Chondroitin sulfates enriched in the developing rat hindbrain restrict commissural projections of vestibular neurons

Abstract

In vitro studies have shown that chondroitin sulfate proteoglycans are inhibitory to axon outgrowth and constitute barriers to pathfinding axons. To assess if chondroitin sulfates (CSs) are involved in limiting axonal projections in the early embryonic hindbrain, chondroitinase ABC was delivered into the 4th ventricles of rat embryos (E11.5–E13.5) in culture (24 h, 37 ° C) to digest away CS moieties in the nearby hindbrain matrix. Controls were treated instead with the PBS-vehicle or the heat-inactivated enzyme. The resulting commissural projections after the treatments were mapped with DiI which was introduced into the vestibular nuclear complex (VNC) near the VIIIth cranial nerve entry zone. At E11.5 (+1DIV) , ^ 5 neurites could be traced extending half of the way towards the midline. With the removal of CSs, a robust outgrowth of axons extended more than half of the way towards the midline. This suggests that CSs limit axonal outgrowth in early embryos. At E12.5 (+1 DIV) , the commissural projections assumed fascicles that reached the midline in the controls. In the enzyme-treated embryos, the axons however were unfasciculated as they extended normal to the midline. This suggested that CSs are important in limiting and guiding the course of these pioneering axons. By E13.5 (+1 DIV) , enzyme treatment apparently did not affect the pioneer axons that had advanced as thick fascicles normal to the midline. However, later axonal projections from the VNC that took place after the injection of enzyme were defasciculated as they traversed the enzyme-treated matrix. They were diverted from the course of the pioneers at various angles and positions when crossing the midline. This suggests that CSs also limit the course of the later projections which otherwise would be attracted to alternative targets. Recovery of proteoglycans from the early hindbrain and analysis of the CS components found enrichment in 6-sulfated isoforms of chondroitin as early as E11.5. These results provide in vivo evidence for possible contributions of 6-sulfated chondroitins to limit stray outgrowth and foster axonal fasciculation as vestibular neurons project across the midline towards the contralateral target.