A pan-mammalian map of interhemispheric brain connections predates the evolution of the corpus callosum

Abstract

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.

Keywords: anterior commissure; claustrum; corpus callosum; cortical connectome; diffusion tensor MRI.

Fig. 1.

Evolution of cortical commissures. (A) Cladogram of birds and mammals showing the origin of the neocortex (NCx) and the corpus callosum (cc). (B) Coronal brain schematics. The anterior commissure (ac) of pigeons contains mostly unidirectional projections from the ventral arcopallium (Av) to contralateral homotopic and heterotopic targets, including the mesopallium (M), nidopallium (N), and hyperpallium intercalatum (HI). In eutherians, callosal axons are topographically segregated and connect mostly homotopic regions, whereas the ac carries axons from the piriform cortex (Pir) and lateral NCx. Homologous circuits in noneutherians are carried exclusively through the ac.

Fig. 2.

Segregation of commissural fibers by cortical areas in marsupials. (A) HARDI tractography of the dunnart brain (marsupial) using a spherical ROI at the anterior commissure (ac, cyan) and inclusion ROIs at seven cortical regions (spheres), reveal segregated tracts into color-coded homotopic domains. (B) Midsagittal dunnart MRI scan showing five hand-drawn ROIs within the anterior commissure (Inset) that generate the color-coded tracts in C. (C) Tracts generated by ROIs in B reveal contralateral homotopy of the main cortical areas. (D and E) Coronal sections of dunnart brains after ex vivo injections of DiI (red) and DiD (green) in the regions indicated by asterisks. (F) Comparable coronal views of fiber segregation within the ac, as revealed by MRI and histology (from top down, high-magnification views of the areas shown in A, D, and E, respectively). AON, anterior olfactory nucleus; Cg/M, cingulate/motor cortex; Ent, entorhinal cortex; Fr, frontal cortex; ic, internal capsule; NCx, neocortex; OB, olfactory bulb; Olf, olfactory branch; Pir, piriform cortex; S1, primary somatosensory cortex; S1HL, S1 hindlimb; S1Md, S1 mandible; S1My, S1 mystacial whiskers. (Scale bars, 1 mm in D and E and 200 μm in F.)

Fig. 3.

Topography of the anterior commissure of the platypus. (A) T1-weighted coronal series of a fixed platypus brain showing color-coded tracts between cortical ROIs (small spheres) and the anterior commissure (ac, large sphere). (B) Higher-magnification coronal views of the tracts generated in A as viewed from the front (Top) and the back (Bottom). (C) Midsagittal view of the platypus brain and the anterior commissure (Inset) showing the parcellation of ROIs that generate the tracts in D. (D) Interhemispheric tracts across ROIs of C, showing color-coded homotopic domains. A, anterior; D, dorsal; Ent, entorhinal cortex; L, lateral; Pir, piriform cortex; R, rostral somatosensory cortex; Rhs, R head-shield; Rub, R upper-bill; S1cub, primary somatosensory cortex central upper-bill; S1ub, primary somatosensory cortex upper-bill. (Scale bar, 4 mm.)

Fig. 4.

Homotopic and heterotopic commissural neurons in marsupials resemble the callosal connectome of eutherians. (A) Coronal sections of dunnart brains showing a summary of retrograde tracer injection sites (DAPI and color-coded in the Left) and the contralateral position of cell bodies (dots). (B) Connectivity matrix combining injection sites (columns; approximate brain positions on Top Left) and percentage of labeled neurons per brain area (rows). aMed, anterior medial cortex; aSom, anterior somatosensory cortex; Fr/Orb, frontal/orbital cortices; Lat, lateral cortex; pMed, posterior medial cortex; pSom, posterior somatosensory cortex; Olf/Pir, olfactory/piriform cortices; Occip, occipital cortex. (Scale bar, 1 mm.)

Fig. 5.

Heterotopic claustro-cortical commissural connections in dunnarts. (A) A posteromedial CTB injection and contralaterally projecting neurons across the rostrocaudal extent (B) in mm from Bregma. (C–E) Insets of the regions highlighted in B include neurons in the posterior cingulate (pCg), retrosplenial (RS), and motor (M) cortices in C, the claustrum (Cl) and insula (Ins) in D, and the frontal (Fr) and orbital cortices (Orb) in E. (Scale bars: 1 mm in B, and 250 μm in C and D.)

Fig. 6.

Anterograde mapping of contralateral projections in dunnarts reveals a conserved interhemispheric connectome. (A–F) In-pouch electroporated neurons are shown in the left hemisphere and their axon terminals in the right hemisphere in six different cases. Homotopic targets are shown with arrowheads, and Insets show heterotopic terminals in the contralateral motor/cingulate cortex. To reveal the cell bodies and their axons, the hemispheres have different brightness/contrast levels in A–C and F. AON/OB, anterior olfactory nucleus/olfactory bulb; FrA, anterior frontal cortex; PRh/Ent, perirhinal/entorhinal cortex; S1/M, primary somatosensory/motor cortex; S2/Aud, secondary somatosensory/auditory cortex. (Scale bar, 1 mm.)

Fig. 7.

Intermingled populations of homotopic and heterotopic commissural neurons in dunnarts. (A and B) Double retrograde injections with CTB647 and CTB555 and/or DiI and DiD in the medial and lateral cortices result in intermingled cell bodies in the contralateral homotopic cortices (C and D) with very few double-labeled cells (arrowheads). (E) The proportion of cells that project exclusively to homotopic (Hom), heterotopic (Het) and to both targets (double-labeled cells) over the total number of cells labeled in the section, differ between the medial and lateral cortices (mean + SEM, n = 5; *P < 0.026). (Scale bars, 1 mm in B, 100 μm in H.)

Fig. 8.

Heterotopic circuits and a pan-mammalian connectome. (A) Coronal dunnart brain section stained against Nurr1 (red), Ctip2 (green), and DAPI (blue) outline the claustrum (Cl) from the striatum (St), white matter (wm), and insula (Ins). (B and C) Axon terminals in the claustrum (Cl) were differentially found after electroporations in the perirhinal (PRh, B) or somatosensory (S1, C) cortices of the contralateral hemisphere. (D) Schematic of a coronal brain section representing the features of an interhemispheric connectome likely conserved by all mammals. Neocortical (NCx) neurons from layers 2/3 and 5 project to contralateral homotopic regions (1), following a topographic arrangement of axons across the midline (2), whereas hyperconnected and heterotopic circuits include the cingulate-motor (Cg/M) and insular (Ins) cortices, as well as the claustrum (3). rf, rhinal fissure. (Scale bars, 250 μm.)