Effect of serotonin on ciliary beating and intracellular calcium concentration in identified populations of embryonic ciliary cells

Abstract

SUMMARYEmbryos of the pond snail Helisoma trivolvis express three known subtypes of ciliary cells on the surface of the embryo early in development:pedal, dorsolateral and scattered single ciliary cells (SSCCs). The pedal and dorsolateral ciliary cells are innervated by a pair of serotonergic sensory-motor neurons and are responsible for generating the earliest whole-animal behavior, rotation within the egg capsule. Previous cell culture studies on unidentified ciliary cells revealed that serotonin(5-hydroxytryptamine; 5-HT) produces a significant increase in the ciliary beat frequency (CBF) in a large proportion of ciliary cells. Both Ca2+ influx and a unique isoform of protein kinase C (PKC) were implicated in the signal transduction pathway underlying the cilio-excitatory response to 5-HT. The goal of the present study was to characterize the anatomical and physiological differences between the three known populations of superficial ciliary cells. The pedal and dorsolateral ciliary cells shared common structural characteristics, including flat morphology, dense cilia and lateral accessory ciliary rootlets. By contrast, the SSCCs had a cuboidal morphology, reduced number of cilia, increased ciliary length and absence of lateral accessory rootlets. In cultures containing unidentified ciliary cells,the calcium/calmodulin-dependent enzyme inhibitor calmidazolium (2 μmol l–1) blocked the stimulatory effect of 5-HT (100 μmol l–1) on CBF. In addition, 50% of unidentified cultured cells responded to 5-HT (100 μmol l–1) with an increase in[Ca2+]i. To facilitate the functional analyses of the individual populations, we developed a method to culture identified ciliary subtypes and characterized their ciliary and calcium responses to 5-HT. In cultures containing either pedal or dorsolateral ciliary cells, 5-HT (100μmol l–1) produced a rapid increase in CBF and a slower increase in [Ca2+]i in all cells examined. By contrast,the CBF and [Ca2+]i of SSCCs were not affected by 100μmol l–1 5-HT. Immunohistochemistry for two putative 5-HT receptors recently cloned from Helisoma revealed that pedal and dorsolateral ciliary cells consistently express the 5-HT1Helprotein. Intense 5-HT7Hel immunoreactivity was observed in only a subset of pedal and dorsolateral ciliary cells. Cells neighboring the SSCCs,but not the ciliary cells themselves, expressed 5-HT1Hel and 5-HT7Hel immunoreactivity. These data suggest that the pedal and dorsolateral ciliary cells, but not the SSCCs are a homogeneous physiological subtype that will be useful for elucidating the signal transduction mechanisms underlying 5-HT induced cilio-excitation.

Neural control of the velum in larvae of the gastropod,Ilyanassa obsoleta

“…This suggests that pre-oral ciliary cells are inhibited by catecholamines, but postoral ciliated cells may be unresponsive to dopamine or may in fact respond to catecholamines with an increase in ciliary beating, although this hypothesis must be tested by observations of post-oral CBF under normal conditions. Similar variations in the responsiveness of different populations of ciliated cells have been reported for serotonin in H. trivolvis (Doran et al, 2004) and neuropeptides and dopamine in the clam Mercenaria mercenaria (Gainey et al, 1999), suggesting that a different catecholamine receptor or even a lack thereof on the post-oral ciliated cells is plausible.…”

Nervous control of ciliary beating by Cl-, Ca2+ and calmodulin inTritonia diomedea

“…Could there be different pathways controlling coordination and CBF increase? We also provide evidence that IP 3 may not be a significant player in CBF excitation, but that does not rule out a PLC dependent pathway using PKC, as found in Helisoma embryos (Christopher et al, 1999;Doran et al, 2004), nor the adenylate cyclase/cAMP/ PKA pathway found to be necessary in Mytilus (Stommel and Stephens, 1985;Aiello, 1990) (preliminary findings suggest that cAMP plays no role in CPE cells; O. M. Woodwood, unpublished observation), nor other non-calmodulin dependent Ca 2+ actions (Salathe and Bookman, 1999). Electrically induced excitation of CPE cells in Tritonia diomedea is consistent with their role as primary locomotory effectors, and like muscle, their RyR-gated stores and voltage-gated Ca 2+ channels may be an adaptation permitting parallel CNS control.…”

“…Ciliary beating may be under hormonal control (Verdugo, 1980;Korngreen and Priel, 1996;Korngreen et al, 1998;Lieb et al, 2002;Barrera et al, 2004) or nervous control via dopamine and neuropeptides (Willows et al, 1997;Woodward and Willows, 2006), serotonin (Goldberg et al, 1994;Christopher et al, 1996;Willows et al, 1997;Nguyen et al, 2001; Doran et al, 2004), acetylcholine (ACh) (Salathe and Bookman, 1999;Zagoory et al, 2001;Zagoory et al, 2002) or depolarization (Aiello and Guideri, 1964;Mackie et al, 1969;Mackie et al, 1976;Murakami and Takahashi, 1975). Work permitting any distinction between these control mechanisms is sparse and this may be due, in part, to technical challenges presented by vertebrate models.…”

A novel serotonin-secreting cell type regulates ciliary motility in the mucociliary epidermis of Xenopus tadpoles

“…Serotonin has previously been demonstrated to regulate CBF in a number of systems (Doran et al, 2004;Nguyen et al, 2001;Wada et al, 1997). To test whether loss of serotonin synthesis affected cilia-driven fluid flow in the larval skin, flow velocity was analyzed in PCPA-treated specimens.…”

“…Serotonin-mediated CBF regulation differs between species and tissues, both with respect to receptor type and source of ligand (Doran et al, 2004;König et al, 2009;Longley and Peterman, 2012;Nguyen et al, 2001). Experimental analysis of mammalian brain and airway MCEs is further hampered by the use of organ cultures from biopsies, which prevents monitoring of long-term effects or complex functional manipulation of multiple pathways.…”