Journal
JOURNAL OF EXPERIMENTAL BIOLOGY
Volume 207, Issue 8, Pages 1415-1429Publisher
COMPANY OF BIOLOGISTS LTD
DOI: 10.1242/jeb.00924
Keywords
serotonin; 5-hydroxytryptamme; pond snail; Helisoma trivolvis; intracellular calcium; ciliary beating; cilia; serotonin receptor
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Embryos 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 behaviour, 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 mumol 1(-1)) blocked the stimulatory effect of 5-HT (100 mumol 1(-1)) on CBF. In addition, 50 % of unidentified cultured cells responded to 5-HT (100 mumol 1(-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 (100 mumol 1(-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 100 mumol 1(-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-HT1Hel protein. 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.
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