The brain of mammals differs from that of all other vertebrates, in having a six-layered neocortex that is extensively interconnected within and between hemispheres. Interhemispheric connections are conveyed through the anterior commissure in egg-laying monotremes and marsupials, whereas eutherians evolved a separate commissural tract, the corpus callosum. Although the pattern of interhemispheric connectivity via the corpus callosum is broadly shared across eutherian species, it is not known whether this pattern arose as a consequence of callosal evolution or instead corresponds to a more ancient feature of mammalian brain organization. Here we show that, despite cortical axons using an ancestral commissural route, monotremes and marsupials share features of interhemispheric connectivity with eutherians that likely predate the origin of the corpus callosum. Based on ex vivo magnetic resonance imaging and tractography, we found that connections through the anterior commissure in both fat-tailed dunnarts (Marsupialia) and duck-billed platypus (Monotremata) are spatially segregated according to cortical area topography. Moreover, cell-resolution retrograde and anterograde interhemispheric circuit mapping in dunnarts revealed several features shared with callosal circuits of eutherians. These include the layered organization of commissural neurons and terminals, a broad map of connections between similar (homotopic) regions of each hemisphere, and regions connected to different areas (heterotopic), including hyperconnected hubs along the medial and lateral borders of the cortex, such as the cingulate/motor cortex and claustrum/insula. We therefore propose that an interhemispheric connectome originated in early mammalian ancestors, predating the evolution of the corpus callosum. Because these features have been conserved throughout mammalian evolution, they likely represent key aspects of neocortical organization.

哺乳动物的大脑与所有其他脊椎动物的大脑不同，其具有六层新皮质，在半球内部和半球之间广泛互连。产卵的单孔类动物和有袋动物的半球间连接是通过前连合传递的，而真兽类则进化出一个单独的连合束，即胼胝体。尽管通过胼胝体的半球间连接模式在真兽类物种中广泛存在，但尚不清楚这种模式是胼胝体进化的结果，还是对应于哺乳动物大脑组织的更古老的特征。在这里，我们表明，尽管皮质轴突使用祖先的连合路线，但单孔类动物和有袋动物与真兽类共享半球间连接的特征，这些特征可能早于胼胝体的起源。基于离体磁共振成像和纤维束成像，我们发现肥尾袋鼠（Marsupialia）和鸭嘴兽（Monotremata）通过前连合的连接根据皮质区域地形在空间上分离。此外，邓纳特动物的细胞分辨率逆行和顺行半球间回路映射揭示了与真兽类胼胝体回路共有的几个特征。这些包括连合神经元和末梢的分层组织、每个半球的相似（同位）区域之间的广泛连接图，以及连接到不同区域（异位）的区域，包括沿皮质内侧和外侧边界的超连接中枢，例如如扣带皮层/运动皮层和屏状核/岛叶。因此，我们认为半球间连接组起源于早期哺乳动物祖先，早于胼胝体的进化。由于这些特征在哺乳动物的进化过程中一直得到保留，因此它们可能代表了新皮质组织的关键方面。