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First published online February 1, 2008
Journal of Experimental Biology 211, 630-641 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.008565

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Swelling-activated chloride channels in leech Retzius neurons

Philippe Coulon^*, Hans-Joachim Wüsten, Peter Hochstrate and Paul Wilhelm Dierkes

Institut für Neurobiologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany

View larger version (72K): [in this window] [in a new window]   Fig. 1. Confocal images of leech Retzius neurons under isotonic and anisotonic conditions. Retzius neurons were loaded iontophoretically with the fluorescent dye Oregon Greens® 488 BAPTA-1. Under isotonic conditions (Control), the cell bodies of the neurons were ball shaped. Under hypertonic conditions (+85 mmol l–1 NaCl, A), the neurons shrank and showed membrane invaginations. Under hypotonic conditions (–59 mmol l–1 NaCl, B), the neurons swelled and showed evaginations (`blebs'). Images were taken after 5 min under anisotonic conditions.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Volume changes of Retzius neurons under anisotonic conditions. Volume changes were measured via the fluorescence intensity of Fura-2 excited at its isosbestic wavelength (see text for details). (A) Hypertonic solution (+85 mmol l–1 NaCl, 0.5 0/exp) induced cell shrinkage. In 24 out of a total of 84 cells regulatory volume increase (RVI) was observed, during which the cell volume partly recovered from a minimum of 77±7% of the control value in standard leech saline (SLS) to 82±6%. (B) Raising the extracellular osmolality caused decreases in the cell volume that were significantly smaller than expected for an ideal osmometer (broken line). Note that the cell shrinkage was not dependent on whether the extracellular osmolality was increased by adding NaCl, which also raises the osmotic strength of the extracellular solution, or by adding carbohydrates such as sorbitol or glucose, which leaves the ionic strength constant. (C) Hypotonic solution (–59 mmol l–1 NaCl, 2.4 0/exp) induced cell swelling. A regulatory volume decrease (RVD) was never observed (N=75, –40 to –81 mmol l–1 NaCl). (D) Reducing the extracellular osmolality caused cell volume increases that were significantly smaller than expected for an ideal osmometer (broken line). Volrel, relative cell volume. V0 and Vexp, cell volume at osmolality 0 and exp, with index `0' indicating isotonic conditions (SLS) and index `exp' indicating experimentally changed, anisotonic conditions. The volume of an ideal osmometer is inversely proportional to the osmolality of the surrounding medium. This relationship is indicated by the broken line in B and D. Data in B and D are means ± s.d. of N=2–84 experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Effect of reducing the extracellular NaCl concentration on Em, Rin and the generation of action potentials. (A) Continuous recording showing the effect of reducing the extracellular NaCl concentration by 40 mmol l–1 for 5 min. Upward deflections are spontaneous action potentials, downward deflections were caused by the injection of negative current (–5 nA, duration 1 s, pulse interval 10 s). (B) Segment of the trace in A on an expanded time scale, shortly before and after the reduction of the extracellular NaCl concentration, as indicated. Action potentials truncated due to high gain. Note the partial recovery of the current-induced hyperpolarization (`depolarizing sag', arrow), which is due to the activation of hyperpolarization-activated (Ih) channels. (C) Rin calculated from the current-induced hyperpolarization shown in B.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Effect of the extracellular osmolality on Em and Rin. The extracellular osmolality was varied by increasing or decreasing the NaCl concentration of the bath solution, and the changes in Em (A) and Rin (B) were determined 5 min later. For comparison, volume changes (Volrel) as a percentage of control values are shown (grey squares, right-hand axes, mean values of N=2–84 experiments, see Fig. 2B,D). Data are means ± s.d.; number of experiments is given next to each data point. Asterisks indicate significant differences from the data in SLS (*P<0.05, **P<0.01). Osmolalities of the anisotonic solutions were normalized to SLS (Relative osmolality, see Fig. 2).

View larger version (15K): [in this window] [in a new window]   Fig. 5. Effect of the membrane potential on cell volume changes. (A) Cell volume was measured using ion-sensitive microelectrodes filled with Corning 477317, which monitored the concentration of the volume marker TMA+. In parallel, Em was clamped to a fixed holding potential (here –60 mV) and the clamp current (IVC) was recorded. Hypotonic conditions (–40 mmol l–1 NaCl) caused a reversible swelling and a transient outward current, while hypertonic conditions (+40 mmol l–1 NaCl) caused a reversible shrinkage and a transient inward current. (B) Cell volume changes (Volrel) recorded under anisotonic conditions at Em=–40 to –70 mV. Cell shrinkage did not change significantly with the holding potential, while cell swelling was almost twice as large at –70 mV as at –40 mV (P<0.05). Data are means ± s.d.; number of experiments is given beside the data bars (bar at +40 mmol l–1 NaCl, –70 mV results from a single experiment).

View larger version (21K): [in this window] [in a new window]   Fig. 6. Membrane currents under isotonic and hypotonic conditions. (A) Superimposed membrane currents upon shifting Em for 1 s from –50 mV to values ranging from –120 to –40 mV (10 mV increments, 5 s intervals) under isotonic conditions (SLS) and after 5 min in hypotonic solution (–40 mmol l–1 NaCl). Subtraction of the current needed under isotonic conditions from the respective current under hypotonic conditions isolated the swelling-activated membrane current (`Difference'). The dependence of this current on Em is shown in B. The traces are means of 34 experiments. Before averaging, the single traces were filtered through an 8-pole Bessel filter (100 Hz). The performance of the voltage-clamp protocol took 1 min; in the meantime, the recording system was in the current-clamp mode. (B) Voltage dependence of the averaged swelling-activated membrane current under control conditions (see A), in Cl––free solution and in the presence of 0.5 mmol l–1 DIDS. Data are means ± s.d. of N=47 (control, included are 13 experiments with a clamp duration of only 0.5 s), N=7 (Cl– free) and N=11 experiments (DIDS). Arrowhead indicates the reversal potential of the swelling-activated current under control conditions.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Time course of activation of the swelling-induced membrane current. (A) Dependence of the swelling-activated membrane current on Em at different times after reducing the extracellular osmolality (–40 mmol l–1 NaCl). Retzius neurons were clamped from a holding potential of –50 mV to –100, –80, –60 or –40 mV for 1 s with an interstimulus interval of 2 s (see Fig. 6A). This protocol was applied in SLS as well as 50, 100... 300 s after changing the extracellular osmolality to obtain the presented I–V relationships. Data are means ± s.d. of N=5 experiments. (B) Time course of the activation and deactivation of the swelling-induced membrane current, as derived from the data shown in A, as well as from current recordings after return to SLS. Activation and deactivation of the swelling-induced current occurred with a similar time course to the changes in cell volume (compare Fig. 2C, Fig. 5A, Fig. 10A,B).

View larger version (8K): [in this window] [in a new window]   Fig. 8. Effect on Em and Rin of reducing the extracellular osmolality in the presence of Cl– channel blockers and in Cl–-free solution. The preparations were exposed to the Cl– channel blocker DIDS (0.5 mmol l–1) or NPPB (50 µmol l–1), or to isotonic Cl–-free solution (Cl– replaced by gluconate), and after 5 min Em and Rin were measured. Subsequent reduction of the extracellular osmolality by omitting 40 mmol l–1 NaCl or sodium gluconate from the bath solution caused changes in Em (A) and Rin (B) that were determined after 5 min. Significant differences from the control data (taken from Fig. 4, broken line) are indicated by asterisks (**P<0.01).

View larger version (10K): [in this window] [in a new window]   Fig. 9. Effect of colchicine on the swelling-induced changes in Rin and membrane current. The swelling-induced changes in Rin (A) and membrane current (B) were measured 5 min after reducing the extracellular osmolality (–40 mmol l–1 NaCl), either under control conditions or after incubation of the preparations in SLS plus 25 µmol l–1 colchicine for 1 h. After colchicine application the swelling-induced changes in Rin and membrane current were reduced. Data are means ± s.d. of N=5 experiments.

View larger version (32K): [in this window] [in a new window]   Fig. 10. Role of the cytoskeleton in cell volume changes. Using Fura-2 as a volume marker, cell volume changes were monitored in the presence of substances that affect the assembly of microtubules or actin filaments. (A,B) The tubulin polymerization inhibitors colchicine (25 µmol l–1) and vinblastine (200 µmol l–1) had little effect on the cell shrinkage under hypertonic conditions (+85 mmol l–1 NaCl) but caused an increased swelling under hypotonic conditions (–59 mmol l–1 NaCl; 5 min each). The effects were abolished in the presence of the tubulin polymerization enhancer paclitaxel (30 nmol l–1). The simultaneously measured cytosolic Ca2+ concentration was hardly affected by the various drugs and/or changes in the extracellular osmolality. (C) Statistical analysis (means ± s.d., number of experiments below data bars) of experiments under hypotonic conditions with colchicine, vinblastine, paclitaxel, and the actin polymerization inhibitors cytochalasine B and D. Cell swelling increased when tubulin polymerization was inhibited (P<0.01) but not when actin polymerization was inhibited.