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Spermine and other endogenous polyamines potentiate, block and permeate the N -methyl- d -aspartate receptor channel. To identify structural determinants of the N -methyl- d -aspartate channel that mediate spermine's actions, we generated mutant receptors with asparagine (N) to glutamine (Q) or arginine (R) substitutions in the selectivity filter of the channel. We demonstrate that mutation of the three critical asparagines in this domain differentially affects block by phencyclidine and both potentiation and block by spermine. N-to-Q and N-to-R mutations in the N site of the NR1 subunit (N598 in NR1 011 , N619 in NR1 100 ) and N-to-Q mutations in the N and N+1 sites (N595 and N596 in NR2A, respectively) of the NR2 subunit (Q/NN, R/NN, N/QN, N/NQ, Q/QN and Q/NQ receptors) reduced affinity for phencyclidine. The Q/NN receptor showed markedly reduced potentiation by spermine, with little or no change in spermine block. The R/NN receptor showed markedly reduced spermine potentiation and affinity for spermine at its block site. The N/QN, N/NQ and Q/QN mutant receptors showed somewhat enhanced spermine block, while the Q/NQ double mutant exhibited significantly more enhanced spermine block. Thus, the asparagine residues critical to Ca 2+ permeability and Mg 2+ block of N -methyl- d -aspartate channels are also critical to block by spermine and phencyclidine. To examine the interaction of spermine and phencyclidine within the channel, we performed competition studies. Spermine appeared to compete with phencyclidine for binding to the receptor; however, blocks by phencyclidine and by spermine were not additive. The findings suggest that spermine can bind to a site in the external vestibule of the channel to impede phencyclidine binding, but allow Na + influx.

The effects of serotonin on pyramidal cells of layer V of the medial prefrontal cortex were examined using intracellular recording techniques in rat brain slices in vitro. Bath administration of serotonin (0.3-100 microM) produced two distinct responses which could be differentiated physiologically and pharmacologically. The first of these responses was a membrane hyperpolarization. This effect of serotonin was associated with a decrease in input resistance and was independent of the transmembrane chloride gradient, suggesting that it was mediated by an increase in potassium conductance. The ability of serotonin to induce a hyperpolarization was mimicked by (+/-)-8-hydroxy-dipropylaminotetralin hydrobromide and was blocked by BMY 7378 and spiperone but not by ketanserin, indicating that it was mediated by the activation of receptors of the 5-hydroxytryptamine1A subtype. The second response to serotonin involved a membrane depolarization, the replacement of the afterhyperpolarization that follows a burst of spikes in these cells by a slow depolarizing afterpotential, and a decrease in spike frequency accommodation. These effects were mimicked by 4-bromo-2,5-dimethoxyphenyl-isopropylamine and antagonized by ketanserin and by low concentrations of spiperone, indicating that they were mediated by the activation of 5-hydroxytryptamine2 receptors. Interestingly, qualitatively identical responses could be elicited in these cells by activation of muscarinic and alpha 1-adrenergic receptors suggesting that 5-hydroxytryptamine2, muscarinic and alpha 1-adrenergic receptors converge onto a common set of membrane mechanisms to increase cellular excitability. Although 5-hydroxytryptamine1A and 5-hydroxytryptamine2 receptors mediated opposing effects on membrane excitability, most pyramidal neurons appeared to express both receptor subtypes on their membrane surface. The coactivation of both receptor subtypes resulted in a selective enhancement of responsiveness to strong excitatory stimuli with little effect on weaker stimuli. The paradoxical presence of two serotonin receptors mediating opposite effects on membrane excitability in the same cell provides a flexible mechanism by which serotonin might regulate how pyramidal neurons encode incoming excitatory stimuli onto firing activity.