Brain Aromatase Modulates Serotonergic Neuron by Regulating Serotonin Levels in Zebrafish Embryos and Larvae.

Zulvikar Syambani Ulhaq,Mitsuyo Kishida

doi:10.3389/fendo.2018.00230

Abstract

Teleost fish are known to express two isoforms of P450 aromatase, a key enzyme for estrogen synthesis. One of the isoforms, brain aromatase (AroB), cyp19a1b, is highly expressed during early development of zebrafish, thereby suggesting its role in brain development. On the other hand, early development of serotonergic neuron, one of the major monoamine neurons, is considered to play an important role in neurogenesis. Therefore, in this study, we investigated the role of AroB in development of serotonergic neuron by testing the effects of (1) estradiol (E2) exposure and (2) morpholino (MO)-mediated AroB knockdown. When embryos were exposed to E2, the effects were biphasic. The low dose of E2 (0.005 µM) significantly increased serotonin (5-HT) positive area at 48 hour post-fertilization (hpf) detected by immunohistochemistry and relative mRNA levels of tryptophan hydroxylase isoforms (tph1a, tph1b, and tph2) at 96 hpf measured by semi-quantitative PCR. To test the effects on serotonin transmission, heart rate and thigmotaxis, an indicator of anxiety, were analyzed. The low dose also significantly increased heart rate at 48 hpf and decreased thigmotaxis. The high dose of E2 (1 µM) exhibited opposite effects in all parameters. The effects of both low and high doses were reversed by addition of estrogen receptor (ER) blocker, ICI 182,780, thereby suggesting that the effects were mediated through ER. When AroB MO was injected to fertilized eggs, 5-HT-positive area was significantly decreased, while the significant decrease in relative tph mRNA levels was found only with tph2 but not with two other isoforms. AroB MO also decreased heart rate and increased thigmotaxis. All the effects were rescued by co-injection with AroB mRNA and by exposure to E2. Taken together, this study demonstrates the role of brain aromatase in development of serotonergic neuron in zebrafish embryos and larvae, implying that brain-formed estrogen is an important factor to sustain early development of serotonergic neuron.

Highlights

Biosynthesis of estrogen is catalyzed by the action of cytochrome P450 aromatase, a product of cyp19a1 gene [1, 2]
We demonstrated that exogenous administration of E2 biphasically affected parameters such as 5-HT-positive areas, relative expression of tph isoforms, heart rate and thigmotactic behavior with stimulation and suppres sion of serotonin system at the low dose and the high dose, Figure 4 | AroB MO-mediated effect on serotonergic neuron. 5-HT-positive neuron area was measured in 2-dpf embryos; (A) injection of AroB MO and control MOs (Std MO, inverted AroB MO (InvB MO) and AroA MO); (B,C) co-injection of AroB mRNA and exposure to E2, respectively, to rescue the effect of AroB MO; (D) co-injection of p53 MO to test off-target effect of AroB MO. (E) Semi-quantitative PCR measurement of expression of tph isoforms in 6-dpf larvae injected with AroB MO with and without exposure to E2
Activities of serotonergic neurons were suppressed by AroB MO-mediated knockdown, suggesting that brain-formed E2 in early develop ment stimulates serotonergic neurons, which is in accordance with the results of the low-dose E2

Summary

Introduction

Biosynthesis of estrogen is catalyzed by the action of cytochrome P450 aromatase, a product of cyp19a1 gene [1, 2]. Wide spread proliferation zones are detected in zebrafish brain [6, 9], while only limited areas such as subependymal and subgranular zones exhibit proliferation in mammals [7]. Such high neurogenic activity in teleost fish may be attributed to increased synthesis of estrogen due to the elevated expression of brain aromatase. Developmental studies in zebrafish show that expression of brain aromatase in embryos increases rapidly after 12 hour post-fertilization (hpf), and is regulated by posi tive feedback loop through its own product, estrogen, acting on estrogen response element of cyp19a1b [3, 13, 14]. The zebrafish model expressing elevated levels of brain aromatase in early development is suitable to investigate the functional sig nificance of aromatase and neural estrogen in developing brain

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