Actin filaments mediate mechanical gating during osmosensory transduction in rat supraoptic nucleus neurons

Abstract

Osmosensory transduction is a bidirectional process displayed by neurons involved in the control of thirst and antidiuretic hormone release, and is therefore crucial for body fluid homeostasis. Although this mechanism is known to involve the activation of nonselective cation channels during hypertonicity-evoked shrinking, and the inhibition of these channels during hypotonicity-evoked swelling, the basis for this regulation is unknown. Here, we investigated this process using whole-cell patch-clamp recordings from neurons acutely isolated from the supraoptic nucleus of adult rats. The mechanosensitivity index, defined as the ratio of conductance change to normalized volume change, was quantitatively equivalent whether cell volume was increased or decreased by changes in extracellular fluid osmolality, or by changes in pipette pressure. Moreover, responses induced by hyperosmotic or hypo-osmotic media could be reversed by increasing or decreasing pipette pressure, respectively. The mechanosensitivity index was significantly reduced in neurons treated with cytochalasin-D, a compound that promotes the depolymerization of actin filaments. Conversely, cells treated with jasplakinolide, a compound that promotes actin polymerization, showed a significant increase in mechanosensitivity index. Finally, the depolarizing and excitatory effects of hypertonic stimuli were significantly enhanced by jasplakinolide and reduced by cytochalasin-D. We conclude that osmosensory transduction in these neurons is a reversible mechanical process that depends on an intact actin cytoskeleton, and the sensitivity of the transducer appears to vary in proportion with the density of actin filaments.

Figure 1.

Volume modulation of cation current by osmotic and hydrostatic pressure. A, Steady-state I–V relationships measured from isolated MNCs (V_H = −70 mV) exposed to hypotonic (left) and hypertonic solutions (right). Note that the stimuli respectively reduce and increase slope conductance. B, Relationships between changes in slope conductance (ΔG) and volume decrease (1 − nV) during osmotic stimulation. Each point depicts simultaneous measures of ΔG and 1 − nV taken at several time points during hypotonic (bottom left quadrant) or hypertonic stimuli (top right quadrant). Solid line is a linear regression fit of the data (slope = 4.1 nS/nΔV; n = 22; r = 0.6847). C, Steady-state I–V relationships measured from cells inflated by positive pressure in the patch pipette (left) and shrunken by negative pressure (right). Note that the stimuli respectively reduce and increase slope conductance. D, Relationship between ΔG and 1 − nV during pressure-evoked responses. Points depict simultaneous measures of ΔG and 1 − nV taken at various times during application of positive pressure (bottom left quadrant) or negative pressure (top right quadrant). The solid line is a linear regression fit of the data (slope = 3.9 nS/nΔV; r = 0.602; n = 21). Mean values of the reversal potentials of the ionic currents modulated by hypo-osmolality (−36 ± 3 mV), hyperosmolality (−31 ± 4 mV), positive pressure (−33 ± 5 mV), and negative pressure (−33 ± 4 mV) were not significantly different from one another (p = 0.83; one-way ANOVA), and were consistent with the involvement of SIC channels in all of these responses. The slopes of the regression lines shown in B and D are not different (p > 0.05).

Figure 2.

Reversal of osmosensory transduction by hydrostatic pressure. A, Plot showing values of membrane conductance (G) and nV observed in an isolated MNC exposed to a sustained hypotonic stimulus (−60 mosmol/kg, gray area). After 110 s, negative pressure (bar, −50 mmHg) was applied to the recording pipette. Note that this procedure reversed the effects of the hypotonic stimulus on nV and G. B, Plot showing values of G and nV observed in a cell exposed to a sustained hypertonic stimulus (+60 mosmol/kg, gray area). After 100 s, positive pressure (bar, +30 mmHg) was applied to the recording pipette. Note that this procedure reversed the effects of the hypertonic stimulus on nV and G. C, Bar graphs showing mean values of transducer sensitivity observed when hydrostatic pressure and osmotic stimuli were used to initiate (onset, open bars) or reverse (reversal, filled bar) the modulation of membrane cation conductance by changes in cell volume. No significant difference was observed (p > 0.05; two-way ANOVA).

Figure 3.

Role of F-actin in mechanotransduction. A, Whole-cell current recordings (V_H = −60 mV) from isolated MNCs exposed to Cyt-D (middle), vehicle (control, top), or JSK (bottom). Vertical deflections are current responses to hyperpolarizing steps (−20 mV) applied every 5 s to monitor G. In each case, negative pressure (∼−100 mmHg) was applied to the patch pipette (bar) to induce a decrease in cell volume of ∼10%. Note that Gd³⁺ (250 μm) was applied near the end of each recording to confirm that the current induced was caused by the activation of SIC channels. B, Bar graph showing mean (±SEM) values of transducer mechanosensitivity measured in each group (*p < 0.05; ANOVA).

Figure 4.

F-actin is required for osmosensory transduction. A, Whole-cell current-clamp recordings from isolated MNCs subjected to hypertonic stimuli (bar; +60 mosmol/kg). The traces show representative recordings taken from cells treated with vehicle (control, top), Cyt-D (middle), and JSK (bottom). In all traces, the spikes are truncated and the dashed line indicates −55 mV. B, Bar graphs showing that mean (±SEM) osmoreceptor potentials (depolarization measured at 60 s; middle) and excitation (increase in firing observed between 50 and 70 s compared with control; right) were significantly reduced by Cyt-D and significantly enhanced by JSK, whereas no difference was noted in the amount of shrinking evoked by the osmotic stimulus (p = 0.923; measured 60 s after stimulus onset; left). *p < 0.05.