Protocol to map multi-transmitter neurons in the mouse brain using intersectional strategy

Although it is now known that certain neurons can produce, store, and release multiple neurotransmitters, their locations, abundance, and functions remain elusive. We developed intersectional genetic strategies to identify multi-transmitter neurons based on the expression of neurotransmitter-specific genes. Here we present our procedures for whole-brain mapping of GABA/glutamate co-releasing neurons. We also detail our technique for labeling GABA/glutamate neurons in specific brain regions with adeno-associated virus (AAV). Our protocol can be readily extended to other types of multi-transmitter neurons. For complete details on the use and execution of this protocol, please refer to Xu et al. (2022).¹.