Supplementary Material for: Vocal Parameters That Indicate Threat Level Correlate with FOS Immunolabeling in Social and Vocal Control Brain Regions

Transmitting information via communicative signals is integral to interacting with conspecifics, and some species achieve this task by varying vocalizations to reflect context. Although signal variation is critical to social interactions, the underlying neural control has not been studied. In response to a predator, black-capped chickadees (Poecile atricapilla) produce mobbing calls (chick-a-dee calls) with various parameters, some of which convey information about the threat stimulus. We predicted that vocal parameters indicative of threat would be associated with distinct patterns of neuronal activity within brain areas involved in social behavior and those involved in the sensorimotor control of vocal production. To test this prediction, we measured the syntax and structural aspects of chick-a-dee call production in response to a hawk model and assessed the protein product of the immediate early gene FOS in brain regions implicated in context-specific vocal and social behavior. These regions include the medial preoptic area (POM) and lateral septum (LS), as well as regions involved in vocal motor control, including the dorsomedial nucleus of the intercollicular complex and the HVC. We found correlations linking call rate (previously demonstrated to reflect threat) to labeling in the POM and LS. Labeling in the HVC correlated with the number of D notes per call, which may also signal threat level. Labeling in the call control region dorsomedial nucleus was associated with the structure of D notes and the overall number of notes, but not call rate or type of notes produced. These results suggest that the POM and LS may influence attributes of vocalizations produced in response to predators and that the brain region implicated in song control, the HVC, also influences call production. Because variation in chick-a-dee call rate indicates predator threat, we speculate that these areas could integrate with motor control regions to imbue mobbing signals with additional information about threat level.通过通讯信号传递信息是与同种个体互动的核心环节，部分物种通过调整发声以匹配不同情境来完成这一过程。尽管信号变异对社会互动至关重要，但其背后的神经调控机制尚未得到充分研究。当遭遇捕食者时，黑顶山雀（Poecile atricapilla）会发出具有多种参数的驱赶鸣啭（chick-a-dee呼叫），其中部分参数可传递有关威胁刺激的信息。我们推测，能够指示威胁程度的发声参数，会与参与社会行为的脑区以及参与发声的感觉运动调控的脑区内的特定神经元活动模式相关联。为验证这一推测，我们针对苍鹰模型的刺激，测量了黑顶山雀发出chick-a-dee呼叫的句法与结构特征，并对参与情境特异性发声与社会行为的脑区内的即刻早期基因FOS（immediate early gene FOS）蛋白产物进行了检测。这些脑区包括内侧视前区（medial preoptic area, POM）、外侧隔核（lateral septum, LS），以及参与发声运动调控的丘间复合体背内侧核与HVC。我们发现，呼叫速率（此前研究已证实其可反映威胁程度）与内侧视前区和外侧隔核内的标记信号存在相关性。HVC内的标记信号与每通呼叫中的D音节数量相关，而该参数同样可能指示威胁等级。发声调控脑区丘间复合体背内侧核内的标记信号，则与D音节的结构以及总音节数相关，但与呼叫速率或发出的音节类型无关。上述结果表明，内侧视前区与外侧隔核可能会调控捕食者诱发性发声的各项特征，而此前被认为参与鸣啭调控的脑区HVC同样会对呼叫产生影响。鉴于chick-a-dee呼叫的速率变化能够反映捕食者的威胁程度，我们推测上述脑区可与运动调控脑区协同工作，为驱赶鸣啭赋予更多有关威胁等级的额外信息。