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 acid (NMDA) was studied using a rapid perfusion system and whole-cell voltage-clamp recording from rat hippocampal neurons in dissociated culture. 2. Concentration jump responses to 100 mum NMDA in the presence of 10 mum glycine revealed potentiation by 3 mM spermine at a membrane potential of + 60 mV, but depression at - 120 mV; the degree of potentiation at + 60 mV was variable from cell to cell while marked depression 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 recorded at + 60 mV in the presence of 10 mum glycine revealed a half-maximal effect at 125 mum. Under the same conditions, but at - 60 mV, analysis 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 activation of NMDA receptors at + 60 mV revealed a 3-5-fold increase in apparent affinity for glycine in the presence of 1 mm spermine. This increase 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 receptors, while the rate constant for dissociation of NMDA was not reduced. 5. In the presence of non-saturating concentrations of glycine, spermine-induced potentiation at + 60 mV developed with two exponential components: 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 glycine, amplitude = 22.6 +/- 7-1 %, tau = 45 +/- 13 ms), and a faster component (tau < 20 ms at all concentrations of glycine), the amplitude of which varied from cell to cell, and which became larger with increase 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 guanidine derivative arcaine, and the polyamines diethylenetriamine and 1,10-diaminodecane also produced voltage-dependent block of responses to NMDA, with apparent equilibrium dissociation constants at 0 mV of 0.75, 2.93 and 4.79 mm, respectively. None of these compounds appeared to act as potent antagonists at the site(s) at which spermine induced potentiation of responses to NMDA, nor did they block the effects of spermine on the rate of onset of the glycine-sensitive component of desensitization. 7. Our results suggest that spermine acts at multiple sites on NMDA receptors to produce potentiation and block. Potentiation results from both an increase in apparent affinity for glycine as well as an increase in maximum amplitude of responses to NMDA recorded in the presence of a saturating concentration of glycine; we propose that separate mechanisms underlie each effect. Variation in the degree to which these effects occur in individual neurons suggest the occurrence of subpopulations of NMDA receptors. Block is voltage dependent, and most likely due to binding of polyamines to sites within the ion channel.