Immediate Early Gene (ZENK, Arc) Expression in the Auditory Forebrain of Female Canaries Varies in Response to Male Song Quality Stefan Leitner,1,2 Cornelia Voigt,2 Reinhold Metzdorf,3 Clive K. Catchpole1 1
School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, United Kingdom
2
Max-Planck-Institute for Ornithology, D-82319 Seewiesen, Germany
3
Department of Developmental and Behavioral Neurobiology, Faculty of Life Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands
Received 23 September 2004; accepted 4 January 2005
ABSTRACT: In male songbirds, the song control pathway in the forebrain is responsible for song production and learning, and in females it is associated with the perception and discrimination of male song. However, experiments using the expression of immediate early genes (IEGs) reveal the activation of brain regions outside the song control system, in particular the caudomedial nidopallium (NCM) and the caudomedial mesopallium (CMM). In this study on female canaries, we investigate the role of these two regions in relation to playback of male songs of different quality. Male canaries produce elaborate songs and some contain syllables with a more complex structure (sexy syllables) that induce females to perform copulation solicitation displays (CSD) as an invitation to mate. Females were first exposed to playback of a range of songs of different quality, before they were finally tested with playback of songs containing either sexy or nonsexy syllables. We
then sectioned the brains and used in situ hybridization to reveal brain regions that express the IEGs ZENK or Arc. In CMM, expression of ZENK mRNA was significantly higher in females that last heard sexy syllables compared to those that last heard nonsexy syllables, but this was not the case for NCM. Expression of Arc mRNA revealed no differences in either CMM or NCM in both experimental groups. These results provide evidence that in female canaries CMM is involved in female perception and discrimination of male song quality through a mechanism of memory reconsolidation. The results also have further implications for the evolution of complex songs by sexual selection and female choice. ' 2005 Wiley Periodicals, Inc. J Neurobiol 64: 275–284, 2005
Keywords: song; brain; female canary; caudomedial mesopallium (CMM, formerly CMHV); caudomedial nidopallium (NCM); HVC; immediate early gene; ZENK; Arc
INTRODUCTION
Correspondence to: S. Leitner (
[email protected]). Contract grant sponsor: BBSRC; contract grant number: S11403 (C.K.C.). ' 2005 Wiley Periodicals, Inc. Published online 16 May 2005 in Wiley InterScience (www.interscience. wiley.com). DOI 10.1002/neu.20135
The songs of male songbirds are essential for female attraction and stimulation, and in a variety of species female songbirds prefer more complex songs or songs containing more attractive syllables (Catchpole and Slater, 1995). Females who select males according to these traits will drive the evolution of song complexity through sexual selection and female choice. Song production and learning is controlled by the song con275
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trol system in the forebrain, which is larger in males than in females, an exception being species where both sexes sing (reviewed in DeVoogd and Lauay, 2001; Gahr et al., 2002). Besides this neuronal network, it is now well established that the expression of immediate early genes (IEGs) also plays an important role in song production and song perception (reviewed in Ball and Balthazart, 2001; Bolhuis and Eda-Fujiwara, 2003). IEGs are activity-dependent genes that are expressed in neural circuits in response to various stimuli (Morgan and Curran, 1989). In singing males we now find expression of IEGs, such as c-fos and ZENK, in the song control nuclei HVC (High Vocal Center) and RA (robust nucleus of the arcopallium) (Jarvis and Nottebohm, 1997). In contrast, when hearing songs, the caudomedial mesopallium (CMM), formerly caudomedial hyperstriatum ventrale (CMHV), and the caudomedial nidopallium, formerly caudomedial neostriatum (NCM) show an elevated IEG expression (Mello and Clayton, 1994). For information about the new brain nomenclature, see Reiner et al. (2004). Although there has been much less work on the smaller female pathway, it has now been shown to be involved in song perception and song discrimination (e.g., Leitner and Catchpole, 2002). In female songbirds, song-induced IEG expression is also present in NCM and CMM. Several IEG studies concerning song discrimination in female birds have now been conducted in the starling (Gentner et al., 2001; Sockman et al., 2002), the zebra finch (Bailey et al., 2002), and in the budgerigar (Eda-Fujiwara et al., 2003). So far only one study has addressed this issue in female canaries (Ribeiro et al., 1998). Female canaries provide a useful model for song discrimination, as they respond differentially to certain syllable types within the male song. These are called ‘‘sexy syllables’’ and are characterized by having a complex structure, rapid frequency modulations, high repetition rate, and wide frequency range (Vallet et al., 1998). Females react with sexual display to playback of these syllables (Vallet et al., 1998), and also show an increased response in HVC neurons (Del Negro et al., 2000). In an earlier study we showed that females that discriminated most between sexy and nonsexy playback also had a larger HVC (Leitner and Catchpole, 2002). In this study we aim to investigate the expression of the two IEGs ZENK and Arc (activity-regulated cytoskeleton-associated protein) in NCM and CMM in relation to playback of songs with different quality containing either sexy or nonsexy syllables. ZENK is an IEG that belongs to the functional class of regulatory transcription factors and has been used in a num-
ber of studies, where it has been shown to be a reliable marker of IEG activity in both mammals and birds (e.g., Cole et al., 1989; Mello et al., 1992). Arc represents an IEG of the effector protein class that is able to directly modulate cell function. Its RNA and protein products are localized in neuronal soma and dendrites (Lyford et al., 1995). Both ZENK and Arc have been associated with synaptic plasticity and modulation of long-term memory in mammals (Guzowski et al., 2000; Jones et al., 2001). In recent studies, Arc has been shown to be involved in learning abilities and long-term potentiation (LTP) in rats (Guzowski et al., 2001; Yin et al., 2002), but has not yet been tested in birds. In songbirds, the neural representations that are associated with song learning and song discrimination processes are not well described. As vocal recognition in songbirds is linked to experience-dependent memorization, synaptic plasticity as revealed by Arc expression may be important for these processes. Our present study has two main objectives. First, we compare the expression pattern of ZENK with the expression pattern of Arc in female canaries at the mRNA level, which may help to discriminate between different auditory processing mechanisms. We then test whether CMM and NCM in females are involved in aspects of song discrimination by exposing them to playback of sexy and nonsexy syllables and studying the resulting patterns of IEG expression.
METHODS Subjects The subjects were 10 1-year-old adult female canaries (Serinus canaria) of the local domesticated strain from our breeding colony at Seewiesen, Germany. The canaries were kept throughout the year under natural light conditions in an outdoor aviary with indoor shelter. Behavior was monitored daily and natural breeding activity started at the end of March. After completion of their first clutch, 10 breeding females were selected for the experiment. They were transferred to a soundproof room at the beginning of May and kept on a 15:9 L/D photoperiod. The females were housed in cages (56 27.5 37 cm) with two females per cage, separated by a partition wall. Females could hear but not see each other. They were kept in the soundproof room during the entire test period (6 weeks) and allowed to nest and lay eggs in a plastic nest bowl.
Experimental Songs Canary songs (Fig. 1) consist of distinct, individual syllables repeated as a phrase, and a song consists of a series of
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Figure 1 (a) Computer-edited song stimuli used in the playback experiments. Example of sexy syllables (A,B) and nonsexy syllables (C,D). For playback both were embedded in a whistlelike control. (b) Example of a computer-edited song (duration 15.4 s). A canary syllable (either sexy or nonsexy) was embedded within a synthetic control note that is not attractive to females.
different phrases. Previous experimental studies (e.g., Vallet et al., 1998) have shown that some syllables with a particular structure are particularly potent to females, who respond with copulation solicitation displays (CSD) at a higher rate when compared to other syllables. This is particularly useful as hormone implantation is not necessary to obtain a CSD response in canaries. These ‘‘sexy syllables’’ share a number of characteristics, including a more complex two-note structure, high repetition rate, rapid frequency-modulations (more than 40 Hz per ms), and short intervals between syllables. Figure 1(a) shows examples of sexy and nonsexy syllables, and how these are embedded in our computer-edited songs. The sexy syllables used in this experiment have proved to be ‘‘sexy’’ in previous experiments as they elicited a larger number of CSDs than control syllables (Leitner et al., 2001; Leitner and Catchpole, 2002). Five synthetic songs were created for our experiments using either sexy or nonsexy syllables. We used five different syllable types extracted from the normal songs of five different adult males. We then created five computer-edited songs, using two analysis and synthesis software packages
(Sound edit and Sound editPro Macromedia for Macintosh computers; all sounds were sampled at 22 kHz). Each synthetic song was constructed from one adult male canary syllable (0.8 s), either sexy, nonsexy, or control, followed by repetitions of the same control note (6 s), which is not attractive to females. To construct a song, this pattern was repeated two times, after a 1 s control note to start the song and another canary syllable (0.8 s) to finish it. Thus, each of the five experimental songs had a total duration of 15.4 s [see Fig. 1(b)]. To simulate the normal pattern of canary song, the songs were repeated in bouts of three that were separated by 9 s of silence. This also allowed the female time to respond with CSD. The total duration of any one song bout was therefore 64.2 s (see also Leitner and Catchpole, 2002).
Playback The protocol of the playback experiment followed described procedures previously used to successfully elicit CSD in female canaries (e.g., Leitner et al., 2001; Leitner
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and Catchpole, 2002). It was designed to expose each female to the same song stimuli in random order for the same period of time. During each test session, all five song bouts were presented in random order, and this was then repeated three times, separated by intervals of 2 min. This procedure was carried out once daily (just over 15 min) for each female for the 6 weeks of the experiment. At the last day, the 10 birds were assigned to two experimental groups. One group heard an experimental song containing only sexy syllables (referred to as ‘‘sexy group’’), the other group heard an experimental song containing nonsexy syllables (referred to as ‘‘nonsexy’’ group).
TG(CT) TT(AG) AA(CT) TCC CAC CA(CT) TT(CT) AA-30 . Following PCR, amplified fragments were purified, blunt-ended, and a product of 440 bp cloned into the SmaI site of pGEM7 (Promega, Madison, WI). Clones were sequenced to verify the authenticity of the amplification. The revealed zebra finch Arc nucleotide sequence (GenBank accession number AY792623) is 89% identical to the chicken counterpart (nt 440–nt 835; GenBank accession number NM204432).
Morphometric Analysis Brain Histology Thirty to forty minutes after the last playback, all birds were killed with an overdose of chloroform and perfused transcardially, first with 0.9% saline, followed by phosphate buffered 4% formaldehyde. Brains were postfixed in 4% phosphate-buffered formaldehyde and their weight recorded. One-half of each brain was immersed in RNAse free 10% and 30% phosphate-buffered sucrose and cut with a freezing microtome into 40 m parasagittal sections. For collection, the sections went into PBS (phosphate buffered saline) and alternate sections were mounted onto Superfrost Plus microscope slides (Menzel Gla¨ser, Braunschweig, Germany). The ZENK and Arc mRNA expression on the brain sections was detected with cRNA probes labeled with 35SCTP of the zebra finch using the in situ hybridization. This technique followed a previously described protocol for the androgen receptor mRNA (Metzdorf et al., 1999). This method was used to monitor brain regions and to identify distinct areas such as NCM and CMM. Slides were counterstained with thionin and coverslipped. The in situ slides were analyzed under darkfield illumination with a microscope (Leitz Aristoplan).
For morphometric analysis, brain regions were video-digitized on a PC equipped with an image analysis system (MetaMorph 4.6; Visitron Systems, Germany). Anatomical extension of NCM and CMM was located according to previously described studies (e.g., Gentner et al., 2001; Sockman et al., 2002), and additionally we were also scanning other telencephalic areas. The ZENK and Arc mRNA expression patterns were calculated by estimating the density of the silver grains on the tissues. The video-digitized images were converted to grayscale in order to quantify the level of mRNA expression in a selected area (5211 m2). On these images, a threshold level was adjusted to separate the silver grains from the background. The thresholded area was calculated by a built-in function of the software and was expressed as a fractional area covered by silver grains. These measurements revealed the mRNA expression level that is the mean of three measurements per region (i.e., counting frames) of interest. In NCM we sampled from a dorsal, a ventral, and an intermediate part, considering its large extension (see also Fig. 2). To correct for different intensities of background labeling due to different sets of the in situ hybridization method, we measured the intensity of silver grains in an area that lacked specific labeling. Such an area was represented by the tractus septopallio-mesence-
Cloning of the cDNA The cloning and characterization of a region of the zebra finch ZENK gene (1.1 kb) has been described previously (Long and Salbaum, 1998; GenBank no. AF026084). The coding sequences from canary and zebra finch have 96% identity (Long and Salbaum, 1998). The same homology was found in the 30 untranslated area. This high homology allows the use of this fragment for the localization of ZENK mRNA in the canary. Based on sequence information from mammalian species, we used PCR to amplify fragments of Arc from the zebra finch. The mRNA was prepared from brain tissue by using the Dynabeads mRNA DIRECT kit (Deutsche Dynal, Hamburg, Germany). The synthesis of first-strand cDNA was done with SUPERSCRIPT II Reverse Transcriptase (Life Technologies, Bethesda, MD) and oligo (dT)-primer. The resulting RNA-DNA hybrids were subsequently used in PCRs to generate pieces of the Arc gene. For Arc, the forward primer was 50 -AA(AG) (AC)G(AGCT) GA(AG) ATG CA(CT) GT(AGCT) TGG-30 and the reverse primer was 50 -
Figure 2 Schematic drawing of the three sampling locations within caudomedial mesopallium (CMM) and caudomedial nidopallium (NCM), respectively. Dorsal is at the top and caudal is on the right.
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Figure 3 Darkfield photomicrographs of ZENK and Arc mRNA expression in the caudomedial mesopallium (CMM) and the caudomedial nidopallium (NCM) in relation to the two experimental groups. Dorsal is at the top and caudal is on the right in these sagittal sections. Scale bar ¼ 300 m. phalicus (TSM). Thus, we corrected the intensity values by subtracting the mean values of TSM from the mean values of NCM and CMM, respectively. The resulting values were used for comparisons of ZENK and Arc mRNA expression levels.
hoc comparisons using StatView 5.0 software. Values shown are means 6 SE.
RESULTS
Statistical Analysis
Expression Patterns of ZENK and Arc
The expression levels in the brain regions were compared by means of a paired t test. Treatment groups were compared using a repeated measures ANOVA following post-
We found ZENK mRNA and Arc mRNA expression throughout the investigated brain regions CMM and NCM (Fig. 3). Generally, expression intensity was
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Figure 4 Comparison of ZENK and Arc mRNA expression in High Vocal Center (HVC) and robust nucleus of the arcopallium (RA) of one experimental female. Arrows indicate the borders of the brain regions. Dorsal is at the top and caudal is on the right in these sagittal sections. Scale bar ¼ 300 m.
larger in ZENK compared to Arc, both in CMM [F(1, 8) ¼ 7.81; p ¼ 0.012] and NCM [F(1, 8) ¼ 24.91; p < 0.001]. We further compared the intensity of expression in these two brain regions. The Arc mRNA expression level revealed a higher intensity in CMM compared to NCM (t ¼ 4.00, df ¼ 9, p ¼ 0.003), whereas with ZENK mRNA there was no difference in expression intensity between these two brain regions (t ¼ 0.008, df ¼ 9, p ¼ 0.994). No expression was found in areas belonging to the song control system, except in one female that was part of the nonsexy experimental group. This individual had an elevated expression of ZENK mRNA and, to a lesser extent, Arc mRNA expression in the song control regions HVC and RA, thus allowing a clear identification of these areas (Fig. 4).
Effect of Song Playback on IEG Expression The expression of ZENK mRNA and Arc mRNA was present in all experimental groups. With ZENK, there was an overall effect of treatment on the analyzed brain regions, with significantly more expression in response to sexy syllables [F(1, 8) ¼ 12.52; p ¼ 0.008]. However this was largely due to CMM, and did not hold for both areas when analyzed separately. Expression of ZENK mRNA was higher in females
hearing sexy syllables compared to those hearing nonsexy syllables in CMM (p ¼ 0.0004), but not in NCM (p ¼ 0.172). Expression of Arc mRNA revealed no significant differences between the two experimental groups [F(1, 8) ¼ 0.07; p ¼ 0.800], either in CMM (p ¼ 0.856) or in NCM (p ¼ 0.378; see also Figs. 3 and 5).
DISCUSSION Our results reveal an overall difference in ZENK expression when females hear the more complex sexy syllables, but this is largely due to CMM rather than NCM. Although we used the classical marker (ZENK) we were able to compare it with a marker not previously used in avian research (Arc). ZENK mRNA and Arc mRNA distribution did not reveal an identical pattern of expression intensity, which might reflect their classification into different functional classes of IEGs. Our finding of a higher ZENK expression in CMM after playback of sexy syllables compared to nonsexy syllables is similar to the pattern found by Sockman et al. (2002) in the starling, although in contrast to their study, we did not find differences in NCM. Further, in our study there was no difference in overall ZENK expression levels between these two brain regions. This would suggest a slightly different mech-
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Figure 5 Expression of ZENK mRNA and Arc mRNA in response to playback of sexy and nonsexy songs. ZENK mRNA expression in the sexy playback group was higher compared to the nonsexy group in CMM but not in NCM. Expression of Arc mRNA revealed no significant differences in the two experimental groups.
anism in female canaries, as they react differentially to special sexy syllables. In the study by Sockman et al. (2002), starlings were exposed to different song experiences in the different experimental groups: one group heard short-bout songs and the other group heard long-bout songs, both for more than 5 h per day. The authors then found that ZENK response toward long-bout song was larger in the group with long-bout experience, suggesting an experiencedependent forebrain response bias. Another experience-dependent neuronal representation of song memory has been suggested in a recent study on song recognition in male zebra finches (Terpstra et al., 2004). In our study, females experienced both types of song, sexy and nonsexy, before they were tested with playback of either sexy or nonsexy songs. However, there was a robust effect of sexy songs on the ZENK expression level that corresponds to the effect of long-bout song in the starling study. In contrast to female starlings, the female canaries in our study did not require long-term priming with a certain type of stimulus (e.g., either sexy or nonsexy syllables) in order to obtain a differential effect in their IEG
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response. However, as adult female canaries are able to modify song preferences that they experienced in early life (Nagle and Kreutzer 1997a,b), they may be able to adjust their preferences according to the experimental conditions, as they had access to sexy as well as nonsexy syllables before the final testing. Further, in an electrophysiological study in starlings, Gentner and Margoliash (2003) found an experiencedependent representational plasticity in CMM. An interesting result of different auditory processing mechanisms in CMM and NCM was revealed by a recent study on male starlings (Gentner et al., 2004). These authors found that CMM expression was influenced by both the novelty of the song and the persisting maintenance of existing representations, that is, recognition of familiar songs, whereas a differential expression in NCM was significantly elevated only during the acquisition of novel song discriminations. Our results in female canaries point in the same direction, as the exposure to a familiar song playback, that is, the songs that have been perceived during the preceding playback sessions, resulted in a differential expression only in CMM, but not in NCM, and thus can be attributed to a mechanism of memory reconsolidation. Alternatively, our results could suggest that in female canaries, CMM may be more important than NCM in integrating songs of different quality. In female zebra finches, lesions of CMM lead to a disruption of their song discrimination ability (MacDougall-Shackleton et al., 1998). In a number of studies, although CMM was involved in the integration of song stimuli as well, NCM was the area that showed greater differences in IEG expression (Gentner et al., 2001; Eda-Fujiwara et al., 2003; but see Bailey et al., 2002; Sockman et al., 2002). Both NCM and field L (the primary auditory projection region) have reciprocal connections with CMM, which could lead to a shift in neuronal integration according to different experimental conditions during auditory processing. Concerning auditory representation patterns in the avian forebrain, it is important to emphasize that in a number of studies, NCM was subdivided into different subregions, for example, in a dorsal and ventral part (Gentner et al., 2001; Eda-Fujiwara et al., 2003; Maney et al., 2003), in a medial and lateral part (Terpstra et al., 2004), and most interestingly, Ribeiro et al. (1998) found a specific pattern of activation within NCM according to different auditory stimuli and even syllables in canaries. Therefore, due to the diversity of applied NCM subdivisions and in light of the previous results found in the canary we found it not advisable to predispose an arbitrary division of NCM for analysis.
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In our study, Arc mRNA expression was higher in CMM compared to NCM, although Arc expression did not reveal a difference in our two experimental groups. In a study on rats (Guzowski et al., 2001), Arc RNA levels in the dorsal hippocampus were higher in those individuals that had been trained in water tasks compared to cage controls. Being an effector IEG, it was argued that Arc serves to task-relevant neural encoding processes during spacial learning. The results of our study could hint to a different mechanism of Arc RNA induction. Although we did find expression of Arc RNA in the female canary brain, there was no effect of sexually attractive versus unattractive stimuli. Thus, a possible differential expression of Arc may not be related to processes that are associated with an auditory response to stimuli related to sexual behavior. Consequently, if Arc is dependent on the intensity of learning processes and memory formation, and if auditory preference for syllables with a broad bandwidth is innate (Draganoiu et al., 2002), it is likely that the canaries in our experiment did not show differences in Arc expression, as the birds were exposed to both types of stimuli before, that is, sexy and nonsexy syllables. Thus, the lack of a differential Arc expression hints to an auditory processing system that is well activated, as reflected in a differential ZENK expression, but only in a certain context of memory formation and not in a context of evaluating song quality. Moreover, in a recent study Arc was involved in the consolidation of memories via modulation of brain-derived neurotrophic factor (BDNF) in a contextual fear conditioning experiment in rats, whereas memory reconsolidation required the transcription factor Zif268 (ZENK) in the hippocampus (Lee et al., 2004). Therefore, the lack of Arc-up-regulation in our study suggests that a differential response to sexy syllables does not involve synaptic plasticity, as syllable recognition would then lead to memory reconsolidation processes. An isolated but interesting result was the clear expression of our two IEG markers in the main song control nuclei of one experimental female. This is the first occurrence of IEG expression in an experiment concerned with song perception. According to our behavioral observations, conducted between the last playback and sacrifice, we had no indication that this female might have shown singing behavior during the experiment, and have no clear explanation to offer. However, in a recent experiment investigating IEG response to song playback in white-crowned sparrows (Zonotrichia leucophrys oriantha), Maney et al. (2003) found ZENK expression in HVC, RA, and Area X in one female that was singing a number of songs in response to the playback. They further reported that this pattern of ZENK induction was similar to that of singing males. In our study, the distribu-
tion of the IEG expression in HVC and RA clearly corresponded to other robust markers of the song control system, such as androgen receptor mRNA or Nissl stain (personal observation). However, so far IEG expression in song control nuclei has not been found in any other studies that were concerned with song perception (Ball and Gentner, 1998; Bolhuis and Eda-Fujiwara, 2003). In a previous experiment on female canaries, we found that those individuals that responded and discriminated more between male songs of different quality had a larger HVC (Leitner and Catchpole, 2002). Moreover, lesions of HVC lead to a loss of the females’ discrimination ability between various conspecific songs (Del Negro et al., 1998; Halle et al., 2002). Since the first investigations of Brenowitz (1991) concerning the role of the song control system in female perception, all these studies suggest that HVC somehow is involved in the females’ perception of male song. Moreover, HVC receives input from CMM via the nucleus interfacialis (Nif). Therefore, in light of all these results, both HVC and CMM may play an important role in differentiation of learned song patterns. In terms of stimulus presentation, our data suggest that the more complex sexy syllables could act to prevent a habituation effect in CMM. Generally, a monotonous stimulus containing few syllables is thought to lead to a habituation of the neuronal response to this stimulus (Searcy, 1992). In an electrophysiological experiment, Chew et al. (1995) found that in NCM the neuronal habituation after repeated presentation of a stimulus is specific to that stimulus, and that the presentation of a novel stimulus initially elicits a large response before habituation occurs again after repeated presentation. It was argued that there is a correlation between the expression of ZENK and the long-term modification of neuronal responsiveness (Chew et al., 1995). With a possible scenario of the maintenance of long-term plasticity in NCM, the repeated presentation of sexy syllables to female canaries would not cause changes in IEG expression. Further experiments concerning the role of song elements that are tightly connected to reproductive behavior are needed to elucidate the role of gene expression and their products in the mediation of auditory song memories. We thank Michael Salbaum for providing zebra finch ZENK plasmid DNA.
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