THE GENETICS OF SOUND INDUCED SEIZURE IN INBRED MICE'

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1 THE GENETICS OF SOUND INDUCED SEIZURE IN INBRED MICE' K. SCHLESINGER, R. C. ELSTON AND W. BOGGAN Departments of Psychology and Biostatistics, and the Genetics Curriculum, Uniuersity of North Carolina, Chapel Hill Received January 13, 1966 USCEPTIBILITY to audiogenic seizure in mice (HALL 1947; MILLER, GINS- BURG and POTAS 1952) and rats (MAIER and GLASER 1940; MAIER 1943) has been shown to depend on the genotype of the animal. Different investigators have suggested different modes of inheritance for sound induced seizure including single-gene ( WITT and HALL 1949), two-gene ( GINSBURG and MILLER 1963) and polygenic modes of inheritance (FULLER and THOMPSON 1960). SCHLESINGER, BOGGAN and FREEDMAN (1965) have suggested that some of these discrepancies may depend on the particular phenotype used as an index of seizure, i.e., whether wild running, clonic, or tonic seizures are used as indices. Another variable that could determine the outcome of such analyses is the exact age of the animal at the time of the seizure test, since age is known to be a major factor in determining susceptibility within a genotype (FULLER 1962; SCHLESINGER et al. 1965). The purpose of the experiments reported here is to investigate further the mode of inheritance of susceptibility to audiogenic seizure, using statistical methods developed by ELSTON (1966) and paying close attention to these two variables, i.e., to the phenotype used as an index and to the age of the animals. HUFF and HUFF (1962) and HUFF and FULLER (1964) have suggested that the dilute gene (d) might be involved in determining susceptibility to audiogenic seizure in the mouse. COLEMAN (1960) has postulated a mechanism that might account for the effects of the d locus on audiogenic seizures: dilute strains of mice are deficient in phenylalanine hydroxylase activity and excrete abnormal phenylalanine metabolites such as phenylacetic acid. Since phenylacetic acid inhibits decarboxylating reactions ( SANDLER and CLOSE 1959; HANSON 1958) d/d strains of mice might be expected to have low levels of gamma-aminobutyric acid, norepinephrine, and serotonin in the brain; and this might, in turn, account for the high incidence of seizures typically observed in these mice. SCHLESINGER et al. (1965), in an attempt to test this hypothesis, were able to show that DBA/2J mice, a d/d strain with high seizure-incidence at a certain age, had lower levels of norepinephrine and serotonin in brain than nonsusceptible mice, but only at an age which corresponded to the period of maximal seizure risk for the DBA/BJ mice. A secondary purpose of these experiments, therefore, was to test whether the d locus is indeed important in audiogenic seizure; for this purpose single-gene mutants on DBA/2J background were also used. (Breeding pairs of this mutant stock were kindly supplied by DR. E. RUSSELL, The Jackson Laboratory, Bar This reseaich waa suppoi ted in part by Research Grant MH from the National Institute of Mental Health Genetics 54: 05-3 July 1966.

2 96 K. SCHLESINGER et al. Harbor, Maine. This mutant stock consists of DBA/2J (d/d) mice in which a mutation (reversion) to full coat color () had occurred.) MATERIALS AND METHODS Subjects: Breeding pairs from strains DBA/2J (d/d) and C57BL/6J (D/D), purchased from The Jackson Laboratory, Bar Harbor, Maine, were used. The origin and degree of inbreeding of these mice has been already described (JAY 1963). These animals were bred in our laboratories, and F,, F,, and backcross generation animals were obtained. Birth dates were noted and all animals used in these experiments were either 21 or 28 days of age to the nearest day. Experimental Method. Animals were tested for susceptibility to audiogenic seizure using methods previously described (SCHLESINGER et al. 1965). They were taken from their home cages one at a time, brought to another room, placed into a large chromatography jar (height, 17 inches; diameter, 11 inches), and given seconds to adapt. A five-inch electric bell mounted over the chromatography jar was sounded for 90 seconds. The animals were observed during this period, and records made of the incidences of the wild running phase, and clonic and tonic seizures. In those cases in which the full tonic seizure occurred the bell was immediately turned off, and in the later experiments records were made of whether the seizure was lethal. RESULTS The basic data, consisting of the results of three different measures of the phenotype at two different ages at which the response was measured, are given in Tables 1 and 2. First analysis: For each of the phenotypes the method described by ELSTON (1966) was used to determine whether a one-locus hypothesis was compatible with the data. If only one locus is involved, and if the two parent strains are homozygous at that locus, only three genotypes are possible: genotype for C57BL/6J, having a probability a, of responding; genotype for F,, having a probability a, of responding; genotype for DBA/2J, having a probability a2 of responding. The modified minimum x2 estimates of a,, a,, and a2 for each of the phenotypes are given in Table 3; any estimate less than 0.01 has been replaced TABLE 1 Numbers of 21-day old mice responding by wild running, clonic, or tonic seizure Genotype Number tested DBA/2J 37 DBA/2J x F, 60 F, F2 60 C57BL/6J X F, C57BL/6J Wild running -~ 36 (37.00) 54 (50.52) 12 (.26) 4.0 (35.52) 3 (.26) 0 (0) Number responding' Clonic Tonic 33 (37.00) 34 (35.) 47 (33.84) 39 (.87) 3 (1.92) 3 (1.25) 18 (18.84) (16.65) 0 (1.92) 0 (1.25) 0 (0) 0 (0) Chi-square for goodness of fit+ 11.6(3df).5(3df), 12.5(2df) 9.8(3df), 5.6(ldf) Probability on a one-locus hypothesis P <.01 P <.005 P <,005 P <.05 P <.05 ' Numbers in parentheses are expected values under a one-locus, two-allele, hypothesis. Italicized numbers are small expected values that tend to inflate the chi-square value. t Numbers in parentheses are degrees of freedom. Where two chi-square values are given, the first includes and the second excludes the classes where the expected numbers are italicized.

3 SOUND INDUCED SEIZURE IN MICE 97 TABLE 2 Numbers of 28-day old mice responding by wild running, clonic, or tonic seizure Number responding; Genotype Number tested Wild running Clonic Tonic DBA/2J (34.48) 18 (21.) 6 (7.49) DBA/W x F, (58.29) 23 (20.64) (8.88) Fl 13 (.37) 0 (1.28) 0 (1.23) F (H.57) 19 (12.54) (6.54) C57BL/6J x F, 16 (.42) 3 (2.22) 3 (2.) C57BL/6J 0 (1.085) 0 (0.94.) 0 (0.87) Chi-square for goodness of fit+ 39.0(3df), 9.1 (Idf) 8.5(3df) 5.3(3df) Probability on a one-locus hypothesis P<,001 P<.oo5 P<.M P >.1 Numbers in parentheses are expected values under a one-locus, two-allele, hypothesis. Italicized numbers are expected values (corresponding to small expected numbers not responding) that tend to inflate the chi-square value. + Numbers m parentheses are degrees of freedom. Where two chi-square values are given, the first includes and the second excludes the classes where the expected numbers are italicized. by 0, and any estimate greater than 0.99 has been replaced by 1. These estimates were used to obtain the expected values indicated in Tables 1 and 2, and in each case a x2 goodness of fit test was performed, resulting in a xz value with 3 degrees of freedom. In some cases the high value of x2 obtained might be attributed to a small expected frequency for one or more particular classes, and in these cases a x' value with fewer degrees of freedom was calculated, eliminating the offending classes; these are given at the bottom of Tables 1 and 2. (In view of the work of LANCASTER (1949) uncorrected x2 values have been used in each case.) It is seen that in all but one of the six cases, even discounting the possibility of a high x2 value due to low expected frequencies, the fit is significantly poor. The one exception is that of tonic seizure at 28 days old; in the other five cases the data are not compatible with the hypothesis that the observed phenotype is determined by genes at one locus. Second analysis: At 21 days of age we have three different degrees of response, none of which is alone controlled by one locus. The question then arises as to whether we cannot, in some 'way, by looking at more than one response at a time, TABLE 3 Modified minimum xz estimates of the probabilities a,, a,, and a, of responding Type of response Age Parameter Tonic seizure Clonic seizure Wild running 21 days a a a o 28 days a a a

4 98 K. SCHLESINGER et al. determine a phenotypic measure which is controlled by a single locus. One way of doing this is to try and discover a most heritable score for each individual on the basis of how he responds. Each individual can be classified into one of four mutually exclusive groups depending on the degree o his response: (1) No response at all; (a) wild running but no seizure; (3) clonic but no tonic seizure; (4) tonic seizure. Suppose, since the origin for such a scale is arbitrary, a score of 0 is given to any individual in group 1 ; and suppose individuals in groups 2, 3, and 4 are scored x, y, and z respectively. Then those values of x, y, and z are determined which maximize the following ratio when an analysis of variance is performed on the individual scores: (sum of squcrres among the six mating types)/(totaz sum of squares among all individuals). This procedure has been described by FISHER (1950) and the computations are simplified by using HEALY S (1964) results. Arbitrarily, the largest value of x, y, and z is made equal to unity, since the absolute scale of the scores is not relevant, and when this is done the following scores are obtained: group (1) 0; group (2) -0.; group (3) 0.05; group (4) 1. Thus at 21 days of age the scores are virtually 0 for the first three groups and unity for the fourth group-in other words if the phenotypes are described by a simple dichotomy, the most heritable phenotype is obtained by classifying each individual as either responding by a tonic seizure or not so responding. But this response, as has been shown, cannot be due to a single locus. The same analysis was performed for the responses at 28 days of age, and the following scores obtained: group (1) 0; group (2) 1; group (3) 0.66; group (4) It is interesting to note that the four scores are virtually equally spaced on the unit interval, but that their rank sequence does not correspond to the rank sequence expected for the four groups on the basis of the presumed strength of the response: the highest score is that for group 2, the next highest that for group 3, and then comes group 4, and finally group 1. This suggests that if we are to consider a simple dichotomy it should be one of the following: (a) group 2, versus groups 1, 3, and 4; (b) groups 2 and 3, versus groups 1 and 4; or (c) groups 2,3, and 4, versus group 1. None of these dichotomies corresponds to considering as the response tonic seizure alone, which fits the one-locus hypothesis. The last of these three dichotomies is the same as considering wild running as the response, and we have already seen this does not fit the one-locus hypothesis. When, however, the first two dichotomies are subjected to the same analysis, we find that in both cases there is a very good fit to a one-locus hypothesis (see Table 4); the fit is considerably better than when tonic seizure alone is considered, as is evidenced by the smaller x2 values. The parameter estimates found for the first dichotomy, i.e. where the response is wild running but no seizure, are: a. = 0.037, a, = and az = 0.339; those for the second dichotomy, i.e.

5 ~~ SOUND INDUCED SEIZURE IN MICE 99 TABLE 4 Numbers of 28-day old mice responding by wild running but (A) no seizure or (B) no tonic seizure Number responding' Genotype No. tested DBA/2J 35 DBA/2J x F, 60 Fl F, 60 C57BL/6J x F, C57BL/6J Chi-equare for goodness of fit (3df) (A) Wild running but no seizure (B) Wild running but no tonic seizure 13 (11.87) 25 (24.68) 32 (35.46) 46 (47.58) 13 (12.65) 13 (13.22) 33 (.93) 42 (37.62) 13 (13.20) 13 (13.83) 0 (0.56) 0 (0.62) * Numbers in parentheses are expected values under a one-locus, two-allele hypothesis. where the response is wild running but no tonic seizure, are: a, = 0.041, a, = and a, = Lethal response: In our previous analyses a fourth feature of the response has been overlooked; seizures may be either lethal or not lethal. Since, in our original experiments no records of this feature of the response had been kept for the two parental strains and for the F, hybrid group, new experiments were carried out for these three groups of mice. This feature of the response was observed in 21 -day old mice only, since seizures are almost never lethal at 28 days of age. The results of these experiments, together with the goodness of fit statistic, are given in Table 5. It can be seen that the data fit a one-locus hypothesis; the parameter estimates found for this case are a, = 0, a, = and a2 = The dilute locus: Two tests were performed in an attempt to determine whether or not the dilute locus contributes significantly to susceptibility to audiogenic seizure. The first test utilized DBA/BJ mice in which a mutation (reversion) to full coat-color had occurred. DBA/2J mice homozygous dilute (d/d) were compared to DBA1.J mice either homozygous or heterozygous non-dilute, i.e.. TABLE 5 Numbers of 21-day old mice responding by lethal seizures Genotype Number tested DBA/2J 31 DBA/2J x F, 60 Fl 68 F* 60 C57BL/6J x F, C5 7BL/6J Chi-square for goodness of fit (3df) Probability on a one-locus hypothesis Number lethal seizures (20.68) 20 (21.08) 7 (4.01) 8 (11.77) 0 (0.89) 0 (0) 5.8 P >.1 * Numbers in parentheses are expected ralues under a one-locus, two-allele hypothesis

6 0 K. SCHLESINGER et al. TABLE 6 Eflect of the dilute locus on audiogenic seizures at five different ages Number responding Age Genotype Number tested Wild running Clonic Tonic days d/d 21 days d/d 28 days d/d 35 days d/d 42 days d/d either D/D or D/d. The data obtained in these experiments are summarized in Table 6. Dilute and non-dilute DBA/2J mice were compared for wild running, clonic, and tonic seizures at 5 ages. All relevant 2 x 2 tables were examined (using FISHER S exact tests where appropriate) to evaluate the data statistically; at no age, and for no phenotype, were dilute mice found to be significantly different from non-dilute mice. The second test utilized F, and F, x DBA/2J cross animals. In both of these crosses four coat-color phenotypes are recovered. These are black, chocolate brown, dilute black, and dilute brown mice; the first two phenotypes are genetically either D/d or D/D, and the second two phenotypes are genetically d/d. The data obtained for these experiments are presented in Table 7. A statistical examination gives no indication that audiogenic seizure is associated with coat color. TABLE 7 Effect of the diluie locus on audiogenic seizure in 21-day old mice with black or brown coat color Number responding Phenotype Genotype Number tested Wild running Clonic Tonic Mice derived from F, Black o/- Chocolate brown Dilute black d/d Dilute brown d/d Mice derived from DBA/2J x F, Black Chocolate brown Dilute black d/d Dilute brown d/d

7 SOUND INDUCED SEIZURE IN MICE 1 DISCUSSION There is no doubt that the overall syndrome of audiogenic seizure is polygenically determined. Our attempt here has been to try and discover whether certain aspects of this overall syndrome might not be attributable to differences at single loci. Two points should be stressed concerning the analysis reported here: First, any agreement found between the data and a one-locus hypothesis needs to be confirmed by breeding tests before the possibility of a polygenic mode of inheritance can be ruled out; and secondly, data of the type we have here, i.e. where only six genotypically different classes are examined, can never distinguish with certainty between the involvement of two loci and more than two loci; for if two loci are involved nine different genotypes are possible. Furthermore the following assumption, as yet untested, underlies the whole analysis and conclusions: it is assumed that each of the parental strains is homozygous with respect to all the loci involved in the response measured. The experiments we have performed differ from previous experiments reported by others who have attempted to elucidate the genetics of audiogenic seizure in two important respects: the age of the animals has been better controlled and specific responses distinguished in the analyses. Since age is known to have an important effect on susceptibility, valid inferences can be made only if either (1) the age distribution is the same within each of the genotypic classes or (2) appropriate randomization is performed. The second alternative is an impossibility, since neither genotype nor age can be randomly assigned; we have therefore attempted to secure the first alternative by ensuring that in each experiment all animals are the same age to the nearest day. In the analyses, each specific phenotype has first been examined separately; then certain composite phenotypes, suggested by the scores found appropriate for maximizing the genotypic variances, have been subjected to a similar analysis. The results indicate that in 21-day old mice no phenotypic response that we have examined can be due to a genetic difference at one locus, except the probability of a lethal response. The fact that the probabilities for the three genotypes are not all zero or one indicates that something else, either environmental or in the genetic background, also affects the lethal response; but there may be one gene, incompletely penetrant and usually recessive, that has a major effect in determining it. The lethal effect of this gene virtually disappears by 28 days of age, at which time it is possible that various other types of response are determined in the main by genes at single loci. It could be that this same gene that shows a lethal effect at 21 days of age causes, with very low penetrance, the tonic response at 28 days of age; and this is a response for which the gene has a more or less additive effect rather than being recessive. The best fit to a one-locus hypothesis, however, is arrived at by considering, in 28-day old mice, the response of wild running alone (or possibly followed by a clonic, but not tonic, seizure) ; and the penetrance of this gene is greater when it occurs as a single dose in the heterozygote. If we consider the various responses in 28-day old animals, we can postulate

8 1 02 K. SCHLESINGER et al. that there are at least two separate genetic systems at work, and each may be largely dominated by one major gene: one system determines whether there will be wild running, the other whether there will be tonic seizure; and if an animal seizes because of his genotypic constitution with respect to the latter system, then it is virtually certain to run wild also, either of physiological necessity or because the two genetic systems are closely linked. Finally, we note that our data show no evidence at all for the involvement of the dilute locus in the responses we have examined. HUFF and HUFF (19612) present data on the basis of which they tentatively suggest that the dilute locus is involved in audiogenic seizures of DBA/l mice, HUFF and FULLER (1964) concluded that the d gene has little effect on susceptibility within the particular genetic and environmental background with which they were working. SUMMARY The mode of inheritance of sound induced seizure (audiogenic seizure) was studied in DBA/2J, C57BL/6J, PI, F,, and backcross mice. Close attention was given ( 1 ) to the phenotype used as an index of seizure susceptibility, i.e., whether the wild running phase, clonic, tonic, or lethal seizures were used as indices, and (2) to the exact age of the animals at the time of the test. Results indicate that a one-locus hypothesis does not fit the data except for the following phenotypes: lethal seizure at 21 days of age, tonic seizure at 28 days of age and wild running but no seizure at 28 days of age. Tests with single gene mutants at the dilute locus do not indicate that this gene is involved in susceptibility to audiogenic seizures. LITERATURE CITED COLEMAN, D. L., 1960 Phenylalanine hydroxylase activity in dilute and nondilute strains of mice. Arch. Biochem. Biophys. 91 : 0-6. ELSTON, R. C., 1966 On testing whether one locus can account for the genetic difference in susceptibility between two homozygous lines. Genetics 4 : FISHER, R. A., 1950 Statistical Methods for Research Workers. 11th Edition. Oliver and Boyd, Edinburgh. (Section 49.2.) FULLER, J. L., 1962 Effect of drugs on psychological development. Ann. N. Y. Acad. Sci. 96: FULLER, J. L., and W. R. THOMPSON, 1960 Behauior Genetics. Wiley, New York. GINSBURG, B. E., and D. S. MILLER, 1963 Genetic factors in audiogenic seizures. Psychophysiologie neuropharmacologie et biochimie de la crise audioghe. Centre national de la recherche scientifique, Paris. HALL, C. S., 1947 Genetic differences in fatal audiogenic seizures between two inbred strains of house mice. J. Heredity 38: 2-6. HANSON, A., 1958 Inhibition of brain glutamic acid decarboxylase by phenylalanine metabolites. Natunvissenschaften 45: 423. HEALY, M. J. R., A property of the multinomial distribution and the determination of appropriate scores. Biometrika 51 : 265. HUFF, S. D., and J. L. FULLER, Audiogenic seizures, the dilute locus and phenylalanine hydroxylase in DBA/1 mice. Science 4: 4-5.

9 SOUND INDUCED SEIZURE IN MICE 3 HUFF, S. D.. and R. L. HUFF, 1962 Dilute locus and audiogenic seizures in mice. Science 136: JAY, G. E., JR., 1963 Genetic strains and stocks, pp Methodology in Mammalian Genetics. Edited by W. J. BURDETTE. Holden-Day, San Francisco. LANCASTER, H. O., 1949 The combination of probabilities arising from data in discrete distributions. Biometrika 36: MAIER, N. R. F., 1943 Studies of abnormal behavior in the rat. XIV. Strain differences in the inheritance of susceptibility to convulsions J. Comp. Psychol. 35: MAIER, N. R. F., and N. M. GLASER, 1940 Studies of abnormal behavior in the rat. I. The inheritance of the neurotic pattern. J. Comp. Psychol. : MILLER, D. S., B. E. GINSBERG, and H. S. POTAS, 1952 Inheritance of seizure susceptibility in the house mouse Mus musculus. Genetics 37: SANDLER, M., and H. CLOSE, 1959 Biochemical effect of phenylacetic acid in a patient with 5-hydroxytryptophan secreting carcinoid tumour. Lancet 277: SCHLESINGER, K., W. BOGGAN, and D. X. FREEDMAN, 1965 Genetics of audiogenic seizures: I. Relation to brain serotonin and norepinephrine in mice. Life Sciences 4: WITT, G., and C. S. HALL, 1949 The genetics of audiogenic seizures in the house mouse. J. Comp. Physiol. Psychol. 42: