Hypolocomotion, asymmetrically directed behaviors (licking, lifting, flinching, and shaking) and dynamic weight bearing (gait) changes are not measures of neuropathic pain in mice

<p>Abstract</p> <p>Background</p> <p>Spontaneous (non-evoked) pain is a major clinical symptom of neuropathic syndromes, one that is understudied in basic pain research for practical reasons and because of a lack of consensus over precisely which behaviors reflect spontaneous pain in laboratory animals. It is commonly asserted that rodents experiencing pain in a hind limb exhibit hypolocomotion and decreased rearing, engage in both reflexive and organized limb directed behaviors, and avoid supporting their body weight on the affected side. Furthermore, it is assumed that the extent of these positive or negative behaviors can be used as a dependent measure of spontaneous chronic pain severity in such animals. In the present study, we tested these assumptions via blinded, systematic observation of digital video of mice with nerve injuries (chronic constriction or spared nerve injury), and automated assessment of locomotor behavior using photocell detection and dynamic weight bearing (i.e., gait) using the CatWalk^®system.</p> <p>Results</p> <p>We found no deficits in locomotor activity or rearing associated with neuropathic injury. The frequency of asymmetric (ipsilaterally directed) behaviors were too rare to be seriously considered as representing spontaneous pain, and in any case did not statistically exceed what was blindly observed on the contralateral hind paw and in control (sham operated and unoperated) mice. Changes in dynamic weight bearing, on the other hand, were robust and ipsilateral after spared nerve injury (but not chronic constriction injury). However, we observed timing, pharmacological, and genetic dissociation of mechanical allodynia and gait alterations.</p> <p>Conclusions</p> <p>We conclude that spontaneous neuropathic pain in mice cannot be assessed using any of these measures, and thus caution is warranted in making such assertions.</p

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

Serotonin-Induced Hypersensitivity via Inhibition of Catechol O-Methyltransferase Activity

Abstract 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 β2- and β3-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

A biopsychosocial formulation of pain communication

We present a detailed framework for understanding the numerous and complicated interactions among psychological and social determinants of pain through examination of the process of pain communication. The focus is on an improved understanding of immediate dyadic transactions during painful events in the context of broader social phenomena. Fine-grain consideration of social transactions during pain leads to an appreciation of sociobehavioral events affecting both suffering persons as well as caregivers. Our examination considers knowledge from a variety of perspectives, including clinical health psychology, social and developmental processes, evolutionary psychology, communication studies, and behavioral neuroscience

Structural and functional interactions between six-transmembrane mu-opioid receptors and beta(2)-adrenoreceptors modulate opioid signaling

The primary molecular target for clinically used opioids is the mu-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 beta(2)-adrenergic receptors (beta(2)-ARs) through an interaction with the fifth and sixth helices of beta(2)-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 beta(2)-antagonist in BE(2)-C cells, and in trigeminal and dorsal root ganglia. Co-administration of 6TM-MOR and beta(2)-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 beta(2)-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 beta(2)-AR antagonists, providing a new avenue for opioid therapy.Peer reviewe

Loss of neuronal potassium/chloride cotransporter 3 (KCC3) is responsible for the degenerative phenotype in a conditional mouse model of hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum

Disruption of the potassium/chloride cotransporter 3 (KCC3), encoded by the SLC12A6 gene, causes hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum (HMSN/ACC), a neurodevelopmental and neurodegenerative disorder affecting both the peripheral nervous system and CNS. However, the precise role of KCC3 in the maintenance of ion homeostasis in the nervous system and the pathogenic mechanisms leading to HMSN/ACC remain unclear. We established two Slc12a6 Cre/LoxP transgenic mouse lines expressing C-terminal truncated KCC3 in either a neuron-specific or ubiquitous fashion. Our results suggest that neuronal KCC3 expression is crucial for axon volume control. We also demonstrate that the neuropathic features of HMSN/ACC are predominantly due to a neuronal KCC3 deficit, while the auditory impairment is due to loss of non-neuronal KCC3 expression. Furthermore, we demonstrate that KCC3 plays an essential role in inflammatory pain pathways. Finally, we observed hypoplasia of the corpus callosum in both mouse mutants and a marked decrease in axonal tracts serving the auditory cortex in only the general deletion mutant. Together, these results establish KCC3 as an important player in both central and peripheral nervous system maintenance

Molecular genetic mechanisms of allelic specific regulation of murine Comt expression

A functional allele of the mouse catechol-O-methyltransferase (Comt) gene is defined by the insertion of a B2 short interspersed repeat element in its 3′-untranslated region (UTR). This allele has been associated with a number of phenotypes, such as pain and anxiety. In comparison with mice carrying the ancestral allele (Comt+), ComtB2i mice show higher Comt mRNA and enzymatic activity levels. Here, we investigated the molecular genetic mechanisms underlying this allelic specific regulation of Comt expression. Insertion of the B2 element introduces an early polyadenylation signal generating a shorter Comt transcript, in addition to the longer ancestral mRNA. Comparative analysis and in silico prediction of Comt mRNA potential targets within the transcript 3′ to the B2 element was performed and allowed choosing microRNA (miRNA) candidates for experimental screening: mmu-miR-3470a, mmu-miR-3470b, and mmu-miR-667. Cell transfection with each miRNA downregulated the expression of the ancestral transcript and COMT enzymatic activity. Our in vivo experiments showed that mmu-miR-667-3p is strongly correlated with decreasing amounts of Comt mRNA in the brain, and lentiviral injections of mmu-miR-3470a, mmu-miR-3470b, and mmu-miR-667 increase hypersensitivity in the mouse formalin model, consistent with reduced COMT activity. In summary, our data demonstrate that the Comt+ transcript contains regulatory miRNA signals in its 3′-untranslated region leading to mRNA degradation; these signals, however, are absent in the shorter transcript, resulting in higher mRNA expression and activity levels

Pain modality- and sex-specific effects of COMT genetic functional variants

The enzyme catechol-O-methyltransferase (COMT) metabolizes catecholamine neurotransmitters involved in a number of physiological functions including pain perception. Both human and mouse COMT genes possess functional polymorphisms contributing to inter-individual variability in pain phenotypes such as sensitivity to noxious stimuli, severity of clinical pain and response to pain treatment. In this study, we found that the effects of Comt functional variation in mice are modality-specific. Spontaneous inflammatory nociception and thermal nociception behaviors were correlated the most with the presence of the B2 SINE transposon insertion residing in the 3’UTR mRNA region. Similarly, in humans, COMT functional haplotypes were associated with thermal pain perception and with capsaicin-induced pain. Furthermore, COMT genetic variations contributed to pain behaviors in mice and pain ratings in humans in a sex-specific manner. The ancestral Comt variant, without a B2 SINE insertion, was more strongly associated with sensitivity to capsaicin in female versus male mice. In humans, the haplotype coding for low COMT activity increased capsaicin-induced pain perception in women, but not men. These findings reemphasize the fundamental contribution of COMT to pain processes, and provide a fine-grained resolution of this contribution at the genetic level that can be used to guide future studies in the area of pain genetics