Endomorphins-2 (EM-2) could decrease both the frequency and amplitude of the sEPSC of the motoneurons in lamina IX, which was reversed by the MOR antagonist CTOP. EM-2-IR fibers originated from primary afferent fibers form symmetric synaptic connections with motoneurons innervating skeletal muscles of the lower limbs in lamina IX of the spinal ventral horn and EM-2 might exert inhibitory effects on the activities of these motoneurons through both presynaptic and postsynaptic mechanisms.

After surviving for 3 days, all the rats retrograde labeled by WGA-HRP were deeply anesthetized by injection of an overdose of sodium pentobarbital (100 mg/kg), and then perfused with 100 ml of 0.01 M PBS (pH 7.4), followed by 500 ml of a fixative consisting of 4% paraformaldehyde and 0.01% glutaraldehyde in 0.1 M PB (pH 7.4). The spinal cord were removed and stored in 0.1 M PB (pH 7.4) containing of 4% paraformaldehyde at 4°C for 2–4 h. The intact sections were chose for TMR and Endomorphin-2(EM-2) or MOR double staining. The sections were blocked with 10% normal goat serum in 0.01 M PBS (pH 7.4) and then incubated for 24 h with guinea pig anti-TMR (1:500) and rabbit anti-EM-2 (1:200) or rabbit anti-MOR (1:500) antibodies at room temperature (RT). After washing in 0.01 M PBS (pH 7.4), sections were incubated for 8 h at RT with: (1) biotinylated donkey anti-rabbit IgG (1:500) and Alexa594-conjugated donkey anti-guinea pig IgG (1:500) diluted with PBS-NDS (for TMR and EM-2 staining); (2) biotinylated donkey anti-rabbit IgG (1:500) and Alexa488-conjugated donkey anti-guinea pig IgG (1:500) diluted with PBS-NDS (for TMR and MOR staining). Then, after washing in 0.01 M PBS (pH 7.4), sections were incubated with FITC-Avidin (1:1,000) (for EM-2 staining) or Alexa594-Avidin (1:1,000) (for MOR staining) in 0.01 M PBS containing 0.5% Triton X-100 (PBS-X) for 3 h at RT. Finally, the sections were rinsed with 0.01 M PBS, mounted onto clean glass slides, air-dried and cover slipped with a mixture of 0.05 M PBS containing 50% (v/v) glycerin and 2.5% (w/v) triethylenediamine. Images were taken using a laser-scanning confocal microscope.