BEATING OF GILL LATERAL CELL CILIA OF Crassostrea virginica INVOLVES NEURONAL INNERVATION AND THE PRESENCE OF GAP JUNCTIONS

Nicole Dobey*, Reniece Buchanan, Dane Frank1, Margaret A. Carroll and Edward J. Catapane
 
Department of Biology 
1Behavioral Instruments
Medgar Evers College 
5 Jill Court, Suite 1
Brooklyn, NY, 11225 USA 
Hillsborough, NJ 08844 USA
catapane@mec.cuny.edu

Suspension feeding bivalve molluscs employ a ciliary pump driven by tracts of lateral cilia located between gill filaments. Coordinated beating of gill lateral cell cilia provides the water current responsible for feeding, respiration and waste elimination. These cells in Crassostrea virginica are controlled by a serotonergic-dopaminergic innervation from their ganglia via the branchial nerve. Serotonin is the neurotransmitter that increases cilia beating rates, while dopamine decreases it. These lateral cells of many other bivalves are similarly innervated. The cellular mechanism regulating lateral cell cilia beating and water pumping is not fully explained. It has long been known second messenger systems are involved. While evidence shows the gill filaments receive projections from the branchial nerve, it is still not known if each lateral cell is individually innervated. It is known that the lateral cells have gap junctions and questions arise as to whether the gap junctions are involved in controlling the rate or coordination of beating. We hypothesize that these gap junctions are involved in the cellular mechanism regulating lateral cell cilia beating rate and/or coordination. To study this we made immunohistological preparations using antibodies to visualize and confirm the presence of the gap junctions, and used the Sihler Whole Mount method to visualize the nerves in the gill filaments. We also set up preparations in which gill filaments were divided into proximal and distal sections by a petroleum jelly barrier, which prevented diffusion of drugs from one side to the other. In these preparations, the branchial nerve entering the proximal end of the filaments was stimulated (5 hz, 2 msec, 10 - 20 volts) with suction electrodes, which is known to cause a release of serotonin and increase beating rates. Stroboscopic microscopy was used to measure cilia beating rates. Drugs were applied to distal ends of preparations and lateral cell cilia beating rates of both proximal and distal ends were compared. Applying procaine, a local anesthetic, to the distal end prevented electrical stimulation from increasing cilia beating rates, while the cilia beating rate at the proximal end increased, showing that nerve impulse propagation is necessary for ciliary response. Using the same procedure, we also tested 3 gap junction blockers, each with different mechanisms of action: lindane, diphenylborinic anhydride, and mefloquine. In response to branchial nerve stimulations, mefloquine had no effect on cilia beating rates, but lindane and diphenylborinic anhydride both produced statistically significant reductions in beating rates at the distal ends where they were applied. Lindane had the stronger effect, causing a dose-dependent (10-7 - 10-4M) blockage of cilia beating, while diphenylborinic anhydride only blocked at the high dose of 10-4M. The results showed neuronal innervation to the filaments in conjunction with functioning gap junctions in the cells are necessary to increase cilia beating rates. Disrupting either one of these aspects interfered with the normal response. Lindane is reported to reduce gap junction permeability and increase intracellular Ca2+ levels; either one or both of these actions may be responsible for the results we obtained. This work was supported by 2R25GM06003 of NIGMS and 0516041071 of NYSDOE.