Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL 62794-9626, USA.
J Neurosci Methods. 2012 Jan 30;203(2):344-53. doi: 10.1016/j.jneumeth.2011.09.020. Epub 2011 Oct 1.
Toward the goal of reproducible live neuronal networks, we investigated the influence of substrate patterns on neuron compliance and network activity. We optimized process parameters of micro-contact printing for reproducible geometric patterns of 10 μm wide lines of polylysine with 4, 6, or 8 connections at a constant square array of nodes overlying the recording electrodes of a multielectrode array (MEA). We hypothesized that an increase in node connections would give the network more inputs resulting in higher neuronal outputs as network spike rates. We also chronically stimulated these networks during development and added astroglia to enhance network activity. Our results show that despite frequent localization of neuron somata over the electrodes, the number of spontaneously active electrodes was reduced 3-fold compared to random networks, independent of pattern complexity. Of the electrodes active, the overall spike rate was independent of pattern complexity, consistent with homeostasis of activity. Lower mean burst rates were seen with higher levels of pattern complexity; however, burst durations increased 1.6-fold with pattern complexity (n=6027 bursts, p<0.001). Inter-burst interval and percentage of active electrodes displaying bursts also increased with pattern complexity. The extra-burst (non-burst or isolated) spike rate increased 4-fold with pattern complexity, but this relationship was reversed with either chronic stimulation or astroglia addition. These studies suggest for the first time that patterns which limit the distribution of branches and inputs are deleterious to activity in a hippocampal network, but that higher levels of pattern complexity promote non-burst activity and favor longer lasting, but fewer bursts.
为了实现可重现的活神经元网络的目标，我们研究了基底模式对神经元顺应性和网络活动的影响。我们优化了微接触印刷工艺参数，以在多电极阵列（MEA）的记录电极上重现具有 4、6 或 8 个连接的 10μm 宽聚赖氨酸线的几何图案，这些图案具有恒定的正方形节点阵列。我们假设节点连接的增加会给网络提供更多的输入，从而导致更高的神经元输出，即网络尖峰率。我们还在发育过程中对这些网络进行了慢性刺激，并添加了星形胶质细胞以增强网络活动。我们的结果表明，尽管神经元胞体经常定位于电极上，但与随机网络相比，自发活跃电极的数量减少了 3 倍，而与图案复杂性无关。在活跃的电极中，整体尖峰率与图案复杂性无关，与活动的自我平衡一致。具有更高图案复杂性的情况下，平均爆发率较低；然而，爆发持续时间增加了 1.6 倍（n=6027 个爆发，p<0.001）。爆发之间的间隔和显示爆发的活跃电极的百分比也随着图案复杂性的增加而增加。爆发外（非爆发或孤立）尖峰率随着图案复杂性增加了 4 倍，但这种关系与慢性刺激或星形胶质细胞添加相反。这些研究首次表明，限制分支和输入分布的模式对海马网络中的活动是有害的，但更高水平的模式复杂性会促进非爆发活动，并有利于更长时间但更少的爆发。
