Belgian Waterslager Research Articles

Strain differences in song and hearing in canaries (Sernius canarius)

Breeders have bred canaries either for specific song characteristics (song canaries) or morphology/plumage (type canaries) for centuries. Type canaries (e.g., Border and Gloster strains) retain song characteristics that are typically quite similar to those of wild canaries. By contrast, song canaries (e.g., Belgian Waterslager and Roller strains) have been selected for song types pleasing to the human ear, resulting in songs that, in most cases, are less complex, lower pitched, and narrower in a frequency range than songs from wild canaries. We now suspect that song selection in the Belgian Waterslager song canary has either directly or indirectly resulted in high-frequency hearing loss associated with hair cell abnormalities. Here, we compare hearing in the Belgian Waterslager and several other type and song canaries including the American Singer Canary. Though bred only since the 1930s, American Singer canaries also have a high-frequency hearing loss that looks very similar to that of the Belgian Waterslager and may have similar pathologies. Illumina whole genome sequencing has preliminarily identified a number of high-impact SnpEff variants in Belgian Waterslager and American Singer Canaries, some of which are related to deafness genes in mammals.

Strain differences in hearing in song canaries.

Belgian Waterslager song canaries, bred for hundreds of years for a low-pitched song, have also acquired an inherited high-frequency hearing loss associated with hair cell abnormalities. Here, auditory thresholds measured using auditory brainstem responses and psychophysical methods in three different strains of canaries are compared: Belgian Waterslagers, American Singers, and Borders. Border canaries have not been bred for song characteristics while American Singer canaries have been bred for song only since the 1930s. Results show that American Singer canaries also have elevated high frequency thresholds that are similar to those of the Belgian Waterslager, while Border canaries have normal thresholds. These results strengthen the case that song canary breeders in selecting for song characteristics may have inadvertently selected for hearing abnormalities.

Perceptual perseverance in a passerine with permanent papillar impairment

The Belgian Waterslager canary is unique for its loud, low-pitched song which is accompanied by a hereditary pathology involving missing and damaged hair cells in the basal end of the papilla. These birds were been bred for hundreds of years for loud, low-pitched song. Breeders likely selected for high frequency hearing loss. In spite hair cell regeneration, the papillae in these birds never approaches that of normal-hearing canaries. Auditory nerve and brainstem responses are also diminished, and auditory brainstem nuclei show reduced cell size. And these birds show a suite of psychoacoustic deficits consistent with impaired active processing as seen in humans with hearing loss. It is rather remarkable, then, that Belgian Waterslagers are able to learn, discriminate, and produce complex, species-specific sounds with such impaired frequency selectivity and phase processing. This feat, in the presence of severe peripheral auditory damage, underscores the importance of temporal information in the avian auditory perception and vocal learning. The obvious genetic basis of this pathology places the Belgian Waterslager canary in another unique position of being the only nonhuman organism which must navigate through vocal development and vocal learning in the face of an inherited developmental peripheral auditory pathology.

Electrophysiological and morphological development of the inner ear in Belgian Waterslager canaries

Belgian Waterslager (BW) canaries have an inherited hearing loss due to missing and abnormal hair cells, but it is unclear whether the loss is congenital or developmental. We used auditory brainstem responses and scanning electron microscopy to describe the development of auditory sensitivity and hair cell abnormalities in BW and non-BW canaries. In both strains, adult ABR thresholds were higher than behavioral thresholds, but BW canaries exhibited higher thresholds than non-BW canaries across all frequencies. Immediately post-hatch, ABR thresholds and hair cell numbers were similar in both strains. Two weeks later, thresholds were significantly higher in BW canaries, and hair cell number progressively decreased as the birds aged. These data show that in BW canaries: the peripheral auditory system is functionally similar to non-BW canary from hatch to 2 weeks, ABR thresholds improve during this developmental period, actually becoming better than those of adults, but then worsen as the bird continues to age. Hair cell number and appearance is similar to non-BW canaries at hatch but progressively declines after 30 days of age. These data show that the hearing loss characteristic of BW canaries is, at least in part, developmental and is established by the time song learning begins.

Psychophysical evidence of damaged active processing mechanisms in Belgian Waterslager Canaries.

Belgian Waterslager canaries (BWC), bred for a distinct low-pitched song, have an inherited high-frequency hearing loss associated with hair cell abnormalities. Hair cells near the abneural edge of the papilla, which receive primarily efferent innervation in normal birds, are among the most severely affected. These cells are thought to support nonlinear active processing in the avian ear, though the mechanisms are poorly understood. Here we present psychophysical evidence that suggests degraded active processing in BWC compared to normal-hearing non-BWC. Critical ratios, psychophysical masking patterns and phase effects on masking by harmonic complexes were measured in BWC and non-BWC using operant conditioning procedures. Critical ratios were much larger in BWC than in non-BWC at high frequencies. Psychophysical tuning curves derived from the masking patterns for BWC were broadened at high frequencies. BWC also showed severely reduced phase effects on masking by harmonic complexes compared to non-BWC. As has been hypothesized previously for hearing-impaired humans, these results are consistent with a loss of active processing mechanisms in BWC.

WDR1 presence in the songbird basilar papilla

WD40 repeat 1 protein (WDR1) was first reported in the acoustically injured chicken inner ear, and bioinformatics revealed that WDR1 has numerous WD40 repeats, important for protein–protein interactions. It has significant homology to actin interacting protein 1 (Aip1) in several lower species such as yeast, roundworm, fruitfly and frog. Several studies have shown that Aip1 binds cofilin/actin depolymerizing factor, and that these interactions are pivotal for actin disassembly via actin filament severing and actin monomer capping. However, the role of WDR1 in auditory function has yet to be determined. WDR1 is typically restricted to hair cells of the normal avian basilar papilla, but is redistributed towards supporting cells after acoustic overstimulation, suggesting that WDR1 may be involved in inner ear response to noise stress. One aim of the present study was to resolve the question as to whether stress factors, other than intense sound, could induce changes in WDR1 presence in the affected avian inner ear. Several techniques were used to assess WDR1 presence in the inner ears of songbird strains, including Belgian Waterslager (BW) canary, an avian strain with degenerative hearing loss thought to have a genetic basis. Reverse transcription, followed by polymerase chain reactions with WDR1-specific primers, confirmed WDR1 presence in the basilar papillae of adult BW, non-BW canaries, and zebra finches. Confocal microscopy examinations, following immunocytochemistry with anti-WDR1 antibody, localized WDR1 to the hair cell cytoplasm along the avian sensory epithelium. In addition, little, if any, staining by anti-WDR1 antibody was observed among supporting cells in the chicken or songbird ear. The present observations confirm and extend the early findings of WDR1 localization in hair cells, but not in supporting cells, in the normal avian basilar papilla. However, unlike supporting cells in the acoustically damaged chicken basilar papilla, the inner ear of the BW canary showed little, if any, WDR1 up-regulation in supporting cells. This may be due to the fact that the BW canary already has established hearing loss and/or to the possibility that the mechanism(s) involved in BW hearing loss may not be related to WDR1.

Detection and discrimination of simple and complex sounds by hearing-impaired Belgian Waterslager canaries

Belgian Waterslager canaries (BWC) are bred to produce a distinctive low-pitched song with energy restricted to a lower range of frequencies than in other types of canaries. Previous studies have shown a high frequency hearing loss primarily above 2000 Hz that is related to hair cell abnormalities in BWC, but little is known about auditory perception in these birds. Here, frequency, duration, and intensity discrimination, temporal integration, gap detection, and discrimination of temporally reversed harmonic complexes in BWC were measured and compared to normal-hearing non-BWC. BWC had excellent frequency discrimination ability at 1000 Hz, but showed poor frequency discrimination compared to non-BWC at frequencies in the region of hearing loss. Duration and intensity discrimination were not adversely affected in BWC. Temporal integration was reduced in BWC, except at 2000 Hz. Gap detection and discrimination of temporally reversed stimuli were somewhat better in BWC than in non-BWC. Those tests that relied primarily on temporal processing were less affected by the cochlear damage in BWC than tests that probably relied more on audibility and spectral analysis. Thus, despite significant high frequency hearing loss and extensive damage along the basilar papilla, BWC retain relatively good hearing abilities under many conditions.

Impaired frequency resolution in canaries with hereditary high-frequency hearing loss

Belgian Waterslager canaries (BWS), bred for a distinct low-pitched song, have an inherited high-frequency hearing loss related to hair cell abnormalities. Many hair cells are missing or damaged, and many of the remaining hair cells have abnormal stereocilia bundles. To investigate how the hair cell abnormalities affect frequency tuning along the BWS basilar papilla, we measured psychophysical tuning curves (PTCs) in BWS and normal-hearing non-BWS canaries using operant conditioning procedures. Non-BWS canaries showed relatively symmetrical tuning curves that became more narrowly tuned with increasing center frequency. Despite near-normal hearing at low frequencies, PTCs in BWS were elevated for all center frequencies tested. The sharpness of tuning was normal in BWS for PTCs with a center frequency of 1000 Hz and broader than normal at higher frequencies. The shapes of the PTCs were abnormal in BWS, particularly at higher frequencies. The abnormal tuning properties in the BWS are consistent with changes in the impedance characteristics of the basilar papilla due to structural damage. [Work supported by NIH DC01372 and DC04664.]

Strain differences in auditory sensitivity in canaries bred for song and plumage

Canaries have been domesticated for well over 100 years. They have been bred for specific characteristics such as song, plumage, or body shape. Here we measured audiograms in several canary strains using both behavioral (operant conditioning) and physiological (auditory brainstem responses) methods. Overall, there was a good correspondence between behavioral and ABR audiograms, but there were differences in audiogram shape and sensitivity between strains. Belgian Waterslager canaries bred for low-pitched song had elevated high frequency thresholds, while Spanish Timbrado canaries bred for high-pitched song had slightly better high frequency thresholds than canaries that were not bred for song. Other strains showed intermediate sensitivity. These results suggest there can be more variability in hearing within a species than previously thought and that these differences are correlated with other behavioral or morphological traits. [Work supported by DC00198, DC001372, DC04664-01A2, and DC005450.]

Sex–linked inheritance of hearing and song in the Belgian Waterslager canary

Belgian Waterslager canaries have less sensitive hearing at high frequencies and produce songs with more energy at low frequencies than wild-type canaries. A backcross pedigree between Belgian Waterslager canaries and a domestic strain with wild-type song revealed inheritance patterns consistent with a factor of major effect located on the Z sex chromosome affecting both poor high-frequency hearing at 4 kHz and the relative energy in the spectra of the learned songs of males. Hearing thresholds at 4 kHz were significant predictors of the relative amount of song energy at 4 kHz for individual males. One hypothesis for the mechanistic basis of this correlation between hearing and song abnormalities is that a reduction in the ability to hear higher-frequency songs biases males towards learning lower-frequency songs.

Reduced temporal integration in Belgian Waterslager canaries

Belgian Waterslager canaries (BWS) are bred for their low-frequency song and have been shown to have hair cell abnormalities in the inner ear that result in elevated thresholds at higher frequencies. Previous results show that resolution of temporal fine structure, or the ability to resolve rapidly occurring changes in complex sounds, is enhanced in BWS canaries. In a continuing effort to assess the effects of the BWS inner ear pathology on hearing, we here investigate the ability to integrate acoustic information over longer periods of time. Absolute thresholds for 1.0-, 2.0-, and 4.0-kHz pure tones were measured in BWS and normal-hearing nonBWS canary strains for durations ranging from 5 to 480 ms using operant conditioning methods. NonBWS canaries showed a decrease in threshold of approximately 10–15 dB with increasing tone duration for all frequencies. In contrast, BWS showed almost no change in threshold across the range of durations tested for all frequencies. The reduced temporal integration in BWS canaries with hair cell abnormalities parallels similar findings in humans with cochlear damage. [Work supported by NIH DC01372 to RJD and DC05450 to AML.]

Frequency selectivity in canaries with a hereditary hearing loss

Sensorineural hearing loss is associated with reduced frequency selectivity due to the broadening of the auditory filters in mammals. In European starlings, hearing loss caused by the ototoxic drug kanamycin results in the widening of auditory filters at 5 kHz [Marean et al. (1998)]. In the present study, we examine frequency selectivity in a bird with a permanent hereditary hearing impairment, the Belgian Waterslager (BWS) canary. This strain of canary has long been bred for its loud, low-frequency song, and has been shown to have a hearing loss primarily at higher frequencies (2 kHz and above). Using operant conditioning and the method of constant stimuli, thresholds for detecting pure tones in flat-spectrum broadband noise were measured in BWS and non-BWS canaries. Critical ratios were calculated for comparison with other species of birds. At higher frequencies, critical ratios for BWS canaries were much larger than those of non-BWS canaries and other birds, suggesting reduced frequency selectivity in the region of the birds’ hearing loss. [Work supported by NIDCD R01DC001372 to RJD and Brenda M. Ryals.]

Nucleus magnocellularis and nucleus laminaris in Belgian Waterslager and normal strain canaries

Belgian Waterslager (BWS) canaries are characterized by a mean 30% loss of hair cells in the basilar papilla compared to other canaries, and a corresponding increase in behavioral auditory thresholds. In spite of the large number of missing and damaged sensory cells, there is on average only a 12% reduction in the number of fibers in the VIIIth nerve. In this study, we examined cell number and size, and volume of auditory nuclei, specifically in nucleus magnocellularis and nucleus laminaris in Belgian Waterslager canaries. While the overall anatomical structure and organization of these nuclei and the total number of cells in the non-BWS and BWS canaries were comparable, BWS canaries showed a significant decrease in the volume of the auditory nuclei that was attributed to a reduction in cell size. These results provide further evidence in favor of a role of the sensory epithelium in the maintenance of central auditory structures.

Hair Cell Death in a Hearing-Deficient Canary

Cell death has been documented in bird auditory inner ear epithelia after induced damage. This cell death is quickly followed by an increase in supporting cell division and regeneration of the epithelium, thereby suggesting a possible relationship between these two processes. However, aspects of this relationship still need to be better understood. The Belgian Waterslager (BWS) canary is an ideal system in which to study cell death and subsequent cell division. In contrast to mixed breed (MB) canaries, cell division normally occurs in the auditory end organ of the BWS without any external manipulation. In addition, some of the cells in the auditory epithelium may be dying through an apoptotic-like process. In the present study two methods were used to quantify dying cells in the BWS and MB canary auditory epithelia: morphological criteria and TUNEL. Results confirm that some of the abnormal hair cells in the BWS auditory epithelium are apoptotic-like. The presence of both cell death and cell division indicates that these processes act concurrently in the adult end organ. Future studies are needed to determine if cell death is a stimulus for the observed cell division.

A quantitative analysis of the nerve fibers in the VIIIth nerve of Belgian Waterslager canaries with a hereditary sensorineural hearing loss

The number of auditory nerve fibers was determined for non-Belgian Waterslager canaries (non-BWS) and Belgian Waterslager canaries (BWS) that are affected by a sensorineural high frequency hearing loss and a 30% reduction in the number of auditory hair cells. Counts were obtained from semithin cross sections of the Durcupan-embedded auditory nerve at the level of the internal auditory meatus. In addition, the number of lagenar fibers was determined from cross sections near the apical end of the cochlear duct in order to separate them from the total number of auditory nerve fibers. The mean number of auditory nerve fibers was 6076 in non-BWS and 5363 in BWS canaries, representing a 12% reduction in BWS. This small reduction in the number of auditory nerve fibers, as compared to the larger reduction in hair cell number, might be explained by a predominant loss of abneural hair cells in BWS, since it has been shown for other species that a large proportion of abneural hair cells are devoid of afferent innervation. In addition, we observed that despite the prominent hair cell pathologies documented for BWS canaries, the mean diameter of auditory nerve fibers from non-BWS canaries (2.22±0.81 μm) did not differ from those of BWS canaries (2.21±0.96 μm).

Neither endocochlear potential nor Tegmentum vasculosum are affected in hearing impaired Belgian Waterslager canaries

We previously showed that the Belgian Waterslager canary strain is affected by a hereditary hearing loss that is associated with a reduced number of hair cells and hair cell pathologies in the basilar papilla. Since hair cell pathologies were also present in the sacculus, Weisleder et al. (1994) suggested that these birds are afflicted by Scheibe’s like dysplasia, a cochleo-saccular defect. In mammals, cochleo-saccular defects are characterized primarily by the lack of an endocochlear potential and abnormalities in the Stria vascularis which only secondarily lead to hair cell loss (Steel and Bock, 1983; Steel, 1994; 1995). Here we report the endocochlear potential of six ears from three non-Belgian Waterslager canaries and three ears of two Belgian Waterslager canaries to decide if Waterslager canaries are affected by a cochleo-saccular or by a neuroepithelial defect. The mean endocochlear potential was 17.6±2.5 mV in the non-Waterslager canaries and 20.3±0.6 mV in Waterslager canaries. In addition, and consistent with the presence of a normal endocochlear potential, light microscopy of the tegmentum vasculosum provided no evidence for pathology. These data show that Belgian Waterslager canaries are affected by a neuroepithelial rather than a cochleo-saccular inner ear defect.

Vestibular function in Belgian Waterslager canaries ( Serinus canarius)

The purpose of this study was to measure vestibular function in Belgian Waterslager canaries using short latency vestibular evoked potentials (VsEPs) elicited by linear acceleration stimuli. Responses were recorded with vertex to mastoid leads using traditional signal averaging. Response thresholds, latencies, and amplitudes were quantified and compared to non-Waterslager controls. Cochlear and vestibular organs were also processed for scanning electron microscopy. Results indicated that vestibular response thresholds were slightly, but significantly, higher for Belgian Waterslager canaries and response amplitudes at 0 dBre:1.0 g/ms were significantly reduced compared to non-Waterslagers. Response peak latencies were not significantly different. The most striking morphological finding was that the stereociliary bundles of Waterslager saccular hair cells showed no common orientation. Previous reports have also found significantly less hair cells in Waterslager saccules (Weisleder and Park, Hear. Res. 80 (1994) 64–70); however, the present study did not confirm this finding. The utricle and ampullae appeared normal. The present results indicate that vestibular neural function, as measured with VsEPs, is affected in Belgian Waterslager canaries. The results also suggest that one structural correlate of the functional loss is the disordered stereociliary bundles in the sacculus.

Hair cells of a congenitally hearing impaired canary have abnormal distribution of filamentous proteins

Previous investigations into the anatomy of the inner ear of Belgian Waterslager canaries (BWC) have demonstrated myriad malformations associated with dysgenesis of the pars inferior of the otocyst. These malformations are associated with a congenital hearing impairment primarily affecting mid to high frequencies. Specific hair cell abnormalities include irregular cell boundaries, deformed cuticular plate, and multiple and deformed stereocilia tufts. This constellation of structural deformities lead us to hypothesize that the content of non-muscle filamentous protein in BWC hair cells need be abnormal. As a first step in exploring this hypothesis, we have used immunocytochemical techniques to assess the presence and distribution of the major components of the actin microfilament system, i.e. actin, tropomyosin and α -actinin, at the apical end of hair cells from the BWC basilar papilla. Our results show qualitative differences between control and BWC hair cells. First, label of the BWC cuticular plate was scattered and ill-matched with that of control birds for each of the three proteins examined. Second, labeling of BWC cilia revealed abnormal distribution of the assessed proteins. Specifically, anti-actin and anti- α -actinin antibodies were clustered over the cilia, while immunostaining against tropomyosin was not seen. These findings support a link between the proteins associated with cochlear hair cell structural integrity and the dysmorphologies associated with the congenital hearing impairment identified in the BWC model.

Evidence for supporting cell proliferation and hair cell differentiation in the basilar papilla of adult Belgian Waterslager canaries (Serinus canarius).

We used the bromodeoxyuridine technique to study the proliferative activity in the basilar papilla of normal and Belgian Waterslager canaries with and without preceding sound trauma. Without sound trauma, there were, on average, six supporting cell divisions per day in the basilar papilla of Waterslager canaries. This rate of supporting cell proliferation corresponds well with estimates of the rate of hair cell differentiation derived from counts of immature-appearing hair cells obtained by using scanning electron microscopy of the Waterslager basilar papilla. Thus, supporting cell division appeared correlated with hair cell differentiation in Waterslager canaries. Bromodeoxyuridine labeling of cells in undamaged non-Waterslager canaries also indicated a very low rate of supporting cell division. In contrast with Waterslager canaries, this low rate of proliferation was not associated with a measurable rate of hair cell differentiation. In both normal and Waterslager canaries, exposure to traumatizing sound induced a dramatic increase in the rate of cell proliferation. These data show that a very low rate of supporting cell proliferation is normally present in birds, but it is not associated with a corresponding rate differentiation of hair cells. Only an increase above this low ambient rate of supporting cell proliferation, such as that following loss of hair cells, induces the differentiation of new hair cells in birds. The reason why Waterslager canaries do not completely compensate for their inherited hair cell deficit of 30% is not clear, when they can clearly respond to additional cochlear trauma from noise exposure with an increase in proliferation rate.

Anatomical basis of a congenital hearing impairment: basilar papilla dysplasia in the Belgian Waterslager canary.

Recent investigations into the anatomy of the inner ear of Belgian Waterslager canaries (BWC) have demonstrated myriad malformations associated with dysgenesis of the pars inferior of the otocyst. In those studies, the surface anatomy of BWC's basilar papilla and sacculus was examined utilizing scanning electron microscopy. In the present investigation, we utilized both light microscopy and transmission electron microscopy to describe the cross sectional anatomical pathology of the BWC's basilar papilla. Examination of the BWC's organ of Corti revealed numerous dysmorphologies: 1) hair cells from the tall hair cell region appeared broad and stunted, with deformed cuticular plates, abnormal stereocilia, and recognizable microvilli; 2) quantitative analysis of these hair cells revealed disproportionately large nuclei and abnormally short stereocilia; 3) hair cells from the short hair cell region of the papilla were absent, replaced by a population of large cells with electron-lucent cytoplasm; and 4) the tectorial membrane in the BWC papilla was narrow, covering only the area where the deformed tall hair cells were found. The malformations appeared to be more severe at the apex and midsection of the basilar papilla than at the base. These observations allow us to suggest a hypothesis to account for the distinct anatomofunctional hearing deficit observed in these birds. In addition, they further support our hypothesis that the inner ear of BWC is afflicted by a disorder similar to Scheibe's dysplasia, the most common inner ear defect associated with congenital hearing loss in humans.