本地CA1γ振荡通过强直刺激生成
Summary
振荡是基本的网络性能和疾病和药物调节。研究脑切片振荡允许在受控条件下隔离网络的表征。提供用于制备急性脑片的CA1唤起γ振荡协议。
Abstract
神经网络振荡是在健康和疾病的大脑活动的重要特征，并且可以通过一系列临床使用药物进行调制。的协议提供生成模型为研究CA1γ振荡（20 – 80赫兹）。这些γ振荡是稳定至少30分钟，取决于兴奋性和抑制性突触活动除了激活起搏器电流。 Tetanically刺激振荡具有许多重复且容易量化的特性，包括穗计数，振荡持续时间，延迟和频率时的网络状态报告。的电刺激振荡的优点包括稳定性，再现性和偶发采集使网络功能的鲁棒表征。可用于CA1γ振荡的这种模型来研究细胞机制，并系统地研究网络如何神经元活动的改变在疾病和毒品。疾病状态药理可通过使用脑切片的从转基因或介入的动物模型可容易地并入到能够选择的药物特异性靶向疾病的机制。
Introduction
这关联到行为状态不同频带内的脑网络的振荡出现。在啮齿类动物，海马θ振荡（5 – 10赫兹）在探索行为1,2观察，而γ振荡（20 – 80赫兹）与相关联的各种认知过程，包括感知和关注3,4。同步γ网络活动也牵连在病症如癫痫和精神分裂症5,6的病理。例如，γ振荡被认为对应于皮质癫痫病灶5,7,8的领域，并可以作为pharmacosensitivity或电阻，调查在癫痫研究9的两个重要方面的标记。
海马脑切片是已经广泛地用于研究网络活动10-12的模型。各种协议已被开发，以产生在脑切片γ振荡通常我nvolve药理调节，如低镁离子，4-氨基吡啶（4AP），荷包牡丹和红藻氨酸12-17。药理触发振荡的缺点是，他们随机出现后用药，不可靠产生或保持稳定的时间。电触发γ振荡克服许多这样的问题，也有被暂时锁定到刺激事件允许偶发记录和分析的优点。这里的协议描述为通过提供强直刺激的地层东方明珠Oriens海马切片CA1产生γ振荡。
Protocol
在老鼠身上所有实验均批准弗洛里学院动物伦理委员会。 1.设置切削脑片制备由（mM计）125胆碱氯，2.5氯化钾，0.4氯化钙2，6的MgCl 2，1.25的NaH 2 PO 4，26的NaHCO 3的切削液，20 D-葡萄糖饱和卡波金气（95％O 2 -5由（毫米）125氯化钠，2.5氯化钾，2-氯化钙2，2的MgCl 2，1.25的NaH 2 PO 4，26的NaHCO …
Representative Results
地层东方明珠Oriens的强直刺激产生的可再生的γ振荡（35.4±2.2赫兹）， 见图3B。为了证明是本地网络CA1区CA3从投入是用弯曲32克针切割片在CA2区域内切断产生的振动。在切割片的振荡性质没有从未切割切片差异（P = 0.85;切割片6.16±1.1尖峰，每组6;未切割切片5.89±0.8尖峰，N = 6），表明该振荡机产生的。 这种方法的一个重要优点是记录的稳定性。当破伤风交付在5分钟…
Discussion
一个强大的方法来产生急性脑片CA1γ振荡描述。出现所产生的振动从本地电路能够控制和了解网络振荡12的神经生理学基础上更好的机会。 AMPA受体，GABA A受体，I H和T型Ca 2+通道所需的所有在此模型γ振荡。而这里介绍的地方CA1振荡可以产生强劲，这是依赖于确保脑片是健康的。一个关键步骤是快速去除从头骨的大脑，小心去除，然后迅速浸入冰冷的溶液中不会穿透…
Declarações
The authors have nothing to disclose.
Acknowledgements
Supported by APA to RJH, NHMRC program grant 400121 to SP, and NMHRC fellowship 1005050 to SP. CAR acknowledges the support of the ARC (FT0990628) and the DOWD fellowship scheme. The Florey Institute of Neuroscience and Mental Health is supported by Victorian State Government infrastructure funds.
Materials
| 4-(N-Ethyl-N-phenylamino)-1,2- dimethyl-6-(methylamino) pyrimidinium chloride (ZD7288) | Sigma-Aldrich | Z3777 | |
| Biuculline | Sigma-Aldrich | 14340 | |
| 6-cyano-7-nitroquinoxa- line-2,3-dione (CNQX) | Sigma-Aldrich | C127 | |
| Nickel | Sigma-Aldrich | 266965 | |
| Carbamazepine | Sigma-Aldrich | C4024 | |
| (2R)-amino-5-phosphonopentano-ate (APV) | Tocris Bioscience | 0105 | |
| Retigabine | ChemPacific | 150812-12-7 | |
| Choline-Cl | Sigma Aldrich | C1879-5KG | |
| KCl | Sigma Aldrich | P9333-500G | |
| NaH2PO4 | Sigma Aldrich | S9638-250G | |
| NaHCO3 | Sigma Aldrich | S6297-250G | |
| NaCl | Sigma Aldrich | S7653-5KG | |
| Glucose | Sigma Aldrich | G8270-1KG | |
| CaCl2.2H2O | Sigma Aldrich | 223506-500G | |
| MgCl2.6H2O | Sigma Aldrich | M2670-500G | |
| Electrode glass | Harvard Apparatus | GC150F-10 | |
| Concentric bipolar stimulating metal electrode | FHC | CBBPF75 | |
| Digital Isolator | Getting Instruments | Model BJN8-9V1 | |
| Model 1800 amplifier | A-M systems | Model 1800 amplifier | |
| Digitizer | National Intruments | NI USB-6211 | |
| Vibrotome | Leica | VT1200s |
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Tags
CA1 Oscillations947; OscillationsTetanic StimulationNeuronal NetworkBrain ActivityPacemaker CurrentsExcitatory Synaptic ActivityInhibitory Synaptic ActivitySpike CountOscillation DurationLatencyFrequencyNetwork FunctionDisease State PharmacologyGenetically Modified Animal ModelsInterventional Animal Models
Citar este artigo
Hatch, R. J., Reid, C. A., Petrou, S. Generation of Local CA1 γ Oscillations by Tetanic Stimulation. J. Vis. Exp. (102), e52877, doi:10.3791/52877 (2015).