Hyperexcitability of spinal reflexes and reduced synaptic inhibition are commonly associated with spasticity after spinal cord injury (SCI). In adults, the activation of gamma-aminobutyric acid(A) (GABAA) and glycine receptors inhibits neurons as a result of low intracellular chloride (Cl-) concentration, which is maintained by the potassium-chloride cotransporter KCC2 (encoded by Slc12a5). We show that KCC2 is downregulated after SCI in rats, particularly in motoneuron membranes, thereby depolarizing the Cl- equilibrium potential and reducing the strength of postsynaptic inhibition. Blocking KCC2 in intact rats reduces the rate-dependent depression (RDD) of the Hoffmann reflex, as is observed in spasticity. RDD is also decreased in KCC2-deficient mice and in intact rats after intrathecal brain-derived neurotrophic factor (BDNF) injection, which downregulates KCC2. The early decrease in KCC2 after SCI is prevented by sequestering BDNF at the time of SCI. Conversely, after SCI, BDNF upregulates KCC2 and restores RDD. Our results open new perspectives for the development of therapeutic strategies to alleviate spasticity.

In healthy adults, activation of γ-aminobutyric acid (GABA) A and glycine receptors inhibits neurons as a result of low intracellular chloride concentration ([Cl -] i ), which is maintained by the potassium-chloride cotransporter KCC2. A reduction of KCC2 expression or function is implicated in the pathogenesis of several neurological disorders, including spasticity and chronic pain following spinal cord injury (SCI). Given the critical role of KCC2 in regulating the strength and robustness of inhibition, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance. We show that activation of 5-hydroxytryptamine (5-HT) type 2A receptors to serotonin hyperpolarizes the reversal potential of inhibitory postsynaptic potentials (IPSPs), E IPSP , in spinal motoneurons, increases the cell membrane expression of KCC2 and both restores endogenous inhibition and reduces spasticity after SCI in rats. Up-regulation of KCC2 function by targeting 5-HT 2A receptors, therefore, has therapeutic potential in the treatment of neurological disorders involving altered chloride homeostasis. However, these receptors have been implicated in several psychiatric disorders, and their effects on pain processing are controversial, highlighting the need to further investigate the potential systemic effects of specific 5-HT 2A R agonists, such as (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2). ] i (depolarizing shift of the chloride equilibrium potential, E Cl ) dramatically compromises the inhibitory control of firing rate and excitatory inputs (5-7). Given the role of KCC2 in regulating the strength of inhibitory synaptic transmission, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance.Spasticity is a disabling complication affecting individuals with spinal cord injury (SCI) and is characterized by a velocity-dependent increase in muscle tone resulting from hyperexcitable stretch reflexes, spasms, and hypersensitivity to normally innocuous sensory stimulations (8, 9). Down-regulation of KCC2 after SCI in rats is implicated in the development of spasticity (10) and chronic pain (11,12). Notably, the expression of KCC2 in the motoneuron membrane is reduced, and, concomitantly, the density of cytoplasmic clusters is higher, suggesting that the surface stability of the transporter is reduced in these pathological conditions (10).Mounting evidence indicates that phosphorylation of KCC2 in the C-terminal intracellular domain dynamically regulates its activity and surface expression (1). In particular, phosphorylation by protein kinase (PK)C, enhances KCC2 activity and reduces endocytosis (13). Interestingly, activation of 5-hydroxytryptamine type 2 receptors (5-HT 2 Rs) to serotonin stimulates PKC and strengthens the left-right alternation of motor bursts observed during locomotion (14-16), which rely on reciprocal inhibition (17, 18). ...

A number of noradrenaline and serotonin agonists were tested to investigate which of them replicate the depressive actions of monoamines on transmission from group II muscle afferents in the cat spinal cord. The agonists were applied ionophoretically at the two sites at which maximal monosynaptic focal field potentials are evoked from group II afferents-in the intermediate zone and the dorsal horn of the 4th and 5th lumbar segments. Their effects were estimated from changes in the amplitude of the field potentials. The compounds tested fell into three categories according to the site at which they depressed transmission from group II afferents: one category with highly selective actions in the intermediate zone, a second category with similarly selective actions in the dorsal horn, and a third category with non-selective actions. Drugs in the first category included three noradrenaline agonists (tizanidine, B-HT 933 and clonidine), included in the second were five serotonin agonists (8-OH-DPAT, 5-methoxytryptamine, alpha-methyl serotonin, DOI and 2-methyl-serotonin), and in the third two noradrenaline agonists (phenylephrine and isoproterenol) and two serotonin agonists (RU 24969 and 5-carboxamidotryptamine). Field potentials evoked by group I afferents remained unaffected by all but one compound (8-OH-DPAT). Effects of one noradrenaline agonist and one serotonin agonist (tizanidine and 5-methoxytryptamine) were also tested on responses of single extracellularly recorded neurons. Tizanidine depressed responses induced by stimulation of group II afferents in intermediate zone interneurons, but not in dorsal horn neurons, while 5-methoxytryptamine depressed activation of the latter. Tizanidine had no effect on responses evoked by group I afferents, either in intermediate zone interneurons or in the dorsal spino-cerebellar tract neurons of Clarke's column. It is hypothesized that noradrenaline and serotonin released by descending monoaminergic neurons differ in the potency with which they depress transmission from group II afferents to different functional types of neuron. The results suggest that this depression may involve different membrane receptors at different locations, primarily alpha2 adrenoceptors in the intermediate zone/ventral horn and 5-HT1A serotonin receptors in the dorsal horn.