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The periaqueductal gray (PAG)-rostral ventromedial medulla (RVM)-spinal dorsal horn (SDH) connection has been characterized anatomically and physiologically as an endogenous analgesic pathway in the central nervous system.Neurotensin (NT) and neurotensin receptor 1 (NTR1) play important roles for this descending pain regulating system.However, there is still lacking morphological evidence for the neurochemical properties of the synapses on such an NTergic pathway.The aim of the present study was to investigate the involvement of NTergic neurons in the PAG-RVM-SDH descending pathway.Retrograde tracer Fluoro-gold (FG) was injected into the SDH and anterograde tracer biotinylated dextran amine (BDA) was injected into the PAG, respectively.The immunofluorescence histochemistry combined with tract tracing methods indicated that NTRl-immunoreactive (IR) neurons in the RVM which received NT-IR projections from PAG also sent projections to the SDH.To provide potent morphological evidence, we combined tract tracing with pre-embedding electron microscopic (EM) study and found: (a)Dual immunohistochemistry revealed that NT-IR terminals made asymmetric synapses on NTR1-IR dendrites and cell bodies;(b) after BDA was injected into the PAG and horseradish peroxidase (HRP) into the unilateral SDH, synaptic connections between BDA anterogradely labeled terminals and HRP retrogradely labeled neurons were observed in the RVM under the EM;(c) BDA-labeled terminals formed synapses on NTR1-IR dendrites as well as HRP-labeled dendrites;(d) triple labeling further revealed that NT-IR terminals made synapses on HRP-labeled dendrites showing NTRl-immunoreactivities;(e) BDA-labeled terminals made synapses on HRP-labeled dendrites exhibiting NTRl-immunoreactivities;(f)BDA-labeled terminals containing NT made synapses on HRP-labeled dendrites.The present data suggest that most PAG projecting axon terminals contain NT and form asymmetric synapses on NTR1-IR neurons in the RVM which send descending fibers to the SDH.Our study offers morphological evidence for NTergic PAG-RVM-SDH pathway and provides new clues for the mechanisms of NT-induced analgesia.