Pascale Boulenguez

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 γ-aminobutyric acidA (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.

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), EIPSP, 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-HT2A 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-HT2AR agonists, such as (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2).

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.

Biochemical and Pharmacological Characterization of Serotonin‐O‐Carboxymethylglycyl[125I]Iodotyrosinamide5 a New Radioiodinated Probe for 5‐HT1B and 5‐HT1D Binding Sites

Abstract: There is a lack of radioactive probes, particularly radioiodinated probes, for the direct labeling of serotonin‐1B (5‐HT1B) and serotonin‐ID (5‐HT1D) binding sites. Serotonin‐0‐carboxymethylglycyltyrosinamide (S‐CM‐GTNH2) was shown previously to be specific for these two subtypes; we, therefore, linked a 125I to its tyrosine residue. Biochemical and pharmacological properties of S‐CM‐G[125I]TNH2‐binding sites were studied by quantitative au‐toradiography on rat and guinea pig brain sections. S‐CM‐G[I25I]TNH2 binding is saturable and reversible with a KD value of 1.3 nM in the rat and 6.4 nM in the guinea pig. Binding is heterogeneous, paralleling the anatomical distribution of 5‐HT1B sites in the rat and of 5‐HTD sites in the guinea pig. The binding of 0.02 nM S‐CM‐G[125I]TNH2 was inhibited by low concentrations of 5‐HT, S‐CM‐GTNH2, CGS 12066 B, 5‐methoxytryptamine, and tryptamine in both species. Propranolol inhibited the radioligand binding with a greater affinity in the rat than in the guinea pig. Conversely, 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin inhibited S‐CM‐G[125I]TNH2 binding with a greater affinity in the guinea pig than in the rat. Other competitors, specific for 5‐HT,c, 5‐HT2, 5‐HT3, and adrenergic receptors, inhibited S‐CM‐G[125I]TNH2 binding in rat and guinea pig substantia nigra and in other labeled structures known to contain these receptors, but only at high concentrations. S‐CM‐G[I25I]TNH2 is then a useful new probe for the direct study of 5‐HT1B and 5‐HT1D binding sites.

Subcellular localization of 5‐HT1B binding sites in the stratum griseum superficiale of the rat superior colliculus: An electron microscopic quantitative autoradiographic study

Previous works have shown the potential location of 5‐HT1B binding sites on the retinal afferents to the superior colliculus in the rat. In order to confirm this hypothesis, the distribution of 5‐HT1B binding sites, labelled by S‐CM‐G[125I]TNH2, was analysed by quantitative autoradiography at both light and electron microscopic levels in the upper stratum griseum superficiale (SGS) of the superior colliculus, 5 days after unilateral eye ablation. At the light microscopic level, no 5‐HT1B sites were found to be associated with capillaries, glial, or neuronal cell bodies, but the fine neuropile was specifically labelled. At the electron microscopic level, the quantitative analysis performed with 50% probability circles showed that classical dendritic processes, presynaptic dendritic processes, and processes containing flat synaptic vesicles were not labelled. In the SGS ipsilateral to the eye ablation, silver grains were specifically associated with processes containing round and pleomorphic vesicles and with non‐synaptic contacts between those processes and dendritic or other non identified neuronal processes. In the deafferented contralateral SGS, 5‐HT1B receptors were associated with degenerating retinal terminals, with processes containing round vesicles and with non‐synaptic contacts between those two tissue compartments. This is the first direct demonstration of the existence of 5‐HT1B receptors in non‐synaptic contacts and in non‐serotonergic terminals. The existence of 5‐HT1B terminal heteroreceptors localised on primary visual afferents shows that serotonin might modulate the transmission of visual messages to the superior colliculus.

Effects of retinal deafferentation on serotonin receptor types in the superficial grey layer of the superior colliculus of the rat

The effects of retinal axon terminal degeneration on the serotonin-1A, -1B, -2, nuerokinin-1 and gamma-amionobutyric acid-A high affinity binding sites in the superficial grey layer of the superior colliculus were tested with quantitative autoradiography on rat brain sections. The binding to serotonin-2, neurokinin-1 and gamma-aminobutyric acid-A high affinity receptors was not changed in the deafferented superficial grey layer of the superior colliculus after unilateral enucleation. By contrast, we demonstrate that the previously described 21% decrease in the binding of [3H]serotonin to serotonin-1 receptors observed in the deafferented superficial grey layer of the superior colliculus after enucleation, was not due to a decrease in the affinity of the serotonin-1 receptors for the radioligand, but to a decrease in the number of binding sites. Of the different serotonin-1 receptor subtypes, only the serotonin-1B was lost. This signifies that these receptors are probably located on the optic fibre terminals. Visual cortex lesion caused no apparent regulation of the serotonin-1 binding sites in the superficial grey layer of the superior colliculus. A bilateral enucleation produced a smaller decrease in serotonin-1 receptor density than that observed after unilateral enucleation, suggesting the existence of a compensatory mechanism.

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

Upregulation of the persistent sodium current (INaP) in motoneurons contributes to the development of spasticity after spinal cord injury (SCI). We investigated the mechanisms that regulate INaP 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 INaP in motoneurons. Similarly, the calpain-dependent cleavage of Nav1.6 channels expressed in human embryonic kidney (HEK) 293 cells caused the upregulation of INaP. The pharmacological inhibition of calpain activity by MDL28170 reduced the cleavage of Nav channels, INaP in motoneurons and spasticity in rats with SCI. Similarly, the blockade of INaP 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 INaP and spasticity.

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.

8 - Computer-Assisted Quantitative Receptor Autoradiography

Publisher Summary This chapter describes the components of a computer device for quantitative autoradiography and the characteristics and properties of each component. It also describes a program for image analysis and a rationale for its utilization. The chapter highlights a typical application of this method for analysis of the distribution of specific binding sites of neurotransmitters. It is important to understand which technical characteristics are essential for a convenient device and how the system should be used. The detection of neurotransmitter receptor binding sites by autoradiography is important for the study of the function of receptors in the physiology and pathology of the brain. Such work can be done only with the help of quantitative analysis to determine the biochemical, pharmacological, and anatomical properties of the receptors. The computer is essential to assist the manual work in studying receptor density distribution. There are several commercially available setups for such analyses, and software programs are available individually, allowing the construction of a system with a personal computer and camera. Such a system may also be used in immunohistochemistry or in situ hybridization of mRNA either at the macroscopic or microscopic level.