Abstract-The apoE knockout (E0) mouse is one of the most widely used animal models of atherosclerosis, and there may be similarities to chylomicron remnant-induced atherosclerosis in humans. Although the lesions of these mice contain large numbers of cholesteryl ester (CE)-laden macrophages (foam cells), E0 plasma lipoproteins are relatively weak inducers of cholesterol esterification in macrophages. Previous in vivo work has suggested that arterial wall sphingomyelinase (SMase) may promote atherogenesis in the E0 mouse, perhaps by inducing subendothelial lipoprotein aggregation and subsequent foam cell formation. The goal of the present study was to test the hypothesis that the modification of E0 lipoproteins by SMase converts these lipoproteins into potent inducers of macrophage foam cell formation. When dϽ1.063 E0 lipoproteins were pretreated with SMase and then incubated with E0 macrophages, cellular CE mass and stimulation of the cholesterol esterification pathway were increased Ϸ5-fold compared with untreated lipoproteins. SMase-treated E0 lipoproteins were more potent stimulators of cholesterol esterification than either E0 lipoproteins in the presence of lipoprotein lipases or oxidized E0 lipoproteins. The uptake and degradation of SMase-treated E0 lipoproteins by macrophages were saturable and specific and substantially inhibited by partial proteolysis of cell-surface proteins. Uptake and degradation were diminished by an anti-apoB antibody and by competition with human S f 100-400 hypertriglyceridemic VLDL, raising the possibility that a receptor that recognizes apoB-48 might be involved. In conclusion, SMase-modification of E0 lipoproteins, a process previously shown to occur in lesions, may be an important mechanism for foam cell formation in this widely studied model of atherosclerosis. Moreover, the findings in this report may provide important clues regarding the atherogenicity of chylomicron remnants in humans.

Sensors for imaging brain activity have been under development for almost 50 years. The development of some of these tools is relatively mature, whereas qualitative improvements of others are needed and are actively pursued. In particular, genetically encoded voltage indicators are just now starting to be used to answer neurobiological questions and, at the same time, more than 10 laboratories are working to improve them. In this Biophysical Perspective, we attempt to discuss the present state of the art and indicate areas of active development.

The yeast α-factor pheromone receptor (Ste2) belongs to the family of G protein-coupled receptors (GPCRs) that contain seven transmembrane domains. To define the residues that are accessible to the cytoplasmic G protein, Cys scanning mutagenesis was carried out in which each of the residues that span the intracellular loops and the cytoplasmic end of transmembrane domain 7 were substituted with Cys. The 90 different Cys-substituted residues were then assayed for reactivity with MTSEAbiotin (2-([biotinoyl] amino) ethyl methanethiosulfonate), which reacts with solvent accessible sulfhydryl groups. As part of these studies we show that adding free Cys to stop the MTSEA-biotin reactions has potential pitfalls in that Cys can rapidly undergo disulfide exchange with the biotinylated receptor proteins at pH ≥7. The central regions of the intracellular loops of Ste2 were all highly accessible to MTSEA-biotin. Residues near the ends of the loops typically exhibited a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. Interestingly, these boundary residues were enriched in hydrophobic residues, suggesting that they may form a hydrophobic pocket for interaction with the G protein. Comparison with accessibility data from a previous study of the extracellular side of Ste2 indicates that the transmembrane domains vary in length, consistent with some transmembrane domains being tilted relative to the plane of the membrane as they are in rhodopsin. Altogether, these results define the residues that are accessible to the G protein and provide an important structural framework for the interpretation of the role of Ste2 residues that function in G protein activation.The S. cerevisiae α-factor pheromone receptor (Ste2) belongs to the large family of Gproteincoupled receptors (GPCRs) that transduce the signals for light, taste, olfaction, and many biomedically important hormones. Although GPCRs are quite diverse in sequence, they function in a similar manner to activate the α subunit of heterotrimeric G proteins to bind GTP and they also show similar structural architecture in that they are composed of a bundle of seven transmembrane domains (TMDs) connected by extracellular loops and intracellular loops (1,2). Ste2 shows overall similarity to many mammalian GPCRs in that the core region containing the TMDs is involved in signal transduction and the C terminal tail is a target for † This work was supported by National Institutes of Health Grant GM55107 awarded to J.B.K. post-translational modifications that regulate receptor desensitization and endocytosis (3). Ste2 activates a heterotrimeric G protein in which the α subunit shows about 45% identity with mammalian Gα proteins and is most closely related to the Gi subfamily (4). In addition, comparison of Ste2 with rhodopsin, a member of the large class A subfamily of mammalian GPCRs, indicates that there are similar microdomains in these divergent receptors (5). These results suggest that there are underlying ...

Human neuroblastoma cells (IMR32) respond to treatment with either dibutyryl-cAMP or nerve factor by acquiring a neuronal phenotype which is accompanied by a marked increase in the density of neuronal (N-type) VDCC currents. Using IMR32 cells as a model for neuronal differentiation, we were interested in examining possible changes in the level of expression of the K K1B subunit of N-type calcium channels as well as beta subunit isoforms. Upon differentiation with dibutyryl-cAMP and 5-bromo-2-deoxyuridine for 16 days, we observed a dramatic increase in K K1B protein which initiated between day 8 and 10. Day 10 evidenced maximal expression of K K1B protein, which was followed by an interval of relatively constant expression of K K1B (day 12 to day 16). Monitoring beta subunit expression using a pan specific anti-beta antibody (Ab CW20), we observed an increase in expression of a single 82 kDa beta subunit. The predominant 82 kDa beta subunit expressed throughout the course of differentiation was identified as the L L1b isoform using a panel of beta subunit specific antibodies. Of significance, neither the L L2 nor L L3 isoforms were detected in full differentiated IMR32 cells. Contrary to a previous report on the absence of neurotypic expression of VDCC beta subunits in a second model for in vitro differentiation, NGF-treated rat pheochromocytoma cells (PC12 cells) [1], we report the regulated expression of the L L1b protein in differentiated IMR32 cells suggesting a cell specific function for this beta subunit which parallels the acquisition of the neuronal phenotype. The restrictive expression of the L L1b in IMR32 cells may reflect a cell-type specific function that extends beyond its role as an auxiliary subunit of VDCC complexes.z 1997 Federation of European Biochemical Societies.

Genetically encoded voltage indicators (GEVIs) are fluorescent protein reporters of membrane potential. These tools can, in principle, be used to monitor the neural activity of genetically distinct cell types in the brain. Although introduced in 1997, they have been a challenge to use to study intact neural circuits due to a combination of small signal-to-noise ratio, slow kinetics, and poor membrane expression. New strategies have yielded novel GEVIs such as ArcLight, which have improved properties. Here, we compare the in vivo properties of ArcLight with Genetically Encoded Calcium Indicators (GECIs) in the mouse olfactory bulb. We show how voltage imaging can be combined with organic calcium sensitive dyes to measure the input-output transformation of the olfactory bulb. Finally, we demonstrate that ArcLight can be targeted to olfactory bulb interneurons. The olfactory bulb contributes substantially to the perception of the concentration invariance of odor recognition.

Voltage imaging was first conceived in the late 1960s and efforts to find better organic voltage sensitive dyes began in the 1970s and continue until today. At the beginning it was difficult to measure an action potential signal from a squid giant axon in a single trial. Now it is possible to measure the action potential in an individual spine. Other chapters will discuss advances in voltage imaging technology and applications in a variety of biological preparations. The development of genetically encoded voltage sensors has started. A genetically encoded sensor could provide cell type specific expression and voltage recording (see Chap. 20). Optimizing the signal-to-noise ratio of an optical recording requires attention to several aspects of the recording apparatus. These include the light source, the optics and the recording device. All three have improved substantially in recent years. Arc lamp, LED, and laser sources are now stable, more powerful, and less expensive. Cameras for recording activity have frames rates above 1 kHz and quantum efficiencies near 1.0 although they remain expensive. The sources of noise in optical recordings are well understood. Both the apparatus and the noise sources are discussed in this chapter.

Voltage-gated calcium channels (VDCC) are essential to neuronal maturation and differentiation. It is believed that important signaling information is encoded by VDCC-mediated calcium influx that has both spatial and temporal components. VDCC are multimeric complexes comprised of a pore-forming alpha1 subunit and auxiliary beta and alpha2/delta subunits. Changes in the fractional contribution of distinct calcium conductances to the total calcium current have been noted in developing and differentiating neurons. These changes are anticipated to reflect the differential expression and localization of the pore-forming alpha1 subunits. However, as in vitro studies have established that beta regulates the channel properties and targeting of alpha1, attention has been directed toward the developmental expression and assembly of beta isoforms. Recently, changes in the beta component of the omega-conotoxin GVIA (CTX)-sensitive N-type VDCC have indicated differential assembly of alpha1B with beta in postnatal rat brain. In addition, unique properties of beta4 have been noted with respect to its temporal pattern of expression and incorporation into N-type VDCC complexes. Therefore, the expression and assembly of specific alpha1/beta complexes may reflect an elaborate cellular strategy for regulating VDCC diversity. The importance of these developmental findings is bolstered by a recent study which identified mutations in the beta4 as the molecular defect in the mutant epileptic mouse (lethargic; lh/lh). As beta4 is normally expressed in both forebrain and cerebellum, one may consider the impact of the loss of beta4 upon VDCC assembly and activity. The importance of the beta1b and beta4 isoforms to calcium channel maturation and assembly is discussed.

Pilocarpine-induced rat epilepsy model is an established animal model that mimics medial temporal lobe epilepsy in humans. The purpose of this study was to investigate neuroimaging abnormalities in various stages of epileptogenesis and to correlate them with seizure severity in pilocarpine-induced rat epilepsy model. Fifty male Sprague-Dawley rats were subject to continuous video and electroencephalographic monitoring after inducing status epilepticus (SE) and seizure severity was estimated by frequency and total durations of class 3 to 5 spontaneous recurrent seizures (SRS) by modified Racine's classification. The 7.0 Tesla magnetic resonance imaging (MRI) with high resolution flurodeoxyglucose positron emission tomography (FDG-PET) was performed at 3 hours, 1, 3, 7 days and 4 weeks after the initial insult. The initial SRS was observed 9.7±1.3 days after the pilocarpine injection. MRI revealed an abnormal T2 signal change with swelling in both hippocampi and amygdala in acute (day 1 after injection) and latent phases (days 3 and 7), in association with PET hypometabolism in these areas. Interestingly, the mean frequency of class 3 to 5 SRS was positively correlated with abnormal T2 signals in hippocampal area at 3 days. SRS duration became longer with more decreased glucose metabolism in both hippocampi and amygdala at 7 days after pilocarpine injection. This study indicates that development and severity of SRS at chronic phase could be closely related with structural and functional changes in hippocampus during the latent period, a pre-epileptic stage.