Hot receptors in the brain

Background

The complex neuronal circuitry of the dorsal horn of the spinal cord is as yet poorly understood. However, defining the circuits underlying the transmission of information from primary afferents to higher levels is critical to our understanding of sensory processing. In this study, we have examined phosphodiesterase 1C (Pde1c) BAC transgenic mice in which a green fluorescent protein (GFP) reporter gene reflects Pde1c expression in sensory neuron subpopulations in the dorsal root ganglia and spinal cord.

Results

Using double labeling immunofluorescence, we demonstrate GFP expression in specific subpopulations of primary sensory neurons and a distinct neuronal expression pattern within the spinal cord dorsal horn. In the dorsal root ganglia, their distribution is restricted to those subpopulations of primary sensory neurons that give rise to unmyelinated C fibers (neurofilament 200 negative). A small proportion of both non-peptidergic (IB4-binding) and peptidergic (CGRP immunoreactive) subclasses expressed GFP. However, GFP expression was more common in the non-peptidergic than the peptidergic subclass. GFP was also expressed in a subpopulation of the primary sensory neurons immunoreactive for the vanilloid receptor TRPV1 and the ATP-gated ion channel P2X₃. In the spinal cord dorsal horn, GFP positive neurons were largely restricted to lamina I and to a lesser extent lamina II, but surprisingly did not coexpress markers for key neuronal populations present in the superficial dorsal horn.

Conclusion

The expression of GFP in subclasses of nociceptors and also in dorsal horn regions densely innervated by nociceptors suggests that Pde1c marks a unique subpopulation of nociceptive sensory neurons.     

Specific involvement of atypical PKCζ/PKMζ in spinal persistent nociceptive processing following peripheral inflammation in rat

Background

Central sensitization requires the activation of various intracellular signalling pathways within spinal dorsal horn neurons, leading to a lowering of activation threshold and enhanced responsiveness of these cells. Such plasticity contributes to the manifestation of chronic pain states and displays a number of features of long-term potentiation (LTP), a ubiquitous neuronal mechanism of increased synaptic strength. Here we describe the role of a novel pathway involving atypical PKCζ/PKMζ in persistent spinal nociceptive processing, previously implicated in the maintenance of late-phase LTP.

Results

Using both behavioral tests and in vivo electrophysiology in rats, we show that inhibition of this pathway, via spinal delivery of a myristoylated protein kinase C-ζ pseudo-substrate inhibitor, reduces both pain-related behaviors and the activity of deep dorsal horn wide dynamic range neurons (WDRs) following formalin administration. In addition, Complete Freund's Adjuvant (CFA)-induced mechanical and thermal hypersensitivity was also reduced by inhibition of PKCζ/PKMζ activity. Importantly, this inhibition did not affect acute pain or locomotor behavior in normal rats and interestingly, did not inhibited mechanical allodynia and hyperalgesia in neuropathic rats. Pain-related behaviors in both inflammatory models coincided with increased phosphorylation of PKCζ/PKMζ in dorsal horn neurons, specifically PKMζ phosphorylation in formalin rats. Finally, inhibition of PKCζ/PKMζ activity decreased the expression of Fos in response to formalin and CFA in both superficial and deep laminae of the dorsal horn.

Conclusions

These results suggest that PKCζ, especially PKMζ isoform, is a significant factor involved in spinal persistent nociceptive processing, specifically, the manifestation of chronic pain states following peripheral inflammation.

     

Significant changes in mitochondrial distribution in different pain models of mice

Mitochondria play an important role in pathophysiology of inflammatory and neuropathic pain but the mechanism is unclear. So far no comprehensive study exists that evaluates the changes of mitochondrial dynamics following the pain. In this study, we detected the mitochondrial distribution and subcellular morphology by using intrathecal injection of mitochondrial marker, Mitotracker Red® CM-H2XRox (Mito-Red) and confocal microscopic analysis in models of formalin-induced acute inflammatory pain, Complete Freund's Adjuvant (CFA)-induced persistent pain and spared nerve injury (SNI)-induced neuropathic pain. The results demonstrated that subcutaneous formalin injection did not affect the number of Mito-Red cells within the spinal dorsal horn at both acute and tonic phases, but significantly increased the number of cluster type mitochondria in superficial spinal dorsal horn (laminas I–II) at tonic phase. Differently, the number of Mito-Red cells significantly increased in superficial and deep spinal dorsal horn (laminas III–V) following persistent CFA and SNI neuropathic pain. Moreover, both CFA and SNI remarkably increased the number of cluster type mitochondria and decreased the number of granule type mitochondria, in both superficial and deep spinal dorsal horn. So we concluded that abnormal mitochondrial distribution contributes to neuropathic and some forms of inflammatory pain.     

Differential expression of microRNAs in mouse pain models

Background

MicroRNAs (miRNAs) are short non-coding RNAs that inhibit translation of target genes by binding to their mRNAs. The expression of numerous brain-specific miRNAs with a high degree of temporal and spatial specificity suggests that miRNAs play an important role in gene regulation in health and disease. Here we investigate the time course gene expression profile of miR-1, -16, and -206 in mouse dorsal root ganglion (DRG), and spinal cord dorsal horn under inflammatory and neuropathic pain conditions as well as following acute noxious stimulation.

Results

Quantitative real-time polymerase chain reaction analyses showed that the mature form of miR-1, -16 and -206, is expressed in DRG and the dorsal horn of the spinal cord. Moreover, CFA-induced inflammation significantly reduced miRs-1 and -16 expression in DRG whereas miR-206 was downregulated in a time dependent manner. Conversely, in the spinal dorsal horn all three miRNAs monitored were upregulated. After sciatic nerve partial ligation, miR-1 and -206 were downregulated in DRG with no change in the spinal dorsal horn. On the other hand, axotomy increases the relative expression of miR-1, -16, and 206 in a time-dependent fashion while in the dorsal horn there was a significant downregulation of miR-1. Acute noxious stimulation with capsaicin also increased the expression of miR-1 and -16 in DRG cells but, on the other hand, in the spinal dorsal horn only a high dose of capsaicin was able to downregulate miR-206 expression.

Conclusions

Our results indicate that miRNAs may participate in the regulatory mechanisms of genes associated with the pathophysiology of chronic pain as well as the nociceptive processing following acute noxious stimulation. We found substantial evidence that miRNAs are differentially regulated in DRG and the dorsal horn of the spinal cord under different pain states. Therefore, miRNA expression in the nociceptive system shows not only temporal and spatial specificity but is also stimulus-dependent.

     

Chromatin and siRNA pathways cooperate to maintain DNA methylation of small transposable elements in Arabidopsis

Background

DNA methylation occurs at preferred sites in eukaryotes. In Arabidopsis, DNA cytosine methylation is maintained by three subfamilies of methyltransferases with distinct substrate specificities and different modes of action. Targeting of cytosine methylation at selected loci has been found to sometimes involve histone H3 methylation and small interfering (si)RNAs. However, the relationship between different cytosine methylation pathways and their preferred targets is not known.

Results

We used a microarray-based profiling method to explore the involvement of Arabidopsis CMT3 and DRM DNA methyltransferases, a histone H3 lysine-9 methyltransferase (KYP) and an Argonaute-related siRNA silencing component (AGO4) in methylating target loci. We found that KYP targets are also CMT3 targets, suggesting that histone methylation maintains CNG methylation genome-wide. CMT3 and KYP targets show similar proximal distributions that correspond to the overall distribution of transposable elements of all types, whereas DRM targets are distributed more distally along the chromosome. We find an inverse relationship between element size and loss of methylation in ago4 and drm mutants.

Conclusion

We conclude that the targets of both DNA methylation and histone H3K9 methylation pathways are transposable elements genome-wide, irrespective of element type and position. Our findings also suggest that RNA-directed DNA methylation is required to silence isolated elements that may be too small to be maintained in a silent state by a chromatin-based mechanism alone. Thus, parallel pathways would be needed to maintain silencing of transposable elements.     

Purinergic mechanosensory transduction and visceral pain

Background

Little is known about whether peripheral nerve injury during the early postnatal period modulates synaptic efficacy in the immature superficial dorsal horn (SDH) of the spinal cord, or whether the neonatal SDH network is sensitive to the proinflammatory cytokine TNFα under neuropathic conditions. Thus we examined the effects of TNFα on synaptic transmission and intrinsic membrane excitability in developing rat SDH neurons in the absence or presence of sciatic nerve damage.

Results

The spared nerve injury (SNI) model of peripheral neuropathy at postnatal day (P)6 failed to significantly alter miniature excitatory (mEPSCs) or inhibitory (mIPSCs) postsynaptic currents in SDH neurons at P9-11. However, SNI did alter the sensitivity of excitatory synapses in the immature SDH to TNFα. While TNFα failed to influence mEPSCs or mIPSCs in slices from sham-operated controls, it significantly increased mEPSC frequency and amplitude following SNI without modulating synaptic inhibition onto the same neurons. This was accompanied by a significant decrease in the paired-pulse ratio of evoked EPSCs, suggesting TNFα increases the probability of glutamate release in the SDH under neuropathic conditions. Similarly, while SNI alone did not alter action potential (AP) threshold or rheobase in SDH neurons at this age, TNFα significantly decreased AP threshold and rheobase in the SNI group but not in sham-operated littermates. However, unlike the adult, the expression of TNFα in the immature dorsal horn was not significantly elevated during the first week following the SNI.

Conclusion

Developing SDH neurons become susceptible to regulation by TNFα following peripheral nerve injury in the neonate. This may include both a greater efficacy of glutamatergic synapses as well as an increase in the intrinsic excitability of immature dorsal horn neurons. However, neonatal sciatic nerve damage alone did not significantly modulate synaptic transmission or neuronal excitability in the SDH, which could reflect a relatively weak expression of TNFα in the injured spinal cord at early ages. The above data suggest that although the sensitivity of the SDH network to proinflammatory cytokines after nerve injury is present from the first days of life, the profile of spinal cytokine expression under neuropathic conditions may be highly age-dependent.     

Sickle cell mice exhibit mechanical allodynia and enhanced responsiveness in light touch cutaneous mechanoreceptors

Background

Interleukin-8 (IL-8) is known for its roles in inflammation and plays critical roles in the development of pain. Its expression increases in the brain after peripheral inflammation. Prefrontal cortex, including the anterior cingulate cortex (ACC), is a forebrain structure known for its roles in pain transmission and modulation. Painful stimuli potentiate the prefrontal synaptic transmission, however, little is known about the expression of IL-8 and its role in the enhanced ACC synaptic transmission in animals with persistent inflammatory pain.

Findings

In the present study, we examined IL-8 expression in the ACC, somatosensory cortex (SSC), and the dorsal horn of lumbar spinal cord following hind-paw administration of complete Freund's adjuvant (CFA) in mice and its effects on the ACC synaptic transmission. Quantification of IL-8 at protein level (by ELISA) revealed enhanced expression in the ACC and spinal cord during the chronic phases of CFA-induced peripheral inflammation. In vitro whole-cell patch-clamp recordings revealed that IL-8 significantly enhanced synaptic transmission through increased probability of neurotransmitter release in the ACC slice. ACC local infusion of repertaxin, a non-competitive allosteric blocker of IL-8 receptors, notably prolonged the paw withdrawal latency to thermal radian heat stimuli bilaterally in mice.

Conclusions

Our findings suggest that up-regulation of IL-8 in the ACC partly attributable to the enhanced prefrontal synaptic transmission in the mice with persistent inflammatory pain.     

Chronic morphine administration enhances nociceptive sensitivity and local cytokine production after incision

Background

Recent evidence suggests that oxytocin (OT), secreted in the superficial spinal cord dorsal horn by descending axons of paraventricular hypothalamic nucleus (PVN) neurons, produces antinociception and analgesia. The spinal mechanism of OT is, however, still unclear and requires further investigation. We have used patch clamp recording of lamina II neurons in spinal cord slices and immunocytochemistry in order to identify PVN-activated neurons in the superficial layers of the spinal cord and attempted to determine how this neuronal population may lead to OT-mediated antinociception.

Results

We show that OT released during PVN stimulation specifically activates a subpopulation of lamina II glutamatergic interneurons which are localized in the most superficial layers of the dorsal horn of the spinal cord (lamina I-II). This OT-specific stimulation of glutamatergic neurons allows the recruitment of all GABAergic interneurons in lamina II which produces a generalized elevation of local inhibition, a phenomenon which might explain the reduction of incoming Aδ and C primary afferent-mediated sensory messages.

Conclusion

Our results obtained in lamina II of the spinal cord provide the first clear evidence of a specific local neuronal network that is activated by OT release to induce antinociception. This OT-specific pathway might represent a novel and interesting therapeutic target for the management of neuropathic and inflammatory pain.     

In vivo evidence that truncated trkB.T1 participates in nociception

Background

Clearance of synaptically released glutamate, and hence termination of glutamatergic neurotransmission, is carried out by glutamate transporters, most especially glutamate transporter-1 (GLT-1) and the glutamate-aspartate transporter (GLAST) that are located in astrocytes. It is becoming increasingly well appreciated that changes in the function and expression of GLT-1 and GLAST occur under different physiological and pathological conditions. Here we investigated the plasticity in expression of GLT-1 and GLAST in the spinal dorsal horn using immunohistochemistry following partial sciatic nerve ligation (PSNL) in rats.

Results

Animals were confirmed to develop hypersensitivity to mechanical stimulation by 7 days following PSNL. Baseline expression of GLT-1 and GLAST in naive animals was only observed in astrocytes and not in either microglia or neurons. Microglia and astrocytes showed evidence of reactivity to the nerve injury when assessed at 7 and 14 days following PSNL evidenced by increased expression of OX-42 and GFAP, respectively. In contrast, the total level of GLT-1 and GLAST protein decreased at both 7 and 14 days after PSNL. Importantly, the cellular location of GLT-1 and GLAST was also altered in response to nerve injury. Whereas activated astrocytes showed a marked decrease in expression of GLT-1 and GLAST, activated microglia showed de novo expression of GLT-1 and GLAST at 7 days after PSNL and this was maintained through day 14. Neurons showed no expression of GLT-1 or GLAST at any time point.

Conclusion

These results indicate that the expression of glutamate transporters in astrocytes and microglia are differentially regulated following nerve injury.     

Involvement of lysophosphatidic acid in bone cancer pain by potentiation of TRPV1 via PKCϵ pathway in dorsal root ganglion neurons

Background

Our previous study demonstrated that nitric oxide (NO) contributes to long-term potentiation (LTP) of C-fiber-evoked field potentials by tetanic stimulation of the sciatic nerve in the spinal cord in vivo. Ryanodine receptor (RyR) is a downstream target for NO. The present study further explored the role of RyR in synaptic plasticity of the spinal pain pathway.

Results

By means of field potential recordings in the adult male rat in vivo, we showed that RyR antagonist reduced LTP of C-fiber-evoked responses in the spinal dorsal horn by tetanic stimulation of the sciatic nerve. Using spinal cord slice preparations and field potential recordings from superficial dorsal horn, high frequency stimulation of Lissauer's tract (LT) stably induced LTP of field excitatory postsynaptic potentials (fEPSPs). Perfusion of RyR antagonists blocked the induction of LT stimulation-evoked spinal LTP, while Ins(1,4,5)P3 receptor (IP₃R) antagonist had no significant effect on LTP induction. Moreover, activation of RyRs by caffeine without high frequency stimulation induced a long-term potentiation in the presence of bicuculline methiodide and strychnine. Further, in patch-clamp recordings from superficial dorsal horn neurons, activation of RyRs resulted in a large increase in the frequency of miniature EPSCs (mEPSCs). Immunohistochemical study showed that RyRs were expressed in the dorsal root ganglion (DRG) neurons. Likewise, calcium imaging in small DRG neurons illustrated that activation of RyRs elevated [Ca²⁺]_i in small DRG neurons.

Conclusions

These data indicate that activation of presynaptic RyRs play a crucial role in the induction of LTP in the spinal pain pathway, probably through enhancement of transmitter release.     

Comparison of central versus peripheral delivery of pregabalin in neuropathic pain states

Background

Although pregabalin therapy is beneficial for neuropathic pain (NeP) by targeting the CaV??₂??-1 subunit, its site of action is uncertain. Direct targeting of the central nervous system may be beneficial for the avoidance of systemic side effects.

Results

We used intranasal, intrathecal, and near-nerve chamber forms of delivery of varying concentrations of pregabalin or saline delivered over 14 days in rat models of experimental diabetic peripheral neuropathy and spinal nerve ligation. As well, radiolabelled pregabalin was administered to determine localization with different deliveries. We evaluated tactile allodynia and thermal hyperalgesia at multiple time points, and then analyzed harvested nervous system tissues for molecular and immunohistochemical changes in CaV??₂??-1 protein expression. Both intrathecal and intranasal pregabalin administration at high concentrations relieved NeP behaviors, while near-nerve pregabalin delivery had no effect. NeP was associated with upregulation of CACNA2D1 mRNA and CaV??₂??-1 protein within peripheral nerve, dorsal root ganglia (DRG), and dorsal spinal cord, but not brain. Pregabalin's effect was limited to suppression of CaV??₂??-1 protein (but not CACNA2D1 mRNA) expression at the spinal dorsal horn in neuropathic pain states. Dorsal root ligation prevented CaV??₂??-1 protein trafficking anterograde from the dorsal root ganglia to the dorsal horn after neuropathic pain initiation.

Conclusions

Either intranasal or intrathecal pregabalin relieves neuropathic pain behaviours, perhaps due to pregabalin's effect upon anterograde CaV??₂??-1 protein trafficking from the DRG to the dorsal horn. Intranasal delivery of agents such as pregabalin may be an attractive alternative to systemic therapy for management of neuropathic pain states.     

Role of spinal cord alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in complete Freund's adjuvant-induced inflammatory pain

Background

Honeybee's sting on human skin can induce ongoing pain, hyperalgesia and inflammation. Injection of bee venom (BV) into the intraplantar surface of the rat hindpaw induces an early onset of spontaneous pain followed by a lasting thermal and mechanical hypersensitivity in the affected paw. The underlying mechanisms of BV-induced thermal and mechanical hypersensitivity are, however, poorly understood. In the present study, we investigated the role of mitogen-activated protein kinase (MAPK) in the generation of BV-induced pain hypersensitivity.

Results

We found that BV injection resulted in a quick activation of p38, predominantly in the L4/L5 spinal dorsal horn ipsilateral to the inflammation from 1 hr to 7 d post-injection. Phosphorylated p38 (p-p38) was expressed in both neurons and microglia, but not in astrocytes. Intrathecal administration of the p38 inhibitor, SB203580, prevented BV-induced thermal hypersensitivity from 1 hr to 3 d, but had no effect on mechanical hypersensitivity. Activated ERK1/2 was observed exclusively in neurons in the L4/L5 dorsal horn from 2 min to 1 d, peaking at 2 min after BV injection. Intrathecal administration of the MEK inhibitor, U0126, prevented both mechanical and thermal hypersensitivity from 1 hr to 2 d. p-ERK1/2 and p-p38 were expressed in neurons in distinct regions of the L4/L5 dorsal horn; p-ERK1/2 was mainly in lamina I, while p-p38 was mainly in lamina II of the dorsal horn.

Conclusion

The results indicate that differential activation of p38 and ERK1/2 in the dorsal horn may contribute to the generation and development of BV-induced pain hypersensitivity by different mechanisms.