Single cell RNA-sequencing in the mouse central amygdala (CEA)

The central amygdala (CEA) is a brain region that consists of primarily GABAergic neurons. It has been widely investigated for its role in many innate and adaptive behaviors, such as appetitive and defensive behaviors. Despite the complex functions of CEA, the molecular diversity of CEA neurons has not been systemically examined. Here, we performed single-cell RNA-sequencing (scRNA-Seq) in the CEA to classify the molecularly defined neuron types in this region. Single-cell RNA sequencing was focused on the central amygdala (CEA). For visually guided dissection of the CEA, we used the fluorescent boundaries of the robust PBN to CEA axon projection. This specifically labels the CEA but not surrounding nuclei without directly expressing a transgene in neurons to be analyzed by scRNA-Seq. For this, male C57Bl/6J mice were bilaterally injected with AAV2/1-CAG-GFP into the parabrachial nucleus (PBN). Four weeks after PBN injection, mice were sacrificed, and the CEA was identified and dissected based on specific labeling of afferent fibers from the PBN. The manual single-cell sorting procedure to isolate non-fluorescent cell bodies from micro-dissected brain slices was similar to a previously described protocol (Hempel et al., 2007). Briefly, mice were deeply anesthetized with isoflurane and decapitated to collect 300 µm coronal brain slices. The CEA was manually dissected with spring scissors using the GFP signal as guidance. Two to three tissue sections from each animal were taken to cover the entire CEA and subjected to protease digestion, after which cells were dissociated. Intact neurons were manually selected into individual wells. Neurons collected from seven animals were pooled for sequencing.