A high-throughput method for generating uniform microislands for autaptic neuronal cultures.
A high-throughput method for generating uniform microislands for autaptic neuronal cultures.
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DOI：
10.1016/j.jneumeth.2011.04.012
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发表时间：
2011-06-15
影响因子：
3
通讯作者：
Bajjalieh, Sandra M.
中科院分区：
文献类型：
--
作者：
Sgro, Allyson E.;Nowak, Amy L.;Austin, Naola S.;Custer, Kenneth L.;Allen, Peter B.;Chiu, Daniel T.;Bajjalieh, Sandra M.
通过喷雾应用聚-D赖氨酸在盖玻片上产生培养基质的微岛是用于培养形成自身（自适应）突触的分离的神经元的常用方法。这种制备方法对于孤立地研究突触传递具有多种优势;然而，通过喷雾产生微岛会产生大小不均匀的岛，因此培养物中含有单个神经元的岛的数量变化很大。为了解决这些问题，我们开发了一种高通量的方法，用于可靠地产生均匀形状的微岛的培养基质。用圆形阵列制造由聚二甲基硅氧烷（PDMS）形成的印模模具，并用于生成由9.2%琼脂糖制成的印模。琼脂糖印模能够加载足够的聚D-赖氨酸和溶于乙酸的胶原蛋白，以快速产生每个盖玻片含有至少64个微岛的盖玻片。当海马神经元在这些盖玻片上培养时，每个盖玻片上有明显更多的单神经元岛。我们注意到，单个神经元倾向于形成三种不同的神经突乔木形态之一，这与岛的大小和细胞体在岛上的位置不同。令我们惊讶的是，每个自适应神经元的突触数量与乔木形状或岛屿大小无关，这表明其他因素调节了孤立神经元形成的突触数量。我们报告的冲压方法可用于增加每个培养物的单神经元岛的数量，并有助于微岛的快速可视化。
Generating microislands of culture substrate on coverslips by spray application of poly-D lysine is a commonly used method for culturing isolated neurons that form self (autaptic) synapses. This preparation has multiple advantages for studying synaptic transmission in isolation; however, generating microislands by spraying produces islands of non-uniform size and thus cultures vary widely in the number of islands containing single neurons. To address these problems, we developed a high-throughput method for reliably generating uniformly-shaped microislands of culture substrate. Stamp molds formed of poly(dimethylsiloxane) (PDMS) were fabricated with arrays of circles and used to generate stamps made of 9.2% agarose. The agarose stamps were capable of loading sufficient poly D-lysine and collagen dissolved in acetic acid to rapidly generate coverslips containing at least 64 microislands per coverslip. When hippocampal neurons were cultured on these coverslips, there were significantly more single-neuron islands per coverslip. We noted that single neurons tended to form one of three distinct neurite-arbor morphologies, which varied with island size and the location of the cell body on the island. To our surprise, the number of synapses per autaptic neuron did not correlate with arbor shape or island size, suggesting that other factors regulate the number of synapses formed by isolated neurons. The stamping method we report can be used to increase the number of single-neuron islands per culture and aid in the rapid visualization of microislands.
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影响因子：
2.9
作者：
Vogt, AK;Lauer, L;Offenhäusser, A
通讯作者：
Offenhäusser, A
DOI：
10.1073/pnas.73.11.4225
发表时间：
1976-01-01
影响因子：
11.1
作者：
FURSHPAN, EJ;MACLEISH, PR;POTTER, DD
通讯作者：
POTTER, DD
影响因子：
3.4
作者：
Mayer, M;Yang, J;Whitesides, GM
通讯作者：
Whitesides, GM
影响因子：
7.4
作者：
Takano, H;Sul, JY;Porter, MD
通讯作者：
Porter, MD
影响因子：
3.2
作者：
Branch, DW;Corey, JM;Wheeler, BC
通讯作者：
Wheeler, BC