海马胰岛素显微注射和<em在体内</em>微透析过程中空间记忆测试
Summary
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>微透析意识，表现动物的代谢产物。
Abstract
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。
Protocol
1。术前准备处理。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…
Representative Results
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?…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院/ NIDDK（DK077106到ECM）。
Materials
The majority of reagents are standard laboratory grade and can be obtained from a supplier of choice. Similarly, equipment such as syringe pumps and tubing can be used from any of several manufacturers. Specific items used here for which details are important include: |
|||
| CMA 12 microdialysis probes | CMA/ Microdialysis | CMA-12-XXX | These are available in various membrane lengths and cutoffs, indicated by specific codes in the ‘XXX.’ |
| Human insulin (Humulin) | Eli Lilly | N/a | |
| Liquid swivel | Instech | 375/D/22QM | This specific swivel has very low torque and internal volume, as well as a nonreactive quartz lining. |
References
- McNay, E. C., Fries, T. M., Gold, P. E. Decreases in rat extracellular hippocampal glucose concentration associated with cognitive demand during a spatial task. Proc. Natl. Acad. Sci. U.S.A. 97 (6), 2881 (2000).
- McNay, E. C., Gold, P. E. Age-related differences in hippocampal extracellular fluid glucose concentration during behavioral testing and following systemic glucose administration. J. Gerontol. A. Biol. Sci. Med. Sci. 56 (2), 66 (2001).
- McNay, E. C., McCarty, R. C., Gold, P. E. Fluctuations in brain glucose concentration during behavioral testing: dissociations between brain areas and between brain and blood. Neurobiol. Learn. Mem. 75 (3), 325 (2001).
- McNay, E. C., Gold, P. E. Extracellular glucose concentrations in the rat hippocampus measured by zero-net-flux: effects of microdialysis flow rate. 72 (2), 785 (1999).
- McNay, E. C., Sherwin, R. S. Effect of recurrent hypoglycemia on spatial cognition and cognitive metabolism in normal and diabetic rats. Diabetes. 53 (2), 418 (2004).
- McNay, E. C., et al. Hippocampal memory processes are modulated by insulin and high-fat-induced insulin resistance. Neurobiology of Learning and Memory. 93 (4), 546 (2010).
- McNay, E. C., McCarty, R. C., Gold, P. E. Fluctuations in brain glucose concentration during behavioral testing: dissociations between brain areas and between brain and blood. Neurobiology of Learning & Memory. 75 (3), 325 (2001).
- McNay, E. C., Williamson, A., McCrimmon, R. J., Sherwin, R. S. Cognitive and neural hippocampal effects of long-term moderate recurrent hypoglycemia. Diabetes. 55 (4), 1088 (2006).
- McNay, E. C., Sherwin, R. S. From artificial cerebro-spinal fluid (aCSF) to artificial extracellular fluid (aECF): microdialysis perfusate composition effects on in vivo brain ECF glucose measurements. Journal of Neuroscience Methods. 132 (1), 35 (2004).
- McNay, E. C., et al. Hippocampal memory processes are modulated by insulin and high-fat-induced insulin resistance. Neurobiology of Learning and Memory. 93 (4), 546 (2010).
- Benveniste, H., Drejer, J., Schousboe, A., Diemer, N. H. Regional cerebral glucose phosphorylation and blood flow after insertion of a microdialysis fiber through the dorsal hippocampus in the rat. Journal of Neurochemistry. 49 (3), 729 (1987).
- Benveniste, H., Diemer, N. H. Cellular reactions to implantation of a microdialysis tube in the rat hippocampus. Acta Neuropathologica. 74 (3), 234 (1987).
- McNay, E. C., Fries, T. M., Gold, P. E. Decreases in rat extracellular hippocampal glucose concentration associated with cognitive demand during a spatial task. Proceedings of the National Academy of Sciences of the United States of America. 97 (6), 2881 (2000).
- McNay, E. C., et al. Hippocampal memory processes are modulated by insulin and high-fat-induced insulin resistance. Neurobiol. Learn Mem. 93 (4), 546 (2010).
- Lonnroth, P., Jansson, P. -. A., Smith, U. A microdialysis method allowing characterization of intercellular water space in humans. American Journal of Physiology. 1987, E228 (1987).
- McNay, E. C., Gold, P. E. Extracellular glucose concentrations in the rat hippocampus measured by zero-net-flux: effects of microdialysis flow rate. 72 (2), 785 (1999).
- Lalonde, R. o. b. e. r. t. The neurobiological basis of spontaneous alternation. Neuroscience & Biobehavioral Reviews. 26 (1), 91 (2002).
- Richman, C., Dember, W., Kim, P. Spontaneous alternation behavior in animals: A review. Current Psychology. 5 (4), 358 (1986).
- McNay, E. C., Canal, C. E., Sherwin, R. S., Gold, P. E. Modulation of memory with septal injections of morphine and glucose: effects on extracellular glucose levels in the hippocampus. Physiol. Behav. 87 (2), 298 (2006).
- McNay, E. C., Gold, P. E. Memory modulation across neural systems: intra-amygdala glucose reverses deficits caused by intraseptal morphine on a spatial task but not on an aversive task. Journal of Neuroscience. 18 (10), 3853 (1998).
- Rex, A., Bert, B., Fink, H., Voigt, J. P. Stimulus-dependent changes of extracellular glucose in the rat hippocampus determined by in vivo microdialysis. Physiol. Behav. 98 (4), 467 (2009).
Tags
Hippocampal Insulin MicroinjectionIn Vivo MicrodialysisSpatial Memory TestingGlucose MetabolismNeural Activity Marker2-deoxyglucoseFunctional Magnetic Resonance ImagingAnesthesiaBehavioral MeasuresBrain State AlterationContinuous MeasurementBrain Extracellular Fluid ConcentrationsLactateMetabolitesAwake AnimalsUnrestrained AnimalsTasks Designed For Specific Brain RegionsAcute Pharmacological ManipulationHippocampal MeasurementsSpatial Working Memory TaskSpontaneous AlternationEnhanced GlycolysisIntrahippocampal Insulin AdministrationMemory ImprovementHippocampal GlycolysisMicrodialysis Probe DeliveryMetabolite MeasurementSpontaneous Alternation TaskHippocampal Function Measure
Cite This Article
McNay, E. C., Sandusky, L. A., Pearson-Leary, J. Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing. J. Vis. Exp. (71), e4451, doi:10.3791/4451 (2013).