Localization of the GM1 ganglioside in the vestibular system using cholera toxin

Cholera toxin is an ubiquitous activator of intracellular adenylate cyclase and is divided in two major components: A and B. The B-component consists of several subunits that specifically bind to the external cell membrane. The receptor for the toxin, the GM1 ganglioside, is concentrated in nervous tissues. The B subunit of the cholera toxin, conjugated to different molecules (i.e., choleragenoid) is therefore a sensitive anatomical tracer and has been used to detect the presence of GM1 in mammalian tissues. Using choleragenoid, unlabeled and labeled with FITC, we have determined the distribution of the GM1 ganglioside in the vestibular system of the chinchilla. Vestibular tissues were fixed in 4% paraformaldehyde in phosphate buffer, decalcified in 10% EDTA and prepared as either whole-mount, surface-preparations, or for radial cryosections. Positive control tissue consisted of binding to normal brain tissues. Negative controls consisted of several treatments: masking of the GM1 receptors with unlabeled choleragenoid, tissue extraction of GM1 using ethanol, and preabsorbing the choleragenoid with bovine GM1. In addition, to exclude staining of glycoproteins that may have a carbohydrate structure similar to GM1, tissues were digested with trypsin prior to choleragenoid exposure. In the vestibular system, a strongly positive reaction was observed in: the sensory stereocilia and supporting cells of the maculae and cristae, epithelial cells of the planum semilunatum, and polygonal cells of the semicircular canal. Positive but less strong reactivity was observed in the sensory cell body of maculae and cristae, nerve fibers, epithelial cells of utricle and ampulla walls and flattened epithelial cells of the semicircular canals. No reactivity was present in the supporting connective tissue cells and fibrils, blood vessels, gelatinous cupula of the cristae ampullaris and statoconial membranes. Brain tissue showed strong choleragenoid reactivity. The negative controls showed no or greatly reduced reactivity to choleragenoid. Trypsin digestion did not decrease reactivity to choleragenoid.