Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark.
J Neurosci Methods. 2010 Jul 15;190(2):205-13. doi: 10.1016/j.jneumeth.2010.05.011. Epub 2010 May 19.
The study of visually identified neurons in slice preparations from the central nervous system offers considerable advantages over in vivo preparations including high mechanical stability in the absence of anaesthesia and full control of the extracellular medium. However, because of their relative thinness, slices are not appropriate for investigating how individual neurons integrate synaptic inputs generated by large numbers of neurons. Here we took advantage of the exceptional resistance of the turtle to anoxia to make slices of increasing thicknesses (from 300 to 3000 microm) from the lumbar enlargement of the spinal cord. With a conventional upright microscope in which the light condenser was carefully adjusted, we could visualize neurons present at the surface of the slice and record them with the whole-cell patch clamp technique. We show that neurons present in the middle of the preparation remain alive and capable of generating action potentials. By stimulating the lateral funiculus we can evoke intense synaptic activity associated with large increases in conductance of the recorded neurons. The conductance increases substantially more in neurons recorded in thick slices suggesting that the size of the network recruited with the stimulation increases with the thickness of the slices. We also find that that the number of spontaneous excitatory postsynaptic currents (EPSCs) is higher in thick slices compared with thin slices while the number of spontaneous inhibitory postsynaptic currents (IPSCs) remains constant. These preliminary data suggest that inhibitory and excitatory synaptic connections are balanced locally while excitation dominates long-range connections in the spinal cord.
在中枢神经系统切片标本中观察到的神经元的研究，相对于在体标本具有很大的优势，包括在没有麻醉的情况下具有较高的机械稳定性，以及完全控制细胞外介质。然而，由于其相对较薄，切片不适合研究单个神经元如何整合由大量神经元产生的突触输入。在这里，我们利用海龟对缺氧的异常抵抗力，从脊髓腰膨大区制作了越来越厚的切片（从 300 微米到 3000 微米）。在传统的直立显微镜中，仔细调整聚光器，我们可以观察到切片表面存在的神经元，并使用全细胞膜片钳技术对其进行记录。我们表明，位于中间部分的神经元仍然存活并能够产生动作电位。通过刺激外侧束，我们可以诱发强烈的突触活动，导致记录神经元的电导显著增加。在厚切片中记录到的神经元的电导增加幅度明显更大，这表明与刺激相关的网络募集大小随切片厚度的增加而增加。我们还发现，与薄切片相比，厚切片中自发性兴奋性突触后电流（EPSC）的数量更高，而自发性抑制性突触后电流（IPSC）的数量保持不变。这些初步数据表明，在脊髓中，抑制性和兴奋性突触连接在局部是平衡的，而兴奋在长程连接中占主导地位。
