Biotin staining in the giant fiber systems of the lobster

The avidin-biotin-complex method is a popular immunocytochemical technique. This method labels consistently a group of neurons in the lobster ventral nerve cord in the absence of primary antibodies. The specific staining is due to a relatively high level of endogenous biotin (or biocytin) in these neurons. These biotin-positive neurons are located in the supraesophageal, thoracic, and abdominal ganglia. Intraaxonal injection of Lucifer yellow followed by Texas red-conjugated streptavidin staining reveals that the neurons are members of the medial giant (MG) and lateral giant (LG) systems, which are important in mediating rapid tail flipping during escape maneuvers. In neuronal somata, staining is restricted to the cytoplasm. Within MG axons, staining appears as punctate, subaxolemmal structures. Preincubating nerve cords in biocytin or direct intraaxonal injection of biocytin enhances staining of these punctate organelles. In LG axons, staining is localized to fragments of braided filamentous structures that also appear to be associated with the axolemma. Preincubation of ventral nerve cords in various concentrations of biocytin results in the appearance of additional groups of stained neurons, suggesting that there are subsets of neurons with specific biocytin-uptake or -retention mechanisms. In the crayfish, biotin-positive staining is confined to the MG neurons; the LG neurons are not stained. In the earthworm, no staining is observed in the MG and LG axon escape systems. In the goldfish, no biotin-staining is seen in the Mauthner neurons and their axons. The significance of specific localization of biotin or biocytin to subsets of neurons is unclear. It may reflect the presence of high levels of biocytin moieties on biotin-dependent enzymes. Biotin is an important cofactor in the catalytic functions of several decarboxylases crucial in energy production and lipogenesis. Axons of the giant fiber systems in lobsters and crayfish may have high energy and fatty acid synthesis requirements. Increased levels of biotin accumulation may also be related to other functions of the giant axon systems, such as the formation of electrical synapses among themselves and with phasic motoneurons.