Effects of estrogen on the neuromuscular system in the embryonic zebrafish (Danio rerio)

Estrogen (E₂) has been shown to play an important role in maintaining central nervous system (CNS) axonal growth, synapse formation, and neurotransmitter release; however, there is less direct evidence for a similar role in the peripheral nervous system (PNS). In a previous study we have shown that when E₂ was removed from embryonic zebrafish (Danio rerio) system using the aromatase inhibiter (AI) 4-hydroxyandrostenedione (4-OH-A) fish did not developmentally express normal sensory-motor (S-M) functions such as tactile, vestibular, and swimming behaviors, creating a condition called 'listless.' These findings led to speculation that E₂ deprivation, under these conditions, caused a neuromuscular-like "denervation" resulting in the 'listless' condition. Morphometric data analysis reported in this study indicated that there was an absence of vesicular acetylcholine transporter (VAChT) staining in the primary motor neurons as a result of AI treatment compared to controls. In contrast, E₂ co-treatment with AI (E₂+AI) rescued a significant number of VAChT stained nerve endings and treatment of fish with E₂ alone exhibited a significantly higher number of VAChT profiles than in control fish. In addition, in the AI treated group znp-1 antibody staining of the primary motor neurons demonstrated: 1) diminished axon branching; 2) shorter primary axons; and 3) an absence in the posterior trunk regions of fish. In turn, trunk muscles were significantly diminished in size and less organized when treated with AI when compared to controls and E₂+AI treatment restored myotome width and height accompanied by some dramatic changes in the α-bungarotoxin-labeled ACh post-synaptic receptor elements of the trunk skeletal muscles. Data from this study suggest that treatment with the AI 4-OH-A essentially denervates the zebrafish trunk skeletal muscles, most likely by compromising the development of the vesicular transport system for ACh preventing it from acting at the synaptic terminals. These findings begin to demonstrate the prominent role that E₂ plays in the developing zebrafish PNS, particularly at the neuromuscular level.