Gene expression of circadian pacemaker hormone PDF in the brain of honey bee, Apis mellifera

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Abstracts

peptide of the prosobranch. AAamide induced a partial contraction to esophagus. It also augmented the TEP (Thais excitatory peptide)-induced contraction of penial complex. However, the peptide had little actions on the heartbeat and the motility of reproductive glands. Immunohistochemistry on paraffin-thin sections with a specific anti-AAamide antibody visualized the AAamide containing somata in circum-esophageal ganglia. There was a tendency that most of the positive neurons were found in the supra-esophageal ganglia such as cerebral ganglion, while positive processes were distributed in the neuropile in every ganglia. These results suggest that AAamide is a neuropeptide of T. clavigera, mediating the neural regulation of digestive system.

synaptic plasticity in the CGC, under the control of the cAMP signaling cascade. These results indicate that LymSERT could be the candidates which regulate the synaptic transmission related to CTA learning.

doi:10.1016/j.cbpb.2006.10.047

Masahiro Sokabe a,b,c, Ling Chena b, Xiao-Niu Dai a, aDept. Physiol., Nagoya Univ. Gad. Sch. Med., Nagoya 466-8560, Japan; bICORP/SORST Cell Mechanosensing, JST, Nagoya 466-8560, Japan; cDept. Mol. Physiol. Natl. Inst. Physiol. Sci., NINS, Okazaki 444-8585, Japan

doi:10.1016/j.cbpb.2006.10.049

24. Neurosteroid DHEAS facilitates the induction of hippocampal longterm potentiation via sigma-1 receptor

22. Blockade of FMRFamide-gated Na channel by divalent cations Yasuo Furukawa, Laboratory of Neurobiolgy, Grad. Sch. Integrated Arts and Sci., Hiroshima Univ., Higashi-Hiroshima, Hiroshima 739-8526, Japan FMRFamide-gated Na + channel (FaNaC) is an only peptide gated ion channel whose primary structure is known. Structurally, FaNaC is a member of the ENaC/DEG family. To elucidate the structure-function relationship of this peptide-gated channel, we cloned an Aplysia FaNaC (AkFaNaC) and examined its electrophysiological characteristics in Xenopus oocytes [1]. AkFaNaC is blocked voltage-dependently by amiloride as is often documented in other ENaC/DEG family channels. By contrast, an inorganic cation, Ca 2+, blocked the channel voltage-independently, suggesting that the blocking site is outside the membrane electric field. Mg 2+ did not show such blocking action. The EC50 of Ca 2+ block was around 0.6 mM. We further investigated the effect of other divalent cations. Because oocytes develop leakage currents in the absence of Ca 2+, we examined the action of other divalent cations in a physiological solution for oocytes which contains 1.8 mM CaCl2. Under this condition, Ni 2+ and Mn 2+ were found to block AkFaNaC at millimolar range (EC50 was around 1–3 mM). Cd 2+ showed similar but more potent blocking action with EC50 of around 0.07 mM. Thus, the order of blocking potency in the presence of 1.8 mM Ca 2+ was Cd 2+ ≫ Ni 2+ = Mn 2+. [1] Furukawa, Miyawaki, Abe (2006), Pflugers Arch, 451:646–656.

Dehydroepiandrosterone sulfate (DHEAS), one of the most abundant neurosteroids synthesized de novo in the brain, has been known to improve cognitive performances the aged and to reverse β-Amyloid (A β)-induced deficits in memory. However, little is known about the underlying molecular mechanisms. We report here that chronically administered DHEAS (20 mg/kg) for 7 days lowered the threshold to induce activity-dependent long-term potentiation (LTP) in rat hippocampal CA1 synapses. While tetanus of at least 50 pulses (at 100 Hz) was required to induce LTP in control rats, only 20 pulses were needed in DHEAS-treated rats. DHEAS did not alter the presynaptic glutamate release in response to both test and tetanic stimulations. In contrast, in postsynaptic (pyramidal) neurons obtained from the DHEAS-treated rats, Src tyrosine phosphorylation levels and NMDA-induced postsynaptic Ca 2+ transients were significantly potentiated. Interestingly, the Src family kinase inhibitor PP2 attenuated the potentiated NMDA-induced Ca 2+-transient that was essential for the potentiation of Src phosphorylation and the DHEAS-facilitated LTP induction. The sigma-1 receptor antagonists, haloperidol or NE100, completely inhibited the DHEAS-facilitated LTP and the potentiation of NMDA-induced Ca 2+ transient. This novel postsynaptic sigma-1 receptor-mediated signal amplification through the “NMDAr-Ca 2+→Src→NMDAr-Ca 2+” cycle may play a pivotal role in the DHEAS-facilitated LTP induction. doi:10.1016/j.cbpb.2006.10.050

doi:10.1016/j.cbpb.2006.10.048 25. No abstract submitted

23. The learning-associated gene expression of serotonin transporter in Lymnaea stagnalis Hisayo Sadamoto, Etsuro Ito, Facl. Pharm. Sci., Kagawa Campus, Tokushima Bunri Univ., 1314-1 Shido, Sanuki, Kagawa 769-2193, Japan

IN the pond snail Lymnaea stagnalis, one of the serotonergic neuron, the cerebral giant cell (CGC) was isolated and examined its electrical properties. Especially, based on the behavioral experiments for conditioned taste aversion (CTA) leaning, the neuromodulatory model including the CGC was proposed. To investigate the regulatory mechanism of synaptic plasticity in CTA learning, we focused on serotonin transporter (SERT). First, we isolated SERT homologue from Lymnaea cDNA library (LymSERT). LymSERT encodes 631 amino acids and the deduced amino acid sequence is highly identical to mammalian SERT in the functional region. We next performed real-time RTPCR experiments and showed that LymSERT mRNA level in trained animals was significantly higher than that in control animals. Further, we confirmed that several cyclic AMP-responsive element (CRE) sequences existed close to the transcription start site of LymSERT. Previously, we found that cyclic AMPresponsive element binding proteins (CREB) function as necessary players in

doi:10.1016/j.cbpb.2006.10.051

26. Do tyraminergic neurons exist in the rat brain? Akira Komatsu a, Airi Yamaguchi b, Noriko Makikusa b, Osamu Koizumi b, a Dept. Physiol., Tokyo Women's Med. Univ., Sch. Med., Tokyo 162-8666, Japan; bNeurosci. Lab., Fukuoka Women's Univ. Fukuoka 813-8529, Japan

Trace amine receptors were discovered in mammals, but their endogenous ligands have not yet been found. To search for them, we developed a new method to make antibodies against monoamines for immunohistochemistry (IHC). Tyramine (TA) was conjugated to a keyhole limpet hemocyanine (KLH) using an imidoester cross-linker, dimethyl suberimidate (DMS). Rabbits were immunized by the conjugated macromolecule. The obtained anti-TA antibodies (α-TAs) were assayed by ELISA and competitive ELISA techniques to check their antibody titer and specificity respectively. The antibodies recognized the monoamine-DMS part within the conjugate. The α-TAs recognized specifically TA compared to its related amines such as dopamine (DA). The crossreactivity

Abstracts of one of the α-TAs with DA was 1/3,000. For IHC, the rat brain was perfused by 2% DMS, post-fixed by 4% formaldehyde and then frozen-sectioned. After incubating in the α-TA solution, the sections were processed by ABC method. The α-TA staining successfully revealed neurons that correspond to dopaminergic neurons. The α-TA absorbed with a TA-DMS-BSA conjugate failed to stain the dopaminergic neurons. On the other hand, the α-TA absorbed with a DA-DMS-BSA conjugate stained them. These results suggest that the dopaminergic neurons have TA which functions as a possible neurotransmitter/modulator. doi:10.1016/j.cbpb.2006.10.052

27. Fruitless specifies sexually dimorphic neural circuitry in the Drosophila brain Ken-ichi Kimura a, Tatsunori Tazawa a, Tomoaki Hachiya a, Daisuke Yamamoto b, Hokkaido Univ. of Edu. Iwamizawa Campus, Iwamizawa 068-8642d, Japan; b Grad. Sch. of Life Sci., Touhoku Univ., Sendai 980-8578, Japan a

The Drosophila fruitless (fru) gene product Fru has been postulated to be a neural sex determination factor that directs [KK1] development of the CNS, producing male-typical courtship behaviour. Male-specific Fru protein is expressed in small groups of neurons scattered throughout the CNS of males, but not of females. These observations collectively suggest that Fru masculinizes certain neurons thereby establishing the neural substrates for male-typical behaviour. However, it is totally unknown what difference is actually produced in neurons by the presence or absence of Fru. By experiments that label individually the neurons that express Fru, we have identified a subset of brain interneurons that display marked sexual dimorphism in their number and projection pattern. We have revealed that programmed cell death is involved in formation of sexual dimorphism in the neural circuit. Here, we demonstrate that Fru supports the development of those neurons with male-specific dendritic fields, whereas these neurons are programmed to die during development in females as a result of the absence of Fru. Thus the Fru specifies sexually dimorphic neural circuitry in the Drosophila brain. doi:10.1016/j.cbpb.2006.10.053

28. Neuronal mechanisms for photic and thermoperiodic entrainment of Drosophila circadian locomotor rhythms Yoko Miyasako, Kenji Tomioka, Grad. Sch. Nat. Sci. Tech., Okayama Univ., Okayama 700-8530, Japan

Daily behavioral rhythms are precisely timed by environmental light and temperature cycles. This is accomplished through photic and thermal synchronization of an underlying endogenous circadian system consisting of multiple oscillators. Whereas molecular mechanisms for light and temperature entrainment have been so far proposed, how multiple oscillators orchestrate behavioral rhythms synchronizing to natural environmental cycles remains elusive. Drosophila melanogaster has an advantage to address this general issue, because, like mammals, it shows bimodal circadian locomotor activity with two peaks around light-on (morning peak) and before light-off (evening peak) and because the cells driving the peaks have been identified. In this study we show, by behavioral and immunohistochemical assays, that separate groups of clock neurons, either lightentrainable or temperature-entainable, form a functional system. The laterally located protocerebral neurons (LN) are preferentially light-entrainable and the dorsally located neurons and lateral posterior neurons are temperature-entrainable. Further behavioral studies suggested that one group of LN cells have a strong impact to regulate the phase of the temperature-entrainable cells. The two sets of

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clock cells with different entrainability to light and temperature and the coupling between them enable the Drosophila circadian activity to keep a proper phase relationship to the natural daily light and temperature cycles. doi:10.1016/j.cbpb.2006.10.054

29. Diversely regulated transcripts of the circadian clock gene period in the cricket Gryllus bimaculatus Yukimasa Takeda a , Akane Iwasaki a, Ayami Matsushima a , Yasuyuki Shimohigashi a, Miki Shimohigashi b, aDept. of Chem., Fac. of Sci., Kyushu Univ., Fukuoka 812-8581, Japan; bDept. of Biol., Fac. Sci., Fukuoka Univ., Fukuoka 814-0180, Japan

The molecular basis of the circadian clock indicates that the clock is an autoregulatory feedback loop in which the PAS domain-containing protein PERIOD periodically inhibits transcription of its own period gene. We have identified a distinct alternative splicing mechanism in several insect species and propose that period splicing variants play key physiological roles in the circadian clock system. In the present study, to explore the possibility of a general splicing mechanism amongst insects, we attempted to clone the period mRNA genes in the cricket Gryllus bimaculatus. To identify preserved alternative splicing sites, a specific PCR-based cloning method was performed for cDNAs prepared from mRNAs isolated from brains and optic lobes. 3′ RACE was performed especially to find out the sites of alternative polyadenylation. Eventually, we identified five alternative splicing sites and three polyA addition sites. One of the splicing sites was exactly the same site as reported for period mRNA isoforms in the honey-bee. The genomic DNA sequence readily explained a splicing mechanism based on the splicing consensus sequences in the exon/intron structure. The present result strongly suggests the presence of general post-transcriptional events for period transcripts. doi:10.1016/j.cbpb.2006.10.055

30. Gene expression of circadian pacemaker hormone PDF in the brain of honey bee, Apis mellifera Miho Sumiyoshi a, Seiji Sato b, Yukimasa Takeda b, Keita Koga b, Kazunori Sumidab, Yasuyuki Shimohigashi b, Miki Shimohigashi a, aDept. Biol, Fac. Sci., Fukuoka Univ., Fukuoka 814-0180, Japan; bDept. Chem., Fac. of Sci., Kyushu Univ., Fukuoka 812-8581, Japan

Pigment-dispersing factor (PDF), an 18-amino acid neuropeptide, is a pacemaker hormone that plays an important role in the output circuit from the biological clock in the locomotor rhythm in insects. In the honeybee Apis mellifera, the young bee of nurse does not exhibit any clear locomotor rhythms, while the old bee forager exhibits a distinct locomotor rhythm. In the present study, we carried out the cDNA cloning and the quantitative real-time PCR (QRT-PCR) to explore the relationship between the pdf mRNA expression and the locomotor rhythm. The results of detailed cDNA cloning revealed that PDF has a characteristic amino acid substitution (Asp→Asn) at position 17 as compared with other ordinary PDF peptides. Furthermore, we identified a unique alternative splicing site at 5′UTR and three polyA-addition sites at 3′UTR in pdf mRNA. QRT-PCR revealed that the total pdf mRNA level has a daily fluctuation. We also performed in situ hybridization and immunocytochemistry in order to clarify the cells expressing pdf mRNA and PDF peptide. These anatomical analyses explored approximately 14 lateral neurons showing both signals in the optic lobes. doi:10.1016/j.cbpb.2006.10.056