The songbird lateral habenula projects to dopaminergic midbrain and is important for normal vocal development

Reviewer #1 (Public Review):

Reinforcement mechanisms play a central role in learning structured behaviors, and recent studies in the songbird have shown that reinforcement learning is also integral to the imitation of the internally motivated singing behavior of songbirds. In this study, Roeser, Teoh et al. investigate the role of the lateral habenula in this process. The lateral habenula is thought to signal unexpected aversive outcomes, like reward omission, and inhibit dopaminergic neurons in the ventral tegmental area (VTA) via direct synaptic projections. Thus, the lateral habenula could logically play a key role in the trial-and-error learning of song by signaling worse performance outcomes (as evaluated by comparing to a memory of the tutor song) as birds practice copying their father's song.

The authors show that both the anatomical and functional connectivity of the lateral habenula in songbirds resembles what has been described in other vertebrates, including in afferent inputs from the ventral pallidum and efferent projections to the VTA that suppresses activity of putative dopaminergic neurons. Additionally, they show the lateral habenula circuits appear to be integrated with circuits known to be important for learning song, including receiving input from an auditory region, AIV, thought to be important in relaying song evaluation signals and providing inputs to VTA that overlap with neurons projecting to areas of the striatum essential for vocal learning (VTA-Area X neurons). They conclude that lesions of the lateral habenula early in song development do not disrupt a bird's ability to accurately imitate the song of their tutor but result in either the retention or development of unusual vocalizations that have qualities observed in the songs of zebra finches that have been experimentally raised without having access to a song tutor. The analysis of the adult song behavior is particularly compelling and provides novel approaches for identifying outlier vocalizations. Lastly, the authors show that birds will include these isolate-like syllables during courtship behaviors and that lesions of the lateral habenula do lead to disruptions in adult birds.

The conclusions stemming from the analysis of habenula connectivity require stronger support, and incomplete evidence is provided to link lesions of the lateral habenula to the observed disruptions in song learning.

This study has several strengths. First, the goal of understanding the role of the lateral habenula in natural learning of a complex behavior, like birdsong, is a valuable research avenue that can ultimately better link how natural learning of intrinsically rewarded behaviors may (or may not) harness similar learning mechanisms that have been well delineated in laboratory trained and externally reinforced behaviors. Second, the computational approaches brought to bear on the analysis of song, including variational autoencoders to help define the range of control song syllables from abnormal song syllables and anomaly scores, help provide a good framework for examining and conveying disruptions in behavior that might be associated with lesions of the lateral habenula. Lastly, the manuscript is well-written and clearly presented, and the authors do acknowledge some of the weaknesses mentioned below.

The major weakness of the article is that the authors do not verify the completeness (i.e., how much of the lateral habenula is lesioned in individual animals) or the extent (if neuronal regions adjacent to the lateral habenula neural are also lesioned) of their lesions. It is argued that this is not possible because of the timeframe (long survival times) of the experiments. However, there are standard ways of addressing this technical hurdle. One simple approach would be to first examine the correlation in the number of retrogradely labeled neurons in LHb, VP, and Area X following injections of tracer into VTA. For convergent anatomical pathways, there is typically a strong positive correlation across input circuits. Therefore, given the number of retrogradely labeled neurons in VP and Area X following VTA injections, one can make reasonable predictions for how many retrogradely labeled neurons would be expected in LHb. Using tracer injections at the end of the experiments and quantification of the retrograde labeling would allow the authors to reasonably estimate the completeness of their lesions.

This unfortunate problem with the design of the experiments significantly weakens any interpretations for the role of the lateral habenula in song learning. This is particularly important because the lateral habenula is a small area that has several adjacent brain structures that could also play significant roles in song development, most of which have not been well studied in this context. These include the medial habenula, the thalamic nuclei DMP and UVA, and forebrain axons from RA, as well as axons flowing into, out of, and interconnecting the structures previously mentioned. Additional tracer injections with different color tracers could be used to provide reasonable assurance that these other adjacent circuits are still intact at the end of each lesion experiment.

There are two weaknesses with the assessment of the functional connectivity of the lateral habenula. First, the anatomical tracing experiments are not particularly compelling. Very little data is shown and there is no quantification of any of the results. In the inset for retrograde labeling of VP-LHb and VP-VTA neurons, it is unclear that neurons of either population are shown in that image. Likewise, terminals from LHb in VTA are very sparse and it is not clear how well they overlap with VTA-X neurons which are intermingled with dopaminergic neurons projecting to other areas of the brain. The images shown seem out of focus and blurry. Although the electrophysiological experiments provide better assurance of these pathways, the sample sizes in these neurophysiology experiments seem preliminary. Stronger evidence in both regards would provide better assurance of LHb circuitry.

The interpretations and theoretical implications of these results are unclear. This is in part because it is not possible to fully tie behavioral outcomes specifically to lesions of the lateral habenula, but also because, albeit interesting, the behavioral results are somewhat confusing. The developmental lesions did not impact the ability of zebra finches to learn how to copy the song of their tutor over development, indicating, in a strict sense, this circuit is not needed for vocal imitation of a social model. However, birds clearly exhibit unusual song syllables that they throw into their song bouts, even when singing in courtship displays. What this may reflect is not addressed in this study. It could be that lesions disrupt a bird's ability to prune away poor syllables over development, and/or that lesions result in birds being unable to suppress unwanted vocal behaviors during performances. Analysis of song over development could provide insights into these possibilities and help provide a better understanding of what the lateral habenula contributes to the song-learning process.

Reviewer #2 (Public Review):

Summary:

This work presents a previously undescribed neuroanatomical and neurophysiological analog between mammals and songbirds. Juvenile zebra finches learn to sing by memorizing an adult song and then, through practice, converging to a close copy of the stored template. Previous work identified pathways emanating from the avian auditory cortical regions (AIV) and basal ganglia that, through ventral pallium (VP), and the subthalamic nucleus, innervate the finches' ventral tegmental area (VTA). As in mammals, the dopaminergic projections of the VTA onto the avian striatopallidal nucleus, area X, deliver a prediction error signal. This signal encodes a surprisingly better or worse performance of the ongoing song and therefore allows the birds to improve.

In mammals, lateral Habenula (LHb) neurons contribute to learning by signaling disappointing trial outcomes or aversive stimuli. Using viral tract tracing Roesner et al. identify projections from the zebra finch VP and AIV to the LHb as well as from the LHb to the VTA. The authors use functional mapping to show that the VP activates the LHb and that the LHb suppresses the Area X-projecting VTA neurons. Then, the authors show that lesioning the LHb in juvenile finches does not prevent them from copying their tutor's song but still leads to worse performance than controls due to the production of highly abnormal vocalizations, peppered in both lone and female-directed songs. In contrast, lesioning the LHb in adult finches has no effect on the song. Together, these findings suggest that the LHb may be part of a song evaluation system and may participate in learning by signaling vocalizations that deviate from the desired tutor template.

The LHb is an evolutionarily conserved structure that connects the forebrain and midbrain with the epithalamus in vertebrates. By identifying the LHb as a component in song learning, the authors lay the grounds for a trove of new research into the various emotional, biophysical, memory, and sensory processes that contribute to learning within and through the LHb. Most conclusions of this paper are well supported by data, but some conceptual and analytic aspects require framing with respect to methodological limitations.

Strengths:
The use of both anatomical tracing and functional circuit mapping is a uniquely-powerful approach to addressing the main line of inquiry in this work. Specifically, collision testing and antidromic identification allow identifying LHb-->VTA and VTA-->X projecting neurons and therefore testing the response of these specific learning-related projections to stimulation in VP and LHb (respectively).

The evaluation of abnormal vocalizations using a variational autoencoder (VAE) is a particularly strong approach that is immune to observer biases. By training this artificial neural network model with sham or pre-lesion animals, the authors clearly distinguish abnormal syllables because of their significantly poorer reconstruction through the VAE. This approach allowed the authors to provide strong quantitative support to the effect of LHb lesion in juvenile finches on their adult song.

Weaknesses:
The lesions in juveniles, as the authors discuss, were histologically examined at the end of the song development, months after their creation. The authors mention not being able to rule out damage to the medial part of the Hb. But the effect of the lesions could perhaps be mediated by damage to other brain regions, such as DLM, or passing fibers (when using electrolytic lesions).

Additionally, the effect on learning could also be mediated indirectly. In mammals, the outputs of the LHb target dopaminergic regions, serotonergic regions, and a cholinergic region. In birds, the LHb may also have a diverse impact on neuromodulators and therefore an impact on behavior states and on sleep. Disrupted behavior states may lead to poorer or less frequent practice and indirectly to abnormal results that do not stem from erroneous performance evaluation.