JoVE Journal > Neuroscience
Summary
Abstract
Introduction
Protocol
Results
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Acknowledgements
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신경과학

成人脑切片长期抑郁症诱导评估

Published: October 16, 2019
doi:
10.3791/59859
,
1Laboratory for Behavioral Genetics, Center for Brain Science,RIKEN, 2Senior Adviser’s Office, Center for Brain Science,RIKEN

Summary

在一些基因操纵的动物中，使用单一协议可能无法诱导小脑Purkinje细胞中的LTD，并且LTD和运动学习之间可能存在差异。在基因操纵的动物中，需要多种协议来评估LTD诱导。显示了标准协议。

Abstract

突触可塑性为学习和记忆提供了一种机制。对于小脑运动学习，从平行纤维（PF）到Purkinje细胞（PC）的突触透射的长期凹陷（LTD）被认为是运动学习的基础，并且观察到各种基因操纵的动物。常用的运动学习集，如光动反射（OKR）、前庭-眼反射（VOR）和轮状测试等，用于评估运动学习能力。然而，从GluA2-Carboxy终点体改进的撞鼠获得的结果显示，尽管缺乏PF-LTD，但VOR和OKR的正常适应。在该报告中，LTD的诱导只尝试在室温下使用一种刺激方案。因此，在接近生理温度下使用各种方案在同一敲锥突变体中探索了诱导小脑LTD的条件。最后，我们发现了刺激方案，通过这种刺激方案，可以在这些基因操纵的小鼠中诱导LTD。在这项研究中，提出了一套对LTD归纳进行评估的协议，这将更准确地用于检查LTD与电机学习之间的因果关系。总之，在评价基因操纵小鼠的LTD时，实验条件至关重要。

Introduction

小脑皮层的精心设计的神经元网络的突触组织，由PC、分子层内神经元（篮子和硬质细胞）、Golgi细胞、颗粒细胞的PF、青苔纤维和攀爬纤维（CFs）组成，已经得到阐明。在激发/抑制和发散/收敛方面，组织良好的电路图表明小脑是一个”神经机”1，尽管以前不知道这个”机器”的目的。后来Marr提出，PC的PC输入构成一个三层关联学习网络2。他还建议，每个CF传达一个大脑指令的元素运动2。他假设同时激活 PF 和 CF 将增强 PF-PC 突触活动，并导致 PF-PC 突触的长期强权 （LTP）。另一方面，Albus 假设 PF 和 CF 的同步激活导致 PF-PC 突触3的 LTD。上述两项研究均将小脑解释为一种独特的记忆装置，将小脑并入小脑皮质网络导致Marr_Albus模型学习机模型的形成。

根据这些理论预测，两条证据线表明小脑中存在突触可塑性。第一线的证据是由絮凝器的解剖组织建议的;这里前庭器官起源的MF通路和视网膜起源的CF通路收敛在PC4上。这种独特的收敛模式表明，在絮凝中发生的突触可塑性导致前视反射的显著适应性。第二，记录花团的PC反应和絮状物的病变也支持了上述假设5，6，7。此外，在猴子手部运动8的适应过程中，PC放电模式支持了突触可塑性假说，尤其是阿不思的LTD-假说3。

为了直接确定突触可塑性的性质，一束PF的重复结合刺激（Cjs）和CF，专门在体内对PC进行内联作用，被证明诱导LTD对PF_PC突触的传输功效9， 10，11.在随后使用小脑切片12和培养 PC 的体外探索中，共培养的颗粒细胞刺激和橄榄细胞刺激13或电磷基质和体细胞结合去极化14，15引起 LTD.使用体外制剂16、17，对LTD诱导背后的信号转导机制也进行了深入的研究。

VOR 和 OKR 的适应通常用于定量评估基因操作对小脑运动学习的影响，因为前庭-小脑皮层被证明是 VOR18适应性学习的基本来源，19、20和 OKR19、21 LTD 诱导失败与行为运动学习障碍之间的相关性已被视为 LTD 在电机中起着重要作用的证据学习机制22.这些观点统称为运动学习的LTD假说，或马尔-阿布斯-伊托假说23，24，25，26。

使用类似的方案测量了眼动的自适应学习，同时利用各种实验条件诱导在切片制备27、28、29、30、31中诱导LTD.最近，Schonewille等人26日报告说，一些基因操纵的小鼠表现出正常的运动学习，但小脑切片没有表现出LTD，因此得出结论，LTD对运动学习来说并非必要。然而，在室温下只尝试使用一种类型的协议来诱导LTD。因此，我们在30°C左右的记录条件下使用几种LTD诱导协议，并证实在接近生理温度32的温度下使用这些协议在基因操纵的小鼠中可靠地诱导了LTD。

然而，关于结合刺激的基本特性，仍然存在一些问题。第一个是复杂尖峰的形状和LTD的振幅之间的关系。其次，结合PF刺激和躯体去极化，是否有必要使用刺激的数量是难以捉摸的。在本研究中，用野生型（WT）小鼠调查了这些问题。

Protocol

所有实验程序都通过了RIKEN在实验中照顾和使用动物委员会。老鼠被关在RIKEN脑科学中心的动物设施中，温度（23-25°C）和湿度（45%~65%）良好控制。条件。使用雄性小鼠和雌性WT小鼠（C57BL/6，3~6个月）。 1. 实验中使用的解决方案的制备 注：所有溶液均应在不含金属（电阻率 > 18.2 MΩ）和其他杂质（总有机碳 （TOC） < 5.0 ppb）的超纯水中制成。工?…

Representative Results

本研究使用了四种方案来诱导小脑LTD。在前两个协议（协议1和2）中，在电流夹紧条件下应用了PF刺激和CF刺激的结合。在其他两个协议（协议3和4）中，体体去极化被取代在电压夹条件下的CF刺激。比较了结合刺激过程中的电压轨迹或电流轨迹（图2）。 在电流夹合条件下（协议-1）中，1个PF刺激和1个CF刺激的结合通常用于切片制备26，27。</s…

Discussion

四个协议之间的差异

在LTD诱导协议1和2中，Cjs在1Hz时300次足以诱导小脑LTD. CF的刺激频率似乎在生理范围内，因为警报成年小鼠（P60）的复杂尖峰发射速率报告为1.25赫兹36。然而，单单CF刺激并没有导致PF-CF突触的长期可塑性，如协议1和2（图4，图5）中使用的，虽然仅CF刺激在较高频率诱导LTD24。协?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢奥巴的技术援助。这项研究得到了对K.Y的科研援助资助（C）17K01982的部分支持。

Materials

Amplifier Molecular Devices-Axon Multiclamp 700B
Borosilicate glass capillary Sutter BF150-110-10
Digitizer Molecular Devices-Axon Digidata1322A
Electrode puller Sutter Model P-97
Isoflurane FUJIFILM Wako Pure Chemical 26675-46-7
Isolator A.M.P.I. ISOflex
Linear slicer Dosaka EM PRO7N
Microscope NIKON Eclipse E600FN
Peristaltic pump Gilson MP1 Single Channel Pump
Picrotoxin Sigma-Aldrich P1675
Pure water maker Merck-Millipore MilliQ 7000
Software for experiment Molecular probe-Axon pClamp 10
Software for statistics KyensLab KyPlot 5.0
Stimulator WPI DS8000
Temperature controller Warner TC-324B
Tetrodotoxin Tocris 1078
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References

  1. Eccles, J. C., Ito, M., Szentagothai, J. . The Cerebellum as a Neuronal Machine. , (1967).
  2. Marr, D. A theory of cerebellar cortex. Journal of Physiology. 202 (2), 437-470 (1969).
  3. Albus, J. S. Theory of cerebellar function. Mathematical Biosciences. 10 (1), 25-61 (1971).
  4. Maekawa, K., Simpson, J. I. Climbing fiber responses evoked in vestibulocerebellum of rabbit from visual system. Journal of Neurophysiology. 36 (4), 649-666 (1973).
  5. Ito, M., Shiida, T., Yagi, N., Yamamoto, M. Visual influence on rabbit horizontal vestibulo-ocular reflex presumably effected via the cerebellar flocculus. Brain Research. 65 (1), 170-174 (1974).
  6. Ghelarducci, B., Ito, M., Yagi, N. Impulse discharge from flocculus Purkinje cells of alert rabbits during visual stimulation combined with horizontal head rotation. Brain Research. 87 (1), 66-72 (1975).
  7. Robinson, D. A. Adaptive gain control of vestibulo-ocular reflex by the cerebellum. Journal of Neurophysiology. 39 (5), 954-969 (1976).
  8. Gilbert, P. F. C., Thach, W. T. Purkinje cell activity during motor learning. Brain Research. 128 (2), 309-328 (1977).
  9. Ito, M., Sakurai, M., Tongroach, P. Climbing fibre induced depression of both mossy fibre responsiveness and glutamate sensitivity of cerebellar Purkinje cells. Journal of Physiology. 324, 113-134 (1982).
  10. Ito, M., Kano, M. Long-lasting depression of parallel fiber-Purkinje cell transmission induced by conjunctive stimulation of parallel fibers and climbing fibers in the cerebellar cortex. Neuroscience Letters. 33 (3), 253-258 (1982).
  11. Ekerot, C. F., Kano, M. Long-term depression of parallel fibre synapses following stimulation of climbing fibres. Brain Research. 342 (2), 357-360 (1985).
  12. Sakurai, M. Synaptic modification of parallel fibre-Purkinje cell transmission in in vitro guinea-pig cerebellar slices. Journal of Physiology. 394, 462-480 (1987).
  13. Hirano, T. Depression and potentiation of the synaptic transmission between a granule cell and a Purkinje cell in rat cerebellar culture. Neuroscience Letters. 119 (2), 141-144 (1990).
  14. Linden, D. J. A long-term depression of AMPA currents in cultured cerebellar purkinje neurons. Neuron. 7 (1), 81-89 (1991).
  15. Linden, D. J., Connor, J. A. Participation of postsynaptic PKC in cerebellar long-term depression in culture. Science. 254 (5038), 1656-1659 (1991).
  16. Ito, M. Cerebellar long-term depression: characterization, signal transduction and functional roles. Physiological Reviews. 81 (3), 1143-1195 (2001).
  17. Ito, M. The molecular organization of cerebellar long-term depression. Nature Reviews Neuroscience. 3, 896-902 (2002).
  18. Ito, M., Jastreboff, P. J., Miyashita, Y. Specific effects of unilateral lesions in the flocculus upon eye movements in albino rabbits. Experimental Brain Research. 45 (1-2), 233-242 (1982).
  19. Nagao, S. Effects of vestibulocerebellar lesion upon dynamic characteristics and adaptation of vestibulo-ocular and optokinetic responses in pigmented rabbits. Experimental Brain Research. 53 (1), 36-46 (1983).
  20. Watanabe, E. Neuronal events correlated with long-term adaptation of the horizontal vestibulo-ocular reflex in the primate flocculus. Brain Research. 297 (1), 169-174 (1984).
  21. van Neerven, J., Pompeiano, O., Collewijn, H. Effects of GABAergic and noradrenergic injections into the cerebellar flocculus on vestibulo-ocular reflexes in the rabbit. Progress in Brain Research. 88, 485-497 (1991).
  22. Ito, M. Mechanism of motor learning in the cerebellum. Brain Research. 886, 237-245 (2000).
  23. De Schutter, E. Cerebellar long-term depression might normalize excitation of Purkinje cells: a hypothesis. Trends in Neurosciences. 18 (7), 291-295 (1995).
  24. Hansel, C., Linden, D. J. Long-term depression of the cerebellar climbing fiber-Purkinje neuron synapse. Neuron. 26 (2), 473-482 (2000).
  25. Safo, P., Regehr, W. G. Timing dependence of the induction of cerebellar LTD. Neuropharmacology. 54 (1), 213-218 (2007).
  26. Schonewille, M., et al. Reevaluating the role of LTD in cerebellar motor learning. Neuron. 70 (1), 43-500 (2011).
  27. Karachot, L., Kado, T. R., Ito, M. Stimulus parameters for induction of long-term depression in in vitro rat Purkinje cells. Neuroscience Research. 21 (2), 161-168 (1994).
  28. Hartell, N. A. Induction of cerebellar long-term depression requires activation of glutamate metabotropic receptors. Neuroreport. 5, 913-916 (1994).
  29. Aiba, A., et al. Deficient cerebellar long-term depression and impaired motor learning in mGluR1 mutant mice. Cell. 79, 377-388 (1994).
  30. Steinberg, J. P., et al. Targeted in vivo mutations of the AMPA receptor subunit GluR2 and its interacting protein PICK1 eliminate cerebellar long-term depression. Neuron. 46 (6), 845-860 (2006).
  31. Koekkoek, S. K., et al. Deletion of FMR1 in Purkinje cells enhances parallel fiber LTD, enlarges spines, and attenuates cerebellar eyelid conditioning in Fragile X syndrome. Neuron. 47 (3), 339-352 (2005).
  32. Yamaguchi, K., Itohara, S., Ito, M. Reassessment of long-term depression in cerebellar Purkinje cells in mice carrying mutated GluA2 C terminus. Proceedings of the National Academy of Sciences of the United States of America. 113 (36), 10192-10197 (2016).
  33. De Schutter, E., Bower, J. M. An active membrane model of the cerebellar Purkinje cell II. Simulation of synaptic responses. Journal of Neurophysiology. 71 (1), 401-419 (1994).
  34. Swensen, A. M., Bean, B. Ionic mechanisms of burst firing in dissociated Purkinje neurons. Journal of Neuroscience. 23 (29), 9650-9663 (2003).
  35. Fukuda, J., Kameyama, M., Yamaguchi, K. Breakdown of cytoskeletal filaments selectively reduces Na and Ca spikes in cultured mammal neurones. Nature. 294 (5836), 82-85 (1981).
  36. Arancillo, M., White, J. J., Lin, T., Stay, T. L., Silltoe, R. V. In vivo analysis of Purkinje cell firing properties during postnatal mouse development. Journal of Neurophysiology. 113, 578-591 (2015).
  37. Ishikawa, T., Shimuta, M., Häusser, M. Multimodal sensory integration in single cerebellar granule cell in vivo. eLife. 4, e12916 (2015).
  38. Tempia, F., Minlaci, M. C., Anchisi, D., Strata, P. Postsynaptic current mediated by metabotropic glutamate receptors in cerebellar Purkinje cells. Journal of Neurophysiology. 80, 520-528 (1998).
  39. Wang, S. S., Denk, W., Häusser, M. Coincidence detection in single dendritic spines mediated by calcium release. Nature Neuroscience. 3, 1266-1273 (2000).
  40. Kuroda, S., Schweighofer, N., Kawato, M. Exploration of signal transduction pathways in cerebellar long-term depression by kinetic simulation. Journal of Neuroscience. 21 (15), 5693-5702 (2001).
  41. Wang, W., et al. Distinct cerebellar engrams in short-term and long-term motor learning. Proceedings of the National Academy of Sciences of the United States of America. 111 (1), E188-E193 (2014).
  42. Inoshita, T., Hirano, T. Occurrence of long-term depression in the cerebellar flocculus during adaptation of optokinetic response. eLife. 27, 36209 (2018).
  43. Belmeguenai, A., et al. Intrinsic plasticity complements long-term potentiation in parallel fiber input gain control in cerebellar Purkinje cells. Journal of Neuroscience. 30 (41), 13630-13643 (2010).
  44. Ohtsuki, G., Piochon, C., Adelman, J. P., Hansel, C. SK2 channel modulation contributes to compartment specific dendritic plasticity in cerebellar Purkinje cells. Neuron. 75, 108-120 (2012).

Tags

LTD InductionCerebellar SlicesGene ManipulationMotor LearningACSFTetrodotoxinBrain DissectionPurkinje CellsCerebellum SlicingCutting ACSFOxygen-carbon Dioxide Gas
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Yamaguchi, K., Ito, M. Assessment of Long-term Depression Induction in Adult Cerebellar Slices. J. Vis. Exp. (152), e59859, doi:10.3791/59859 (2019).
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