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Experience with songs in adulthood reduces song-induced gene expression in songbird auditory forebrain
Neurobiology of Learning and Memory 86 (2006) 330–335 www.elsevier.com/locate/ynlme
Experience with songs in adulthood reduces song-induced gene expression in songbird auditory forebrain Tammy L.B. McKenzie ¤, Alexandra M. Hernandez, Scott A. MacDougall-Shackleton Department of Psychology, University of Western Ontario, London, Ont., Canada Received 6 March 2006; revised 17 May 2006; accepted 17 May 2006 Available online 27 June 2006
Abstract Male songbirds learn to produce song within a limited phase early in life; however they continue to learn to recognize songs in adulthood. Studies looking at Zenk activation after exposure to songs learned early in life for song production and songs learned in adulthood show opposite patterns of activation, suggesting distinct neural mechanisms may be involved in these two forms of learning. In this study, we look at IEG Zenk activation in auditory regions NCM and CMM of song sparrows (Melospiza melodia) to see whether recent exposure to song in adulthood leads to greater or decreased Zenk activation upon hearing that song versus a novel song. We found signiWcantly lower activation in birds exposed to previously heard songs versus novel songs in vNCM but not dNCM, though further analysis suggest an overall trend in NCM. We found no signiWcant diVerence in the amount of activation to previously heard songs vs. novel songs in CMM. These results support previous Wndings suggesting that activation is reduced to learned stimuli; we discuss possible implications of these Wndings in relation to song production learning early in life and song recognition learning in adulthood. © 2006 Elsevier Inc. All rights reserved. Keywords: Zenk; Egr-1; Melospiza melodia; Birdsong
1. Introduction In most species male songbirds learn to produce song within a limited time frame early in life, yet there is evidence that males also learn to recognize songs in adulthood, and can do so rapidly (e.g., Stripling, Milewski, Kruse, & Clayton, 2003). Song sparrows, Melospiza melodia, provide a case study. Males in this species learn to produce songs early in life (Marler & Peters, 1987). Male song sparrows learn to imitate songs they hear during their Wrst summer and autumn (Nordby, Campbell, & Beecher, 2001) and once song is crystallized new songs are not added to the adult repertoire (Nordby, Campbell, & Beecher, 2002). Although song sparrows are clearly age-limited learners in regard to imitative vocal learning, they may, however, retain the ability to learn to recognize new songs into adulthood. For example, individ* Corresponding author. Present address: Department of Psychology, Brandon University, 270-18th Street, Brandon, Man., Canada R7A 6A9. E-mail address: [email protected] (T.L.B. McKenzie).
1074-7427/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nlm.2006.05.002
ual recognition by vocalization is widespread in songbirds (Lambrechts & Dhondt, 1995; Stoddard, 1996). Individual recognition by voice is typically inferred by diVerential responses to playback of a neighboring male’s song from the appropriate or an inappropriate territory boundary, or through neighbor/stranger discrimination. Male song sparrows can discriminate a neighbor’s from a stranger’s song on the basis of a single song from each (Stoddard, Beecher, Horning, & Campbell, 1991). In some populations, neighboring males share many song types and use shared songs interactively during counter-singing (e.g., Beecher & Campbell, 2005). Presumably adult males can learn to recognize the songs of new neighbors in adulthood. Supporting this, adult song sparrows, and many other species, can learn to discriminate among novel, unfamiliar conspeciWc songs in laboratory operant procedures (e.g., Reeves, Beecher, & Brenowitz, 2003). Thus, despite age limitations in imitative vocal learning, song recognition learning likely extends throughout life. Neural activation in auditory forebrain regions, both electrophysiological and immediate-early gene (IEG)
T.L.B. McKenzie et al. / Neurobiology of Learning and Memory 86 (2006) 330–335
activation, habituate in response to repeated song presentation (Chew, Vicario, & Nottebohm, 1996; Mello, Nottebohm, & Clayton, 1995; Stripling, Volman, & Clayton, 1997). Chew et al. (1996) showed a reduction in electrophysiological response in caudal medial nidopallium (NCM) in male zebra Wnches, Taeniopygia guttata, following the repeated presentation of a song, with dishabituation occurring upon presentation of a novel song. These authors suggested that NCM is specialized for remembering the calls and songs of individuals (Chew et al., 1996). Similarly, Mello et al. (1995) showed lower activation of the IEG ZENK in NCM following the repeated presentation of a song; this activation increased once again with the presentation of a diVerent song. These Wndings suggest that NCM is important in auditory processing and may be important in song recognition learning. In this study, we examine Zenk activation in NCM and caudal medial mesopallium (CMM), another auditory region important in song processing (Mello, Vicario, & Clayton, 1992), to determine whether recent exposure to song in adulthood leads to greater or decreased Zenk activation upon hearing that song versus a novel song. Mello et al. (1995) found decreased activation in auditory regions when birds heard learned songs, whereas Bolhuis, Zijlstra, den Boer-Visser, and Van der Zee (2000) found higher activation to playback of learned songs. In the present study, we examined the eVects of adult experience with song on Zenk activation in male song sparrows. We tested whether repeated experience with songs (akin to repeated exposure to a neighboring male’s songs) would modify the Zenk response in the auditory forebrain to those songs in comparison to the eVects of novel songs (akin to the songs of a strange male seeking to establish a new territory). To examine this, we collected birds as adults from the wild and repeatedly exposed them to a set of conspeciWc songs for a week. Following this extended period of exposure, birds were acoustically isolated for an extended period of two days then presented either with the same songs or novel songs and IEG Zenk activation in auditory regions were examined. 2. Methods 2.1. Animals Thirteen wild-caught adult male song sparrows, M. melodia, were used in this study. Birds were captured near London, Ontario, Canada, between 15 June and 17 July 2003 and between 28 May and 18 June 2004, thus all birds captured were in breeding condition. Birds were captured by luring them into mist nets using a simulated territorial intrusion. Sex was conWrmed by the presence of a cloacal protuberance and later by post-mortem examination of gonads. All birds were housed individually in cages and maintained on a diet of Mazuri small bird maintenance, millet, and water ad libitum and periodically supplemented with spinach.
2.2. Song playback stimuli Song stimuli used in this experiment were recorded from a population approximately 500 km northeast of London, Ontario. It is thus very
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unlikely that the birds used in the current study would have been exposed to these songs prior to the experimental procedures. Songs were recorded on a Marantz PMD-222 recorder using a Senneheiser ME-66 microphone. Songs were selected randomly and based on quality of recordings. Three songs from each of 12 diVerent male song sparrows were selected, resulting in 36 diVerent songs for song playbacks. Song playback stimuli were prepared using Canary 1.2.1 sound software (Cornell Laboratory of Ornithology). Song recordings digitized at 44.1 kHz and 16 bits, were Wltered to remove background noise and peak pressure normalized across all songs to standardize peak amplitude during playback. Three sets of 12 songs (Set A, B, and C) were created by randomly selecting one of the three songs from each of the 12 male song sparrows. Thus, each set contained one song from each of the 12 male song sparrows. For each set, 10 diVerent 30-min playback sequences were created with song order determined randomly. Each male song sparrow’s song was repeated consecutively for 2.5 min at a rate of 4 songs/min, mimicking singing rates in the wild. With each of 12 songs being played for 2.5 min each resulted in the 30-min of playback. For each stimulus set, Wve 1 h CDs were made by randomly assigning two 30-min tracks to each CD.
2.3. Procedure 2.3.1. Apparatus Each bird was placed into a sound attenuation chamber (70 £ 70 £ 50 cm interior) on the afternoon of day 1 (the Wrst day following capture) and housed individually in a 30 £ 30 £ 40 cm cage. Each chamber was lit with a 29 W compact Xuorescent bulb. Cages were equipped with two perches, a water bottle, and a food dish. Birds were maintained on a long day photoperiod matching the natural photoperiod at the time of capture. Each chamber was equipped with an Optimus Pro X44AV Speaker that played the song stimuli and a microphone used to monitor the birds during playback test sessions (see below). Songs were played at with peak amplitude of 70–72 dB SPL as measured from the center of the cage. 2.3.2. Song playback Each bird was exposed to song playback in two phases; Wrst a 7-day period of song pre-exposure, then an acute playback test session used to induce Zenk expression. From day 2 through day 8 (pre-exposure period) birds were exposed to 5 h of song playback from 06:00 to 11:00 h daily. During the pre-exposure period, all of the birds were exposed to long day photoperiod. The order of play of each 1 h CD was determined randomly each day. On day 9 there was no song playback and on day 10 the playback test session was conducted (approximately 48 h after the last playback exposure). During this test session birds were exposed to either 30 min of songs that they heard over the previous week (Same Song group) or 30 min of songs they had not heard previously (Novel Song group). All song sets were used as pre-exposure stimuli and test stimuli, and were counterbalanced across birds. That is, any given song set was used as preexposure song for some of the birds, test song for some of the birds, and as both pre-exposure and test song for some of the birds. We recorded the birds during the test session to determine if birds sang in response to the playback. For the Wrst six birds, the chamber lights remained on during the test session playback. Two of these birds sang during playback and so were excluded from further analyses. For the remainder of the birds the lights were turned oV during the playback test sessions and none of the remaining birds sang during these test sessions. We controlled for potential eVects of lights being on or oV during playback by including this as a variable during statistical analyses. The Wnal sample size thus was 11 males (Same Song Group n D 4; Novel Song Group n D 7). Assignment to the two treatment groups was balanced across the two years of the study. Following 60 min of silence at the end of the test playback sessions birds were deeply anesthized using ketamine and xylazine, and then transcardially perfused using 0.1 M phosphate-buVered saline (PBS; pH 7.5) and 4% buVered paraformaldehyde, and brains were extracted. Brains were further Wxed in 4% buVered paraformaldehyde overnight, then cryoprotected in 30% sucrose solution for 24 h. Brains were then frozen on powdered dry ice and placed in a ¡80 °C freezer until later processing.
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2.3.3. Immunocytochemistry Brains were cut in the sagittal plane at 40 m using a cryostat. Every second section was collected into tissue wells containing 0.1 M PBS. We ran 2 or 4 brains in each run of immunocytochemistry, so that each treatment group was equally represented in each run. Sections were washed in 0.1 M PBS two times and incubated in 0.5% H2O2. All washes were a minimum of 5 min. Sections were then washed in 0.1 M PBS three times and incubated in 10% normal goat serum (Vector) in 0.3% Triton in PBS (PBS/T) for 1 h. This was followed by a 20 h incubation in the primary antibody (Egr-1 antibody sc-189, Santa Cruz Biotechnology) at 1:4000 in 0.3% PBS/T. Sections were then washed three times in 0.1% PBS/T and incubated in biotinylated secondary antibody (goat antirabbit IgG 1:250; Vector) for 1 h. This was followed by three washes in 0.1% PBS/T. Sections were then incubated in avidin–biotin horseradishperoxidase complex (Vectastain ABC, Elite kit, Vector) 1:2000 for 1 h and then washed two times in 0.1% PBS/T. Sections were visualized with DAB (Sigma Fast-DAB), mounted onto gelatin-coated slides, dehydrated in ethanol and cleared in Citrosol (Fisher), and then cover-slipped using Permount (Fisher). 2.3.4. Zenk quantiWcation Our quantiWcation protocol followed that of several previous studies (Avey, Phillmore, & MacDougall-Shackleton, 2005; Gentner, Hulse, DuVy, & Ball, 2000; Hernandez & MacDougall-Shackleton, 2004). We quantiWed the level of Zenk activation in three telencephalic areas illustrated in Fig. 1A: caudal medial mesopallium (CMM), dorsal caudal nidopallium (dNCM), and ventral caudal medial nidopallium (vNCM). These two regions of the NCM have previously been shown to exhibit diVerential IEG expression in response to song playback (e.g., Gentner et al., 2000). We began to quantify where the NCM became attached to the rest of the brain and Weld L was visible as a region of non-immunoreactivity. We quantiWed eight sections per region per hemisphere (thus spanning 640 m). The exact sampling location within each brain area was selected centrally within each structure, as deWned by Zenk immunoreactivity, to ensure we were sampling well within the border of the desired area (Fig. 1A). The observer was blind to the birds’ playback group during quantiWcation. Images of each sample location (area 0.39 £ 0.29 mm) were captured using a digital camera (Spot Insight, Diagnostic Instruments Inc., USA) mounted on a Zeiss, Axiophot microscope. Inclusion limits for cell size and optical density were used to count Zenk immunoreactive (Zenkir) cells using SigmaScan Pro software (SPSS Science). The average minimum optical density threshold of immunoreactive cells was determined on a sample-by-sample basis. This value identiWed the minimum optical density that was counted as a stained cell on the top focal layer of the tissue for each sample. The inclusion limits for cell size were 7.7–34.5 m2 as veriWed in a previous study (Avey et al., 2005).
3. Results A 3 £ 2 repeated measures ANOVA with brain region (CMM, vNCM, and dNCM) as a within-subjects factor and light exposure (Lights On, Lights OV during playback) as a between-subjects factor was conducted to examine whether having lights on or oV during playbacks had an eVect on levels of Zenk immunoreactivity. The main eVect of Light Exposure was not signiWcant F (1, 9) D 1.12, p D 0.32, and the brain region £ light exposure interaction was also not signiWcant, F (2, 18) D 1.60, p D 0.23. Therefore, the data from birds experiencing lights on during the Zenk test session playback were combined with birds that had lights oV during playback in all further analyses. A 2 £ 3 repeated-measures ANOVA with treatment group (Same Song, Novel Song) as a between-subjects factor and brain region (CMM, vNCM, and dNCM) as a
within-subject factor was conducted to examine the eVect of hearing previously heard songs (Same Song Group) versus novel songs (Novel Song Group) on Zenk immunoreactivity in auditory regions (Fig. 1B). The main eVects of treatment group and brain region were not signiWcant, F (1, 9) D 1.29, p D 0.29 and F (2, 18) D 1.06, p D 0.37, respectively. However, there was a signiWcant group £ region interaction, F (2, 18) D 10.47, p D 0.001 (Fig. 2). Post hoc tests were conducted to further examine this interaction. Individual one-way ANOVAs, with group (Same Song, Novel Song) as a between subjects factor, were conducted on each of the three auditory areas. There was no diVerence in Zenk immunoreactivity between the groups in CMM, F (1, 9) D 1.85, p D 0.21. There was, however, a signiWcant diVerence in Zenk immunoreactivity between the Experimental Groups in vNCM, F (1, 9) D 8.41, p D 0.02, with birds exposed to previously heard songs (Same Song Group) showing lower activation to songs than birds exposed to new songs (Novel Song Group). While not signiWcant, Zenk immunoreactivity in dNCM diVered between the groups in the same direction, F (1, 9) D 3.25, p D 0.10. To further explore potential diVerences between groups in Zenk immunoreactivity in NCM we ran a 2 £ 2 repeated measures ANOVA with group (Same Song, Novel Song) as a between-subjects factor and NCM subregion (vNCM, dNCM) as a within-subject factor. There was a signiWcant main eVect of experimental group F (1, 9) D 6.00, p D 0.04, with birds from the Same Song Group showing lower activation (M D 133.19) to songs than birds from the Novel Song Group (M D 188.19). There was no signiWcant main eVect of NCM subregion, F (1, 9) D 1.86, p D 0.21 and there was no signiWcant interaction, F (1, 9) D 0.36, p D 0.56. Together these results suggest birds exposed to previously heard songs (Same Song Group) had lower activation in NCM overall to songs played than birds exposed to new songs (Novel Song Group). 4. Discussion We found greater Zenk immunoreactivity in the auditory forebrain of male song sparrows exposed to novel songs then those exposed to previously heard songs. This eVect was statistically detectable in vNCM in an analysis that included all three sampled brain regions. In a post hoc analysis examining NCM alone, the eVect was present in both ventral and dorsal regions of NCM. Because we used a fully balanced design, this eVect can be attributed to song experience per se rather than particular acoustic features of particular songs. Moreover, the eVect of prior experience was evident despite 48 h of acoustic isolation since the previous song playback. Our results add to a growing body of evidence indicating that song-induced neural activity in the auditory forebrain of songbirds—as assessed by immediate-early gene induction—is modulated by previous experience with songs. That is, the Zenk response to song in these auditory
T.L.B. McKenzie et al. / Neurobiology of Learning and Memory 86 (2006) 330–335
333
A CMM
L dNCM vNCM
B
Same Song
Novel Song
CMM
dNCM
vNCM
Fig. 1. (A) Illustration of locations sampled to measure Zenk immunoreactivity. Photomicrograph is a parasagittal section of song sparrow brain with rostral left and dorsal up. The line drawing illustrates the caudal telencephalon containing auditory regions sampled in this study. Boxes indicate regions sampled for CMM, dNCM and vNCM. Dashed line indicates primary auditory region Field L. Scale bar in line drawing is 0.5 mm. (B) Example photomicrographs of Zenk immunoreactivity in birds exposed to novel songs or the same songs, in each of the three regions illustrated in Fig. 1A.
regions is modiWed by memories of songs heard earlier. The evidence for these experience-dependent eVects is based on (i) studies of habituation of the auditory respon-
siveness to song, (ii) studies of the eVect of song exposure early in life, and (iii) studies of song exposure in adulthood.
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T.L.B. McKenzie et al. / Neurobiology of Learning and Memory 86 (2006) 330–335 250
Same Song Novel Song
*
Cell Count
200
150
100
50
0 CMM
dNCM
vNCM
Auditory Area
Fig. 2. Mean (§SE) cell count in auditory areas CMM, dNCM, and vNCM for birds exposed to the same songs versus birds exposed to novel songs during playback.
Early studies of the auditory Zenk response in NCM noted that ZENK expression was higher following playback of conspeciWc song than to heterospeciWc song or other sounds (Mello et al., 1992). Shortly thereafter it was noted that the Zenk response disappears following repetitious playback of the same song, but is again reinstated upon presentation of a novel song (Mello et al., 1995). Electrophysiological studies conWrmed the habituation of NCM to repeated song exposure and indicated that habituation may last up to 20 h (Chew et al., 1996). Recently it has been shown that the habituation of the Zenk response is context-speciWc in addition to being song-speciWc (Kruse, Stripling, & Clayton, 2004). Thus, experience with song modiWes neural responsiveness to song in NCM, and these eVects have been shown to last for up to a day. Experience with songs has also been shown to aVect Zenk responses in the auditory forebrain on longer time scales. Song experience, both early in life and in adulthood, inXuences levels of song-induced Zenk expression in the auditory forebrain. For example, tutor songs induce greater Zenk response in zebra Wnches than non-tutor songs, and there is a correlation between this Zenk response and how well the tutor song matches the learned song (Bolhuis et al., 2000, Bolhuis, Hetebrij, Den Boer-Visser, De Groot, & Zijlstra, 2001). European starling females prefer longer songs to shorter songs, and have greater immediate-early gene response in NCM to longer songs (Gentner & Hulse, 2000; Gentner et al., 2000). However, pre-exposure to long or short songs modulates this diVerential immediate-early gene response (Sockman, Gentner, & Ball, 2005). Thus, song experience modiWes the Zenk response in NCM on time scales longer than typically attributed to habituation. Our results corroborate studies on the habituation of the Zenk response (Kruse et al., 2004; Mello et al., 1995), but on
a time scale more similar to that used by Sockman et al. (2005). Here we demonstrate that exposure to a set of 12 songs for a week led to reduced Zenk response in NCM to those same songs following 2 days of acoustic isolation, compared to the response to a novel set of songs. This eVect can be attributed to formation of auditory memories in adulthood, as none of the sparrows in our study would have heard the test songs prior to this experiment. Our experiment was designed to mimic the auditory experience of an adult male sparrow who is exposed to a new territorial neighbor, and must learn to recognize that neighbor’s songs. Although song sparrows do not learn to produce new songs in adulthood (Marler & Peters, 1987; Nordby et al., 2002) our results indicate that they can retain auditory memories of novel songs in adulthood that last for at least 2 days. Kruse et al. (2004) have proposed an attentional model for enhancement of Zenk response in NCM. They found that the Zenk response to song could be dishabituated by changing the context of song playback without changing the song heard. Similarly, Vignal, Andru, and Mathevon (2005) found that social context -housed alone or in a group-modiWed Zenk response to playback of female calls. Our results are consistent with this attentional model. In the wild, song sparrows and other songbirds learn to recognize their neighbors’ songs and respond less aggressively to them than to the songs of strangers (e.g., Stoddard et al., 1991; Wilson & Vehrencamp, 2001). Novel songs indicate territory instability or an invading unfamiliar male and demand an aggressive response. Thus, novel territorial songs are likely more salient and capture more attention than songs heard repeatedly during the preceding week. Song-induced Zenk response may thus reXect the salience of the acoustic stimulus and this salience may often result from previous experience.
T.L.B. McKenzie et al. / Neurobiology of Learning and Memory 86 (2006) 330–335
Acknowledgments We thank Jeremy PfaV for assistance in Weld recording. Funding was provided by NSERC Canada and a Premier’s Research Excellence Award from the Government of Ontario. References Avey, M. T., Phillmore, L. S., & MacDougall-Shackleton, S. A. (2005). Immediate early gene expression following exposure to acoustic and visual components of courtship in zebra Wnches. Behavioural Brain Research, 165, 247–253. Beecher, M. D., & Campbell, E. (2005). The role of unshared songs in singing interactions between neighbouring song sparrows. Animal Behaviour, 70, 1297–1304. Bolhuis, J. J., Hetebrij, E., Den Boer-Visser, A. M., De Groot, J. H., & Zijlstra, G. G. O. (2001). Localized immediate early gene expression related to the strength of song learning in socially reared zebra Wnches. European Journal of Neuroscience, 13, 2165–2170. Bolhuis, J. J., Zijlstra, G. G. O., den Boer-Visser, A. M., & Van der Zee, E. A. (2000). Localized neuronal activation in the zebra Wnch brain is related to the strength of song learning. Proceedings of the National Academy of Sciences United States of America, 97, 2282–2285. Chew, S. J., Vicario, D. S., & Nottebohm, F. (1996). A large-capacity memory system that recognizes the calls and songs of individual birds. Proceedings of the National Academy of Sciences United States of America, 93, 1950–1955. Gentner, T. Q., & Hulse, S. H. (2000). Female European starling preference and choice for variation in conspeciWc male song. Animal Behaviour, 59, 443–458. Gentner, T. Q., Hulse, S. H., DuVy, D., & Ball, G. F. (2000). Response biases in auditory forebrain regions of female songbirds following exposure to sexually relevant variation in male song. Journal of Neurobiology, 46, 48–58. Hernandez, A. M., & MacDougall-Shackleton, S. A. (2004). EVects of early song experience on song preferences and song control and auditory brain regions in female house Wnches (Carpodacus mexicanus). Journal of Neurobiology, 59, 247–258. Kruse, A. A., Stripling, R., & Clayton, D. F. (2004). Context-speciWc habituation of the zenk gene response to song in adult zebra Wnches. Neurobiology of Learning and Memory, 82, 99–108.
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Lambrechts, M. M., & Dhondt, A. A. (1995). Individual voice discrimination in birds. Current Ornithology, 12, 115–139. Marler, P., & Peters, S. (1987). A sensitive period for song acquisition in the song sparrow, Melospiza melodia: a case of age-limited learning. Ethology, 76, 89–100. Mello, C., Nottebohm, F., & Clayton, D. (1995). Repeated exposure to one song leads to a rapid and persistent decline in an immediate early gene’s response to that song in zebra Wnch telencephalon. Journal of Neuroscience, 15, 6919–6925. Mello, C. V., Vicario, D. S., & Clayton, D. F. (1992). Song presentation induces gene expression in the songbird forebrain. Proceedings of the National Academy of Sciences United States of America, 89, 6818– 6822. Nordby, J. C., Campbell, E., & Beecher, M. D. (2001). Late song learning in song sparrows. Animal Behaviour, 61, 835–846. Nordby, J. C., Campbell, E., & Beecher, M. D. (2002). Adult song sparrows do not alter their song repertoires. Ethology, 108, 39–50. Reeves, B. J., Beecher, M. D., & Brenowitz, E. A. (2003). Seasonal changes in avian song control circuits do not cause seasonal changes in song discrimination in song sparrows. Journal of Neurobiology, 57, 119–129. Sockman, K. W., Gentner, T. Q., & Ball, G. F. (2005). Complementary neural systems for the experience-dependent integration of mate-choice cues in European starlings. Journal of Neurobiology, 62, 72–81. Stoddard, P. K. (1996). Vocal recognition of neighbors by territorial passerines. In D. E. Kroodsma & E. H. Miller (Eds.), Ecology and evolution of acoustic communication in birds (pp. 356–374). Ithaca: Cornell University Press. Stoddard, P. K., Beecher, M. D., Horning, C. L., & Campbell, S. E. (1991). Recognition of individual neighbors by song in the song sparrow, a species with song repertoires. Behavioral Ecology and Sociobiology, 29, 211–215. Stripling, R., Milewski, L., Kruse, A. A., & Clayton, D. F. (2003). Rapidly learned song-discrimination without behavioral reinforcement in adult male zebra Wnches (Taeniopygia guttata). Neurobiology of Learning and Memory, 79, 41–50. Stripling, R., Volman, S., & Clayton, D. F. (1997). Response modulation in the zebra Wnch caudal neostriatum: relationship to nuclear gene regulation. Journal of Neuroscience, 17, 3883–3893. Vignal, C., Andru, J., & Mathevon, A. (2005). Social context modulates behavioural and brain immediate early gene responses to sound in male songbird. European Journal of Neuroscience, 22, 949–955. Wilson, P. L., & Vehrencamp, S. L. (2001). A test of the deceptive mimicry hypothesis in song-sharing song sparrows. Animal Behaviour, 62, 1197–1205.
Experience with songs in adulthood reduces song-induced gene expression in songbird auditory forebrain Tammy L.B. McKenzie ¤, Alexandra M. Hernandez, Scott A. MacDougall-Shackleton Department of Psychology, University of Western Ontario, London, Ont., Canada Received 6 March 2006; revised 17 May 2006; accepted 17 May 2006 Available online 27 June 2006
Abstract Male songbirds learn to produce song within a limited phase early in life; however they continue to learn to recognize songs in adulthood. Studies looking at Zenk activation after exposure to songs learned early in life for song production and songs learned in adulthood show opposite patterns of activation, suggesting distinct neural mechanisms may be involved in these two forms of learning. In this study, we look at IEG Zenk activation in auditory regions NCM and CMM of song sparrows (Melospiza melodia) to see whether recent exposure to song in adulthood leads to greater or decreased Zenk activation upon hearing that song versus a novel song. We found signiWcantly lower activation in birds exposed to previously heard songs versus novel songs in vNCM but not dNCM, though further analysis suggest an overall trend in NCM. We found no signiWcant diVerence in the amount of activation to previously heard songs vs. novel songs in CMM. These results support previous Wndings suggesting that activation is reduced to learned stimuli; we discuss possible implications of these Wndings in relation to song production learning early in life and song recognition learning in adulthood. © 2006 Elsevier Inc. All rights reserved. Keywords: Zenk; Egr-1; Melospiza melodia; Birdsong
1. Introduction In most species male songbirds learn to produce song within a limited time frame early in life, yet there is evidence that males also learn to recognize songs in adulthood, and can do so rapidly (e.g., Stripling, Milewski, Kruse, & Clayton, 2003). Song sparrows, Melospiza melodia, provide a case study. Males in this species learn to produce songs early in life (Marler & Peters, 1987). Male song sparrows learn to imitate songs they hear during their Wrst summer and autumn (Nordby, Campbell, & Beecher, 2001) and once song is crystallized new songs are not added to the adult repertoire (Nordby, Campbell, & Beecher, 2002). Although song sparrows are clearly age-limited learners in regard to imitative vocal learning, they may, however, retain the ability to learn to recognize new songs into adulthood. For example, individ* Corresponding author. Present address: Department of Psychology, Brandon University, 270-18th Street, Brandon, Man., Canada R7A 6A9. E-mail address: [email protected] (T.L.B. McKenzie).
1074-7427/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nlm.2006.05.002
ual recognition by vocalization is widespread in songbirds (Lambrechts & Dhondt, 1995; Stoddard, 1996). Individual recognition by voice is typically inferred by diVerential responses to playback of a neighboring male’s song from the appropriate or an inappropriate territory boundary, or through neighbor/stranger discrimination. Male song sparrows can discriminate a neighbor’s from a stranger’s song on the basis of a single song from each (Stoddard, Beecher, Horning, & Campbell, 1991). In some populations, neighboring males share many song types and use shared songs interactively during counter-singing (e.g., Beecher & Campbell, 2005). Presumably adult males can learn to recognize the songs of new neighbors in adulthood. Supporting this, adult song sparrows, and many other species, can learn to discriminate among novel, unfamiliar conspeciWc songs in laboratory operant procedures (e.g., Reeves, Beecher, & Brenowitz, 2003). Thus, despite age limitations in imitative vocal learning, song recognition learning likely extends throughout life. Neural activation in auditory forebrain regions, both electrophysiological and immediate-early gene (IEG)
T.L.B. McKenzie et al. / Neurobiology of Learning and Memory 86 (2006) 330–335
activation, habituate in response to repeated song presentation (Chew, Vicario, & Nottebohm, 1996; Mello, Nottebohm, & Clayton, 1995; Stripling, Volman, & Clayton, 1997). Chew et al. (1996) showed a reduction in electrophysiological response in caudal medial nidopallium (NCM) in male zebra Wnches, Taeniopygia guttata, following the repeated presentation of a song, with dishabituation occurring upon presentation of a novel song. These authors suggested that NCM is specialized for remembering the calls and songs of individuals (Chew et al., 1996). Similarly, Mello et al. (1995) showed lower activation of the IEG ZENK in NCM following the repeated presentation of a song; this activation increased once again with the presentation of a diVerent song. These Wndings suggest that NCM is important in auditory processing and may be important in song recognition learning. In this study, we examine Zenk activation in NCM and caudal medial mesopallium (CMM), another auditory region important in song processing (Mello, Vicario, & Clayton, 1992), to determine whether recent exposure to song in adulthood leads to greater or decreased Zenk activation upon hearing that song versus a novel song. Mello et al. (1995) found decreased activation in auditory regions when birds heard learned songs, whereas Bolhuis, Zijlstra, den Boer-Visser, and Van der Zee (2000) found higher activation to playback of learned songs. In the present study, we examined the eVects of adult experience with song on Zenk activation in male song sparrows. We tested whether repeated experience with songs (akin to repeated exposure to a neighboring male’s songs) would modify the Zenk response in the auditory forebrain to those songs in comparison to the eVects of novel songs (akin to the songs of a strange male seeking to establish a new territory). To examine this, we collected birds as adults from the wild and repeatedly exposed them to a set of conspeciWc songs for a week. Following this extended period of exposure, birds were acoustically isolated for an extended period of two days then presented either with the same songs or novel songs and IEG Zenk activation in auditory regions were examined. 2. Methods 2.1. Animals Thirteen wild-caught adult male song sparrows, M. melodia, were used in this study. Birds were captured near London, Ontario, Canada, between 15 June and 17 July 2003 and between 28 May and 18 June 2004, thus all birds captured were in breeding condition. Birds were captured by luring them into mist nets using a simulated territorial intrusion. Sex was conWrmed by the presence of a cloacal protuberance and later by post-mortem examination of gonads. All birds were housed individually in cages and maintained on a diet of Mazuri small bird maintenance, millet, and water ad libitum and periodically supplemented with spinach.
2.2. Song playback stimuli Song stimuli used in this experiment were recorded from a population approximately 500 km northeast of London, Ontario. It is thus very
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unlikely that the birds used in the current study would have been exposed to these songs prior to the experimental procedures. Songs were recorded on a Marantz PMD-222 recorder using a Senneheiser ME-66 microphone. Songs were selected randomly and based on quality of recordings. Three songs from each of 12 diVerent male song sparrows were selected, resulting in 36 diVerent songs for song playbacks. Song playback stimuli were prepared using Canary 1.2.1 sound software (Cornell Laboratory of Ornithology). Song recordings digitized at 44.1 kHz and 16 bits, were Wltered to remove background noise and peak pressure normalized across all songs to standardize peak amplitude during playback. Three sets of 12 songs (Set A, B, and C) were created by randomly selecting one of the three songs from each of the 12 male song sparrows. Thus, each set contained one song from each of the 12 male song sparrows. For each set, 10 diVerent 30-min playback sequences were created with song order determined randomly. Each male song sparrow’s song was repeated consecutively for 2.5 min at a rate of 4 songs/min, mimicking singing rates in the wild. With each of 12 songs being played for 2.5 min each resulted in the 30-min of playback. For each stimulus set, Wve 1 h CDs were made by randomly assigning two 30-min tracks to each CD.
2.3. Procedure 2.3.1. Apparatus Each bird was placed into a sound attenuation chamber (70 £ 70 £ 50 cm interior) on the afternoon of day 1 (the Wrst day following capture) and housed individually in a 30 £ 30 £ 40 cm cage. Each chamber was lit with a 29 W compact Xuorescent bulb. Cages were equipped with two perches, a water bottle, and a food dish. Birds were maintained on a long day photoperiod matching the natural photoperiod at the time of capture. Each chamber was equipped with an Optimus Pro X44AV Speaker that played the song stimuli and a microphone used to monitor the birds during playback test sessions (see below). Songs were played at with peak amplitude of 70–72 dB SPL as measured from the center of the cage. 2.3.2. Song playback Each bird was exposed to song playback in two phases; Wrst a 7-day period of song pre-exposure, then an acute playback test session used to induce Zenk expression. From day 2 through day 8 (pre-exposure period) birds were exposed to 5 h of song playback from 06:00 to 11:00 h daily. During the pre-exposure period, all of the birds were exposed to long day photoperiod. The order of play of each 1 h CD was determined randomly each day. On day 9 there was no song playback and on day 10 the playback test session was conducted (approximately 48 h after the last playback exposure). During this test session birds were exposed to either 30 min of songs that they heard over the previous week (Same Song group) or 30 min of songs they had not heard previously (Novel Song group). All song sets were used as pre-exposure stimuli and test stimuli, and were counterbalanced across birds. That is, any given song set was used as preexposure song for some of the birds, test song for some of the birds, and as both pre-exposure and test song for some of the birds. We recorded the birds during the test session to determine if birds sang in response to the playback. For the Wrst six birds, the chamber lights remained on during the test session playback. Two of these birds sang during playback and so were excluded from further analyses. For the remainder of the birds the lights were turned oV during the playback test sessions and none of the remaining birds sang during these test sessions. We controlled for potential eVects of lights being on or oV during playback by including this as a variable during statistical analyses. The Wnal sample size thus was 11 males (Same Song Group n D 4; Novel Song Group n D 7). Assignment to the two treatment groups was balanced across the two years of the study. Following 60 min of silence at the end of the test playback sessions birds were deeply anesthized using ketamine and xylazine, and then transcardially perfused using 0.1 M phosphate-buVered saline (PBS; pH 7.5) and 4% buVered paraformaldehyde, and brains were extracted. Brains were further Wxed in 4% buVered paraformaldehyde overnight, then cryoprotected in 30% sucrose solution for 24 h. Brains were then frozen on powdered dry ice and placed in a ¡80 °C freezer until later processing.
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2.3.3. Immunocytochemistry Brains were cut in the sagittal plane at 40 m using a cryostat. Every second section was collected into tissue wells containing 0.1 M PBS. We ran 2 or 4 brains in each run of immunocytochemistry, so that each treatment group was equally represented in each run. Sections were washed in 0.1 M PBS two times and incubated in 0.5% H2O2. All washes were a minimum of 5 min. Sections were then washed in 0.1 M PBS three times and incubated in 10% normal goat serum (Vector) in 0.3% Triton in PBS (PBS/T) for 1 h. This was followed by a 20 h incubation in the primary antibody (Egr-1 antibody sc-189, Santa Cruz Biotechnology) at 1:4000 in 0.3% PBS/T. Sections were then washed three times in 0.1% PBS/T and incubated in biotinylated secondary antibody (goat antirabbit IgG 1:250; Vector) for 1 h. This was followed by three washes in 0.1% PBS/T. Sections were then incubated in avidin–biotin horseradishperoxidase complex (Vectastain ABC, Elite kit, Vector) 1:2000 for 1 h and then washed two times in 0.1% PBS/T. Sections were visualized with DAB (Sigma Fast-DAB), mounted onto gelatin-coated slides, dehydrated in ethanol and cleared in Citrosol (Fisher), and then cover-slipped using Permount (Fisher). 2.3.4. Zenk quantiWcation Our quantiWcation protocol followed that of several previous studies (Avey, Phillmore, & MacDougall-Shackleton, 2005; Gentner, Hulse, DuVy, & Ball, 2000; Hernandez & MacDougall-Shackleton, 2004). We quantiWed the level of Zenk activation in three telencephalic areas illustrated in Fig. 1A: caudal medial mesopallium (CMM), dorsal caudal nidopallium (dNCM), and ventral caudal medial nidopallium (vNCM). These two regions of the NCM have previously been shown to exhibit diVerential IEG expression in response to song playback (e.g., Gentner et al., 2000). We began to quantify where the NCM became attached to the rest of the brain and Weld L was visible as a region of non-immunoreactivity. We quantiWed eight sections per region per hemisphere (thus spanning 640 m). The exact sampling location within each brain area was selected centrally within each structure, as deWned by Zenk immunoreactivity, to ensure we were sampling well within the border of the desired area (Fig. 1A). The observer was blind to the birds’ playback group during quantiWcation. Images of each sample location (area 0.39 £ 0.29 mm) were captured using a digital camera (Spot Insight, Diagnostic Instruments Inc., USA) mounted on a Zeiss, Axiophot microscope. Inclusion limits for cell size and optical density were used to count Zenk immunoreactive (Zenkir) cells using SigmaScan Pro software (SPSS Science). The average minimum optical density threshold of immunoreactive cells was determined on a sample-by-sample basis. This value identiWed the minimum optical density that was counted as a stained cell on the top focal layer of the tissue for each sample. The inclusion limits for cell size were 7.7–34.5 m2 as veriWed in a previous study (Avey et al., 2005).
3. Results A 3 £ 2 repeated measures ANOVA with brain region (CMM, vNCM, and dNCM) as a within-subjects factor and light exposure (Lights On, Lights OV during playback) as a between-subjects factor was conducted to examine whether having lights on or oV during playbacks had an eVect on levels of Zenk immunoreactivity. The main eVect of Light Exposure was not signiWcant F (1, 9) D 1.12, p D 0.32, and the brain region £ light exposure interaction was also not signiWcant, F (2, 18) D 1.60, p D 0.23. Therefore, the data from birds experiencing lights on during the Zenk test session playback were combined with birds that had lights oV during playback in all further analyses. A 2 £ 3 repeated-measures ANOVA with treatment group (Same Song, Novel Song) as a between-subjects factor and brain region (CMM, vNCM, and dNCM) as a
within-subject factor was conducted to examine the eVect of hearing previously heard songs (Same Song Group) versus novel songs (Novel Song Group) on Zenk immunoreactivity in auditory regions (Fig. 1B). The main eVects of treatment group and brain region were not signiWcant, F (1, 9) D 1.29, p D 0.29 and F (2, 18) D 1.06, p D 0.37, respectively. However, there was a signiWcant group £ region interaction, F (2, 18) D 10.47, p D 0.001 (Fig. 2). Post hoc tests were conducted to further examine this interaction. Individual one-way ANOVAs, with group (Same Song, Novel Song) as a between subjects factor, were conducted on each of the three auditory areas. There was no diVerence in Zenk immunoreactivity between the groups in CMM, F (1, 9) D 1.85, p D 0.21. There was, however, a signiWcant diVerence in Zenk immunoreactivity between the Experimental Groups in vNCM, F (1, 9) D 8.41, p D 0.02, with birds exposed to previously heard songs (Same Song Group) showing lower activation to songs than birds exposed to new songs (Novel Song Group). While not signiWcant, Zenk immunoreactivity in dNCM diVered between the groups in the same direction, F (1, 9) D 3.25, p D 0.10. To further explore potential diVerences between groups in Zenk immunoreactivity in NCM we ran a 2 £ 2 repeated measures ANOVA with group (Same Song, Novel Song) as a between-subjects factor and NCM subregion (vNCM, dNCM) as a within-subject factor. There was a signiWcant main eVect of experimental group F (1, 9) D 6.00, p D 0.04, with birds from the Same Song Group showing lower activation (M D 133.19) to songs than birds from the Novel Song Group (M D 188.19). There was no signiWcant main eVect of NCM subregion, F (1, 9) D 1.86, p D 0.21 and there was no signiWcant interaction, F (1, 9) D 0.36, p D 0.56. Together these results suggest birds exposed to previously heard songs (Same Song Group) had lower activation in NCM overall to songs played than birds exposed to new songs (Novel Song Group). 4. Discussion We found greater Zenk immunoreactivity in the auditory forebrain of male song sparrows exposed to novel songs then those exposed to previously heard songs. This eVect was statistically detectable in vNCM in an analysis that included all three sampled brain regions. In a post hoc analysis examining NCM alone, the eVect was present in both ventral and dorsal regions of NCM. Because we used a fully balanced design, this eVect can be attributed to song experience per se rather than particular acoustic features of particular songs. Moreover, the eVect of prior experience was evident despite 48 h of acoustic isolation since the previous song playback. Our results add to a growing body of evidence indicating that song-induced neural activity in the auditory forebrain of songbirds—as assessed by immediate-early gene induction—is modulated by previous experience with songs. That is, the Zenk response to song in these auditory
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A CMM
L dNCM vNCM
B
Same Song
Novel Song
CMM
dNCM
vNCM
Fig. 1. (A) Illustration of locations sampled to measure Zenk immunoreactivity. Photomicrograph is a parasagittal section of song sparrow brain with rostral left and dorsal up. The line drawing illustrates the caudal telencephalon containing auditory regions sampled in this study. Boxes indicate regions sampled for CMM, dNCM and vNCM. Dashed line indicates primary auditory region Field L. Scale bar in line drawing is 0.5 mm. (B) Example photomicrographs of Zenk immunoreactivity in birds exposed to novel songs or the same songs, in each of the three regions illustrated in Fig. 1A.
regions is modiWed by memories of songs heard earlier. The evidence for these experience-dependent eVects is based on (i) studies of habituation of the auditory respon-
siveness to song, (ii) studies of the eVect of song exposure early in life, and (iii) studies of song exposure in adulthood.
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Same Song Novel Song
*
Cell Count
200
150
100
50
0 CMM
dNCM
vNCM
Auditory Area
Fig. 2. Mean (§SE) cell count in auditory areas CMM, dNCM, and vNCM for birds exposed to the same songs versus birds exposed to novel songs during playback.
Early studies of the auditory Zenk response in NCM noted that ZENK expression was higher following playback of conspeciWc song than to heterospeciWc song or other sounds (Mello et al., 1992). Shortly thereafter it was noted that the Zenk response disappears following repetitious playback of the same song, but is again reinstated upon presentation of a novel song (Mello et al., 1995). Electrophysiological studies conWrmed the habituation of NCM to repeated song exposure and indicated that habituation may last up to 20 h (Chew et al., 1996). Recently it has been shown that the habituation of the Zenk response is context-speciWc in addition to being song-speciWc (Kruse, Stripling, & Clayton, 2004). Thus, experience with song modiWes neural responsiveness to song in NCM, and these eVects have been shown to last for up to a day. Experience with songs has also been shown to aVect Zenk responses in the auditory forebrain on longer time scales. Song experience, both early in life and in adulthood, inXuences levels of song-induced Zenk expression in the auditory forebrain. For example, tutor songs induce greater Zenk response in zebra Wnches than non-tutor songs, and there is a correlation between this Zenk response and how well the tutor song matches the learned song (Bolhuis et al., 2000, Bolhuis, Hetebrij, Den Boer-Visser, De Groot, & Zijlstra, 2001). European starling females prefer longer songs to shorter songs, and have greater immediate-early gene response in NCM to longer songs (Gentner & Hulse, 2000; Gentner et al., 2000). However, pre-exposure to long or short songs modulates this diVerential immediate-early gene response (Sockman, Gentner, & Ball, 2005). Thus, song experience modiWes the Zenk response in NCM on time scales longer than typically attributed to habituation. Our results corroborate studies on the habituation of the Zenk response (Kruse et al., 2004; Mello et al., 1995), but on
a time scale more similar to that used by Sockman et al. (2005). Here we demonstrate that exposure to a set of 12 songs for a week led to reduced Zenk response in NCM to those same songs following 2 days of acoustic isolation, compared to the response to a novel set of songs. This eVect can be attributed to formation of auditory memories in adulthood, as none of the sparrows in our study would have heard the test songs prior to this experiment. Our experiment was designed to mimic the auditory experience of an adult male sparrow who is exposed to a new territorial neighbor, and must learn to recognize that neighbor’s songs. Although song sparrows do not learn to produce new songs in adulthood (Marler & Peters, 1987; Nordby et al., 2002) our results indicate that they can retain auditory memories of novel songs in adulthood that last for at least 2 days. Kruse et al. (2004) have proposed an attentional model for enhancement of Zenk response in NCM. They found that the Zenk response to song could be dishabituated by changing the context of song playback without changing the song heard. Similarly, Vignal, Andru, and Mathevon (2005) found that social context -housed alone or in a group-modiWed Zenk response to playback of female calls. Our results are consistent with this attentional model. In the wild, song sparrows and other songbirds learn to recognize their neighbors’ songs and respond less aggressively to them than to the songs of strangers (e.g., Stoddard et al., 1991; Wilson & Vehrencamp, 2001). Novel songs indicate territory instability or an invading unfamiliar male and demand an aggressive response. Thus, novel territorial songs are likely more salient and capture more attention than songs heard repeatedly during the preceding week. Song-induced Zenk response may thus reXect the salience of the acoustic stimulus and this salience may often result from previous experience.
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