We measured the long term spontaneous electrical activity of neuronal networks with different sizes, grown on lithographically prepared substrates and recorded with multi-electrode-array technology. The time sequences of synchronized bursting events were used to characterize network dynamics. All networks exhibit scale-invariant Lévy distributions and long-range correlations. These observations suggest that different-size networks self-organize to adjust their activities over many time scales. As predictions of current models differ from our observations, this calls for revised models.

SUMMARY1. Responses to N-methyl-D-aspartic acid (NMDA) were recorded from mouse embryonic hippocampal neurones in dissociated culture, using whole-cell patchclamp recording. A fast perfusion system, with an exchange time constant of less than 10 ms, was used to study modulation of NMDA receptor desensitization by glycine.2. The onset of NMDA receptor desensitization was well fitted by a singleexponential function; with 30 nM-glycine the time constant was 250 ms, corresponding to a rate of 4 s-1. The rate of onset of desensitization became faster with increasing glycine concentration, with a slope of 0-87 x 107 M-1 s-1. Recovery from desensitization, studied with a twin-pulse technique, was also well fitted by a singleexponential function; with 30 nM-glycine the time constant of recovery was 1-95 s-'. The rate of recovery from desensitization became faster with increasing glycine concentration, with a slope of 0-76 x 107 M-1 s-1. These results are consistent with a model in which the effect of glycine occurs via an increase in the rate constant for recovery from desensitization, with little effect on the rate constant for onset of desensitization. Over the range 30-300 nM-glycine, the ratio of the rate constants calculated for recovery and onset of desensitization was a good predictor of the degree of desensitization recorded at equilibrium.3. Concentration jump experiments with glycine were performed with 100 /LM-NMDA present continuously, and for a single binding site model gave estimates of the association (1l1 x 107 m-l s-') and dissociation (3-1 s-1) rate constants for interaction of glycine with the NMDA receptor. In the presence of NMDA, concentration jumps from 3 ,tM-glycine to lower concentrations gave relaxations which became slower with decreasing glycine concentration over the range 1 uM-30 nM. A similar slowing of desensitization occurred when the glycine concentration was altered over the same range. 3l. BENVVEXISTE AXD OTHERS 4. Glycine analogues of lower affinity produced desensitization with faster kinetics. D-Alanine, 150 nm, produced desensitization with a time constant of 175 ms, faster than recorded with an equipotent concentration of glycine (50 nm, time constant 259 ms). Responses of similar peak amplitude, recorded with 60 JM-Lalanine, and 500 /M-D,L-homoserine, did not produce strong desensitization, consistent with desensitization too rapid to resolve in our experiments. Concentration jump experiments with L-alanine and D,L-homoserine confirmed that the dissociation rate constants for these analogues (42 and 53 s-) were much faster than those obtained for glycine and D-alanine (341 and 4-9 s-').5. Numerical simulations were developed to test possible kinetic schemes for NMDA receptor activation and desensitizatiohn. Together with the experimental data they suggest that transitions to the open state do not occur unless glycine has first bound to a closed state of the NMDA receptor. Potentiation of NMDA responses by glycine results from the absolute requirement for glycine in promoting tra...

SUMMARY1. Responses to N-methyl-D-aspartic acid (NMDA) were recorded from mouse embryonic hippocampal neurones in dissociated culture, using the tight-seal, wholecell, patch-clamp technique for voltage clamp. A rapid perfusion system, with an exchange time constant of less than 10 ms, was used to apply NMDA under conditions which minimized slow, calcium-sensitive desensitization. With no added glycine, responses to 100 ,uM-NMDA applied for 1-5 s declined by greater than 90%, due to an additional component of desensitization of time constant 250 ms.2. Adding glycine to the extracellular solution, over the range 30 nm to 3/tM, both potentiated responses to NMDA and to L-glutamate, and reduced fast desensitization. In the presence of 3 ,uM-glycine responses to NMDA declined by only 10 %. Similar potentiation and reduction of desensitization was obtained with 3 /tM concentrations of the glycine analogues D-alanine and D-serine.3. Analysis of dose-response curves for the action of glycine on responses to 100 ,tM-NMDA revealed a 3-fold higher potency of glycine for potentiation of peak versus steady-state responses, with concentrations for half-activation of 134 and 382 nm, respectively. The competitive glycine antagonist 7-chlorokynurenic acid produced a similar shift of both the peak and steady-state dose-response curves for glycine, consistent with an equilibrium dissociation constant of 280 nm for interaction of 7-chlorokynurenic acid with the glycine binding site on NMDA receptors.4. In the presence of 100 nM-glycine, 10 ,tM-7-chlorokynurenic acid produced nearly complete block of the response to 3 mM-NMDA, suggesting that glycine is absolutely required for activation of the NMDA receptor channel complex.5. In some neurones responses to NMDA showed essentially no desensitization in the presence of 3 /tM-glycine. Under these conditions, 7-chlorokynurenic acid produced a concentration-dependent block of both the initial and equilibrium response to NMDA, with a 4-fold greater sensitivity for block of the steady-state 6. Our results show that glycine-evoked potentiation of NMDA receptor activity is accompanied by reduced desensitization. Because responses to NMDA measured at equilibrium require higher concentrations of glycine for expression of activity than responses measured early after the application of agonist, the reduction of desensitization by glycine is an important mechanism for promoting responses to sustained applications of NMDA receptor agonists.