Djurisic Maja
Department of Biology, Neurobiology, and Bio-X, Stanford University;
J Vis Exp. 2020 Jul 2(161). doi: 10.3791/61377.
Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol's critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.
急性海马切片使一代又一代的神经科学家能够详细且高保真地探索突触、神经元和神经回路的特性。如果没有这种经典的制备方法，对长时程增强（LTP）和长时程抑制（LTD）机制、单个神经元树突计算以及神经回路中依赖经验的变化的探索将是不可能的。然而，除了少数例外，大多数使用急性海马切片的基础研究都是使用来自相对年轻年龄（约P20 - P40）啮齿动物的切片进行的，尽管突触和内在兴奋性机制有很长的发育后期，一直持续到P60之后。使用年轻海马切片的主要吸引力在于其对缺氧损伤具有更高的耐受性，有助于保持神经元健康。然而，有必要了解发育更成熟阶段的神经元功能，各种神经退行性疾病动物模型的发展进一步凸显了这一需求，这些模型需要老龄大脑的制备。在这里，我们描述了一种对急性海马切片制备方法的改进，该方法能够可靠地从成年和老龄小鼠海马中获取健康的切片。该方案的关键步骤是经心灌注和用冰冷的无钠NMDG-aSCF进行切片。这些步骤共同作用，减轻了断头后缺氧诱导的ATP下降以及被动钠通量引起的细胞毒性水肿。我们展示了如何使用振动切片机切取海马加皮层的横向切片。通过这种方式获得的急性海马切片在数小时的记录过程中都能可靠地保持健康，适用于场电位记录和靶向膜片钳记录，包括对荧光标记神经元的靶向记录。
