Contributions of Tutor and Bird’s Own Song Experience to Neural Selectivity in the Songbird Anterior Forebrain

Songs of ts cut birds at 60 d

Bilateral NXIIts (ts) transections do not disturb the respiratory outputs involved in song production; thus, birds receiving ts cuts at ∼30 d of age readily sang, but because they could not control their syringeal musculature, they produced extremely abnormal songs by 60 d. These birds sang a series of simple syllables consisting of harmonically related notes. These “harmonic stack” syllables had little amplitude modulation (Fig.3A), and the frequencies of the stacks often fluctuated, giving the song a wavery quality. The song of this ts cut bird (tsBOS) was very different from its tutor song (Fig. 3B). Although the syllables of the tsBOS shown were longer than normal, the average syllable and interval durations in ts cut song were not significantly different from those of normal adult or 60 d song (p > 0.635 for all comparisons, unpaired t tests). The song of a normal 60 d sibling of the bird in Figure 3A is shown for comparison (Fig.3C). Although this normal 60 d song had immature features such as noisier syllables and a longer song duration than the tutor, it had clear similarity in syllable morphology and timing to the tutor song. Thus, the ts cut manipulation produced songs that were considerably simpler than normal plastic song, and dramatically reduced the similarity between BOS and tutor song that can occur by 60 d.

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Fig. 3.

NXIIts nerve transections minimized the similarity between BOS and tutor song at 60 d. A, Sonogram and oscillogram of the song of a ts cut bird at 60 d, which underwent nerve transections at 29 d. Sonograms plot frequency versus time, and the energy of each frequency band is indicated by itsdarkness; oscillograms plot the amplitude of the song waveform versus time. Calibration: A–C, 500 msec.B, Tutor song of the ts cut bird in A. Introductory notes (I) and syllables (A, B) are labeled. C, Song of a 60 d juvenile whose tutor song is also shown in B. Syllables that resemble those in the tutor song are labeled (i.e., syllable b in the juvenile song is similar to syllableB in the tutor song). D, Measures of similarity to tutor song from the matching task shown for different bird groups. Black circles show the mean percentage of observers that matched a song to the correct tutor song (left ordinate). This mean averages the frequency of matching across all songs in each song type. There is no percentage for random matches, because their correct tutor was never present among the tutor song choices. The mean spectral (white circles) and temporal (white triangles) similarity scores are plotted along the right ordinate. Error bars indicate SEM.

We quantified the decrease in similarity between tsBOS and tutor songs using multiple methods of song analysis. In a matching task, observers tried to match an experimental song (ts cut 60 d, normal 60 d, normal adult, or random) with that of its tutor, which was present in a group of possible tutors (see Materials and Methods). Songs from ts cut birds were correctly matched to their tutor song significantly less frequently than were songs from normal 60 d birds (Fig.3D) (unpaired t test, p < 0.002). Because NXIIts transections in adult birds are known to preserve the overall timing of song but to eliminate normal spectral features (Simpson and Vicario, 1990; Williams and McKibben, 1992), observers also scored separately the spectral and the temporal similarity between each experimental song and the chosen tutor song (see Materials and Methods). The resulting mean spectral similarity score and the mean temporal similarity score for ts cut songs were significantly lower than those for normal 60 d songs (Fig.3D) (unpaired t test, p < 0.0001 for spectral similarity; p < 0.001 for temporal similarity). For reference, the mean spectral and temporal similarity scores for randomly matched songs (songs whose actual tutor song was not present among the choices; for details, see Materials and Methods) and for normal adult songs are also shown. Note that ts cut songs had significantly lower spectral similarity scores than did the randomly matched songs (unpaired t test, p < 0.002).

Comparison of LMAN neural responses with tsBOS and tutor song

Extracellular recordings of 52 LMAN neurons from 16 ts cut birds revealed selectivity for tsBOS. Figure4A shows a neuron that responded substantially more to tsBOS than to tutor song, adult conspecific song, and reverse tsBOS (a “mirror image” reversed song in which both entire syllables and syllable sequence are reversed). Thus, this neuron was sensitive to the spectral and temporal properties of tsBOS, despite its simple structure. Many other neurons showed a strong preference for tsBOS over tutor song. We quantified the preference for tsBOS over tutor song for each neuron with ad′_{tsBOS–tutor} value (see Materials and Methods); neurons with d′_{tsBOS–tutor} values ≥0.5 were considered to prefer tsBOS over tutor song, and neurons withd′_{tsBOS–tutor} values of −0.5 or less were considered to prefer tutor over tsBOS. Classified in this way, 28% of LMAN neurons preferred tsBOS over tutor song, and only 5% of neurons preferred tutor over tsBOS (Fig. 4B). These strong preferences for the abnormal tsBOS over tutor song demonstrate the ability of BOS experience to shape LMAN neuron properties.

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Fig. 4.

LMAN selectivity for tsBOS at 60 d.A, PSTHs show the greater response of a single LMAN neuron to tsBOS than to tutor song, reverse tsBOS, and adult conspecific song; 20 trials of each song were presented. For this neuron, d′_{tsBOS–tutor} = 1.50;d′_tsBOS–rev = 1.11; andd′_{tsBOS–adult con} = 1.41. B, The cumulative distribution of tsBOS versus tutor song preferences for all LMAN neurons recorded, as quantified withd′_{tsBOS–tutor} values, is shown withwhite circles. For comparison, the distribution ofd′_BOS–tutor values from normal 60 d birds is shown with black circles. Gray shading highlights those cells considered to respond equally well to both songs. Inset, Mean RS of all LMAN neurons recorded to BOS and tutor song for both ts cut (white circles) and normal (black circles) 60 d birds. Error bars indicate SEM.

Unexpectedly, many LMAN neurons responded equally well to tsBOS and tutor song, despite the large acoustic differences between these two songs. Figure 5A shows an example of such a neuron, which came from a ts cut bird whose song was matched to the correct tutor song by only one of nine observers. This type of neuron represented a substantial proportion of LMAN neurons recorded (Fig. 4B): 67% of the neurons hadd′_{tsBOS–tutor} values between −0.5 and 0.5, thus classifying them as neurons with equivalent responses to both tsBOS and tutor song. Overall, the mean ofd′_{tsBOS–tutor} values of neurons from ts cut birds was not significantly different from that obtained from normal 60 d birds (Fig. 4B) (unpaired ttest, p = 0.089; normal 60 d data from Solis and Doupe, 1997). On average, tsBOS elicited a greater response than tutor song, as was true for LMAN neurons from normal 60 d birds (Fig.4B, inset; paired t test,p < 0.004 for neurons from ts cut birds;n = 46).

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Fig. 5.

Equivalent responses to tsBOS and tutor song.A, PSTHs show the responses of a single LMAN neuron to 13 presentations of each song. Although this neuron responded equally well to tsBOS and tutor song (d′_{tsBOS–tutor} = 0.14), it did not respond well to adult conspecific song (d′_{tsBOS -adult con} = 1.13; d′_{tutor–adult con} = 1.08) or to reverse tutor song (d′_tutor–rev= 1.08). B, The tsBOS versus tutor song preference of each LMAN neuron is compared with its selectivity by plottingd′_{tsBOS–tutor} values againstd′_tsBOS–rev (black circles) and d′_tutor–rev (open circles) values. Gray shading indicates those neurons that responded equally well to tsBOS and tutor song. Thedashed vertical line marks the criterion for selectivity (d′ = 0.5) C, This histogram shows the number of LMAN neurons classified as selective (black bars) and unselective (hatched bars) in the three different tsBOS versus tutor song preference categories.D, For those neurons responding equally well to both tsBOS and tutor song, histograms show paired comparisons between the mean RS to tsBOS (black bars) or tutor song (white bars) and the mean RS to adult conspecific song.E, For those neurons responding equally well to tsBOS and tutor song, histograms show paired comparisons between the mean RS to tsBOS (black bars) or tutor song (white bars) and the mean RS to their corresponding reverse songs. InD and E, error bars indicate SEM, andasterisks denote significant differences between each pair of stimuli.

Neurons with equivalent responses to acoustically dissimilar tsBOS and tutor songs might indicate that both song experiences shape the selectivity of single neurons. There are alternative explanations for such neurons, however. First, these neurons might not have exhibited a stronger preference for tsBOS because they were tested with a version of tsBOS that was not optimal for eliciting responses; the variability of plastic song at 60 d makes this possible. Second, it is possible that neurons with similar responses to tsBOS and tutor song are simply immature: younger neurons from 30 d birds respond equally well to all song stimuli (Doupe, 1997). Third, the equivalent responses to tsBOS and tutor song could be attributable to residual similarities between the two songs. Although song analysis revealed that, on average, tsBOS songs share little similarity with tutor song, it is important to compare each bird’s neural properties with the similarity between its tsBOS and its tutor song. The first two alternative explanations are discussed immediately below; the third possibility will be examined in the last section of Results, using detailed song analysis.

Plastic song renditions elicited equivalent neural responses

Because of plastic song variability normally present at 60 d, it seemed possible that neurons without a strong tsBOS preference had been presented with a version of plastic song to which neurons were less responsive. To assess this, LMAN neurons were presented with three different renditions of tsBOS in eight experiments. Many neurons responded equally well to all three renditions, whereas others responded more to the tsBOS version most frequently produced by the bird. This version was always used as the primary tsBOS, to which all other songs were compared when measuring selectivity. Overall, there was no significant difference in the responses elicited by the three versions of tsBOS (ANOVA, p = 0.954; n= 21). Thus, it is unlikely that selectivity measurements were biased by inappropriate tsBOS presentation.

LMAN neurons with equivalent responses to tsBOS and tutor song were not simply immature

Because AF neuron selectivity increases between 30 d and adulthood (Doupe, 1997), selectivity can be used to assay neuronal maturity. Two types of selectivity were analyzed to determine whether neurons were immature. First, neural responses to tsBOS and tutor song were compared with those to adult conspecific songs. Second, neural responses to tsBOS and tutor song were compared with those to reversed versions of these songs; for such reversed stimuli, both the entire syllables and the sequence of syllables within the song were reversed. Immature neurons would respond equally well to all of these stimuli (Fig. 1C). When we analyzed the selectivity of individual neurons with similar responses to tsBOS and tutor song, however, it was clear that these neurons were not simply immature. For example, although the neuron in Figure 5A responded strongly to both tsBOS and tutor song, it did not respond well to adult conspecific or reverse tutor song. Figure 5B further illustrates this selectivity by plotting the tsBOS versus tutor song preference of each neuron (indicated by its d′_{tsBOS–tutor} value) against a measure of selectivity (d′_tsBOS–revand d′_tutor–rev). Many neurons responding equally well to tsBOS and tutor song had d′ values exceeding 0.5 for these measures of selectivity, indicating that they responded substantially more to tsBOS and tutor song than to reverse songs (Fig.5B, points that lie within the gray zoneand to the right of the dashed vertical line). Similarly, neurons with equivalent responses to tsBOS and tutor songs still discriminated between these songs and adult conspecific song (data not shown). Figure 5C shows the result of classifying neurons as selective or unselective. We considered a neuron to be selective if it had a d′ value ≥0.5 for any one of four selectivity categories: tsBOS–adult conspecific, tutor–adult conspecific, tsBOS–reverse, and tutor–reverse. Classified in this way, 66% of neurons responding equally well to tsBOS and tutor song were selective. In comparison, 68% of this neuron type were classified as selective in normal 60 d birds (Solis and Doupe, 1997). Only 8 of 52 LMAN cells in the ts cut birds resembled 30 d neurons, with similar responses to every song stimulus, and seven of these came from the same animal.

Another measure of maturity is to consider the selectivity of a population of neurons by averaging their responses to different song stimuli. It is possible for individual neurons that do not themselves meet the d′ criterion for selectivity but whose responses are slightly biased toward selectivity to contribute to the selectivity of an entire population of cells. As a population, LMAN neurons with similar responses to tsBOS and tutor song had greater RS on average to tsBOS and tutor song than to adult conspecific (Fig. 5D) and reverse songs (Fig. 5E) (paired t test, for tsBOS–adult conspecific, p < 0.0001;n = 27; for tutor–adult conspecific, p< 0.004; n = 27; for tsBOS–reverse, p< 0.0001; n = 26; for tutor–reverse,p < 0.011; n = 21). Thus, using both individual neuron and population measures, neurons with equivalent responses to tsBOS and tutor song exhibited selectivity, unlike immature neurons.

Alternative methods of measuring neural selectivity

In the previous analyses, comparisons of neural responses to different stimuli can be affected by stimulus duration. A neuron’s RS to a stimulus was calculated by normalizing the number of spikes fired during the stimulus by the stimulus duration. If neural responses fatigue during presentation of a long stimulus, then this method will result in an RS that is less than the neuron’s initial firing rate to the stimulus. This phenomenon can complicate comparisons between responses to two songs when the song durations differ substantially. For example, if two songs with large duration differences elicit the same number of spikes from a cell, then the RS to the longer song will be much less than that to the shorter song; d′ measures, which compare RS to two stimuli, would tend to favor the shorter of the two stimuli. In this study, 7 of 19 experiments had substantial differences between tsBOS and tutor song duration, in which one song was at least twice as long as the other song. An example of the effect of normalizing by song duration is shown in Figure6A; thed′_{tsBOS–tutor} value obtained indicates a strong preference for the shorter tutor song, yet the PSTHs show qualitatively similar responses of an LMAN neuron to tsBOS and tutor song. When thed′_{tsBOS–tutor} values of individual neurons were compared with the relative difference in duration between tsBOS and tutor song, as expressed by the ratio (duration_tsBOS − duration_tutor)/(duration_tsBOS + duration_tutor), a strong correlation resulted (r² = 0.584; p < 0.0001); d′_{tsBOS–tutor} values reflected a preference for the shorter of the two songs.

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Fig. 6.

Stimulus duration can influence the quantification of tsBOS versus tutor song preference. A, PSTHs show the responses of a single LMAN neuron to 12 presentations of tsBOS and a short tutor song (602 msec). Although the responses appear equivalent, the d′_{tsBOS–tutor} value indicates a preference for the shorter tutor song. B, The distributions of peak d′_{tsBOS–tutor} values are shown for all LMAN (black) and X (open) cells, regardless of stimulus duration.C, For those cells responding equally well to tsBOS and tutor song (according to their peakd′_{tsBOS–tutor} values), histograms show the mean peak RS to different stimuli for LMAN (left panel) and X (right panel) cells. Paired comparisons show that tsBOS (black bars) and tutor song (white bars) elicited greater average responses than did adult conspecific song; asterisksdenote significant differences, and error bars indicate SEM.

To investigate the impact of this duration effect on the results so far described, data from experiments in which tsBOS and tutor song had similar durations (difference in duration was less than twice the shorter song) were analyzed separately (30 LMAN neurons from 10 experiments). Within this data subset, the properties described for the whole population persisted; some LMAN neurons preferred tsBOS over tutor song, whereas others responded equally well to these two songs. Among LMAN neurons responding similarly to tsBOS and tutor song, 93% (13 of 14) were classified as selective, and on average this type of neuron responded more to tsBOS and tutor song than to adult conspecific and reverse songs (data not shown) (paired t test, for tsBOS–adult conspecific song, p < 0.001;n = 14; for tutor–adult conspecific song,p < 0.008; n = 14; for tsBOS–reverse,p < 0.009; n = 13; for tutor–reverse,p < 0.031; n = 11). Thus, the neuronal properties present for the whole data set also described the subset of data collected from experiments with similar tsBOS and tutor song durations.

Another method of removing stimulus duration bias from selectivity measures is to obtain a peak firing rate for each stimulus. Peak firing rate assesses a neuron’s maximum response during a stimulus, regardless of where it occurs in time. For each LMAN neuron, the maximum firing rate occurring within a sliding 500 msec window was used to calculate a peak RS to each stimulus (see Materials and Methods); thus, every response was normalized by 500 msec, regardless of stimulus duration. Peak d′ values were then calculated using the peak RS to different stimuli. The resulting peakd′_{tsBOS–tutor} values indicated that there were still neurons that responded equally well to tsBOS and tutor song (53%), and neurons that preferred tsBOS over tutor song (47%) (Fig.6B). Of the neurons responding equally well to tsBOS and tutor song, 63% were selective, as determined from their peakd′ values in the four selectivity categories. In addition, the population of neurons with similar responses to tsBOS and tutor song were also selective when their responses were measured using peak RS; neurons responded on average significantly more to tsBOS and tutor song than to adult conspecific (Fig. 6C) and reverse (data not shown) songs (paired t tests: for tsBOS–adult conspecific, p < 0.0001; n = 19; for tutor–adult conspecific, p < 0.006; n= 19; for tsBOS–reverse, p < 0.0004;n = 18; for tutor–reverse, p < 0.017;n = 16). Thus, using peak RS and peak d′values, neurons that responded similarly to tsBOS and tutor song were still selective. Although peak d′_{tsBOS–tutor}values reclassified 39% of LMAN neurons in terms of their tsBOS and tutor song preferences, the overall distribution was only slightly shifted toward tsBOS preference relative to the originald′ _{tsBOS–tutor} values (mean difference ind′_{tsBOS–tutor} = 0.22; paired t test,p < 0.002; n = 43). Because there is no duration difference between forward and reverse versions of the same song, the maintenance of significant response differences between forward and reverse versions of song with peak RS also indicates that the 500 msec time window chosen was not too small to detect differences between responses to different stimuli.

Thus, LMAN properties in ts cut birds were the same when (1) the measurement of RS originally used in this and other studies was applied to the whole data set, (2) the original RS was used for a data subset comprising neurons collected from experiments without large duration differences between tsBOS and tutor song, and (3) peak RS was used to measure responses of the whole data set. For all three analyses, neurons that preferred tsBOS over tutor song and neurons that responded equally well to tsBOS and tutor song were apparent. The latter neurons were also selective. The original measurement of RS will be used to describe further LMAN properties in this study, because it has been used in previous studies of AF neurons.

Selectivity of the entire population of LMAN neurons

Song and order selectivity

We also examined in detail the song and order selectivity of the entire population of LMAN neurons, regardless of their tsBOS versus tutor song preferences. By definition, song-selective neurons respond more to tsBOS or tutor song than to other song stimuli, such as adult conspecific and heterospecific songs. For the entire population of LMAN cells recorded, song selectivity was apparent for both tsBOS and tutor song. On average, both tsBOS and tutor song produced significantly stronger responses than adult conspecific (Fig.7A) and heterospecific songs (Fig. 7B) (paired t tests: p < 0.0001 for tsBOS–adult conspecific; n = 45; and tsBOS-heterospecific; n = 47; p < 0.004 for tutor–adult conspecific; n = 43;p < 0.010 for tutor–heterospecific; n= 47). The song selectivity of individual LMAN neurons is illustrated with scatterplots comparing each neuron’s RS to tsBOS (Fig.7D) or tutor song (Fig. 7E) with its RS to adult conspecific song. In both plots, the majority of cells lie below the diagonal line, indicating their stronger responses to tsBOS or tutor song than to adult conspecific song. The percentages of selective LMAN cells in each song selectivity category are listed in Table1.

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Fig. 7.

Song selectivity of the entire population of LMAN neurons recorded in ts cut birds. Paired comparisons of mean RS show that neurons responded more to tsBOS and tutor song than to adult conspecific (A) and heterospecific song (B). C, Paired comparisons also show greater responses to tsBOS than to ts cut and normal 60 d songs. In A–C, error bars indicate SEM, andasterisks mark significant differences between song pairs. D, The mean RS to tsBOS of each neuron is plotted against its mean RS to adult conspecific song (adult con). The diagonal line marks where cells lie if the RS to the two stimuli were equal. Black circlesindicate those neurons with significantly greater responses to the stimulus on the abscissa (p< 0.05, unpaired t test between abscissastimulus trials and all adult conspecific trials). E, The mean RS to tutor song of each neuron is plotted against the mean RS to adult conspecific song. Conventions are as inD.

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Table 1.

Frequencies of selective cells

To test whether neurons were tuned specifically to tsBOS, rather than to the noisy, immature features common to all plastic songs, other plastic songs of ts cut and normal 60 d birds were presented. On average, neurons responded more to tsBOS than to other plastic songs; however, this reached statistical significance for only the tsBOS–normal plastic song comparison (Fig. 7C) (pairedt test, p < 0.0001 for tsBOS–normal plastic; n = 32; p = 0.055 for tsBOS–ts cut plastic; n = 28). Thus, LMAN neurons were tuned to features specific to tsBOS.

As a population, LMAN neurons from ts cut birds were also order-selective. A neuron is considered order-selective when it responds significantly more to forward song than to a song that is completely reversed (see labels in Fig. 8A). On average, LMAN neurons responded significantly more to tsBOS and tutor song than to reversed versions of these songs (Fig.8A) (pairedt test, p < 0.002 for tsBOS–reverse;n = 42; p < 0.013 for tutor–reverse;n = 30). The order selectivity of individual LMAN neurons is shown by plotting each neuron’s RS to tsBOS (Fig.8D) or tutor song (Fig. 8E) against its RS to the corresponding reverse song. In these scatterplots, many cells lie below the diagonal line, indicating their stronger responses to tsBOS or tutor song than to the corresponding reverse song stimuli.

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Fig. 8.

Order selectivity of the population of LMAN neurons recorded from ts cut birds. A, Paired comparisons of mean RS show that neurons responded more to tsBOS and tutor song in the forward direction than to their respective reverse songs. B, The mean RS to tsBOS and tutor song were greater than those to reverse order versions of these songs.C, The mean RS to tsBOS was greater than to the syllable reverse version of tsBOS. In A–C, error bars indicate SEM, and asterisks mark significant differences between song pairs. D, The mean RS to tsBOS of each neuron is plotted against its mean RS to reverse tsBOS. The diagonal line shows where cells lie when they respond equally to the two stimuli compared. Black circles indicate those cells that had significantly greater RS to the stimulus on theabscissa than to the reverse manipulation (p < 0.05, unpaired t test between forward song trials and corresponding reverse song trials).E, The mean RS to tutor song of each neuron is plotted against its mean RS to reverse tutor song. Conventions are as inD.

Comparison of X neural responses to tsBOS and tutor song

X is the first nucleus in the AF pathway; it receives inputs from HVc and itself projects to DLM, which in turn goes to LMAN. In addition, X receives feedback via projections from LMAN. To understand the circuitry underlying AF selectivity and potential interactions between LMAN and X, 64 single X neurons were also recorded from 19 ts cut birds.

As in LMAN, some X neurons responded more to tsBOS than to tutor song. The neuron in Figure 9A not only strongly preferred tsBOS over tutor song, but it also preferred tsBOS over adult conspecific song and reverse tsBOS. In addition, many X neurons responded equally well to tsBOS and tutor song, despite the acoustic dissimilarity of these songs; an example of such a neuron is illustrated in Figure10A. The distribution of d′_{tsBOS–tutor} values from individual X neurons is shown in Figure 9B: 37% of X neurons recorded preferred tsBOS over tutor song; 35% responded equally well to tsBOS and tutor song; and 28% preferred tutor song over tsBOS. This distribution did not differ significantly from that obtained from X neurons in normal 60 d birds (unpaired t test,p = 0.711; normal 60 d data from Solis and Doupe, 1997). On average, in ts cut birds, neural responses to tsBOS were not significantly different from those to tutor song (Fig. 9B, inset) (paired t test, p = 0.862;n = 63).

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Fig. 9.

Selectivity for tsBOS in X. A, PSTHs show the responses of a single X neuron to 20 presentations of each stimulus. This neuron responded more to tsBOS than to tutor song, reverse tsBOS, and an adult conspecific song (d′_{tsBOS–tutor} = 1.52;d′_tsBOS–rev = 0.80; andd′_{tsBOS–adult con} = 2.35). Thedashed white line indicates the neuron’s average spontaneous firing rate. Note that the ordinate of the PSTHs begins at 10 spikes/sec. B, Cumulative distributions of d′_{tsBOS–tutor} values of individual X neurons from ts cut birds (white circles) and normal 60 d birds (black circles).Inset, Mean RS to BOS and tutor song of the population of X neurons recorded from ts cut (white circles) and normal (black circles) 60 d birds. Error bars indicate SEM.

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Fig. 10.

Some X neurons responded equally well to tsBOS and tutor song. A, PSTHs made from the responses of a single X neuron to 10 presentations of each stimulus. This neuron responded more to tsBOS and tutor song than to reverse tsBOS or adult conspecific. For the responses shown,d′_{tsBOS–tutor} = 0.43;d′_tsBOS–rev = 1.43;d′_{tsBOS–adult con} = 1.06; andd′_{tutor–adult con} = 0.96. The white dashed line indicates the neuron’s average spontaneous firing rate. Note that the ordinate of the PSTHs begins at 10 spikes/sec. This particular tsBOS was matched to the correct tutor song by only one of nine observers. B, Thed′_{tsBOS–tutor} value of each X neuron is plotted against two measures of selectivity:d′_{tsBOS–adult con} (black circles) and d′_{tutor–adult con}(open circles). The gray regionhighlights those neurons considered to have responded equally well to tsBOS and tutor song. The dashed vertical line marks the criterion for selectivity (d′ = 0.5). C, Number of X neurons classified as selective (black bars) and unselective (hatched bars) in the three different tsBOS versus tutor song preference categories. D, For those neurons responding equally well to both tsBOS and tutor song, histograms show paired comparisons of the mean RS to tsBOS (black bars) or tutor song (white bars) to adult conspecific song. E, For those neurons responding equally well to tsBOS and tutor song, histograms show paired comparisons of the mean RS to tsBOS (black bars) or tutor song (white bars) and their corresponding reverse songs. In D and E, error bars indicate SEM, and asterisks denote significant differences between each pair of stimuli.

Song and order selectivity of the entire population of X neurons

When the entire population of X neurons recorded was considered, regardless of their tsBOS versus tutor song preferences, they were song selective for both tsBOS and tutor song. Using the original RS values normalized by stimulus duration, X neurons responded significantly more to tsBOS and tutor song than to adult conspecific (Fig.11A) and heterospecific song (Fig. 11B) (paired ttest, p < 0.0001 for tsBOS–conspecific;n = 63; tsBOS–heterospecific; n = 64; and tutor–heterospecific; n = 63; p < 0.0004 for tutor–conspecific; n = 62). The song selectivity of individual X neurons is illustrated with scatterplots, which compare the RS to adult conspecific song of each neuron with its RS to tsBOS (Fig. 11D) and to tutor song (Fig.11E).

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Fig. 11.

Song selectivity of the entire population of X neurons recorded in ts cut birds. Paired comparisons of mean RS show that neurons responded more to tsBOS and tutor song than to adult conspecific (A) and heterospecific (B) song.C, Paired comparisons also show greater responses to tsBOS than to ts cut and normal 60 d songs. InA–C, error bars indicate SEM, andasterisks indicate significant differences between song pairs. D, The mean RS to tsBOS of each neuron is plotted against its mean RS to adult conspecific song (adult con). The diagonal line marks where cells lie if the RS to each stimulus were equal. Black circlesindicate those neurons with significantly greater responses to stimulus on the abscissa (p < 0.05, unpaired t test between abscissa stimulus trials and all adult conspecific trials). E, The mean RS to tutor song of each neuron is plotted against the mean RS to adult conspecific song. Conventions are as in D.

Further tests of tsBOS song selectivity indicated that neurons were tuned to tsBOS specifically, rather than to features common to plastic songs. Responses to tsBOS were significantly greater than responses to plastic songs of ts cut and normal 60 d birds (Fig.11C) (paired t test, p < 0.0005 for tsBOS–ts cut; n = 49; p < 0.0002 for tsBOS–normal; n = 51). The percentages of selective X cells in each song selectivity category are listed in Table1.

The X neurons recorded were also order-selective for tsBOS (Fig.12A). On average, X neurons responded significantly more to tsBOS than to reverse tsBOS (paired t test, p < 0.0001;n = 54). Although the average response to tutor song was slightly more than to that to reverse tutor song, this difference was not statistically significant (paired t test,p = 0.144; n = 45). The order selectivity of individual neurons is shown by plotting each neuron’s mean RS to tsBOS (Fig. 12D) and to tutor song (Fig.12E) against its mean RS to the corresponding reverse stimulus.

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Fig. 12.

Order selectivity of the entire population of X neurons recorded in ts cut birds. Paired comparisons of mean RS show greater responses to tsBOS and tutor song than to reverse (A) and reverse order (B) versions of these songs. C, Paired comparisons show greater RS to tsBOS than to syllable reverse tsBOS. InA–C, error bars indicate SEM, andasterisks mark significant differences between song pairs. D, The mean RS of each X neuron to tsBOS is plotted against its mean RS to reverse tsBOS. The diagonal line indicates where cells would lie if their responses to the two stimuli were equal. Black circles indicate those cells whose responses to the stimulus on the abscissawere significantly greater than its responses to theordinate stimulus (p < 0.05, unpaired t test). E, The mean RS of each X neuron to tutor song is plotted against its mean RS to reverse tutor song. Conventions are as in D.

To test the importance of syllable sequence on X neuron order selectivity, reverse order stimuli were presented (Fig.12B). In contrast to LMAN, X neurons did not discriminate between tsBOS and reverse order tsBOS (paired ttest, p = 0.411; n = 48). This suggests that these neurons become selective for syllable identity first and then later for the syllable sequence within song. However, X neurons did respond significantly more to forward than to reverse order tutor song (p < 0.001; n = 45), indicating that they were sensitive to the syllable sequences within tutor song. This difference between forward and reverse order tutor song was small, however, and similar to that between forward and reverse tutor song. This suggests that discrimination between the two reverse manipulation types was not really that different for X cells; consistent with this, the percentage of selective neurons in the tutor–reverse category was similar to neurons in the tutor–reverse order category (Table 1). Finally, to assay the contribution of temporal features within tsBOS syllables to X responses, syllable reverse stimuli were presented. Neurons responded significantly more to forward tsBOS than to syllable reverse tsBOS, indicating a sensitivity to the temporal structure within a syllable (Fig. 12C) (paired t test, p < 0.029;n = 17). The percentages of selective X cells in each order selectivity category are listed in Table 1.

Comparisons of LMAN and X neurons

X neurons were more broadly responsive than LMAN neurons; they responded readily to broad-band noise bursts, tone bursts,and “nonpreferred” song stimuli (i.e., those not eliciting the largest RS). In X, 53% (34 of 64) of cells recorded responded significantly to all nonpreferred stimuli presented, whereas in LMAN, only 4% (2 of 52) of neurons did. When both nuclei were sampled in an individual bird (13 experiments), mean selectivity values for each nucleus were calculated. When selectivity was measured by the ratio of response magnitudes to two stimuli (SI; see Materials and Methods), LMAN was significantly more selective than X for only two categories of selectivity: tsBOS–tutor song and tsBOS–adult conspecific song comparisons (data not shown; paired t test,p < 0.006 and p < 0.002, respectively). In contrast, comparisons of mean d′ values across nuclei yielded no significant differences in selectivity. Thus, in general, LMAN and X shared similar degrees of selectivity to song stimuli.

Comparisons between neural properties of ts cut birds and those of normal juvenile birds

Although the distribution of their responses to tsBOS and tutor song were similar to those obtained from normal 60 d birds (Figs.4B, 9B), AF neurons from ts cut birds were less selective than neurons from normal 60 d birds. The meand′ values for different selectivity categories are compared between ts cut and normal 60 d birds in Figure13 (normal 60 d data from Solis and Doupe, 1997). The mean d′ values of LMAN neurons from ts cut birds were significantly lower than those from normal 60 d birds when tsBOS selectivity was analyzed (unpaired t tests,p < 0.0001 for tsBOS–reverse; p < 0.001 for tsBOS–adult conspecific). The mean d′ values of X neurons from ts cut birds were significantly lower than those from normal 60 d birds when order selectivity was examined (unpairedt test, p < 0.009 for tsBOS–reverse;p < 0.002 for tutor–reverse). This lower selectivity relative to normal 60 d birds was also maintained when only data from ts cut and normal birds with the same tutor (different clutches) were compared (data not shown). Thus, the lower selectivity is probably not attributable to differences in tutor bird efficacy between the normal and ts cut studies. Furthermore, this lower selectivity was apparent when the percentage of selective neurons in ts cut birds was compared with that found in normal 60 d birds. In LMAN, the percentages of selective neurons in the tsBOS > tutor and the tsBOS > reverse tsBOS categories were significantly lower in ts cut birds. In X, the percentages of selective neurons in the tsBOS > reverse tsBOS, tutor > reverse tutor, and tsBOS > reverse order tsBOS categories were significantly lower in ts cut birds (Table 1).

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Fig. 13.

The degree of selectivity of AF neurons from ts cut 60 d birds (white circles) is less than that observed in normal 60 d neurons (black circles). The mean d′ values for each selectivity category (listed on the ordinate) are plotted along theabscissas; error bars indicate SEM.Asterisks denote significant differences between ts cut and normal 60 d birds.

Despite this lower selectivity, neurons from ts cut birds were clearly selective relative to neurons from normal 30 d birds; as seen in Figures 7, 8, 11, and 12, neurons from ts cut 60 d birds on average discriminated tutor song from adult conspecific song and reverse tutor song (except in X), which is not true for 30 d neurons. In addition, when classified according to tutor song categories of selectivity only (i.e., d′_{tutor–adult con} and d′_tutor–rev), ts cut birds had significantly more selective LMAN and X neurons than did 30 d birds (30 d data from Doupe, 1997; χ² tests,p < 0.0004 for both comparisons).

RA volumes were not affected by NXIIts transections

Transecting the NXIIts nerve might have caused neuronal atrophy or death in upstream nuclei, which could lead to nonspecific changes in LMAN or X selectivity. To estimate potential retrograde effects of the nerve transections, RA volumes were measured. In ts cut birds, the mean ± SD RA volume (0.244 ± 0.053 mm³) was within the range of RA volumes previously reported for normal adults (0.220–0.372 mm³; Gurney, 1981). Also, although syrinx weight of ts cut birds was significantly less than that of normal 60 d birds (unpaired t test,p < 0.0001), the normalized RA volume was not significantly different between the same two groups of birds (unpairedt test, p = 0.300); this indicates that nerve transection did not affect RA volume. Finally, the mean RA volume of a ts cut bird did not correlate with its meand′_{tsBOS–tutor} value from LMAN or X (Table2). Thus, possible retrograde effects of nerve transection, as measured here with RA volume, do not account for the difference in selectivity between ts cut and normal 60 d birds or for the range of tsBOS versus tutor song preferences.

Differences between individual ts cut birds

Neurons recorded from the same bird had similar tsBOS versus tutor song preferences (Fig. 14). Experimental d′_{tsBOS–tutor} values of LMAN neurons clustered in nine of nine birds in which multiple cells were recorded (see Materials and Methods). This frequency of clustering was greater than expected by chance (sign test, p < 0.002). Similarly, the d′_{tsBOS–tutor} values of X neurons within a bird were also clustered more frequently than expected by chance (12 of 14 birds; sign test, p < 0.006). The d′_{tsBOS–tutor} values obtained from both LMAN and X cells in a single bird were also more similar than expected (15 of 17 birds were clustered; p < 0.001). The similarity of d′_{tsBOS–tutor} values for neurons from the same bird suggests that factors specific to the experiment or to the bird could account for the responses to these two songs.

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Fig. 14.

Clustering ofd′_{tsBOS–tutor} values within each bird. The individual d′_{tsBOS–tutor} values obtained from each LMAN (open circles) and X (gray circles) cell recorded from the same bird are shown; eachnumber on the ordinate refers to an individual bird.

A bird’s neural preference for tsBOS versus tutor song was not readily explained by conditions that varied between experiments (Table 2). Potential measures of anesthesia depth, such as the spontaneous rate, maximum RS of a neuron, or time at which each neuron was recorded relative to anesthesia administration, were not well correlated to the neuron’s d′_{tsBOS–tutor} value. Anatomical location within the nucleus did not predict thed′_{tsBOS–tutor} value of a neuron in the anteroposterior, mediolateral, or dorsoventral dimension. Neither slight intensity differences between tsBOS and tutor song stimuli nor the ages of individual birds correlated well with the meand′_{tsBOS–tutor} value from each bird. To weigh the contribution that each bird made to these correlations by the number of cells recorded from it, these last two comparisons were also made using the d′ values of individual neurons; this did not improve their correlations (Table 2). Furthermore, no significant correlations were found among these variables when only data from experiments with equivalent tsBOS and tutor song durations were considered or when peak d′_{tsBOS–tutor} values were used (Table 2). The inclusion of immature neurons in these correlations could obscure a relation between the variables tested andd′_{tsBOS–tutor} values, because these cells would have equivalent responses regardless of any experimental condition tested here. Thus, these correlations were recalculated with the 10 unselective neurons excluded from the data set; this still did not reveal any significant correlations (data not shown). Thus, a bird’s neural preference for tsBOS versus tutor song did not seem to depend on conditions that varied between experiments.

Acoustic similarity between the tsBOS and tutor song does not correlate with neural responses to these two song stimuli

Because the equivalent responses to tsBOS and tutor song were not the result of inappropriate tsBOS stimulus choice nor of neuronal immaturity, we considered another possible explanation for such responses. In principle, residual acoustic similarities between tsBOS and tutor song might have produced similar responses to tsBOS and tutor song. Although the average similarity between tsBOS and tutor songs was lower than normal (Fig. 3D), it remained possible that this similarity for an individual ts cut bird predicted the song preference of that bird’s neurons. If the d′_{tsBOS–tutor}values obtained from a bird reflect residual acoustic similarity between tsBOS and tutor songs, then neurons with equivalent responses to tsBOS and tutor song should come from birds with tsBOS similar to tutor song. As similarity decreases, neurons would have strong preferences for either tsBOS or tutor song, depending on the experience shaping selectivity (Fig.15A, top panel). This “similarity hypothesis” predicts a negative correlation between the absolute value ofd′_{tsBOS–tutor} values (‖d′_{tsBOS–tutor}‖) and the measured similarity between tsBOS and tutor song (Fig. 15A, bottom panel).

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Fig. 15.

A, If residual acoustic similarity between tsBOS and tutor song accounts for the neural tsBOS versus tutor song preferences, then the trends shown with the gray dashed lines in the top panel are expected. For both scenarios, high similarity between tsBOS and tutor song is associated with low d′_{tsBOS–tutor} values. If tsBOS shapes neural selectivity, then low similarity should be associated with strong tsBOS preference over tutor song; conversely, if tutor song shapes selectivity, then low similarity should be associated with strong tutor song preference. The bottom panel combines these two trends by plotting the absolute values ofd′_{tsBOS–tutor} against similarity. According to this similarity hypothesis, large mean ‖d′_{tsBOS–tutor}‖ values should occur in birds with low similarity to tutor song, and smaller mean ‖d′_{tsBOS–tutor}‖ values should occur in those with higher similarity to tutor song. B, The percent of observers who correctly matched the tsBOS song to the tutor song is plotted against the mean ‖d′_{tsBOS–tutor}‖ values obtained for each bird. The regression coefficient (slope) is −0.219 ± 0.853 (±95% confidence intervals). Points to theright of the dashed line refer to birds whose frequency of correct matches by nine observers was greater than expected by chance (p < 0.05, sign test). In B–D, error bars indicate SEM, and the thick black line is the linear least squares fit of the data.C, The mean spectral similarity score for each ts cut bird is plotted against the mean ‖d′_{tsBOS–tutor}‖ value for neurons recorded from that bird; the regression coefficient is 0.869 ± 2.781. The dashed line marks the mean spectral similarity score given to randomly matched songs. For theabscissa, the error bars refer to the variance in observer scoring; because of normalization, a value of 0.4 refers to low similarity, and a value of 2 refers to high similarity.D, The mean temporal similarity score for each ts cut bird is compared with the mean ‖d′_{tsBOS–tutor}‖ value for neurons recorded from that bird. The regression coefficient is −0.388 ± 1.062. The dashed line marks the mean temporal similarity score given to randomly matched songs; the normalized scale of similarity is as described in C.

Cross-correlations

Because some similarities may have been too subtle for detection by human observers, we also used cross-correlation methods to analyze song pairs. Three types of cross-correlation measures were calculated between each tsBOS and its tutor song. First, each tsBOS spectrogram was cross-correlated with the corresponding tutor song spectrogram. Second, to analyze spectral similarity alone, all syllables from each tsBOS were isolated from the song and individually cross-correlated with the isolated syllables of the tutor song. Third, to compare temporal similarity, the amplitude envelopes of each song were cross-correlated. Figure16A shows the mean cross-correlation measures obtained from tsBOS and tutor song comparisons. For reference, mean cross-correlation measures were also calculated between tutor song and other song types, including normal adult, normal 60 d, and randomly matched songs. Of these measures, only the syllables-only cross-correlation measures distinguished differences between these song types. Songs from ts cut 60 d birds had less similarity with their tutor songs than did normal 60 d songs (unpaired t test, p < 0.004); however, the similarity between normal 60 d songs and tutor song was not significantly different from that between normal adult–tutor song pairs. In contrast, cross-correlation measures for the entire spectrogram and temporal envelope comparisons were uniformly low for all song types; no song type’s mean cross-correlation measure was significantly different from that obtained from randomly matched song pairs. Thus, the entire spectrogram and temporal envelope cross-correlations were not sensitive enough to detect similarities that were apparent to humans (Fig. 3D). When the most sensitive syllables-only cross-correlation measure of similarity was compared with the mean ‖d′_{tsBOS–tutor}‖ value from LMAN neurons, no strong correlation resulted (Fig.16B; r² = 0.128). This was also true when mean peak ‖d′_{tsBOS–tutor}‖ values were used (r² = 0.021).

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Fig. 16.

Automated measures of similarity between experimental and tutor song pairs. A, Mean cross-correlation measures (left ordinate) for each song group are shown, obtained from the entire spectrogram (white circles), syllables only (black squares), and temporal envelope (white trian-gles) comparisons. In addition, mean overlap values (right ordinate) are plotted for song–song overlap (white squares) and motif–song overlap (black triangles) values. Error bars indicate SEM. B, Each bird’s mean ‖d′_{tsBOS–tutor}‖ value of LMAN neurons is plotted against the syllable-only cross-correlation measures obtained from comparisons of tsBOS to tutor song. Thethick black line is the least squares fit of the data, the vertical dashed line indicates the mean obtained for randomly matched songs, and error bars indicate SEM. The regression coefficient (slope) is −2.437 ± 3.816 (±95% confidence intervals). C, Each bird’s mean ‖d′_{tsBOS–tutor}‖ value of LMAN neurons is plotted against the motif–song overlap values, which measure temporal similarity between tsBOS and tutor song. Conventions are as inB. The regression coefficient is 0.714 ± 0.595.

Song stereotypy shows little correlation with neuronal song preference

Rather than reflecting the acoustic properties of songs, the tsBOS versus tutor song preferences found in each bird might instead reflect its stage of song maturity. Although a weak correlation existed between mean d′_{tsBOS–tutor} values and age, the variability in rate of song development between birds makes age a less reliable indicator of song maturity. Because song stereotypy also increases with song development, we used it as a more direct measure of song maturity. Song stereotypy was estimated by analyzing the similarity between multiple (usually 10) song samples from each bird. Stereotypy was measured using human subjective scoring, syllables-only cross-correlations, and motif–song overlap analyses (see Materials and Methods). Only the human scores and the overlap measures distinguished among all three song types (Fig.17A). Songs from ts cut birds were significantly less stereotyped than normal 60 d songs (unpaired t test, p < 0.005 for human scores; p < 0.003 for overlap values). As expected, normal 60 d songs were less stereotyped than normal adult songs (unpaired t test, p < 0.001 for human scores; p < 0.024 for overlap values). The syllables-only cross-correlation measures did not find significant differences between adult and juvenile song stereotypy, regardless of whether adult song was compared with ts cut or normal 60 d songs (unpaired t tests, p = 0.148 andp = 0.062, respectively).

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Fig. 17.

Song stereotypy analysis. A, Three measures of stereotypy are compared for different song groups. The mean syllables only cross-correlation measures (white circles) and motif–song overlap values (black triangles) are shown on the left ordinate, and the mean human stereotypy scores are shown on the right ordinate. Error bars indicate SEM and in one case is smaller than the symbol itself. B, The meand′_{tsBOS–tutor} values of LMAN neurons from each ts cut bird are compared with the human scores of stereotypy. Because individual scores were normalized by the observer’s mean score, a value of 0.3 refers to low stereotypy, and a value of 1.4 refers to high stereotypy. Thethick black lines are least squares fit of the data, and error bars indicate SEM. The regression coefficient is 1.133 ± 1.64 (±95% confidence intervals). C, The meand′_{tsBOS–tutor} values of LMAN neurons from each ts cut bird are compared with the motif–song overlap measure of stereotypy. The regression coefficient is −0.328 ± 1.42. Conventions are as in B.

When we compared the mean d′_{tsBOS–tutor} values obtained from LMAN of each bird to the two sensitive measures of song stereotypy, no strong correlations were found (Fig. 17B,C) (r² = 0.147 for human scores;r² = 0.019 for motif–song overlap). These stereotypy measures also failed to predict the meand′_{tsBOS–tutor} values obtained from X (data not shown) (r² = 0.027 for human scores; r² = 0.012 for motif–song overlap). When mean peak d′_{tsBOS–tutor} values of LMAN cells were compared with the human stereotypy scores, a weak positive correlation resulted (r² = 0.206; p = 0.089), suggesting that an increase in stereotypy was related to an increased neural preference for tsBOS. Otherwise, no strong correlations occurred using mean peakd′_{tsBOS–tutor} values in either LMAN or X (in LMAN, r² = 0.012 for motif–song overlap; in X, r² = 0.014 for human scores;r² = 0.017 for motif–song overlap). In addition, comparing the individuald′_{tsBOS–tutor} values of each cell with these stereotypy measures did not improve these correlations (Table 3), nor did excluding data from the bird contributing the majority of unselective neurons (data not shown).

NXIIts cut song shows evidence of poor temporal and spectral learning

The effects of NXIIts transections on song in this study indicate the importance of respiratory and vocal muscle interactions during song development. The timing of syllable occurrence within song depends on the patterns of airflow through the syrinx, which are controlled by both syringeal and respiratory musculature (Hartley and Suthers, 1989;Vicario, 1991; Goller and Suthers, 1996; Suthers, 1997). The song system coordinates these muscle groups during song through RA, which projects both to motor neurons of syringeal muscles contained in nXIIts and to respiratory premotor nuclei such as RAm and PAm (Wild, 1993,1997; Reinke and Wild, 1998). In adults, NXIIts transections transform syllables into harmonic stacks but maintain the timing of syllable occurrence within song (Simpson and Vicario, 1990; Williams and McKibben, 1992); thus, in NXIIts-transected adults, the respiratory pathway alone can maintain the temporal structure of song. In contrast, NXIIts transections made in juveniles resulted in songs that consisted mainly of harmonic stacks but that shared little spectral or temporal similarity with the tutor song. Thus, for these juveniles, the connection from RA to respiratory centers was not sufficient to produce timing similar to the tutor song. Because RA volume was unaffected by these transections, it seems unlikely that retrograde effects of NXIIts transection on RA resulted in improper signaling to the respiratory centers. Instead, syringeal control through NXIIts appears to participate in learning song timing.

Of the song analysis methods used here, the human matching task was the most sensitive to similarities between songs from different birds. For example, humans readily detected similarities between normal 60 d songs and tutor songs that the cross-correlation and overlap measures did not. The insensitivity of some of the automated methods was surprising; some cross-correlation analyses and overlap values measured the similarity apparent to humans between normal adult song and their tutor songs to be as low as that between randomly matched songs. These automated measures may be more suitable for quantifying similarity between songs from the same bird; indeed, in the stereotypy analysis, the motif–song overlap measure was as sensitive as human scoring. Despite the subjectivity inherent in human judgment of song qualities, these methods are currently the most sensitive and remain the standard for song analysis (Eales, 1985; Williams, 1990; Scharff and Nottebohm, 1991; Nordeen and Nordeen, 1992). An important step in the birdsong field will be the development of algorithms capable of detecting and quantifying similarities between songs from different birds at least as well as humans.

Contributions of two song experiences to AF selectivity

Many AF neurons responded equally well to tsBOS and tutor song, despite the acoustic differences between these songs. These neurons were also song- and order-selective, which demonstrates that they were not indiscriminately responding to any stimulus. Thus, such neurons support the idea that both tsBOS and tutor song can shape the selectivity of single AF neurons. These neurons might be useful for comparisons of tsBOS and tutor song, which must occur behaviorally during song learning (Konishi, 1965; Price, 1979). The AF could be involved in calculating an error signal that measures the difference between tsBOS and the tutor song template; this error signal could then guide the modification of plastic song during learning.

Examples of single-neuron selectivity for two different stimuli have been found in other neural systems. Some neurons in the bat auditory cortex are tuned to two different durations, depending on whether neurons are responding to biosonar pulses or communication calls (Ohlemiller et al., 1996). Dual selectivity for more complex stimuli has been found in the inferior temporal visual cortex. There, neurons selectively respond to dissimilar fractal patterns that had been consecutively presented during training (Miyashita, 1988). This dual selectivity may reflect an association made between two stimuli consistently experienced together. Similarly, the selectivity for both tsBOS and tutor song could reflect experience of both songs during learning.

Other types of neurons that we observed also supported roles for tsBOS and tutor song experience in shaping AF selectivity. Neurons with strong preferences for the abnormal tsBOS over tutor song clearly indicate that, rather than reflecting poor tutor song copying, BOS experience itself can shape AF selectivity. The sensitivity of these neurons to tsBOS makes them well suited to provide information about the state of plastic song during sensorimotor learning, perhaps participating in the evaluation of BOS. Neurons that preferred tutor song over tsBOS, although rare, are also consistent with an influence of tutor song experience on AF selectivity. Such neurons could represent the stored memory of tutor song. Their rarity, however, combined with the sizable population of neurons responding to both tsBOS and tutor, raises the possibility that tutor song information is primarily found in neurons that also respond to BOS rather than residing in neurons dedicated solely to encoding tutor song. Alternatively, the rarity of neurons with strong tutor song preferences might indicate that tutor song information is distributed sparsely or is encoded differently or elsewhere in the brain (Doupe and Solis, 1997).

Recordings from normal 60 d birds had already suggested a contribution of both BOS and tutor song experience to AF selectivity (Solis and Doupe, 1997). Thus, the tuning of these neurons to two stimuli may be a normal feature of learning in songbirds. As birds mature, tutor song responses may eventually be lost, leaving selectivity only for BOS. Consistent with this idea, the average response of LMAN neurons to tutor song was less than that to tsBOS, and in general, selectivity for tutor song over other stimuli was weaker than that for tsBOS. A transition in selectivity also occurs in the barn owl optic tectum, in which tuning to two different auditory cues precedes the establishment of selectivity for only one cue during the recalibration of auditory–visual maps (Brainard and Knudsen, 1995). Thus, raising ts cut birds to adulthood might reveal a strong preference for BOS over tutor song. Furthermore, the lower selectivity of ts cut birds relative to normal birds could reflect the delayed song development in ts cut birds, which was evident in their low song stereotypy. Again, ts cut birds raised to adulthood may show an increase in AF selectivity.

Measuring neural responses in LMAN and X

The differences in duration between tsBOS and tutor song stimuli in some experiments compelled us to evaluate different methods of measuring neural responses. The appropriate way to measure a neural response depends on how neurons downstream from LMAN and X respond to their inputs. If downstream neurons integrate afferent activity only for a short time, then the peak RS is more appropriate. However, if they integrate incoming signals for the duration of each song stimulus, then the original RS is appropriate. Longer integration times seem more suitable for analyzing these neurons; for a sample of AF neurons, the best discrimination between two stimuli occurred for window sizes >500 msec (our unpublished data). Other clues may also come from behavioral studies. If song duration has behavioral significance in zebra finches, then it is reasonable to measure responses over an entire song stimulus, whatever its duration. Some species respond differently to a song when its duration is varied by altering the number of repeated motifs (Kroodsma, 1976; Becker, 1982). Nevertheless, the peak RS and peak d′ values gave results similar to the original measures of neural response, indicating the robust nature of the selectivity found in this study.

Song experience shaping HVc, the source of AF input

Neurons in HVc also become selective during song learning (Volman, 1993). Studies in both adult (Margoliash, 1986) and juvenile (Volman, 1993) white-crowned sparrows have shown that HVc neurons are selective for BOS. In both studies a subset of birds had songs different from the tutor song, and their neurons showed strong preferences for BOS over tutor song. The weak tutor song responses in these birds might seem inconsistent with the significant tutor song responses found in the AF in this study, because HVc is the source of auditory inputs to the AF. It is possible, however, for AF neurons to derive their selectivity differently from their HVc inputs. HVc comprises two populations of neurons, one projecting to the AF, the other to RA (Sohrabji et al., 1989). X-projecting neurons could be shaped by both tutor song and BOS, whereas RA-projecting neurons could be shaped by BOS alone. Alternatively, tutor song responses may have been overlooked in these HVc studies. First, both used multiunit recordings, which could miss other kinds of selective neurons if they are few. Second, the strong BOS preferences of HVc neurons in this subset of birds could reflect poor copying of the tutor song model. In one study, the birds with songs unlike the tutor song had been tutored with an abnormal tutor song model, unlike natural white-crowned sparrow song (Margoliash, 1986). As juveniles, the birds may have had difficulty memorizing such an abnormal song, potentially leading to storage of something other than the tutor song model as the template. In contrast, the NXIIts transections used in this study reduced the similarity between BOS and tutor song by manipulating the juvenile song itself rather than the tutor song model. Thus, the stronger tutor song responses found here may reflect greater agreement between what was presented as the tutor song and what was stored as the template.

Range of tsBOS versus tutor song preferences among AF neurons

It remains intriguing that there is a variety of tsBOS versus tutor song preferences in ts cut birds, ranging mainly from neurons that strongly prefer tsBOS over tutor song to neurons that respond equally well to both songs. Although not found here, it is theoretically possible that a different method of song analysis, perhaps one that exclusively measures those elements salient to the birds themselves, could find residual similarities between tsBOS and tutor song that were responsible for the equivalent responses to these songs. Alternatively, the state-dependence of auditory responses in neurons of the song system might have influenced the preferences recorded (Hessler and Doupe, 1997; Dave et al., 1998; Schmidt and Konishi, 1998). Although measures of anesthesia depth did not correlate with d′_{tsBOS–tutor} values, it remains possible that subtle differences in arousal state may have differentially emphasized BOS or tutor song responses in neurons actually capable of responding equally well to both stimuli. A third possibility is that the d′_{tsBOS–tutor} values from a bird could reflect its maturity. One measure of song similarity weakly supported this idea: as motif–song overlap values increased (which presumably happens as a bird matures), neural preference for BOS also increased. In contrast, neither the age nor song stereotypy of a bird predicted the d′_{tsBOS–tutor} values obtained from its neurons.

Finally, differences in the accuracy of learning from a tutor bird may have contributed to the range of d′_{tsBOS–tutor}values. In another system, differences between individual animals in neural processing of a task resulted from slightly different behavioral strategies for solving that task (Seidemann et al., 1998). In our study it is difficult to know whether the tutor song presented during the experiment was similar to what the bird had memorized. To favor similarity between the tutor song and the template, juveniles were housed in conditions that maximize copying from the tutor bird (see Materials and Methods). Also, the great degree of similarity between songs from normal adults and their tutor songs shows that there is a high incidence of tutor song copying in the colony used in this study. Nonetheless, because there is currently no direct way to assay what has been stored as the template, it remains possible that all neurons are actually equally shaped by tsBOS and tutor song; neurons with strong preferences for BOS may have been presented with a tutor song that did not match the template.

Possible mechanisms underlying dual selectivity

Although the site of plasticity initially giving rise to selectivity is not addressed in this study, possible mechanisms for mediating selectivity to two different songs exist within the AF. Both LMAN and X have complex intrinsic circuitry that could form multiple, differentiated populations of synapses onto single neurons, enabling them to process tsBOS and tutor song separately (Sohrabji et al., 1993;Vates and Nottebohm, 1995; Farries and Perkel, 1998; Luo and Perkel, 1999). Indeed, synapses onto single LMAN neurons differ markedly in their pharmacological and temporal properties (Boettiger and Doupe, 1998). Moreover, in addition to their auditory responses, LMAN and X neurons are strongly active during singing (Dave et al., 1997;Hessler and Doupe, 1999). This raises the possibility that tuning to different stimuli occurs in different behavioral states, such as singing or listening. The motor-related activity in AF neurons, which could represent efference copy signals (Troyer et al., 1996), might actually contribute to the auditory tuning of these cells. Finally, the acoustical basis of the dual responses to tsBOS and tutor song could be determined in experiments that systematically decompose tsBOS and tutor song stimuli, thus revealing the song components essential for a neural response. Such experiments would contribute to our understanding of a single neuron’s selectivity for two different stimuli.