NOS and aggression

T.R. Vidyasagar et al. – Orientation selectivity in visual cortex

6 Henry, G.H., Dreher, B. and Bishop, P.O. (1974) J. Neurophysiol. 37, 1394–1409 7 Morrone, M.C., Burr, D.C. and Maffei, L. (1982) Proc. R. Soc. London Ser. B 216, 335–354 8 Sillito, A.M. (1975) J. Physiol. 250, 305–329 9 Sillito, A.M. et al. (1980) Brain Res. 194, 517–520 10 Tsumoto, T., Eckart, W. and Creutzfeldt, O.D. (1979) Exp. Brain Res. 34, 351–365 11 Creutzfeldt, O.D., Kuhnt, U. and Benevento, L.A. (1974) Exp. Brain Res. 21, 251–274 12 Heggelund, P. (1981) Exp. Brain Res. 42, 99–107 13 Levick, W.R. and Thibos, L.N. (1980) Nature 286, 389–390 14 Vidyasagar, T.R. and Urbas, J.V. (1982) Exp. Brain Res. 46, 157–169 15 Vidyasagar, T.R. and Heide, W. (1984) Exp. Brain Res. 57, 196–200 16 Soodak, R.E., Shapley, R.M. and Kaplan, E. (1987) J. Neurophysiol. 58, 267–275 17 Shou, T.D. and Leventhal, A.G. (1989) J. Neurosci. 9, 4287– 4302 18 Vidyasagar, T.R. (1987) Biol. Cybern. 57, 11–23 19 Ferster, D. (1986) J. Neurosci. 6, 1284–1301 20 Douglas, R.J., Martin, K.A.C. and Whitteridge, D. (1991) J. Physiol. 440, 659–696 21 Berman, N.J. (1991) J. Physiol. 440, 697–721 22 Douglas, R.J. and Martin, K.A.C. (1991) J. Physiol. 440, 735–769 23 Pei, X. et al. (1991) NeuroReport 2, 485–488 24 Ferster, D. and Jagadeesh, B. (1992) J. Neurosci. 12, 1262–1274 25 Volgushev, M. et al. (1993) Visual Neurosci. 10, 1151–1155 26 Pei, X. et al. (1994) J. Neurosci. 14, 7130–7140 27 Volgushev, M., Vidyasagar, T.R. and Pei, X. (1995) Visual Neurosci. 12, 621–628 28 Freund, T.F. et al. (1985) J. Comp. Neurol. 242, 275–291 29 Huguenard, J.R., Hamill, O.P. and Prince, D.A. (1989) Proc. Natl Acad. Sci. USA 86, 2473–2477 30 Stuart, G.J. and Sakmann, B. (1994) Nature 367, 69–72 31 Stuart, G.J. and Sakmann, B. (1995) Neuron 15, 1065–1076 32 Schwindt, P.C. and Crill, W.E. (1995) J. Neurophysiol. 74, 2220–2224 33 Volgushev, M. et al. (1992) NeuroReport 3, 679–682 34 Ts’o, D.Y., Gilbert, C.D. and Wiesel, T.N. (1986) J. Neurosci. 6, 1160–1170

35 Gilbert, C.D. and Wiesel, T.N. (1989) J. Neurosci. 9, 2432–2442 36 Matsubara, J.A., Cynader, M.S. and Swindale, N.V. (1987) J. Neurosci. 7, 1428–1446 37 Eysel, U.T., Crook, J.M. and Machemer, H.F. (1990) Exp. Brain Res. 80, 626–630 38 Hata, Y. et al. (1988) Nature 335, 815–817 39 Carandini, M. and Heeger, D.J. (1994) Science 264, 1333–1336 40 Woergoetter, D.A. and Koch, C. (1991) J. Neurosci. 11, 1959–1979 41 Douglas, R.J. et al. (1995) Science 269, 981–988 42 Somers, D.C., Nelson, S.B. and Sur, M. (1995) J. Neurosci. 15, 5448–5465 43 Ben-Yishai, R., Lev Bar-Or, R. and Sompolinsky, H. (1995) Proc. Natl Acad. Sci. USA 92, 3844–3848 44 Michalski, A., Wimborne, B.M. and Henry, G.H. (1993) J. Physiol. 466, 133–156 45 Tanaka, K. (1983) J. Neurophysiol. 49, 1303–1318 46 Friedlander, M.J. et al. (1981) J. Neurophysiol. 46, 80–129 47 Lee, B.B., Cleland, B.G. and Creutzfeldt, O.D. (1977) Exp. Brain Res. 30, 527–538 48 Rose, D. (1977) J. Physiol. 271, 1–23 49 Bullier, J., Mustari, M.J. and Henry, G.H. (1982) J. Neurophysiol. 47, 417– 438 50 Vidyasagar, T.R. (1985) J. Physiol. 358, 15P 51 Vidyasagar, T.R. (1989) in Seeing Contour and Colour (Kulikowski, J.J., Dickinson, C.M. and Murray, I.J., eds), pp. 66–73, Pergamon Press 52 Henry, G.H., Goodwin, A.W. and Bishop, P.O. (1978) Exp. Brain Res. 32, 245–266 53 Nelson, S. et al. (1994) Science 265, 774–776 54 LeVay, S. and Nelson, S.B. (1991) in Vision and Visual Dysfunction (Vol. 4) (Cronly-Dillon, J.R., ed.), pp. 266–315, Macmillan Press 55 Leventhal, A.G. (1983) J. Comp. Neurol. 220, 476– 483 56 Vidyasagar, T.R. and Henry, G.H. (1990) Visual Neurosci. 5, 565–569 57 Vidyasagar, T.R. (1992) NeuroReport, 3, 185–188 58 Peters, A. and Yilmaz, E. (1993) Cereb. Cortex 3, 49–68 59 Peters, A. and Payne, B.R. (1993) Cereb. Cortex 3, 69–78 60 von der Malsburg, C. (1973) Kybernetik 14, 85–100 61 Ferster, D., Chung, S. and Wheat, H. (1996) Nature 380, 249–252

LETTERS NOS and aggression In the March issue of TINS, Mark Good1 reviewed the controversy regarding the role of nitric oxide (NO) as a retrograde messenger in hippocampal LTP, highlighting recent studies using mice lacking the gene for the neuronal NO synthase (nNOS). Perhaps the most pertinent information concerning the relevance of the nNOS knockout mouse2 to this problem is that in both normal and mutant mice, CA1 pyramidal neurones (in which LTP was assessed) express the ‘endothelial’ NOS (eNOS) isoform3 and might still synthesize NO. Analysis of the characteristics of LTP in vivo in a double nNOS/eNOS knockout mouse might be necessary to establish the role of NO in this form of synaptic plasticity. However, behavioural analysis of the existing nNOS knockout mice, demonstrating increased male aggressive and sexual behaviour, points to new and unanticipated roles for NO in brain function4. Or does it? Good1 cites instances of increased aggressiveness described in other knockout mice, and suggests ‘it does raise the

possibility that abnormal aggression and sexual behaviour is a fairly common behavioural phenotype with knockout mice’. Or could such behaviour in some cases be an artefact of the experimental design? The latter possibility arises from the complex genetic makeup of knockout mice, that affects not only the targeted gene but the whole genome. The genetic analysis of behavioural traits repeatedly demonstrates the importance of interactions (epistatic effects) between a gene and other alleles located elsewhere in the genome5,6. Appropriate genetic controls are essential if one is to conclude that a particular phenotype is due to the lack of the targeted gene product. The derivation of the nNOS knockout mouse2 followed a standard pattern: the 129/SV strain of embryonic stem (ES) cells in which the gene encoding nNOS was inactivated were fused with blastocysts, and the resulting chimeric mice crossed with C57BL/6 mice. The offspring of this F1 129/SV 3 C57BL/6 generation were intercrossed to obtain the F2 recombinant generation, a quarter of which, following Mendel’s law, were homozygotic (–/–) for the inactivated gene encoding nNOS (Ref. 2), a quarter homozygous for the wild-type (+/+) gene encoding nNOS, with the remainder

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TO THE EDITOR

heterozygous (+/–) at this locus. At all loci not linked to nNOS, however, these mice contain random combinations of 129/SV and C57BL/6J alleles, segregating independently. Common practice has been to compare the phenotype of mice homozygotic (–/–) and wild type (+/+) for the targeted gene in the F2 recombinant generation. Any phenotypic effect found to segregate with the targeted gene against the systematically randomized genetic background of these F2 mice is assumed to be due to the absence of that gene product. The experimental design is to randomize variables that cannot be controlled (here independently segregating genes) to allow effects associated with the controlled variable (the targeted gene) to emerge. In the behavioural study of the nNOSinactivated mice4, however, (and in an increasing number of similar studies) a ‘knockout colony’ was established by inbreeding nNOS –/– mice of the F2 recombinant generation. Unfortunately, there are no control mice against which the phenotype of such colonies can be assessed. Those used (inbred mice of the parental 129/SV and C57BL/6 strains, and the offspring of a cross between the knockout mice and inbred strains) differ systematically TINS Vol. 19, No. 7, 1996

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TO THE EDITOR from mice of the knockout colony. Randomizing the behavioural data by pooling the data obtained from each of the two inbred strains seems questionable given the genetic and related environmental differences between the mice. Indeed, mice of an inbred line that have been bred separately for more than 10 generations (about three years) diverge genetically to such an extent they become substrains7. Sufficient time elapsed in this study (and in many others) between isolation of the ES cells8 and behavioural analysis of the knockout colony4 to ensure that a mouse generated from the ES cells would be a different substrain to mice of the parental 129/SV strain. Our own experience in the analysis of mice in which the Thy-1 gene has been inactivated9 underlines these concerns. The Thy-1 knockout mice, also derived from a 129/SV ES cell line10 and outbred to C57BL/6 mice, were inbred like the nNOS mice to produce a ‘knockout’ colony. We then crossed these Thy-1 –/– mice with normal 129/SV mice, and interbred the offspring to obtain a second F2 recombinant generation. The failure of the Thy-1 –/– mice to support LTP in the dentate gyrus in vivo, first noticed in mice of the knockout colony, was found in all F2 Thy-1 –/– mice9. However, Thy-1 –/– mice of the knockout colony showed other effects (male aggression and weight gain, openfield activity) that were not recovered in the F2 recombinant generation. The ability to inactivate targeted genes in mice has revolutionized the study of the biological role of proteins in mammals. A living mouse, however, is a complex test system that poses problems for analysis. It seems essential that the practice of comparing –/– and +/+ littermates of an F2 recombinant generation be taken as the minimum acceptable control in assessing the consequences of gene inactivation. More sophisticated controls such as transgenic rescue of the inactivated gene11 and inducible gene inactivation12 are available, although these too are not without their pitfalls. These problems should not detract from the potential of this method to disclose new and unexpected aspects of how molecules make the mouse, but not every intriguing phenotype need be due to the lack of the targeted gene product.

Note added in proof Since submitting this correspondence, the issue of knockout genotype has been discussed in a most welcomed TINS debate13; several points discussed therein are exemplified by the analysis of the nNOS-deficient mouse. Roger Morris UMDS Guy’s Hospital, London Bridge, London, UK SE1 9RT.

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Marika Nosten-Bertrand LGN-CNRS UMR C9923, Hôpital de la Pitié Salpêtrière, 83 Bd de l’hôpital, Paris 75013, France. References

7

8

1 Good, M. (1996) Trends Neurosci. 19, 83–84 2 Huang, P.L. et al. (1993) Cell 75, 1273–1286 3 O’Dell, T.J. et al. (1994) Science 265, 542–546 4 Nelson, R.J. et al. (1995) Nature 378, 383–386 5 Plomin, R., Owen, M.J. and McGuffin, P. (1994) Science 264, 1733–1739 6 Takahashi, J.S., Pinto, J.H. and

Reply Morris and Nosten-Bertrand raise an interesting point regarding the use of transgenic knockout mice to study the role of genes in physiological and behavioral processes. A number of knockout mice have a mixed genetic background due to the breeding scheme employed to maintain the targeted allele. The first available animals are often heterozygous F1 mice between two inbred strains, 129/SV and C57BL/6. Most breeding schemes take advantage of the hybrid vigor of the F1 generation, and breed these heterozygous animals to produce the F2 generation. The F2 offspring will be segregating for all alleles that differ between the two parent strains, thus the mutant phenotype might be attributable to unknown, segregating modifier genes rather than the mutation alone1. As such, concerns are often raised about appropriate control mice. The ideal control is to breed chimeric mice to the original 129/SV founder strain, to produce mice that have an identical genetic composition to the 129/SV strain except for the lack of the gene of interest. Crossing chimeric mice to the 129/SV founder strain has it own problems. Many embryonic stem (ES) cells were derived several years ago from substrains that are not readily available today, or that might have genetically diverged from the ES cells in the intervening years. An added problem is that some 129 substrains are poor breeders and are thus difficult to maintain. Attempts to breed chimeric mice lacking the gene encoding neuronal nitric oxide synthase (nNOS) to the 129/SV founder strain were without success (P. Huang and M. Fishman, pers. commun.). Alternatively, one can transfer the mutant allele to another inbred background by backcrossing to an inbred strain. Heterozygous progeny from this mating are then backcrossed to mice of the inbred strain. This is repeated for several cycles. The more times this cycle is repeated, the more uniform the genetic background becomes. Ten generations of

9 10 11 12 13

Vitaterna, M.H. (1994) Science 264, 1724–1733 Hedrich, H.J. (1981) in The Mouse in Biomedical Research (Vol. I) (Foster, H.L., Small, J.D. and Fox, J.G., eds), pp. 159–176, Academic Press Li, E., Bestor, T.H. and Jaenisch, R. (1992) Cell 69, 915–926 Nosten-Bertrand, M. et al. (1996) Nature 379, 826–829 Stewart, C.L. et al. (1992) Nature 359, 76–79 Whittington, M.A. et al. (1995) Nat. Genet. 9, 197–201 Mayford, M., Abel, T. and Kandel, E.R. (1995) Curr. Opin. Neurobiol. 5, 141–148 Gerlai, R. (1996) Trends Neurosci. 19, 177–181

backcrossing are required for the strains to be considered congenic, and backcrossing must be continued for another 10–12 generations to ensure genetic uniformity for closely linked loci. In lieu of waiting three to four years before performing behavioral studies in the nNOS knockouts we opted for the next best control mice. As indicated by Morris and Nosten-Bertrand, one can generate a second F2 recombinant generation by crossing knockout mice with one of the parental strains. Any phenotype that was due to the original breeding scheme should segregate independently from the targeted allele, except for the most tightly linked loci. We did this in the case of the nNOS knockout mice, as indicated in Fig. 1 of our original report of these findings2. nNOS knockout mice of this F2 recombinant generation behave in a manner indistinguishable from the inbred nNOS knockout strain. Thus, the behavioral phenotype of the nNOS knockout mice is most likely to be due to the absence of the gene encoding nNOS, and not another segregating allele. Ted M. Dawson Depts of Neurology and Neuroscience, Johns Hopkins University, Baltimore, MD, USA.

Randy J. Nelson Dept of Psychology, Behavioral Neuroendocrinology Group, Johns Hopkins University, Baltimore, MD, USA.

Solomon H. Snyder Depts of Neuroscience, Pharmacology and Molecular Sciences, and Psychiatry and Behavioral Sciences, Johns Hopkins University, Baltimore, MD, USA. Selected references 1 Joyner, A.L., ed. (1993) Gene Targeting, A Practical Approach, Oxford University Press 2 Nelson, R.J. et al. (1995) Nature 378, 383–386

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