ACS Chem Neurosci. 2018 Aug 15;9(8):2089-2096. doi: 10.1021/acschemneuro.8b00163. Epub 2018 Jul 3.
Single-cell mass spectrometry has become an established technique to study specific molecular properties such as the neuropeptide complement of identified neurons. Here, we describe a strategy to characterize, by MALDI-TOF mass spectrometry, neurochemical composition of neurons that were identified by their electrophysiological and neuroanatomical characteristics. The workflow for the first time combined perforated patch clamp recordings with dye loading by electroporation for electrophysiological and neuroanatomical characterization as well as chemical profiling of somata by MALDI-TOF mass spectrometry with subsequent immunohistochemistry. To develop our protocol, we used identified central olfactory neurons from the American cockroach Periplaneta americana. First, the combined approach was optimized using a relative homogeneous, well-characterized neuron population of uniglomerular projection neurons, which show acetylcholine esterase immunoreactivity. The general applicability of this approach was verified on local interneurons, which are a diverse neuron population expressing highly differentiated neuropeptidomes. Thus, this study shows that the newly established protocol is suitable to comprehensively analyze electrophysiological, neuroanatomical, and molecular properties of single neurons. We consider this approach an important step to foster single-cell analysis in a wide variety of neuron types.
单细胞质谱分析已成为一种研究特定分子特性的成熟技术，例如鉴定神经元的神经肽组成。在这里，我们描述了一种通过 MALDI-TOF 质谱分析鉴定神经元神经化学组成的策略，这些神经元是通过其电生理和神经解剖学特征来鉴定的。该工作流程首次将穿孔膜片钳记录与电穿孔染料加载相结合，以进行电生理和神经解剖学特征分析，以及通过 MALDI-TOF 质谱分析和随后的免疫组织化学对体细胞进行化学特征分析。为了开发我们的方案，我们使用了来自美洲大蠊 Periplaneta americana 的鉴定中央嗅觉神经元。首先，使用具有乙酰胆碱酯酶免疫反应性的单神经球投射神经元的相对同质且特征明确的神经元群体来优化组合方法。该方法的通用性在局部中间神经元上得到了验证，局部中间神经元是一个具有高度分化神经肽组的多样化神经元群体。因此，本研究表明，新建立的方案适合全面分析单个神经元的电生理、神经解剖和分子特性。我们认为这是促进各种神经元类型中单细胞分析的重要步骤。
