这项工作得到了美国国立卫生研究院/ NIDDK（DK077106到ECM）。

1。术前准备<ol><li>处理。动物（最常用的是，大鼠或小鼠，虽然主要是使用任何所需的特定物种适应需要一般1微透析的方法，和组合与行为测试， 例如用于麻醉）至少为10分钟/天的最小处理手术的前两天。广泛的处理已被证明在测试的时候，让动物在无应力状态，以避免可能的混淆2,4,5。我们将用大鼠在本协议作为一个例子物种。处理必须做到无应力引起的拉毛皮， 例如从乳胶手套或丁腈手套：这可以最好的实现用裸露的双手，在设施的兽医和雇员的卫生官员的协商。如果这是不可能的，柔软的棉手套应戴过垒手套，以避免拉扯对老鼠的皮毛。 </li><li>无菌现场准备工作。之前开始手术，surgic的的制备人区域，周围的立体定位的装置和对整个装置下无菌被单制备无菌字段，覆盖的加热垫，用来维持大鼠体温。提供准确的恒温器的循环温水垫使用的动物，以防止过热。 </li><li>麻醉诱导。异氟醚蒸发器检查包含异氟醚的最佳水平，并连接到感应室。连接进行检查，以确认一个闭环。空气处理真空系统进行检查，操作。异氟醚汽化接通和动物放入感应室使用5％的异氟醚的氧组合：即加入5％异氟烷成100％的氧气流，并传送到感应室。 </li><li>放置到设备。动物固定在立体定位仪使用earbars和齿杆。的正确earbar下配售结果在一个不动的头和平整的沿着earbars的耳朵。动物正在迅速固定和鼻子插入一个专门设计的麻醉鼻锥;交付汽化的异氟醚是从感应室切换的nosecone和调整，以提供2-3％的异氟烷​​入氧气流去的nosecone麻醉组合。 </li><li>麻醉确认。一种外科手术的麻醉平面证实了将硬捏脚和一团空气的眼睛，也不应该造成任何回应。在整个手术大约每隔15分钟重复这些测试，以确认维修的外科麻醉平面。头发从之前的切口部位进入无菌区。 </li></ol> 2。手术<ol><li>动物初始治疗。眼软膏被施加到每个眼球，以防止干燥。 1毫升无菌生理盐水给出SC，以防止在手术过程中的任何脱水，并用无菌的悬垂体覆盖。聚维酮碘涂在头皮上，并从中心擦洗出，用棉签，70％乙醇擦洗同样，和两个拭子步骤重复两次以上，以确保适当的切口部位。聚乙烯吡咯酮碘和酒精的皮肤接触的时间应该是至少3分钟前切口。维持麻醉，并定期检查整个手术;卡布洛芬5克/公斤）的一个单一的皮下注射给发起的镇痛作用，并使用无菌技术。镇痛麻醉诱导后，以尽量减少任何压力注射。 </li><li>切口和颅骨准备。 A 3-4厘米处剪下了得sagitally中心的头骨。 1:1组合的布比卡因肾上腺素局部应用提供进一步的镇痛，减少出血。头皮是从切口处采用手术的剪辑和无菌棉签是用来清除覆盖膜，从头骨。用于钻井的坐标测量使用囟作为一个参考点，标志着使用无菌的钻头（或，可替换地，一个手持烧灼德维CE），并重新确认的准确性钻前开始。使用脑图谱确定为特定的大脑区域的利益（ 如海马）的坐标。海马微透析，我们使用了演练现场5.6毫米后的前囟门，+5.0横向，和3.0腹侧硬膜。 </li><li>钻井。通过颅骨钻三个孔，关心的问题是只适用于很小的力，创伤硬脑膜及相关膜是很少或没有。在测量现场的套管插入一个孔，另外两个被定位为方便插入颅骨螺钉。目的尺寸的螺钉（ 例如 1.17毫米自攻螺钉;精细科学工具）插入这些孔而不影响下面大脑，并使用作为随后的牙科用粘固剂应用程序的锚点。 </li><li>套管的位置和关闭。被定位在所述套管插入坐标，这是重新确认的部位的孔钻出，然后慢慢地降低到目标深度。一旦正确地定位，套管代替用牙科用粘固剂固定。如果需要，一个单一的无菌手术用缝合线用于闭合伤口。钢丝插入的导管保持通畅。在这个协议中，我们使用一个CMA12（CMA /微透析）探针和导管的。 </li><li>急性手术后的护理。 3毫升无菌生理盐水SC继续水化，一个单一的皮下注射卡洛芬5克/公斤）也启动镇痛。动物从设备中删除，并放置在加热恢复室，在一个干净的笼子里，和监控，直到它们完全从麻醉中恢复。评估完全恢复恢复翻正反射和正常的运动。动物，然后返回自己的家乡笼，并定期举行房间。 </li><li>短期后续治疗。动过手术的动物的笼子都标有日期的手术。动物每天至少监测一次，至少THREE天手术后，以下两个天手术后，每一个卡洛芬咀嚼​​片（2毫克）的日子。如果动物不消耗卡洛芬，可注射的卡洛芬可能被以确保有足够的镇痛作用。监控的整体健康的动物和国家机构动物护理指导原则和寻求援助或从动物保健工作人员或兽医的意见，如果需要的话，伤口部位的感染，红肿等。更重要的是，要注意适当的处理和驯化的实验者是必不可少的：动物应该处理广泛，包括操作的套管，直到没有任何迹象仍然是当被实验者处理时的紧张或压力。 </li><li>随后的动物护理和治疗。根据批准的协议，以及各自具体的实验组，的动物将按照适当的测试和安乐死程序。 </li></ol> 3。微透析（MD） <ol><li> Perfus吃的准备。人工细胞外液（AECF）153.5毫米的Na，K，4.3毫米0.41 mM的镁，钙0.71毫米，139.4毫米氯，葡萄糖1.25毫米，在pH值7.4 6缓冲。 注意 ，准确的流体成分是必不可少的：不准确或使用，生理体液，如林格氏液微透析PBS将导致明显错误的结果6。要特别注意，离子组成的细胞外液（ECF）是不一样的CSF，我们发现在2004年的详细研究海马ECF 6。在一天的测试，牛血清白蛋白（BSA）应在2％重量/重量加入，并完全溶解，这减少了损失的肽，如胰岛素从遵守油管，并且也降低了风险的流体损失（超滤）在探头的膜。准备完毕后，灌流液应通过一个0.2微米的过滤器过滤。 </li><li>如果是被传递到胸罩如胰岛素治疗在感兴趣的区域（这里的海马），准备使用的等分试样的制备AECF这种治疗与适当的药物浓度。胰岛素的浓度为400 nM（66.7μU个/μl）已被证明影响通过反向微透析交付海马代谢和认知功能7。请注意，将所得的组织浓度胰岛素不测量这里仍然不明。 </li><li>设置MD探头和线。准备一个新鲜的微透析探针和线测试的前一天。创建两条线“流入”和“流出”。使用PE50管材连接两个1米长的的FEP管件，并确保有最小的死线之间的空间。连接的流入管连接到一个1毫升的Hamilton注射器充满无菌过滤，用去离子水（dH的2 0 2 0），然后将附加到探针。 </li><li>微透析旋转。为了让免费动物的运动进行测量时，连接液体旋转的inflow和流出线，关闭泵，使用额外的FEP管。记得要考虑这个旋转和管内部容积在考虑采样时间（见下文）。 </li><li> MD泵。打开MD泵和1.5μl/ min的运行，直到您看到DH 2 0退出流出管探头上。然后，当泵关闭时，流出管和一个样品收集管之间的连接的其他行。运行5毫升，通过该管，并把探头于小瓶中含有无菌蒸馏水2 0过夜;确保探针针尖始终保持湿润。 </li><li>预探测测试对象。在测试前24小时，从大鼠的头移除虚设钢丝，并插入一个：毫达西探针（仅用于此目的，而不是为采样）10分钟。然后替换虚拟钢丝和到它的笼子里，把老鼠。此过程是为了尽量减少任何影响胶质细胞增生测试8日，在我们的手中，这提供了良好的resulTS其他技术相匹配的数据，似乎反映了海马激活2,5,7,9;他人已经使用了类似的方法，离开探头在24小时之前测量，这也是一个很好的方法，如果损坏可避免探针过夜。 </li><li>探头平衡。在当天的微透析技术，填补了汉密尔顿注射器和过滤AECF闪烁小瓶和泵通过平衡1小时。如果需要的话，填充第二个Hamilton注射器与制备的治疗（例如，胰岛素AECF）和放入注射器泵。 </li><li>探针插入。平衡是完成时，删除虚拟钢丝和通过导管轻轻插入的平衡MD探测到老鼠的大脑中。将大鼠成一个透明的塑料盒，其中包含一些老鼠的家居床上用品。 ，以抗衡确保管：连接一个1.6毫升的离心管中装满水的管笼子外面在这样一种方式，重力保持microdi“辩线unkinked，但不紧。让探针平衡2小时大鼠。在这一时期开始，确认，灌流液的流动是畅通的，这是最容易做到在规定的时间内，通过收集灌流液流出，称量样品，以确认预期的体积退出系统。任何产量预期量的90％，和不正确的拆卸和重新插入后，应更换探头（否则在探针水肿会导致）。如果流量持续低或没有，检查各连接处的渗漏，做不到这一点，断开管道的阶段，看是否可以隔离堵塞。如果没有发现问题，更换探头，如果这个问题不解决的话，可能需要重新开始新的探头和线第3.3节。在两小时的平衡期后允许探头周围的重新密封，避免探针插入的急性影响任何的血 - 脑屏障。 </li><li>采集样本。确保合作diesel。关联关系的样品透析（大脑）和收集（管），精确计算。比如，使用2米转体和探头之间的FEP管，有30微升的总体积之间的探头和收集，因此它需要20分钟，在1.5微升/分钟的流速样本通过探头，油管，连接器和旋转的管道收集到了最后。收集样品时确保你收集到足够的量有您的分析所必需的浓度。在这里，我们将使用5分钟取样桶，从而在每个收集管收集的透析液7.5μL。 </li><li>基线样本。一旦平衡后，开始收集样品。收集至少三个样品，而大鼠家庭室中处于静止状态时，建立了稳定的基线测量</li><li>治疗时机。小心计算所需的时间开始治疗之前的实验：请记住，只是有一个时间滞后之间的透析和样品收集，离开注射器和到达动物的海马的灌流液之间有一个滞后（通常是相同​​的）。因此，在我们30μl的注射器和探头之间的音量设置，改变注射器含治疗灌流前20分钟你想治疗开始到达的海马。 </li><li>改变注射器。如果需要的话，可以使用一种液体开关，但不是必需的：在适当的时间，只需断开控制灌流液的注射器的流入线路从和快速连接到治疗灌流液的注射器。这应该不超过5秒，以避免重大中断的灌注流量。 注意 ，此过程所需剂量已交付后，如果提供治疗所需的时间有限，应予以冲回。可选地，处理可以继续采样的持续时间（参见图2）。不应引入进线切换时的气泡注射器，因为它们可能会积聚在透析膜和降低探头效率。 </li><li>行为测试。如果执行行为的任务，按照该程序（见下文第4节）。 注意协调提供治疗的海马是很重要的。例如，胰岛素交付时间应发生前10分钟开始测试10。因此，含胰岛素灌注的开关应该发生的行为测试前30分钟（20分钟灌流通过线加上10分钟的测试所需的交付时间提前）。 </li><li>完成样品采集。收集所需的样品后，轻轻取出，将探针从动物的头，再次记住透析和样品采集到之间的时间间隔。动物的饲养笼，并密切观察任何实验后的健康或行为的变化，直到动物被杀害，并且在大脑中删除，确认无误探头的位置。如果牺牲不会立即发生，返回假心针的套管，以避免任何引进外资的材料。 </li><li>分析。的分析的方法会有所不同，取决于感兴趣的分析物（s）的。由于透析取样不允许进行完整的分析在探头膜的平衡，样品浓度应予以纠正利用零净通量法11,12给ECF浓度的。 </li></ol> 4。行为测试<ol><li>放置在迷宫中。在基线样本（ 即至少三个样本后，已经完成了透析，但仍可能在的流出管收集），轻轻移动老鼠到行为测试设备。可以使用任何合适的任务，在图1中的数据，收集使用的四臂加形的迷宫和测量自发交替，它是衡量空间工作记忆：大鼠最初放置在中心，后的迷宫，并允许自由探索9,13。由于可以使用任何行为测试，此协议的焦点是不作为这里的一个例子（其他部分详述9,14,15），但简单的具体任务，动物被允许探索迷宫（期间最近被浏览的灰色方块图1），使用hippocampally依赖过程保留这些武器的内存。 </li><li>透析管在测试过程中的运动。握住管子，使得它可自由移动，既不影响大鼠的运动也被允许在前面的动物移动和分散。留在原地，并尽量减少自己的动作，这样你就不会影响老鼠的行为。 </li><li>续样品的采集。在基线期间，流出管移动到新的收集管中，每5分钟。 </li><li>交替测试。让动物自由地探索迷宫20分钟。记录的顺序和时间的手臂条目，或者使用视频记录或由专人为以后的工作性能分析9,15。 </li></ol> 5。测试后<ol><li>最后的样本集合。以下测试，取出老鼠轻轻地从迷宫和返回控制腔。继续收集微透析样品至少有四个样本，涵盖的时间执行任务的复苏。 </li><li>探针清除。所有样品已收集后，轻轻地取下探头从动物的头部和地点到存储瓶，返回它的首页笼的动物。 </li><li>探针存储。回国后，动物的饲养笼和完成任何剩余透析液的收集，彻底清洗电极，用dH 2 0和存储在闪烁瓶用dH 2 0填充和封口膜覆盖。冲洗管，通过切换到一个注射器中含有的溶液为1:10,000的Kathon dH的2 0，以防止微生物的生长。探头可以重新使用，只要它们保持良好的流动性，并具有无损伤膜护理这应定期在超过10用途。 </li><li>组织学研究。杀死的动物和消除大脑。片上低温恒温器，并使用标准的组织学技术（ 如甲酚紫染色），以确认正确的探头的位置。 </li></ol>

<ol>
	<li>McNay, E. C., Fries, T. M., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreases+in+rat+extracellular+hippocampal+glucose+concentration+associated+with+cognitive+demand+during+a+spatial+task.">Decreases in rat extracellular hippocampal glucose concentration associated with cognitive demand during a spatial task.</a> Proc. Natl. Acad. Sci. U.S.A. 97 (6), 2881 (2000).</li><li>McNay, E. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+differences+in+hippocampal+extracellular+fluid+glucose+concentration+during+behavioral+testing+and+following+systemic+glucose+administration.">Age-related differences in hippocampal extracellular fluid glucose concentration during behavioral testing and following systemic glucose administration.</a> J. Gerontol. A. Biol. Sci. Med. Sci. 56 (2), 66 (2001).</li><li>McNay, E. C., McCarty, R. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+in+brain+glucose+concentration+during+behavioral+testing:+dissociations+between+brain+areas+and+between+brain+and+blood.">Fluctuations in brain glucose concentration during behavioral testing: dissociations between brain areas and between brain and blood.</a> Neurobiol. Learn. Mem. 75 (3), 325 (2001).</li><li>McNay, E. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+glucose+concentrations+in+the+rat+hippocampus+measured+by+zero-net-flux:+effects+of+microdialysis+flow+rate.">Extracellular glucose concentrations in the rat hippocampus measured by zero-net-flux: effects of microdialysis flow rate.</a> 72 (2), 785 (1999).</li><li>McNay, E. C., Sherwin, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+recurrent+hypoglycemia+on+spatial+cognition+and+cognitive+metabolism+in+normal+and+diabetic+rats.">Effect of recurrent hypoglycemia on spatial cognition and cognitive metabolism in normal and diabetic rats.</a> Diabetes. 53 (2), 418 (2004).</li><li>McNay, E. 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=Hippocampal+memory+processes+are+modulated+by+insulin+and+high-fat-induced+insulin+resistance.">Hippocampal memory processes are modulated by insulin and high-fat-induced insulin resistance.</a> Neurobiology of Learning and Memory. 93 (4), 546 (2010).</li><li>McNay, E. C., McCarty, R. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+in+brain+glucose+concentration+during+behavioral+testing:+dissociations+between+brain+areas+and+between+brain+and+blood.">Fluctuations in brain glucose concentration during behavioral testing: dissociations between brain areas and between brain and blood.</a> Neurobiology of Learning &amp; Memory. 75 (3), 325 (2001).</li><li>McNay, E. C., Williamson, A., McCrimmon, R. J., Sherwin, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+and+neural+hippocampal+effects+of+long-term+moderate+recurrent+hypoglycemia.">Cognitive and neural hippocampal effects of long-term moderate recurrent hypoglycemia.</a> Diabetes. 55 (4), 1088 (2006).</li><li>McNay, E. C., Sherwin, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+artificial+cerebro-spinal+fluid+(aCSF)+to+artificial+extracellular+fluid+(aECF):+microdialysis+perfusate+composition+effects+on+in+vivo+brain+ECF+glucose+measurements.">From artificial cerebro-spinal fluid (aCSF) to artificial extracellular fluid (aECF): microdialysis perfusate composition effects on in vivo brain ECF glucose measurements.</a> Journal of Neuroscience Methods. 132 (1), 35 (2004).</li><li>McNay, E. 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=Hippocampal+memory+processes+are+modulated+by+insulin+and+high-fat-induced+insulin+resistance.">Hippocampal memory processes are modulated by insulin and high-fat-induced insulin resistance.</a> Neurobiology of Learning and Memory. 93 (4), 546 (2010).</li><li>Benveniste, H., Drejer, J., Schousboe, A., Diemer, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cerebral+glucose+phosphorylation+and+blood+flow+after+insertion+of+a+microdialysis+fiber+through+the+dorsal+hippocampus+in+the+rat.">Regional cerebral glucose phosphorylation and blood flow after insertion of a microdialysis fiber through the dorsal hippocampus in the rat.</a> Journal of Neurochemistry. 49 (3), 729 (1987).</li><li>Benveniste, H., Diemer, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+reactions+to+implantation+of+a+microdialysis+tube+in+the+rat+hippocampus.">Cellular reactions to implantation of a microdialysis tube in the rat hippocampus.</a> Acta Neuropathologica. 74 (3), 234 (1987).</li><li>McNay, E. C., Fries, T. M., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreases+in+rat+extracellular+hippocampal+glucose+concentration+associated+with+cognitive+demand+during+a+spatial+task.">Decreases in rat extracellular hippocampal glucose concentration associated with cognitive demand during a spatial task.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (6), 2881 (2000).</li><li>McNay, E. 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=Hippocampal+memory+processes+are+modulated+by+insulin+and+high-fat-induced+insulin+resistance.">Hippocampal memory processes are modulated by insulin and high-fat-induced insulin resistance.</a> Neurobiol. Learn Mem. 93 (4), 546 (2010).</li><li>Lonnroth, P., Jansson, P. -. A., Smith, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microdialysis+method+allowing+characterization+of+intercellular+water+space+in+humans.">A microdialysis method allowing characterization of intercellular water space in humans.</a> American Journal of Physiology. 1987, E228 (1987).</li><li>McNay, E. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+glucose+concentrations+in+the+rat+hippocampus+measured+by+zero-net-flux:+effects+of+microdialysis+flow+rate.">Extracellular glucose concentrations in the rat hippocampus measured by zero-net-flux: effects of microdialysis flow rate.</a> 72 (2), 785 (1999).</li><li>Lalonde, R. o. b. e. r. t. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurobiological+basis+of+spontaneous+alternation.">The neurobiological basis of spontaneous alternation.</a> Neuroscience &amp; Biobehavioral Reviews. 26 (1), 91 (2002).</li><li>Richman, C., Dember, W., Kim, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+alternation+behavior+in+animals:+A+review.">Spontaneous alternation behavior in animals: A review.</a> Current Psychology. 5 (4), 358 (1986).</li><li>McNay, E. C., Canal, C. E., Sherwin, R. S., Gold, P. E. <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+memory+with+septal+injections+of+morphine+and+glucose:+effects+on+extracellular+glucose+levels+in+the+hippocampus.">Modulation of memory with septal injections of morphine and glucose: effects on extracellular glucose levels in the hippocampus.</a> Physiol. Behav. 87 (2), 298 (2006).</li><li>McNay, E. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+modulation+across+neural+systems:+intra-amygdala+glucose+reverses+deficits+caused+by+intraseptal+morphine+on+a+spatial+task+but+not+on+an+aversive+task.">Memory modulation across neural systems: intra-amygdala glucose reverses deficits caused by intraseptal morphine on a spatial task but not on an aversive task.</a> Journal of Neuroscience. 18 (10), 3853 (1998).</li><li>Rex, A., Bert, B., Fink, H., Voigt, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus-dependent+changes+of+extracellular+glucose+in+the+rat+hippocampus+determined+by+in+vivo+microdialysis.">Stimulus-dependent changes of extracellular glucose in the rat hippocampus determined by in vivo microdialysis.</a> Physiol. Behav. 98 (4), 467 (2009).</li></ol>

没有利益冲突的声明。

微透析中使用的所有的解决方案应该被过滤在即将使用前，使用一个0.2微米的过滤器。后插入导管的探讨，观察，以确认流的畅通和样品采集。如果流插入后停止，最可能的原因是在探头的膜护理不足所造成的插入损坏，也必须以一个新的探头。 如上所述，这些方法的主要优点是缺乏的混淆，允许在清醒，自由活动动物行为和神经化学的措施：利用测试之前，广泛的处理是?...

<table border="1"><tbody><tr><td></td><td></td><td></td><td>大多数试剂的标准实验室级，并可以从供应商，给出以下方式获得。同样，如注射器泵和管道的设备，可以使用任何几个厂家。这里使用的细节是重要的具体项目包括： </td></tr><tr><td> CMA 12个微透析探针</td><td> CMA /微透析</td><td> CMA-12-XXX </td><td>这些都是在各种膜的长度和临界值，表示特定代码在“XX​​X”。 </td></tr><tr><td>人胰岛素（优泌林） </td><td>礼来公司</td><td> N / A </td><td></td></tr><tr><td>液体旋转</td><td> Instech </td><td> 375/D/22QM </td><td>此特定的旋转接头具有非常低扭矩和低的内部容积，以及一个nonrea莫如石英衬。 </td></tr></tbody></table>

hippocampal insulin microinjection and in vivo microdialysis during spatial memory testing

葡萄糖代谢是一个有用的标记为本地的神经活动，如2 - 脱氧葡萄糖和功能磁共振成像的方法形成的基础。然而，使用这样的方法在动物模型中需要麻醉，因此既改变了大脑状态并防止行为的措施。另一种方法是利用体内微透析，采取连续测量脑细胞外液浓度的葡萄糖，乳酸和相关的代谢物清醒，奔放的动物。这种技术是与任务相结合，依赖于特定的大脑区域和/或急性药理操作时特别有用，例如在空间工作记忆任务中（自发的交替）的测量表明，海马浸在细胞外葡萄糖和乳酸上升，暗示增强糖酵解1,2和海马胰岛素给药，提高记忆力和增加海马乙二醇ysis 3。 （如胰岛素）的物质，可以通过相同的微透析探针用于测量代谢物传递到海马。作为衡量的海马功能的自发交替使用，目的是为了避免任何混淆紧张的激励（ 例如电击），约束，或奖励（ 如食品），所有这一切都可以改变任务的性能和代谢的任务也提供了一个衡量非特异性治疗的效果，可以控制电机的活动。 ，这些方法可直接测量的神经化学和代谢调节行为的变量。

大鼠应迅速从手术中恢复，并提高警觉，麻醉停止后30分钟内能动的和积极的。插管帽应该是最小的影响，如果在手术进行清洁和最小的牙科用粘固剂使用。手术后的监测过程中发现任何感染迹象，或以任何方式的痛苦或不适的迹象，立即终止实验的老鼠，这应该是极为罕见的。在处理测试前，动物应警惕，可以自由活动的，友好和好奇的。基本上应该有良好的处理的动物从手术，手术后的恢复?...

Watch this Scientific Journal Video about Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing at JoVE.com

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

调制的hippocampally依赖空间工作记忆直接海马显微注射，其次是<em在体内</em&gt;微透析意识，表现动物的代谢产物。

海马胰岛素显微注射和<em在体内</em&gt;微透析过程中空间记忆测试

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

hippocampal-insulin-microinjection-vivo-microdialysis-during-spatial

Research

JoVE Journal

Neuroscience

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

16.4K 次观看.  University at Albany. 调制的hippocampally依赖空间工作记忆直接海马显微注射，其次是

视频: Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

科研

神经科学

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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

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

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

微透析的乙醇操作性乙醇的自我管理和乙醇的测定气相色谱

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

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 ...

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 ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

高分辨率<em&gt;在体内</em&gt;使用3T磁共振成像手动分割协议为人类海马子字段

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

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 ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

评估空间学习记忆小鳞类爬行动物

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

用于测试胰岛素模拟化合物的蛋模型

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...