The excitatory and inhibitory modulation of primary afferent fibre-evoked responses of ventral roots in the neonatal rat spinal cord exerted by nitric oxide

Abstract

1 We investigated the role of nitric oxide (NO) in modulating spinal synaptic responses evoked by electrical and noxious sensory stimuli in the neonatal rat spinal cord in vitro. 2 Potentials were recorded extracellularly from a ventral root (L3-L5) of the isolated spinal cord preparation or spinal cord-saphenous nerve-skin preparation of 0- to 2-day-old rats. Spinal reflexes were elicited by electrical stimulation of the ipsilateral dorsal root or by noxious skin stimulation. 3 In the spinal cord preparation, single shock stimulation of a dorsal root at C-fibre strength induced mono-synaptic reflex followed by a slow depolarizing response lasting about 30 s (slow ventral root potential; slow VRP) in the ipsilateral ventral root of the same segment. Bath-application of NO gas-containing medium (10-4-10-2 dilution of saturated medium) and NO donors, 1-hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-1-triazene (NOC12, 3-300 μM), S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 3-300 μM) and S-nitroso-L-glutathione (GSNO, 3-300 μM), produced an inhibition of the slow VRP and a depolarization of ventral roots. Another NO donor, 3-morpholinosydononimine (SIN-1, 30-300 μM), also depressed the slow VRP but did not depolarize ventral roots. These agents did not affect the mono-synaptic reflex. 4 In the spinal cord-saphenous nerve-skin preparation, application of capsaicin (0.1-0.2 μM) to skin evoked a slow depolarizing response of the L3 ventral root. This slow VRP was depressed by NOC12 (10-300 μM) and SIN-1 (100-300 μM). When the concentration of NOC12 was increased to 1 mM, spontaneous synaptic activities were augmented and the depressant effect of NOC12 on the slow VRP became less pronounced. 5 A NO-scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO, 100-300 μM) prevented the depressant effect on the dorsal root-evoked slow VRP and ventral root depolarizing effects of NO donors. Carboxy-PTIO increased spontaneous synaptic activities and markedly potentiated the slow VRP. A NO synthase (NOS) inhibitor, N(ω)-nitro-L-arginine methyl ester (L-NAME, 0.03-1 μM), but not D-NAME (0.03-1 μM), also markedly potentiated the slow VRP and this effect was reversed by L-arginine (300 μM). 6 8-Bromo-cyclic guanosine 3':5'-monophosphate (8-Br-cyclic GMP, 100-300 μM) produced both an inhibition of the slow VRP and a depolarization of ventral roots. A cyclic GMP-dependent protein kinase inhibitor, KT5823 (0.3 μM), partly inhibited the depressant effects of NO donors and 8-Br-cyclic GMP on the dorsal root-evoked slow VRP. In contrast, KT5823 did not inhibit the depolarizing effects of NO donors. 7 Perfusion of the spinal cord with medium containing tetrodotoxin (0.3 μM) and/or low Ca2+ (0.1 mM)-high Mg2+ (10 mM) markedly potentiated the depolarizing effect of NO donors. The SNAP-evoked depolarization in the tetrodotoxin-containing low Ca2+-high Mg2+ medium was significantly inhibited by excitatory amino acid receptor antagonists D-(-)-2-amino-5-phosphonovaleric acid (30 μM) and 6-cyano-7-nitroquinoxaline-2,3-dione (10 μM). 8 The present study suggests that inhibitory and excitatory mechanisms meditated by the NO-cyclic GMP cascade are involved in the primary afferent fibre-evoked nociceptive transmission in the neonatal rat spinal cord. The inhibitory mechanism, but not the excitatory mechanism, appears to be partly mediated by cyclic GMP-dependent protein kinase. It is also suggested that Ca2+-independent release of excitatory amino acid neurotransmitters contributes to the depolarizing response to NO of ventral roots.