Interstitial branch formation within the red nucleus by deep cerebellar nuclei‐derived commissural axons during target recognition

Abstract

Target recognition by developing axons is one of the fundamental steps for establishing the proper pattern of neuronal connectivity during development. However, knowledge of the mechanisms that underlie this critical event is still limited. In this study, to examine how commissural axons in vertebrates recognize their targets after crossing the midline, we analyzed in detail the behavior of postcrossing commissural axons derived from the deep cerebellar nuclei (DCN) in the developing mouse cerebellum. For this, we employed a cell-type-specific genetic labeling approach to selectively visualize DCN axons during the time when these axons project to the red nucleus (RN), one of the well-characterized targets of DCN axons. We found that, when DCN axons initially entered the RN at its caudal end, these axons continued to grow rostrally through the RN without showing noticeable morphological signs of axon branching. Interestingly, after a delay, DCN axons started forming interstitial branches from the portion of the axon shaft selectively within the RN. Because commissural axons acquire responsiveness to several guidance cues when they cross the midline, we further addressed whether midline crossing is a prerequisite for subsequent targeting by using a Robo3 knockdown strategy. We found that DCN axons were still capable of forming interstitial branches within the RN even in the absence of midline crossing. These results therefore suggest that the mechanism of RN recognition by DCN axons involves a delayed interstitial branching, and that these axons possess an intrinsic ability to respond to the target-derived cues irrespective of midline crossing.

Normal-Appearing Cerebellar Damage in Neuromyelitis Optica Spectrum Disorder

“…Spinal neurons can directly connect to cerebellar nuclei, especially the interposed nucleus, via the spinocerebellar tracts and spinoreticulocerebellar tracts. 23 Moreover, the cerebellar nuclei also indirectly connect with spinal neurons via the cerebellar cortices 24 and brain stem nuclei, such as the inferior olive, 25 lateral reticular nucleus, 26 red nucleus, 27 and vestibular nuclei. 28 Based on the direct and indirect connections between the cerebellar nuclei and spinal neurons, the axonal degeneration secondary to spinal cord damage may explain why the NAGM volume of the cerebellar nuclei is reduced in NMOSD.…”

Post‐crossing segment of dI1 commissural axons forms collateral branches to motor neurons in the developing spinal cord

“…To visualize the trajectory of dI1 commissural axons with GFP, in vivo electroporation was performed as described (Saito, ; Inamata & Shirasaki, ; Hara et al, ). Briefly, we used exo utero procedure for in vivo electroporation in order to inject plasmid vectors precisely into the central canal of the spinal cord.…”

“…To address this issue, we analyzed in detail the behavior of post‐crossing dI1 axons in mice, using the Atoh1 enhancer element‐based conditional expression system that enables selective and sparse labeling of individual post‐crossing dI1 axons (Hara, Kaneyama, Inamata, Onodera, & Shirasaki, ). Moreover, we combined this genetic labeling approach with Hb9 and ChAT immunohistochemistry for precise identification of MNs in the developing spinal cord (e.g., Phelps et al, ; Arber et al, ; Thaler et al, ).…”

Anatomical development of the cerebellothalamic tract in embryonic mice

“…5). At E14.5 the CbT did not yet reach diencephalic structures (data not shown; (Hara et al, 2016). At E15.5 and E16.5 the CbT is positioned dorsally in the mesencephalic curvature and has progressed beyond the red nucleus to reach the prethalamus (Fig.…”

“…Although it is well understood that cerebellar nuclei (CN) neurons are the sole source of the CbT tract, there are few studies available on the development of their axonal connections to the thalamic complex. Some sparse reports on a developing CbT tract in the post-natal opossum (Martin et al, 1987), rat (Cholley et al, 1989;Shirasaki et al, 1995) and a single report in mouse embryo (Hara et al, 2016) describe the timing of CN innervation of midbrain nuclei, such as the parabrachial and red nuclei, and subsequently commence to the thalamic primordium, but no data on the ontogeny of cerebellar innervation of the thalamic complex is currently available. Given that early synaptic afferents have recently been shown to modulate thalamic activity patterns, gene expression profiles and the thalamocortical connectivity (Mire et al, 2012;Chou et al, 2013;Moreno-Juan et al, 2017) it is of utmost importance to elucidate at what embryonic stage cerebellar axons start to innervate the developing thalamus.…”