Oxytocin (Oxt) neurons regulate diverse physiological responses via direct connections with different neural circuits. However, the lack of comprehensive input-output wiring diagrams of Oxt neurons and their quantitative relationship with Oxt receptor (Oxtr) expression presents challenges to understanding circuit-specific Oxt functions. Here, we establish a whole-brain distribution and anatomic connectivity map of Oxt neurons, and their relationship with Oxtr expression using high-resolution 3D mapping methods in adult male and female mice. We use a flatmap to describe Oxt neuronal expression in four hypothalamic domains including under-characterized Oxt neurons in the tuberal nucleus (TU). Oxt neurons in the paraventricular hypothalamus (PVH) broadly project to nine functional circuits that control cognition, brain state, and somatic visceral response. In contrast, Oxt neurons in the supraoptic (SO) and accessory (AN) nuclei have limited central projection to a small subset of the nine circuits. Surprisingly, quantitative comparison between Oxt output and Oxtr expression showed no significant correlation across the whole brain, suggesting abundant indirect Oxt signaling in Oxtr-expressing areas. Unlike output, Oxt neurons in both the PVH and SO receive similar monosynaptic inputs from a subset of the nine circuits mainly in the thalamic, hypothalamic, and cerebral nuclei areas. Our results suggest that PVH-Oxt neurons serve as a central modulator to integrate external and internal information via largely reciprocal connection with the nine circuits while the SO-Oxt neurons act mainly as unidirectional Oxt hormonal output. In summary, our Oxt wiring diagram provides anatomic insights about distinct behavioral functions of Oxt signaling in the brain.

SIGNIFICANCE STATEMENT Oxytocin (Oxt) neurons regulate diverse physiological functions from prosocial behavior to pain sensation via central projection in the brain. Thus, understanding detailed anatomic connectivity of Oxt neurons can provide insight on circuit-specific roles of Oxt signaling in regulating different physiological functions. Here, we use high-resolution mapping methods to describe the 3D distribution, monosynaptic input and long-range output of Oxt neurons, and their relationship with Oxt receptor (Oxtr) expression across the entire mouse brain. We found Oxt connections with nine functional circuits controlling cognition, brain state, and somatic visceral response. Furthermore, we identified a quantitatively unmatched Oxt-Oxtr relationship, suggesting broad indirect Oxt signaling. Together, our comprehensive Oxt wiring diagram advances our understanding of circuit-specific roles of Oxt neurons.

催产素 (Oxt) 神经元通过与不同神经回路的直接连接调节不同的生理反应。然而，缺乏 Oxt 神经元的综合输入输出接线图及其与 Oxt 受体 (Oxtr) 表达的定量关系对理解电路特定的 Oxt 功能提出了挑战。在这里，我们建立了 Oxt 神经元的全脑分布和解剖连接图，以及它们与 Oxtr 表达的关系，使用成年雄性和雌性小鼠的高分辨率 3D 映射方法。我们使用平面图来描述四个下丘脑区域的 Oxt 神经元表达，包括结节核 (TU) 中未充分表征的 Oxt 神经元。室旁下丘脑 (PVH) 中的 Oxt 神经元广泛投射到九个控制认知、大脑状态、和躯体本能反应。相比之下，视上核 (SO) 和辅助核 (AN) 中的 Oxt 神经元将中央投射限制在九个回路的一小部分。令人惊讶的是，Oxt 输出和 Oxtr 表达之间的定量比较显示整个大脑没有显着相关性，表明 Oxtr 表达区域存在丰富的间接 Oxt 信号。与输出不同，PVH 和 SO 中的 Oxt 神经元从主要位于丘脑、下丘脑和大脑核区的九个回路的一个子集中接收相似的单突触输入。我们的结果表明，PVH-Oxt 神经元作为一个中央调制器，通过与九个回路的主要相互连接来整合外部和内部信息，而 SO-Oxt 神经元主要作为单向 Oxt 激素输出。总之，

重要性声明催产素 (Oxt) 神经元通过大脑中的中央投射调节从亲社会行为到痛觉的各种生理功能。因此，了解 Oxt 神经元的详细解剖连接性可以深入了解 Oxt 信号在调节不同生理功能中的电路特异性作用。在这里，我们使用高分辨率映射方法来描述 Oxt 神经元的 3D 分布、单突触输入和远程输出，以及它们与整个小鼠大脑中 Oxt 受体 (Oxtr) 表达的关系。我们发现 Oxt 与控制认知、大脑状态和躯体内脏反应的九个功能回路有关。此外，我们确定了一个数量上不匹配的 Oxt-Oxtr 关系，表明广泛的间接 Oxt 信号。一起，