Activity-dependent redistribution of Kv2.1 ion channels on rat spinal motoneurons

Abstract

Homeostatic plasticity occurs through diverse cellular and synaptic mechanisms, and extensive investigations over the preceding decade have established Kv2.1 ion channels as key homeostatic regulatory elements in several central neuronal systems. As in these cellular systems, Kv2.1 channels in spinal motoneurons (MNs) localize within large somatic membrane clusters. However, their role in regulating motoneuron activity is not fully established in vivo. We have previously demonstrated marked Kv2.1 channel redistribution in MNs following in vitro glutamate application and in vivo peripheral nerve injury (Romer et al., 2014, Brain Research, 1547:1-15). Here, we extend these findings through the novel use of a fully intact, in vivo rat preparation to show that Kv2.1 ion channels in lumbar MNs rapidly and reversibly redistribute throughout the somatic membrane following 10 min of electrophysiological sensory and/or motor nerve stimulation. These data establish that Kv2.1 channels are remarkably responsive in vivo to electrically evoked and synaptically driven action potentials in MNs, and strongly implicate motoneuron Kv2.1 channels in the rapid homeostatic response to altered neuronal activity.

Keywords: C‐boutons; Kv2.1; voltage‐gated ion channels, activity dependent; α‐motoneuron.

Figure 1

Reorganization of Kv2.1‐IR clusters on rat lumbar α‐motoneurons is activity dependent. The sciatic nerve was stimulated in vivo at 150 Hz and 4x tetanic threshold for 10 min. Motoneurons displayed in (A and B) are from the same tissue slice with fixed imaging parameters. Scale bars are 10 μm. (A) Micrograph of a confocal stack (33 × 1.0 μm z‐steps) of a contralateral “control” motoneuron showing Kv2.1‐IR (red). (B) Micrograph of a confocal stack (30 × 1.0 μm z‐steps) of an MG α‐motoneuron following 10 min of sciatic nerve stimulation showing reduced Kv2.1‐IR (red) macrocluster areas compared to the unstimulated motoneuron in panel A. (C) Quantitative analysis of reduced Kv2.1‐IR soma macrocluster areas on medial gastrocnemius α‐motoneurons following 10 min of sciatic nerve stimulation in all 3 rats sampled. (D) Pooled quantitative analysis of Kv2.1‐IR following 0.5 μmol/L tetrodotoxin (TTX) showing no significant changes compared to the absence of TTX application. (C and D) N = the number of Kv2.1 macroclusters sampled. Significance (P < 0.05) is indicated with asterisk and determined with Mann–Whitney and data are presented ± SD.

Figure 2

Kv2.1‐IR macroclusters on medial gastrocnemius α‐motoneurons recover to control sizes by 2 h following sciatic nerve stimulation in all 3 rats sampled. Absence of significance was determined with Mann–Whitney T‐test and data are presented ± SD. N = number of Kv2.1 macroclusters sampled.

Figure 3

Kv2.1‐IR macroclusters on α‐motoneurons significantly reduce when motor axons are stimulated. (A) Quantitative analysis of reduced Kv2.1‐IR soma macrocluster areas on lumbar α‐motoneurons following sciatic nerve stimulation with dorsal rhizotomy in all 3 rats sampled. The impact of dorsal rhizotomy on Kv2.1‐IR macrocluster areas on lumbar α‐motoneurons was quantified in 3 rats. N = the number of Kv2.1 macroclusters sampled. Significance (P < 0.05) is indicated with asterisk and determined with Mann–Whitney T‐test and data are presented ± SD. (B) Electromyography (EMG) of medial gastrocnemius (MG) muscle indicates that the injury discharge during dorsal root cuts did not cause motor output activity in the muscle. The MG nerve (MGN) itself was stimulated before and after the dorsal roots were cut to confirm EMG recordings.

Figure 4

Kv2.1‐IR macroclusters on α‐motoneurons significantly reduce when sensory afferent circuitry is driven through dorsal root stimulations. Quantitative analysis of Kv2.1‐IR on medial gastrocnemius α‐motoneuron somas in all three animals samples demonstrates significant decrease in area. N = the number of Kv2.1 macroclusters sampled. Significance (P < 0.05) is indicated with asterisk and determined with Mann–Whitney T‐test and data are presented ± SD.

Figure 5

Summary of spinal motoneuron Kv2.1 ion cluster responses to various in vivo stimuli. Kv2.1 ion channels significantly reduce by 30% following sciatic nerve stimulation (sciatic stimulation), an effect inhibited by the application of tetrodotoxin proximal to the stimulation site (TTX + stimulation) that results in an 4% increase in cluster size. Channel cluster sizes are restored to the original sizes, with a 2% reduction, 2 h following the sciatic nerve stimulation (2 h poststimulation). A significant 28% reduction in cluster size was produced when the effect of the sciatic nerve stimulation was isolated to just antidromic activation of motor axons through dorsal rhizotomy (sciatic stimulation with dorsal rhizotomy). However, the dorsal rhizotomy itself, without nerve stimulation, also induced a significant 11% reduction cluster areas (dorsal rhizotomy without stimulation). Finally, the sensory‐evoked synaptic activity, through dorsal root stimulations (dorsal root stimulation), caused a significant 19% reduction in cluster areas.