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Previous Article | Next Article
The Journal of Neuroscience, June 1, 1999, 19(11):4559-4584
Contributions of Tutor and Bird's Own Song Experience to Neural Selectivity in the Songbird Anterior Forebrain
Michele M. Solis and Allison J. Doupe Keck Center for Integrative Neuroscience and Neuroscience Graduate Program, Departments of Physiology and Psychiatry, University of California, San Francisco, San Francisco, California 94143-0444
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Auditory neurons of the anterior forebrain (AF) of zebra finches become selective for song during song learning. In adults, these neurons respond more to the bird's own song (BOS) than to the songs of other zebra finches (conspecifics) or BOS played in reverse. In contrast, AF neurons from young birds (30 d) respond equally well to all song stimuli. AF selectivity develops rapidly during song learning, appearing in 60-d-old birds. At this age, many neurons also respond equally well to BOS and tutor song. These similar neural responses to BOS and tutor song might reflect contributions from both song experiences to selectivity, because auditory experiences of both BOS and tutor song are essential for normal song learning. Alternatively, they may simply result from acoustic similarities between BOS and tutor song. Understanding which experience shapes selectivity could elucidate the function of song-selective AF neurons.
To minimize acoustic similarity between BOS and tutor song, we induced juvenile birds to produce abnormal song by denervating the syrinx, the avian vocal organ, before song onset. We recorded single neurons extracellularly in the AF at 60 d, after birds had had substantial experience of both the abnormal BOS (tsBOS) and tutor song. Some neurons preferred the unique tsBOS over the tutor song, clearly indicating a role for BOS experience in shaping neural selectivity. In addition, a sizable proportion of neurons responded equally well to tsBOS and tutor song, despite their acoustic dissimilarity. These neurons were not simply immature, because they were selective for tsBOS and tutor song relative to conspecific and reverse song. Furthermore, their similar responses to tsBOS and tutor song could not be attributed to residual acoustic similarities between the two stimuli, as measured by several song analyses. The neural sensitivity to two very different songs suggests that single AF neurons may be shaped by both BOS and tutor song experience.
Key words: auditory selectivity; song selectivity; experience-dependent plasticity; NXIIts transections; LMAN; Area X; zebra finch
INTRODUCTION
Songbirds, much like humans, depend on auditory experience during early life to learn their vocal behavior. This learning occurs in two stages, called the sensory and sensorimotor phases (Fig. 1A). During the sensory phase, a young bird listens to and memorizes the song of its tutor; this memory is called the "template." The sensorimotor phase begins with the onset of singing; using auditory feedback, the juvenile compares its immature vocalizations with the tutor song template and gradually modifies the plastic song until it produces a mature "crystallized song," which is highly stereotyped and resembles the tutor song. Thus, experience of both the tutor song and the bird's own song (BOS) is required for normal song learning (Konishi, 1965
; Price, 1979
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Figure 1. A, Zebra finches learn to sing in two overlapping phases. The sensory phase ends at ~60 d; the sensorimotor phase begins at ~30 d and continues until 90 d. B, Anatomy of the song system. Motor pathway nuclei are gray, and the AF nuclei are black. C, AF neurons develop selectivity for song during development. At 30 d, LMAN neurons have equal RS to tutor song (TUT), conspecific song (CON), and reverse tutor song (REV). At 60 d, these neurons respond significantly more to TUT than to CON or to REV. In addition, BOS elicits a stronger RS than CON or reverse BOS (REV). In adults, LMAN neurons are extremely selective for BOS. D, At 60 d, there is a range of BOS versus tutor song preferences among LMAN neurons. The cumulative distribution of preferences is shown, as quantified with a d'BOS-tutor value for each neuron (see Materials and Methods). Neurons with values
0.5 or less are considered to prefer tutor over BOS. Gray shading highlights those values for which there was no strong preference for one song over the other (
Likely candidates for circuits involved in processing BOS and tutor song experience during learning lie within the song system, a group of nuclei dedicated to song learning and production (Fig. 1B). The motor pathway, which is necessary for normal song production throughout life, includes HVc, the robust nucleus of the archistriatum (RA), and the tracheosyringeal portion of the hypoglossal nucleus (nXIIts). The nXIIts contains the motor neurons innervating the muscles of the syrinx, the avian vocal organ. RA also projects to a group of nuclei associated with respiration, such as nucleus retroambigualis (RAm) and nucleus paraambigualis (PAm) (Wild, 1993
Consistent with an auditory role for the AF during learning, AF neurons in adult, anesthetized birds are auditory and respond selectively to BOS (Doupe and Konishi, 1991
Determining the experience responsible for AF neuron selectivity could elucidate AF function during song learning. For example, neurons tuned by BOS experience could provide feedback about the current state of BOS, whereas those tuned by tutor song experience could store tutor song information. When neural responses to BOS and tutor song are compared at 60 d, a range of preferences for one song over another is evident (Fig. 1D; adapted from Solis and Doupe, 1997
If neurons with similar responses to BOS and tutor song result from acoustic similarities between the two songs, then it is unclear which song experience is responsible for neural selectivity. Inducing a juvenile bird to produce an abnormal song could resolve this issue, because it would reduce similarity between BOS and tutor song (Fig. 2A). If neurons with equivalent responses to BOS and tutor song result from the similarities between these two songs, then such neurons should not exist in birds with songs very different from their tutor song (Fig. 2B, solid line). Alternatively, if such neurons reflect the contributions of both song experiences, then neurons with similar responses to the abnormal song and tutor song should persist (Fig. 2B, dashed line).
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Figure 2. Consequences of decreasing similarity between BOS and tutor song. A, When a juvenile stores a good copy of the tutor song (A) as its template (A) and accurately models its own song after the template, the resulting BOS (A) will highly resemble the tutor song. Thus, if a neuron is tuned by BOS experience only, it could also respond well to tutor song when the two songs are similar enough. This ambiguity could be resolved by making the BOS very different (B) from the tutor song. B, Decreasing the similarity between BOS and tutor song has two predicted outcomes on the distribution of d'BOS-tutor values. If BOS experience shapes some neurons, and tutor song experience shapes others, then the distribution should be split in two, with some neurons preferring tsBOS over tutor song and others preferring tutor over tsBOS but none responding equally well to both (solid line). Alternatively, if both tsBOS and tutor song influence the neural properties of single neurons, then neurons with equivalent responses should persist (dotted line). C, If a poor copy of the tutor song (A) is stored as the template (a) and then a good copy of the template is produced, then the resulting BOS (a) is a better model of the template than the tutor song itself. In this case, neurons preferring BOS would nonetheless reflect tutor song experience. Inducing an abnormal BOS by disrupting sensorimotor learning (B) should decrease the similarity between BOS and a song resulting from poor memorization of the tutor song.
Birds producing abnormal songs could also clarify the experience responsible for neurons that prefer BOS over tutor song in normal 60 d birds. The simplest interpretation is that these neurons are shaped by BOS experience. If, however, a bird has poorly copied the tutor song during the sensory phase, then these neurons might instead represent the template. This possibility is schematized in Figure 2C; if a bird stores a poor copy of the tutor (A) as its template (a) and models its own song accurately after the template (a), then BOS itself is a better representation of the template than the tutor song. This issue could be resolved with birds induced to produce very abnormal songs; if neurons preferring BOS over tutor song persist in such birds, then it is likely that they result from experience of the song unique to that bird.
In this study, we minimized the similarity between the songs of juvenile birds and their tutors by transecting the tracheosyringeal portion of the hypoglossal nerve [NXIIts (ts)], which innervates the syringeal muscles, before song onset. Extracellular recordings of single LMAN and X neurons in these birds at 60 d showed that, although the BOS and tutor song were now acoustically very different, many neurons still responded equally well to both stimuli. This result is similar to that found in normal 60 d birds and suggests a role for both song experiences in shaping AF selectivity.
MATERIALS AND METHODS
Animals. Experiments used male juvenile zebra finches (Taeniopygia guttata). The care and treatment of experimental animals was reviewed and approved by an university animal care and use committee at University of California, San Francisco (UCSF). Birds were raised in individual cages, with their parents and siblings from the same clutch. Opaque dividers between cages visually isolated birds from other conspecifics in the colony. Because juvenile birds shared a cage with a single adult male tutor and were visually isolated from other conspecifics within earshot, their learning should have been restricted to the tutor in their cage (Immelmann, 1969
Surgery. When birds were 26-33 d old (mean ± SD, 28 ± 2 d), the tracheosyringeal portion of the hypoglossal nerve (NXIIts) was transected bilaterally under isofluorane anesthesia [0.5-1.5% (v/v); Abbott Laboratories, North Chicago, IL]. The nerves were exposed by an incision along the skin of the neck, where lidocaine had been injected subcutaneously (2% solution; Elkins-Sinn, Cherry Hill, NJ). The NXIIts nerve was dissected away from the trachea at the proximal end of the incision and cut; dissection then continued along the length of the neck, and the nerve was pulled to remove the distal end. This removed ~1 cm of nerve. After bilateral transections, the skin was closed with skin adhesive (Krazy Glue; Borden, Columbus, OH). The ts cut birds were returned to their home cages until they were 60-d-old.
Two days before the experiment, we prepared birds for recording by affixing a head post to the skull and marking the location of the song nuclei on the skull (for details, see Solis and Doupe, 1997
Stimuli. One to 2 d before the experiment, the songs of the ts cut bird and its tutor were recorded. Each bird was placed in a sound-attenuated chamber (Acoustic Systems, Austin, TX) connected to an automatically triggered audio system. Approximately 90 min of bird sounds were recorded and then scanned for song. A typical plastic song rendition was usually chosen after listening to at least 25 songs and looking at several song spectrograms; a typical song was considered to be the song most frequently sung. A typical tutor song was chosen after listening to 10 songs. Songs were digitized at 32 kHz and stored on a SPARC (Sun Microsystems, Palo Alto, CA) IPX computer at similar peak intensity levels (range, 64-73 dB; software by Michael Lewicki and Larry Proctor, California Institute of Technology, Pasadena, CA). In 15 experiments, three different plastic song renditions from a bird were stored for presentation during the experiment. The durations of tsBOS and tutor songs ranged from 602 to 2461 msec.
During electrophysiological recording, acoustic stimuli were presented by a speaker 25 cm away from the bird, inside a double-walled anechoic sound-attenuated chamber (Acoustic Systems, Austin, TX). The frequency response measured at the bird's location inside the chamber was flat (±5.0 dB) between 500 Hz and 8 kHz. The stimuli included songs of the ts cut juvenile (tsBOS), its tutor song, reverse manipulations of tsBOS and tutor song, the songs of other zebra finches (conspecific), the acoustically similar songs of other species of estrildid finches (heterospecific), broad-band noise bursts, and tone bursts. Stimuli were presented in a random, interleaved manner. An effort was made to present each neuron with 15-20 trials of each stimulus type: tsBOS, reverse tsBOS, reverse order tsBOS, tutor, reverse tutor, reverse order tutor, at least two adult conspecific songs, at least two heterospecific songs, at least two juvenile conspecific songs, at least two ts cut juvenile conspecific songs, broad-band noise bursts, and tone bursts; however, some neurons were lost before characterization was completed.
Electrophysiology. Extracellular neuronal signals were amplified and filtered between 300 Hz and 10 kHz (A-M Systems, Everett, WA). To locate auditory neurons, search stimuli included tsBOS, tutor song, adult conspecific song, heterospecific song, broad-band noise bursts, and tone bursts. Most neurons were isolated with a window discriminator (UCSF Physiology Shop). Twelve units were isolated offline using spike-sorting software (Lewicki, 1994
Anatomy. At the end of an experiment, the bird was deeply anesthetized with Metofane (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 and cut in 40 µm sections with a freezing microtome. Sections were stained with cresyl violet, and electrode tracks and lesions were identified. Only neurons histologically confirmed to be in LMAN or X were used; their specific location within each nucleus was also documented.
RA volumes were measured for each ts cut bird and for normal 60 d birds, recorded in a previous study. Measurements were made blind to the experimental condition. The Nissl-defined boundaries of RA were traced at 80 µm intervals, and the resulting area was calculated using an image analysis program (NIH Image). The total area was multiplied by section thickness and then by the total number of sections to give a final volume. Because of individual differences in post-fixation time, each RA volume was normalized by the volume of the nucleus pretectalis (PT), which is unrelated to the song system. Final RA/PT ratios were compared between ts cut and normal birds. When measurements from both hemispheres were available, the mean RA volume and mean PT volume were used. For nine ts cut birds, PT volume was not available. Thus, RA volumes alone were also compared within all ts cut birds for which post-fixation times were equivalent.
The syrinx of each ts cut bird was also dissected after perfusion. Each syrinx was cut 1 mm distal and 4 mm proximal of the bifurcation of the bronchi and then weighed to assess relative muscle mass, a marker of denervation success.
Data analysis. We quantified responses to an acoustic stimulus during the period of stimulus presentation, offset by an estimate of the latency. The latency of each neuron was measured by examining its responses to a broad-band or tone burst stimulus with a peristimulus time histogram (PSTH) divided into 5 or 10 msec bins. The latency was defined as the onset of the first of two consecutive bins during the stimulus that had at least twice as many spikes as the mean number of spikes per bin during the background. LMAN neurons often did not respond to broad-band noise or tone bursts. For these cases, the latency of another neuron from the same bird was used; if there was none, then the neuron was assigned a latency characteristic of neurons from normal 60 d birds (65 msec; from Solis and Doupe, 1997
To be considered auditory and included for analysis, a neuron had to have an average firing rate during one of the stimuli that was significantly different from the background rate (two-tailed paired t test, p < 0.05). The firing rate during a stimulus was obtained by normalizing the number of spikes elicited during the stimulus by the duration of the stimulus. The background rate was calculated by averaging the firing rate of the neuron from two different periods: 2 sec preceding stimulus onset and 2-3 sec beginning 1 sec after the end of the stimulus. The response strength (RS) of a neuron to a stimulus was the difference between the firing rate during the stimulus (offset by the latency) and the background rate. The RS was measured for each stimulus trial and then averaged across trials to get the neuron's RS to that stimulus, expressed in spikes per second. Data for different stimuli but of the same stimulus type were also averaged in this way to get an RS for a stimulus type; e.g., to obtain the RS for adult conspecific song, the RS values for each trial of two different adult conspecific song stimuli were averaged together.
The selectivity of an individual neuron for one stimulus (A) over another (B) was quantified using the d'A-B measure (Green and Swets, 1966
In this equation,
and
are the mean RS to stimulus A and B, respectively, and
2 is the variance of each RS. If d'A-B is positive, then stimulus A elicited a greater response; if it is negative, then stimulus B elicited a greater response. Values of d'A-B close to 0 indicate no difference in the RS elicited by the two stimuli. A particular d' value was calculated only for neurons that had a significant response to at least one of the two stimuli compared. A neuron was considered selective for stimulus A over stimulus B if it had a d'A-B value
To convey the magnitude of the difference between the RS elicited by two different stimuli, the selectivity index (SI) was also calculated (Volman, 1996
When comparing RS to two stimuli with large differences in song duration, normalizing spike counts elicited by the two stimuli by stimulus duration may bias comparisons of the RS. For example, if two stimuli, one short and one long, elicit a similar response in which the neuron initially fires strongly and then fatigues, then normalizing by song duration will give a substantially decreased RS for the long stimulus relative to the shorter stimulus; this in turn will result in a d' value that prefers short stimuli over long stimuli. Because large differences in song duration occurred in several experiments, a peak RS was also calculated to remove bias attributable to varying song durations in the comparisons of a neural response. First, a maximum firing rate during the stimulus was found using a 500 msec sliding window, which moved across a response in 1 msec increments. Second, the maximum background rate was also found using a 500 msec window. Third, the peak RS was calculated by taking the difference between the maximum firing rate during the stimulus and the maximum background rate; this peak measurement removes duration bias because it normalizes every spike count by 500 msec, regardless of the stimulus duration. Finally, peak d' values were also calculated using the peak RS obtained from the 500 msec window. A 500 msec window was chosen for two reasons. First, it was shorter than the shortest song stimulus (602 msec). Second, for a subset of neurons (five from LMAN and five from X), a series of sliding windows (10-2000 msec) were used to calculate the peak RS and resulting peak d' values. Among those windows <600 msec, the 500 msec window gave the largest peak d' values between two stimuli of similar durations. For some cells, windows >500 msec resulted in d' values higher than those for short windows (our unpublished data); this indicates that peak d' measures can underestimate the selectivity of a cell.
Cluster analysis. We tested whether the d'tsBOS-tutor values of neurons recorded from each bird were more similar than expected by chance. To do this, the variance of the d'tsBOS-tutor values obtained experimentally from each bird was compared with a simulated distribution of variances created from the data from all birds. This distribution was determined from 1000 Monte Carlo simulations; each simulation randomly selected n d'tsBOS-tutor values from the pool of all experimental d'tsBOS-tutor values (includes all cells from all birds) and calculated their variance (n equals the number of cells recorded in each bird). The median of the resulting distribution of simulated variances was compared with each bird's experimental variance. If the experimental variance was significantly less than the median of the simulated distribution (one-sample sign test, p < 0.05), the d'tsBOS-tutor values from that bird were considered clustered. A sign test determined whether the frequency of clustering in the group of birds studied was greater than expected by chance. This procedure was completed for d'tsBOS-tutor values from LMAN neurons alone, X neurons alone, and both neuron types together.
Song analysis: similarity. Once electrophysiology experiments were completed, we analyzed the tsBOS and tutor songs themselves using several methods. Song is composed of syllables, which are continuous acoustical signals, 10-200 msec in duration. Syllables are separated from other syllables by a sudden fall in amplitude to near zero or by brief silent intervals. Syllables are composed of smaller continuous signals called "notes." A repeated sequence of syllables is a "motif." A song "bout" consists of introductory notes followed by one or more motifs (for detailed song descriptions, see Price, 1979
The first song analysis was a matching task, completed by nine human observers familiar with zebra finch song but blind to the neural properties of each bird. Observers tried to match each experimental song with that of its tutor, which was present among a group of six potential tutors. The observers listened to and looked at sonograms and oscillograms of the songs before selecting the tutor song that best matched the experimental song. Thus, the percentage of observers that correctly matched the experimental song to its tutor song indicated the overall similarity between tsBOS and tutor song; this measure was called the "percent correctly matched." After selecting a "best match" tutor song, observers scored the song pair on spectral similarity and on temporal similarity using a scale from 1 to 5. For spectral similarity, observers only considered syllable morphology and sequence. A score of 1 referred to a song pair for which no elements in the experimental song resembled anything in the best match tutor song; 2 was given to a song pair when some notes in the experimental song resembled notes present in the best match song; 3 designated a song pair in which one or more syllables of the experimental song resembled distinctive syllables of the best match song; 4 referred to a song pair for which several experimental song syllables resembled those of the best match song, and the syllable sequences were somewhat similar; and 5 was given to a song pair when the experimental song resembled the best match song in both syllable morphology and sequence, making it a good copy of the best match song.
To judge temporal similarity, observers disregarded the spectral features of song and considered only the durations of syllables and intervals and their patterns, or rhythm, within the songs. Each song pair was scored on a scale of 1 to 5. A score of 1 referred to a song pair for which a timing similarity between the experimental song and the best match song could not be detected; 2 indicated a song pair for which the relative durations of at least two syllables and the interval between them in the experimental song resembled timing in the best match song (e.g., doublets or triplets were heard in both songs); 3 was given to a song pair when combinations of doublets or triplets in the experimental song resembled the timing structures of the best match song; 4 was given to a song pair when many syllables and intervals of the experimental song had relatively similar duration and patterning as those in the best match song; and 5 indicated a song pair for which the timing of the experimental song was highly similar to that of the best match song, although differences in speed may have been apparent.
Songs of non-ts cut birds were also included among the experimental songs for analysis, and their respective tutor songs were also present among the possible tutor choices; this provided references against which ts cut song similarity scores could be compared. Normal 60 d song (n = 16), normal adult song (n = 9), and randomly matched song (songs for which the correct tutor was not present among the possible tutor choices; n = 6) were also matched to a tutor song and scored for spectral and temporal similarity. Scores given to normal adult songs provided an upper bound of similarity between songs from normal adults and their tutors, whereas scores given to randomly matched songs provided a lower bound of similarity. Randomly matched songs included those from two normal adult, two normal 60 d, and two ts cut 60 d birds.
To control for slight scoring differences between observers, we normalized each observer's score for a song by the observer's mean score for all songs. Thus, if an observer scored the spectral similarity of a song pair as a 5, but the observer's mean score was a 3, then the score for this particular song pair was 5/3 = 1.7. The normalized scores for birds ranged from 0.30 to 2.43. The final score for each song was the average of each observer's normalized score. This final score included scores given to incorrect experimental-tutor song matches. Scores calculated with incorrect matches excluded were not significantly different (paired t test, p < 0.05); this indicates that incorrectly chosen tutor songs were as dissimilar from the experimental song as the tutor song itself. The mean score for song type (i.e., ts cut, normal 60 d, adult control, and randomly matched) was calculated from the final scores for each song belonging to the song type.
The similarity between each experimental song (ts cut, normal 60 d, normal adult, and randomly matched songs) and its tutor song was also measured with a cross-correlation algorithm (Theunissen and Doupe, 1998
To measure overall similarity, the entire spectrogram of an experimental song was cross-correlated to the entire spectrogram of the tutor song. To measure spectral similarity, the "syllables-only" cross-correlation measure was calculated for each song pair. For this, each isolated syllable of the experimental song was compared with each isolated syllable of the tutor song. The cross-correlation measure was calculated for each comparison, and the maximum was taken as the best match for the syllable. The resulting maxima were then averaged to produce the syllables-only cross-correlation measure. To measure temporal similarity, each song waveform was rectified and low-pass filtered at 62.5 Hz. The filtered versions of experimental song and tutor song were then cross-correlated to give the "temporal envelope" cross-correlation measure.
To further compare temporal features of song, overlap values were calculated between these song pairs (program by Michael Brainard, UCSF). For this, the syllables of each song were replaced with square pulses of equal amplitude. The resulting square pulse strings preserved syllable and interval durations and their patterns found in the original songs. The square pulse string of an entire experimental song was then compared with that of the entire tutor song by calculating the percent overlap between syllables and intervals. The proportion of overlap between experimental syllables and tutor song syllables was calculated separately from the proportion of overlap between experimental intervals and tutor song intervals. The mean of the syllable and interval overlap values was the "song-song overlap" value.
In addition, a "motif-song overlap" value was calculated, which maximized the chance of overlap. The song-song overlap measure described above could miss timing similarities between motifs of two songs, if there were different intervals between multiple motifs within a song. To avoid this, the motif-song overlap value compared a string based on a single motif of the experimental song with a string based on the entire tutor song. In addition, the song-song overlap measure could miss timing similarities if there were differences in song speed; thus, the motif-song overlap calculations allowed the motif string to stretch proportionately 80-120% of its original length, in 2% increments. The percent overlap between each stretched version of the motif string and the tutor song string was calculated, and the maximum was taken as the "maximum overlap" value. Finally, overlap values are sensitive to the complexity of the motif string of the experimental bird. For example, a simple motif comprising only two syllables is likely to give a high maximum overlap value for both the tutor song and a random song. To correct for this, the maximum overlap value was normalized by how well the motif overlapped with random songs. To obtain a measure for random overlap, the maximum overlap value was determined between the motif string and 20 randomly chosen, normal adult song strings. The mean of the 20 maximum overlap values gave the "random overlap" value. This random overlap value was used to normalize the maximum overlap value obtained from the comparison of the motif and tutor song strings, such that:
Song analysis: stereotypy. We measured song stereotypy of each bird in three ways: human subjective scoring, syllables-only cross-correlations, and motif-song overlap analysis. For reference, songs of normal adult and normal 60 d birds were included in the stereotypy test. For each bird, 10 song bouts were randomly selected for analysis (except in four cases: two normal 60 d birds had two songs each; one normal 60 d bird had five songs; and one ts cut 60 d bird had only three songs).
Three observers rated how consistently a particular motif was present in each song sample from a single bird on a scale from 1 to 5. They listened to each song sample and looked at their accompanying sonograms and oscillograms before deciding on the score. Both spectral and temporal pattern repeats contributed to the score. A score of 1 referred to a group of songs that were not at all stereotyped: short syllable sequences and small temporal patterns were rarely, if at all, repeated in the song samples. A score of 2 indicated that a particular syllable sequence or brief temporal pattern was repeated in half or fewer of the song samples. A 3 was given when a short syllable sequence or temporal pattern was repeated in almost all or all song samples. Alternatively, a 3 was given if an entire motif structure was repeated in only half of the song samples. The syllables outside of the repeated structures could vary in identity and ordering. A score of 4 was given when an entire motif structure was apparent in most or all of the song samples; however, some variability remained, with syllables added or dropped from the motif in different renditions. A score of 5 was given when identical motifs were found in every song sample. Each score was normalized by the observer's mean score, as described for the similarity scoring in the matching task. Normalized stereotypy scores ranged from 0.26 to 1.40.
To isolate spectral stereotypy, we used cross-correlations to measure how consistently the syllables in one song were present in the other song samples. Spectral stereotypy was calculated in the same manner as the syllables-only cross-correlation measure of similarity described above, except that the cross-correlations were done between syllables from songs of the same bird. The mean of the resulting syllables-only cross-correlation measures (usually nine coefficients) gave the spectral stereotypy measure for the bird.
Motif-song overlap analysis was used to measure temporal stereotypy. To measure how consistently temporal patterns were repeated in song samples from a bird, a motif-song overlap value was obtained for an experimental motif string and each song sample string (usually nine sample strings), as described above. These were also normalized by a random overlap value, which was obtained by calculating the maximum overlap between the motif string and nine randomly chosen songs from all the experimental groups (adult control, 60 d control, and 60 d ts cut). The normalized motif-song overlap values for each comparison between songs from the same bird were then averaged to give an overlap stereotypy measure.
RESULTS
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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Figure 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 its darkness; 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 syllable B 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
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. Figure 4A 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 a d'tsBOS-tutor value (see Materials and Methods); neurons with d'tsBOS-tutor values
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Figure 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; and d'tsBOS-adult con = 1.41. B, The cumulative distribution of tsBOS versus tutor song preferences for all LMAN neurons recorded, as quantified with d'tsBOS-tutor values, is shown with white circles. For comparison, the distribution of d'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 had d'tsBOS-tutor values between
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Figure 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 plotting d'tsBOS-tutor values against d'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. The dashed 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. In D and E, error bars indicate SEM, and asterisks 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
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
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 Figure 6A; the d'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 the d'tsBOS-tutor values of individual neurons were compared with the relative difference in duration between tsBOS and tutor song, as expressed by the ratio (durationtsBOS
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 peak d'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 peak d' 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 original d' tsBOS-tutor values (mean difference in d'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.
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Figure 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 peak d'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; asterisks denote significant differences, and error bars indicate SEM.
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 Table 1.
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Figure 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, and asterisks 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 circles indicate those neurons with significantly greater responses to the 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.
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) (paired t 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) (paired t 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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Table 1. Frequencies of selective cells
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Figure 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 the abscissa 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 in D.
Features important to order selectivity
To test the importance of syllable sequence within a song for order selectivity, "reverse order" stimuli were presented. Reverse order songs maintain the temporal order within individual syllables but reverse the syllable sequence within a song (see labels in Fig. 8B). On average, cells responded significantly more to forward tsBOS and tutor song than to reverse order versions of these songs (Fig. 8B) (paired t test, for tsBOS-reverse order, p < 0.010; n = 29; for tutor-reverse order, p < 0.050; n = 33). Thus, cells were sensitive to the syllable sequences within tsBOS and tutor song. Because of the simple harmonic stack structure of syllables in many tsBOS, it seemed possible that neurons would be insensitive to reversal of the temporal structure within syllables from tsBOS. To test the contribution of individual syllable structure to order selectivity for tsBOS, we also presented "syllable reverse" stimuli. Syllable reverse stimuli maintain the correct syllable sequence within a song but reverse the individual syllables (see labels in Fig. 8C). On average, cells responded significantly more to forward tsBOS than to syllable reverse tsBOS (Fig. 8C) (paired t test, p < 0.003; n = 13). Thus, cells were also sensitive to the temporal structure within the simpler tsBOS syllables. The percentage of selective neurons in each order selectivity category is listed in Table 1.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 Figure 10A. 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
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Figure 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; and d'tsBOS-adult con = 2.35). The dashed 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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Figure 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; and d'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, The d'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 region highlights 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.
Plastic song renditions and neuronal maturity
Neurons that did not strongly prefer tsBOS were unlikely to have resulted from inappropriate tsBOS choice; X neurons responded equally well to three different renditions of tsBOS (ANOVA, p = 0.079; n = 38). Furthermore, neurons with similar responses to tsBOS and tutor song were also selective, indicating that they were not immature. For example, the neuron in Figure 10A responded strongly to both tsBOS and tutor song but substantially less to conspecific song and reverse tsBOS. The song selectivity of neurons that responded equivalently to tsBOS and tutor song was examined by plotting the d'tsBOS-tuto
