title: Fast neuronal labeling in live tissue using a biocytin conjugated fluorescent probe.
abstract: BACKGROUND
  Biocytin 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.
  
  
  METHODS
  We demonstrate that TMR biocytin, a conjugate of biocytin and a rhodamine fluorophore, is an effective neuronal tracer in live tissue when applied by electroporation.
  
  
  RESULTS
  The initial fiber transport velocity of TMR biocytin is high-5.4mm/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 10min. TMR biocytin is fixable, stable during methyl salicylate clearing, and can be visualized deep in nervous tissue.
  
  
  METHODS
  Retrograde 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.
  
  
  CONCLUSIONS
  TMR 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.
authors.affiliation: Department of Neuroscience and Pharmacology, Copenhagen University, Panum Institute, 12.3, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Mads
authors.initials: M
authors.lastname: Harsløf
authors.affiliation: Department of Neuroscience and Pharmacology, Copenhagen University, Panum Institute, 12.3, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Felix C
authors.initials: FC
authors.lastname: Müller
authors.affiliation: Department of Neuroscience and Pharmacology, Copenhagen University, Panum Institute, 12.3, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Julie
authors.initials: J
authors.lastname: Rohrberg
authors.affiliation: Department of Neuroscience and Pharmacology, Copenhagen University, Panum Institute, 12.3, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. Electronic address: jrekling@sund.ku.dk.
authors.forename: Jens C
authors.initials: JC
authors.lastname: Rekling
mesh_terms: D000704:Analysis of Variance; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001933:Brain Stem / Q000166:cytology*; D002118:Calcium; D018274:Electroporation; D005456:Fluorescent Dyes / Q000378:metabolism*; D066298:In Vitro Techniques; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D051379:Mice; D009434:Neural Pathways / Q000502:physiology*; D009474:Neurons / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism; D014966:Xanthenes / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; D005456:Fluorescent Dyes; D012235:Rhodamines; D014966:Xanthenes; C561295:fluo-8 AM; C005358:tetramethylrhodamine; C013411:biocytin; D008239:Lysine; D002118:Calcium
keywords: Anterograde; Biocytin; Conjugated probes; Neuronal tracer; Retrograde; TMR biocytin