A biotin-containing compound N-(2-aminoethyl)biotinamide for intracellular labeling and neuronal tracing studies: comparison with biocytin

Abstract

The hydrochloride salt of a new, small molecular weight (M.W. = 286) biotin-containing compound referred to as biotinamide (N-(2-aminoethyl)biotinamide) was compared with biocytin (M.W. = 372) for its use in intracellular labeling of neurons and in neuronal tracing experiments using avidin conjugates for histochemical detection. The DC resistance and current passing ability of electrodes filled with 1-2 M potassium chloride, potassium acetate or potassium methylsulfate and containing 1-4% of these compounds were compared. Although differences were observed due to the electrolyte, with KCl electrodes being the least resistant, no electrode differences could be attributed to the concentration or type of tracer. However, whereas biocytin could be electrophoresed with either positive or negative current with roughly similar facility, biotinamide was selectively ejected with positive current. This would be beneficial to electrophysiologists using hyperpolarizing current to stabilize the membrane potential of neurons prior to recording. In addition, biotinamide-HCl could be dissolved at concentrations of 2-4% in either 1 or 2 M salt without precipitation, whereas biocytin precipitated in some of these solutions. Both compounds were equally useful for neuronal tracing experiments with survival times of 2 days, but labeling was much weaker with longer survival times. There was also little difference in the ability to histochemically localize these compounds using avidin conjugates, including avidin-biotin-horseradish peroxidase complex. In conclusion, biotinamide shares many of the useful features of biocytin, but can be selectively electrophoresed with positive current and can be dissolved at higher concentrations with little detriment in the electrical properties of the recording electrode.