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We recently demonstrated, in a rat model of chemotherapy-induced peripheral neuropathy (CIPN), that there is a significant decrease in the duration of the depolarization-evoked Ca(2+) transient in isolated somata of putative nociceptive afferents innervating the glabrous skin of the hindpaw, but no change in transient magnitude or the resting concentration of intracellular Ca(2+) ([Ca(2+)]i). Because the Na(+)-Ca(2+) exchanger (NCX) only contributes to the regulation of the duration of the evoked Ca(2+) transient, in putative nociceptive dorsal root ganglion (DRG) neurons, we hypothesized that an increase in NCX activity underlies the CIPN-induced change in this subpopulation of neurons. Acutely dissociated retrogradely labeled sensory neurons from naïve, vehicle-, and paclitaxel-treated rats were studied with fura-2 based Ca(2+) imaging. There was no difference in the relative level of NCX activity between glabrous neurons from paclitaxel-treated or control rats. However, in contrast to the relatively large and long lasting Ca(2+) transients needed to evoke NCX activity in neurons from naïve rats, there was evidence of resting NCX activity in glabrous neurons from both vehicle- and paclitaxel-treated rats. More interestingly, there was a paclitaxel-induced increase in NCX activity in putative nociceptive neurons innervating the thigh, neurons in which there is no evidence of a change in the depolarization-induced Ca(2+) transient, or a body site in which there was a change in nociceptive threshold. Furthermore, while the majority of NCX activity in glabrous neurons is sensitive to the NCX3-preferring blocker KB-R7943, the increase in NCX activity in thigh neurons was resistant to KB-R7943 but sensitive to the NCX1-preferring blocker SEA0400. These results suggest that a mechanism(s) other than NCX underlies the paclitaxel-induced decrease in the duration of the evoked Ca(2+) transient in putative nociceptive glabrous skin neurons. However, the compensatory response to paclitaxel observed may also explain why only subpopulations of sensory neurons are impacted by paclitaxel, raising the intriguing possibility that CIPN is due to the failure of injured neurons to appropriately compensate for the deleterious consequences of this compound.

The purpose of the present study was to characterize the properties of A-type GABA receptor (GABAA receptor) currents in human sensory neurons. Neurons were obtained from adult organ donors. GABAA currents were recorded in isolated neurons. Both large inactivating low-affinity currents and smaller persistent high-affinity currents were present in all of the 129 neurons studied from 15 donors. The kinetics of human GABAA currents were slower than those in rat sensory neurons. GABA currents were completely blocked by bicuculline (10 μM), and persistent currents were activated by the δ-subunit-preferring agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP). The GABA current equilibrium potential was ∼ 20 mV more hyperpolarized than in rat neurons. Both low- and high-affinity currents were increased by inflammatory mediators but via different second messenger pathways. These results highlight potentially important species differences in the properties of ion channels present in their native environment and suggest the use of human sensory neurons may be a valuable tool to test compounds prior to use in humans.

While debate continues over whether somatosensory information is transmitted via labeled line, population coding, frequency coding, or some combination therein, researchers have begun to address this question at the level of the primary afferent by using optical approaches that enable the assessment of neural activity in hundreds to even thousands of neurons simultaneously. However, with limited availability of tools to optically assess electrical activity in large populations of neurons, researchers have turned to genetically encoded Ca(2+) indicators (GECIs) including GCaMP to enable the detection of increases in cytosolic Ca(2+) concentrations as a correlate for neuronal activity. One of the most widely used GECIs is GCaMP6, which is available in three different versions tuned for sensitivity (GCaMP6s), speed (GCaMP6f), or a balance of the two (GCaMP6m). In order to determine if these issues were unique to GCaMP6 itself, or if they were inherent to more than one generation of GCaMP, we also characterized jGCaMP7. In the present study, we sought to determine the utility of the three GCaMP6 isoforms to detect changes in activity in primary afferents at frequencies ranging from 0.1 to 30 Hz. Given the heterogeneity of sensory neurons, we also compared the performance of each GCaMP6 isoform in subpopulations of neurons defined by properties used to identify putative nociceptive afferents: cell body size, isolectin B4 (IB4) binding, and capsaicin sensitivity. Finally, we compared results generated with GCaMP6 with that generated from neurons expressing the newest generation of GCaMP, jGCaMP7s and jGCaMP7f. A viral approach, with AAV9-CAG-GCaMP6s/m/f, was used to drive GECI expression in acutely dissociated rat trigeminal ganglion (TG) neurons, and neural activity was driven by electrical field stimulation. Infection efficiency with the AAV serotype was high >95%, and the impact of GCaMP6 expression in TG neurons over the period of study (90% of neurons. However, the utility of the GCaMP6 isoforms to enable an assessment of the firing frequency let alone changes in firing frequency of each neuron was relatively limited and isoform specific: GCaMP6s and 6m had the lowest resolution, enabling detection of spikes at 3 Hz in 15% and 32% of neurons respectively, but it was possible to resolve discrete single spikes up to 10 Hz in 36% of GCaMP6f neurons. Unfortunately, using other parameters of the Ca(2+) transient, such as magnitude of the transient or the rate of rise, did not improve the range over which these indicators could be used to assess changes in spike number or firing frequency. Furthermore, in the presence of ongoing neural activity, it was even more difficult to detect a change in firing frequency. The frequency response relationship for the increase in Ca(2+) was highly heterogeneous among sensory neurons and was influenced by both the GCaMP6 isoform used to assess it, the timing between the delivery of stimulation trains (inter-burst interval), and afferent subpopulation. Notably, the same deficiencies were observed with jGCaMP7s and 7f in resolving the degree of activity as were present for the GCaMP6 isoforms. Together, these data suggest that while both GCaMP6 and jGCaMP7 are potentially useful tools in sensory neurons to determine the presence or absence of neural activity, the ability to discriminate changes in firing frequency ≥ 3 Hz is extremely limited. As a result, GECIs should probably not be used in sensory neurons to assess changes in activity within or between subpopulations of neurons.

The ability to modulate cellular electrophysiology is a fundamental aspect for investigation of tissue development, maturation and function. Currently, there is a need for remote, non-genetic, light-induced control of cellular activity in native-like state, three-dimensional (3D) tissues such as spheroids and organoids. Here, we report a breakthrough hybrid-nanomaterial for remote, non-genetic photostimulation of both two-dimensional (2D) and 3D neural cellular systems. We combine one-dimensional (1D) nanowires (NWs) and 2D graphene flakes grown out-of-plane for highly controlled photostimulation at subcellular precision with laser energies lower than hundred nanojoules, 1-2 orders of magnitude lower than Au-, C- and Si-based photostimulation. Photostimulation using NW-templated 3D fuzzy graphene (NT-3DFG) is flexible due to its broadband absorption and does not generate cellular stress. Therefore, it serves a novel powerful toolset for studies of cell signaling within and between tissues and can enable new therapeutic interventions.

A search for the exotic meson X(5568) decaying into the B_{s}^{0}π^{±} final state is performed using data corresponding to 9.6 fb^{-1} from pp[over ¯] collisions at sqrt[s]=1960 GeV recorded by the Collider Detector at Fermilab. No evidence for this state is found and an upper limit of 6.7% at the 95% confidence level is set on the fraction of B_{s}^{0} produced through the X(5568)→B_{s}^{0}π^{±} process.

Without reliable tools to record activity in large populations of neurons, investigators have turned to genetically-encoded Ca2+ indicators such as GCaMP, assuming that changes in cytosolic Ca2+ ([Ca2+]c) can be used as an indirect measure of neural activity. Three versions of GCaMP6 have been tuned for sensitivity (GCaMP6s), speed (GCaMP6f), or a balance between the two (GCaMP6m) by manipulating Ca2+binding kinetics. We recently presented preliminary data suggesting that heterogeneity among primary afferents in firing frequency-dependent [Ca2+]c regulation does, in fact, impact GCaMP6’s ability to encode neural activity. Here, we expanded upon this work to assess the relationship between neural activity and [Ca2+]c as well as the utility of GCaMP6s/m/f to detect activity changes in trigeminal sensory neuron subpopulations. To drive GCaMP6 expression in acutely dissociated rat trigeminal ganglion neurons, we used AAV9-CAG. Neurons were then stimulated using different protocols and frequencies. First, we measured infection efficiency of AAV9-CAG-GCAMP6(s/m/f). Then, comparing isoforms and neuronal subpopulations, we assessed the (i) ability to detect a single spike, (ii) ability to resolve single spikes in a spike train at different frequencies, (iii) dynamic range, and (iv) optimal firing frequency. Using Fura-2, we measured [Ca2+]c in GCaMP6-expressing neurons at rest and following stimulation. GCaMP6 expression was detected within 6 days, and results were collected 6-10d post-infection. Infection efficiency is >80% with all three isoforms in all neuron subpopulations. All three GCaMP6 isoforms detect a single spike in 84-98% of neurons in all subpopulations. However, GCaMP6s's dynamic range is greater than 6m's or 6f's. We confirmed our initial observation that there is an optimal coding frequency that is isoform- and neuronal subpopulation-dependent, which is often below 30 Hz. These data suggest that care should be taken in selection, use, and interpretation of experiments that use GCaMP6 as a proxy for peripheral neurons activity.

Migraine is a common, disruptive, and often debilitating neurological disorder that the World Health Organization classifies in the highest disability category. Migraine is up to three times more common in women than men. Attacks are commonly triggered by stress, and many migraineurs show signs of sympathetic dysregulation. While the vascular hypothesis for migraine has largely fallen from favor, there are still several lines of evidence to suggest that dysregulation of the dural vasculature may contribute to the initiation of a migraine attack. These include the evidence that migraine pain arises from dural nociceptors, that the dural vasculature is heavily innervated by both nociceptors and sympathetic post-ganglionic neurons, that vascular endothelial cells may be a source of inflammatory mediators contributing to the sensitization and activation of dural nociceptors, and that the vasculature is a primary target for CGRP, a peptide that appears to play a central role in both the initiation and maintenance of pain associated with a migraine attack. We hypothesized that stress would result in a sex-dependent loss of sympathetic control over dural vasculature. Male and female rats were randomized to one of three groups: naive, acute stress (studied right after stress) or recovery (studied 24 hrs after stress). Stress consisted of a 30-minute immobilization restraint. A bipolar electrode was placed in the superior cervical ganglion (SCG) and the skull was thinned over the middle meningeal artery. FITC-dextran was used to visualize dural blood vessels while the SCG was stimulated at differing frequencies. Vasopressin and nitroprusside were used to establish the minimum and maximum vessel diameters, respectively, per rat. Preliminary results suggest that the SCG stimulation frequency range of vessel regulation is sex-dependent and may be shifted in stressed animals. A stress-induced loss of sympathetic regulation of dural vessels could contribute to migraine initiation.

The purpose of the present study was to test the prediction that the unique manifestation of chemotherapeutic-induced peripheral neuropathy (CIPN) would be reflected in a specific pattern of changes in the regulation of the intracellular Ca(2+) concentration ([Ca(2+)]i) in subpopulations of cutaneous neurons. To test this prediction, we characterized the pattern of changes in mechanical nociceptive threshold associated with paclitaxel administration (2mg/kg, iv, every other day for four days), as well as the impact of target of innervation and paclitaxel treatment on the regulation of [Ca(2+)]i in subpopulations of putative nociceptive and non-nociceptive neurons. Neurons innervating the glabrous and hairy skin of the hindpaw as well as the thigh were identified with retrograde tracers, and fura-2 was used to assess changes in [Ca(2+)]i. Paclitaxel was associated with a persistent decrease in mechanical nociceptive threshold in response to stimuli applied to the glabrous skin of the hindpaw, but not the hairy skin of the hindpaw or the thigh. However, in both putative nociceptive and non-nociceptive neurons, resting [Ca(2+)]i was significantly lower in neurons innervating the thigh after treatment. The magnitude of the depolarization-evoked Ca(2+) transient was also lower in putative non-nociceptive thigh neurons. More interestingly, while paclitaxel had no detectable influence on either resting or depolarization-evoked Ca(2+) transients in putative non-nociceptive neurons, in putative nociceptive neurons there was a subpopulation-specific decrease in the duration of the evoked Ca(2+) transient that was largely restricted to neurons innervating the glabrous skin. These results suggest that peripheral nerve length alone, does not account for the selective distribution of CIPN symptoms. Rather, they suggest the symptoms of CIPN reflect an interaction between the toxic actions of the therapeutic and unique properties of the neurons deleteriously impacted.

Background Migraine attacks are associated with sterile inflammation of the dura. Immune cells are a primary source of inflammatory mediators, and we therefore sought to further explore the link between dural immune cells and migraine. Objective Based on the observations that migraine is more common in women than in men, stress is the most common trigger for a migraine attack, and sympathetic post-ganglionic innervation of the dura enables local control of dural immune cells, we hypothesized that stress shifts the balance of inflammatory mediator expression in dural immune cells toward those that trigger a migraine attack, where these changes are larger in females and dependent, at least in part, on sympathetic post-ganglionic innervation of the dura. Our objective was to test this hypothesis. Methods Dura were obtained from naive or stressed, intact or surgically sympathectomized, adult male and female rats. Dura were assessed immediately or 24 hours after termination of 4 continuous days of unpredictable, mild stressors. Following enzymatic digestion of each dura, myeloid and lymphoid-derived dural immune cells were isolated by fluorescence-activated cell sorting for semi-quantitative polymerase chain reaction analysis. Results In myeloid-derived dural immune cells, there was an increase in pro-inflammatory mediator mRNA following stress, particularly in females, which remained elevated with a 24-hour delay after stress. There was a stress-induced decrease in anti-inflammatory mediator mRNA immediately after stress in females, but not males. The stress-induced changes were attenuated in sympathectomized females. In lymphoid-derived dural immune cells, there was a persistent increase in pro-inflammatory mediator mRNA following stress, particularly in females. A stress-induced increase in anti-inflammatory mediator mRNA was also observed in both males and females, and was further attenuated in sympathectomized females. Conclusions Consistent with our hypothesis, there is a stress-induced shift in the balance of pro- and anti-inflammatory mediator expression in dural immune cells that is more pronounced in females, and is dependent, at least in part, on sympathetic post-ganglionic innervation in females. This shift in the balance of inflammatory mediator expression may not only play an important role in triggering migraine attacks, but also suggests it may be possible, if not necessary, to employ different strategies to most effectively treat migraine in men and women.

Persistent inflammation results in an increase in the amplitude and duration of depolarization-evoked Ca2+transients in putative nociceptive afferents. Previous data indicated that these changes were the result of neither increased neuronal excitability nor an increase in the amplitude of depolarization. Subsequent data also ruled out an increase in voltage-gated Ca2+currents and recruitment of Ca2+-induced Ca2+release. Parametric studies indicated that the inflammation-induced increase in the duration of the evoked Ca2+transient required a relatively large and long-lasting increase in the concentration of intracellular Ca2+implicating the Na+/Ca2+exchanger (NCX), a major Ca2+extrusion mechanism activated with high intracellular Ca2+loads. The contribution of NCX to the inflammation-induced increase in the evoked Ca2+transient in rat sensory neurons was tested using fura-2 AM imaging and electrophysiological recordings. Changes in NCX expression and protein were assessed with real-time PCR and Western blot analysis, respectively. An inflammation-induced decrease in NCX activity was observed in a subpopulation of putative nociceptive neurons innervating the site of inflammation. The time course of the decrease in NCX activity paralleled that of the inflammation-induced changes in nociceptive behavior. The change in NCX3 in the cell body was associated with a decrease in NCX3 protein in the ganglia, an increase in the peripheral nerve (sciatic) yet no change in the central root. This single response to inflammation is associated with changes in at least three different segments of the primary afferent, all of which are likely to contribute to the dynamic response to persistent inflammation.

While the safety and efficacy of peripheral nerve blocks for postoperative pain management has been established in several well controlled prospective trials, the local anesthetic (LA) concentration and volume used in these studies was associated with a significant increase muscle weakness due to motor nerve block. The purpose of the present retrospective study of patients undergoing total knee arthroplasty was to assess the relative analgesic efficacy and functional outcomes of the low concentration, low volume of LA used in peripheral nerve blocks for postoperative pain management.Twenty-four months of deidentified patient data were extracted from an electronic medical record system. All patients received opioids with or without continuous femoral and sciatic nerve block infusions for postoperative analgesia. Pain (resting and with activity), cumulative opioid and LA use were primary endpoints, participation in physical therapy (PT), muscle strength deficits and length of hospital stay (LOS) were secondary endpoints.Postoperative pain and opioid use were significantly lower in patients with peripheral nerve blocks (n = 1,329) than those with opioids alone (n = 439). There was no detectable decrease in strength associated with nerve blocks, while a significantly greater proportion of patients with nerve blocks were able to participate in PT on postoperative day 1 (96.4% vs 57.1%). These differences were not due to the impact of the surgeon per se, but whether or not the surgeon used nerve blocks for pain management. There was a small but statistically significant decrease in the average LOS in patients with blocks.This analysis supports the use of low concentration, low volume of LA based peripheral nerve blocks for postoperative pain management.

Objective The objective of this study was to determine the extent to which altered loading in the temporomandibular joint (TMJ), as might be associated with a malocclusion, drives degeneration of articulating surfaces in the TMJ. We therefore sought to quantify the effects of altered joint loading on the mechanical properties and biochemical content and distribution of TMJ fibrocartilage in the rabbit. Design Altered TMJ loading was induced with a 1 mm splint placed unilaterally over the maxillary and mandibular molars for 6 weeks. At that time, TMJ fibrocartilage was assessed by compression testing, biochemical content (collagen, glycosaminoglycan (GAG), DNA) and distribution (histology), for both the TMJ disc and the condylar fibrocartilage. Results There were no changes in the TMJ disc for any of the parameters tested. The condylar fibrocartilage from the splinted animals was significantly stiffer and the DNA content was significantly lower than that in control animals. There was significant remodeling in the condylar fibrocartilage layers as manifested by a change in GAG and collagen II distribution and a loss of defined cell layers. Conclusions A connection between the compressive properties of TMJ condylar fibrocartilage after 6 weeks of splinting and the changes in histology was observed. These results suggest a change in joint loading leads to condylar damage, which may contribute to pain associated with at least some forms of TMJ disease.

The goals of the present study were to determine (1) the properties of the nicotinic acetylcholine receptor (nAChR) currents in rat cutaneous dorsal root ganglion (DRG) neurons; (2) the impact of nAChR activation on the excitability of cutaneous DRG neurons; and (3) the impact of inflammation on the density and distribution of nAChR currents among cutaneous DRG neurons. Whole-cell patch-clamp techniques were used to study retrogradely labeled DRG neurons from naive and complete Freund’s adjuvant inflamed rats. Nicotine-evoked currents were detectable in ∼70% of the cutaneous DRG neurons, where only one of two current types, fast or slow currents based on rates of activation and inactivation, was present in each neuron. The biophysical and pharmacological properties of the fast current were consistent with nAChRs containing an α7 subunit while those of the slow current were consistent with nAChRs containing α3/β4 subunits. The majority of small diameter neurons with fast current were IB4− while the majority of small diameter neurons with slow current were IB4+. Preincubation with nicotine (1 μM) produced a transient (1 min) depolarization and increase in the excitability of neurons with fast current and a decrease in the amplitude of capsaicin-evoked current in neurons with slow current. Inflammation increased the current density of both slow and fast currents in small diameter neurons and increased the percentage of neurons with the fast current. With the relatively selective distribution of nAChR currents in putative nociceptive cutaneous DRG neurons, our results suggest that the role of these receptors in inflammatory hyperalgesia is likely to be complex and dependent on the concentration and timing of acetylcholine release in the periphery.

Available evidence indicates voltage-gated Na+ channels (VGSCs) in peripheral sensory neurons are essential for the pain and hypersensitivity associated with tissue injury. However, our understanding of the biophysical and pharmacological properties of the channels in sensory neurons is largely based on the study of heterologous systems or rodent tissue, despite evidence that both expression systems and species differences influence these properties. Therefore, we sought to determine the extent to which the biophysical and pharmacological properties of VGSCs were comparable in rat and human sensory neurons. Whole cell patch clamp techniques were used to study Na+ currents in acutely dissociated neurons from human and rat. Our results indicate that while the two major current types, generally referred to as tetrodotoxin (TTX)-sensitive and TTX-resistant were qualitatively similar in neurons from rats and humans, there were several differences that have important implications for drug development as well as our understanding of pain mechanisms. DOI: http://dx.doi.org/10.7554/eLife.23235.001

Because of the importance of intracellular Ca2+ to processes ranging from protein-interactions to cell death, the concentration of cytosolic Ca2+ ([Ca2+]c) is tightly regulated by a complex array of pumps, transporters, exchangers, and binding proteins. We have recently demonstrated that the Na+/Ca2+ exchanger (NCX), in particular NCX3, plays an important role in attenuating large-increases in [Ca2+]c in a subpopulation of putative nociceptive rat dorsal root ganglion (DRG) neurons. Interestingly, we subsequently demonstrated that inflammation-induced dysregulation of [Ca2+]c in this subpopulation of putative nociceptors was due, at least in part, to a decrease in NCX activity in the sensory neuron cell body. Given the potential importance of this exchanger to nociceptive processing as well as injury-induced changes in nociceptive processing, in the face of a growing list of differences between rodent and human sensory neurons, the purpose of the present study was to characterize NCX activity in human sensory neurons. We employed electrical field stimulation to evoke Ca2+ transients in the presence and absence of LiCl or Choline-Cl bath to block NCX activity. Interestingly, electrical field stimulation of human DRG neurons, at the same parameters used in rat, evoked a much larger Ca2+ transient in both magnitude and duration. We obtained evidence of NCX activity in the majority of neurons tested. Similar to the role of NCX in rat DRG neurons, block of NCX in human DRG neurons increased the duration, but not the magnitude of the electrically evoked Ca2+ transient. However, in contrast to the rat, there appeared to be constitutive NCX activity. Consistent with a number of recent studies, these results highlight potentially important similarities and differences between rodent and human sensory neurons, underscoring the importance of human sensory neurons as a pre-clinical screen of novel therapeutics targeting the primary afferent. Research funded by NIH T32NS073548, 1R01NS083347, and R01DK107966.

We proposed recently that induction of delayed activation of trigeminovascular neurons by cortical spreading depression (CSD) can explain the delayed onset of headache after the migraine aura ("aura"). This prompted us to search for ways to block the neuronal activation by CSD - a preclinical correlate of an attempt to find a drug that can block the initiation of headache when administered shortly after onset of aura (i.e., preemptively). Because migraine headache and epileptic seizures are comorbid chronic neurological disorders characterized by hyperexcitable brain networks, we began the search for such goal with an M-type potassium channel opener. We opted to use ezogabine, recently approved by the FDA as adjunctive treatment of partial onset seizures in adults, because it is a selective KCNQ2/3 channel opener. When CSD was induced before ezogabine injection (8.25 mg/kg, i.p.), 40% (6/15) of the units doubled their firing rate about 45 min later for about 95 min. Similarly, when CSD was induced before vehicle was injected (4% DMSO, 0.5% methylcellulose), 50% (3/6) of the units doubled their firing rate about 30 min later for about 120 min. When CSD was triggered 1h after ezogabine injection, it activated only 8% of the units. By itself, ezogabine injection resulted in a 30% attenuation of ongoing firing in all 10 control units. Thus, activation of KCNQ2/3 channels during the aura is unlikely to preempt the onset of headache but may reduce the incidence of migraine if given during prodromes that precede the headache by hours. Given the mechanistic similarities between migraine aura and epileptic seizures, it may be worthwhile to determine whether preemptive administration of ezogabine can prevent oncoming seizures in patients whose warning signs precede their seizures by more than an hour.

We present a measurement of the total WW and WZ production cross sections in pp collision at s=1.96 TeV, in a final state consistent with leptonic W boson decay and jets originating from heavy-flavor quarks from either a W or a Z boson decay. This analysis uses the full data set collected with the CDF II detector during Run II of the Tevatron collider, corresponding to an integrated luminosity of 9.4 fb-1. An analysis of the dijet mass spectrum provides 3.7σ evidence of the summed production processes of either WW or WZ bosons with a measured total cross section of σWW+WZ=13.7±3.9 pb. Independent measurements of the WW and WZ production cross sections are allowed by the different heavy-flavor decay patterns of the W and Z bosons and by the analysis of secondary-decay vertices reconstructed within heavy-flavor jets. The productions of WW and of WZ dibosons are independently seen with significances of 2.9σ and 2.1σ, respectively, with total cross sections of σWW=9.4±4.2 pb and σWZ=3.7-2.2+2.5 pb. The measurements are consistent with standard-model predictions.

We have recently demonstrated that in a rat model of chemotherapy-induced peripheral neuropathy (CIPN), there is a significant decrease in the duration of the depolarization-evoked Ca2+ transient in small diameter, IB4+, and capsaicin-responsive neurons innervating the glabrous skin of the hindpaw. This change was specific to the transient duration and significantly smaller if not undetectable in neurons innervating the dorsal skin of the hindpaw or the skin of the inner thigh. Given the importance of mitochondria in intracellular Ca2+ regulation and the findings of chemotherapy-associated increase in mitotoxicity along the sensory neuron axons, we hypothesized that CIPN is due to both increases and decreases in mitochondria function, with changes manifest in distinct subpopulations of afferents. To begin to test this hypothesis, we used confocal microscopy and Ca2+ imaging in combination with pharmacological manipulations to study paclitaxel-induced changes in retrograde tracer-labeled neurons from naïve, vehicle-treated, and paclitaxel-treated rats. Paclitaxel treatment was not associated with decreased mitochondrial membrane potential or increased superoxide levels in the somata of putative nociceptive glabrous skin neurons. However, it was associated with significant increases in the relative contribution of mitochondria to the control of the evoked Ca2+ transient duration in putative nociceptive glabrous skin neurons, as well as increases in mitotracker and Tom20 staining which reflected an increase in mitochondrial volume. Furthermore, the relative contribution of the sarco-endoplasmic reticulum Ca2+ ATPase to the regulation of the duration of the depolarization evoked Ca2+ transient was also increased in this subpopulation of neurons from paclitaxel treated rats. Our results indicate that the paclitaxel-induced decrease in the duration of the evoked Ca2+ transient is due to both direct and indirect influences of mitochondria. It remains to be determined if and how these changes contribute to the manifestation of CIPN.

Citation: Aaltonen, T., Amerio, S., Amidei, D., Anastassov, A., Annovi, A., Antos, J., . . . Zucchelli, S. (2016). Measurement of sin2 ?eff lept using e+e- pairs from ??/Z bosons produced in pp collisions at a center-of-momentum energy of 1.96 TeV. Physical Review D - Particles, Fields, Gravitation and Cosmology, 93(11). doi:10.1103/PhysRevD.93.112016

et al., A search for a Higgs boson with suppressed couplings to fermions, hf, assumed to be the neutral, lower-mass partner of the Higgs boson discovered at the Large Hadron Collider, is reported. Such a Higgs boson could exist in extensions of the standard model with two Higgs doublets, and could be produced via pp¯→H±hf→W∗hfhf→4γ+X, where H± is a charged Higgs boson. This analysis uses all events with at least three photons in the final state from proton-antiproton collisions at a center-of-mass energy of 1.96 TeV collected by the Collider Detector at Fermilab, corresponding to an integrated luminosity of 9.2 fb−1. No evidence of a signal is observed in the data. Values of Higgs-boson masses between 10 and 100 GeV/c2 are excluded at 95% Bayesian credibility., This work was supported by the U.S. Department of Energy and National Science Foundation; the Italian Istituto Nazionale di Fisica Nucleare; the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Natural Sciences and Engineering Research Council of Canada; the National Science Council of the Republic of China; the Swiss National Science Foundation; the A. P. Sloan Foundation; the Bundesministerium für Bildung und Forschung, Germany; the Korean World Class University Program, the National Research Foundation of Korea; the Science and Technology Facilities Council and the Royal Society, United Kingdom; the Russian Foundation for Basic Research; the Ministerio de Ciencia e Innovación, and Programa Consolider-Ingenio 2010, Spain; the Slovak R&D Agency; the Academy of Finland; the Australian Research Council (ARC); and the EU community Marie Curie Fellowship Contract No. 302103.

Aim of investigation Due to compelling evidence in support of links between sex, stress, sympathetic post-ganglionic innervation, dural immune cells, and migraine, our aim was to characterize the impacts of these factors on the type and proportion of immune cells in the dura. Methods Dural immune cells were obtained from naïve or stressed adult male and female Sprague Dawley rats for flow cytometry. Rats with surgical denervation of sympathetic post-ganglionic neurons of the dura were also studied. Results Immune cells comprise ∼17% of all cells in the dura. These included: macrophages/granulocytes (“Macs”; 63.2% of immune cells), dendritic cells (0.88%), T-cells (4.51%), natural killer T-cells (0.51%), natural killer cells (3.08%), and B-cells (20.0%). There were significantly more Macs and fewer B- and natural killer T-cells in the dura of females compared with males. Macs and dendritic cells were significantly increased by stress in males, but not females. In contrast, T-cells were significantly increased in females with a 24-hour delay following stress. Lastly, Macs, dendritic cells, and T-cells were significantly higher in sympathectomized-naïve males, but not females. Conclusions It may not only be possible, but necessary to use different strategies for the most effective treatment of migraine in men and women.

To account for benzodiazepine-induced spinal analgesia observed in association with an inflammation-induced shift in the influence of the GABAAreceptor antagonist gabazine on nociceptive threshold, the present study was designed to determine whether persistent inflammation is associated with the upregulation of high-affinity GABAAreceptors in primary afferents. The cell bodies of afferents innervating the glabrous skin of the rat hind paw were retrogradely labeled, acutely dissociated, and studied before and after the induction of persistent inflammation. A time-dependent increase in GABAAcurrent density was observed that was more than twofold by 72 h after the initiation of inflammation. This increase in current density included both high- and low-affinity currents and was restricted to neurons in which GABA increased intracellular Ca2+. No increases in GABAAreceptor subunit mRNA or protein were detected in whole ganglia. In contrast, the increased current density was completely reversed by 20-min preincubation with the tyrosine kinase inhibitor genistein and partially reversed with the Src kinase inhibitor PP2. Genistein reversal was partially blocked by the dynamin inhibitor peptide P4. Changes in nociceptive threshold following spinal administration of genistein and muscimol to inflamed rats indicated that the pronociceptive actions of muscimol observed in the presence of inflammation were reversed by genistein. These results suggest that persistent changes in relative levels of tyrosine kinase activity following inflammation provide not only a sensitive way to dynamically regulate spinal nociceptive signaling but a viable target for the development of novel therapeutic interventions for the treatment of inflammatory pain.

International audience; This document was approved by the European Heart Rhythm Association, a registered branch of the European Society of Cardiology (ESC), the Heart Rhythm Society, the American Heart Association, the American Society of Echocardiography, the Heart Failure Society of America, the American Heart Association Science Advisory and Coordinating Committee, the European Association of Echocardiography of the ESC, and the Heart Failure Association of the ESC. This article is co published in HeartRhythm. The Heart Rhythm Society requests that this document be cited as follows: 2012 EHRA/HRS expert consensus statement on cardiac resynchronization therapy in heart failure: implant and follow-up recommendations and management. Copies: This document is available on the World Wide Web sites of the European Heart Rhythm Association (www.escardio.org/communities/EHRA), and the Heart Rhythm Society (www.hrsonline.org). For copies of this document, please contact Sonja Olson at the Heart Rhythm Society solson@hrsonline. org. Permissions: Modification, alteration, enhancement, and/or distribution of this document are not permitted without the express permission of the European Heart Rhythm Association or the Heart Rhythm Society.

Persistent inflammation is associated with a shift in spinal GABA(A) signaling from inhibition to excitation such that GABA(A)-receptor activation contributes to inflammatory hyperalgesia. We tested the hypothesis that the primary afferent is the site of the persistent inflammation-induced shift in GABA(A) signaling which is due to a Na(+)-K(+)-Cl(-)-co-transporter (NKCC1)-dependent depolarization of the GABA(A) current equilibrium potential (E(GABA)). Acutely dissociated retrogradely labeled cutaneous dorsal root ganglion (DRG) neurons from naïve and inflamed (3 days after a subcutaneous injection of complete Freund's adjuvant) adult male rats were studied with Ca(2+) imaging, western blot and gramicidin-perforated patch recording. GABA evoked a Ca(2+) transient in a subpopulation of small- to medium-diameter capsaicin-sensitive cutaneous neurons. Inflammation was associated with a significant increase in the magnitude of GABA-induced depolarization as well as the percentage of neurons in which GABA evoked a Ca(2+) transient. There was no detectable change in NKCC1 protein or phosphoprotein at the whole ganglia level. Furthermore, the increase in excitatory response was comparable in both HEPES- and HCO(3)(-)-buffered solutions, but was only associated with a depolarization of E(GABA) in HCO(3)(-)-based solution. In contrast, under both recording conditions, the excitatory response was associated with an increase in GABA(A) current density, a decrease in low threshold K(+) current density, and resting membrane potential depolarization. Our results suggest that increasing K(+) conductance in afferents innervating a site of persistent inflammation may have greater efficacy in the inhibition of inflammatory hyperalgesia than attempting to drive a hyperpolarizing shift in E(GABA).

Introduction: We sought community opinion on consent alternatives when linking childhood immunisation and hospital attendance records for the purpose of vaccine safety surveillance. Methods: We conducted computer-assisted telephone interviewing (CATI) of a sample of rural and metropolitan residents of South Australia in 2011. Results: Of 2002 households interviewed (response rate 55.6%), 96.4% supported data linkage for postmarketing surveillance of vaccines; very few were completely opposed (1.5%) or undecided (2.2%). The majority (75.3%) trusted the privacy protections used in data linkage and most wished to have minimal or no direct involvement, preferring either opt-out consent (40.4%) or no consent (30.6%). A quarter of respondents (24.6%) favoured opt-in consent, but their preferences were divergent; half requested consent be sought prior to every use (11.4%) while the remainder preferred to give broad consent just once (3.4%) or renewed at periodic intervals (9.8%). Over half of the respondents gave higher priority to rapid vaccine safety surveillance (56.5%) rather than first seeking parental consent (26.6%) and one in seven was undecided (14.5%). Although 91.6% of respondents believed childhood vaccines are safe, over half (53.1%) were very or somewhat concerned that a vaccine could cause a serious reaction. Nevertheless, 92.4% of the parents in the sample (556/601) reported every child in their care as being fully immunised according to the National Immunisation Program schedule. Only 3.7% of parents (22/601) reported one or more children as under immunised, and 3.9% (23/601) reported that none of their children were immunised. Conclusions: This survey demonstrates that data linkage for vaccine safety surveillance has substantial community support and that a system utilising opt-out consent or no consent was preferred to one using opt-in consent. These findings should inform public health policy and practice; data linkage should be established where feasible to address limitations in passive surveillance systems Refereed/Peer-reviewed

Despite evidence that high-affinity GABA(A) receptor subunit mRNA and protein are present in dorsal root ganglia (DRG), low-affinity currents dominate those detected in acutely dissociated DRG neurons in vitro. This observation raises the possibility that high-affinity receptors are normally trafficked out of the DRG toward central and peripheral terminals. We therefore hypothesized that with time in culture, there would be an increase in high-affinity GABA(A) currents in DRG neurons. To test this hypothesis, we studied dissociated DRG neurons 2 h (acute) and 24 h (cultured) after plating with whole-cell patch-clamp techniques, Western blot, and semiquantitative reverse transcriptase polymerase chain reaction (sqRT-PCR) analysis. GABA(A) current density increases dramatically with time in culture in association with the emergence of two persistent currents with EC50's of 0.25±0.01 μM and 3.2±0.02 μM for GABA activation. In a subpopulation of neurons, there was also an increase in the potency of GABA activation of the transient current from an EC50 of 78.16±10.1 μM to 9.56±1.3 μM with time in culture. A fraction of the high-affinity current was potentiated by δ-subunit agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP). δ-subunit immunoreactivity was largely restricted to the cytosolic fraction in acute, but the membrane fraction in cultured, DRG neurons, with no detectable change in δ-subunit mRNA. However, the emergence of a high-affinity current blocked by THIP and insensitive to bicuculline was detected in a subpopulation of cultured neurons as well in association with an increase in ρ2- and ρ3-subunit mRNA in cultured DRG neurons. Our results suggest that high-affinity δ-subunit-containing GABA(A) receptors are normally trafficked out of the DRG where they are targeted to peripheral and central processes. They also highlight that the interpretation of data obtained from cultured DRG neurons should be made with caution.

Facial pain is a common and nonspecific symptom that is associated with known and unknown etiologies. Because the most effective therapeutic interventions address a disorder’s etiopathogenesis, it is important, when possible, to properly classify patients with different etiologies of facial pain. This is particularly true for trigeminal neuralgia (TN), because of the intensity of the pain associated with this disorder. Historically, the term TN has been used to refer to several different conditions. Taken in its most literal and general form, trigeminal neuralgia denotes pain that occurs within the dermatomal distribution of the trigeminal nerve. Many clinicians, however, reserve the term, TN, to signify a more specific disorder, which manifests as attacks of sudden, unilateral, and lancinating facial pain with characteristic triggers (e.g., light touch, cold air). These attacks may result from vascular compression of the trigeminal nerve near its entry into the brainstem (Jannetta 1967; Gardner and Miklos 1959). Vascular compression as the etiopathogenesis of TN, however, occurs in a minority of patients with facial pain. Furthermore, facial pain that does not fit this description completely may also be associated with probable incidental vascular compression.

Background Experimental data suggest general anesthetics preferring γ-aminobutyric acid receptor type A may increase postoperative pain in patients with persistent inflammation. The current study was designed to begin to test this hypothesis. Methods Groups of rats were defined by the presence of inflammation, surgical intervention, and/or the type of general anesthetic used for a 3-h period of anesthesia. Persistent inflammation was induced with complete Freund adjuvant. The surgical intervention was a plantar incision. Three mechanistically distinct general anesthetics were used: pentobarbital, ketamine/xylazine, and isoflurane. Ongoing pain and hypersensitivity were assessed with guarding behavior analysis and the von Frey test, respectively. Results There was no influence of general anesthetic type on the magnitude or time course of recovery from postoperative hypersensitivity in the absence of persistent inflammation. However, in the presence of persistent inflammation, recovery from hypersensitivity was significantly slower in the pentobarbital group than in the ketamine/xylazine or isoflurane groups. The pentobarbital effect was significant within 3 days of surgery and persisted through the remainder of the testing period. A comparable delay in recovery was observed in pentobarbital-anesthetized inflamed rats not subjected to hind paw incision. The time to 50% recovery in the pentobarbital-treated inflamed groups was almost double that in the other groups. No differences were observed between ketamine/xylazine and isoflurane. Pentobarbital exposure did not increase guarding scores. Conclusions These results suggest that general anesthetics preferring γ-aminobutyric acid receptor type A may have deleterious consequences when used in the presence of persistent inflammation.

Approximately 20% of the adult population suffers from migraine. This debilitating pain disorder is three times more prevalent in women than in men. To begin to evaluate the underlying mechanisms that may contribute to this sex difference, we tested the hypothesis that there is a sex difference in the inflammatory mediator (IM)-induced sensitization of dural afferents. Acutely dissociated retrogradely labeled dural afferents from adult Sprague-Dawley rats were examined with whole cell patch-clamp recordings. Baseline passive and active electrophysiological properties of dural afferents from both sexes were comparable. However, while IM-induced increases in the excitability of dural afferents from male and female rats were also comparable, the proportion of dural afferents from female rats sensitized by IM (∼100%) was significantly greater than that of dural afferents from male rats (∼50%). This appeared to be due to differences downstream of IM receptors, as tetrodotoxin-resistant sodium current was increased by IM in a majority of male dural afferents (13/14). These data indicate that there are both quantitative and qualitative differences in the IM-induced sensitization of dural afferents that may contribute to the sex difference in the manifestation of migraine.

Persistent pain remains a tremendous health problem due to both its prevalence and dearth of effective therapeutic interventions. To maximize pain relief while minimizing side effects, current gene therapy-based approaches have mostly exploited the expression of pain inhibitory products or interfered with pronociceptive ion channels. These methods do not enable control over the timing or duration of analgesia, nor titration to analgesic efficacy. Here, we describe a gene therapy strategy that potentially overcomes these limitations by providing exquisite control over therapy with efficacy in clinically relevant models of inflammatory pain. We utilize a herpes simplex viral (HSV) vector (vHGlyRα1) to express a ligand-regulated chloride ion channel, the glycine receptor (GlyR) in targeted sensory afferents; the subsequent exogenous addition of glycine provides the means for temporal and spatial control of afferent activity, and therefore pain. Use of an endogenous inhibitory receptor not normally present on sensory neurons both minimizes immunogenicity and maximizes therapeutic selectivity.

Chronic pain is a debilitating clinical condition associated with a variety of disease entities including diabetic neuropathy, postherpetic neuralgia, low back pathology, fibromyalgia, and neurological disorders. For many general practitioners and specialists, managing chronic pain has become a daunting challenge. As a modality of multidisciplinary chronic pain management, medications are often prescribed in combinations, an approach referred to as combination drug therapy (CDT). However, many medications for pain therapy, including antidepressants and opioid analgesics, have significant side effects that can compound when used in combination and impact the effectiveness of CDT. To date, clinical practice of CDT for chronic pain has been based largely on clinical experiences. In this article, we will focus on (1) the scientific basis and rationales for CDT, (2) current clinical data on CDT, and (3) the need for more clinical studies to establish a framework for the use of CDT. Perspective More preclinical, clinical, and translational studies are needed to improve the efficacy of combination drug therapy that is an integral part of a comprehensive approach to the management of chronic pain.

The impact of persistent inflammation on voltage-activated Ca(2+) 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 Ca(2+) 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 (Ca(V)2.2) mRNA. An increase in both α2δ1 and Ca(V)2.2 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 Cd(2+) (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 Ca(2+) 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.

The incidence of chronic pain is estimated to be 20–25% worldwide. Few patients with chronic pain obtain complete relief from the drugs that are currently available, and more than half report inadequate relief. Underlying the challenge of developing better drugs to manage chronic pain is incomplete understanding of the heterogeneity of mechanisms that contribute to the transition from acute tissue insult to chronic pain and to pain conditions for which the underlying pathology is not apparent. An intact central nervous system (CNS) is required for the conscious perception of pain, and changes in the CNS are clearly evident in chronic pain states. However, the blockage of nociceptive input into the CNS can effectively relieve or markedly attenuate discomfort and pain, revealing the importance of ongoing peripheral input to the maintenance of chronic pain. Accordingly, we focus here on nociceptors: their excitability, their heterogeneity and their role in initiating and maintaining pain.

Primary afferents are known to be inhibited by kappa opioid receptor (KOR) signaling. However, the specific types of somatosensory neurons that express KOR remain unclear. Here, using a newly developed KOR-cre knockin allele, viral tracing, single-cell RT-PCR, and ex vivo recordings, we show that KOR is expressed in several populations of primary afferents: a subset of peptidergic sensory neurons, as well as low-threshold mechanoreceptors that form lanceolate or circumferential endings around hair follicles. We find that KOR acts centrally to inhibit excitatory neurotransmission from KOR-cre afferents in laminae I and III, and this effect is likely due to KOR-mediated inhibition of Ca2+ influx, which we observed in sensory neurons from both mouse and human. In the periphery, KOR signaling inhibits neurogenic inflammation and nociceptor sensitization by inflammatory mediators. Finally, peripherally restricted KOR agonists selectively reduce pain and itch behaviors, as well as mechanical hypersensitivity associated with a surgical incision. These experiments provide a rationale for the use of peripherally restricted KOR agonists for therapeutic treatment.

Migraineurs experience debilitating headaches that result from neurogenic inflammation of the dura and subsequent sensitization of dural afferents. Given the importance of inflammatory mediator (IM)-induced dural afferent sensitization to this pain syndrome, the present study was designed to identify ionic mechanisms underlying this process. Trigeminal ganglion neurons from adult female Sprague Dawley rats were acutely dissociated 10-14 d after application of retrograde tracer DiI onto the dura. Modulation of ion channels and changes in excitability were measured in the absence and presence of IMs (in mum: 1 prostaglandin, 10 bradykinin, and 1 histamine) using whole-cell and perforated-patch recordings. Fura-2 was used to assess changes in intracellular Ca(2+). IMs modulated a number of currents in dural afferents, including those both expected and/or previously described [i.e., an increase in tetrodotoxin-resistant voltage-gated Na(+) current (TTX-R I(Na)) and a decrease in voltage-gated Ca(2+) current] as well currents never before described in sensory neurons (i.e., a decrease in a Ca(2+)-dependent K(+) current and an increase in a Cl(-) current), and produced a sustained elevation in intracellular Ca(2+). Although several of these currents, in particular TTX-R I(Na), appear to contribute to the sensitization of dural afferents, the Cl(-) current is the primary mechanism underlying this process. Activation of this current plays a dominant role in the sensitization of dural afferents because of the combination of the density and biophysical properties of TTX-R I(Na), and the high level of intracellular Cl(-) in these neurons. These results suggest novel targets for the development of antimigraine agents.

According to the positive illusions model (Murray, Holmes, & Griffin, 1996a), people in romantic relationships are more satisfied when they view their partners more favorably than the partners see themselves. By contrast, shared reality theory (Hardin & Conley, 2001) emphasizes the benefits of perceiving a partner as the partner sees himself or herself. We analyzed archived data from the American Couples Study (Blumstein & Schwartz, 1983) to test the applicability of the positive illusions model to gay and lesbian relationships. Structural equation models demonstrated that the positive illusions model effectively explains relationships among lesbian, gay, heterosexual cohabitating, and married couples.

The dihydropyridines nifedipine, nimodipine and Bay K 8644 are widely used as pharmacological tools to assess the contribution of L-type voltage-gated Ca(2+) channels to a variety of neuronal processes including synaptic transmission, excitability and second messenger signaling. These compounds are still used in neuronal preparations despite evidence from cardiac tissue and heterologous expression systems that they block several voltage-dependent K(+) (Kv) channels. Both because these compounds have been used to assess the relative contribution of L-type Ca(2+) channels to several different processes in dorsal root ganglion (DRG) neurons and because a relatively wide variety of Kv channels present in other neuronal populations is present in DRG neurons, we determined the extent to which dihydropyridines block Kv currents in these neurons. Standard whole cell patch clamp techniques were used to study acutely disassociated adult rat DRG neurons. All three dihydropyridines tested blocked Kv currents in DRG neurons; IC(50) values (concentration resulting in an inhibition that is 50% of maximum) for nifedipine and nimodipine-induced block of sustained Kv currents were 14.5 and 6.6 microM, respectively. The magnitude of sustained current block was 44+/-1.6%, 60+/-2%, and 56+/-2.9% with 10 microM nifedipine, nimodipine and Bay K 8644, respectively. Current block was occluded by neither 4-aminopyridine (5 mM) nor tetraethylammonium (135 mM). Dihydropyridine-induced block of Kv currents was not associated with a shift in the voltage-dependence of current activation or inactivation, the recovery from inactivation, or voltage dependent block. However, there was a small use-dependence to the dihydropyridine-induced block. Our results suggest that several types of Kv channels in DRG neurons are blocked by mechanisms distinct from those underlying block of Kv channels in cardiac myocytes. Importantly, our results suggest that if investigators wish to explore the contribution of L-type Ca(2+) channels to neuronal function, they should consider alternative strategies for the manipulation of these channels than the use of dihydropyridines.

Chronic pain is a serious medical condition with millions of sufferers for whom long-term therapies are either lacking or inadequate. Here we review the use of herpes simplex virus vectors as therapeutic tools to treat chronic pain by gene therapy. We describe an approach to inhibit chronic pain signaling whereby vector-mediated genes transferred to sensory nerves will modify the primary afferent nociceptor to prevent pain signaling to second-order nerves in the spinal cord. This approach may be used to reverse the chronic pain state of the nociceptor and could affect downstream pain-related changes in the central nervous system.

Melzack and Wall's Gate Control Theory of Pain laid the theoretical groundwork for a role of spinal inhibition in endogenous pain control. While the Gate Control Theory was based on the notion that spinal inhibition is dynamically regulated, mechanisms underlying the regulation of inhibition have turned out to be far more complex than Melzack and Wall could have ever imagined. Recent evidence indicates that an exquisitely sensitive form of regulation involves changes in anion equilibrium potential (E(anion)), which subsequently impacts fast synaptic inhibition mediated by GABA(A), and to a lesser extent, glycine receptor activation, the prototypic ligand gated anion channels. The cation-chloride co-transporters (in particular NKCC1 and KCC2) have emerged as proteins that play a critical role in the dynamic regulation of E(anion) which in turn appears to play a critical role in hyperalgesia and allodynia following peripheral inflammation or nerve injury. This review summarizes the current state of knowledge in this area with particular attention to how such findings relate to endogenous mechanisms of hyperalgesia and allodynia and potential applications for therapeutics based on modulation of intracellular Cl(-) gradients or pharmacological interventions targeting GABA(A) receptors.

Aims The presence of muscarinic receptors on bladder urothelial cells (BUC), suggests BUC may be a target for antimuscarinics. This study determined whether human BUC are responsive to a muscarinic agonist and if so, whether responses are altered in interstitial cystitis (IC) BUC. Methods Primary urothelial cell cultures were established from cystoscopic biopsies. Normal (NB) and IC BUC were studied using calcium imaging techniques as a means to monitor the response to muscarinic receptor activation with the agonist, carbachol (CCh). Changes in intracellular Ca2+ concentration ([Ca2+]i) were measured with fura-2 ratiometric microfluorimetry. Dose–response curves (CCh dose vs. [Ca2+]i) were measured in IC and NB BUC. Tolterodine was used to confirm the specificity (muscarinic versus nicotinic) of CCh evoked increases in [Ca2+]i. Results CCh induced a dose-dependent increase in [Ca2+]i. Potency and efficacy of CCh was significantly greater in IC BUC. The maximal increase in [Ca2+]i was 136.3 ± 5.1% over baseline in 78 cells from 4 IC patients versus 92.4 ± 4.8% over baseline in 67 cells from 4 NB subjects (P

The objective of this study was to assess the impact of persistent inflammation on spinal γ-aminobutyric acid-A (GABA-A) receptor–mediated modulation of evoked nociceptive behavior in the adult rat. Nocifensive threshold was assessed with von Frey filaments applied to the dorsal surface of the hind paw. The GABA-A receptor agonist muscimol, the antagonist gabazine, the benzodiazepine receptor agonist midazolam, and antagonists PK11195 and flumazenil were administered spinally in the presence and absence of complete Freund's adjuvant (CFA)-induced inflammation. In naive rats, muscimol increased and gabazine decreased nociceptive threshold. After CFA, the effects of these compounds were reversed: Low doses of muscimol exacerbated the inflammation-induced decrease in nociceptive threshold and gabazine increased nociceptive threshold. Midazolam increased nociceptive threshold both in the presence and absence of inflammation. Flumazenil but not PK11195 blocked the analgesic effects of midazolam. These findings indicate that inflammation-induced changes in GABA-A signaling are complex and are likely to involve several distinct mechanisms. Rectifying the changes in GABA-A signaling may provide effective relief from hypersensitivity observed in the presence of inflammation. Perspective An inflammation-induced shift in spinal GABA-A receptor signaling from inhibition to excitation appears to underlie inflammatory pain and hypersensitivity. Use of GABA-A receptor selective general anesthetics in association with therapeutic interventions may be contraindicated. More importantly, rectifying the changes in GABA-A signaling may provide effective relief from inflammatory hypersensitivity.

The concentration of intracellular Ca 2+ ([Ca 2+ ] i ) influences neuronal properties ranging from excitability to neurotransmitter release. Persistent inflammation is associated with changes in the properties of primary afferent neurons ranging from excitability to transmitter release. The purpose of the present study was to determine whether previously described inflammation-induced changes in excitability and transmitter release are associated with changes in the regulation of [Ca 2+ ] i . Acutely dissociated dorsal root ganglion (DRG) neurons harvested from adult rats 3 days following a hind-paw injection of complete Freund's adjuvant (CFA) or naive controls, were stimulated with 30 mM K + (High K + ). High K + evoked changes in [Ca 2+ ] i were assessed with fura-2 ratiometric microfluorimetry. Subpopulations of DRG neurons were defined by cell body diameter, isolectin B4 (IB4) binding, capsaicin (CAP) sensitivity and target of innervation (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbo-cyanine perchlorate labeling). Inflammation was associated with significant increases in resting [Ca 2+ ] i and increases in the magnitude and decreases in the decay, of the evoked increase in [Ca 2+ ] i . The changes in evoked transients were larger in neurons innervating the site of inflammation. Furthermore, there were differences among subpopulations of DRG neurons with respect to changes in magnitude and/or decay of the evoked transient such that the increase in magnitude was larger in small- and medium-diameter neurons than in large diameter neurons while the decrease in the decay was greater in CAP responsive, IB4 positive, small- and medium-diameter neurons than in CAP unresponsive, IB4 negative and/or large-diameter neurons. These changes in the regulation of [Ca 2+ ] i were not due to inflammation-induced changes in passive or active electrophysiological properties. Importantly, an inflammation-induced increase in evoked Ca 2+ transients in putative nociceptive afferents may contribute to the pain and hyperalgesia associated with persistent inflammation via facilitation of transmitter release from these afferents.

Because the pulp tissue extirpated during root canal procedures might serve as a valuable resource with which to assess underlying mechanisms of persistent pain, we sought to determine whether standard Western blotting techniques could be used to quantify neural proteins in pulp extirpated from teeth with irreversible pulpitis. Pulp harvested from healthy intact teeth extracted for orthodontic reasons was used for comparison. The neural marker PGP9.5 was detectable in all samples tested. A membrane enrichment protocol enabled detection of even low abundance, high molecular weight proteins such as the sodium channel alpha-subunit NaV1.8. Importantly, it was possible to quantify a approximately 6-fold increase in the relative density of NaV1.8 in inflamed pulp compared with control pulp. Our results suggest that it should be possible to use extirpated tooth pulp to validate mechanisms of persistent pain implicated in preclinical studies as well as evaluate the therapeutic efficacy of novel antinociceptive interventions.

In September 2006, members of the Sex, Gender and Pain Special Interest Group of the International Association for the Study of Pain met to discuss the following: (1) what is known about sex and gender differences in pain and analgesia; (2) what are the "best practice" guidelines for pain research with respect to sex and gender; and (3) what are the crucial questions to address in the near future? The resulting consensus presented herein includes input from basic science, clinical and psychosocial pain researchers, as well as from recognized experts in sexual differentiation and reproductive endocrinology. We intend this document to serve as a utilitarian and thought-provoking guide for future research on sex and gender differences in pain and analgesia, both for those currently working in this field as well as those still wondering, "Do I really need to study females?"

Aims: To determine whether behavioral, anatomical, and physiologic endpoints widely used to infer the presence of pain in rodent models of temporomandibular disorders (TMD) were applicable to the rabbit model of TMD associated with altered joint loading. Methods: Unilateral molar dental splints were used to alter temporomandibular joint (TMJ) loading. Changes in nociceptive threshold were assessed with a mechanical probing of the TMJ region on nine splinted and three control rabbits. Fos-like immunoreacitivty in the trigeminal subnucleus caudalis was assessed with standard immunohistochemical techniques from three splinted and six control animals. Retrogradely labeled TMJ afferents were studied with patch-clamp electrophysiologic techniques from three splinted and three control animals. Remodeling of TMJ condyles was assessed by histologic investigations of three splinted and three control animals. A Student t test or a Mann-Whitney U test was used with significance set at P < .05 to compare splinted to control samples. Results: While variable, there was an increase in mechanical sensitivity in splinted rabbits relative to controls. The increase in Fos+ cells in splinted rabbits was also significant relative to naive controls (86 ± 8 vs 64 ± 15 cells/section, P < .05). The rheobase (364 ± 80 pA) and action potential threshold (–31.2 ± 2.0 mV) were higher in TMJ afferents from splinted rabbits compared to controls (99 ± 22 pA and –38.0 ± 2.0 mV, P < .05). There was significant remodeling in the condylar fibrocartilage layers as manifested by a change in glycosaminoglycan distribution and a loss of defined cell layers. Conclusion: Behavioral and anatomical results were consistent with an increase in nociceptive signaling in concert with condylar remodeling driven by altered TMJ loading. Changes in excitability and action potential waveform were consistent with possible compensatory changes of TMJ afferents for an overall increase in afferent drive associated with joint degeneration. These compensatory changes may reflect pain-adaption processes that many patients with TMJ disorders experience.