The incorporation of poly(ethylene glycol) (PEG)-conjugated lipids in lipid-based carriers substantially prolongs the circulation lifetime of liposomes. However, the mechanism(s) by which PEG-lipids achieve this have not been fully elucidated. It is believed that PEG-lipids mediate steric stabilization, ultimately reducing surface-surface interactions including the aggregation of liposomes and/or adsorption of plasma proteins. The purpose of the studies described here was to compare the effects of PEG-lipid incorporation in liposomes on protein binding, liposome-liposome aggregation and pharmacokinetics in mice. Cholesterol-free liposomes were chosen because of their increasing importance as liposomal delivery systems and their marked sensitivity to protein binding and aggregation. Specifically, liposomes containing various molecular weight PEG-lipids at a variety of molar proportions were analyzed for in vivo clearance, aggregation state (size exclusion chromatography, quasi-elastic light scattering, cryo-transmission and freeze fracture electron microscopy) as well as in vitro and in vivo protein binding. The results indicated that as little as 0.5 mol% of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) modified with PEG having a mean molecular weight of 2000 (DSPE-PEG(2000)) substantially increased plasma circulation longevity of liposomes prepared of 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). Optimal plasma circulation lifetimes could be achieved with 2 mol% DSPE-PEG(2000). At this proportion of DSPE-PEG(2000), the aggregation of DSPC-based liposomes was completely precluded. However, the total protein adsorption and the protein profile was not influenced by the level of DSPE-PEG(2000) in the membrane. These studies suggest that PEG-lipids reduce the in vivo clearance of cholesterol-free liposomal formulations primarily by inhibition of surface interactions, particularly liposome-liposome aggregation.

A silicon mask-etching technique was used to prepare grooved surfaces that control the direction of outgrowths of human gingival explants. The method used to produce the grooves is excellent in terms of both the uniformity of the grooves and the control with which surfaces of the desired specifications can be obtained.

We have tested the hypothesis that functional differences between synapses are associated with ultrastructure in cultured cortical neurons. Using Ca(2+) imaging, we measured NMDA receptor-mediated miniature synaptic calcium transients attributed to the spontaneous release of single transmitter quanta. After imaging, the identified neurons were processed for serial transmission electron microscopy. At sites of quantal NMDA receptor-dependent Ca(2+) transients, we confirmed the presence of excitatory synapses and measured spine size and synaptic contact area. Our results demonstrate that synapse size correlates positively with the amplitude of the NMDA receptor-mediated postsynaptic response, suggesting that larger synapses express a greater number of NMDA receptors. Therefore, regulation of quantal amplitude may involve processes that alter synapse size.

Abstract-We tested the hypothesis that heterogeneity in the frequency of miniature synaptic activity reflects differences in the number of vesicles present in presynaptic terminals. Using imaging techniques, we measured dendritic miniature synaptic calcium transients attributed to the spontaneous release of single transmitter quanta. Following imaging, the identified neurons were processed for serial transmission electron microscopy. At sites of quantal Ca 2ϩ transients mediated by N-methyl-d-aspartate receptors, we confirmed the presence of excitatory synapses and measured the total number of vesicles and the number of docked vesicles.We observed no correlation between the frequency of spontaneous miniature activity and either the total vesicle number or the number of docked vesicles. We conclude that the presynaptic vesicle complement as measured by ultrastructural analysis does not necessarily determine the frequency of spontaneous activity at synapses mediated by N-methyl-d-aspartate receptors. ᭧ 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved.Key words: NMDA, quantal, EPSC, release, ultrastructure, electron microscopy.Studies using electron microscopy (EM) have indicated that the morphology of cortical excitatory glutamatergic synapses is highly variable. 19 Bouton size, vesicle number, vesicle diameter and postsynaptic spine volume vary widely within a relatively homogenous population of neurons. 36,41 It has been hypothesized that structural differences between synapses underlie some of the functional differences that are observed between neurons. 6,11,24 Accordingly, it is necessary to assess both synaptic structure and function at single CNS synapses.We have recently described techniques to compare structure and function at multiple synapses along a region of dendrite. 26 Using Ca 2ϩ imaging, we measured the Nmethyl-d-aspartate (NMDA) receptor-mediated component of spontaneous miniature excitatory postsynaptic currents (mEPSCs), termed the miniature synaptic Ca 2ϩ transient (MSCT). 31,32 Calcium imaging provides a method to localize synaptic responses to particular dendritic spines. 29,31,34 Sensitivity to the NMDA receptor antagonist d,l-APV suggested that under the conditions we have used, Ca 2ϩ transients associated with miniature synaptic activity are largely attributed to NMDA receptors. 31,48,34 We have previously reported a positive correlation between the MSCT amplitude and mEPSC amplitude, 32 indicating that Ca 2ϩ imaging can be used to evaluate the local characteristics of synaptic events. Following MSCT imaging, we performed serial reconstruction of transmission EM images to identify synapses at the origins of the Ca 2ϩ transients. This enabled the ultrastructural characterization of the specific synapses where MSCTs were measured. Using these techniques, we have previously reported that synapse size correlated positively with the amplitude of the NMDA component of the quantal postsynaptic response, suggesting that synapse size may place a limit on the number of postsynaptic...