International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)

H3. Mouse Genetics as a Tool to Study Nociceptive Signaling

Andreas Zimmer
Department of Molecular Neurobiology, University of Bonn and Laboratory of Genetics, National Institute of Mental Health

We are using genetics to study the molecular mechanisms involved in nociceptive signaling. We have started out with a reverse genetics approach from genes that were thought to be involved in the transmission of noxious signals. We have thus generated mouse mutants which cannot produce the neuropeptides enkephalin1, substance K or substance P2, or the neuromodulatory cannabinoid receptor CB13,4. All mutant mouse strains are viable, although CB1 knockouts show a dramatically increased mortality.

The behavioral analysis revealed distinct and characteristic behavioral alterations in each strain: Substance P knockout mice are hypoalgesic, while enkephalin knockout mice are hyperalgesic. These behavioral alterations reflect the antagonistic roles of these neuropeptides in the transmission of nociceptive signals. Enkephalin knockout mice also display altered 'emotional' behaviors, suggesting the possibility of an imbalance of limbic functions. Indeed, we found striking molecular changes in limbic structures of enkephalin knockout mice, including an up-regulation of opioid receptor gene expression5. CB1 knockout mice were hypoalgesic and hypoactive. Because CB1 cannabinoid receptors are expressed at high levels in the basal ganglia, a brain structure critical for sensorimotor and motivational aspects of behavior, we studied the expression of various neuropeptides and transmitter-related enzymes in basal ganglia neurons. CB1 mutants display significantly increased expression of substance P, dynorphin, enkephalin, and GAD 67 in neurons of the two output pathways of the striatum that project to the substantia nigra and the globus pallidus, thus indicating a critical role of the CB1 receptor in normal basal ganglia function.

The analysis of congenic C57BL/6 and DBA/2 mouse strains with the enkephalin mutation showed that the effect of the mutation on nociceptive signaling are strongly influenced by the genetic background. Interestingly, these strain effects are specific for the nociceptive test. Mutant C57BL/6 congenic mice are hyperalgesic in the hotplate test and more sensitive in tests for visceral pain. Mutant DBA/2 congenics display increased stress-induced analgesia. It should be possible to utilize these strain difference to identify modifier genes that modulate the effects of the enkephalin mutation on specific nociceptive signaling pathways.

We are also using a forward genetic approach in order to identify novel genes involved in nociceptive signaling. For this purpose, we are participating in a large scale ethylnitrosourea (ENU)-mutagenesis program which is sponsored by the German Human Genome Project and carried out at the GSF - National Research Center in Neuherberg, Germany6. ENU, an alkylating agent, is one of the most powerful mutagens for the production of mutations in mice. It creates mainly point mutations, as well as small intragenic lesions7. Systematic ENU-mutagenesis screens have been successfully performed in Drosophila melanogaster, Caenorhabditis elegans, and in zebrafish. Male mice are injected with ENU and mated to females to produce mutant F1 offspring. These F1 animals are then analyzed for dominant traits, or bred further to screen for recessive phenotypes. We have screened over 1600 F1 animals for nociceptive behaviors using the hotplate test. Mice that deviate in three subsequent tests (performed at weekly intervals) by more than two standard deviations from the strain means, are considered variants. All variants are crossed back to wild type mice and the F2 offspring of this backcross are analyzed in the hotplate test. If this analysis indicates Mendelian (autosomal dominant) transmission of the trait, then the variant is called a mutant. To this date, we have identified over 30 variants and 5 mutants.

1. Konig, M. et al. Pain responses, anxiety and aggression in mice deficient in pre- proenkephalin. Nature 383, 535-538 (1996).

2. Zimmer, A. et al. Hypoalgesia in mice with a targeted deletion of the tachykinin 1 gene. Proc Natl Acad Sci U S A 95, 2630-2635 (1998).

3. Zimmer, A., Zimmer, A.M., Hohmann, A.G., Herkenham, M. & Bonner, T.I. Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice [see comments]. Proc Natl Acad Sci U S A 96, 5780-5785 (1999).

4. Steiner, H., Bonner, T.I., Zimmer, A.M., Kitai, S.T. & Zimmer, A. Altered gene expression in striatal projection neurons in CB1 cannabinoid receptor knockout mice [see comments]. Proc Natl Acad Sci U S A 96, 5786-5790 (1999).

5. Brady, L.S. et al. Region-specific up-regulation of opioid receptor binding in enkephalin knockout mice. Brain Res Mol Brain Res 68, 193-197 (1999).

6. Hrabé de Angelis, M. et al. Genome wide large scale production of mutant mice by ENU mutagenesis. Nature Genetics in press(2000).

7. Hrabé de Angelis, M. & Balling, R. Large scale ENU screens in the mouse: genetics meets genomics. Mutat. Res. 400, 24-32 (1998).

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