Laser ablation reveals regulation of ciliary activity by serotonergic neurons in molluscan embryos

Abstract

Early in embryonic development, the pond snail Helisoma trivolvis exhibits a rotational behavior that is generated by beating of cilia in the dorsolateral and pedal bands. Although previous anatomical and pharmacological studies provided indirect evidence that a pair of serotonergic neurons, Embryonic Neurons C1 (ENC1s), is involved in regulating embryonic rotation, direct evidence linking ENC1 to ciliary function is still lacking. In the present study, we used laser microbeams to perturb ENC1 in vivo while monitoring ciliary activity in identified ciliary bands. A laser treatment protocol to specifically ablate ENC1 without damaging the surrounding cells was established. Unilateral laser treatment of ENC1 caused transient increases in the activity of the pedal and ipsidorsolateral cilia, lasting 30-50 min. In contrast, activity of cilia that were not anatomically associated with ENC1 was unaffected by laser treatment. Mianserin, an effective serotonin antagonist in Helisoma ciliated cells, decreased the overall CBF of pedal and dorsolateral cilia by reducing the occurrence of spontaneous CBF surges in these cilia. Finally, the cilioexcitatory action of ENC1 laser treatment was mimicked by serotonin and reduced in the presence of mianserin. These results suggest that laser treatment provokes a release of serotonin from ENC1, resulting in a prolonged elevation of activity in the target ciliary cells. We conclude that, in addition to their previously established role in regulating neurodevelopment, ENC1s also function as serotonergic motor neurons to regulate ciliary activity, and therefore the rotational behavior of early embryos.

Identification and evolutionary implications of neurotransmitter–ciliary interactions underlying the behavioral response to hypoxia inLymnaea stagnalisembryos

“…Perhaps the prominent surges revealed in the analysis of rotational dynamics (Fig.2A) are dopamine-induced events associated with bursts of activity in the TAC neurons. These data are consistent with the TAC neurons of L. stagnalis functioning very similarly to their ENC1 homologs in H. trivolvis (Kuang and Goldberg, 2001), despite having a different primary neurotransmitter.…”

“…Acetylated tubulin immunofluorescence revealed that L. stagnalis embryos contained the same ciliated structures on the embryonic surface as those previously described in H. trivolvis (Kuang and Goldberg, 2001;Doran et al, 2004), including two dorsolateral bands of cilia, a pedal band of cilia, the dorsal stomodeum and scattered single ciliary cells (Fig.1). Lymnaea stagnalis embryos also contained the ciliated apical plate (Fig.1), which is absent in H. trivolvis.…”

“…For example, the embryonic neurons thought to be the primary neural control elements of the behavior have diverged significantly. The ENC1 neurons of H. trivolvis are sensory-motor neurons that function both in hypoxia sensation and cilioexcitation (Kuang and Goldberg, 2001;Kuang et al, 2002). These serotonergic neurons have a short apical dendrite tipped with a sensory specialization on the embryonic surface, as well as a primary descending axon that innervates both the dorsolateral and pedal bands of cilia (Diefenbach et al, 1998;Koss et al, 2003).…”

“…Although the pedal cilia are active and capable of responding to both serotonin and dopamine in L. stagnalis embryos, these cilia are not the primary drivers of embryonic rotation. In future studies, in situ analysis of apical plate and pedal cilia could reveal whether the pedal cilia display surges in activity in parallel with those displayed by the apical plate cilia (Kuang and Goldberg, 2001). This would determine whether dopamine released from the TAC neurons also causes paracrine activation of the pedal cilia as a secondary pathway driving the embryonic rotation behavior.…”

“…ENC1s are sensorimotor neurons that release serotonin onto postsynaptic ciliary cells in response to hypoxia, resulting in faster ciliary beating and embryonic rotation Kuang and Goldberg, 2001;Koss et al, 2003). Hypoxia elicits sustained, dose-dependent and reversible accelerations of embryonic rotation in control embryos, whereas this response is absent in embryos that have undergone bilateral laser ablation of their ENC1s (Kuang et al, 2002).…”