Pascal Darbon

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.

Mechanisms controlling bursting activity induced by disinhibition in spinal cord networks

Disinhibition reliably induces regular synchronous bursting in networks of spinal interneurons in culture as well as in the intact spinal cord. We have combined extracellular multisite recording using multielectrode arrays with whole cell recordings to investigate the mechanisms involved in bursting in organotypic and dissociated cultures from the spinal cords of embryonic rats. Network bursts induced depolarization and spikes in single neurons, which were mediated by recurrent excitation through glutamatergic synaptic transmission. When such transmission was blocked, bursting ceased. However, tonic spiking persisted in some of the neurons. In such neurons intrinsic spiking was suppressed following the bursts and reappeared in the intervals after several seconds. The suppression of intrinsic spiking could be reproduced when, in the absence of fast synaptic transmission, bursts were mimicked by the injection of current pulses. Intrinsic spiking was also suppressed by a slight hyperpolarization. An afterhyperpolarization following the bursts was found in roughly half of the neurons. These afterhyperpolarizations were combined with a decrease in excitability. No evidence for the involvement of synaptic depletion or receptor desensitization in bursting was found, because neither the rate nor the size of spontaneous excitatory postsynaptic currents were decreased following the bursts. Extracellular stimuli paced bursts at low frequencies, but failed to induce bursts when applied too soon after the last burst. Altogether these results suggest that bursting in spinal cultures is mainly based on intrinsic spiking in some neurons, recurrent excitation of the network and auto‐regulation of neuronal excitability.

INaP underlies intrinsic spiking and rhythm generation in networks of cultured rat spinal cord neurons

We have shown previously that rhythm generation in disinhibited spinal networks is based on intrinsic spiking, network recruitment and a network refractory period following the bursts. This refractory period is based mainly on electrogenic Na/K pump activity. In the present work, we have investigated the role of the persistent sodium current (INaP) in the generation of bursting using patch‐clamp and multielectrode array recordings. We detected INaP exclusively in the intrinsic spiking cells. The blockade of INaP by riluzole suppressed the bursting by silencing the intrinsic spiking cells and suppressing network recruitment. The blockade of the persistent sodium current produced a hyperpolarization of the membrane potential of the intrinsic spiking cells, but had no effect on non‐spiking cells. We also investigated the involvement of the hyperpolarization‐activated cationic current (Ih) in the rhythmic activity. The bath application of ZD7288, a specific Ih antagonist, slowed down the rate of the bursts by increasing the interburst intervals. Ih was present in ∼ 70% of the cells, both in the intrinsic spiking cells as well as in the non‐spiking cells. We also found both kinds of cells in which Ih was not detected. In summary, in disinhibited spinal cord cultures, a persistent sodium current underlies intrinsic spiking, which, via recurrent excitation, generates the bursting activity. The hyperpolarization‐activated cationic current contributes to intrinsic spiking and modulates the burst frequency.

Fast non-genomic effects of progesterone-derived neurosteroids on nociceptive thresholds and pain symptoms.

Abstract Fast Inhibitory controls mediated by glycine (GlyRs) and GABAA receptors (GABAARs) play an important role to prevent the apparition of pathological pain symptoms of allodynia and hyperalgesia. The use of positive allosteric modulators of these receptors, specifically expressed in the spinal cord, may represent an interesting strategy to limit or block pain expression. In this study, we have used stereoisomers of progesterone metabolites, acting only via non‐genomic effects, in order to evaluate the contribution of GlyRs and GABAARs for the reduction of mechanical and thermal heat hypernociception. We show that 3α neurosteroids were particularly efficient to elevate nociceptive thresholds in naive animal. It also reduced mechanical allodynia and thermal heat hyperalgesia in the carrageenan model of inflammatory pain. This effect is likely to be mediated by GABAA receptors since 3β isomer was inefficient. More interestingly, 3α5β neurosteroid was only efficient on mechanical allodynia while having no effect on thermal heat hyperalgesia. We characterized these paradoxical effects of 3α5β neurosteroid using the strychnine and bicuculline models of allodynia. We clearly show that 3α5β neurosteroid exerts an antinociceptive effect via a positive allosteric modulation of GABAARs but, at the same time, is pronociceptive by reducing GlyR function. This illustrates the importance of the inhibitory amino acid receptor channels and their allosteric modulators in spinal pain processing. Moreover, our results indicate that neurosteroids, which are synthesized in the dorsal horn of the spinal cord and have limited side effects, may be of significant interest in order to treat pathological pain symptoms.

Antinociceptive action of oxytocin involves inhibition of potassium channel currents in lamina II neurons of the rat spinal cord

BackgroundGrowing evidence in the literature shows that oxytocin (OT) has a strong spinal anti-nociceptive action. Oxytocinergic axons originating from a subpopulation of paraventricular hypothalamic neurons establish synaptic contacts with lamina II interneurons but little is known about the functional role of OT with respect to neuronal firing and excitability.ResultsUsing the patch-clamp technique, we have recorded lamina II interneurons in acute transverse lumbar spinal cord slices of rats (15 to 30 days old) and analyzed the OT effects on action potential firing ability. In the current clamp mode, we found that bath application of a selective OT-receptor agonist (TGOT) reduced firing in the majority of lamina II interneurons exhibiting a bursting firing profile, but never in those exhibiting a single spike discharge upon depolarization. Interestingly, OT-induced reduction in spike frequency and increase of firing threshold were often observed, leading to a conversion of the firing profile from repetitive and delayed profiles into phasic ones and sometimes further into single spike profile. The observed effects following OT-receptor activation were completely abolished when the OT-receptor agonist was co-applied with a selective OT-receptor antagonist. In current and voltage clamp modes, we show that these changes in firing are strongly controlled by voltage-gated potassium currents. More precisely, transient IA currents and delayed-rectifier currents were reduced in amplitude and transient IA current was predominantly inactivated after OT bath application.ConclusionThis effect of OT on the firing profile of lamina II neurons is in good agreement with the antinociceptive and analgesic properties of OT described in vivo.

Contributions of NMDA receptors to network recruitment and rhythm generation in spinal cord cultures

N‐methyl‐d‐aspartic acid (NMDA) receptors are implicated in fictive locomotion; however, their precise role there is not clear. In cultures of dissociated cells from foetal rat spinal cord, synchronous bursting (but not fictive locomotion) can be induced by disinhibition, which is produced by blocking glycinergic and γ‐aminobutyric acid (GABA)A‐dependent synaptic conductances. In this study, we investigate the role of NMDA‐R in rhythm generation during disinhibition with multielectrode arrays and patch‐clamp. We previously determined that bursting activity is generated by repetitive recruitment of a network through recurrent excitation. Blocking NMDA‐R with d(−)‐2‐amino‐5‐phosphonopentanoic acid (APV) decreased the burst duration, suggesting a role of such receptors in the maintenance of high network activity during the bursts. In addition, APV reduced burst rate in about a third of the experiments, suggesting a contribution of NMDA‐R in network recruitment. When (±)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid hydrate (AMPA)/kainate receptors were blocked with 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) in the presence of disinhibition, the burst rate was reduced and burst onset was slowed in two‐thirds of the experiments. In the remaining experiments, bursting ceased completely with CNQX. Neither APV nor CNQX changed the spatial patterns of activity in the network, suggesting a co‐operation of both receptors in rhythm generation. While NMDA alone was not able to create a rhythm, it accelerated bursting in the presence of disinhibition, made it more regular and slowed down network recruitment. These effects were most likely due to the depolarization of the interneurons in the network. We conclude that NMDA‐R contribute to rhythm generation in spinal cultures by supporting recurrent excitation and network recruitment and by depolarizing the network.

Etifoxine analgesia in experimental monoarthritis: a combined action that protects spinal inhibition and limits central inflammatory processes.

Summary Etifoxine long‐lasting analgesia is mediated by a general reduction in spinal inflammation. In addition to a direct increase in gamma‐aminobutyric acid (GABA)‐ergic inhibition, glycinergic disinhibition is prevented. ABSTRACT Inflammatory and degenerative diseases of the joint are major causes of chronic pain. Long‐lasting pain symptoms are thought to result from a central sensitization of nociceptive circuits. These processes include activation of microglia and spinal disinhibition. Using a monoarthritic rat model of pain, we tried to potentiate neural inhibition by using etifoxine (EFX), a nonbenzodiazepine anxiolytic that acts as an allosteric‐positive modulator of gamma‐aminobutyric acid type A (GABAA) receptor function. Interestingly, EFX also can bind to the mitochondrial translocator protein (TSPO) complex and stimulate the synthesis of 3&agr;‐reduced neurosteroids, the most potent positive allosteric modulator of GABAA receptor function. Here we show that a curative and a preventive treatment with 50 mg/kg of EFX efficiently reduced neuropathic pain symptoms. In the spinal cord, EFX analgesia was accompanied by a reduction in microglial activation and in the levels of proinflammatory mediators. Using electrophysiological tools, we found that EFX treatment not only amplified spinal GABAergic inhibition, but also prevented prostaglandin E2–induced glycinergic disinhibition and restored a “normal” spinal pain processing. Because EFX is already distributed in several countries under the trade name of Stresam for its anxiolytic actions in humans, new clinical trials are now required to further extend its therapeutic indications as pain killer.

Rhythm Generation in Spinal Cultures: Is It the Neuron or the Network?

Neural networks of many regions of the CNS are able to generate synchronized rhythmic activity. In humans, rhythmic cortical activity has been recorded for years with electroencephalography (EEG). The various frequency bands that are observed in these recordings are associated with different states of consciousness (Steriade, 2001). In the hippocampus, rhythmic activity has been related to longterm potentiation and memory functions (Vertes and Kocsis, 1997). The release of neuropeptides in the hypothalamus is controlled through rhythmically active neural networks (Kwiecien and Hammond, 1998). Finally, repetitive muscle contractions that occur during respiration, locomotion, or scratching are controlled by rhythmically active neural networks in brainstem and spinal cord (Grillner et al., 1998; Rekling and Feldman, 1998). All these examples show that rhythmic activity in neural networks underlies many of the specific CNS functions and also suggest that the capability for rhythm generation must be a fundamental property of neural networks. In principle there are two ways a network can generate rhythms: rhythm may be produced by a welldefined circuit, usually composed of excitatory and inhibitory cells, or rhythm generation depends mainly on the cellular properties of certain neurons in the network and the circuit structure is of lesser importance. A mechanism of the second type is thought to underlie population bursting: a network is activated through the positive feedback of recurrent excitation and silenced by one or several accommodation mechanisms. It is the aim of this chapter to present some results concerning the mechanisms involved in rhythm generation, which occurs in networks of cultured spinal neurons. Such networks are at least partially grown in vitro: in the case of dissociated cell cultures, they are entirely regrown from randomly seeded cells. In the case of organotypic slice cultures, networks develop out of premature

Corticosterone analgesia is mediated by the spinal production of neuroactive metabolites that enhance GABAergic inhibitory transmission on dorsal horn rat neurons.

Corticosterone (CORT) is a glucocorticoid produced by adrenal glands under the control of the hypothalamic–pituitary–adrenal axis. Circulating CORT can enter the central nervous system and be reduced to neuroactive 3α5α‐reduced steroids, which modulate GABAA receptors. In the dorsal spinal cord, GABAergic transmission modulates integration of nociceptive information. It has been shown that enhancing spinal inhibitory transmission alleviates hyperalgesia and allodynia. Therefore, the spinal neuronal network is a pivotal target to counteract pain symptoms. Thus, any increase in spinal 3α5α‐reduced steroid production enhancing GABAergic inhibition should reduce nociceptive message integration and the pain response. Previously, it has been shown that high levels of plasma glucocorticoids give rise to analgesia. However, to our knowledge, nothing has been reported regarding direct non‐genomic modulation of neuronal spinal activity by peripheral CORT. In the present study, we used combined in vivo and in vitro electrophysiology approaches, associated with measurement of nociceptive mechanical sensitivity and plasma CORT level measurement, to assess the impact of circulating CORT on rat nociception. We showed that CORT plasma level elevation produced analgesia via a reduction in C‐fiber‐mediated spinal responses. In the spine, CORT is reduced to the neuroactive metabolite allotetrahydrodeoxycorticosterone, which specifically enhances lamina II GABAergic synaptic transmission. The main consequence is a reduction in lamina II network excitability, reflecting a selective decrease in the processing of nociceptive inputs. The depressed neuronal activity at the spinal level then, in turn, leads to weaker nociceptive message transmission to supraspinal structures and hence to alleviation of pain.

Effects of brain-derived neurotrophic factor (BDNF) on activity mediated by NMDA receptors in rat spinal cord cultures.

Brain-derived neurotrophic factor (BDNF) is involved in the differentiation and the survival of neurons. It has also been shown to be associated with the regrowth of neurons of damaged spinal cord and the modulation of ionic currents by acting on sodium channels and NMDA receptors through tyrosine kinase B (TrkB) receptors. We investigated the effects of BDNF on rhythm generation induced by disinhibition in dissociated cultures from embryonic rat spinal cord (E14), with extracellular multisite recordings (MultiElectrode Arrays, MEAs) or intracellular patch-clamp recordings. Exogenous BDNF had only minor effects on the bursting by increasing the activity during the burst. This increase of activity is suggested to be mediated by a potentiation of the postsynaptic NMDA receptors because it has been found that BDNF potentiates the NMDA-evoked depolarization in cultures incubated with BDNF for 10 min. Possible direct effects of BDNF on sodium channels were also investigated by local application of BDNF to the soma of patched neurons but no depolarization was observed. Long-term application of BDNF strongly decreased the activity during the burst and also the number of active electrodes, possibly due to a decrease in network density.