A psychophysical approach to measuring the threshold for acoustic stimulation in zebrafish (Danio rerio).

Zebrafish are a popular vertebrate animal model for biomedical research including investigations of the auditory system. Responses to acoustic stimulation have been a challenge to carefully measure in zebrafish. Here, the authors have developed a procedure for measuring hearing sensitivity in adult zebrafish using an appetitive automated Go/No Go task. In this task, a trial is initiated when a fish passes through an observing gate. In a sound trial, the fish is reinforced by an automated food delivery system when it enters the reinforcement compartment. If the fish enters the reinforcement compartment during a no-sound trial, a timeout is implemented. Zebrafish successfully learned this task in a median of about ten days of daily training. Zebrafish were most sensitive at a frequency of 800 Hz, which corresponds well with sensitivity reported from physiological methods. As far as the authors know, the present study is the first to provide hearing thresholds for zebrafish using a conventional combination of operant conditioning and psychophysical procedures. This could open the door to other kinds of tests using acoustic stimuli as are commonly conducted in many other laboratory animals.

Differential reinforcement of an approach response in zebrafish (Danio rerio).

Five zebrafish were trained to approach a target using a fully automated training procedure. During a training session, if the distance between the fish and the target was closer than an arbitrarily set distance, the approach response was reinforced by food. The fish continued to respond under this reinforcement contingency and the distance criterion could be shortened up to eighty times within a 1h session. The initial distance limit was then shortened for the next test training session. Once the initial distance criterion was reduced to a final minimum distance, the distance criterion was fixed at this value for the next nine successive sessions. In a second experiment using different fish, we manipulated approach distances in three conditions. The first condition was identical to the changing criterion training as in Experiment 1. In the second condition, only response distances under a distance criterion were reinforced. And in the last condition, only response distances over the distance criterion were reinforced. Results show that zebrafish can control the distance between themselves and a target. In other words, zebrafish are sensitive to the spatial consequences of their behavior. The present results show that a differential reinforcement paradigm can be successfully applied to zebrafish which therefore enhances their value as a vertebrate model for studies of complex behavior including visuomotor learning.

An automated device for appetitive conditioning in zebrafish (Danio rerio).

An automated device and a procedure for the operant conditioning individual zebrafish were developed. The key feature of this procedure was the construction of a simple, inexpensive feeder that can deliver extremely small amounts of food, thus preventing rapid satiation. This allows the experimenter to run multiple trails in a single test session and multiple sessions in one day. In addition, small response keys made from acryl rods and fiber sensors were developed that were sufficiently sensitive to detect fish contact. To illustrate the efficiency and utility of the device for traditional learning paradigms, we trained zebrafish in a fixed ratio schedule where subjects were reinforced with food after 10 responses. Zebrafish reliably responded on the response key for sessions that lasted as long 80-reinforcements. They also showed the traditional "break and run" response pattern that has been found in many species. These results show that this system will be valuable for behavioral studies with zebrafish, especially for experiments that need many repeated trials using food reinforcer in a session. The present system can be used for sensory and learning investigations, as well applications in behavioral pharmacology, behavioral genetics, and toxicology where the zebrafish is becoming the vertebrate model of choice.

Amphibious auditory responses of the American alligator (Alligator mississipiensis).

Animals that thrive both on land and underwater are faced with the task of interpreting stimuli in different media. This becomes a challenge to the sensory receptors in that stimuli (e.g., sound, motion) may convey the same type of information but are transmitted with different physical characteristics. We used auditory brainstem responses to examine hearing abilities of a species that makes full use of these two environments, the American alligator (Alligator mississipiensis). In water, alligators responded to tones from 100 Hz to 2,000 Hz, with peak sensitivity at 800 Hz. In air, they responded to tones from 100 Hz to 8,000 Hz, with peak sensitivity around 1,000 Hz. We also examined the contribution to hearing of an air bubble that becomes trapped in the middle ear as the animal submerges. This bubble has been previously implicated in underwater hearing. Our studies show that the trapped air bubble has no affect on auditory thresholds, suggesting the bubble is not an important adaptation for underwater hearing in this species.

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.

Masking by harmonic complexes in birds: behavioral thresholds and cochlear responses.

Thresholds for pure tones embedded in harmonic complexes were measured behaviorally and physiologically for three species of birds, and physiologically in gerbils. The harmonic maskers were generated using the Schroeder-phase algorithm, characterized by monotonically increasing or decreasing phase across frequency. Previous work has shown that these stimuli produce large differences in masking in humans but not budgerigars. In this study, we show that for two additional species of birds, the patterns of masking were similar to those shown for budgerigars, with masking differing only slightly for the two Schroeder-phase waveforms, and in the opposite direction from that demonstrated in humans. Amounts of masking among species corresponded qualitatively to differences in their critical ratios. Evoked potential measurements in birds and gerbils indicated responses that were consistent with the behaviorally measured thresholds in birds and humans. Results are interpreted in light of differences in frequency selectivity and cochlear temporal processing across species.

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 microm) did not differ from those of BWS canaries (2.21+/-0.96 microm).

Frequency discrimination in budgerigars (Melopsittacus undulatus): effects of tone duration and tonal context.

Studies of frequency resolving power in budgerigars (Melopsittacus undulatus) have shown that this species has excellent discrimination abilities for both simple and complex sounds falling in the region of 2 to 4 kHz--the frequency range of their contact call. In four experiments, frequency discrimination by budgerigars of short tones similar to elements found in the contact call was examined. Frequency difference limens (FDLs) for simple pure tones at 2.86 kHz were constant for tone durations above 20 ms but higher for shorter tones. Budgerigars generally showed larger FDLs for shorter duration 1-, 2-, and 4-kHz pure tones. FDLs in budgerigars for 20-ms tones embedded in a sequence of six other tones were similar to FDLs measured for tones of the same frequency presented in isolation. Moreover, there was no effect of introducing trial-by-trial variation in the location of the frequency change in the seven-tone complexes for budgerigars, a condition for which humans showed a large decrement in performance. Taken together, these results suggest budgerigars possess enhanced spectral resolving power for short duration pure tones when they are embedded in contact call-like tonal patterns.

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.

Masking by harmonic complexes in budgerigars (Melopsittacus undulatus).

In humans, masking by harmonic complexes is dependent not only on the frequency content of the masker, but also its phase spectrum. Complexes that have highly modulated temporal waveforms due to the selection of their component phases usually provide less masking than those with flatter temporal envelopes. Moreover, harmonic complexes that are created with negative Schroeder phases (component phases monotonically decreasing with increasing harmonic frequency) may provide more masking than those created with positive Schroeder phases (monotonically increasing phase), even though both temporal envelopes are equally flat. To date, there has been little comparative work on the masking effectiveness of harmonic complexes. Using operant conditioning and the method of constant stimuli, masking of pure tones by harmonic complexes was examined in budgerigars at several different masker levels for complexes constructed with two different fundamental frequencies. In contrast to humans, thresholds in budgerigars differed very little for the two Schroeder-phase waveforms. Moreover, when there was a difference in masking by these two waveforms, the positive Schroeder was the more effective masker--the reverse of that described for humans. Control experiments showed that phase selection was relevant to the masking ability of harmonic complexes in budgerigars. Release from masking occurred when the components were in coherent phase, compared with a complex with random phases selected for each component. It is suggested that these psychoacoustic differences may emerge from structural and functional differences between the avian and mammalian peripheral auditory systems involving traveling wave mechanics and spectral tuning characteristics.

Avian species differences in susceptibility to noise exposure.

Previous studies of hair cell regeneration and hearing recovery in birds after acoustic overstimulation have involved relatively few species. Studies of the effects of acoustic overexposure typically report high variability. Though it is impossible to tell, the data so far also suggest there may be considerable species differences in the degree of damage and the time course and extent of recovery. To examine this issue, we exposed four species of birds (quail, budgerigars, canaries, and zebra finches) to identical conditions of acoustic overstimulation and systematically analyzed changes in hearing sensitivity, basilar papilla morphology, and hair cell number. Quail and budgerigars showed the greatest susceptibility to threshold shift and hair cell loss after overstimulation with either pure tone or bandpass noise, while identical types of overstimulation in canaries and zebra finches resulted in much less of a threshold shift and a smaller, more diffuse hair cell loss. All four species showed some recovery of threshold sensitivity and hair cell number over time. Canary and zebra finch hearing and hair cell number recovered to within normal limits while quail and budgerigars continued to have an approximately 20 dB threshold shift and incomplete recovery of hair cell number. In a final experiment, birds were exposed to identical wide-band noise overstimulation under conditions of artificial middle ear ventilation. Hair cell loss was substantially increased in both budgerigars and canaries suggesting that middle ear air pressure regulation and correlated changes in middle ear transfer function are one factor influencing susceptibility to acoustic overstimulation in small birds.

Effects of deafening on the calls and warble song of adult budgerigars (Melopsittacus undulatus).

Budgerigars are small Australian parrots that learn new vocalizations throughout adulthood. Earlier work has shown that an external acoustic model and auditory feedback are necessary for the development of normal contact calls in this species. Here, the role of auditory feedback in the maintenance of species-typical contact calls and warble song in adult budgerigars is documented. Deafened adult birds (five male, one female) vocalized less frequently and showed both suprasegmental and segmental changes in their contact calls and warble song. Contact calls of all adult-deafened budgerigars showed abnormalities in acoustic structure within days to a few weeks following surgery. Within 6 months of surgery, nearly all contact calls produced by deafened birds were strikingly abnormal, showing highly variable patterns of frequency modulation and duration. The warble song of deafened male budgerigars also differed significantly from that of normal budgerigars on several acoustic measures. These results show that auditory feedback is necessary for the maintenance of a normal, species-typical vocal repertoire in budgerigars.

Detection of modulation in spectral envelopes and linear-rippled noises by budgerigars (Melopsittacus undulatus).

Budgerigars were trained to discriminate complex sounds with two different types of spectral profiles from flat-spectrum, wideband noise. In one case, complex sounds with a sinusoidal ripple in (log) amplitude across (log) frequency bandwidth were generated by combining 201 logarithmically spaced tones covering the frequency region from 500 Hz to 10 kHz. A second type of rippled stimulus was generated by delaying broadband noise and adding it to the original noise in an iterative fashion. In each case, thresholds for modulation depth (i.e., peak-to-valley in dB) were measured at several different ripple frequencies (i.e., cycles/octave for logarithmic profiles) or different repetition pitches (i.e., delay for ripple noises). Budgerigars were similar to humans in detecting ripple at low spatial frequencies, but were considerably more sensitive than humans in detecting ripples in log ripple spectra at high spatial frequencies. Budgerigars were also similar to humans in detecting linear ripple in broadband noise over a wide range of repetition pitches. Taken together, these data show that the avian auditory system is at least as good, if not better, than the human auditory system at detecting spectral ripples in noise despite gross anatomical differences in both the peripheral and central auditory nervous systems.

Detection of changes in timbre and harmonicity in complex sounds by zebra finches (Taeniopygia guttata) and budgerigars (Melopsittacus undulatus).

Thresholds for detecting alterations in the timbre and harmonicity of complex harmonic signals were measured in zebra finches, budgerigars, and humans. The stimuli used in this experiment were designed to have particular salience for zebra finches by modeling them after natural zebra finch calls. All 3 species showed similar abilities for detecting an amplitude decrement in a single component of a harmonic complex. However, zebra finches and budgerigars were extraordinarily sensitive to the mistunings of single harmonics and exhibited significantly lower thresholds compared with humans at 2 different fundamental frequencies, 570 Hz and 285 Hz. Randomizing relative phases of components in a harmonic stimulus resulted in a significant increase in threshold for detecting mistunings in zebra finches but not in humans. Decreasing the duration of mistuned harmonic stimuli resulted in higher thresholds for both birds and humans. The overall superiority of birds in discriminating inharmonicity suggests that birds and mammals may use different strategies in processing these complex harmonic sounds.

Hearing and vocalizations of wild-caught Australian budgerigars (Melopsittacus undulatus).

This study examined the hearing and contact calls of wild-caught Australian budgerigars (Melopsittacus undulatus) and compared these data to hearing and vocalizations in the much more extensively studied domesticated budgerigar. The spectral energy in the contact calls of both wild-caught and domesticated budgerigars falls almost exclusively in the frequency of 2-4 kHz. Absolute and masked thresholds were similar in both groups of birds. Similar to the results found in domesticated birds, critical ratio functions for the wild-caught budgerigars decreased at frequencies of 1.0 kHz-2.86 kHz and then increased again dramatically at frequencies above 2.86 kHz.

Control of vocal intensity in budgerigars (Melopsittacus undulatus): differential reinforcement of vocal intensity and the Lombard effect.

Call production in budgerigars was studied using operant conditioning. In several experiments, budgerigars were reinforced with food for producing calls that were above or below a criterion level of intensity. This differential reinforcement procedure was successful in controlling vocal intensity in both directions showing that the intensity with which budgerigars produce vocalizations is under voluntary control. In additional experiments, call intensity maintained by food reinforcement was measured both in the quiet and in the presence of various levels of broadband noise. Call intensity in budgerigars increased significantly in noise, paralleling the well-known Lombard effect in humans which is the reflexive increase in speech intensity during communication in noise. Call intensity was measured in broadband noise and in a notched noise (no energy between 1.5 and 4.5 kHz) with the same overall level. Results show that noise in the spectral region of contact calls is most effective in causing an increase in vocal intensity. In aggregate, these experiments show that budgerigars have voluntary control over the intensive aspect of their vocalizations, that they normally monitor their vocal output though external auditory feedback, and, like humans, they exhibit the Lombard effect.

Vocal development in budgerigars (Melopsittacus undulatus): contact calls.

Budgerigars have a complex vocal repertoire, some of which develops through learning. The authors examined the course of vocal development in budgerigars from hatching to about 4 weeks postfledging (approximately 85 days old). Food-begging calls showed changes in duration, peak frequency, bandwidth, and frequency modulation with age. Within a week of fledging, each bird produced a contact call bearing a strong resemblance to a shortened version of its patterned food-begging call. By 4 weeks postfledging, budgerigar contact call repertoires often contained more than one call type, and there was clear evidence of sharing and imitation among the calls of parents, fledglings, and other social companions. Perceptual testing showed that whereas acoustic variation in the structure of developing calls decreased both within and between nestling birds, the discrimination of these calls was easier for adult birds as young birds matured. These results suggest parallels with certain aspects of language development in humans.

Perception of synthetic /ba/-/wa/ speech continuum by budgerigars (Melopsittacus undulatus).

Other than humans, extensive vocal learning has only been widely demonstrated in birds. Moreover, there are only a handful of avian species that are known to be good mimics of human speech. One such species is the budgerigar (Melopsittacus undulatus), which is a popular mimic of human speech and learns new vocalizations throughout adult life. Using operant conditioning procedures with a repeating background task, we tested budgerigars on the discrimination of tokens from two synthetic /ba/-/wa/ speech continua that differed in syllable, but not transition, duration. Budgerigars showed a significant improvement in discrimination performance on both continua near the phonetic boundary for humans. Budgerigars also showed a shift in the location of the phonetic boundary with a change in syllable length, similar to what has been described for humans and other primates. These results on a nonmammalian species provide support for the operation of a general, nonphonetic, auditory process as one mechanism which can lead to the well-known stimulus-length effect in humans.

Mechanisms of vocal production in budgerigars (Melopsittacus undulatus).

Songbirds vocalizing in helium show a change in the spectral quality of their vocalizations. This effect is due to an increase in the speed of sound in helium that in turn alters the resonance properties of the vocal tract. Here, this approach is extended to a psittacine, the budgerigar (Melopsittacus undulatus), whose syringeal anatomy and innervation differ from that of a songbird. Contact calls from birds vocalizing in heliox (70/30 helium/oxygen environment) showed an overall increase in the amount of energy at frequencies above the fundamental, slight changes in the frequency of the fundamental and harmonics, and some change in the level of harmonics. Calls produced by a syringeally denervated bird showed more dramatic changes. Recordings from live birds were compared with sounds produced by various simple "artificial" tracheal and syringeal models. Results suggest that budgerigars produce contact calls using the syringeal membranes as a unitary sound source which produces acoustic energy in a narrow frequency band whose fundamental frequency is matched to the resonant frequency of the trachea. The syrinx is not normally coupled to the tracheal resonator, and resonances probably play only a minor role in shaping the spectrum of contact calls.