Supplementary Material for: β-Adrenergic Signaling Regulates Evolutionarily Derived Sleep Loss in the Mexican Cavefish

Sleep is a fundamental behavior exhibited almost universally throughout the animal kingdom. The required amount and circadian timing of sleep differs greatly between species in accordance with habitats and evolutionary history. The Mexican blind cavefish, <i>Astyanax mexicanus, </i>is a model organism for the study of adaptive morphological and behavioral traits. In addition to loss of eyes and pigmentation, cave populations of <i>A. mexicanus </i>exhibit evolutionarily derived sleep loss and increased vibration attraction behavior, presumably to cope with a nutrient-poor environment. Understanding the neural mechanisms of evolutionarily derived sleep loss in this system may reveal critical insights into the regulation of sleep in vertebrates. Here we report that blockade of β-adrenergic receptors with propranolol rescues the decreased-sleep phenotype of cavefish. This effect was not seen with α-adrenergic antagonists. Treatment with selective β1-, β2-, and β3-antagonists revealed that the increased sleep observed with propranolol could partially be explained via the β1-adrenergic system. Morphological analysis of catecholamine circuitry revealed conservation of gross catecholaminergic neuroanatomy between surface and cave morphs. Taken together, these findings suggest that evolutionarily derived changes in adrenergic signaling underlie the reduced sleep of cave populations.

睡眠是几乎遍及动物界的核心行为之一。不同物种的睡眠需求量与昼夜节律时序，会随栖息环境及演化历程呈现显著差异。墨西哥盲洞鲈（*Astyanax mexicanus*）是研究适应性形态与行为性状的经典模式生物。除眼部与色素缺失外，该物种的洞穴种群还演化出睡眠缺失与振动趋性行为增强的特征，推测这是为适应营养匮乏的洞穴环境所形成的适应性性状。解析该物种中演化而来的睡眠缺失的神经机制，或可为脊椎动物的睡眠调控研究提供关键洞见。本研究发现，使用普萘洛尔阻断β肾上腺素能受体（β-adrenergic receptors），可逆转洞穴鲈的睡眠减少表型；而使用α肾上腺素能拮抗剂（α-adrenergic antagonists）则未观测到该效应。通过选择性β1、β2、β3肾上腺素能拮抗剂开展的实验显示，普萘洛尔所诱导的睡眠增加，可部分通过β1肾上腺素能受体系统得以解释。对儿茶酚胺（catecholamine）神经环路的形态学分析表明，地表型与洞穴型墨西哥盲洞鲈的整体儿茶酚胺能神经解剖结构具有保守性。综上，本研究结果提示，肾上腺素能信号通路的演化改变，是洞穴种群睡眠减少的潜在分子基础。