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Previous Article | Next Article
The Journal of Neuroscience, April 1, 2001, 21(7):2501-2517
Postlearning Consolidation of Birdsong: Stabilizing Effects of Age and Anterior Forebrain Lesions
Michael S. Brainard and Allison J. Doupe Keck Center for Integrative Neuroscience, Departments of Physiology and Psychiatry, University of California San Francisco, San Francisco, California 94143-0444
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Birdsong is a learned, sequenced motor skill. For the zebra finch, learned song normally remains unchanging beyond early adulthood. However, stable adult song will gradually deteriorate after deafening (Nordeen and Nordeen, 1992
), indicating an ongoing influence of auditory feedback on learned song. This plasticity of adult song in response to deafening gradually declines with age (Lombardino and Nottebohm, 2000
Key words: basal ganglia; song learning; motor learning; memory consolidation; timing; language; speech; auditory feedback; hearing loss; deafness
INTRODUCTION
Birdsong, like human speech, is a complex learned vocal behavior. For many songbird species, song is learned in two stages (Marler, 1970
The susceptibility of adult song to deterioration after deafening decreases with age (Lombardino and Nottebohm, 2000
The songbird anterior forebrain pathway (AFP) (Fig. 1, filled) may play an important role in regulating song plasticity. A variety of evidence suggests that the AFP is homologous to mammalian basal ganglia cortical circuits (Bottjer and Johnson, 1997
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Figure 1. Schematic sagittal view of song system nuclei. The "motor pathway" (shaded) is required throughout life for song production and includes nucleus HVc, the robust nucleus of the archistriatum (RA), and brainstem nuclei that control vocal and respiratory musculature, including the tracheosyringeal subdivision of the hypoglossal nucleus (nXIIts). The "anterior forebrain pathway" (solid) forms a basal ganglia-forebrain loop that leaves the motor pathway at the level of HVc and passes successively through a nucleus of the basal ganglia (Area X), the thalamus [medial subdivision of the dorsolateral thalamus (DLM)], and the anterior forebrain (LMAN) before returning to the motor pathway at the level of RA. Field L provides auditory input to the song system via indirect connections to nucleus HVc.
These observations raise the question of how similar the "artificial" stabilization of song by AFP lesions is to the natural stabilization of song that occurs with aging, and whether these two phenomena are related. Here we investigate this issue by characterizing the deterioration of song that occurs after deafening in young adult birds and the degree to which that deterioration is reduced in birds that either received lesions of the AFP before deafening or that were deafened at greater ages. For most aspects of deafening-induced changes to song, lesions of the AFP provided a greater degree of song stabilization than did aging. However, an acceleration of song tempo that followed deafening in young birds was largely prevented by aging but not by lesions of the AFP.
MATERIALS AND METHODS
Animals
The subjects for the experiments were 45 male zebra finches (Taeniopygia guttata; Table 1). Birds were raised in individual breeding cages with their parents and siblings until at least 60 d of age. Throughout the duration of the experiments, birds were housed with other male finches, except during song recording sessions or recovery from surgery. All birds were maintained on a 14/10 light/dark cycle throughout the experiments.
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Table 1. Postlearning consolidation of birdsong Surgery
Before all surgical procedures, birds were deprived of food for 1 hr and then anesthetized with either Equithesin (2 ml/kg, i.m.; 0.85 gm of chloral hydrate, 0.21 gm of pentobarbital, 0.42 gm of MgSO4, 2.2 ml of 100% ethanol, and 8.6 ml of propylene glycol to a total volume of 20 ml with water) or isoflurane (by inhalation; 2% v/v in O2). After surgery, all skin incisions were sealed with cyanoacrylate surgical glue. Birds were then transferred to individual cages equipped with heat lamps where they were monitored until full recovery before returning them to their home cages. Birds were deafened by bilateral cochlear removal (Konishi, 1965Anatomy
After the final song recording session, birds were deeply anesthetized with Metofane (by inhalation; Pitman-Moore, Mundelein, IL) and transcardially perfused with 0.9% saline, followed by 3.7% formalin in 0.025 M phosphate buffer. Brains were post-fixed for 4 hr, then sunk in sucrose overnight and cut coronally in 40 µm sections with a freezing microtome. Alternate sections were stained with cresyl violet acetate or reacted with an antibody to CGRP (Bottjer et al., 1997Song analysis
Sound recording. All song was recorded from birds that were isolated in sound attenuating chambers and therefore was "undirected." A computerized, song-activated recording system was used to detect and digitize song for later off-line analysis using software written in the Matlab programming language (Mathworks). For each bird and time point,10 bouts of song were recorded for analysis. Song structure. "Bouts" of song were defined as periods of singing surrounded by silent intervals at least 2 sec in duration. "Syllables" were defined as song elements that were separated from each other by silent intervals exceeding 5 msec in duration. Typical bouts of song started with a variable number of short introductory syllables followed by one or more "motifs", consisting of stereotyped sequences of more complex syllables. For some birds, there were one or two variants on a basic motif; these typically differed in the presence or absence of optional syllables, usually occurring at the end of a conserved "core" sequence. We defined a single characteristic motif for each bird as the motif variant that occurred most frequently across all recorded songs. We defined the "repertoire" of syllables for a bird as all structurally distinct syllables (including introductory syllables) that were present in at least 50% of a bird's song bouts. Syllable similarity. To characterize changes to the structure of individual syllables, we used a subjective scoring procedure similar to that used in previous studies of birdsong (Nordeen and Nordeen, 1992
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Figure 2. Example illustrating the stable song of a normal adult zebra finch (zfa12). A shows a spectrographic representation of song recorded from a normal zebra finch at 103 d of age. B shows an example of the same bird's song recorded at 540 d of age. Individual syllables of the bird's repertoire are labeled above each song; boxes enclose the stereotyped sequence of syllables, or motif, that characterized the bird's song. Both the repertoire of syllables and the order in which they were sung remained essentially unchanged, although the rate of song delivery increased.
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Figure 3. Example illustrating one of the larger changes to song observed after deafening (zfa4), in which the original song degraded to an unrecognizable state. A, Song at 105 d of age, immediately before deafening. B, Song from 3 d after deafening. C, Expanded time axis showing an example of syllables "b" and "c" recorded before (top) and 3 d after (bottom) deafening. After deafening the previously silent interval between the syllables was often "voiced" (arrow). D, Song from 26 d after deafening. None of the original repertoire of syllables remained recognizable at this date. E, Song from 475 d after deafening. Syllables had further degraded and now included features not normally observed in zebra finch songs from our colony. F, Expanded time axis showing examples of some abnormal syllables from songs recorded 475 d after deafening. These included upwardly sweeping frequency components (solid arrows) and brief silent intervals intercalated within short duration syllables (open arrows).
Observers also scored syllable waveriness on a 0-3 scale. This measure was intended to document what appeared to be abnormally rapid fluctuations in frequency of some syllables sung by birds after deafening. Syllables that received a waveriness score of 0 had component stacks or sweeps for which the fundamental frequency changed smoothly over time (e.g., syllables "a" and "b" in Fig. 5a). In contrast, those syllables that received a waveriness score of 3 had abnormally rapid fluctuations in fundamental frequency over the course of component stacks or sweeps (e.g., syllable "d" in Fig. 4c). Judgments of waveriness were based only on those portions of syllables in which the fundamental frequency was best defined; hence, for very noisy syllables in which the fundamental frequency was not apparent, no score was assigned.
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Figure 4. Example illustrating one of the smaller changes to song observed after deafening (zfa7), in which all original syllables remained recognizable but were spectrally degraded, and in which abnormal vocalizations were introduced into the song. A, Song at 103 d of age, immediately before deafening. B, Song from 7 d after deafening. The original syllables were preserved, but variable, low-amplitude vocalizations were also introduced into the song (*). C, Song from 41 d after deafening. In addition to the presence of abnormal vocalizations (*), the spectral structure of original syllables had begun to degrade. D, Song from 203 d after deafening. The spectral structure of original syllables had further degraded, and syllables "c" and "d" had become merged.
Changes to the temporal pattern of song. To assess temporal stability, songs were represented by the timing of syllables and intervals, whereas the spectral structure of individual syllables was ignored. For each bird the timing pattern representing the most common motif before any experimental manipulation was identified. We then searched song bouts from later recording sessions, using a cross-correlation-based technique, for the pattern of syllables that provided the closest temporal match to the initial motif: at each possible alignment (in increments of 5 msec) between motif and song bout, the degree of temporal match was quantified as the average of the percentage overlap of syllables and the percentage overlap of intervals. Interval overlap and syllable overlap were calculated separately and then averaged to increase the sensitivity of the measure to the pattern of song: intervals constitute only a small fraction of the overall song motif (and hence would influence a simple cross-correlation of syllables and intervals combined only minimally), but their location is a critical component of the temporal pattern. For each bird, the maximal overlap was calculated for 10 song bouts and then averaged to provide a measure ("xcorr") of how well the temporal pattern of song was conserved after experimental manipulation. Because the delivery of some songs speeded up "proportionately" over time (i.e., with no apparent change in the relative durations of syllables and intervals), we allowed for proportional changes in the temporal pattern of the song (ranging from 75 to 110% of initial song duration) in searching for the maximal overlap. To control partially for the varying complexity of the motifs sung by different birds, the same method was used to calculate the maximal overlaps between each motif and one song bout from each of 20 different unrelated birds; these 20 different values of maximum overlap were averaged to give a measure ("rand") of the expected similarity between each motif and random, unrelated songs. The temporal stability of song was then expressed as a normalized value ranging from 0 (indicating no more preservation of temporal pattern than random) to 1 (indicating perfect preservation of temporal pattern):
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Figure 5. Another example illustrating one of the smaller changes to song observed after deafening (zfm1), in which some original syllables remained recognizable but exhibited changes both to spectral structure and to sequencing. A, Songs recorded at 395 d of age, before deafening. B, Songs from 4 d after deafening. C, Songs from 34 d after deafening. Syllable "g" was frequently dropped from the song (arrows) and, when sung, was spectrally degraded. D, Songs from 94 d after deafening. In addition to further degradation and dropping of syllable "g", songs at this date also included other abnormalities of sequence (arrows) and introduction of abnormal syllables (*). E, Songs from 256 d after deafening. Songs exhibited a further degradation of spectral structure, more new transitions between recognizable syllables (arrows), and continued presence of abnormal syllables (*).
The baseline motif for one bird from the deafened group and one bird from the lesioned-deafened group had a very simple temporal pattern (three syllables) and consequently showed a high degree of temporal similarity to all songs (including those from unrelated birds). These two birds were therefore excluded from further analysis of changes in temporal pattern. Syllable and interval durations and song tempo. At least two methods have previously been used to characterize changes to syllable and interval durations. Several studies have first defined the repertoire of syllables for each set of songs, and then defined the mean syllable duration as the average of a single instance of each (unique) element of the repertoire (Price, 1979
RESULTS
The deterioration of song after deafening has been previously described, particularly with respect to syllable changes (Nordeen and Nordeen, 1992
Control birds
Manipulations were performed on adult male zebra finches that were ~100 d of age or older. Previous studies of the development of zebra finch song have reported that by this age individual birds have learned a fairly stable and stereotyped song ("crystallized song") that they will maintain with little modification throughout life (Immelmann, 1969
For control birds, the initial syllable repertoire and characteristic sequencing of syllables generally changed little over time. Figure 2b shows a segment of song recorded at 540 d of age from the same bird. Although >1 year had elapsed from the initial recording, each of the syllables that was initially present was still clearly identifiable and there were at most only subtle changes in the structure of individual syllables. Likewise, the typical sequence in which the syllables were sung remained unchanged.
Although the syllable structure and syllable sequencing of control songs recorded at ~100 d of age generally remained unchanged at later ages, there were for some birds more subtle changes in song. For example, over time the frequency structure of some individual syllables appeared to become less noisy and variable, suggesting that control of the detailed structure of syllables continued to improve with age. Likewise, in some birds there were small changes with age in the relative probability with which different syllable sequences were sung. Finally, in many birds, there was a speeding up of song production with age so that the same syllables were delivered more rapidly. This is apparent for the songs in Figure 2, in which the total duration of the sequence "abcdabc" decreased from 867 msec (SD = 14 msec) at 103 d of age to 736 msec (SD = 5 msec) at 540 d of age. Apart from these sorts of subtle changes, songs of control birds were largely unchanging beyond ~100 d of age.
Young deafened birds
In contrast, the songs of adult birds that were deafened could change dramatically. As in humans, however, the degree of change in vocal output after deafening was quite variable across individuals. Figure 3 illustrates one of the more extreme examples of deafening-induced changes in song, observed in a bird that was deafened at 105 d of age. Immediately before deafening, the bird had a well defined repertoire of six different syllables (including introductory syllables) that were generally sung in a stereotyped sequence ("iabbcd"; Fig. 3a). Three days after deafening, the bird's song was largely unchanged (Fig. 3b); each of the syllables that was present initially was retained recognizably with little or no modification. However, the syllable "e" which, before deafening, was usually interposed between occurrences of the sequence "iabbcd", was rarely sung after deafening. Furthermore, the interval between syllables "b" and "c", which had originally been silent, was frequently "voiced" after deafening (Fig. 3c), suggesting a possible disruption in the coordination of vocal and respiratory musculature. The lack of dramatic changes in the song of this bird immediately after deafening was typical of birds in this study.
By 26 d after deafening, the original repertoire of syllables sung by this bird was no longer apparent (Fig. 3d). Conspicuously absent were the distinctive high-frequency syllables "b" and "e" as well as the complex syllable "d". Most of the syllables present at this point did not have clear antecedents in the predeafening song, and many of the syllables appeared to be noisy, with poorly defined harmonic structure. Because we could not establish correspondences between the syllables that were sung before and after deafening, it was not possible to assess the degree to which there had been further changes in the sequencing of syllables. The detailed temporal pattern of the song, however, appeared to differ qualitatively from that present before deafening, suggesting that the changes in song reflected more than just an alteration of the spectral structure of individual syllables.
By the final recording from this bird, at more than a year after deafening (Fig. 3e), the syllables had deteriorated further, and many exhibited features that were not found in normal syllables recorded from birds in our colony (Fig. 3f). These abnormal features included frequency components that swept upwards (solid arrows) and brief silent intervals (<5 msec duration) that split short duration syllables into two separate components (open arrows). The overall temporal pattern of the song had also changed further to a series of relatively uniform, short syllables.
The rapid and dramatic changes in song that were seen for this bird were shared by several of the other birds that were deafened at ~100 d of age. For these birds, the syllables originally present in the song were rapidly obscured by deafening-induced changes, so that they were no longer recognizable in songs recorded as early as a few weeks after deafening.
At the opposite extreme, there were birds that exhibited comparatively minor changes in song and retained recognizable versions of their original syllables even months after deafening. Figure 4 illustrates changes in song for one such bird. This bird was deafened at 105 d of age. Before deafening, the bird regularly sang the four syllable sequence "abcd". At 7 and 41 d after deafening, the bird still sang a relatively unchanged and easily recognizable version of this sequence (Fig. 4b,c). Indeed, even at 6 months after deafening, the bird continued to produce a modified but recognizable version of the sequence, in which the most notable change was that the terminal two syllables ("c" and "d") were merged (Fig. 4d).
Despite the preservation of this characteristic syllable sequence, there were nevertheless a variety of appreciable changes to the bird's song that followed deafening. These included the introduction of syllables to song that had not been present in the initial repertoire (*). These syllables often had an abnormal structure and were of lower amplitude than the more normal and recognizable syllables. These introduced syllables were often separated by intervals of longer duration than typically occur within the context of a motif. Unlike the syllables of control songs, these added syllables also tended to be variable from one rendition to the next and could not be readily characterized by a small and fixed set of exemplars.
Introduction of abnormal material into the songs of deafened birds was observed in approximately half of the cases in which there was also some recognizable preservation of the original syllables. In these cases, individual bouts of song were often initiated with the most normal vocalizations, and abnormal syllables only subsequently appeared in the bout (as in the examples of Fig. 4). This gave the impression that songs became increasingly disrupted as singing progressed. The stretches of disorganized and abnormal vocalizations were often interrupted by relatively loud, call-like vocalizations, after which another comparatively normal sequence of syllables could be produced (Fig. 4c).
Another feature that characterized song changes after deafening was that the frequency control of individual syllables appeared to deteriorate; the fundamental frequency wavered abnormally rapidly, and the overall appearance of syllables was noisier. This was especially clear for some syllables that remained recognizable after deafening and therefore could be followed over time. For example, the terminal component of syllable "d" in Figure 4a was a harmonic stack with a smoothly varying fundamental frequency before deafening. At 41 d after deafening this same component of the syllable exhibited rapid fluctuations in the fundamental frequency (and its harmonics) so that it had a more "wavery" appearance in the spectrogram (Fig. 4c). By the final recording from this bird, at 203 d after deafening, this waveriness was even more pronounced, and the harmonics were less clearly separated, so that the syllable had a noisier overall appearance (Fig. 4d). Although it was especially clear in cases in which individual syllables could be followed, this type of deterioration of frequency control was a general feature of the changes in song that followed deafening. That is, there was an apparent tendency for the overall quality of the song to become more noisy and for individual syllables to exhibit more waveriness after deafening.
As observed for several of the younger control birds, this bird's song also speeded up. The total duration of the sequence "abcd" decreased from 578 msec (SD = 10) at 103 d of age to 535 msec (SD = 8) 41 d after deafening and 489 msec (SD = 15) in the final recording ~6 months later. The speeding up was generally more pronounced in the deafened birds than in the controls (see below for quantification), suggesting that the underlying mechanisms for the speeding up of song not only did not require auditory feedback but were actually accentuated in the absence of feedback.
Old deafened birds
Another example of comparatively moderate changes in song that occurred after deafening of an older bird is shown in Figure 5. This bird was deafened at 395 d of age. Before deafening (Fig. 5a), the bird had a very stereotyped motif ("bcdefg") that was typically sung twice in immediate succession. Shortly after deafening (4 d post), the song was essentially unchanged (Fig. 5b). Similarly, at 34 d after deafening, the bird continued to produce all of the syllables of its initial repertoire (Fig. 5c). However, the syllable "g" was only very rarely produced, so that most motifs ended abnormally early (Fig. 5c, arrows; the probability of a "g" after an "f" had dropped from an initial value of 100% to a value of 25%). Moreover, when the syllable "g" was produced, its spectral structure was noticeably degraded. Also at this time, the song no longer linked together two motifs in immediate succession; instead there were often pauses of up to several hundred milliseconds, further suggesting some difficulty in proceeding normally through the terminal portion of the motif. By 94 d after deafening (Fig. 5d), this apparent difficulty was even more pronounced; the bird continued to produced a truncated motif (lacking the last syllable "g" in >95% of motifs), and on the rare occasions when the syllable "g" was produced, it was further degraded, exhibiting a noisy spectral structure. The truncated motif was almost always followed by a series of abnormal, low-amplitude, and variable syllables (*), before another partial motif was initiated. These stretches of abnormal vocalizations often included syllables with unusually short durations and intervals with unusually long durations, relative to less disrupted portions of song. Other abnormalities of sequence (arrows) were also apparent at this time; for example, syllables occasionally were produced in sequences that were never observed before deafening (e.g., the transition from "c" to "e"). In the final recording from this bird at 256 d after deafening, these types of changes in song were further accentuated (Fig. 5e). Although some of the original syllables remained essentially unchanged, some (e.g., "c" and "g") were never observed, and others (e.g., "f") were appreciably altered. There also continued to be stretches of abnormal, short-, and low-amplitude syllables (*) interposed between sequences of recognizable syllables. Furthermore, the sequences in which the remaining recognizable syllables were sung frequently included additional transitions that were never observed before deafening (e.g., "a" to "d", "b" to "d", "c" to "d", and "i" to "b", indicated by Fig. 5e, arrows).
Summary of qualitative changes to song after deafening
The preceding examples illustrate the range of effects on song that we observed after deafening. The changes in song were clearly quite variable across birds. In the most extreme cases, all recognizable aspects of the original song were obliterated within the first few weeks after deafening (Fig. 3). In other cases, songs retained some or all of their original syllables in recognizable form for as long as birds were followed after deafening (up to ~1 year; Figs. 4, 5). However, even the least seriously affected birds eventually developed conspicuous abnormalities in their songs. These often included a deterioration in the spectral structure of recognizable syllables such that they became more noisy (with a more uniform distribution of energy across frequency) or developed rapid fluctuations in frequency (i.e., syllable "d" in Fig. 4, and syllables "g" and "f" in Fig. 5). These abnormalities could also include changes to the temporal structure of the song, such as the introduction of variable new syllables (*), as well as the introduction of atypical transitions between recognizably retained syllables.
Stabilization of song by AFP lesions and age
In the following sections of Results, we characterize more systematically the changes in song that occurred for the entire population of birds in this study, and we examine whether and how the degree of song changes varied with the age of deafening and with lesions of the anterior forebrain pathway. This characterization is divided into two components, one focusing on changes to the individual syllables of the bird's repertoire, and the other on changes to the overall temporal pattern of the song. This division was chosen both to make analysis of the data more tractable and because physiological experiments suggest that different components of the song motor control system govern these two aspects of song in zebra finches (Vu et al., 1994
Changes to the structure of individual syllables
To grossly assess changes to syllables after deafening, we scored the extent to which individual syllables that were initially present in each bird's repertoire were retained in subsequent recording sessions, using a method similar to that previously used in studies of song (Nordeen and Nordeen, 1992
We then asked whether the syllable deterioration at 1 month of after deafening continued to progress with time and whether any age-dependent differences persisted over long periods after deafening. We also wanted to compare the degree of protection from deterioration provided by age with that imparted by AFP lesions. For the analyses that follow, we therefore divided the deafened birds into three groups: "young-deafened" birds, deafened between 100 and 130 d of age (n = 11), "old-deafened" birds, deafened at >165 d of age (n = 8), and "lesioned-deafened" birds, lesioned immediately before deafening, between 100 and 130 d of age (n = 6). For these groups, we quantified song changes at two time points: an early time point (~1 month after deafening), and a late time point, representing the final recording from each bird (at least 6 months after deafening). Because recordings were not available from all birds at both time points, the numbers for each analysis and experimental group varied as noted in Table 1. This table also summarizes the values of a variety of measures, discussed in further detail below, that were applied to the experimental groups and indicates differences between groups that were significant. Figure 7 shows changes in syllable similarity for each experimental group. An ANOVA confirmed that there were significant effects of both age at deafening and time after deafening on the amount of syllable deterioration. At 1 month after deafening, young-deafened adults exhibited a significant reduction in syllable similarity relative to age-matched controls. In contrast, old-deafened birds had not yet deteriorated significantly relative to controls. For both groups of birds, however, there was a highly significant decrease in syllable similarity between 1 month after deafening and the final post-deafening recording. Indeed, for each intact deaf individual in the study, syllable similarity decreased over this period. These data indicate that syllables continue to deteriorate over a period of months after deafening. By the final recording, syllable similarity for young-deafened birds was no longer significantly different from the baseline value of 0.96 for randomly matched songs (dashed line). For old-deafened birds syllable similarity had also decreased significantly relative to age-matched controls but remained significantly greater than the value for young deafened birds. These data indicate that there was a larger and more rapid deterioration of syllables in young-deafened birds than in old-deafened birds and that the difference between these groups persisted for at least 6 months after deafening.
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Figure 6. Changes to syllable similarity after deafening. Summary for all birds of the average similarity between syllables initially present in each bird's repertoire and syllables present 1 month after deafening (solid circles) or, for control birds (open points), 1 month after the initial recording. Data are plotted as a function of the age at which birds were deafened or, for control birds, as a function of the age at which song was initially recorded.
In contrast, lesioned-deafened birds (young adult birds that received bilateral lesions of LMAN just before deafening) were strikingly different from both young and old-deafened birds. The right panel of Figure 6b shows that at 1 month after deafening syllables were as well preserved in the lesioned birds as in hearing controls and were significantly better preserved than in young-deafened birds, consistent with a previous report (Brainard and Doupe, 2000
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Figure 7. Overall summary of changes to syllable similarity of song at 1 month and final time points for each experimental group. Each point corresponds to an individual bird and indicates the average similarity (see Materials and Methods) between syllables from the repertoire present in the initial recording session and syllables present ~1 month or >6 months after the indicated manipulations. Bars indicate group means and SEs. Significant differences between groups are indicated in Table 1. Control birds (cont) and deafened birds (deaf) were each divided into two groups, based on age at initial recording: young adults (Y) were between 100 and 130 d of age, and old adults (O) were between 165 d and 2 years of age. Data from the young-deafened birds are indicated by filled bars, and data from the old-deafened birds are indicated by striped bars. Birds in the "lesion" group (shaded bars) received bilateral electrolytic lesions of nucleus LMAN immediately before deafening. Birds in the "sham" group received lesions outside of the song system before deafening. All of the lesioned birds except for one (indicated by the square) were young adults at the time of the manipulation. Data from two other groups are shown for comparison: the random group indicates the average similarity present between the syllables of unrelated birds, and the nerve cut group indicates the average similarity between syllables sung before and a few days after denervation of the bird's vocal organ by bilateral transection of the tracheosyringeal nerve (Fig. 1). These two groups provide measures of the minimal expected degree of syllable similarity (dashed line).
The nature of changes to syllables
Although the "gestalt" syllable similarity measures used here and by others (Nordeen and Nordeen, 1992
We also used a subjective scoring procedure to assess the impression that syllable frequency structure became noisier after deafening. Observers who were familiar with zebra finch song but blind to the treatment of birds scored individual syllables for their overall degree of noisiness (see Materials and Methods). This procedure was applied to a random sampling of syllables from each bird and recording session. It therefore differed from the quantitative procedure above in that it could be applied to cases in which individual syllables were no longer recognizable and was also sensitive to the introduction of new syllables to song. Figure 8b shows the average change in noise scores between the first recording session (immediately before any manipulation) and the final recording session (>6 months later) for the different groups. The control birds showed no significant change in noisiness of syllables, although there was again a trend for these measures to decrease slightly in the young controls. In contrast, deafening caused a large and significant increase in this measure. These two measures thus confirm the qualitative impression from the examples shown earlier that deafening increases the noisiness of syllables. A second change to syllables that followed deafening was an increase in the waveriness of spectral structure; that is there was a tendency to produce syllables with abnormally rapid fluctuations in frequency. Figure 8c summarizes changes to the waveriness of syllables (see Materials and Methods) for each of the experimental groups. Control birds again tended to sing slightly less wavery syllables as they got older, although this improvement was not significant, whereas deafening caused a large and significant deterioration of this aspect of frequency control. A third attribute of song that was scored by observers was whether individual syllables were abnormal in structure. Syllables that were scored as abnormal included ones with conspicuous upwardly sweeping frequency components (Fig. 3f, bold arrows), very short duration interruptions (Fig. 3f, open arrows), high levels of noise, and extreme waveriness. The percentage change in abnormal syllables for each experimental group is shown in Figure 8d. Again, as suggested by the examples shown earlier, there was a significant introduction of abnormal syllables only to the songs of the deafened birds. Finally, one aspect of human speech that has been reported to change systematically after profound hearing loss in adulthood is pitch (Leder et al., 1987a
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Figure 8. Nature of changes to syllables after deafening. Each bar indicates the mean change (±SE) between the first and final recording session (>6 months later) for all birds in the indicated groups. A, Changes in syllable noisiness as characterized by depth of modulation of harmonic spectral structure (see Materials and Methods) for syllables that remained identifiable over time. Five young-deafened birds and one old-deafened bird were excluded because of a lack of retained syllables. B, C, Changes in noisiness and waveriness scores of spectral structure (see Materials and Methods). These measures were applied to a random sample of 50-100 syllables from each recording session. D, Changes in percentage of syllables that were judged to be abnormal based on their spectral structure. E, Changes in fundamental frequency. Asterisks indicate significant differences from age-matched controls and from birds that received lesions before deafening.
Changes to the temporal structure of song
In addition to the acoustic structure of syllables, song, like speech, has numerous global temporal features, including the sequence and temporal pattern of syllables, the duration of individual syllables and intervals, and the overall rate of delivery of the song ("tempo"). These aspects of song may be controlled by brain regions different from those controlling syllable structure. Indeed, the effects of AFP lesions on deafening-induced changes in temporal structure did not exactly parallel the effects of these lesions on changes in syllables.Changes to temporal patterning
To assess changes to the patterned temporal structure of the song of all birds, even when syllables were too deteriorated after deafening to be recognized for sequence analysis, we used a quantitative measure based on cross-correlation analysis (see Materials and Methods). This measure assessed the degree to which the initial timing of syllables within the bird's motif was preserved in later songs and explicitly ignored any changes to the spectral structure of individual syllables. Moreover, because some songs speeded up over time (see below), this measure allowed for "proportional" changes in the temporal pattern of motifs (i.e., without change in the relative durations of syllables and intervals). This measure showed that the temporal structure of song deteriorated after deafening in a manner similar to that for syllable structure (Fig. 9). At 1 month after deafening, changes were highly variable among intact deaf individuals; some birds exhibited no more change in temporal pattern than was present for controls, whereas others already exhibited a maximal degree of deterioration. However, as a group, young-deafened birds exhibited significant deterioration of temporal pattern relative to controls, whereas old-deafened birds did not. By the final recording session, the temporal pattern for both young- and old-deafened groups had further deteriorated and no longer preserved greater temporal similarity to predeafening songs than to random songs. In contrast, birds that had been lesioned as well as deafened were still not significantly different from controls, indicating that LMAN lesions stabilized the temporal pattern of song to a greater extent than did age.
In principle, changes to the temporal pattern of song can occur independently of deterioration of syllable structure. This occurs, for example, after transection of the tracheosyringeal nerve, which is essential for controlling the structure of individual syllables but not for the timing of expiration during song. Such nerve transection in two birds caused an acute loss of syllable similarity without grossly affecting the temporal pattern of song (Figs. 7, 9; "nerve cut"). Despite this dissociability of song changes, there was nevertheless a correlation between the degree of deterioration of syllables (as assessed by the syllable similarity measure) and the degree of deterioration of temporal pattern in the songs of birds after deafening (R2 = 0.4; p < 0.05 that the slope of the regression is 0). These results indicate that, like the structure of individual syllables, the temporal pattern of song deteriorates in an age-dependent manner after deafening. Birds deafened as young adults exhibited larger and more rapid deterioration of temporal pattern than did birds deafened at greater ages. However, deaf birds of all ages eventually exhibited significant deterioration of temporal pattern relative to controls, and this deterioration was prevented in birds that received lesions of the AFP.
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Figure 9. Summary of changes to the temporal pattern of song. Each point corresponds to an individual bird and indicates the similarity between the temporal pattern (see Materials and Methods) of songs from the initial recording session and songs recorded ~1 month or >6 months after the indicated manipulations. Conventions are as in Figure 7.
Changes to average syllable and interval durations
The data above demonstrate that the temporal pattern of the bird's original motif can be disrupted after deafening. However, this measure explicitly ignores changes to the tempo of song elements (see Materials and Methods) and also does not characterize changes to the temporal structure of song that occur outside the context of the motif, such as the introduction of short duration syllables to songs in which the original motif is well preserved (Fig. 4) or the lengthening of pauses that occur between motifs or within stretches of abnormal vocalizations (Figs. 3-5). As a gross measure of the nature of overall changes to the temporal structure of song, we therefore also measured changes in the average durations of syllables and intervals. This was a population measure of "unmatched" song elements in which all incidences of all syllables (or intervals) were included (see Materials and Methods). Figure 10 shows the percentage changes in mean syllable and interval durations between the first and final recording sessions for each group of birds. For the control birds, there was little change in the average duration of syllables or intervals, although there was a tendency for these elements to shorten in young control birds. In contrast, for both young- and old-deafened birds there was a large decrease in syllable durations and a large increase in interval durations, and these changes were significantly greater than those observed for age-matched controls. The direction and magnitude of these deafening-induced changes were qualitatively in agreement with a previous study that used the same measure to characterize changes to the songs of adult birds after deafening (Wang et al., 1999
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Figure 10. Average change in duration between the first and the final recording session for unmatched syllables (A) and intervals (B). For each bird and recording session the average duration of all syllables from at least 10 songs was calculated as well as the average duration of all intervals <500 msec in duration. The average duration from the final recording session was expressed as a percentage of the average duration from the initial recording session for each bird. Bars indicate the mean (±SE) change in durations for all birds in each group.
Changes to song tempo
A variety of different factors may have contributed to changes to mean unmatched syllable and interval durations. For example, mean syllable durations could decrease either if individual syllables were sped up (Fig. 2, control bird; Fig. 4, deafened bird) or if more short duration syllables were sung relative to the number of long duration syllables (Fig. 5, deafened bird). To determine whether changes to song tempo contributed to changes in the average durations of all (unmatched) syllables and intervals, we measured changes in the duration of matched sequences of syllables that were retained between initial recordings and recordings made at ~1 month and >6 months after experimental manipulations (Fig. 11). Five young-deafened birds and one old-deafened bird were excluded from this analysis because original sequences of syllables were not sufficiently preserved. The young control birds exhibited a small but significant increase in song tempo. By 1 month after the initial recording, the durations of matched sequences were shortened on average by 1.5% of their initial values, and by the final recording session the durations had shortened by 7% of their initial values. This finding is consistent with the observation of Arnold (1975)
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Figure 11. Change in duration of matched sequences of syllables. Points correspond to individual birds and indicate the durations of identified sequences of syllables at the 1 month and final time points as a percentage of the durations of those same sequences at the initial recording. Bars indicate mean (±SE) values for all birds from each group. For comparison, data are shown illustrating the average decrease in duration of identified sequences for songs sung to another bird (directed) versus songs sung in isolation (undirected). These data derive from (1) eight birds in a published study (Sossinka and Bohner, 1980
The speeding up of song was accentuated in young-deafened birds. By 1 month after deafening the average duration of identified sequences had shortened by 6%, and by the final recording session it had shortened by 18%. This increase in tempo was significantly greater than that in young control birds, indicating that removal of auditory feedback contributed to the acceleration of song. A couple of the old-deafened birds also exhibited large increases in tempo. However, on average, deafening did not significantly speed up the songs of old-deafened birds relative to age-matched controls. This indicates that aging decreases the effects on song tempo of removing auditory feedback. In contrast, lesions of the AFP did not prevent increases in song tempo; lesioned-deafened birds exhibited increases in tempo that were as great as those that occurred in intact young-deafened birds and that were significantly greater than those that occurred for control groups or for old-deafened birds.
Changes to the tempo of matched sequences could reflect changes to the durations of the syllables that make up those sequences, the intervals within the sequences, or both. We therefore separately analyzed percentage changes to these two components of the total duration of the sequences used to assess tempo (matched syllables and matched intervals). For control birds, there was a significant correlation between changes to matched syllable durations and corresponding sequence durations (Fig. 12a; R2 = 0.77) as well as between changes to matched interval durations and sequence durations (Fig. 12b; R2 = 0.52). These data indicate that changes to both syllables and intervals contributed to the overall acceleration of song tempo in control birds. For deafened birds, there was also a significant correlation between changes to syllable durations and sequence durations (Fig. 12c; R2 = 0.86). However, there was not a correlation between changes to interval durations and sequence durations for these birds (Fig.
