Potter S M, Pine J, Fraser S E
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
Scanning Microsc Suppl. 1996;10:189-99.
We are developing a multielectrode silicon "neuroprobe" for maintaining a long-term, specific, two-way electrical interface with nervous tissue. Our approach involves trapping a neuron (from an embryonic rat hippocampus) in a small well with a stimulation/recording electrode at its base. The well is covered with a grillwork through which the neuron's processes are allowed to grow, making synaptic contact with the host tissue, in our case a cultured slice from a rat hippocampus. Each neuroprobe can accommodate 15 neurons, one per well. As a first step in studying neurite outgrowth from the neuroprobe, it was necessary to develop new staining techniques so that neurites from the probe neurons can be distinguished from those belonging to the host, without interference from non-specific background staining. We virtually eliminated background staining through a number of innovations involving dye solubility, cell washing, and debris removal. We also reduced photobleaching and phototoxicity, and enhanced imaging depth by using a 2-photon laser-scanning microscope. We focused on using the popular membrane dye, DiI, however a number of other membrane dyes were shown to provide clear images of neural processes using pulsed illumination at 900 nm. These techniques will be useful to others wishing to follow over time the growth of neurons in culture of after transplantation in vivo, in a non-destructive way.
我们正在研发一种多电极硅“神经探针”，用于与神经组织维持长期、特定的双向电接口。我们的方法包括将一个（来自胚胎大鼠海马体的）神经元捕获在一个小凹槽中，在其底部设置一个刺激/记录电极。凹槽上覆盖着一个格栅，神经元的突起可以通过格栅生长，与宿主组织建立突触联系，在我们的案例中，宿主组织是大鼠海马体的培养切片。每个神经探针可以容纳15个神经元，每个凹槽一个。作为研究神经探针上神经突生长的第一步，有必要开发新的染色技术，以便探针神经元的神经突能够与宿主的神经突区分开来，不受非特异性背景染色的干扰。我们通过一系列涉及染料溶解性、细胞清洗和碎片清除的创新方法，几乎消除了背景染色。我们还通过使用双光子激光扫描显微镜减少了光漂白和光毒性，并提高了成像深度。我们专注于使用流行的膜染料DiI，然而，研究表明，使用其他一些膜染料，在900nm的脉冲照明下也能提供神经突起的清晰图像。这些技术将对其他希望以非破坏性方式长期跟踪培养中的神经元或体内移植后神经元生长情况的人有用。
