Biotin staining in the giant fiber systems of the lobster

Abstract

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.

Biotin is endogenously expressed in select regions of the rat central nervous system

“…Biotin labeling has also been reported in cerebellar Purkinje cells, although this was in pigs fed a biotin-supplemented diet (Cooper et al, 1997). These latter results are not easily compared with endogenous biotin in rats maintained on a normal diet, as artificial increases in biotin (as exemplified by ventricular injection of biocytin) leads to widespread labeling of cells that are ordinarily biotin-negative (McDonald et al, 1992;Ma, 1994).…”

“…Biotin has been identified in the lobster giant axon (Ma, 1994) and in neurosecretory cells of the insect Manduca sexta (Ziegler et al, 1995). Biotin was identified in a subset of neurons from the turtle spinal cord (Berkowitz, 2002), in axons and synaptic boutons in numerous regions of frog brain (Eisner et al, 1996), in salamander and goldfish retina (Bhat- tacharjee et al, 1997), and in two nuclei following song learning in birds (Johnson et al, 2000).…”

“…The idea that significant amounts of biotin might be concentrated in systems with similar functional purposes or energetic demands was explored by Ma (1994). That work demonstrated that significant concentrations of biotin are present in lobster giant axons, although in other species with giant axon escape systems biotin was not detectable (Ma, 1994).…”

“…High levels of biotin are present in kidney and liver cells (Bhagavan and Coursin, 1970;Wood and Warnke, 1981;Wang and Pevsner, 1999), and in select cells of nonmammalian nervous systems (Ma, 1994;Ziegler et al, 1995; hearing loss, ataxia, and tremor (Wolf and Feldman, 1982;Suchy et al, 1985;Wolf et al, 1983Wolf et al, , 1985Wolf, 2001). Second, active carrier-mediated transport mechanisms raise biotin in CSF to levels 50 -250% greater than plasma, and maintain the activity of biotin-dependent enzymes in brain even during challenge with low dietary biotin (Bhagavan and Coursin, 1970;Chiang and Mistry, 1974;Sander et al, 1982;Mock, 1987, 1988;Lo et al, 1991).…”

Concentration and storage of biotin in the amphibian brain

“…However, McDonald et al (1992) reported that specific sets of neurons in rat brain are stained in histochemical preparations for biotin at an intensity well above background when biocytin (biotinyl-lysine) is injected into the lateral ventricle or brain parenchyma. Ma (1994) documented the selective staining of endogenous biotin or biotin-associated compounds in certain neurons in ganglia of the lobster ventral nerve cord. Although the significance of these effects has not been elucidated, these investigators have suggested that particular neurons may have an elevated requirement for biotin or its derivatives for a specialized function.…”