Hisayo Sadamoto

De novo sequencing and transcriptome analysis of the central nervous system of mollusc Lymnaea stagnalis by deep RNA sequencing.

The pond snail Lymnaea stagnalis is among several mollusc species that have been well investigated due to the simplicity of their nervous systems and large identifiable neurons. Nonetheless, despite the continued attention given to the physiological characteristics of its nervous system, the genetic information of the Lymnaea central nervous system (CNS) has not yet been fully explored. The absence of genetic information is a large disadvantage for transcriptome sequencing because it makes transcriptome assembly difficult. We here performed transcriptome sequencing for Lymnaea CNS using an Illumina Genome Analyzer IIx platform and obtained 81.9 M of 100 base pair (bp) single end reads. For de novo assembly, five programs were used: ABySS, Velvet, OASES, Trinity and Rnnotator. Based on a comparison of the assemblies, we chose the Rnnotator dataset for the following blast searches and gene ontology analyses. The present dataset, 116,355 contigs of Lymnaea transcriptome shotgun assembly (TSA), contained longer sequences and was much larger compared to the previously reported Lymnaea expression sequence tag (EST) established by classical Sanger sequencing. The TSA sequences were subjected to blast analyses against several protein databases and Aplysia EST data. The results demonstrated that about 20,000 sequences had significant similarity to the reported sequences using a cutoff value of 1e-6, and showed the lack of molluscan sequences in the public databases. The richness of the present TSA data allowed us to identify a large number of new transcripts in Lymnaea and molluscan species.

Altered gene activity correlated with long-term memory formation of conditioned taste aversion in Lymnaea

The pond snail Lymnaea stagnalis is capable of learning conditioned taste aversion (CTA) and then consolidating that learning into long‐term memory (LTM) that persists for at least 1 month. LTM requires de novo protein synthesis and altered gene activity. Changes in gene activity in Lymnaea that are correlated with, much less causative, memory formation have not yet been identified. As a first step toward rectifying this situation, we constructed a cDNA microarray with mRNAs extracted from the central nervous system (CNS) of Lymnaea. We then, using this microarray assay, identified genes whose activity either increased or decreased following CTA memory consolidation. We also identified genes whose expression levels were altered after inhibition of the cyclic AMP response element‐binding protein (CREB) that is hypothesized to be a key transcription factor for CTA memory. We found that the molluscan insulin‐related peptide II (MIP II) was up‐regulated during CTA‐LTM, whereas the gene encoding pedal peptide preprohormone (Pep) was down‐regulated by CREB2 RNA interference. We next examined mRNAs of MIP II and Pep using real‐time RT‐PCR with SYBR Green. The MIP II mRNA level in the CNS of snails exhibiting “good” memory for CTA was confirmed to be significantly higher than that from the CNS of snails exhibiting “poor” memory. In contrast, there was no significant difference in expression levels of the Pep mRNA between “good” and “poor” performers. These data suggest that in Lymnaea MIP II may play a role in the consolidation process that forms LTM following CTA training.

Involvement of Insulin-Like Peptide in Long-Term Synaptic Plasticity and Long-Term Memory of the Pond Snail Lymnaea stagnalis

The pond snail Lymnaea stagnalis is capable of learning taste aversion and consolidating this learning into long-term memory (LTM) that is called conditioned taste aversion (CTA). Previous studies showed that some molluscan insulin-related peptides (MIPs) were upregulated in snails exhibiting CTA. We thus hypothesized that MIPs play an important role in neurons underlying the CTA–LTM consolidation process. To examine this hypothesis, we first observed the distribution of MIP II, a major peptide of MIPs, and MIP receptor and determined the amounts of their mRNAs in the CNS. MIP II was only observed in the light green cells in the cerebral ganglia, but the MIP receptor was distributed throughout the entire CNS, including the buccal ganglia. Next, when we applied exogenous mammalian insulin, secretions from MIP-containing cells or partially purified MIPs, to the isolated CNS, we observed a long-term change in synaptic efficacy (i.e., enhancement) of the synaptic connection between the cerebral giant cell (a key interneuron for CTA) and the B1 motor neuron (a buccal motor neuron). This synaptic enhancement was blocked by application of an insulin receptor antibody to the isolated CNS. Finally, injection of the insulin receptor antibody into the snail before CTA training, while not blocking the acquisition of taste aversion learning, blocked the memory consolidation process; thus, LTM was not observed. These data suggest that MIPs trigger changes in synaptic connectivity that may be correlated with the consolidation of taste aversion learning into CTA–LTM in the Lymnaea CNS.

Identification and expression analysis of the genes involved in serotonin biosynthesis and transduction in the field cricket Gryllus bimaculatus

Serotonin (5‐HT) modulates various aspects of behaviours such as aggressive behaviour and circadian behaviour in the cricket. To elucidate the molecular basis of the cricket 5‐HT system, we identified 5‐HT‐related genes in the field cricket Gryllus bimaculatus DeGeer. Complementary DNA of tryptophan hydroxylase and phenylalanine‐tryptophan hydroxylase, which convert tryptophan into 5‐hydroxy‐L‐tryptophan (5‐HTP), and that of aromatic L‐amino acid decarboxylase, which converts 5‐HTP into 5‐HT, were isolated from a cricket brain cDNA library. In addition, four 5‐HT receptor genes (5‐HT1A, 5‐HT1B, 5‐HT2α, and 5‐HT7) were identified. Expression analysis of the tryptophan hydroxylase gene TRH and phenylalanine‐tryptophan hydroxylase gene TPH, which are selectively involved in neuronal and peripheral 5‐HT synthesis in Drosophila, suggested that two 5‐HT synthesis pathways co‐exist in the cricket neuronal tissues. The four 5‐HT receptor genes were expressed in various tissues at differential expression levels, suggesting that the 5‐HT system is widely distributed in the cricket.

Direct Observation of Dimerization between Different CREB1 Isoforms in a Living Cell

Cyclic AMP-responsive element binding protein 1 (CREB1) plays multiple functions as a transcription factor in gene regulation. CREB1 proteins are also known to be expressed in several spliced isoforms that act as transcriptional activators or repressors. The activator isoforms, possessing the functional domains for kinase induction and for interaction with other transcriptional regulators, act as transcriptional activators. On the other hand, some isoforms, lacking those functional domains, are reported to be repressors that make heterodimers with activator isoforms. The complex and ingenious function for CREB1 arises in part from the variation in their spliced isoforms, which allows them to interact with each other. To date, however, the dimerization between the activator and repressor isoforms has not yet been proved directly in living cells. In this study, we applied fluorescence cross-correlation spectroscopy (FCCS) to demonstrate direct observation of dimerization between CREB1 activator and repressor. The FCCS is a well established spectroscopic method to determine the interaction between the different fluorescent molecules in the aqueous condition. Using differently labeled CREB1 isoforms, we successfully observed the interaction of CREB1 activator and repressor via dimerization in the nuclei of cultured cells. As a result, we confirmed the formation of heterodimer between CREB1 activator and repressor isoforms in living cells.

Learning-dependent gene expression of CREB1 isoforms in the molluscan brain

Cyclic AMP-responsive element binding protein1 (CREB1) has multiple functions in gene regulation. Various studies have reported that CREB1-dependent gene induction is necessary for memory formation and long-lasting behavioral changes in both vertebrates and invertebrates. In the present study, we characterized Lymnaea CREB1 (LymCREB1) mRNA isoforms of spliced variants in the central nervous system (CNS) of the pond snail Lymnaea stagnalis. Among these spliced variants, the three isoforms that code a whole LymCREB1 protein are considered to be the activators for gene regulation. The other four isoforms, which code truncated LymCREB1 proteins with no kinase inducible domain, are the repressors. For a better understanding of the possible roles of different LymCREB1 isoforms, the expression level of these isoform mRNAs was investigated by a real-time quantitative RT-PCR method. Further, we examined the changes in gene expression for all the isoforms in the CNS after conditioned taste aversion (CTA) learning or backward conditioning as a control. The results showed that CTA learning increased LymCREB1 gene expression, but it did not change the activator/repressor ratio. Our findings showed that the repressor isoforms, as well as the activator ones, are expressed in large amounts in the CNS, and the gene expression of CREB1 isoforms appeared to be specific for the given stimulus. This was the first quantitative analysis of the expression patterns of CREB1 isoforms at the mRNA level and their association with learning behavior.

Molecular basis of the dopaminergic system in the cricket Gryllus bimaculatus

In insects, dopamine modulates various aspects of behavior such as learning and memory, arousal and locomotion, and is also a precursor of melanin. To elucidate the molecular basis of the dopaminergic system in the field cricket Gryllus bimaculatus DeGeer, we identified genes involved in dopamine biosynthesis, signal transduction, and dopamine re-uptake in the cricket. Complementary DNA of two isoforms of tyrosine hydroxylase (TH), which convert tyrosine into l-3,4-dihydroxyphenylalanine, was isolated from the cricket brain cDNA library. In addition, four dopamine receptor genes (Dop1, Dop2, Dop3, and DopEcR) and a high-affinity dopamine transporter gene were identified. The two TH isoforms contained isoform-specific regions in the regulatory ACT domain and showed differential expression patterns in different tissues. In addition, the dopamine receptor genes had a receptor subtype-specific distribution: the Dop1, Dop2, and DopEcR genes were broadly expressed in various tissues at differential expression levels, and the Dop3 gene was restrictedly expressed in neuronal tissues and the testicles. Our findings provide a fundamental basis for understanding the dopaminergic regulation of diverse physiological processes in the cricket.

Excitatory Effects of GABA on Procerebrum Neurons in a Slug

Classical neurotransmitters, such as glutamate and γ-aminobutyric acid (GABA), often have different actions on invertebrate neurons from those reported for vertebrate neurons. In the terrestrial mollusk Limax, glutamate was found to function as an inhibitory transmitter in the procerebrum (PC), but it has not yet been clarified how GABA acts in the PC. We thus examined what effects GABA exerts on PC neurons in the present study. For this purpose, we first applied GABA to isolated PC preparations and recorded postsynaptic currents and potentials in PC neurons. The GABA application reduced the amplitude of inhibitory postsynaptic currents and depolarization-induced outward currents recorded in nonbursting neurons and increased the number of spontaneous spikes of nonbursting neurons. However, direct GABA-induced currents were not observed in either bursting or nonbursting neurons. These results suggest a potential direct effect of GABA on outward currents resulting in enhanced excitability of PC neurons. Next, we measured the change in [Ca(2+)](i) in cultured PC neurons by application of GABA. The GABA application increased spontaneous Ca(2+) events in cultured neurons. These Ca(2+) events were ascribable to the influx of extracellular Ca(2+). We then confirmed the presence of GABA and GABA receptors in the PC. The GABA-like immunoreactivity was observed in the neuropil layers of the PC, and the mRNAs for both GABA(A) and GABA(B) receptors were expressed in the PC. In particular, GABA(B) receptor mRNA, rather than GABA(A), was found to be more abundantly expressed in the PC. These results suggest that GABA functions as an excitatory modulator for PC neurons via mainly GABA(B) receptors.

Glutamate transporters in the central nervous system of a pond snail

Previous studies on glutamate (GLU) and its receptors in the pond snail Lymnaea stagnalis have suggested that GLU functions as a neurotransmitter in various behaviors, particularly for generation of feeding rhythm. The uptake mechanism of GLU is not yet known in Lymnaea. In the present study, we characterized the GLU transporters and examined their functions in the feeding circuits of the central nervous system (CNS) in Lymnaea. First, measurement of the accumulation of 3H‐labeled GLU revealed the presence of GLU transport systems in the Lymnaea CNS. The highest accumulation rate was observed in the buccal ganglia, supporting the involvement of GLU transport systems in feeding behavior. Second, we cloned two types of GLU transporters from the Lymnaea CNS, the excitatory amino acid transporter (LymEAAT) and the vesicular GLU transporter (LymVGLUT). When we compared their amino acid sequences with those of mammalian EAATs and VGLUTs, we found that the functional domains of both types are well conserved. Third, in situ hybridization revealed that the mRNAs of LymEAAT and LymVGLUT are localized in large populations of nerve cells, including the major feeding motoneurons in the buccal ganglia. Finally, we inhibited LymEAAT and found that changes in the firing patterns of the feeding motoneurons that have GLUergic input were similar to those obtained following stimulation with GLU. Our results confirmed the presence of GLU uptake systems in the Lymnaea CNS and showed that LymEAAT is required for proper rhythm generation, particularly for generation of the feeding rhythm.

Fluorescence Cross-correlation Spectroscopy (FCCS) to Observe Dimerization of Transcription Factors in Living Cells.

Fluorescence cross-correlation spectroscopy (FCCS) is an established spectroscopic method to observe the interaction between the different fluorescent molecules. Using FCCS, researchers can assess the interaction of target molecules in the aqueous condition, and can apply the technique in cultured cells. Here, we describe the method of FCCS to demonstrate direct observation of dimerization between transcription factors in a living cell.