Chemical properties of the extracellular matrix of the snail nervous system: a comprehensive study using a combination of histochemical techniques

Abstract

The extracellular matrix (ECM) consists of various types of protein and carbohydrate polymers with red-ox and acid-base properties that have a crucial impact on tissue homeostasis. In the present study, a combination of both frequently applied and also specialized histochemical staining methods were used to reveal the chemical properties of the ECM of the snail central nervous system (CNS) which has a long been favored experimental model for comparative neurobiologists. Reactions such as silver ion reduction to label oxidative elements and different protein fibers, visible and fluorescent periodic-Schiff (PAS) reaction for the detection of unbranched chain of carbohydrates, and cationic dyes (acridine orange and alcian blue) for differentiating acidic carbohydrates were used. Illumination of sections stained with toluidine blue at pH 4.0 by a fluorescent light (lambda ex546/em580 nm), visualized components of the extraneural space (ECM molecules and glial cells) of the adult and also the developing CNS. Silver, toluidine blue and azure A were used to detect specific molecule bands in CNS extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Some molecules showed both negative character and had carbohydrate side chains revealed by the Solanum tuberosum lectin probe. In a comparison of a freshwater aquatic (Lymnaea stagnalis) and a terrestrial (Helix pomatia) species, the ECM showed similarities in the composition of the periganglionic sheath and interperikaryonal space. The sheath was rich in alcian blue-positive sulfated proteoglycans infiltrated the space between collagen and reticular fibers, whereas in the interperikaryonal space PAS- and acridine orange-positive neutral and weakly acidic carbohydrates were detected. The ganglionic neuropil was mostly filled with PAS-positive material, but negatively charged sulfated and carboxylated molecules detected by acridine orange and alcian blue were present only in Helix. A low carbohydrate content was also found in the neuropil of both adult and developing Lymnaea, but most of the ECM components appeared only during the postembryonic juvenile stages. Comparing the SDS-PAGE of the periganglionic sheath and neural tissue extracts, toluidine blue (pH 4.0) and azure A (pH 2.0) revealed negatively charged molecules; some were found in both fractions. These results show, for the first time, the general chemical characteristics of the ECM of the snail CNS, indicating differences in the composition of the ganglion neuropil between aquatic and terrestrial species. Hence, a different strategy for retaining water by the neural tissue is suggested in species living in different environments.