Systems genetic and pharmacological analysis identifies candidate genes underlying mechanosensation in the von Frey test.

Mechanical sensitivity is commonly affected in chronic pain and other neurological disorders. To discover mechanisms of individual differences in punctate mechanosensation, we performed quantitative trait locus (QTL) mapping of the response to von Frey monofilament stimulation in BXD recombinant inbred (BXD) mice. Significant loci were detected on mouse chromosome (Chr) 5 and 15, indicating the location of underlying polymorphisms that cause heritable variation in von Frey response. Convergent evidence from public gene expression data implicates candidate genes within the loci: von Frey thresholds were strongly correlated with baseline expression of Cacna2d1, Ift27 and Csnk1e in multiple brain regions of BXD strains. Systemic gabapentin and PF-670462, which target the protein products of Cacna2d1 and Csnk1e, respectively, significantly increased von Frey thresholds in a genotype-dependent manner in progenitors and BXD strains. Real-time polymerase chain reaction confirmed differential expression of Cacna2d1 and Csnk1e in multiple brain regions in progenitors and showed differential expression of Cacna2d1 and Csnk1e in the dorsal root ganglia of the progenitors and BXD strains grouped by QTL genotype. Thus, linkage mapping, transcript covariance and pharmacological testing suggest that genetic variation affecting Cacna2d1 and Csnk1e may contribute to individual differences in von Frey filament response. This study implicates Cacna2d1 and Ift27 in basal mechanosensation in line with their previously suspected role in mechanical hypersensitivity. Csnk1e is implicated for von Frey response for the first time. Further investigation is warranted to identify the specific polymorphisms involved and assess the relevance of these findings to clinical conditions of disturbed mechanosensation.

Comt1 genotype and expression predicts anxiety and nociceptive sensitivity in inbred strains of mice.

Catechol-O-methyltransferase (COMT) is a ubiquitously expressed enzyme that maintains basic biologic functions by inactivating catechol substrates. In humans, polymorphic variance at the COMT locus has been associated with modulation of pain sensitivity and risk for developing psychiatric disorders. A functional haplotype associated with increased pain sensitivity was shown to result in decreased COMT activity by altering mRNA secondary structure-dependent protein translation. However, the exact mechanisms whereby COMT modulates pain sensitivity and behavior remain unclear and can be further studied in animal models. We have assessed Comt1 gene expression levels in multiple brain regions in inbred strains of mice and have discovered that Comt1 is differentially expressed among the strains, and this differential expression is cis-regulated. A B2 short interspersed nuclear element (SINE) was inserted in the 3'-untranslated region (3'-UTR) of Comt1 in 14 strains generating a common haplotype that correlates with gene expression. Experiments using mammalian expression vectors of full-length cDNA clones with and without the SINE element show that strains with the SINE haplotype (+SINE) have greater Comt1 enzymatic activity. +SINE mice also exhibit behavioral differences in anxiety assays and decreased pain sensitivity. These results suggest that a haplotype, defined by a 3'-UTR B2 SINE element, regulates Comt1 expression and some mouse behaviors.

High-throughput behavioral phenotyping in the expanded panel of BXD recombinant inbred strains.

Genetic reference populations, particularly the BXD recombinant inbred (BXD RI) strains derived from C57BL/6J and DBA/2J mice, are a valuable resource for the discovery of the bio-molecular substrates and genetic drivers responsible for trait variation and covariation. This approach can be profitably applied in the analysis of susceptibility and mechanisms of drug and alcohol use disorders for which many predisposing behaviors may predict the occurrence and manifestation of increased preference for these substances. Many of these traits are modeled by common mouse behavioral assays, facilitating the detection of patterns and sources of genetic coregulation of predisposing phenotypes and substance consumption. Members of the Tennessee Mouse Genome Consortium (TMGC) have obtained phenotype data from over 250 measures related to multiple behavioral assays across several batteries: response to, and withdrawal from cocaine, 3,4-methylenedioxymethamphetamine; "ecstasy" (MDMA), morphine and alcohol; novelty seeking; behavioral despair and related neurological phenomena; pain sensitivity; stress sensitivity; anxiety; hyperactivity and sleep/wake cycles. All traits have been measured in both sexes in approximately 70 strains of the recently expanded panel of BXD RI strains. Sex differences and heritability estimates were obtained for each trait, and a comparison of early (N = 32) and recent (N = 37) BXD RI lines was performed. Primary data are publicly available for heritability, sex difference and genetic analyses using the MouseTrack database, and are also available in GeneNetwork.org for quantitative trait locus (QTL) detection and genetic analysis of gene expression. Together with the results of related studies, these data form a public resource for integrative systems genetic analysis of neurobehavioral traits.

Transgenic studies of pain and analgesia: mutation or background genotype?

The application of transgenic (knockout) technology to the study of pain is rapidly expanding. Despite its power, this technique has several shortcomings that complicate the interpretation of the data obtained. Although compensation by other genes is a well recognized problem, issues related to the background genotype of the mutant mice are less well appreciated. This review describes these confounds as they apply to studies of pain and pain inhibition. We show that the 129 and C57BL/6 mouse strains, which provide the default genetic background on which null mutants are constructed, display significant and sometimes extreme phenotypic differences in many assays of nociception, hypersensitivity, and analgesia. Although problems related to the differential responsiveness of the two strains are minimized by placing knockouts onto "pure" 129 and/or C57BL/6 backgrounds, we also illustrate that neither of these strains are particularly representative of inbred mice in general. Procedures to reduce confounds and converging evidence must be used to accurately determine the functions of the targeted genes in pain-related phenomena.

The bee venom test: a new tonic-pain test.

The present study describes a new test of tonic pain to be used as an animal model of persistent pain. First, pain responses and edema produced by subcutaneous injection of increasing doses of honey bee venom into the hind paw of the rat were quantified. Second, the effect of morphine and aspirin on the pain responses was investigated. Finally, the response to concurrent injections of bee venom and formalin was examined. Subcutaneous injection of bee venom produced local inflammation, tonic-pain responses lasting from 10 min to more than 1 h, and marked edema lasting from 3 h to more than 48 h. Increasing doses of bee venom produced higher mean pain scores and increased durations of responding. The time course of the edema did not follow the time course of the pain responses. Analgesia was produced by morphine and aspirin, indicating that the bee venom test can be used to test analgesic drugs. Concurrent administration of bee venom and formalin produced pain responses similar to formalin alone, with a less profound interphase depression and a longer duration. The data suggest that the bee venom test is a valid animal model of experimental tonic pain.

Hypophysectomy produces analgesia and paraventricular lesions have no effect on formalin-induced pain.

Chemical destruction of the pituitary gland has been shown to alleviate severe cancer pain in a substantial proportion of patients. The underlying mechanisms, however, remain controversial. The present study investigated the effects of hypophysectomy in the formalin test, which provides an animal model of tonic pain, and attempted to determine a possible neural mechanism to explain the effectiveness of the procedure. Hypophysectomized rats displayed significantly less pain behavior in the formalin test than control rats that underwent a sham hypophysectomy, implicating the pituitary gland in the modulation of tonic pain. Since the paraventricular nucleus of the hypothalamus (PVN) provides a major source of input to the pituitary gland, the effects of electrolytic lesions of the PVN on tonic pain were examined. The results failed to show a significant effect of PVN lesions on pain responses in the formalin test. The results suggest that the pituitary gland modulates tonic pain in the formalin test and that the test provides an animal model to study possible mechanisms which underlie the relief of severe cancer pain by hypophysectomy. However, since PVN lesions did not affect the response of rats in the formalin test, it is possible that the PVN is only one of multiple interacting neural and endocrine structures that influence the functions of the pituitary.