BEHAVIORAL BIOLOGY, 13, 413-423 (1975), Abstract No. 4225
The Response to Selection for Altered Conduction Velocity In Mice
JOSEPH P. HEGMANN 1
Zoology Department, University of Iowa, Iowa City, Iowa 52242 Conduction velocities from caudal nerves of 5318 mice resulting from bidirectional selection imposed on three different base populations show immediate response to selection which is asymmetrical after a few generations of selection pressure. Realized heritability estimates from the response to selection were similar to previous estimates (from parent offspring regression) and response was realized without alteration of either body weights or tail lengths in the lines. The physiological basis of the response and behavioral differences between the lines will be investigated. Since individual differences in function of the nervous system should influence many aspects o f behavior, gene differences which modify that function are o f special significance to the study of behavior. Within populations such gene differences should contribute not only to genetic variance for each o f the behaviors dependent on the measured function but also to genetic covariance among those behaviors. Differences between populations should indicate different behavioral interrelationships and different potentialities for behavioral response, in a multivariate sense, to changing environmental circumstances. The selection experiments discussed here were designed to provide detailed information regarding the nature of gene differences modifying peripheral nerve conduction velocity and to develop lines of animals different at loci modifying this aspect o f function and ideal for analysis o f behavioral variance and covariance imposed b y gene differences influencing conduction velocity. MATERIALS AND METHODS Subjects Bi-directional, within family selection for altered conduction velocity in peripheral nerves, was imposed on three base populations of mice. One 1Supported in part by NINDS Grants NS09536, and NS70099. 413 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
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population was composed of 20 litters of mice (176 individuals) resulting from randomly mating individuals from the F2 generation of a two-way cross between the C3H/HeJ and DBA/1J inbred strains. These strains were known to display extremely different conduction velocities (see Hegmann, 1972). After eight generations control line animals derived from this same population were allowed to produce second litters (120 animals) which served as a base population for replicate selected lines staggered in time from the initialselected lines but with common origin and controls. For the two-way cross animals this procedure allowed between generation differences in common environmental fluctuations to be observed without confounding generation of selection. At the same time this replicate was derived, ten litters (125 individuals) descended by random mating from the eight-way cross among inbred strains described by McClearn, Wilson, and Meredith (1970) provided the third base population. All animals in each generation and line were housed in stainless steel cages in 16L:8D light cycle with food and water available ad lib. Litters were housed with parents to the age of 25 days when they were weaned and caged separately by sex. Data Collection
Caudal nerve conduction velocity for each of the 5318 animals in these experiments was assessed (with mice anesthetized by 0.06 mg pentabarbitol sodium per gram body wt) when individuals were 45+2 days (range) old. Tails were pierced with three pairs of stainless steel electrodes rigidly mounted in Plexiglas. Two pairs, differential recording electrodes, were separated by 15 mm and the third pair, used for stimulus delivery, pierced the tail 10 mm proximal to the recording pairs. Stimulus delivery (0.025 msec duration supramaximal pulse delivered orthodromic to motor nerves) activated the dual traces of a storage oscilloscope employed to monitor electrical activity at the sites of the recording electrodes. Compound action potentials were amplified and stored with a sweep speed of 5 msec per division. Time elapsed from the peak of the compound action potential at the first recording site to the peak at the second site was used, with the distance between sites, to calculate caudal nerve conduction velocities (Hegmann, 1972). Body weights and tail lengths for all animals were assessed at the time conduction velocity was determined. Selection Procedures
Parents of the first generation of selection for high conduction velocity in the two-way cross (H1) and those of the first generation of the low line (L1) were full sibs taken from ten of the 20 litters in the base population.
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The male and female with highest velocity from each litter and those with lowest conduction velocity were chosen and mated at random within lines to establish H1 and L1.Parents of the control line for these animals (C1) were a male and a female taken at random from each of the other ten litters. Subsequent generations of H1 were produced using the highest male and female from each family in that line as parents of the next generation and those of L1 using the lowest velocity male and female from each family in that line. Mating within lines was at random with regard to families. Contemporaneous control line generations resulted from randomly mating 20 animals chosen each generation at random with regard to conduction velocity but with the restriction that each family in the line contribute a male and a female to the parents of the next generation. Parents of the replicate selected lines (H2 and L2) from the two-way cross were the highest and lowest conducting male and female mouse from each second litter produced by the control line (C 1) in generation 8. Selection and mating of parents of subsequent generations in these lines proceeded exactly as described for H1 and L1. C1 animals served as controls for both of these sets of selected lines. Selection from the eight-way cross population was initiated at the same time H2 and L2 were begun and selection procedures were identical except for the choice of parents for the first generation. In this experiment one male and one female from each of the ten litters were chosen at random to serve as control line (C) parents prior to the selection, from the same litters, of the highest and lowest pair from each litter to serve as parents of the high (H) and low (L) selected lines.
Data Analysis Scores for subjects in each generation of selection and in each experiment were subjected to analysis of variance to assess the significance of differences due to sex and to selection. Sums of squares and degrees of freedom between lines (2) were partitioned to yield single degree of freedom contrasts testing high line against control and low lines against control. Least square effects generated by these contrasts are not orthogonal but they are especially useful since they equal the response of the line to selection (Falconer, 1960) and allow direct testing of the significance of differences imposed by selection. Body weight and tail length were included as dependent variables in initial analyses and, in subsequent analyses of covariance, variance in conduction velocity due to variance in these morphological characters was removed both stepwise and simultaneously to allow examination of their role in the response to selection for conduction velocity. Due to the presence of unequal subclass numbers, analyses of variance and covariance employed the generalized procedures discussed by Harvey
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(1960). Procedures for genetic analysis of within family selection are those discussed by Falconer (1960).
RESULTS The response of the three populations to selection for altered conduction velocity is illustrated in Fig. 1 where means for animals in each line and generation are presented. Animals in the H1 and L1 lines show conduction velocities which diverge from controls in the expected direction in all generations. This direct response to selection is documented in detail in Table 1 which displays least squares estimates of the effects of sex and selection on conduction velocity. Sex differences for conduction velocity in these lines reach significance in only four o f the 13 generations but after the third generation they are uniformly negative, indicating that conduction velocities in females are consistently higher. Linear contrasts directly estimating the effect of selection for high conduction velocity (H-C) are positive (indicating that the high line animals average higher conduction velocity) in all generations and reach significance in 11 of the 13 contrasts available. Effects of selection for low conduction velocity are uniformly negative and significant in 12 of the 13 generations. Two aspects of the effects shown in Table 1 suggest that response to selection in this population is not symmetrical. First, effects of selection for
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low conduction velocity increase rather consistently as generation number increases. Response in the high line is variable, increasing in the early generations but falling in generations 6 and 7 to the point that H 1 and C 1 are not significantly different. Further, for the last 8 generations the effects of selection in the low direction exceed those measuring high line response. Both body weights and tail lengths of animals in these generations display consistent and significant sex differences (reflecting the generally larger body size of males) but there is no evidence for correlated response of these morphological variables to selection for conduction velocity. Thus, for body weight, H1 animals are heavier than control mice in seven generations and lighter in six generations. Similarly, low line subjects are lighter in eight generations and heavier in five, than control animals. Although tail lengths for H1 mice are consistently greater than those of C1 animals (and significantly so in eight of the 13 generations) L1 animals also display longer tails in six generations and especially in later generations when H1 and L1 are most widely separated in average conduction velocities. The effects of sex and selection for generations of the other two populations are presented in Table 2. Females in both of these populations also average consistently higher conduction velocities than males and the sex difference for both morphological characters is again obvious in both populations. Response to selection in the replicate lines (H2 and L2) is similar to that in the original lines from the two-way cross: High and low selected lines separate rapidly primarily due to low lines deviating from controls. Thus, both H2 and L2 display lower conduction velocities than do C1 animals in three of the five generations tested. In generations 2 and 4 of this replicate, H2 and C1 animals were virtually indistinguishable in average conduction velocity while in generations 3 and 5, separation was in the predicted direction. Effects of selection for low speed of conduction are uniformly negative, larger than those for high velocity in four of the five generations, and statistically significant in the last two generations. Selection for conduction velocity produced no consistant differences in body weights and tail lengths among the lines. Direct response to selection in the eight-way cross is also obvious. Effects of selection for high conduction velocity are uniformly positive and significant for four of five generations of selection; those for low selection are all negative and significant in the last three of five generations (Table 2). Once more there is some evidence for asymmetrical response to selection. The response to selection for high conduction velocity falls, following initial separation from the control line, to the point where H animals are not significantly different from C line mice. Though response to high selection recovers, the steadily increasing divergence of L animals from controls exceeds divergence in the high direction by generation 5.
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Body weights and tail lengths of animals in selected generations of this population give a slight suggestion of correlated response to selection. Thus, over the five generations body weights for H animals exceed those of the control line and mice in the low line tend to be lighter than C animals. A similar pattern is displayed in the later selected generations by tail lengths of animals in these lines. Each of the three sets of lines shown in Fig. 1 show substantial differences between generations which influence conduction velocities by all lines (controls and selected lines) in the same generation in the same direction. This situation is most obvious in the earliest generations of HI, L1, and CI where scores in all lines increase over base population values, then decrease to points substantially below the base population, and then increase rapidly for the next two generations. Peripheral nerve conduction velocity assessed by the procedures used here is sensitive to temperature (Hegmann, 1972) and to developmental levels of the mice employed (White and Hegmann, 1974)which may differ between generations due to changes in test room temperature and seasonal variation in developmental rates. While effects of this sort are not unusual in selection experiments (c.f., DeFries, et aL, in press) the present study provides an unusual opportunity to examine these influences for their dependence on generation of selection and on base population. Generations 8-13 of the initial two-way cross population, 0-5 of the replicate lines from that base population and 0-5 from the eight-way cross population were all reared in the same laboratory at the same times. Fluctuations between generation which influence HI, L1, and C1 in the same direction produce similar common effects on H2 and L2 and on the H, L, and C lines. Thus, these differences between generations are independent of degree of previous selection and of the base population on which selection is imposed. Calculation of response to selection as a deviation from contemporaneously reared control line scores (as in the H-C and L-C contrasts in Tables 1 and 2) provides measures of response free from these between generation differences common to all lines. Together with measures of selection intensity the estimates of response from controls were used to assess the realized heritability of conduction velocity in these populations and to examine the causes of the asymmetry of response for the high and low lines. Since selection procedures employed within litter deviations, selection differentials (scores of selected parents as deviations from the population from which they were chosen) were calculated on a within litter basis and summed for the litters in a line for each generation. Response of each line to selection is shown as a function of cumulative (across generations) selection intensity in Fig. 2. The asymmetrical response to selection is clearly not the result of different selection intensities in the high and low lines since response was greatest in the low direction but selection intensity was greatest in the high lines of each of the three
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Fig. 2. Response of caudal nerve conduction velocities for selected mice in three selection experiments from their control lines as a function of cumulative selection differential. Selection differentials shown are sums of deviations of selected parents from their litter means weighted according to the relative number of offspring contributed to the new generation. populations. Realized heritability estimates calculated from the information displayed in Fig. 2 for each of these lines (see DeFries and Hegmann, 1970) reflect this asymmetry. Regressions of response on cumulative selection differentials were calculated for each selected line from each base population. These regressions provide direct estimates of the heritability of within family deviations for conduction velocity and were corrected using the intraclass correlations among full sibs in the base populations (0.64, 0.64, and 0.54 for H1 and L1, H2 and L2, and H and L, respectively) and the coefficients of relationship (0.5 in all cases) to gain estimates of the heritability of conduction velocity. Estimates from L1, L2, and L were 0 . 1 6 + .04, 0.16 -+ .08; and 0.22 + .04 while those from H I , H2, and H were 0.05 -+ .03, 0.03 + .04, and 0.05 + .14. One additional result deserves comment. From examination of Fig. 1 it is obvious that all lines derived from the eight-way cross population display substantially higher conduction velocities than contemporaneously reared animals from t h e t w o - w a y cross. Thus, scores from H, L, and C animals of generations 0-5 are uniformly higher than their H I , L1, and C1 counterparts in generations 8-13. In fact, they are significantly higher than H2 and L2 animals which have been subjected to the same number of generations of selection (t(9) = 9.1). This result is in agreement with observations of higher conduction velocity for the more heterogeneous population reported by White
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and Hegmann (1974). The overall difference in conduction velocity between the two populations may be due to heterosis (Hegmann, 1972) or to the fact that the strains in the two-way cross, though phenotypically diverse, provide a restricted sample of genotypes at loci relevant to conduction velocity.
DISCUSSION Genetic variance influencing differences in conduction velocity was reported by Hegmann (1972) and analyzed in more detail in subsequent breeding work (Hegmann, White, and Kater, 1973). In the latter research, character heritability from parent-offspring regression was reported to be 0.09 +- .04 for the two-way cross population used here, and 0.18 +- .04 for the eight-way cross population. Realized heritabilities from the results of selection are quite close to those estimates. Falconer (1960) reasons that with asymmetrical response to selection the mean of the realized heritabilities in the two directions might correspond to a good predicted value. Here, the means for the two-way cross experiments are 0.10-+ .04 and 0.09-+ .06 and, from the eight-way cross population, 0.14 + .09. In spite of this relatively low character heritability, careful selection procedures have produced lines differing by more than 20% (4.82 msec) of the highest line. Asymmetrical response to selection may be due to unequal selection intensities in the two directions or to genetic asymmetry with regard either to allelic dominance (with alleles yielding differences in one direction consistently dominant over alleles influencing the character in the other direction) or to frequency (with the most frequent alleles at loci consistently those which influence the character in one direction). Figure 2 clearly demonstrates that the asymmetrical response observed here is not due to different selection intensities. The evidence, independent of the asymmetrical response to selection, for strong directional dominance (Hegmann, 1972) supports the conclusion that dominant alleles at most loci which influence conduction velocity are those which increase speed of conduction. It is interesting to note that Falconer (1960) mentions that neither directional dominance nor directional gene frequencies should yield asymmetry in the first few generations of selection. Figure 2 shows that asymmetry in the conduction velocity lines is primarily evident after the first two generations of selection. Among the possible physiological bases for the observed response (for instance, altered body temperatures, differential resistance to anesthetic, changed distribution of motor and sensory fibers, or altered membrane or axoplasmic resistances) the imposition, through selection of differences between the lines for axon diameters seems most likely. Clearly, divergence in conduction velocity has not been accomplished through altered body size and, one could reason, therefore not through changed gross morphology of the
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nervous system. Such reasoning depends on the assumption that gene differences influencing the size of axons must do so with attendant effects on body size. The recent demonstration by White and Hegmann (1974) of gene differences altering rate of development of conduction velocity independently of effects on body size development suggests caution regarding that assumption. Direct measurement of differences in axon diameter is difficult because the nerve is compound and, conduction velocity differences observed might require a modal fiber diameter difference of less than a micrometer. Detailed investigation of the basis of the response will be undertaken when the response appears to plateau. Behavioral differences between the lines are of great interest regardless of the physiological basis of the response and behavioral testing is in progress. Robb and Hegmann (1974) have presented tentative evidence from inbred strain comparisons suggesting that leg reflex speed is influenced by gene differences modifying caudal nerve conduction. If that indication is correct then the effects of selection reported here may have general influence on peripheral function and substantial impact on behavior.
REFERENCES DeFries, J. C., and Hegmann, J. P. (1970). Genetic analysis of open-field behavior. In G. Lindzey and D. D. Thiessen (Eds.), "Contributions to behavior-genetic analysis: The mouse as a prototype, pp. 23-56. New York: Appleton~entury-Crofts. DeFries, J. C., Hegmann, J. P., and Halcomb, R. A. (1974). The response to 20 generations of selection for open-field activity in mice. Behav. Biol. 1 I, 481-495. Falconer, D. S. (1960). "Introduction to Quantitative Genetics." Edinburgh and London: Oliver and Boyd Ltd. Harvey, W. R. (1960). Least Squares Analysis of Data with Unequal Subclass Numbers, Publication N ARS-20-8, United States Department of Agriculture, Washington, DC. Hegmann, J. P. (1972). Physiological function and behavioral genetics. 1. Genetic variance for peripheral conduction velocity in mice. Behav. Genet. 2, 55-67. Hegmann, J. P., White, J. E., and Kater, S. B. (1973). Physiological function and behavioral genetics. II. Quantitative genetic analysis of conduction velocity of caudal nerves of the mouse, Mus musculus. Behav. Genet. 3, 2, 121-131. McClearn, G. E., Wilson, J. R., and Meredith, W. (1970). The use of isogenic and heterogenic mouse stocks in behavioral research. In G. Lindzey and D. D. Thiessen (Eds.), "Contributions to Behavior43enetic Analysis: The Mouse as a Prototype, pp. 3-22. New York: Appleton-Century-Crofts. Robb, N. G., and J. P. Hegmann. (1974). Nervous system function: behavioral influences of gene effects on peripheral nerve conduction velocity in mice. Behav. Biol. 11, 281-283. White, J. E., and Hegmann, J. P. (1974). Development of nervous system function in mice: normative data and gene effects. Behav. Biol. l i , 83-88.