The anatomy, organisation and development of contralateral callosal projections of the mouse somatosensory cortex

Abstract

Background: Alterations in the development of neuronal connectivity can result in dramatic outcomes for brain function. In the cerebral cortex, most sensorimotor and higher-order functions require coordination between precise regions of both hemispheres through the axons that form the corpus callosum. However, little is known about how callosal axons locate and innervate their contralateral targets.

Methods: Here, we use a combination of in utero electroporation, retrograde tracing, sensory deprivation and high-resolution axonal quantification to investigate the development, organisation and activity dependence of callosal axons arising from the primary somatosensory cortex of mice.

Results: We show that distinct contralateral projections arise from different neuronal populations and form homotopic and heterotopic circuits. Callosal axons innervate the contralateral hemisphere following a dorsomedial to ventrolateral and region-specific order. Furthermore, we identify two periods of region- and layer-specific developmental exuberance that correspond to initial callosal axon innervation and subsequent arborisation. Early sensory deprivation affects only the latter of these events.

Conclusion: Taken together, these results reveal the main developmental events of contralateral callosal targeting and may aid future understanding of the formation and pathologies of brain connectivity.

Keywords: Corpus callosum; activity dependence; contralateral targeting; cortical development.

Figure 1.

A characterisation of the callosal projections that result from L2/3 S1 in utero electroporation at E15.5. (a) Labelling these cells and examining the brain in the coronal plane at P10 reveals (a′) diffuse labelling of S1, (a′′) a dense projection into the S1/S2 border region, (a′′′) diffuse labelling in S2 and a dense projection into the Ins/PRh cortex. (b)–(b′′′) This pattern is maintained into adulthood (P50). Tangential sections through the (c) electroporated and (d) contralateral hemispheres of a P10 brain confirm a projection to the (c′) ipsilateral Ins/Prh region and that the S1/S2 projection extends along a longer anterior–posterior axis than the Ins/PRh projection in the (d′) contralateral hemisphere. n = 3 animals for each condition. Scale bars: 500 µm for (a) and (b) and (c′) and (d′), 250 µm for (a) and (b) insets, 1000 µm for (c) and (d).

Figure 2.

Cellular origins of S1/S2 and Ins/PRh callosal projections. Retrograde tracing experiments reveal the organisation of the S1/S2 and Ins/PRh projections. (a) DiI/DiD (red/green) injections into the S1/S2 and Ins/PRh cortex showed a generally homotopic pattern of contralaterally labelled cell bodies and (a′) a segregated arrangement within the white matter, where axons projecting to S1/S2 are located more dorsomedially than axons projecting to Ins/PRh. There are, however, some retrogradely labelled cell bodies in the heterotopic location and these predominantly do not colocalise with homotopically labelled cells (a′′ and a′′′; arrowheads). These results are also reflected upon quantification of labelled cell number (b) in different areas and (c) degree of colocalisation. n = 3 animals for each condition. Scale bars: 1000 µm for (a), 500 µm for (a′) and 100 µm for (a′′) and (a′′′).

Figure 3.

The development of contralateral axonal innervation of L2/3 S1 callosal neurons. Photomicrographs and densitometric line scans of axonal fluorescence intensity through S1 (yellow), S1/S2 (red), S2 (purple) and Ins/PRh (green) of mouse brains electroporated into L2/3 of S1 at E15.5 and collected at (a) P4 and (b and b′) P5. A detailed examination of the process of contralateral callosal targeting throughout development was obtained by examining stages (c) P6, (d and d′) P7, (e and e′) P8, (f) P10, (g) P12, (h) P15 and (i) P20. The graphs represent the entire cortical thickness of each layer are divided into six cortical layers that apply to S1, S1/S2 and S2 but not Ins/PRh, which is not a six-layered structure. f/f_{bg tissue}: axonal fluorescence/fluorescence of background tissue. Data are represented as mean ± SEM. n ⩾ 7 animals per age. Scale bars: 1000 µm for (a)–(i) and 100 µm for insets.

Figure 4.

Two periods of temporally, regionally, and layer-specific developmental exuberance in L2/3 S1 callosal neuron projections. Densitometric fluorescence analyses of labelled axons were assessed over nine developmental stages to obtain average traces for each age across the cortical plate in (a) S1, (b) S1/S2, (c) S2 and (d) Ins/PRh across different stages of development. (e)–(h) The average fluorescence intensity of each layer reveals a distinct peak of fluorescence at P7 (red) in L6 of all neocortical areas and possibly lower layers of Ins/PRh. The future sparsely innervated regions (S1 and S2) generally have a linear increase in fluorescence intensity in (i) and (k) upper cortical layers, whereas the densely innervated regions trend towards a peak of fluorescence at P10, followed by (j) and (l) slight retraction. f/f_{bg tissue}: axonal fluorescence/fluorescence of background tissue. Data are represented as mean ± SEM. n ⩾ 7 animals per age.

Figure 5.

Unilateral sensory deprivation inhibits developmental exuberance of axonal arbours but not initial axon targeting. (a)–(d) Unilateral cauterisation at P3 results in no change in axonal innervation in any cortical layer of S1/S2 by P7. (e)–(h) However, the same manipulation causes a significant change in axonal presence in L2/3 and 5 (but not L6) when examined at P10. f/f_{bg tissue}: axonal fluorescence/fluorescence of background tissue. Data are represented as mean ± SEM. n ⩾ 7 animals per condition. Scale bars: 250 µm.