Objective To explore the morphological features of the neural projection pathway from the dorsal raphe nucleus (DRN) to the claustrum (CLA) in mice.  Methods After injection of the retrograde tracer 594 retrobeads into the right CLA (n=3) or the anterograde tracer biotinylated dextran amine(BDA) into the DRN (n=3),  the distribution and the neurochemical feature of the retrogradely labeled neurons in the DRN, as well as the locations of nerve fibers and terminals throughout the brain and within the CLA, were observed by a combined immunofluorescent staining for 5-hydrozytryptamine(5-HT). The downstream target of the DRN-CLA projection was studied by injection of rabies retrograde monosynaptic virus (RV) into calcium ion/calmodulin dependent protein kinaeⅡ(CaMKⅡ)-Cre or glutamate decarboxylase 67(GAD67)-Cre mice. Results Retrograde labeled neurons were observed in the rostral, middle, and caudal segments of the DRN, with those in the middle segment and in the ventral part of the nucleus (DRV) the densest. Over 90% of the retrograde labeled neurons were 5-HT positive. After injection of BDA into the DRN, dense fiber projections were observed in the ventral tegmental area, parafascicular nucleus, ventral pallidum, and central amygdala nucleus. In comparison, DRN derived fibers and terminals in the CLA were relatively sparse. After injection of RV trans-synaptic virus into the CLA of CaMKⅡ-Cre and GAD67-Cre mice, respectively, dense RV-labeled presynaptic neurons were observed in the DRN of CaMKⅡ-Cre mice, while those in the DRN of GAD67-Cre mice were rather sparse.Conclusion The DRN-CLA projection pathway is characterized by the dense 5-HTergic neurons in the DRV, the relatively sparse fibers or terminals in the CLA, and a primary synaptic connection with CaMKⅡ positive neurons in the CLA. 

Dorsal raphe nucleus, Claustrum, 5-hydroxytryptamine ergic neuron, Calcium ion/calmodulin dependent protein kinaseⅡ positive neuron, Nerve tract tracing, Immunofluorescence, Mouse