EXCITATORY AMINO ACID-INDUCED CURRENTS IN RAT SEPTAL CHOLINERGIC NEURONS IN CULTURE

Whole-cell voltage-clamp recordings were used to study excitatory amino acid-induced currents in neurons isolated from the septum of fetal rat brains. The neurons were cultured for more than four weeks on a feeder layer composed of glial cells obtained from the septal region. Septal neurons were either fusiform, triangular or multipolar and 83% of cells showed acetylcholinesterase activity. L-Glutamate, kainate, quisqualate and alpha-amino-3-hydroxy-5-methpl-4-isoxazolepropionate (AMPA) applied by local in amplitude with increasing concentration of agonist; they desensitized when induced at higher concentrations except for the I-kai. The EC(50) for the peak I-kai and sustained I-ghu, I-quis and I-AMPA were 55, 13, 0.39 and 3.5 mu M, respectively. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) depressed I-kai and I-AMPA evoked at a concentration of 10 mu M (IC(50)s: 0.58 and 0.84 mu M, respectively). Schild analysis For the CNQX action on I-kai gave a dissociation constant of 0.27 mu M for CNQX. n-Methyl-D-aspartate (NMDA) (with glycine, 3 mu M) produced an inward current (I-NMDA) at -44 mV whose peak amplitude enhanced with increased concentrations (EC(50) = 32 mu M). I-NMDA was potentiated by glycine (EC(50) = 0.15 mu M) and inhibited by D-2-amino-5-phosphovalerate (IC50 = 9.9 mu M for I-NMDA evoked at a concentration of 50 mu M). MK-801 (0.1-10 mu M) inhibited I-NMDA in a dose- and use-dependent manner. I-NMDA was potentiated by spermine (EC(50) = 247 mu M; 91% increase at 1 mM) in a manner independent of holding potential (V-H). I-NMDA was inhibited by Mg2+ and Zn2+ (IC50 = 673 and 39 mu M, respectively, at -44 mV) in a manner dependent on V-H; the magnitudes of a depolarization required for an e-fold increase in their IC(50)s in a range of -64 to -24 mV were 16 and 22 mV, respectively. The action of Zn2+ was independent of V-H > -24 mV. Current-voltage relations for I-kai, I-quis and I-AMPA exhibited outward rectification, while that of I-NMDA showed a region of negative conductance at V-H < -30 mV, which disappeared in a Mg2+-free solution. Reversal potentials for I-kai, I-quis, I-AMPA and I-NMDA were close to 0 mV, indicating the involvement of non-specific cation channels. Increasing extracellular Ca2+ concentration from 2.4 to 30 mM did not affect the I-kai and I-quis, reversal potential showing negligible Ca2+ component, but shifted I-NMDA reversal potential to a more positive potential, yielding a ratio of Ca2+ permeability to that of monovalent cation to be 13. Cholinergic septal neurons in culture express non-NMDA-(AMPA/kainate-) and NMDA-type of glutamate receptor channels. Their properties were quantitatively similar to those of glutamate receptor channels on other types of neurons in the brain except for the actions of endogenous neuromodulators (Mg2+, Zn2+ and spermine) on NMDA receptor channels. It is suggested that NMDA receptor channels on different types of neurons may play a distinct role depending on a difference in the actions of these neuromodulators.