Etsuro Ito

Tokushima Bunri University, Shido, Kagawa, Japan

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Publications (214)516.36 Total impact

  • Journal of Neuroscience Methods 11/2015; DOI:10.1016/j.jneumeth.2015.10.008 · 2.05 Impact Factor
  • S Kojima · H Sunada · K Mita · M Sakakibara · K Lukowiak · E Ito ·
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    ABSTRACT: Insulin is well known as a hormone regulating glucose homeostasis across phyla. Although there are insulin-independent mechanisms for glucose uptake in the mammalian brain, which had contributed to a perception of the brain as an insulin-insensitive organ for decades, the finding of insulin and its receptors in the brain revolutionized the concept of insulin signaling in the brain. However, insulin's role in brain functions, such as cognition, attention, and memory, remains unknown. Studies using invertebrates with their open blood-vascular system have the promise of promoting a better understanding of the role played by insulin in mediating/modulating cognitive functions. In this review, the relationship between insulin and its impact on long-term memory (LTM) is discussed particularly in snails. The pond snail Lymnaea stagnalis has the ability to undergo conditioned taste aversion (CTA), that is, it associatively learns and forms LTM not to respond with a feeding response to a food that normally elicits a robust feeding response. We show that molluscan insulin-related peptides are up-regulated in snails exhibiting CTA-LTM and play a key role in the causal neural basis of CTA-LTM. We also survey the relevant literature of the roles played by insulin in learning and memory in other phyla.
    Journal of Comparative Physiology 08/2015; 201(10). DOI:10.1007/s00359-015-1032-5 · 2.04 Impact Factor
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    ABSTRACT: To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10-18 moles of the p24/assay corresponds to ca. 103 copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (102 copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.
    PLoS ONE 06/2015; 10(6):e0131319. DOI:10.1371/journal.pone.0131319 · 3.23 Impact Factor
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    ABSTRACT: In Lymnaea, experiments showed that one-day food deprivation before aversive classical conditioning results in optimal conditioned taste aversion (CTA) and long-term memory (LTM), whereas 5-day food deprivation before training did not. We hypothesized that snails do in fact learn and form LTM when trained after prolonged food deprivation, but that severe food deprivation blocks their ability to express memory. We trained 5-day food-deprived snails under various conditions, and found that memory was indeed formed but overwhelmed by severe food deprivation. Moreover, CTA-LTM was context-dependent and could be observed only when the snails were in a context similar to that in which the training occurred. © 2015. Published by The Company of Biologists Ltd.
    Journal of Experimental Biology 04/2015; 218(11). DOI:10.1242/jeb.120329 · 2.90 Impact Factor
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    ABSTRACT: Behavioural/motivational state is known to influence nearly all aspects of physiology and behaviour. The cellular basis of behavioural state control is only partially understood. Our investigation, performed on the pond snail Lymnaea stagnalis whose nervous system is useful for work on completely isolated neurons, provided several results related to this problem. First, we demonstrate that the behavioural state can produce long-term changes in individual neurons that persist even after neuron isolation from the nervous system. Specifically, we found that pedal serotonergic neurons that control locomotion show higher activity and lower membrane potential after being isolated from the nervous systems of hungry animals. Second, we show that the modulatory state (the chemical neuroactive microenvironment of the central ganglia) changes in accord with the nutritional state of an animal and produces predicted changes in single isolated locomotor neurons. Third, we report that observed hunger induced effects can be explained by the increased synthesis of serotonin in pedal serotonergic neurons, which impacts the electrical activity of isolated serotonergic neurons and the intensity of extrasynaptic serotonin release from the pedal ganglia. © 2015. Published by The Company of Biologists Ltd.
    Journal of Experimental Biology 02/2015; 218(8). DOI:10.1242/jeb.111930 · 2.90 Impact Factor
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    Etsuro Ito · Yusuke Ikemoto · Tohru Yoshioka ·
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    ABSTRACT: The activity of thermo-transient receptor potential (TRP) channels is highly dependent on temperature, and thus thermo-TRP reactions have a high temperature coefficient Q10. In thermodynamics, a high value of Q10 indicates the existence of a large activation energy (i.e., a large enthalpy) over a short period during the transition process between the closed and open states of the channels. The Gibbs free energy equation shows that a large entropy is required to compensate for this large enthalpy and permit activation of the channels, suggesting a large conformational change of the channels. These large values of enthalpy and entropy seem to be a match for the values of the unfolding process of globular proteins. We outline these thermodynamic issues in thermo-TRPs.
    BIOPHYSICS 01/2015; 11:33-38. DOI:10.2142/biophysics.11.33
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    ABSTRACT: The involvement of serotonin in mediating hunger-related changes in behavioral state has been described in many invertebrates. However, the mechanisms by which hunger signals to serotonergic cells remain unknown. We tested the hypothesis that serotonergic neurons can directly sense the concentration of glucose, a metabolic indicator of nutritional state. In the snail Lymnaea stagnalis, we demonstrate that completely isolated pedal serotonergic neurons that control locomotion changed their biophysical characteristics in response to glucose application by lowering membrane potential and decreasing the firing rate. Additionally, the excitatory response of the isolated serotonergic neurons to the neuroactive microenvironment of the pedal ganglia was significantly lowered by glucose application. Because hunger has been reported to increase the activity of select neurons and their responses to the pedal ganglia microenvironment, these responses to glucose are in accordance with the hypothesis that direct glucose signaling is involved in the mediation of the hunger-related behavioral state.
    BIOPHYSICS 01/2015; 11:55-60. DOI:10.2142/biophysics.11.55

  • 01/2015; 12:79-86. DOI:10.2142/biophysico.12.0_79
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    ABSTRACT: Serotonin (5-hydroxytryptamine: 5-HT) is a multimodal transmitter that controls both feeding response and heart- beat in snails. However, the effects of 5-HT on the hunger state are still unknown. We therefore examined the rela- tion among the hunger state, the heartbeat rate and the 5-HT action in food-starved snails. We found that the hunger state was significantly distinguished by the heart- beat rate in snails. The heartbeat rate was high in the food-satiated snails, whereas it was low in the food- starved snails. An increase in 5-HT concentration in the body boosted the heartbeat rate in the food-starved snails, but did not affect the rate in the food-satiated snails. These results suggest that 5-HT application may mimic the change from a starvation to a satiation state normally achieved by direct ingestion of food.
    BIOPHYSICS 01/2015; 11:1-5. DOI:10.2142/biophysics.11.1
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    ABSTRACT: The pond snail Lymnaea stagnalis can learn conditioned taste aversion and then consolidate it into long-term memory (LTM). A high voltage electric shock was used as the unconditioned stimulus (US), whereas we previously used KCl. We varied both the strength of the conditioned stimulus (CS) and US to determine if the so-called Yerkes-Dodson law prevailed. This is an empirical relationship between the state of arousal and LTM formation, showing that there is an optimal level of arousal leading to memory formation. However, too little or too much arousal results in poorer LTM. We found here that the most appropriate stimuli to use in taste aversion training in Lymnaea were a 10 mmol l(-1) sucrose solution as the CS and a 3-s electric shock as the US.
    Journal of Experimental Biology 12/2014; 218(3). DOI:10.1242/jeb.113266 · 2.90 Impact Factor
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    ABSTRACT: Conditioned taste aversion (CTA) in Lymnaea is brought about by pairing a sucrose solution (the conditioned stimulus, CS) with an electric shock (the unconditioned stimulus, US). Following repeated CS-US pairings, CTA occurs and it is consolidated into long-term memory (LTM). The best CTA is achieved, if snails are food-deprived for 1day before training commences. With a longer period of food deprivation (5days), learning and memory formation does not occur. It has been hypothesized that the levels of insulin in the central nervous system (CNS) are very important for CTA to occur. To test his hypothesis, we injected insulin directly into 5-day food-deprived snails. The injection of insulin, as expected, resulted in a decrease in hemolymph glucose concentration. Consistent with our hypothesis with insulin injection, learning and memory formation of CTA occurred. That is, the 'insulin spike' is more important than an increase in hemolymph glucose concentration for CTA-LTM. If we injected an insulin receptor antibody into the snails before the insulin injection, learning was formed but memory formation was not, which is consistent with our previous study. Therefore, a rise in the insulin concentration (i.e., insulin spike) in the CNS is considered to be a key determining factor in the process of CTA-LTM. Copyright © 2014 Elsevier Inc. All rights reserved.
    Neurobiology of Learning and Memory 10/2014; 116. DOI:10.1016/j.nlm.2014.10.006 · 3.65 Impact Factor
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    ABSTRACT: Acetylcholine plays various important roles in the central nervous system of invertebrates as well as vertebrates. In the olfactory center of the terrestrial slug Limax, the local field potential (LFP) oscillates, and the change in its oscillatory frequency is thought to correlate with the detection of odor that potentially changes an ongoing behavior of the animal. Acetylcholine is known to up-regulate the frequency of the LFP oscillation, and is one of the candidates for the neurotransmitters that are involved in such higher cognitive functions. However, there have been no histological data on the cholinergic system in gastropods, nor are there data on the receptors that are responsible for the up-regulation of the oscillatory frequency of LFP due to the lack of analytical tools (such as antibodies or cDNA sequence information of cholinergic system-related genes). Here we cloned the cDNAs of choline acetyltransferase (ChAT), acetylcholinesterase, vesicular acetylcholine transporter, and several nicotinic acetylcholine receptors (nAChRs), and investigated their localization in the brain of Limax. We also generated a polyclonal antibody against ChAT to examine its localization, and investigated pharmacologically the involvement of nAChRs in the LFP oscillation. Our data showed: (1) dense distribution of the neurons expressing mRNAs of ChAT and vesicular acetylcholine transporter in the olfactory center; (2) spatially unique expression patterns of different nAChRs in the olfactory center; (3) involvement of nAChRs in the up-regulation of the oscillation; (4) localization of ChAT protein in nerve fibers and/or terminals; and (5) the presence of cholinergic nerves in the tentacles. J. Comp. Neurol., 2014. © 2014 Wiley Periodicals, Inc.
    The Journal of Comparative Neurology 09/2014; 522(13). DOI:10.1002/cne.23559 · 3.23 Impact Factor
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    ABSTRACT: The honeybee waggle dance communicates the location of profitable food sources, usually with a certain degree of error in the directional information ranging from 10-15° at the lower margin. We simulated one-day colonial foraging to address the biological significance of information error in the waggle dance. When the error was 30° or larger, the waggle dance was not beneficial. If the error was 15°, the waggle dance was beneficial when the food sources were scarce. When the error was 10° or smaller, the waggle dance was beneficial under all the conditions tested. Our simulation also showed that precise information (0-5° error) yielded great success in finding feeders, but also caused failures at finding new feeders, i.e., a high-risk high-return strategy. The observation that actual bees perform the waggle dance with an error of 10-15° might reflect, at least in part, the maintenance of a successful yet risky foraging trade-off.
    Scientific Reports 02/2014; 4:4175. DOI:10.1038/srep04175 · 5.58 Impact Factor
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    ABSTRACT: An ultrasensitive method for the determination of proteins is described that combines an enzyme-linked immunosorbent assay (ELISA) and a thionicotinamide-adenine dinucleotide (thio-NAD) cycling method. A sandwich method using a primary and a secondary antibody for antigens is employed in an ELISA. An androsterone derivative, 3α-hydroxysteroid, is produced by the hydrolysis of 3α-hydroxysteroid 3-phosphate with alkaline phosphatase linked to the secondary antibody. This 3α-hydroxysteroid is oxidized to a 3-ketosteroid by 3α-hydroxysteroid dehydrogenase (3α-HSD) with a cofactor thio-NAD. By the opposite reaction, the 3-ketosteroid is reduced to a 3α-hydroxysteroid by 3α-HSD with a cofactor NADH. During this cycling reaction, thio-NADH accumulates in a quadratic function-like fashion. Accumulated thio-NADH can be measured directly at an absorbance of 400 nm without any interference from other cofactors. These features enable us to detect a target protein with ultrasensitivity (10–19 mol/assay) by measuring the cumulative quantity of thio-NADH. Our ultrasensitive determination of proteins thus allows for the detection of small amounts of proteins only by the application of thio-NAD cycling reagents to the usual ELISA system.
    BIOPHYSICS 01/2014; 10:49-54. DOI:10.2142/biophysics.10.49
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    ABSTRACT: The pond snail Lymnaea stagnalis is capable of being classically conditioned to avoid food and to consolidate this aversion into a long-term memory (LTM). Previous studies have shown that the length of food deprivation is important for both the acquisition of taste aversion and its consolidation into LTM, which is referred to as conditioned taste aversion (CTA). Here we tested the hypothesis that the hemolymph glucose concentration is an important factor in the learning and memory of CTA. One-day food deprivation resulted in the best learning and memory, whereas more prolonged food deprivation had diminishing effects. Five-day food deprivation resulted in snails incapable of learning or remembering. During this food deprivation period, the hemolymph glucose concentration decreased. If snails were fed for 2 days following the 5-day food deprivation, their glucose levels increased significantly and they exhibited both learning and memory, but neither learning nor memory was as good as with the 1-day food-deprived snails. Injection of the snails with insulin to reduce glucose levels resulted in better learning and memory. Insulin is also known to cause a long-term enhancement of synaptic transmission between the feeding-related neurons. On the other hand, injection of glucose into 5-day food-deprived snails did not alter their inability to learn and remember. However, if these snails were fed on sucrose for 3 minutes, they then exhibited learning and memory formation. Our data suggest that hemolymph glucose concentration is an important factor in motivating acquisition of CTA in Lymnaea and that the action of insulin in the brain and the feeding behavior are also important factors.
    Neurobiology of Learning and Memory 10/2013; 107. DOI:10.1016/j.nlm.2013.10.013 · 3.65 Impact Factor
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    ABSTRACT: The brain of gastropod mollusks contains many giant neurons with polyploid genomic DNAs. Such DNAs are generated through repeated DNA endoreplication during body growth. However, it is not known what triggers DNA endoreplication in neurons. There are two possibilities: (1) DNAs are replicated in response to some unknown molecules in the hemolymph that reflect the nutritive status of the animal; or (2) DNAs are replicated in response to some unknown factors that are retrogradely transported through axons from the innervated target organs. We first tested whether hemolymph with rich nutrition could induce DNA endoreplication. We tested whether the transplanted brain exhibits enhanced DNA endoreplication like an endogenous brain does when transplanted into the homocoel of the body of a slug whose body growth is promoted by an increased food supply. However, no enhancement was observed in the frequency of DNA endoreplication when we compared the transplanted brains in the growth-promoted and growth-suppressed host slugs, suggesting that the humoral environment is irrelevant to triggering the body growth-dependent DNA endoreplication. Next, we tested the requirement of target innervation by surgically dissecting a unilateral posterior pedal nerve of an endogenous brain. Substantially lower number of neurons exhibited DNA endoreplication in the pedal ganglion ipsilateral to the dissected nerve. These results support the view that enhanced DNA endoreplication is mediated by target innervation and is not brought about through the direct effect of humoral factors in the hemolymph during body growth. © 2013 Wiley Periodicals, Inc. Develop Neurobiol, 2013.
    Developmental Neurobiology 08/2013; 73(8). DOI:10.1002/dneu.22087 · 3.37 Impact Factor
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    ABSTRACT: Fluorescence correlation spectroscopy (FCS) is a technique in which measurement of fluorescence intensity fluctuations is used to clarify dynamic molecular interactions within a very small space in a solution containing a small number of fluorescent molecules. The FCS-based analysis gives the average number and average diffusion time of the fluorescent molecules during their passage through a very small space. One advantage of FCS is that physical separation between free and bound fluorescent probes is not required because the properties of fluorescence fluctuations are accounted for. Therefore, when fluorescent probes are bound with proteins by peroxidase and hydrogen peroxide (H2O2), FCS enables us to detect H2O2 with high sensitivity. In addition, because H2O2 is generated by oxidase-catalyzed reactions, a highly sensitive method for detecting H2O2 is applicable to the measurement of low levels of various oxidases and their substrates, such as glucose. We here describe the protocol of a de novo, highly sensitive method for the measurement of H2O2 and glucose using FCS.
    Methods in enzymology 06/2013; 526:135-43. DOI:10.1016/B978-0-12-405883-5.00008-9 · 2.09 Impact Factor
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    E Ito · S Kojima · K Lukowiak · M Sakakibara ·
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    ABSTRACT: The neural circuitry comprising the central pattern generator (CPG) that drives feeding behavior in the great pond snail (Lymnaea stagnalis (L., 1758)) has been worked out. Because the feeding behavior undergoes associative learning and long-term memory (LTM) formation, it provides an excellent opportunity to study the causal neuronal mechanisms of these two processes. In this review, we explore some of the possible causal neuronal mechanisms of associative learning of conditioned taste aversion (CTA) and its subsequent consolidation processes into LTM in L. stagnalis. In the CTA training procedure, a sucrose solution, which evokes a feeding response, is used as the conditioned stimulus (CS) and a potassium chloride solution, which causes a withdrawal response, is used as the unconditioned stimulus (US). The pairing of the CS-US alters both the feeding response of the snail and the function of a pair of higher order interneurons in the cerebral ganglia. Following the acquisition of CTA, the polysynaptic inhibitory synaptic input from the higher order interneurons onto the feeding CPG neurons is enhanced, resulting in suppression of the feeding response. These changes in synaptic efficacy are thought to constitute a "memory trace" for CTA in L. stagnalis.
    Canadian Journal of Zoology 06/2013; 91(6):405-412. DOI:10.1139/cjz-2012-0292 · 1.30 Impact Factor
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    ABSTRACT: The pond snail Lymnaea stagnalis learns taste aversion and consolidates it into long-term memory (LTM). This is referred to as conditioned taste aversion (CTA). The superfusion of molluscan insulin-related peptides (MIPs) over the isolated snail brain causes a long-term enhancement of synaptic input between the cerebral giant cell and the B1 buccal motor neuron. This enhancement is hypothesized to underlie CTA. The synaptic enhancement caused by the superfusion of MIPs can be blocked by the application of human insulin receptor antibody, which recognizes the extracellular domain of human insulin receptor and acts as an antagonist even for MIP receptors. An injection of the human insulin receptor antibody into the abdominal cavity of trained snails blocks the consolidation process leading to LTM, even though the snails acquire taste aversion. Here, we examined whether or not taste-aversion training changes the mRNA expression level of MIP receptor in the snail brain and found that it does not. This result, taken together with previous findings, suggest that the MIPs' effect on synaptic function in the snail brain is attributable to a change in the MIP concentration, and not to a change in the mRNA expression level of MIP receptor, which is thought to reflect the number of MIP receptors.
    Communicative & integrative biology 05/2013; 6(3):e23955. DOI:10.4161/cib.23955
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    ABSTRACT: A simple spectrophotometric method for the assay of steroid 5α-reductase (5α-SR) was developed in which 5α-dihydrotestosterone (5α-DHT) and 5α-androstane-3α,17β-diol (5α-diol), metabolites formed in the NADPH-dependent reduction of testosterone with enzyme sources of 5α-SR, were measured by enzymatic cycling using 3α-hydroxysteroid dehydrogenase in the presence of excess thionicotinamide-adenine dinucleotide (thio-NAD) and NADH. It was found that 5α-SR activity was proportional to the accumulated thio-NADH having an absorption maximum at 400 nm. Because of the high cycling rate (> 600 cycle per min) and no interference from testosterone, enzymatic cycling can determine the sum of 5α-DHT and 5α-diol at the picomole level without separation from excess testosterone. The present method was readily applicable to the assay of 5α-SR activity of rat liver and prostate microsomes as well as to the assay of inhibitory activity of finasteride, a synthetic inhibitor of 5α-SR.
    Analytical Sciences 04/2013; 29(4):455-9. DOI:10.2116/analsci.29.455 · 1.39 Impact Factor

Publication Stats

3k Citations
516.36 Total Impact Points


  • 2006-2015
    • Tokushima Bunri University
      • • Kagawa School of Pharmaceutical Sciences
      • • Faculty of Pharmaceutical Sciences
      Shido, Kagawa, Japan
  • 1994-2008
    • Hokkaido University
      • • Division of Biological Sciences (Biology)
      • • Graduate School of Science
      Sapporo, Hokkaidō, Japan
  • 1991-2008
    • Waseda University
      • • Department of Physics
      • • School of Human Sciences
      • • Advanced Research Center for Human Sciences
      Tokyo, Tokyo-to, Japan
  • 2001
    • Sapporo Medical University
      • Department of Physiology
      Sapporo, Hokkaidō, Japan
  • 1999
    • University of Münster
      • Institute of Zoophysiology (IZP)
      Muenster, North Rhine-Westphalia, Germany