PLoS One. 2014 Dec 29;9(12):e114328.

Down-regulation of KCC2 expression and phosphorylation in motoneurons, and increases the number of in primary afferent projections to motoneurons in mice with post-stroke spasticity.

Toda T1, Ishida K1, Kiyama H2, Yamashita T3, Lee S1.
  • 1Department of Physical and Occupational Therapy, Graduate School of Medicine, Nagoya University, Nagoya, Japan.
  • 2Department of Functional Anatomy and Neuroscience, Graduate School of Medicine, Nagoya University, Nagoya, Japan.
  • 3Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, Osaka, Japan.

 

Abstract

Spasticity obstructs motor function recovery post-stroke, and has been reported to occur in spinal cord injury and electrophysiological studies. The purpose of the present study was to assess spinal cord circuit spasticity in post-stroke mice. At 3, 7, 21, and 42 d after photothrombotic ischemic cortical injury in C57BL/6J mice, we observed decreased rate-dependent depression (RDD) of the Hoffmann reflex (H reflex) in the affected forelimb of mice compared with the limbs of sham mice and the non-affected forelimb. This finding suggests a hyper-excitable stretch reflex in the affected forelimb. We then performed immunohistochemical and western blot analyses to examine the expression of the potassium-chloride cotransporter 2 (KCC2) and phosphorylation of the KCC2 serine residue, 940 (S940), since this is the main chloride extruder that affects neuronal excitability. We also performed immunohistochemical analyses on the number of vesicular glutamate transporter 1 (vGluT1)-positive boutons to count the number of Ia afferent fibers that connect to motoneurons. Western bolts revealed that, compared with sham mice, experimental mice had significantly reduced KCC2 expression at 7 d post-stroke, and dephosphorylated S940 at 3 and 7 d post-stroke in motoneuron plasma membranes. We also observed a lower density of KCC2-positive areas in the plasma membrane of motoneurons at 3 and 7 d post-stroke. However, western blot and immunohistochemical analyses revealed that there were no differences between groups 21 and 42 d post-stroke, respectively. In addition, at 7 and 42 d post-stroke, experimental mice exhibited a significant increase in vGluT1 boutons compared with sham mice. Our findings suggest that both the down-regulation of KCC2 and increases in Ia afferent fibers are involved in post-stroke spasticity.

PMID: 25546454

 

Supplements:

We tried to confirm whether KCC2 (potassium-chloride cotransporter 2) expression decreases in motoneurons after stroke. KCC2 downregulation is well known as one of mechanism of spasticity.

KCC2 serves to extrude chloride from neurons, while Na-K-Cl cotransporter 1 (NKCC1) is an active transporter of sodium, potassium, and chloride into and out of cells. Normally, neurons maintain low intracellular chloride levels (Supplement Figure 1). Therefore, both gamma-aminobutyric acid (GABA)A and glycine receptors work in an inhibitory fashion in order to maintain low chloride in the cytosol and high chloride extracellularly. However, in brain and spinal cord injuries, the KCC2 expression decrease in plasma membrane of neuron. These inhibitory receptors in the neurons work in excitatory fashion.

 

supFig1

Supplement Figure 1. Down-regulation of KCC2 induces loss of inhibitory neuron

 

Intracellular chloride ions in neurons keep low by functions of KCC2 and NKCC1. Therefore, both gamma-aminobutyric acid (GABA)A and glycine receptors, are able to work as inhibition for neurons. However, intracellular chloride ions are upregulated for down-regulation of KCC2.

 

supFig2

Supplement Figure 2. Methods of photothrombotic stroke

 

To make ischemia to restricted cortex area, we attached an aluminum tip to a fiber optic bundle and covered the skull other than target area with aluminum-tape.

 supFig3

Supplement Figure 3. Change of KCC2 expression on plasma membrane of motoneuron

 

Immunohistochemistry images show KCC2 expression in ventral spinal cord at each days after stroke. KCC2 (green) was detected dendrite and soma of neurons in ventral horn. To assess the function of KCC2, we focused on KCC2 expression on motoneuron.

KCC2 expression which located on the edge of motoneuron decreased. And KCC2 distribution was changed to intermittent from continuous (White arrowhead in pictures). Scale bar shows 20 μm.

 supFig4Supplement Figure 4. Summary of results

 

Results in this study showed decrease of KCC2 expression on the motoneuron membrane in early phase (3 and 7 days after stroke) and increase of the vGLUT1-positive boutons to project on the motoneuron until chronic phase (7 and 42 days after stroke). The excitability of neuronal circuit in affected side, which was assessed by RDD of H-reflex, kept high level from 3 days to 42 days after stroke. Interestingly, these results showed the mechanism of hyper-excitability of motoneuron inducing spasticity was different every phase of stroke. Furthermore, down-regulation of KCC2 may triggered off malaplasticity such as increase of vGLUT1 boutons, which occurred the negative effect for functional recovery after stroke.

 

Takuya Toda, Main researcher in this study

Sachiko Lee, Coresponding author, (Lab HP: http://www.lee-lab.com/)

 

 

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