Basic neuroscienceFast neuronal labeling in live tissue using a biocytin conjugated fluorescent probe

- TMR biocytin is a very effective retrograde neuronal tracer when applied by electroporation.
- The initial fiber transport velocity of TMR biocytin is high-5.4 mm/h.
- TMR biocytin can be used with standard AM calcium dyes to dual label live neurons.
- TMR biocytin is fixable, and stable after methyl salicylate tissue clearing.

BackgroundBiocytin has found numerous uses as a neuronal tracer, since it shows both antero- and retrograde transport in neuronal tracts. The main advantage of biocytin lies in the comprehensive intracellular distribution of the molecule, and in effective detection using avidin-based reactions. The main drawback is that biocytin cannot be visualized in live tissue.New methodWe demonstrate that TMR biocytin, a conjugate of biocytin and a rhodamine fluorophore, is an effective neuronal tracer in live tissue when applied by electroporation.ResultsThe initial fiber transport velocity of TMR biocytin is high-5.4 mm/h. TMR biocytin can be used in conjunction with AM calcium dyes to label neuronal somas from distances of several millimetres, and record calcium transients during the course of a few hours. Juxtacellular application of TMR biocytin leads to fast anterograde transport with labeling of local synapses within 10 min. TMR biocytin is fixable, stable during methyl salicylate clearing, and can be visualized deep in nervous tissue.Comparison with existing methodsRetrograde labeling with TMR biocytin enables long-range neuronal visualization and concurrent calcium imaging after only a few hours, which is substantially faster than other fluorescence-based tracers. The green emitting Atto 488 biotin is also taken up and transported retrogradely, but it is not compatible with standard green emitting calcium dyes.ConclusionsTMR biocytin is an attractive neuronal tracer. It labels neurons fast over long distances, and it can be used in conjunction with calcium dyes to report on neuronal activity in retrogradely labeled live neurons.

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