tetramethylrhodamine and biocytin

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.. 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.. 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.. 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.. 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.

Topics: Analysis of Variance; Aniline Compounds; Animals; Animals, Newborn; Brain Stem; Calcium; Electroporation; Fluorescent Dyes; In Vitro Techniques; Lysine; Mice; Neural Pathways; Neurons; Rhodamines; Xanthenes

Single-molecule orientational imaging using total internal reflection fluorescence microscopy has been employed to investigate the dynamics of a protein-ligand system. Emission patterns from single tetramethylrhodamine (TMR)-biocytin molecules bound to streptavidin show that the TMR dipole adopts a limited number of favored orientations. The angular trajectories of individual dipoles exhibit remarkably similar patterns that are characteristic of single TMR molecules interacting with a relatively homogeneous population of nanoenvironments. Analysis of the polar and azimuthal angle distributions reveals a tendency for the dipole to assume three primary and two secondary orientations. Autocorrelation analysis of the dipole trajectories shows a predominantly bimodal behavior in the reorientation rates with the slow and fast components corresponding to the primary and secondary orientations, respectively. A number of mechanisms by which the observed orientations might be stabilized have been considered, in particular specific interactions between the zwitterionic TMR probe and charged residues on the streptavidin surface. Variations in the reorientation rates have been discussed in terms of local thermal fluctuations in the protein.

Topics: Crystallography, X-Ray; Kinetics; Ligands; Lysine; Models, Molecular; Molecular Conformation; Protein Binding; Proteins; Rhodamines; Streptavidin

Intercellular communication through gap junctions is vital for many developmental processes, including cell division and synaptogenesis. This study is the first demonstration that olfactory organ cells are functionally coupled by gap junctions. Cell coupling was examined during development in the olfactory organ using gap junction permeable dyes in live zebrafish embryos. At 1 day post-fertilization (dpf), cells of the olfactory organ were not coupled by gap junctions. At 2 and 3 dpf, olfactory organ cells passed dye from one cell to another, indicating functional coupling via gap junctions. Coupled cell cohorts included combinations of all three olfactory cell types: basal cells, support cells, and olfactory receptor cells. As the olfactory organ matured, the number of cells per coupled cell cohort increased. Gap junctional coupling corresponded with maturation of the olfactory organ and indicates that functional gap junctions may be involved in proper development of the olfactory organ.

Topics: Animals; Anti-Ulcer Agents; Carbenoxolone; Embryo, Nonmammalian; Embryonic and Fetal Development; Fluorescent Dyes; Gap Junctions; Immunohistochemistry; Lysine; Olfactory Bulb; Rhodamines; Time Factors; Zebrafish

Retrogradely labeled cells in superficial and deep layers of the entorhinal cortex (EC) were analyzed following application of the fluorescent tracer rhodamine-dextran-amine in different sites of the hippocampal formation in a slice preparation. The results demonstrate a strong projection from layer IV/V to the dentate gyrus, that is in slices significantly stronger than that from layer II. In deep layers a large number of multipolar cells were found which were only labeled by dye application to the subiculum. Patch-clamp recordings from these cells revealed intrinsic low threshold membrane potential oscillations, suggesting their possible contribution to oscillatory network activity of the EC and subiculum.

Topics: Animals; Axons; Biological Clocks; Cell Size; Dendrites; Dextrans; Entorhinal Cortex; Fluorescent Dyes; Hippocampus; Lysine; Membrane Potentials; Neural Pathways; Organ Culture Techniques; Rats; Rhodamines

By using an in vitro tract-tracing technique, the neural connections between two diencephalic cell groups, the posterior subdivision of the nucleus preopticus periventricularis (PPp) and the preglomerular nucleus (PG), was examined in the weakly electric gymnotiform fish Apteronotus leptorhynchus. Neurons of the PPp project to one area within PG, the ventromedial cell group of the medial subdivision of the preglomerular nucleus (PGm-vmc). Axons of these cells reach the ipsilateral PGm-vmc via the basic hypothalamic tract, while collaterals decussate via the postoptic commissure to innervate the contralateral PGm-vmc. We hypothesize that those neurons within PPp that project to the PGm-vmc are homologous to neurons of the medial preoptic area of mammals. As part of an elaborate circuit, PPp and PG may participate, as in mammals, in the control of complex social behavior patterns.

Topics: Animals; Brain Mapping; Coloring Agents; Dextrans; Diencephalon; Drug Combinations; Electric Fish; Lysine; Neurons; Rhodamines; Synaptic Transmission