ª Federation of European Neuroscience Societies

European Journal of Neuroscience, Vol. 22, pp. 949–955, 2005

Social context modulates behavioural and brain immediate early gene responses to sound in male songbird Cle´mentine Vignal,1,2 Julie Andru,1 and Nicolas Mathevon1 1

Equipe ‘Communications Acoustiques’, NAMC CNRS UMR 8620, Universite´ Paris-XI-Orsay, & ENES, Universite´ Jean Monnet, 23 rue Docteur Paul Michelon, 42023 Saint-Etienne Cedex 2, France 2 Laboratoire Traitement du Signal et Instrumentation, Universite´ Jean Monnet, 10 ruebarroin 42000 Saint-Etienne, France Keywords: auditory perception, immediate early gene, social environment, zebra finch, ZENK, Zif-268

Abstract Although it is well known that brain sensory information processing is a highly modulated phenomenon, how this brain function is shaped by experience and social context remains a question to explore. In this paper, we present the first attempt to investigate this problem using a songbird acoustic communication paradigm. Social context is well known to influence acoustic communicating behaviours in birds. The present paper investigates whether brain processing of auditory inputs can be modulated by this ‘audience effect’. Given that call-based communication is known to be highly context-dependent, we focused on the response of male zebra finches (Taeniopygia guttata) to female calls. We tested to see if the current social context surrounding the hearing bird can modify a sound-induced immediate early gene (IEG) activation in the specific region of the caudomedial nidopallium (NCM), a songbird brain analogous to the superficial layers of the mammalian primary auditory cortex. Our results show that the expression of the sound-induced immediate early gene ZENK in the NCM is considerably enhanced when the hearing bird is in the presence of conspecifics, compared to when he is alone. This context-dependent increase of a sound-induced immediate early gene expression can be correlated with the differential behavioural response of males to the playback of the same acoustic stimulus as a function of social context.

Introduction Besides their elaborate songs emitted mainly for courtship and territory defence, songbirds use a repertoire of calls with simpler acoustic structure. Calls provide information about factors such as the bird’s identity or the presence of different important stimuli in the bird’s environment such as food or predators (Marler, 2004). They thus often exhibit a highly variable pattern of production. Emotional state has been implicated in the regulation of call emission; stressful situations like predation are known to modify call signalling behaviour in songbirds (McGregor et al., 2000). At the receiver’s end, it has also been shown that behavioural response to calls can be modified by the current audience – the so-named ‘audience effect’ (Baltz & Clark, 1997; Evans & Evans, 1999; Vignal et al., 2004b). Songbird’s callbased acoustic communication may thus represent a good paradigm to investigate the question of if and how social context may modify neural processing of sound stimuli. One of the striking aspects of neural responses to acoustic stimuli in the bird brain is the sound-induced activation of immediate early genes (IEGs). ZENK, the avian homologue of zif-268, is a neural activitydependent IEG induced in cells of song control nuclei during singing behaviour [e.g. in high vocal center (HVC) and robust nucleus of the arcopallium (RA), Jarvis & Nottebohm, 1997; see also Reiner et al., 2004 for recent avian nomenclature], and in areas of central auditory pathways during sound hearing [e.g. in caudomedial nidopallium (NCM), Field L1 and L3, caudomedial mesopallium (CMM), reviewed in Mello, 2002]. Remarkably, exposure to conspecific song leads to more robust ZENK expression in the NCM than does the presentation of

heterospecific signals (Mello et al., 1992; Mello & Ribeiro, 1998; Stripling et al., 2001). NCM thus appears to be a possible area for extraction of biologically relevant information in the acoustic signal (Ball & Gentner, 1998; Mello, 2002; Vignal et al., 2004a). Thus, the magnitude of the ZENK response could depend on the relevance of the information, which can be defined by the stimulus type (Ribeiro et al., 1998; Gentner et al., 2001; Eda-Fujiwara et al., 2003; Maney et al., 2003), its recent familiarity (Mello et al., 1995; Sockman et al., 2002), the individual experience of the bird (Bolhuis et al., 2000; Hernandez & MacDougall-Shackleton, 2004; Sockman et al., 2005), the bird’s sex (Duffy et al., 1999; Bailey & Wade, 2003; Phillmore et al., 2003), but also by the context in which the stimulus appeared. Indeed, the specificity of the ZENK response in the NCM is altered by physical restraint (Park & Clayton, 2002) and thus may be modulated by environmental factors associated with arousal and stress. Although previous works have shown that social context could influence singingrelated neural activity in motor control nuclei (Jarvis et al., 1998; Hessler & Doupe, 1999), no study has yet investigated the impact of social factors on the brain sensory processes. The present paper addresses this issue by investigating whether current social context can alter the behavioural responses and the brain immediate early gene responses of male to female distance calls in the zebra finch (Taeniopygia guttata).

Materials and methods Subjects

Correspondence: Dr C. Vignal, as above. E-mail: [email protected] Received 21 February 2005, revised 17 May 2005, accepted 18 May 2005

doi:10.1111/j.1460-9568.2005.04254.x

Thirty-six adult male zebra finches (Taeniopygia guttata) served as the subjects for the experiments and were naive to all testing procedures. These birds were bred in our aviary (12L ⁄ 12D photoperiod with

950 C. Vignal et al. adapted wavelengths; food and water ad libitum; temperature between 23 and 25
C). All experiments occurred between 08:00 and 09:00 h. During isolation and stimulation periods, conditions of temperature, food and water were the same as in the aviary. The experimental protocols were approved by the Jean Monnet University’s animal care committee.

Sound stimuli We recorded distance calls from four female zebra finches bred in our aviary (using a SENNHEISER MD42 microphone placed 0.3 m above the cage and connected to a MARANTZ PMD690 ⁄ W1B recorder with 22 050 Hz sampling rate). A synthetic copy of one distance call of each female was created with Avisoft-SAS LaboratoryPro software (version 4.16, 2002) in order to have experimental signals with neither any background noise nor sound degradation. We used the graphic synthetizer of Avisoft-SAS Laboratory-Pro software, which allows first to accurately reproduce the modulations of the original fundamental frequency and second to create the associated harmonic series, the whole signal presenting the same energy spectrum as the one of the natural call. We took particular care to obtain high-quality synthetic signals. All the copies used for our experiments were accurately mimicking the acoustic structure of the originals in both temporal and frequency domains. According to preliminary experiments and previous behavioural results (e.g. Vignal et al., 2004b), these synthetic calls could not be perceptually discriminated from the natural calls by the tested bird. These synthetic female calls were used for playback tests; zebra finch distance calls have been shown to induce specific response as songs do (Chew et al., 1996). We constructed four different playback signals, each composed of calls of one out of the four recorded females. One of these four playback signals was randomly chosen and was broadcast in each playback experiment, avoiding the pseudo-replication problem.

Neurobiological experiments The subjects were divided into two experimental groups defined by their social context: (i) isolated birds (n ¼ 12) – each subject was put in complete social isolation in an experimental cage placed in an acoustic isolated chamber during the whole period of the experiment and (ii) grouped birds (n ¼ 12) – each subject was accompanied by another birds cage placed near the experimental cage in the chamber. This companion cage contained two male birds, which constituted the ‘audience’. These companions were different individuals for each trial. Because they were all bred in the same aviary, all audience birds were familiar to the subjects. The playback procedure was as follows. One sound stimulus was composed of 60 repetitions of a series of ten identical calls (approximately 1 call per second, i.e. natural call rate of a female, with an interstimuli interval randomly chosen from 0.5 to 1 s). Two consecutive series were separated by 20 s of silence. Consequently, the total duration of the stimulus was 30 min. Each tested bird was acoustically and visually isolated from the aviary, either alone (Isolated) or accompanied by its audience (Grouped), for 24 h prior to the start of stimulus presentation. The tested bird and the audience (if any) were thus housed in an experimental cage and in a separate cage, respectively, both placed in a soundproof chamber on a 12L ⁄ 12D photoperiod, except in the last six hours and during the stimulation period where they were in darkness in order to avoid any uncontrolled stimulation of the NCM by spontaneous singing behaviour. In preliminary experiments, tested birds accompanied with

an audience were recorded with a video recorder (SONY DCRTRV33) in order to confirm that the darkness avoided any vocalizations either in response to playback or spontaneously. All the birds remained silent during the IEG playback using this design. The emission chain was composed of two high fidelity speakers (TRIANGLE COMETE 202) placed at either end of the experimental cage connected to a DAT recorder (SONY DTC-ZE 700) and an amplifier (Yamaha AX-396). During each test only one randomly chosen speaker emitted the playback stimulus (sound level 60 dB at 1 meter). After the 24 h isolation period, the tested bird was presented with 30 min of one acoustic stimulus. The acoustic stimulation was followed immediately by 30 min of silence, during which the animal was kept inside the isolation chamber. In each of the two experimental groups (Isolated and Grouped; n ¼ 12 birds per group), six birds were challenged with a sound stimulus (six isolated and challenged birds; six grouped and challenged birds) and six birds were kept all the time in silence as control (six isolated and in silence birds; six grouped and in silence birds). The tissue preparation and the immunocytochemistry (ICC) protocol were as follows. The bird was killed by decapitation one hour after the start of the stimulation period, which is within the time of peak ZENK protein expression (Mello et al., 1992). The brain was then frozen at )30
C during 1min in 2-methylbutane (Sigma–Aldrich Chemie GmbH) on dry ice and then stored at )80
C. Brain sections (14 lm) were performed in the sagittal plane using a cryostat in the left hemisphere. The sections were mounted onto slides (Superfrost plus, Mewzel glaser GmbH) and stored at )20
C. Each of the following steps was followed by three washes (10 min each) in 0.01 m phosphate buffer saline (PBS 0.01 m Sigma Laboratories # P-3813). First, slides were fixed in 4% paraformaldehyde for one night at 4
C. Next, slides were incubated as follows: (i) 30 min at room temperature (RT) in blocking solution (BS; 0.5% albumin and 0.3% Triton X-100 in PBS 0.01 m); (ii) biotin blocking treatment (Dako # X0590) at RT; (iii) one night at 4
C in a commercially available Egr-1 antibody (Santa Cruz Biotechnology, catalogue # sc-189) diluted at 1 : 1000 in BS; (iv) 2 h at RT in biotinylated goat antirabbit IgG (Sigma Laboratories # B7389) diluted at 1 : 200 in BS and (v) 1 h 30 min at RT in avidin-biotin peroxidase complex (Sigma Laboratories # E-2886) diluted at 1 : 150 in 0.01 m PBS, followed by incubation in 3,3¢-diaminobenzidine (DAB; Sigma Laboratories PAST 3,3¢ DAB Tablet sets). Reaction time in the DAB was held constant at 10 min across all different ICC runs. Controls were run by omitting the primary Egr-1 antibody used in step (ii). Finally, the sections were dehydrated and coverslipped with Entellan. In order to quantify the level of ZENK expression in each subject according to experimental condition, we took three section levels in the NCM, situated at 432, 628 and 824 lm of the midline. The images of the sections were captured in grey levels via a numeric photographer (NIKON Coolpix 4500) mounted on top of a microscope (LEICA DMLB, 5· objective, 10· eyepiece). We quantified the NCM’s labelling into a sampling area, with boundaries defined as follows (Fig. 1). We cut the captured image following the ventricle separating the NCM from hippocampus, then we separated then the NCM from the more rostral auditory areas Field L and CMM (caudomedial mesopallium, see Reiner et al., 2004 for a revision of avian brain nomenclature) by defining a rostral border of the NCM straight before the band of very light ZENK expression defining Field L. For the more lateral section level, this chosen rostral boundary could include part of the area Field L3 in the sampling area. Because previous studies (Mello & Clayton, 1994) observed that Field L3 showed increased ZENK expression in response to conspecific song as the NCM does, we can consider this region as

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 949–955

Social context and sound-induced immediate early gene expression 951 homogeneous for the response to acoustic stimuli. This chosen sampling area allowed us to probe ZENK expression in comparable regions across the medio-lateral structure of the NCM. In order to quantify the spatial repartition of ZENK expression, this sampling area was then divided in four topographical zones for quantification of ZENK expression, as shown in Fig. 1. We counted the number of ZENK immunoreactive cells (NI) within these reference boundaries using NIH Image 2000 software (Scion corporation Beta 4.0.2). Using the density slice function, we defined a threshold corresponding to the grey level above which a cluster of pixels was considered signal, and below which it was considered noise (Gentner et al., 2001). The threshold used for each sample is defined from the maximum grey level observed on control section (ICC omitting the primary Egr-1 antibody). We defined the size of a cluster of pixels corresponding to the size of the nucleus of NCM cells. This threshold size was used in the particles counting procedure of NIH Image 2000 software. The experimenter who counted the immunoreactive cells was blind to all experimental conditions. To avoid any effect of the surface of the sampling area, the NI was normalized (that is the density of counted cells inside the zone expressed in cells ⁄ mm2). The NI for each zone in each section was then transformed using the NI in order to homogenize the variances and to obtain a gaussian distribution. Differences between NI-values were examined using an analysis of variance for repeated measures, with topographical zones as within group factor and three between groups factors: social context (Grouped vs. Isolated), acoustic stimulus (Challenged with calls vs. in Silence) and section level (repeated-measures anova, P ¼ 0.05, Statistica Software version 6). The anova was followed by a Fisher PLSD posthoc test (P ¼ 0.05). All means were expressed with their corresponding standard errors (SEM).

Behavioural experiments As in the neurobiological experiments, the subjects (n ¼ 12) were divided into two experimental groups defined by their social context. (i) Isolated birds (n ¼ 6) – each subject was placed in complete social isolation during the all time of the experiment. (ii) Grouped birds (n ¼ 6) – each subject was accompanied by a companion cage containing two male birds. These companions were different individuals during each trial. Because they were all bred in the same aviary, all audience birds were familiar to the subjects. The playback procedure was as follows. The playback stimulus was composed by ten repetitions of a series of ten identical calls (approximately 1 call per second, i.e. natural call rate of a female, with an interstimuli interval randomly chosen from 0.5 to 1 s). Two consecutive series were separated by 20 s of silence. Consequently, the total duration of the stimulus was 5 min. Each tested bird was placed in the experimental cage 24 h prior to the start of stimulus presentation. This cage (and the companion cage for grouped birds) was placed in a soundproof chamber under a 12L ⁄ 12D photoperiod. The experimental cage (240 · 50 · 50 cm) was equipped with roosts. Two speakers connected to a DAT recorder (same equipment as for the neurobiological experiments) was placed at either end of the

Fig. 1. (A) Parasagittal section of the right hemisphere of the zebra finch brain, stained with cresyl violet, showing the boundaries of the NCM. The NCM is observed in front of the cerebellum (cb). The captured images of sections treated for ZENK-ICC labelling (B) are cut following the ventricle separating the NCM from hippocampus (Hp) and from the more rostral auditory areas Field L and CMM (caudomedial mesopallium). This sampling area was then divided into four topographical zones of quantification of ZENK expression as shown in B. ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 949–955

952 C. Vignal et al. experimental cage. During each test, only one randomly chosen speaker emitted the playback stimulus (sound level 60 dB at 1 meter). During the playback test, the vocal and locomotor activities of the tested bird were recorded with a video recorder (SONY DCR-TRV33). Male zebra finches respond to the playback of conspecific calls by producing vocalizations, e.g. song and two different types of calls (Zann, 1996; Vignal et al., 2004b). To assess the bird’s response during our playback tests, we thus counted the three types of vocalizations likely to be emitted by the tested birds. (i) The distance calls, equally known as ‘long calls’ (Zann, 1984; Vicario et al., 2001) are the most frequently sounds emitted by both males and females zebra finches. The distance call is the longest and loudest call and is used by birds to remain in contact when the flock is foraging or feeding and especially when the birds lose visual contact with each other (Zann, 1984). The male distance call is a complex sound with an elevated fundamental frequency (600–1000 Hz) associated with several harmonics. It is typically a frequency modulated downsweep and presents a short duration of around 100 ms (Zann, 1984; Vicario et al., 2001). (ii) The contact calls, although known as ‘soft calls’ or ‘tets’ (Zann, 1996) are emitted during hopping movements and are not directed at one particular individual. Contact calls have harmonic structure too but present lower fundamental frequency (400–500 Hz) and shorter duration (50 ms) than distance calls. Contrary to distance calls, contact calls are relatively unmodulated in frequency. (iii) The songs are more or less elaborated combinations of several notes and calls, emitted mainly by the male to the female during precopulatory courtship (Zann, 1996). For each tested male, the three types of vocalizations emitted during playback were identified and counted in recordings using Syntana software (Aubin, 1994) and Goldwave 4.26. Three behavioural parameters reflecting the vocal activity of the tested bird were thus measured: the number of distance calls (NDC), the number of contact calls (NCC) and the number of songs (NS). Basal activity of each bird was also assessed by measuring the vocal activity during one minute prior to the beginning of playback broadcasting. As our sample failed normality and homogeneity, we performed nonparametric statistical tests. The effect of the social context (Isolated or Grouped) on the bird’s basal activity was analysed with a nonparametric Mann–Whitney U-test (P ¼ 0.05, Statistica Software version 6) for each parameter (NDC, NCC and NS). The effect of the social context (Isolated vs. Grouped) on the bird’s vocal activity in response to playback was measured using a nonparametric Kolmogorov–Smirnov two-sample test (P ¼ 0.05, Statistica Software version 6). The NDC, NCC and NS were thus compared between Isolated and Grouped conditions.

Results Neurobiological experiments We observed a statistically significant effect of acoustic stimulation on the ZENK-ICC labelling of NCM’s cells (parameter NI) (repeatedmeasures anova, P < 0.001). Acoustic stimulation with female calls (C, Fig. 2) increased ZENK-ICC labelling in comparison with silence condition (S, Fig. 2; P < 0.001). Social context also significantly modified the ZENK-ICC labelling, which is greater in grouped birds (G, Fig. 2) in comparison with isolated birds (I, Fig. 2; P < 0.001). We observed a significant effect of the interaction between acoustic stimulation and social context (P ¼ 0.018): whereas the ZENK-ICC labelling in silence did not depend on social context (GS not significantly different from IS; Fig. 2; Fisher PLSD P ¼ 0.098), the ZENK-ICC labelling induced by acoustic stimulation is strongly

Fig. 2. ZENK-ICC labelling (A) in the zone 1 of the NCM of grouped birds challenged with calls (GC), grouped birds in silence (GS), isolated birds challenged with calls (IC) and isolated birds in silence (IS). (B) Acoustic stimulation increased ZENK-ICC labelling in comparison with silence in both social contexts (C groups significantly higher than in S groups, Fisher PLSD ***P < 0.001). Whereas ZENK-ICC labelling in silence did not depend on social context (GS not significantly different from IS, Fisher PLSD P ¼ 0.098), ZENK-ICC labelling in response to calls is strongly enhanced in the Grouped context vs. the Isolated one (GC significantly higher than IC, Fisher PLSD ***P < 0.001). Boxes represent standard errors and bars correspond to standard deviation.

enhanced in the Grouped context vs. the Isolated one (GC significantly higher than IC; Fig. 2; Fisher PLSD P < 0.001). Thus, whereas the basal level of ZENK expression in silence is not modified by social context, the magnitude of the ZENK-ICC labelling in response to acoustic stimulation is much increased in the presence of a conspecific audience. We also measured a statistically significant effect of section level on ZENK-ICC labelling (repeated-measures anova, P ¼ 0.018). ZENKICC labelling decreased from the midline to the lateral part of the NCM. There was no effect of interaction between section level and social context (Grouped or Isolated) on the ZENK-ICC labelling (P ¼ 0.93) and between section level and acoustic stimulus (challenged with calls or in silence) on the ZENK-ICC labelling (P ¼ 0.64). The ZENK-ICC labelling decrease from midline to lateral NCM does not depend on social context or on acoustic stimulus. Besides this result, the repeated-measures anova shows a statistically significant effect of topographical zones of quantification of

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 949–955

Social context and sound-induced immediate early gene expression 953

Fig. 3. Median number of vocalizations emitted during playback test by grouped birds (open bars) and isolated birds (filled bars). Points are medians, boxes represent quartiles and bars correspond to minimum–maximum values. Three types of vocalizations are counted: Contact Calls (CC), Distance Calls (DC) and Songs (S). Social context significantly modifies the vocal response to playback; whereas isolated birds use significantly more Contact Calls, grouped birds emit significantly more Distance Calls and Songs (nonparametric Kolmogorov–Smirnov two-sample test ***P ¼ 0.001).

ZENK expression as within-factor (P < 0.001). Zone 1 presents a significantly higher ZENK-ICC labelling than the other three (Ficher PLSD P < 0.001); conversely, zone 4 presents a significantly lower ZENK-ICC labelling than the other three (Ficher PLSD P < 0.001). The NCM thus shows a topography of ZENK expression. There is no effect of interaction between the topographical zones of quantification and the factors social context (P ¼ 0.053) and acoustic stimulus (P ¼ 0.151) on the ZENK-ICC labelling. The topography of ZENKICC labelling in NCM is not modified by social context or by acoustic stimulus. There is no effect of interaction between section level and topographical zones of quantification (P ¼ 0.679). The topography of ZENK-ICC labelling in NCM is maintained by ZENK-ICC labelling decrease from midline to lateral NCM. There is no effect of interaction between section level, topographical zones of quantification, social context and acoustic stimulus (P ¼ 0.31).

Behavioural experiments Social context did not modify the basal activity of the bird (nonparametric Mann–Whitney U-test; NDC P ¼ 0.63, NCC P ¼ 0.63 and NS P ¼ 0.15). Conversely, social context significantly influenced the vocal response to playback: contrary to grouped birds which emit significantly more distance calls and songs in response to playback (nonparametric Kolmogorov-Smirnov two-sample test for NDC P ¼ 0.001 and NS P ¼ 0.001, Fig. 3), isolated birds used significantly more contact calls (nonparametric Kolmogorov-Smirnov two-sample test for NCC P ¼ 0.001, Fig. 3). Social context thus modifies the behavioural response of males to conspecific females calls; whereas grouped males use numerous calls of strong intensity (distance calls) and songs, isolated males show weaker vocal responses constituted by more calls of lower intensity (contact calls).

Discussion To our knowledge, the present study is the first to demonstrate unequivocally the influence of a conspecific audience on the ZENK gene activation linked to acoustic perception in the songbird brain.

Our results emphasize that a genic expression induced by a sensory stimulus (here female calls) can be modulated by the social context occurring during stimulus processing. Previous works have shown that the presence of conspecifics can influence brain ZENK expression in motor control nuclei during song production (Jarvis et al., 1998; Hessler & Doupe, 1999) but this effect has never been investigated in auditory processing areas. In this study, we show that the same acoustic stimulus – conspecific female calls – induces a higher ZENK response in NCM when the tested male is in the presence of two conspecific males than when he is alone. This context-dependent increase of ZENK response can be correlated with the differential behavioural response of males to the playback of female calls as a function of social context. A grouped male uses mainly calls of high intensity (distance calls) and songs in response to the playback of female calls, whereas a male alone shows a behavioural response typical of a gregarious bird isolated from its colony with more calls of low intensity (contact calls) (Zann, 1996). Conversely, the bird’s basal state, i.e. ZENK expression in silence as well as vocal activity before playback, is not influenced by social context. Whereas our behavioural experiments were realized in normal light conditions, the playbacks for ZENK experiment were performed in darkness in order to avoid any uncontrolled stimulation of the NCM by the spontaneous singing behaviour of the tested bird. Because the isolation period began 24 h prior to the start of stimulus presentation, the tested bird in the grouped condition could interact with his companions during one whole day before the playback. This could explain the reality of the influence of the social context in the ZENK experiment. Previous studies have observed that environmental factors could selectively enhance or attenuate the genomic response to particular stimuli. Indeed, the magnitude of the IEG expression bias in response to acoustic stimuli is known to be regulated by a bird’s recent experience (Sockman et al., 2002; Sockman et al., 2005) or by a bird’s experience early in life (Jin & Clayton, 1997; Bolhuis et al., 2000; Hernandez & MacDougall-Shackleton, 2004). Nevertheless, few studies have investigated the influence of immediate experience on the selectivity of the IEG response. Kruse and colleagues (Kruse et al., 2004) demonstrated that the abolished NCM ZENK response, due to repeated exposure to one given song, can be reinduced by changing auditory context, like speaker location, playback pressure level, or visual context. Moreover, genomic response in NCM is known to be influenced by somatosensory information like aversive footshock (Jarvis et al., 1995) or physical restraint (Park & Clayton, 2002). In our study, the ZENK expression is greater in response to conspecific vocalizations than in silence regardless of social context, but the magnitude of this expression bias in response varies with the immediate social context. However, it remains to be explored if social context facilitates the magnitude of ZENK expression in response to any acoustic stimuli or if this socially induced increase of brain IEG expression is strictly specific to conspecific vocalizations. Previous studies have demonstrated anatomical variations in IEG expression in response to different acoustic stimuli (Ribeiro et al., 1998; Gentner et al., 2001), but to our best knowledge no variation of this topography has been observed as a function of bird experience (Sockman et al., 2002; Phillmore et al., 2003; Hernandez & MacDougall-Shackleton, 2004; Sockman et al., 2005). Accordingly, our results show an anatomical topography of the NCM ZENK-ICC labelling in response to conspecific female calls, which is not modified by social context. Our results show that ZENK expression in NCM during auditory processing is modulated by the presence of conspecifics, whereas a previous study reported an influence of social context on ZENK expression in song motor pathway during song production (Jarvis

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954 C. Vignal et al. et al., 1998). One interesting point is that in our results, the presence of conspecifics increased the ZENK expression bias in response to conspecific vocalizations compared to ZENK expression in silence, whereas ZENK expression in song motor pathway is lower in males singing song directed towards a conspecific female compared to males singing alone (Jarvis et al., 1998). The regulation by the presence of conspecifics of ZENK expression thus seems to be different in auditory and in song motor brain areas. The causes of this difference remain unknown, but one possible basis of the context-dependent regulation of song-induced IEG expression could be the emotional state of the bird. Indeed, enhanced vigilance (Park & Clayton, 2002; Kruse et al., 2004) and arousal in the presence of conspecifics could explain this increased ZENK expression. On the contrary, social isolation could be a situation of stress (Wolfer et al., 2004) in comparison to the natural context where the zebra finch lives in large groups and experiences permanent social interactions (Zann, 1996). Indeed social isolation from familiar conspecifics is known to elicit glucocorticoid release in zebra finches (Remage-Healey et al., 2003). This physiological stress reaction could modulate cerebral IEG expression. Another possible explanation is that the presence of two male conspecifics in our experiments could be a context of particular relevance for the bird; the other males could appear as competitors in front of a potential sexual partner, i.e. the playback female. This kind of context-dependent differential eliciting value of the stimulus has previously been observed in zebra finches, e.g. the male pays attention to the mating status of conspecific pairs and uses this information to control its behaviour towards its female partner (Vignal et al., 2004b). As it is likely to be modulated by the socially driven emotional state of the bird, the NCM ZENK response can be seen as an integration of several stimulating factors of the environment. Thus, as the same playback stimulus broadcast in two different social contexts might not have the same salience for the zebra finch, it could elicit a different state of arousal or stress and consequently induce a differential ZENK response. This hypothesis meets the proposal that IEG induction corresponds to the ‘ethological sign’ of the stimulus (Ball & Balthazart, 2001; Kruse et al., 2004), this ‘sign’ being constantly fitted by the emotional state of the bird. Studies in mammals underlined that environmental stimuli regulate the expression of transcription factors, which subsequently alter gating of behavioural responses to emotional stimuli (Barrot et al., 2002). ZENK in NCM could appear as such a ‘gate’ between emotional acoustic stimuli and behavioural responses. It is to be noticed that this context-dependant ZENK activation may bring potential limitations about the use of the magnitude of the ZENK response in NCM as an index of auditory selectivity (Mello et al., 2004). ZENK induction has often been linked to the phenomenon of activity-dependent synaptic plasticity (Mello, 2002). Neuronal depolarization implies the expression of the immediate early gene ZENK, which represents an early regulatory event in a genomic cascade leading to long-lasting changes in neuronal cells (Mello, 2002). The ZENK gene encodes a zinc-finger transcription factor that binds to a DNA motif present in the promoter region of several genes. This genomic cascade potentially results in plasticity events in songactivated cells (Mello, 2002). The present results can provide some support to this view. The social regulation of sound-induced ZENK expression in the NCM might play a role in the plasticity necessary to the processing of conspecific vocalizations. The integration of the social context in the evaluation by the bird of the relevance of the acoustic stimulus can rely on synaptic plasticity events in secondary auditory brain areas like the NCM.

What are the neurophysiological pathways and processes supporting the social-dependent ZENK activation? Clearly, more investigations are needed. One argument in support of a potential role of arousal and attention in regulation of NCM activity is that this telencephalic area receives dopaminergic and noradrenergic innervations (Mello et al., 1998; Durstewitz et al., 1999; Pinaud et al., 2004) known to modulate alertness. Modulatory effects of environmental context on several physiological parameters are thought to affect the saliency of the primary stimulus by altering the sensitivity of neural pathways to their initial inputs. Other brain regions might be involved in context-dependent acoustic processing; for instance lMAN and area X are known to have a context specific neuronal firing (Hessler & Doupe, 1999) and ZENK expression level (Jarvis et al., 1998) associated to song production, as well as brain areas implicated in sexual motivation like POM (medial preoptic nucleus), which shows a context-dependent ZENK and FOS activation (Riters et al., 2004). The present study prepares the ground by investigating the influence of social context on call-induced ZENK expression in the NCM area. No doubt, further investigations considering different social conditions, other brain regions, and using various behavioural and neurobiological approaches will progressively bring a full understanding of how songbirds integrate social factors during their processing of acoustic signals.

Acknowledgements We are grateful to Colette Bouchut, Corinne Eyzac, Laurent Legendre, Michae¨l Ogier, Sabine Palle. We thank Clarisse Stouvenot and Sheetal Peetal for improving the English. This work was supported by the Interdisciplinary Program ‘Cognition and Information Processing’ (CTI 02–19) of the French Centre National de la Recherche Scientifique (CNRS) and by the Program ‘Emergence’ of the Re´gion Rhoˆne-Alpes. C.V. is supported by the French Ministry of National Education.

Abbreviations ICC, immunocytochemistry; IEG, immediate early gene; NCC, number of contact calls; NCM, caudomedial nidopallium; NDC, number of distance calls; NI, number of ZENK immunoreactive cells; NS, number of songs.

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