An M-like outward current regulates the excitability of spinal motoneurones in the adult turtle

Abstract

The excitatory action of muscarine on spinal motoneurones was investigated with intracellular recordings in a slice preparation from adult turtles. In these cells muscarine is known to facilitate a persistent inward current mediated by L-type Ca(2+) channels. When this effect was blocked by nifedipine, muscarine still increased the excitability. In voltage clamp, a slowly activating outward current, generated during depolarizing voltage commands and deactivating as a tail current on return to the holding voltage, was reduced by muscarine. This outward current was activated when the voltage was stepped to potentials positive to -60 mV, was voltage sensitive and had a deactivation time constant of approximately 80 ms. These findings are compatible with an M-current. This possibility was also supported by the finding that the current was reduced by XE-991 - a selective blocker of the KCNQ potassium channels underlying M-currents in other cell types. Our findings suggest that an M-like current, mediated by a KCNQ channel, contributes to the intrinsic response properties of motoneurones in the adult spinal cord by increasing adaptation of repetitive firing and decreasing the slope of the frequency-current relation.

Figure 1. Multiple excitatory effects of muscarine

A, response of a motoneurone to a depolarizing current pulse. Muscarine (12.5 μm) increased early spiking, induced discharge acceleration and afterdepolarization. Addition of nifedipine (20 μm) blocked discharge acceleration and afterdepolarization, but did not affect the increase in excitability. Atropine (3.5 μm) partially blocked excitatory effect of muscarine. B, instantaneous spike frequency as a function of time (same data as in A). All recordings from the same cell.

Figure 2. Muscarine-sensitive tail current

In the presence of nifedipine, depolarizing voltage steps activated a voltage-sensitive slowly activating non-inactivating outward current relaxing as a tail (A, left). Addition of muscarine (12.5 μm) reduced this current (A, right). Insets: tail currents at a higher resolution. Voltage sensitivity of the tail current was not affected by muscarine (B). Action potentials were blocked by TTX.

Figure 3. Block of KCNQ channels increases excitability of motoneurones

In current clamp, addition of XE991 (1 μm) enhanced the response to a suprathreshold depolarizing current pulse (A). XE991 reduced the early adaptation of firing frequency (B), shifting the steady state f-I relation to the left and increasing its steepness (C). The data in B were obtained in response to a 1.4 nA depolarizing current pulse lasting 3s. Recordings from the same cell, in the presence of nifedipine.

Figure 4. Block of KCNQ channels reduces the tail current

In voltage clamp, addition of XE991 (1 μm) reduced the voltage-sensitive tail current (arrows in A). A similar voltage-sensitive tail current present in cobalt Ringer (B and C) was reduced by XE991 (5 μm) (C). B and C are from different motoneurones. The ionic basis of the XE991 sensitive current was investigated by applying hyperpolarized voltage steps of increasing amplitude (protocol illustrated in D) in the presence of ZD 7288 (100 μm). The amplitude of the tail current was measured as a function of the potential before and after addition of XE991 (5 μm) (D). The 2 curves were crossing at −86 mV, value which indicates the reversal potential for ions carrying the tail current. Action potentials were blocked by TTX. A and D in the presence of nifedipine.