5-HT1A receptors increase excitability of spinal motoneurons by inhibiting a TASK-1-like K+ current in the adult turtle

Abstract

The modulatory effects of serotonin mediated by 5-HT1A receptors in adult spinal motoneurons were investigated by intracellular recordings in a slice preparation from the turtle. In current-clamp mode, activation of 5-HT1A receptors by 8-OH-DPAT led to depolarization and an increase in input resistance in most motoneurons but caused hyperpolarization and a decrease in input resistance in the remaining smaller fraction of cells. When slices were preincubated in medium containing the 5-HT1A receptor antagonist WAY-100635, 8-OH-DPAT had no effect. In voltage-clamp mode, with 1 mM CsCl in the bathing medium, 8-OH-DPAT consistently inhibited a leak current that was sensitive to extracellular acidification and anandamide, a TASK-1 channel blocker. In medium with a low pH, as in the presence of anandamide, 8-OH-DPAT had no effect. Our results show that activation of 5-HT1A receptors contributes to the excitatory effect of serotonin on spinal motoneurons by inhibition of a TASK-1 potassium channel leading to depolarization and increased input resistance.

Figure 3. 8-OH-DPAT blocks a leak conductance

Experiments in voltage-clamp mode in the presence of TTX (1 μm) and CsCl (1 mm). A, current response to 1 s voltage steps of incrementing amplitude. B, response to the same protocol in the presence of 8-OH-DPAT (10 μm). Note the inhibition of a voltage-insensitive current (see the dots linked by lines in the I–V plot in C). C, I–V plot from the data from the step protocol (lines + dots) and from a voltage ramp before (black line) and after addition of 8-OH-DPAT (red line). The two protocols produced similar results.

Figure 1. Activation of 5-HT receptors induces heterogeneous effects in spinal motoneurons

A, response of a motoneuron to a hyperpolarizing current pulse before (black trace) and after addition of 5-HT (10 μm; red trace). 5-HT hyperpolarized the cell from −61 to −68 mV and decreased the input resistance from 32 to 25 MΩ (−21.9 %). B, response to positive depolarizing current pulses. In 5-HT (red), a positive bias current was injected in the cell to adjust the membrane potential to the same value as in control. Note the decrease in excitability. C, response of another motoneuron to hyperpolarizing current pulses. 5-HT (red) depolarized the cell from −70 to −68 mV and increased the input resistance from 18 to 20 MΩ (+11.1 %). D, response to positive depolarizing current pulses. Note the increase in excitability induced by 5-HT (red). No bias current was injected. A and B from one motoneuron, C and D from another motoneuron. In all experiments synaptic potentials were blocked by CNQX (25 μm), AP7 (25 μm) and strychnine (10 μm).

Figure 2. Activation of 5-HT_1A receptors induces various effects in spinal motoneurons

Data in A–C and D–F were from two different motoneurons. In all experiments synaptic potentials were blocked by CNQX (25 μm), AP5 (50 μm) and strychnine (10 μm). I_h was blocked by ZD7288 (100 μm). A, response to a hyperpolarizing current pulse. 8-OH-DPAT (10 μm; red trace) depolarized the cell from −66 to −61 mV and increased input resistance from 25 to 27.5 MΩ (+10 %). B, subthreshold response to a depolarizing current pulse in control medium. C, suprathreshold response to the same current pulse in the presence of 8-OH-DPAT; resting potential normalized with negative bias current (i.e. −66 mV). D, response of another motoneuron to a hyperpolarizing current pulse. Addition of 8-OH-DPAT (10 μm; red trace) hyperpolarized the cell from −70 to −73 mV, and decreased its input resistance from 49 to 46 MΩ (−6 %). E and F, response to a depolarizing current pulse before (E) and after (F) addition of 8-OH-DPAT. With the membrane potential normalized to −70 mV with depolarizing bias current, the same current pulse as in E generated one rather than two spikes. Inset: in a motoneuron from a slice preincubated in WAY-100635 (10 μm), 8-OH-DPAT (red trace) did not change the membrane potential or input resistance.

Figure 4. 8-OH-DPAT inhibits a TASK-1-like channel

Experiments in voltage-clamp mode in the presence of TTX (1 μm) and CsCl (1 mm). A, I–V plot from the ramp protocol before (black) and after addition of 8-OH-DPAT (10 μm; red). The 8-OH-DPAT sensitive component (grey) showed mild outward rectification. B, normalized and averaged I–V plot from the ramp protocol before (black) and after addition of 8-OH-DPAT (10 μm; red) (n = 4). Inset: normalized current measured at −110 mV. The inhibition was statistically significant. C, normalized and averaged I–V plot before (black) and after changing to medium with low pH (green) (n = 7). The inhibition was significant (*). Subsequent addition of 8-OH-DPAT (10 μm; red) had no additional effect. D, normalized and averaged I–V plot before (black) and after addition of anandamide (blue) (n = 4). The inhibition was significant (*). Subsequent addition of 8-OH-DPAT (10 μm) had no additional effect (inset; red).

Figure 5. 5-HT increases motoneuron excitability by inhibiting TASK-1 channels

A, mean input resistance of motoneurons (n = 4). First column: control conditions. Second column: after addition of 5-HT (10 μm). Third column: after wash out of 5-HT. Fourth column: in low pH Ringer solution (6.4). Fifth column: after addition of 5-HT (10 μm) in the low pH Ringer solution. The input resistance was estimated as the voltage change induced by small hyperpolarizing current pulses applied from resting membrane potential divided by the amount of current injected. B, in normal medium, 5-HT induced an increase in input resistance of 15.7 ± 7.4 %. In low pH medium, 5-HT increased the input resistance by 8.8 ± 6.6 %, a value significantly lower than the one measured in normal medium (P < 0.01; paired t test). Synaptic potentials were blocked by CNQX (25 μm), AP7 (25 μm), strychnine (10 μm) and bicuculline (20 μm).