Behavior Genetics, Vol. 5, No. 4, 1975
Albinism and Auditory Function in the Laboratory Mouse. II. Effects of Acoustic Priming and CrossFostering Mark M. Haythorn t and Kenneth R. Henry 1 Received 5 Aug. 1974--Final 21 Oct. 1974
Albino (c/c) and nonalbino ( + / c and + / + ) congenie C57BL/6J mice were examined for the effects of acoustic prestimulation (priming) on audiogenic seizures. While no genotypic-specific effects were noticed 1 day after priming, major effects were observed in separate groups of mice tested 5 days after priming. The c/c mice were most susceptible to audiogenic seizures, and no differences were observed between + / c and + / + mice. While cross-fostering did not change this relationship, it provided protection for mice of all three genotypes. The interpretation that melanin offers protection from acoustic trauma is considered inconclusive because of the interaction of innate- and priming-induced audiogenic seizures in the + / c and c/c mice. KEY W O R D S : audiogenic seizure; albinism; heterosis; cross-fostering; mouse.
INTRODUCTION Many studies have observed behavioral differences between albino and pigmented mammals. Several of these studies have suggested that a major portion of this effect may result from an alteration in either the albino's sensitivity to sensory input or its behavioral responsiveness (McClearn, 1960; DeFries et al., 1966; Thiessen et al., 1970). Of particular interest to our research are those studies which have described a differential recovery in albinos exposed to acoustic trauma. Supported by N S F Grant GB31921 and PHS Grant NS11565-01 to the second author. Department of Psychology, University of California, Davis, California. 321 (~) 1975 Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher.
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Galioto and Bonaccorsi (1965) and Crifo (1973) both reported a weaker resistance of the albino guinea pig's ear to acoustic trauma, although at normal intensities the albino guinea pig had a better auditory capacity than the pigmented one. According to Bonaccorsi (1963), the inner ear pigment, melanin, is involved in the respiratory chain of the inner ear. This involvement of melanin led to the hypothesis that the pigment could function as a source of metabolic energy for the inner ear under conditions of hypoxia caused by acoustic trauma (Cherubino, 1968). Since the wild-type (+) gene displays complete dominance over the albino (c) allele for the phenotype of coat color, the assumption could be made that + / + and +/c animals would be similar in their behavioral response to manipulations affected by the presence of melanin. However, since we have found differences in the auditory development of + / + and +/c C57BL/6J mice (Henry and Haythorn, 1975), we decided to extend these findings to include the effects of postnatal manipulations on auditory responsiveness in this same congenic strain of mice. Acoustic deprivation has been found to induce susceptibility to audiogenic seizures in mice which are otherwise nonsusceptible to this behavior. Elevation of the absolute auditory threshold, and thus deprivation, can result from an intense acoustic exposure (Henry, 1967; Willott and Henry, t974), from conductive hearing loss (Chen et al., 1973; McGinn et al., 1974), or from an apparent genetic/developmental interaction (Henry and Haythorn, 1974, 1975). The present paper further extends the examination of possible genetic/developmental manipulations influencing the occurrence of audiogenic seizures. Congenic C57BL/6J mice of +/+, +/c, and c/c genotypes, differing only at the locus affecting melanin formation, were used in order to assess the possible pleiotropic effects of the albino gene on the development of audiogenic seizures following acoustic trauma. Two previous studies have examined audiogenic seizures in both albino and pigmented littermate mice. Gutherz and Thiessen (1966) exposed albino and nonalbino F2 offspring from an AJ • DBA/1J cross to 60 sec of daily acoustic stimulation beginning on postpartum day 14. The pigmented mice exhibited audiogenic seizures from 1 to 2 days earlier than the albino mice. The second study (Henry and Haythorn, 1975), part of which was run concurrently with the present experiments, examined audiogenic seizures in +/+, +/c, and c/c congenic C57BL/6J mice during their initial acoustic exposure on day 16. Only c/c and +/c genotypes exhibited audiogenic seizures, a finding which was positively correlated with the retarded auditory development associated with the c gene. These two studies could be interpreted as yielding contradictory results about the influence of the albino gene on seizure susceptibility. The earlier appearance of seizures for the pigmented mice in the Gutherz and Thiessen
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(1966) study suggests a lower susceptibility for albino mice. But the results of the two Studies are not directly comparable due to the difference in the first sound presentation. The mice in the earlier study were first exposed to the acoustic stimulus on day 14 postpartum, whereas in the latter study the first exposure was at day 16 postpartum. Since during this developmental period the mouse's auditory system first begins to function, and since differences have been found in the auditory development of C57BL/6J mice (Henry and Haythorn, 1975), the time of the first auditory exposure may be crucial in the interpretation of the results of the two studies. In addition, in the Gutherz and Thiessen (1966) study, the grandparents of the nonalbino mice were susceptible but the albino grandparents were not, suggesting the strong possibility of linkage persisting to the F~ generation and complicating any interpretation of their data in exclusive terms of the c locus. The present study controlled for this difficulty by examining nonsusceptible congenic C57BL/6J mice which differed genetically only in their number of c genes.
EXPERIMENT 1 Methods
Subjects. Mice of the C57BL/6J strain were used. The high degree of inbreeding resulted in their being nearly isogenic, with the exception of known differences at the albino locus. Mice of +/c and c/c genotypes were obtained from +/c x c/c or c/c x c/+ matings, while + / + genotypes were obtained from + / + x + / + matings. Apparatus. Mice were acoustically primed and tested for audiogenic seizures with a 114-db (re 0.0002 dynes cm-~), 6-inch electric bell, mounted atop a 30-cm-tall by 30-cm-wide glass jar. A rubber gasket between the bell housing and the jar reduced the vibration transmitted to the subject at the bottom of the container. Procedure. After being reared by their natural parents, 22 c/c, 28 +/c, and 22 + / + mice were acoustically stressed at 16 days of age. This was achieved by individually exposing them to the sound of the bell for 30 sec. This initial exposure, termed "acoustic priming," has been repeatedly found to induce susceptibility to audiogenic seizures when it is performed at this age (Henry, 1967; Boggan et al., 1971). These subjects were then returned to their home cages. One day later, they were reexposed to the same acoustic source for 60 sec, during which time their latencies to wild running and their incidence of the successive stages of the audiogenic seizure syndrome were recorded.
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Statistical tests were performed on the total seizure severity score obtained for each animal by equally weighing the four components of the audiogenic seizure (see Henry and Bowman, 1970, for statistical justification). Mice of all three genotypes were susceptible to audiogenic seizures 24 hr after acoustic priming. Statistics for the seizure severity scores (Table I) revealed no significant difference among the recorded indices as a function of genotype. These 17-day-old +/+, +/c, and c/c mice exhibit audiogenic seizures to a degree which was previously found only in nonprimed albino C57BL/6J mice at a comparable age. Earlier, Henry and Haythorn (1975) had found + / + and +/c C57BL/6J to be resistant to seizures on the first exposure, while albino C57BL/6Js had an average seizure score of 24.0. This was somewhat lower than the scores of 42.0 (c/c), 48.3 (+/c), and 48.5 ( + / + ) found in the present study. Therefore, 24 hr after the priming exposure the pigmented mice show an identically large increase in audiogenic seizures, whereas the albino mice have a lesser increase. This failure of the 17-day-old albino mice to exhibit as great a change in seizure severity as a result of acoustic priming might have been due to their innately elevated auditory thresholds, presumably a result of their retarded auditory development (Henry and Haythorn, 1975). Therefore, if tests for audiogenic seizures were to be conducted at an age when the auditory development is complete for mice of all three genotypes, differences might be observed between +/+, +/c, and c/c mice.
Table I, Audiogenic Seizures in 17-Day-01d C57BL/6J Mice 24 hr After Acoustic Priming Stages of audiogenic seizuresGenotype
(N)
Latencyb
Wild running
-~/-k
(22) (28) (22)
26.7 28.7 30.5
59 61 54
-k/c c/c
Clonic Tonic 59 61 50
59 50 50
Death
Severity score~
27 21 14
48.5 48.3 42.0
In percentages. Latency to wild running, in seconds. A composite score, the average of the percentages of the four separate stages of the seizure syndrome.
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EXPERIMENT 2 As was earlier determined by the input-output function and the absolute threshold of the auditory evoked potential, the auditory systems of +/+, +/c, and c/c C57BL/6J mice are mature by 21 days of age. Therefore, the second experiment tested mice at this age. Since the + / + mice were obtained from different parents than were the +/c and c/c mice, it was also considered necessary to determine whether postnatal maternal effects were responsible for any of the obtained results. To achieve this end, the second experiment also utilized cross-fostering. Methods Mice were obtained from the same matings described in Experiment 1. Acoustic priming was also performed at 16 days postpartum, in the way previously described. One group of 25 + / + , 19 +/c, and 14 c/c mice remained with their parents until 21 days of age, being removed only for 30 sec of priming. At 21 days of age, they were weighed and then tested for audiogenic seizures. Sixteen + / + , 31 +/c, and 9 c/c littermate controls were cross-fostered on the first day postpartum; the c/c and +/c pups were transferred from their c/c • +/c or +/c • c/c parents to + / + • + / + foster parents, while the + / + pups were transferred from their + / + x + / + parents to c/c • +/c or +/c • c/c foster parents. These mice remained with their foster parents until weighing and testing at 21 days postpartum with the exception of a brief removal at age 16 days for 30 sec of acoustic priming. Results Marked differences were observed among the genotypes as a result of the increased delay between priming and testing (Table II and Fig. 1). When compared to the younger mice of Experiment 1, both the + / + and c/c mice displayed an increased audiogenic seizure severity score (for + / + mice, 17 vs. 21 days, 48.5 vs. 65.0, t = 3.08, df = 45, p < 0.01; for r mice, 17 vs. 21 days, 42.0 vs. 86.0, t = 2.80, df = 2 1 , p < 0.02). This effect was not observed in mice of the +/c genotype (for 17 vs. 21 days, 48.3 vs. 46.0, t = 0.158). Latencies to wild running showed similar significant patterns. By 21 days of age, albino mice exhibited more severe audiogenic seizures than nonalbinos (Table II and Fig. 1). This was verified by the significant t test for mice reared with their natural parents (t = 2.15, df = 56,
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Table II. Audiogenic Seizures and Body Weights in Cross-Fostered and Non-CrossFostered 21-Day-Old C57BL/6J Mice 5 Days After Acoustic Priming Stages of audiogenic seizuresb Rearing conditions and genotype
(N)
LaWild Severity Weight~ tency c running Clonic Tonic Death score
Non-cross-fostered +/+ Non-cross-fostered
(25)
10.78
19.8
72
64
64
60
65
(19)
8.34
24.6
63
47
42
32
46
Non-cross-fostered
(14)
8.08
12.2
86
86
86
86
86
Cross-fostered +/+ Cross-fostered
(16)
9.93
23.3
69
12
12
6
24.75
(31)
9.71
33.0
48
13
13
3
19.25
Cross-fostered
(9)
8.20
16.4
78
78
78
33
58.5
+/c
c/c
+/c
c/c
Body weight in grams. b In percentages. c Latency to wild running, in seconds. p < 0.05) and for those foster-reared (t = 3.92, df = 53, p < 0.001). The + gene was dominant for the phenotype of protection from audiogenic seizures under both rearing conditions, as demonstrated by the significant quadratic trends (for non-cross-fostered mice, F = 5.65, df = 1/55, p < 0.025; for cross-fostered mice, F = 11.3, df = 1/53, p < 0.005). In view of the facts that both + / + and +/c mice exhibited protection and that there was no significant difference between their scores, we conclude that the + gene is completely dominant to t h e c gene, with no demonstratable heterosis. The trends described above for the seizure severity scores were also observed for latencies to wild running. Rearing conditions had a large effect on audiogenic seizures. The net effect of cross-fostering was protective (F = 14.99, df = 1/108, p < 0.0001). This was the case for mice of all three genotypes, as revealed by the nonsignificant genotype • cross-fostering interaction (F = 0.34). Again, the latencies displayed the same pattern as the seizure severity scores.
Discussion Regardless of rearing conditions, primed 21-day-old C57BL/6J c/c mice exhibited more frequent and more severe seizures with shorter
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latencies than + / + and +/c mice. The complete dominance of the + gene for the phenotype of priming-induced audiogenic seizures in the present study is consistent with the complete dominance of this same gene for melanin. These results are also consistent with the hypothesis that melanin pigment in the inner ear aids in the functional recuperation of hearing following intense acoustic stimulation (Cherubino, 1968). Presumably, the elevation of the absolute auditory threshold in the c/c mouse would require a longer recuperative period than that of either the +/c or the + / + mouse; as a result, the period of auditory deprivation would be longer. This would be expressed by a greater rebound behavioral supersensitivity (audiogenic seizure) in albino mice compared to the pigmented mice. This interpretation is consistent with several studies which show that lightly pigmented races of man suffer more hearing loss than do darkly pigmented races (see Karsai et al., 1972). But the data of the present study reflect the effects of both innate- and priming-induced audiogenic seizures, and the agreement with Cherubino's (1968) hypothesis could be entirely artifactual. In an attempt to determine the increase in susceptibility due exclusively to acoustic priming, the seizure severity scores from the nonprimed, 100"
75"
Q
50 9 U
..N ul
~5"
.).
.),
,),
Genotype Fig. 1. Mean severity of audiogenic seizures in congenic 21-day-old C57BL/6J crossfostered (cf) and non-cross-fostered (n-cf) mice as a function of single gene changes at the albino locus.
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non-cross-fostered 21 day-old mice of a concurrently run study (Henry and Haythorn, 1975) were subtracted from the severity scores of the primed, non-cross-fostered mice of the present study. This manipulation assumed that the effects of innate- and priming-induced audiogenic seizures are additive, an assumption which may or may not be justified. Nonetheless, these differences in scores produced a profile which was quite different from the data seen in Fig. 1; the increases in severity were 65.0, 25.5, and 43.75 for + / + , + / c and c/e genotypes, respectively. By this comparison, then, one might conclude that the albino is more resistant to the effects of acoustic trauma, an interpretation opposite to the one first offered. These differences in scores also suggest that the + / c mouse might display heterosis for this effect. We are presently attempting to resolve this paradox by examining acoustically stressed mice of all three genotypes for acoustic startle thresholds, audiograms, cochlear morphology, and cochlear biochemistry at an age when innate audiogenic seizures are no longer seen. Although we did not expect to find such a large cross-fostering effect, the obtained results are not too surprising. While nonacoustic manipulations have been found incapable of priming the C57BL/6J mouse, the susceptibility of acoustically primed mice can be greatly altered by manipulations such as increased levels of pyridoxine (Henry and Bowman, 1968) and altered levels of neurotransmitters (Boggan et al., 1971), No consistent changes in body weights were observed as a function of cross-fostering, so it is unlikely that global developmental differences were responsible for the protection offered by cross-fostering. REFERENCES Boggan, W. O., Freedman, D. X., Lovell, R. A., and Schlesinger, K. (1971). Studies in audiogenic seizure susceptibility. Psychopharmacologia 20:48-56. Bonaccorsi, P. (1963). Comportamento delle barriere emolabirintica, liquorale ed oftalmiea nell' albinismo.Ann. Laringol. Otol. Rinol. Faringol. 62:432-456. Chen, C.-S., Gates, G. R., and Bock, G. R. (1973). Effect of priming and tympanic membrane destruction on development of audiogenic seizure susceptibility in BALB/e mice. Exp. Neurol. 39:277 284. Cherubino, M. (1968). I melanaciti dell' orecchio interno. In Crifo (1973). Crifo, S. (1973). Shiver-audiometry in the conditioned guinea-pig (simplified Anderson-Wedenberg test), Acta Oto-Laryngol. 75:38-44. DeFries, J. C., Hegmann, J. P., and Weir, M. W. (1966). Open-field behavior in mice: Evidence for a major gene effect mediated by the visual system. Science 154:1577 1579. Galioto, G. B., and Bonaccorsi, P. (1965). I1 fattore constituzionale del trauma acuatico individuato sperimentalmente nella diversa coneentrazione di melania nella strai vaacolare. In Crifo (1973). Gutherz, K., and Thiessen, D. D. (1966). Albinism and audiogenic seizures in the mouse. Psychon. Sci. 6:97-98. Henry, K. R. (1967). Audiogenic seizure susceptibilityinduced in C57BL/6J mice by prior auditory exposure. Science 158:938-940.
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Henry, K. R., and Bowman, R. E. (1968). The dilute locus, pyridoxine deficiency, and audiogenic seizures in mice. Proc. Soc. Exp. Biol. Med. 128:635-638. Henry, K. R., and Bowman, R. E. (1970). Behavior-genetic analysis of the ontogeny of acoustically primed audiogenic seizures in mice. J. Comp. Physiol. Psychol. 70:235-241. Henry, K. R., and Haythorn, M. M. (1974). Similarities in auditory processing associated with auditory deprivation in the DBA/2J and the acoustically primed C57BL/6J mouse. J. Cornp. Physiol. Psychol., in press. Henry, K. R., and Haythorn, M. M. (1975). Albinism and auditory function in the laboratory mouse. I. Effects of single-gene substitutions on auditory physiology, audiogenic seizures, and developmental processes. Behav. Genet. 5:137-149. Karsai, L. K., Bergman, M., and Bin Choo, Y. (1972). Hearing in ethnically different longshoremen. Arch. Otolargyngol. 96:499-504. McCtearn, G. E. (1960). Strain differences in activity of mice: Influence of illumination. J. Comp. Physiol. Psychol. 53:142-143. McGinn, M. D., Willott, J. F., and Henry, K. R. (1974). Effects of conductive hearing loss on auditory evoked potentials and audiogenic seizures in mice. Nature New Biol. 244:255-256. Savin, C. (1965). The blood vessels and pigmentary cells of the inner ear. Ann. Otol. 74:611 622. Sze, P. Y. (1970). Neurochemical factors in auditory stimulation and development of susceptibility to audiogenic seizures. In Welch, B. L., and Welch, A. S. (eds.), Physiological EJfects of Noise, Plenum Press, New York, pp. 259-270. Thiessen, D. D., Owen, K., and Whitsett, M. (1970). Chromosome mapping of behavioral activities. In Lindzey, G., and Thiessen, D. D. (eds.), Contributions to Behavior-Genetic Analysis: The Mouse as a Prototype, Appleton-Century-Crofts, New York. Willott, J. F., and Henry, K. R. (1974). Auditory evoked potentials: Developmental changes of threshold and amplitude following early acoustic trauma. J. Comp. Physiol. Psychol. 86:1-7.
Albinism and Auditory Function in the Laboratory Mouse. II. Effects of Acoustic Priming and CrossFostering Mark M. Haythorn t and Kenneth R. Henry 1 Received 5 Aug. 1974--Final 21 Oct. 1974
Albino (c/c) and nonalbino ( + / c and + / + ) congenie C57BL/6J mice were examined for the effects of acoustic prestimulation (priming) on audiogenic seizures. While no genotypic-specific effects were noticed 1 day after priming, major effects were observed in separate groups of mice tested 5 days after priming. The c/c mice were most susceptible to audiogenic seizures, and no differences were observed between + / c and + / + mice. While cross-fostering did not change this relationship, it provided protection for mice of all three genotypes. The interpretation that melanin offers protection from acoustic trauma is considered inconclusive because of the interaction of innate- and priming-induced audiogenic seizures in the + / c and c/c mice. KEY W O R D S : audiogenic seizure; albinism; heterosis; cross-fostering; mouse.
INTRODUCTION Many studies have observed behavioral differences between albino and pigmented mammals. Several of these studies have suggested that a major portion of this effect may result from an alteration in either the albino's sensitivity to sensory input or its behavioral responsiveness (McClearn, 1960; DeFries et al., 1966; Thiessen et al., 1970). Of particular interest to our research are those studies which have described a differential recovery in albinos exposed to acoustic trauma. Supported by N S F Grant GB31921 and PHS Grant NS11565-01 to the second author. Department of Psychology, University of California, Davis, California. 321 (~) 1975 Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher.
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Galioto and Bonaccorsi (1965) and Crifo (1973) both reported a weaker resistance of the albino guinea pig's ear to acoustic trauma, although at normal intensities the albino guinea pig had a better auditory capacity than the pigmented one. According to Bonaccorsi (1963), the inner ear pigment, melanin, is involved in the respiratory chain of the inner ear. This involvement of melanin led to the hypothesis that the pigment could function as a source of metabolic energy for the inner ear under conditions of hypoxia caused by acoustic trauma (Cherubino, 1968). Since the wild-type (+) gene displays complete dominance over the albino (c) allele for the phenotype of coat color, the assumption could be made that + / + and +/c animals would be similar in their behavioral response to manipulations affected by the presence of melanin. However, since we have found differences in the auditory development of + / + and +/c C57BL/6J mice (Henry and Haythorn, 1975), we decided to extend these findings to include the effects of postnatal manipulations on auditory responsiveness in this same congenic strain of mice. Acoustic deprivation has been found to induce susceptibility to audiogenic seizures in mice which are otherwise nonsusceptible to this behavior. Elevation of the absolute auditory threshold, and thus deprivation, can result from an intense acoustic exposure (Henry, 1967; Willott and Henry, t974), from conductive hearing loss (Chen et al., 1973; McGinn et al., 1974), or from an apparent genetic/developmental interaction (Henry and Haythorn, 1974, 1975). The present paper further extends the examination of possible genetic/developmental manipulations influencing the occurrence of audiogenic seizures. Congenic C57BL/6J mice of +/+, +/c, and c/c genotypes, differing only at the locus affecting melanin formation, were used in order to assess the possible pleiotropic effects of the albino gene on the development of audiogenic seizures following acoustic trauma. Two previous studies have examined audiogenic seizures in both albino and pigmented littermate mice. Gutherz and Thiessen (1966) exposed albino and nonalbino F2 offspring from an AJ • DBA/1J cross to 60 sec of daily acoustic stimulation beginning on postpartum day 14. The pigmented mice exhibited audiogenic seizures from 1 to 2 days earlier than the albino mice. The second study (Henry and Haythorn, 1975), part of which was run concurrently with the present experiments, examined audiogenic seizures in +/+, +/c, and c/c congenic C57BL/6J mice during their initial acoustic exposure on day 16. Only c/c and +/c genotypes exhibited audiogenic seizures, a finding which was positively correlated with the retarded auditory development associated with the c gene. These two studies could be interpreted as yielding contradictory results about the influence of the albino gene on seizure susceptibility. The earlier appearance of seizures for the pigmented mice in the Gutherz and Thiessen
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(1966) study suggests a lower susceptibility for albino mice. But the results of the two Studies are not directly comparable due to the difference in the first sound presentation. The mice in the earlier study were first exposed to the acoustic stimulus on day 14 postpartum, whereas in the latter study the first exposure was at day 16 postpartum. Since during this developmental period the mouse's auditory system first begins to function, and since differences have been found in the auditory development of C57BL/6J mice (Henry and Haythorn, 1975), the time of the first auditory exposure may be crucial in the interpretation of the results of the two studies. In addition, in the Gutherz and Thiessen (1966) study, the grandparents of the nonalbino mice were susceptible but the albino grandparents were not, suggesting the strong possibility of linkage persisting to the F~ generation and complicating any interpretation of their data in exclusive terms of the c locus. The present study controlled for this difficulty by examining nonsusceptible congenic C57BL/6J mice which differed genetically only in their number of c genes.
EXPERIMENT 1 Methods
Subjects. Mice of the C57BL/6J strain were used. The high degree of inbreeding resulted in their being nearly isogenic, with the exception of known differences at the albino locus. Mice of +/c and c/c genotypes were obtained from +/c x c/c or c/c x c/+ matings, while + / + genotypes were obtained from + / + x + / + matings. Apparatus. Mice were acoustically primed and tested for audiogenic seizures with a 114-db (re 0.0002 dynes cm-~), 6-inch electric bell, mounted atop a 30-cm-tall by 30-cm-wide glass jar. A rubber gasket between the bell housing and the jar reduced the vibration transmitted to the subject at the bottom of the container. Procedure. After being reared by their natural parents, 22 c/c, 28 +/c, and 22 + / + mice were acoustically stressed at 16 days of age. This was achieved by individually exposing them to the sound of the bell for 30 sec. This initial exposure, termed "acoustic priming," has been repeatedly found to induce susceptibility to audiogenic seizures when it is performed at this age (Henry, 1967; Boggan et al., 1971). These subjects were then returned to their home cages. One day later, they were reexposed to the same acoustic source for 60 sec, during which time their latencies to wild running and their incidence of the successive stages of the audiogenic seizure syndrome were recorded.
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Statistical tests were performed on the total seizure severity score obtained for each animal by equally weighing the four components of the audiogenic seizure (see Henry and Bowman, 1970, for statistical justification). Mice of all three genotypes were susceptible to audiogenic seizures 24 hr after acoustic priming. Statistics for the seizure severity scores (Table I) revealed no significant difference among the recorded indices as a function of genotype. These 17-day-old +/+, +/c, and c/c mice exhibit audiogenic seizures to a degree which was previously found only in nonprimed albino C57BL/6J mice at a comparable age. Earlier, Henry and Haythorn (1975) had found + / + and +/c C57BL/6J to be resistant to seizures on the first exposure, while albino C57BL/6Js had an average seizure score of 24.0. This was somewhat lower than the scores of 42.0 (c/c), 48.3 (+/c), and 48.5 ( + / + ) found in the present study. Therefore, 24 hr after the priming exposure the pigmented mice show an identically large increase in audiogenic seizures, whereas the albino mice have a lesser increase. This failure of the 17-day-old albino mice to exhibit as great a change in seizure severity as a result of acoustic priming might have been due to their innately elevated auditory thresholds, presumably a result of their retarded auditory development (Henry and Haythorn, 1975). Therefore, if tests for audiogenic seizures were to be conducted at an age when the auditory development is complete for mice of all three genotypes, differences might be observed between +/+, +/c, and c/c mice.
Table I, Audiogenic Seizures in 17-Day-01d C57BL/6J Mice 24 hr After Acoustic Priming Stages of audiogenic seizuresGenotype
(N)
Latencyb
Wild running
-~/-k
(22) (28) (22)
26.7 28.7 30.5
59 61 54
-k/c c/c
Clonic Tonic 59 61 50
59 50 50
Death
Severity score~
27 21 14
48.5 48.3 42.0
In percentages. Latency to wild running, in seconds. A composite score, the average of the percentages of the four separate stages of the seizure syndrome.
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EXPERIMENT 2 As was earlier determined by the input-output function and the absolute threshold of the auditory evoked potential, the auditory systems of +/+, +/c, and c/c C57BL/6J mice are mature by 21 days of age. Therefore, the second experiment tested mice at this age. Since the + / + mice were obtained from different parents than were the +/c and c/c mice, it was also considered necessary to determine whether postnatal maternal effects were responsible for any of the obtained results. To achieve this end, the second experiment also utilized cross-fostering. Methods Mice were obtained from the same matings described in Experiment 1. Acoustic priming was also performed at 16 days postpartum, in the way previously described. One group of 25 + / + , 19 +/c, and 14 c/c mice remained with their parents until 21 days of age, being removed only for 30 sec of priming. At 21 days of age, they were weighed and then tested for audiogenic seizures. Sixteen + / + , 31 +/c, and 9 c/c littermate controls were cross-fostered on the first day postpartum; the c/c and +/c pups were transferred from their c/c • +/c or +/c • c/c parents to + / + • + / + foster parents, while the + / + pups were transferred from their + / + x + / + parents to c/c • +/c or +/c • c/c foster parents. These mice remained with their foster parents until weighing and testing at 21 days postpartum with the exception of a brief removal at age 16 days for 30 sec of acoustic priming. Results Marked differences were observed among the genotypes as a result of the increased delay between priming and testing (Table II and Fig. 1). When compared to the younger mice of Experiment 1, both the + / + and c/c mice displayed an increased audiogenic seizure severity score (for + / + mice, 17 vs. 21 days, 48.5 vs. 65.0, t = 3.08, df = 45, p < 0.01; for r mice, 17 vs. 21 days, 42.0 vs. 86.0, t = 2.80, df = 2 1 , p < 0.02). This effect was not observed in mice of the +/c genotype (for 17 vs. 21 days, 48.3 vs. 46.0, t = 0.158). Latencies to wild running showed similar significant patterns. By 21 days of age, albino mice exhibited more severe audiogenic seizures than nonalbinos (Table II and Fig. 1). This was verified by the significant t test for mice reared with their natural parents (t = 2.15, df = 56,
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Table II. Audiogenic Seizures and Body Weights in Cross-Fostered and Non-CrossFostered 21-Day-Old C57BL/6J Mice 5 Days After Acoustic Priming Stages of audiogenic seizuresb Rearing conditions and genotype
(N)
LaWild Severity Weight~ tency c running Clonic Tonic Death score
Non-cross-fostered +/+ Non-cross-fostered
(25)
10.78
19.8
72
64
64
60
65
(19)
8.34
24.6
63
47
42
32
46
Non-cross-fostered
(14)
8.08
12.2
86
86
86
86
86
Cross-fostered +/+ Cross-fostered
(16)
9.93
23.3
69
12
12
6
24.75
(31)
9.71
33.0
48
13
13
3
19.25
Cross-fostered
(9)
8.20
16.4
78
78
78
33
58.5
+/c
c/c
+/c
c/c
Body weight in grams. b In percentages. c Latency to wild running, in seconds. p < 0.05) and for those foster-reared (t = 3.92, df = 53, p < 0.001). The + gene was dominant for the phenotype of protection from audiogenic seizures under both rearing conditions, as demonstrated by the significant quadratic trends (for non-cross-fostered mice, F = 5.65, df = 1/55, p < 0.025; for cross-fostered mice, F = 11.3, df = 1/53, p < 0.005). In view of the facts that both + / + and +/c mice exhibited protection and that there was no significant difference between their scores, we conclude that the + gene is completely dominant to t h e c gene, with no demonstratable heterosis. The trends described above for the seizure severity scores were also observed for latencies to wild running. Rearing conditions had a large effect on audiogenic seizures. The net effect of cross-fostering was protective (F = 14.99, df = 1/108, p < 0.0001). This was the case for mice of all three genotypes, as revealed by the nonsignificant genotype • cross-fostering interaction (F = 0.34). Again, the latencies displayed the same pattern as the seizure severity scores.
Discussion Regardless of rearing conditions, primed 21-day-old C57BL/6J c/c mice exhibited more frequent and more severe seizures with shorter
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latencies than + / + and +/c mice. The complete dominance of the + gene for the phenotype of priming-induced audiogenic seizures in the present study is consistent with the complete dominance of this same gene for melanin. These results are also consistent with the hypothesis that melanin pigment in the inner ear aids in the functional recuperation of hearing following intense acoustic stimulation (Cherubino, 1968). Presumably, the elevation of the absolute auditory threshold in the c/c mouse would require a longer recuperative period than that of either the +/c or the + / + mouse; as a result, the period of auditory deprivation would be longer. This would be expressed by a greater rebound behavioral supersensitivity (audiogenic seizure) in albino mice compared to the pigmented mice. This interpretation is consistent with several studies which show that lightly pigmented races of man suffer more hearing loss than do darkly pigmented races (see Karsai et al., 1972). But the data of the present study reflect the effects of both innate- and priming-induced audiogenic seizures, and the agreement with Cherubino's (1968) hypothesis could be entirely artifactual. In an attempt to determine the increase in susceptibility due exclusively to acoustic priming, the seizure severity scores from the nonprimed, 100"
75"
Q
50 9 U
..N ul
~5"
.).
.),
,),
Genotype Fig. 1. Mean severity of audiogenic seizures in congenic 21-day-old C57BL/6J crossfostered (cf) and non-cross-fostered (n-cf) mice as a function of single gene changes at the albino locus.
328
Haythorn and Henry
non-cross-fostered 21 day-old mice of a concurrently run study (Henry and Haythorn, 1975) were subtracted from the severity scores of the primed, non-cross-fostered mice of the present study. This manipulation assumed that the effects of innate- and priming-induced audiogenic seizures are additive, an assumption which may or may not be justified. Nonetheless, these differences in scores produced a profile which was quite different from the data seen in Fig. 1; the increases in severity were 65.0, 25.5, and 43.75 for + / + , + / c and c/e genotypes, respectively. By this comparison, then, one might conclude that the albino is more resistant to the effects of acoustic trauma, an interpretation opposite to the one first offered. These differences in scores also suggest that the + / c mouse might display heterosis for this effect. We are presently attempting to resolve this paradox by examining acoustically stressed mice of all three genotypes for acoustic startle thresholds, audiograms, cochlear morphology, and cochlear biochemistry at an age when innate audiogenic seizures are no longer seen. Although we did not expect to find such a large cross-fostering effect, the obtained results are not too surprising. While nonacoustic manipulations have been found incapable of priming the C57BL/6J mouse, the susceptibility of acoustically primed mice can be greatly altered by manipulations such as increased levels of pyridoxine (Henry and Bowman, 1968) and altered levels of neurotransmitters (Boggan et al., 1971), No consistent changes in body weights were observed as a function of cross-fostering, so it is unlikely that global developmental differences were responsible for the protection offered by cross-fostering. REFERENCES Boggan, W. O., Freedman, D. X., Lovell, R. A., and Schlesinger, K. (1971). Studies in audiogenic seizure susceptibility. Psychopharmacologia 20:48-56. Bonaccorsi, P. (1963). Comportamento delle barriere emolabirintica, liquorale ed oftalmiea nell' albinismo.Ann. Laringol. Otol. Rinol. Faringol. 62:432-456. Chen, C.-S., Gates, G. R., and Bock, G. R. (1973). Effect of priming and tympanic membrane destruction on development of audiogenic seizure susceptibility in BALB/e mice. Exp. Neurol. 39:277 284. Cherubino, M. (1968). I melanaciti dell' orecchio interno. In Crifo (1973). Crifo, S. (1973). Shiver-audiometry in the conditioned guinea-pig (simplified Anderson-Wedenberg test), Acta Oto-Laryngol. 75:38-44. DeFries, J. C., Hegmann, J. P., and Weir, M. W. (1966). Open-field behavior in mice: Evidence for a major gene effect mediated by the visual system. Science 154:1577 1579. Galioto, G. B., and Bonaccorsi, P. (1965). I1 fattore constituzionale del trauma acuatico individuato sperimentalmente nella diversa coneentrazione di melania nella strai vaacolare. In Crifo (1973). Gutherz, K., and Thiessen, D. D. (1966). Albinism and audiogenic seizures in the mouse. Psychon. Sci. 6:97-98. Henry, K. R. (1967). Audiogenic seizure susceptibilityinduced in C57BL/6J mice by prior auditory exposure. Science 158:938-940.
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Henry, K. R., and Bowman, R. E. (1968). The dilute locus, pyridoxine deficiency, and audiogenic seizures in mice. Proc. Soc. Exp. Biol. Med. 128:635-638. Henry, K. R., and Bowman, R. E. (1970). Behavior-genetic analysis of the ontogeny of acoustically primed audiogenic seizures in mice. J. Comp. Physiol. Psychol. 70:235-241. Henry, K. R., and Haythorn, M. M. (1974). Similarities in auditory processing associated with auditory deprivation in the DBA/2J and the acoustically primed C57BL/6J mouse. J. Cornp. Physiol. Psychol., in press. Henry, K. R., and Haythorn, M. M. (1975). Albinism and auditory function in the laboratory mouse. I. Effects of single-gene substitutions on auditory physiology, audiogenic seizures, and developmental processes. Behav. Genet. 5:137-149. Karsai, L. K., Bergman, M., and Bin Choo, Y. (1972). Hearing in ethnically different longshoremen. Arch. Otolargyngol. 96:499-504. McCtearn, G. E. (1960). Strain differences in activity of mice: Influence of illumination. J. Comp. Physiol. Psychol. 53:142-143. McGinn, M. D., Willott, J. F., and Henry, K. R. (1974). Effects of conductive hearing loss on auditory evoked potentials and audiogenic seizures in mice. Nature New Biol. 244:255-256. Savin, C. (1965). The blood vessels and pigmentary cells of the inner ear. Ann. Otol. 74:611 622. Sze, P. Y. (1970). Neurochemical factors in auditory stimulation and development of susceptibility to audiogenic seizures. In Welch, B. L., and Welch, A. S. (eds.), Physiological EJfects of Noise, Plenum Press, New York, pp. 259-270. Thiessen, D. D., Owen, K., and Whitsett, M. (1970). Chromosome mapping of behavioral activities. In Lindzey, G., and Thiessen, D. D. (eds.), Contributions to Behavior-Genetic Analysis: The Mouse as a Prototype, Appleton-Century-Crofts, New York. Willott, J. F., and Henry, K. R. (1974). Auditory evoked potentials: Developmental changes of threshold and amplitude following early acoustic trauma. J. Comp. Physiol. Psychol. 86:1-7.
