masterflex perfusion pump</td>
			<td>Cole-Parmer; Vernon Hills, IL</td>
			<td>7524-10</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>Masterflex platinum-cured silicone tubing</td>
			<td>Cole-Parmer; Vernon Hills, IL</td>
			<td>96410-15</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>Formalin (10% buffered solution; 4 L)</td>
			<td>Sigma-Aldrich; St. Louis, MO</td>
			<td>HT501128</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich; St. Louis, MO</td>
			<td>S0389</td>
			<td>Cryoprotection</td>
		</tr>
	</tbody>
</table>

quantifying acute changes in renal sympathetic nerve activity in response to central nervous system manipulations in anesthetized rats

肾交感神经活动 (北美放射学会) 和平均动脉压是心血管和自主研究的重要参数;然而, 在测量和分析这些变量的技术上, 有有限的资源指导科学家。本协议描述了麻醉大鼠北美放射学会和平均动脉压力的测量方法。该议定书还包括在中枢神经系统 (CNS) 操作北美放射学会录音期间访问大脑的方法。北美放射学会记录技术与中枢神经系统的药理、optogenetic 或电刺激兼容。当研究人员在非生存实验中测量短期 (最小到 h) 自主性反应时, 这种方法是有用的, 以解剖与中枢神经系统核相关。该方法不打算用于获得北美放射学会在大鼠的慢性 (生存) 记录。北美放射学会的放电, 平均矫正北美放射学会, 平均动脉压力可以量化和分析进一步使用参数统计试验。本文还介绍了获得静脉通路、记录平均动脉压 telemetrically 和脑内固定的方法。

图 1显示了一个戊巴比妥麻醉鼠的样本北美放射学会和 BP 记录。静脉注射肾上腺素被用来诱发平均动脉压力的增加, 并唤起反射和瞬态 sympathoinhibition4,6。为量化北美放射学会, 对未重叠的十年代段的原始北美放射学会进行了矫正和平均;从每个段中减去噪声估计值。
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Watch this Scientific Journal Video about Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats at JoVE.com

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

麻醉大鼠中枢神经系统操作对肾交感神经活动的急性变化量化

测量中枢神经系统 (CNS) 的交感神经和心血管反应的方法对于推进神经科学是很重要的。该协议的制定, 以帮助科学家测量和量化的急性肾脏交感神经活动 (北美放射学会) 在麻醉大鼠 (不存活) 的变化。

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are ...

quantifying-acute-changes-renal-sympathetic-nerve-activity-response

Research

JoVE Journal

Neuroscience

7.7K 次观看.  University of Illinois at Chicago. 测量中枢神经系统 (CNS) 的交感神经和心血管反应的方法对于推进神经科学是很重要的。该协议的制定, 以帮助科学家测量和量化的急性肾脏交感神经活动 (北美放射学会) 在麻醉大鼠 (不存活) 的变化。

视频: Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

视神经横断:一个成人在中枢神经系统的神经元凋亡模型

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

科研

神经科学

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

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 ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

调查中枢神经系统修复中受伤范式<em&gt;果蝇</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

在中枢神经系统Translatome由多核糖体分馏体内拷问

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

急性<em&gt;体内</em&gt;麻醉大鼠超导途径局部场电位和多单位活动的电生理记录

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

第三星果蝇中枢神经系统活动的电生理记录

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

麻醉大鼠中枢神经系统操作对肾交感神经活动的急性变化量化

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