MULTIPLE EFFECTS OF SPERMINE ON N-METHYL-D-ASPARTIC ACID RECEPTOR RESPONSES OF RAT CULTURED HIPPOCAMPAL-NEURONS

1. The modulation by polyamines of responses to N-methyl-D-aspartic ac id (NMDA) was studied using a rapid perfusion system and whole-cell vo ltage-clamp recording from rat hippocampal neurons in dissociated cult ure. 2. Concentration jump responses to 100 mum NMDA in the presence o f 10 mum glycine revealed potentiation by 3 mM spermine at a membrane potential of + 60 mV, but depression at - 120 mV; the degree of potent iation at + 60 mV was variable from cell to cell while marked depressi on at - 120 mV was observed in all cells. The depression of responses to NMDA by spermine was highly voltage dependent (zdelta = 1.17) with an apparent equilibrium dissociation constant for block at 0 mV of 27 mm. 3. Analysis of spermine dose-potentiation curves for responses rec orded at + 60 mV in the presence of 10 mum glycine revealed a half-max imal effect at 125 mum. Under the same conditions, but at - 60 mV, ana lysis of spermine-evoked depression was performed for cells with less than 5 % potentiation at + 60 mV, and revealed half-maximal inhibition at 344 mum. 4. Dose-response analysis for the glycine-sensitive activ ation of NMDA receptors at + 60 mV revealed a 3-5-fold increase in app arent affinity for glycine in the presence of 1 mm spermine. This incr ease in affinity for glycine was accompanied by a 3.3-fold decrease in the rate of development of glycine-sensitive desensitization, and a 2 .4-fold decrease in the rate of dissociation of glycine from NMDA rece ptors, while the rate constant for dissociation of NMDA was not reduce d. 5. In the presence of non-saturating concentrations of glycine, spe rmine-induced potentiation at + 60 mV developed with two exponential c omponents: a slow glycine-sensitive component, the amplitude and time constant of which decreased with increasing glycine concentration (30 nm glycine, amplitude = 80.2 +/- 5.1 %, tau = 780 +/- 79 ms; 3 /mum gl ycine, amplitude = 22.6 +/- 7-1 %, tau = 45 +/- 13 ms), and a faster c omponent (tau < 20 ms at all concentrations of glycine), the amplitude of which varied from cell to cell, and which became larger with incre ase in concentration of glycine. When responses to the application of spermine were measured in the presence 10 muM L-alanine instead of 100 nm glycine, the slow component of potentiation was absent. 6. The gua nidine derivative arcaine, and the polyamines diethylenetriamine and 1 ,10-diaminodecane also produced voltage-dependent block of responses t o NMDA, with apparent equilibrium dissociation constants at 0 mV of 0. 75, 2.93 and 4.79 mm, respectively. None of these compounds appeared t o act as potent antagonists at the site(s) at which spermine induced p otentiation of responses to NMDA, nor did they block the effects of sp ermine on the rate of onset of the glycine-sensitive component of dese nsitization. 7. Our results suggest that spermine acts at multiple sit es on NMDA receptors to produce potentiation and block. Potentiation r esults from both an increase in apparent affinity for glycine as well as an increase in maximum amplitude of responses to NMDA recorded in t he presence of a saturating concentration of glycine; we propose that separate mechanisms underlie each effect. Variation in the degree to w hich these effects occur in individual neurons suggest the occurrence of subpopulations of NMDA receptors. Block is voltage dependent, and m ost likely due to binding of polyamines to sites within the ion channe l.