Fig. 4.

Intracellular Cl⁻ concentration ([Cl⁻]_i) in DRG neurons of identified phenotype. A: differential interference contrast (DIC) image of acutely dissociated rat DRG neurons (P12). B: isolectin B4 (IB4) fluorescent labeling (FITC) of the cells in A. IB4 appears as a white halo around the surface of the cells that bind it. IB4⁺ cells are of small size [soma cross-sectional area (CSA) ∼450 μm²]. The only large neuron (1,506 μm²) in the field (arrowhead) is IB4⁻. Small colored circles in A and B delimit the digital pinhole areas from which MQAE fluorescence was collected. C: frequency distribution of dissociated DRG neurons according to cell size (soma area) and IB4 labeling (n = 695). Red bars represent IB4⁺ cells and black bars IB4⁻ cells. All IB4⁺ cells are small or medium size, whereas IB4⁻ cells are small or medium to large size (note skewed tail toward large sizes). D: typical intracellular Cl⁻ transient in response to isosmotic removal and readdition of external Cl⁻ used to measure basal [Cl⁻]_i and Cl⁻ equilibrium potential (E_Cl) in single-identified DRG neurons loaded with MQAE. The neuron was initially equilibrated in isosmotic control solution (ISO). On removal of external Cl⁻ (0 Cl⁻), the cell was depleted of Cl⁻. On restoration of external Cl⁻ (131 mM), [Cl⁻]_i recovered to initial values. All the solutions in this group of cells were buffered with HEPES. The basal [Cl⁻]_i (measured as the difference indicated by the bracketed arrow labeled with a red asterisk) in this neuron (IB4⁻, 309 μm² CSA, P12) was 60 mM, and E_Cl = −22.2 mV. Inset: calibration plot of relative MQAE-emitted fluorescence (F_o/F_t) as a function of [Cl⁻] in the calibration solutions. The slope of this relation is the Stern-Volmer constant (K_sv in Eq. 1), which in this case was 19.2 M⁻¹. E: changes in [Cl⁻]_i following isosmotic removal and readdition of external Cl⁻, in the absence and presence of external Na⁺. All the solutions were buffered with CO₂/HCO₃⁻. On removal of external Cl⁻ (0 Cl⁻), the cell (IB4⁺, CSA 556 μm², P0) was depleted of Cl⁻. On restoration of external Cl⁻ (113 mM) in the absence of external Na⁺ (0 Na⁺) there was a small increase in [Cl⁻]_i that reached steady state at the expected electrochemical equilibrium (E_Cl = −70.8 mV; [Cl⁻]_i = 7.2). This is the sodium-independent (SI) component of Cl⁻ accumulation. On exposure to the ISO control solution, [Cl⁻]_i recovered to initial values (44 mM, measured as indicated by red asterisk; E_Cl = 24.2 mV). The later recovery is the sodium-dependent (SD) component of Cl⁻ accumulation. Scale bar in B applies to A.