5-HT2A-mediated signaling regulates spontaneous waves and 5-HT cell number in the embryonic mouse hindbrain

Abstract

The mouse hindbrain experiences waves of spontaneous depolarizations early in the development of the nervous system. Hindbrain cells along the ventral midline of the neural tube participate in waves for three days with no known functional purpose. The window of activity temporally coincides with the neurogenesis of the hindbrain’s serotonergic population. The Bosma lab has previously implicated serotonin (5-HT) signaling in regulating hindbrain activity. The present study has verified through calcium imaging the central role of 5-HT2A signaling in the initiation of spontaneous events. Additionally, in attempt to discover a functional role for spontaneous activity (SA) in hindbrain development, hindbrains were subjected to 24-hours of 5-HT2A receptor antagonists to disrupt SA in vitro. On average, a 40% reduction in the number of 5-HT+ cells was found in brains cultured with 5-HT2A receptor antagonists. Interestingly, 5-HT+ cell numbers were only reduced when cultured during the window of neurogenesis; cultures during the 24-hours post-specification exhibited no change in 5-HT+ cell numbers. To determine whether this reduction was resultant of disrupted SA or disrupted serotonergic signaling, hindbrains were cultured in mibefradil, a T-type calcium channel antagonist known to stop SA. No decreases in 5-HT+ cell numbers were observed, suggesting signaling via 5-HT2A receptors regulates 5-HT cell numbers. No changes were detected in the amount of the other neural population generated in the same progenitor domain during the same developmental window, the visceral motor neurons. Collectively, these data suggest 5-HT2A signals during neurogenesis are important for the appropriate development of 5-HT neurons.