n-(ethoxycarbonylmethyl)-6-methoxyquinolinium and fluorexon

Adult primary afferent neurons are depolarized by GABA throughout their entire surface, including their somata located in dorsal root ganglia (DRG). Primary afferent depolarization (PAD) mediated by GABA released from spinal interneurons determines presynaptic inhibition, a key mechanism in somatosensory processing. The depolarization is due to Cl(-) efflux through GABA(A) channels; the outward Cl(-) gradient is generated by a Na+,K+,2Cl(-) cotransporter (NKCC) as first established in amphibians. Using fluorescence imaging microscopy we measured [Cl(-)]i and cell water volume (CWV) in dissociated rat DRG cells (P0-P21) loaded with N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide and calcein, respectively. Basal [Cl(-)]i was 44.2 +/- 1.2 mM (mean +/- SE), Cl(-) equilibrium potential (E Cl) was -27.0 +/- 0.7 mV (n = 75). This [Cl(-)]i is about four times higher than electrochemical equilibrium. On isosmotic removal of external Cl(-), cells lost Cl(-) and shrank. On returning to control solution, cells reaccumulated Cl(-) and recovered CWV. Cl(-) reaccumulation had Na+-dependent (SDC) and Na+-independent (SIC) components. The SIC stabilized at [Cl(-)]i = 13.2 +/- 1.2 mM, suggesting that it was passive (E(Cl) = -60.5 +/- 3 mV). Bumetanide blocked CWV recovery and most (65%) of the SDC (IC50 = 5.7 microM), indicating that both were mediated by NKCC. Active Cl(-) uptake fell with increasing [Cl(-)]i and became negligible when [Cl(-)]i reached basal levels. The kinetics of active Cl(-) uptake suggests a negative feedback system in which intracellular Cl(-)regulates its own influx thereby keeping [Cl(-)]i constant, above electrochemical equilibrium but below the value that would attain if NKCC reached thermodynamic equilibrium.

Topics: Animals; Animals, Newborn; Bumetanide; Chlorides; Dose-Response Relationship, Drug; Extracellular Fluid; Fluoresceins; Fluorescent Dyes; Ganglia, Spinal; Neurons, Afferent; Quinolinium Compounds; Rats; Sodium; Sodium Potassium Chloride Symporter Inhibitors; Sodium-Potassium-Chloride Symporters; Thermodynamics

The morphology of viable taste disks of the frog was explored with multi-photon microscopy. In order to identify single sensory or supporting cells within the tissue, we searched for fluorescent dyes that stained subsets of the cell population or possibly cell types. Some cell types indeed stained preferentially with certain fluorescent dyes. A subset of glia-like cells (type Ic) stained with BCECF, a H+-sensitive dye, and indo-1, a Ca2+-sensitive dye, both presented in the membrane-permeant ester form. BCECF-ester also stained the dendrites of type III receptor cells, but indo-1 ester did not. Receptor cells of type II stained with MQAE, a positively charged Cl- -sensitive dye. A subset of type II cells accumulated amiloride, a positively charged fluorescent diuretic. Certain supporting cells, i.e., wing cells (type Ib) and glia-like cells (type Ic), were labeled by negatively charged dyes, e.g., calcium green-1 dextran. Mucus cells (type Ia) were stained with only two of the 19 dyes examined, and Merkel-like basal cells (type IV) were stained only with a membrane-labeling voltage-sensitive dye, presumably by endocytosis. No dye was found which would stain all types of cells or all receptor cells. This finding reveals a potential problem for future functional imaging aiming at population responses, as the responses of unstained cells then would remain unobserved. Specificity of dyes with respect to cell types was sufficient to identify supporting cells and receptor cells. Cell shape could then be reconstructed, using optical slicing and rendering techniques. Thus populations of dye-loaded elongated cells, especially types Ic, II and III, could for the first time be visualized in three dimensions.

Topics: Acridines; Amiloride; Animals; Cell Membrane; Colforsin; Dextrans; Diffusion; Fluoresceins; Fluorescent Dyes; Fura-2; Imaging, Three-Dimensional; Indoles; Iontophoresis; Microscopy, Confocal; Microscopy, Fluorescence, Multiphoton; Organic Chemicals; Pyridinium Compounds; Quaternary Ammonium Compounds; Quinolinium Compounds; Rana esculenta; Rana ridibunda; Staining and Labeling; Taste Buds