长时程增强一个非常稳定的和可重复的记录和保存海马鼠标片制备工艺的改进
Summary
本文提出了一种完整的方法来准备和保存<em>在体外</em>急性成年小鼠海马脑片。该协议允许非常稳定持久的长时程增强（LTP）的记录超过8小时，95％的成功率。
Abstract
长时程增强（LTP）是一种类型的神经元突触可塑性，其特征在于由突触强度的增加，被认为参与记忆编码。 LTP诱导急性海马切片的CA1区中已被广泛研究。然而，维护阶段这种现象背后的分子机制仍然知之甚少。这可能是部分原因是由于不同的实验室所使用的各种实验条件。事实上，对LTP的维持阶段是强烈地依赖于充氧，温度和湿度等外部参数。它也是依赖于内部参数，如解剖后切片平面和切片活力的方向。
所有这些参数的优化使一个高度可再现性和非常稳定的长时程增强的诱导。这种方法提供了可能的分子机制，进一步探索参与稳定增长海马脑片突触强度。这也凸显了在体外研究的神经生理现象的实验条件的重要性。
Introduction
如今，有多么复杂的存储和调用记忆在神经回路水平的了解有限。然而，记忆存储是一个统一的假设和广泛接受的记忆存储在中枢神经系统的神经元之间的突触连接强度的变化。就其本身而言，对突触可塑性的研究已经在很大程度上得益于两个突破性的发现。 （1）在一个开创性的实验，极乐和Lomo 1，使用完整的麻醉兔，结果发现提供一个短暂的高频率（1秒，100赫兹）刺激海马穿路径造成一个长期持久的（几个小时）增加相关的突触连接。这迷人的现象被称为“长时程增强”或LTP由道格拉斯和戈达德在1975年2。 （2）随后，我们发现了类似的现象可能会触发在脑切片（0.4毫米）的人为保持的活体外 </eM>。研究得最广泛的LTP是通过提供一个或多个破伤风记录产生的场兴奋性突触电位在所谓的CA1区锥体细胞诱发的成捆的轴突（所谓的谢弗络），而在体外观察到。诱导LTP的机制在很大程度上被显露出来。基本上，Ca 2 +的涌入通过NMDA受体激活酶有两个后果：AMPA受体的磷酸化（提高效率）和加入额外的AMPA受体在突触后膜3。 LTP的维持阶段的机制，与此相反，在很大程度上是未知的，特别是因为它是实验更难以维持健康的许多小时，比30至60分钟切片。
大量的研究，一直致力于为LTP机制的理解和有趣的理论已经阐述多年来4-11。但联合国直到现在，精确的分子突触强度的稳定增长机制尚未阐明。这可能是部分原因是由于难以再现先前结果在不同实验室使用不同的技术来制备和海马切片的维护。在他们的方法纸，Sajikumar 等12强调的实验条件下对大鼠海马脑片的制备和记录LTP稳定的重要性。在这段视频中，我们介绍了多年来在我们的实验室开发的，能够记录一个非常稳定的小鼠海马脑片LTP的优化步骤。
这种优化已经开发并成功使用小鼠13只大鼠和11其他实验室研究LTP机制的协议。它允许有经验的研究人员，以诱导和记录一个很长的持久LTP具有很高的成功率，在成年小鼠。在p诱导LTP hysiological基础进行了仔细的检查，并展示了14。在这种方法中纸，我们展示的任何修改的实验条件下，可以产生深远的影响，如温度或氧LTP维护解剖过程，同时可以深入修改切片兴奋。还必须强调的是，所有这些参数的精确控制需要几个月的培训新手同学。
Protocol
按照与国立卫生法规的护理和使用动物的研究，并与当地伦理委员会的协议，所有动物的程序进行。 1。人工脑脊髓液的制备同一介质，则用于解剖，切割和灌注片（1毫升/分钟），在休息期间和电生理记录。这种介质组成的124 mM氯化钠，氯化钾4.4毫米，26毫米的NaHCO 3，1毫米的NaH 2 PO 4，1.3毫米，2.5毫米的 CaCl 2，用M…
Representative Results
此方法已被用来分析性能的持久的成年C57BL/6J小鼠（SAS JANVIER，法国）14在急性海马脑片长时程增强诱导。令人惊讶的是，实验条件的改善已导致了一种新的方式寻找在LTP。我们发现，持久的突触强度的增加并不需要新的蛋白质合成。 在这里，我们将展示，诱导LTP取决于片上的生存能力和兴奋。当夹层的海马速度太慢或太有害，切片兴奋性增加，可观察到多突触反应LTP…
Discussion
我们在我们的实验室已经开发相结合的方法开发和使用的其他实验室有一个大的专业知识在LTP录音11,17的协议。此协议适合于成年小鼠海马，并可以用在任何年龄段的任何背景基因型的动物。它还允许在转基因小鼠的神经退行性疾病如阿尔茨海默病18,19分析LTP。
利用大鼠海马脑片这个协议必须适应。例如，大多数的大鼠进行研究，使用温度为32°C，而不是28°C?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢巴纳德Foucart的技术援助。这项工作是由比利时科学研究基金（FRS-FNRS）和伊丽莎白女王医学研究基金的支持。艾格尼丝维莱是研究员在比利时科学研究基金。
Materials
| Reagent/Material | |||
| NaCl | Sigma – Aldrich | S7653 | |
| NaHCO3 | Sigma – Aldrich | S8875 | |
| KCl | Sigma – Aldrich | P9333 | |
| D-glucose | Sigma – Aldrich | G7528 | |
| NaH2PO4 | Sigma – Aldrich | S9638 | |
| MgSO4 1M | Sigma – Aldrich | 63126 | |
| CaCl2 | Sigma – Aldrich | C4901 | |
| Carbogen | Air Liquide (Belgium) | ||
| Capillaries | WPI, Inc. (UK) | TW150-4 | |
| Stimulating Electrodes | FHC (USA) | CE2B30 | |
| Surgical tools | FST (Germany) | ||
| Filter paper 84 g/m2 | Sartorius | FT-3-105-110 | |
| Mesh | Lycra | 15 den | |
| Glue | UHU | plus endfest300 | |
| Instrument | |||
| Amplifier | WPI, Inc. (UK) | ISO-80 | |
| Interface recording chamber | FST (Germany) | ||
| Peristaltic pumps | Gilson (USA) | Minipuls 3 | |
| Temperature controller | University of Edinburgh | www.etcsystem.com | |
| Tissue Chopper | Mcllwain | ||
| Stimulators | Grass (USA) | S88X + SIU-V | |
| Program analysis | WinLTP | www.winltp.com | |
| Micromanipulators | Narishige | MM-3 and MMO-220A | |
| Surgical microscope | Leica Microsystem | ||
| A/D converter | National Instruments | NIPCI-6229 M-series |
References
- Bliss, T. V., Lomo, T. Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J. Physiol. 232 (2), 331-356 (1973).
- Douglas, R. M., Goddard, G. V. Long-term potentiation of the perforant path-granule cell synapse in the rat hippocampus. Brain Res. 86, 205-215 (1975).
- Squire, L., Kandel, E. . Memory: From mind to molecules. , (1999).
- Nguyen, P. V., Abel, T., Kandel, E. R. Requirement of a critical period of transcription for induction of a late phase of LTP. Science. 265 (5175), 1104-1107 (1994).
- Frey, U., Morris, R. G. Synaptic tagging and long-term potentiation. Nature. 385 (6616), 533-536 (1038).
- Bortolotto, Z. A., Collingridge, G. L. A role for protein kinase C in a form of metaplasticity that regulates the induction of long-term potentiation at CA1 synapses of the adult rat hippocampus. Eur. J. Neurosci. 12 (11), 4055-4062 (2000).
- Migues, P. V., Hardt, O., et al. PKMzeta maintains memories by regulating GluR2-dependent AMPA receptor trafficking. Nat. Neurosci. 13 (5), 630-634 (2010).
- Ehlers, M. D., Heine, M., Groc, L., Lee, M. -. C., Choquet, D. Diffusional trapping of GluR1 AMPA receptors by input-specific synaptic activity. Neuron. 54 (3), 447-460 (2007).
- Vickers, C. A., Dickson, K. S., Wyllie, D. J. A. Induction and maintenance of late-phase long-term potentiation in isolated dendrites of rat hippocampal CA1 pyramidal neurones. J. Physiol. 568 (3), 803-813 (2005).
- Fonseca, R. Activity-dependent actin dynamics are required for the maintenance of long-term plasticity and for synaptic capture. Eur. J. Neurosci. 35 (2), 195-206 (2012).
- Redondo, R. L., Okuno, H., Spooner, P. A., Frenguelli, B. G., Bito, H., Morris, R. G. M. Synaptic tagging and capture: differential role of distinct calcium/calmodulin kinases in protein synthesis-dependent long-term potentiation. J. Neurosci. 30 (14), 4981-4989 (2010).
- Sajikumar, S., Navakkode, S., Frey, J. U. Protein synthesis-dependent long-term functional plasticity: methods and techniques. Curr. Opin. Neurobiol. 15 (5), 607-613 (2005).
- Connor, S. A., Wang, Y. T., Nguyen, P. V. Activation of beta-adrenergic receptors facilitates heterosynaptic translation-dependent long-term potentiation. J. Physiol. 589 (17), 4321-4340 (2011).
- Villers, A., Godaux, E., Ris, L. Long-lasting LTP requires neither repeated trains for its induction nor protein synthesis for its development. PLoS One. 7 (7), e40823 (2012).
- Capron, B., Sindic, C., Godaux, E., Ris, L. The characteristics of LTP induced in hippocampal slices are dependent on slice-recovery conditions. Learn. Mem. 13 (3), 271-277 (2006).
- Villers, A., Godaux, E., Ris, L. Late phase of L-LTP elicited in isolated CA1 dendrites cannot be transferred by synaptic capture. Neuroreport. 21, 210-215 (2010).
- Nguyen, P. V., Kandel, E. R. Brief theta-burst stimulation induces a transcription-dependent late phase of LTP requiring cAMP in area CA1 of the mouse hippocampus. Learn. Mem. 4 (2), 230-243 (1997).
- Dewachter, I., Ris, L., et al. Modulation of synaptic plasticity and Tau phosphorylation by wild-type and mutant presenilin1. Neurobiol. Aging. 29 (5), 639-652 (2008).
- Dewachter, I., Filipkowski, R. K., et al. Deregulation of NMDA-receptor function and down-stream signaling in APP[V717I] transgenic mice. Neurobiol. Aging. 30 (2), 241-256 (2009).
- Mathis, D. M., Furman, J. L., Norris, C. M. Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology. J. Vis. Exp. (49), e2330 (2011).
- Kirov, S. A., Sorra, K. E., Harris, K. M. Slices have more synapses than perfusion-fixed hippocampus from both young and mature rats. J. Neurosci. 19 (8), 2876-2886 (1999).
- Bourne, J. N., Kirov, S. A., Sorra, K. E., Harris, K. M. Warmer preparation of hippocampal slices prevents synapse proliferation that might obscure LTP-related structural plasticity. Neuropharmacology. 52, 55-59 (2007).
- Alger, B. E., Dhanjal, S. S., Dingledine, R., Garthwaite, J., Henderson, G., King, G. L., Dingledine, R., et al. Appendix: Brain slice methods. Brain Slices. , 381-437 (1984).
- Watson, P. L., Weiner, J. L., Carlen, P. L. Effects of variations in hippocampal slice preparation protocol on the electrophysiological stability, epileptogenicity and graded hypoxia responses of CA1 neurons. Brain Res. 775, 134-143 (1997).
- Frey, U., Krug, M., Reymann, K. G., Matthies, H. Anisomycin, an inhibitor of protein synthesis, blocks late phases of LTP phenomena in the hippocampal CA1 region in vitro. Brain Res. 452 (1-2), 57-65 (1988).
- Kandel, E. R. The molecular biology of memory storage: a dialogue between genes and synapses. Science. 294 (5544), 1030-1038 (2001).
- Fonseca, R., Nägerl, U. V., Bonhoeffer, T. Neuronal activity determines the protein synthesis dependence of long-term potentiation. Nat. Neurosci. 9 (4), 478-480 (2006).
- Rudy, J. W. Is there a baby in the bathwater? Maybe: some methodological issues for the de novo protein synthesis hypothesis. Neurobiol. Learn. Mem. 89 (3), 219-224 (2008).
- Sharma, A. V., Nargang, F. E., Dickson, C. T. Neurosilence: Profound Suppression of Neural Activity following Intracerebral Administration of the Protein Synthesis Inhibitor Anisomycin. J. Neurosci. 32 (7), 2377-2387 (2012).
- Volianskis, A., Jensen, M. S. Transient and sustained types of long-term potentiation in the CA1 area of the rat hippocampus. J. Physiol. 550 (2), 459-492 (2003).
- Ris, L., Villers, A., Godaux, E. Synaptic capture-mediated long-lasting long-term potentiation is strongly dependent on mRNA translation. Neuroreport. 20 (17), 1572-1576 (2009).
- Abbas, A. -. K., Dozmorov, M., et al. Persistent LTP without triggered protein synthesis. Neurosci. Res. 63 (1), 59-65 (2009).
- Ho, O. H., Delgado, J. Y., O’Dell, T. J. Phosphorylation of proteins involved in activity-dependent forms of synaptic plasticity is altered in hippocampal slices maintained in vitro. J. Neurochem. 91, 1344-1357 (2004).
- Whittingham, T. S., Lust, W. D., Christakis, D. A., Passonneau, J. V. Metabolic stability of hippocampal slice preparations during prolonged incubation. J. Neurochem. 43, 689-696 (1984).
- Dunlop, D. S., van Elden, W., Lajtha, A. Optimal conditions for protein synthesis in incubated slices of rat brain. Brain Res. 99, 303-318 (1975).
- Taubenfeld, S. M., Stevens, K. A., Pollonini, G., Ruggiero, J., Alberini, C. M. Profound molecular changes following hippocampal slice preparation: loss of AMPA receptor subunits and uncoupled mRNA/protein expression. J. Neurochem. 81 (6), 1348-1360 (2002).
- Gruart, A., Munoz, M. D., Delgado-Garcia, J. M. Involvement of the CA3-CA1 synapse in the acquisition of associative learning in behaving mice. J. Neurosci. 26 (4), 1077-1087 (2006).
- Whitlock, J. R., Heynen, A. J., Shuler, M. G., Bear, M. F. Learning induces long-term potentiation in the hippocampus. Science. 313, 1093-1097 (2006).
- Grant, S. G. N., Silva, A. J. Targeting learning. TINS. 17 (2), 71-75 (1994).
- Izquierdo, I., Medina, J. H., Vianna, M. R. M., Izquierdo, L. A., Barros, B. M. Separate mechanisms for short- and long-term memory. Behav. Brain Res. 103, 1-11 (1999).
- Morice, E., Andreae, L. C., Cooke, S. F., Vanes, L., Fisher, E. M. C., Tybulewicz, V. L. J., Bliss, T. V. P. Preservation of long-term memory and synaptic plasticity despite short-term impairments in the Tc1 mouse model of Down syndrome. Learn Mem. 15 (7), 492-500 (2008).
- Dunlop, D. S., van Elden, W., Plucinska, I., Lajtha, A. Brain Slice Protein Degradation and Development. J. Neurochem. 36, 258-265 (1981).
- Izquierdo, I. Long-term potentiation and the mechanisms of memory. Drug Dev. Res. 30, 1-17 (1993).
Citer Cet Article
Villers, A., Ris, L. Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation. J. Vis. Exp. (76), e50483, doi:10.3791/50483 (2013).