NMDA-INDUCED BURST DISCHARGE IN GUINEA-PIG TRIGEMINAL MOTONEURONS IN-VITRO

Abstract

1. The responses of guinea pig trigeminal motoneurons (TMNs) to N-methyl-D,L-aspartate (NMA) were studied using brain stem slice preparations and whole cell patch-clamp (n = 89) or conven tional microelectrode (n = 22) recording techniques. The primary goals of this study were to determine whether N-methyl-D-aspartate (NMDA) receptor activation would produce spontaneous bursting activity in TMNs and, if so, the underlying mechanisms responsible for the generation of these bursts. 2. Bath-applied NMA (100-300 mu M, n = 80) in standard per fusion medium elicited depolarization, increase in apparent input resistance (R(inp)), and rhythmic burst discharges (1-90 a in duration) from TMNs. These effects were blocked by the NMDA receptor antagonist DL-2-amino-5-phosphonopentanoic acid (AP5, 30 mu M, n = 6), but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5-10 mu M, n = 10). Furthermore, the burst-inducing effect of NMA was not mimicked by the non-NMDA receptor agonists kainate (KA, 5-10 mu M, n = 6) and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA, 5-10 mu M, n = 5). 3. In tetrodotoxin (TTX) treatment conditions (n = 13), NMA elicited depolarization, an increase in apparent R(inp), and rhythmic membrane potential oscillations without action potential bursts (i.e., plateau potentials), suggesting that the effects of NMA observed in the TTX-free condition resulted from activation of postsynaptic NMDA receptors. 4. Graded depolarization of neurons (n = 20) by intracellular direct current injection generally led to a graded increase in frequency and duration of the NMA-induced bursts and plateau potentials until these rhythmic events eventually became transformed into continuous spike discharge and maintained depolarization, respectively. Removal of Mg2+ from the perfusion medium (n = 11) also turned the bursts and plateau potentials into continuous spike discharge and maintained depolarization, respectively. 5. The effects of NMA on the current-voltage (I-V) curve after a depolarizing ramp voltage-clamp command (15-20 mV/s) were examined (n = 40). Under NMA (100-300 mu M) conditions, the I-V relationship exhibited a region of negative slope conductance (NSC) between -60 and -35 mV, thus making the I-V relationship N-shaped. The NSC was abolished by AP5 (30 mu M, n = 8), but not by CNQX (5-10 mu M, n = 6). The I-V relationship in AMPA (3-10 mu M n = 5) or KA (3-10 mu M, n = 5) was almost linear between -80 and -30 mV. In perfusion medium lacking Mg2+, the NMA-induced NSC was abolished, leaving the I-V relationship linear in the region between -80 and -30 mV (n = 6). 6. The role of Ca2+ in the generation and termination of individual NMA-induced bursts was examined (n = 13). Application of zero- or low-Ca2+ medium either 1) blocked completely the generation of NMA-induced plateau potentials (n = 6), 2) increased the duration of plateau potentials (n = 5), or 3) prevented the termination (i.e., repolarization) of plateau potentials (n = 2). The I-V curves examined in low-Ca2+ medium were almost linear in the vast majority of the cases (7 of 8) because of the reduction of NMA-induced inward current in the region of the NSC. 7. The role of Ca2+-dependent K+ currents in terminating individual NMA-induced bursts was assessed using the bee venom apamin and the scorpion venom iberiotoxin, which block the SK and BK types of K+ channels, respectively. Although apamin (200 nM, n = 5) completely blocked the afterhyperpolarization after each action potential, it (100 nM, n = 2; 200 nM, n = 3) failed to prevent the termination of NMA-induced bursts; only a slight to moderate increase of the duration and the underlying depolarization of the bursts was apparent during application of apamin. Iberiotoxin (30 nM) had no obvious effects on either the bursts or the action potential waveform (n = 5). 8. The data from this study demonstrate that selective activation of postsynaptic NMDA receptors induces rhythmic burst discharges in TMNs. Additionally, the data suggest that the NMA-induced bursts arise from activation of an Mg2+- and voltage-dependent NMDA conductance that is manifest as a region of NSC in the NMA I-V relationship. Furthermore, the bursts are dependent on the presence of Ca2+ in the ACSF in a subpopulation of TMNs. 9. Therefore it is concluded that any models of rhythmic oral-motor activity must include the unique properties of the NMDA channel.