Despite the importance of postsynaptic inhibitory circuitry targeted by mid/long-range projections (e.g., top-down projections) in cognitive functions, its anatomical properties, such as laminar profile and neuron type, are poorly understood owing to the lack of efficient tracing methods. To this end, we developed a method that combines conventional adeno-associated virus (AAV)-mediated transsynaptic tracing with a distal-less homeobox (Dlx) enhancer-restricted expression system to label postsynaptic inhibitory neurons. We called this method “Dlx enhancer-restricted Interneuron-SpECific transsynaptic Tracing” (DISECT). We applied DISECT to a top-down corticocortical circuit from the secondary motor cortex (M2) to the primary somatosensory cortex (S1) in wild-type mice. First, we injected AAV1-Cre into the M2, which enabled Cre recombinase expression in M2-input recipient S1 neurons. Second, we injected AAV1-hDlx-flex-green fluorescent protein (GFP) into the S1 to transduce GFP into the postsynaptic inhibitory neurons in a Cre-dependent manner. We succeeded in exclusively labeling the recipient inhibitory neurons in the S1. Laminar profile analysis of the neurons labeled via DISECT indicated that the M2-input recipient inhibitory neurons were distributed in the superficial and deep layers of the S1. This laminar distribution was aligned with the laminar density of axons projecting from the M2. We further classified the labeled neuron types using immunohistochemistry and in situ hybridization. This post hoc classification revealed that the dominant top-down M2-input recipient neuron types were somatostatin-expressing neurons in the superficial layers and parvalbumin-expressing neurons in the deep layers. These results demonstrate that DISECT enables the investigation of multiple anatomical properties of the postsynaptic inhibitory circuitry.

中文翻译：

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通过增强子限制的突触追踪对皮质自上而下受体抑制电路的解剖学识别

尽管中/远程预测（例如自上而下的预测）所针对的突触后抑制电路在认知功能中很重要，但由于缺乏有效的追踪方法，对其解剖学特性（例如层状分布和神经元类型）知之甚少。 。为此，我们开发了一种方法，将传统的腺相关病毒（AAV）介导的突触追踪与远端无同源盒（Dlx）增强子限制表达系统相结合来标记突触后抑制神经元。我们将此方法称为“Dlx 增强子限制的中间神经元特异性跨突触追踪”(DISECT)。我们将 DISECT 应用于野生型小鼠中从次级运动皮层 (M2) 到初级体感皮层 (S1) 的自上而下的皮质回路。首先，我们将 AAV1-Cre 注射到 M2 中，从而使 Cre 重组酶在 M2 输入受体 S1 神经元中表达。其次，我们将 AAV1-hDlx-flex-green 荧光蛋白 (GFP) 注射到 S1 中，以 Cre 依赖性方式将 GFP 转导到突触后抑制神经元中。我们成功地专门标记了 S1 中的受体抑制神经元。通过 DISECT 标记的神经元的层状分布分析表明，M2 输入受体抑制神经元分布在 S1 的浅层和深层。这种层流分布与从 M2 突出的轴突的层流密度一致。我们使用免疫组织化学进一步对标记的神经元类型进行分类就地杂交。这事后分类显示，主要的自上而下 M2 输入受体神经元类型是表层表达生长抑素的神经元和深层表达小白蛋白的神经元。这些结果表明 DISECT 能够研究突触后抑制电路的多种解剖特性。