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Department of Neurobiology, Duke University School of Medicine, Durham, North Carolina 27710
Submitted 5 May 2004; accepted in final form 22 June 2004
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ABSTRACT |
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INTRODUCTION |
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; Knudsen 1998; Tees and Werker 1984; Udin 1983). Unlike most animals, songbirds learn their vocal repertoires by imitating vocalizations heard during a juvenile sensitive period (Doupe and Kuhl 1999; Marler and Peters 1987). Of many models heard, some are imitated only transiently whereas others persist in the adult repertoire (Marler and Peters 1981). This study addresses whether songs transiently learned by juvenile zebra finches evoke auditory responses in a nucleus important to song learning.
The telencephalic nucleus lateral magnocellular nucleus of the anterior nidopallium (LMAN) is necessary for juvenile song learning (Bottjer et al. 1984; Scharff and Nottebohm 1991) and adult forms of audition-dependent vocal plasticity (Brainard and Doupe 2000) (Fig. 1). LMAN neurons exhibit both auditory and vocal-related activity (Doupe and Konishi 1991; Hessler and Doupe 1999), and juvenile auditory and vocal experience influences the structure and function of LMAN neurons. Tutor song experience acts on or through LMAN because raising birds in isolation from a tutor slows LMAN synaptic maturation (Livingston et al. 2000; Wallhausser-Franke et al. 1995) and blocking N-methyl-D-aspartate (NMDA) receptors in LMAN during tutoring impairs copying (Basham et al. 1996). Auditory and vocal experience also strongly influences the response properties of LMAN neurons, which are highly selective for playback of the bird's own song (BOS) and tutor song (TS) versus other conspecific songs (CON) (Doupe and Konishi 1991; Solis and Doupe 1999). Auditory experience of TS and auditory–vocal experience of BOS shapes these responses because juveniles that sing disrupted songs as a result of vocal nerve section contain LMAN neurons responsive to both TS and BOS (Solis and Doupe 1999). Notably, after these birds mature, BOS and TS playback largely fail to evoke suprathreshold responses from LMAN neurons, even though these stimuli activate areas presynaptic to LMAN (i.e., area X) (Solis and Doupe 2000). In other systems, synapses patterned by early experience are functionally suppressed when in conflict with subsequent experience (Brickley et al. 1994; Zheng and Knudsen 1999), raising the possibility that LMAN auditory responses remain subthreshold when the BOS conflicts with the stored model.
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). However, prior studies of LMAN auditory tuning subjected birds to a single tutor, leaving unresolved LMAN's auditory properties when birds copy multiple tutors. We sequentially exposed juvenile zebra finches to 2 tutors (Fig. 1) to test whether early experience of self-generated vocalizations or a model song is permanently engrained in the auditory responses of LMAN neurons. We used in vivo intracellular recordings from juvenile and adult LMAN neurons to detect sub- and suprathreshold responses to transiently experienced songs. We find that sequential copying of 2 different models is paralleled in LMAN by transiently expressed auditory responses to the first TS and the bird's copy of this model, which are lost or overwritten as the bird learns a new song. |
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METHODS |
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; Rosen and Mooney 2000) and so are described more briefly here. All experiments were performed in accordance with a protocol approved by the Duke University Instructional Animal Care and Use Committee. Sequential tutoring protocol
Thirty-four male zebra finches (Taeniopygia guttata) were reared using a protocol that carefully controls auditory and social exposure to adult tutors, and that has been shown previously to successfully extend sensory acquisition beyond posthatch day (PHD) 60 (Livingston et al. 2000). Twenty-three of these experimental birds were hatched and reared until PHD 22–36 (28 ± 0.82 PHD, n = 23) with their father (zebra finch–fathered birds: ZF-birds), mother, and siblings in a sound isolation chamber (Industrial Acoustic, Bronx, NY). Eleven of the 34 experimental birds were reared initially by Bengalese finch (Lonchura striata) foster "parents" until PHD 25–35 (Bengalese finch–fathered birds: BF-birds; 31 ± 1.3 days; n = 11 BF birds). The Bengalese finch song differs from zebra finch song in numerous ways, including shorter duration of individual syllables, a tendency to produce notes with broadly spaced harmonic elements, a higher incidence of single-syllable repeats, and a variable syllable sequence. Therefore by using a Bengalese finch as a foster father we could enlarge the acoustical differences between the first and second tutor songs (TS1 and TS2), making it easier to assess which tutor supplied material copied in the adult song of the sequentially tutored bird. Beginning PHD 22 to 36, individual birds were placed in a small cage, with 2 to 6 of these cages housed in a sound isolation chamber. During this period, BF birds and ZF birds were never housed in the same isolation chamber. Opaque partitions were placed between individual cages. Juveniles housed in this manner could hear but not see each other, and were totally isolated from adult birds of either sex. Under these conditions, juvenile zebra finches do not copy from each other (Livingston et al. 2000; Morrison and Nottebohm 1993). Although birds were isolated from the first tutor across a fairly wide age range (i.e., PHD 22–36), no correlation was detected between the age of isolation from the first tutor and the acoustic similarity of the BOS at PHD60 and TS1 using either Sound Analysis or subjective scoring methods (data not shown; see following text for song analysis methods). Birds remained in visual isolation until PHD 60–65, when they were either used for experiments or subjected to a second tutor. Twelve birds were either used for electrophysiology at PHD 60 (7 ZF and 1 BF), or otherwise were not subjected to a second tutor, and used strictly for behavioral analysis (4 BF birds). The remaining 22 birds were paired with a second tutor (an adult male zebra finch distinct from the biological or foster father) starting at PHD 60 (mean = 61.3 ± 0.42) for 20–34 days (mean 24.1 ± 0.94 days), and then isolated again until used for electrophysiological recordings after PHD 115 (mean = 139 ± 4.8) (Fig. 1A). Adult birds that had been sequentially tutored by 2 zebra finches are termed "ZZ" birds (n = 16), whereas adult birds initially tutored by a Bengalese finch and then by a zebra finch are termed "BZ" (n = 6) birds. See Table 1 for the behavioral analysis of these birds.
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Songs were recorded at approximately 1-wk intervals beginning just before introduction of the second tutor (
PHD 60) for 1 mo, and then approximately biweekly continuing up to the day before electrophysiological recording. This recording schedule was followed because we observed that the juvenile bird's songs changed dramatically in the first 2 wk of the second tutoring period, and then changed more gradually toward the end of this tutoring period and throughout the second isolation period. To obtain song, the bird was placed in a small recording chamber with an adult female except during the second tutoring period, when the bird was recorded in the presence of its tutor. We observed that the time juveniles started to sing after introduction to the recording chamber could be quite variable, from several hours to more than a day. Therefore we recorded the bird's song during the second tutoring period in the presence of the second tutor, to minimize interindividual variation in the amount of total tutor exposure. All songs were recorded using a Shure SM-57 microphone placed about 25 cm in front of the bird's cage, the signal from which was low-pass filtered at 10 kHz, sampled at 22.05 kHz using a data-acquisition card (National Instruments AT MIO 16E Series), and written directly to disk using software written in the LabView environment by R. Balu. The songs from the tutor birds, as well as other unrelated conspecific songs (CON), were recorded in the presence of an adult female. Before electrophysiological experiments in adult birds, songs were recorded at least twice over an interval of one or more weeks to ensure that birds were singing stable songs. Final songs were recorded within 24 h preceding the experiment.
We used "Sound Analysis" (Tchernichovski et al. 2000) to measure the overall similarity of the BOS at the final adult point (BOSAdult) (i.e., at the time of electrophysiological recordings we made in adult birds, range 117–205 PHD; mean = 139 ± 4.8; n = 22 birds subjected to electrophysiological recording) to juvenile versions of the BOS [i.e., BOS at PHD 60 (BOSPHD60)] or to either of the tutor songs (TS1 and TS2), as well as CON songs. When comparing the song of a subject zebra finch to another zebra finch song, we recorded more than 10 renditions of each song and picked the most frequently repeated single motif. We compared single motifs from each of the 2 songs (excluding introductory notes). The various parts of a given motif were identified automatically by periods of silence >70 ms, and then were compared with the parts of the other motif (Sound Analysis settings: "chunk-to-chunk" mode, threshold 81%, interval 70 ms). In Sound Analysis, song similarity is automatically calculated by estimating and comparing 4 acoustic parameters in the 2 songs: pitch, entropy, frequency modulation (FM), and continuity. Here we report the "similarity" scores based on these 4 parameters. For comparisons of juvenile zebra finch songs in which the first tutor was a Bengalese finch, we estimated the average similarity between a BOSPHD60 and each of 3 renditions of the Bengalese tutor song, because the motif was not clearly defined in Bengalese finch song. In addition, because Bengalese finch songs constitute more tonal syllables of short duration compared with the zebra finch, we used different settings for the interval (39 ms), although the same threshold setting (81%).
Sound Analysis is insufficient by itself to assess low levels of copying, given that 2 songs could achieve a low similarity score either because they were unrelated (i.e., not derived from the same tutor) or because one was modeled only in part after the other. Therefore to better assess copying, we used subjective scoring by 4 observers experienced in birdsong analysis who were asked to compare each of the 2 tutor songs with BOSPHD60 and BOSAdult. The observers were blind to the order of the 2 tutors (i.e., which was TS1 or TS2). Observers were instructed to inspect sonograms and listen to each of the 4 songs as needed, then score those syllables of the BOSAdult or BOSPHD60, if any, that were similar to those found in either of the tutor songs. The criterion for deeming that a syllable had been "copied" was that at least 3 of the 4 observers scored a syllable of BOSAdult or BOSPHD60 as copied from either of the 2 tutors. Percentage similarity of song was calculated from the number of syllables copied from a given tutor divided by the total number of syllables in the BOSAdult.
Song stimuli for electrophysiological recordings
All songs were low-pass filtered at 10 kHz, digitized at 22.05 kHz, and edited to include 2 or 3 motifs for each song. Song stimuli included the BOSAdult, BOSPHD60, TS1, and TS2. Most cells were presented these 4 songs in both the forward and reverse (Song-REV) direction, and also presented a CON song. We used a total of 5 different CON songs for all of the electrophysiological experiments performed here, although we used the same CON song consistently throughout a single experiment. Each stimulus was played through a speaker facing the bird at a distance of about 25 cm and was 1.5–2.5 s in duration, with an average sound pressure level of 70 dB (RMS, A-weighting). The various song stimuli were presented in a fixed sequence during a recording from a given cell.
In vivo electrophysiology
Of the 34 birds used in this project, 22 (16 ZZ and 6 BZ; 1 ZZ bird failed to yield any responsive LMAN neurons) were used for electrophysiological recordings after PHD 115, and 8 (7 ZF and 1 BF) were used for electrophysiological recordings at the end of the first isolation period (PHD 62–69; i.e., they were not exposed to a second tutor). On the day of the electrophysiological experiment, the bird was injected intramuscularly with 20% urethane in dH2O (90–100 µl total; Sigma, St. Louis, MO) in 30- to 40-µl doses at 30-min intervals, further anesthetized with inhalation anesthetics as needed, and then equipped with a stainless steel post mounted on its head with dental acrylic and cyanoacrylate.
Sharp electrodes (borosilicate glass, BF100-50-10; Sutter Instrument, Navato, CA) were pulled to 60–200 M
when filled with 2 M K-acetate, 5% neurobiotin, and placed into the nucleus using stereotaxic coordinates and a hydraulic microdrive (Soma Scientific). An AxoClamp 2B intracellular amplifier (Axon Instruments) recorded intracellular potentials in the bridge mode. The recorded potentials were low-pass filtered at 3 kHz, digitized at either 10 or 11.05 kHz (National Instruments PCI MIO 16E series data-acquisition board) and stored on a PC. In the first several experiments, we used intracellular staining with neurobiotin to confirm post hoc that we were indeed recording from LMAN neurons. In subsequent experiments, LMAN neurons were identified on-line by their characteristic spike shape and firing patterns (Livingston and Mooney 1997; Rosen and Mooney 2000). If the impaled LMAN neuron had a resting membrane potential negative of –60 mV, and also displayed robust spontaneous synaptic activity, we then probed its auditory response properties using the panel of song stimuli mentioned above. In some cases, tonic negative or positive currents were injected through the electrode to shift the membrane potentials of the neurons, to accentuate evoked synaptic or action potential activity, respectively. In these cases, comparisons of responses to different stimuli were made at the same membrane potential. Ten to 90 iterations of each auditory stimulus, including 2 s of silence pre- and poststimulus, were delivered at 5-s intervals to the adults and 10- to 12-s intervals to the young birds by a speaker about 25 cm in front of the bird's head. Typical experiments lasted about 12 h; although robust responses usually persisted over this duration, experiments were discontinued if responsiveness to the BOS diminished or disappeared.
Analysis of electrophysiological data
For analyzing electrophysiological data, we used methods that have been described in previously published studies (Rosen and Mooney 2000). The suprathreshold responsiveness of neurons was calculated by response strength (RS) = SFR – BFR, where SFR and BFR are the firing rates during each whole song stimulus presentation and during the baseline period before each song stimulus presentation, respectively. To determine whether a suprathreshold response was significant for each cell, we used a paired t-test to compare mean firing rates during each song stimulus presentation and during a prestimulus baseline period of equal duration, and P < 0.05 as the criterion for responsiveness. For measuring the subthreshold response, we calculated a z-score for positive area (Zarea): the area measurements were given by the difference between the average area below median-filtered traces during song stimulus presentation (Sarea) and during the period immediately before each song stimulus presentation (Barea), each calculated relative to the baseline membrane potential value estimated by the mode of the prestimulus resting potential
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To maximize the possibility of detecting phasic responses to song playback, we also calculated the Zarea of the response peak: a window (200–800 ms wide) was centered about the highest peak during song presentation, and a window of equivalent size was centered about the largest prestimulus peak. The largest peaks in the median-filtered trace averages were determined by visual inspection. To determine whether a neuron was responsive to a given song at the subthreshold level, we used a paired t-test to compare average area during the peak of each song stimulus presentation and during the peak of a prestimulus baseline period of equal duration, and P < 0.05 as the criterion for significance. To compare the relative response bias to BOS versus one of the other song stimuli [reversed BOS (REV), BOSPHD60, TS1, TS2, and CON], we used a d' statistic, which estimates the discriminability between 2 stimuli for an ideal observer (Green and Swets 1966)
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2 is the response variance for the given stimulus. A d' > 0.5 reflects a net bias to the BOS, whereas a d' = 0 reflects no net bias. This measure is superior to a ratio measure of response strengths because it takes into account the variance of the responses and can report negative values (Solis and Doupe 1997). Statistical tests
In those cases where multiple comparisons were made, an ANOVA was used followed by a post hoc mean comparison using Tukey's test, with significance determined at P < 0.05. Other specific tests used to determine significance are given in the RESULTS and/or figure legends. All values reported are means ± SE, unless otherwise noted.
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RESULTS |
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Behavior
SONGS OF PHD 60 JUVENILES RAISED IN A SEQUENTIAL TUTORING PROTOCOL.
To distinguish whether copying from TS1 had occurred at the end of the first isolation period (PHD 60), we analyzed song of 15 birds where another zebra finch was the tutor during the first posthatch month ("ZF" birds; note that the song of one ZF bird was not recorded at PHD 60, so this bird was used only for the adult song comparisons). Although song structure is only roughly formed at this stage, complicating subjective analysis, blind observers still identified song syllables similar to syllables in the songs of the first tutor in 3 of the 15 juveniles (Fig. 2; Table 1; mean % similarity = 4.1 ± 2.2%, n = 15 birds; copied syllable range 0–1, mean 0.2 ± 0.11; total syllable range 2–9 syllables, mean 4.5 ± 0.42 syllables). Incontrast, blind observers never scored songs of PHD 60 birds as similar to the second tutor, a bird to which the PHD 60 juveniles had not yet been exposed (Fig. 2C; note that for subjective scoring at this stage the "CON" songs were TS2). A further indication of copying from the first tutor was provided by percentage similarity scores calculated using Sound Analysis, an automated routine that estimates similarity between 2 motifs using 4 acoustic parameters: entropy, pitch, FM, and continuity (Tchernichovski et al. 2000). We found the similarity between BOSPHD60 and TS1 was significantly higher than that between BOSPHD60 and TS2 (i.e., a zebra finch song to which they had not yet been exposed) (BOSPHD60 and TS1 50 ± 6.9%, BOSPHD60 and TS2, 35 ± 6.3%, P < 0.02, paired t-test; n = 15). In addition, the mean similarity between the BOSPHD60 and TS1 was slightly but not significantly greater than the mean similarity between the BOSPHD60 and any of 4 different CON songs (Fig. 2C; P < 0.05, ANOVA). Thus analysis of song before introduction of the second tutor provides evidence of partial copying from the first tutor, at least in some birds.
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ADULT SONGS OF SEQUENTIALLY TUTORED BIRDS.
Adult birds that had been sequentially tutored as juveniles by 2 birds showed strong evidence of copying from the most recent tutor (TS2), and little or no sign of copying from TS1 (Figs. 3A and 4A). We exposed a total of 22 zebra finches to a second (zebra finch) tutor starting at 60 days [mean 61.3 ± 0.42 PHD; 16 birds were initially tutored by another zebra finch (ZZ birds), and another 6 birds were initially tutored by a Bengalese finch (BZ birds)]. Syllable copying from the second tutor, as assessed by subjective scoring, was observed in 14/16 adult ZZ and 4/6 adult BZ birds (Fig. 4B). For all 22 birds, we found that on average 2.9 ± 0.4 out of 6.2 ± 0.4 BOS syllables were copied from TS2 (Table 1), yielding a mean subjective similarity score of 48.7 ± 7.0% (Fig. 4). Automated scoring of similarity between BOSAdult and TS2, using Sound Analysis, showed that the BOSAdult was significantly more similar to TS2 than to the songs of nontutor (CON) zebra finches (61.5 ± 5.6% vs. 36.3 ± 2.2%; each BOSAdult was compared with the songs of 4 nontutor zebra finches from our colony). The similarity scores between BOSAdult to TS2 obtained using Sound Analysis (>60% similarity) were comparable to the similarity scores others have obtained when comparing an adult bird's song to that of its tutor (Tchernichovski et al. 2001).
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; Eales 1985; Livingston et al. 2000; Morrison and Nottebohm 1993). Although the number of syllables copied from TS2 varied across birds (as estimated by subjective scoring), 5 (4 ZZ and 1 ZZ) of the 20 birds copied almost all (>80%) of their syllables from their second tutor (see examples in Figs. 3A and 4A). In those birds that copied 2 or more of their syllables from TS2, the syllable sequences were never altered from those of the tutor (Figs. 3A and 4A). The amounts of copying as well as the tendency to maintain syllable sequences in their original order are features typical of the song-learning process in zebra finches (Immelmann 1969). We found little evidence that adult birds retained syllables from their first tutor after exposure to a second tutor. Over 80% (18/22) of the sequentially tutored adult birds showed no evidence of syllables copied from TS1 (Fig. 4B, right; 4/22 birds retained syllables from TS1: one ZZ bird copied one syllable, one BZ bird copied 2 syllables, and 2 BZ birds copied one syllable; based on subjective scoring). Furthermore, estimates of song similarity calculated using Sound Analysis indicated that the mean similarity between BOSAdult and TS1 was not significantly different from the similarity between BOSAdult and CON (48.7 ± 7.0% vs. 36.3 ± 2.2%; NS, ANOVA). However, the incidence of high-similarity scores (i.e., >50% similarity) in the BOSAdult and TS1 comparisons (8/22 birds) was slightly higher than the range of high-similarity scores in other BOSAdult and CON comparisons (BOSAdult >50% similarity to 4 CON songs ranged from 3 to 7 out of the 22 birds, using Sound Analysis). Overall, the BOSAdult tended to resemble the TS2 and not the TS1, as reflected in the mean similarity scores obtained using both subjective scoring and Sound Analysis (Subjective: BOSAdult and TS2, 48.7 ± 7.0%; BOSAdult and TS1, 3.2 ± 1.6%; Sound Analysis: BOSAdult and TS2, 61.5 ± 5.6%; BOSAdult and TS1, 42.0 ± 5.2%).
The similarity to TS2 and not TS1 also characterized the songs of the subset of adult birds that previously had shown clear evidence of copying from their first tutor at PHD 60, based on subjective analysis [n = 7; 3 ZZ and 4 BZ birds; 6/7 adult birds showed copying from TS2; average similarity for all 7 birds' songs to TS2 using Sound Analysis was 59.8 ± 11.9%; note that 2 of these birds' adult songs were judged by subjective scoring to be slightly similar to TS1 (25 and 11%)]. These results indicate that the protocol used in these experiments produces adults with songs copied from the most recent tutor (TS2), with little or no copying from the first tutor to whom they were exposed (TS1). This tendency to copy from the most recent tutor is a trait also manifested in zebra finches tutored serially in a contiguous manner [i.e., without an intervening isolation period; see Eales (1985)].
To further quantify how the BOS changed over development in relation to each of the birds' 2 tutors, we estimated the change in similarity between the BOS and a given tutor song from PHD 60 to after PHD 115 (Fig. 5A). These developmental changes in individual birds' songs were estimated by subtracting the similarity score between the BOSPHD60 and a given tutor song (i.e., TS1 or TS2) from the similarity score between the BOSAdult and the same tutor song. Automated similarity score differences showed that the songs of 16/21 birds became more similar to TS2 after exposure to the second tutor (Fig. 5A, left). Similarly, subjective similarity score differences revealed that songs of 17/21 sequentially tutored birds became more like TS2 over this period (Fig. 5A, right; note that song from one bird was not recorded before the second tutoring period). In contrast, subjective analysis indicated that similarity to TS1 either stayed the same or decreased in 18/21 birds over this same period (Fig. 5A, right). Comparisons of Sound Analysis similarity score differences also revealed that the majority (13/21) of birds' songs decreased in similarity to TS1 over development (Fig. 5A, left). Ultimately, the mean similarity score of the BOS to TS2 estimated using Sound Analysis increased substantially between PHD 60 and 115 (33.5 ± 4.8% similarity between BOSPHD60 and TS2 vs. 61.5 ± 5.6% between BOSAdult and TS2, P = 0.003, paired t-test), whereas over the same period the BOS decreased slightly but not significantly in similarity to TS1 (48.6 ± 6.4% between BOSPHD60 and TS1 vs. 42.0 ± 5.2% between BOSAdult and TS1; NS, P = 0.3, paired t-test). Therefore during development, the BOS increased in similarity to TS2, and most often decreased in similarity to TS1. This result suggests that sequentially tutored birds lost or overwrote their old songs as they learned new songs.
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PHD 90) (Zann 1996), and we confirmed that song was stable after PHD 115 in these sequentially tutored birds by comparing the song at the time of sacrifice with songs produced over the prior one to 3 wk. We noted that all birds' songs changed rapidly in the first 2 to 4 wk after the introduction of the second tutor, and were stable by the end of the 2nd mo after beginning exposure to the second tutor (the current BOS exceeded 90% similarity to the BOSAdult 53.3 ± 5.4 days after the beginning of exposure to the second tutor). Mean similarity scores between the current BOS and the BOSAdult increased from 36.4 ± 4.9% at the day before the beginning of the second tutor period (n = 21 birds; one bird was not recorded before the second tutoring period and 2 birds were not recorded during the second tutoring period), to 91 ± 3.9% 53 days after the beginning of the second tutor period (average of 11 birds whose songs were recorded between 49 and 56 days from the beginning of second tutor). Therefore vocal changes including copying from TS2 occur rapidly after exposure to TS2. Electrophysiological properties of LMAN neurons
JUVENILE LMAN NEURONAL RESPONSIVENESS.
Prior studies suggest that LMAN neurons in juvenile zebra finches raised with normal access to a tutor are already partially selective for the bird's own song and the tutor song by PHD 60 (Solis and Doupe 1997). We were curious to know whether LMAN neurons in PHD 60 birds subjected to more restricted tutor access (i.e., for only the first 35 PHD), as in the first phase of the sequential tutoring protocol used here, exhibited selectivity for their own song as well as the TS1. In the event that responses to each song might manifest themselves at only a subthreshold synaptic level, and not in the action potential discharge of LMAN neurons, we used in vivo intracellular recordings and signal-averaging methods to detect both sub- and suprathreshold activity (Fig. 1B; see METHODS).
We recorded LMAN neuronal activity from a set of juvenile birds at the end of their first isolation period (mean = PHD 63 ± 0.98, n = 8 birds, 7 ZF and 1 BF). At PHD 60, we found that BOSPHD60 and the tutor's song (i.e., TS1) could elicit significant suprathreshold responses in LMAN neurons (Fig. 6A; see METHODS). All 37 LMAN neurons we sampled at this time point responded to the BOSPHD60, and almost half presented with TS1 responded to this stimulus (12/25 neurons; 12 neurons in 3 birds were not tested with TS1) (Fig. 6A and Table 2). Four of 5 birds tested contained TS1-responsive cells; 3 of these 4 birds copied some material from TS1, whereas the one bird that did not contain any TS1-responsive cells also did not copy from TS1, as assessed by subjective scoring. The mean response strength was greater for the current BOS (i.e., BOSPHD60) than that for all other stimuli tested (Fig. 6B; see figure for statistical comparisons). We also assessed the neuronal response at a subthreshold level, by calculating positive area z-scores over the duration of the entire song stimulus (Zarea; see METHODS). In general, the subthreshold response properties were the same as those at the suprathreshold level (Fig. 6A). We encountered a total of 35 neurons that showed significant subthreshold responses. All 35 neurons responded to BOS and almost half presented with TS1 responded to this stimulus (12/25 cells; 12 neurons in 3 birds were not tested with TS1). The mean subthreshold response z-score was greatest for the BOS and the rank order of stimulus-evoked subthreshold response strengths was identical to the pattern seen with suprathreshold response strength measurements (Fig. 6C). These results indicate that the current BOS and TS1 are effective at driving sub- and suprathreshold responses in LMAN neurons of PHD 60 zebra finches tutored with this protocol, and that CON, BOS–REV, and TS1–REV are largely ineffective stimuli.
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The juvenile responsiveness to TS1 could reflect either broadly tuned properties of relatively nonselective neurons or instead narrowly tuned properties of selective neurons. As noted previously, LMAN neurons at PHD 60 as a population exhibited strong responsiveness to the BOSPHD60 and TS1, but failed to respond to CON, BOSPHD60–REV, and TS1–REV, hinting that these neurons were already moderately selective in their response properties (Fig. 6B). We used the d' metric to determine whether LMAN neurons at PHD 60 were selective for forward playback of the current BOS versus other songs (see METHODS; when using the BOS as the referent, d' values > 0.5 reflect a biased response to the BOS over the comparison song). These d' values indicated that LMAN neurons at PHD 60 were strongly selective for the BOSPHD60 versus CON and BOSPHD60–REV (Fig. 6, E and F: d' BOSPHD60–CON = 1.14 ± 0.16, n = 23, d' BOSPHD60–REV =1.03 ± 0.14, n = 37). We also noted that LMAN neurons in these juvenile birds were selective for BOSPHD60 versus TS1 (d' BOSPHD60–TS1 = 0.83 ± 0.13, n = 25). Although the mean d' value for this comparison was lower than BOSPHD60 versus CON, this difference was not significant (Fig. 6E). Furthermore, the cumulative distributions of d' values comparing BOSPHD60 to CON, TS1, and REV were not significantly different from one another [Kolmogorov–Smirnov (K-S) test], suggesting that LMAN neurons in juvenile birds were equivalently selective for the current BOS versus these other stimuli (Fig. 6F). However, TS1-responsive neurons were selective for forward over reverse TS1 playback (Fig. 6E; d' TS1–TS1 REV = 0.61 ± 0.15, n = 25). Therefore LMAN neurons in these birds were already highly selective for the current BOS over other song stimuli and showed selectivity for forward over reverse playback of the first tutor song. These results indicate that LMAN neurons in PHD 60 zebra finches reared with limited exposure to a tutor nonetheless exhibit narrowly tuned responses to their own song and the tutor song.
LMAN NEURONAL RESPONSES TO SONG PLAYBACK IN ADULT SEQUENTIALLY TUTORED BIRDS.
In other systems, patterns of early sensory experience that conflict with later experience can be maintained in a latent manner at the synaptic level (Feldman and Knudsen 1997; Udin 1983). We tested whether LMAN neurons in sequentially tutored adult birds (n = 22) exhibited any responsiveness to the songs of the first tutor or to the transient copies that the juvenile had made of its first tutor's song. In general, we found that LMAN neurons in sequentially tutored adult birds exhibited sub- and suprathreshold responses to the current BOSAdult and/or the TS2, but responded less often or not at all to either TS1 or the BOSPHD60 (Fig. 7; Table 2).
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We investigated whether "latent" synaptic responses to the TS1 or juvenile versions of the BOS might be maintained at a subthreshold level. The results of this subthreshold analysis closely paralleled the results of the suprathreshold analysis, in that both the BOSAdult and TS2 evoked a strong response, whereas the TS1 and BOSPHD60 did not (Fig. 9A; see legend for statistical comparisons). As with the suprathreshold analysis, we did not detect any subthreshold responses to TS1 in adult birds that had copied one or more syllables from TS1 at PHD 60 (Fig. 9B).
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200–800 ms) bracketing the peak response evoked by the tutor's song. Using such a peak subthreshold analysis, we found only 2 additional TS1-responsive neurons, both of which came from one of the 3 birds in which we previously found TS1-responsive cells using suprathreshold criteria (Fig. 9C; i.e., a bird that had not copied from TS1 at PHD 60, as assessed by subjective scoring). In contrast, the same analysis detected an additional 22 cells that were responsive to TS2 (Fig. 9C). Therefore LMAN neurons in adult sequentially tutored birds rarely respond to the transiently copied tutor song, whereas a substantial portion responds to the most recent tutor song. CORRELATION BETWEEN SONG SIMILARITY AND RESPONSE STRENGTH. We hypothesized that stronger suprathreshold responses may be evoked in LMAN neurons of adult sequentially tutored birds by those tutor songs or juvenile versions of the BOS acoustically more similar to the BOSAdult. In this case, the response strength of LMAN neurons to a given song should correlate with the acoustic similarity between the BOS and that song. To investigate this possibility, we plotted the response strength of individual LMAN neurons to TS1, TS2, or BOSPHD60 as a function of the percentage similarity of the BOS to the given song, as measured with either Sound Analysis or subjective methods (Fig. 10). These plots revealed a significant correlation between acoustic similarities and response strength for TS2 and BOSPHD60 but not for TS1 (Fig. 10). In the case of TS1, the absence of a correlation could be accounted for by the fact that only a small fraction of LMAN neurons responded to this stimulus. However, we also noted that the acoustic similarities between the BOSAdult and TS1 estimated using Sound Analysis spanned a range greater than that for the similarities between the BOSAdult and the BOSPHD60 (compare top and bottom left, Fig. 10), suggesting that the lack of responses to TS1 was not simply attributed to acoustic dissimilarities between the BOSAdult and TS1. Therefore acoustic similarity as measured with these 2 techniques can account for LMAN responsiveness for only the most recent tutor song and juvenile versions of the BOS, but not for a tutor song copied only transiently earlier in development.
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; Doupe and Konishi 1991). We were curious to know whether LMAN neurons in sequentially tutored adult birds exhibited auditory response properties like those in normal birds, or were more broadly responsive to songs other than the BOSAdult because of their experience with multiple tutors. Therefore we plotted cumulative distributions of suprathreshold d' values for all 84 song-responsive LMAN neurons that we recorded in sequentially tutored adult zebra finches, comparing individual neuronal responses to the BOSAdult versus either TS1, TS2, BOSPHD60, or CON (Fig. 11A). These cumulative distributions indicated that the majority of LMAN neurons were strongly selective for the BOSAdult over all of the other song stimuli (i.e., a majority of cells showed d' > 0.5). Furthermore, the d' distribution for the BOSAdult versus TS1 completely overlapped the distribution for BOSAdult versus CON (P = 0.94, K-S test), whereas the distribution for the BOSAdult versus TS2 comparison was shifted significantly leftward (i.e., in the direction of decreased selectivity for BOSAdult). The mean d' values revealed equivalently strong selectivity for BOSAdult over TS1, BOSPHD60, or CON, whereas the mean selectivity for BOSAdult versus TS2 was significantly lower than these other values (Fig. 11B; see legend for statistical comparisons). In addition, LMAN neurons were strongly selective for the forward over reverse playback of BOSAdult (d' BOSAdult–REV: 1.45 ± 0.14, n = 27), and the mean d' value for this comparison was almost identical to that obtained from a large sample of LMAN neurons recorded intracellularly in normal adult zebra finches [d' BOSAdult–REV: 1.41 ± 0.08; n = 235 cells; (Rosen and Mooney 2000)]. The subset of LMAN neurons responsive to TS2 was also selective for forward over reverse playback of this song (d' TS2–TS2-REV: 1.51 ± 0.14, n = 22 cells). In summary, LMAN neurons in sequentially tutored adult zebra finches are highly selective for the BOSAdult over other song stimuli, with diminished selectivity for BOSAdult versus the second of the 2 tutor songs to which the bird was exposed during development.
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DISCUSSION |
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Behavior of sequentially tutored birds
The sequential tutoring approach provided juveniles with 2 tutors, affording them an opportunity to copy 2 song models. Birds tutored in this manner could copy from each tutor, overwriting the copy of the first model as the bird mimicked the second tutor. Just before the introduction of the second tutor, the BOS exhibited features indicative of partial copying from TS1. This early copying was most apparent in juvenile zebra finches first exposed to a Bengalese finch tutor, but could also be seen in some juvenile birds raised initially by another conspecific. In contrast, sequentially tutored adults produced songs with copied syllables derived largely from the second and not the first tutor, although more subtle effects of TS1 may have persisted in the adult song of some birds, as indicated by the elevation in Sound Analysis similarity scores >50%. Certain songbird species, including swamp and song sparrows, copy from several different tutors, then undergo selective attrition, retaining only a subset of these vocalizations to form a more limited suite of adult song types (Marler and Peters 1981, 1982a; Nordby et al. 2000). In their capacity to mimic multiple vocal models and retain only a subset of these juvenile imitations in the adult vocal repertoire, sequentially tutored zebra finches roughly resemble these other songbirds. One issue emerging from our results is whether the serial "bilingual" birds we studied retain permanent auditory or motor memories of the songs they transiently learn. In swamp sparrows, song material deleted in the first year can be reexpressed during periods of plastic song production in subsequent springs, implying retention of auditory or motor memories of the "lost" songs (Marler and Peters 1982b). If such auditory memories persist, then one may wonder whether early experience, especially of other species' songs, extends perceptual performance, as for humans with early experience of a language distinct from their native tongue (Tees and Werker 1984).
Sequential tutoring may more closely approximate some aspects of zebra finch song learning in the wild than protocols using only a single tutor. Juvenile zebra finches become independent about PHD 35 (i.e., slightly after exposure to TS1 in these experiments), potentially exposing them to the songs of other conspecifics in addition to their father's song (Immelmann 1969; Zann 1996). Social interactions are especially important in determining which of several potential tutor songs a juvenile subsequently copies, including feeding by adult males (Immelmann 1969) and tutor aggression (Clayton 1987). The importance of extended social interactions increases the likelihood that birds other than the father will serve as the tutor. Indeed, songs experienced later in the sensory acquisition phase (i.e., PHD 35–60) seem to be especially important in shaping the final BOS: juvenile zebra finches sequentially tutored without an intervening isolation period and introduced to the second tutor at PHD 35 develop adult songs copied predominantly from this second tutor (Eales 1985). Although juvenile copying from the first tutor was not addressed in this earlier study, we report here that early models are imitated transiently and that adult sequentially tutored birds copied almost exclusively from the second tutor, rather than fusing material from the 2 sources. The tendency to learn material from a single tutor may be a zebra finch specialization (Bohner 1983; Clayton 1987), and clearly differs from songbirds with multiple song types.
Although isolation extended the bird's capacity to memorize a new model beyond PHD 60, it did not delay song stabilization, which was achieved by about PHD 100, when zebra finches normally crystallize their songs (Immelmann 1969; Zann 1996). This pattern of condensed motor learning resembles other delayed-learning protocols (Morrison and Nottebohm 1993; Tchernichovski et al. 2001). Because some studies suggest that crystallization is chronologically invariant (Funabiki and Konishi 2003; Pytte and Suthers 2000), more complete copying might have occurred after an earlier introduction of the second tutor, as in other sequential-tutoring studies (Eales 1985).
Electrophysiological correlates of sequential learning
Given the capacity for serial copying, one possibility is that areas important to song learning retain vestiges of early auditory/vocal experience, even when this experience differs from the current song. In other systems, including visually calibrated auditory space maps in owl midbrain (Feldman and Knudsen 1997) and areas of binocular convergence in the frog optic tectum (Udin 1983), distinct synaptic projections patterned by conflicting types of juvenile experience are maintained into adulthood. One area of the songbird brain influenced strongly by experience of the BOS and the TS is LMAN: developmental changes in structure and function of synapses within LMAN are retarded in birds raised without a tutor (Livingston et al. 2000; Wallhausser-Franke et al. 1995), and LMAN neurons of late juvenile (i.e., PHD 60) and adult zebra finches are strikingly selective to playback of the BOS (Doupe and Konishi 1991; Solis and Doupe 1997). These features make LMAN a likely site to search for persistent changes wrought by early song-learning experience.
Consistent with the behavioral evidence of transient copying of TS1, the initial song-learning experience clearly influenced juvenile LMAN response properties. We found that LMAN neurons in juvenile birds subjected to only the first tutoring and isolation phases responded strongly to the juvenile BOS and to TS1. Rather than reflecting properties of poorly tuned neurons, these responses were highly selective for playback of the juvenile BOS and the TS1 over either of these songs played in reverse, and for the BOS versus other conspecific songs. Therefore even at a stage of development where copying was rudimentary, and despite a prolonged period of isolation from the tutor, LMAN neurons displayed selective responses to both the BOS and TS, resembling LMAN neurons in PHD 60 birds reared with unrestricted tutor access (Doupe and Konishi 1991; Solis and Doupe 1997). Therefore early experience of the juvenile BOS and/or TS1 influences auditory properties of LMAN neurons, raising the possibility that such effects persist in the adult LMAN.
In contrast to this idea, LMAN neurons in the adult do not maintain responsiveness to songs that the bird transiently produced or to the tutor songs on which these ephemeral vocalizations are based. Despite the development of selective BOS- and TS1-evoked responses in the juvenile LMAN, no sub- or suprathreshold auditory responses were evoked in LMAN by TS1 or BOSPHD60 in those adult birds that had copied previously from the first of their 2 tutors. Although a small fraction of LMAN neurons in all of the adult birds we recorded from did respond selectively to TS1 in the adult, these neurons were obtained from birds exhibiting no clear evidence of early copying based on subjective analysis. Throughout development, the response profiles of LMAN neurons shift to the current song (and the most recent TS on which it is based), but highly selective responses for the juvenile BOS are not maintained in the adult. Together with the electrophysiological data in juveniles, these results indicate that selective responses to the current BOS and the first tutor are present at PHD 60, but are lost or overwritten as the bird copies from the second tutor. Therefore auditory tuning in LMAN differs from sensory tuning in the midbrain of the barn owl or the frog, where 2 conflicting sensory representations formed during early development are maintained in the adult brain, albeit with one representation persisting as latent synaptic connections (Brickley et al. 1994; Zheng and Knudsen 1999).
Unlike neurons in primary sensory areas, LMAN neurons exhibit song-related motor and auditory activity (Hessler and Doupe 1999). Therefore LMAN tuning could be influenced by auditory experience, by song motor activity, or by combinations of factors, including auditory experience of one's own song in the context of either the memorized model or song motor activity. The most compelling evidence that the BOS evokes LMAN activity in the context of the memorized model is that LMAN neurons in adult birds with songs disrupted developmentally by tracheosyringeal nerve section are largely unresponsive to BOS playback (Solis and Doupe 2000). In this study, acoustic similarity to the BOS may account for TS2- but not TS1-evoked responses in LMAN neurons of the adult (see Fig. 10). The findings reported here indicate that auditory and/or motor experience of the BOS in the context of the current tutor model influences LMAN auditory tuning, consistent with prior evidence that self-generated vocalizations are the key determinant of auditory selectivity in the song system (Doupe 1997; Volman 1993). One prediction of a model where self-generated experience dictates LMAN responsiveness is that tracheosyringeal nerve section in the adult will induce LMAN neuronal retuning. Finally, if songbirds that learn transiently retain "discarded" auditory or vocal motor memories, rather than losing or overwriting them with their current song, these memories are stored in other areas of the songbird's brain or in forms other than BOS-selective neurons.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. Mooney, Dept. of Neurobiology, Duke University School of Medicine, Box 3209, Durham, NC 27710 (E-mail: mooney{at}neuro.duke.edu).
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ABSTRACT
INTRODUCTION
