studies have indicated that at least one species that does not learn vo-calizations has the capacity to entrain to a beat (Cook et al., 2013;Wilson and Cook 2016). While the extent to which vocal learning isrequired for rhythmic entrainment is still unclear (Ten Cate et al.,2016), a far greater number of vocal-learning species than non-vocallearners have demonstrated a capacity to entrain to a beat (Schachneret al., 2009). As one of the few of these species amenable to breeding inlaboratory conditions, the zebrafinch is a good model to study thejuvenile development of rhythm perception. Their songs, which areused for nest site defense and courtship, are highly rhythmic (Nortonand Scharff, 2016; Zann, 1996). A zebrafinch song bout consists of asequence of repetitive introductory notes, followed by repetitions of anordered set of syllables called a motif. The intervals between notes inthese songs are very regular (Fig. 1). The natural rhythmic structure ofzebrafinch song adds to their value as a model species to improve ourunderstanding of how rhythm perception develops in a vocal-learningspecies.Whether zebrafinches and other birds are capable of perceivinghigher order rhythms or are simply focused on local timing elements isan issue still under debate (Benichov et al., 2016; Ten Cate et al., 2016).In a go/no go paradigm where birds were asked to discriminate be-tween regular and irregular beat patterns, zebrafinches seemed todefault to making decisions based on local temporal structure, such asduration of notes or inter-onset intervals, but also appeared capable ofdetecting broader temporal structure, encompassing multiple shorterintervals (Ten Cate et al., 2016). A similar study judged birds’capacityto discriminate between isochronous and irregular tone sequences (vander Aa et al., 2015). While zebrafinches could discriminate between thetraining stimuli, they were not able to generalize to isochronousrhythms at different tempi, which was interpreted to mean that theywere only able to attend to local timing features (van der Aa et al.,2015). In addition, both of these studies utilize stimuli composed ofpure tones to assess rhythmic discrimination capabilities in zebrafin-ches. NCM and CMM do not respond as strongly to tones as conspecificvocalizations (Stripling et al., 2001; Mello et al., 1992; Bailey et al.,2002; Bailey and Wade, 2003, 2006), thus the capacity to processglobal rhythms may be different if the sound pattern is composed ofnatural zebrafinch sounds which induce more activity in auditoryprocessing areas of the brain. The overall capacity of zebrafinches toattend to different levels of timing and rhythm requires further in-vestigation. We used the immediate early gene ZENK with relativelynatural song stimuli to assess differences in neural responses to ecolo-gically relevant rhythmic and arrhythmic stimuli.Expression of this gene and/or its protein product is frequently usedto study neural activity in zebrafinches. ZENK is an acronym for themultiple names that have been assigned to this evolutionarily conservedprotein, zif-268 (Christy et al., 1988), egr-1 (Sukhatmeet al., 1988),NGFI-A (Milbrandt, 1987), and Krox-24 (Lemaire et al., 1988). ZENK isinvolved in learning and synaptic plasticity (Mello et al., 2004), and canact through regulation of other genes through a DNA binding site(Christy and Nathans, 1989). Inhibition of ZENK expression in juvenilezebrafinches during tutor song exposure prevents normal song learning(London and Clayton, 2008).A previous study in our lab focused on rhythm effects on ZENKexpression in the adult zebrafinch brain (Lampen et al., 2014). ZENKexpression was assessed in the caudomedial nidopallium (NCM), thecaudomedial mesopallium (CMM), which while anatomically distinct inthe zebrafinch brain, are both considered analogous to the auditoryassociation cortex in humans (Bolhuis and Gahr, 2006), and nucleustaeniae (Tn) which is analogous to the mammalian amygdala (Riterset al., 2004). NCM is likely the location where the learned song tem-plate is stored in the brain (London and Clayton, 2008; Gobes et al.,2010; Yanagihara and Yazaki-Sugiyama, 2016). We hypothesize thatthis template may contain information about the proper timing ofsongs, allowing for discrimination of timing and rhythmicity. In ourstudy on adults (Lampen et al., 2014), birds exposed to song that wasmodified to disrupt its natural rhythmic structure had significantlymore ZENK expression in all three brain regions compared to birdsexposed to song with the same syllables presented with the original(unmodified) rhythm. These different levels of activity in secondaryauditory areas may suggest that birds perceive errors in the arrhythmicsong relative to the learned template. The increased activity in Tn,which is involved in pair bonding and mate selection (Riters et al.,2004; Dios et al., 2013; Svec et al., 2009), may indicate an aversion tothe disrupted rhythm and an assessment of poor quality as a potentialmate.The general timeline of vocal development in zebrafinches is agreedupon, but the exact ages at which specific milestones occur is still de-bated to some degree (Doupe, 1993; Doupe and Solis, 1997; Mooney,2009; Tomaszycki et al., 2009). Template formation occurs betweenapproximately post-hatching days 20 and 60 (Tomaszycki et al., 2009;Mooney, 2009; Doupe and Solis, 1997). In males, vocalizations begin asfood begging, then develop into subsong, an immature form of vocali-zation that is quiet and contains poorly formed notes with greatlyvariable structure and sequence (Zann, 1996). As the males mature,these songs are practiced and modified to relatively closely match thelearned template. This phase of sensorimotor integration occurs be-tween about post hatching day 25 and sexual maturity which occursaround 90 days of age (Doupe and Solis, 1997; Mooney, 2009;Tomaszycki et al., 2009). After sensorimotor integration, males have acrystalized song that will not undergo significant changes throughoutthe rest of their lives under normal circumstances (Doupe,1993). Inaddition to learning to produce specific song syllables, juvenile malesalso learn a specific grammar to structure their song (Menyhart et al.,2015).Understanding the developmental trajectory of neural responses torhythm is useful in elucidating their relationship to function. We ex-posed birds to rhythmic or arrhythmic song prior to and during thetemplate formation period, as well as during early sensorimotor in-tegration. Differences in neural activity following stimulation with thetwo types of songs prior to acquisition of a template could indicate aninnate capacity to perceive song-related rhythms. Discriminationduring and after template formation, but not before, would suggest thatcharacteristics of rhythm are learned. If rhythm discrimination emergesduring sensorimotor integration in males, it would suggest that a motorcomponent is required for distinct neural responses to rhythmicity.Differences between the sexes may be informative here as well, as bothmales and females appear to form templates (Lauay et al., 2004), eventhough only males engage in the production of song (Zann, 1996).2. Material and methods2.1. SubjectsZebrafinches hatched and were reared in large, walk-in aviariescontaining 5–7 adult male and female pairs and their offspring. On dayof hatching, toes were clipped as a means of unique identification.Tissue from the toes was used to identify the sex of the birds throughFig. 1.A natural zebrafinch song used to generate one of the stimuli from the presentstudy is depicted. Introductory notes are labeled with I. A, B, and C labels indicate se-parate syllables in the song, with each repetition of the three syllables constituting amotif.Modified from Fig. 1 in (Lampen et al., 2014).J. Lampen et al.Behavioural Processes 163 (2019) 45–5246