Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan.
Department of Genetics, SOKENDAI (The Graduate University for Advanced Studies), Mishima, Shizuoka, 411-8540, Japan.
BMC Biol. 2018 Apr 25;16(1):45. doi: 10.1186/s12915-018-0502-y.
Fear conditioning is a form of learning essential for animal survival and used as a behavioral paradigm to study the mechanisms of learning and memory. In mammals, the amygdala plays a crucial role in fear conditioning. In teleost, the medial zone of the dorsal telencephalon (Dm) has been postulated to be a homolog of the mammalian amygdala by anatomical and ablation studies, showing a role in conditioned avoidance response. However, the neuronal populations required for a conditioned avoidance response via the Dm have not been functionally or genetically defined.
We aimed to identify the neuronal population essential for fear conditioning through a genetic approach in zebrafish. First, we performed large-scale gene trap and enhancer trap screens, and created transgenic fish lines that expressed Gal4FF, an engineered version of the Gal4 transcription activator, in specific regions in the brain. We then crossed these Gal4FF-expressing fish with the effector line carrying the botulinum neurotoxin gene downstream of the Gal4 binding sequence UAS, and analyzed the double transgenic fish for active avoidance fear conditioning. We identified 16 transgenic lines with Gal4FF expression in various brain areas showing reduced performance in avoidance responses. Two of them had Gal4 expression in populations of neurons located in subregions of the Dm, which we named 120A-Dm neurons. Inhibition of the 120A-Dm neurons also caused reduced performance in Pavlovian fear conditioning. The 120A-Dm neurons were mostly glutamatergic and had projections to other brain regions, including the hypothalamus and ventral telencephalon.
Herein, we identified a subpopulation of neurons in the zebrafish Dm essential for fear conditioning. We propose that these are functional equivalents of neurons in the mammalian pallial amygdala, mediating the conditioned stimulus-unconditioned stimulus association. Thus, the study establishes a basis for understanding the evolutionary conservation and diversification of functional neural circuits mediating fear conditioning in vertebrates.
恐惧条件反射是一种对动物生存至关重要的学习形式，被用作研究学习和记忆机制的行为范式。在哺乳动物中，杏仁核在恐惧条件反射中起着关键作用。在硬骨鱼中，背侧端脑的内侧区（Dm）通过解剖学和消融研究被推测为哺乳动物杏仁核的同源物，在条件性回避反应中发挥作用。然而，通过 Dm 进行条件性回避反应所需的神经元群体尚未在功能或遗传上定义。
我们旨在通过在斑马鱼中进行遗传方法来确定恐惧条件反射所必需的神经元群体。首先，我们进行了大规模的基因陷阱和增强子陷阱筛选，并创建了转基因鱼系，在大脑的特定区域表达 Gal4FF，这是 Gal4 转录激活子的工程版本。然后，我们将这些表达 Gal4FF 的鱼与携带 botulinum 神经毒素基因的效应系进行杂交，该基因位于 Gal4 结合序列 UAS 的下游，然后分析双转基因鱼的主动回避恐惧条件反射。我们在各种脑区发现了 16 条表达 Gal4FF 的转基因鱼系，它们在回避反应中的表现降低。其中两条鱼在 Dm 的亚区中具有 Gal4 表达的神经元群体，我们将其命名为 120A-Dm 神经元。抑制 120A-Dm 神经元也会导致 Pavlovian 恐惧条件反射表现降低。120A-Dm 神经元主要是谷氨酸能神经元，并且具有投射到其他脑区的投射，包括下丘脑和腹侧端脑。
在此，我们确定了斑马鱼 Dm 中对恐惧条件反射必不可少的神经元亚群。我们提出，这些是哺乳动物大脑皮层杏仁核中神经元的功能等效物，介导条件刺激-非条件刺激关联。因此，该研究为理解脊椎动物恐惧条件反射的功能神经回路的进化保守性和多样性奠定了基础。
