Labeling of the serotonergic neuronal circuits emerging from the raphe nuclei via some retrograde tracers

Abstract Serotonin (5‐hydroxytryptamine, 5‐HT) is a very important neurotransmitter emerging from the raphe nuclei to several brain regions. Serotonergic neuronal connectivity has multiple functions in the brain. In this study, several techniques were used to trace serotonergic neurons in the dorsal raphe (DR) and median raphe (MnR) that project toward the arcuate nucleus of the hypothalamus (Arc), dorsomedial hypothalamic nucleus (DM), lateral hypothalamic area (LH), paraventricular hypothalamic nucleus (PVH), ventromedial hypothalamic nucleus (VMH), fasciola cinereum (FC), and medial habenular nucleus (MHb). Cholera toxin subunit B (CTB), retro‐adeno‐associated virus (rAAV‐CMV‐mCherry), glycoprotein‐deleted rabies virus (RV‐ΔG), and simultaneous microinjection of rAAV2‐retro‐Cre‐tagBFP with AAV‐dio‐mCherry in C57BL/6 mice were used in this study. In addition, rAAV2‐retro‐Cre‐tagBFP was microinjected into Ai9 mice. Serotonin immunohistochemistry was used for the detection of retrogradely traced serotonergic neurons in the raphe nuclei. The results indicated that rAAV2‐retro‐Cre‐tagBFP microinjection in Ai9 mice was the best method for tracing serotonergic neuron circuits. All of the previously listed nuclei exhibited serotonergic neuronal projections from the DR and MnR, with the exception of the FC, which had very few projections from the DR. The serotonergic neuronal projections were directed toward the Arc by the subpeduncular tegmental (SPTg) nuclei. Moreover, the RV‐ΔG tracer revealed monosynaptic non‐serotonergic neuronal projections from the DR that were directed toward the Arc. Furthermore, rAAV tracers revealed monosynaptic serotonergic neuronal connections from the raphe nuclei toward Arc. These findings validate the variations in neurotropism among several retrograde tracers. The continued discovery of several novel serotonergic neural circuits is crucial for the future discovery of the functions of these circuits. Research Highlights Various kinds of retrograde tracers were microinjected into C57BL/6 and Ai9 mice. The optimum method for characterizing serotonergic neuronal circuits is rAAV2‐retro‐Cre‐tagBFP microinjection in Ai9 mice. The DR, MnR, and SPTg nuclei send monosynaptic serotonergic neuronal projections toward the arcuate nucleus of the hypothalamus. Whole‐brain quantification analysis of retrograde‐labeled neurons in different brain nuclei following rAAV2‐retro‐Cre‐tagBFP microinjection in the Arc, DM, LH, and VMH is shown. Differential quantitative analysis of median and dorsal raphe serotonergic neurons emerging toward the PVH, DM, LH, Arc, VMH, MHb, and FC is shown.