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Hair cell regeneration Part 2 Brenda M. Ryals, Ph.D. JJames M Madison di U University i it Robert Dooling, Ph.D. University of Maryland Academy of Doctors of Audiology November 4-6, 2010 With special appreciation to Dr. Robert Dooling, University of Maryland Co-investigator – Functional aspects of Hair cell Regeneration Work supported by NIDCD grants DC01245 DC1372, DC00198 and DC00353 and The Comparative and Evolutionary Biology of Hearing Training Grant (DC00046) And to Dr. Beth Brittan-Powell, University of Maryland for all of the ABR results Work supported by NIDCD grants DC01245 DC1372, DC00198 and DC00353 and The Comparative and Evolutionary Biology of Hearing Training Grant (DC00046) 1 Will Hair Cell Regeneration Cure Hearing Loss? • Will regenerated hair cells result in the return of normal hearing (audiogram), complex auditory perception (speech understanding)? Will Hair Cell Regeneration Cure Hearing Loss? •Does the onset and etiology of hearing loss matter? •Pre-lingual/post-lingual •Congenital/genetic •Acquired Birds offer a unique animal model of complex auditory perception, vocal learning and vocal production 2 Birds offer a unique animal model of complex auditory perception, vocal learning and vocal production AND..they regenerate hair cells Effect of Early Deafening on Warble Song Normal Budgerigar Deafened Budgerigar Effect of Adult Deafening on Contact Calls Heaton, et al, 1999 3 Vocal Learning in Budgerigars Farabaugh, et al, 1994 Birds naturally regenerate and re-innervate hair cells after acquired injury Birds naturally use hearing to modify vocal production What does the world sound like after hair cell regeneration? Does it make a difference to vocal production? 4 Hearing Sensitivity Recovers to near normal within 8 weeks 125 10 20 30 250 Frequency in Hz 500 1000 X X X 2000 4000 8000 X = pre-injection X X X X X X X X 40 X = 2 weeks post injection X X X= 8 weeks post injection 50 X 70 X Absolute Threshold dB SPL (re 20 pa) 60 80 90 100 80 Injections 70 60 50 40 30 PTS of 23 dB 20 10 0 0 5 10 15 20 25 30 Days Dooling, Ryals and Manabe 1997 Hearing Resolution returns to normal even sooner A 30 F (Hz) 1.0 kHz 2.86 kHz 20 10 0 Pre 4-6 8-10 12+ Pre 4-6 8-10 12+ Weeks B 6 1.0 kHz 2.86 kHz 4 I (dB) Hearing Level in dB SPL 0 2 0 Pre 4-6 8-10 12+ Pre 4-6 8-10 12+ Weeks Dooling, Ryals and Manabe 1997 5 Does Hair Cell Regeneration Cure Hearing Loss? Yes. Behavioral audiograms show 40-50dB recovery of sensitivity for even the most severely damaged regions. Difference limens for frequency and intensity return to normal. Human pigeon What does Wh d the brain have to do with anything? What is the effect of regeneration on perception and production of relevant, complex acoustical stimuli? 6 Perceptual Maps of Budgerigar Contact Calls Before Kanamycin (VAF = 79%) 28-Day Recovery (VAF = 78%) Dimension 2 0.50 D D' 0.25 B B' DB'B D D' A' E E' 0.00 C' C -0.25 E'E A' A -0.50 -0.50 -0.25 0.00 0.25 C'C 0.50 -0.50 -0.25 0.00 0.25 0.50 Dimension 1 Recovery was back to pre-injection levels by 23 weeks Dooling, Ryals, Dent and Reid, 2006 7 Will Hair Cell Regeneration Cure Hearing Loss? When the papilla first becomes repopulated, the world sounds different. Absolute sensitivity and simple p discrimination recovers within weeks of regeneration while more complex recognition tasks may take several months to recover. Full regeneration, maturation and re-innervation of hair cells occurs well before full recovery of function. Vocal production in budgerigars Kazu Manabe, Michael Osmanski, Ashwin Plachikkat, Manjit Sahota Training birds to produce specific vocalizations by operant conditioning Relattive Similarity Effect of Kanamycin (200mg/kg/day) on Vocal Precision 1.0 0.9 0.8 0.7 Injections 0.6 0 5 10 15 20 25 Days 8 Relative Similarity 1.1 1.0 0.9 0.8 0.7 0.6 0.5 A Injections 0.0 -5 0 5 10 15 20 25 15 20 25 30 Days Sequence Stereotyp py 1.1 10 1.0 0.9 Injections 0.8 0.7 0.6 0.5 B 0.0 -5 0 5 10 30 Days Woolley and Rubel 2002 Can regenerated hair cells support normal vocal production? The precision of vocal production is only temporarily disrupted and only y when hearing g loss is the most severe Does the onset and etiology of hearing loss matter? Belgian Waterslager Canaries – a model of prelingual, hereditary deafness i conjunction in j i with i h ongoing hair cell regeneration 9 Canary Domestication •Initial domestication over 500 years ago by Spanish monks •Breeding by miners in Tyrol and the Hartz mountains •Developed first song strain: Hartz Rollers Canary Song Strains • Dozens of canary strains bred for color, plumage style, conformation • Several strains bred specifically for song: German (Hartz) Rollers, Belgian Waterslagers, Spanish Timbrados, Russian Singers, American Singers, Borders. Absolute Threshold in dB SPL (re 20Pa) • Earliest known reference to Belgian Waterslager is 1713, near Malinois Belgium, possible strain bottleneck around turn of the century 90 Behavioral Audiograms 80 Belgian Waterslager 70 Canary 60 50 40 30 20 10 0.1 Non-Belgian Waterslager Canary 1 10 Frequency (kHz) 10 What is the Inheritance Pattern? Absolute Threshold in dB SPL (re 20Pa) Auditory Sensitivity in Hybrid Canaries 90 Belgian Waterslager Canary 80 70 Symbols = F1 Hybrids 60 50 40 30 20 10 Non-Belgian Waterslager Canary 0.1 1 10 Frequency (kHz) Okanoya, et al, 1987 Canary ABR Thresholds Threshold in dB SPL(re 20 uPa) 110 100 90 Waterslagers 80 70 60 Non-Waterslagers 50 Only Waterslager Z's (n=6) 40 At least 1 Border Z (n=11) 30 20 500 1000 1500 2000 2860 4000 5700 8000 Frequency (Hz) Does the onset and etiology of hearing loss matter? BWS have genetic inner ear abnormality (Wright et al 2004) -non-syndromic, non syndromic recessive, rapidly progressive, sex-linked 1 V) 8 4 2 0 -4 Amplitude ( -8 0 2 4 6 8 10 Time (ms) stimulus reaches bird's ear (.88 ms delay) Brittan-Powell, Dooling and Ryals 2008 11 Can BWS canaries regenerate hair cells? 27 hours after BrdU injection 136 hours after BrdU injection Gleich, et al, 1997 After 10 days of tritiated thymidine injections Yes, regeneration rate in BWS canaries ~ 6 cells/day Ryals, et al 1999 Not only are new hair cells being “born” every day, but other hair cells are dying every day. Wilkins et al 2001 Does the onset and etiology of hearing loss matter? So hair cell abnormalities increase and cells begin dying soon after hatching (~20 days). ABR thresholds in BWS never achieve normal sensitivity 12 Does this constant turn-over of hair cells affect the auditory nerve? The cochlear nucleus? •Number of afferent synapses are reduced from 4-5/HC to ~2/HC in BWS •Efferent synapses are similar in BWS and normal canary ~1-2 per HC There is a corresponding •There average 12% loss of neural fibers – BUT the loss of HC in adults is 30% or more •Cell size but not number is reduced in BWS at first order nuclei Kubke et al 2002; Gleich Dooling and Ryals 2001; Ryals and Dooling 2008 •Could we “cure” hearing loss in BWS by stimulating the remaining precurser cells (supporting cells) to divide and repopulate the epithelium? 13 Threshold sh hift (dB) Recovery from Kanamycin (200mg/kg/day) 50 1000 Hz 40 BWS canaries (n=4) Normal canaries (n=4) Pre-injection threshold 2000 Hz 2860 Hz 4000 Hz 50 40 30 30 20 20 10 10 0 0 -10 -10 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12 Weeks following injections What have we learned from birds? •Verifying the influence of regenerated hair cells on hearing •We We must measure more than the pure tone audiogram if we want to know what regeneration means to hearing •We see two time courses of recovery – Perception of complex vocal calls returns much more slowly than simple auditory sensitivity. What is the role of experience/training? What have we learned from birds? – Onset and Etiology of Hearing Loss – Genetic bases of Hearing Loss •Small songbirds such as budgerigars and canaries are essentially “deaf” in the first two weeks post-hatch. This offers a “window” for studies of the influence of regeneration on early onset, “prelingual” deafness. •Because these are songbirds, we may also be able to gain some insight into the influence of regeneration on early vocal learning 14 What have we learned from birds? •Issues of Candidacy – Onset and Etiology of Hearing Loss – Genetic bases of Hearing Loss •At least for the Belgian Waterslager Canary, genetic inner ear abnormalities at the level of the hair cell do not preclude the hair cell regeneration •Stimulation of increased hair cell regeneration, in this case of genetic abnormality, results in improved – but not normal- auditory sensitivity With appreciation to my Co-Author Dr. Robert Dooling and to students and collaborators: Auditory Research Lab James Madison University Edwina Westbrook; Tracy Tucker; Vince Hottinger; Ericka Mills; Kristin Wehman; Rhonda Snorrason; WC Sheets; Kelly Moran, Tracy Tucker, Deb Leap, Katie Roper, Heather Bennett Laboratory of Comparative Psychoacoustics University of Maryland Beth Brittan-Powell; Micheal Dent; Otto Gleich;Tracy Kidd; George Klump; Melonie Mavilia; Kazuo Okanoya; Thomas Park;Joelle Presson; Richard Sleboda; Heather Wilkins; Elena Sanovich, Timothy Wright Work supported by NIDCD grants DC01245 DC1372, DC00198 and DC00353 and The Comparative and Evolutionary Biology of Hearing Training Grant (DC00046) 15