Yves De Koninck

BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain

Neuropathic pain that occurs after peripheral nerve injury depends on the hyperexcitability of neurons in the dorsal horn of the spinal cord. Spinal microglia stimulated by ATP contribute to tactile allodynia, a highly debilitating symptom of pain induced by nerve injury. Signalling between microglia and neurons is therefore an essential link in neuropathic pain transmission, but how this signalling occurs is unknown. Here we show that ATP-stimulated microglia cause a depolarizing shift in the anion reversal potential (Eanion) in spinal lamina I neurons. This shift inverts the polarity of currents activated by GABA (γ-amino butyric acid), as has been shown to occur after peripheral nerve injury. Applying brain-derived neurotrophic factor (BDNF) mimics the alteration in Eanion. Blocking signalling between BDNF and the receptor TrkB reverses the allodynia and the Eanion shift that follows both nerve injury and administration of ATP-stimulated microglia. ATP stimulation evokes the release of BDNF from microglia. Preventing BDNF release from microglia by pretreating them with interfering RNA directed against BDNF before ATP stimulation also inhibits the effects of these cells on the withdrawal threshold and Eanion. Our results show that ATP-stimulated microglia signal to lamina I neurons, causing a collapse of their transmembrane anion gradient, and that BDNF is a crucial signalling molecule between microglia and neurons. Blocking this microglia–neuron signalling pathway may represent a therapeutic strategy for treating neuropathic pain.

Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain

Modern pain-control theory predicts that a loss of inhibition (disinhibition) in the dorsal horn of the spinal cord is a crucial substrate for chronic pain syndromes. However, the nature of the mechanisms that underlie such disinhibition has remained controversial. Here we present evidence for a novel mechanism of disinhibition following peripheral nerve injury. It involves a trans-synaptic reduction in the expression of the potassium–chloride exporter KCC2, and the consequent disruption of anion homeostasis in neurons of lamina I of the superficial dorsal horn, one of the main spinal nociceptive output pathways. In our experiments, the resulting shift in the transmembrane anion gradient caused normally inhibitory anionic synaptic currents to be excitatory, substantially driving up the net excitability of lamina I neurons. Local blockade or knock-down of the spinal KCC2 exporter in intact rats markedly reduced the nociceptive threshold, confirming that the reported disruption of anion homeostasis in lamina I neurons was sufficient to cause neuropathic pain.

Expression of CCR2 in both resident and bone marrow-derived microglia plays a critical role in neuropathic pain.

Neuropathic pain resulting from damage to or dysfunction of peripheral nerves is not well understood and difficult to treat. Although CNS hyperexcitability is a critical component, recent findings challenge the neuron-centric view of neuropathic pain etiology and pathology. Indeed, glial cells were shown to play an active role in the initiation and maintenance of pain hypersensitivity. However, the origins of these cells and the triggers that induce their activation have yet to be elucidated. Here we show that, after peripheral nerve injury induced by a partial ligation on the sciatic nerve, in addition to activation of microglia resident to the CNS, hematogenous macrophage/monocyte infiltrate the spinal cord, proliferate, and differentiate into microglia. Signaling from chemokine monocyte chemoattractant protein-1 (MCP-1, CCL2) to its receptor CCR2 is critical in the spinal microglial activation. Indeed, intrathecal injection of MCP-1 caused activation of microglia in wild-type but not in CCR2-deficient mice. Furthermore, treatment with an MCP-1 neutralizing antibody prevented bone marrow-derived microglia (BMDM) infiltration into the spinal cord after nerve injury. In addition, using selective knock-out of CCR2 in resident microglia or BMDM, we found that, although total CCR2 knock-out mice did not develop microglial activation or mechanical allodynia, CCR2 expression in either resident microglia or BMDM is sufficient for the development of mechanical allodynia. Thus, to effectively relieve neuropathic pain, both CNS resident microglia and blood-borne macrophages need to be targeted. These findings also open the door for a novel therapeutic strategy: to take advantage of the natural ability of bone marrow-derived cells to infiltrate selectively affected CNS regions by using these cells as vehicle for targeted drug delivery to inhibit hypersensitivity and chronic pain.

Spatial and temporal relationship between monocyte chemoattractant protein‐1 expression and spinal glial activation following peripheral nerve injury

Peripheral nerve injury can induce spinal microglial/astrocyte activation. Substances released by activated glial cells excite spinal nociceptive neurons. Pharmacological disruption of glial activation or antagonism of substances released by activated glia prevent or reverse pain hypersensitivity. It is not known, however, what causes spinal cord glia to shift from a resting to an activated state. In an attempt to understand the potential role of monocyte chemoattractant protein‐1 (MCP‐1) in triggering spinal glial activation and its contribution to the development of neuropathic pain, we investigated the effect of peripheral nerve injury on MCP‐1 expression in dorsal root ganglia (DRG) and the spinal cord, and established its temporal relationship with activation of spinal microglia and astrocytes. We observed that MCP‐1 was induced by chronic constriction of the sciatic nerve in DRG sensory neurons, spinal cord motor neurons and in the superficial dorsal horn, ipsilateral to the injury. Neuronal MCP‐1 induction was followed by surrounding microglial activation. After peaking at day 7 after injury, MCP‐1 levels began to decline rapidly and had returned to baseline by day 150. In contrast, microglial activation peaked by day 14 and declined afterwards to reach a lower, yet significantly raised level beyond day 22 and remained increased until the end of the test period. Astrocyte activation became detectable later, progressed more slowly and also remained increased until the end of the test period, in parallel with a decreased nociceptive threshold. Our results suggest that neuronal MCP‐1 may serve as a trigger for spinal microglial activation, which participates in the initiation of neuropathic pain. Delayed, sustained astrocyte activation may participate with microglia in the persistent phase of pain hypersensitivity.

Junctional versus Extrajunctional Glycine and GABAAReceptor-Mediated IPSCs in Identified Lamina I Neurons of the Adult Rat Spinal Cord

Colocalization of GABA and glycine in synaptic terminals of the superficial dorsal horn raises the question of their relative contribution to inhibition of different classes of neurons in this area. To address this issue, miniature IPSCs (mIPSCs) mediated via GABAA receptors (GABAARs) and glycine receptors (GlyRs) were recorded from identified laminae I-II neurons in adult rat spinal cord slices. GABAAR-mediated mIPSCs had similar amplitude and rise times, but significantly slower decay kinetics than GlyR-mediated mIPSCs. Lamina I neurons appeared to receive almost exclusively GlyR-mediated mIPSCs, even after application of hypertonic solutions. Yet, all neurons responded to exogenous applications of both GABA and glycine, indicating that they expressed both GABAARs and GlyRs. Given that virtually all glycinergic interneurons also contain GABA, the possibility was examined that GABAARs may be located extrasynaptically in lamina I neurons. A slow GABAAR-mediated component was revealed in large, but not minimally evoked monosynaptic IPSCs. Administration of the benzodiazepine flunitrazepam unmasked a GABAAR component to most mIPSCs , suggesting that both transmitters were released from the same vesicle. The isolated GABAAR component of these mIPSCs had rising kinetics 10 times slower than that of the GlyR component (or of GABAAR mIPSCs in lamina II). The slow GABAAR components were prolonged by GABA uptake blockers. It is concluded that, whereas GABA and glycine are likely released from the same vesicle of transmitter in lamina I, GABAARs appear to be located extrasynaptically. Thus, glycine mediates most of the tonic inhibition at these synapses. This differential distribution of GABAARs and GlyRs confers distinct functional properties to inhibition mediated by these two transmitters in lamina I.

Gain control of firing rate by shunting inhibition: Roles of synaptic noise and dendritic saturation

Adjusting input–output gain is crucial for information processing by the brain. Gain control of subthreshold depolarization is commonly ascribed to increased membrane conductance caused by shunting inhibition. But contrary to its divisive effect on depolarization, shunting inhibition on its own fails to divisively modulate firing rate, apparently upsetting a critical tenet of neural models that use shunting inhibition to achieve gain control. Using a biophysically realistic neuron model, we show that divisive modulation of firing rate by shunting inhibition requires synaptic noise to smooth the relation between firing rate and somatic depolarization; although necessary, noise alone endows shunting inhibition with only a modest divisive effect on firing rate. In addition to introducing noise, synaptic input is associated with a nonlinear relation between somatic depolarization and excitation because of dendritic saturation; this nonlinearity dramatically enhances divisive modulation of firing rate by shunting inhibition under noisy conditions. Thus, shunting inhibition can act as a mechanism for firing rate gain control, but its modulatory effects (which include both divisive and subtractive components) are fully explained only when both synaptic noise and dendritic saturation are taken into account.

Morphine hyperalgesia gated through microglia-mediated disruption of neuronal Cl- homeostasis

A major unresolved issue in treating pain is the paradoxical hyperalgesia produced by the gold-standard analgesic morphine and other opiates. We found that hyperalgesia-inducing treatment with morphine resulted in downregulation of the K+-Cl− co-transporter KCC2, impairing Cl− homeostasis in rat spinal lamina l neurons. Restoring the anion equilibrium potential reversed the morphine-induced hyperalgesia without affecting tolerance. The hyperalgesia was also reversed by ablating spinal microglia. Morphine hyperalgesia, but not tolerance, required μ opioid receptor–dependent expression of P2X4 receptors (P2X4Rs) in microglia and μ-independent gating of the release of brain-derived neurotrophic factor (BDNF) by P2X4Rs. Blocking BDNF-TrkB signaling preserved Cl− homeostasis and reversed the hyperalgesia. Gene-targeted mice in which Bdnf was deleted from microglia did not develop hyperalgesia to morphine. However, neither morphine antinociception nor tolerance was affected in these mice. Our findings dissociate morphine-induced hyperalgesia from tolerance and suggest the microglia-to-neuron P2X4-BDNF-KCC2 pathway as a therapeutic target for preventing hyperalgesia without affecting morphine analgesia.

Chemokines and pain mechanisms.

The development of new therapeutic approaches to the treatment of painful neuropathies requires a better understanding of the mechanisms that underlie the development of these chronic pain syndromes. It is now well established that astrocytic and microglial cells modulate the neuronal mechanisms of chronic pain in spinal cord and possibly in the brain. In animal models of neuropathic pain following peripheral nerve injury, several changes occur at the level of the first pain synapse between the central terminals of sensory neurons and second order neurons. These neuronal mechanisms can be modulated by pro-nociceptive mediators released by non neuronal cells such as microglia and astrocytes which become activated in the spinal cord following PNS injury. However, the signals that mediate the spread of nociceptive signaling from neurons to glial cells in the dorsal horn remain to be established. Herein we provide evidence for two emerging signaling pathways between injured sensory neurons and spinal microglia: chemotactic cytokine ligand 2 (CCL2)/CCR2 and cathepsin S/CX3CL1 (fractalkine)/CX3CR1. We discuss the plasticity of these two chemokine systems at the level of the dorsal root ganglia and spinal cord demonstrating that modulation of chemokines using selective antagonists decrease nociceptive behavior in rodent chronic pain models. Since up-regulation of chemokines and their receptors may be a mechanism that directly and/or indirectly contributes to the development and maintenance of chronic pain, these molecular molecules may represent novel targets for therapeutic intervention in sustained pain states.

Transformation of the output of spinal lamina I neurons after nerve injury and microglia stimulation underlying neuropathic pain

BackgroundDisinhibition of neurons in the superficial spinal dorsal horn, via microglia – neuron signaling leading to disruption of chloride homeostasis, is a potential cellular substrate for neuropathic pain. But, a central unresolved question is whether this disinhibition can transform the activity and responses of spinal nociceptive output neurons to account for the symptoms of neuropathic pain.ResultsHere we show that peripheral nerve injury, local spinal administration of ATP-stimulated microglia or pharmacological disruption of chloride transport change the phenotype of spinal lamina I output neurons, causing them to 1) increase the gain of nociceptive responsiveness, 2) relay innocuous mechanical input and 3) generate spontaneous bursts of activity. The changes in the electrophysiological phenotype of lamina I neurons may account for three principal components of neuropathic pain: hyperalgesia, mechanical allodynia and spontaneous pain, respectively.ConclusionThe transformation of discharge activity and sensory specificity provides an aberrant signal in a primarily nociceptive ascending pathway that may serve as a basis for the symptoms of neuropathic pain.

Four cell types with distinctive membrane properties and morphologies in lamina I of the spinal dorsal horn of the adult rat

Lamina I of the spinal dorsal horn plays an important role in the processing and relay of nociceptive information. Signal processing depends, in part, on neuronal membrane properties. Intrinsic membrane properties of lamina I neurons were therefore investigated using whole cell patch clamp recordings in a slice preparation of adult rat spinal cord. Based on responses to somatic current injection, four cell types were identified: tonic, which fire comparatively slowly but continuously throughout stimulation; phasic, which fire a high frequency burst of variable duration; delayed onset, which fire irregularly and with a marked delay to the first spike; and single spike, which typically fire only one action potential even when strongly depolarised. Classification by spiking pattern was further refined by identification of characteristic stimulus‐response curves and quantification of several response parameters. Objectivity of the classification was confirmed by cluster analysis. Responses to stimulus trains and synaptic input as well as the kinetics of spontaneous synaptic events revealed differences in the signal processing characteristics of the cell types: tonic and delayed onset cells appeared to act predominantly as integrators whereas phasic and single spike cells acted as coincidence detectors. Intracellular labelling revealed a significant correlation between morphological and physiological cell types: tonic cells were typically fusiform, phasic cells were pyramidal, and delayed onset and single spike cells were multipolar. Thus, there are multiple physiological cells types in lamina I with specific morphological correlates and distinctive signal processing characteristics that confer significant differences in the transduction of input into spike trains.