Abnormal Behavior Associated with a Point Mutation in the Structural ...

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Genetic and metabolic studies have been done on a large kindred in which several males are affected by a syndrome of borderline mental retardation and ...
Copyright © 1993 by the American Association for the Advancement of Science Volume 262(5133)

22 October 1993

pp 578-580

Abnormal Behavior Associated with a Point Mutation in the Structural Gene for Monoamine Oxidase A [Reports] Brunner, H. G.*; Nelen, M.; Breakefield, X. O.; Ropers, H. H.; van Oost, B. A. H. G. Brunner, Department of Human Genetics, University Hospital Nijmegen, Geert Grooteplein 20, 6525 GA Nijmegen, the Netherlands. M. Nelen, H. H. Ropers, B. A. van Oost, Department of Human Genetics, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, the Netherlands. X. O. Breakefield, Neuroscience Center, Massachusetts General Hospital, Charlestown, MA 02129. *To whom correspondence should be addressed.

Abstract Genetic and metabolic studies have been done on a large kindred in which several males are affected by a syndrome of borderline mental retardation and abnormal behavior.The types of behavior that occurred include impulsive aggression, arson, attempted rape, and exhibitionism. Analysis of 24-hour urine samples indicated markedly disturbed monoamine metabolism. This syndrome was associated with a complete and selective deficiency of enzymatic activity of monoamine oxidase A (MAOA). In each of five affected males, a point mutation was identified in the eighth exon of the MAOA structural gene, which changes a glutamine to a termination codon. Thus, isolated complete MAOA deficiency in this family is associated with a recognizable behavioral phenotype that includes disturbed regulation of impulsive aggression.

Studies of aggressive behavior in animals and humans have implicated altered metabolism of serotonin [1-7], and to a lesser extent dopamine [4,8,9], and noradrenaline [3-5,10-12]. These observations suggest that genetic defects in the metabolism of these neurotransmitters may affect aggressive behavior, but such mutations have not yet been reported. We have described a large kindred in which several males are affected by a syndrome of borderline mental retardation and exhibit abnormal behavior,

including disturbed regulation of impulsive aggression [13]. Obligate female carriers in this family have normal intelligence and behavior. The genetic defect for this condition was assigned to the p11-p21 region of the X chromosome, in the vicinity of the genes for MAOA and monoamine oxidase B (MAOB). Because MAOA and MAOB are known to metabolize serotonin, dopamine, and noradrenaline, we evaluated these patients for MAO deficiency. The MAOB activity is normal in affected males from this family [13]. To test the hypothesis that affected males in this family have selective MAOA deficiency, we established skin fibroblast cultures from three clinically affected males, two carrier females, and one noncarrier female [14]. Cultured human skin fibroblasts from normal individuals express both MAOA and MAOB activity in a ratio of about 80 to 90% to 10 to 20%, respectively, and the amounts of activity are stable from passage to passage during the proliferative growth phase [15]. Treatment of fibroblast cultures with dexamethasone produces a 6- to 14- fold increase in MAOA activity and a 2- to 3-fold increase in MAOB activity [16]. We assessed MAO activity in homogenates from skin fibroblast strains with a common substrate, tryptamine, at a concentration that favors MAOA measurement [17]. Strains from two normal controls were used that represent very low (strain GM2037) and moderately high (strain HF24) amounts of activity, on the basis of previous analyses of more than 30 control strains with activity amounts that spanned a range of 1 to 100 pmol/min per milligram of protein [14,18]. These controls were grown in parallel with fibroblasts from family members to minimize activity differences due to serum components [19]. Negligible amounts of apparent MAO activity were found in strains from three affected males in the presence or absence of dexamethasone Table 1. The amounts of activity in two carrier females and in one noncarrier female from the same family were in the low to moderate control range and, as in control strains, were increased by treatment with dexamethasone and inhibited by more than 90% by the selective MAOA inhibitor, clorgyline.

Table 1 .

MAO activity in cultured skin fibroblasts. For the detection of MAOA activity, cells were harvested at confluency (minus DEX) or after an additonal 7 to 9 days of exposure to 50 nM dexamethasone (plus DEX) as described [15]. Activity amounts for the affected males were all below detection limits (less than 30% above a blank that had no homogenate). All values are given as the average plus minus SD with the number of assays in parentheses.

To establish whether the lack of MAOA activity was caused by a mutation in the MAOA structural gene, we determined the coding sequence of the mRNA for MAOA by first-strand complementary DNA (cDNA) synthesis, polymerase chain reaction (PCR) amplification, and direct sequencing [20]. Four base substitutions were detected, three of which were neutral polymorphisms (G to T at position 941, T to A at position 1077, and T to C at positio n 1460). However, a nonconservative C to T mutation was found at position 936. This mutation changes a glutamine (CAG) codon to a termination (TAG) codon at position 296 of the deduced amino acid sequence [21] Figure 1. Amplification and sequencing of the eighth exon [22], which contains nucleotides 846 to 1005, confirmed the presence of the C to T mutation at nucleotide 936 in each of five clinically affected males and in two

obligatory heterozygotes. In contrast, the mutation could be excluded in 12 unaffected males in this family Figure 1. Two-point linkage calculations [23] between the clinical phenotype and the mutation in the MAOA gene reported here yield a lod score (logarithm of the likelihood ratio for linkage) of 3.55 without recombination.

Figure 1 .

Segregation of a mutation in the MAOA structural gene in a family with X-linked borderline mental retardation and prominent behavioral disturbance. All affected males and obligate carriers have a C to T mutation at nucleotide position 936. In 12 normal males, only the normal C is present.

These results document complete and selective deficiency of MAOA in affected males. Interestingly, MAOA activity in two carrier females was not different from that of a noncarrier female and two unrelated controls. Therefore, carrier females are not detectable by enzymatic activity in cultured fibroblasts. Whether this is due to high activity of the normal allele, incomplete X-inactivation, or other factors is unknown. Selective MAOA deficiency in this family results in a marked disturbance of monoamine metabolism. Increased urinary excretion of normetanephrine and tyramine and decreased concentrations of 5- hydroxyindole3-acetic acid (5-HIAA), homovanillic acid (HVA), and vanillylmandelic acid (VMA) have been documented by analysis of 24-hour urine samples [13]. Although measurements of cerebrospinal fluid metabolites are not available for this family, the urinary findings presumably reflect altered central neurotransmitter metabolism. Selective inhibition of MAOA in male rats has been shown to increase concentrations of noradrenaline, dopamine, and serotonin in the brain [24].

Five patients with X chromosomal deletions including MAOA and MAOB as well as the Norrie disease gene have been described that had severe mental retardation [25]. The relatively mild symptoms in males with selective MAOA deficiency, and the absence of psychiatric symptoms or mental retardation in two brothers with a complex deletion involving the Norrie disease gene and part of the MAOB structural gene that leaves MAOA intact [26], may reflect the overlapping substrate specificities and tissue distribution of the MAOA and MAOB isozymes. The behavioral phenotype in this family is characterized by borderline mental retardation and a tendency toward aggressive outbursts, often in response to anger, fear, or frustration. These behavioral responses have been noted in each of eight affected males for whom clinical data are available and have occurred in affected subjects living in different parts of the country at different times [13]. It should be stressed that the aggressive behavior varied markedly in severity and over time, even within this single pedigree. Other types of impulsive behavior that occurred in individual cases included arson, attempted rape, and exhibitionism. It has been postulated that aggression in animals can be subdivided into several subtypes [1]. In humans, impulsive aggression rather than premeditated aggression and violence has been linked to low concentrations of 5-HIAA in cerebrospinal fluid [2]. This observation is usually taken to indicate a reduction in central serotonergic function in impulsive aggression. Our data suggest that reduced 5HIAA concentrations may also be caused by absent MAOA activity in these subjects. Further studies are required to determine whether complete isolated MAOA deficiency is associated with similar behavioral patterns in other families, or even in animal models. Also, it is presently unclear whether all of the bioche mical alterations caused by the MAOA deficiency state are required to cause the apparent increase in liability to impulsive aggressive behavior. The inhibition of MAO has not been reported to cause aggressive behavior in adult humans [27] but deficiencies throughout life might have different consequences. Only limited data are available on MAO activity and aggression regulation in animals. MAO inhibition increased shockinduced aggression in male rats in one study [28]. Other studies of aggressive behavior have stressed the importance of reduced serotonergic transmission [1-7], increased dopaminergic transmissio n [4,8,9], or increased noradrenergic transmission [3-5,11,12] in animals as well as in humans. Another factor that could be involved in causing increased impulsive aggression is rapid eye movement (REM) sleep deprivation. MAOA inhibitors have been shown to suppress REM sleep in human subjects [29], whereas REM sleep deprivation increases shock- induced fighting in rats, especially in combination with dopaminergic stimulation [9]. Taken together, data obtained in this family suggest a relation between isolated complete deficiency of MAOA activity and abnormal aggressive behavior in affected males. This observation raises a number of important questions. First, the

frequency of MAOA deficiency in the population has to be determined. Second, given the wide range of variation of MAOA activity in the normal population [18], one could ask whether aggressive behavior is confined to complete MAOA deficiency. Third, animal models could help to determine the various neurochemical alterations that are induced by selective MAO A deficiency and their secondary effects on the organism. Such studies might also suggest possibilities for treatment of the metabolic disturbance caused by the MAOA deficiency state. Finally, the possibility of hypertensive crises in selective MAOA deficiency through increased sensitivity to dietary and pharmacologic amines has not yet been investigated.

REFERENCES AND NOTES 1. L. Valzelli, Pharmacol. Res. Commun. 14, 1 (1982); Psychobiology of Aggression and Violence (Raven, New York, 1981). 2. E. F. Coccaro, Br. J. Psychiatry 155 (suppl. 8), 52 (1989). 3. J. D. Higley et al., Arch. Gen. Psychiatry 49, 436 (1992). 4. L. R. P. Troncone and S. Tufik, Physiol. Behav. 50, 173 (1991). 5. G. L. Brown, F. K. Goodwin, J. C. Ballenger, F. P. Goyer, L. F. Major, Psychiatry Res. 1, 131 (1979). 6. M. Linnoila et al., Life Sci. 33, 2609 (1983); K. M. Kantak, L. R. Hegstrand, J. Whitman, B. Eichelman, Pharmacol. Biochem. Behav. 12, 173 (1980); M. J. P. Kruesi et al., Arch. Gen. Psychiatry 47, 419 (1990). 7. N. K. Popova, A. V. Kulikov, E. M. Nikulina, E. Y. Kozlachkova, G. B. Maslova, Aggressive Behav. 17, 207 (1991). 8. E. M. Nikulina, D. F. Avgustinovich, N. K. Popova, ibid. 18, 65 (1992). 9. S. Tufik, C. J. Lindsey, E. A. Carlini, Pharmacology 16, 98 (1978). 10. D. J. Reis, Assoc. Res. Nerv. Ment. Disord. 50, 266 (1972). 11. B. Eichelman and N. B. Thoa, Biol. Psychiatry 6, 143 (1973). 12. J. T. Winslow and K. A. Miczek, Psychopharmacology 81, 286 (1983). 13. H. G. Brunner et al., Am. J. Hum. Genet. 52, 1032 (1993). 14. After informed consent was obtained, skin fibroblasts were established from punch biopsies of family members. Normal control cell lines were chosen on the basis of very low activity (GM2037) and moderately high activity (HF24) *RF 18*. All strains were in the proliferative stage of growth and were grown in parallel in Dulbecco's modified essential medium with 10% fetal calf serum, penicillin, and streptomycin (Gibco, Paisley, Scotland). For measurement of MAO activity, cells were harvested at confluency or after an additional 7 to 9 days of exposure to 50 nM dexamethasone, as described *RF 15*. Cell homogenates from two or more harvests were sonicated, and the protein was determined by the method of Bradford *RF 30*.

15. S. B. Edelstein, C. M. Castiglione, X. O. Breakefield, J. Neurochem. 31, 1247 (1978); X. O. Breakefield, S. B. Edelstein, M. H. Grossman, J. P. Schwartz, in Genetic Research Strategies in Psychobiology and Psychiatry, E. S. Gershon, S. Matthysse, X. O. Breakefield, R. D. Ciaranello, Eds. (Boxwood, Pacific Grove, CA, 1981), pp. 129-142. 16. S. B. Edelstein and X. O. Breakefield, Cell. Mol. Neurobiol. 6, 121 (1986). 17. We measured MAO activity by the toluene extraction procedure in fibroblast homogenates with 100 mu g of protein per assay *RF 16*. All assays were done in triplicate within the range of linearity for time. A buffer blank was used routinely, but in some cases additional blanks of 10 sup minus 6 M clorgyline (Sigma, St. Louis, MO) or 10 sup minus 6 M deprenyl were included. For a common substrate we used 30 mu M [ethyl-sup 3 H]tryptamine (35 Ci/mmol, New England Nuclear, Boston, MA) for both MAOA and MAOB activity, although at this concentration MAOA is favored by its higher substrate affinity. Values obtained in these experiments for control cell strains were about sevenfold those of previous studies *RF 14,31*. This is probably because of the different lots of serum used and the effects of hormones contained in serum on MAO activity *RF 19*. 18. G. S. Hotamisligil and X. O. Breakefield, Am. J. Hum. Genet. 49, 383 (1991). 19. S. B. Edelstein and X. O. Breakefield, Biochem. Biophys. Res. Commun. 98, 836 (1981). 20. We isolated RNA from fibroblasts by acid guanidinium thiocyanate-phenol-chloroform extraction *RF 32* with a commercially available kit (Campro Scientific, Elst, the Netherlands). First-strand cDNA was synthesized from 2.5 mu g of RNA with oligo-(dT) and random primers with the GeneAmp kit (Perkin-Elmer, Branchburg, NJ). Two overlapping PCR fragments were prepared essentially as described *RF 18*. Both strands from the 5 prime 867-base pair (bp) PCR fragment were sequenced as described *RF 33*, with the PCR primers and two internal primers, corresponding to nucleotide positions (np) 483 to 502 of the MAOA cDNA sequence *RF 21* in both directions. The 1010-bp 3 prime PCR fragment was sequenced with the PCR primers and four internal primers [np 1177 to 1206 in both the 5 prime-3 prime and the 3 prime-5 prime directions, np 1382 to 1401 (forward), and np 1485 to 1505 (reverse)]. The sequence was determined on both strands except for the 300 3 prime base pairs of the 1010-bp PCR fragment. 21. Y.-P. P. Hsu et al., Neurochemistry 51, 1321 (1988); A. W. J. Bach et al., Proc. Natl. Acad. Sci. U.S.A. 85, 4934 (1988); Z.-Y. Chen et al., Nucleic Acids Res. 19, 4537 (1992). 22. Genomic DNA was amplified with primers corresponding to np 874 to 892 (forward) and 987 to 1004 (reverse). Both primer sequences are derived from exon 8 *RF 21*, and both strands were sequenced. 23. We calculated the genetic linkages with the MLINK program from the Linkage program package (version 5. 03) *RF 34*. 24. A. J. Sleight, C. A. Marsden, K. F. Martin, M. G. Palfreyman, Br. J. Pharmacol. 93, 303 (1988); P. Blier, C. De Montigny, A. J. Azzaro, J. Pharmacol. Exp. Ther. 237, 987 (1986); A. J. Greenshaw, T. S. Rao, A. J. Nazarali, G. B. Baker, R. T. Coutts, Biol. Psychiatry 25, 1014 (1989); B. Morden et al., Physiol. Behav. 3, 425 (1968). 25. A. de la Chapelle, E. M. Sankila, M. Lindl"f, P. Aula, R. Norio, Clin. Genet. 28, 317 (1985); E. M. Bleeker-Wagemakers, I. Zweije-Hofman, A. Gal, Ophthalmic Paediatr. Genet. 9, 137 (1988); D. Donnai, R. C. Mountford, A. P. Read, J. Med. Genet. 25, 73 (1988); D. Zhu et al., Am. J. Med. Genet. 33, 485 (1989); F. C. Collins et al., ibid. 42, 127 (1992). 26. W. Berger et al., Nat. Genet. 1, 199 (1992).

27. D. L. Murphy, N. A. Garrick, R. M. Cohen, in Antidepressants, G. D. Burrows, T. R. Norman, B. Davies, Eds. (Elsevier Science, New York, 1983), pp. 209-227; D. Pickar, D. L. Murphy, R. M. Cohen, I. C. Campbell, S. Lipper, Arch. Gen. Psychiatry 39, 535 (1982). 28. B. Eichelman and J. Barchas, Pharmacol. Biochem. Behav. 3, 601 (1975). 29. R. M. Cohen et al., Psychopharmacology 78, 137 (1982). 30. M. M. Bradford, Anal. Biochem. 72, 248 (1976). 31. K. B. Sims et al., Neuron 2, 1069 (1989). 32. P. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156 (1987). 33. A. M. W. van den Ouweland et al., Nat. Genet. 2, 99 (1992). 34. C. M. Lathrop and J. M. Lalouel, Am. J. Hum. Genet. 36, 460 (1984). 35. We are grateful to J. Knoll for supplying deprenyl and B. Jansen and C. Fleet for technical assistance. We also thank Z. Y. Chen, C. Shalish, and B. Tivol of X.O.B.'s laboratory. Supported by NIH grant NS 21921 (X.O.B.).

Copyright © 1995 by the American Association for the Advancement of Science Volume 268(5218)

23 June 1995

pp 1763-1766

Aggressive Behavior and Altered Amounts of Serotonin and Norepinephrine in Mice Lacking MAOA

Brain

[Reports] Cases, Olivier; Seif, Isabelle*; Grimsby, Joseph; Gaspar, Patricia; Chen, Kevin; Pournin, Sandrine; Muller, Ulrike; Aguet, Michel; Babinet, Charles; Shih, Jean Chen; De Maeyer, Edward O. Cases, I. Seif, E. De Maeyer, Centre National de la Recherche Scientifique (CNRS), Unite de Recherche Associee (URA) 1343, Institut Curie, 91405 Orsay, France. J. Grimsby, K. Chen, J. C. Shih, Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, CA 90033, USA. P. Gaspar, Institut National de la Sante et de la Recherche Medicale, Unite 106, H"pital de la Salpntriere, 75651 Paris, France. S. Pournin and C. Babinet, CNRS, URA 361, Institut Pas teur, 75724 Paris, France. U. Muller, Institute of Molecular Biology I, University of Zurich, 8093 Zurich, Switzerland. M. Aguet, Genentech, South San Francisco, CA 94080, USA. *To whom correspondence should be addressed.

Abstract Deficiency in monoamine oxidase A (MAOA), an enzyme that degrades serotonin and norepinephrine, has recently been shown to be associated with aggressive behavior in men of a Dutch family.A line of transgenic mice was isolated in which transgene integration caused a deletion in the gene encoding MAOA, providing an animal model of MAOA deficiency. In pup brains, serotonin concentrations were increased up to ninefold, and serotonin- like immunoreactivity was present in catecholaminergic neurons. In pup and adult brains, norepinephrine concentrations were increased up to twofold, and cytoarchitectural changes were observed in the somatosensory cortex. Pup behavioral alterations, including trembling, difficulty in righting, and fearfulness were reversed by the serotonin synthesis inhibitor parachlorophenylalanine. Adults manifested a distinct behavioral syndrome, including enhanced aggression in males.

In the debate surrounding advances in genetic research on aggressive behavior [1], transgenic animal models carrying single gene defects are of critical importance. We describe transgenic mice lacking MAOA as a result of the integration of an interferon beta (IFN-beta) transgene into the gene encoding MAOA, and report

that MAOA-deficient males show increased aggressiveness. MAOA and monoamine oxidase B (MAOB) are mitochondrially located enzymes with overlapping substrate specificities and tissue distributions. They inactivate neuroactive amines such as serotonin, dopamine, and norepinephrine. MAOA and MAOB are encoded by separate genes that are closely linked on the X chromosome, and they share 70% similarity in amino acid sequence [2]. The loss of both MAO genes may be implicated in the severe mental retardation of some patients with Norrie disease [3], and recently a family has been described in which a point mutation in the gene encoding MAOA abolishes MAOA catalytic activity and is associated with impulsive aggression [4]. We generated mice transgenic for IFN-beta by injecting an IFN-beta minigene into one-cell embryos of C3H/HeJ (C3H) mice. The minigene was devised to evaluate the potential of the gene encoding IFN-beta for antiviral gene therapy. We describe the transgenic line Tg(H2-IFN-beta)8 (Tg8), which was initially characterized by the X- linked recessive abnormal behavior of mouse pups. By Southern (DNA) blot hybridization and polymerase chain reaction (PCR), a single minigene copy was found in Tg8 DNA [5]. This transgene was transmitted by X- linked inheritance. By reverse transcriptase- mediated PCR (RT-PCR), IFNbeta RNA was detected in the testis and spleen but not in the brain. Because this suggested that the abnormal behavior of Tg8 mice was not due to IFN-beta, we examined the behavior of the F2 progeny of Tg8 females mated to knockout males lacking the IFN-beta receptor [6]. F2 and F3 pups having the IFN-beta transgene but no IFN-beta receptor displayed the same abnormal behavior as Tg8 pups and were used to rule out the possibility of IFN-beta affecting the phenotypic features of Tg8 mice. By inverse PCR [7] done on Tg8 DNA with transgene primers, we obtained 2.0 kb of the two genomic regions flanking the transgene. We then isolated the junctions by inverse PCR done on C3H DNA with primers derived from the flanking regions, and it appeared that transgene integration had caused a genomic deletion of at least 1 kb. The 2-kb flanking sequences and 1-kb deleted sequences were not found in the GenBank and European Molecular Biology Laboratory databases but were mapped to a 3- million-base pair (bp) X-chromosome region containing several genes [8], including the genes encoding MAOA and MAOB. RT-PCR done on total RNA from Tg8 spleen or testes, with two primers derived from rat MAOA exons 1 and 8 [9], showed that the MAOA RNA of Tg8 mice, instead of occurring as a single species as did the MAOA RNA of C3H mice, consisted of at least four smaller species Figure 1A. PCR on Tg8 DNA indicated that exons 2 and 3 were missing. The deletion encompassing MAOA exons 2 and 3 was estimated by Southern blot analysis to span 17 kb.

Figure 1. MAOA RNA of Tg8 mice. (A) Representation of the Tg8 gene encoding MAOA between exons 1 and 8 (the gene for MAOA probably has 15 exons) and structure of the four species of MAOA RNA detected by RT -PCR. The species containing IFN-beta sequences (an exon of 199 bp) results from splicing events between legitimate MAOA splice sites and cryptic IFN-beta splice sites (SA and SD, acceptor and donor sites present in antisense IFN-beta RNA). (B) Sequence of SA and SD, and sequence of MAOA exon 2 acceptor splice site. The MAOA exon 2 acceptor has a strong putative branch site sequence (TGTTAAC), as opposed to the cryptic acceptor SA. (C) C3H MAOA sequences obtained by RT-PCR between exons 1 and 8. The Tg8 RNA splice between exons 1 and 4 causes a reading frame shift, and the IFN-beta exon of the hybrid RNA species does not restore the normal reading frame. Internal initiation of translation may occur at ACAATG within exon 4.

Any truncated polypeptide that could be synthesized from the various Tg8 MAOA RNA species (Fig. 1C) was expected to be devoid of catalytic activity, because MAO exon 2 encodes part of the beta alpha beta unit that is required to position the cofactor FAD [10]. Using serotonin as a substrate, we found that MAOA activity was abolished in the brain and liver Table 1. In contrast, MAOB activity on phenylethylamine was not altered [11].

Table 1. MAOA activity in the brain and liver of Tg8 and C3H mice. MAOA activity (in nanomoles per 20 min per milligram of protein) was determined by use of14 C-labeled serotonin (5-HT) as substrate [23] and, when necessary, by addition of 10minus 6 M L-deprenyl (dep) to inhibit interference by MAOB, which was abundant in liver tissue (as compared with MAOA, MAOB has less affinity for 5-HT and greater affinity for L-deprenyl). Each value corresponds to one mouse (average of a duplicated sample, with less than 10% variation). Similar results were obtained with other sets of mice of various ages (8 to 400 days old).

All Tg8 pups displayed the same pattern of altered behavior, which varied with age. Chronic administration of the serotonin synthesis inhibitor parachlorophenylalanine (PCPA) [300 mg per kilogram of body weight per day, subcutaneous (SC)] reversed to normal all the behavioral traits of Tg8 pups, whereas none of the traits were modified by the catecholamine synthesis inhibitor alpha-methylparatyrosine (300 mg/kg per day, SC). In newborns, the first sign of abnormal behavior was intense head nodding. Between days 5 and 10, the pattern included (i) trembling upon locomotion and suspension by the tail, (ii) prolonged righting Figure 2A, (iii) moving backward instead of pivoting when placed on a new surface, and (iv) prolonged and stronger reactions to pinching. These behaviors could be reproduced in C3H pups, but with less intensity, by daily injection of the MAOA inhibitor clorgyline (30 mg/kg per day, SC). Between days 11 and 16, Tg8 mice showed (i) frantic running and falling over, jumping, or prompt digging to hide under woodshavings in response to moderate sound and movement; (ii) sleep accompanied by violent shaking and jumps causing frequent dispersion of littermates; (iii) propensity to bite the experimenter; (iv) hunched posture; and (v) bat and ball postures with hindlimb crossing upon suspension by the tail [12]. Postural traits were well reproduced in 12-day-old C3H pups by a single injection of clorgyline, whereas the other behaviors, particularly auditory startle, were not fully obtained even when clorgyline was administered from birth (30 mg/kg per day).

Figure 2. Behavioral alterations of Tg8 mice. (A) Righting response. Pups were separated from their dam, isolated in a bare cage at 20 degrees C for 3 min to wake them up, and placed on their backs. The graph gives the mean time for the pups to turn over with an upper limit of 1 min (mean of the mean of five consecutive time points for each pup plus minus SEM). Tg8 pups turned over with forceful movements and excitation, whereas C3H pups acted calmly. (B) Aggression between Tg8 male cage mates. The plot represents a 1-day survey of skin wounds in 2- to 7-month-old males housed in groups from the time of weaning (12 cages; in the one cage of unwounded 4-month-old mice, two individuals were wounded 2 weeks later). In the control survey, no wounded individuals were found among 2- to 8-month-old C3H males housed in groups from the time of weaning (173 males in 22 cages). Skin wounds were not present in Tg8 and C3H female groups. (C) Latency to the first appearance of biting attack in resident-intruder tests after a long period of breeding. Each 6-month-old resident was given a single 10-min encounter in his home cage with a 2-month-old C3H intruder. The 30 Tg8 residents and 30 C3H residents were housed with a female from the age of 2 months and reared several litters. The 60 intruders were housed in groups of 10 from the time of weaning; attack latency (mean plus minus SEM) of Tg8 and C3H residents was 74 plus minus 10 s and 237 plus minus 18 s, respectively (t test; t sub (58) equals 7.90, P less than 0.0001). (D) Latency to the first appearance of biting attack in resident-intruder tests after a period of isolation. Tg8 and C3H males were individually housed for 5 weeks beginning at weaning and were given single 3-min encounters (a short time to allow further testing) in their home cages with 2-month-old C3H intruders. Attack latency of Tg8 and C3H residents was 110 plus minus 12 s and 172 plus minus 4 s, respectively (P less than 0.0001). One month later, the residents were given single 10-min encounters in their home cages with 2-month-old BALB/cJ intruders: Attack latency of Tg8 and C3H residents was 27 plus minus 6 s and 252 plus minus 35 s, respectively (P less than 0.0001).

Adult mice had a different pattern of behavioral alterations. Males housed in groups from the time of weaning showed signs of offensive aggressive behavior [13] (Fig. 2B), notably bite wounds on genitals and rump, which were most apparent from the age of 3 months (areas scabbed over, with fur missing; in 6- to 12-week-old individuals, wounds were detected by palpation). We investigated aggression in Tg8 males in two different resident-intruder tests (Fig. 2, C and D). Tg8 resident males attacked the intruder faster than did C3H residents. In the interval preceding the onset of attack, C3H residents displayed intense social investigation and home cage checking, whereas Tg8 residents adopted a static, hunched, fluffed-fur posture after the first olfactory stimulus. Alterations were also seen in mating behavior. When 2- month-old virgin Tg8 males were tested individually for 30 min with a nonreceptive virgin C3H female, the courtship differed from that of C3H males in that it was disrupted by episodes of grasping [12 plus minus 2 grasping events (mean plus minus SEM, n equals 5)] that were reflected by increased frequency and intensity of female squeaking (113 plus minus 20 versus 28 plus minus 8 squeaks, P less than 0.001). In the Porsolt's swim test [14], Tg8 adults, instead of mostly floating, made persistent attempts to escape, which corresponds to what is observed when normal mice receive MAO inhibitors and other antidepressants. For 9-week-old mice, the time spent immobile in water, in a test of 4 min (after 2 min for habituation), was 200 plus minus 7 s for C3H females (mean plus minus SEM, n equals 15), 84 plus minus 12 s for Tg8 females, 156 plus minus 12 s for C3H males, and 37 plus minus 8 s for Tg8 males (P less than 0.001). In the open field test as described by Chen et al. [1], Tg8 adults stayed a longer time in the center, with much hesitation as to which direction to take, and it remains to be determined whether this behavior corresponds to sensory or cognitive deficits or to reduced fear. For 12-week-old mice, the time spent in the center of the field was 10 plus minus 2 s for C3H females (mean plus minus SEM, n equals 13), 93 plus minus 34 s for Tg8 females, 20 plus minus 4 s for C3H males, and 118 plus minus 27 s for Tg8 males (P less than 0.001). In the beam-walking test, Tg8 adults grasped the edge of the beam with hindlimbs while walking [15], whereas C3H adults were sure- footed (it should be noted that adult Tg8 and C3H mice are blind, as they carry a retinal degeneration gene). A particular behavioral test to distinguish MAOA-deficient adults from normal adults consisted in giving an injection of L-deprenyl [15 mg/kg, intraperitoneal (IP)] or lazabemide (60 mg/kg, IP) [16], two MAOB inhibitors differing in several pharmacological properties. These treatments did not overtly affect C3H behavior but caused, in Tg8 adults, restlessness and attentional deficit, which disrupted social interaction, feeding, and self- grooming, beginning within 2 hours after injection and lasting for at least 3 hours [17]. In the rodent brain, MAOB activity is low at birth and increases during the first month (partly due to gliogenesis), whereas MAOA activity is close to adult

amounts from the first week on [18]. This developmental difference may explain why the phenotype of Tg8 mouse pups was more severe than that of adults, and why L-deprenyl did not aggravate the behavioral traits of 12-day-old Tg8 mice. Comparison of the amount of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) in Tg8 and C3H brains showed an increase in all three amines in Tg8 brains Figure 3. The elevation in DA was slight, although the DA metabolite dihydroxyphenylacetic acid was markedly decreased (3.5 times less at 3 months). The amount of 5-HT was considerably increased in Tg8 pups (ninefold at day 1 and sixfold at day 12) and returned to normal in older mice, whereas the amount of the 5-HT metabolite 5-HIAA (probably produced by MAOB) was considerably decreased in Tg8 pups and returned to normal in older mice. The MAOB inhibitor L-deprenyl (15 mg/kg, IP) elicited within 3 hours a greater increase in 5-HT in Tg8 than in C3H brains (2.5- fold versus 1.2-fold in 4-monthold males) and caused the near disappearance of 5-HIAA in Tg8 brains but no decrease in 5-HIAA in C3H brains. This suggests that the role of MAOB in 5-HT oxidation in the brain in vivo is more important than can be predicted from in vitro tests Table 1.

Figure 3. Amounts of 5-HT, 5-HIAA, DA, and NE in whole brains from Tg8 and C3H mice. Values from HPLC assays [24] are expressed in picogram per milligram of wet brain and represent the mean plus minus

SEM (n equals 4, 5, 7, 8, 4, 4, and 2). Mice were of both sexes and various ages (except for the 3-month-old mice, which were males only) and were housed in groups according to sex beginning at weaning (except for the 7-month-old mice, which were breeding pairs).

In 7-day-old Tg8 pups, the density of fibers stained by 5-HT immunochemistry was enhanced in several brain regions (such as the striatum, cerebral cortex, and the hilus of the dentate gyrus), and 5-HT- like staining was abnormally present in catecholaminergic neurons of the locus coeruleus and nigral complex (A8, A9, and A10) Figure 4, D and E). These neurons showed no immunoreactivity to the 5HT-synthesizing enzyme tryptophan hydroxylase (TPH), and during chronic administration of the TPH inhibitor PCPA (300 mg/kg per day, SC), they did not remain immunoreactive to antibody to 5-HT. Thus, the presence of 5-HT immunoreactivity probably corresponds to 5-HT uptake [19] and the likely absence of MAOB in this category of neurons [20]. In contrast, in 10-week-old Tg8 mice, the distribution and density of 5-HT- immunoreactive cell bodies and fibers appeared normal. One of the possibilities to explain the difference between pups and adults is that 5-HT may be captured by MAOB-rich cells in adults (such as mature glial cells) [20].

Figure 4. Abnormal 5-HT immunostaining in 7-day -old Tg8 brains. (A through C) Coronal sections of C3H brain. (D through F) Corresponding sections of Tg8 brain. 5-HT polyclonal antibody was used at a dilution of 1:15,000 and was revealed by a streptavidin-biotin-peroxidase complex and by diaminobenzidine. In the locus coeruleus (lc) [(A) and (D)], substantia nigra (sn), and ventral tegmental area (vta) [(B) and (E)], 5-HTlike immunoreactivity is present in terminal fibers and medial forebrain bundle (mfb) in C3H and Tg8 mice but is abnormally present in cell bodies in Tg8 mice [arrows in (D) and (E)]. Four different 5-HT antisera (three polyclonal and one monoclonal) revealed the same staining pattern. Double immunofluorescent staining for 5-HT and the catecholamine synthesizing enzyme tyrosine hydroxylase showed that these 5-HTlike immunoreactive cell bodies were catecholaminergic. In the somatosensory cortex [(C) and (F)], clusters of 5-HT-immunoreactive fibers delineate barrels in layer IV in C3H mice (C), whereas they form a continuous band in layer IV in Tg8 mice (F). Scale bar, 225 mu m.

It is well documented that in the somatosensory cortex of normal pups, serotonergic afferents show a pattern that coincides with cylindrical aggregates (barrels) of granule cells in layer IV [21]. Instead of this disjunctive pattern (Fig. 4C), the somatosensory cortex of Tg8 pups showed a continuous band of 5-HT immunostaining (Fig. 4F). In Tg8 pups and adults, Nissl and cytochrome oxidase chemistry revealed a complete absence of barrelfield in the somatosensory cortex, whereas barrelettes were present in the trigeminal and thalamic nuclei bridging the sensory periphery to the cortex [11]. Neonatal administration of PCPA (300 mg/kg per day, SC) partly restored the capacity to form cortical barrels. A role for serotonin in barrelfield formation could be considered because, for example, thalamic afferents in the barrels of normal pups express large amounts of 5-HT1B receptors [22]. Tg8 pup cortices that were stained for the 5-HT (1B) receptors did not show the barrel pattern that was found in C3H cortices [11]. It will be interesting to check for the presence of the barrelfield in the progeny of Tg8 mice mated to diverse 5-HT receptor knockouts. This study shows that MAOA-deficient mouse pups have a dramatically altered serotonin metabolism and severe behavioral alterations, both phenomena being linked. The behavioral traits of adults may be related to persisting defects in monoamine metabolism or to structural alterations such as the one we demonstrated in the cerebral cortex, an issue that pharmacological interventions may help to clarify. The finding that MAOA-deficient males with a C3H/HeJ genetic background display enhanced aggression under standard rearing conditions supports the idea that the particularly aggressive behavior of the few known human males lacking MAOA is not fostered by an unusual genetic background or complex psychosocial stressors but is a more direct consequence of MAOA deficiency.

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b

6. U. Muller et al., Science 264, 1918 (1994). 7. H. Ochman, A. S. Gerber, D. L. Hartl, Genetics 120, 621 (1988). Inverse PCR was done without prior size fractionation and isolation of restriction fragments.

8. This is a region between the DXHS32 and A-raf loci [S. D. M. Brown et al., Mamm. Genome 4, S269 (1993)]. 9. T. Kuwahara, S. Takamoto, A. Ito, Agric. Biol. Chem. 54, 253 (1990). 10. J. Grimsby et al., Proc. Natl. Acad. Sci. U.S.A. 88, 3637 (1991); R. K. Wierenga et al., J. Mol. Biol. 187, 101 (1986). 11. O. Cases et al., unpublished data. 12. I. Q. Whishaw, T. Schallert, B. Kolb, J. Comp. Physiol. Psychol. 95, 85 (1981). 13. S. C. Maxson, in Techniques for the Genetic Analysis of Brain and Behavior: Focus on the Mouse, D. Goldowitz, D. Wahlstein, R. E. Wimer, Eds. (Elsevier, Amsterdam, 1992), pp. 349-373. 14. R. D. Porsolt, A. Bertin, M. Jalfre, Arch. Int. Pharmacodyn, 229, 327 (1977); F. Borsini and A. Meli, Psychopharmacology 94, 147 (1988). 15. B. Kolb and I. Q. Whishaw, Can. J. Psychol. 37, 211 (1983). 16. W. E. Haefely et al., Adv. Neurol. 53, 505 (1990). 17. S. C. Gerson and R. J. Baldessarini, Life Sci. 27, 1435 (1980). 18. D. Tsang, K. P. Ho, H. L. Wen, Dev. Neurosci. 8, 243 (1986); M. Strolin Benedetti, P. Dostert, K. F. Tipton, Dev. Pharmacol. Ther. 18, 191 (1992); Y. Koide and K. Kobayashi, Neurochem. Res. 9, 595 (1984). 19. W. Lichtensteiger et al., J. Neurochem. 14, 489 (1967); E. Shaskan and S. H. Snyder, J. Pharmacol. Exp. Ther. 175, 404 (1970); H. W. M. Steinbusch, A. A. J. Verhofstad, H. W. J. Joosten, M. Goldstein, in Cytochemical Methods in Neuroanatomy, V. Chan-Palay and S. L. Palay, Eds. (Liss, New York, 1982), pp. 407-421; J. A. Wallace et al., Brain Res. Bull. 9, 117 (1982). 20. P. Levitt, J. E. Pintar, X. O. Breakefield, Proc. Natl. Acad. Sci. U.S.A. 79, 6385 (1982); K. N. Westlund, R. M. Denney, L. M. Kochersperger, R. M. Rose, C. W. Abell, Science 230, 181 (1985). In the normal brain, most catecholaminergic neurons of the locus coeruleus and nigral complex are rich in MAOA. 21. T. Woolsey and H. Van der Loos, Brain Res. 17, 205 (1970); R. J. D'Amato et al., Proc. Natl. Acad. Sci. U.S.A. 84, 4322 (1987); R. W. Rhoades et al., J. Comp. Neurol. 293, 190 (1990); M. C. OsterheldHass et al., Dev. Brain Res. 77, 189 (1994). 22. C. A. Bennett-Clarke et al., Proc. Natl. Acad. Sci. U.S.A. 90, 153 (1993). 23. H.-F. Wu, K. Chen, J. C. Shih, Mol. Pharmacol. 43, 888 (1993). 24. For high-performance liquid chromatography (HPLC) assays, whole brain was homogenized by sonication in 0.1 M HCIO containing 0.2% Na S O .In the first assay, the HPLC system was composed of a 5-mu m Ultrosphere column (Beckman, Fullerton, CA) and an LC-2A electrochemical detector (Bioanalytical Systems, West Lafayette, IN), and the mobile phase was an 80:20 mixture (pH 2.9) of 100 mM Na H PO and methanol, with 2.75 mM octane sulphonate, 0.1 mM EDTA, and 0.25 mM triethylamine. In the second assay (to refine NE determination), the HPLC system was a Coulochem II ESA with an HR-80 catecholamine column (ESA, Bedford, MA), and the mobile phase 4

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was an 88:6:6 mixture (pH 5.2) of 75 mM Na H PO , methanol, and acetonitrile, with 2.75 mM octane sulphonate, 0.02 mM EDTA, and 0. 7 mM triethylamine. 2

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25. We thank D. Herva, J.-P. Tassin, J. Adam, B. Courtier, L. Eusebe, and D. Haranger for participation; P. Avner for the DNA backcross panel; M. Da Prada for lazabemide; and R. Hen, M. Jouvet, J. P. Changeux, I. Gresser, L. Abbott, E. Lauret, V. Vieillard, R. Zawatzky, and J. De Maeyer-Guignard for discussion. Supported by CNRS, the Agence Nationale de Recherches sur le Sida, the Association pour la Recherche sur le Cancer, and National Institute of Mental Health grants RO1 MH 37020, R37 MH 39085, and K05 MH 00796.