Since the initial identification of song control nuclei2,30
, songbirds have been widely embraced as models for studying the neural basis of vocal learning; studies of songbird vocal learning can have wider implications for understanding human speech learning31
. With their large size and discrete boundaries, the song control nuclei make compelling targets for physiological investigation. Functional roles for some song nuclei have now been established in adult song production4,32
and developmental song learning26,33–35
. However, attempts to locate the tutor song template, the basis for the emergent song structure, in the song control nuclei have been much less rewarding36
On the basis of evidence from auditory perception studies in adult songbirds5–13
, we hypothesized that molecular processing in the auditory forebrain would be necessary for tutor song memorization in juveniles. Specifically, our experiment focused on the ERK signaling cascade in auditory lobule. The ERK pathway is essential for learning and memory formation in a number of model systems14,15,17
, and changes in ERK and zenk
activities have been correlated with adult song-recognition learning in adult zebra finches5,7,8,37
. The auditory lobule is a brain area that has no known direct role in motor behavior. It is not considered part of the traditional song system, but its two main components, NCM and CMM38
, are highly specialized to process complex sounds, especially conspecific birdsongs (for example, see refs. 6–8,12,13,39
). We used a well-established pharmacological inhibitor of ERK activation to achieve a transient, reversible disruption of memory processes in the auditory lobule. Our experiment targeted the sensory processes of developmental song learning by manipulating the auditory lobule only during tutor exposure and before the young birds had developed their own songs. Using adult song structure as a read-out of the fidelity of tutor song memorization, our results establish that ERK signaling in the auditory lobule is required for tutor song memorization. Although the ERK signaling cascade is surely not the only one involved in developmental song learning, our study demonstrates that it is essential for accurate tutor song copying, and, to our knowledge, this is the first functional demonstration that a brain area outside of the song control system is required for developmental song learning.
We carried out a series of control experiments to exclude alternatives to our conclusion that ERK interference in the auditory lobule selectively disrupted tutor song memorization. First, we considered the possibility that U0126 infusion into the auditory lobule acutely disrupted the ability of the birds to hear and recognize song. Arguing against this, all of the birds in the U0126 group vocalized in response to the tutor's vocalizations during tutor sessions. Three control experiments further excluded a major disruptive effect of U0126 on hearing. First, regions of the auditory lobule surrounding the U0126 injection site showed a zenk
response to song stimulation, a response that occurs only when the bird hears a complex auditory stimulus8
. Second, song playbacks continued to evoke complex auditory potentials at the injection site after infusion of U0126. Third, birds infused with U0126 were still able to perform an operant song discrimination task that they had previously learned. These control experiments were not designed as fine-grained dissections of perceptual discrimination, and, in any case, it is not yet understood how song experience is represented in the auditory lobule6,9
. However, across three levels of analysis (molecular, neurophysiological and behavioral), these control experiments all indicated that birds are able to hear and recognize songs after U0126 infusion into the auditory lobule.
Next, we considered the possibility that U0126 infusion in the auditory lobule could have somehow altered the birds' rate of singing during the tutor sessions or later in vocal development and that this difference could affect the accuracy of tutor song copying. Although we did not carry out an exhaustive analysis of singing rates or song structure across development, birds in all groups responded with calls to the other birds during tutor sessions and they all sang and called during adulthood. Also, when measured early in vocal song development, the singing patterns of all groups were equally similar to the tutor song. Notably, the birds in the U0126/live with group produced accurate tutor song copies. Collectively, these behaviors suggest that overt motor deficits did not occur in U0126 birds compared to the other groups and that U0126 injections did not functionally alter the rate of vocal practice.
We also considered the possibility that U0126 treatment could have caused a more general disruption of auditory lobule development and function that is not necessarily linked to tutor song memorization. To test this, we injected young control birds with U0126, but then allowed them to experience the tutor at times when the drug was not active. As adults, these birds produced accurate copies of the tutor song. It is formally possible that U0126 disrupted auditory lobule development in a way that was overcome by extended live with tutor exposure, but not by the tutor/CD experience. However, there is no evidence that the live with condition provides stronger tutoring than the tutor/CD condition (), and a previous study demonstrated that more tutor exposure does not increase the extent of tutor song copying22
Finally, we considered the possibility that tutoring would be less effective in birds that had just been handled and infused (for example, see refs. 40,41
), especially given evidence that restraint and isolation42
and stimulus context37
can have major effects on the normal molecular response to hearing song in adult birds. To control for this, we included a group that was infused on the same schedule as the U0126 birds but was administered U0124, an inactive compound that is structurally similar to U0126. The U0124-treated birds produced songs that were as similar to the tutor song as those produced by unmanipulated birds exposed to the tutor. Therefore, cannula insertion, stress of handling and drug injection cannot account for the deficits in U0126 bird's tutor song copying.
Both U0126 and isolate groups produced songs that were equally poor copies of tutor song as assessed by quantitative song analysis, but direct comparison of U0126 and isolate songs suggested that they were also different from each other. It may be that U0126 birds acquired some limited information from tutor experience that was not fully captured by the analysis algorithm. For example, five of the seven U0126 birds had an element that by eye is similar to syllable E in the tutor song ( and Supplementary Fig. 2
). Perhaps the U0126 birds formed a partial tutor song memory because U0126 infusions did not spread throughout the entire auditory lobule. In addition, the half-life of the U0126 injections was 2 h (Supplementary Methods
), permitting some ERK signaling recovery as the tutor sessions progressed. Furthermore, it may be that, unlike the isolate birds, the U0126 birds gained nonauditory information from their social interactions during tutor sessions that could have influenced the development of U0126 songs by, for example, demonstrating body position and rhythm during singing. To further investigate these mechanisms, it would be interesting to test whether reversible deafening during tutoring (for example, lidocaine infusion into Field L) has the same effect on song development as social isolation.
The auditory lobule is an intricate neuroanatomical structure38
, and distinct functions have been ascribed to subregions on the basis of molecular and physiological responses to tutor and other songs10–13,27
. Our analysis did not reveal a substantial effect of cannula placement on tutor song copying, but it is possible that future experiments injecting smaller amounts of U0126 into specific auditory lobule subregions would uncover an effect of spatial location on tutor song memorization. The current results, however, are not entirely surprising, given the extensive interconnections between auditory lobule subregions38
, and are consistent with an earlier study that showed that localized inhibition of caspase-3 activity in the auditory lobule blocks song-specific zenk
response habituation across the extent of auditory lobule43
Our results demonstrate that memory-related functions in the auditory lobule are required for tutor song memorization, but the auditory lobule is clearly not the only brain area involved in developmental song learning. Previous studies of song development focused on the nuclei of the traditional song control system26,33–35
, a neural circuit that seems to have evolved concomitantly with the ability of birds to produce learned vocalizations44
. These studies showed that two major song nuclei, area X and the lateral magnocellular nucleus of the anterior medial nidopallium (LMAN), must be intact for a normal song to develop, but are not immediately required for song production in adulthood33,34
. Furthermore, disruption of NMDA receptors26
in these areas causes deficits in tutor song copying. Thus, complete developmental song learning must rely on the coordinated functions of several brain areas.
How might the auditory lobule and the song control circuit work together during developmental song learning? On the basis of our current results, we propose that the tutor song is initially memorized via activities in the auditory lobule during sensory learning. The representation in the auditory lobule is sensory5–13,27
, but it can influence nuclei controlling song production via established efferent projections from the auditory lobule to the song system38
. Integration of tutor song memories and the bird's own vocal performance is probably mediated by the basal ganglia feedback pathway, leading through area X and LMAN3
. These nuclei are necessary for developmental song learning33,34
and their neurophysiological activities can influence motor output3,45–48
; they are less clearly influenced by auditory feedback47,49
. It may be that a representation of the tutor song passes from the auditory lobule to the area X and LMAN pathway, where it is required for the subsequent integration of sensory and motor information during song rehearsal. Without this tutor song representation, the sensorimotor error correction mechanisms necessary for accurate tutor song copying are deficient. Therefore, disruptions of either the auditory lobule or area X and LMAN26,33–35
during tutoring prevent the development of an accurate copy of the tutor's song.
Our study shows that the ERK function in the auditory lobule is required for the complex process of developmental song learning. To fully understand developmental song learning, it will be necessary to investigate brain areas outside the evolutionarily unique song control nuclei44
and to determine how their functions are coordinated with those of the song system. Although this may seem to further complicate what was once considered a ‘simple’ model of neural circuit design, it may be essential for uncovering how important species-specific behaviors emerge from multiple interacting systems in the brain.