Song and brain development in canaries raised under ...

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Song and Brain Development in Canaries Raised Under Different Conditions of Acoustic and Social Isolation Over Two Years Stefan Leitner,1,2 Clive K. Catchpole1 1

School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 OEX, United Kingdom

2

Department Neurobiology of Behaviour, Max Planck Institute for Ornithology, D-82319 Seewiesen, Germany

Received 23 January 2007; accepted 15 March 2007

ABSTRACT: Early isolation experiments indicate that male songbirds learn their songs during an early sensitive period, although later work has shown that some open-ended learners modify songs in later years. Recent isolation experiments suggest that in some species song has a stronger genetic basis than previously thought. This study raised domestic canaries under different combinations of acoustic and social isolation and followed song development into the second year. Males raised alone in acoustic isolation developed songs with normal syllables, but larger repertoires and also produced syllables with lower repetition rates when compared to controls. The smallest repertoire occurred in males raised in a peer group. Isolate males had a smaller song control nucleus HVC than controls, but there was no effect on nucleus RA or on brain weight in general. In the second year, after introduction into a large nor-

INTRODUCTION Experience of social and environmental cues during a discrete period after birth can inﬂuence the development of distinct behaviors. One such behavior is the Correspondence to: S. Leitner ([email protected]). Contract grant sponsor: BBSRC; contract grant number: BBC5002601. ' 2007 Wiley Periodicals, Inc. Published online 24 May 2007 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/dneu.20521

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mal colony, isolate and peer group males adjusted their syllable repertoire to normal size. In particular, the isolates reduced their repertoire even though the size of HVC showed a signiﬁcant increase in volume. However, songs of isolate and peer group males still differ in repetition rate and number of single syllables in the common aviary. In contrast, control males showed low syllable turnover and no signiﬁcant change in repertoire size. Nor did they show any signiﬁcant change in the volumes of song control nuclei. It seems that complete isolation affects only some aspects of song and brain development, and later socialization corrects some but not all of these in the second year. ' 2007 Wiley Periodicals, Inc. Develop Neurobiol 67: 1478–1487, 2007

Keywords: song development; song learning; social interaction; acoustic isolation; brain differentiation; song control system

song of songbirds, where learning from adults is important in the development of complex songs (Catchpole and Slater, 1995). Closed-ended learners usually acquire new songs during a period early in life and are represented by the zebra ﬁnch (Taeniopygia guttata) (Eales, 1985), and the chafﬁnch (Fringilla coelebs) (Slater and Ince, 1982). In open-ended learners such as the canary (Serinus canaria) the ability to modify the content of the bird’s song persists throughout life (Nottebohm and Nottebohm, 1978; Nottebohm et al., 1986). The social context of song learning is also important. It has been shown that live tutors have a signiﬁ-

Song and Brain Development in Social Isolation

cantly higher impact on the strength of song learning compared to learning from tapes (Baptista and Petrinovitch, 1984; Chaiken et al., 1993), suggesting that the social context is important for song learning. Clayton (1987) showed in the zebra ﬁnch that social inﬂuences also guided the choice of which male to learn from. Poirier et al. (2004) found in starlings (Sturnus vulgaris) that direct social contact is important for song development, and overrides auditory information acquired earlier in life. Hearing song from adults was thought to be essential in order to develop species-speciﬁc song (Thorpe, 1958; Nottebohm, 1968). In a study on black-capped chickadees (Parus atricapillus), Kroodsma et al. (1995) found that handraising tape-tutored males can result in an enhanced repertoire over that shown by wild birds. In contrast, there is evidence that young songbirds raised in acoustic isolation have the potential to develop quite normal song (gray catbird Dumetella carolinensis, Kroodsma et al., 1997; sedge warbler Acrocephalus schoenobaenus, Leitner et al., 2002). However, these birds developed larger repertoires than birds that have been exposed to playback. Recently, Gardner et al. (2005) suggested that certain innate rules must govern song development in canaries, as the typical adult song structure is present in birds raised in acoustic isolation. Song production and song learning is controlled by the song control system in the forebrain and some studies have found a clear relationship between song complexity (syllable repertoire size) and the volume of the song control nucleus HVC (reviewed in Garamszegi and Eens, 2004), but see Leitner and Catchpole (2004). In a study on sedge warblers, volumes and dendritic spine density of HVC and RA did not differ between birds that have been hand-raised in acoustic isolation and those raised with song playback exposure (Leitner et al., 2002) suggesting that song and neural substrate can develop independently of song exposure during ontogeny. Early investigations on male canary song development were conducted on different canary strains where males reared in acoustic isolation developed normal song structure (Metfessel, 1935; Poulsen, 1959). Since the 1970s juvenile and adult song development has been studied in some detail (e.g. Waser and Marler, 1977; Nottebohm and Nottebohm, 1978; Leitner and Catchpole, 2004). Gardner et al. (2005) suggested a time segregation in the song learning process with different learning strategies in young and adult canaries. Lehongre et al. (2006) recently showed that male canaries reared in group isolation could not readjust syllable abnormalities when introduced to normal males later in life. The aim of the

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present study was to investigate the song development of domesticated canaries much further under different social and acoustic conditions, and also to examine to what extent their songs change in later life. We further investigated whether changes in song development corresponded to changes in the morphology of the song control system, focusing on HVC and RA which are particularly important in song production.

MATERIALS AND METHODS Animals We selected eight breeding pairs from our colony of common outbred domesticated canaries (n ¼ 61) kept at Seewiesen/Germany (478580 N, 118140 E) in a large outdoor aviary with indoor shelter on a natural photoperiod. First, these pairs each raised a complete clutch in the aviary and were then transferred into cages (56 3 28 3 37 cm3) that were placed inside sound-proof chambers (65 3 43 3 56 cm3 inside, 103 3 57 3 101 cm3 outside) on a 14:10 L/D photoperiod. Each pair was provided with plastic nest bowls and nesting material (as in the aviary) that allowed females to build a nest and lay eggs. One to two days after hatching, the male was removed from the cage and females fed the young on their own. After 5 weeks, sibling juveniles were transferred into another sound-proof chamber, keeping juveniles of one clutch per chamber together. Clutches comprised on average 3.0 6 1.2 juveniles. The juveniles stayed there for 1 week before they were randomly assigned to the experimental groups. At that time, a small blood sample (10–30 lL) was collected from the wing vein of each juvenile and stored in queens lysis buffer (0.01 M Tris-HCl, 0.01 M NaCl, 0.01 M Na-EDTA (pH8.0), 1% (v/v) n-lauroylsarcosine, pH 8) at room temperature until analysis for molecular sexing. Group 1: Isolate Males. From the age of 6 weeks, six juvenile males were kept singly in cages in the sound-proof chambers. Between 2–12 months of age they received a daily 30 min playback of a mixture of control natural sounds (water ripple, sea rush, wind). We adjusted a seasonal photoperiod that was gradually altered from 14:10 L/ D in spring when birds were juvenile to 9:15 L/D in winter and back to 14:10 L/D in the next spring when birds approached the age of 1 year, thus simulating a natural seasonal change in photoperiod. When birds were 11–12 months old, their songs were recorded using Vivanco EM 216 Condenser microphones (Vivanco GmbH, Ahrensburg, Germany) and a Sony TC-D5M cassette recorder (Sony, Tokyo, Japan). Afterwards, three birds were randomly chosen and sacriﬁced for neuroanatomical analysis. The remaining three birds were brought into a normal acoustic environment by introducing them into the canary colony. This was done by ﬁrst placing these birds together in a small aviary for 1 week and then releasing them into the Developmental Neurobiology. DOI 10.1002/dneu

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large colony. They were allowed to stay there for 1 year on a natural photoperiod and their songs were recorded again at an age of 23–24 months and then sacriﬁced for neuroanatomical analysis. Group 2: Peer Group Males. Six juvenile males were selected for this experimental group. From the age of 6 weeks, these birds were kept together in a separate indoor aviary without acoustic contact to other birds. This aviary had a normal acoustic environment, in contrast to the impoverished conditions within a sound proof chamber, and was only lacking the vocalizations of other species and adult canaries. They remained in the aviary until they were 12 months old. Again, we adjusted a seasonal photoperiod that was gradually altered from 14:10 L/D in spring when birds were juvenile to 9:15 L/D in winter and back to 14:10 L/D in the next spring when birds approached the age of 1 year, thus simulating a natural seasonal change in photoperiod. For recording at 12 months they were then placed in cages (56 3 28 3 37 cm3) and moved into a large recording room (330 3 264 3 236 cm3). Songs were recorded using a Sennheiser ME 67 microphone (Sennheiser electronic, Wedemark, Germany) and a Sony TC-5M cassette recorder. Afterwards, three birds were randomly chosen and sacriﬁced for neuroanatomical analysis. The remaining three birds were transferred to a normal acoustic environment by introducing them into the main canary colony. The males were allowed to stay for one further year in the aviary on a natural photoperiod, and their songs were recorded again at the age of 24 months. They were then sacriﬁced for neuroanatomical analysis. Group 3: Control Males. Six males that were transferred into the large communal aviary were chosen as the main control. They stayed in the aviary with the population of adult birds and their songs were recorded when they were 1 year old. Recording procedures were the same as for Group 2, and three males were then randomly chosen and sacriﬁced for neuroanatomical analysis. The songs of the remaining three birds were recorded again when they were 2 years old, and the birds were then sacriﬁced for neuroanatomical analysis. Birds were kept on a natural photoperiod.

Song Analysis Sonographic analysis used Canary 1.2.4 software (Cornell Laboratory of Ornithology, Ithaca, NY) on a Power Macintosh computer. Sampling rate was at a frequency of 22 kHz with a 16 bit sample size. The frequency/time resolution was set at 342 Hz with a frame length of 256 FFT. Song analysis was ﬁrst performed on spectrograms by visual inspection with special focus on measuring syllable repertoire size, syllable repetitions, and number of \sexy syllables" (for deﬁnition, see below). In addition, a catalogue of the different syllables per individual bird in each year was prepared in order to identify identical syllables within and between years. The syllable repertoire is the number of different syllables that a bird uses to construct songs and our Developmental Neurobiology. DOI 10.1002/dneu

protocol followed previously described methods to quantify canary songs (Leitner et al., 2001; Leitner and Catchpole, 2004). We obtained a minimum sample of 350 s of song from each bird within each year. This is sufﬁcient to estimate the repertoire size, as a cumulative plot of new syllables reaches an asymptote well before (Halle et al., 2003). A song is deﬁned as a syllable sequence longer than 1.5 s that contain intervals that are not longer than 0.4 s (see also Leitner et al., 2001). Single syllables are syllables without repetition. Repetition rate was counted per repeated syllable (produced in a tour), as the number of syllable repetitions per second (Hz). We also counted the \sexy syllables" that have a characteristic complex (more than one element) structure both with rapid frequency modulation and high repetition rate (Vallet and Kreutzer, 1995; Vallet et al., 1998). We used the term \sexy syllables" for all complex syllables which possess the physical characteristics that have been experimentally shown to induce sexual displays in females (see Vallet et al., 1998; Leitner and Catchpole, 2002). The proportion of single syllables (%) and the proportion of sexy syllables (%) were deﬁned as the number of these syllables divided by the total syllable repertoire size. Song analysis was done blind to rearing condition.

Sex Determination DNA was extracted with the GFX Genomic Blood DNA Puriﬁcation Kit (Amersham Biosciences) according to manufacturers instructions except for using 50 lL blood/Queens mix as starting material (instead of 5 lL whole blood). Sex determination was carried out according to Grifﬁths et al. (1998) using the primers P2 and P8, with a maximum of 50 ng DNA per sample. After the PCR, DNA fragments were separated by size on a 2% agarose gel containing ethidium bromide, and visualized under UV light. Images were then photographed with a Polaroid GelCam camera (Polaroid Corp., Dumbarton, UK) for later analysis and the sexing technique conﬁrmed by inclusion of samples from individuals of known sex.

Brain Analysis Birds of the three groups were sacriﬁced with an overdose of chloroform when they were 1 year (n ¼ 9) or two years old (n ¼ 9). Birds were perfused transcardially with 0.9% saline followed by 4% phosphate-buffered formaldehyde solution (FPBS). Brains were post-ﬁxed in FPBS and their weight recorded. Testes were removed, stored in FPBS, and weighed. One half of each brain was immersed in 10%, followed by 30% phosphate-buffered sucrose. Brains were then sectioned parasagittally on a freezing microtome (Leica CM 3080, Leica Microsystems Bensheim, Germany) at 30 lm, collected in phosphate buffered saline and alternate sections were mounted onto Superfrost Plus microscope slides (Menzel Gla¨ser, Braunschweig, Germany) for Nissl staining. Slides were analyzed under brightﬁeld with a Leitz Aristoplan microscope (Leitz Wetzlar, Germany). The areas of the brain regions HVC and RA were video-

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Figure 1 Song structure in one year old experimental canaries. (a) The individual syllable repertoire sizes were clearly different between the birds in the three experimental groups. (b) The number of single syllables within the repertoires was highest in isolate and peer group birds. (c) Sexy syllables occurred only rarely in isolates and peer group birds. (d) The repetition rate (Hz) of repeated syllables was highest in control birds. digitized using a PC equipped with an image analysis system (MetaMorph 4.6, Visitron Systems, Germany). Volumes of brain regions were analyzed blind to rearing condition and were calculated from every third section as the sum of the area sizes multiplied by 120 lm (section interval 3 section thickness).

Statistical Analysis Comparisons between the treatment groups within 1 year were performed by means of a one-way ANOVA, comparisons between years with a repeated measures ANOVA, and brain measurements with a one-way ANOVA, followed by post-hoc comparisons using Stat View 5.0 software. Values shown are means 6 SD.

RESULTS Song Structure in One Year Birds The three different conditions had a signiﬁcant effect on the syllable repertoire size of adult males. All three experimental groups could be clearly distinguished by total syllable repertoire size in the ﬁrst year (F2,15 ¼ 31.27, p < 0.0001). Males kept in isola-

tion had the highest repertoire size, followed by aviary control birds, whereas males of the peer group had the smallest repertoire [Fig. 1(a)]. The highest repertoire size of an isolate bird was 62, in a control Bird 49 and in a bird of the peer group 21. In canary songs, the proportion of single syllables is a crucial estimate for the ability to form phrases as these require syllable repetitions. The proportion of single syllables differed between experimental groups (F2,15 ¼ 47.17, p < 0.0001) and was larger in isolate and peer group males, both signiﬁcantly different from the control birds (p < 0.0001). A comparison between the proportion of single syllables between isolates and peer group just failed to attain signiﬁcance (p ¼ 0.0504), see Figure 1(b). With the proportion of sexy syllables in the repertoire, we found signiﬁcant differences across experimental groups (F2,15 ¼ 15.12, p ¼ 0.0003). None were found in the peer group birds and the three groups can clearly be distinguished by the proportion of sexy syllables within the syllable repertoire [post-hoc tests, all p < 0.05, Fig. 1(c)]. Song length did not differ between the three groups (F2,15 ¼ 0.765, p ¼ 0.484), but there was a signiﬁcant difference in syllable repetition rate (F2,15 ¼ 6.66, p ¼ 0.009). Birds reared in isolation Developmental Neurobiology. DOI 10.1002/dneu

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Figure 2 Changes in song structure from the ﬁrst year to the second year in experimental canaries transferred to a social environment. Open boxes: isolate birds, open circles: peer group birds, full circles: control birds. (a) Changes in repertoire size in isolates and peer group birds, but control birds did not change their repertoire size. (b) Changes in the number of single syllables within the repertoire. (c) Changes in the number of sexy syllables. (d) Repetition rate (Hz) was still reduced in isolates and peer group birds in the second year.

and in the peer group had a reduced syllable repetition rate compared to control birds [post-hoc tests, p < 0.05 and p < 0.01, respectively, see Fig. 1(d)].

Changes in Song Structure from the First to the Second Year The second year birds signiﬁcantly changed the total number of syllables after being in the aviary (F2,6 ¼ 12.07, p ¼ 0.008). This was clearly an effect of treatment (F2,6 ¼ 21.82, p ¼ 0.002), but not of year (F1,6 ¼ 1.63, p ¼ 0.249). Post-hoc comparisons revealed that isolates can clearly be distinguished from control and peer group birds (p ¼ 0.010; p ¼ 0.0004, respectively). More speciﬁcally, isolate birds reduced repertoire size (t ¼ 4.39, n ¼ 3, p ¼ 0.048), whereas birds of the peer group increased repertoire size (t ¼ 4.60, n ¼ 3, p ¼ 0.044). No change in repertoire size occurred in the control birds (t ¼ 1.51, n ¼ 3, p ¼ 0.269), see Figure 2(a). In the comparison between years there is a strong overall difference in the proDevelopmental Neurobiology. DOI 10.1002/dneu

portion of single syllables (F2,6 ¼ 33.70, p ¼ 0.0005), and this is mainly due to a general decline in the proportion of single syllables as the birds get older, from the ﬁrst to the second year [F1,6 ¼ 53.19, p ¼ 0.0003, Fig. 2(b)]. We also found signiﬁcant differences in the proportion of sexy syllables [F2,6 ¼ 24.07, p ¼ 0.001, Fig. 2(c)]. Only one syllable that fulﬁlled the criteria for a sexy syllable was found in the repertoires of the previous peer group birds. However, there was no overall effect of year on the amount of sexy syllables (F1,6 ¼ 4.80, p ¼ 0.071). There was also no difference in song length between years (F1,6 ¼ 0.069, p ¼ 0.802). Birds reared in isolation and in the peer group still had a lower repetition rate of repeated syllables compared to controls as we did not ﬁnd a signiﬁcant difference when comparing the ﬁrst and second year [F2,6 ¼ 2.47, p ¼ 0.165, Fig. 2(d)]. Syllable repertoire composition clearly differed between the experimental groups (F2,6 ¼ 11.87, p ¼ 0.008). Syllable turnover (i.e. exchange of syllables) changed dramatically from the ﬁrst to the second year both in isolates and the peer group and was not signif-

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Figure 3 Percentage of identical syllables between ﬁrst year and second year in a new social environment. Relatively few syllables from the ﬁrst year are still present in the repertoires of isolates and peer group birds in the second year.

icantly different between these two groups (p ¼ 0.813). Only 20% of syllables were retained in the second year repertoire, whereas control birds retained on average 69% of their repertoire. This was signiﬁcantly different from isolates (p ¼ 0.005) and the peer group (p ¼ 0.006), see Figure 3. Figure 4 shows examples of songs from the three experimental groups within the ﬁrst year, and then after introduction to the aviary in the second year.

Morphological Measurements There was no signiﬁcant effect of the experimental treatment on brain weight (F5,12 ¼ 1.81, p ¼ 0.185), but there was a signiﬁcant effect on the volume of HVC (F5,12 ¼ 3.16, p ¼ 0.048). This was mainly due to a difference in the isolate birds, which had a significantly smaller HVC after 1 year compared to the second year when translocated into the large communal aviary (p ¼ 0.009). First year isolates also had a smaller HVC than birds in the other groups (p < 0.05 for all comparisons). There were no differences between ﬁrst and second year birds in the peer group or the control group (all p > 0.05, Fig. 5) and there was no effect of experimental treatment on RA size in general (F5,12 ¼ 1.54, p ¼ 0.251). Finally, testes size did not show signiﬁcant differences between the experimental groups (F5,12 ¼ 2.87, p ¼ 0.063, isolates: 1st year: 92 6 20, 2nd year: 235 6 71; peer

Figure 4 Examples of sonagrams of three birds from the different experimental treatments (a–c). Songs of the same birds within a group are shown in the ﬁrst year and in the second year, after introduction into a new social environment. Developmental Neurobiology. DOI 10.1002/dneu

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Figure 5 Neuroanatomical measurements. Volumes (mm3) of the song control nuclei HVC (a), and RA (b), in 1 and 2 year birds as revealed by Nissl staining. One year old isolated males have a signiﬁcantly smaller HVC compared to males in the other groups. Photomicrographs of HVC (c), and RA (d), of a 1 year old isolated male, and of HVC (e), and RA (f), of an isolated male that has been kept in a communal aviary in the second year. Scale bar ¼ 100 lm.

group: 1st year: 141 6 46, 2nd year 109 6 27; control: 1st year: 125 6 56, 2nd year: 169 6 73, values in mg).

DISCUSSION Our study conﬁrms the striking result we found in the sedge warbler (Leitner et al., 2002), that males raised in acoustic isolation can still produce quite normal songs and can have a larger syllable repertoire size. We also show that, in the canary, isolates can make substantial changes in song structure when brought into a normal environment in the second year. The remarkable differences in song produced by birds in the Developmental Neurobiology. DOI 10.1002/dneu

different experimental conditions in the ﬁrst year could be partly readjusted when the birds were brought into the large colony. There were also some changes in the brain as the volume of HVC increased in those birds that were introduced to conspeciﬁcs after spending their ﬁrst year in isolation. This is quite different from the results of Thorpe (1958) who hand-reared isolated chafﬁnches and found that they were entirely lacking the normal features of chafﬁnch song. He also grouped birds and raised them in isolation together. These birds built up a common song pattern in each group. A similar effect could be observed in our peer group canaries that developed songs with a smaller repertoire size. Unlike the canary, the chafﬁnch is a closed-ended

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Table 1 Examples of Studies Investigating Song Development of Male Birds in the Absence of an Adult Song Model Species Chafﬁnch (Fringilla coelebs) Domesticated Canary (Serinus canaria)

Sedge warbler (Acrocephalus schoenobaenus) Grey catbird (Dumetella carolinensis) Swamp sparrow (Melospiza georgiana) Song sparrow (Melospiza melodia)

Type of Treatment

Effect on Repertoire Size

Isolation Peer group Isolation Isolation Deafening Deafening Peer group Isolation Peer group

? ? Reduced Reduced No Enlarged Reduced

Yes Yes No No Yes Yes Yes Yes Yes

Isolation Isolation Isolation Isolation

Enlarged Enlarged Reduced Reduced

No No Yes Yes

learner and Thorpe (1958) stated that \it is almost impossible for a chafﬁnch to learn any new songs or indeed to modify its old ones after it has reached the age of about 13 months". In our canaries we were clearly able to monitor song changes during the second year and see how they depend upon social context. Indeed, our peer group canaries did change their songs, but in a less distinct manner when compared to isolates. This suggests that the birds in the peer group had already elaborated their songs during their ﬁrst year, albeit less strongly compared to the controls. However, despite song adjustment towards a more \normal" canary song, males also retained some abnormalities in the second year. A very striking result is that the isolated males developed larger repertoires than the controls and that peer group males developed smaller repertoires than controls. Larger repertoire sizes, observed in isolate males conﬁrmed the results of our previous study in the sedge warbler (Leitner et al., 2002). In the canary, some earlier studies found a smaller repertoire size in birds that were deprived from imitation or hearing (Marler and Waser, 1977; Gu¨ttinger, 1981). In contrast, in a recent study the repertoire size of isolation reared peer-grouped males was similar compared to control birds (Lehongre et al., 2006), see Table 1 for a summary. However in our study, the songs of isolates and peer group birds can be distinguished from normal canary song by three main features. First, there is a signiﬁcant difference in repertoire size and second, the percentage of single, nonrepeated syllables is almost three times larger in isolate and peer group birds. One possible consequence is that, there are less sexy syllables in the repertoires of isolate and peer group birds. Also, the songs of isolate and peer group birds had a lower syl-

No No

Effect on Song Structure

Reference Thorpe, 1958 Metfessel, 1935 Poulsen, 1959 Marler and Waser, 1977 Gu¨ttinger, 1981 Lehongre et al., 2006 This study

Leitner et al., 2002 Kroodsma et al., 1997 Marler and Sherman, 1985 Marler and Sherman, 1985

lable repetition rate. These ﬁndings differ from the study in the sedge warbler, as apart from syllable repertoire size, there was no signiﬁcant difference in song structure between isolate birds and their control siblings (Leitner et al., 2002). Thus the enhanced repertoire of the canaries housed in isolation could be caused by a lack of attrition of syllables formed in subsong. In contrast, the reduced repertoire of the peer group canaries could then be due to attrition, leading to convergence on the subset of syllables that all birds were able to produce. The song control system was clearly affected by housing the birds in isolation as they developed a smaller HVC. After keeping isolate birds yet one more year in an aviary where they could interact with other canaries, these nuclei had increased in volume. There were many opportunities for interactions, ranging from chasing other males at the feeding site to ﬁnding a mate. That the increase in HVC volume is more likely due to social interactions and not just due to exposure to song can be deduced from the sedge warbler study. In this study there were no size differences in HVC between isolate birds and birds subjected to playback (Leitner et al., 2002). These birds did not have contact with adult birds and were not kept in a large aviary as was the case in our canaries. Neuroanatomical changes related to social parameters mainly concern dominance hierarchies in other animal groups. For example, in the plainﬁn midshipman (Porichthys notatus), so-called type I and type II male ﬁsh perform a different vocal courtship that is correlated with differences in motor neuron structures controlling the sonic muscles (Bass, 1992). Very recently, Boseret et al. (2006) found a different HVC size of testosterone-implanted male canaries in relation to social context. In their study, HVC volume Developmental Neurobiology. DOI 10.1002/dneu

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was larger in males that were housed with a female compared with males that were housed with birds of the same sex. In our study, the previously isolated birds experienced a completely new social environment interacting with a large number of individuals. Isolates were exposed to not only the presence of singing males as well as females, but also to a wealth of new somatosensory senses that need to be integrated. Remarkably, while both isolates and peer group birds changed their repertoire in the new environment by about 75%, we only observed an increase in HVC volume in isolate birds. This strengthens the hypothesis that a new social environment also stimulated neural growth in HVC. In these birds, there was no increase in RA size, which indicates a rather conservative modulation of sub-syllable motor units, as RA functions in that particular motor task (Yu and Margoliash, 1996). The peer group birds, although they also encountered a new situation in the aviary were used to the same canary interactions. Although they had to cope with new songs no increase in HVC volume appeared to be necessary. Isolates and peer group birds retained the potential to change their songs in the second year in both syllable repertoire size and turnover. These birds adjusted their song according to the new situation they encountered in the aviary. Remarkably, they retained only 10%–20% of their syllables that they used in the ﬁrst year. This was mainly due to the change of single syllables to new repeated syllables that were presented in a phrase, thus more resembling normal canary song. In contrast, males in the control group showed a rather low syllable turnover and no signiﬁcant change in repertoire size in the second year. Age-dependent plasticity in the reprogramming of songs were also found by Gardner et al. (2005) who tape-tutored isolated Waterslager canaries with canary songs that lacked the typical phrasing. After treatment with testosterone, they developed a normal song structure. Lehongre et al. (2006) could not observe an alteration of syllable ﬁne structure in their isolation reared peer-grouped males when they have been kept with normal birds of the same sex from 1– 5 years of age. Therefore, canaries might have some innate template of normal song that can be activated by physiological or social stimuli. Our experiments clearly show that social interactions with both males and females are very important for song development. Baptista and Gaunt (1997) classiﬁed the canary as a facultative social learner. Birds of this category are able to learn from a tape; however the presence of a live tutor enhances the accuracy of learning. This is in contrast to obligate social learners that are unable to learn only from Developmental Neurobiology. DOI 10.1002/dneu

tapes. Another important aspect is learning from peers and not from adults. There is evidence that na¨ıve hand-reared juveniles deprived of contact with adult conspeciﬁcs tend to produce abnormal songs (Baptista, 1996). Peer-grouped birds can sometimes delay the sensory learning phase and then exposing them to model song will result in the development of a normal species song. Our results point in the same direction, as our peer groups developed a restricted song repertoire, but were able to modify their songs later in life. In contrast, isolate birds had to reduce their large improvised repertoire and also exchange a number of syllables later. Yet the isolates and peer group birds in our experiment still had more in common than control males concerning changes in repertoire composition, as these birds have not heard the novel syllables before that are then acquired through auditory as well as motor learning. The failure of complete restoration of normal canary song structure may well be due to the lack of a song model in the ﬁrst year, despite the strong impact of the later social environment and the consequent enlargement of HVC in previously isolate birds. We thank Sylvia Kuhn for help with molecular sexing and Roswitha Brighton for animal care. We are grateful to Cornelia Voigt for help with ﬁgure preparation.

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Developmental Neurobiology. DOI 10.1002/dneu