BACKGROUND:Neuroethologists study many non-model animals. The development of techniques for precise and standardized histological brain analysis is key for understanding neural mechanisms across species. NEW METHOD:Here we present a novel cost-effective approach to generate species-specific brain matrices for precise and reproducible trimming, blocking, and sectioning of brain tissue. To produce the matrix, we took high-quality photographs of two male CP brains and then used the open-source 3D graphics suite Blender and its inexpensive photogrammetry plug-in SnapMesh to generate the 3D brain surface model. The brain matrix was then modeled in Blender, and 3D printed. RESULTS:Using this approach, we produced the first 3D brain surface model and brain matrix for Seba's short-tailed fruit bat Carollia perspicillata, CP, and assessed its quality using CP brains. Our brain matrix facilitates and standardizes trimming or blocking of brains, providing consistent access to brain regions of CP in the sagittal and coronal planes. This workflow should be suitable for most vertebrate brains. COMPARISON WITH EXISTING METHODS:Photogrammetry offers a viable, inexpensive alternative to 3D- or CT-scanners. Our workflow is not only cheaper than alternatives, but does not require blocking the brain during preparation of the 3D surface model, resulting in no tissue loss. CONCLUSION:The cost-effectiveness and the tissue-preserving features will benefit researchers globally, particularly those with limited financial means and/or few valuable specimens. By providing an accessible, customizable and reproducible workflow, our study represents a significant step toward democratizing advanced neuroscience across diverse species.

IntroductionGenetic manipulation of murine retinal tissue through ocular administration of adeno-associated viruses (AAVs) has become a standard technique to investigate a multitude of mechanisms underlying retinal physiology. Resultantly, developments of recombinant viral vectors with improved transduction efficiency and further methodological improvements have mostly focused on murine tissue, whereas AAVs successfully targeting avian retinae have remained scarce.MethodologyUsing a custom-designed injection setup, we identified a viral serotype with the capability to successfully induce widespread transduction of the bird retina.ResultsIntravitreal administration of an AAV type 2/9 encoding for enhanced green fluorescent protein (EGFP) in night-migratory European robins (Erithacus rubecula) resulted in transduction coverages of up to 60% within retinal tissue. Subsequent immunohistochemical analyses revealed that the AAV2/9-EGFP serotype almost exclusively targeted photoreceptors: rods, various single cones (UV, blue, green, and red cones), and both (accessory and principal) members of double cones.DiscussionThe consistently high and photoreceptor-specific transduction efficiency makes the AAV2/9 serotype a powerful tool for carrying out genetic manipulations in avian retinal photoreceptors, thus opening a wealth of opportunities to investigate physiological aspects underlying retinal processing in birds, such as physiological recordings and/or post-transductional behavioural readouts for future vision-related research.

Rare disruptions of the transcription factor FOXP1 are implicated in a human neurodevelopmental disorder characterized by autism and/or intellectual disability with prominent problems in speech and language abilities. Avian orthologues of this transcription factor are evolutionarily conserved and highly expressed in specific regions of songbird brains, including areas associated with vocal production learning and auditory perception. Here, we investigated possible contributions of FoxP1 to song discrimination and auditory perception in juvenile and adult female zebra finches. They received lentiviral knockdowns of FoxP1 in one of two brain areas involved in auditory stimulus processing, HVC (proper name) or CMM (caudomedial mesopallium). Ninety-six females, distributed over different experimental and control groups were trained to discriminate between two stimulus songs in an operant Go/Nogo paradigm and subsequently tested with an array of stimuli. This made it possible to assess how well they recognized and categorized altered versions of training stimuli and whether localized FoxP1 knockdowns affected the role of different features during discrimination and categorization of song. Although FoxP1 expression was significantly reduced by the knockdowns, neither discrimination of the stimulus songs nor categorization of songs modified in pitch, sequential order of syllables or by reversed playback were affected. Subsequently, we analyzed the full dataset to assess the impact of the different stimulus manipulations for cue weighing in song discrimination. Our findings show that zebra finches rely on multiple parameters for song discrimination, but with relatively more prominent roles for spectral parameters and syllable sequencing as cues for song discrimination.NEW & NOTEWORTHY In humans, mutations of the transcription factor FoxP1 are implicated in speech and language problems. In songbirds, FoxP1 has been linked to male song learning and female preference strength. We found that FoxP1 knockdowns in female HVC and caudomedial mesopallium (CMM) did not alter song discrimination or categorization based on spectral and temporal information. However, this large dataset allowed to validate different cue weights for spectral over temporal information for song recognition.

The zebra finch has become a paradigmatic model system to mechanistically investigate vocal communication from genes to behavior. Here, we report 1) a comprehensive map of the polyadenylated fraction of the transcriptome of the male zebra finch telencephalon at two ages, during the song learning phase at 50 days and when fully adult at 2 years as well as 2) as an expanded and refined annotation of the zebra finch genome based on our transcriptome data. Using high‐throughput next generation sequencing of paired‐end strand‐ specific cDNA fragments, we detected over 50% of the annotated protein coding transcripts (ENSEMBL v.55) in the telencephalon at both stages. We exploited the information gained from the paired‐end sequence reads and from reads falling onto splice junctions to update the existing annotation (ENS55) with new transcript structures and alternative splicing events. The dataset allowed to refine 2822 of the available gene models by extending them in the 3’‐UTR and to detect 11391 undiscovered transcriptional units in non‐coding regions. Our results illustrate the value of continuously incorporating new transcriptional evidence into existing annotations.### Competing Interest StatementThe authors have declared no competing interest.

The search for molecular underpinnings of human vocal communication has focused on genes encoding forkhead-box transcription factors, as rare disruptions of FOXP1, FOXP2, and FOXP4 have been linked to disorders involving speech and language deficits. In male songbirds, an animal model for vocal learning, experimentally altered expression levels of these transcription factors impair song production learning. The relative contributions of auditory processing, motor function or auditory-motor integration to the deficits observed after different FoxP manipulations in songbirds are unknown. To examine the potential effects on auditory learning and development, we focused on female zebra finches (Taeniopygia guttata) that do not sing but develop song memories, which can be assayed in operant preference tests. We tested whether the relatively high levels of FoxP1 expression in forebrain areas implicated in female song preference learning are crucial for the development and/or maintenance of this behavior. Juvenile and adult female zebra finches received FoxP1 knockdowns targeted to HVC (proper name) or to the caudomedial mesopallium (CMM). Irrespective of target site and whether the knockdown took place before (juveniles) or after (adults) the sensitive phase for song memorization, all groups preferred their tutor's song. However, adult females with FoxP1 knockdowns targeted at HVC showed weaker motivation to hear song and weaker song preferences than sham-treated controls, while no such differences were observed after knockdowns in CMM or in juveniles. In summary, FoxP1 knockdowns in the cortical song nucleus HVC were not associated with impaired tutor song memory but reduced motivation to actively request tutor songs.

Visual (and probably also magnetic) signal processing starts at the first synapse, at which photoreceptors contact different types of bipolar cells, thereby feeding information into different processing channels. In the chicken retina, 15 and 22 different bipolar cell types have been identified based on serial electron microscopy and single‐cell transcriptomics, respectively. However, immunohistochemical markers for avian bipolar cells were only anecdotally described so far. Here, we systematically tested 12 antibodies for their ability to label individual bipolar cells in the bird retina and compared the eight most suitable antibodies across distantly related species, namely domestic chicken, domestic pigeon, common buzzard, and European robin, and across retinal regions. While two markers (GNB3 and EGFR) labeled specifically ON bipolar cells, most markers labeled in addition to bipolar cells also other cell types in the avian retina. Staining pattern of four markers (CD15, PKCα, PKCβ, secretagogin) was species‐specific. Two markers (calbindin and secretagogin) showed a different expression pattern in central and peripheral retina. For the chicken and European robin, we found slightly more ON bipolar cell somata in the inner nuclear layer than OFF bipolar cell somata. In contrast, OFF bipolar cells made more ribbon synapses than ON bipolar cells in the inner plexiform layer of these species. Finally, we also analyzed the photoreceptor connectivity of selected bipolar cell types in the European robin retina. In summary, we provide a catalog of bipolar cell markers for different bird species, which will greatly facilitate analyzing the retinal circuitry of birds on a larger scale.

Singing in birds is accompanied by beak, head and throat movements. The role of these visual cues has long been hypothesised to be an important facilitator in vocal communication, including social interactions and song acquisition, but has seen little experimental study. To address whether audio‐visual cues are relevant for birdsong we used high‐speed video recording, 3D scanning, 3D printing technology and colour‐realistic painting to create RoboFinch, an open source adult‐mimicking robot which matches temporal and chromatic properties of songbird vision. We exposed several groups of juvenile zebra finches during their song developmental phase to one of six singing robots that moved their beaks synchronised to their song and compared them with birds in a non‐synchronised and two control treatments. Juveniles in the synchronised treatment approached the robot setup from the start of the experiment and progressively increased the time they spent singing, contra to the other treatment groups. Interestingly, birds in the synchronised group seemed to actively listen during tutor song playback, as they sung less during the actual song playback compared to the birds in the asynchronous and audio‐only control treatments. Our open source RoboFinch setup thus provides an unprecedented tool for systematic study of the functionality and integration of audio‐visual cues associated with song behaviour. Realistic head and beak movements aligned to specific song elements may allow future studies to assess the importance of multisensory cues during song development, sexual signalling and social behaviour. All software and assembly instructions are open source, and the robot can be easily adapted to other species. Experimental manipulations of stimulus combinations and synchronisation can further elucidate how audio‐visual cues are integrated by receivers and how they may enhance signal detection, recognition, learning and memory.

In many songbird species, young birds learn their song from adult conspecifics. Like much animal communication, birdsong is multimodal: singing is accompanied by beak and body movements. We hypothesized that these visual cues could enhance vocal learning thus partly explaining the reduced learning from unimodal audio playbacks compared to multimodal live social tutoring observed in many birdsong studies. To test this, juvenile zebra finches, Taeniopygia guttata, were tutored in a yoked design where replicate tutoring groups of three male-female dyads were exposed to the same live tutor simultaneously in three different ways. (1) Tutees were housed with the tutor in a central compartment; hence they could hear, see and interact with their tutor ('live'). (2) Tutees placed in one of two adjacent compartments could hear but not see the same tutor from behind a black loudspeaker cloth ('audio only'). (3) Tutees could likewise hear the tutor through loudspeaker cloth but could also see the tutor through a one-way mirror ('audiovisual'). Comparisons of subadult and adult song showed more changes in the audio-only than in the audiovisual or live tutored tutees, suggesting the audio-only group's song development was delayed. According to (blinded) human observer similarity scoring, the audio-only tutees' singing was least similar and the live tutees' singing most similar to their tutor's singing, while the audiovisual tutees showed an intermediate level of similarity, but the between-treatment differences in similarity were not significant. Conversely, the audio-only group showed the highest similarity values with their father's song, which they only heard before the experimental tutoring. Given that the quantity and quality of the tutor song input were the same across treatments within tutoring groups, the results support the hypothesis that visual in addition to auditory exposure to a tutor can affect the timing and possibly also the amount of vocal learning.(c) 2022 The Author(s). Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour.

Bird song and human speech are learned early in life and for both cases engagement with live social tutors generally leads to better learning outcomes than passive audio-only exposure. Real-world tutor–tutee relations are normally not uni- but multimodal and observations suggest that visual cues related to sound production might enhance vocal learning. We tested this hypothesis by pairing appropriate, colour-realistic, high frame-rate videos of a singing adult male zebra finch tutor with song playbacks and presenting these stimuli to juvenile zebra finches ( Taeniopygia guttata ). Juveniles exposed to song playbacks combined with video presentation of a singing bird approached the stimulus more often and spent more time close to it than juveniles exposed to audio playback only or audio playback combined with pixelated and time-reversed videos. However, higher engagement with the realistic audio–visual stimuli was not predictive of better song learning. Thus, although multimodality increased stimulus engagement and biologically relevant video content was more salient than colour and movement equivalent videos, the higher engagement with the realistic audio–visual stimuli did not lead to enhanced vocal learning. Whether the lack of three-dimensionality of a video tutor and/or the lack of meaningful social interaction make them less suitable for facilitating song learning than audio–visual exposure to a live tutor remains to be tested.

Vögel in Kunst und Literatur - spannende Begegnungen. Ob es sich beim Gesang der Vögel um eine Form von »Naturmusik« oder gar um eine Sprache handelt, wird aktuell in verschiedenen naturtheoretischen, philosophischen, zoomusikologischen und von den animal studies inspirierten Kontexten diskutiert. Unstrittig ist neben dem reinen Faktum der Stimmenvielfalt die Fülle der Referenzen auf Stimme, Gestalt und Verhalten der Vögel in bildender Kunst, Literatur und Musik. Vögel sind jedoch nicht nur inhaltlich Thema, sondern zugleich immer auch ein Natur-/Kulturgrenzen überschreitender, selbstreflexiver Spiegel, eine epistemische Figur. Der Band bringt Beiträge von OrnithologInnen, VerhaltensbiologInnen, Literatur-, Kunst- und MusikwissenschaftlerInnen und gliedert sich in vier Bereiche: »Aesthetical Birding, ornithologische Poiesis« verfolgt eine literarische Vogelkunde; »Von Subsong bis Territorialgesang« fragt nach der Musik der Vögel; »Warnruf, Lockruf, Kontaktruf« untersucht, ob Vogelgesang (eine) Sprache ist; »Alle Vögel sind nicht mehr da« thematisiert das avifaunische Artensterben - in Artefakten und als Frage des Naturschutzes. Mit Beiträgen von Frieder von Ammon, Henrik Brumm, Michael Eggers, Ludwig Fischer, Tanja van Hoorn, Norbert Hummelt, Julin Lee, Manfred Lütkepohl, Wolfgang Rathert, Constance Scharff, Christian Schmitt, Monika Schmitz-Emans und Jessica Ullrich.

The transcription factor FOXP2 is crucial for the formation and function of cortico-striatal circuits. FOXP2 mutations are associated with specific speech and language impairments. In songbirds, experimentally altered FoxP2 expression levels in the striatal song nucleus Area X impair vocal learning and song production. Overall FoxP2 protein levels in Area X are low in adult zebra finches and decrease further with singing. However, some Area X medium spiny neurons (MSNs) express FoxP2 at high levels (FoxP2 high MSNs) and singing does not change this. Because Area X receives many new neurons throughout adulthood, we hypothesized that the FoxP2 high MSNs are newly recruited neurons, not yet integrated into the local Area X circuitry and thus not active during singing. Contrary to our expectation, FoxP2 protein levels did not predict whether new MSNs were active during singing, assayed via immediate early gene expression. However, new FoxP2 high MSNs had more complex dendrites, higher spine density and more mushroom spines than new FoxP2 low MSNs. In addition, FoxP2 expression levels correlated positively with nucleus size of new MSNs. Together, our data suggest that dynamic FoxP2 levels in new MSNs shape their morphology during maturation and their incorporation into a neural circuit that enables the maintenance and social modulation of adult birdsong.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The flexible control of sequential behavior is a fundamental aspect of speech, enabling endless reordering of a limited set of learned vocal elements (syllables or words). Songbirds are phylogenetically distant from humans but share both the capacity for vocal learning and neural circuitry for vocal control that includes direct pallial-brainstem projections. Based on these similarities, we hypothesized that songbirds might likewise be able to learn flexible, moment-by-moment control over vocalizations. Here, we demonstrate that Bengalese finches (Lonchura striata domestica), which sing variable syllable sequences, can learn to rapidly modify the probability of specific sequences (e.g. 'ab-c' versus 'ab-d') in response to arbitrary visual cues. Moreover, once learned, this modulation of sequencing occurs immediately following changes in contextual cues and persists without external reinforcement. Our findings reveal a capacity in songbirds for learned contextual control over syllable sequencing that parallels human cognitive control over syllable sequencing in speech. eLife digest Human speech and birdsong share numerous parallels. Both humans and birds learn their vocalizations during critical phases early in life, and both learn by imitating adults. Moreover, both humans and songbirds possess specific circuits in the brain that connect the forebrain to midbrain vocal centers. Humans can flexibly control what they say and how by reordering a fixed set of syllables into endless combinations, an ability critical to human speech and language. Birdsongs also vary depending on their context, and melodies to seduce a mate will be different from aggressive songs to warn other males to stay away. However, so far it was unclear whether songbirds are also capable of modifying songs independent of social or other naturally relevant contexts. To test whether birds can control their songs in a purposeful way, Veit et al. trained adult male Bengalese finches to change the sequence of their songs in response to random colored lights that had no natural meaning to the birds. A specific computer program was used to detect different variations on a theme that the bird naturally produced (for example, "ab-c" versus "ab-d"), and rewarded birds for singing one sequence when the light was yellow, and the other when it was green. Gradually, the finches learned to modify their songs and were able to switch between the appropriate sequences as soon as the light cues changed. This ability persisted for days, even without any further training. This suggests that songbirds can learn to flexibly and purposefully modify the way in which they sequence the notes in their songs, in a manner that parallels how humans control syllable sequencing in speech. Moreover, birds can learn to do this 'on command' in response to an arbitrarily chosen signal, even if it is not something that would impact their song in nature. Songbirds are an important model to study brain circuits involved in vocal learning. They are one of the few animals that, like humans, learn their vocalizations by imitating conspecifics. The finding that they can also flexibly control vocalizations may help shed light on the interactions between cognitive processing and sophisticated vocal learning abilities. Introduction A crucial aspect of the evolution of human speech is the development of flexible control over learned vocalizations (Ackermann et al., 2014; Belyk and Brown, 2017). Humans have unparalleled control over their vocal output, with a capacity to reorder a limited number of learned elements to produce an endless combination of vocal sequences that are appropriate for current contextual demands (Hauser et al., 2002). This cognitive control over vocal production is thought to rely on the direct innervation of brainstem and midbrain vocal networks by executive control structures in the frontal cortex, which have become more elaborate over the course of primate evolution (Hage and Nieder, 2016; Simonyan and Horwitz, 2011). However, because of the comparatively limited flexibility of vocal production in nonhuman primates (Nieder and Mooney, 2020), the evolutionary and neural circuit mechanisms that have enabled the development of this flexibility remain poorly understood. Songbirds are phylogenetically distant from humans, but they have proven a powerful model for investigating neural mechanisms underlying learned vocal behavior. Song learning exhibits many parallels to human speech learning (Doupe and Kuhl, 1999); in particular, juveniles need to hear an adult tutor during a sensitive period, followed by a period of highly variable sensory-motor exploration and practice, during which auditory feedback is used to arrive at a precise imitation of the tutor song (Brainard and Doupe, 2002). This capacity for vocal learning is subserved by a well-understood network of telencephalic song control nuclei. Moreover, as in humans, this vocal control network includes strong projections directly from cortical (pallial) to brainstem vocal control centers (Doupe and Kuhl, 1999; Simonyan and Horwitz, 2011). These shared behavioral features and neural specializations raise the question of whether songbirds might also share the capacity to learn flexible control over syllable sequencing. Contextual variation of song in natural settings, such as territorial counter-singing or female-directed courtship song, indicate that songbirds can rapidly alter aspects of their song, including syllable sequencing and selection of song types (Chen et al., 2016; Heinig et al., 2014; King and McGregor, 2016; Sakata et al., 2008; Searcy and Beecher, 2009; Trillo and Vehrencamp, 2005). However, such modulation of song structure is often described as affectively controlled (Berwick et al., 2011; Nieder and Mooney, 2020). For example, the presence of potential mates or rivals elicits a global and unlearned modulation of song intensity (James et al., 2018) related to the singer's level of arousal or aggression (Alcami et al., 2021; Heinig et al., 2014; Jaffe and Brainard, 2020). Hence, while prior observations suggest that a variety of ethologically relevant factors can be integrated to influence song production in natural settings, it remains unclear whether song can be modified more flexibly by learned or cognitive factors. Here, we tested whether Bengalese finches can learn to alter specifically targeted vocal sequences within their songs in response to arbitrarily chosen visual cues, independent of social or other natural contexts. Each Bengalese finch song repertoire includes ~5–12 acoustically distinct elements ('syllables') that are strung together into sequences in variable but non-random order. For a given bird, the relative probabilities of specific transitions between syllables normally remain constant over time (Okanoya, 2004; Warren et al., 2012), but previous work has shown that birds can gradually adjust the probabilities of alternative sequences in response to training that reinforces the production of some sequences over others. In this case, changes to syllable sequencing develop over a period of hours to days (Warren et al., 2012). In contrast, we investigate here whether birds can learn to change syllable sequencing on a moment-by-moment basis in response to arbitrary visual cues that signal which sequences are adaptive at any given time. Our findings reveal that songbirds can learn to immediately, flexibly, and adaptively adjust the sequencing of selected vocal elements in response to learned contextual cues. Results Bengalese finches can learn context-dependent syllable sequencing For each bird in the study, we first identified variably produced syllable sequences that could be gradually modified using a previously described aversive reinforcement protocol ('single context training'; Tumer and Brainard, 2007; Warren et al., 2012). For example, a bird that normally transitioned from the fixed syllable sequence 'ab' to either 'c' or 'd' (Figure 1A,B, sequence probability of ~36% for 'ab-c' and ~64% for 'ab-d') was exposed to an aversive burst of white noise (WN) feedback immediately after the 'target sequence' 'ab-d' was sung. In response, the bird learned over a period of days to gradually decrease the relative probability of that sequence in favor of the alternative sequence 'ab-c' (Figure 1C). This change in sequence probabilities was adaptive in that it enabled the bird to escape from WN feedback. Likewise, when the sequence, 'ab-c' was targeted, the probability of 'ab-d' increased gradually over several days of training (Figure 1D). These examples are consistent with prior work that showed such sequence modifications develop over a period of several days, with the slow time course suggesting a gradual updating of synaptic connections within syllable control networks in response to performance-related feedback (Warren et al., 2012). In contrast, the ability to immediately and flexibly reorder vocal elements in speech must reflect mechanisms that enable contextual factors to exert moment-by-moment control over selection and sequencing of alternative vocal motor programs. Having identified sequences for each bird for which the probability of production could be gradually modified in this manner, we then tested whether birds could be trained to rapidly switch between those same sequences in a context-dependent manner. Figure 1 Download asset Open asset Bengalese finches can learn context-dependent sequencing. (A) Example spectrogram highlighting points in song with variable sequencing. Syllables are labeled based on their spectral structure, target sequences for the different experiments (ab-c and ab-d) are marked with colored bars. Y-axis shows frequency in Hz. (B) Transition diagram with probabilities for sequences ab-c and ab-d. The sequence probability of ab-d (and complementary probability ab-c) stayed relatively constant over five days. Shaded area shows 95% confidence interval for sequence probability. Source data in Figure 1—source data 3. (C) Aversive reinforcement training. Schematic showing aversive WN after target sequence ab-d; spectrogram shows WN stimulus, covering part of syllable d. WN targeted to sequence ab-d led to a gradual decrease in the probability of that sequence over several days, and a complementary increase in the probability of ab-c. (D) WN targeted to ab-c led to a gradual increase in the sequence probability of ab-d. Source data in Figure 1—source data 2. (E) Schematic of the contextual learning protocol, with target for WN signaled by colored lights. (F) Left: Two example days of baseline without WN but with alternating blocks of green and yellow context. Colors indicate light context (black indicates periods of lights off during the night), error bars indicate SEM across song bouts in each block. Right: Average sequence probability in yellow and green blocks during baseline. Open circles show individual blocks, error bars show SEM across blocks. (G) Left: Two example days after training (WN on). Right: Average sequence probability in yellow and green blocks after training. (H) Contextual difference in sequence probability for eight trained birds before and after training (**p<0.01 signed rank test). Source data in Figure 1—source data 1. Figure 1—source data 1 Switch magnitude during baseline and after training for all birds, to generate Figure 1H, and plots like Figure 1F,G for all birds. https://cdn.elifesciences.org/articles/61610/elife-61610-fig1-data1-v1.mat Download elife-61610-fig1-data1-v1.mat Figure 1—source data 2 Sequence data for the example bird during single-context training, to generate Figure 1C,D. https://cdn.elifesciences.org/articles/61610/elife-61610-fig1-data2-v1.mat Download elife-61610-fig1-data2-v1.mat Figure 1—source data 3 Sequence data for the example bird during baseline, to generate Figure 1B. https://cdn.elifesciences.org/articles/61610/elife-61610-fig1-data3-v1.mat Download elife-61610-fig1-data3-v1.mat To determine whether Bengalese finches can learn to flexibly select syllable sequences on a moment-by-moment basis, we paired WN targeting of specific sequences with distinct contextual cues. In this context-dependent training protocol, WN was targeted to defined sequences in the bird's song as before, but the specific target sequence varied across alternating blocks, signaled by different colored lights in the home cage (see Materials and methods). Figure 1E shows an example experiment, with 'ab-d' targeted in yellow light, and 'ab-c' in green light. At baseline, without WN, switches between yellow and green contexts (at random intervals of 0.5–1.5 hr) did not lead to significant changes in the relative proportion of the target sequences, indicating that there was no inherent influence of the light cues on sequence probabilities (Figure 1F, p(ab-d) in yellow vs. green context was 67 ± 1.6% vs. 64 ± 1.5%, p=0.17, rank-sum test, n = 53 context blocks from baseline period). Training was then initiated in which WN was alternately targeted to each sequence, over blocks that were signaled by light cues. After 2 weeks of such context-specific training, significant sequencing differences developed between light contexts that were appropriate to reduce aversive feedback in each context (Figure 1G, p(ab-d) in yellow vs. green context shifted to 36.5 ± 4.8% vs. 83.1 ± 3.5%, p<0.01, rank-sum test, n = 22 context blocks, block duration between 1 and 2.5 hr). Likewise, for all birds trained on this protocol (n = 8), context-dependent sequencing differences developed in the appropriate direction over a period of weeks (27 ± 6% difference in probabilities between contexts after a mean of 33 days training, versus 1% ± 2% average difference in probabilities at baseline; p<0.01, n = 8, signed rank test, Figure 1H). Thus, Bengalese finches are able to learn context-specific modifications to syllable sequencing. Syllable sequencing shifts immediately following switches in context Contextual differences between different blocks could arise through an immediate shift in sequence probabilities upon entry into a new context and/or by rapid learning within each block. We examined whether trained birds exhibited any immediate shifts in their syllable sequencing when entering a new light context by computing the average probability of target sequences across songs aligned with the switch between contexts (Figure 2A,B, example experiment). This 'switch-triggered average' revealed that across all birds, switches to the yellow context were accompanied by an immediate decrease in the probability of the yellow target sequence, whereas switches out of the yellow context (and into the green context) led to an immediate increase in the yellow target sequence (Figure 2C,D, p<0.05, signed rank test comparing first and last song, n = 8). To quantify the size of these immediate shifts, we calculated the difference in sequence probability from the last five songs in the previous context to the first five songs in the current context; this difference averaged 0.24 ± 0.06 for switches to green light and −0.22 ± 0.06 for switches to yellow light (Figure 2E,F). These results indicate that birds could learn to immediately recall an acquired memory of context-appropriate sequencing upon entry into each context, even before having the chance to learn from reinforcing feedback within that context. Figure 2 Download asset Open asset Sequence probabilities shift immediately following a switch in context. (A, B) Average sequence probability per song for example Bird 1 aligned to switches from green to yellow context (A) and from yellow to green context (B). Error bars indicate SEM across song bouts (n = 35 switches (A), n = 33 switches (B)). (C) Changes in sequence probability from the last song in green context to the first song in yellow context for all eight birds. Example bird in (A, B) highlighted in bold. **p<0.01 signed rank test. (D) Changes in sequence probability from the last song in yellow context to the first song in green context. *p<0.05 signed rank test. (E) Shift magnitudes for all birds, defined as the changes in sequence probability from the last five songs in the green context to the first five songs in the yellow context. Open circles show individual birds, error bars indicate SEM across birds. (F) Same as (E) for switches from yellow to green. Source data in Figure 2—source data 1. (G) Shift magnitudes over training time for the example bird (11 days and 49 context switches; seven of the original 56 context switches are excluded from calculations of shift magnitudes because at least one of the involved blocks contained only one or two song bouts.). (H) Trajectory of switch-aligned sequence probabilities for the example bird early in training (red) and late in training (blue). Probabilities are normalized by the sequence probability in preceding block, and plotted so that the adaptive direction is positive for both switch directions (i.e. inverting the probabilities for switches to yellow.) (I) Slopes of fits to the sequence probability trajectories over song bouts within block. Units in change of relative sequence probability per song bout. (K) Intercepts of fits to sequence probability trajectories over song bouts within block. Units in relative sequence probability. (L) Changes in slopes and changes in intercepts for five birds over the training process, determined as the slopes of linear fits to curves as in (I and K) for each bird. Source data in Figure 2—source data 2. Figure 2—source data 1 Switch magnitude between all contexts after training, to generate Figures 2C–F and 3E–H. https://cdn.elifesciences.org/articles/61610/elife-61610-fig2-data1-v1.mat Download elife-61610-fig2-data1-v1.mat Figure 2—source data 2 Summary of training data, to generate Figure 2L. https://cdn.elifesciences.org/articles/61610/elife-61610-fig2-data2-v1.mat Download elife-61610-fig2-data2-v1.mat We next asked whether training additionally led to an increased rate of learning within each context, which also might contribute to increased contextual differences over time. Indeed, such faster re-learning for consecutive encounters of the same training context, or 'savings', is sometimes observed in contextual motor adaptation experiments (Lee and Schweighofer, 2009). To compare the magnitude of the immediate shift and the magnitude of within-block learning over the course of training, we plotted the switch-aligned sequence probabilities at different points in the training process. Figure 2G shows for the example bird that the magnitude of the shift (computed between the first and last five songs across context switches) gradually increased over 11 days of training. Figure 2H shows the switch-aligned sequence probability trajectories (as in Figure 2A,B) for this bird early in training (red) and late in training (blue), binned into groups of seven context switches. Qualitatively, there was both an abrupt change in sequence probability at the onset of each block (immediate shift at time point 0) and a gradual adjustment of sequence probability within each block (within-block learning over the first 80 songs following light switch). Over the course of training, the immediate shift at the onset of each block got larger, while the gradual change within blocks stayed approximately the same (learning trajectories remained parallel over training, Figure 2H). Linear fits to the sequence probabilities for each learning trajectory (i.e. the right side of Figure 2H) reveal that, indeed, the change in sequence probability at the onset of blocks (i.e. intercepts) increased over the training process (Figure 2K), while the rate of change within blocks (i.e. slopes) stayed constant (Figure 2I). To quantify this across birds, we measured the change over the course of learning in both the magnitude of immediate shifts (estimated as the intercepts from linear fits) and the rate of within-block learning (estimated as the slopes from linear fits). As for the example bird, we found that the rate of learning within each block stayed constant over time for all five birds (Figure 2L). In contrast, the magnitude of immediate shifts increased over time for all birds (Figure 2L). These analyses indicate that adjustments to sequence probability reflect two dissociable processes, an immediate cue-dependent shift in sequence probability at the beginning of blocks, that increases with contextual training, and a gradual adaptation of sequence probability within blocks, that does not increase with contextual training. Visual cues in the absence of reinforcement are sufficient to evoke sequencing changes The ability of Bengalese finches to implement an immediate shift in sequencing on the first rendition in a block – and thus before they have a chance to learn from reinforcing feedback – argues that they can maintain context-specific motor memories and use contextual visual cues to anticipate correct sequencing in each context. To explicitly test whether birds can flexibly switch between sequencing appropriate for distinct contexts using only visual cues, we included short probe blocks which presented the same light cues without WN stimulation. Probe blocks were interspersed in the sequence of training blocks so that each switch between types of blocks was possible and, on average, every third switch was into a probe block (see Materials and methods). Light switches into probe blocks were associated with similar magnitude shifts in sequence probability as switches into WN blocks of the corresponding color (−0.22 ± 0.06 to both yellow WN and yellow probe blocks from green WN blocks, p=0.94, signed rank test; 0.24 ± 0.06 to green WN and 0.23 ± 0.07 to green probe blocks from yellow WN blocks, p=0.64, signed rank test). As the most direct test of whether light cues alone evoke adaptive sequencing changes, we compared songs immediately before and after switches between probe blocks without intervening WN training blocks (probe-probe switches). Figure 3A,B shows song bouts for one example bird (Bird 2) which were sung consecutively across a switch from yellow probe to green probe blocks. In the first song following the probe-probe switch, the yellow target sequence ('f-ab') was more prevalent, and the green target sequence ('n-ab') was less prevalent, and such an immediate effect was also apparent in the average sequence probabilities for this bird aligned to probe–probe switches (Figure 3C,D). Similar immediate and appropriately directed shifts in sequencing at switches between probe blocks were observed for all eight birds (Figure 3E,F, p<0.05 signed rank test, n = 8), with average shifts in sequence probabilities of −0.21 ± 0.09 and 0.17 ± 0.08 (Figure 3G,H). The presence of such changes in the first songs sung after probe–probe switches indicates that visual cues alone are sufficient to cause anticipatory, learned shifts between syllable sequences. Figure 3 with 1 supplement see all Download asset Open asset Contextual cues alone are sufficient to enable immediate shifts in syllable sequencing. (A,B) Examples of songs sung by Bird 2 immediately before (A) and after (B) a switch from a yellow probe block to a green probe block (full song bouts in Figure 3—figure supplement 1). Scale for x-axis is 500 ms; y-axis shows frequency in Hz. (C, D) Average sequence probability per song for Bird 2 aligned to switches from green probe to yellow probe blocks (C) and from yellow probe to green probe blocks (D). Error bars indicate SEM across song bouts (n = 14 switches (C), 11 switches (D)). (E, F) Average sequence probabilities for all eight birds at the switch from the last song in green probe context and the first song in yellow probe context, and vice versa. Example Bird 2 is shown in bold. *p<0.05 signed rank test. (G, H) Shift magnitudes for probe–probe switches for all birds. Open circles show individual birds; error bars indicate SEM across birds. Source data in Figure 2—source data 1. Contextual changes are specific to target sequences A decrease in the probability of a target sequence in response to contextual cues must reflect changes in the probabilities of transitions leading up to the target sequence. However, such changes could be restricted to the transitions that immediately precede the target sequence, or alternatively could affect other transitions throughout the song. For example, for the experiment illustrated in Figure 1, the prevalence of the target sequence 'ab-d' was appropriately decreased in the yellow context, in which it was targeted. The complete transition diagram and corresponding transition matrix for this bird (Figure 4A,B) reveal that there were four distinct branch points at which syllables were variably sequenced (after 'cr', 'wr', 'i', and 'aab'). Therefore, the decrease in the target sequence 'ab-d' could have resulted exclusively from an increase in the probability of the alternative transition 'ab-c' at the branch point following 'aab'. However, a reduction in the prevalence of the target sequence could also have been achieved by changes in the probability of transitions earlier in song such that the sequence 'aab' was sung less frequently. To investigate the extent to which contextual changes in probability were specific to transitions immediately preceding target sequences, we calculated the difference between transition matrices in the yellow and green probe contexts (Figure 4C). This difference matrix indicates that changes to transition probabilities were highly specific to the branch point immediately preceding the target sequences (specificity was defined as the proportion of total changes which could be attributed to the branch points immediately preceding target sequences; specificity for branch point 'aab' was 83.2%). Such specificity to branch points that immediately precede target sequences was typical across experiments, including cases in which different branch points preceded each target sequence (Figure 4D–F, specificity 96.9%). Across all eight experiments, the median specificity of changes to the most proximal branch points was 84.95%, and only one bird, which was also the worst learner in the contextual training paradigm, had a specificity of less than 50% (Figure 4G). Hence, contextual changes were specific to target sequences and did not reflect the kind of global sequencing changes that characterize innate social modulation of song structure (Sakata et al., 2008; Sossinka and Böhner, 1980). Figure 4 with 1 supplement see all Download asset Open asset Contextual changes are local to the target sequences. (A) Transition diagram for the song of Bird 6 (spectrogram in Figure 1) in yellow probe context. Sequences of syllables with fixed transition patterns (e.g. 'aab') as well as repeat phrases and introductory notes have been summarized as single states to simplify the diagram. (B) Transition matrix for the same bird, showing same data as in (A). (C) Differences between the two contexts are illustrated by subtracting the transition matrix in the yellow context from the one in the green context, so that sequence transitions which are more frequent in green context are positive (colored green) and sequence transitions which are more frequent in yellow are negative (colored yellow). For this bird, the majority of contextual differences occurred at the branch point ('aab') which most closely preceded the target sequences ('ab-c' and 'ab-d'), while very little contextual difference occurred at the other three branch points ('i', 'wr', 'cr'). (D–F) Same for Bird 2 for which two different branch points ('f' and 'n') preceded the target sequences ('f-abcd' and 'n-abcd') (spectrogram in Figure 3). (G) Proportion of changes at the branch point(s) most closely preceding the target sequences, relative to the total magnitude of context differences for each bird (see Materials and methods). Most birds exhibited high specificity of contextual changes to the relevant branch points. Source data in Figure 4—source data 1. Figure 4—source data 1 Overview of different experimental parameters and song features for each bird, to generate (Figure 4G, Figure 4—figure supplement 1). https://cdn.elifesciences.org/articles/61610/elife-61610-fig4-data1-v1.mat Download elife-61610-fig4-data1-v1.mat Distinct sequence probabilities are specifically associated with different visual cues Our experiments establish that birds can shift between two distinct sequencing states in response to contextual cues. In order to test whether birds were capable of learning to shift to these two states from a third neutral context, we trained a subset of three birds with three different color-cued contexts. For these birds, after completion of training with WN targeted to distinct sequences in yellow and green contexts (as described above), we introduced interleaved blocks cued by white light in which there was no reinforcement. After this additional training, switches from the unreinforced context elicited changes in opposite directions for the green and yellow contexts (example bird Figure 5A). All birds (n = 3) showed adaptive sequencing changes for the first song bout in probe blocks (Figure 5B,C) as well as immediate shifts in the adaptive directions for all color contexts (Figure 5D, 0.11 ± 0.04 and 0.19 ± 0.05 for switches to green WN and green probe blocks, respectively; −0.15 ± 0.06 and −0.09 ± 0.02 for switches to yellow WN and yellow probe blocks, respectively). While additional data would be required

Understanding animal behaviour through psychophysical experimentation is often limited by insufficiently realistic stimulus representation. Important physical dimensions of signals and cues, especially those that are outside the spectrum of human perception, can be difficult to standardize and control separately with currently available recording and displaying techniques (e.g. video displays). Accurate stimulus control is in particular important when studying multimodal signals, as spatial and temporal alignment between stimuli is often crucial. Especially for audiovisual presentations, some of these limitations can be circumvented by the employment of animal robots that are superior to video presentations in all situations requiring realistic 3D presentations to animals. Here we report the development of a robotic zebra finch, called RoboFinch, and how it can be used to study vocal learning in a songbird, the zebra finch.

Mutations in the transcription factors FOXP1 and FOXP2 are associated with speech impairments. FOXP1 is additionally linked to cognitive deficits, as is FOXP4. These FoxP proteins are highly conserved in vertebrates and expressed in comparable brain regions, including the striatum. In male zebra finches, experimental manipulation of FoxP2 in Area X, a striatal song nucleus essential for vocal production learning, affects song development, adult song production, dendritic spine density, and dopamine-regulated synaptic transmission of striatal neurons. We previously showed that, in the majority of Area X neurons FoxP1, FoxP2, and FoxP4 are coexpressed, can dimerize and multimerize with each other and differentially regulate the expression of target genes. These findings raise the possibility that FoxP1, FoxP2, and FoxP4 (FoxP1/2/4) affect neural function differently and in turn vocal learning. To address this directly, we downregulated FoxP1 or FoxP4 in Area X of juvenile zebra finches and compared the resulting song phenotypes with the previously described inaccurate and incomplete song learning afterFoxP2knockdown. We found that experimental downregulation ofFoxP1andFoxP4led to impaired song learning with partly similar features as those reported forFoxP2knockdowns. However, there were also specific differences between the groups, leading us to suggest that specific features of the song are differentially impacted by developmental manipulations ofFoxP1/2/4expression in Area X.SIGNIFICANCE STATEMENTWe compared the effects of experimentally reduced expression of the transcription factors FoxP1, FoxP2, and FoxP4 in a striatal song nucleus, Area X, on vocal production learning in juvenile male zebra finches. We show, for the first time, that these temporally and spatially precise manipulations of the three FoxPs affect spectral and temporal song features differentially. This is important because it raises the possibility that the different FoxPs control different aspects of vocal learning through combinatorial gene expression or by acting in different microcircuits within Area X. These results are consistent with the deleterious effects of humanFOXP1andFOXP2mutations on speech and language and addFOXP4as a possible candidate gene for vocal disorders.

Rhythm is an essential component of human speech and music but very little is known about its evolutionary origin and its distribution in animal vocalizations. We found a regular rhythm in three multisyllabic vocalization types (echolocation call sequences, male territorial songs and pup isolation calls) of the neotropical bat Saccopteryx bilineata. The intervals between element onsets were used to fit the rhythm for each individual. For echolocation call sequences, we expected rhythm frequencies around 6–24 Hz, corresponding to the wingbeat in S. bilineata which is strongly coupled to echolocation calls during flight. Surprisingly, we found rhythm frequencies between 6 and 24 Hz not only for echolocation sequences but also for social vocalizations, e.g. male territorial songs and pup isolation calls, which were emitted while bats were stationary. Fourier analysis of element onsets confirmed an isochronous rhythm across individuals and vocalization types. We speculate that attentional tuning to the rhythms of echolocation calls on the receivers' side might make the production of equally steady rhythmic social vocalizations beneficial.

Many mating signals consist of multimodal components that need decoding by several sensory modalities on the receiver's side. For methodological and conceptual reasons, the communicative functions of these signals are often investigated only one at a time. Likewise, variation of single signal traits are frequently correlated by researchers with senders' quality or receivers' behavioral responses. Consequently, the two classic and still dominating hypotheses regarding the communicative meaning of multimodal mating signals postulate that different components either serve as back-up messages or provide multiple meanings. Here we discuss how this conceptual dichotomy might have hampered a more integrative, perception encompassing understanding of multimodal communication: neither the multiple message nor the back-up signal hypotheses address the possibility that multimodal signals are integrated neurally into one percept. Therefore, when studying multimodal mating signals, we should be aware that they can give rise to multimodal percepts. This means that receivers can gain access to additional information inherent in combined signal components only ("the whole is something different than the sum of its parts"). We review the evidence for the importance of multimodal percepts and outline potential avenues for discovery of multimodal percepts in animal communication.

Mutations in the transcription factors FOXP1, FOXP2, and FOXP4 affect human cognition, including language. The FoxP gene locus is evolutionarily ancient and highly conserved in its DNA-binding domain. In Drosophila melanogaster FoxP has been implicated in courtship behavior, decision making, and specific types of motor-learning. Because honeybees (Apis mellifera, Am) excel at navigation and symbolic dance communication, they are a particularly suitable insect species to investigate a potential link between neural FoxP expression and cognition. We characterized two AmFoxP isoforms and mapped their expression in the brain during development and in adult foragers. Using a custom-made antiserum and in situ hybridization, we describe 11 AmFoxP expressing neuron populations. FoxP was expressed in equivalent patterns in two other representatives of Apidae; a closely related dwarf bee and a bumblebee species. Neural tracing revealed that the largest FoxP expressing neuron cluster in honeybees projects into a posterior tract that connects the optic lobe to the posterior lateral protocerebrum, predicting a function in visual processing. Our data provide an entry point for future experiments assessing the function of FoxP in eusocial Hymenoptera.