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First published online March 9, 2004
Journal of Experimental Biology 207, 1415-1429 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00924
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Effect of serotonin on ciliary beating and intracellular calcium concentration in identified populations of embryonic ciliary cells

Shandra A. Doran, Ron Koss, Cam Ha Tran, Kimberly J. Christopher, Warren J. Gallin and Jeffrey I. Goldberg*

Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9



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  		Fig. 1. Isolation of identified ciliary cells from Helisoma trivolvis embryos. Schematic of a side view of a stage E30 Helisoma trivolvis embryo reveals the location of the various ciliary cells and embryonic neuron C1 (ENC1). A suction pipette was used to remove identified pieces of tissue from the surface of the embryo. DLB, dorsolateral ciliary band; PB, pedal ciliary band; SSC, scattered single ciliary cell.

 


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  		Fig. 2. Whole-mount scanning electron micrographs (SEM) and transmission electron micrographs (TEM) showing the overall external and internal anatomy of the three ciliary subtypes of a stage E28 embryo. (A) Left lateral view SEM showing the position of the scattered single ciliary cells (sc) relative to the dorsolateral ciliary bands (dl) and pedal ciliary band (p). The shell (sh) and mouth (mo) are also shown. Scale bar, 25 µm. (B) SEM showing the configuration and density of cilia of a dorsolateral ciliary cell. Scale bar, 1 µm. (C) SEM showing the configuration and density of cilia of a pedal ciliary cell. Scale bar, 1 µm. (D) TEM of a dorsolateral ciliary cell showing numerous mitochondria (mi) and the nucleus (n). Electron-translucent regions are designated by an arrow. The cilia (c) with basal body (bb), ciliary rootlet (cr) and accessory ciliary rootlet (acr) are also shown. Scale bar, 1 µm. (E) SEM showing the configuration and density of cilia of one of the scattered single ciliated cells. Scale bar, 1 µm. (F) TEM of a scattered single ciliated cell showing the cytoplasm. Note the cilia (c) with basal body (bb), ciliary rootlet (cr), less prominent mitochondria (mi) and the nucleus (n). Scale bar, 1 µm. Inset: cross section through a cilium showing the (9x2)+2 doublets of microtubules. Scale bar, 0.5 mm.

 


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  		Fig. 3. Effect of the calmodulin inhibitor calmidazolium on ciliary beat frequency (CBF) in unidentified ciliary cells in culture. Application of 100 mmol l–1 5-hydroxytryptamine (5-HT) produced a significant increase in CBF versus the saline control (asterisk: P<0.05, N=7 cells). Application of either 0.1% DMSO (N=7 cells) or 2 mmol l–1 calmidazolium (Cal; N=12 cells) caused no change in CBF. When 2 mmol l–1 Cal was co-applied with 100 mmol l–1 5-HT, the calmodulin inhibitor blocked the stimulatory effect of 5-HT (N=9 cells).

 


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  		Fig. 4. Effect of 5-hydroxytryptamine (5-HT) and ionomycin on the intracellular Ca2+ concentration in unidentified ciliary cells in culture. Ciliary cells microinjected with fura-2 dextran were imaged at 1-min intervals in the presence of saline (HS), 5-HT or ionomycin. (A) Representative trace of a ciliary cell that was not affected by a 15 min application of 100 mmol l–1 5-HT but displayed a rapid rise in the 340/380 ratio in response to 10 µmol l–1 ionomycin. (B) Representative trace of an unidentified ciliary cell responding to both 100 mmol l–1 5-HT application and 10 mmol l–1 ionomycin with an increase in the 340/380 ratio. Dotted lines represent the time at which drug treatments were initiated.

 


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  		Fig. 5. Effect of 5-hydroxytryptamine (5-HT) and ionomycin on ciliary beat frequency (CBF) in pedal, dorsolateral and scattered single ciliary cells. Perfusion with 100 µmol l–1 5-HT resulted in an approximate doubling in the rate of ciliary beating in pedal (A), fura dextran-loaded pedal (B) and dorsolateral (C) ciliary cells. This 5-HT-stimulated increase in CBF was statistically significant in all cases. The CBF partially recovered when cells were perfused for 5 min with Helisoma saline (HS), whereas perfusion with 10 mmol l–1 ionomycin further stimulated CBF in pedal (A), fura dextran-loaded pedal (B) and dorsolateral (C) ciliary cells. The scattered single ciliary cells did not display cilio-excitatory responses to 5-HT or ionomycin (D). Measurements were taken once every minute, N=6 cells for A, C and D and N=4 cells for B. Dotted lines represent the time when the specified treatments were initiated.

 


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  		Fig. 9. 5-HT1Hel receptor immunoreactivity in identified Helisoma ciliary cells. Nomarski differential interference contrast (DIC) images (left panels) and corresponding fluorescence micrographs (right panels) of tissue explants containing identified ciliary cells that were processed for 5-HT1Hel immunoreactivity. Arrows indicate the apical surface of the ciliary cells. (A) Immunopositive pedal ciliary cell. (B) Immunoreactivity in multiple dorsolateral ciliary cells. (C) Immunoreactivity restricted to the non-ciliary epithelial cells (asterisks) surrounding an unstained single ciliary cell. (D) A control explant containing pedal ciliary cells was exposed to preimmune serum instead of primary antibody. Scale bars, 10 µm.

 


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  		Fig. 10. 5-HT7Hel receptor immunoreactivity in identified Helisoma ciliary cells. Nomarski differential interference contrast (DIC) images (left panels) and corresponding fluorescence micrographs (right panels) of tissue explants containing identified ciliary cells that were processed for 5-HT7Hel immunoreactivity. Arrows indicate the apical surface of the ciliary cells. (A) Immunopositive pedal ciliary cells. (B) Immunonegative pedal ciliary cells. (C) Immunopositive dorsolateral ciliary cells. (D) Immunonegative dorsolateral ciliary cells. (E) As with 5-HT1Hel immunoreactivity, 5-HT7Hel immunoreactivity was restricted to non-ciliary epithelial cells (asterisks) surrounding the scattered single ciliary cell. Scale bars, 10 µm.

 


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  		Fig. 6. Effect of 5-hydroxytryptamine (5-HT) and ionomycin on intracellular Ca2+ concentration ([Ca2+]i) in pedal ciliary cells. Pedal ciliary cells were microinjected with fura-2 dextran and imaged once every 15 s. Application of 100 µmol l–1 5-HT generated two different [Ca2+]i profiles – an early peak in [Ca2+]i followed by a sustained plateau (A; N=5 cells) or a gradual rise in [Ca2+]i over the course of drug application (B; N=5 cells). In both cases, the [Ca2+]i did not recover within 5 min of exposure to Helisoma saline (HS), whereas 10 µmol l–1 ionomycin stimulated a large increase in the [Ca2+]i. The inset in A shows the experiment on a different scale to illustrate the magnitude of the increase in [Ca2+]i in response to ionomycin application. Dotted lines represent the time when the specified treatments were initiated.

 


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  		Fig. 7. Effect of 5-hydroxytryptamine (5-HT) and ionomycin on [Ca2+]i in dorsolateral ciliary cells. Dorsolateral ciliary cells were microinjected with fura-2 dextran and imaged once every 15 s.Application of 100 µmol l–1 5-HT generated two different types of changes in [Ca2+]i, an early peak followed by a sustained plateau (A; N=4 cells) or a gradual rise in [Ca2+]i over the course of 5-HT perfusion (B; N=6 cells). In all cells, regardless of the response, 10 µmol l–1 ionomycin produced a variable increase in [Ca2+]i. Dotted lines represent the time when the specified treatments were initiated.

 


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  		Fig. 8. Effect of 5-hydroxytryptamine (5-HT) and ionomycin on [Ca2+]i in scattered single ciliary cells. Scattered single ciliary cells were microinjected with fura-2 dextran and imaged once every 15 s. (A) A representative trace of an isolated tuft cell that did not respond to 100 µmol l–1 5-HT and exhibited only a weak increase in [Ca2+]i in response to 10 mmol l–1 ionomycin. (B) A representative trace of an isolated tuft cell that displayed oscillations in [Ca2+]i. Dotted lines represent the time when the specified treatments were initiated.

 

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© The Company of Biologists Ltd 2004