Abstract
Many studies of axon regeneration in the lamprey focus on 18 pairs of large identified reticulospinal (RS) neurons, whose regenerative abilities have been individually quantified. Their axons retract during the first 2 weeks after transection (TX), and many grow back to the site of injury by 4 weeks. However, locomotor movements begin before 4 weeks and the lesion is invaded by axons as early as 2 weeks post-TX. The origins of these early regenerating axons are unknown. Their identification could be facilitated by studies in central nervous system (CNS) wholemounts, particularly if spatial resolution and examination by confocal microscopy were not limited by light scattering. We have used benzyl alcohol/benzyl benzoate (BABB) clearing to enhance the resolution of neuronal perikarya and regenerated axons by confocal microscopy in lamprey CNS wholemounts, and to assess axon regeneration by retrograde and anterograde labeling with fluorescent dye applied to a second TX caudal or rostral to the original lesion, respectively. We found that over 50% of the early regenerating axons belonged to small neurons in the brainstem. Some propriospinal neurons located close to the TX also contributed to early regeneration. The number of early regenerating propriospinal neurons decreased with distance from the original lesion. Descending axons from the brainstem were labeled anterogradely by application of tracer to a second TX close to the spinal–medullary junction. This limited contamination of the data by regenerating spinal axons whose cell bodies are located rostral or caudal to the TX and confirmed the regeneration of many small RS axons as early as 2 weeks post-TX. Compared with the behavior of axotomized giant axons, the early regenerating axons were of small caliber and showed little retraction, probably because they resealed rapidly after injury.
Highlights
The lamprey central nervous system (CNS) has been used extensively to study the mechanisms of axon regeneration after spinal cord (SC) injury, in part because it has identified neurons and neuron types that can be imaged in vivo
Lampreys were deeply anesthetized by immersion in saturated aqueous benzocaine until motionless to tail pinch, pinned onto a Sylgard-coated dissecting dish filled with ice-cold lamprey Ringer [14]
After fluorescent images were captured with widefield microscopy, benzyl alcohol/benzyl benzoate (BABB) was removed by rehydration in increasing concentrations of ethanol (70%, 80%, 90%, 95%, and 100%)
Summary
The lamprey central nervous system (CNS) has been used extensively to study the mechanisms of axon regeneration after spinal cord (SC) injury, in part because it has identified neurons and neuron types that can be imaged in vivo. The lamprey’s normal symmetrical body locomotion is controlled by central pattern generators in the SC, which are activated by reticulospinal (RS) projections from brainstem [1]. Because these connections are lost after SC transection (TX), the body below the TX site becomes paralyzed. Cells 2020, 9, 2427 these neurons have suggested that their axons retract during the first 2 weeks after TX, grow back toward the TX site. They reach the glial “scar” by 3 weeks, enter the lesion site by 4 weeks, and regenerate into the caudal stump thereafter [7,8,9]. Physiological studies showed that, immediately after SC TX, locomotor muscle activity can be recorded only from segments rostral to the lesion, the activity can already be recorded from segments just caudal to the TX by 2 weeks post-TX; and with increasing recovery times, at progressively more caudal levels [13]
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