Verstraelen Peter, Pintelon Isabel, Nuydens Rony, Cornelissen Frans, Meert Theo, Timmermans Jean-Pierre
Laboratory of Cell Biology & Histology, University of Antwerp, Groenenborgerlaan 171, 2020, Antwerp, Belgium.
Cell Mol Neurobiol. 2014 Jul;34(5):757-76. doi: 10.1007/s10571-014-0057-6. Epub 2014 Apr 19.
Mental disorders, such as schizophrenia or Alzheimer's disease, are associated with impaired synaptogenesis and/or synaptic communication. During development, neurons assemble into neuronal networks, the primary supracellular mediators of information processing. In addition to the orchestrated activation of genetic programs, spontaneous electrical activity and associated calcium signaling have been shown to be critically involved in the maturation of such neuronal networks. We established an in vitro model that recapitulates the maturation of neuronal networks, including spontaneous electrical activity. Upon plating, mouse primary hippocampal neurons grow neurites and interconnect via synapses to form a dish-wide neuronal network. Via live cell calcium imaging, we identified a limited period of time in which the spontaneous activity synchronizes across neurons, indicative of the formation of a functional network. After establishment of network activity, the neurons grow dendritic spines, the density of which was used as a morphological readout for neuronal maturity and connectivity. Hence, quantification of neurite outgrowth, synapse density, spontaneous neuronal activity, and dendritic spine density allowed to study neuronal network maturation from the day of plating until the presence of mature neuronal networks. Via acute pharmacological intervention, we show that synchronized network activity is mediated by the NMDA-R. The balance between kynurenic and quinolinic acid, both neuro-active intermediates in the tryptophan/kynurenine pathway, was shown to be decisive for the maintenance of network activity. Chronic modulation of the neurotrophic support influenced the network formation and revealed the extreme sensitivity of calcium imaging to detect subtle alterations in neuronal physiology. Given the reproducible cultivation in a 96-well setup in combination with fully automated analysis of the calcium recordings, this approach can be used to build a high-content screening assay usable for neurotoxicity screening, target identification/validation, or phenotypic drug screening.
精神障碍，如精神分裂症或阿尔茨海默病，与突触发生受损和/或突触通讯障碍有关。在发育过程中，神经元组装成神经网络，这是信息处理的主要超细胞介质。除了精心编排的基因程序激活外，自发电活动和相关的钙信号已被证明在这种神经网络的成熟过程中起着关键作用。我们建立了一个体外模型，该模型概括了神经网络的成熟过程，包括自发电活动。接种后，小鼠原代海马神经元长出神经突，并通过突触相互连接，形成一个培养皿范围内的神经网络。通过活细胞钙成像，我们确定了一个有限的时间段，在此期间神经元之间的自发活动同步，这表明功能性网络的形成。网络活动建立后，神经元长出树突棘，其密度被用作神经元成熟度和连接性的形态学指标。因此，对神经突生长、突触密度、自发神经元活动和树突棘密度的量化，使得我们能够从接种当天到成熟神经网络出现时研究神经网络的成熟过程。通过急性药理学干预，我们表明同步网络活动是由NMDA受体介导的。犬尿氨酸和喹啉酸（色氨酸/犬尿氨酸途径中的两种神经活性中间体）之间的平衡被证明对维持网络活动起决定性作用。神经营养支持的慢性调节影响了网络形成，并揭示了钙成像检测神经元生理学细微变化的极端敏感性。鉴于在96孔板中可重复培养，并结合对钙记录的全自动分析，这种方法可用于构建一种高内涵筛选检测方法，可用于神经毒性筛选、靶点鉴定/验证或表型药物筛选。
