- Open Access
The chemerin receptor 23 agonist, chemerin, attenuates monosynaptic C-fibre input to lamina I neurokinin 1 receptor expressing rat spinal cord neurons in inflammatory pain
© Dickie and Torsney; licensee BioMed Central Ltd. 2014
Received: 22 November 2013
Accepted: 1 April 2014
Published: 9 April 2014
Recent evidence has shown that the chemerin receptor 23 (ChemR23) represents a novel inflammatory pain target, whereby the ChemR23 agonists, resolvin E1 and chemerin, can inhibit inflammatory pain hypersensitivity, by a mechanism that involves normalisation of potentiated spinal cord responses. This study has examined the ability of the ChemR23 agonist, chemerin, to modulate synaptic input to lamina I neurokinin 1 receptor expressing (NK1R+) dorsal horn neurons, which are known to be crucial for the manifestation of inflammatory pain.
Whole-cell patch-clamp recordings from pre-identified lamina I NK1R+ neurons, in rat spinal cord slices, revealed that chemerin significantly attenuates capsaicin potentiation of miniature excitatory postsynaptic current (mEPSC) frequency, but is without effect in non-potentiated conditions. In tissue isolated from complete Freund’s adjuvant (CFA) treated rats, chemerin significantly reduced the peak amplitude of monosynaptic C-fibre evoked excitatory postsynaptic currents (eEPSCs) in a subset of lamina I NK1R+ neurons, termed chemerin responders. However, chemerin did not alter the peak amplitude of monosynaptic C-fibre eEPSCs in control tissue. Furthermore, paired-pulse recordings in CFA tissue demonstrated that chemerin significantly reduced paired-pulse depression in the subset of neurons classified as chemerin responders, but was without effect in non-responders, indicating that chemerin acts presynaptically to attenuate monosynaptic C-fibre input to a subset of lamina I NK1R+ neurons.
These results suggest that the reported ability of ChemR23 agonists to attenuate inflammatory pain hypersensitivity may in part be due to a presynaptic inhibition of monosynaptic C-fibre input to lamina I NK1R+ neurons and provides further evidence that ChemR23 represents a promising inflammatory pain target.
Inflammatory pain is a common clinical problem, however many currently used treatments, such as opioids or cyclooxygenase (COX) inhibitors, lack efficacy and/or exhibit undesirable side effects [1, 2]. Therefore, the development of new analgesics that are both efficacious and lack side effects is a key challenge for pain research.
Lamina I neurokinin 1 receptor expressing (NK1R+) neurons, a significant proportion of which are projection neurons [3–6], are known to be crucial for the manifestation of inflammatory pain hypersensitivity. Selective ablation of these neurons by spinal administration of a substance P–saporin conjugate significantly attenuates hyperalgesia and allodynia in inflammatory pain models . C-fibre nociceptors also play an essential role in the development of inflammatory pain. Transgenic mice in which Na v 1.8 expressing nociceptors, which includes the majority of C-fibres, have been selectively eliminated, fail to develop inflammatory pain hypersensitivity . Lamina I NK1R+ neurons predominantly receive monosynaptic C-fibre input [9–11] and some of this input may be potentiated in inflammatory pain . Therefore, exploring potential strategies to modulate C-fibre input to these neurons could offer insights into novel inflammatory pain treatments.
Recent evidence has suggested that the chemerin receptor 23 (ChemR23), a G α i associated G-protein coupled receptor (GPCR), may be a promising target for the development of novel inflammatory pain treatments. The lipid mediator, resolvin E1 (RvE1), which acts via ChemR23 , has been shown to reduce thermal and mechanical hypersensitivity in the complete Freund’s adjuvant (CFA) and carrageenan inflammatory pain models . Furthermore, RvE1 attenuates the second phase of the formalin response, in a manner that is comparable to morphine and NS-398, a commonly used COX-2 inhibitor, but at a substantially reduced dose . Chemerin, a natural ChemR23 ligand [15, 16], has also been shown to inhibit the second phase of the formalin test . Interestingly, basal mechanical and thermal thresholds and the first phase of the formalin test are unaltered by RvE1 administration, suggesting that strategies which target ChemR23 may promisingly attenuate maladaptive/chronic pain without altering acute protective pain.
ChemR23 agonists have been found to have both peripheral and central actions. In the periphery, RvE1 reduces carrageenan-induced oedema, neutrophil infiltration and proinflammatory cytokine and chemokine expression . Electrophysiological study of central actions, in spinal cord slices, have established that RvE1 and chemerin can ‘normalise’ potentiated spinal cord responses, without altering basal synaptic transmission in the dorsal horn. Specifically, application of chemerin or RvE1 abolishes capsaicin potentiation of spontaneous excitatory postsynaptic current (sEPSC) frequency, while RvE1 has also been shown to inhibit TNF- α mediated potentiation of sEPSC frequency and NMDA currents, in unidentified lamina II neurons . However, as these findings were obtained using sEPSC recordings from unidentified neurons, it is not known where in the spinal cord network or upon which neuronal subtype these central effects are mediated.
Anatomical studies in mice demonstrate that ChemR23 is expressed both peripherally, in dorsal root ganglia (DRG) and centrally, on the central terminals of primary afferent fibres and also on spinal cord neurons . Microarray studies, performed using rat tissue, also report ChemR23 mRNA expression in DRG neurons and in the dorsal horn, with expression levels being unaltered in CFA inflammation . In mice, almost one third of DRG neurons express ChemR23 and there is a large degree of overlap between ChemR23 and transient receptor potential subtype vanilloid 1 (TRPV1) channel expression, with ∼45% of ChemR23 expressing neurons also expressing TRPV1 and ∼60% of TRPV1 expressing (TRPV1+) neurons also expressing ChemR23 . In lamina I of the dorsal horn, ChemR23 is expressed on the central terminals of substance P containing (substance P+) afferents . Lamina I NK1R+ neurons are known to be targeted by both TRPV1+ [18–20] and substance P+ afferents [18, 21], with many C-fibres, which are the predominant type of input received by these neurons [9–11], expressing TRPV1 and/or substance P [22–28]. Therefore, given the co-expression of ChemR23 with TRPV1 and substance P, it is likely that some of the monosynaptic C-fibre input to lamina I NK1R+ neurons will also express ChemR23. As such, it is possible that the ability of ChemR23 agonists to attenuate inflammatory pain hypersensitivity could in part be due to an inhibition of C-fibre input to these key spinal cord output neurons.
In this study we aimed to determine whether the ChemR23 agonist, chemerin, could attenuate capsaicin potentiation of miniature excitatory postsynaptic current (mEPSC) frequency in lamina I NK1R+ neurons. Furthermore, we have investigated whether chemerin can modulate monosynaptic C-fibre input to these neurons during inflammatory pain. While much of the interest in targeting ChemR23 has revolved around the use of RvE1, it should be noted that RvE1 is not commercially available, therefore it was not possible to investigate the effect of this compound upon the synaptic input to lamina I NK1R+ neurons.
Chemerin attenuates capsaicin potentiation of mEPSC frequency in lamina I NK1R+ neurons, but is without effect in non-potentiated conditions
Characterisation of monosynaptic C-fibre input to lamina I NK1R+ neurons
Chemerin attenuates monosynaptic C-fibre input to a subset of lamina I NK1R+ neurons in inflammatory pain
Chemerin presynaptically inhibits monosynaptic C-fibre input to a subset of lamina I NK1R+ neurons in inflammatory pain
It is known that ChemR23 agonists can alleviate hypersensitivity in animal models of inflammatory pain through both peripheral and central mechanisms . These include a reduction in peripheral inflammation and a normalisation of potentiated spinal cord responses, respectively. In terms of central mechanisms, electrophysiological recordings from unidentified lamina II dorsal horn neurons have demonstrated that the ChemR23 agonists, RvE1 and chemerin, attenuate capsaicin potentiation of sEPSC frequency. However, as these findings were obtained using sEPSC recordings in unidentified neurons it is not known where in the spinal cord network or upon which neuronal subtypes that these effects are mediated. In this study we have investigated the ability of chemerin to modulate excitatory input to lamina I NK1R+ neurons following capsaicin potentiation and CFA inflammation. Our results have novelly revealed that chemerin can attenuate capsaicin potentiation of mEPSC frequency in lamina I NK1R+ neurons and presynaptically reduce monosynaptic C-fibre input to a subset of these neurons in inflammatory pain. Notably, chemerin was without effect in non-potentiated/control conditions. Given the essential role of lamina I NK1R+ neurons in the manifestation of inflammatory pain , which is driven by C-fibres , the chemerin attenuation of monosynaptic C-fibre input to these neurons suggests that the reported ability of ChemR23 agonists to attenuate inflammatory pain hypersensitivity may in part be due to presynaptic inhibition of monosynaptic C-fibre input to these key spinal cord output neurons.
We have demonstrated that chemerin can significantly reduce, but not eliminate, capsaicin potentiation of mEPSC frequency in rat lamina I NK1R+ neurons. However, Xu et al.  report that chemerin, at the same dose used here, can completely prevent capsaicin potentiation of sEPSC frequency in unidentified mouse lamina II neurons. This difference likely reflects the different concentrations of capsaicin employed, 1 μ M in the present study, which resulted in a ∼44-fold increase in frequency, vs. 100 nM used by Xu et al. , which increased frequency by only 2-fold. We did investigate use of 100 nM capsaicin, but found that this concentration did not reliably potentiate mEPSC frequency in lamina I NK1R+ neurons (data not shown). This dissimilarity may also reflect the different species employed, rat vs. mouse, different cell types targeted, lamina I NK1R+ neurons vs. unidentified lamina II neurons or the different recording approach, mEPSC vs. sEPSC recording, employed. Other groups have demonstrated that 100 nM capsaicin can potentiate mEPSC frequency in unidentified lamina I/II neurons [31, 32], however potentiation of mEPSCs in lamina I NK1R+ neurons has only been reported using 1 μ M capsaicin . Interestingly, Labrakakis and MacDermott  report that 73% of lamina I NK1R+ neurons display an increase in mEPSC frequency in response to 1 μ M capsaicin, which is comparable to our findings, that 83% of neurons had capsaicin-sensitive input.
On the basis of anatomical expression data  we hypothesised that ChemR23 would be expressed on a subset of monosynaptic C-fibre inputs to lamina I NK1R+ neurons. Indeed we provide supporting functional evidence for this expression pattern through our novel demonstration that chemerin presynaptically attenuates monosynaptic C-fibre input to a subset of these neurons in inflammatory pain. To unequivocally demonstrate that the chemerin effects reported here are mediated via ChemR23, we would ideally have shown blockade by a ChemR23 antagonist, but this was not possible as no such ligand is commercially available. Pertussis toxin (PTX), an inhibitor of G α i coupled GPCRs, the receptor family to which ChemR23 belongs, has been used by others to inhibit the RvE1 attenuation of capsaicin potentiated input in unidentified lamina II neurons . While this approach could have been used to provide additional confirmation that the actions of chemerin were mediated by ChemR23, PTX inhibition of the chemerin response would only indicate that chemerin acted via a G α i coupled GPCR and not ChemR23 specifically.
ChemR23 agonists are proposed to reduce inflammatory pain in part by normalising potentiated spinal cord responses [14, 33]. In the present data, chemerin reduced monosynaptic C-fibre input to a subset (∼44%) of lamina I NK1R+ neurons in CFA tissue but was without effect in control tissue. Interestingly, electrical stimulation of monosynaptic C-fibre input to lamina I projection neurons, that are likely to be NK1R+ neurons, in a manner which mimics the spontaneous firing pattern seen during inflammatory pain, results in the potentiation of C-fibre input to only a subset of these neurons . It could therefore be hypothesised that neurons classified as chemerin responders were the subset that received potentiated C-fibre input. We therefore investigated whether there was a correlation between the initial peak amplitude of monosynaptic C-fibre eEPSCs (with greater amplitudes possibly signifying potentiated inputs) and the magnitude of the chemerin mediated change in peak amplitude. Our results found there to be no correlation between the initial eEPSC peak amplitude and the amplitude change (data not shown), however it should be recognised that this finding cannot confirm or refute the possibility that neurons classified as chemerin responders were those that received potentiated input, as it is not possible to directly distinguish potentiated inputs in this kind of population comparison study. Moreover, when the peak amplitude of monosynaptic C-fibre eEPSCs are compared between control and CFA tissue there is no significant potentiation observed overall (data not shown) as previously reported .
It has previously been established that ChemR23 agonists do not alter acute pain sensitivity and have no effect upon sEPSC frequency, in unidentified lamina II neurons, in non-potentiated conditions . In accordance, we have shown that chemerin does not alter basal mEPSC frequency or amplitude in lamina I NK1R+ neurons or the peak amplitude of monosynaptic C-fibre eEPCSs in these neurons in control tissue. It is proposed that ChemR23 activation reduces inflammatory pain hypersensitivity by normalising potentiated spinal cord responses, as opposed to a general reduction in sensory transmission [14, 33], for which our findings provide additional support.
ChemR23 activation is proposed to normalise spinal cord inflammatory pain potentiation by inhibition of the extracellular signal-regulated kinase (ERK) pathway, which is key for central sensitisation , both presynaptically in the central terminals of primary afferents and postsynaptically in dorsal horn neurons . Inhibition of ERK, with the mitogen-activated protein kinase kinase inhibitors PD98059 and U0126, prevents capsaicin potentiation of sEPSCs, while application of RvE1 reduces capsaicin/TNF- α driven ERK phosphorylation in DRG cultures . Therefore, the chemerin attenuation of capsaicin potentiated mEPSC frequency and chemerin mediated presynaptic inhibition of monosynaptic C-fibre inputs in inflammatory pain, reported here, may result from blockade of ERK mediated glutamate release from central terminals. Chemerin may also mediate its effects via inhibition of TRPV1, which is crucial for inflammatory pain [35, 36], as RvE1 is a highly potent inhibitor of TRPV1, with different resolvins interestingly differentially modulating different TRP channels via GPCR activation [37, 38].
Chemerin is a natural ChemR23 ligand [15, 16] and while it is currently unclear which cell types are responsible for its endogenous production and release, endothelial cells, keratinocytes, chondrocytes, platelets and osteoclasts have all been proposed as possible sources [39–44]. Chemerin plays a key role in a number of physiological processes including adipocyte generation and metabolism  and the chemotaxis of macrophages and dendritic cells . However, it is not currently known whether endogenous chemerin plays any role in the modulation of inflammatory pain.
It is worth noting that chemerin is known to display high affinity binding with receptors other than ChemR23, namely the chemokine (C-C motif) receptor-like 2 (CCRL2) and G protein-coupled receptor 1 (GPR1), however these receptors are thought to play a limited role in cell signalling. Current evidence suggests that CCRL2 is not involved in cell signalling but may play a functional role in presenting chemerin to ChemR23 [40, 46, 47]. Interestingly, CCRL2 is known to be expressed in the spinal cord and wider CNS, predominantly in microglia . Binding of chemerin to GPR1 results in receptor internalisation and signalling, but this signalling is weak  and it has been speculated that GPR1 may act as a decoy receptor . There is evidence that GPR1 is expressed in the CNS [50–53], however spinal cord expression has not been investigated. It is possible, therefore, that inflammation-induced changes in the expression of either of these receptors could have influenced the results presented here.
While the evidence presented here supports the work by Xu et al.  which revealed that drugs which target ChemR23 may be effective in the treatment of inflammatory pain, it is worth noting that chemerin or RvE1 may have limited therapeutic potential given that they are not metabolically stable and are rapidly inactivated in vivo [33, 54, 55]. Interestingly, a stable analogue of RvE1, 19-(p-fluorophenoxy)-RvE1, has been shown to effectively reduce thermal hypersensitivity in the CFA inflammatory pain model for an extended time period compared to RvE1 . Stable chemerin analogues have been developed , however their use has not yet been investigated in models of inflammatory pain. Further research into the use of these or new stable analogues of ChemR23 agonists should further establish ChemR23 as a promising target for the treatment of inflammatory pain.
This study demonstrates that the ChemR23 agonist, chemerin, can attenuate capsaicin potentiation of mEPSC frequency in lamina I NK1R+ neurons, but is without effect in non-potentiated conditions. Furthermore, chemerin presynaptically inhibits monosynaptic C-fibre input to a subset of these neurons in inflammatory pain, but does not alter C-fibre eEPSCs in control conditions. These findings suggest that the reported ability of ChemR23 agonists to attenuate inflammatory pain hypersensitivity could in part be due to the inhibition of C-fibre input to these key spinal cord output neurons and provides further evidence that ChemR23 represents a promising target for the development of novel inflammatory pain treatments.
All procedures were approved by the University of Edinburgh Ethical Review Committee and carried out in accordance with the UK Animals (Scientific Procedures) Act 1986. Juvenile Sprague Dawley rats of both sexes (approximately postnatal day 21 [P21]; the University of Edinburgh Biological Research Resources, Edinburgh UK) were used in all experiments. No differences were found between male and female rats so all data presented are a combination of both sexes. Animals were housed in cages at 21°C and 55% relative humidity, with a 12 h light-dark cycle. Food and water were provided ad libitum.
Inflammatory pain model
Complete Freund’s adjuvant (CFA, 0.5 mg/ml saline) was injected into the intraplantar surface of the left hindpaw (1 μ l/g body weight) under isoflurane anaesthesia at ∼P18, 2–6 days prior to electrophysiological recordings at ∼P21. This procedure has been shown to produce persistent peripheral inflammation and behavioural hypersensitivity in juvenile rats .
Spinal cord slice preparation
CFA treated or naïve untreated (control) rats were anaesthetised with isoflurane and decapitated. Spinal cords, in some cases with dorsal roots attached, were removed and were initially placed in ice-cold dissection solution. The lumbar (L4/L5) segment was embedded in an agarose block and cut into transverse slices (350 μ m). Where slices with dorsal roots attached were cut, dorsal root ganglia were removed and only the left dorsal roots were used in CFA treated rats. Slices were incubated at 36–37°C for 1 h in oxygenated recovery solution after which they were incubated at room temperature for 30 mins with 35 nM tetramethylrhodamine conjugated substance P (TMR-SP), as described previously [10, 11, 19]. Slices were allowed to recover for a further 1 h at room temperature prior to recording. Slices were transferred to the recording chamber of an upright microscope (Zeiss), equipped with fluorescence for the identification of TMR-SP labelled (TMR-SP+) neurons and infrared-differential interference contrast (IR-DIC) for electrophysiological recordings and were continually perfused with oxygenated Krebs solution (1–2 ml/min) at room temperature. The composition of the 95% O2/5% CO2 saturated Krebs solution is as follows (in mM): 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 25 glucose, 1 MgCl2, 2 CaCl2, pH7.4. Recovery solution was identical to Krebs but with 6 mM MgCl2 and 1.5 mM CaCl2. Dissection solution was the same as recovery but with the addition of 1 mM kynurenic acid.
Our technique for pre-identifying NK1R+ neurons is expected to have little impact on the synaptic activity we are studying. TMR-SP is one of the least biologically active of fluorescence conjugated substance P analogues and does not inhibit neuronal M-type K+ current at the nanomolar concentration used in our study, although it does elevate calcium in Chinese hamster ovary cells expressing NK1 receptor . Moreover, this TMR-SP labelling approach has been validated in a previous study of spinal NK1R+ neurons . However, we cannot rule out the possibility that this labelling approach may interfere with the response of these neurons to chemerin. Retrograde labelling of these likely projection neurons [12, 58] is an alternative approach that could be employed to address this potential issue.
Whole-cell patch-clamp recordings were made from ‘identified’ neurons in the lamina I region of the dorsal horn. All recordings were made at a holding potential of -70 mV and junction potential was corrected prior to recording. Data were recorded and acquired with an Axopatch 200B amplifier and pClamp 10 software (Molecular Devices). Data were filtered at 5 kHz and digitised at 10 kHz.
mEPSCs were recorded from lamina I NK1R+ neurons in spinal slices from control tissue with dorsal roots removed, in the presence of 0.5 μ M TTX, 10 μ M bicuculline and 1 μ M strychnine. The intracellular solution composition was as follows (in mM); 110 K-methanesulfonate, 10 NaCl, 10 EGTA, 1 CaCl2, 10 HEPES, 5 Mg2+-ATP, 0.5 Na+-GTP, pH adjusted to 7.2 with KOH, osmolarity 290 mOsm. 1 μ M Alexa Fluor 488 hydrazide was also included in the recording pipette to confirm that the TMR-SP+ neuron detected under fluorescence was the same as that targeted for recording under IR-DIC.
Baseline mEPSCs were recorded for 5 mins, following which the TRPV1 agonist, capsaicin (1 μ M), was bath applied for 5 mins to pharmacologically potentiate excitatory input . Potentiation was assessed by comparing mEPSCs recorded at baseline (final 2 mins) and during capsaicin application (final 2 mins). To assess whether chemerin modulates this capsaicin potentiation, chemerin (100 ng/ml) was bath applied for 10 mins prior to and throughout capsaicin application, in a separate group of lamina I NK1R+ neurons. Two separate neuronal populations were used, rather than a within cell comparison, because capsaicin potentiation of mEPSC frequency does not return to baseline, even following a long wash period (data not shown), as is reported elsewhere  and it is known that repeated capsaicin applications results in a progressive reduction in capsaicin response [31, 60]. The effect of chemerin upon mEPSCs in non-potentiated conditions was similarly assessed by applying chemerin alone. In all cases the final 2 mins of baseline/drug application was analysed using Mini Analysis (Synaptosoft), mEPSC events were automatically detected by the software and were then accepted or rejected following further visual examination.
eEPSCs were recorded from lamina I NK1R+ neurons in spinal cord slices, with dorsal roots attached, from control and CFA treated rats. Monosynaptic C-fibre input was identified by stimulation of the dorsal root with a suction electrode, as described previously [10, 11]. The dorsal root was stimulated (3 times) at low frequency (0.05 Hz), using an ISO-Flex stimulus isolator (A.M.P.I Intracel), at 20, 100 and 500 μ A (0.1 ms stimulus duration) to activate A β-, A δ- and C-fibre inputs, respectively. C-fibre eEPSCs were identified as longer latency components evident at 500 μ A (but not 20 or 100 μ A) stimulation intensity. C-fibre eEPSCs were considered monosynaptic if when stimulated (20 times) at the higher frequency of 1 Hz they displayed no synaptic failures, regardless of whether there was latency variability . A 500 μ A stimulation intensity was employed for C-fibres, as compound action potential recordings demonstrated that the slow C-fibre component appeared to be maximally recruited at this intensity (Figure 3C). It is possible that higher stimulation intensities could recruit additional C-fibres, but this seems unlikely given that this 500 μ A intensity reveals monosynaptic C-fibre input to a similar proportion of these neurons  as that observed in experiments employing stimulation intensities greater than 800 μ A . The intracellular solution was composed of the following (in mM): 120 Cs-methylsulfonate, 10 Na-methylsulfonate, 10 EGTA, 1 CaCl2, 10 HEPES, 5 QX-314-Cl [2(triethylamino)-N-(2,6-dimethylphenyl) acetamine chloride] and 2 Mg2+-ATP, pH adjusted to 7.2 with CsOH, osmolarity 290 mOsm. Additionally, 1 μ M Alexa Fluor 488 hydrazide was included in the recording pipette.
To assess the ability of chemerin to modulate monosynaptic C-fibre input to lamina I NK1R+ neurons, C-fibre EPSCs were evoked at 500 μ A (0.05 Hz, 0.1 ms stimulus duration) for 10 mins (‘baseline’), followed by a further 15 mins in the presence of either chemerin (100 ng/ml) or vehicle (Krebs only). Peak monosynaptic C-fibre eEPSC amplitude was measured for each sweep and the mean peak amplitude per minute was calculated. All data were normalised to minute 2 because in many neurons there was a large degree of run-down in the eEPSC peak amplitude between minute 1 and minute 2, after which the response generally stabilised (data not shown).
We hypothesised that chemerin would modulate primary afferent input to a subset of lamina I NK1R+ neurons, given that ChemR23 is only expressed on a subset of TRPV1+ and a subset of substance P+ afferents . To identify this subgroup, linear regression analysis was performed on vehicle data to calculate 95% prediction bands. A neuron was classified as a responder if the eEPSC peak amplitude fell below the lower limit of the 95% prediction bands for at least the final 5 mins of chemerin application. To evaluate the validity of this classification method and to assess whether responders and non-responders were two distinct subpopulations, frequency histograms of normalised mean peak amplitude in the final 5 mins of chemerin/vehicle application were plotted. Additionally, histograms of actual mean peak amplitude in the 5 mins prior to and the last 5 mins of chemerin application were plotted for both chemerin responders and non-responders.
To determine the pre/postsynaptic nature of chemerin effects, paired-pulse recordings were conducted in a subset of neurons in CFA tissue. Monosynaptic C-fibre eEPSCs were recorded at baseline and in the presence of chemerin as described above, however rather than a single stimulus being applied every 20 seconds (0.05 Hz) the dorsal root was stimulated twice in close succession (500 ms interstimulus duration) every 20 seconds. PPR, of the monosynaptic C-fibre component, was calculated for the 5 mins prior to (‘baseline’) and the final 5 mins of chemerin application, as PPR = mean eEPSC peak amplitude 2/mean eEPSC peak amplitude 1, so as to correct for misleading facilitation that can be caused by random amplitude fluctuations .
Isolated dorsal root preparation
Spinal cords were removed from control rats, in the manner described above and lumbar (L4/L5) dorsal roots, with dorsal root ganglia removed, were cut near to the dorsal root entry zone and placed in oxygenated recovery solution, at a temperature of 36–37°C, for 1 h and were then maintained at room temperature prior to recording.
Compound action potential recording
Compound action potential recordings were made from isolated dorsal roots using two glass suction electrodes, placed at either end of the dorsal root. To determine the electrical activation threshold of the different primary afferent components, dorsal roots were stimulated 10 times at a frequency of 0.2 Hz (0.1 ms duration) with an ISO-flex stimulus isolator (A.M.P.I. Intracel) at the following intensities (in μ A): 1, 2, 3, 4, 5, 7.5, 10, 15, 20, 25, 30–100 (in 10 μ A steps) and 150–500 μ A (in 50 μ A steps) . The main components of the compound action potentials were differentiated as fast (A β), medium (A δ) and slow (C) conducting components, each with a characteristic triphasic (positive-negative-positive) response (Figure 3A). Threshold stimulus intensities for the components were defined as the lowest stimulation intensity at which the negative component of the triphasic response was first clearly identifiable. The conduction velocity of each component was calculated based on the latency to the negative peak of the response, at 20, 100 and 500 μ A for A β-, A δ- and C-fibres, respectively. The amplitude of the C-fibre component was measured as the distance between the negative and positive peaks, at 500 μ A. To assess latency prolongation in the A δ- and C-fibre components, dorsal roots were stimulated 20 times at 1 and 2 Hz (500 μ A intensity, 0.1 ms duration) and the difference between the latency measured at stimulus 1 and stimulus 20 was calculated. Data were acquired and recorded using a Cygnus ER-1 differential amplifier (Cygnus Technologies Inc.) and pClamp 10 software (Molecular Devices).
All data were assessed for normality using D’Agostino and Pearson omnibus normality tests, to determine whether it was appropriate to use parametric or non-parametric statistical tests and are presented as mean ± SEM. All statistical analysis was performed using Prism 6 (Graphpad Software).
All chemicals were obtained from Sigma except; bicuculline (Tocris), TMR-SP (Enzo Life Sciences), Alexa Fluor 488 hydrazide (Molecular Probes), chemerin (R&D Systems), QX-314-Cl and TTX (Alomone Labs).
This work was supported by a PhD scholarship from the College of Medicine and Veterinary Medicine, The University of Edinburgh, funded by the Medical Research Council.
- Schnitzer TJ: Update on guidelines for the treatment of chronic musculoskeletal pain. Clin Rheumatol 2006, 25 Suppl 1: S22–29.PubMedView ArticleGoogle Scholar
- Scholz J, Woolf CJ: Can we conquer pain? Nat Neurosci 2002, 5 Suppl: 1062–1067.PubMedView ArticleGoogle Scholar
- Al-Khater KM, Kerr R, Todd AJ: A quantitative study of spinothalamic neurons in laminae I, III, and IV in lumbar and cervical segments of the rat spinal cord. J Comp Neurol 2008, 511: 1–18. 10.1002/cne.21811PubMed CentralPubMedView ArticleGoogle Scholar
- Marshall GE, Shehab SA, Spike RC, Todd AJ: Neurokinin-1 receptors on lumbar spinothalamic neurons in the rat. Neuroscience 1996, 72: 255–263. 10.1016/0306-4522(95)00558-7PubMedView ArticleGoogle Scholar
- Spike RC, Puskár Z, Andrew D, Todd AJ: A quantitative and morphological study of projection neurons in lamina I of the rat lumbar spinal cord. Eur J Neurosci 2003,18(9):2433–2448. 10.1046/j.1460-9568.2003.02981.xPubMedView ArticleGoogle Scholar
- Todd AJ, McGill MM, Shehab SA: Neurokinin 1 receptor expression by neurons in laminae I, III and IV of the rat spinal dorsal horn that project to the brainstem. Eur J Neurosci 2000,12(2):689–700. 10.1046/j.1460-9568.2000.00950.xPubMedView ArticleGoogle Scholar
- Nichols ML, Allen BJ, Rogers SD, Ghilardi JR, Honore P, Luger NM, Finke MP, Li J, Lappi DA, Simone DA, Mantyh PW: Transmission of chronic nociception by spinal neurons expressing the substance P receptor. Science 1999,286(5444):1558–1561. 10.1126/science.286.5444.1558PubMedView ArticleGoogle Scholar
- Abrahamsen B, Zhao J, Asante CO, Cendan CM, Marsh S, Martinez-Barbera JP, Nassar MA, Dickenson AH, Wood JN: The cell and molecular basis of mechanical, cold, and inflammatory pain. Science 2008,321(5889):702–705. 10.1126/science.1156916PubMedView ArticleGoogle Scholar
- Ruscheweyh R, Ikeda H, Heinke B, Sandkühler J: Distinctive membrane and discharge properties of rat spinal lamina I projection neurones in vitro. J Physiol 2004,555(2):527–543. 10.1113/jphysiol.2003.054049PubMed CentralPubMedView ArticleGoogle Scholar
- Torsney C, MacDermott AB: Disinhibition opens the gate to pathological pain signaling in superficial neurokinin 1 receptor-expressing neurons in rat spinal cord. J Neurosci 2006,26(6):1833–1843. 10.1523/JNEUROSCI.4584-05.2006PubMedView ArticleGoogle Scholar
- Torsney C: Inflammatory pain unmasks heterosynaptic facilitation in lamina I neurokinin 1 receptor-expressing neurons in rat spinal cord. J Neurosci 2011,31(13):5158–5168. 10.1523/JNEUROSCI.6241-10.2011PubMedView ArticleGoogle Scholar
- Ikeda H, Stark J, Fischer H, Wagner M, Drdla R, Jäger T, Sandkühler J: Synaptic amplifier of inflammatory pain in the spinal dorsal horn. Science 2006,312(5780):1659–1662. 10.1126/science.1127233PubMedView ArticleGoogle Scholar
- Arita M, Bianchini F, Aliberti J, Sher A, Chiang N, Hong S, Yang R, Petasis NA, Serhan CN: Stereochemical assignment, antiinflammatory properties, and receptor for the omega-3 lipid mediator resolvin E1. J Exp Med 2005,201(5):713–722. 10.1084/jem.20042031PubMed CentralPubMedView ArticleGoogle Scholar
- Xu ZZ, Zhang L, Liu T, Park JY, Berta T, Yang R, Serhan CN, Ji RR: Resolvins RvE1 and RvD1 attenuate inflammatory pain via central and peripheral actions. Nat Med 2010,16(5):592–597. 10.1038/nm.2123PubMed CentralPubMedView ArticleGoogle Scholar
- Meder W, Wendland M, Busmann A, Kutzleb C, Spodsberg N, John H, Richter R, Schleuder D, Meyer M, Forssmann WG: Characterization of human circulating TIG2 as a ligand for the orphan receptor ChemR23. FEBS Lett 2003,555(3):495–499. 10.1016/S0014-5793(03)01312-7PubMedView ArticleGoogle Scholar
- Wittamer V, Franssen JD, Vulcano M, Mirjolet JF, Le Poul E, Migeotte I, Brezillon S, Tyldesley R, Blanpain C, Detheux M, Mantovani A, Sozzani S, Vassart G, Parmentier M, Communi D: Specific recruitment of antigen-presenting cells by chemerin, a novel processed ligand from human inflammatory fluids. J Exp Med 2003,198(7):977–985. 10.1084/jem.20030382PubMed CentralPubMedView ArticleGoogle Scholar
- Rodriguez Parkitna J, Korostynski M, Kaminska-Chowaniec D, Obara I, Mika J, Przewlocka B, Przewlocki R: Comparison of gene expression profiles in neuropathic and inflammatory pain. J Physiol Pharmacol 2006,57(3):401–414.PubMedGoogle Scholar
- Hwang SJ, Burette A, Valtschanoff JG: VR1-positive primary afferents contact NK1-positive spinoparabrachial neurons. J Comp Neurol 2003,460(2):255–265. 10.1002/cne.10647PubMedView ArticleGoogle Scholar
- Labrakakis C, MacDermott AB: Neurokinin receptor 1-expressing spinal cord neurons in lamina I and III/IV of postnatal rats receive inputs from capsaicin sensitive fibers. Neurosci Lett 2003,352(2):121–124. 10.1016/j.neulet.2003.08.042PubMedView ArticleGoogle Scholar
- Tong CK, MacDermott AB: Both Ca 2+ -permeable and -impermeable AMPA receptors contribute to primary synaptic drive onto rat dorsal horn neurons. J Physiol 2006, 575: 133–144. 10.1113/jphysiol.2006.110072PubMed CentralPubMedView ArticleGoogle Scholar
- Todd AJ, Puskar Z, Spike RC, Hughes C, Watt C, Forrest L: Projection neurons in lamina I of rat spinal cord with the neurokinin 1 receptor are selectively innervated by substance P-containing afferents and respond to noxious stimulation. J Neurosci 2002,22(10):4103–4113.PubMedGoogle Scholar
- Amaya F, Oh-hashi K, Naruse Y, Iijima N, Ueda M, Shimosato G, Tominaga M, Tanaka Y, Tanaka M: Local inflammation increases vanilloid receptor 1 expression within distinct subgroups of DRG neurons. Brain Res 2003,963(1–2):190–196. 10.1016/S0006-8993(02)03972-0PubMedView ArticleGoogle Scholar
- Kobayashi K, Fukuoka T, Obata K, Yamanaka H, Dai Y, Tokunaga A, Noguchi K: Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with A δ /C-fibers and colocalization with Trk receptors. J Comp Neurol 2005,493(4):596–606. 10.1002/cne.20794PubMedView ArticleGoogle Scholar
- Lawson SN, Perry MJ, Prabhakar E, McCarthy PW: Primary sensory neurones: neurofilament, neuropeptides, and conduction velocity. Brain Res Bull 1993,30(3–4):239–243. 10.1016/0361-9230(93)90250-FPubMedView ArticleGoogle Scholar
- Lawson SN, Crepps BA, Perl ER: Relationship of substance P to afferent characteristics of dorsal root ganglion neurones in guinea-pig. J Physiol 1997, 505: 177–191. 10.1111/j.1469-7793.1997.00177.xPubMed CentralPubMedView ArticleGoogle Scholar
- McCarthy PW, Lawson SN: Cell type and conduction velocity of rat primary sensory neurons with substance P-like immunoreactivity. Neuroscience 1989,28(3):745–753. 10.1016/0306-4522(89)90019-5PubMedView ArticleGoogle Scholar
- Michael GJ, Priestley JV: Differential expression of the mRNA for the vanilloid receptor subtype 1 in cells of the adult rat dorsal root and nodose ganglia and its downregulation by axotomy. J Neurosci 1999,19(5):1844–1854.PubMedGoogle Scholar
- Yu L, Yang F, Luo H, Liu FY, Han JS, Xing GG, Wan Y: The role of TRPV1 in different subtypes of dorsal root ganglion neurons in rat chronic inflammatory nociception induced by complete Freund’s adjuvant. Mol Pain 2008, 4: 61. 10.1186/1744-8069-4-61PubMed CentralPubMedView ArticleGoogle Scholar
- Baba H, Doubell TP, Woolf CJ: Peripheral inflammation facilitates A β fiber-mediated synaptic input to the substantia gelatinosa of the adult rat spinal cord. J Neurosci 1999,19(2):859–867.PubMedGoogle Scholar
- Nakatsuka T, Ataka T, Kumamoto E, Tamaki T, Yoshimura M: Alteration in synaptic inputs through C-afferent fibers to substantia gelatinosa neurons of the rat spinal dorsal horn during postnatal development. Neuroscience 2000,99(3):549–556. 10.1016/S0306-4522(00)00224-4PubMedView ArticleGoogle Scholar
- Baccei ML, Bardoni R, Fitzgerald M: Development of nociceptive synaptic inputs to the neonatal rat dorsal horn: glutamate release by capsaicin and menthol. J Physiol 2003, 549: 231–242. 10.1113/jphysiol.2003.040451PubMed CentralPubMedView ArticleGoogle Scholar
- Spicarova D, Palecek J: The role of the TRPV1 endogenous agonist N-Oleoyldopamine in modulation of nociceptive signaling at the spinal cord levelleoyldopamine in modulation of nociceptive signaling at the spinal cord level. J Neurophysiol 2009, 102: 234–243. 10.1152/jn.00024.2009PubMedView ArticleGoogle Scholar
- Ji RR, Xu ZZ, Strichartz G, Serhan CN: Emerging roles of resolvins in the resolution of inflammation and pain. Trends Neurosci 2011,34(11):599–609. 10.1016/j.tins.2011.08.005PubMed CentralPubMedView ArticleGoogle Scholar
- Ji RR, Baba H, Brenner GJ, Woolf CJ: Nociceptive-specific activation of ERK in spinal neurons contributes to pain hypersensitivity. Nat Neurosci 1999,2(12):1114–1119. 10.1038/16040PubMedView ArticleGoogle Scholar
- Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, Koltzenburg M, Basbaum AI, Julius D: Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000,288(5464):306–313. 10.1126/science.288.5464.306PubMedView ArticleGoogle Scholar
- Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, Harries MH, Latcham J, Clapham C, Atkinson K, Hughes SA, Rance K, Grau E, Harper AJ, Pugh PL, Rogers DC, Bingham S, Randall A, Sheardown SA: Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature 2000,405(6783):183–187. 10.1038/35012076PubMedView ArticleGoogle Scholar
- Bang S, Yoo S, Yang TJ, Cho H, Kim YG, Hwang SW: Resolvin D1 attenuates activation of sensory transient receptor potential channels leading to multiple anti-nociception. Br J Pharmacol 2010,161(3):707–720. 10.1111/j.1476-5381.2010.00909.xPubMed CentralPubMedView ArticleGoogle Scholar
- Park CK, Xu ZZ, Liu T, Lü N, Serhan CN, Ji RR: Resolvin D2 is a potent endogenous inhibitor for transient receptor potential subtype V1/A1, inflammatory pain, and spinal cord synaptic plasticity in mice: distinct roles of resolvin D1, D2, and E1. J Neurosci 2011,31(50):18433–18438. 10.1523/JNEUROSCI.4192-11.2011PubMed CentralPubMedView ArticleGoogle Scholar
- Berg V, Sveinbjörnsson B, Bendiksen S, Brox J, Meknas K, Figenschau Y: Human articular chondrocytes express ChemR23 and chemerin; ChemR23 promotes inflammatory signalling upon binding the ligand chemerin 21–157 . Arthritis Res Ther 2010,12(6):R228. 10.1186/ar3215PubMed CentralPubMedView ArticleGoogle Scholar
- Bondue B, Wittamer V, Parmentier M: Chemerin and its receptors in leukocyte trafficking, inflammation and metabolism. Cytokine Growth Factor Rev 2011,22(5–6):331–338. 10.1016/j.cytogfr.2011.11.004PubMedView ArticleGoogle Scholar
- Du XY, Zabel BA, Myles T, Allen SJ, Handel TM, Lee PP, Butcher EC, Leung LL: Regulation of chemerin bioactivity by plasma carboxypeptidase N, carboxypeptidase B (activated thrombin-activable fibrinolysis inhibitor), and platelets. J Biol Chem 2009,284(2):751–758. 10.1074/jbc.M805000200PubMed CentralPubMedView ArticleGoogle Scholar
- Luangsay S, Wittamer V, Bondue B, De Henau O, Rouger L, Brait M, Franssen JD, de Nadai P, Huaux F, Parmentier M: Mouse ChemR23 is expressed in dendritic cell subsets and macrophages, and mediates an anti-inflammatory activity of chemerin in a lung disease model. J Immunol 2009,183(10):6489–6499. 10.4049/jimmunol.0901037PubMedView ArticleGoogle Scholar
- Nagpal S, Patel S, Jacobe H, DiSepio D, Ghosn C, Malhotra M, Teng M, Duvic M, Chandraratna RA: Tazarotene-induced gene 2 (TIG2), a novel retinoid-responsive gene in skin. J Invest Dermatol 1997, 109: 91–95. 10.1111/1523-1747.ep12276660PubMedView ArticleGoogle Scholar
- Vermi W, Riboldi E, Wittamer V, Gentili F, Luini W, Marrelli S, Vecchi A, Franssen JD, Communi D, Massardi L, Sironi M, Mantovani A, Parmentier M, Facchetti F, Sozzani S: Role of ChemR23 in directing the migration of myeloid and plasmacytoid dendritic cells to lymphoid organs and inflamed skin. J Exp Med 2005,201(4):509–515. 10.1084/jem.20041310PubMed CentralPubMedView ArticleGoogle Scholar
- Goralski KB, McCarthy TC, Hanniman EA, Zabel BA, Butcher EC, Parlee SD, Muruganandan S, Sinal CJ: Chemerin, a novel adipokine that regulates adipogenesis and adipocyte metabolism. J Biol Chem 2007,282(38):28175–28188. 10.1074/jbc.M700793200PubMedView ArticleGoogle Scholar
- Yoshimura T, Oppenheim JJ: Chemokine-like receptor 1 (CMKLR1) and chemokine (C-C motif) receptor-like 2 (CCRL2); Two multifunctional receptors with unusual properties. Exp Cell Res 2010,317(5):674–684.PubMed CentralPubMedView ArticleGoogle Scholar
- Zabel BA, Nakae S, Zúñiga L, Kim JY, Ohyama T, Alt C, Pan J, Suto H, Soler D, Allen SJ, Handel TM, Song CH, Galli SJ, Butcher EC: Mast cell-expressed orphan receptor CCRL2 binds chemerin and is required for optimal induction of IgE-mediated passive cutaneous anaphylaxis. J Exp Med 2008,205(10):2207–2220. 10.1084/jem.20080300PubMed CentralPubMedView ArticleGoogle Scholar
- Brouwer N, Zuurman MW, Wei T, Ransohoff RM, Boddeke HWGM, Biber K: Induction of glial L-CCR mRNA expression in spinal cord and brain in experimental autoimmune encephalomyelitis. Glia 2004, 46: 84–94. 10.1002/glia.10352PubMedView ArticleGoogle Scholar
- Barnea G, Strapps W, Herrada G, Berman Y, Ong J, Kloss B, Axel R, Lee KJ: The genetic design of signaling cascades to record receptor activation. Proc Natl Acad Sci USA 2008, 105: 64–69. 10.1073/pnas.0710487105PubMed CentralPubMedView ArticleGoogle Scholar
- Croitoru-Lamoury J, Guillemin GJ, Boussin FD, Mognetti B, Gigout LI, Chéret A, Vaslin B, Le Grand R, Brew BJ, Dormont D: Expression of chemokines and their receptors in human and simian astrocytes: evidence for a central role of TNF α and IFN γ in CXCR4 and CCR5 modulation. Glia 2003,41(4):354–370. 10.1002/glia.10181PubMedView ArticleGoogle Scholar
- Marchese A, Docherty JM, Nguyen T, Heiber M, Cheng R, Heng HH, Tsui LC, Shi X, George SR, O’Dowd BF: Cloning of human genes encoding novel G protein-coupled receptors. Genomics 1994,23(3):609–618. 10.1006/geno.1994.1549PubMedView ArticleGoogle Scholar
- Marchese A, Cheng R, Lee MC, Porter CA, Heiber M, Goodman M, George SR, O’Dowd BF: Mapping studies of two G protein-coupled receptor genes: an amino acid difference may confer a functional variation between a human and rodent receptor. Biochem Biophys Res Commun 1994,205(3):1952–1958. 10.1006/bbrc.1994.2899PubMedView ArticleGoogle Scholar
- Shimizu N, Soda Y, Kanbe K, Liu HY, Jinno A, Kitamura T, Hoshino H: An orphan G protein-coupled receptor, GPR1, acts as a coreceptor to allow replication of human immunodeficiency virus types 1 and 2 in brain-derived cells. J Virol 1999,73(6):5231–5239.PubMed CentralPubMedGoogle Scholar
- Arita M, Oh SF, Chonan T, Hong S, Elangovan S, Sun YP, Uddin J, Petasis NA, Serhan CN: Metabolic inactivation of resolvin E1 and stabilization of its anti-inflammatory actions. J Biol Chem 2006,281(32):22847–22854. 10.1074/jbc.M603766200PubMedView ArticleGoogle Scholar
- Guillabert A, Wittamer V, Bondue B, Godot V, Imbault V, Parmentier M, Communi D: Role of neutrophil proteinase 3 and mast cell chymase in chemerin proteolytic regulation. J Leukoc Biol 2008,84(6):1530–1538. 10.1189/jlb.0508322PubMedView ArticleGoogle Scholar
- Shimamura K, Matsuda M, Miyamoto Y, Yoshimoto R, Seo T, Tokita S: Identification of a stable chemerin analog with potent activity toward ChemR23. Peptides 2009,30(8):1529–1538. 10.1016/j.peptides.2009.05.030PubMedView ArticleGoogle Scholar
- Bennett VJ, Simmons MA: Analysis of fluorescently labeled substance P analogs: binding, imaging and receptor activation. BMC Chem Biol 2001, 1: 1. 10.1186/1472-6769-1-1PubMed CentralPubMedView ArticleGoogle Scholar
- Lu Y, Perl ER: Modular organization of excitatory circuits between neurons of the spinal superficial dorsal horn (laminae I and II). J Neurosci 2005,25(15):3900–3907. 10.1523/JNEUROSCI.0102-05.2005PubMedView ArticleGoogle Scholar
- Yang K, Kumamoto E, Furue H, Yoshimura M: Capsaicin facilitates excitatory but not inhibitory synaptic transmission in substantia gelatinosa of the rat spinal cord. Neurosci Lett 1998,255(3):135–138. 10.1016/S0304-3940(98)00730-7PubMedView ArticleGoogle Scholar
- Sikand P, Premkumar LS: Potentiation of glutamatergic synaptic transmission by protein kinase C-mediated sensitization of TRPV1 at the first sensory synapse. J Physiol 2007,581(2):631–647. 10.1113/jphysiol.2006.118620PubMed CentralPubMedView ArticleGoogle Scholar
- Kim J, Alger BE: Random response fluctuations lead to spurious paired-pulse facilitation. J Neurosci 2001,21(24):9608–9618.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.