A conditional strategy for cell-type-specific labeling of endogenous excitatory synapses in Drosophila.
A conditional strategy for cell-type-specific labeling of endogenous excitatory synapses in Drosophila.
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DOI：
10.1016/j.crmeth.2023.100477
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发表时间：
2023-05-22
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化学神经传递发生在神经递质释放机制与神经递质受体的特殊接触处，神经递质受体是神经回路功能的基础。一系列复杂的事件是突触前和突触后蛋白质向神经元连接募集的基础。为了更好地研究单个神经元的突触发育，我们需要细胞类型特异性策略来可视化内源性突触蛋白。尽管存在突触前策略，但由于缺乏细胞类型特异性试剂，突触后蛋白质的研究仍然较少。为了研究兴奋性突触后的细胞类型特异性，我们设计了dlg1[4K]，这是果蝇兴奋性突触后密度的条件标记物。在二元表达系统中，dlg1[4K]标记了幼虫和成虫的中枢和外周突触后。使用dlg1[4K]，我们发现在成年神经元中突触后组织有不同的规则，多个二进制表达系统可以以细胞类型特异性的方式同时标记突触前和突触后，神经元dlg1有时可以定位突触前。这些结果验证了我们的有条件突触后标记策略，并展示了突触组织的原理。dlg1[4K]是一种体内细胞类型特异性内源性兴奋性突触后标记dlg1[4K]标记多种内源性中枢和外周突触后亚型，dlg1[4K]标记允许定量突触组织/对应分析，与GAL4/QF/lexA系统一起使用可以并发进行突触前和突触后标记。在果蝇中，通过优秀的细胞类型特异性突触前标记来理解突触前组织已经取得了相当大的进展。但该领域缺乏一般的细胞类型特异性突触后标签。dlg1[4K]是一种可靠的、细胞类型特异性的、普遍的兴奋性突触后标签，用于果蝇的电路定位，是突触前标签的重要对应物。dlg1[4K]与多个突触一起工作，使定量电路水平和突触结构分析能够更好地理解神经发育和神经解剖学。这项工作是用于细胞类型特异性标记的遗传工具的关键进步，填补了神经元研究技术的空白。研究神经元回路需要在确定的神经元亚群中同时进行突触前和突触后标记。Parisi等人创建了dlg1[4K]，这是一种经过crispr修饰的dlg1位点，通过细胞类型特异性二进制表达在体内兴奋性突触后有条件地标记dlg1。dlg1[4K]标记使以前不可用的突触后标记和增强定量电路分析成为可能。
Chemical neurotransmission occurs at specialized contacts where neurotransmitter release machinery apposes neurotransmitter receptors to underlie circuit function. A series of complex events underlies pre- and postsynaptic protein recruitment to neuronal connections. To better study synaptic development in individual neurons, we need cell-type-specific strategies to visualize endogenous synaptic proteins. Although presynaptic strategies exist, postsynaptic proteins remain less studied because of a paucity of cell-type-specific reagents. To study excitatory postsynapses with cell-type specificity, we engineered dlg1[4K], a conditionally labeled marker of Drosophila excitatory postsynaptic densities. With binary expression systems, dlg1[4K] labels central and peripheral postsynapses in larvae and adults. Using dlg1[4K], we find that distinct rules govern postsynaptic organization in adult neurons, multiple binary expression systems can concurrently label pre- and postsynapse in a cell-type-specific manner, and neuronal DLG1 can sometimes localize presynaptically. These results validate our strategy for conditional postsynaptic labeling and demonstrate principles of synaptic organization. dlg1[4K] is an in vivo cell-type-specific endogenous excitatory postsynapse label dlg1[4K] labels multiple subtypes of endogenous central and peripheral postsynapses The dlg1[4K] label permits quantitative synaptic organization/apposition analyses Use with GAL4/QF/lexA systems enables concurrent pre- and postsynaptic labeling In Drosophila, considerable advances have been made to understand presynaptic organization via excellent cell-type-specific presynaptic labels, but the field suffered from a lack of general cell-type-specific postsynaptic labels. dlg1[4K] is a reliable, cell-type-specific, general excitatory postsynaptic label for circuit mapping in Drosophila and an essential counterpoint to presynaptic labels. dlg1[4K] works with multiple synapses to enable quantitative circuit-level and synaptic architectural analysis to better understand neurodevelopment and neuroanatomy. The work is a critical advance in genetic tools for cell-type-specific labeling and fills a gap in the technologies available for neuronal study. Studying neuronal circuits requires concurrent pre- and postsynaptic labeling in identified subsets of neurons. Parisi et al. create dlg1[4K], a CRISPR-modified dlg1 locus that conditionally labels DLG1 at excitatory postsynapses in vivo using cell-type-specific binary expression. The dlg1[4K] label enables previously unavailable postsynaptic labeling and enhances quantitative circuit analysis.