Anterograde Signalling by Nitric Oxide: Characterisation and In Vitro Reconstitution of an Identified Nitrergic Synapse

Nitric oxide (NO) is recognized as a signaling molecule in the CNS where it is a candidate retrograde neurotransmitter. Here we provide direct evidence that NO mediates slow excitatory anterograde transmission between the NO synthase (NOS)-expressing B2 neuron and an NO-responsive follower neuron named B7nor. Both are motoneurons located in the buccal ganglia of the snail Lymnaea stagnalis where they participate in feeding behavior. Transmission between B2 and B7nor is blocked by inhibiting NOS and is suppressed by extracellular scavenging of NO. Furthermore, focal application of NO to the cell body of the B7nor neuron causes a depolarization that mimics the effect of B2 activity. The slow interaction between the B2 and B7nor neurons can be re-established when the two neurons are cocultured, and it shows the same susceptibility to NOS inhibition and NO scavenging. In cell culture we have also examined spatial aspects of NO signaling. We show that before the formation of an anatomical connection, the presynaptic neuron can cause depolarizing potentials in the follower neuron at distances up to 50 micro(m). The strength of the interaction increases when the distance between the cells is reduced. Our results suggest that NO can function as both a synaptic and a nonsynaptic signaling molecul

Associative memory stored by functional novel pathway rather than modifications of preexisting neuronal pathways

Associative conditioning involves changes in the processing pathways activated by sensory information to link the conditioned stimulus (CS) to the conditioned behavior. Thus, conditioning can recruit neuronal elements to form new pathways for the processing of the CS and/or can change the strength of existing pathways. Using a behavioral and systems level electrophysiological approach on a tractable invertebrate circuit generating feeding in the mollusk Lymnaea stagnalis, we identified three independent pathways for the processing of the CS amyl acetate used in appetitive conditioning. Two of these pathways, one suppressing and the other stimulating feeding, mediate responses to the CS in naive animals. The effects ofthese two pathways on feeding behavior are unaltered by conditioning. In contrast, the CS response ofa third stimulatory pathway is significantly enhanced after conditioning, becoming an importantcontributor to the overall CS response. This is unusual because, in most of the previous examples in which naive animals already respond to the CS, memory formation results from changes in the strength of pathways that mediate the existing response. Here, we show that, in the molluscan feeding system, both modified and unmodified pathways are activated in parallel by the CS after conditioning, and it is their integration that results in the conditioned respons

Timed and targeted differential regulation of nitric oxide synthase (NOS) and anti-NOS genes by reward conditioning leading to long-term memory formation

In a number of neuronal models of learning, signaling by the neurotransmitter nitric oxide (NO), synthesized by the enzyme neuronal NO synthase (nNOS), is essential for the formation of long-term memory (LTM). Using the molluscan model system Lymnaea, we investigate here whether LTM formation is associated with specific changes in the activity of members of the NOS gene family: Lym-nNOS1, Lym-nNOS2, and the antisense RNA-producing pseudogene (anti-NOS). We show that expression of the Lym-nNOS1 gene is transiently upregulated in cerebral ganglia after conditioning. The activation of the gene is precisely timed and occurs at the end of a critical period during which NO is required for memory consolidation. Moreover, we demonstrate that this induction of the Lym-nNOS1 gene is targeted to an identified modulatory neuron called the cerebral giant cell (CGC). This neuron gates the conditioned feeding response and is an essential part of the neural network involved in LTM formation. We also show that the expression of the anti-NOS gene, which functions as a negative regulator of nNOS expression, is downregulated in the CGC by training at 4 h after conditioning, during the critical period of NO requirement. This appears to be the first report of the timed and targeted differential regulation of the activity of a group of related genes involved in the production of a neurotransmitter that is necessary for learning, measured in an identified neuron of known function. We also provide the first example of the behavioral regulation of a pseudogene

A P2X receptor from the tardigrade species Hypsibius dujardini with fast kinetics and sensitivity to zinc and copper

<p>Abstract</p> <p>Background</p> <p>Orthologs of the vertebrate ATP gated P2X channels have been identified in <it>Dictyostelium </it>and green algae, demonstrating that the emergence of ionotropic purinergic signalling was an early event in eukaryotic evolution. However, the genomes of a number of animals including <it>Drosophila melanogaster </it>and <it>Caenorhabditis elegans</it>, both members of the Ecdysozoa superphylum, lack P2X-like proteins, whilst other species such as the flatworm <it>Schistosoma mansoni </it>have P2X proteins making it unclear as to what stages in evolution P2X receptors were lost. Here we describe the functional characterisation of a P2X receptor (<it>Hd</it>P2X) from the tardigrade <it>Hypsibius dujardini </it>demonstrating that purinergic signalling is preserved in some ecdysozoa.</p> <p>Results</p> <p>ATP (EC₅₀~44.5 μM) evoked transient inward currents in <it>Hd</it>P2X with millisecond rates of activation and desensitisation. <it>Hd</it>P2X is antagonised by pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid (IC₅₀15.0 μM) and suramin (IC₅₀22.6 μM) and zinc and copper inhibit ATP-evoked currents with IC₅₀values of 62.8 μM and 19.9 μM respectively. Site-directed mutagenesis showed that unlike vertebrate P2X receptors, extracellular histidines do not play a major role in coordinating metal binding in <it>Hd</it>P2X. However, H306 was identified as playing a minor role in the actions of copper but not zinc. Ivermectin potentiated responses to ATP with no effect on the rates of current activation or decay.</p> <p>Conclusion</p> <p>The presence of a P2X receptor in a tardigrade species suggests that both nematodes and arthropods lost their P2X genes independently, as both traditional and molecular phylogenies place the divergence between Nematoda and Arthropoda before their divergence from Tardigrada. The phylogenetic analysis performed in our study also clearly demonstrates that the emergence of the family of seven P2X channels in human and other mammalian species was a relatively recent evolutionary event that occurred subsequent to the split between vertebrates and invertebrates. Furthermore, several characteristics of <it>Hd</it>P2X including fast kinetics with low ATP sensitivity, potentiation by ivermectin in a channel with fast kinetics and distinct copper and zinc binding sites not dependent on histidines make <it>Hd</it>P2X a useful model for comparative structure-function studies allowing a better understanding of P2X receptors in higher organisms.</p

Suppression of nitric oxide (NO)-dependent behavior by double-stranded RNA-mediated silencing of a neuronal NO synthase gene

We have used double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) to disrupt neuronal nitric oxide (NO) synthase (nNOS) gene function in the snail Lymnaea stagnalis and have detected a specific behavioral phenotype. The injection of whole animals with synthetic dsRNA molecules targeted to the nNOS-encoding mRNA reduces feeding behavior in vivo and fictive feeding in vitro and interferes with NO synthesis by the CNS. By showing that synthetic dsRNA targeted to the nNOS mRNA causes a significant and long-lasting reduction in the levels of Lym-nNOS mRNA, we verify that specific RNAi has occurred. Importantly, our results establish that the expression of nNOS gene is essential for normal feeding behavior. They also show that dsRNA can be used in the investigation of functional gene expression in the context of whole animal behavior, regardless of the availability of targeted mutation technologies

P2x receptor function in Lymnaea stagnalis and other lower organisms

The mollusc Lymnaea stagnalis has a relatively simple central nervous system (CNS) consisting of large and easily identifiable neurons. This feature together with well characterized neuronal circuitry for important physiological processes and an established adenosine 5’-triphosphate (ATP) release system puts Lymnaea forward as an attractive model to examine the CNS function of P2X receptors. Furthermore, lower organism P2X receptors may provide structure function insights by virtue of conservation of functionally important domains. Following the identification of a P2X receptor-like sequence (LymP2X) in the Lymnaea CNS, the cloned complementary deoxyribonucleic acid encoding LymP2X when heterologously expressed in Xenopus oocytes exhibited ATP-evoked inward currents (EC50: 6.2 µM) with slow desensitisation. 2’, 3’-O-(4-Benzoylbenzoyl) adenosine 5’-triphosphate (BzATP) was a partial agonist (EC50: 2.4 µM), whilst pyridoxalphosphate-6-azophenyl-2', 5'-disulphonic acid (PPADS) and suramin were antagonists (IC50: 8.1 and 27.4 µM respectively). A P2X receptor from another invertebrate Hypsibius Dujardini (HdP2X) was also characterised in a comparative study. This tardigrade receptor displayed ATP-evoked currents (EC50: 28.5 µM) with extremely rapid activation and desensitization kinetics, which were concentration-dependently inhibited by copper and zinc. Histidine 306 in HdP2X was found to have a minor role in copper inhibition. Quantitative reverse transcription-polymerase chain reaction and in situ hybridization detected LymP2X expression in all CNS ganglia, including buccal suggesting a possible role in the feeding network. Intracellular recording of motoneuron activity demonstrated that application of ATP (1 mM) to buccal ganglia increased the rate of fictive feeding whereas PPADS abolished this response. The application of BzATP, in an attempt to selectively activate P2X receptors, did not trigger fictive feeding, suggesting that LymP2X activation alone does not initiate the feeding rhythm. BzATP however did cause a significant hyperpolarisation of a key feeding motoneuron, suggesting that LymP2X activation may be required for the latter phase in the ATP-evoked feeding cycle.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Extrinsic modulation and motor pattern generation in a feeding network: a cellular study

Systems level studies have shown that the paired serotonergic cerebral giant cells (CGCs) of gastropod mollusks have important extrinsic modulatory actions on the central pattern generator (CPG) underlying rhythmic ingestion movements. Here we present the first study that investigates the modulatory actions of the CGCs and their released transmitter 5-HT on the CPG at the cellular level. In the snail, Lymnaea, motoneurons such as the B4, B8, and B4CL cells are part of the feeding CPG and receive serotonergic synaptic inputs from CGCs. These motoneurons were used to investigate the effect of serotonergic modulation on endogenous cellular properties of CPG neurons. Cells were isolated from the intact nervous system, and their properties were examined by pharmacological methods in cell culture. Motoneurons were also grown in coculture with CGCs to compare 5-HT effects with CGC stimulation. Three distinc

Modulation of serotononergic neurotransmission by nitric oxide

Nitric oxide (NO) and serotonin (5-HT) are two neurotransmitters with important roles in neuromodulation and synaptic plasticity. There is substantial evidence for a morphological and functional overlap between these two neurotransmitter systems, in particular the modulation of 5-HT function by NO. Here we demonstrate for the first time the modulation of an identified serotonergic synapse by NO using the synapse between the cerebral giant cell (CGC) and the B4 neuron within the feeding network of the pond snail Lymnaea stagnalis as a model system. Simultaneous electrophysiological recordings from the pre- and postsynaptic neurons show that blocking endogenous NO production in the intact nervous system significantly reduces the B4 response to CGC activity. The blocking effect is frequency dependent and is strongest at low CGC frequencies. Conversely, bath application of the NO donor DEA/NONOate significantly enhances the CGC-B4 synapse. The modulation of the CGC-B4 synapse is mediated by the soluble guanylate cyclase (sGC)/cGMP pathway as demonstrated by the effects of the sGC antagonist 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). NO modulation of the CGC-B4 synapse can be mimicked in cell culture, where application of 5-HT puffs to isolated B4 neurons simulates synaptic 5-HT release. Bath application of diethylamine NONOate (DEA/NONOate) enhances the 5-HT induced response in the isolated B4 neuron. However, the cell culture experiment provided no evidence for endogenous NO production in either the CGC or B4 neuron suggesting that NO is produced by an alternative source. Thus we conclude that NO modulates the serotonergic CGC-B4 synapse by enhancing the postsynaptic 5-HT response. Copyright © 2007 The American Physiological Society

Pharmacological characterisation of a P2X receptor cloned from the central nervous system of Lymnaea stagnalis.

Pharmacological characterisation of a P2X receptor cloned from the central nervous system of Lymnaea stagnalis

Consistency management with repair actions

Comprehensive consistency management requires a strong mechanism for repair once inconsistencies have been detected. In this paper we present a repair framework for inconsistent distributed documents. The core piece of the framework is a new method for generating interactive repairs from full first order logic formulae that constrain these documents. We present a full implementation of the components in our repair framework, as well as their application to the UML and related heterogeneous documents such as EJB deployment descriptors. We describe how our approach can be used as an infrastructure for building higher-level, domain specific frameworks and provide an overview of related work in the database and software development environment community