Olfactory exposure to males, including men, causes stress and related analgesia in rodents.

We found that exposure of mice and rats to male but not female experimenters produces pain inhibition. Male-related stimuli induced a robust physiological stress response that results in stress-induced analgesia. This effect could be replicated with T-shirts worn by men, bedding material from gonadally intact and unfamiliar male mammals, and presentation of compounds secreted from the human axilla. Experimenter sex can thus affect apparent baseline responses in behavioral testing.

Remote optogenetic activation and sensitization of pain pathways in freely moving mice.

We report a novel model in which remote activation of peripheral nociceptive pathways in transgenic mice is achieved optogenetically, without any external noxious stimulus or injury. Taking advantage of a binary genetic approach, we selectively targeted Nav1.8(+) sensory neurons for conditional expression of channelrhodopsin-2 (ChR2) channels. Acute blue light illumination of the skin produced robust nocifensive behaviors, evoked by the remote stimulation of both peptidergic and nonpeptidergic nociceptive fibers as indicated by c-Fos labeling in laminae I and II of the dorsal horn of the spinal cord. A non-nociceptive component also contributes to the observed behaviors, as shown by c-Fos expression in lamina III of the dorsal horn and the expression of ChR2-EYFP in a subpopulation of large-diameter Nav1.8(+) dorsal root ganglion neurons. Selective activation of Nav1.8(+) afferents in vivo induced central sensitization and conditioned place aversion, thus providing a novel paradigm to investigate plasticity in the pain circuitry. Long-term potentiation was similarly evoked by light activation of the same afferents in isolated spinal cord preparations. These findings demonstrate, for the first time, the optical control of nociception and central sensitization in behaving mammals and enables selective activation of the same class of afferents in both in vivo and ex vivo preparations. Our results provide a proof-of-concept demonstration that optical dissection of the contribution of specific classes of afferents to central sensitization is possible. The high spatiotemporal precision offered by this non-invasive model will facilitate drug development and target validation for pain therapeutics.

Serotonin-induced hypersensitivity via inhibition of catechol O-methyltransferase activity.

The subcutaneous and systemic injection of serotonin reduces cutaneous and visceral pain thresholds and increases responses to noxious stimuli. Different subtypes of 5-hydroxytryptamine (5-HT) receptors are suggested to be associated with different types of pain responses. Here we show that serotonin also inhibits catechol O-methyltransferase (COMT), an enzyme that contributes to modultion the perception of pain, via non-competitive binding to the site bound by catechol substrates with a binding affinity comparable to the binding affinity of catechol itself (K(i) = 44 µM). Using computational modeling, biochemical tests and cellular assays we show that serotonin actively competes with the methyl donor S-adenosyl-L-methionine (SAM) within the catalytic site. Binding of serotonin to the catalytic site inhibits the access of SAM, thus preventing methylation of COMT substrates. The results of in vivo animal studies show that serotonin-induced pain hypersensitivity in mice is reduced by either SAM pretreatment or by the combined administration of selective antagonists for ß(2)- and ß(3)-adrenergic receptors, which have been previously shown to mediate COMT-dependent pain signaling. Our results suggest that inhibition of COMT via serotonin binding contributes to pain hypersensitivity, providing additional strategies for the treatment of clinical pain conditions.

The Rat Grimace Scale: a partially automated method for quantifying pain in the laboratory rat via facial expressions.

We recently demonstrated the utility of quantifying spontaneous pain in mice via the blinded coding of facial expressions. As the majority of preclinical pain research is in fact performed in the laboratory rat, we attempted to modify the scale for use in this species. We present herein the Rat Grimace Scale, and show its reliability, accuracy, and ability to quantify the time course of spontaneous pain in the intraplantar complete Freund's adjuvant, intraarticular kaolin-carrageenan, and laparotomy (post-operative pain) assays. The scale's ability to demonstrate the dose-dependent analgesic efficacy of morphine is also shown. In addition, we have developed software, Rodent Face Finder®, which successfully automates the most labor-intensive step in the process. Given the known mechanistic dissociations between spontaneous and evoked pain, and the primacy of the former as a clinical problem, we believe that widespread adoption of spontaneous pain measures such as the Rat Grimace Scale might lead to more successful translation of basic science findings into clinical application.

Learning-related plasticity of temporal coding in simultaneously recorded amygdala-cortical ensembles.

Emotional learning requires the coordinated action of neural populations in limbic and cortical networks. Here, we performed simultaneous extracellular recordings from gustatory cortical (GC) and basolateral amygdalar (BLA) neural ensembles as awake, behaving rats learned to dislike the taste of saccharin [via conditioned taste aversion (CTA)]. Learning-related changes in single-neuron sensory responses were observed in both regions, but the nature of the changes was region specific. In GC, most changes were restricted to relatively late aspects of the response (starting approximately 1.0 s after stimulus administration), supporting our hypothesis that in this paradigm palatability-related information resides exclusively in later cortical responses. In contrast, and consistent with data suggesting the amygdala's primary role in judging stimulus palatability, CTA altered all components of BLA taste responses, including the earliest. Finally, learning caused dramatic increases in the functional connectivity (measured in terms of cross-correlation peak heights) between pairs of simultaneously recorded BLA and GC neurons, increases that were evident only during taste processing. Our simultaneous assays of the activity of single neurons in multiple relevant brain regions across learning suggest that the transmission of taste information through amygdala-cortical circuits plays a vital role in CTA memory formation.

Epiregulin and EGFR interactions are involved in pain processing.

The EGFR belongs to the well-studied ErbB family of receptor tyrosine kinases. EGFR is activated by numerous endogenous ligands that promote cellular growth, proliferation, and tissue regeneration. In the present study, we have demonstrated a role for EGFR and its natural ligand, epiregulin (EREG), in pain processing. We show that inhibition of EGFR with clinically available compounds strongly reduced nocifensive behavior in mouse models of inflammatory and chronic pain. EREG-mediated activation of EGFR enhanced nociception through a mechanism involving the PI3K/AKT/mTOR pathway and matrix metalloproteinase-9. Moreover, EREG application potentiated capsaicin-induced calcium influx in a subset of sensory neurons. Both the EGFR and EREG genes displayed a genetic association with the development of chronic pain in several clinical cohorts of temporomandibular disorder. Thus, EGFR and EREG may be suitable therapeutic targets for persistent pain conditions.

Optogenetic Silencing of Nav1.8-Positive Afferents Alleviates Inflammatory and Neuropathic Pain.

We report a novel transgenic mouse model in which the terminals of peripheral nociceptors can be silenced optogenetically with high spatiotemporal precision, leading to the alleviation of inflammatory and neuropathic pain. Inhibitory archaerhodopsin-3 (Arch) proton pumps were delivered to Nav1.8(+) primary afferents using the Nav1.8-Cre driver line. Arch expression covered both peptidergic and nonpeptidergic nociceptors and yellow light stimulation reliably blocked electrically induced action potentials in DRG neurons. Acute transdermal illumination of the hindpaws of Nav1.8-Arch(+) mice significantly reduced mechanical allodynia under inflammatory conditions, while basal mechanical sensitivity was not affected by the optical stimulation. Arch-driven hyperpolarization of nociceptive terminals was sufficient to prevent channelrhodopsin-2 (ChR2)-mediated mechanical and thermal hypersensitivity in double-transgenic Nav1.8-ChR2(+)-Arch(+) mice. Furthermore, prolonged optical silencing of peripheral afferents in anesthetized Nav1.8-Arch(+) mice led to poststimulation analgesia with a significant decrease in mechanical and thermal hypersensitivity under inflammatory and neuropathic conditions. These findings highlight the role of peripheral neuronal inputs in the onset and maintenance of pain hypersensitivity, demonstrate the plasticity of pain pathways even after sensitization has occurred, and support the involvement of Nav1.8(+) afferents in both inflammatory and neuropathic pain. Together, we present a selective analgesic approach in which genetically identified subsets of peripheral sensory fibers can be remotely and optically inhibited with high temporal resolution, overcoming the compensatory limitations of genetic ablations.

Structural and functional interactions between six-transmembrane µ-opioid receptors and ß2-adrenoreceptors modulate opioid signaling.

The primary molecular target for clinically used opioids is the µ-opioid receptor (MOR). Besides the major seven-transmembrane (7TM) receptors, the MOR gene codes for alternatively spliced six-transmembrane (6TM) isoforms, the biological and clinical significance of which remains unclear. Here, we show that the otherwise exclusively intracellular localized 6TM-MOR translocates to the plasma membrane upon coexpression with ß2-adrenergic receptors (ß2-ARs) through an interaction with the fifth and sixth helices of ß2-AR. Coexpression of the two receptors in BE(2)-C neuroblastoma cells potentiates calcium responses to a 6TM-MOR ligand, and this calcium response is completely blocked by a selective ß2-antagonist in BE(2)-C cells, and in trigeminal and dorsal root ganglia. Co-administration of 6TM-MOR and ß2-AR ligands leads to substantial analgesic synergy and completely reverses opioid-induced hyperalgesia in rodent behavioral models. Together, our results provide evidence that the heterodimerization of 6TM-MOR with ß2-AR underlies a molecular mechanism for 6TM cellular signaling, presenting a unique functional responses to opioids. This signaling pathway may contribute to the hyperalgesic effects of opioids that can be efficiently blocked by ß2-AR antagonists, providing a new avenue for opioid therapy.

The nicotinic α6 subunit gene determines variability in chronic pain sensitivity via cross-inhibition of P2X2/3 receptors.

Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6's role in analgesia is at least partially due to direct interaction and cross-inhibition of α6* nAChRs with P2X2/3 receptors in DRG nociceptors. Finally, we establish the relevance of our results to humans by the observation of genetic association in patients suffering from chronic postsurgical and temporomandibular pain.

Broad-spectrum analgesic efficacy of IBNtxA is mediated by exon 11-associated splice variants of the mu-opioid receptor gene.

µ-Opioids remain vastly important for the treatment of pain, and would represent ideal analgesics if their analgesic effects could be separated from their many side effects. A recently synthesized compound, iodobenzoylnaltrexamide (IBNtxA), acting at 6-transmembrane (6-TM) splice variants of the µ-opioid receptor gene, was shown to have potent analgesic actions against acute, thermal pain accompanied by a vastly improved side-effect profile compared to 7-TM-acting drugs such as morphine. Whether such analgesia can be seen in longer-lasting and nonthermal algesiometric assays is not known. The current study demonstrates potent and efficacious IBNtxA inhibition of a wide variety of assays, including inflammatory and neuropathic hypersensitivity and spontaneous pain. We further demonstrate the dependence of such analgesia on 6-TM µ-opioid receptor variants using isobolographic analysis and the testing of Oprm1 (the µ-opioid receptor gene) exon 11 null mutant mice. Finally, the effect of nerve damage (spared nerve injury) and inflammatory injury (complete Freund's adjuvant) on expression of µ-opioid receptor variant genes in pain-relevant central nervous system loci was examined, revealing a downregulation of the mMOR-1D splice variant in the dorsal root ganglion after spared nerve injury. These findings are supportive of the potential value of 6-TM-acting drugs as novel analgesics.

Using the Mouse Grimace Scale to reevaluate the efficacy of postoperative analgesics in laboratory mice.

Postoperative pain management in animals is complicated greatly by the inability to recognize pain. As a result, the choice of analgesics and their doses has been based on extrapolation from greatly differing pain models or the use of measures with unclear relevance to pain. We recently developed the Mouse Grimace Scale (MGS), a facial-expression-based pain coding system adapted directly from scales used in nonverbal human populations. The MGS has shown to be a reliable, highly accurate measure of spontaneous pain of moderate duration, and therefore is particularly useful in the quantification of postoperative pain. In the present study, we quantified the relative intensity and duration of postoperative pain after a sham ventral ovariectomy (laparotomy) in outbred mice. In addition, we compiled dose-response data for 4 commonly used analgesics: buprenorphine, carprofen, ketoprofen, and acetaminophen. We found that postoperative pain in mice, as defined by facial grimacing, lasts for 36 to 48 h, and appears to show relative exacerbation during the early dark (active) photophase. We find that buprenorphine was highly effective in inhibiting postoperative pain-induced facial grimacing in mice at doses equal to or lower than current recommendations, that carprofen and ketoprofen are effective only at doses markedly higher than those currently recommended, and that acetaminophen was ineffective at any dose used. We suggest the revision of practices for postoperative pain management in mice in light of these findings.