Quantitative immunoelectron microscopy and tannic acid study of dynamics of neurohaemal and non-synaptic peptide release by the caudodorsal cells of Lymnaea stagnalis

Abstract

The caudodorsal cells (CDCs) of the freshwater snail Lymnaea stagnalis control egg laying and egg-laying associated behaviours by releasing various peptides including the ovulation hormone CDCH. Previously it has been shown that release occurs (1) into the haemolymph from neurohaemal axon terminals in the outer compartment of the cerebral commissure, and (2) into the intercellular space of the central nervous system from non-synaptic release sites of axon collaterals in the inner compartment of the commissure. Outer and inner compartments are separated by a sheath of glial cells. In the present study the secretory dynamics of neurohaemal and collateral release have been studied. Immunoelectron microscopy with an antibody against a synthesized fragment of the egg-laying hormone [CDCH (21–36)] indicates that CDCH is released by exocytosis from both sites: positive immunoreaction was found for the contents of secretory granules and contents that underwent exocytosis, and furthermore in the intercellular spaces of the inner and outer compartments. Quantitative (immunogold) electron microscopy combined with either the tannic acid-glutaraldehyde-osmium tetroxide (TAGO) method or the tannic acid-Ringer incubation (TARI) method for the visualization and quantification of exocytosis of CDCH, shows different dynamics of neurohaemal and collateral CDCH release. Neurohaemal release is strongly increased during electrical activity of the CDCs (active state). This increase does not only appear from an increased number of (immunopositive) exocytoses (3×) but also from increases in (1) the percentages of all stationary and all exocytosing granule contents that are immunopositive (both increase from 70% to 85%), (2) the degree of immunopositivity per exteriorized granule content (2×) and (3) the degree of immunopositivity in the intercellular space of the neurohaemal area (5×). Collaterals show a different picture: CDCH release particularly occurs during electrical silence (resting and inhibited states). No effect was noted of the electrical state of the CDCs on the percentages of CDCH-immunoreactive stationary or exteriorized granule contents, nor on the degree of immunopositivity of the exteriorized contents. Furthermore, the degree of immunopositivity in the intercellular space of the inner compartment is drastically decreased (8×). Finally, both in the resting and the active state, the percentage of CDCH-positive exocytosing contents in the collaterals is smaller than that of CDCH-positive stationary contents, whereas in the neurohaemal area these percentages do not differ. It is concluded that the CDCs establish dual secretory dynamics in the neurohaemal and collateral system, not only with respect to the intensity of exocytosis but also with respect to the amount of CDCH released per secretory granule. Possible cellular mechanisms underlying these dynamics are discussed. The dual secretory capacity may be crucial for the CDCs in timing and coordinating the activities of reproductive organs (by peptide release from the neurohaemal area) and of central neurons that control egg-laying-associated behaviours (by peptides released non-synaptically from the collaterals).