Cingulate cortex shapes early postnatal development of social vocalizations

Reviewer #1 (Public Review):

Summary:

This study seeks to quantify changes in vocal behavior during development in marmosets with bilateral anterior cingulate cortex (ACC) lesions. The ACC and its role in social vocal behaviors are of great interest given previous literature on its involvement in the initiation of vocalizations, processing emotional content, and its connectivity to two other critical nodes in the vocal network, the amygdala and the PAG. The authors seek to test the hypothesis that the ACC contributes to the development of mature vocal behaviors during the first few weeks of life by disrupting this process with neonatal ACC lesions. Imaging and histological analyses confirm the extent of the lesion and suggest downstream effects in connected regions. Analysis of call rates and call type proportions show no or slight differences between lesioned and controlled animals. Additional analyses on the proportion of grouped 'social' calls and certain acoustic features of a particular call, the phee, reveal more distinct differences between the groups.

Strengths:

The authors have identified that ACC lesions in early life have no or little influence on certain aspects of vocal behavior (e.g. call rate, call intervals) but larger impacts on other aspects (e.g. acoustic features of phee calls). This data is a valuable addition to the literature on the effects of the ACC on vocal production.

The histological methods and resulting quantification of neural changes in the lesioned area and in downstream areas of interest are intriguing given the large time gap between the lesion and these analyses.

Weaknesses:

The article emphasizes vocal social behavior but none of the experiments involve a social element. Marmosets are recorded in isolation which could be sufficient for examining the development of vocal behavior in that particular context. However, the early-life maturation of vocal behavior is strongly influenced by social interactions with conspecifics. For example, the transition of cries and subharmonic phees which are high-entropy calls to more low-entropy mature phees is affected by social reinforcement from the parents. And this effect extends cross-context where differences in these interaction patterns extend to vocal behavior when the marmosets are alone. From the chord diagrams, cries still consist of a significant proportion of call types in lesioned animals. Additionally, though it is an intriguing finding that the infants' phee calls have acoustic differences being 'blunted of variation, less diverse and more regular,' the suggestion that the social message conveyed by these infants was 'deficient, limited, and/or indiscriminate' is not but can be tested with, for example, playback experiments.

The manuscript would benefit from the addition of more details to be able to better determine if the conclusions are well supported by the data. Understanding that this is very difficult data to get, the number of marmosets and some variability in the collection of the data would allow for the plotting of each individual across figures. For example, in the behavioral figures, which is the marmoset that is in the behavioral data that has a sparing of the ACC lesion in one hemisphere? Certain figures, described below in the recommendations for the authors, could also do with additional description.

Reviewer #2 (Public Review):

Summary:

Nagarajan et al. investigate the role of the anterior cingulate cortex (ACC) in vocal development of infant marmoset monkeys using lesions in this brain area. Many previous studies show that ACC plays an important role in volitional and emotion-driven vocal behavior in mammals. The experiments Nagarajan et al. performed strengthen the long-standing hypothesis that ACC influences the development of social-vocal behavior in non-human primates. Furthermore, their anatomical studies support the idea of cortical structures exerting cognitive control over subcortical networks for innate vocalization, and thus, enabling mammals to perform flexible social-vocal communication.

Strengths:

Many invasive behavioral studies in monkeys often times use 2-3 animals. The authors used a sufficiently high number of animals for their experiments. This increases the power of their conclusions.
The study also investigates the impact of ACC lesions on downstream areas important for innate vocal production. This adds further evidence to the role of ACC in influencing these subcortical regions during vocal development and vocal behavior in general.

Weaknesses:

The authors state that the integrity of white matter tracts at the injection site was impacted but do not show data.

The study only provides data up to the 6th week after birth. Given the plasticity of the cortex, it would be interesting to see if these impairments in vocal behavior persist throughout adulthood or if the lesioned marmosets will recover their social-vocal behavior compared to the control animals.

Even though this study focuses entirely on the development of social vocalizations, providing data about altered social non-vocal behaviors that accompany ACC lesions is missing. This data can provide further insights and generate new hypotheses about the exact role of ACC in social-vocal development. For example, do these marmosets behave differently towards their conspecifics or family members and vice versa, and is this an alternate cause for the observed changes in social-vocal development?

Reviewer #3 (Public Review):

Summary:

In this manuscript, Nagarajan et al. study the impact of early damage to the anterior cingulate cortex (ACC) on the vocal development of marmoset monkeys. AAC lesions were performed on neonatal marmosets and their vocal patterns and the spectrotemporal features of their calls were analyzed compared to control groups during the first six weeks of life. While the vocal repertoire was not significantly affected by ACC lesions, the authors described notable differences in the social contact call, the phee call. Marmosets with ACC damage made fewer social contact calls, and when they did, these calls were shorter, louder, and monotonic. Additionally, the study revealed that ACC damage in infancy led to permanent alterations in downstream brain areas involved in social vocalizations, such as the amygdala and periaqueductal gray.

Strengths:

This study suggests that the ACC plays a crucial role in the normal development of social vocal behavior in infant marmosets. Studying vocal behavior in marmosets can provide insights into the neural mechanisms underlying human speech and communication disorders due to their similarity in brain structure and social behavior.

The methods are robust and reliable with precise localization of the lesions with neuroimaging and histological examination.

Weaknesses:

It is striking to find that the vocal repertoire of infant marmosets was not significantly affected by ACC lesions. During development, the neural circuits are still maturing and the role of different brain regions may evolve over time. While the ACC likely contributes to vocalizations across the lifespan, its relative importance may vary depending on the developmental stage. In neonates, vocalizations may be more reflexive or driven by physiological needs. At this stage, the ACC may play a role in basic socioemotional regulation but may not be as critical for vocal production. Since the animals lived for two years, further analysis might be helpful to elucidate the precise role of ACC in the vocal behavior of marmosets.

- Figure 3D. According to the Introduction "...infant ACC lesions abolish the characteristic cries that infants normally issue when separated from its mother". Are the present results in marmosets showing the opposite effect? Please discuss.

- Figure 3E and Discussion. Phees are mature contact calls and cries immature contact calls (Zhang et al, 2019, Nat Commun). Therefore, I would rather say that the proportion of immature (cries) contact calls increases vs the mature (phee, trill, twitters) contact calls in the ACC group. Cries are also "isolated-induced contact calls" to attract the attention of the caregivers.

- Figure 4D. Animal location and head direction within the recording incubator can have significant effects on the perceived amplitude of a call. Were these factors taken into account?

- Figure 4E. When a phee call has a higher amplitude, as is the case for the ACC group (Figure 4D), the energy of the signal will be concentrated more strongly at the phee call frequency ~8KHz. This concentration of the energy reduces the variability in the frequency distribution, leading to lower entropy. The interpretation of the results should be reconsidered. A faint call (control group) can exhibit more variability in the frequency content since the energy is distributed across a wider range of frequencies contributing to higher entropy. It can still be "fixed, regular, and stereotyped" if the behavior is consistent or predictable with little variation. Also, to define ACC calls as "monotonic" I would rather search for the lack of frequency modulation, amplitude variation, or narrower bandwidth.

- Apart from the changes in the vocal behavior, did the AAC lesions manifest in any other observable cognitive, emotional, or social behavior? ACC plays a role in processing pain and modulating pain perception. Could that be the reason for the observed increase in the proportion of cries in the ACC group and the increase in the phee call amplitude? Did the cries in the ACC group also display a higher amplitude than the cries in the control group?

- Discussion. Louder calls have the potential to travel longer distances compared to fainter calls, possess higher energy levels, and can propagate through the environment more effectively. If the ACC group produced louder phee syllables, how could be the message conveyed over long distances "deficient, limited, and/or indiscriminate"?