Alteration of glycinergic receptor expression in lumbar spinal motoneurons is involved in the mechanisms underlying spasticity after spinal cord injury.

Spasticity is a disabling motor disorder affecting 70% of people with brain and spinal cord injury. The rate-dependent depression (RDD) of the H reflex is the only electrophysiological measurement correlated with the degree of spasticity assessed clinically in spastic patients. Several lines of evidence suggest that the mechanism underlying the H reflex RDD depends on the strength of synaptic inhibition through GABAA (GABAAR) and glycine receptors (GlyR). In adult rats with spinal cord transection (SCT), we studied the time course of the expression of GABAAR and GlyR at the membrane of retrogradely identified Gastrocnemius and Tibialis anterior motoneurons (MNs) 3, 8 and 16 weeks after injury, and measured the RDD of the H reflex at similar post lesion times. Three weeks after SCT, a significant decrease in the expression of GABAA and GlyR was observed compared to intact rats, and the H-reflex RDD was much less pronounced than in controls. Eight weeks after SCT, GlyR values returned to normal. Simultaneously, we observed a tendency to recover normal RDD of the H reflex at higher frequencies. We tested whether an anti-inflammatory treatment using methylprednisolone performed immediately after SCT could prevent alterations in GABAA/glycine receptors and/or the development of spasticity observed 3 weeks after injury. This treatment restored control levels of GlyR but not the expression of GABAAR, and it completely prevented the attenuation of RDD. These data strongly suggest that alteration of glycinergic inhibition of lumbar MNs is involved in the mechanisms underlying spasticity after SCI.

Activation of 5-HT2A Receptors Restores KCC2 Function and Reduces Neuropathic Pain after Spinal Cord Injury.

Downregulation of the potassium chloride cotransporter type 2 (KCC2) after a spinal cord injury (SCI) disinhibits motoneurons and dorsal horn interneurons causing spasticity and neuropathic pain, respectively. We showed recently (Bos et al., 2013) that specific activation of 5-HT2A receptors by TCB-2 [(4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide] upregulates KCC2 function, restores motoneuronal inhibition and reduces SCI-induced spasticity. Here, we tested the potential analgesic effect of TCB-2 on central (thoracic hemisection) and peripheral [spared nerve injury (SNI)] neuropathic pain. We found mechanical and thermal hyperalgesia reduced by an acute administration of TCB-2 in rats with SCI. This analgesic effect was associated with an increase in dorsal horn membrane KCC2 expression and was prevented by pharmacological blockade of KCC2 with an intrathecal injection of DIOA [(dihydroindenyl)oxy]alkanoic acid]. In contrast, the SNI-induced neuropathic pain was not attenuated by TCB-2 although there was a slight increase of membrane KCC2 expression in the dorsal horn ipsilateral to the lesion. Up-regulation of KCC2 function by targeting 5-HT2A receptors, therefore, has therapeutic potential in the treatment of neuropathic pain induced by SCI but not by SNI.

Cleavage of Na(+) channels by calpain increases persistent Na(+) current and promotes spasticity after spinal cord injury.

Upregulation of the persistent sodium current (I(NaP)) in motoneurons contributes to the development of spasticity after spinal cord injury (SCI). We investigated the mechanisms that regulate I(NaP) and observed elevated expression of voltage-gated sodium (Nav) 1.6 channels in spinal lumbar motoneurons of adult rats with SCI. Furthermore, immunoblots revealed a proteolysis of Nav channels, and biochemical assays identified calpain as the main proteolytic factor. Calpain-dependent cleavage of Nav channels after neonatal SCI was associated with an upregulation of I(NaP) in motoneurons. Similarly, the calpain-dependent cleavage of Nav1.6 channels expressed in human embryonic kidney (HEK) 293 cells caused the upregulation of I(NaP). The pharmacological inhibition of calpain activity by MDL28170 reduced the cleavage of Nav channels, I(NaP) in motoneurons and spasticity in rats with SCI. Similarly, the blockade of I(NaP) by riluzole alleviated spasticity. This study demonstrates that Nav channel expression in lumbar motoneurons is altered after SCI, and it shows a tight relationship between the calpain-dependent proteolysis of Nav1.6 channels, the upregulation of I(NaP) and spasticity.

Activation of 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2.

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).

Chapter 1--importance of chloride homeostasis in the operation of rhythmic motor networks.

GABA and glycine are classically called "inhibitory" amino acids, despite the fact that their action can rapidly switch from inhibition to excitation and vice versa. The postsynaptic action depends on the intracellular concentration of chloride ions ([Cl(-)](i)), which is regulated by proteins in the plasma membrane: the K(+)-Cl(-) cotransporter KCC2 and the Na(+)-K(+)-Cl(-) cotransporter NKCC1, which extrude and intrude Cl(-) ions, respectively. A high [Cl(-)](i) leads to a depolarizing (excitatory) action of GABA and glycine, as observed in mature dorsal root ganglion neurons and in motoneurons both early during development and in several pathological conditions, such as following spinal cord injury. Here, we review some recent data regarding chloride homeostasis in the spinal cord and its contribution to network operation involved in locomotion.

Distinct expression of c-Jun and HSP27 in axotomized and spared bulbospinal neurons after cervical spinal cord injury.

In several populations of adult central nervous system neurons, axon damage can lead to an up-regulation of some transcription factors among which is c-Jun, known to be a key regulator of neuron cell body response to injury and of its intrinsic potential for axon regeneration. Thus, cervical spinal hemisection leads to c-Jun up-regulation in bulbospinal and rubrospinal axotomized neurons. The aims of the present study were to specify, after a unilateral cervical spinal cord injury, the expression of another marker of the neuronal stress response, heat shock protein 27 (HSP27) in axotomized neurons of the medulla (labeled by fluorogold retrograde tracer), and to compare it to that of c-Jun. In the medulla of injured rats, HSP27 and phospho-HSP27 were expressed in sub-populations of axotomized neurons, principally in the rostral ventral respiratory group (rVRG) (20%), the dorsal part of the gigantocellularis (Gi) (50%), and vestibular nucleus, but seldom in the ventral Gi and raphe nucleus, indicating a heterogeneous post-lesion cell body response between these different neuron populations. By contrast, phospho-c-Jun was up-regulated in axotomized neurons in all nuclei containing bulbospinal neurons, including the rVRG and Gi. In these areas, phospho-c-Jun was also up-regulated in uninjured bulbospinal neurons which project caudal to the spinal cord injury (labeled by fluorogold retrograde tracer). In contrast to phospho-c-Jun, HSP27 immunoreactivity was generally not present in neurons with spared axons. Our results show that various bulbospinal neuron populations react differentially to the injury and that spinal cord injury affects also bulbospinal neurons that are spared by the injury. However, the molecular cell body response of spared neurons is distinct from that of axotomized neurons since they can up-regulate c-Jun but not HSP27.

Down-regulation of the potassium-chloride cotransporter KCC2 contributes to spasticity after spinal cord injury.

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.

Strategies to restore motor functions after spinal cord injury.

This review presents recent advances in the development of strategies to restore posture and locomotion after spinal cord injury (SCI). A set of strategies focusing on the lesion site includes prevention of secondary damages, promotion of axonal sprouting/regeneration, and replacement of lost cells. Other strategies focus on spinal central pattern generators (CPGs). Training promotes functional recovery by enhancing the plasticity of CPGs and these sublesional networks can be reactivated by means of pharmacological or electrical stimulation. It is now clear that substantial functional recovery will require a combination of strategies adapted to each phase following SCI. Finally, improvements in the understanding of the mechanisms underlying spasticity may lead to new treatments of this disabling complication affecting patients with SCI.

Restorative respiratory pathways after partial cervical spinal cord injury: role of ipsilateral phrenic afferents.

After disruption of the descending respiratory pathways induced by unilateral cervical spinal cord injury (SCI) in rats, the inactivated ipsilateral (ipsi) phrenic nerve (PN) discharge may partially recover following some specific experimental procedures [such as contralateral (contra) phrenicotomy (Phx)]. This phrenic reactivation involves normally silent contra pathways decussating at the level of the phrenic nucleus, but the mechanisms of this crossed phrenic activation are still poorly understood. The present study investigates the contribution of sensory phrenic afferents to this process by comparing the acute effects of ipsi and contra Phx. We show that the phrenic discharge (recorded on intact PNs) was almost completely suppressed 0 h and 3 h after a lateral cervical SCI, but was already spontaneously reactivated after 1 week. This ipsi phrenic activity was enhanced immediately after contra Phx and was completely suppressed by an acute contra cervical section, indicating that it is triggered by crossed phrenic pathways located laterally in the contra spinal cord. Ipsi phrenic activity was also abolished immediately after ipsi Phx that interrupts phrenic sensory afferents, an effect prevented by prior acute ablation of the cervical dorsal root ganglia, indicating that crossed phrenic activation depends on excitatory phrenic sensory afferents but also putatively on inhibitory non-phrenic afferents. On the basis of these data, we propose a new model for crossed phrenic activation after partial cervical injury, with an essential role played by ipsi-activating phrenic sensory afferents.

Specific and artifactual labeling in the rat spinal cord and medulla after injection of monosynaptic retrograde tracers into the diaphragm.

The use of fluorescent dyes has been a major improvement for paths tracing studies. However, these tracers present different properties and have to be chosen carefully. The present study compares the ability of different tracers to specifically label phrenic motoneurons (PMNs) innervating the rat diaphragm. The administration of fluorogold (FG) from the transected phrenic nerve specifically labeled PMNs in the ipsilateral spinal cord. However, when FG was injected into one hemidiaphragm, in addition with ipsilateral PMNs, a less intense artifactual labeling was observed in the spinal cord (mainly in contralateral PMNs) and in the medulla oblongata (mainly in the area postrema and cranial motor nuclei). Similar results were observed using horseradish peroxidase, while no labeling was observed after injection of nuclear yellow or diamidino yellow into the diaphragm. By contrast, the dextran amine fluororuby (FR) and the carbocyanine DiAsp selectively and exclusively labeled ipsilateral PMNs 2 or 3 weeks after injection into the diaphragm, respectively. The lipophilic properties of DiAsp and the high molecular weight of FR may prevent their diffusion to adjacent tissues and into the blood stream which seems to account for the artifactual labeling observed with the other tracers. The higher homogeneity and quality of the labeling observed with FR compared to DiAsp make it the most appropriate tracer for the specific monosynaptic fluorescent labeling of PMNs after injection into the diaphragm.

Respiratory neuron subpopulations and pathways potentially involved in the reactivation of phrenic motoneurons after C2 hemisection.

Lateral hemisection of the cervical (C2) spinal cord in the rat interrupts ipsilateral bulbospinal respiratory pathways arising mainly from the rostral ventral respiratory group (rVRG) and Bötzinger complex and projecting to phrenic motoneurons in C3-C5. Deafferented phrenic motoneurons can be reactivated via previously silent contralateral pathways, a process called the crossed phrenic phenomenon (CPP). In order to further characterise the neuronal bases of the CPP and quantify the neurons involved, respiratory neurons projecting to the ipsilateral phrenic nucleus in hemisected rats were labelled by injection of the monosynaptic retrograde tracer fluorogold (FG) in ipsilateral C3-C4 metamers. Respectively 36% and 23% neurons were labelled in the contralateral and ipsilateral rVRG in hemisected rats compared to controls, and respectively 26% and 2% in the contralateral and ipsilateral Bötzinger complex. This shows that phrenic motoneurons located under the C2 hemisection may still be activated by axons or collaterals of contralateral respiratory premotoneurons located in the rVRG and Bötzinger complex which cross the spinal cord midline at the level of the phrenic nuclei, and also by axon collaterals of ipsilateral rVRG premotoneurons which cross the midline both in the brainstem and in the spinal cord. Neurons with double crossing axons were twice as many in the caudal part of the rVRG (38%) compared to the part located rostrally to the area postrema (20%), which further argues in favour of a subdivision of this nucleus. These pathways may be involved in the CPP and could be differentially activated in acute or chronic lesioned rats.

Axotomized bulbospinal neurons express c-Jun after cervical spinal cord injury.

In several central nervous system neuronal populations, axotomy triggers the upregulation of regeneration-associated genes such as c-Jun, which determines neurons ability to regenerate axon in a growth-permissive environment. We analyzed the expression of c-Jun in rat ventral medullary neurons after cervical hemisection in order to investigate their intrinsic regenerative potential. Maximal expression of c-Jun was observed 7 days after injury mainly in axotomized medullary neurons located in the gigantocellularis nucleus, the raphe nucleus and, although less intensively, in the rostral ventral respiratory group. This suggests that after high cervical injury, a large number of medullary neurons projecting to the spinal cord become competent for axonal regeneration, although this regenerating potential may not be equivalent between the various neuronal populations.