SK channel inhibition mediates the initiation and amplitude modulation of synchronized burst firing in the spinal cord

Abstract

Burst firing in motoneurons represents the basis for generating meaningful movements. Neuromodulators and inhibitory receptor blocker cocktails have been used for years to induce burst firing in vitro; however, the ionic mechanisms in the motoneuron membrane that contribute to burst initiation and amplitude modulation are not fully understood. Small conductance Ca²⁺-activated potassium (SK) channels regulate excitatory inputs and firing output of motoneurons and interneurons and therefore, are a candidate for mediating bursting behavior. The present study examines the role of SK channels in the generation of synchronized bursting using an in vitro spinal cord preparation from adult mice. Our results show that SK channel inhibition is required for both initiation and amplitude modulation of burst firing. Specifically, administration of the synaptic inhibition blockers strychnine and picrotoxin amplified the spinal circuit excitatory drive but not enough to evoke bursting. However, when SK channels were inhibited using various approaches, the excitatory drive was further amplified, and synchronized bursting was always evoked. Furthermore, graded SK channel inhibition modulated the amplitude of the burst in a dose-dependent manner, which was reversed using SK channel activators. Importantly, modulation of neuronal excitability using multiple approaches failed to mimic the effects of SK modulators, suggesting a specific role for SK channel inhibition in generating bursting. Both NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptors were found to drive the synchronized bursts. The blocking of gap junctions did not disturb the burst synchrony. These results demonstrate a novel mechanistic role for SK channels in initiating and modulating burst firing of spinal motoneurons.NEW & NOTEWORTHY This study demonstrates that cholinergic inhibition or direct blockade of small conductance Ca²⁺-activated potassium (SK) channels facilitates burst firing in spinal motoneurons. The data provide a novel mechanistic explanation for synchronized bursting initiation and amplitude modulation through SK channel inhibition. Evidence also shows that synchronized bursting is driven by NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptors and that gap junctions do not mediate motoneuron synchronization in this behavior.

Keywords: SK channels; motor output; spinal motoneurons; synchronized bursting.

Fig. 1.

Pharmacological inhibitors of SK channels initiate synchronized bursting in the disinhibited spinal cord. A: the application of strychnine (STR) and bicuculline (BIC) induces spontaneous bursts in the spinal cord. The bursts are synchronized on both sides of the cord, as shown at the S3 segment [right (R S3) and left (L S3) ventral roots]. The inset shows a single burst of activity on each side of the cord on a smaller time scale. B: strychnine and picrotoxin (PTX) did not induce bursting until apamin was added. Inset shows the spikes and firing rates recorded intracellularly from a motoneuron during spontaneous bursting and the activity recorded simultaneously from the segmentally aligned ventral root. C: a different SK channel blocker (d-tubocurarine) also initiated synchronized bursting in the disinhibited spinal cord. D: reduction of extracellular free Ca²⁺ through the application of the Ca²⁺ chelator EGTA similarly evokes bursting. Inset (C and D) show a single burst on each side.

Fig. 2.

Muscarinic inhibition of SK channels facilitates bursting. A: the muscarinic agonist oxotremorine induces bursting in the presence of strychnine (STR) and picrotoxin (PTX). This bursting was inhibited by the SK channel activator, CyPPA. The preapplication of either the selective M₂ receptor antagonist methoctramine (B) or the selective M₁ receptor antagonist telenzepine (C) does not inhibit oxotremorine-induced bursting. D: the combined M₁ and M₂ receptors blockade completely inhibits the oxotremorine effect; however, the application of the direct SK channel blocker apamin induces synchronized bursts.

Fig. 3.

Amplification of the excitatory drive by apamin vs. strychnine/picrotoxin (STR/PTX). A and B: average ventral root response to single pulses of dorsal root stimulation at 1 × T, before (black) and after (gray) bath application of either apamin (A) or STR/PTX (B). In these experiments, each ventral root response in presence of the drug(s) is normalized to its control response to account for the variability among preparations. C and D: ventral root response to 5 pulses of dorsal root stimulation at 50 Hz with intensity of 10 × T before (black) and after (gray) bath application of apamin (C) or STR/PTX (D). In these experiments, the coAP amplitudes are normalized to the amplitude of the first pulse recorded in nACSF without any drugs. Quantification of the effect of apamin or STR/PTX was done from multiple experiments. Data represented as the grand means ± SD. Asterisks indicate statistically significant differences (*P < 0.05; **P < 0.01). P1–P5, pulses 1–5.

Fig. 4.

SK channel inhibition is necessary for burst initiation. A: application of the Ca²⁺ PIC blocker nimodipine does not change the synchronized bursting. B: a summary of the effect of nimodipine on the burst characteristics. Data plotted as the grand means obtained from multiple experiments, and error bars represent SD. C: intracellular recording from an S3 motoneuron after the application of serotonin (20 µM) in the presence of STR and PTX. In response to ramp current injection (bottom), the cell fires repetitively and continues to fire at lower current values (ΔI = 1.3 nA) on the descending ramp (middle), indicating that persistent inward currents are activated. The top shows the instantaneous firing frequency. D: synchronized bursts induced by serotonin in the presence of STR and PTX are not changed by the application of nimodipine, whereas CyPPA eliminates the bursting. Asterisk indicates statistically significant difference (*P < 0.05).

Fig. 5.

The modulation of the level of SK channel inhibition grades the burst amplitude. A: bursts in the ventral roots increase in amplitude with increasing apamin concentrations in a dose-dependent fashion. B: the burst peak amplitude is correlated with apamin concentration. C: oxotremorine induces a similar graded response, which is inhibited by CyPPA in a dose-dependent manner. D: similar to apamin, oxotremorine concentration determines the burst amplitude. Note that the concentrations of oxotremorine were first converted to nanomolar values; then the logarithms of those values were used. E: ventral root responses to electrical stimulation of the ipsilateral dorsal roots at 1 ×, 1.5 ×, 2 ×, 5 ×, and 10 × T in the presence of STR/PTX and apamin. The response has 2 components: a compound action potential (arrowhead in the 1 × T response), followed by a burst (circles). The inset shows the 1 × T response at a less-compressed time scale. F: at high apamin concentration (100 nM), the burst amplitude does not change with the stimulus intensity; however, the relationship is steeper at lower apamin concentrations (5 nM). The burst amplitudes were normalized to that obtained at the highest drug concentration or stimulation intensity (i.e., 10 × T). Each data point on the graph is the grand mean obtained from multiple experiments, and error bars represent SD. Asterisks indicate statistically significant difference from the preceding data point (*P < 0.05).

Fig. 6.

Synchronized bursts are synaptically driven through glutamate receptors. A: the blocking of the NMDA receptors with APV does not eliminate the spontaneous bursting but decreases its frequency. B: a summary of the effect of APV on the burst characteristics. Data plotted as the grand means obtained from multiple experiments, and error bars represent SD. C: DNQX, the AMPA receptor blocker, completely stops the bursting; however, electrical stimuli to dorsal roots (arrows, left inset) can still trigger bursting. When NMDA is added to the recording solution, bursting can be resumed while AMPA receptors are still blocked. Furthermore, the addition of APV inhibited this effect. Electrical stimulation of the dorsal roots no longer evokes bursting when both AMPA and NMDA receptors are blocked (right inset). Asterisks indicate statistically significant differences (*P < 0.05).

Fig. 7.

Synchronization is not mediated via gap junctions. A: at different segments on the same side of the cord, the bursts and their subcomponents are time locked (arrows) with ~10 ms delays (see expanded time scale, inset). B: at the same segment, bursts and subcomponents are synchronized with a shorter delay (~5 ms; see inset). Bottom shows intracellular recording from a motoneuron at the same segment, with the firing behavior matching the root discharge on both sides of the cord. C: the gap junction blocker carbenoxolone does not change the bursting synchrony. D: a summary of the effects of carbenoxolone on the burst characteristics. Data plotted as the grand means obtained from multiple experiments, and error bars represent SD. Asterisk indicates statistically significant differences (*P < 0.05).