Ceng Luo

Synaptic scaffolding protein Homer1a protects against chronic inflammatory pain.

Glutamatergic signaling and intracellular calcium mobilization in the spinal cord are crucial for the development of nociceptive plasticity, which is associated with chronic pathological pain. Long-form Homer proteins anchor glutamatergic receptors to sources of calcium influx and release at synapses, which is antagonized by the short, activity-dependent splice variant Homer1a. We show here that Homer1a operates in a negative feedback loop to regulate the excitability of the pain pathway in an activity-dependent manner. Homer1a is rapidly and selectively upregulated in spinal cord neurons after peripheral inflammation in an NMDA receptor–dependent manner. Homer1a strongly attenuates calcium mobilization as well as MAP kinase activation induced by glutamate receptors and reduces synaptic contacts on spinal cord neurons that process pain inputs. Preventing activity-induced upregulation of Homer1a using shRNAs in mice in vivo exacerbates inflammatory pain. Thus, activity-dependent uncoupling of glutamate receptors from intracellular signaling mediators is a novel, endogenous physiological mechanism for counteracting sensitization at the first, crucial synapse in the pain pathway. Furthermore, we observed that targeted gene transfer of Homer1a to specific spinal segments in vivo reduces inflammatory hyperalgesia. Thus, Homer1 function is crucially involved in pain plasticity and constitutes a promising therapeutic target for the treatment of chronic inflammatory pain.

Synaptic plasticity in pathological pain

Chronic pain represents a major challenge to clinical practice and basic science. Excitatory neurotransmission in somatosensory nociceptive pathways is predominantly mediated by glutamatergic synapses. A key feature of these synapses is their ability to adapt synaptic strength in an activity-dependent manner. Such disease-induced synaptic plasticity is paramount to alterations in synaptic function and structure. Recent work has recognized that synaptic plasticity at both excitatory and inhibitory synapses can function as a prime mechanism underlying pathological pain. In this review, cellular and molecular mechanisms underlying synaptic plasticity in nociceptive pathways will be reviewed and discussed. New insights derived from these advances are expected to expedite development of novel interventional approaches for treatment of pathological pain.

Presynaptically localized cyclic GMP-dependent protein kinase 1 is a key determinant of spinal synaptic potentiation and pain hypersensitivity.

Electrophysiological and behavioral experiments in mice reveal that a cGMP-dependent kinase amplifies neurotransmitter release from peripheral pain sensors, potentiates spinal synapses, and leads to exaggerated pain.

Activity-dependent potentiation of calcium signals in spinal sensory networks in inflammatory pain states

Abstract The second messenger calcium is a key mediator of activity‐dependent neural plasticity. How persistent nociceptive activity alters calcium influx and release in the spinal cord is not well‐understood. We performed calcium‐imaging on individual cell bodies and the whole area within laminae I and II in spinal cord slices from mice in the naïve state or 24 h following unilateral hindpaw plantar injection of complete Freund’s adjuvant. Calcium signals evoked by dorsal root stimulation at varying strengths displayed a steep rise and slow decay over 15–20 s and increased progressively with both increasing intensity and frequency of stimulation in naïve mice. Experiments with pharmacological inhibitors revealed that both ionotropic glutamate receptors and intracellular calcium stores contributed to maximal calcium signals in laminae I and II evoked by stimulating dorsal roots at 100 Hz frequency. Importantly, as compared to naïve mice, we observed that in mice with unilateral hindpaw inflammation, calcium signals were potentiated to 159 ± 10% in the ipsilateral dorsal horn and 179 ± 8% in the contralateral dorsal horn. In addition to the contribution from NMDA receptors, GluR‐A‐containing AMPA receptors were found to be critically required for the above changes in spinal calcium signals, as revealed by analysis of genetically modified mouse mutants, whereas intracellular calcium release was not required. Thus, these results suggest that there is an important functional link between calcium signaling in superficial spinal laminae and the development of inflammatory pain. Furthermore, they highlight the importance of GluR‐A‐containing calcium‐permeable AMPA receptors in activity‐dependent plasticity in the spinal cord.

Peripheral calcium-permeable AMPA receptors regulate chronic inflammatory pain in mice

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type (AMPA-type) glutamate receptors (AMPARs) play an important role in plasticity at central synapses. Although there is anatomical evidence for AMPAR expression in the peripheral nervous system, the functional role of such receptors in vivo is not clear. To address this issue, we generated mice specifically lacking either of the key AMPAR subunits, GluA1 or GluA2, in peripheral, pain-sensing neurons (nociceptors), while preserving expression of these subunits in the central nervous system. Nociceptor-specific deletion of GluA1 led to disruption of calcium permeability and reduced capsaicin-evoked activation of nociceptors. Deletion of GluA1, but not GluA2, led to reduced mechanical hypersensitivity and sensitization in models of chronic inflammatory pain and arthritis. Further analysis revealed that GluA1-containing AMPARs regulated the responses of nociceptors to painful stimuli in inflamed tissues and controlled the excitatory drive from the periphery into the spinal cord. Consequently, peripherally applied AMPAR antagonists alleviated inflammatory pain by specifically blocking calcium-permeable AMPARs, without affecting physiological pain or eliciting central side effects. These findings indicate an important pathophysiological role for calcium-permeable AMPARs in nociceptors and may have therapeutic implications for the treatment chronic inflammatory pain states.

Genetic deletion of synapsin II reduces neuropathic pain due to reduced glutamate but increased GABA in the spinal cord dorsal horn.

Abstract The synaptic vesicle protein synapsin II is specifically expressed in synaptic terminals of primary afferent nociceptive neurons and regulates transmitter release in the spinal cord dorsal horn. Here, we assessed its role in nerve injury‐evoked molecular and behavioral adaptations in models of peripheral neuropathic pain using mice genetically lacking synapsin II. Deficiency of synapsin II resulted in reduced mechanical and cold allodynia in two models of peripheral neuropathic pain. This was associated with decreased glutamate release in the dorsal horn of the spinal cord upon sciatic nerve injury or capsaicin application onto the sciatic nerve and reduced calcium signals in spinal cord slices upon persistent activation of primary afferents. In addition, the expression of the vesicular glutamate transporters, VGLUT1 and VGLUT2, was strongly reduced in synapsin II knockout mice in the spinal cord. Conversely, synapsin II knockout mice showed a stronger and longer‐lasting increase of GABA in lamina II of the dorsal horn after nerve injury than wild type mice. These results suggest that synapsin II is involved in the regulation of glutamate and GABA release in the spinal cord after nerve injury, and that a dysbalance between glutamatergic and GABAergic synaptic transmission contributes to the manifestation of neuropathic pain.

A role for Kalirin-7 in nociceptive sensitization via activity-dependent modulation of spinal synapses

Synaptic plasticity is the cornerstone of processes underlying persistent nociceptive activity-induced changes in normal nociceptive sensitivity. Kalirin-7 is a multifunctional guanine-nucleotide-exchange factor (GEF) for Rho GTPases that is characterized by its localization at excitatory synapses, interactions with glutamate receptors and its ability to dynamically modulate the neuronal cytoskeleton. Here we show that spinally expressed Kalirin-7 is required for persistent nociceptive activity-dependent synaptic long-term potentiation as well as activity-dependent remodelling of synaptic spines in the spinal dorsal horn, thereby orchestrating functional and structural plasticity during the course of inflammatory pain.

Cyclic GMP-dependent protein kinase-I localized in nociceptors modulates nociceptive cortical neuronal activity and pain hypersensitivity

Chronic pain represents a frequent and poorly understood public health issue. Numerous studies have documented the key significance of plastic changes along the somatosensory pain pathways in chronic pain states. Our recent study demonstrated that the cGMP-dependent protein kinase I (PKG-I) specifically localized in nociceptors constitutes a key mediator of hyperexcitability of primary sensory neurons and spinal synaptic plasticity after inflammation. However, whether PKG-I in nociceptors further affects the cortical plasticity in the ascending pain pathways under pathological states has remained elusive. The immediate-early gene c-fos and phosphorylated ERK1/2 (pERK1/2) are considered reliable indicators for the neuronal activation status and it permits a comprehensive and large-scale observation of nociceptive neuronal activity along the ascending pain pathways subjected to tissue injury. In the present study, we systemically demonstrated that peripheral injury in PKG-Ifl/fl mice produced a significant upregulation of c-Fos or pERK1/2 over from the periphery to the cortex along the pain pathways, including dorsal root ganglion, spinal dorsal horn, ventral posterolateral thalamus, primary somatosensory hindlimb cortex, anterior cingulate cortex, basolateral amygdala, periaqueductal gray, and parabrachial nucleus. In contrast, very few cells in the above regions showed c-Fos or pERK1/2 induction in nociceptor-specific knockout mice lacking PKG-I (SNS-PKG-I−/− mice). Our results indicate that PKG-I expressed in nociceptors is not only a key determinant of dorsal root ganglion hyperexcitability and spinal synaptic plasticity but also an important modulator of cortical neuronal activity in pathological pain states and represent what we believe to be novel targets in the periphery for pain therapeutics.

Presynaptic cGMP-dependent protein kinase-I mediates synaptic potentiation in spinal amplification of pain

Background Activity-dependent facilitation of pain is functionally linked to plasticity at synapses between peripheral sensory afferents and spinal projection neurons. However, the underlying cellular and molecular mechanisms are not well-understood [1]. We observed that long-term potentiation at these synapses involves a presynaptic mechanism comprising activity-induced decrease in synaptic failures. This process involves activation of the cGMP-dependent protein kinase-I (PKG-I) in presynaptic terminals of nociceptive afferents and potentiation of vesicular transmitter release via modulation of IP3 receptors and myosin light chains. Mice lacking PKG-I specifically in nociceptors did not develop spinal long-term potentiation and showed marked defects in pathological pain in vivo.

353 SYNAPTIC SCAFFOLDING PROTEIN HOMER1A PROTECTS AGAINST INFLAMMATORY PAIN

was performed to isolate spinal cord from supraspinal structures. Noxious stimuli were applied to peripheral receptive field to evoke responses from isolated wide dynamic range (WDR) neurones in 4min cycles. CP-101,606 (iv) was then administered cumulatively (8min cycles) until >50% reduction in responses were observed and ED50 values calculated. Results: In animals with inflammation and an intact spinal cord, CP-101,606 (25–400mmol/kg) dose-dependently inhibited noxious stimuli evoked firing, with an ED50 of 188mmol/kg. In animals with a transected spinal cord, CP-101,606 (100–600mmol/kg) had no effect. In animals with neuropathy, CP-101,606 (20–320mmol/kg) produced dosedependant inhibition of noxious stimuli evoked firing in animals with an intact and transected spinal cord; ED50 values were 195 mmol/kg and 148mmol/kg respectively. Conclusions: Spinal cord NR2B containing NMDA receptors contribute to the antinociceptive activity of CP-101,606 in model of neuropathic, but not inflammatory, pain demonstrating differential roles for spinal NR2B containing NMDA receptors in these models.