In vitro neuronal network activity in NMDA receptor encephalitis.

BACKGROUND: Anti-NMDA-encephalitis is caused by antibodies against the N-methyl-D-aspartate receptor (NMDAR) and characterized by a severe encephalopathy with psychosis, epileptic seizures and autonomic disturbances. It predominantly occurs in young women and is associated in 59% with an ovarian teratoma. RESULTS: We describe effects of cerebrospinal fluid (CSF) from an anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis patient on in vitro neuronal network activity (ivNNA). In vitro NNA of dissociated primary rat cortical populations was recorded by the microelectrode array (MEA) system.The 23-year old patient was severely affected but showed an excellent recovery following multimodal immunomodulatory therapy and removal of an ovarian teratoma. Patient CSF (pCSF) taken during the initial weeks after disease onset suppressed global spike- and burst rates of ivNNA in contrast to pCSF sampled after clinical recovery and decrease of NMDAR antibody titers. The synchrony of pCSF-affected ivNNA remained unaltered during the course of the disease. CONCLUSION: Patient CSF directly suppresses global activity of neuronal networks recorded by the MEA system. In contrast, pCSF did not regulate the synchrony of ivNNA suggesting that NMDAR antibodies selectively regulate distinct parameters of ivNNA while sparing their functional connectivity. Thus, assessing ivNNA could represent a new technique to evaluate functional consequences of autoimmune encephalitis-related CSF changes.

Communication with emblematic gestures: shared and distinct neural correlates of expression and reception.

Emblematic (or symbolic) gestures allow individuals to convey a variety of thoughts and emotions ranging from approval to hostility. The use of such gestures involves the execution of a codified motor act by the addresser and its perception and decoding by the addressee. To examine underlying common and distinct neural correlates, we used fMRI tasks in which subjects viewed video clips of emblematic one-hand gestures. They were asked to (1) take the perspective of the addresser and imagine executing the gestures ("expression" condition), and to (2) take the perspective of the addressee and imagine being confronted with the gestures ("reception" condition). Common areas of activation were found in inferior frontal, medial frontal, and posterior temporal cortices with left-hemispheric predominance as well as in the cerebellum. The distinction between regions specifically involved in the expression or reception condition partly resembled the dorsal and ventral stream dichotomy of visual processing with junctions in inferior frontal and medial prefrontal cortices. Imagery of gesture expression involved the dorsal visual stream as well as higher-order motor areas. In contrast, gesture reception encompassed regions related to semantic processing, and medial prefrontal areas known to be involved in the process of understanding the intentions of others. In conclusion, our results provide evidence for a dissociation in representations of emblematic gesture processing between addresser and addressee in addition to shared components in language-related areas.

On the neural networks of empathy: A principal component analysis of an fMRI study.

BACKGROUND: Human emotional expressions serve an important communicatory role allowing the rapid transmission of valence information among individuals. We aimed at exploring the neural networks mediating the recognition of and empathy with human facial expressions of emotion. METHODS: A principal component analysis was applied to event-related functional magnetic imaging (fMRI) data of 14 right-handed healthy volunteers (29 +/- 6 years). During scanning, subjects viewed happy, sad and neutral face expressions in the following conditions: emotion recognition, empathizing with emotion, and a control condition of simple object detection. Functionally relevant principal components (PCs) were identified by planned comparisons at an alpha level of p < 0.001. RESULTS: Four PCs revealed significant differences in variance patterns of the conditions, thereby revealing distinct neural networks: mediating facial identification (PC 1), identification of an expressed emotion (PC 2), attention to an expressed emotion (PC 12), and sense of an emotional state (PC 27). CONCLUSION: Our findings further the notion that the appraisal of human facial expressions involves multiple neural circuits that process highly differentiated cognitive aspects of emotion.

Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor.

Although their contribution remains unclear, lipids may facilitate noncanonical routes of protein internalization into cells such as those used by cell-penetrating proteins. We show that protein C inhibitor (PCI), a serine protease inhibitor (serpin), rapidly transverses the plasma membrane, which persists at low temperatures and enables its nuclear targeting in vitro and in vivo. Cell membrane translocation of PCI necessarily requires phosphatidylethanolamine (PE). In parallel, PCI acts as a lipid transferase for PE. The internalized serpin promotes phagocytosis of bacteria, thus suggesting a function in host defense. Membrane insertion of PCI depends on the conical shape of PE and is associated with the formation of restricted aqueous compartments within the membrane. Gain- and loss-of-function mutations indicate that the transmembrane passage of PCI requires a branched cavity between its helices H and D, which, according to docking studies, precisely accommodates PE. Our findings show that its specific shape enables cell surface PE to drive plasma membrane translocation of cell-penetrating PCI.

Differential lipid packing abilities and dynamics in giant unilamellar vesicles composed of short-chain saturated glycerol-phospholipids, sphingomyelin and cholesterol.

The ability of membrane components to arrange themselves heterogeneously within the bilayer induces the formation of microdomains. Much work has been devoted to mimicking domain-assembly in artificial bilayers and characterizing their physico-chemical properties. Ternary lipid mixtures composed of unsaturated phospholipids, sphingomyelin and cholesterol give rise to large, round domains. Here, we replaced the unsaturated phospholipid in the ternary mixture with sphingomyelin and cholesterol by saturated glycero-phospholipids of different chain length and characterized the critical role of cholesterol in sorting these lipids by confocal imaging and fluorescence correlation spectroscopy (FCS). More cholesterol is needed to obtain phase segregation in ternary mixtures, in which the unsaturated phospholipid is replaced by a saturated one. Finally, lipid dynamics in distinct phases is very low and astonishingly similar, thereby suggesting the poor ability of cholesterol in sorting short-chain saturated glycero-phospholipids and sphingomyelin.

Characterization of interaction between cationic lipid-oligonucleotide complexes and cellular membrane lipids using confocal imaging and fluorescence correlation spectroscopy.

Complexes formed by cationic liposomes and single-strand oligodeoxynucleotides (CL-ODN) are promising delivery systems for antisense therapy. ODN release from the complexes is an essential step for inhibiting activity of antisense drugs. We applied fluorescence correlation spectroscopy and confocal laser scanning microscopy to monitor CL-ODN complex interaction with membrane lipids leading to ODN release. To model cellular membranes we used giant unilamellar vesicles and investigated the transport of Cy-5-labeled ODNs across DiO-labeled membranes. For the first time, we directly observed that ODN molecules are transferred across the lipid bilayers and are kept inside the giant unilamellar vesicles after release from the carriers. ODN dissociation from the carrier was assessed by comparing diffusion constants of CL-ODN complexes and ODNs before complexation and after release. Freely diffusing Cy-5-labeled ODN (16-nt) has diffusion constant D(ODN) = 1.3 +/- 0.1 x 10(-6) cm2/s. Fluorescence correlation spectroscopy curves for CL-ODN complexes were fitted with two components, which both have significantly slower diffusion in the range of D(CL-ODN) = approximately 1.5 x 10(-8) cm2/s. Released ODN has the mean diffusion constant D = 1.1 +/- 0.2 x 10(-6) cm2/s, which signifies that ODN is dissociated from cationic lipids. In contrast to earlier studies, we report that phosphatidylethanolamine can trigger ODN release from the carrier in the full absence of anionic phosphatidylserine in the target membrane and that phosphatidylethanolamine-mediated release is as extensive as in the case of phosphatidylserine. The presented methodology provides an effective tool for probing a delivery potential of newly created lipid formulations of CL-ODN complexes for optimal design of carriers.

Fluorescence correlation spectroscopy relates rafts in model and native membranes.

The lipid raft model has evoked a new perspective on membrane biology. Understanding the structure and dynamics of lipid domains could be a key to many crucial membrane-associated processes in cells. However, one shortcoming in the field is the lack of routinely applicable techniques to measure raft association without perturbation by detergents. We show that both in cell and in domain-exhibiting model membranes, fluorescence correlation spectroscopy (FCS) can easily distinguish a raft marker (cholera toxin B subunit bound to ganglioside (GM1) and a nonraft marker (dialkylcarbocyanine dye diI)) by their decidedly different diffusional mobilities. In contrast, these markers exhibit only slightly different mobilities in a homogeneous artificial membrane. Performing cholesterol depletion with methyl-beta-cyclodextrin, which disrupts raft organization, we find an analogous effect of reduced mobility for the nonraft marker in domain-exhibiting artificial membranes and in cell membranes. In contrast, cholesterol depletion has differential effects on the raft marker, cholera toxin B subunit-GM1, rendering it more mobile in artificial domain-exhibiting membranes but leaving it immobile in cell membranes, where cytoskeleton disruption is required to achieve higher mobility. Thus, fluorescence correlation spectroscopy promises to be a valuable tool to elucidate lipid raft associations in native cells and to gain deeper insight into the correspondence between model and natural membranes.

Lipid domain formation and dynamics in giant unilamellar vesicles explored by fluorescence correlation spectroscopy.

Lipids in eukaryotic cell membranes have been shown to cluster in "rafts" with different lipid/protein compositions and molecular packing. Model membranes such as giant unilamellar vesicles (GUVs) provide a key system to elucidate the physical mechanisms of raft assembly. Despite the large amount of work devoted to the detection and characterization of rafts, one of the most important pieces of information still missing in the picture of the cell membrane is dynamics: how lipids organize and move in rafts and how they modulate membrane fluidity. This missing element is of crucial importance for the trafficking at and from the periphery of the cell regulated by endo- and exocytosis and, in general, for the constant turnover which redistributes membrane components. Here, we review studies of combined confocal fluorescence microscopy and fluorescence correlation spectroscopy on lipid dynamics and organization in rafts assembled in GUVs prepared from various lipid mixtures which are relevant to the problem of raft formation.

Lipid dynamics and domain formation in model membranes composed of ternary mixtures of unsaturated and saturated phosphatidylcholines and cholesterol.

In recent years, the implication of sphingomyelin in lipid raft formation has intensified the long sustained interest in this membrane lipid. Accumulating evidences show that cholesterol preferentially interacts with sphingomyelin, conferring specific physicochemical properties to the bilayer membrane. The molecular packing created by cholesterol and sphingomyelin, which presumably is one of the driving forces for lipid raft formation, is known in general to differ from that of cholesterol and phosphatidylcholine membranes. However, in many studies, saturated phosphatidylcholines are still considered as a model for sphingolipids. Here, we investigate the effect of cholesterol on mixtures of dioleoyl-phosphatidylcholine (DOPC) and dipalmitoyl-phosphatidylcholine (DPPC) or distearoyl-phosphatidylcholine (DSPC) and compare it to that on mixtures of DOPC and sphingomyelin analyzed in previous studies. Giant unilamellar vesicles prepared from ternary mixtures of various lipid compositions were imaged by confocal fluorescence microscopy and, within a certain range of sterol content, domain formation was observed. The assignment of distinct lipid phases and the molecular mobility in the membrane bilayer was investigated by fluorescence correlation spectroscopy. Cholesterol was shown to affect lipid dynamics in a similar way for DPPC and DSPC when the two phospholipids were combined with cholesterol in binary mixtures. However, the corresponding ternary mixtures exhibited different spatial lipid organization and dynamics. Finally, evidences of a weaker interaction of cholesterol with saturated phosphatidylcholines than with sphingomyelin (with matched chain length) are discussed.

Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy.

Confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS) have been employed to investigate the lipid spatial and dynamic organization in giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol. For a certain range of cholesterol concentration, formation of domains with raft-like properties was observed. Strikingly, the lipophilic probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C18) was excluded from sphingomyelin-enriched regions, where the raft marker ganglioside GM1 was localized. Cholesterol was shown to promote lipid segregation in dioleoyl-phosphatidylcholine-enriched, liquid-disordered, and sphingomyelin-enriched, liquid-ordered phases. Most importantly, the lipid mobility in sphingomyelin-enriched regions significantly increased by increasing the cholesterol concentration. These results pinpoint the key role, played by cholesterol in tuning lipid dynamics in membranes. At cholesterol concentrations >50 mol%, domains vanished and the lipid diffusion slowed down upon further addition of cholesterol. By taking the molecular diffusion coefficients as a fingerprint of membrane phase compositions, FCS is proven to evaluate domain lipid compositions. Moreover, FCS data from ternary and binary mixtures have been used to build a ternary phase diagram, which shows areas of phase coexistence, transition points, and, importantly, how lipid dynamics varies between and within phase regions.