‘r tit-5‘6 i lllllllllllllllllllllllllllllllllll '“ll'ilill 73”” “93> 3 1293 (ENC-639 2860 LIBRARY Michigan State L University This is to certify that the dissertation entitled Effects of Wh—Gene on the BSERs of Hamsters using Auditory Brain—Stem Evoked Potentials presented by Geoffrey Kwabla Pilot Amedofu has been accepted towards fulfillment of the requirements for Ph.D. degreein Audiology & Speech Sciences .—-.--.W “(u/VFW """ CSSOI‘ Date July 24 1989 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE lN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative Action/Equal Opportunity Institution l. lllllul ll‘ili EFFECTS OF THE GENE HQ ON THE HEARING OF HAMSTERS USING AUDITORY BRAIN-STEM EVOKED RESPONSES BY Geoffrey Kwabla Pilot Amedofu, M.A. A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Audiology and Speech Sciences 1989 9001135 ABSTRACT EFFECTS OF wngENE ON THE BSERS OF HAMSTERS USING AUDITORY BRAIN—STEM EVOKED POTENTIALS BY Geoffrey Kwabla Pilot Amedofu The gene, anophthalmic white (Eh) in the Syrian hamster is an autosomal semidominant with pleiotropic effects leading to abnormalities eye development, pigmentation and hearing. While several investigations have been conducted on the morphologic, physiologic and behavioral abnormalities caused by this mutation, there is a complete lack of information on auditory brain—stem response of the genotypes currently available in the AN/As— Wh strain. Five sets of genotypes from the AN/As— Wh strain were used. Stimulus intensity was presented from 25—75 dB nHL (~ 50 to 100 dB peSPL). Filtered clicks with a major spectra at 2000 Hz and a repetition rate of 11.1/sec were presented to both ears of each animal. We demonstrate that genotypes differ in their responses to increasing intensity levels. As such, genotypes can be classified as normal, moderate—to—severe and profound with regard to hearing deficits. DEDICATION To my parents, Amegah (bereaved) and Kesevi, my wife, Doris, and children, Mawuli and Sitsophe. ii ACKNOWLEDGEMENTS I wish to express my sincere appreciation to my major professor and chairman of my dissertation committe, Dr. Ernest J., Moore for his advice, guidance and encouragement during the planning, execution and writing of this dissertation, as well as his unflinching support during my stay at Michigan State University. My sincere gratitude is extended to Dr. James H. Asher Jr., for his expertise on hereditary deafness, and the use of animals. I also extend my appreciation to other members of my dissertation committee, Dr. Leo V. Deal, Dr. Linda L. Smith and Dr. Yash Y. Kapur for their invaluable support, encouragement and guidance throughout my academic program, and during the period of this study. Special thanks are extended to Mr. Randy Robb for his expert technical assistance. My gratitude also goes to the two students in Dr. Asher's laboratory, Casey Miller and Tracy Johnson for the care of the hamsters used in this study, and for their time in making them available. Further appreciation is extended to Michigan State University, particularly, the Department of Audiology and Speech Sciences for making it possible for me to receive financial support for my studies and for making space, equipment and resources available to me. Finally, my heart-felt acknowledgement goes to my wife, Doris, and my two children, Mawuli and Sitsophe, for enduring the hardships, yet providing support during my absence. iv TABLE OF CONTENTS List of Figures ........ V ........................................... ix List of Tables ..................................................... xii CHAPTER I. BACKGROUND AND PURPOSE ........................ 1 CHAPTER II. REVIEW OF LITERATURE ........................ 13 Introduction. . o o o o o o u o a o o o 0c- ooooooooooooo 13 CHAPTER III. CHAPTER IV. CHAPTER V. CHAPTER VI. REFERENCES.. APPENDICES.. Appendix A. -Genotypes and phenotypes of the Anophtha‘ lmic White and a second Locus Cream (ef.vl4 Effects of the wg— gene in tne'namSter...l6 Deafness of Genetic Origin ............... 23 The Waardenberg Syndrome ................. 26 The use of ABR in Hearing Evaluation ..... 31 Human Studies .................. . ......... 35 Animal Studies of ABR .................... 45 ABR in the Syrian hamster ................ 52 INSTRUMENTATION AND PROCEDURE .............. 56 Stimulus Generating System....;... ....... 56 Electrophysiologic Recording System ...... 59 Calibration of System .................... 60 Materials ....... ... ...................... 64 Procedure .............. a ................. 65 Data reduction and Statistical Analysis..66 RESULTS .............................. ' ...... 70 DISCUSSION ................................ 155 SUMMARY AND CONCLUSIONS .................. .173' Suggestions for additional research.... .179 ........................................... .182 ........................................... .204 .204 Definition of terms ................ Appendix Appendix APPENDIX Appendix APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX F. Auditory brain—stem evoked responses of five sets of genotypes to clicks ............. . ................. 209 Latency and amplitude values for the Agouti for waves I—IV ................ 235 Latency and amplitude values for the Cream for waves I-IV ................. 244 Latency and amplitude values for the Black—eyed White for waves I-IV ...... 253 Latency and amplitude values for the White—belly Agouti for waves I-IV....262 Mean latency values for waves I—IV for all animals right and left ears) ................. . .............. 271 Differences between Agouti and Cream with respect to latency as given by F—values ............................. 276 Summary of genotype (Agouti and Cream) x intensity x ANOVA X BSER latency for‘ right and left ears ................... 278 Duncan's test for latency for waves I—IV at various intensity levels ............................. ..281 Mean ILDs for BSER waves I-IV for the Agouti and Cream ..................... 290 Summary of Ear x latency x ANOVA for BSER waves I-IV of Agouti and Cream..292 Summary of genotype (BEW and WBA) x intensity x ANOVA x BSER latency for right and left ears .................. 295 Mean ILDs for BSER waves I—IV for the BEW and BA ........................... 298 Summary of Ear x intensity x latency x ANOVA for BSER waves I—IV of BEW and NBA .............................. 300 vi APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX 21 Linear regression latency values for waves I-IV for both ears of all genotypes ............................ 303 Individual and mean thresholds for waves I—IV for all genotypes ............................ 305 Differences between Wh—locus and E—locus with regard to wave latency as given by Chi—square ............... 309 Mean amplitude values for waves I—IV for all genotypes .................... 311 Differences between Agouti and Cream with respect to amplitude as given by F—values ............................. 316 Summary of genotype (Agouti and Cream) x intensity x amplitude x ANOVA for BSER waves I-IV for right and left ears ................................. 318 Duncan's test for amplitude of waves I—IV at various intensity levels ..... 321 Summary of Ear x intensity x amplitude x ANOVA for BSER waves I—IV of Agouti and Cream ............. 330 Summary of genotype (BEW and WBA) x intensity x amplitude x ANOVA for waves I—IV for right and left ears ................................. 333 Summary of Ear x intensity x amplitude X ANOVA for BSER aneS I-IV of BEW and NBA .................................. 336 Linear regression latency values for waves I—IV for both ears of all genotypes ............................ 339 Relative amplitude ratio for waves III/I for all genotypes .............. 342 vii APPENDIX 2. Differences between Wh-locus and g-locus with respect to wave amplitude as revealed by Chi—square ........................... 344 viii III—l. III—2. IV—1. IV-2. IV-3. IV-4. IV-5. IV—6. IV-7. IV-8. IV—9. IV—lO. IV-ll IV—IZ. LIST OF FIGURES Block Diagram of Instrumentation ................ 57 Block Diagram of Calibration equipment .......... 61 Input—output latency functions for Agouti ....... 75 Input—output latency functions for the Cream....77 Input—output functions for waves I and II for the Black-eyed White .......................... 81 Input—output functions for waves III and IV for the Black—eyed White .......................... 83 Input—output functions for the White—belly Agouti ............................... . ........ 85 Composite data points of all genotypes for latency as a function of stimulus intensity for wave I .................................... 89 Composite data points for all genotypes for latency as a function of stimulus intensity for wave II ................................... 91 Composite data points for all genotypes for latency as a function of stimulus intensity for wave III .................................. 93 Composite data points for all genotypes for latency as a function of stimulus intensity for wave IV ................................... 95 Input—output amplitude functions for wave I of the Agouti ................................. 100 Input—output amplitude functions for wave II of the Agouti ................................. 102 Input—output amplitude functions for wave III of the Agouti .................................... 104 ix IV-l3. IV—l4 IV-15. IV-l6. IV-l7. IV-18. IV-19. IV‘ZO. IV-21. IV-22. IV-23. IV—24. IV-25. IV—26. IV—27. Input—output amplitude functions for wave IV of the Agouti ................................... 106 Input—output amplitude functions for wave I of the Cream .......................................... 108 Input-output amplitude functions for wave II of the Cream .................................... 110 Input—output amplitude functions for wave III of the Cream ................................. 112 Input—output amplitude functions for wave IV of the Cream ................................. 114 Input-output amplitude functions for wave I of the BEW ...................................... 117 Input—output amplitude functions for wave II of the BEW ...................................... 119 Input—output amplitude funcitons for wave III of the BEW ...................................... 121 Input—output amplitude functions for wave IV of the BEW ................................... 123 'Input—output amplitude functions for wave I of the WBA ................................... 125 Input—output amplitude functions for wave II of the WBA ................................... 127 Input—output amplitude functions for wave III of the WBA ................................... 129 Input—output amplitude functions for wave IV of the WBA ................................... 131 Composite data points for the amplitude of wave I (R/E) as a function of stimulus intensity and genotype ....................... 136 Composite data points for the amplitude of wave II (R/E) as a function of stimulus intensity and genotype ....................... 138 IV—28 IV-29. IV-30. IV-3l. IV-32. IV-33. Composite data points for the amplitude of wave III (R/E) as a function of stimulus intensity .................................... 140 Composite data points for the amplitude of wave IV (R/E) as a function of stimulus intensity .................................... 142 Composite data points for the amplitude of wave I (L/E) as a function of stimulus intensity and genotype ....................... 144 Composite data points for amplitude of wave II (L/E) as a function of stimulus intensity and genotype ..................................... 146 Composite data points for amplitude of wave III as a function of stimulus intensity and genotype ..................................... 148 Composite data points for amplitude of wave IV as a function of stimulus intensity and genotype ..................................... 150 xi TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE 03. O4. 05. 06. 07. 08. 09. 10. 11. LIST OF TABLES Mutations Affecting Coat Color ................ 4 Mutations Affecting the Hair .................. 5 Mutations Affecting the Nervous System ........ 7 Genotypes and Phenotypes of Hamsters in the AN/As— Wh- and AN/AS— ~E strain ............. 8 Possible Generators of ABR waves I — V in Human and rat at ........................ p..33 Latency of Wave I from 25-75 dB nHL for Agouti (R/E) .............................. 236 Amplitude of Wave I from 25—75 dB nHL for Agouti (R/E) .............................. 236 Latency of Wave I from 25-75 dB nHL for Agouti (L/E) .............................. 237 Amplitude of Wave I from 25—75 dB nHL for Agouti (L/E) .............................. 237 Latency of Wave II from 25—75 dB nHL for Agouti (R/E) .............................. 238 Amplitude for Wave II from 25—75 dB nHL for Agouti (R/E) ------------------------------ 238 Latency of Wave II from 25—75 dB nHL for Agouti (L/E) .............................. 239 Amplitude of Wave II from 25—75 dB nHL for Agouti (L/E) .............................. 239 Latency of Wave III from 25—75 dB nHL for Agouti (R/E) .............................. 240 Amplitude of Wave III from 25—75 dB nHL for Agouti (R/E) .............................. 240 Latency of Wave III from 25—75 dB nHL for Agouti (L/E) .............................. 241 TABLE 17. TABLE 18. TABLE 19. TABLE 20. TABLE 21. TABLE 22. TABLE 23. TABLE 24. TABLE 25. TABLE 26. TABLE 27. TABLE 28. TABLE 29. TABLE 30. TABLE 31. TABLE 32. Amplitude of Wave III from 25—75 dB H Agouti (L/E) ...................... ?.L for 241 Latency for Wave IV from 25-75 dB nHL for Agouti (R/E) ......................... 242 Amplitude for Wave IV from 25—75 dB nHL for Agouti (R/E) ........................ 242 Latency for WaVe IV from 25—75 dB nHL for Agouti (L/E) ......................... 243 Amplitude for Wave IV from 25—75 dB nHL Agouti (L/E) ......................... for 243 Latency for WAVE I from 25—75 dB nHL for Cream (R/E) ......................... 245 Amplitude for Wave I from 25—75 dB nHL for Cream (R/E) ......................... 245 Latency for Wave I from 25—75 dB nHL for Cream (L/E) --------------------------- 246 Amplitude for Wave I from 25—75 dB nHL for Cream (L/E) .......................... 246 Latency for Wave II from 25—75 dB nHL for Cream (R/E) .......................... 247 Amplitude for Wave II from 25—75 dB nHL for Cream (R/E) ............... . ......... 247 Latency for Wave II from 25-75 dB nHL for Cream (L/E) ------------------------ 248 Amplitude for Wave II from 25—75 dB nHL for Cream (L/E) ........................ 248 Latency for Wave III from 25—75 dB nHL for Cream (R/E) ------------------------- 249 Amplitude for Wave III from 25—75 dB nHL for Cream (R/E) .......................... 249 Latency for Wave III from 25—75 dB nHL for Cream (L/E) .......................... 250 xiii TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE Table TABLE TABLE TABLE TABLE TABLE TABLE 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. Amplitude for Wave III from 25—75 dB nHL for Cream (L/E) ............................... 250 Latency for Wave IV from 25—75 dB nHL for Cream (R/E) ............................... 251 Amplitude for wave IV from 25—75 dB nHL for Cream (R/E) ............................... 251 Latency for Wave IV from 25—75 dB nHL for Cream (L/E) ............................... 252 Amplitude for Wave IV from 25—75 dB nHL for Cream (L/E) ............................... 252 Latency for Wave I from 25—75 dB nHL for BEW (R/E) ................................. 254 Amplitude for Wave I from 25—75 dB nHL for BEW (R/E) ................................. 254 Latency for Wave 1 from 25—75 dB nHL for BEW (L/E) ................................. 255 Amplitude for Wave I from 25—75 dB nHL for BEW (L/E) ................................. 255 Latency for Wave II from 25—75 dB nHL for BEW (R/E) ................................. 256 Amplitude for Wave II from 25—75 as nHL for BEW (R/E) ................................. 256 Latency for Wave II from 25—75 dB nHL for BEW (L/E) ................................. 257 Amplitude for Wave II from 25—75 dB nHL for BEW (L/E) ................................. 257 Latency for Wave III from 25—75 dB nHL for BEW (R/E) ................................. 258 Amplitude for Wave III from 25—75 dB nHL for BEW (R/E) ................................. 258 Latency for Wave III from 25—75 dB nHL for BEW (L/E) ................................. 259 TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. Amplitude for Wave III from 25— BEW (L/E) .................... ??.?é.nHL for259 Latency for Wave IV from 25—75 dB BEW (R/E, ....................... I“? f” 260 Amplitude for Wave IV from 25—75 BEW (R/E) ...................... 99.?HL for 260 Latency for Wave IV from 25—75 dB BEW (L/E) ....................... II? for 261 Amplitude for Wave IV from 25-75 dB BEW (L/E) ......................... DHL for 261 Latency for Wave I from 25—75 as nH L (R/E) .............................. for wBA263 Amplitude for Wave I from 25~75 dB WBA (R/E) ........................ 3?? for 263 Lafency for Wave I from 25-75 dB nHL for WBA L E) ................. .................... 264 Amplitude for Wave I from 25—75 dB WBA (L/E) ........................ TIL for 264 Latency for Wave II from 25—75 dB I WBA (R/E) ....................... II? for 265 Amplitude for Wave II from 25—75 WBA (R/E) ............... . ...... 3?.IHL for 265' Latency for Wave II from 25—75 dB WBA (L/E) ....................... II? for 266 Amplitude for Wave II from 25—75 d WBA (L/E) ....................... 3.3HL for 266 Latency for Wave III from 25—75 d B WBA (R/E) ........................ 33L for 267 Amplitude for Wave III from 25—75 WBA (R/E) ....................... 3?.HHL for267 Latency for Wave IV from 25—75 dB WBA (L/E) ....................... II? for 268 TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE 65. 66. 67. 70. 71. 72. 73. 74. 75. 76. 77. Amplitude for Wave IV from 25—75 dB for WBA (L/E) ................................... 268 Latency for Wave IV from 25—75 dB nHL for WBA (R/E) ................................. 269 Amplitude for Wave IV from 25-75 dB nHL for WBA (R/E) ................................. 269. Latency for Wave IV from 25—75 dB nHL for WBA (L/E) ................................. 270 Amplitude for Wave IV from 25—75 dB nHL for WBA (L/E) ................................. 270 Latency for Wave I from 25-75 dB nHL for all animals (R/E) ......................... 272 Latency for Wave II from 25-75 dB nHL for all animals (R/E) ....................... 272 Latency for Wave III from 25—75 db nHL for all animals (R/E) ....................... 273 Latency for Wave IV from 25—75 dB nHL for all animals (R/E) ....................... 273 Latency for Wave I from 25—75 dB nHL for all animals ........................... 274 Latency for Wave II from 25—75 dB nHL for all animals (L/E) ....................... 274 Latency for Wave III from 25—75 dB nHL for all animals (L/E) ...................... 275 Latency for Wave IV from 25—75 dB nHL for all animals (L/E) ...................... 275 Differences between Agouti and Cream with respect to wave latency as given by F~va1ues ............................ 277 Summary of genotype (Agouti and Cream) x intensity x ANOVA X BSER latency for Right Ear .............................. 279 l 3.4;; TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE 80. 81. 82. 83. 84. 85. 86. 88. 89. 90. 91. Summary of genotype (Agouti and Cream) x intensity x ANOVA x BSER latency for the Left Ear .................................. 280 Mean ILDs for all genotypes for waves I—IV for Agouti and Cream ...................... 291 Summary of Ear x intensity x latency x ANOVA for BSER waves I-IV of Agouti ............. 293 Summary of Bar x intensity x latency x ANOVA of BSER waves I—IV of Cream ......... 294 Differences between BEW and WBA with respect to latency as given by F—values. . .............................. 296 Summary of genotype (BEW and WBA) X intensity x ANOVA of BSER latency of . waves I—IV for the right ear .............. 297 Summary of genotype (BEW and WBA) x intensity x ANOVA of BSER latency for waves I—IV of the left ear ................ 299 ILDs for BEW and WBA ........................ 301 Summary of Ear x intensity x latency x ANOVA of BSER waves I—IV for the BEW ....................................... 302 Summary of Ear x intensity x latency x ANOVA of BSER waves I—IV of the WBA ....................................... 304 Correlation latency values for waves I—IV for both ears of all genotypes ....... 304 Interceps of latency values for waves I—IV for all genotypes .................... 304 Slopes of latency values for waves I—IV for all genotypes .................... 306 Individual thresholds for wave I for all genotypes ................................. 306 xvii TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE 94. 97. 98. 100. 101. 102. 103. 104. 105. 106. 107. 108. Individual thresholds for wave II for all genotypes ................................. 307 Individual thresholds for wave III for all genotypes ................................. 307 Individual thresholds fer wave IV for all genotypes ................................. 308 Mean thresholds for waves I—IV for all genotypes (R/E) ........................... 308 Mean thresholds for waves I—IV for all genotypes (L/E) ........................... 310 Difference between Wh-locus and E—locus with regard to latency as given by Chi—square ................................ 312 Amplitude (NV) for wave I from 25—75 dB nHL for all animals (R/E) ................. 312 Amplitude (NV) for wave II from 25—75 dB nHL for all animals (R/E) ................. 313 Amplitude (NV) for wave III from 25—75 dB nHL for all animals (R/E) ................. 313 Amplitude (NV) for wave IV from 25—75 dB nHL for all animals (R/E).. ............... 314 Amplitude (NV) for wave I from 25-75 dB nHL for all animals (L/E) ................. 314 Amplitude (NV) for wave II from 25—75 dB nHL for all animals (L/E) ................. 315 Amplitude (NV) for wave III from 25—75 dB nHL for all animals (L/E) ................. 315 Amplitude (NV) for wave IV from 25—75 dB nHL for all animals (L/E) ................. 317 Difference between Agouti and Cream with regard to amplitude as given by F—value ................................... 319 xviii TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE 109. 110. 111. 112. 113. 114. 117. 118. 119. Summary of genotype (Agouti and Cream) x intensity x ANOVA for BSER amplitudes for waves I—IV (R/E) ...................... 320 Summary of Ear x intensity x amplitude x ANOVA of BSER waves I-IV for the Agouti .................................... 331 Summary of Ear x intensity x amplitude x ANOVA of BSER waves I— IV of the Cream ..................................... 332 Summary of genotype (BEW and WBA) x intensity x ANOVA of BSER amplitudes of waves I-IV (R/E) .......................... 334 Summary of genotype (BEW and WBA) x intensity x ANOVA of BSER for waves I—IV (L/E) ................................ 335 Summary of Ear x intensity x amplitude x ANOVA of BSER for waves I—IV of BEW ....................................... 337 Summary of Bar x intensity x amplitude x ANOVA of BSER of waves I—IV of WBA ....................................... 338 Correlation values for amplitude for all waves (R/E) ........................... 340 Intercepts for waves I-IV for all genotypes (R/E) ..................................... 340_ Slope for wave I—IV for all genotypes (R/E).340 Correlation for waves I-IV for all genotypes (L/E) ..................................... 341 Intercepts for waves I—IV for all genotypes (L/E) ..................................... 341 Slope for waves I—IV for all genotypes (L/E) ..................................... 341 III/I amplitude ratio for all genotypes (R/E) ..................................... 343 TABLE 123 TABLE 124. III/I amplitude ratio for all genotypes (L/E) ................................ 343 Differences between Wh-locus and E—locus with respect to wave amplitude as revealed by Chi—square ....................... 345 XX CHAPTER I BACKGROUND AND PURPOSES The golden Syrian hamster, (Mesocritus Auratus), was originally named Critus Auratus. It is believed that all Syrian hamsters currently being used as laboratory animals in Europe and the United States descended from three siblings, one male and two females, captured from Syria in 1930 (Adler, 1948). Since the time in which they were introduced to the laboratory setting, a number of mutations have been described and the genetics of several of these mutations have been investigated. As a result, at least two autosomal linkages have been established, in addition to sex—linkage. Several of these linkages are explained in Tables I-l, I-2 and I-3. It can be seen in Table I—1 that at least 16 mutations affecting coat color are known. Seven of these are of the recessive trait, while four are dominant. At least ten mutations affecting traits other than coat color are also known (see Tables I—2 and I-4). Of these, five affect hair, four appear to affect the nervous system and one affects the muscular system. 2 The gene anophthalmic white (W2) (Table I—4) is one of the mutations that affect the nervous system. According to Asher (1968), this gene is perhaps one of the most potent in the Syrian hamster, as well as other mammals as related to disorders of the auditory system. This mutant gene was first described by Knapp and Polivanov (1958) as an autosomal recessive gene, inherited independently of the partial albino 98 mutation. Beher and Beher (1959) demonstrated that the WE mutation is dominant since the heterozygotes may be distinguished from the homozygotes. On this premise they suggested that the gene acted as a partial dominant. Based on this notion, they proposed the symbol Wh for the mutant gene. Heterozygous hamsters (EB/EB) were found to be agouti, but posessed white belly fur as opposed to the pale cream fur of the wild type. Robinson (1962, 1964) described the Wh gene as incompletely dominant and indicated that animals homozygous for the mutant showed a complete absence of coat and skin pigmentation, while aplasia of the eyes resulted in extreme anophthalmia (see Appendix A for Definition of Terms). Studies of the mutant Wh in the AN/AS—Wh strain (Asher, 1980) have revealed a strong interaction between Wh and cream (9). The expression of Wh is enhanced in the presence 3 of_e_and the homozygous cream (e/e) hamsters appear to be more active than the contrasting wild—type golden hamsters. In addition to the W2 and g, a mutation of the wild—type ‘ allele E, the extension locus show a strong epistatic interaction such that Wh/wh;E/E hamsters are white bellied agouti (the so—called imperial hamsters), while the Wh/wh; e/e hamsters are black—eyed whites (see table 4). At the Biological Research Center at Michigan State University, inbred lines are maintained by full sibling matings. These inbred lines were designed to reveal several genetically—based differencies which provide several lines of experimental materials for bio-medical research. The strain contains the following genotypes segregating on a single genetic background: (1) wh/wh; E/E (agouti), (2) wh/wh;§/g (cream), (3) Whiwh;§£e (white bellied agouti), (4) flfi/W§:§/g/ (black-eyed white) and (5) Wh/WQ: -- (anophthalmic white) (see table 4). There are three major aspects of the gene that are well known: (1) The homozygotes Wh/Wh lack all melanocyte derived pigmentation, (2) they have severe optic degeneration but, for the purposes of this investigation, (3) they are deaf (Robinson, 1962, 1964; Yoon, 1973, 1975; Asher, 1981; and Asher and James, 1982). In fact, the gene WE (anophthalmic white) of the Syrian hamster is known to be 4 Table 1. Mutations affecting coat color in the Syrian hamster (Yoon, 1973) Gene Mode of Name Symbol inheritance Description Acromelanic white gd Autosomal recessive. White pelage with dark pinna Brown b Autosomal recessive Amber in _ colour CreaM g Autosomal recessive Rich creamy yellow Dark gray gg Autosomal recessive Dark gray with less brown Dermal pigmentation — — Dermic . melanism surpressed Dominant spotting Es Autosomal dominant Irregular patches Frost eye Lethal gray Light undercolor Motled white Piebald white Ruby eye Rust Tortoise shell Autosomal dominant S—linked dominant. Autosomal recessive Autosomal recessive Autosomal recessive S—linked semidominant of white fur over the back and sides homozygoteS' are lethal Gray hairs, occassional nomalies Gray fur; lethal when homozygous, Whitish undercolour heterozygous females have white areas Irregular patches Dilute coat colour Dark—brown Yellow in males and homozygous females, yellow patches in heterozygous females Table 1 (continued) Tawny T — Light agouti White band Ba Autosomal dominant White band in trunk region Table 2. Mutations Affecting Hair in the Syrian Hamster (Yoon, 1973) Gene Mode of Name Symbol Inheritance Description Hairless hr Autosomal recessive Sparse hair Long hair I Autosomal recessive Long hair Naked N Autosomal semidominant Heterozygous have sparse hair; homozygous are devoid of hair Rex EE Autosomal recessive Wavy hair Satin Sa Autosomal semidominant Hair has __ satiny sheen in hete— rozygous, and is satiny thin in .homozygotes. Table 3. 1973) Gene Name Anophthalmia Hind—leg paralysis Hydrocephalus Seizure Mode of Symbol Inheritance in 29 13X Autosomal semidominant S—linked Autosomal recessive Mutations affecting the nervous system (Yoon, Description Homozygotes show acromia and anophthalmia and anomalous optic nerve and hearing; hetero— zygotes have diminished ventral hair recessive paralysed hind-legs Displacement of brain structures Frequent seizures The Syrian hamster from the AN/As- Wh and AN/As— E Table 4. Strain Gene Name Symbol Description Agouti wh/wh;§/§ Agouti on dorsal and pale yellow on ventrum Agouti wh/wh;§/§ Agouti on dorsal and pale yellow on ventrum Cream wh/wh;e/e Dark yellow on dorsum and pale yellow on ventrum some white spots on ventrum ‘ Imperial Wh/wh;§/§ White ventrum hamster agouti on dorsal sprink white hairs Imperial Wh/wh;§/e White ventrum hamster agouti on dorsal Black—eyed Wh/W§;§/§ hamster Anophthalmic Wh/Wh;—— White with black eye Lack all pigment, white blind and deaf. 9 a highly pleiotropic mutation, causing several morphologic, physiologic and behavioral abnormalities (Asher, 1968; Pratt, 1979, 1982; Hagen and Asher, 1983). The morphologic effects of the WW mutation are to cause homozygotes to be deaf, blind and white. Further, a careful examination of the cochlea of the hamsters using light and electron microscopy reveal that the gene causes degeneration of the tectorial membrane, which becomes very apparent between 10- and 15 days of neonatal Iife (Asher, 1988). Other well known deleterious effects caused by this mutation include, infertility, small adrenal glands, growth retardation, reduced growth rates, increased metabolic rate with concomitant increase in food and water consumption, and altered plasma amino acid pools, (Asher, 1968, 1981). While several studies have been conducted which reveal the morphologic, physiologic and behavioral abnormalities caused by the gene Wh, there is a complete lack of empirical data in the results of the audiology literature on the effects of the WW gene on the hearing of hamsters. Mutations homologous to WW are not unique to hamsters. That is, the Wh gene is believed to be homologous to the mouse mutant Wi% and the Waardenberg syndrome WSI in man. Fundamental to this notion is that the WW mutation showed a full range of phenotypic effects observed in the Waardenberg syndrome. Thus, Waardenberg patients are recognized by the following 10 characteristics: lateral displacement of inner canthi, a high broad nasal root, confluent eyebrows, heterochromia iridum, a white forelock or early greying, and congenital deafness of one or both ears. Despite the documented significance of the WW gene as it relates to the Waardenberg syndrome in humans, however, an investigation to evaluate the hearing sensitivity of hamsters with this mutation using auditory brain—stem evoked responses has not been completed. We know that the ABR can be used to effectively evaluate and monitor the hearing status of human infants (Hecox and Galambos, 1974; Salamy, Mckean and Buda, 1975; Schulman— Galambos and Galambos, 1975; Salamy and Mckean, 1976; Teas, 1982; among others). In the same vein, the ABR has also been investigated in other animal species including the cat (Jewett and Romano, 1972; Shipley, Buchwald and Norman, 1980; Laukli and Mair, 1982; Walsh, McGee and Javel, 1986a), rat (Jewett and Romano, 1972), mouse (Henry and Lepkowski, 1978; Shnerson and Pujol, 1982) and gerbil (Woolf and Ryan, 1985b). Thus, while the hamster has been chosen as a model for the study of the developing capabilities of the peripheral auditory apparatus (Pujol, Abonnenc and Rebillard, 1975; Pujol, Carlier and Lenoir, 1980; Stonek, 1977; Book and Seiter, 1978; Relkin, Saunders and Konkle, 1979; Relkin and Saunders, 1980), very little is known about the hearing sensitivity of the hamster. A thorough review of 11 the results of the literature revealed that there has not been any investigations conducted to systematically evaluate the hearing of the hamsters. There is a need to conduct an investigation into the hearing of the hamsters using the ABR technique. Such an investigation may lead to a better understanding of the hearing patterns associated with the WW gene and may provide insight into the possible commonality between the WW gene and the Waardenberg syndrome in humans. Therefore, the primary purpose of this investigation is to assess the hearing of hamsters in the AN/As—WW strain. This study may assist subsequent studies currently being designed to clone and sequence the normal and mutant genes from humans (W81) and hamsters (Asher, personal communication). In the long run, the genetic studies may determine how the primary defect of these genes cause the multitude of phenotypes. The logical sequel of this finding will be, perhaps, the development of a diagnostic procedure to identify affected WW hamsters, and eventually, WSl individuals in utero. With studies designed to assess the integrity of the auditory system of the AN/As hamster, one may determine the effect of the mutation on the hearing of the animals. Specifically, this experimental investigation was designed to test the following null hypothesis: The genotype WW/WW;E/e (Agouti) has no effect on the morphology, latency and amplitude of waves I—IV 12 of the auditory brain—stem response at varying intensities. The genotype EB/227349 (Cream) has no effect on the morphology, latency and amplitude of waves I—IV of the auditory brain—stem response at varying intensities. The genotype WW/WW;e/g (Black—eyed white) has no effect on the morphology, latency and amplitude of waves I-IV of the auditory brain—stem response at varying intensities. ‘ The genotype WW/WW, E/e (White—belly Agouti) has no effect on the morphology, latency and amplitude of waves I—IV of the auditory brain-stem response at varying intensities. The genotype WW/WW;-— (Anophthalmic white) has no effect on the morphology, latency and amplitude of waves I—IV of the auditory brain—stem response at varying intensities. CHAPTER II REVIEW OF LITERATURE Introduction The Syrian hamster (Mesocritus Auratus) is a Small rodent that has been used increasingly in biomedical research since its capture from Syria in 1930 (Adler and Theodore, 1931; Adler, 1948). One of the mutations that affect the nervous system, the WW gene (anophthalmic white), of the Syrian hamster was discovered by Knapp and Polivanov (1958). Investigations (Robinson, 1962; Asher, 1968, 1981; James et al, 1980) have shown that the E mutation is a highly pleiotropic gene causing deleterious effects upon eye development, pigmentation and reproduction. While the WW mutation causes deafness, apparently, very little is known about the details of the integrity of the auditory system of the genotypes. This is important since the WW gene is believed to be homologous to the WSl gene that causes deafness in humans associated with the Waardenberg syndrome. We do know that the ABR is an invaluable, non—invasive technique that has proved especially useful in assessing the hearing capabilities of both humans (Jewett and Williston, 1971) and animals (Jewett and Romano, l3 14 1972). As such, studies using ABR to evaluate the auditory system of the genotypes resulting from the WW mutation may prove fruitful. In the following review, I will dicuss the phenotypic effects of the WW mutation, and the use of the ABR in assessing hearing sensitivity in humans and animals. Genotypes and Phenotypes of the Anophthalmic White (WW) mutation and a mutation at a Second Locus Cream (e) As already noted, the WW mutation has three obvious characteristics, the complete suppresSion of pigment from the pelage so that the animal bears a remarkable resemblance to albinos, degeneration of the eye which results in anophthalmia (Robison, 1962; Asher, 1968), and the degeneration of the tectorial membrane (Asher, 1988). Thus, the homozygote, WW/WW; is devoid of pigment, is anophthalmic and is deaf. The eye structures are rudimentary and often the eyelids are sealed by an exudate (Robinson, 1964; Yoon, 1973; Asher and James, 1982). The heterozygote, WW/WW;§/— is superficially, a normal agouti. On.a closer observation, however, the ventral surface is white instead of the usual pale yellow. The difference of pigmentation readily provides a distinguishing characteristic of the WW/WW phenotype from the others and occurs between 8th and 12th day of life. The mutant gene epistatically interacts 15 with mutants at another locus g to produce other phenotypes such as WW/WW; g/g is cream colored, while WW/WW;e/g is black-eyed white (Robinson, 1958, 1959, 1962, 1964; Asher, 1968) (see table 4). The E mutation is known to inhibit the formation of eumelanin (black pigment). Individuals homozygous for the mutant are referred to as "cream". The mutant is inherited as an autosomal recessive gene. The heterozygote, e/g, is of normal variability and fertility. The coat color varies from the straw—yellow to rich apricot yellow. Although eumelanin is removed from the hair, the eyes are dark and some pigment is observed around the genitalia of both sexes. The AN/As strain is homozygous for e. The normal allele W was then backcrossed onto the AN/As strain. The WW/WW;g/g, is a normal hamster with an agouti coloration on the dorsal fur and pale yellow on the ventrum. Dermal melanocytes are found in the hairy skin. The eyes are black (Robinson, 1958; Ghadially and Baker, 1960; Rapppaport, et a1. 1963). The WW/WW; e/g, is similar to WW/WW;g/e, in phenotype, even though the combination of the extension genes E and e are different. The WW/WW; e/g, hamsters have dark yellow fur on the dorsum and pale yellow fur on the ventrum with ventral white spotting (Robinson, 1955; Illman and Ghadially, 1960; Pratt, 1979). The compbund mutant hamsters, WW/WWie/e, on the contrary have almost white or completely white fur. Pigmentation, when it is present in the fur, is an extremely pale yellow and is usually noticeable on top 16 of the head and shoulders as descrete small patches. The ears have only patchy black pigmentation; while the eyes are dark but lighter in color than the eyes of WW/WW;e/e, or WW/WW; g/W (Pratt, 1979). Finally, WW/WW72/e have a white ventral fur, and a sprinkling of white hairs on the dorsum (Robinson, 1962, 1964). The WW/WW;e/§, are similar to WW/WW; e/g, in phenotype. Effects of the WW Gene in the Hamster Attempts to examine the effects of the WW gene in the hamster is not recent. The mutation was first described by Knapp and 'Polivanov (1958) as an autosomal recessive gene and was given the symbol WW. Since that time, several investigators have attempted to describe the deleterious effects of the mutation in the hamster. Robinson (1962, 1964) described the WW mutant as an incompletely dominant gene which produced in the homozygous condition a hamster which is completely devoid of pigment and is anophthalmic. Additionally, he observed that the gene appeared as an interspecies mutant similar to WW” (microphthalmia white) of the house mouse. Asher (1968) investigated the AN/As—WW strain of hamsters to determine the phenotypic and genotypic characteristics of the WW gene. Specifically, the purpose of his study was to describe the morphologic effects of the gene WW and to 17 describe some of its biochemical activities. The characteristics that were measured on 45 adult (130 days) hamsters included total body weight, adrenal weights, uterine weights, plasma total proteins, plasma cholesterol and free plasma amino acid. General observations were made with respect to gross anatomy and behavior. Further, normal embryos were collected from day 11—0 through birth. Gross embryonic anatomy was examined along with total fetal weights. Observations that were made on the embryos included number of normal embryos, number of abnormal embryos, relative position of each embryo in utero with respect to ovaries and genotypes of embryos. Histological examinations were also made of eyeless and normal embryos, as well as testicular material from eyeless and normal adult males. Finally, gross anatomical observation. were made upon each adult hamster sacrificed. It was observed that there were no apparent differences when the weights of the normal embryos (wh/WW) were compared with the weights of the eyeless embryos (WW/WW). This indicated that the WW mutation appeared to act after birth. In addition, histological examination revealed an absence of pigment as well as an apparent detachment of the retina in a single eyeless embryo at days 13. It was observed that this defect may be related to the failure of the closure of the 18 choroid fissure. In adult hamsters, it was observed that the WW gene affected pigmentation, eye development, degree of "nervousness", soundness of sleep, sexual differentiation, fur texture and the general growth. Pigmentation was absent from the fur and skin of the heterozygous WW/WW hamsters with a slight reduction in retinal and ear pigmentation. On the contrary, pigmentation was completely absent from the WW/WW hamsters. Section of the orbital contents of a single adult eyeless WW/WW hamsters showed that adults had severe microthalmia rather‘ than anophthalmia. Observation. of a single slide of a normal (WW/WW) and eyeless (WW/WW) testicle revealed that the eyeless male lacked spermatozoa. Data from the biochemical analysis suggested that the primary biochemical lesion caused by WW is related to the urea cycle. Since eyeless WW/WW hamsters have elevated arginine levels and lowered citrulline levels, the "metabolic" bloc then appears at arginase (an enzyme in the liver) which converts arginine to ornithine and releases urea. It was proposed that the elevated levels of plasma arginine along with a possible NH3 elevation could act as a metabolic inhibitor of fundamental biochemical and developmental processes. The differences noted in the adrenal size, sex differenciation and general growth between normal and eyeless hamsters could be explained by abnormal pituitary function. Malfunction of the pituitary could be explained by elevated levels of NH3, arginine, or 19 citrulline 1J1 the cerebrospinal fluid. Pigmentation abnormalities could arise by two mechanisms, namely, inhibition of ndgration or differentiation of neural crest tissue. The abnormal eye development appeared to arise from improper closure of the choroid fissure. James et al (1980) investigated the effects of the WW mutation on reproduction in the Syrian hamster. The phenotypes used in the study included normal (WW/WW;g/e), heterozygous (WW/WW;e_/_e_),and homozygous (WW/WW;_e_/_e) from the AN/As—WW strain (Asher, 1968) (see table I-4). This strain. was maintained by a full sibling mating where at least one parent of each generation was heterozygous for the WW gene. Ten sets of the normal, homozygous and.heterozygous hamsters were used. After the animals were anesthetized, the testicular tissue of both normal and eyeless hamsters were collected, and further processed for comparison by both light and electron microscopy. It was found that testicular tissue from several mutant animals approached the normal phenotype, due to variable expression of the gene. Most testes from homozygous mutant hamsters, however, were found ix>lme hypoplastic and aspermic. Since the primary function of the gene was unknown at the time, it was suggested that the mutation either acted directly to alter pituitary function.or that the abnormalities in reproduction were due to the failure of eye development and subsequent lack of function of the visual pathway. 20 Asher (1981) measured ten physiologic parameters controlled by the hypothalamic axis on males of three genotypes: WW/WW; g/e, WW/WW; e/g, and WW/WW; 9/3. The physiologic parameters measured included body weight, metabolic rate, fasting weight loss, body temperature, feed consumed, water consumed, urine volume, urinary pH, respiratory rate and heart rate. The results showed.a reduction in body temperature and respiratory rate, and an elevation in metabolic rate, drinking rate, urinary pH and urinary volume. This indicated that the WW/WW mutation altered independently, six parameters known to be regulated by six different sites in the hypothalamus. The union of these six pleiotropic effects led to the conclusion that the development of the pituitary and hypothalamus (the location of the pituitary regulatory centers) in concert with the eyes are altered. In View of the fact that the biological intersection of the eye, the hypothalamus and the pituitary is the embryonic diencephalous, then, it was reasonable to propose that the primary action of the WW gene is to alter all structures whose development and function are dependent on the embryonic diencephalon. James and Asher (1981) compared the hypophysis from ten heterozygous, ten homozygous mutants and five normal enucleated hamsters from the AN/As—WW strain using light and electron microscopy. The purpose of the study was to 21 determine whether morphologic abnormalities existed in the classes of hamsters. As expected, pituitaries from hamsters homozygous for the WW gene showed far different morphologic characteristics from the normal heterozygous and normal enucleated animals. The investigators postulated that the WW mutation either altered cellular differentiation of the embryonic hypophysis or caused an abnormal differentiation in the adult hamster. Asher and James (1982) performed an ultrastructural analysis of embryonic eyes on three genotypes, namely, WW/WW;e/g, WW/WW; g/g, and WW/WW;g/e, using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). They found that the primary defect caused by the WW mutation is the abnormal retention of cilia by embryonic cells. Since the normal sequence of events is to lose the cilia, Asher and James (1982) concluded that the WW gene blocked the resorptive process. These retained cilia are proposed to interfere with normal cell-cell interactions and subsequent cell differen— tiations. It was then argued that the abnormal eye development III the WW} homozygous If; associated vditi the presence of cilia on both epithelial cell layers of the optic cup and within the epithelium of the lense vessicle. Hagen and Asher (1983) employed genetically normal (WW/WW), genetically normal and blinded (WW/WW— B) and mutant eyeless 22 hamsters (WW/WW) in order to determine whether the WW gene by itself influences testicular differentiation, and also whether removal of the pineal gland will restore fertility in these classes of hamsters. Fundamental to this idea is that, male hamsters homozygous for this gene are usually sterile. Secondly, both WW and the pineal organ are known to suppress reproductive functions. Here again, it was shown that testes from the experimentally blinded (WW/WW B) and 70% of the WW/WW hamsters degenerated to less than one-tenth of their normal size. With these properties known, it was surmised that the atrophy of the testes from WW/WW hamsters is a pineal-mediated phenomenon due to failure of eye development and the subsequent lack of a functional visual pathway. This hypothesis was demostrated.to be true when genetically eyeless males had normal fertility after pinealectomy. Asher (1988) explored the effect of the WW mutation on the cochlea using light and electron microscopy. It was found that the WW gene caused the degeneration of the tectorial membrane and that this became apparent between the tenth and fifteenth day of neonatal life. It turns out that this morphology is associated with the failure of the tectorial membrane to detach from the future inner sulcus cells of the organ of (knfiju The tectorial membrane becomes hylane in nature and then disappears. The appearance of cilia, however, were not observed in any unusual places during inner ear 23 development. As such, the primary action of the gene WW cannot be to inhibit resorption of cilia. Thus, the microscopic examinations demonstrated the effect of the WW mutation on the cochlea of the hamster. No one at this point, however, has evaluated the auditory system of the genotypes found in the AN/As—WW strain of hamsters using the ABR. Deafness of Genetic Origin Deafness of genetic origin may be present at birth or may manifest itself in infancy or later in life. Hereditary hearing loss may be classified into three types, namely, aplasia, hedero—degenerations and chromosomal abberations. Aplasia is described as incomplete or arrested development of the inner ear and is always congenital. There are various types of aplasia based on the degree of development. Thus, the time at which development was arrested determines the ultimate structural appearance of the ear (Ormerod, 1960). The various types of aplasia are named after those who first described them in the results of the literature. Thus, the most commonly occuring type is that described by Scheibe (1892). In this type of aplasia, the bony labyrinth is fully developed but the cochlea and the saccule are malformed. The next in order of frequency of occurence was described by Mondini (1791), and it is characterised by incomplete development of the bony and membraneous labyrinth. Another 24 group of aplasias is the Michel type (1863) and is characterized by lack of development of the petrous portion of the temporal bone. Heredogenerations is associated with deafness which may occur alone or in combination with other abnormalities (Johnson, 1952; Ormerod, 1960). Chromosomal abberations refer to anomalies due to the presence of an extra chromosome. It is responsible for a number of severe abnormalities (e.g. trisomy D), including deafness. There is a controversy about the classification. of the heredo-degenerations. Goodhill (1950) inns classified this hereditary type of deafness into infantile and adult types, however, Cawthorn and Hinchcliff (1957) proposed a continuous distribution for the age of onset. Johnson (1952) reported that familial nerve deafness is transmited by a dominant gene. Ersner and Saltzman (1941) reporetd a family with sex—linked recessive inherited deafness, while, Ford (1952) described two families with progressive nerve deafness due to recessive inheritance. This type of deafness may occur alone or in combination with other abnormalities, in which case, they are known as syndromes. Syndromes aux; classified jinx) mesodermal, ectodermal and neuro-ectodermal syndromes. Mesodermal syndromes consist of varying, but generally consistent patterns of middle layer disorders CHE the central nervous sysytem, associated with 25 deafness. An example of this is the Marfans syndrome, usually characterized by dislocation.of the lens, deafness and laxity of the joints. Ectodermal syndromes, on the other hand, consist of the Waardenberg syndrome, Usher's syndrome, which is characterized by retinitis pigmentosa, deafness and mental disorder. Neuroectodermal syndromes are characterized by subcutaneous tumors, auditory nerve tumors, pigmentary changes and deafness (Schuknecht, 1967). Chromosomal abberations including trisomy (an extra chromosome) that causes anomalies associated with deafness. An. example is trisomy' D which has the following characteristics, absence of the external auditory‘ canal, absence of the middle ear, microphthalmia and cataracts. Interestingly, of all the syndromes discussed above, the Waardenberg syndrome was found tx>lme homologous to the WW gene; the focus of this study. Thus, the following review is a detailed description of the characteristics of the Waardenberg's syndrome. 26 The Waardenberg Syndrome The Waardenberg syndrome is an inherited dominant condition. This mutant gene shows a marked pleiotrophy observed both between and within families (Waardenberg, 1951; Preus, Linstrom, Polomeno, et al, 1983; Arias, 1984; Mckusick, 1986). According to Wang, Karmodi, and Pashayan (1981), the syndrome affects 18 specific characteristics, six of which were listed in the original descriptions. The six original characteristics are: lateral displacement of medial canthi of the eyes and of inferior lacrymal puncta (distopia canthorum), a high broad nasal root, hyperplasia of the medial portion of the eye-brows and their confluence (confluence of the eye—brows) , heterochromia iridis (different colored eyes), and congenital total or varying degrees of partial deafness (Bwibo and Mkono, 1970; Arias, 1971; Hageman and Delleman, 1977). According to Waardenberg (1951), deafness is the most obviours manifestation of the syndrome; occuring in about 20% of the 1,050 patients at institutes of the deaf in Holland. Since the original description, over 1,200 cases of the syndrome have been reported, not only in patients of Dutch origin, but also in EMglish, American, African, and Asian peoples (Wang, Karmodiet and Pashayani, 1981). These additional reports have helped to explain further the manifestations of the syndrome originally described by Waardenberg znui have led 1X) the description of additional 27 characteristics. These include abnormal pigmentation of the skin, premature graying of hair, peculiar facial appearance and pigmentary changes of the fundi, full lips, cleft palate, cardiac murmur and vestibular abnormality. Other isolated findings have been described, such as meningocele and atresia of the esophagus, but these have not been firmly established as characteristics of the syndrome (Zerlig, 1961; Bwibo and Mkono, 1970). Fisch (1959) reported the audiologic findings of Waardenberg syndrome patients, describing two types of hearing loss. These are almost total bilateral deafness with some residual hearing in the low frequencies, and a unilateral moderate hearing loss with uniform loss for the lower and middle frequencies with an improvement of hearing in the higher frequencies. Fisch (1959) provided the only historic description of the temporal bone in a patient with the syndrome. He found total absence of the organ of Corti and atrophy of the spiral ganglion. The saccule and the utricle, however, were found to be normal. Hageman (1977) studied 34 patients and noted that dominant hereditary deafness as part of the Waardenberg syndrome was found in 35% of the patients. Audiometric examination at the maximum output of the audiometer (110 dB HL) on eleven patients indicated that five of them had total bilateral 28 deafness, while six patients had unilateral hearing loss. From these examination, he described four types of hearing loss. These are profound bilateral hearing loss (type I), severe bilateral hearing loss (type II), profound unilateral hearing loss (III) and moderate unilateral hearing loss, particularly in the low frequencies (type IV). Owing to the heterogenity.of the Waardenberg syndrome, some investigators (Arias, 1971; Hageman and Delleman, 1977; Hageman, 1977) posited that the syndrome seemed to consist of two genetically distinct entities that can be differentiated clinically. These are type I Waardenberg syndrome with dystopia canthorum, and type II Waardenberg syndrome without dystopia canthorum. Both types have an autosomal dominant mode of transmission. Hageman and Delleman (1977) made an extensive literature review of more than 1,000 patients with the Waardenberg syndrome. They found that deafness in both ears (the most serious expression of the syndrome) occured in about 25% of patients with Waardenberg's syndrome type I and in 50% of patients with type II. This striking difference is connected with the difference between deafness and pigmentary disorders. Klein (1983) provided evidence for another category of Waardenberg syndrome, known variously as, Waardenberg syndrome type III, Klein-Waardenberg syndrome, or Waardenberg syndrome 29 with upper limb abnormalities. In his studies on a French family, Klein (1983) reported a case in which the father had a complete Klein-Waardenberg syndrome including muscular and skeletal defects of the upper limbs; while his 12 year old son had the classical Waardenberg syndrome without upper limb abnormalities. It would appear that this mutation acts as an autosomal dominant type without linkage assignment. Patients with this disorder have the classical WSI phenotype an association with the abnormalities of upper limbs, hypoplasia of the musculoskeletal system and microcephally, at least in some patients. Richieri-Costa, Gollop anxi Otto (1983) provided the description of another sub-division of patients initially classified by Waardenberg (1951) as WSI. He noted in his genetic study that five children of two Brazillian families had anophthalmia and multiple congenital abnormalities. Among the four affected children, three had bilateral while one had unilateral anophthalmia. He surmised that this mutation behaved as an autosomal recessive inheritance without linkage assignment. This syndrome was designated as Waardenberg Anophthalmia Syndrome (McKusick, 1986). Another type of the Waardenberg syndrome (WSI) was described by Shah, Subhash, Desai et a1 (1981). In their study of twelve babies of five families in Bombay, India, they found 30 that these babies had intestinal obstructions in addition to having the classical WSI phenotype characteristics. On the basis of their study, they proposed that this combination may be inherited.as an autosomal recessive traitn McKusick (1986) designated this variant of Waardenberg syndrome as Waardenberg~Shah—Syndrome. The foregoing review demonstrates that there j£3£3 complex heterogeneity in this syndrome both within and between families. Within this frame of reference, and in view of the fact that Waardenberg Syndrome is homologous to the WW— mutation found.in hamsters, Asher and Friedman (1988) gave the following three genetic explainations of the complex variation of phenotypes associated with the Waardenberg syndrome. These are (1) different mutant alleles at a single locus, (2) mutant alleles at more than one locus affecting the same developmental processes, and (3) a single mutant locus with alleles interacting with genes at other loci which vary among different families. It can be argued that additional information is needed to enable investigators to continue with an analysis of the WSI phenotype. This information include (1) the number of loci responsible for the Waardenberg syndrome, (2) number of mutant alleles at a given locus, (3) the primary function of the loci involved, and (4) the mechanisms by which alterations of these 31 primary functions produce abnormalities of the eye, ear, pigmentation and skeleton.‘ In order to obtain this information, it will be necessary to use an animal model which exhibit the phenotypic effects of the Waardenberg syndrome. We know that the WW syndrome in the Syrian hamster covers an enormous range of phenotypic variations observed among the Waardenberg syndrome patients. .As such, it would.be possible to use the hamster model to clone the normal and mutant genes of WSI and WW- so as to develop a diagnostic procedure for identifying affected individuals in utero. We also know that the major abnormality caused by the Waardenberg syndrome is total deafness (Hageman and Delleman, 1977). Therefore, a starting point for meaningful research that needs to be conducted in cloning the WW gene would be to assess the hearing sensitivity of samples of the genotypes and phenotypes of Syrian hamsters from the AN/As- WW strain using the ABR. These considerations constitute the underpinnings of ‘the present study. The Use of ABR in Hearing Evaluation In 1970, Jewett and hulliston recorded far—field auditory evoked potentials from the human brain stem (brain stem auditory evoked response, BAER) tn] using scalp electrodes (Jewett and Williston, 1971). BAERs are produced by the electrical activity in the peripheral and central nervous system in response to sound stimuli. In recording these 32 potentials, the electrodes are affixed to the scalp at three positions. The positive electrode is placed at the vertex (Cz), the indifferent electrode over the test ear's mastoid and the ground electrode over the forehead (FPz). The components of the auditory brain—stem response waves have been shown by dissection studies (Buchwald and Huang, 1975) to be associated with the following anatomical locations: wave I cochlear nerve; wave II cochlear nucleus; wave III, superior olivary complex; wave IV, ventral nucleus of the lateral lemniscus, and V, the inferior colliculus. The exact location of wave IV is disputed (Stockard and Rossiter, 1977) and often appears to be fused with wave V (Starr and Achor, 1975). The location of wave VI may‘ be the medial geniculate body (Stockard and Rossiter, 1977) but its location along with that of wave VII is still disputed (Brackman and Selters, 1978). On the other hand, Hashimoto et al (1981) claim that wave I originates from the distal portion of the cochlear nerve, while wave 11 originates from the proximal portion of the cochlear nerve and perhaps the pons. Waves III and IV are from the pons and the lateral lemniscus, and wave V from the lateral lemniscus. Moller and Janetta (1985) agreed with Hashimoto et al (1981) on the generators of waves I and II; and noted further that wave III originates from the cochlear nucleus, while wave IV originates from the superior olivary complex and the lateral lemniscus. Moller and Janetta (1985), posited, however, that the neural generators of waves V, VI 33 and VII are exceedingly complex in that more than one anatomical structure contributes to each peak and that each anatomical structure contributes to more than one peak (see table 5). The possibility that the ABR might provide useful information for estimating thresholds in man and animals has been suggested (Jewett and Williston, 1971). Thus, for children and adults unable to cooperate during standard audiological procedures, such aniobjective physiologic measure could provide invaluable information about the integrity of the peripheral system. We also know that the ABR procedure can be reliably used in assessing the hearing sensitivity of animals (Jewett and Romano, 1972), and particularly in the hamster, and under anesthesia (Schweitzer, 1987). It has been shown that the threshold of the ABR is near the behavioral threshold for the same signal in human subjects. In addition, it has been posited that as the intensity decreases, the amplitude of the ABR waves decreases, while latency increases (Picton, Hillyard, and Kraus et a1, 1974; Hecox and Galambos, 1974: Yamada, Yamane and Kodera, 1977). These promising results enouraged clinicians to include the ABR test in the clinical armamentarium, as will be justified in the following review of the results of the literature in both humans and animals. 34; Table 5. Generator sources of ABR in humans and in the rat Human Possible Generator Rat Possible Generator I Distal portion P1 _Auditory Nerve of Cochlea Nerve II Proximal portion P2 Cochlear Nucleus of Cochlea Nerve III - Cochlear Nucleus P3 Superior Olivary Complex IV Superior Olivary Complex P4 Lateral Lemniscus V Lateral Lemniscus P5 Inferior Colliculus Hecox and to 3 yea: (60, 40 5 levels of for the 2 responses square u; that the stimulus Stillman 49 subje. ten were having a duration IUthsit 0€termin intensit a deCrea normal h most Sui 35 Human Studies of ABR Hecox and Galambos (1974) recorded the ABR in infants (3 weeks to 3 years of age) and adults, using three intensity levels (60, 40 and 20 dB SL) in varying order for the former and six levels of attenuation (-60, ~50, —40, —30, -20, and -10 dB SL) for the latter. Tracings were made On all subjects by summing responses presented to the monoaural ear using a 0.1 msec square wave generated at a rate of 30 per second. They found that the amplitudes and latencies of ABR waves vary with stimulus intensity. This demonstrated reliability and limited variability of the ABR waves, providing the basis for an optimistic estimate of their usefulness as anmobjective method of assessing hearing in infants and adults. Stillman (1976) employed 500 Hz tone bursts to record ABR from 49 subjects. Twenty one of these subjects had normal hearing, ten were hearing-impaired, while eighteen were suspected of having a hearing impairment. He used a tone busrt of 4 msec duration, 1 msec rise/fall and a repetition rate of 15/sec. Intensity was varied to the point where threshold could be determined. Here, again, it was found that successive intensity increments resulted in an increase in amplitude and a decrease in latency. Additionally, ABR threshold for all normal hearing subjects occured between 10 and 30 dB SL, with most subjects exhibiting threshold at 2M) dB SL. For the hearing- were for values Further, a functi latency fun :10: recuire NOller Patient had sy aSsymet Symmetr a gradu audiogr with dr repetit d8 HL: aflat1 the sub grOGUal 36 hearing-impaired individuals, however, response thresholds were found to be higher than for normals, and that specific values 'were related_ to the extent of the hearing loss. Further, changes in the amplitude and waveform responses as a function.of stimulus intensity were generally unlike changes in the response for normal subjects. As expected, mean latency for normal subjects was found to decrease as a function of stimulus intensity. For hearing—impaired subjects, (all of whom had moderate to severe hearing loss at 500 Hz) the latency-intensity functions were displaced to the right on the abscissa; and at this point, an intense stimulus was required to evoke a response. M¢ller and Blegvad (1976) used unflitered clicks to study 60 patients with sensorineural hearing loss. Of this number, 48 had symmetrical bilateral hearing loss, while 12 had assymetrical hearing-impairment. Among subjects with symmetrical hearing loss, 25% had a flat audiogram, 25% had a gradually slopping audiogram, 25% qui a steeply slopping audiogram while the remaining 25% consisted of a mixed group with diverse types of audiograms. Filtered clicks at varying repetition rates of 12 and 16/sec were presented at 10 to 90 dB HL in 10 dB steps. The results showed that patients with a flat hearing loss produced ABR responses that are closer to the subjective threshold than was the case in subjects with gradually slopping or steeply slopping audiograms. Interest- ingly, i better i to be sh hearing steeply Coats an frequenc presente d8 steps and cor Procedu: Dr0jecte POints c with big additio: the ABR phase fo the wave Were n01 1088. Yamada patholog freqUenr SenSOIix 37 ingly, in patients with flat hearing loss, all of whom had better hearing in the higher frequencies, latency was found to be shorter than in patients with pronounced high frequency hearing loss (that is, groups with gradually slopping and steeply slopping audiograms). Coats and Martin (1977) studied 16 normal subjects and.35 high frequency hearing loss subjects, using filtered clicks presented at varying intensity levels of 10 to 90 dB HL in 10 dB steps. At each intensity level, responses to rarefaction and condensation clicks were plotted seperately. The procedure adopted to obtain thresholds was a straight line projected to visually fit to the lowest three suprathreshold points on the input-output curve. It was found that subjects with high-frequency hearing loss affected ABR waveforms. In addition, such subjects showed prolonged latency of wave V of the ABR. Further, ABR peaks prior to wave V appear out of phase for condensation and rarefaction stimuli. Interestingly, the wave I to IV condensation -rarefaction polarity reversals were not present in all patients with high-frequency hearing lOSS. Yamada et al (1979) examined the effects of inner ear pathology on wave V of the ABR in 12 patients with flat, low frequency, severe high frequency and gradual high frequency sensorineural hearing loss. .Auditory stimuli consisted of Wai— ~G benc'l' hearin 38 clicks presented at 90 to 10 dB SL in 10 dB steps. The results showed that in patients with flat and low frequency sensorineural hearing loss, the latencies of wave V at intensities of 4-10 dB greater than their response thresholds were approximately the same as those in normal subjects. It was also noted that in patients with high frequency sensorineural hearing loss, the latencieS'were always delayed; compared.with those with.normal hearing. Finally; in patients with. gradually' slopping' high-frequency' hearing' loss, the latency of wave V was delayed according to the degree of hearing loss, as determined by the pure—tone audiogram. Jerger and Mauldin (1978) measured ABR threshold and latency from 275 ears of 185 patients with sensorineural hearing loss. The acoustic signal used was a half cycle of a 3000 Hz sinusoid at the rate of 20/sec. Each subject was tested at the intensity levels of 100 dB HL in 10 to 20 dB decrements until ABR waves were no longer discernible. The lowest intensity level at which a repeatable response was observed was defined as ABR threshold. In addition to the ABR thresholds and latency measures, a number of pure tone indices were also obtained from the patients' clinical audiograms. These consisted of pure tone averages at 500, 1000 and 2000 Hz; 1000, 2000 and 4000 Hz; and thresholds at 2000 and 4000 Hz. Correlational analysis of the ABR thresholds with the audiometric indices of sensorineural hearing loss indicated the tug region. threshol 0.48) we tone thr predicte reaffirm audiomet Kev nagh had sens loss and Clicks ; intensit Stimulus Evaluati measurem ABR, lat determin the best found to hEaring among SL Patholog 39 the highest correlation in the 2000 to 4000 Hz threshold region. The highest correlation was with the 4000 Hz threshold (r = 0.49), but the correlation with the PTA (r = 0.48) was almost as high. It was also shown that the pure tone thresholds at 1000, 2000 and 4000 Hz was most accurately predicted by multiplying ABR threshold by 0.6. The study also reaffirmed.the prolongation of ABR latency with.down-slopping audiometric configurations. Kavanagh and Beardsley (1979) tested 33 subjects, 24 of who had sensorineural hearing loss, eight had conductive hearing loss and one had a mixed type of hearing impairment. Filtered clicks were presented at a rate of 31/sec and at varying intensity levels. The threshold for ABRs was defined as the stimulus intensity at which waves were first discernible. In evaluating the ABR to determine hearing sensitivity, three measurements were made. These included the threshold of the ABR, latency and amplitude of wave V. It turned out that in determining the degree of hearing loss, wave V threshold was the best index. Wave latency at high intensity levels was found to have little correspondence to degree of sensorineural hearing loss. Wave V amplitude was found to be highly variable among subjects, but still a useful indicator for detecting pathology. _ __. _.-.—_._i. _ Fria and 10 patien Tone pip: Wave V la A horizor to the p. the LIF x From thi patients less th; threshol Rosenham SUbjects Cemprise (56.1 ye years). of ~80, 22~5/See latenciel latEnCie: between other tr shOrter betWeen Furtherm 40 Fria and Sabo (1980) compared ABR and audiometric findings in 10 patients using latency intensity function (LIF) techniques. Tone pips varied from 10 to 90 dB HL were used as stimuli. Wave V latency was plotted and compared with the normal LIF. A horizontal line was drawn at 20 dB from the normal latency to the patients LIF. The point where this line intersected the LIF was the estimated conductive hearing loss component. From this analysis, it was found that seven of the ten patients had predictable conductive hearing loss that were less than 15 dB different from the 4000 Hz pure tone threshold. Rosenhammer et al (1980) recorded ABR from 62 normal hearing subjects, grouped according to gender and age. The groups comprised young females (mean age of 26.8 years), old females (56.1 years), young males (59.3 years) and old males (59.3 years). Filtered clicks were presented at attenuation levels of -80, —60, and —40 dB SPL, and at a repetition rate of 22.5/sec. The ABRs were measured with respect to peak latencies and interpeak intervals. With regard to peak latencies, highly significant differencies were established between the group of young females on the one hand, and the other three groups on the other; with females exhibiting shorter latencies of the order of 0.2 msec. Differencies between the other three groups were less significant. Furthermore, in old subjetcs, but not in the young ones, the individua with redu order of were seen and 4000 with ccch duration criteria absolute latency 0 £353 'n'avef and 4000 It was ot increa5ed Presumabl response. followS. resWises Compared sensitivi at 35 dB hEal—ing l HZ Sensit 41 individual III—V interval exhibited.21 significant increase with reduction of click intensity from 80 to 60 dB SL of the order of 0.1 msec. Variances of interpeak interval measures were seen not to differ significantly. Bauch and Olsen (1986) investigated the effects of 2000, 3000 and 4000 Hz hearing sensitivity on the ABR from 458 patients with cochlear hearing loss. Rarefaction clicks with 100 us duration and a repetition rate of 30/sec were used. The criteria used for abnormal ABR responses were defined as: (l) absolute latency of wave V exceeding 6.20 msec (i.e. delayed latency of wave V > 6.2 msec), and (2) absence of repeatable ABR waveform. As expected, hearing sensitivity at 2000, 3000 and 4000 Hz was found to have an influence on ABR waveforms. It was observed that the percentage of abnormal ABR results increased with the severity of the hearing loss. This was due, presumably, to cochlear' dysfunction and. reduced. cochlear responseu The salient features of the results were as follows. First, hearing sensitivity at 2000 Hz influenced responses more than sensitivity at 4000 Hz, when both were compared at similar intensity levels. When 2000 Hz hearing sensitivity was normal, and 3000 and 4000 Hz thrsholds were at 35 dB HL or better, ABR results were normal for cochlear hearing loss patients, at least 94% of the time. When 2000 Hz sensitivity was normal, but thresholds for 3000 and 4000 Hz were at 40 dB HL or poorer, ABR results were abnormal for 15 to BC depending occurence 2000 Hz 1 Hz threst Bauch and to 4000 H at 2000, and Olsex results three-:1“ Prosserg V of AB] hearing normal, Patients DUlses 0 (181) an Subjects in 5 as at a fi caICUIat where LP‘ at 90 dB 42 15 to 80% of the patients with cochlear hearing losses, depending on the severity of the hearing deficit. The occurence of the abnormal ABR reuslts increased markedly when 2000 Hz thresholds reached 65 to 70 dB HL and 3000 and 4000 Hz thresholds were in the 50 to 70 dB HL range. Bauch and Olsen (1987) further analyzed the influence of 2000 to 4000 Hz hearing sensitivity on.ABRs by averaging thresholds at 2000, 3000 and 4000 Hz derived from a previous study (Bauch and Olsen, 1986). They found that the percent of normal ABR results decreased quite systematically as the averaged three—frequency hearing loss increased. Prosser and.Arslan (1987) collected three sets of data on wave V of ABR on normal subjects and those with sensorineural hearing loss. The three sets of data were comprised of 10 normal, 56 patients with sensori—neural hearing loss and 32 patients suffering from surgically confirmed CPA tumors. Tone pulses of 0.1 msec duration, 75 msec interstimulus interval (181) and alternating polarity was used. The normal hearing subjects were stimulated at intensities from 90 to 0 dB nHL in 5 dB steps. The hearing-impaired patients were stimulated at a fixed intensity of 90 dB nHL. The V index was calculated according to the formula V = LP(90) — Ln(90-X), where LP(90) represents wave V latency of the pathological ear at 90 dB nHL; Ln(90—X) represents wave V latency as predicted from norm patients frequenci cochlear were p101 threshold retrococh with no 3 HZ) thres Jerger ar gender, absolute hearing retrococt DOlarity auditOry determinG Was quan hearing 1 again, u Systemat: trend Was ShQWed l: to 60 dB further : 43 from normal intensity ~latency function; and X represents the patients pure—tone hearing loss as the average of the frequencies at 2000 and 4000 Hz. It. was found that for cochlear patients,' V was negative. When the absolute values were plotted against the PTA (2000 and 4000 Hz) hearing threshold, a linear relationship was found (r = 0.94). For retrocochlear patients, however, the V was mostly positive, with no significant ralationship with the PTA (2000 and 4000 Hz) threshold (r = 0.61). Jerger and Johnson (1988) studied the interactive effects of gender, age, and degree of sensorineural hearing on the absolute latency of wave V 'in 325 subjects with cochlear hearing loss and 87 subjets with surgically confirmed retrocochlear disease. Filtered clicks with alternating polarity and a repetition rate of 21.1/sec were used as auditory stimului. The click presentation level was determined by the degree of high frequency hearing loss. This was quantified as the average of the pure-tone threshold hearing levels (HTLs) at 1000, 2000 and 4000 Hz. Here again, the results showed that wave V latency increased systematically'as high frequency hearing loss increased. fifima trend was not, however, linear. For example, wave V latency showed little change with degree of hearing loss up to the 50 to 60 dB region,_but increased in a linear fashion with a further increase in hearing loss. In summa technique conductii which ob; cochlear informat: include‘ latencie the resul hearing hearing- than for to the 9 measures decrease the lat! subjects greater et all 1. also Pro‘ III~V an with Coc Stimuli meaSUrab the ABR V 44 In summary, it can be observed that the ABR is a useful technique to use in the differential diagnosis of both conductive and sensorineural hearing loss. It provides data which objectively evaluates the site~of~the—lesions, whether cochlear or retrocochlear. We do note that this diagnostic information could be drawn from three main sources. These include the threshold of the ABR waves and the amplitudes and latencies of waves I, III and V. With regard to threshold, the results of the literature showed that threshold for normal hearing subjects occured. between» 10 to 30 dB 5M1. For hearing-impaired subjects, response thresholds were higher than for normal subjects, and the specific values are related to the extent of the hearing loss. In the case of latency measures, the mean latency of normal hearing subjects decreased as a function of stimulus intensity. As expected, the latency input—output functions for hearing—impaired subjects were displaced to the right on the abscissa, as greater stimuli were required to evoke a response (Stillman et al, 1976). By the same token, diagnostic information was also provided by interpeak latency intervals, such as, I~III, III-V and I-V. Thus it was clearly shown that in patients with cochlear hearing loss, the ABR evoked by high intensity stimuli showed a I—V interval quite similar to those measurable in individuals with normal hearing. Amplitude of the ABR was shown to be the least useful index in determining hearing 5 usefulnes by Kavana intensiti amplitude observati in neasur \ . ' YY Fax/P. - M: The use ( paralled use of t) that are Similar humans. n0tahle ( used in COnVEntic ABRS are When r8c< presente( 8Y3t8m_ that it ( high rel differen< 45 hearing sensitivity, due to its large standard deviation, The usefulness of amplitude in establishing threshold was noted by Kavanagh and Beardsley (1979), who reported that at high intensities, when the latency of wave V is normal, the amplitude is often abnormal. Thus, these fundamental observations formed the basis of the criteria to be utilzed in measuring the hearing sensitivity of the hamsters in the AN/As- WE strain. Animal Studies of ABR The use of ABR in animal studies over the past 25 years has paralled the significant increase in experimental and clinical use of this procedure in humans. To be true, the techniques that are used to non—invasively record ABRs from animals are similar in most respects to those used for recordings in lunmnui, Under typical operating conditions, however, some notable differences can be found in the procedures commonly used in recording ABRs from animals. Sui the first place, conventional earphone.headsets, routinely used.to:record.human ABRs are obviously inappropriate for animal use. As such, when recording ABRs from animals, sound stimuli are either presented free-field or, more often, through a closed ear-tube system. Fundamental to the use of the ear—tube is the fact that it can be inserted in the external auditory canal with high reliability of placement so that the test and re—test differences in absolute stimulation levels are minimal. There i cup—shap recordin electroc recordir typical] general cannot however made wi way reg anesthe In anot head 5; favorat audito: non-pr; amplih is tha the 31, detect two di in the 46 There is .another point that bears mentioning. While cup—shaped, or flat disc electrodes typically used in recording human ABRs can also be used with animals, needle electrodes are preferred because they insure a more stable recording with animals. In addition, human ABR recordings are typically done with the subject either awake or sleep. A general anesthesia is administered only when the patient cannot be successfuly tested in any other state. As a rule, however, ABR recordings in animals are almost never: never made with the animal awake or in natural sleep (unless in some way restrained). As such, animals are always sedated or anesthetized during testing. In another vein, in laboratory animals, the smaller absolute head size and the concomitant brain size result in a more favorable situation for recording the responses elicited by auditory stimuli. Therefore, ABRs detected at the scalp in non—primate laboratory animals tend to have relatively large amplitude waves as compared to humans. Still another reason is that a more favorable signal—to-noise ratio is observed in the signal detected from an animal‘s scalp as compared to that detected from the human scalp. The logical sequel for these two differencies is that, the overall amount of gain needed in the biological amplifier system and the amount of averaging necesssa: commonly The reco origin. investig filtered recorded Berlin, Schorn e 1982; C Buchwalc Shipley 1986a, (Henry 1979; H< arQUed Specifi of the; Studies of the 47 necesssary to evoke a clear response may be lower than that commonly used for recordings in humans. The recording of ABRs in laboratory animals is not of recent origin. In point of fact, Jewett (1970) was the first investigator to record ABR from the scalp of the cat using filtered clicks. Since then, ABRs have been extensively recorded in guinea pigs (Dobie and Berlin, 1979b; Gardi and Berlin, 1981; Dun, 1984), rats (Jewett and Romano, 1972; Schorn et a1, 1976; Tokimoto et a1, 1977; Iwasa and Potsil, 1982; Church et a1, 1984), cats (Jewett and Romano, 1972; Buchwald and Huang, 1975; Achor and Starr, 19808, 1980b; Shipley et al, 1980; Laukli and Mair, 1982; Walsh et al, 1986a, 1986b, 1986c), monkeys (Allen and Starr, 1978) mouse (Henry and Lepkowski, 1978; Henry and Haythorn, 1978; Henry, 1979; Henry and Chole, 1979a, 1979b; Shnerson and Pujol, 1982) gerbil (Wolf and Ryan, 1985b; Smith and Kraus, 1987) and hamster (Schweitzer, 1987; Moore et a1, 1988). It can be argued that most of the studies noted above were not directed specifically to the use of ABR in determining the thresholds of the animals investigated. As such, we will not review these studies. In View of this fact, however, the following review of the literature will focus on the use of ABR in evaluating hearing sensitivity in animals. Jewett an: ABR from 1 adult ca anestheti dosage in 30 mg/kg weight up 60 days . rats, rat of click: and from increase latenCy . Henry an intensit 01d and were use mg/kg Sc 0f Clio Stimulus and ~10 Stimulu: 48 Jewett and Romano (1972) explored neonatal development of the ABR from the scalp of 12 adult rats and 124 rat pups and three adult cats together with eight kittens. Animals were anesthetized using sodium pentobarbital. The pentobarbital dosage in cats was 45 mg/kg body weight, and in rats it was 30 mg/kg body weight up to 30 days of age; 45 mg/kg body weight up to 45 days of age; and 60 mg/kg body weight up to 60 days of age. Halothane oxygene was administered to the rats, rat pups, kitten and the adult cats. Stimuli consisted of clicks that were varied from 30 to 85 dB SL for the rats and from 40 to 70 dB for the kittens and cats. It was found that for all the animals, a reduced intensity resulted in an increased latency. At younger ages, the absolute increase in latency was found to be greater. Henry and Haythorn (1978) studied the effects of age and intensity on ABR in the laboratory mouse. Twenty eight 16—day old and twenty eight 35—day old mice of the C/57SBL/6 strain were used in the study. Subjects were anesthetized with 60 mg/kg sodium petobarbitol and were presented with four series of clicks at a repetition rate of 5/sec. Intensity of the stimulus was varied at the attenuation levels of —70, —50, -30 and —10 dB HL. Here again, an inverse relationship between stimulus intensity and latency of the components of the ABR was observed in the mature 35—day old mice. By contrast, the lantencie decline a Henry' an: genetic p the C/57 progressi 8 to 11 n days of sodium pe Waves fr: Viewing revealed deCreaSe age-r913 60 dB SI WEre UO‘ functiOp the ampl t0 Chang a Small 49 lantencies of the ABR waves of the 16-day old mice did not decline as the stimulus intensity was increased. Henry anui Lepkowski (1978) investigated ABR cmurelates of genetic progressive hearing loss in the mouse. They compared the C/57BL/6 mice which displayed genetic sensorineural progressive hearing loss with the CBA/J strain of mice. From 8 to 11 mice of each genotype were tested at 50, 100 and 200 days of age. The animals were anesthetized with 60 mg/kg sodium pentobarbitol and maintained at a room temperature of 37°C, as measured by a microthermister placed against the bulla. Acoustic stimuli consisted of clicks presented at the rate of 20/sec and at the atteuation levels of —100, —60 and —40 dB SPL. It turns out that there were no genotypic differences in the amplitudes and the latencies of the ABR waves from the two groups of mice at 50 and 100 days of age. Viewing the amplitudes of the two genotypes at 60 dB SPL revealed that over 150 day span, amplitude of the ABR waves decreased for the two genotypes. In addition, no significant age—related latency changes in the genotypes were observed at 60 dB SPL. Further, at 50 days, no genotypic differencies were noted in relative amplitude of the ABR waves as a function of stimulus intensity. Interestingly, by 100 days, the amplitude—intensity relationship which.was observed began to change, in that at this age the C/57BL/6 strain of mice had a smaller dynamic range for the amplitude of ABR waves. Again, at were obse increasing Henry (19 inbred CB. with ch10 sodium p maintaine to 64000 rise/deca revealed varied a; input-ou1 with the Curves. Osako et ABR duri: AHESthes a daily Varying Signals. 50 Again, at 200 days of age, pronounced genotypic differences were observed in the rate of relative amplitude change to increasing click intensity. Henry (1979) examined ABR amplitudes and latencies in 18 inbred CBA/J laboratory mice. The animals were anesthetized with chlorphrothixine (0.60 mg/kg) and pre—anesthetized with sodium pentobarbital (60 mg/kg). Body temperature was maintained at 37.8°C. Tone pips at nine frequencies from 4000 to 64000 Hz produced at a repetition rate of 20/sec, 200 us rise/decay time and 1.0 msec duration were used. The results revealed that the amplitude and latency of the ABR waves varied as a function of stimulus frequency. In addition, input-output functions were found to be related to frequency, with the 4000 Hz curves having longer latencies than 8000 Hz curves . Osako et a1 (1979) explored the effects of kanamycin on the ABR during post—natal development of the hearing of 105 rats. Anesthesia was applied (25 mg/kg sodium pentobarbital) after a daily dosage of 400 mg/kg of kanamycin. Tone pips of varying intensities and frequencies were used as input signals. A pronounced suppression of ABR waves and thresholds of the auditory responses were observed in the group of rats to which kanamycin was administered. Church et levels of 8/sec to 4 as a fun latencies with inc: increasir 10 dB is componen‘ Dun (198 guinea p k9). Ac lO/sec a SPL, ran that at1 0f anima Control Potentia three ir Smith ar 0f the , CliCks deliver The SOu 51 Church et a1 (1984) employed clicks presented at attenuation levels of —O, —20, ~40 and -60 dB and a repetition rate of 8/sec to evaluate variations in ABRs of normal laboratory rats as a function of stimulus intensity. They found that the latencies of waves 1, II, III and IV of the ABRs decreased with increasing intensity, while amplitudes increased with increasing stimulus intensity levels. At threshold ( 0 and 10 dB levels) wave II of the ABR was the most prominent component. Dun (1984) studied the postnatal development of ABR in 10 guinea pigs anesthetized with sodium pentobarbital (3 mg/lOO kg). Acoustic clicks were presented at a repetition rate of 10/sec and at the attenuation levels of —20, —40 and —60 dB SPL, ranging in frequency from 500 to 15000 Hz. It was found that at birth, thresholds of the ABR in the experimental group of animals corresponded with those of the adult animals in the control group. Shortly after birth, however, the evoked potentials appeared at significantly longer latencies at all three intensities. Smith and Kraus (1987) investigated the postnatal development of the ABR in 71 unanesthetized mongolian gerbils. Filtered clicks varied front 0 to 100 dB HL in 10 dB steps, were delivered through an ear speculum with an attached sound tube. The sound tube was glued into the external auditory meatus to provide mi lowest in responses threshold During t sensitivj In contr most con: was the deveIOpi It 'as r provides developn time of would a Study oi System AlthOug On the 1 Central Only ax teChnic 52 provide monoaural stimulation. Threshold was defined as the lowest intensity level yielding a replicable ABR. At the highest intensity level used (100 dB HL) no replicable responses were obtained prior to 12th day after birth. An ABR threshold was however, obtained after the 12th day of birth. During this period, the response remained variable in sensitivity, and by day 20, all ABR waves were detectable. In contrast to the adult response in which wave IV was the most consistently detected component at threshold, wave I was the most sensitive indicator of threshold in the developing gerbil. ABR in the Syrian hamster It was reported earlier in this review that the Syrian hamster provides an excellent opportunity for the study of developmental biology because of its relative maturity at the time of birth (Boyer, 1953; Hoffman et-al, 1968). Thus, it would appear that the hamster is a suitable model for the study of developmental capabilities of the peripheral auditory system (Pujol et a1, 1975; Reilkin and Saunders, 1980). Although the hamster has been regarded as a model of research on the peripheral auditory system, not much is known about the central auditory system responses of the hamster. Thus, the only available study on the hamster that employed the ABR technique was that of Schweitzer (1987). Schweitze mixed wi hamsters {dosage, Ills/ks). heating ascendi: at a re} the W8V1 was suf Older, The Sal reliahl SPL. rEIiab: decrees peak‘tc 53 Schweitzer (1987) studied 40 golden Syrian hamsters using a mixed ‘within and between subject design. Forty infant hamsters (13 days after birth) were assigned to an acute condition (between subjects). These were tested once on either postnatal day 14, 16, 18, 20, 22, 24, 30 or 40. Sixteen infant hamsters (used in the acute condition) were again assigned.ix) a chronic condition (within subjects) and were tested on day 16, 30, and four of the other ages studied in the acute condition. Five normal adult hamsters at least 60 days old were used as a control group. The animals were anesthetized with chloropent, a mixture of chloral hydrate (dosage, 150 mg/kg) and sodium pentobarbital (dosage, 30 mg/kg). A body temperature of 37°C was maintained by a heating pad. Threshold was determined with descending and ascending series of click intensities at 5 dB intervals and at a repetition rate of ZO/sec. Latencies were measured using the wave form that was evoked at 50 dB SPL; an intensity that was sufficiently above threshold for all animals 18 days and older. The salient features of the results were that at day 14, no reliable ABRs could be evoked even by clicks as high as 94 dB SPL. By day 16, however, all of the hamsters showed a reliable response 1x3 clicks at SN; dB SPL. .A significant decrease in threshold was noted between days 18 and 20. Peak-to—peak amplitudes were found. to be ‘variable. Neverthe] later pee As expec1 In summa effects been den mutation behavior morpholc homozygc observai hOmOlOgc both ap] d0 note mutatio: humans) the ABR monitOr 0f anir COrrele genotyE threshc genotyx has no 54 Nevertheless, a progressive increase in the amplitude of the later peaks (waves III, IV and.V) occured.with increasing age. As expected, the latencies of all waves decreased with age. In summary, several studies have consistently explored the effects of the Eh mutation on the hamster. Indeed, it has been demostrated that the Eh gene is -a highly pleiotropic mutation causing numerous morphologic, physiologic and behavioral abnormalities. To In; true, time more obviours morphological effects of this gene as noted was to cause homozygotes to be deaf, blind and white. Still another observation was that the Eh gene in the Syrian hamster is homologous to the Waardenberg syndrome in humans, in.that they both appear to affect the same developmental processes. We do note that deafness is the most serious defect of the Eh mutation (in the hamster), and the Waardenberg syndrome (in humans), of the individuals affected. It is well known that the ABR as a non-invasive measure has proved invaluable in monitoring the hearing sensitivities of infants and a variety of animals. ‘RD be true, studies of iflua evoked potential correlates of genetic progressive hearing loss in two genotypes of mice (CS7BL/6 and CBA/J) have shown that hearing thresholds, amplitudes and latencies differed for these two genotypes. We also know that at this point in time, there has not txxnu any investigation conducted 11) determine the hearing sensitivity of the five sets of genotypic hamsters now available to detern deemed in term obje and mutan this StUK procedure preventec at the e investig; phenotyp¢ Present 55 available in the AN/As-Eh strain“ Thus, an.ABR study designed to determine the hearing levels of these Eh genotypes is deemed important, in that it may pave the way for the long term objective of using the hamster as a model for the normal and mutant genes of WSl and Eh deafness. Data generated from this study may assist in the development of a diagnostic procedure to identify individuals affected by this gene in utero, so that hearing loss might be detected and perhaps prevented via genetic engineering (Asher and Friedman, 1988) at the earliest possible age. There is the need, then, to investigate the hearing sensitivity of the Eh genotypes and phenotypes in the AN/As—Eh strain, hence, the impetus for the present study. (3' O) The Sic presenta' one desc CliCkS S to the e (N208) CHAPTER III INSTRUMENTATION AND PROCEDURE Stimulus generating System The basic experimental apparatus employed in the presentation and control of the clicks was similar to the one described by Moore et a1 (1988) and is shown in the block diagram in Figure III—I. The specific components were the following: .One power source (M12100) .One function generator (MI2 208) .One dual attenuator (MI2 108) .One amplifier (Grass PS Series) .One level discriminator (MI2 104) .One data controller timer (MI2 214) Clicks generated by a computer program were routed manually to the ears of each hamster from the function generator (M208), dual attenuator (M108), the amplifier and the filter. 56 57 Figure III—l. Block diagram of instrumentation. n_flwC*Nm _len ILWJIWNCZmZJIBQJOZ \ wczoafiyr oc>r I omzmmhom 1 2.32549 IV, 2% 1.! 35mm J u . I I u nmmocmzo< l' >09..ij mkauIOzm " y ooczamm zozfiom saw/K m WYjfiw wafl_ mm _thfpi " gage." m I 4 oozzmoaoJ * i .. "fiomgrromoonfl i: _ 353mm T_ ooztcamm Itazami F . . _\o zmzom< A>\o . , mom 1 mcflumm A oozZtCEmm 4|; lull. \ EOCEW El; _ZMBEC7sm2d540Z ”be experim Each elect amPlifies animal, f pass frOm Setting, . Electroph GVQrager Summed 2C time Of 1 points. Clicks f: 59 Electrophysiologic Recording Sysytem The experimental equipment employed in the recording of electrophysiologic activity is also shown in figure III-I. The specific components were the following: .Three needle elctrodes (AkAI) .One grass amplifier (Grass P5 Series) .One A/D converter (M12 202) .One I/O Bus (M12 101) .One computer (IBM AT) .One plotter (IBM 6180) .One oscillosc0pe (Textronix D15) .One frequency counter (Hewlett Packard 5314A) Each electrode was connected to the grass amplifier which amplifies the small electrophysiologic signal from the animal. The amplified signal was then filtered with a band pass from 100-3 KHz and the gain was set at 200 K. This setting, of course, resulted in a gain of 106 dB. The electrophysiologic activity was then passed through an averager (A/D converter, memory buffer and I/O Bus) and summed 2048 times, using a sweep time of 10 msec, a dwell time of 10 us, a 100,000 KHz sample rate and 1000 data points. The monitor oscilloscope was used to display the clicks from the function generator, clicks before attenuation, clicks prior to earphone and electrOphysiologic - ' < T. activ1t1- 1 form b‘.’ the I :igure III-2 in the calit microphone v cii k was se attenuator, output from to the scum connected t output sigr a calibrate as the £114 generator. reading of the sine w (filtered pulse, the 111 0‘8 SP1 prOdUCed E SOUnd 19“ msec whiC Computer 60 activity. The summed responses were printed in an analog form by the printer which was connected to the computer. Calibration System Figure III-2 shows the block diagram of the equipment used in the calibration of the earphones. As a first step, the microphone was calibrated using a pistophone. Secondly, a click was sent through the function generator and to the attenuator, the amplifier, and to the headphones. The output from the earphone was routed through the Zoo coupler to the sound level meter. The oscilloscope that is connected to the sound level meter was used to observe the' output signal. In order to establish a peak equivalent SPL, a calibrated sine wave with the same amplitude and frequency as the filterd click was generated using a second function generator. The idea was to make certain that the SPL reading of the click was equivalent to the SPL reading of the sine wave (pure tone). The duration of the pulse ~ (filtered click) was 0.2 msec. We know that for a 0.2 msec pulse, the first cycle must be about 2 kHz. Accordingly, a 111 dB SPL tone burst from the function generator at 2 kHz produced a 1650 Hz signal at 111 dB SPL measured on the sound level meter. The 1650 Hz signal gave us less than 0.5 msec which means that it was the best stimulus to use. A computer program was written so as to make certain that the 61 Figure III—2. Block diagram.of calibration equipment “_OCTNWH ___llN flu>_l_mmN\D,1—I_OZ mz=urfimm FJ nmzmmhom IV >jmzc>aom omzmmhom mnmoamcz >z>rol imam.» noccrmm _quzmm omoFromOObm flocmm Elm o>Cmm>joz m4mam: |\| 111 dB SPL ger equivalent to Durrant (1983 equivalent SP have a freque that of the f a frequency e earphone/sue: calibrated b“. its waveform filtered cli oscilloscope the nature 0 that compute formula: Sin w . where x We do know ‘ a Straight the {umber then, the f GECIEaseS. 63 111 dB SPL generated at 2 kHz at the input level was equivalent to the 1650 Hz at 111 dB SPL at the output level. Durrant (1983) posited that in order to establish the peak equivalent SPL of a click stimulus, the comparison tone must have a frequency whose period is approximately the same as that of the first cycle of the click, i.e., about 3 KHz, or a frequency equal to the resonant frequency of the earphone/speaker, i.e., around 2 KHz. The filtered click was calibrated by first passing it through an oscilloscope and its waveform observed visually. We also monitored the filtered clicks routed to the earphones using the oscilloscope and spectrum analyzer. This judgement about the nature of the filtered click was based on the theory that computer generated filtered clicks are created by the formula: Sin w . Sin ¢ when ¢-= 0 to 180 , w = 0 to X . 360 where X is the number of cycles desired. We do know that an infinite 2000 Hz sine wave would give us a straight line frequency spectrum. We also know that as the number of cycles per second decrease from infinity, then, the frequency bandwidth of the spectrum also decreases. The hamsters E by Asher (196E full-sibling r at the Biolog' This designat rmmenclature thus, by appr gene )h and i background. presence of < The gene g, without appa phenotype, into the AN/ allele was ; COmplete um rational f0] AN/AS‘EE st: into the in. and 8180 ’ t Animals wit gene, in t1" backchSsir 64 Materials The hamsters employed in this study were desinated AN/As-Eh by Asher (1968), and are currently maintained by full—sibling mating (congenic strain maintained by crossing) at the Biology Research Center Michigan State University. This designation was done according to the standard nomenclature for inbred strains of hamster (Asher, 1968). Thus, by appropriate selection, the strain contained the gene Eh and its normal allele wh on a common genetic background. Since the expression of Wh was enhanced by the presence of cream 9, the strain was made homozygous for g. The gene 3, by itself, prevents the production of eumelanin without apparently altering any other aspect of the phenotype. The normal gene E was later incorporated back into the AN/As through repeated backcrossing. This normal allele was placed back into the strain to promote a more complete understanding of the impact of the Eh gene. The rational for adopting the congenic strain in developing the AN/As—Eh strain and the repeated backcrossing of E mutant into the inbred strain was to eliminate genetic variability, and also, to ensure the full manifestation of the Eh gene. Animals with different alleles of the E gene and the Eh gene, in this investigation, were of the seventh generation, backcrossing. All other animals in the AN/As strain are at their 24th generation with at least one parent at each 65 generation being a heterozygote. This degree of inbreeding insures that hamsters from this strain are 99.9% identical for genes other than the Eh. Hamsters were housed in polycarbonate cages with galvanized stainless steel tops, cleaned weekly and provided with water. Lighting of the animals was on a regime of 14 hours of light and ten hours of darkness. Thus, all hamsters were exposed to the same environment so as to control environmental factors that may influence the full expression of our mutation of interest, the Eh gene. The above genetic combinations resulted in the five genotypes of hamsters utilized in this study. Thus, the five groups of hamsters used in the study were selected from the following genotypes: Eh/Ehzh/g (agouti), Eh/Eh; E/E (cream), Eh/Eh;§/§ (white bellied agouti), Eh/Eh;h/h (black-eyed white), and Eh/Eh; (anophthalmic white). Procedure As a first step in the experiment, the animals were anesthetized with rompun (dose = lOmg/kg) and sodium petobarbital (dose = 30 mg/kg). A heating pad was used to maintain body temperature at 37°C. Supplemental dosages of rompun were administered as necessary to maitain a constant background EEG level. Three stainless steel electrodes were inserted subcutaneously, with active on the vertex (Cz), ground at the center of the forehead (FPz) and reference to the pinna ipsilateral to the side of the recording. The 66 Akai "Walkman type" of earphone was used as a transducer (Ai). A Scc syringe tip was cut and attached to the earpiece with parafin. The click was directed through the syringe tip to the ear canal of each animal. Each animal was then put into a specially prepared animal holder made of molded foam and then placed in an stereotaxic restrainer in a sound restricted room. A quiescent state was desirable since it promoted a quieter physiologic background and reduced on-going noise levels (Moore, 1971). Clicks of 0.2 msec duration were presented to the both ears of each hamster. The clicks with rarefaction, condensation and alternating polarity were presented at a repetition rate of 11.1/sec at an intensity level of 100 dB, p.e. SPL, and decreased by increaments of 10 dB until ABR waves were abscent. Threshold was defined as a 1.0 uV between the minimum negative and maximum positive peak amplitudes for waves I, II, III and IV. Data Reduction and Statistical Analysis ABR threshold was defined as the stimulus intensity at which waves can be detected and was measured as a 1.0 uv difference between the minimum negative and maximum positive amplitudes. This definition has been used by several investigators (M¢ller and Blegvad, 1976; Henry, 1979: Kavanagh and Beardsley, 1979; Osako, Tokimoto and Matsura, 1979; Kodera and Yagi, 1979; Jerger and Mauldin, 1978: Dun, 67 1984; Church, Williams and Holloway, 1984; Smith and Kraus, 1987; Schweitzer, 1987). The latencies were measured from the onset of the stimulus to the most prominent peaks of waves I, II, III and IV. Further, peak—to—peak amplitude measurements were made from the first positive peak to the next negative troughs of waves I, II, III and IV. The mean, standard deviation, lepe and intercepts were calculated for the threshold data as well as the amplitude and latency values of waves I, II, III and IV across animal genotypes. Composite data were computed for amplitudes and latencies of waves I, II, III and IV as a function of stimulus intensity. By the same token, amplitude and latency data for waves I, III and IV were compared across genotypes at a specific intensity level. The statistical procedure selected to test for the differencies between the means of the thresholds, amplitude and latency of waves I, II, III, and IV of the various genotypes was the analysis of variance. According to several investigators (Myers, 1979; Winer, 1971; Borg and Gall, 1979) ANOVA statistics are powerful tools that can be used even with small samples. Thus, the two—factor ANOVA design was used to determine the simultaneous effects of intensity and genotype (independent variables), and their combined effects on the dependent measures (amplitude and latency of waves I, II, III and IV of the ABR). The ANOVA 68 is very robust (Silverman, 1985), and thus, if we violate the normality assumption in terms of the sample size in each genotype, we can still be assured of the accuracy of our analysis (Glass and Hopkins, 1984). We also employed the Chi-square to test for differences between the Eh-locus and the g-locus. The Duncan's Multiple Range Test was used to determine which class means contribute significantly to the differences detected by the F-test. The Duncan's test utilizes the error mean square from the analysis of variance as the best estimate of variance of the populations. From the error mean square, the standard error of the mean is obtained. Before this test is performed, one must see that the number of replications for each cell are equal. This post hoc test also utilizes "protected significant studentized ranges". These values represent significant student—t values at the five and one percent levels of significance which have been calculated considering the degree of freedom for the error mean square and the number‘ of means spanned in the comparison. Duncan (1955) has accordingly tabulated values of the "significant studentized ranges" with respect to level of significance (five or one percent), degrees of freedom of the error mean square, and number of means spanned in the comparison. Finally, the "shortest significant range" (Rp) is obtained by multiplying the "significant studentized range" by standard error of the mean. Rp = S.E.m X significant studentized 69 range. In performing the test, all means compared are placed on a single line in ascending order. The values of Rp are then calculated from data available in the analysis of variance table. A different value of Rp exists for different numbers of means spanned. Thus, by comparing the differences between any two means, and the appropriate value of Rp, one may determine whether the means are significantly different at the five or one percent level. That is, by a few simple calculations, one may compare any number of means and determine which are high, low, or intermediate. The “significant studentized ranges" extrapolated from Duncan's (1955) table at P = .05 with 40 degrees of freedom and for 2, 3, 4, 5, 6 means spanned are: 2.86, 3.01, 3.10, 3.17 and 3.22. These values were used in determining differences between means of various intensity levels. By multiplying these values by the appropriate standard error, the shortest significant ranges (R) are obtained. Thus,by perfoyming the Duncan's (1955) test, the effect of treatments are determined when such differences were indicated by the analysis of variance. CHAPTER 4 RESULTS This chapter presents the BSER findings obtained from 20 hamsters. Wherever applicable, descriptive and inferential statistics which were performed so as to provide answers to the various research questions posed. A visual representation of data is also provided by use of tables and figures. The study employed a 5 x 6 and a 2 x 6 factorial design as it sought to determine thresholds, latency and amplitude of BSERs of five sets of genotypes at six intensity levels of 25, 35, 45, 55, 65 and 75 dB nHL for- both right and left ears. Specifically, the study was designed to answer the following null hypothesis: (1) The genotype Eh/Eh, E/g (Agouti) has no effect on the morphology, latency and amplitude of waves I — IV of the auditory brain—stem response at varying intensities. (2) The genotype Eh/Eh, E/E (Cream) has no effect on the morphology, latency and amplitude of waves I—IV of auditory brain-stem response at varying 7O 71 intensities (3) The genotype Eh/Eh, g/g (Black-eyed white) has no effect on the morphology, latency and amplitude of waves I — IV of the auditory brain—stem response at varying intensities. (4) The genotype Eh/Eh, E/E (White—belly Agouti) has no effect on the morphology, latency and amplitude of auditory brain—stem response at varying intensities (5) The genotype Eh/Eh~— has no effect on the morphology, latency and amplitude of auditory brain—stem response at varying intensities. Twenty animals designated as AN/As—Eh by Asher (1968) were used in the study. They were chosen from a total of 30 hamsters so as to constitute a final sample. Animals were anesthetized with rompun (dose = 10 mg/kg) and sodium pentobarbital (dose = 30 mg/kg). Three stainless steel electrodes were inserted subcutaneously, with active on the vertex (Cz), ground at the center of the forehead (FPZ) and reference at the pinna ipsilateral to the side of the recording. Stimulus intensity was presented from 25 — 75 dB nHL (~ 50 — 100 dB p.e. SPL). Clicks with a major Spectral peak at 2000 Hz and a repetition rate of ll-l/sec were presented to both ears of each animal. 72 The typical responses were a series of positive-negative waves. The waves obtained from two sets of genotypes, Agouti (Eh/Eh, E/h) and the Cream (Eh/Eh, h/e) exhibit the same distinct morphological characteristics and the same time of occurence as reported by earlier investigators in several other species (Jewett, 1970; Jewett and Romano, 1972; Dobie and Berlin, 1976; Church et al, 1976; Schweitzer, 1987; and Ahmadizadeh et a1, 1987). The peaks have been designated in this study as waves I—IV (Jewett, 1970). We note that wave III is the most stable, especially at lower intensity levels in most animals; wave II is often fused with wave III and is often clear at higher intensity levels. By contrast, Black-eyed white (Eh/Eh g/g), White— belly agouti (Eh/Eh,§/§) and Anophthalmic white (Eh/Eh-—) present a different morphology of BSER waves. Thus, with the Black—eyed white (BEW) and the White—belly agouti (WBA), we see that at high intensity levels, waves I — IV followed the same morphological patterns as the normal genotypes. However, the waves have a narrow dynamic range compared with the normal genotypes. Additionally, at high intensity levels of 55 — 75 dB nHL waves I—IV are mostly discernible. Below this intensity level, however, the waves often become indistinguishable. We see that in some animals there is a Potential preceding wave I. This potential is referred to I I ' ' d' rn wave V. In 33 I - Again in certain traces, one can isce _ the case of the Anophthalmic white (AW), waves I — IV were 73 totally absent. The analog wave forms for the twenty genotypes can be found in Appendix B and the numerical values are listed in Appendix C through F. Latency for Normal Hearing Genotypes This section describes latency values for the Agouti and the Cream. The latency data from these genotypes for waves I - IV are presented in Appendix G; input - output latency functions can be found in figures 1 and 2. Inspection of Appendix G and figures 1 and 2 revealed that as the intensity of the stimulus is increased from 25—75 dB nHL, all four waves showed a systematic decrease in absolute latency as a function of increasing stimulus intensity. Exceptions were noted however, in the individual data, in that in some animals, latency remained constant as a function of stimulus intensity. This was noted for wave 11 in the right ear of HM33 (Agouti) (p..m%3) at intensity levels of 35 and 45 dB nHL and for wave I of HM21 (Cream) 09.245 ) at the same intensity levels. Observe also that as the intensity is increased, the standard deviation (SD) of the waves decrease. In certain definite regions, however, there is also an increase in SD as intensity is increased. We know that the magnitude of the SD indicates the degree of variability of measures from which it is computed. We also (now that the larger the standard deviation, the more Iariable the measures. The smallest possible SD as we know, 74 is zero, which of course, indicates no variability. Thus, the smaller the SD the more representative the mean. It can be observed in our data that represented in figures 1 and 2 that at each intensity level, the latency values are heterogenous. The factorial analysis revealed that the coefficient of variation in latency for the Agouti and the Cream for waves I - IV is less than 15% This indicates lower variability among the animals. The two-way analysis of variance (ANOVA) was calculated for waves 1, II, III and IV to determine whether the genotypes Efl/EQ E/g (Agouti) and_Eh/Eh E/E (Cream) have any significant effect on latency. It turns out that the latencies of waves I — IV are dependent on genotype. Thus, the two-way ANOVA indicated that there is a genotypic difference with respect to latency. There was no interaction between genotype and latency (Appendices H1 and H2). The ANOVA values for the right ear are: Wave II, [ F(1,5) = 7.16 = P < .05], wave III, [ F(1,5) = 8.32 P < .05] wave IV, [ F(1,5) = 6.42 P < .05]. For the left ear, the two—way ANOVA results are: Wave I, [ F(1,5) = .967 P > .05], wave II, [ F(1,5) = 19.06 P < .05], wave III, [ F(1,5) = 3.46 P > .05] and wave IV, [ F(1,5) = 6.52 P < .05]. We know that the null hypothesis states that no differences will be found between the means compared. If the null hypothesis were true, we will find this large differences 75 Figure IV-l. Input-output latency functions for Agouti (EB/ED, E/g). VJ AGOUTI - LATENCY O_O RIGHT 00— Lair 1 J 1 1 1 1 25 35 45 55 65 75 INTENSITY (dB nHL) Figure IV-2. 77 Input—output latency functions for the Cream (EB/EDI 2/2). 78 CREAM-LATENCY @«—-«. LEI-T l l 4 I 25 35 45 55 INTENSITY (dB nHL) 65 75 79 (H1 and H2) between the sample means only once in twenty experiments. 'Since we have found this large difference, it is probable that the null hypothesis of no difference between the sample means is false. Therefore, we reject the null hypothesis for waves II, III and IV for the right ear, and waves II and IV in the left ear and conclude that genotype Eh/Eh h/g and Eh/Eh 2/2 affect latency differently. Observe in Appendix G that the various intensity levels have significant effect on the latency of the twogenotypes. Duncan's (1955) test was used to determine differences in latency values caused by varying intensity levels (Appendix I). In another vein, it can be observed that at a click intensity level of 75 dB nHL, the inter-aural latency (ILD) was not the same for Agouti and the Cream (Appendix J). In the case of the Agouti, the ILD were: wave I .06, wave II .01, wave III .07 and wave IV .08. The ILD values for the Cream were: wave I .04, wave 11 .01, wave III .17 and wave. IV .21. It can be noted that ILD ranged from .01 ms to .08 for waves I — IV, whereas in the case of the Cream, ILD ranged from .01 ms to .21 ms. Observe that ILD were closer for the two genotypes for waves I and II. The two-way ANOVA Efiufififli no significant difference between right and left ears for the Agouti and the Cream across all intensity -evels for waves I—IV (Appendix K). 80 Latency for Hearing-Impaired This section describes latency reaponses for genotypes Eh/Eh h/h (BEW), Eh/Eh E/E (WBA) and Wh/Wh~~ (AW), for whom some or all of the waves are absent. We see in Appendix G and figures IV-3 through IV—S the composite data and the input~output plots respectively. Inspection of the Appendix and the figures revealed gross differences in normal and hearing-imaired genotypes. It can be seen that for the BEW and WBA the latencies are displaced to the right and as expected, intense stimuli are required to evoke a response. The global picture is that at intensity levels in which responses were obtained for all animals (65—75 dB nHL) lantency decreased as a function of stimulus intensity. Below 65 dB nHL, the pattern appeared different, in that the mean latency values from which points on the graph are derived are from responses of fewer ears. Observe also that SD bars were not ploted on the graphs at some intensity levels. This was the case since a response was obtained from only one ear or where the mean values cancel each other, resulting in zero SD and thus, indicating no variability. Observe also that latency values were not calculated for genotype EhAEh~— in that recordable responses were not obtained even at the highest intensity level of 90 3B nHL. We also note that latency changes differed for the two genotypes. For example, a a gradual-slopping 81 Figure IV-3. Input—output functions for waves 1 and II for the Black-eyed white (Wh/wh, e/e). LATENCY (ms) 82 BLACK-EYED WHITE - LATENCY O—O RIGHT ©—~ LEFT AnimaIs less than IIve I I J I 4 S S 5 6 S 7 f3 INTENSITY ((IU III IL) 83 Figure IV—4. Input—output functions for waves III and IV for the Black—eyed white (Eh/Eh, 2/§)° LATENCY (MS) 84 BLACK—EYED WHITE — LATENCY O—O RIGHT LEI—T * Animals less than five I I I J 45 55 65 75 INTENSITY (dB nHL) Figure IV—5. 85 Input—output for the White-belly Agouti (Eh/ED, E/g). LATENCY(MS) 86 WHITE BELLY AGOUTI — LATENCY 'o.l_\ \.®\ __ \\\I _ 7 I: .®\ __ \\\g ..\ \ \\¢ 63,.-.“ ”Kixgl— _I- «:1 .€¢\\ -1. tr \\ 0 0—0 RIGHT _I_ -—@ LEFT @ . AnimaIs less than three 45 55 65 75 INTENSITY (d8 nHL) 87 configuration of the input-output plot was noted for the BEW for the right ear. The left ear for the same genotype showed somewhat of a flat input—output configuration. With regard to the WBA, the input-output functions for both ears had a gradual slope. The exception noted was for wave I in which the functions became steeper at 65—75 dB nHL for both ears. A non—orthogonal ANOVA was performed comparing mean latency measures for the BEW and the WBA for waves I—IV (Appendice: L). From thiSanalysiS,-we observed that, there was no significant difference between BEW and WBA in latency for all waves, except wave II (left ear) and wave 111 (right ear). There was no genotype-intensity interaction for the two waves. No significant difference was observed between the genotypes for the remaining waves. Appendix M shows the ILD for the BEW and the WBA at 75 dB nHL. It can be observed that ILD for the two genotypes ranged from -.05 to .59 ms. Thus, for the BEW, the ILD values were: Wave 1, —.05 ms, wave II, .59 ms, wave III, .28 ms, and wave IV, .15 ms. In the case of the WBA, the ILDs were: Wave I, .34 ms, wave 11, 1.4, wave III, .35 ms and wave IV, .11 ms. The two—way ANOVA was calculated for aves I—IV to determine whether there are differencies in latency values between the two ears for each genotype across he intensity levels of 55-75 dB nHL. As expected, the 88 results were not statistically significant (Appendix N). intensity levels of 55—75 dB nHL. As expected, the were not statistically significant (Appendix N). resultsthe In another vein, figures IV—6 through IV—9 reveal the composite latency data for all genotypes depicting the degree of interaction among all genotypes across the various intensity levels. In the statistical literature, a distinction is made between two types of interaction, ordinal and disordinal. In the ordinal case, the rank order of one factor on the basis of their dependent variable values is the same within each level of the independent variable. Our latency data as can be seen in figure IV—6 depicts an ordinalinteraction among the genotypes. As can be seen, superiority exists for the Cream in relation to other genotypes at allintensity levels. That is, even though superiority exists, a single statement about the superior treatment can be madewithout qualification or eference to the other genotypes. On the other hand, we see 'n figures IV-7 through IV—9 a graphic representation of isordinal interaction among genotypes. As can be seen, uperiority did not exist for any genotype across the arious intensity levels. In this regard, we cannot make a ingle statement about the superior treatment without ualification. Therefore, the observations regarding a tter treatment depends on the response obtained by a 89 Figure IV-6. Composite data points of all genotypes for latency as a function of stimulus intensity for wave 1. A... ehd I? h‘k Annex/_v \p/Wostni . /\ 90 RT AND LT EAR WAVE I O Agouti D Cream A Black—eyed White O White-belly Agouti (I f .I l! R 2 a: H O a “-3 \O I I I I I I 25 35 45 55 65 75 INTENSITY (dB nHL) 91 Figure IV-7. Composite data points of all genotypes for latency as a function of stimulus intensity for wave II. A: 1.): \fwvsLfl._ /\_ 92 RT AND LT EAR WAVE ll 0 Agouti 1:] Cream A Black-eyed White O White—belly AgOUti INTENSITY (dB nHL) 93 Figure IV—8. Composite data points of all genotypes for latency as a function of stimulus intensity for wave III. TIM—\ZV \.«H v~Lm. _ (I. 94 RT‘AND LT EAR WAVE m 6T 5 _ 4 _ 3 _ \©\U\D O Agouti 2 T [1' Cream A Black-eyed White O White-belly Agouti I I I J I 25 35 45 55 65 75 INTENSITY (dB nHL) 95 Figure IV—9. Composite data points of all genotypes for latency as a function of stimulus intensity for wave IV. PHV f I AmHEV \fflvw/T . h- /\.,. 25 96 RT AND LT EAR WAVE IV C) Agoufi E] Cream A Black-eyed White Q White—beIIy Agouti I I I 35 45 55 INTENSTPKRflBnHL) #— 65 Iii a c 97 particular genotype atia given intensity level. For example,I for wave II in figure IV-7 the BEW showed a higher latency value, whereas at 75 dB nHL the Agouti showed a higher latency value. Similarly, waves III and IV also exhibit a disordinal interaction. We also see in figure IV—6 through IV—9 how the independent variable of intensity and the dependent variable of latency are related for the various genotypes. It can be observed that the relationship between intensity and latency is an inverse function since an increase in intensity tend to be associated with a decrease in latency. -Correlation coefficients were calculated to determine the strength of the relationship between intensity and latency values so that we could determine whether a reliable prediction could be made for latency given a particular intensity level and for a particular genotype. The correlation coefficient was done using the formula: r = nxy — 2xy nx2 — (x)2 nyZ — (y)2 As expected, the correlation values for the latency values of waves I, II, III, and IV showed a strong negative correlation (Appendix 0). The notable exception was the BEW in which it was observed that the latency first decreased and then increased as a function of stimulus intensity. 98 Viewing Appendix P and the figures described above, one finds that thresholds for the Agouti and the Cream (25 dB nHL) are lower than for the BEW and WBA (45 dB nHL and above). It can also be observed that mean thresholds for the right ear are slightly greater for the WBA than the BEW for waves II, III, and IV. The exception noted was for wave I in which mean thresholds are slightly greater for BEW than for WBA. Interestingly, opposite findings were noted in the left ear. As it turned out, mean threshold values were higher for the BEW than for the WBA. The exception noted was for wave IV in which average thresholds are the same for both BEW and WBA. It is noted, of course, that thresholds were not recorded for genotype_Eh/Eh-—, even at the highest intensity level of 75 dB nHL. It was of interest to determine the effect of each gene locus and their possible interaction on latency. Accordingly, the Chi-square was performed comparing the effect of Eh-locus and E/h-locus on latency. Interestingly, there were no statistically significant differences between Eh—locus and E/h-locus with respect to latency (Appendix Q). There was no interaction between the two genes with respect to latency. 99 Amplitude in Normal Genotypes We see in Appendix R the mean amplitude values for the Agouti and Cream. In addition, figures IV—10 through IV—17 depicts input-output plots for the Agouti and Cream. It was observed that as intensity increased, the amplitude of waves I—IV increased with increasing SDs. Exceptions were, noted however, in some individuals for whom amplitude remained constant and even reduced as a function of stimulus intensity. This was noted for wave II in the right ear of the Agouti (HM012) at the intensity levels of 45, 55 and 65 dB nHL. Similar observations were noted in the Cream (HM021). The two—way ANOVA was calculated for the mean amplitudes of waves I, II, III and IV essentially to determine whether there is a significant difference in the Agouti and the Cream with regard to amplitude and whether the various intensity levels have any significant/effects on amplitude of both genotypes. The results indicated significant differences between the Agouti and the Cream (Appendices SI and S2). Appendix 81 summarises the results giving the F—values determined. For example, the statistical differences between the mean values for wave IIIwas as follows: Right ear, [ F(1,5) = 4.30 P < .05] and left ear [ F(1,5) = 50 = P < .05]. No significant difference was noted between genotypes for wave I in the 100 Figure IV—10. Input-output amplitude functions for wave I of the Agouti (Eh[Eh, E/e). T .m J I J a t); /I\ “U 101 AGOUTI - AMPLITUDE Tour 10*- 1.0” .1"— 0—0 RIGHT .OI——- G-G LEFT 25 35 45 55 65 75 INTENSITY (dB nHL) 102 Figure IV-ll. Input-output amplitude functions for wave II of the Agouti (Eh/Eh, g/g). 103 AGOUTI — AMPLITUDE O—o RIGHT .g. LEFT I I I I I 4 25 35 45 55 65 75 INTENSITY (dB nHL) 104 Figure IV-12. Input—output amplitude functions for wave III of the Agouti (wh/Eh!.§/e). AUAOP VA A\/Zv WHOHWJI— _._n.—_\/_/.\ HIVIl—Ll I ULJE \I‘IV) A qu 100 .01 105 AGOUTI - AMPLITUDE I I I 35 45 55 INTENSITY (dB nHL) 106 Figure IV-l3. Input—output amplitude functions for wave IV of the Agouti (wh/Eh, hflg). ....— .- A\I»‘~v _~.~_~‘ ~.~_;Is(» 107 AGOUTI'— AMPLITUDE IOO{' / arr/V .01 0.0 RIGHT ®_@ LEFT I I I I I 25 35 45 55 65 75 INTENSITY (dB nHL) Figure IV—l4. 108 Input-output amplitude functions for wave I of the Cream (wh/wh, e/e). LII III III II. I IiIrII- I I I I III III ‘III IIIIII.I|II ‘ I . fir. III_I L, ---- -. AU «IIJ P N All! I .I.— Tn A\/wtu I...‘.__...._\/./\ 109 CREAM — AMPLITUDE IOOI' IO— 1.0- .I~ .01— O_Q RIGHT ®~-® LEFT I I I I I I 25 35 45 55 65 75 INTENSITY (dB nHL) Figure IV-15. 110 Input-output amplitude functions for wave II of the Cream (Eh/Eh, g/e). .aav. (I. A>~Ev _n_~a.. _...z.)E/\ 111 CREAM — AMPLITUDE 100[ 10— ° __ 3 u 1.0—— .I— .01— L I' I I I I J 25 35 45 55 65 75 INTENSITY (dB nHL) 112 Figure IV—16. Input—output amplitude functions for wave III of the Cream (Eh/Eh, §/§)° 113 CREAM - AMPLITUDE. IOO[‘ 10— 1.0— .1 '01— RIGHT $0 _ LEFT 0 o I I I I I I 25 35 45 55 a5 75 INTENSITY (dB nHL) 114 Figure IV-l7. Input-output amplitude functions for wave IV of the Cream (Eh/Eh, g/g). 03.1 VA a\/Zv when; .Jhk~\7: IHHQJFWLQSER 100T" _L O I .01—— AMPLITUDE (NV) x 100 O I 118 BLACK-EYED WHITE - AMPLITUDE O—O RIGHT @—-® LEFT * Animals less than five I J I I 45 55 65 75 INTENSITY (dB nHL) I8 11.". ‘ OJ .9... I F 119 Figure IV-19. Input-output amplitude functions for wave II of the Black—eyed white (Eh/Eh, §/§)- I00 0 0 I. OOr X A>7MW MIDDLLJLE< IOOI— S I 'o I AMPLITUDE (NV) x 100 T 120 BLACK-EYED WHITE - AMPLITUDE /\\ ®\ / \\ \ng9/9“ 1 0-0 RIGHT cos LEFT * Animals less than five I I I I 45 55 65 75 INTENSIT (dB nHL) 1 b- ure lV-Z .. ’4 (Q 121 Figure IV-20. Input-output amplitude functions for wave 111 of the Black—eyed white (Eh/Eh, g/g). . AU. ‘1 30— VA A>Z _ I v (n.n:4._-.I_»:\c/\ IOOI‘ 10- 3 D >< ’>‘ 1.0- s U 3 2 fi .1— 2 I .01— 122 BLACK-EYED WHITE - AMPLITUDE O~® LEFT * Animals less than five I 45 I 55 INTENSITY (dB nHL) I 65 75 fi"._..._-.. _ ..- _ _. IV- ure Fig 123 Figure IV—21. Input-output amplitude functions for wave IV of the Black—eyed white (Eh/Eh, E/§)° IIIII 0U. I I . OOr X C/ZV MIDDLLJDISTR 100‘ 10 1.0 AMPUTUDE(NV)XTUU .O‘l 124 BLACK-EYED WHITE - AMPLITUDE 00 RIGHT 9"" LEFT . ‘ Animals less than (we L. I I I 45 55 55 \J (fl INTENSITY (d8 nHL) Figure 1‘ 125 Figure IV-22. Input-output amplitude functions for wave 1 of the White-belly Agouti (Eh/Eh, E/h). VIIIIIILIIIIIIIIIIIII coax A>Zcm032412< 5 100(— 10* AMPUTUDE(NV)XIOO 'o l —L O I C —L I 126 WHITE-BELLY AGOUTI - AMPLITUDE (D—CDImGHT ®--:® LEFT Animals less than three I I l l 45 55 65 75 INTENSITY (dB nHL) 31 e TV- '1 t 127 Figure IV-23. Input-output amplitude functions for wave II of the White—belly Agouti (Eh/Eh, E/g). 0 I M III) 0 O .I 4I. . W oorXA>ch03Flu§< 128 WHITE—BELLY AGOUTI - AMPLITUDE 100 (- 10" 81.0—— X 2 __ a! ”F ‘Tw E / LU ./ __ s I 7 / / 5 1/ o. as 2 Zc (HDDDF_I_O:>_< AMPLITUDE (NV) X 100 130 WHITE—BELLY AGOUTI - AMPLITUDE Ioor IO ‘— 5 m — / Q 2*” ” 1 0 — / / 41/ $7 T / .1 ”7 O—O RIGHT '01_ @~® LEFT . Animals less than three I I I J 45 55 65 75 INTENSRY(dBnHL) Figure IV- 131 Figure IV-25. Input-output amplitude functions for wave IV of the White-belly Agouti (EB/EB, E/g). 0 «I. I. 00.. X A>Zc NDJLLJnZZ< l 100 10 132 WHITE-BELLY AGOUTI — AMPLITUDE 1-0 39E? .1._ H” 7 // -OIT‘ (}—C)ImGHT 0.- LEFT . Animals less than three I I I I 45 55 55 75 INTENSITY (dB nHL) observatiOI note that TV from 65 recruitmen increase 8 increased. The two-w; mean ampl various 1 between t statistic inter- an] across t1 nOII-orth. Signific IAppendi FUrtheru input‘OI figures amplitu Waves a the BEW intensi tend tC 133 observations were noted in the BEW for all waves. We do note that amplitude actually decrease for the BEW for wave IV from 65—75 dB nHL. It would appear that the WBA showed a recruitment-like phenomenon, in that amplitude tend to increase abnormally as intensity of the stimulus is increased. The two-way non—orthogonal ANOVA was computed to compare the mean amplitude values of the BEW and the WBA across the various intensity levels. It turns out that differences between the two genotypes with regard to amplitude were not statistically significant (Appendix V). We also compared inter—aural—amplitude differences for the two genotypes across the various intensity levels using as usual, a non-orthogonal ANOVA. Here again, no statistically significant differences were observed between the ears (Appendix W). Furthermore, figures IV-26 through IV—33 display the input—output functions for waves I—IV. We see from these figures that, in general, as intensity is increased, amplitude is increased. It is noted that amplitude of WBA waves are greater than that of all the animals. Curiously, the BEW tends to decrease with increasing stimulus intensity. We also see that functions in these figures tend to intersect, depicting disordinal interaction. As such, SUPE various iI statement genotype For examp interacti Observe, genotype: particul We do kn tells us and the determi: In a 10 Correle are re] straig) Where repres interc Illithi Were 134 such, superiority did not exist for any genotype across the various intensity levels. In view of this, a single statement cannot be made about the superiority of any genotype without reference to a particular intensity level. For example, figures IV—27 and IV—31 display a disordinal interaction for wave II for both right and left ear. Observe, however, that there is more interaction among genotypes in the right than in the left ear for this particular wave. We do know that in a linear—linear plot, the correlation tells us how close the points generated by the fixed factor and the variable factor are to the straight line. This is determined by the equation: Y = mx + b. In a log plot, as shown in in the foregoing figures, the correlation tells us how the variable and the fixed factors are related in a log scale. As such the equation of the straight line is determined by the formula: Logysz‘I'logb where log y is represented by Y-axis, m is the slope, x represents the independent variable, and log b is the intercept. Within this frame of reference, correlation coefficients were computed for the mean values of all genotypes. It w-——.—.._-__p— .—--.- —-.-. .. w turns out ‘ positive c value of t function c In human computed wave III the ratic the relat stimulus ’._l a O o It for the resDonse Y). In 0rde the g5- the Wav the sun Signif: ears w be SEe with r intens 135 turns out that all genotypes except WBA exhibit a strong positive correlation (Appendix X). The negative correlation value of the WBA is indicative of decreasing amplitude as a function of intensity. In human studies the ratio of wave V to that of wave I is computed for amplitude values. In this study we found that wave III was the most robust and most stable. Therefore, the ratio of wave III to wave I was used. As it turned out, the relative amplitude ratio increased with increasing stimulus intensity. Observe that the ratio is greater than 1.0. It is noted also that amplitude ratio is not uniform for the hearing—impaired genotypes. This is because responses are not from all genotypes. As such we cannot make any confident statememnt about these effects (Appendix Y). In order to determine whether there is a difference between the flh-locus and the_§-locus with reSpect to amplitude of the waves, the Chi—square test was performed. Inspection of the summary table (Appendix Z) ndicates that there is a significant difference between yh-locus and g—locus for both ears with regard to amplitude for all waves. Again, it can be seen that there is an interaction between the two genes with regard to amplitude. It can be surmised that at intensity levels at which recordable responses are obtained, Figure 1 136 Figure IV—26. Composite data points for the amplitude of wave I (R/E) as function of stimulus intensity and genotype. 100 {ll 0. ‘1‘ GOP X A>Zc MQULLJQE< 137 RKfliTEARflI 100(— Agoufi 0 Cream Black-eyed white 10"— O White-belly Agouti 1.0—— .1— .O1_ 25 35 45 55 65 75 INTENSITY (dB nHL) Figure I Figure IV-27. 138 Composite data points for the amplitude of wave II (R/E) as function of stimulus intensity and genotype. 100 0. A11 CCr X A\/7.C HODLLJQ§<< 139 RIGHT EAR: WAVE "H IOOf () Agoufi 10— [3 Cream A Black-eyed White . O White-belly A9011“ 1.0—- .1_ 25 35 45 55 55 5 INTENSHY(dBnHL) VW Figure 140 Figure IV-28. Composite data points for the amplitude of wave III (R/E) as function of stimulus intensity and genotype. 100 0 4I 4| 0 . ‘l OOr X A>ZV MODICJQE< 100{ 1.0— .01_ 25 35 45 5 RIGHT EAR: WAVEJI] Agwoufi (3 EC BIrack— meyed White G White— —beIlY Ago”ti I I I U'I _ INTENSITY (dB nHL) Lil‘— Figure I 142 Figure IV-29. Composite data points for the amplitude of wave IV (R/E) as a function of stimulus intensity and genotype. I00 OOr X A>7C WQDLLJQE< AMPLITUDE (NV) x 100 143 ~ RIGHT EAR: WAVE IV ~ IOOI‘ O Agouti Cream . Black—eyed WhIte . 10‘ O White—belly Agou“ I.O»— . E? 4’! A A O -I_. l/! .' U I I I I i 7, F r . J 25 35 45 5.) 65 INTENSITY (dB nHL) Figure 144 Figure IV-30. Composite data points for the amplitude of wave I (L/E) as a function of stimulus intensity and genotype. OOr X A>ZC WOULLJQE< AMPLITUDE (NV) x 100 IOOF .O‘I_ O Agouti {3 Cream 145 LEFT EAR: WAVE I A Black-eyed White O White-belly Agouti 25 35 I I 45 55 INTENSITY (dB nHL) 65 75 :igure Figure IV—31. 146 Composite data points for the amplitude of wave II (L/E) as a function of stimulus intensity and genotype. .IIIIII\IIII 0 I00 0. I OOr X A>Zv mOUFIQE/x 0 AMPUIUDE(NV)XIUU 100 147 LEFT EAR: WAVE TI 0 Agouti [:1 {Cream . A Black-eyed WhIte 10 Q White-belly Agouti 1.0 U .1 r///// ' , ml I I I I I I 55 55 75 2 5 3 5 4 5 INTENSITY (dB nHL) Figure 148 Figure IV—32. Composite data points for the amplitude of wave III (L/E) as a function of stimulus intensity and genotype. _IIIIII LII? IIITIIIIIII IIIIII ._III II I I AI. .I ,A A>~C [5:4I:I_rur2/\ OOE .OI_ 149 LEFT EAR; WAVE III .AQOUII . Cream Black—eyed White While‘bCIIy AQOUII ODDO I ’J/de/Jf—zflfllwu -_ ._ 2'3 35 ”IS {35} (3f) .'. IN I I'I-I:;I I \' ((Il’. ”I II I Figur 150 ‘ Figure IV-33. Composite data points for the amplitude of wave IV (L/E) as a function of stimulus intensity and genotype. :OOI' Li L IIIIIL IliII._| .IIIIII 0 I. 0 OOr X A>ZV NOJFZQE/x 151 LEFT EAR: WAVE IE 1007 O AQouti [:1 Cream 10* A BlaCk‘eYed White O WhIte-beIIy AgOUtI 3 . < E 1.0+— u 3 . I3 3 fl 3 t -I— fi/fl E /D /—O/0 .0L 0 J 4 I I I 25 35 45 55 6f“ INTENSITY (dB nHL) the two I The exce waves II results case is 1.44 = . In summa effects it has pleiotr physiol hamster gene a: and wh in the Syndro same 6 deafne (in t} of th hon~1 heari There Capak fUndz 152 the two genes interact in their effect on the BSER waves. The exception noted was wave I and III in the right ear and waves III and IV in the left ear in which the Chi—square results were not significant. For example the f{ in this case is X2.”df = 1 = 2.57, p = .11 for wave I (R/E) and xflldf = 1.44 = .23 for wave III (L/E). In summary, several studies have consistently explored the effects of the Wh-mutation on the Syrian hamster. Indeed, it has been demonstrated that the Wh-gene is a highly pleiotropic mutation causing numerous morphologic, physiologic and behavioural abnormalities on the Syrian hamster. The more obviours morphological effects of the gene as noted was to cause the homozygotes to be deaf, blind and white. Still another observation was that the Wh-gene in the Syrian hamster is homologous to the Waardenberg syndrome in humans, in that they both appear to affect the same developmental processes. It has been observed that deafness is one of the most serious defects of the Wh—gene (in the hamster), and the Waardenberg syndrome (in humans) of the individuals affected. We do know that BSER as a‘ non—invasive measure has proved invaluable in monitoring the hearing capabilities of infants and in a variety of animals. There has been no investigations conducted to asess the capabilities of the hamsters in the AN/As-Wh strain. These fundamental observations formed the basis of this study. Our result morphologj similar a responses obtained Ahmadizac‘ have norm do note ‘ and inte: and inte in human Signific With IEQ Jthe BEIII differeI latency and the and amp Cream. were or leVeis instan with r reveal EE‘IOC 153 Our results have shown that, in general, wave—form morphology, thresholds, latencies and amplitudes are not similar across genotypes. The results revealed that responses obtained from two genotypes are similar to those obtained by other investigators (Schweitzer, 1987; Ahmadizadeh, et a1 1987). Thus, the Agouti and the Cream have normal thresholds, and normal wave-form morphology. We do note that there is a direct relationship between latency and intensity and an inverse relationship between amplitude and intensity for both genotypes, a not too uncommon finding in humans and animals. The statistical results indicate significant differences between the Agouti and the Cream with regard to latency and amplitude. On the other hand, the BEW, the WBA and the Anophthalmia White (AW) presents a different picture. Thus, wave—form morphology, thresholds, latency and amplitudes were different compared to the Agouti and the Cream. In general, we found input-output-latency and amplitude plots to be unlike those of the Agouti and the Cream. Fundamental to this finding is that the responses were not obtained for all the animals at all intensity levels. The ANOVA results indicate minimal and in some instances no significant difference between the BEW and WBA with regard to latency and amplitude. The study also revealed that there is no significant effect between Wh—locus and E—locus with regard to latency. Interestinly, the X2 res Wh-locus a Suffice i morpholog hamsters different defects I dysfunct. even at did not from n01 the dif: develop such, 9 and prc 154 the thresults showed significant differences between Wh-locus and E-locus. Suffice it to say, that the finding of incomplete waveform morphology and the differential thresholds observed in these hamsters could pave the way for possible detection, differentiation and interpretation of a broad range of defects that may be due to the peripheral auditory dysfunction or brainstem level dysfunction caused by the Wh-mutation. The abberant responses in the Wh/Wh-— genotype even at the highest intensity level of 75 dB nHL and above- did not show any definition of peaks compared to responses from normal-hearing hamsters. Thus, these results indicate the differential effect that the Wh-mutation has on developing hamsters auditory mechanism and brainstem. As such, genotypes can be classified as normal, moderate—severe and profound hearing loss. Xon-invas hrain—ste in assess Latency I the cat rat (Chu 1978). the stin increasr have sh intensi hearing Sindie: Pregre CHAPTER V DISCUSSION Non-invasive hearing testing has revealed that auditory brain-stem evoked potentials BSERs have considerable value in assessing auditory functioning. The amplitude and latency of the BSERs have been thoroughly investigated in the cat (Jewett, 1970, guinea pig (Dobie and Berlin, 1979b), rat (Church et a1, 1984) and mouse (Henry and Lepkowsky, 1978). These studies have shown that as the intensity of the stimulus is increased, the amplitude of the BSER waveS' increases while latency decreases. In addition, studies have shown that BSERs could be obtained at stimulus intensity levels between 10 and 30 dB SL from normal and hearing—impaired subjects (M¢ller and Blegvad, 1976). Studies of the evoked potential correlates of genetic progressive hearing loss in two genotypes of mice C57BL/6 and CBA/J have shown that hearing thresholds differed for the two genotypes. We know that at this point, there has not been any investigation conducted to determine the hearing capabilities of the five sets of genotypic hamsters 155 availabll determinl in the A objectiv in heari function We obse and ampi general Agouti In all, or five differe BSER 0t magnitt third r of the the mo: often levels It was ADOpht It is nHL: V 156 available in the AN/As strain. This study was conducted to determine whether the threshold of hearing of the hamsters in the AN/As strain are normal. Secondly, it was the objective of this investigation to explore the differences in hearing thresholds, latencies and amplitudes as a function of genotype and at varying intensity levels. We observed that waveform morphology, thresholds, latencies and amplitudes are not similar across genotypes. In general, it was noted that the waveform patterns of the Agouti and the Cream are similar to those of other rodents. In all, rodents that were studied exhibited a series of four or five waves. The waveform morphology, however, varied in different species. For example, the first two waves of the BSER obtained from the mouse are of relatively greater magnitude than the later waves. In the rat, the peak of the third wave is usually observed to fall on the downward slope of the second wave. In our study, we found that wave III is the most robust and stable of all the waves; wave II is often fused with wave III and is clear at higher intensity levels for most animals. It was noticed, however, that the BEW, the WBA and the nophthalmic white present different waveform morphology. It is noted that at high intensity levels of 65 and 75 dB HL, waves I-IV can be seen in the BEW and the WBA. Below 45 dB nHL the case absent. morpholo strain? question (1978) t waveform FurtherT absence having one-hal for BSF Posses abnormg In the subjec- that 1 absent absenc nerve Thug, Obsgp of te exDla in th 157 45 dB nHL, however, the waves become indistinguishable. In the case of the Anophthalmic white, waves I-IV were totally absent. Why should there be a difference in the waveform morphology and thresholds for the hamsters in the AN/As—Wh strain? We do not have direct evidence to answer this question, however, we know from the work of Jerger et al (1978) that a high frequency hearing loss can result in poor waveform morphology and even a flat BSER tracing. Furthermore, Selters and Brackmann (1979) reported that the absence of all BSER waves is characteristic of patients having acoustic neuromas. They found that approximately one-half of their acoustic neuroma subjects have no response for BSER. While there is no evidence that these hamsters posses tumors, on—going morphological analysis may reveal abnormalities of coclear and/or retrocochlear structures. In the same vein, Harker (1980) noted that 28% of his subjects did not exhibit BSER. Harker (1980) also observed that if hearing loss is severe enough, there may be an absent BSER without any retrocochlear involvement. Total absence of BSER waves is even more likely to occur in 8th nerve or low brain—stem lesions if there is a hearing loss. Thus, it is possible that the abberant waveform morphology observed in the Anophthalmic white may be due to the absence of tectorial membrane in this particular genotype as explained below. On the other hand, the incomplete waveform in the BEW and WBA can be attributed to either peripheral or retrococ the ED‘Q We know causes ‘ morphol associa detach Corti. membra membra stereo Dallos the s( the d there basil it is at t1 TESL read embe memt a cc fac 158 retrocochlear pathology caused by the variable expression of the Wh-gene. This requires further investigation. We know from the work of Asher (1988) that the Eh gene causes the degeneration of the tectorial membrane. This morphological abberation as noted by Asher (1988), is associated with the failure of the tectorial membrane to detach from the future inner sulcus cells of the organ of Corti. This finding is important since the tectorial membrane is involved in depolarization of hair cells as the membrane exerts a shearing action across the tips of the stereocilia. We also know from the work of Davis (1961) and Dallos (1975) that sound vibrations that are introduced to the scala vestibuli are conducted to the cochlear duct by the deformation of Ressiner's membrane. The endolymph is thereby disturbed, and thus, the vibrations continue. The basilar membrane is coupled to this movement, and therefore, it is similarly displaced, resulting in a pressure release at the round window membrane. Since the organ of Corti resides upon the basilar membrane, the vibrations to it are readily transmitted. As the tips of the hair cells are embedded in the tectorial membrane , when the basilar membrane is displaced upward, the hair cells are sheared in a complex manner. Part of this shearing action is facilitated by the basilar membrane and tectorial membrane having 51 different Our noti derived cells wh potassii 1961; D; upon th f the possibl from.tt 0n-goiI fiMEI 0f ste the cc the SI Changi trans hair nerve such Causr dend QEne hear 159 having slightly different axis of rotation and slide in different directions as they both move up and down. Our notion is that the source of electrical charge is derived within the hair cells. It is known that the hair cells which contain an intracellular fluid are high in potassium and low in sodium. Several investigators (Davis, 1961; Dallos, I975) accept the notion that when the cilium upon the hair cells are deformed, the electrical properties of the hair cell membrane alter, allowing electrical charge possibly related to the movement of potasium ions to enter. from the endolymph. This possibly results in a positive on-going change of the intracellular potential. Within this frame of reference, we could say that it is the modulation of steady potassium flux which results in the generation of the cochlear microphonic (CM) and some of the components of the summating potentials (Dallos, 1975). The electrical changes in the hair cell causes it to release a chemical transmitter which diffuses through the clefts between the hair cell bodies and the afferent endings of the cochlear nerve. The transmitter substance, probably an amino acid, such as glutamate and/or aspartate (Pujol et al, 1980) causes a change in the local membrane permeability of the dendrite and results in the depolarization termed the generator potential. One can perhaps surmise that the hearing loss noted in the BEW, WBA, and the AW, together with the be relate in cochle We also I preceded is a pan particul suggeste the com; Synaptic Hughes z not par- reverse are sug pOSt sy termine the 9V: 1' is 1 defini hamste origin such a WaVeS (Tabb 160 with the abberant waveform morphology in these animals, may be related to the tectorial membrane dysfunction, resulting in cochlear or retrocochlear pathology. We also see that in some animals, the BSER wave I is preceded by a potential referred to as I'. Presently, there is a paucity of information on the origins of this particular wave. Recent studies by Moore et al (1988) suggested that the I' of the BSER and the positive wave of the compound action potential (CAP) recording indicates post synaptic afferent activity of the cochlear afferents. Hughes and Fino (1980) have shown however, that the I' is not part of the cochlear microphonic (CM) since it does not reverse in polarity with clicks of opposite polarity. There are suggestions that the I' may be related to excitatory post synaptic potential (EPSP) arising in the afferent terminals of the eighth nerve. Thus, it can be seen that the evidence regarding cochlear versus neural origin of the I' is still inconclusive. Additionally, there is no definite information on the origins of waves I—V in the hamster. Mflller and Jannetta (1985) reported that the origins of the BSER waves are similar across small animals, such as cats, guinea pigs and rats. It is possible that waves I—IV may have the same origin as reported in the cat (Table 6) thus: wave I, auditory nerve; wave II, cochlear nucleus, wave III; superior olivary complex; wave IV, lateral I observed In anoth some rar in other obsereve possible auditor in man used in small a 0.5 cm differs as the relati Suffic BSERs reseax he 31: can b prOfo Comme theSe there 161 lateral lemniscus; and wave V (in cases where it is observed), inferior colliculus. In another vein, it was observed that there were four and in some rare instances five major BSER peaks in the hamster as in other small rodents. In humans, seven major peaks are obsereved (M¢ller and Jannetta, 1985; Amedofu, 1985). A possible explanation for this difference may be found in the auditory nerve. Lang (1981) noted that the auditory nerve in man is much longer than it is in small animals usually used in auditory research. Thus, the auditory nerve in small animals such us cats, rats, and guinea pigs is 0.3 to 0.5 cm long, while it is about 2.5 cm long in man. Another difference between man and small experimental animals such as the cat is the smaller size of the auditory nuclei in man relative to the head size. Whether this difference is sufficient enough to Cause significant differences in the BSERs recorded from these species is not clear. Further research is needed to determine this. We also noted from the results of this study that genotypes can be classified as normal, moderate—to—severe, and profound hearing loss. Therefore, it is necessary to make comments about the possible pattern of inheritance among these hamsters with regard to hearing loss. We do know that there are three patterns of inheritance; dominant, recessive and X-lin described recessive also knor fih mutat hetrozyg this pre dominant gene as hOmOZYgI and pig extreme Of the incompj homozy while Varyir How a heari Durit Used SUblj SiStI theI homo 162 and X—linked. As already noted, the WE gene was first described by Knapp and Polivanov (1958) as an autosomal recessive gene inherited independently of the albino_C‘i We also know from the work of Beher and Beher (1959) that the Wh mutation has a dominant inheritance in that the hetrozygotes may be distinguished from the homozygotes. On this premise they suggested that the gene acted as a partial dominant. Again, Robinson (1962, 1964) described the WE gene as incompletly dominant and indicated that animals homozygous for the mutant showed a complete absence of coat and pigmentation, while aplasia of the eyes resulted in extreme anophthalmia. We noted that with regard to hearing of the hamsters in the AN/As-Wh strain, the W2 mutation is incompletely dominant. Our research has shown that animals homozygous for the mutant showed a complete lack of hearing, while the heterozygotes showed normal hearing, as well as varying degrees of hearing loss. How do we know that it is the Wh gene that is causing the hearing loss? To answer this question, we need to note the purity of the strains. It is well known that the hamsters used in this study are from a single inbred strain with one subline. To be true, AN/As-Wh has been inbred brother and sister for 27 generations with at least one parent carrying the Wh gene. For this reason, all hamsters are 99.9 percent homozygous with respect to all genes not linked to Eh. With respect 89% homo hamsters must be closely crossed Therefo substra 30 map homozyg respect identir the Wh that i and th them. Me Oh: the 3 t0 de of st animE that bash diff Agou 163 respect to genes within 10 map units of Eh, all hamsters are 89% homozygous. Thus, when making comparisons between hamsters with respect to the Eh gene, any difference found must be caused by the Eh gene, or a gene that is very closely linked. Additionally, the AN/As-E has been back crossed into the pure strain for seven generations. Therefore, with genes unlinked to h, including Eh, the substrain is 99.2 percent homozygous. With respect to genes 30 map units away from h, all animals are 92 percent homozygous. Thus, the comparisons we make in this study with respect to hearing are between hamsters which are nearly identical for all genes with the exception of the genes at the Eh locus and the h locus. As such, any hearing loss that is noted in any genotype must be attributed to the Eh and the E genes, or at least those genes closely linked to them. We observed an inverse relationship between the intensity of the stimulus and latency for the BSERs in that it was seen to decrease with decreasing standard deviation as a function of stimulus intensity. The coefficient of variation between animals was found to be below 159. This points to the fact that we can predict latency values quite accurately on the basis of the changing values of intensity. Significant differences were observed between the latency values of the Agouti and the Cream. Absolute latency values were found to be slig that, t The pos composi homozyg for the Cream 1 destruc It is 2 on the as a r« a resp I‘IBA sh their Cream? invest (1979) abnorn humans wave \ this I rapid an ab they aSSOC 164 be slightly later in the Agouti than the Cream. That is that, the Agouti appeared to be more normal than the Cream. The possible explanation of this finding is the alleleic composition of these two genotypes. The two genotypes are homozygous for the Eh gene and heterozygous or homozygous for the 3 gene. The shorter latency values noted in the Cream is indicative of recruitment and may be due to the destruction of the outer hair cells in the cochlea. It is also noted that for the BEW and the WBA, the latencies on the input—output curves are displaced to the right, and as a result, stimuli of high intensity is required to evoke a response. Why is it that latency of BSERs in the BEW and WBA shift to the right, and why is it that the slope of their responses is steeper than that of the Agouti and the Cream? A possible explanation can be found in the investigations of Hecox and Galambos (1978) and Yamada et al (1979). These investigators identified two types of abnormal latency—intensity functions, at least for wave V in humans. The first type of abnormal function is one in which wave V occurs within normal limits at high intensities. In this case, when intensity is increased, there is either a rapid increase in latency of wave V outside normal limits or an abrupt disappearance of the response. This abnormality, they noted, indicates cochlear hearing loss and is always associated with recruitment. The second type of abnormal latency- function indicate account loss vii amount I Secondl may res this ca of sour wave dI basila rather shift retr0c betwee Therej latent that destr We Th betw surm 165 latency—intensity function is one in which the entire function is skewed to the right on the abscissa, as indicated in our data. There are perhaps, three reasons to account for this observation. Firstly, a conductive hearing loss will displace the latency-intensity function by an amount approximately equivalent to the hearing loss. Secondly, a similar shift of the latency-intensity function may result from a steep high frequency hearing loss. In this case, the shift is not due to the decreased intensity of sound reaching the cochlea but is due to the travelling wave delay to reach a low-frequency response region of the basilar membrane. In a sense, the function is skewed upward rather than to the right. A third cause of this right-ward shift in latency-intensity function is the presence of retrococlear dysfunction that slows neural conduction between the ear and the neural generators of wave V. Therefore, it is reasonable to suggest that the shift in the latency-intensity function for waves I—IV in the BEW and WBA that is noted in our data is possibly due to either the destruction of the outer hair cells caused by the 2 mutation or, the alteration of the number of ganglionic cells by the Eh mutation. We noted also that there was no significant difference between latency values for the BEW and the WBA. We can surmise why this should happen, in that the two genotypes are diffe in that t genotype! data of 1 is const increase and Eggs hearing in the 1 of the expecte taken t basal l have m; such r In the Patter inter; excit; Dictu degre taken We m not , PErh 166 are different. Thus, the following observation may suffice in that the most cogent speculation is that the two genotypes probably have the same type of hearing loss. The data of Selters and Brackmann (1979) suggests that latency is constant for hearing losses of less than 50 dB, but increases for greater losses. We know from the work of Don and Eggermont (1978) and Galambos and Hecox (1978) that hearing loss can be conceived in terms of a filtering action in the pattern of response arising from the various regions of the cochlea. Thus, a high frequency loss might be expected to produce latency delays compatible with the time taken by the cochlear travelling wavefront to traverse the basal region. A precipitous loss above 4000 Hz will not have marked effect on latency because contributions from such regions are masked by that of the 1000-4000 Hz region. In the case of a more gradually slopping hearing loss, the pattern may be difficult to interprete because of the interaction between the intensity-related change in stimulus excitation pattern and the hearing loss. The overall picture is that of extreme complexity, if the variety of degree and slope of hearing loss, and loss etiologies are taken into account. We noted in our data that latency-intensity functions are not uniform, especially for intensities below 65 dB nHL and perhaps due to the differences in the degree of hearing loss among get have the expect a with reg of using Beardslc latency true, t‘ frequen data te to obte Inpect 1V-10 IV-26 betwee (augmt trend remai as a Curic decre that Stim char eta 167 among genotypes. It is possible that the BEW and WBA may have the same slope of hearing loss. As such, we should not expect a significant difference between the two genotypes with regard to latency. In practice, we should be cautious of using latency as an index of hearing loss. Kavanagh and Beardsley (1979) have shown that at high intensity levels, latency have little correlation with hearing loss. To be true, they found that some of their subjects have high frequency hearing loss but have normal latency values. Our data tend to suggest this possibility, but there is a need to obtain more frequency specific data. Inpection of the input—output amplitude plots in figures IV-lO through IV-25 and the composite function in figures IV—26 through IV-33 reveal that a direct relationship exists between the amplitude of the ABR and the stimulus intensity (augmenting phenomenon). There were exceptions to this trend in that there were some animals in whom amplitude remained constant, and, in some cases, amplitude decreased as a function of stimulus intensity (reducing phenomenon). Curiously, too, the amplitude of the BEW as a group tend to decrease as a function of stimulus intensity. It may be that this increase or decrease in amplitude as a funCtion of stimulus intensity might be due to the response characteristics of the inner hair cells. The work of Kiang et al (1965) revealed that while the actual threshold of the receptors the audit intensity nerve f it but went also ten Can we I phenomeI Our dat genotyp finding somato: potent al 198 1985). the he that- heari the n Buchs 0f tt Reva subj ihpu redt 168 receptors is in doubt, there most certainly exist fibers in the auditory system that may respond only to tones of high intensity. Again, Pickles (1982) also reported that some nerve fibers did not saturate in a sharply defined manner, but went on increasing at high intensities. These fibers also tend to have high thresholds than others. Can we term the augmenting/reducing phenomenon a normal phenomenon? The answer to this question is perhaps yes. Our data have shown that the Agouti and the Cream (normal genotypes) exhibit this phenomenon. This is not an uncommon finding, in that the same phenomena has been reported in somatosensory studies (Petrie, 1960), visual evoked potential studies (Buchsbaum and Silverman, 1968; Braden et a1 1983) and brain stem evoked potential studies (Amedofu, 1985). Interestingly, this phenomenon was also observed in the hearing—impaired genotypes. On the other hand, we noted that the reducing phenomenon occured more in the hearing-impaired genotypes, especially within the BEW than the normal animals. This is also not surprising, in that Buchsbaum and Silverman (1968) reported that a larger number of their patients exhibit the reducing phenomenon. Again, Kavanagh and Beardsley (1979) showed that some of their subjects with sensori—neural hearing loss have abnormal input—output amplitude functions similar to that of reducers. It follows that the augmenting —reducing phenome We alsc in our robust hamste wave V consif detect 1984) measu brair CritI acco repe rela The con For gen Cli Sti 169 phenomenon must be considered as pertains to amplitude data. We also determined the amplitude ratio cf wave III to wave I in our study. Wave III was used because it is the most robust and stable of all waveform patterns in the Syrian hamster. In humans, this ratio is simply the amplitude of wave V compared to wave I; and a relative ratio of 3 l is considered normal. This ratio is an important index in detecting hearing loss and retrocochlear lesions (Musiek, 1984). In our study, we found the waves III/I ratio to be > 1 for both normal and abnormal genotypes. Musiek and Gollegly (1985) noted that although amplitude ratio measurements are of value in detecting eighth nerve and brain-stem lesions, they must be used with caution. "Normal criteria for amplitude ratio", they pointed out, "may vary according to instrumentation used, intensity level, repetition rate, and a host of other varables". Hence, relative amplitude of >1 may not be a universal criterion. The limitations inherent in this investigation relate mainly to stimulus parameters that of necessity were either controlled or held constant for the purpose of this study. For example, the utilization of male hamsters may limit our generalization to other subjects. Additionally, the use of clicks may limit our generalization to other types of stimuli. There is a suggestion (Selters and Brackmann, 1977) th have the region c of this derived suggest regions point i towards otoneui (Laukl agreem appfop hearir he dex We 31 evide reasc knOw ihcr decr 11.] deCI 170 1977) that click stimuli at moderate or high intensities have their effective excitation maxima in the 1000-4000 Hz region of the cochlea. It is to be noted that the results of this study are within reason, in that BSER studies using derived narrow band frequencies (Don and Eggermont, 1978) suggest that contributions from high and lower frequency regions do affect BSER waveforms as a whole. Still another point in the use of clicks in this study is that the trend towards the use of more place-specific stimuli for otoneurologic investigations have met with little success (Laukli, 1983). Furthermore, at present there is little agreement about which alternatives to the click are most appropriate. Whatever the choice, a body of data concerning hearing loss effects using other kinds of stimuli remains to be developed (Stapells and Picton, 1981). We also held repetition rate constant at ll.1/sec. There is evidence to suggest that a rate of 11.1/sec is within a reasonable margin of acceptability for obtaining BSERs. We know from the work of others (Moore, 1971; Row, 1980) that increasing the repetition rate also increases latency but decreases the magnitude of the BSER waves. Such an effect is more pronounced for repetition rates greater than ll.1/sec, although a latency increase and an amplitude decrease do not go undetected at rates below ll.1/sec. It is ex morpholI QGDOtYP amplitu BEW and values absence respon: we did white Anothe effect laten< for a stati Eh-lo later under ThuS diff Ihte hang and I ADI imp 171 It is evident from the above findings that waveform morphology, thresholds, and latencies differ across genotypes. Sinificant differences were noted in latency and amplitude for the Agouti and the Cream. The results for the BEW and the WBA are different in that amplitude and latency values were not computed across all intensity levels due to absence of specific waves. Again, for these two genotypes, response thresholds vary between 45—75 dB nHL. As expected, we did not compute any numerical values for the Anophthalmic white because no recordable responses were obtained. Another interesting finding is that there appears to be no effect between the Eh—locus and the_§—locus with regard to latency at the intensity level at which BSERs were obtained for all genotypes in the study. With regard to amplitude, statistically significant differences were noted between the Eh-locus and the h—locus. This finding may conote that latency and amplitude are the result of independent underlying physiologic processes. Thus the results of this study have shown that genotypes differ in their BSERs at different intensity levels. Interestingly, we see from this variability, a corpus of hamsters who can be classified variously as normal (Agouti and Cream), moderate—to-severe (BEW and WBA) and profound (Anophthalmic white) hearing loss. This investigation has important implications for human studies, in that the wh-gene may 9‘ Waardenberg 5 172 Eh-gene may be homologous to the WSl gene that causes Waardenberg syndrome in humans. The gene, I pleiotropi developmen Asher, 196 1983) unde the gene < which beer life. Oti include , It would Waardenbe aPparentl (Waardent know that deafheSS 1981), Only beh know1edg determir CHAPTER VI SUMMARY AND CONCLUSIONS The gene, Anophthalmic white (Eh) in the Syrian hamster is a pleiotropic mutant causing deleterious effects on eye development, pigmentation and deafness (Robinson, 1962; Asher, 1968). Recent investigations of the cochlea (Asher, 1988) under light and electron microscopy have shown that the gene caused degeneration of the tectorial membrane which becomes apparent between 10 to 15 days of neonatal life. Other equally devastating effects caused by this gene include, infertility, growth retardation and metabolic rate. It would appear that the Eh mutation is homologous to the Waardenberg syndrome in humans in that both mutations apparently affect the same developmental processes (Waardenberg, 1951; Preus et al, 1983: Arias, 1984). We know that of all the defects caused by both mutations, deafness is the most pronounced (Asher, 1968; Wang et al, 1981). While deafness has been described in the hamsters, only behavioral observations were conducted. To our knowledge, no investigations have been conducted to determine the hearing of the various genotypes. 173 The BSER caI hearing sta and Mckean, been invest (Jewett and and gerbil, been choseI capabilitil et al, 197 about the 0f the lit investigat hearing 01 investigai Capabilit; the AN/As have nOrm ClasSifie in the St (1) 174 The BSER can be used to effectively evaluate and monitor the hearing status of infants (Hecox and Galambos, 1974; Salamy and Mckean, (1976). In the same vein, the BSER has also been investigated in other animal species including the cat (Jewett and Romano, 1978), mouse (Henry and Lepkowsky, 1978) and gerbil, (Wolf and Ryan, 1985b). While the hamster has been chosen as a model for the study of the developing capabilities of the peripheral auditory apparatus (Relkin, et al, 1979; Stonek, 1977), apparently, very little is known about the Syrian hamster. A thorough review of the results of the literature revealed that there has not been any investigations conducted to systematically evaluate the hearing of various genotypes of hamsters. The present investigation was designed to determine the hearing capabilities of the Eh genotypes and phenotypes observed in the AN/As-Eh strain; and whether there are genotypes which have normal hearing as well as genotypes which can be classified as hearing-impaired. Twenty hamsters were used in the study to test the following null hypothesis: (1) The genotype Eh/whf E/g (Agouti) has no effect on the morphology, threshold, latency and amplitude of waves I—IV of the auditory brain—stem response at varying intensity levels. (2) The genotype wh/Eh,§/§ (Cream) has no effect on the morphology, thresholds, latency and amplitude Animals w Sodium pe of rompur COUstant 598ctra , animal a nHL. Th a COInput reSDOhSe (~3 dB)_ data Doj 175 of waves I-IV of the auditory_brainstem responses at varying intensity levels. (3) The genotype Eh/Eh, h/h (BEW) has no effect on the morphology, thresholds, latency and amplitude of waves I-IV of the auditory brain—stem responses at varying intensity levels. (4) The genotype Eh/Eh,§/§ (WBA) has no effect on the morphology, thresholds, latency and amplitude of waves I—IV of the auditory brain-stem response at varying intensity levels. (5) The genotype Eh/Eh—— (Anophthalmic white) has no effect on the morphology, thresholds, latency and amplitude of waves I-IV of the auditory brain-stem response at varying intensity levels. Animals were anesthetized with rompun (dose = 10 mg/kg) and sodium pentobarbitol (dose = 30 mg/kg). Supplemental dosage of rompun were administered as necessary to maintain a constant background EEG level. Clicks with a maximum spectra at 2000 Hz were presented to both ears of each animal at intensity levels of 25, 35, 45, 55, 65 and 75 dB nHL. The repetition rate was held constant at ll.1/sec with a computer analysis time of 10 ms and an average of 2048 responses. The band pass of the amplifier was 100 - 3 kHz (—3 dB). A dwell time of 10 us was employed using 1000 data points and frequency conversion was 100 kHz. The electrode p as the grou A total of to eliminai minimum in‘ identifiab the Cream thresholds while the recordable Agouti am and laten differenc WW1, E/ different ThresholI WBA. We note rBSpthE Thus, f< Statist; was nOt high in DOSsibl 176 electrode placement was Cz - Ai, with forehead (FPz) serving as the ground. A total of five genotypes were tested in a pilot study so as to eliminate independent and dependent variables having minimum influence on the resultant data. As expected, identifiable BSER responses were obtained for the Agouti and the Cream at 25—75 dB nHL. It turns out that elevated thresholds (45—75 dB nHL) were obtained for the BEW and WBA, while the Anophthalmic white showed complete obscure recordable responses. We observed also that while both the Agouti and the Cream have normal threshold, their amplitude and latency data indicate statistically significant differencecs. Therefore the null hypothesis that genotype. Eh/Eh, h/g (Agouti) and the Eh/Eh, h/g (Cream) are not different is rejected for these genotypes for all waves. Threshold differences were observed between the BEW and the WBA. We note also that even at intensity levels of 45-75 dB nHL, responses from individual animals varied considerably. Thus, for the only two intensity levels for which statistical analysis was possible, no significant difference was noted between the BEW and the WBA. This implies that at high intensity levels at which recordable responses were possible for these genotypes, differences between them were minimal. TI differ, sinI genotypes d amplitude, thresholds It was of : the differr latency an significan that at hi respond tl effects or animals n: is a poss Effect, SiSinifica between I} Waves 1-; them. '1“ IESult 0 Thus, wh between COHIaneI Skin. 5 177 minimal. This does not mean that the two genotypes do not differ, since inspection of data revealed that all genotypes differ with respect to threshold, latency and amplitude, except for the Agouti and Cream for which thresholds were similar. It was of interest to conduct a the Chi-square to determine the difference between Eh-locus and E-locus with regard to latency and amplitude for wave I-IV. No statistically significant difference was noted for latency. This means that at high intensity levels at which all animals can respond the Eh—locus and the E~locus have almost identical effects on latency. This finding is understandable, since animals used in the study are heterozygotes, and thus, there is a possibility that there may be an overlap in gene-locus effect. On the other hand, the Chi-square test of significance revealed that significant differences exist between Eh-locus and h-locus with regard to amplitude for waves I-IV at 75 dB nHL and their is interaction between them. Thus, it may be that latency and amplitude are the result of independent underlying physiologic processes. Thus, while absolute amplitudes reflect both the trade-off between the sizes and orientations of generators, their component neurons, the head volume, and mass of muscle, skin, bone and sinew; latency is dependent on the conduction time along the auditory pathway (Merzenich, et al 1983). For example nerve-condu conduction We also see amplitude I relationsh and the Cr revealed t intensity Therefore amplitude Secondly, does not these twc the wan, latencie: Stimulus tendency amplitud Stimu1Us analygiS reCOrdaI degree 178 For example, a large brain results in larger nerve-conduction pathways, resulting by itself, in slower conduction times. We also see a direct relationship in the group data between amplitude of the BSER and stimulus intensity and an inverse relationship between latency and intensity for the Agouti and the Cream. The results of the statistical analysis revealed that differences exist between the various intensity levels and amplitude for waves 1, II, III, and IV. Therefore the null hypothesis that intensity does not affect amplitude of the Agouti and the Cream is rejected. Secondly, the null hypothesis that the latency of the BSER does not decrease as function of stimulus intensity for these two genotypes is rejected. With regard to the BEW and the WBA, there was no significant difference for the latencies and amplitudes of waves I-IV as function bf stimulus intensity. We observed that these genotypes have a tendency to be reducers more than augmenters, in that the amplitude of their BSER tend to decrease as function of stimulus intensity. We could not compute any statistical analysis for the Anophthalmia white since there were no recordable BSERs. We noted from our results that, while consistent, a certain degree of variability exists, not only between genotypes but also withir We also ot from 25-75 be Eh-gen homozygous hearing wh hearing ca cohort of mnmltAg and prof0‘ context, important manifests geneticaj In view made as (1: 179 also within genotypes with respect to latency and amplitude. We also observed that thresholds vary between genotypes from 25-75 dB nHL. Based on these findings, we can describe the Eh-gene as incompletely dominant since animals homozygous for the mutant showed a complete absence of hearing while the heterozygous showed varying degrees of hearing capability. With these properties in mind, we see a cohort of genotypes whose hearing could be classified as normal (Agouti and Cream), moderate—to—severe (BEW and WBA) and profound hearing loss (Anophthalmic white). Within this context, this animal model of hearing loss may have important implications for individuals exhibiting manifestations of the Waardenberg syndrome, as well as other genetically—based hearing abnormalities. Suggestions for Additional Research In view of these results, the following recommendations are made as areas of additonal investigation: (1) The use of other forms of stimuli with more frequency specificity such as short tone bursts and tone pips using a similar research design is recommended. (2) A study similar to the present investigation should be conducted using different repetition rates so as to determine whether changes in thresholds, latency and amplitude values interact (3) (4) (5) (6) (7) 180 with genotype as a function of various repetition rates. It may be worthwhile to conduct an investigation of this nature on the mouse since the Eh“ in this species provides another suitable model of the Waardenberg syndrome Longitudinal studies using the various genotypes employed in this study should be conducted to investigate whether the variable expression of the Eh-mutation and the E-gene on these genotypes produce progressive hearing loss. A study that differenciates cochlear from retrocochlear pathology such as oto—acoustic emissions (Kemp, 1978) may be warranted in the various genotypes. Histological studies should be conducted on the Anophthalmic white, BEW and the WBA, so as to determine the site-of-lesion of their hearing loss. It follows that a histological examination should also be conducted on the Agouti so as to yield comparative normative data. 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Jewett, D 0f auditc rat and < Jewett, far-fiel 94! 681‘ JOhnson, m KaVanagl auditOr‘ aSSessm O l . .39\£§£ 192 Jerger, J. and Johnson, K. (1988). Interactions of age, gender, and sensorineural hearing loss on ABR latency. Ear and Hearing, 9, 168-175. Jewett, D. L. (1970). ‘Volume—conducted potentials in response to auditory stimuli as detected by averaging in the cat. Electroencephalography and Clinical Neurophysiology, 28, 609-618. Jewett, D. L. and Romano, M. N. (1972). Neonatal development of auditory system potentials averaged from the scalp of the rat and cat. Brain Research, 36, lOl—llS. Jewett, D. L. and Williston, J. S. (1971). Auditory evoked far-fields averaged from the scalp of the human brain” Brain, 94, 681-696. Johnson, S. (1952). The heredity of perceptive deafness. Acta Otolaryngology, 42, 539—552. Kavanagh, 19. T. amui Beardsley, .3. V3 (1979). Brainstem auditory evoked response: II. Clinical applications in the assessment of patients with organic hearing loss. 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Annals of Oto—rhino-Laryngology, 91, 304—309. McKusic, V. A. (1986). Mendelian Inheritance in Man. Johns Hopkins University Press, Baltimore. Merzenich, Animals. Evoked Re: Michel, E de l'orei Lang, J. and variz M Mondini, Bononier m M¢ller, Patient M¢llert auditoz 1% Moore, nerve Ph.D, 194 Merzenich, M. M., Gardi, J. N. and Vivon, M. C. (1983). Animals. In Moore, E. J. (Ed.). Bases of Auditory Brain—stem Evoked Responses, Grune and Stratton, Inc. New York. Michel, E. M. (1896). Memoire sur les anomalies congenitales de l'oreille interne. Gazette Medicine Strasberg, 3, 55—58. Lang, J. (1981). Facial and Vestibular, Topographic Anatomy and variations. In Samii, M. and Jannetta, J. P. (Eds.), The Cranial Nernes. New York, Spriger—Verlag. Mondini, C. (1791). Anatomia surdi nedi sectio. De Bononiensi Scientiarum et Artium Instituto Atque Academi Commentarii, Bologna, 7, 418—443. M¢ller, K. and Blegvad, B. (1976). Brainstem responses in patients with sensorineural hearing loss. Scandinavian Audiology, 5, 115-127. M¢11er, A. and Janetta, P. (1985). Neural generators of the auditory brainstem response. In Jacobson, J. T. (Ed), The Auditory Brainstem response. College-Hill Press, San Diego. Moore, E. J. (1971). Human cochlear microphonics and auditory nerve action potentials from surface electrodesu 'Unpublished Ph.D. Dissertation, University of Wisconsin, Madison, Wisconsin. Moore, E. Hooks, Jr auditory Scandinav Myers, J Allyn an< Ormerod, Journal Osako, s Kanamyc, develop (Stockh Petrie, relief 86, 13 Pickle ACaden 195 Wisconsin. Moore, EL .3., Rakerd, B., Robb, R., Semela, J. J. M., and Hooks, Jr., w. C. (1989). A Microcomputer—based system for auditory evoked potentials . Accepted for publication in, Scandinavian Audiology. Myers, .3. I” (1979). Fundamentals cu? Experimental Design. Allyn and Bacon, Inc., Boston MA. Ormerod, F2 (3. (1960). Pathology (n3 congenital deafness. Journal of Laryngology and Otology, 74, 919-950. Osako, S., Tokimoto, T. and Matsura, S. (1979). Effects of Kanamycin on the auditory evoked responses during postnatal development of the hearing of the rat. Acta Otolaryngologica, (Stockholm) 88(5—6), 359-368. Petrie, A. (1960). Some psychological aspects of pain and relief of suffering. Annals of New York Academy of Sciences, 86, 13-37. Pickles, J. O. (1982). Introduction to physiology'of hearing. Academic Press, New York. Picton, T. Human aut component Neurophys: Pratt, B. State Uni Pratt, B. (Miwh) 0‘ recombin 101. Preus, b Waardenl J°ur\nal Prosser loss. PUjOl, and p apprOa M. (Ed paris. 196 Picton, T. W., Hillyard, H. I., Kraus, H. I., et al (1974). Human auditory evoked potentials. I. Evaluation of components. Electroencephalography enui Clinical Neurophysiology, 36, 179-190. Pratt, B. M. (1979). Unpublished Ph.D. Dissertation, Michigan State University, East Lansing. Pratt, B. M. (1982). Site of gene action of the white allele (Miwh) of the microphthalmia locus: A dermal-epidermal recombination study. Journal.of Experimental Zoology, 220, 93— 101. Preus, M., Linstrom, C., Polomeno, R. C., et a1 (1983). Waardenberg syndrome-penetrance of major signs. American Journal of Medical Genetics, 15, 383-388. Prosser, S. and Arslan, E. (1987). Prediction of auditory brainstem'wave V as a\S\/\//\ ii} a g)? 5 3 €22) 214 I735 - 2332:. mm $00ij wHQIa rm: :_ 1<< m < E 12...... Sx< ,\ <<21§ (i? 2. 2 a\, \f.>«\>>.r/\ \/>S/r\1\/\/\c 5 215 am 2.; _mo.Z< :sm Iiowm - 232:. m5 900ij ow 216 Analog Traces for Wig/Mi, e/e (Cream) 217 IZOEV ~1me E_< Fla: (25% (if; A}: ufi}«f pug -EZEj_QoAODm>§V am 3% : um _ \i ll \.\/\1 \ . (pk om / / .)/ c, \l/ttk .1 [\S.) /, _\.\ golf _/4\ 311.3 / mm / 2 \ x J <\\<\/\>/\\}(}\/R\{\/.\ bm mm zig/(1!\f>/1€2x Iiozm - 22,23. 05 Emmi: r521. 32:. :_ i _< S :F . E loo 72 (\)\/f ow 43m? 218 B\l\/\/‘/\l(/(\I\/\III\/Il\{\/J\( mm r. )\l«\1\/I\\I)/2:I\)\/ll 3512/); pm l\\!\>\a(\/\,.)\7\1(/\a\ll\)2././.llv\:4\/»\v. 220 I7somo -xs:>§V :Hoze _: ___\/ 3%. ' 221 1258. 232:. o5 Aommké Emma 1:012. ___ a m ESP 1 11 / .\/, I P—>(\/\l’\ um 11/\/ E .1. 1 1.. .. KK , /,,/«l\\l\l! .1 . r 1 \ .§§ < 2/\2- \ ( /2\, /K\ mm 5 3 \\//\S\2\/ \\ 2-2K.- 1..\. g; bm \}<\/1\} g; (i( («Ix/\KlIII/{\ \\r(//\lk\2\(I/\J\.\/A{2§I\\f(/«)\/1\(i Mm l<fil/(>\\ 222 Analog Traces for Wh/Wh, e/e (Black-eyed White) 223 IZomm - 1232,}. m5 1wr>OX-mOx-m0x-m|>>>/>1 ..\\ (erflllll.\)\/\;\\z).l\n . IZomm - 123123. o8 1mr>OX-mi \bf fif/i§.§11$\s}\ox.m>\r/lt¢{(l\.>2 228 Analog Traces for Wh/Wh, E/e (White-belly Agouti) 12103 - 532:. m5 1<QOCjV rmmq. WHOIH :1 . am 1:11. 1 : >\/\1/>\/\1M4\\m|'/1\\12 um v\i/ ..... 22)); 100 Z< /\2\ 229 ./\/\/\/.\ {ti/((1. o /)\1/\./\/ .\.\f\\/|/\<>(/J $1222.. 1 <1 {{1/\///\.\\-2-\l\)/21//t 1: - \l; >.|)>\ (\k \ (l\)\.n . \L ) .1 )1 ./\«/.1/P\// /(\\/){).. 1m r/(i2\\) (/ r..2xlr(\( 2.(./2/.-.\> 1210mm - 2312:. m8 1<\\/\>S/\\\/\\S>>>/2 m m $111211} NI} trill/K \/ git/ii; 7c.\./\/./V\l\\/\1.il.l<(//\n\ I.,/.111 E 231 rmma Igomo - 12351:. m5 1<OOCjV sense 111 1 .1 E um am. SSH. 1 <2; d 1.. .\ 1.. mm i\/>>>2-r>/> . wm gift/fist)? om um 232 Analog Traces for Wh/Wh,-- (Anophthalmia White) 233 11.108 2.22:. me $83: ww mm sub 50 Z< . 121m I1<10w1 <<:\<<1.1.:1>ZOUIHI>C<11> <m fig? uw aw DEW wHoma WHozH 2 Iiowm . 2312? m5 1.50511 :33. WHOIH _ 111 1< . .1 1 111. mH. _ . J.(\\/./\\I! um aw D \ .. . :. .1 18 2< . 121m 234 Iiomm - <<:\<<:.: 1>ZOpIfi1>C<11> <m «,1. <1 \Efisgif} \c<\>\1..1 d am 3:. 2:]... L 235 Appendix C. Latency and amplitudes for waves I-IV of Agouti. Table 6. Latency of wave I (ms) from 25-75 dB nHL for Agouti(R/E) Animal 25 35 45 55’ 65 75’ HM012 2.80 2.02 1.98 1.94 1.50 1.45 HM013 2.20 2.10 2.04 1.94 1.68 1.50 HM014 2.10 1.82 1.76 1.56 1.46 1.40 HM016 2.28 2.20 2.08 1.90 1.60 1.52 HMO33 1.96 1.90 1.88 1.74 1.60 1.50 TOTAL 9.90 9.98 9.74 9.08 7.84 7.36 X 1.90 1.80 1.79 1.72 1.56 1.48 SD 0.40_ 0.15 0.30 _0.20 0.09 0.05 Table 7. Amplitude of wave I from 25-75 dB nHL for Agouti(R/E) Animal 25' 35 45 ER? 65 75 HM012 10 3O 65 120 130 300 HM013 20 25 70 195 355 750 HM014 40 50 105 225 140 125 HM016 40 55 25 3O 35 75 HM033 35 50 150 205 300 650 TOTAL 145 210 415 775 960 1920 X 29 42 83 155 192 380 SD 13.4 13-5 47 150 132 310 237 Table 8. Latency of wave I from 25—75 dB nHL for Agouti(L/E) Animal 25 35’ 45* 55 65 75 HM012 2.38 2.32 2.28 2.00 1.94 1.54 HM013 2.18 2.06 1.90 1.88 1.86 1.66 HM014 2.28 2.26 2.24 1.98 1.92 1.60 HM016 2.10 2.02 1.96 1.80 1.56 1.46 HM033 2.00 1.90 1.80 1.62 1.52 1.45 TOTAL 11 10.50 10.18 9.28 8.80 7.37 X 2.20 2.10 2.05 1.86 1.76 1.54 SD 0.16 0.20 0.20 0.20 0.20 0.10 Table 9. Amplitude of wave I from 25—75 dB nHL for Agouti(L/E) Animal 25 35 45 55 65 75 HM012 35 40 115 140 145 170 HM013 20 30 25 50 50 60 HM014 30 45 45 120 190 225 HM016 15 20 45 90 235 350 HM033 35 100 255 575 585 595 TOTAL 135 235 485 975 1205 1400 X 27 47 97 195 241 280 SD 9 31 95 150 .204 204 238 Table 10. Latency for wave II from 25—75 dB nHL for Agouti(R/E) Animal 7255 35 455 ’55 65 755 HM012 2.96 2.86 2.84 2.56 2.34 2.16 HM013 3.38 3.30 3.26 2.90 2.68 2.40 HM014 ' 2.92 2.88 2.86 2.52 2.36 2.20 HM016 2.98 2.90 2.60 2.48 2.36 2.30 HM033 3.20 3.14 3.14 2.98 2.84 2.76 TOTAL 15.50 15.00 14.70~ 13.44 12.58 11.82 X 3.10 3.00 .2.94 2.69 2.60 2.37 SD 0-20 0.19 0.30 0.20 0.20 0.25 Table 11. Amplitude for wave II from 25—75 dB nHL for Agouti (R/E) Animal 25 35 45 55 65’ 475 HM012 10 4O 25 15 15 20 HM013 5 20 45 125 155 200 HM014 20 30 55 210 115 70 HM016 10 50 45 85 200 225 HM033 20 35 45 105 115 220 TOTAL 65 185 215 540 640 735 X 13 35 43 108 118 147 SD 6.7 11 ll 90 70 95.3 239 Table 12. Latency for wave II from 25-75 dB nHL for Agouti(L/E) Animal 25 35 45 55 65’ 075’ HM012 3.38 2.30 3.26 2.94 2.86 2.34 HM013 3.06 3.00 2.96 2.84 2.38 2.28 HM014 3.00 2.98 3.22 3.10 2.86 2.54 HM016 3.40 3.40- 2.84 2.80 2.64 2.12 HM033 3.30 3.20 2.98 2.88 2.66 2.52 TOTAL 16.0 14.8 14.70 14.56 13.40 11.80 x 3.2 2.97 2.90 2.90 2.68 2.36 SD , 0.19 0.40_ 0.20 _0.15 0.20 0.20 Table 13. Amplitude for wave 11 from 25—75 dB nHL for Agouti (L/E) Animal 25’ 35 445 '55 65 75 HM012 10 30 48 66 88 65 HM013 10 20 25 50 150 220 HM014 20 3O 3O 25 45 65 HM016 15 25 25 200 135 48 HM033 10 15 75 185 200 110 TOTAL 65 120 203 526 618 508 X 13 24 40.6 105 124 108 SD 5 6.5 72 85 60 7O 240 Table 14. Latency for wave III from 25-75 dB nHL for Agouti(R/E) Animal 255‘ 35 45 55’ 64* ‘75 HM012 4.68 4.60 4.56 4.04 3.70 3.54 HM013 4.10 4.02 4.00 3.52 3.40 3.28 HM014 4.78 4.76 4.60 3.80 3.12 3.00 HM016 3.48 3.26 3.18 3.08 3.14 3.10 HM033 3.84 3.80 3.74 3.62 3.50 3.42 TOTAL 21 21.5 20.02 18.06 16.86 16.34 x 4.201 4.10, 4.01 3.62 3.40 3.27 SD . 0.55 0.60 _ 0.60 0:36. .30 .0.23 Table 15. Amplitude for wave III from 25—75 dB nHL for Agouti (R/E) Animal 25 35 - 45 55 65 75- HM012 10' 20 70 415 955 1520 HM013 70 100 300 820 950 1565 HM014 20 80 250 255 700 1305 HM016 95 130 175 280 285 330 HM033 40 100 250 350 505 520 TOTAL 235 330 995 2120 3395 5240 X 47 86 199 424 679 1048 SD _ 36 ~ 41 87 220 290 581 241 Table 16. Latency for wave III from 25-75 dB nHL for Agouti (L/E) Animal 25* 35 45 55 65‘ 75 HM012 4.35 4.30 4.20 3.80 3.46 3.06 HM013 4.04 4.00 3.96 3.42 3.36 3.22 HM014 4.13 4.10 4.08 3.74 3.66 3.44 HM016 3.48 3.36 3.28 3.16 3.08 2.98 HM033 3.90 3.86 3.76 3.54 3.38 3.26 TABLE 21 1.92 18.86 17.66 16.94 15.96 x 4.20 3.84 3.72 3.53 3.39 3.20 sp_ 0.22 0.41 0.40 0.30f 0.21 0.20 Table 17. Amplitude for wave III from 25—75 dB nHL for Agouti(L/E) Animal 25 35 45 55 65 75 HM012 7O 55 350 625 125 1295 HM013 30 50 130 145 330 475 HM014 10 45 25 305 800 850 HM016 20 100 480 540 2005 2250 HM033 50 140 550 1850 2000 1005 TOTAL 180 390 1535 3465 6388 5875 X 36 78 307 693 1278 1175 SD 24 41 225. 450 _738 ‘.670 242 Table 18. Latency for wave IV from 25-75 dB nHL for Agouti (R/E) Afiimal 25 35“ 7545 55* 65 ’755 HM012 5.82 5.70 5.60 5.40 5.20 4.90 HM013 4.96 4.95 4.90 4.78 4.56 4.42 HM014 5.36 5.34 5.30 5.20 5.12 4.12 HM016 5.00 4.90 4.86 4.66 4.34 4.04 HM033 5.20 5.0 4.99 4.94 4.74 4.34 TOTAL 27.00 26.00 25.65 24.98 23.96 21.84 X 5.30 5.20 5.13 5.00 4.80 4.37 SD 0.34 0.33. 0.30 0.30 0.40 0,35 Table 19. Amplitude for wave IV from 25—75 dB nHL for Agouti(R/E) Animal 25 35 45 55 65 75 HM012 25 30 15 110 150 350 HM013 30 45 65, 25 65 120 HM014 35 35 55 150 160 225 HM016 20 25 65 80 95 100 HM033 30 40 25 70 90 105 TOTAL 140 175 225 435 560 900 X 28 35 45 87 112 180 SD 6 _ 8 24 75 41 108 243 Table 20. Latency for wave IV from 25-75 dB nHL for Agouti(L/B) Animal 25 35 45 .55 65 ’75 HM012 5.50 5.48 5.02 4.86 4.50 4.24 HM013 4.88 4.82 4.76 4.64 4.38 4.30 HM014 5.40 5.30 5.20 5.10 4.94 4.60 HM016 4.90 4.70 4.52 4.26 4.18 4.00 HM033 5.16 5.10 4.98 4.89 4.84 4.76 TOTAL 26.00 25.40 24.48 23.75 22.84 21.90 X 5.20 5.10 4.89 4.75 4.57 4.38 SD 0.30 0.33 ‘ 0.30 0.31 0.32 0.40 Table 21. Amplitude for wave IV from 25—75 dB nHL for Agouti(L/E) Animal 25 35 45’ 55' 65 75 HM012 20 30 65 85 120 140 HM013 10 20 70 85 265 200 HM014 10 30 15 140 180 750 HM016 15 20 145 185 124 185 HM033 45 50 450 750 1200 1350 TOTAL 100 150 745 1245 1889 2650 X 20 30 149 249 378 525 SD _15 12 175 250 463 524 244 Appendix D. Latency and amplitude values for waves I—IV of Cream. 245 Table 22. Latency for wave I from 25—75 nHL for Cream (R/E) Animal 25 35 45 55 65 75' HM017 2.38 2.30 2.02 1.80 1.56 1.42 HM018 2.04 2.02 2.00 1.62 1.52 1.40 HM019 1.98 1.90 1.86 1.84 1.56 1.50 HM020 1.90 1.84 1.82 1.72 1.62 1.46 HM021 2.20 2.00 2.00 1.98 1.96 1.90 TOTAL 10.00 9.80 9.70 8.96 8.22 7.68 X 2.00 1.98 1.94 1.79 1.65 1.54 SD 0.45 0.18 0.09 0.14 0.18 0.20 Table 23. Amplitude for wave I from 25—75 dB nHL for Creme (R/E) Animal 25 ' 35 45 55 65 75 HM017 45 100 195 200 450 250 HM018 20 45 100 105 495 900 HM019 20 30 100 110 345 350 HM020 10 20 25 25 200 215 HM021 15 15 25 25 150 235 TOTAL 110 190 445 465 1640 1950 X 21 42 89 93 328 390 SD_ 15 34 70 73 152 290 246 Table 24. Latency for wave I from 25-75 dB nHL for Cream (L/E) Animal 55255’ 35 45 55 *565— *75 HM017 2.02 1.98 1.96 1.80 1.66 1.50 HM018 2.30 2.04 1.98 1.86 1.62 1.45 HM019 2.00 1.96 1.94 1.86 1.76 1.40 HMOZO 2.36 2.32 2.30 1.96 1.86 1.65 HM021 2.20 2.04 2.02 1.56 1.54 1.46 TOTAL 10.90 9.50 9.40 9.06 8.44 7.46 x 2.18 1.90 1.98 1.81 1.70 1.50 so 0.17 0.40 0.15 0.15 0.13. 0.15 Table 25. Amplitude for wave I from 25—75 dB nHL for Cream (L/E) Animal 25 35 545 55 65 ’75 HM017 50 60 20 30 50 200 HM018 10 70 130 155 300 550 HM019 45 15 25 35 45 75 HM020 15 20 25 155 125 365 HM021 20 30 75 210 95 255 TOTAL 140 200 275 585 615 1445 X 28 40 55 117 123 289 SD 18_ 24 48 81. 204 180 247 Table 26. Latency for wave II from 25-75 dB nHL for Cream (R/E) Animal 25 35 45 55 65 575 HM017 2.94 2.88 2.64 2.60 2.58 2.14 HM018 2.80 2.64 2.36 2.40 2.54 2.32 HM019 3.00 2.50 2.28 2.08 2.06 2.02 HM020 2.70 2.32 2.30 2.16 2.06 2.00 HM021 3.18 3.16 3.12 3.10 2.92 2.58 TOTAL 14.50 13.50 12.70 12.34 12.16 11.06 x 2.92 2.70 2.54 2.47 2.43 2.20 so 0.19 O 35, 0.35 0.40 0.37 0.24 .. Table 27. Amplitude for wave II from 25975 dB nHL for Cream (R/E) Animal 25 35 “45 55* 65 7'75 HM017 20 30 45 75 90 30 HM018 25 90 150 155 95 120 HM019 20 15 25 25 300 425 HM020 10 20 35 35 50 195 HM021 15 15 60 95 280 325 TOTAL 90 170 310 385 815 1095 X 18 34 62 77 165 219 SD 6 31 51 _52 118‘ 158 248 Table 28. Latency for wave II from 25—75 dB nHL for Cream (L/E) Animal 25 535‘ 45 5555* 65 75 HM017 3.00 2.90 2.86 2.60 2.05 2.00 HM018 3.34 3.00 2.92 2.66 2.58 2.04 HM019 2.70 2.62 2.58 2.44 2.34 2.20 HM020 2.80 2.98 2.86 2.70 2.68 2.24 HM021 2.90 2.70 2.68 2.38 2.28 2.24 TOTAL 14.70 14.10 13.90 12.78 11.93 11.10 x 2.94 2.82 2.78 2.50 2.39 2.21 30 , 0.23 0.16‘ 0.14 0.20f 0.30, 0x20 Table 29. Amplitude for wave II from 25-75 dB nHL for Cream (L/E) Animal 25 35 *45 55 65 in; HM017 25 40 185 25 65 275 HM018 30 60 400 40 145 225 7HM019 10 30 55 155 95 210 HMOZO 45 50 200 225 320 550 HM021 20 45 115 45 110 250 TOTAL 130 225 955 490 735 1510 x 26 45 191 98 147 302 $0 13 11_ 130. 88 101 N141 249 Table 30. Latency for wave III from 25r35 dB nHL for Cream (R/E) Afiimal 25‘ 35 *45* 555* 565 75 HM017 3.92 3.72 3.18 3.08 2.92 2.64 HM018 3.80 3.30 3.28 3.02 2.86 2.74 HM019 3.90 3.60 3.50 3.18 2.94 2.86 HM020 3.40 3.30 3.28 3.00 2.80 2.68 HM021 4.40 4.38 4.38 3.89 3.68 3.54 TOTAL 19.5 18.30 17.62 16.17 15.20 14.46 x 3.9 3.66 3.53 3.24 3.04 2.89 50 . 0.35 0.45 0.49 0.37 0.36_ 0.37 Table 31. Amplitude for wave III from 25-75 dB nHL for Cream (R/E) Animal 25* ’35 45’ 4555 65 75‘ HM017 35 45 225 415 700 1025 HM018 30 100 335 750 865 1500 HM019 45 50 225 420 840 950 HM020 30 45 225 465 800 2500 HM021 15 90 255 820 1500 1700 TOTAL 155 330 1265 2870 4705 7675 X 31 66 253 574 941 1535 SD 11 27 .48 195 319 625 250 ' Table 32. Latency for wave III from 25-75 dB nHL for Cream (L/E) Animal 25 35 45 55 65 75 HM017 3.70 3.68 3.50 3.24 3.16 3.00 HM018 3.66 3.44 3.34 3.06 2.96 2.94 HM019 3.90 3.90 3.72 3.03 3.02 2.96 HM020 4.10 4.08 4.06 4.02 3.80 3.44 HM021 4.04 4.00 3.68 3.48 3.12 3.00 TOTAL 19.40 19.10 18.40 16.83 16.06 13.34 X 3.88 3.82 3.68 3.67 3.22 3.06 SD 0.18 0.30 0.30 0.40 0.34 0.21 Table 33. Amplitude for wave III from -25—75 dB nHL for Cream (L/E) Animal ’25 35 45 ’55 65‘ 75 HM017 150 80 185 215 480 490 HM018 55 100 400 825 1600 2460 HM019 25 30 55 155 285 450 HM020 45 80 200 330 450 575 HM021 30 45 115 250 '335 390 TOTAL 205 330 955 1775 3150 4365 X 41 67 191 355 630 873 SD 13 29 .130 270 .548 890 251 Table 34. Latency for wave IV from 25—75 dB nHL FOR Cream (R/E) Animal ’25 35 45 555‘ 565— 75 HM017 5.20 4.88 4.84 4.26 4.20 4.00 HM018 5.30 4.68 4.46 4.16 4.00 3.84 HM019 4.92 4.80 4.42 4.20 4.10 4.10 HM020 5.32 5.20 4.20 4.02 3.92 3.46 HM021 5.48 5.28 5.25 5.08 4.86 4.56 TOTAL 26.00 24.60 23.17 21.72 21.08 19.96 X 5.24 4.97 4.64 4.34 4.22 3.99 SD .0.22 0.26 0.40 0.42 0.37 0.40 Table 35. Amplitude for wave IV from 25-75 dB nHL for Cream (R/E) Animal 25 35 45 55* 65 575 HM017 30 35 95 110 550 560 HM018 45 50 80 95 125 465 HM019 30 45 65 185 195 200 HM020 10 20» 75 110 225 225 HM021 15 15 40 40 185 110 TOTAL 130 165 355 540 1275 1560 x 26 33 71 108 255 312 SD 14 6 21 52 169 191 252 Table 36. Latency for Wave IV from 25-75 dB nHL for Cream (L/E) Animal 25 35 45 55 65 75 HM017 4.60 4.58 4.54 4.42 4.12 3.82 HM018 4.96 4.92 4.84 4.26 4.22 4.18 HM019 5.20 5.00 4.58 4.42 4.22 4.12 HM020 5.40 5.20 5.00 4.40 4.94 4.68 HM021 5.48 5.40 5.16 4.72 4.48 4.24 TOTAL 26.00 25.10 24.92 23.02 21.98 21.04 x 5.20 5.02 4.98 4.61 4.40 4.20 30 ‘0.35 0.30, 0.32 0.40 0.34 40.31 Table 37. Amplitude for wave IV from 25-75 dB nHl for Cream (L/E) Animal 255 35 *45 55 655 775“ HM017 30 45 48 75 150 300 HM018 25 55 150 165 230 150 HM019 10 30 45 50 75 175 HM020 25 25 55 75 230 300 HM021 35 60 150 135 300 400 TOTAL 140 215 448 500 985 1925 x 25 43 90 100 197 385 30 9.4 15 55 48 77 220 253 Appendix E. Latency and amplitude values for waves I-IV of the Black—eyed white. 254 Table 38. Latency for wave I from 25—75 for Black—eyed whit (R/E) ~ Animal 25 35 45 55 65 75 HM022 NR NR 2.04 2.20 1.74 1.60 HM023 NR NR NR NR 2.04 1.94 HM024 NR NR NR 1.80 1.64 1.58 HMOZS NR NR NR NR 1.94 1.72 HM026 NR NR NR NR 1.68 1.58 TOTAL NR NR 2.04 4.06 9.04 8.42 x NR NR 0.44 0.82 1.81 1.70 SD , NR NR 1.0 1.5 1.50 0.20 Table 39. Amplitude for- wave I from 25—75 for Black-eyed white(R/E) Animal 25 35 45 55 65 75 HM022 NR NR 100 120 300 575 HM023 NR NR NR NR 400 750 HM024 NR NR NR 220 150 55 HM025 NR NR NR NR 255 420 HM026 NR NR NR NR 490 500 TOTAL NR NR 100 340 1595 2300 X NR NR 20 68 319 460 SD NR NR 44 71 131 257 255 Table 40. Latency for wave I from 25~75 for Black—eyed white (L/E) Animal 25 35 45 55 65 75 HM022 NR NR NR NR 2.12 2.02 HM023 NR NR NR NR 1.80 1.62 HM024 NR NR 1.96 1.66 1.50 1.40 HM025 NR NR NR NR 1.78 1.54 HM026 NR NR NR 1.86 1.60 TOTAL NR NR 1.96 1.66 9.06 8.24 X , NR NR 0.40 0.33 1.81 1.65 SD NR NR _ 0.90 - 0.70 0.22 0.20 Table 41. Amplitude for wave I from 25—75 for Black-eyed white (L/E) Animal 25 35 45 55 65 75 HM022 NR NR NR NR 150 200 HM023 NR NR NR NR 500 950 HM024 NR NR 80 750 1050 1300 HM025 NR NR NR NR 35 365 HM026 NR NR NR NR 120 120 TOTAL NR NR 80 750 1855 2935 X NR NR 11 150 371 587 SD NR NR 36 335 419 514 256 Table 42. Latency for wave II from 25-75 dB nHL for Black—eyed (R/E) Animal 25 35 45 755 65 ’75 HM022 NR NR 3.20 3.08 2.94 2.80 HM023 NR NR NR 0 3.08 2.90 HM024 NR NR 3.56 2.52 2.30 2.22 HM025 NR NR NR 0 3.10 2.82 HM026 NR NR NR 2.76 2.62 2.50 TOTAL NR NR 6.76 8.36 14.04 13.24 x NR NR 0.64 1.70 2.81 2.65 50 NR NR 1.90 1.5 0.34 0.28 Table 43. Amplitude for wave II from 25—75 dB nHL for Black—eyed (R/E) Afiimal 25 35 45' 55 65” 75 HM022 NR NR 50* 140 35 35 HM023 NR NR NR NR 55 110 HM024 NR NR NR 120 375 400 HM025 NR NR NR NR 75 75 HM026 NR NR NR 25 125 185 TOTAL NR 50 285 665 805 x 10 57 133 161 $0 22 62 134 145 257 Table 44. Latency for wave II from 25—75 dB nHL for Black—eyed (L/E) .'—v-‘_ Animal ’525 357 45 ’55 65 75 HM022 NR NR NR 2.72 2.54 2.40 HM023 NR NR NR NR 3.14 2.92 HM024 NR NR 2.74 2.50 2.28 2.02 HM025 NR NR NR NR 2.32 2.18 HM026 NR NR NR 3.16 3.00 2.80 TOTAL — - 2.74 8.36 13.28 12.34 X — - 0.60 1.68 2.66 2.06 SD - - 1.20 1.5 0.39 _ 0.38 Table 45. Amplitude for wave II from 25—75 dB nHL for Black-eyed (L/E) Animal 25 35 45 55 65 75 HM022 NR NR NR 25 525 20 HM023 NR NR NR NR 855 110 HMO24 -‘ NR NR 400 220 1500 495 HM025 NR NR NR NR 195 100 HM026 NR NR NR 35 720 385 TOTAL - - 400 280 3795 1110 X - - 80 56 759 222 $0 - _ - 179 110 483 . 206 258 Table 46. Latency for wave III Black-eyed (R/E) from 25-75 dB nHL for Animal 25 ‘35‘ 45‘ 55 65 75 HM022 NR NR 4.02 3.60 3.25 3.24 HM023 NR NR NR 3.10 4.00 3.88 HM024 NR NR 3.56 3.36 3.18 3.10 HM025 NR NR NR 3.19 3.08 3.00 HM026 NR NR 3.82 3.66 3.40 3.16 TOTAL - - 11.40 16.91 17.34 16.16 x - - 2.28 3.39 3.47 3.35 SD - - .2.0 0.25 f 0.33_. 0.34 Table 47. Amplitude for ‘wave III from. 25—75 dB nHL for Black—eyed (R/E) Animal 25 35 45 55 65 75 HM022 NR NR 155 350 550 800 HM023 NR NR NR 25 1300 1900 HM024 NR NR 30 1750 1900 2000 HM025 NR NR NR 100 505 545 HM026 NR NR 300 1050 2500 2750 TOTAL - - 685 3275 6755 7995 X - - 137 655 1351 1599 SD - — 87 719 863 912 259 Table 48. Latency for wave III from 25-75 dB nHL for Black-eyed (L/E) AfiImal 25 ’35 457 55 65 75 HM022 NR NR NR 3.12 2.96 2.80 HM023 NR NR NR 4.00 3.80 3.54 HM024 NR NR 3.50 3.24 3.02 2.88 HM025 NR NR NR NR 3.12 3.04 HM026 NR NR NR 4.10 3.88 3.58 TOTAL — - 3.50 14.66 16.18 15.84 X — - 0.70 2.94 3.36 3.17 SD - - 1.60 1.60 .0.44 .0.36 Table 49. Amplitude for“ wave III from 25-75 dB nHL for Black—eyed (L/E) Animal 25 35 45 55* 65 75 HM022 NR NR NR 400 525 560 HM023 NR NR NR 75 855 1700 HM024 NR NR 400 1005 1500 1495 HM025 NR NR NR NR 195 500 HM026 NR NR NR 390 720 800 TOTAL - — 400 1870 3795 5055 X - — 80 374 759 101 ‘80 .g - 179 389 _483 .y 623 260 Table 50. Latency for wave IV from 25-75 nHL for Black-eyed (R/E) Afiimal 25 ‘35 45 55 65‘ ‘75 HM022 NR NR 5.20 4.96 4.50 4.40 HM023 NR NR NR 4.40 4.26 4.08 HM024 NR NR NR 4.58 4.52 4.42 HM025 NR NR NR NR 4.84 4.54 HM026 1N2 NR 5.00 4.82 4156 "4.36 TOTAL — - 10.20 18.76 23.68 22.80 x - - 2.04 3.75 4.74 4.56 <0 - - 2.7 2.1 0.33 0.30 Table 51. Amplitude for wave Black—eyed (R/E) IV from 25-75 dB nHL for Animal 25 35 545 55 65 75— HM022 NR NR 50 140 150 320 HM023 NR NR NR 200 500 400 HM024 NR NR NR 95 1050 155 HM025 NR NR NR NR 35 300 HM026 NR NR 200 220 120 75 TOTAL - - 250 655 1855 1250 X 1 - 50 131 371 250 $9. — — 87 88 419 132 261 Table 52. Latency for wave IV from 25—75 dB nHL for Blak-eyed (L/E) Animal 25_ 535 45 55' 465‘ 75 HM022 NR NR 4.54 4.34 - 4. 10 4.06 HM023 NR NR NR 5 . 40 5 . 12 S .10 HM024 NR NR 4.86 4.38 4.28 4.12 HM025 NR NR NR NR 4.32 4.10 HM026 - — NR 5.28 4.96 4.64 TOTAL - - 9.40 14.10 23.06 22.04 X - - 1.88 2.82 4.62 4.41 SD 1 - 2.50 2.50 0.54 . 0.45 Table 53. Amplitude for wave IV from 25-75 dB nHL for Black—eyed (L/E) ‘ Animal 25‘ 35 45 55 65 75 HM022 NR NR 25 65 200 250 HM023 NR NR NR NR 510 75 HM024 NR NR 165 400 650 550 RN025 NR NR NR NR 200 360 HD4026 NR NR NR 125 135 145 TOTAL ~ - - 190 590 1695 1380 x — - 38 118 339 276 so _ — — _ _ 71 _,179 . 227 182 262 Appendix F. Latency and amplitude for waves I—IV for the White—belly agouti. 263 Table 54. Latency for Wave I from 25—75 dB nHL for White-belly (R/E) Animal 25 735 45 55 655’ 75 HH027 NR NR NR NR 1.38 1.30 HM028 NR NR NR 1.66 1.54 1.40 HM03O NR NR NR NR 2.04 1.82 TOTAL - — — 1.66 5.96 4.82 X — — — 0.33 1.99 1.20 SD , » — — p p— 0.95 0.34 '0.30 Table 55. Amplitude for wave 1 from 25—75 dB nHL for White—belly (R/E) ’ Animal 25' ’35 45 55 655’ 75 HM027 NR NR NR NR 450 600 HM028 NR NR NR 360 425 500 HM03O NR NR NR NR 500 550 TOTAL — — a 360 1275 1600 X — - — 72 255 330 208 75 50 SD . _ — . .-. 264 Table 56. Latency for wave I from 25-75‘dB nHL for'White—belly (L/E) Animal 25 ’35 45 55 65 75’ HM027 NR NR NR 1.70 1.58 1.52 HM028 NR NR NR 1.66 1.52 1.40 HM03O NR NR NR NR 1.90 1.70 TOTAL — — — 3.36 4.98 4.62 X — - - 1.12 1.66 1.54 SD — .‘ - 0.02 0.20 0.12 Table 57. Amplitude for wave 1 from 25—75 dB for White-belly (L/B) Anifiél 625 35 45 557 65 75 HM027 NR NR NR 185 155 250 HM028 NR NR NR 360 500 750 HM030 NR NR NR NR 120 125 TOTAL - - — 545 755 1125 X — - - 273 258 375 _124 210 330 SD — :4 — 265 Table 58. Latency for wave II from 25—75 dB nHL for White—belly (R/E) Animal 25 35 45’ 55 65 ’75 HM027 NR NR NR NR 2.14 2.12 HM028 NR NR NR NR 2.08 2.02 HM030 NR NR NR NR NR NR TOTAL — — — - 4.22 4.14 X — — — — 1.40 1.38 SD — - — - 0.32 0.30 Table 59. Amplitude for wave II from 25—75 dB nHL for White-belly (R/E) Animal 25 35 45 55 65 75 HM027 NR NR NR NR 250 500 HM028 NR NR NR NR 25 25 HM030 NR NR NR NR NR NR TOTAL — — — — 275 525 X - — — — 91 175 - - 55 105 SD 266 Table 60. Latency for wave 11 from 25—75 dB nHL for white-belly (L/E) Animal 525 35 #45 55 655 75 HM027 NR NR NR NR ' 2.48 2.34 HM028 NR NR , NR NR 2.12 2.02 HM030 - NR 2.90 220 2.52 2.40 TOTAL — — 2.90 220 7.12 6.76 X - - 0.97 0.73 1.43 1.40 SD — — 1.60 1.50 0.20 0.20 Table 61. Amplitude for wave II from 25-75 dB nHL white-belly (L/E) Animal. 25‘ 35 45’ ' 55 65 ‘75 HM027 NR NR NR NR 75 125 HM028 NR NR NR NR 45 125 HM030 NR NR 50 110 250 200 TOTAL - - 50 110 370 450 x - - 17 37 123 150 s0 - — 29 p _ 64 110~ 43 267 Table 62. Latency.for wave III from 25-75 dB nHL white—belly (R/E) Animal 25 35 45 55 65 575 HM027 NR NR NR 3.0 2.92 2.85 HM028 NR NR NR 3.0 2.84 2.72 HMO3O NR NR NR N 3.92 3.64 TOTAL — — - 6.0 9.66 9.21 X — - — 2.0 3.22 3.07 SD — - — 1.7 .61 0.50 Table 63. Amplitude for wave III from 25-75 dB nHL for white-belly (R/E) Animal 25 35 ’45 55 65 7675 HM027 NR NR NR 1005 1100 1300 HM028 NR NR NR 1500 1800 2000 HM030 NR NR NR NR 1500 2000 TOTAL — — — 2505 4400 5300 X — — — 835 1252 1650 SD - — — V764 ‘351 494 268 Table 64. Latency for wave III from 25—75 dB nHL for white-belly (L/E) Animal 25 35 ‘45 ‘55 65 75 HM027 NR NR NR 3.14 3.06 3.02 HM028 NR NR NR 2.94 2.74 2.62 HM030 NR NR 3.72 3.62 2.36 2.93 TOTAL - - 3.72 9.70 8.16 8.90 X — - 1.24 3.23 2.72 2.96 SD - — 2.20~ 0.35 0.35 ‘0.20 Table 65. Amplitude for ‘wave III from 25-75 dB nHL for white-belly (L/E) Animal 25 35 745 55 65 75 HM027 NR NR NR 950 1050 1500 HM028 NR NR NR . 1200 1550 1900 HM030 NR NR 180 200 580 1000 TOTAL — - 180 2350 3180 4400 X — - 60 483 1060 1466 SD - - 104 625 485 451 269 Table 66. Latency for wave IV from 25—75 dB nHL for white-belly (R/E) Animal 25 35 45 55 65 775 HM027 NR NR NR NR 4.00 3.98 HM028 NR NR NR NR 3.82 3.62 HMO3O NR NR NR NR 5.26 5.02 TOTAL — — — — 13.08 2.62 X — — - — 4.36 4.21 SD — — — — 0.80 0.71 Table 67. Amplitude for wave IV from 25—75 dB nHL for white—belly (R/E) Animal ‘725 35 45 55 65 775 HM027 NR NR NR NR 95 340 HM028 NR NR NR NR 500 750 HM030 NR NR NR NR 300 380 TABLE — — - — 895 1470 X — — — — 298 490 — — — 203 226 SD 270 Table 68. Latency for wave IV from 25—75 dB nHL for white—belly (L/E) Animal 25 35 45 55 65 75 HM027 NR NR NR NR 4.08 4.92 HM028 NR NR NR NR 3.94 3.64 HM030 NR NR 5.10 4.80 4.58 3.84 TABLE ~ — 5.10 4.80 12.60 12.40 X — — 1.70 1.60 4.20 4.13 SD — — 2.90 2.80 3.40 0.70 Table 69. Amplitude for wave IV from 25—75 dB nHL for white—belly (L/E) , Animal 25 35 45 55 65 75 HM027 NR NR NR NR 140 200 HM028 NR NR NR NR 220 505 HM030 NR NR 75 45 45 210 TABLE — - 75 45 405 915 X — — 25 15 135 305 SD - — 43 26 88 173 271 Appendix G. Mean latency data of waves I—IV for all genotypes. 272 Table 70. Mean Latency for wave I from 25-75 dB nHL for all animals(R/E) Genotypes 25 35 45 55 65 75 ‘TNT Agouti 1.90 1.80 1.79 1.72 1.56 1.48 5 Cream 2.00 1.98 1.94 1.79 1.65 1 54 5 BEW * * 2.04+ 2.00+ 1.81 1.70 5 WBA * * * 1.66+ 1.99 1.20 3 TOTAL 3.90 3.78 6.13 7.20 7.10 5.95 18 MEAN 1.95 1.89 2.10 1.75 1.75 1.48 ' #_OF ANIMALS 10 10 11 13 18 18 Table 71. Mean Latency for wave 11 from 25—75 dB nHL for all animals (R/E) Genotypes 25 35 45 55 65 75 N Agouti 3.10 3.00 2.94 2.69 2.60 2.37 5 Cream 2.90 2.70 2.54 2.47 2.43 2.20 .5 BEW * * 3.88+ 2.78+ 2.81 2.65 5 WBA * * * * 2.11 2.07 3 TOTAL 6.02 5.70 9.36 7.94 9.95 9.29 18 MEAN 3.01 2.85 3.12 2.70 2.49 2.30 # OF ANIMALS 10 10 12 13_ 18 18 * No response frOm any animal + Response from reduced number of animals 273 Table 72. Mean Latency for wave III from 25—75 dB nHL for all animals (R/E) Genotype 25 35 45 55 65 75 7TNT Agouti 4.20 4.10 4.00 3.62 » 3.40 3.27 5 Cream 3.90 3.66 3.53 3.24 3.04 2.89 5 BEW * * 3.80+ 3.39 3.47 3.35 5 WBA * * * 3.00+ 3.22 3.07 3 TOTAL 8.10 7.76 11.33 13.25 13.13 12.58 18 MEAN 4.50 3.88 3.80 3.30 3.28 3.16 # 0F ANIMALS_ 10 10 -13 17 18 18 Table 73. Mean Latency for wave IV from 25—75 dB nHL for all animals (R/E) Genotypes 25 35 45 55 65 75 (N) Agouti 5.30 5.20 5.13 5.00 4.80 4.30 5 Cream 5.20 4.97 4.64 4.34 4.22 3.99 5 BEW * * 5.10+ 4.65+ 4.74 4.56 5 WBA * * * * 4.36 4.21 3 TOTAL 10.50 10.17 14.87 13.99 18.12 17.06 18 MEAN 5.10 5.08 4.95 4.69 4.53 4.27 # OF ANIMALS 10 10 12 14 p 18 18 * No response from any animal + Response from reduced number of animals. Table 74. Mean Latency for wave animals (L/E) I from 25—75 dB nHL for all Genotypes 25 35 45 55 65 75 (N) Agouti 2.22 2.10 2.05 1.86. 1.76 1.54 5 Cream 2.18 1.90 1.88 1.81 1.70 1.50 5 BEW * * 1.96+ 1.66+ 1.81 1.65 5 WBA * * * 1.68+ 1.66 1.54 3 TOTAL 5.40 4.00 5.89 7.01 6.93 1.56 18 # OF mmlmufi 10 10 14 ]6_ 18 18 Table 75. Mean Latency for wave II fro 25—75 dB nHL for all animals (L/E) Genotypes 25 35 45 55 65 75 (N) Agouti 3.20 2.97 2.90 2.90 2.68 2.36 5 Cream 2.94 2 82 2-78 2.50 2.39 2.21 5 BEW * * 2.74+ 2.78+ 2.66 2.06 5 WBA * * 2.90+ 2.20+ 1.43 1.40 3 TOTAL 6.14 5.79 11.32 10.38 9.16 8.03 18 MEAN 3.07 2.89 2.83 2.60 2.29 2.00 # OF ANIMALS 10 10 13 15 18 18. 7““No response from any animal + Response from reduced number of animals Table 76. Mean Latency for wave III from 25—75 dB nHL for all animals (L/E) Genotype 25 35 45 55 65 75 (N) Agouti 4.20 3.84 3.72 3.53 3.39 3.20 5 Cream 3.28 3 62 3.68 3.67 3.22 3.06 5 BEW * * 3.50+ 3.88+ 3.36 3.17 5 WBA * * 3.72+ 3.23+ 2.72 2.72 3 TOTAL 4.08 7.66 14.62 14.31 12.69 12.15 18 MEAN 2.04 3.83 3.66 3.57 3.17 3.04 # OF ANIMALS 10 10 12 14 18 18 Table 77. Mean Latency for wave IV from 25—75 for animals (L/E) Genotype 25 35 45 55 65 5 (N) Agouti 5.20 5.10 4.89 4.75 4.57 4.38 5 Cream 5.20 5.02 4.98 4.61 4.40 4.20 5 BEN * * 4-70+ 4.85+ 4.62 4.41 5 WBA * * 5.10+ 4.80+ 4.20 4.13 3 TOTAL 10.40 10.12 19.67 19.01 17.79 17.09 18 MEAN 5.20 5.06 4.90 4.69 4.45 4.27 # OF ANIMALS 10 10 13 15 18 18 3“No reépOnse £30m any animal + Response from reduced number of animals 276 Appendix H1. Differences between Agouti and Cream with respect to wave latency as given by F—value. 277 Table 78. Differences between Agouti and Cream with respect to wave latency as given by F—value. Parameter Difference I II III IV R/E Genotype 0.32 7.16* 8.32* 6.49* Latency Intensity 12.26** 6.84** 4.85** 5.54** Interaction 0.52 0.42 0.08 0.95 Parameter Difference I II III IV L/E Genotype 0.967 19.06* 3.46 6.52* Latency Intensity 4.26** 17.47** 10.69** 5.56** Interaction 0.65 0.264 0.04 0.96 .05 with F1,40 = 4.08 .05 with F5,4O = 2.45 *” IndiCates significance at P ** Indicates significance at P 278 Appendix H2. Summary of two—way ANOVA for waves I—IV for genotype (Agouti and Cream) and intensity for right and left ears. 279 Table 79. Summary of genotype (Agouti and Cream) x intensity x ANOVA X ABR latency for the Right Ear Wave ‘A3ource 33 56‘ M3 F_—_ I Between Genotypes 0.01 1 0.01 0.33 Between intensities 1.90 5 0.38 12.26** Genotype-intensity— Interaction 0.08 5 0.016 0.516 Error 1.22 40 0.031 II Between Genotypes 0.68 1 0.68 7.16* Between Intensities 3.27 5 0.65 6.84** Genotype-intensity Interaction 0.20 5 0.04 0.42 Error 3.79 40 0.095 III Between Genotypes 2.00 1 1.99 8.32* Between Intensities 5.79 5 1.16 4.85** Genotype-intensity Interaction 0.09 5 0.018 0.08 Error 9.58 40 0.239 IV Between Genotypes 1.22 1 1.22 6.42* Between Intensities 5.19 5 1.04 5.46** Genotype-Intensity Interaction 0.89 5 0.18 0.94 Error 7,49 40 0.19 “—Indicates significance atT—7= .05 witth,40 = 4.08 ** Indicates significance at P = .05 with.Fg,40 = 2.45 280 Table 80. Summary of genotype (Agouti and Cream) x intensity x ABR latency for the left ear Wave SS DF M3 F I Between Genotypes 0.03 1 0.03 0.967 Between Intensities 0.66 5 0.132 4126** Genotype—Intensity- Interaction 0.01 5 0.002 0.065 Error 1.22 40 0.031 11 Between Genotypes 1.01 1 1.01 19.06* Between Intensities 4.63 5 0.926 IL47** Genotype—intensity— Interaction 0.07 5 0.014 0.264 Error 2.11 40 0.053 III Between Genotypes 0.36 l 0.36 3.46 Between Intensities 5.56 5 1.112 ML69** Genotype—intensity- Interaction 0.02 5 0.004 0.04 Error 4.14 40 0.104 IV Between Genotypes 1.22 1 1.22 6.52** Between Intensities 5.19 5 1.04 5£%** Genotype—intensity Interaction 0.89 5 0.18 0.96 Error 7.49 40 0.19 * Indicates significance at P = .05 with F1,40 = 4.08 ** Indicates significance at P = .05 with F5,40 = 2.45 Appendix I. 281 Duncan's test to determine differences between latencies caused by‘various intensity levels for waves I—IV for right and left ears. Significant studentized ranges are (2) 2.86, (3) 3.01, (4) 3.10, (5) 3.17, (6) 3.22 Means underscored by‘a line are considered equal. W 282 Duncan's test for latency of wave I (R/E). Rp at P = .05 with 40 degree of freedom (No. of means given). Rp (2) 0.22 (3) 0.23, (4) 0.238, (5) 0.24, (6) 0.247 A=75 B=65 C=55 D=45 E=35 F=25 *Means (in ms) 1.51 1.61 1.76 1.86 1.89 1.95 pooled for Agouti & Cream * No genotype—intensity interaction. 283 Duncan's test for latency of wave II (R/E) Rp at P = .05 with 40 degrees of freedom (No. of means given). Rp (2) 0.36, (3) 0.38, (4) 0.39, (5) 0.40, (6) 0.41 A=75 B=65 C=55 D=45 E=35 F=25 * Means (in ms) 2.3 2.51 2.58 2.74 2.85 3.00 pooled for Agouti & Cream * No genotype-intensity interaction 284 Duncan's test for latency of wave III (R/E) Rp at P = .05 with 40 degrees of freedom (No. of means given). RP (2) 0.58, (3) 0.61, (4) 0.63, (5) 0.64, (6) 0.66. A=75 B=65 C=55 D=45 E=35 F=25 * Means (in ms) 3.08 3.22 3.43 3.80 3.88 4.05 pooled for Agouti & Cream * No genotype-intensity interaction 285 Duncan's test for latency of wave IV (R/E) Rp at P = .05 with 40 degrees of freedom (No. of means given). Rp (2) 0.56, (3) 0.57, (4) 0.59, (5) 0.60, (6) 0.61. A=75 B=65 C=55 D=45 E=35 F=25 * Means (in ms) 4.15 4.51 4.67 4.89 5.08 5.25 pooled for Agouti & Cream * No genotype—intensity interaction 286 Duncan's test for latency of wave I (L/E) Rp at P = .05 with 40 degrees of freedom (No. of means given). Rp (2) 0.48, (3) 0.52, (4) 0.54, (5) 0.55, (6) 0.58 A=75 B=65 C=55 D=45 E=35 F=25 * Means (in ms) 1.52 1.73 1.84 1:97 2.00 2.20 pooled for Agouti & Cream * No genotype—intensity interaction .‘ .~ ,4. .1...“ 287 \ Duncan's test for latency of eave II (L/E) Rp at P = .05 with 40 degrees of freedom (No of means given). Rp (2) 0.26, (3) 0.27, (4) 0.28, (5) 0.285, (6) 0.29 A=75 B=65 C=55 D=45 E=35 F=25 * Means (in ms) 2.29 2.54 2.70 2.84 2.89 3.07 pooled for Agouti & Cream * No genotype—intensity interaction 288 Duncan's test for latency of wave III (L/E). Rp at P = .05 with 40 degrees of freedom (No. of means given). RP (2) 0.37, (3) 0.39, (4) 0.40, (5) 0.41, (6) 0.42 A=75 B=65 C=55 _ D=45 E=35 F=25 * Means (in ms) 3.11 3.31 3.60 3.70 3.73 3.74 pooled for Agouti & Cream * No genotype-intensity interaction 289 Duncan's test for latency of wave IV (L/E) Rp at P = .05 with 40 degrees of freedom (No. of means given). Rp (2) 0.54, (3) 0.57, (4) 0.58, (5) 0.60, (6) 0.61. * Means (in ms) 4.29 4.49 4.68 4.94 5.06 5.20 pooled for Agouti & Cream * No genotype-intensity interaction 290 Appendix J. Mean inter-aural latency differencies (ILDs) for ABR waves I—IV all normal genotypes (Agouti and Cream). 291 Table 81. Mean ILDs for all genotypes for waves I-IV for Agouti and Cream Genotype I 11 III IV Agouti 0.06 0.01 0.07 0.08 Cream ‘ 0.04 0.01 0.17 0.21 Appendix K. Summary of two—way ANOVA for differences between ears for waves I—IV across the various intensity levels Agouti and Cream genotypes. 293 Table 82. Summary of Ear x intensity x latency x ANOVA for BSER waves I—IV of Agouti Wave SS DF MS F I Between Ears 0.13 1 0.13 3.33 Between Intensities 2.90 5 0.58 14.87** Ear—intensity— Interaction 0.21 5 0.042 1.08 Error 1.57 40 0.03 II Between Ears 0.00 1 0.00 0.00 Between Intensities 5.68 5 1.14 5 59 Ear—intensity— Interaction 1.66 5 0.33 1.62 Error 8.16 40 0.204 III Between Ears 0.02 l 0.02 0.67 Between Intensities 1.39 5 0.28 9.03** Ear-intensity— Interaction 0.08 5 , 0.16 0.52 Error 1.22 40 0.03 IV Between Ears 0.47 l 0.47 0.61 Between Intensities 2.61 5 0.52 0.675** Ear—intensity— Interaction 2.14 5 0.43 5.58** Error 3.06 40 0.077 * Indicates significance at P = .05 with F1, 40 = 4.08 ** Indicates significance at P = .05 with E5, 40 = 2.45 294 Table 83. Summary of Ear x intensity xx Latency x ANOVA of BSER waves I-IV of the Cream Wave SS DF MS F I Between Ears 0.03 1 0.03 0.0.43 Beween Intensities 2.28 5 0.46 6 6:57** Ear—intensity— ' interaction 0.07 5 0.014 O 0.20 Error 2.94 40 0.07 II Between Ears 0.87 l 0.87 108.7* Between Intensities 0.01 5 0.002 0.25 Error 0.04 5 0.008 III Between Ears 0.00 1 0.00 0.00 Between Intensities 0.45 5 0.09 22.50** Error 0.02 5 0.004 IV Between Ears 3.62 l 3.62 40.22* Between Intensities 5.94 5 1.19 13.22** Ear—intensity— Interaction 5.45 5 1.09 15.33** Error 3.69 40 0.09 * Indicates significance at P = .05 with F1,40 = 4.08 ** Indicates significance at P .05 with F5,40 Appendix L. Summary of two—way ANOVA for latencies of waves I—IV for genotype (BEW and WBA) and intensity for right and left ears. Tab >296 1e 84. Summary of genotype (BEW ANOVA of BSER latency of and WBA) X intensity x waves I—IV for right ear Wave SS DF 1% F P S/NS I Between Genotypes (UC)+ 0.11 1 0.11 Btween Intensities(UC)+ 0.08 1 0.08 Between Genotypes (COR)* 0.09 1 0.09 2.28 0.16 NS Bwteen Intensities (COR)* 0.05 1 0.05 1.34 0.27 NS Interaction I0.00 1 0.00 0.00 II Between Genotypes (UC)* 10.00 I 8.69 Between Intensities (UC)* 3.00 I 3.12 Between Genotypes(COR)+ 8.00 '1 7.83 17.14 0.01 S Between Intensities(COR)+ l I 1.27 2.77 0.11 NS Interaction 1.14 2.50 0.13 NS III Between Genotypes (UC)* 0.14 1 0.14 Between Intensities(UC)* 0.07 1 0.07 Between Genotypes(CORR)+ 0.14 1 0.14 0.63 NS Between Intensities(COR)+ 0.07 l 0.07 0.32 NS Interaction 0.00 1 0.00 0.00 NS IV Between Genotypes(UC)* 0.32 1 0.32 Between Intensities(UC)* 0.28 1 0.28 Between Genotypes (COR)+ 0.38 1 0.38 2.67 0.13 NS Between Intensities(COR)+ 0.34 l 0.34 2.37 0.15 NS Interaction -0.00| 1 0.12 0.82 + Uncorrected * Corrected 8 Indicates significance at P = .05 level NS Indicates non—significance at P = .05 level 297 Table 85. Summary of genotype (BEW and WBA) x intensity x ANOVA of the BSER latency for waves I—IV of the left ear. Wave SS DF ‘MS F P S/NS I Between Genotypes (UC)* 1.00 l 1.00 Between Intensities(UC)* 0.48 1 0.48 Between Genotypes (COR)+ 0.44 l 0.44 2.46 0.14 NS Between Intensities(COR)+ 0.41 1 0.41 2.31 0.15 NS Interaction 0.11 1 0.11 0.64 NS 11 Between Genotypes (UC)* 0.22 l 0.22 Between Intensities(UC)* 0.10 l 0.10 Between Genotypes COR)+ 0.25 1 0,25 2.06 0.18 NS Between Intensities(COR)+ 0.13 1 0.13 1.09 0.32 NS Interaction 0.00 1 0.00 0-00 NS III Between Genotypes (UC)* 3.00 l 3.13 Between Intensities UC)* 0.33 1 0.33 Between Genotypes (COR)+ 3.00 1 3.00 4.41 0.36 S Between Intensities(COR)+ 0.21 l 0.21 0,37 NS Interaction 1 . 03‘ I 1 . 03 1. 85 0 .20 NS IV Between Genotypes (UC)* 0.27 l 0.27 Between Intensities(UC)* 0.26 1 0.26 Between Genotypes (COR)+ 0.25 1 0.25 1.04 0.33 NS Between Genotypes (COR)+ 0.24 1 0.24 1.02 0.34 NS Interaction 0.09 1 0.09. 0.38 NS * Uncorrected + Corrected. S Indicates significance at P = .05 level NS Indicates non—significance at P .05 level 298 Appendix M. Inter—aural latency difference for BEW and WBA for waves I—IV. 299 Table 86. Inter—aural latency difference for BEW and WBA. Genotype I II III IV BEW —0.05 0.59 0.28 0.15 WBA 0.34 1.40 0.35 0.11 300 Appendix N. Summary of ANOVA for latency differences between ears for waves I—IV for BEW and WBA. 301 Table 87. Summary of Bar x intensity x Latency x ANOVA of BSER waves I-IV for the BEW. Wave SS DF MS E I Between Ears 0.04 1 0.04 1.33 Between Intensities 0.03 l 0.03 1.00 Ear—intensity— Interaction 0.00 1 0.00 0.00 Error 0.50 15 0.03 II Between Ears 1 1.00 2.33 Between intensities 1 . 00 1 1 . 04 2 . 42 Ear—intensity— 1‘04 Interaction 0 44 l 0.44 1.03 ' 15 0.43 Error 6.40 ‘ III Between ears 0.33 1 0.33 0.43 Between intensities 0.16 1 0.15 ’ 0.21 Ear—intensity— ' ‘ interaction 0.67 l 0.67 0.86 Error ' 11.67 15 0.78 IV Between ears 0 05 ‘l 0-05 0-08 Between intensities 2124 1 2.24 3.93 Ear—intensity— 7 Interaction ' 1 0.01 0-018 Error 0‘01 15 0.57 8.52 * Indicates significance at P = .05 with F1,15 = 4.54 302 Table 88 Summary of Bar x intensity x latency x ANOVA of BSER waves I-IV of the WBA Wave SS DF MS F I Between ears 0.00 1 0.00 0.00 Between intensities 0.05 1 0.05 0.77 Ear-intensity- Interaction 0.00 1 0.00 0.00 Error 0.52 8 0.065 II Between ears (UC)+ 0.10 1 0.10 Between intensities(UC)+ 0.01 1 0.01 Between ears (CORR)++ 0.10 1 0.10 3.52 Between intensities(0RR)++ 0.02 l 0.02 0.75 Ear-intensity- Interaction 0.02 1. 0.02 0.56 III Between ears 0.39 1 0.39 2.05 Between intensities 0.00 1 0.00 0.00 Ear-intensity- Interaction 0.06 1 0.06 0.315 Error 1.55 8 0.19 IV Between ears 0.04 1 0.04 0.093 Between intensities 0.04 1 0.04 0.093 Ear-intensity— - Interaction 0.01 1 0.006 0.002 Error - 3.46 8 0.43 + Uncorrected ++ Corrected F1,8 = 5.32 at P = .05 303 Appendix 0. Linear regression latency values for waves I-IV for both ears of all genotypes. 304 Table 89. Correlation latency values for waves I—IV of both ears of all genotypes. Genotype I II III IV AG —0.95 -0.95 —0.99 -0.98 CR ' —0.97 -0.99 —0.97 . —0.98 BEW —0.88 —0.96 0.42 -0.94 WBA 0.66 —0.95 —0.90 —0.94 Table 90. Intercept of latency values for waves I—IV for all genotypes. Genotype I II III IV AG 2.89 3.50 4.70 5.80 CR 1.20 3.36 4.50 5.90 BEW 4.6 5.18 3.00 5.30 WBA 1.41 6.00 5.10 7.26 Table 91. Slopes of latency values for waves I-IV for all genotypes Genotype I II III IV AG —0.003 -0.002 -0.002 —0.002 CR —0.002 —0.002 —0.002 —0.002 BEW —0.006 0.004 0.001 -0.001 WBA —0.002 -0.007 0.003 —0.003 305 Appendix P. Individual and mean thresholds for waves I—IV for all genotypes. 306 Table 92. Individual thresholds for wave I for all genotypes Genotype Ear 25 35 45 55 65 75 Agouti R x L x Cream R x L x BEW R x x xxx _ L x x xxx WBA R x xx L xx x Table 93. Individual thresholds for wave II for all genotypes Genotype Ear 25 35 45 55 65 75 Agouti R x L x Cream R x L x BEW R xx x xx L x xx xx WBA R x xx L x xx 307 Table 94. Individual thresholds for wave III for all genotypes Genotype Ear 25 35 45 55 65 75 Agouti R x L x Cream R x L x BEW R xxx xx L x xxx x WBA R xx x L x xx Table 95. Individual thresholds for wave IV for all genotypes Genotype Ear 25 35 45 55 65 75 Agouti R x L x Cream R x L x BEW R XX XX X L xx x xx WBA R XXX L x xx - 308 Table 96. Mean thresholds for waves I-IV for all genotypes (R/E) in dB nHL Genotype I II III IV Agouti 25 25 25 25 Cream 25 25 25 25 BEW 60 55 49 53 WBA 62 65 59 55 Table 97. Mean thresholds for waves I-IV for all genotypes (L/E) in dB nHL Genotype I II III IV Agouti 25 25 25 25. Cream 25 25 25 25 BEW 63 57 55 55 WBA 59 56 52 55 309 Appendix Q. Differences between Wh—locus and E-locus with regard to wave latency as given by Chi—square. 310 * Table 98. Differences between.Wh-locus and E—locus with regard to latency as given by Chi—square. Right Ear Left Ear Parameter X2 P Parameter X2 P Wave I 0.208 0.65 Wave I 0.61 0.44 Wave II 0.53 0.45 Wave II 0.26 0.61 Wave III 0.624 0.80 Wave III 9.11 0.76 Wave IV 0.334 0.56 Wave IV 7.08 0.79 311 Appendix R . Mean amplitude values for waves I-IV for all genotypes. 312 Table 99. Mean amplitude (NV) for wave I from 25—75 dB nHL for all animals (R/E) Genotype 25 35 45 55 65 75 (N) Agouti 29 42 83 155 192 380 5 Cream 21 42 89 93 328 390 5 BEW * * 100+ 170+ 319 460 5 WBA * * * 360+ 257+ 330 3 TOTAL 50 84 272 778 1096 1560 18 MEAN 0F MEANS 25 42 91 195 274 390 ,# OF ANIMALS _10 10»___11 13 .18” 18 Table 100. Mean amplitude (NV) for wave II from 25—75 dB nHL for all animals (R/E) Genotype 25 35 45 55 65 75 (N) Agouti 36 35 43 108 118 147 5 Cream 18 34 62 77 165 219 5 BEW * * 50+ 95+ 133 161 5 WBA * * * * 137 265 3 TOTAL 54 69 155 280 553 792 18 MEAN OF MEANS 27 35 52 93.3 138 198 # OF ANIMALS 10 10 11 _13 17 17 * Indicates no response from any animal + Indicates response from reduced number of animals 313 Table 101. Mean amplitude (NV) for wave III from 25—75 dB nHL for all animals (R/E) Genotype 25 35 45 5 65 75 (N) Agouti 47 86 199 424 679 1048 5 Cream 31 66 253 474 941 1535 5 BEW * * 228+ 655 1351 1599 5 WBA * * * 1253 1252 1650 3 TOTAL 78 152 680 2906 4223 5832 18 MEAN OF MEANS 39 76 227 727 1056 1458 # 0F ANIMALS 10 10 12 17 18 18 Table 102. Mean amplitude (NV) for wave IV from 25—75 dB nHL for all animals (R/E) Genotype 25 35 45 55 65 75 (N) Agouti 28 45 45 87 112 180 '5 Cream 26 71 71 108 255 312 5 BEW * * 125+ 164+ 371 250 5 WBA * * * * 298 490 3 TOTAL 54 116 241 359 1036 1232 18 MEAN OF MEANS 27 58 80 120 259 308 # OF ANIMALS 10 10 12 14 18 18 * Indicates no reponSe from animals + Indicates response from reduced number of animals 314 Table 103. Mean amplitude (NV) for wave I from 25—75 dB nHL for all animals (L/E) Genotype 25 35 45 55 65 75 (N) Agouti 27 47 97 195 241 108 5 Cream 40 40 55 117 123 302 5 BEW * * 80+ 750+ 371 222 5 WBA * * * 273+ 258 150 3 TOTAL 67 87 232 1335 993 1531 18 MEAN OF MEANS 34 44 77 334 248 383 # 0F ANIMALS 10 10 11 13 18 18 Table 104. Mean amplitude (NV) for wave II from 25—75 dB nHL for all animals (L/E) Genotype 25 35 45 55 65 75 (N)7 Agouti 13 24 41 105 124 108 5 Cream 26 45 191 98 147 302 5 BEW * * 400+ 94+ 759 222 5 WBA * * 50+ 110 123 150 3 TOTAL 39 6 682 407 '1153 890 18 MEAN OF MEANS 19 3 171 102 288 223 # 0F ANIMALS 10 1 12 14 18 18 * Indicates no response from animals + Indicates reesponse from reduced number of animals 315 Table 105. Mean amplitude for wave III from 25—75 dB nHL for all animals (L/E) Genotype 25 35 45 .55 65 75 (N) Agouti 25 30 145 249 378 525 5 Cream 20 43 90 100 197 387 5 BEW * * 400+ 468+ 759 1011 5 WBA * * 75 75 135 305 3 TOTAL 45 73 710 892 1469 2228 18 MEAN 0F MEANS 23 37 178 223 367 557 # OF ANIMALS 10 10 12 14 18 18 Table 106. ‘Mean amplitude (NV) for wave IV from 25—75 dB nHL for all animals (L/E) Genotype 25 35 45 55 65 775 (N) Agouti 20 30 145 249 378 525 5 Cream 25 43 90 100 - 194 387 5 BEW * * 95+ 197+ 339 276 5 WBA * * 75+ 75+ 134 305 3 TOTAL 45 73 405 621 1045 1493 18 MEAN 0F MEANS 23 37 101 155 261 ' 373 # OF ANIMALS 10 10 >13 14 18 18 * Indicates no response + Indicates response from less animals 316 Appendix 81. Differences between Agouti and Cream with regard to wave amplitude as given by F-values. 317 Table 107. Difference between Agouti and Cream with regard to wave amplitude as given by F—values Parameter Difference I II III IV Right Ear Genotype 0112 0.14 4.30* 4.13* Amplitude Intensity 8.32** 6.37** 3.70*. 9.95** Interaction 0.45* 0.453 0.96 1.08 Parameter Difference I II III IV Left Ear ‘Genotype 33.28% 9.495 50.95* 4.01 Amplitude ‘ Intensity 50.07** 6.48** 2-45** 9495M Interaction 12.99** 2.63** 1.24 1.06 * Indicates significance at P' :05 with 171,40 ** Indicates significance at P .05 with F5, 40 4.08 2.45 II II n w- .w—-.-~ 'L-Mtz-ur—u.v.‘-_Z_“_.‘ZL_' 318 Appendix S2. Summary of two-way ANOVA for amplitudes of waves I-IV for genotype (Agouti and Cream) and intensity for both ears. — 1w: "' *5": H’-*:'_‘ i _. - -. . - 319 Table 108. Summary of genotype (Agouti and Cream) x intensity x ANOVA for ABR amplitudes for right ear. Wave Source SS DF MS F I Between genotypes 2870.42 1 2870.41 0.12 Between intensities 991768.75 5 198353.75 8.32** Genotype-intensity- interaction 53442.08 5 10688.42 0.45 Error 953150.00 40 238288.8 II Between genotypes 836.27 836.27 0.14 1 Between intensities 188181.33 5 37636.27 6.37** Genotype—intensity- interaction - 13362.33 5 2672.47 0.453 Error 236204.80 40 5905.12 III Between genotypes 438250.42 1 438250.42 4.30* . Between intensitie81883222.08 5 376644.42 3.70** Genotype-intensity (interaction 487782.08 5 97556.42 0.96 Error 4075300.00 40 101882.50 IV Between genotypes‘ 34843.75 1 34843.75 4.13* Between intensities 420228.75 5 84045.75 9.95** Genotype—intensity- interaction 44818.75 5 8963.75 1.08 Error 337550.00 40 8438.75 * Indicates significance at P = .05 with.F1,40 = 4.08 ** Indicates significance at P = .05 with.Fg,40 = 2.45 320 Table 109. Summary' of genotype (Agouti and Cream) x intensity x ANOVA of ABR amplitudes for waves I-IV of the left ear. Wave Source SS DF MS F I Between genotypes 47943.52 1 47943.52 7.328* Between intensities 165796.86 5 33159.37 5.070** Genotype-intensity- interaction 42508.75 5 8501.75 l.29** Error 261691.60 40 6542.4 II Between genotypes 67000.42 1 67000.42 9.49* Between intensities 226199.88 5 45239.98 6.48** Genotype-intensity~ interaction 92973.88 5 18594.77 2.63** Error 282388.40 40 7054.71 III Between genotypes 3255080.44 1 3255080.44 50.95* Between intensities 781894.56 5 156378.91 2.45** Genotype-intensity- interaction 395409.73 5 79081.95 1.24 Error 2555144.40 40 63878.6 IV Between genotypes 33843.75 1 33843.75 4.01 Between intensities 420228.75 5 84045.75 9.95 Genotype-intensity- ” interaction 44818.75 5 8963.75 1.06 Error 337550.00 40 8438.75 * Indicates significance at P = .05 with,F3,40 = 4.08 ** Indicates significance at P = .05 with.Fg,40 = 2.45 Appendix T. 321 Duncan's test to determine the differencies in latencies caused by varying intensity levels for waves I- IV. Significant studentized ranges are (2) 2.86, (3) 3.01, (4) 3.10, (5) 3.17, (6) 3.22. Means underscored by a line are considered equal. 322 Duncan's test for amplitude of wave I (R/E) Rp at P = .05 40 with degrees of freedom (No. of means given). Rp: (2) 197, (3) 208, (4) 214, (5) 218, (6) 222 A=25 E=35 C=45 D=55 B=65 F=75 * Means (in ms) 25 42 . 86 124 260 385 pooled for Agouti & Cream * No genotype—intensity interaction 323 Duncan's test for amplitude of wave II (R/E) Rp at P = .05 with 40 degrees of freedom (No. of means given) RP: (2) 98, (3) 102, (4) 105, (5) 108, (6) 109. A=25 E=35 C=45 D=55 B=65 F=75 * Means (in ms) 27 35 53 93 142 183 pooled for Agouti & Cream * No genotype-intensity interaction 324 Duncan's test for amplitude for wave 111 (R/E). RP at P = .05 with 40 degrees of freedom (No. of means given). Rp: (2) 408, (3) 429, (4) 442, (5) 452, (6) 459. A=25 E=35 C=45 D=55 B=65 F=75 * Means (in ms) 39 76 226 499 810 1292 pooled for Agouti & Cream * No genotype—intensity interaction 325 Duncan‘s test for amplitude for wave IV (R/E). Rp at P = .05 with 40 degrees of freedom (No. of means given). RP: (2) 117.5, (3) 123.4, (4) 127, (5) 129.9, (6) 132 A=25 E=35 C=45 D=55 B=65 F=75 * Means (in ms) 27 58 58 98 184 246 pooled for Agouti & Cream * No genotype-intensity interaction 326 Duncan's test for amplitude of wave I (L/E). Rp at P = .05 with 40 degrees of freedom (No. of means given). Rp: (2) 102, (3) 108, (4) 112, (5) 114, (6) 116. A=25 E=35 C=45 D=55 B=65 F=75 * Means (in ms) 34 44 76 156 182 285 pooled for Agouti & Cream * No genotype-intensity interaction 327 Duncan's test for amplitude of wave II (L/E) Rp at P = .05 with 40 degrees of freedom (No. of means given). Rp: (2) 107, (3) 112.8, (4) 116, (5) 118.8, (6) 120. A=25 E=35 C=55 D=45 B=65 F=75 * Means (in ms) 19 35 102 116 136 205 pooled for Agouti & Cream * No genotype—intensity interaction 328 Duncan's test for amplitude of wave III (L/E). Rp at P = .05 with 40 degrees of freedom (No. of means given). Rp: (2) 323, (3) 340, (4) 350, (5) 358, (6) 363.8. A=35 B=25 C=45 D=55 B=65 F=75 * Means (in ms) 37 39 118 175 288 456 Pooled for Agouti & Cream * No genotype—intensity interaction 329 Duncan‘s test for amplitude of wave IV (L/E). Rp at P = .05 with 40 degrees of freedom (No. of means given) Rp: (2) 117, (3) 123.6, (4) 127, (5) 129.9, (6) 132. A=25 E=35 C=45 D=55 B=65 F=75 * Means (in ms) 23 37 118 175 286 456 pooled for Agouti & Cream * No genotype—ntensity interaction 330 Appendix U. Summary of two—way ANOVA showing differences between ears for in waves I—IV amplitude across various intensity levels for the Cream and Agouti. 331 Table 110. Summary of Bar x intensity x amplitude x ANOVA of BSER waves I-IV for the Agouti. Wave Source SS DF MS F I Between ears 4083 1 4083 0.17 Between intensities 418913 5 83782 3.43** Ear—intensity- interaction 26033 5 5206 0.22 Error 977650 40 24441 II Between ears 400 1 400 0.64 Between intensities 45493 5 9098 1.45 Ear-intensity- ' interaction 13442 5 2688 0.43 Error 250381 40 6260 III Between ears 117483 1 117483 0.577 Between intensities7163757 5 1432751 7JE¢* Ear-intensity— interaction 845183 5 169036 0.83 Error 8133790 40 203344 IV Between ears 129828 1 129828 2.70 Between intensities 583984 5 116796 2.43 Ear—intensity- interaction 282366 5 56473 1.18 Error 1919262 40 4798 .05 with.F1,40 .05 with F5,40 * Indicates significance at P ** Indicates significance at P II II II II tbs O 00 332 Table 111. Summary of Bar x intensity x amplitude x ANOVA of BSERS waves I—IV of the Cream. Wave Source SS DF MS F I Between ears 226 1 226 0.873 Between intensities 19569 5 3913.80 15.13** Ear—intensity— interaction 8631 5 1726.20 6.67** Error 10346 40 258,70 11 Between ears 56733 1 56733 6.20* Between intensities 201558 5 40311 4.40** Ear—intensity— interaction 131448 5. 26289 2.87** Error 365890 40 9147.30 III Between ears 4155 1 4155 2.99 Between intensities 94491 5 18898 l3.61** Ear—intensity— interaction 52142 5 10428.40 7.51** Error 5529 40 1388.20 IV Between ears 4896 1 4896.07 0.52 Between intensities 500682 5 100136.47 10.53** Ear—intensity- ' interaction 29856 5 5971.27 0.63 Error 380103 40 95026.60 * Indicates significance .05 with F1,40 = 4.08 ** Indicates significance = at P = at P = .05 with F5,40 333 Appendix V. Summary of two-way ANOVA for genotype (BEW and WBA) and intensity for waves I—IV of both ears. 334 Table 112. Summary of genotype (BEW and WBA) x intensity x ANOVA of ABR amplitudes of waves I-IV of right ear. Wave Source SS DF MS F P S/NS I Between genotypes 49307 1 49306.64 1.73 0.21 NS Between intensities 60025 1 60024.99 2.11 0.17 NS Genotype-intensity— interaction 2282 1 2281.67 0.08 NS II Between genotypes 28995 1 28994.81 1.52 0.23 NS Between intensities 300 1 300.17 0.02 NS Genotype-intensity- interaction 2400 1 2400.19 0.13 NS 111 Between genotypes 261255 1 261255 0.47 NS Between intensities 44018 1 44017 0.08 NS Genotype-intensity- interaction 445887 1 448557 0.80 NS IV Between genotypes 3291 1 3291.45 0.13 NS Between intensities 41529 1 1528.95 1.60 0.22 NS Genotype—intensity— interaction 148269 1 148268.00 5.70 0.031 S S Indicates significance at P = .05 level NS Indicates nonesignificance at P = .05 level 335 Table 113. Summary of genotype (BEW and WBA) x intensity x ANOVA of BSER for waves I-IV fot the left ear. Wave Source SS DF MS F P S NS I Between genotypes 141172 1 141171 0.75 NS Between intensities 201600 1 201599 1.08 0.32 NS Genotype—intensity— interaction 51450 1 .51450 0.27 NS II Between genotypes 406473 1 406472 4.01 0.07 NS Between intensities 312631 1 312630 3.09 0.10 NS Genotype-intensity- interaction 274694 1 274691 2.71 0.13 NS III Between genotypes 439040 1 439039 1.84 0.20 NS Between intensities 92829 1 92828 0.39 NS Genotype—intensity~ interaction 63431 1 63431 0.27 NS IV Between genotypes 43376 1 43375 0.47 NS Between intensities 97689 1 97689 1.06 0.32 NS Genotype-intensity- interaction 150134 1 150134 1.62 0.22 NS S = Indicates significance at P = .05 level NS = Indicates non-significance at P = .05 level 336 Appendix W. Summary of ANOVA for differences between ears for waves I—IV of the BEW and the WBA. 337 Table 114. Summary of Ear x intensity x amplitude x ANOVA of BSER for waves I-IV of the BEW. Wave Source SS DF MS F I Between ears 7801 1 7801 0.05 Between intensities 261061 1 261061 1.81 Ear—intensity— interaction 38281 1 38281 0.27 Error 2164070 15 144271 II Between ears 433651 1 433651 3.70 Between intensities 197011 1 197011 1.70 Ear-intensity— interaction 272611 1 272611 2.35 Error 1737290 15 115819 III Between ears 8611 1 8611 0.92 Between intensities 2761 1 2761 0.029 Ear—intensity— interaction 22781 1 22781 0.242 Error 1410120 15 94008 IV Between ears 117413 1 117413 2.31 Between intensities 36159 1 36159 0.71 Error 50836 1 50836 Indicates non—significance at P = .05 level with F1,15 — 4.54 338 Table 115. Summary of Bar x intensity x amplitude x ANOVA of BSER of waves I-IV of WBA Wave Source SS DF MS_‘—_“—P——‘_ I Between ears 22968 1 ' 22698 0.66 Between intensities 52 l 52.08 0.008 Ear—intensity- ‘ interaction 37968 1 37968 0.61 Error 494883 8 61860 II Between ears 9627* 1 9626 0.35 Between intensities 10890* 1 10890 0.39 Ear-intensity- interaction 5802 l 5801 0.21 111 Between ears 374533 1 374533 2.07 Between intensities 373536 1 373536 2.06 Ear-intensity— interaction 8533 1 8533 0.05 Error 1450600 8 181325 IV Between ears 91002 1- 91002 2.62 Between intensities 77602 1 77602 2.24 Ear-intensity- interaction 2852 1 2852 0.082 Error 277616 8 34702 Indicates non—significance at P = .05 level with.F3,8 = 5.32 * corrected values since responses are from only two animals. 339 Appendix X. Linear regression amplitude values for waves I, II, III and IV. 340 Table 116. Correlation.values for amplitude for all waves (R/E). Genotype T 'I 5:5 IIV Agouti 0.99 0.94 0.98 . 0.81 Cream 0.98 0.99 0.98 0.97 BEW 0.99 0.96 0.96 0.54 WBA -0.24 0.97 0.86 0.86 Table 117. Intercepts for waves I-IV for all genotypes (R/E). Genotype I II III IV Agouti 7.8 13 12 20 Cream 5.1 5.6 4.8 10 BEW 9.8 10 15 97 WBA 413 12 561 12 Table 118 Slope for waves I—IV for all genotypes (R/E) Genotype I II III IV Agouti 0.02 0.05 0.02 0.009 Cream 0.02 0.02 0.035 0.02 BEW 0.02 0.02 0.03 0.006 WBA 0.002 0.02 0.02 0.02 341 Table 119. Correlation for waves I-IV for all genotypes (L/E). 6enotype ‘T IT T=T 775V _— Agouti 0.98 0.94 0.96 0.96 Cream 0.95 0.96 0.98 0.98 BEW 0.67 0.046 0.92 0.89 WBA 0.91 0.91 0.97 0.93 Table 120. Intercept for waves I-IV for all genotypes (L/E). Eenotype 55 TT‘ 5:5 ‘5TV Agouti 9.2 5 7 4 Cream 12 13 8 7 BEW 15 232 71 18 WBA 6 13 9 7 Table 121. Slope for waves I-IV for all genotypes (L/E)., fienotype 52 I III IV Agouti 0.02 0.02 0.03 0.03 Cream 0.02 0.015 0.03 0.02 BEW 0.02 0.001 0.02 0.02 WBA 0.006 0.001 0.03 0.02 342 Appendix Y. Relative amplitude ratio for waves III/I for all genotypes 343 Table 122. III/I amplitude ratio for all genotypes (R/E) III/I 25 35 45 55 65 75 Agouti 1.62 2.00 2.39 2.73 3.50 2.75 Cream 1.48 1.57 2.84 6.20 2.87 3.90 BEW 2.88 3.85 4.21 3.47 WBA 3.40 4.90 5.00 Table 123. III/I amplitude ratio for all genotypes (L/E) III/I 25 35 45 55 65 75 Agouti 1.03 1.66 3.16 3.59 5.30 4.20 Cream 1.03 1.68 3.47 3.03 5.12 3.02 BEW 5.00 0.49 2.05 1.72 WBA 1.77 4.11 3.90 344 Appendix Z. Difference between Wh-locus and E—locus with respect to wave amplitude as revealed by Chi—square 345 Table 124. Difference between Wh-locus and E—locus with respect to wave amplitude as revealed by Chi-square. Right Ear Left Ear Parameter X2 P Parameter X2 P Wave I 2.57 0.11 Wave I 14.86 0.001* Wave II 37.57 0.00 Wave II 16.62 0.00* Wave III 29.76 0.52 Wave III 1.44 0.23 Wave IV 103.37 0.00 Wave IV 3.49 0.52 * Significant. IIIIIIIIIIIIIIIII LIBRQRIES _._ \HWHHH)NM)1WM)\)\\\\\\\\\\\\\\\)\\ 1 ; 6292860 __