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Our recent work has shown that the early-life administration of vincristine (VNC), commonly used to treat pediatric cancers, evokes mechanical pain hypersensitivity in rats that emerges during adolescence and persists into adulthood. However, the underlying mechanisms remain unclear, as nothing is known about how neonatal VNC treatment influences peripheral and central nociceptive processing at the cellular level. Here, we used in vitro intracellular microelectrode and whole-cell patch-clamp recordings to evaluate the consequences of early-life VNC administration on the intrinsic membrane properties of adolescent dorsal root ganglion and spinal superficial dorsal horn neurons. The results demonstrate that VNC treatment increased the prevalence and rate of repetitive firing in both large- and medium-diameter sensory neurons, while reducing repetitive firing in small-diameter neurons, in comparison with vehicle-treated littermate controls. By contrast, passive membrane properties and peripheral conduction velocities were similar between experimental groups across all classes of primary afferents. Within the adolescent superficial dorsal horn, neonatal VNC exposure significantly enhanced the intrinsic membrane excitability of lamina I spinoparabrachial neurons, as evidenced by a decrease in rheobase and elevation of repetitive firing frequency compared with controls. Meanwhile, putative interneurons within lamina I exhibited a reduction in repetitive action potential discharge after early-life chemotherapy. Collectively, these findings suggest that neonatal VNC treatment evokes cell type-specific changes in intrinsic excitability at multiple levels of the ascending pain pathway. Overall, this work lays an essential foundation for the future exploration of the ionic mechanisms that drive chemotherapy-induced chronic pain in children and adolescents. [ABSTRACT FROM AUTHOR]

Distinct subsets of sensory nerve fibres are involved in mediating mechanical and thermal pain hypersensitivity. They may also differentially respond to analgesics. Heat-sensitive C-fibres, for example, are thought to respond to m-opioid receptor (MOR) activation while mechanoreceptive fibres are supposedly sensitive to d-opioid receptor (DOR) or GABAB receptor (GABABR) activation. The suggested differential distribution of inhibitory neurotransmitter receptors on different subsets of sensory fibres is, however, heavily debated. In this study, we quantitatively compared the degree of presynaptic inhibition exerted by opioids and the GABABR agonist baclofen on (1) vesicular glutamate transporter subtype 3-positive (VGluT31) non-nociceptive primary afferent fibres and (2) putative nociceptive C-fibres. To investigate VGluT31 sensory fibres, we evoked excitatory postsynaptic currents with blue light at the level of the dorsal root ganglion (DRG) in spinal cord slices of mice, expressing channelrhodopsin-2. Putative nociceptive C-fibres were explored in VGluT3-knockout mice through electrical stimulation. The MOR agonist DAMGO strongly inhibited both VGluT31 and VGluT32 C-fibres innervating lamina I neurons but generally had less influence on fibres innervating lamina II neurons. The DOR agonist SNC80 did not have any pronounced effect on synaptic transmission in any fibre type tested. Baclofen, in striking contrast, powerfully inhibited all fibre populations investigated. In summary, we report optogenetic stimulation of DRG neurons in spinal slices as a capable approach for the subtype-selective investigation of primary afferent nerve fibres. Overall, pharmacological accessibility of different subtypes of sensory fibres considerably overlaps, indicating that MOR, DOR, and GABABR expressions are not substantially segregated between heat and mechanosensitive fibres. [ABSTRACT FROM AUTHOR]

Abstract: Sex differences in the spinal processing of somatic and visceral stimuli contribute to greater female sensitivity in many pain disorders. The present study examined spinal mechanisms that contribute to sex differences in visceral sensitivity. The visceromotor response to colorectal distention (CRD) was more robust in normal female rats and after intracolonic mustard oil compared with that in male rats. No sex difference was observed in the CRD-evoked response of lumbosacral (LS) and thoracolumbar (TL) colonic afferents in normal and mustard oil–treated rats, but there was a sex difference in spontaneous activity that was exacerbated by intracolonic mustard oil. The response of visceroceptive dorsal horn neurons to CRD was greater in normal female rats in the LS and TL spinal segments. The effect of intracolonic mustard oil on the CRD-evoked response of different phenotypes of visceroceptive dorsal horn neurons was dependent on sex and segment. The NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (APV) dose-dependently attenuated the visceromotor response in normal rats with greater effect in male rats. Correspondingly, there was greater cell membrane expression of the GluN1 subunit in dorsal horn extracts in female rats. After intracolonic mustard oil, there was no longer a sex difference in the effect of APV nor GluN1 expression in LS segments, but greater female expression in TL segments. These data document a sex difference in spinal processing of nociceptive visceral stimuli from the normal and inflamed colon. Differences in dorsal horn neuronal activity and NMDA receptor expression contribute to the sex differences in the visceral sensitivity observed in awake rats. [Copyright &y& Elsevier]

Abstract: Tissue injury during a critical period of early life can facilitate spontaneous glutamatergic transmission within developing pain circuits in the superficial dorsal horn (SDH) of the spinal cord. However, the extent to which neonatal tissue damage strengthens nociceptive synaptic input to specific subpopulations of SDH neurons, as well as the mechanisms underlying this distinct form of synaptic plasticity, remains unclear. Here we use in vitro whole-cell patch clamp recordings from rodent spinal cord slices to demonstrate that neonatal surgical injury selectively potentiates high-threshold primary afferent input to immature lamina II neurons. In addition, the increase in the frequency of miniature excitatory postsynaptic currents after hindpaw incision was prevented by neonatal capsaicin treatment, suggesting that early tissue injury enhances glutamate release from nociceptive synapses. This occurs in a widespread manner within the developing SDH, as incision elevated miniature excitatory postsynaptic current frequency in both GABAergic and presumed glutamatergic lamina II neurons of Gad-GFP transgenic mice. The administration of exogenous nerve growth factor into the rat hindpaw mimicked the effects of early tissue damage on excitatory synaptic function, while blocking trkA receptors in vivo abolished the changes in both spontaneous and primary afferent-evoked glutamatergic transmission following incision. These findings illustrate that neonatal tissue damage can alter the gain of developing pain pathways by activating nerve growth factor-dependent signaling cascades, which modify synaptic efficacy at the first site of nociceptive processing within the central nervous system. Surgical injury during early life increases the efficacy of lamina II synapses mediated by high-threshold primary afferents, which are sensitive to capsaicin and nerve growth factor. [Copyright &y& Elsevier]

Abstract: Sensitization of primary afferent neurons is one of the most important components of pain hypersensitivity after tissue injury. Insulin-like growth factor 1 (IGF-1), involved in wound repair in injured tissue, also plays an important role in maintaining neuronal function. In the present study, we investigated the effect of tissue IGF-1 on nociceptive sensitivity of primary afferent neurons. Local administration of IGF-1 induced thermal and mechanical pain hypersensitivity in a dose-dependent manner, and was attenuated by IGF-1 receptor (IGF1R) inhibition. Tissue but not plasma IGF-1 levels, as determined by enzyme-linked immunosorbent assay, significantly increased after plantar incision. Immunohistochemistry revealed that IGF1R was predominantly expressed in neurons as well as in satellite glial cells in the dorsal root ganglion (DRG). Double-labeling immunohistochemistry showed that IGF1R expression colocalized with peripherin and TRPV1, but not with NF200 in DRG neurons. The IGF1R inhibitor successfully alleviated mechanical allodynia, heat hyperalgesia, and spontaneous pain behavior observed after plantar incision. Expression of phosphorylated Akt in DRG neurons significantly increased after plantar incision and was suppressed by IGF1R inhibition. These results demonstrate that increased tissue IGF-1 production sensitizes primary afferent neurons via the IGF1R/Akt pathway to facilitate pain hypersensitivity after tissue damage. Insulin-like growth factor 1 is synthesized locally after tissue injury and contributes to the sensitization of primary afferent neurons. [Copyright &y& Elsevier]

Abstract: The impact of persistent inflammation on voltage-activated Ca2+ channels in cutaneous DRG neurons from adult rats was assessed with whole cell patch clamp techniques, sqRT-PCR and Western blot analysis. Inflammation was induced with a subcutaneous injection of complete Freund’s adjuvant (CFA). DiI was used to identify DRG neurons innervating the site of inflammation. Three days after CFA injection, high threshold Ca2+ current (HVA) density was significantly reduced in small and medium, but not large diameter neurons, reflecting a decrease in N-, L- and P/Q-type currents. This decrease in HVA current was associated with an increase in mRNA encoding the α2δ1-subunit complex, but no detectable change in N-type subunit (CaV2.2) mRNA. An increase in both α2δ1 and CaV2.2 protein was detected in the central nerves arising from L4 and L5 ganglia ipsilateral to the site of inflammation. In current clamp experiments on small and medium diameter cutaneous DRG neurons from naïve rats, blocking ∼40% of HVA current with Cd2+ (5μM), had opposite effects on subpopulations of cutaneous DRG neurons (increasing excitability and action potential duration in some and decreasing excitability in others). The alterations in the density and distribution of voltage-activated Ca2+ channels in subpopulations of cutaneous DRG neurons that develop following CFA injection should contribute to changes in sensory transmission observed in the presence of inflammation. [ABSTRACT FROM AUTHOR]

Abstract: Although the formalin test is a widely used model of persistent pain, the primary afferent fiber types that underlie the cellular and behavioral responses to formalin injection are largely unknown. Here we used a combined genetic and pharmacological approach to investigate the effect of ablating subsets of primary afferent nociceptors on formalin-induced nocifensive behaviors and spinal cord Fos protein expression. Intrathecal capsaicin-induced ablation of the central terminals of TRPV1+neurons greatly reduced the behavioral responses and Fos elicited by low-dose (0.5%) formalin. In contrast, genetic ablation of the MrgprD-expressing subset of non-peptidergic unmyelinated afferents, which constitute a largely non-overlapping population, altered neither the behavior nor the Fos induced by low-dose formalin. Remarkably, nocifensive behavior following high-dose (2%) formalin was unchanged in mice lacking either afferent population, or even in mice lacking both populations, which together make up the great majority of C-fiber nociceptors. Thus, at high doses, which are routinely used in the formalin test, formalin-induced “pain” behavior persists in the absence of the vast majority of C-fiber nociceptors, which points to a contribution of a large spectrum of afferents secondary to non-specific formalin-induced tissue and nerve damage. [ABSTRACT FROM AUTHOR]

Abstract: Pain in early life can enhance the response to subsequent injury, but effects are influenced by both the nature and timing of neonatal injury. Using plantar hindpaw incision, we investigated how postnatal age influences the response to repeat surgical injury two weeks later. The degree and time course of behavioural changes in mechanical withdrawal threshold were measured, and injury-related hyperalgesia was further quantified by flexion reflex electromyographic responses to suprathreshold mechanical stimuli 24 h following incision. Plantar hindpaw incision produces acute mechanical hyperalgesia in neonatal and adult rats, but incision in neonatal pups has an additional effect on the response to subsequent injury. With initial incision at postnatal day (P) 3 or 6, the degree of hyperalgesia following repeat incision 2weeks later was greater than in animals having a single incision at the same age. At older ages (initial incision at P10, P21 or P40) responses did not differ in repeat and single incision groups. To test the role of primary afferent activity, levobupivacaine sciatic block was performed prior to P6 plantar incision, and controls received saline or subcutaneous levobupivacaine. Repeat peri-operative, but not a single pre-operative sciatic block, prevented the enhanced response to repeat incision two weeks later. Our results show that the first postnatal week represents a critical period when incision increases hyperalgesia following repeat surgery two weeks later, and effects are initiated by peripheral afferent activity. This has potential therapeutic implications for the type and duration of peri-operative analgesia used for neonatal surgery. [Copyright &y& Elsevier]

Abstract: Peripheral nerve injury promotes an enduring increase in the excitability of the spinal dorsal horn. This change, that likely underlies the development of chronic pain, may be a consequence of prolonged exposure of dorsal horn neurons to mediators such as neurotrophins, cytokines, and neurotransmitters. The long-term effects of such mediators can be analyzed by applying them to spinal neurons in organotypic slice culture. To assess the validity of this approach, we established serum-free, defined-medium organotypic cultures (DMOTC) from E13-14 prenatal rats. Whole-cell recordings were made from neurons maintained in DMOTC for up to 42 days. These were compared with recordings from neurons of similar age in acute spinal cord slices from 15- to 45-day-old rats. Five cell types were defined in acute slices as ‘Tonic’, ‘Irregular’, ‘Delay’, ‘Transient’ or ‘Phasic’ according to their discharge patterns in response to depolarizing current. Although fewer ‘Phasic’ cells were found in cultures, the proportions of ‘Tonic’, ‘Irregular’, ‘Delay’, and ‘Transient’ were similar to those found in acute slices. GABAergic, glycinergic, and ‘mixed’ inhibition were observed in neurons in acute slices and DMOTC. Pure glycinergic inhibition was absent in 7d cultures but became more pronounced as cultures aged. This parallels the development of glycinergic inhibition in vivo. These and other findings suggest that fundamental developmental processes related to neurotransmitter phenotype and neuronal firing properties are preserved in DMOTC. This validates their use in evaluating the cellular mechanisms that may contribute to the development of chronic pain. [Copyright &y& Elsevier]

Abstract: The distribution and modulation of the P2X3 receptor was studied in trigeminal ganglion neurons to provide insight into the role of ATP in craniofacial sensory mechanisms. Binding to the d-galactose specific lectin IB4 was found in 73% of P2X3-positive neurons while only 16% of IB4 neurons expressed P2X3. Neurons expressing P2X3 alone were significantly larger than IB4-or IB4/P2X3-positive neurons. Investigation of target-specificity revealed that 22% of trigeminal ganglion muscle afferent neurons were positive for P2X3 versus 16% of cutaneous afferent neurons. Muscle P2X3 afferents were significantly smaller than the overall muscle afferent population while P2X3 cutaneous afferent neurons were not. Presumptive heteromeric (P2X2/3) muscle afferent neurons were also identified and comprised 77% of the P2X3 muscle afferent population. Muscle afferent neurons co-expressed P2X3 with either calcitonin gene-related peptide (15%) or substance P (4%). The number of P2X3-positive muscle afferent neurons significantly increased one and four days following complete Freund''s adjuvant-induced masseter muscle inflammation, but significantly decreased after 12 days. These results indicate that within trigeminal ganglia: (1) the P2X3 receptor is expressed in both small and medium-sized neurons; (2) the P2X3 receptor is not exclusively expressed in IB4 neurons; (3) P2X3 is co-expressed with neuropeptides; (4) differences in the proportion of cutaneous versus muscle P2X3 afferents are not apparent. Trigeminal P2X3 neurons therefore differ markedly from dorsal root ganglion P2X3 afferents. This study also shows that deep tissue inflammation modulates expression of the P2X3 receptor and thus may warrant exploration as a target for therapeutic intervention. [Copyright &y& Elsevier]

Abstract: Functional and anatomical relationships among primary afferent fibers, blood vessels, and cancers are poorly understood. However, recent evidence suggests that physical and biochemical interactions between these peripheral components are important to both tumor biology and cancer-associated pain. To determine the role of these peripheral components in a mouse model of cancer pain, we quantified the change in nerve and blood vessel density within a fibrosarcoma tumor mass using stereological analysis of serial confocal optical sections of immunostained hind paw. To this end we introduced the Discoma coral-derived red fluorescent protein (DsRed2) into the NCTC 2472 fibrosarcoma line using the Sleeping Beauty transposon methodology, thus providing a unique opportunity to visualize tumor–nerve–vessel associations in context with behavioral assessment of tumor-associated hyperalgesia. Tumors from hyperalgesic mice are more densely innervated with calcitonin gene related peptide (CGRP)-immunoreactive nerve fibers and less densely vascularized than tumors from non-hyperalgesic mice. As hyperalgesia increased from Day 5 to 12 post-implantation, the density of protein gene product 9.5 (PGP9.5)-immunoreactive nerves and CD31-immunoreactive blood vessels in tumors decreased, whereas CGRP-immunoreactive nerve density remained unchanged. Importantly, intra-tumor injection of a CGRP1 receptor antagonist (CGRP 8–37) partially blocked the tumor-associated mechanical hyperalgesia, indicating that local production of CGRP may contribute to tumor-induced nociception through a receptor-mediated process. The results describe for the first time the interaction among sensory nerves, blood vessels and tumor cells in otherwise healthy tissue, and our assessment supports the hypothesis that direct tumor cell-axon communication may underlie, at least in part, the occurrence of cancer pain. [Copyright &y& Elsevier]

The stress-activated mitogen-activated protein kinase (MAPK) p38 is emerging as an important mediator of pain. The present study examined the possible involvement of peripheral and spinal p38 MAPK in capsaicin-induced thermal hyperalgesia. Topical capsaicin produced phosphorylation of p38 MAPK in the skin from the affected hindpaw as well as the corresponding lumbar spinal cord in a time dependent manner. Topical capsaicin produced robust C-fiber mediated thermal hyperalgesia that was inhibited by systemic, local peripheral, or central intrathecal pre-treatment with the p38 MAPK inhibitor, SD-282. Intraperitoneal SD-282 (10–60mg/kg) significantly and dose-dependently attenuated capsaicin-induced C-fiber mediated thermal hyperalgesia. Similarly, 0.1–5mg/kg subcutaneous SD-282 in the hindpaw dose-dependently attenuated capsaicin-induced thermal hyperalgesia. Intrathecal administration of 1μg SD-282 was also anti-hyperalgesic in this model. Functionally, SD-282 decreased capsaicin-induced release of calcitonin gene related peptide in an in vitro skin release assay, consistent with a role for p38 MAPK in peripheral nerve function. These results suggest that p38 MAPK plays a role in the development of hyperalgesic states, exerting effects both centrally in the spinal cord and peripherally in sensory C fibers. [Copyright &y& Elsevier]

Activation of peripheral somatostatin receptors (SSTRs) inhibits sensitization of nociceptors, thus having a short term or phasic effect [Pain 90 (2001) 233] as well as maintaining a tonic inhibitory control over nociceptors [J Neurosci 21 (2001) 4042]. The present study provides several lines of evidence that an important mechanism underlying SSTR modulation of nociceptors is regulation of the transient receptor potential vanilloid 1 ion channel (TRPV1, formerly the VR1 receptor). Double labeling of L5 dorsal root ganglion cells demonstrates that ∼60% of SSTR2a-labeled cells are positive for TRPV1. Conversely, ∼33% of TRPV1-labeled cells are positive for SSTR2a. In vivo behavioral studies demonstrate that intraplantar injection of 20.0 but not 2.0 μM octreotide (OCT, SSTR agonist) significantly reduces capsaicin (CAP, a ligand for TRPV1) -induced flinching and lifting/licking behaviors. This occurs through local activation of SSTRs in the injected hindpaw and is reversed following co-application of the SSTR antagonist cyclo-somatostatin (c-SOM). In vitro studies using a skin-nerve preparation demonstrate that activation of peripheral SSTRs on nociceptors with 20.0 μM OCT significantly reduces CAP-induced activity and can prevent CAP-induced desensitization. Furthermore, blockade of peripheral SSTRs with c-SOM dramatically enhances CAP-induced behaviors and nociceptor activity, demonstrating SSTR-induced tonic inhibitory modulation of TRPV1. Finally, TRPV1 does not appear to be under tonic opioid receptor control since the opioid antagonist naloxone does not change CAP-induced excitation and does not effect OCT-induced inhibition of CAP responses. These data strongly suggest that SSTRs modulate nociceptors through phasic and tonic regulation of peripheral TRPV1 receptors. [Copyright &y& Elsevier]