X Yang1,2, S Zhu1,2, N Jin1,2, Y Li1,2, C Zhen1,2, H Zhang3, A Xu2, M Wang2, C Zheng1. 1. Neurobiology Laboratory, Institute of Physiological Sciences, Wannan Medical College, Wuhu 241002, China. 2. Cell Electrophysiology Laboratory, Institute of Physiological Sciences, Wannan Medical College, Wuhu 241002, China. 3. Psychophysiology Laboratory, Institute of Physiological Sciences, Wannan Medical College, Wuhu 241002, China.
Abstract
OBJECTIVE: To investigate the effect of orexin-A on the functionality of ionotropic γ-aminobutyric acid (GABA) receptors in spinal cord ventral horn neurons and its mechanisms. OBJECTIVE: The spinal cord containing the lumbosacral enlargement was isolated from neonatal SD rats (7-12 days old) and sliced. The slices were digested with papain (in 0.18 g/30 mL artificial cerebrospinal fluid) for 40-60 min, and the ventral horn neurons were separated acutely using fire-polished Pasteur pipettes. After the cells adhered to the bottom of Petri dishes, patch-clamp experiments combined with pharmacological methods were performed to test the effects of orexin-A on GABA currents of the neurons treated with SB334867 (a selective OX1R antagonist), TCSOX229 (a selective OX2R antagonist), Bis-Ⅳ (a PKC inhibitor), PMA (a PKC agonist), Rp-cAMP (a PKA inhibitor), or BAPTA (Ca2+ chelator). OBJECTIVE: The isolated neurons maintained good morphologies with diverse shapes of cell body and long protrusions. Treatment with orexin-A significantly inhibited the amplitude of GABA-induced current (P < 0.001, n=49) with an inhibition rate of (67.48±12.50)%. SB334867 and TCSOX229, when applied simultaneously, completely abolished the suppressive effect of orexin-A on the GABA currents (P=0.93, n=6), and their separate use partially relieved the suppressive effect of orexin-A (P=0.001, n=8; P=0.02, n=8). The application of Bis-Ⅳ also abolished the suppressive effect of orexin-A on GABA currents (P=0.31, n=5). PMA mimicked the effect of orexin-A in these neurons and significantly inhibited GABA currents with an inhibition rate of (60.79±10.94)%, and the application of orexin-A did not cause further suppression of GABA currents in PMA-treated neurons (P=0.15, n=6). Orexin-A was still capable of suppressing GABA currents in Rp-cAMP-treated neurons (P=0.001, n=5). The extracellular Ca2+-free solution (P=0.004, n=8) or the presence of BAPTA (P=0.04, n=7) did not significantly affect the inhibitory effect of orexin-A on GABA currents. OBJECTIVE: Orexin-A inhibits GABA currents in the ventral horn neurons of rat spinal cord probably by activating OX1R, OX2R and Ca2+-independent PKC.
OBJECTIVE: To investigate the effect of orexin-A on the functionality of ionotropic γ-aminobutyric acid (GABA) receptors in spinal cord ventral horn neurons and its mechanisms. OBJECTIVE: The spinal cord containing the lumbosacral enlargement was isolated from neonatal SD rats (7-12 days old) and sliced. The slices were digested with papain (in 0.18 g/30 mL artificial cerebrospinal fluid) for 40-60 min, and the ventral horn neurons were separated acutely using fire-polished Pasteur pipettes. After the cells adhered to the bottom of Petri dishes, patch-clamp experiments combined with pharmacological methods were performed to test the effects of orexin-A on GABA currents of the neurons treated with SB334867 (a selective OX1R antagonist), TCSOX229 (a selective OX2R antagonist), Bis-Ⅳ (a PKC inhibitor), PMA (a PKC agonist), Rp-cAMP (a PKA inhibitor), or BAPTA (Ca2+ chelator). OBJECTIVE: The isolated neurons maintained good morphologies with diverse shapes of cell body and long protrusions. Treatment with orexin-A significantly inhibited the amplitude of GABA-induced current (P < 0.001, n=49) with an inhibition rate of (67.48±12.50)%. SB334867 and TCSOX229, when applied simultaneously, completely abolished the suppressive effect of orexin-A on the GABA currents (P=0.93, n=6), and their separate use partially relieved the suppressive effect of orexin-A (P=0.001, n=8; P=0.02, n=8). The application of Bis-Ⅳ also abolished the suppressive effect of orexin-A on GABA currents (P=0.31, n=5). PMA mimicked the effect of orexin-A in these neurons and significantly inhibited GABA currents with an inhibition rate of (60.79±10.94)%, and the application of orexin-A did not cause further suppression of GABA currents in PMA-treated neurons (P=0.15, n=6). Orexin-A was still capable of suppressing GABA currents in Rp-cAMP-treated neurons (P=0.001, n=5). The extracellular Ca2+-free solution (P=0.004, n=8) or the presence of BAPTA (P=0.04, n=7) did not significantly affect the inhibitory effect of orexin-A on GABA currents. OBJECTIVE: Orexin-A inhibits GABA currents in the ventral horn neurons of rat spinal cord probably by activating OX1R, OX2R and Ca2+-independent PKC.