Uptake, intra-axonal transport and fate of horseradish peroxidase in embryonic spinal neurons of the chick

Abstract

Horseradish peroxidase (HRP) was injected in ovo into the ventral muscle mass of the hind limb of 5- to 7-day-old chick embryos or into the gastrocnemius muscle of 8- to 18-day embryos and localized histochemically. HRP is extensively incorporated via endocytosis into axonal growth cones or presynaptic terminals in the proximity of the injection site. Much of the tracer is taken up in vesicles and small vacuoles. Most of these are smooth-surfaced and only a few are bristle-coated. A small amount of the tracer is also incorporated into the axon terminal through the openings between the axolemma and an intricate membrane channel. The majority of the tracer-laden vesicles and vacuoles rapidly fuse with one another to become large vacuoles, some of which are transformed into multivesicular bodies (MVBs). In axon shafts, many labeled vacuoles and MVBs are transferred to tubule-like organelles, which appear to be the primary carrier for transporting the tracer back to the cell bodies in the lumbar spinal cord. HRP arrives in the sensory ganglia about 0.5-1 hour earlier than in the motoneurons of the lateral motor column. The maximal rate of the retrograde axoplasmic transport is about 3.5 mm/hour. After arriving in the cell bodies, HRP is transferred from tubule-like organelles to discrete vacuoles of various sizes and appearance. Lysosomal dense bodies and HRP-labeled vacuoles can be distinguished ultrastructurally. A fusion of HRP-labeled vacuoles with lysosomal dense bodies or Golgi vesicles was occasionally observed and the density of HRP-labeled vacuoles diminished after 2 to 3 days. Most of the HRP-labeled organelles were found to contain acid phosphatase activity. Therefore, the complete disappearance of HRP by 4 days postinjection is most likely related to lysosomal degradation. Neuronal cell bodies diffusely labeled with HRP were only observed prior to day 6. After day 6, despite various attempts to injure the peripheral axons, only granularly labeled cell bodies were found. This difference may imply that "mature" neurons have a more efficient mechanism for the sequestration of "free" HRP in the cytoplasmic matrix into membrane-bounded organelles. A mature-like retrograde transport mechanism appears to exist at the earliest stages of axonal growth in vivo.