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.

The role of estrogen in the developmental appearance of sensory-motor behaviors in the zebrafish (Danio rerio): the characterization of the "listless" model.

The brain is a steroidogenic organ and is thus dependent on estrogen for many aspects of its development and maintenance in both males and females. The purpose of this study was to develop a model to investigate the effect of estrogen on zebrafish sensory-motor (S-M) maturation through mechanisms found in the central nervous (CNS) and peripheral nervous (PNS) systems. In these experiments the aromatase inhibitor (AI), 4-hydroxy androstenedione (4-OH-A), which blocks estrogen synthesis, was used to diminish estrogen's effects on zebrafish CNS and PNS development. During these various treatments the zebrafish were analyzed for neurological deficits, including tactile response, swimming movements, vestibular behavior, pectoral fin and eye movements. Over a three to five day time period (48-168 h post fertilization), in response to AI treatment, none of these S-M behaviors were developmentally expressed creating a "listless" or non-responding condition. Furthermore, when the AI was removed from the treatment medium the S-M behaviors were fully expressed over a two to three day time period. Most importantly, when estrogen was added at the same time as the AI in a co-treatment paradigm, normal developmental appearance of S-M behaviors was rescued in all neurological parameters measured. Furthermore, the addition of estrogen alone after AI washout accelerated the recovery of the tactile response during the first 24 h of treatment. Treatment of developing zebrafish with the selective estrogen receptor blocker ICI 182,780 mimicked the deficit in S-M behaviors caused by AI treatment. This deficit was overcome by low concentrations of estrogen in a co-treatment paradigm with high ICI levels indicating the possibility of a non-genomic mechanism for estrogen's actions on the developmental expression of these S-M behaviors. Eventually, AI exposed fish died of cardiac arrest 4 to 5 days after the start of treatment; however, AI/estrogen co-treatment allowed for 90-100% survival and the maintenance of normal heart rates during this time period. In conclusion, these studies have demonstrated that the presence of estrogen in the early developing zebrafish embryo is necessary for the proper developmental expression of critical nervous system S-M behaviors necessary for survival, as well as the health of the cardiovascular system. These studies also establish a unique "listless" model for further analysis of estrogen's role in the development of brain, brainstem, and spinal cord circuitry related to the maturation of these behavioral and cardiovascular phenomena.