Correlative light and volume electron microscopy to study brain development.

Recent advances in volume electron microscopy (EM) have been driving our thorough understanding of the brain architecture. Volume EM becomes increasingly powerful when cells and their subcellular structures that are imaged in light microscopy are correlated to those in ultramicrographs obtained with EM. This correlative approach, called correlative light and volume electron microscopy (vCLEM), is used to link three-dimensional ultrastructural information with physiological data such as intracellular Ca2+ dynamics. Genetic tools to express fluorescent proteins and/or an engineered form of a soybean ascorbate peroxidase allow us to perform vCLEM using natural landmarks including blood vessels without immunohistochemical staining. This immunostaining-free vCLEM has been successfully employed in two-photon Ca2+ imaging in vivo as well as in studying complex synaptic connections in thalamic neurons that receive a variety of specialized inputs from the cerebral cortex. In this mini-review, we overview how volume EM and vCLEM have contributed to studying the developmental processes of the brain. We also discuss potential applications of genetic manipulation of target cells using clustered regularly interspaced short palindromic repeats-associated protein 9 and subsequent volume EM to the analysis of protein localization as well as to loss-of-function studies of genes regulating brain development. We give examples for the combinatorial usage of genetic tools with vCLEM that will further enhance our understanding of regulatory mechanisms underlying brain development.

Effects of estradiol on dopaminergic synapse formation in the mouse olfactory bulb.

Olfactory glomeruli are the sites of initial synaptic integration in olfactory information processing. They are surrounded by juxtaglomerular (JG) cells, which include periglomerular, superficial short axon, and external tufted cells. A subpopulation of JG cells expresses the dopamine synthetic enzymes, tyrosine hydroxylase (TH), and aromatic l-amino acid decarboxylase (AADC). TH cells corelease γ-aminobutyric acid (GABA) and their processes extend to multiple glomeruli forming intra- and interglomerular circuits. It is well established that 17ß-estradiol (E2) exerts wide ranging effects in the central nervous system. However, participation of E2 in the modulation of neurotransmission and synaptic plasticity of TH cells in olfactory glomeruli is unknown. To address this, we subcutaneously implanted a 60-day release pellet of E2 or placebo into intact male mice and compared glomerular TH, AADC, and vesicular γ-aminobutyric acid transporter (VGAT) immunoreactivity between them. High-voltage electron microscopy (HVEM) and ultra-HVEM using immunogold revealed significantly increased immunoreactive intensity at the cellular level for TH and AADC after E2 treatment and for VGAT in TH cells. These results indicate that E2 may affect the interplay between dopaminergic and GABAergic systems. Moreover, random-section electron microscopy analysis showed a significant increase in the number of symmetrical synapses from TH cell to mitral/tufted cell dendrites after E2 treatment. This result was supported by quantitative immunofluorescence staining with synapse markers. Together, these data indicate that E2 may regulate inhibition between TH cells and olfactory bulb neurons within the glomerulus via interaction between dopaminergic and GABAergic systems, thereby contributing to neuromodulation of odor information processing.

A Combinatorial Input Landscape in the "Higher-Order Relay" Posterior Thalamic Nucleus.

All pathways targeting the thalamus terminate directly onto the thalamic projection cells. As these cells lack local excitatory interconnections, their computations are fundamentally defined by the type and local convergence patterns of the extrinsic inputs. These two key variables, however, remain poorly defined for the "higher-order relay" (HO) nuclei that constitute most of the thalamus in large-brained mammals, including humans. Here, we systematically analyzed the input landscape of a representative HO nucleus of the mouse thalamus, the posterior nucleus (Po). We examined in adult male and female mice the neuropil distribution of terminals immunopositive for markers of excitatory or inhibitory neurotransmission, mapped input sources across the brain and spinal cord and compared the intranuclear distribution and varicosity size of axons originated from each input source. Our findings reveal a complex landscape of partly overlapping input-specific microdomains. Cortical layer (L)5 afferents from somatosensory and motor areas predominate in central and ventral Po but are relatively less abundant in dorsal and lateral portions of the nucleus. Excitatory inputs from the trigeminal complex, dorsal column nuclei (DCN), spinal cord and superior colliculus as well as inhibitory terminals from anterior pretectal nucleus and zona incerta (ZI) are each abundant in specific Po regions and absent from others. Cortical L6 and reticular thalamic nucleus terminals are evenly distributed across Po. Integration of specific input motifs by particular cell subpopulations may be commonplace within HO nuclei and favor the emergence of multiple, functionally diverse input-output subnetworks.SIGNIFICANCE STATEMENT Because thalamic projection neurons lack local interconnections, their output is essentially determined by the kind and convergence of the long-range inputs that they receive. Fragmentary evidence suggests that these parameters may vary within the "higher-order relay" (HO) nuclei that constitute much of the thalamus, but such variation has not been systematically analyzed. Here, we mapped the origin and local convergence of all the extrinsic inputs reaching the posterior nucleus (Po), a typical HO nucleus of the mouse thalamus by combining multiple neuropil labeling and axon tracing methods. We report a complex mosaic of partly overlapping input-specific domains within Po. Integration of different input motifs by specific cell subpopulations in HO nuclei may favor the emergence of multiple, computationally specialized thalamocortical subnetworks.

The role of snare proteins in cortical development.

Neural communication in the adult nervous system is mediated primarily through chemical synapses, where action potentials elicit Ca2+ signals, which trigger vesicular fusion and neurotransmitter release in the presynaptic compartment. At early stages of development, the brain is shaped by communication via trophic factors and other extracellular signaling, and by contact-mediated cell-cell interactions including chemical synapses. The patterns of early neuronal impulses and spontaneous and regulated neurotransmitter release guide the precise topography of axonal projections and contribute to determining cell survival. The study of the role of specific proteins of the synaptic vesicle release machinery in the establishment, plasticity, and maintenance of neuronal connections during development has only recently become possible, with the advent of mouse models where various members of the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex have been genetically manipulated. We provide an overview of these models, focusing on the role of regulated vesicular release and/or cellular excitability in synaptic assembly, development and maintenance of cortical circuits, cell survival, circuit level excitation-inhibition balance, myelination, refinement, and plasticity of key axonal projections from the cerebral cortex. These models are important for understanding various developmental and psychiatric conditions, and neurodegenerative diseases.

Cross-hierarchical plasticity of corticofugal projections to dLGN after neonatal monocular enucleation.

Perception is the result of interactions between the sensory periphery, thalamus, and cerebral cortex. Inputs from the retina project to the first-order dorsal lateral geniculate nucleus (dLGN), which projects to the primary visual cortex (V1). In return, the cortex innervates the thalamus. While layer 6 projections innervate all thalamic nuclei, cortical layer 5 neurons selectively project to the higher order lateral posterior nucleus (LP) and not to dLGN. It has been demonstrated that a subpopulation of layer 5 (Rbp4-Cre+) projections rewires to dLGN after monocular or binocular enucleation in young postnatal mice. However, the exact cortical regional origin of these projections was not fully determined, and it remained unclear whether these changes persisted into adulthood. In this study, we report gene expression changes observed in the dLGN after monocular enucleation at birth using microarray, qPCR at P6, and in situ hybridization at P8. We report that genes that are normally enriched in dLGN, but not LP during development are preferentially downregulated in dLGN following monocular enucleation. Comparisons with developmental gene expression patters in dLGN suggest more immature and delayed gene expression in enucleated dLGN. Combined tracing and immuno-histochemical analysis revealed that the induced layer 5 fibers that innervate enucleated dLGN originate from putative primary visual cortex and they retain increased VGluT1+ synapse formation into adulthood. Our results indicate a new form of plasticity when layer 5 driver input takes over the innervation of an originally first-order thalamic nucleus after early sensory deficit.

Structural basis for noradrenergic regulation of neural circuits in the mouse olfactory bulb.

Olfactory input is processed in the glomerulus of the main olfactory bulb (OB) and relayed to higher centers in the brain by projection neurons. Conversely, centrifugal inputs from other brain regions project to the OB. We have previously analyzed centrifugal inputs into the OB from several brain regions using single-neuron labeling. In this study, we analyzed the centrifugal noradrenergic (NA) fibers derived from the locus coeruleus (LC), because their projection pathways and synaptic connections in the OB have not been clarified in detail. We analyzed the NA centrifugal projections by single-neuron labeling and immunoelectron microscopy. Individual NA neurons labeled by viral infection were three-dimensionally traced using Neurolucida software to visualize the projection pathway from the LC to the OB. Also, centrifugal NA fibers were visualized using an antibody for noradrenaline transporter (NET). NET immunoreactive (-ir) fibers contained many varicosities and synaptic vesicles. Furthermore, electron tomography demonstrated that NET-ir fibers formed asymmetrical synapses of varied morphology. Although these synapses were present at varicosities, the density of synapses was relatively low throughout the OB. The maximal density of synapses was found in the external plexiform layer; about 17% of all observed varicosities contained synapses. These results strongly suggest that NA-containing fibers in the OB release NA from both varicosities and synapses to influence the activities of OB neurons. The present study provides a morphological basis for olfactory modulation by centrifugal NA fibers derived from the LC.

Maturation of Complex Synaptic Connections of Layer 5 Cortical Axons in the Posterior Thalamic Nucleus Requires SNAP25.

Synapses are able to form in the absence of neuronal activity, but how is their subsequent maturation affected in the absence of regulated vesicular release? We explored this question using 3D electron microscopy and immunoelectron microscopy analyses in the large, complex synapses formed between cortical sensory efferent axons and dendrites in the posterior thalamic nucleus. Using a Synaptosome-associated protein 25 conditional knockout (Snap25 cKO), we found that during the first 2 postnatal weeks the axonal boutons emerge and increase in the size similar to the control animals. However, by P18, when an adult-like architecture should normally be established, axons were significantly smaller with 3D reconstructions, showing that each Snap25 cKO bouton only forms a single synapse with the connecting dendritic shaft. No excrescences from the dendrites were formed, and none of the normally large glomerular axon endings were seen. These results show that activity mediated through regulated vesicular release from the presynaptic terminal is not necessary for the formation of synapses, but it is required for the maturation of the specialized synaptic structures between layer 5 corticothalamic projections in the posterior thalamic nucleus.

Cell-Specific Loss of SNAP25 from Cortical Projection Neurons Allows Normal Development but Causes Subsequent Neurodegeneration.

Synaptosomal associated protein 25 kDa (SNAP25) is an essential component of the SNARE complex regulating synaptic vesicle fusion. SNAP25 deficiency has been implicated in a variety of cognitive disorders. We ablated SNAP25 from selected neuronal populations by generating a transgenic mouse (B6-Snap25tm3mcw (Snap25-flox)) with LoxP sites flanking exon5a/5b. In the presence of Cre-recombinase, Snap25-flox is recombined to a truncated transcript. Evoked synaptic vesicle release is severely reduced in Snap25 conditional knockout (cKO) neurons as shown by live cell imaging of synaptic vesicle fusion and whole cell patch clamp recordings in cultured hippocampal neurons. We studied Snap25 cKO in subsets of cortical projection neurons in vivo (L5-Rbp4-Cre; L6-Ntsr1-Cre; L6b-Drd1a-Cre). cKO neurons develop normal axonal projections, but axons are not maintained appropriately, showing signs of swelling, fragmentation and eventually complete absence. Onset and progression of degeneration are dependent on the neuron type, with L5 cells showing the earliest and most severe axonal loss. Ultrastructural examination revealed that cKO neurites contain autophagosome/lysosome-like structures. Markers of inflammation such as Iba1 and lipofuscin are increased only in adult cKO cortex. Snap25 cKO can provide a model to study genetic interactions with environmental influences in several disorders.

Block Face Scanning Electron Microscopy of Fluorescently Labeled Axons Without Using Near Infra-Red Branding.

In this article, we describe the method that allows fluorescently tagged structures such as axons to be targeted for electron microscopy (EM) analysis without the need to convert their labels into electron dense stains, introduce any fiducial marks, or image large volumes at high resolution. We optimally preserve and stain the brain tissue for ultrastructural analysis and use natural landmarks, such as cell bodies and blood vessels, to locate neurites that had been imaged previously using confocal microscopy. The method relies on low and high magnification views taken with the light microscope, after fixation, to capture information of the tissue structure that can later be used to pinpoint the position of structures of interest in serial EM images. The examples shown here are td Tomato expressing cortico-thalamic axons in the posteromedial nucleus of the mouse thalamus, imaged in fixed tissue with confocal microscopy, and subsequently visualized with serial block-face EM (SBEM) and reconstructed into 3D models for analysis.

Subset of Cortical Layer 6b Neurons Selectively Innervates Higher Order Thalamic Nuclei in Mice.

The thalamus receives input from 3 distinct cortical layers, but input from only 2 of these has been well characterized. We therefore investigated whether the third input, derived from layer 6b, is more similar to the projections from layer 6a or layer 5. We studied the projections of a restricted population of deep layer 6 cells ("layer 6b cells") taking advantage of the transgenic mouse Tg(Drd1a-cre)FK164Gsat/Mmucd (Drd1a-Cre), that selectively expresses Cre-recombinase in a subpopulation of layer 6b neurons across the entire cortical mantle. At P8, 18% of layer 6b neurons are labeled with Drd1a-Cre::tdTomato in somatosensory cortex (SS), and some co-express known layer 6b markers. Using Cre-dependent viral tracing, we identified topographical projections to higher order thalamic nuclei. VGluT1+ synapses formed by labeled layer 6b projections were found in posterior thalamic nucleus (Po) but not in the (pre)thalamic reticular nucleus (TRN). The lack of TRN collaterals was confirmed with single-cell tracing from SS. Transmission electron microscopy comparison of terminal varicosities from layer 5 and layer 6b axons in Po showed that L6b varicosities are markedly smaller and simpler than the majority from L5. Our results suggest that L6b projections to the thalamus are distinct from both L5 and L6a projections.

Loss of X-linked Protocadherin-19 differentially affects the behavior of heterozygous female and hemizygous male mice.

Mutations in the X-linked gene Protocadherin-19 (Pcdh19) cause female-limited epilepsy and mental retardation in humans. Although Pcdh19 is known to be a homophilic cell-cell adhesion molecule, how its mutations bring about female-specific disorders remains elusive. Here, we report the effects of Pcdh19 knockout in mice on their development and behavior. Pcdh19 was expressed in various brain regions including the cerebral cortex and hippocampus. Although Pcdh19-positive cells were evenly distributed in layer V of wild-type cortices, their distribution became a mosaic in Pcdh19 heterozygous female cortices. In cortical and hippocampal neurons, Pcdh19 was localized along their dendrites, showing occasional accumulation on synapses. Pcdh19 mutants, however, displayed no detectable abnormalities in dendrite and spine morphology of layer V neurons. Nevertheless, Pcdh19 hemizygous males and heterozygous females showed impaired behaviors including activity defects under stress conditions. Notably, only heterozygous females exhibited decreased fear responses. In addition, Pcdh19 overexpression in wild-type cortices led to ectopic clustering of Pcdh19-positive neurons. These results suggest that Pcdh19 is required for behavioral control in mice, but its genetic loss differentially affects the male and female behavior, as seen in human, and they also support the hypothesis that the mosaic expression of Pcdh19 in brains perturbs neuronal interactions.

Emerging roles of protocadherins: from self-avoidance to enhancement of motility.

Protocadherins are a group of transmembrane proteins belonging to the cadherin superfamily that are subgrouped into 'clustered' and 'non-clustered' protocadherins. Although cadherin superfamily members are known to regulate various forms of cell-cell interactions, including cell-cell adhesion, the functions of protocadherins have long been elusive. Recent studies are, however, uncovering their unique roles. The clustered protocadherins regulate neuronal survival, as well as dendrite self-avoidance. Combinatorial expression of clustered protocadherin isoforms creates a great diversity of adhesive specificity for cells, and this process is likely to underlie the dendritic self-avoidance. Non-clustered protocadherins promote cell motility rather than the stabilization of cell adhesion, unlike the classic cadherins, and mediate dynamic cellular processes, such as growth cone migration. Protocadherin dysfunction in humans is implicated in neurological disorders, such as epilepsy and mental retardation. This Commentary provides an overview of recent findings regarding protocadherin functions, as well as a discussion of the molecular basis underlying these functions.

Protocadherin-17 mediates collective axon extension by recruiting actin regulator complexes to interaxonal contacts.

In the process of neuronal wiring, axons derived from the same functional group typically extend together, resulting in fascicle formation. How these axons communicate with one another remains largely unknown. Here, we show that protocadherin-17 (Pcdh17) supports this group extension by recruiting actin polymerization regulators to interaxonal contact sites. Pcdh17 is expressed by a subset of amygdala neurons, and it accumulates at axon-axon boundaries because of homophilic binding. Pcdh17 knockout in mice suppressed the extension of these axons. Ectopically expressed Pcdh17 altered the pattern of axon extension. In in-vitro cultures, wild-type growth cones normally migrate along other axons, whereas Pcdh17 null growth cones do not. Pcdh17 recruits the WAVE complex, Lamellipodin, and Ena/VASP to cell-cell contacts, converting these sites into motile structures. We propose that, through these mechanisms, Pcdh17 maintains the migration of growth cones that are in contact with other axons, thereby supporting their collective extension.

Identification of the odour-active cyclic diketone cis-2,6-dimethyl-1,4-cyclohexanedione in roasted Arabica coffee brew.

We investigated odour-active trace compounds in roasted Brazilian Arabica coffee. Aroma dilution extract analysis (AEDA) applied to the volatile oil extracted from roasted coffee brew revealed 34 odour-active compounds. Among these, a pungent-smelling unknown odour-active compound was determined. The volatile oil was fractioned by silica gel column chromatography. Gas chromatography-olfactometry (GC-O) and multidimensional gas chromatography-mass spectrometry (MDGC-MS) of the fraction which contained a significant amount of the target unknown compound revealed the cyclic 1,4-diketone, cis-2,6-dimethyl-1,4-cyclohexanedione, which had a pungent odour, and was thus first identified in roasted coffee. Model experiments revealed that cis-2,6-dimethyl-1,4-cyclohexanedione was formed via thermal degradation of sugars, especially monosaccharides, under alkaline conditions. Further, we demonstrated that 2-hydroxy-3-pentanone and 1-hydroxy-2-propanone, thermal degradation products of monosaccharides, were closely related to the formation of cis-2,6-dimethyl-1,4-cyclohexanedione.

Eucalyptus leaf extract suppresses the postprandial elevation of portal, cardiac and peripheral fructose concentrations after sucrose ingestion in rats.

Overintake of sucrose or fructose induces adiposity. Fructose undergoes a strong Maillard reaction, which worsens diabetic complications. To determine whether Eucalyptus globulus leaf extract (ELE) suppresses the postprandial elevation of serum fructose concentrations (SFCs) in the portal, cardiac, and peripheral blood after sucrose ingestion, we performed gas chromatography/mass spectrometry (GC/MS) and measured SFC without any interference by contaminating glucose in the samples. Fasting Wistar rats were orally administered water (control group) or ELE (ELE group) before sucrose ingestion. Blood was collected from the portal vein, heart, and tail. The increase in the SFCs in the portal and cardiac samples 30 min after sucrose ingestion was lower in the ELE group than in the control group. The coefficient of correlation between the SFCs in the portal and cardiac samples was 0.825. The peripheral SFC in the control group progressively increased and was 146 micromol/L at 60 min. This increase was significantly lower in the ELE group. In contrast, the serum glucose concentrations in the 2 groups were similar. ELE suppressed postprandial hyperfructosemia in the portal, cardiac, and peripheral circulations. ELE may counteract glycation caused by high blood fructose concentrations induced by the consumption of fructose-containing foods or drinks.

Rats fed fructose-enriched diets have characteristics of nonalcoholic hepatic steatosis.

Nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease are increasing in adults and are likely to be increasing in children. Both conditions are hepatic manifestations of metabolic syndrome. Experimental animals fed fructose-enriched diets are widely recognized as good models for metabolic syndrome. However, few reports have described the hepatic pathology of these experimental animals. In this study, 5-wk-old Wistar specific pathogen-free rats, which are a normal strain, were fed experimental diets for 5 wk. We then evaluated the degree of steatohepatitis. The 5 diet groups were as follows: cornstarch (70% wt:wt) [control (C)], high-fructose (70%) (HFr), high-sucrose (70%) (HS), high-fat (15%) (HF), and high-fat (15%) high-fructose (50%) (HFHFr) diets. The macrovesicular steatosis grade, liver:body weight ratio, and hepatic triglyceride concentration were significantly higher in the HFr group than in the other 4 groups. However, the HFr group had a significantly lower ratio of epididymal white fat:body weight than the other 4 groups and had a lower final body weight than the HF and HFHFr groups. The HF group had a greater final body weight than the C, HFr, and HS groups, but no macrovesicular steatosis was observed. The HFr group had a significantly higher grade of lobular inflammation than the other 4 groups. The distribution of lobular inflammation was predominant over portal inflammation, which is consistent with human NASH. In conclusion, rats fed fructose-enriched diets are a better model for NASH than rats fed fat-enriched diets.

Bending-induced switching of dynein activity in elastase-treated axonemes of sea urchin sperm--roles of Ca2+ and ADP.

Flagellar beating is caused by microtubule sliding, driven by the activity of dynein, between adjacent two of the nine doublet microtubules. An essential process in the regulation of dynein is to alternate its activity (switching) between the two sides of the central pair microtubules. The switching of dynein activity can be detected, in an in vitro system using elastase-treated axonemes of sea urchin sperm flagella, as a reversal of the relative direction of ATP-induced sliding between the two bundles of doublets at high Ca(2+) (10(-4) M) at pH 7.8-8.0. The reversal is triggered by externally applied bending of the doublet bundle. However, the mechanism of this bending-induced reversal (or backward sliding) remains unclear. To understand how the switching of dynein activity in flagella can be induced by bending, we studied the roles of ADP, which is an important factor for the dynein motile activity, and of Ca(2+) in the bending-induced reversal of microtubule sliding between two bundles of doublets at pH 7.5 and 7.2. We found that the reversal of sliding direction was induced regardless of the concentrations of Ca(2+) at low pH, but occurred more frequently at low Ca(2+) (<10(-9) M) than at high Ca(2+). At pH 7.5, an application of ADP increased the frequency of occurrence of backward sliding at high as well as low concentrations of Ca(2+). The results indicate that ADP-dependent activation of dynein, probably resulting from ADP-binding to dynein, is involved in the regulation of the bending-induced switching of dynein activity in flagella.

Mechanism of flagellar oscillation-bending-induced switching of dynein activity in elastase-treated axonemes of sea urchin sperm.

Oscillatory movement of eukaryotic flagella is caused by dynein-driven microtubule sliding in the axoneme. The mechanical feedback from the bending itself is involved in the regulation of dynein activity, the main mechanism of which is thought to be switching of the activity of dynein between the two sides of the central pair microtubules. To test this, we developed an experimental system using elastase-treated axonemes of sperm flagella, which have a large Ca(2+)-induced principal bend (P-bend) at the base. On photoreleasing ATP from caged ATP, they slid apart into two bundles of doublets. When the distal overlap region of the slid bundles was bent in the direction opposite to the basal P-bend, backward sliding of the thinner bundle was induced along the flagellum including the bent region. The velocity of the backward sliding was significantly lower than that of the forward sliding, supporting the idea that the dynein activity alternated between the two sides of the central pair on bending. Our results show that the combination of the direction of bending and the conformational state of dynein-microtubule interaction induce the switching of the dynein activity in flagella, thus providing the basis for flagellar oscillation.

Eucalyptus leaf extract inhibits intestinal fructose absorption, and suppresses adiposity due to dietary sucrose in rats.

Sucrose is more lipogenic than starch, and the extreme ingestion of sucrose induces adiposity and obesity. The aim of this study was to examine the effect of the eucalyptus (Eucalyptus globulus) leaf extract (ELE) on adiposity due to dietary sucrose in rats. In addition, in this study, the effect of ELE on intestinal fructose absorption was also examined. Rats were fed a high-sucrose diet (75 % in calorie base) with or without ELE (10 g/kg diet) for 5 weeks. Body weight was lower in the rats receiving ELE than in the controls (342 (sd 37.9) v. 392 (sd 26.0) g (n 7); P<0.05). Furthermore, ELE resulted in decreases in the triacylglycerol concentrations in the plasma (1.44 (sd 0.448) v. 2.79 (sd 0.677) mmol/l (n 7); P<0.05) and liver (19.1 (sd 5.07) v. 44.1 (sd 16.28) micromol/g (n 7); P<0.05). In contrast, ELE did not show any significant effects in the rats fed a starch diet. When rats were orally given ELE 10 min before fructose administration, the intestinal fructose absorption, which was examined by measuring the elevated concentration of fructose in the portal vein at 30 min after the fructose administration, was significantly inhibited in a dose-dependent manner. Furthermore, in rats fed a high-fructose diet, the plasma and hepatic triacylglycerol concentrations were significantly decreased by ELE. These results indicate that ELE, which inhibits the intestinal fructose absorption, can suppress adiposity in rats that ingest large amounts of sucrose or fructose.