Michael O'Shea

NEURONAL EXPRESSION OF NEURAL NITRIC OXIDE SYNTHASE (NNOS) PROTEIN IS SUPPRESSED BY AN ANTISENSE RNA TRANSCRIBED FROM AN NOS PSEUDOGENE

Here, we show that a nitric oxide synthase (NOS) pseudogene is expressed in the CNS of the snail Lymnaea stagnalis. The pseudo-NOS transcript includes a region of significant antisense homology to a previously reported neuronal NOS (nNOS)-encoding mRNA. This suggested that the pseudo-NOS transcript acts as a natural antisense regulator of nNOS protein synthesis. In support of this, we show that both the nNOS-encoding and the pseudo-NOS transcripts are coexpressed in giant identified neurons (the cerebral giant cells) in the cerebral ganglion. Moreover, reverse transcription-PCR experiments on RNA isolated from the CNS establish that stable RNA–RNA duplex molecules do form between the two transcripts in vivo. Using an in vitro translation assay, we further demonstrate that the antisense region of the pseudogene transcript prevents the translation of nNOS protein from the nNOS-encoding mRNA. By analyzing NOS RNA and nNOS protein expression in two different identified neurons, we find that when both the nNOS-encoding and the pseudo-NOS transcripts are present in the same neuron, nNOS enzyme activity is substantially suppressed. Importantly, these results show that a natural antisense mechanism can mediate the translational control of nNOS expression in the Lymnaea CNS. Our findings also suggest that transcribed pseudogenes are not entirely without purpose and are a potential source of a new class of regulatory gene in the nervous system.

Structures of two cockroach neuropeptides assigned by fast atom bombardment mass spectrometry

Amino acid sequences have been assigned to two cockroach neuropeptides (greater than Glu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2, M I, and greater than Glu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH2, M II) by application of fast atom bombardment mass spectrometry, including high resolution and linked scan (metastable) studies. The peptides show considerable homology with two other invertebrate neuropeptides, adipokinetic hormone (AKH, from a locust) and red pigment concentrating hormone (RPCH, from a prawn), whose fast atom bombardment spectra were also studied. M I and M II are thus members of a family of structurally-related invertebrate neuropeptides.

Fitness landscapes and evolvability

In this paper, we develop techniques based on evolvability statistics of the fitness land-scape surrounding sampled solutions. Averaging the measures over a sample of equal fitness solutions allows us to build up fitness evolvability portraits of the fitness land-scape, which we show can be used to compare both the ruggedness and neutrality in a set of tunably rugged and tunably neutral landscapes. We further show that the tech-niques can be used with solution samples collected through both random sampling of the landscapes and online sampling during optimization. Finally, we apply the techniques to two real evolutionary electronics search spaces and highlight differences between the two search spaces, comparing with the time taken to find good solutions through search.

Nitric oxide synthesis and action in an invertebrate brain

Nitric oxide (NO) is synthesized in mammalian neurons by Ca2+/calmodulin activated NO synthase and functions as a signalling molecule by activating soluble guanylyl cyclases in target cells. We demonstrate here that both NO synthase and NO-activated guanylyl cyclase are present in the brain of the locust Schistocerca gregaria. Our observations indicate, for the first time, that the NO-cyclic GMP signalling pathway exists in invertebrate nervous systems.

Better Living Through Chemistry: Evolving GasNets for Robot Control

This paper introduces a new type of artificial neural network (GasNets) and shows that it is possible to use evolutionary computing techniques to find robot controllers based on them. The controllers are built from networks inspired by the modulatory effects of freely diffusing gases, especially nitric oxide, in real neuronal networks. Evolutionary robotics techniques were used to develop control networks and visual morphologies to enable a robot to achieve a target discrimination task under very noisy lighting conditions. A series of evolutionary runs with and without the gas modulation active demonstrated that networks incorporating modulation by diffusing gases evolved to produce successful controllers considerably faster than networks without this mechanism. GasNets also consistently achieved evolutionary success in far fewer evaluations than were needed when using more conventional connectionist style networks.

Anterograde Signaling by Nitric Oxide: Characterization 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 stagnaliswhere 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 μm. 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 molecule.

Molecular characterization of NOS in a mollusc: Expression in a giant modulatory neuron

Here we report on the molecular characterization of the first molluscan NOS in the CNS of the pond snail Lymnaea stagnalis. This Lymnaea NOS (Lym-nNOS) which is expressed preferentially in the CNS is most similar to mammalian neuronal NOS but contains tandem repeats of a seven amino acid motif not found in any other known NOS. We have localized Lym-nNOS to the serotonergic cerebral giant cells (CGCs) which modulate synaptic transmission within a neural network that generates feeding behavior. Our results suggest that the CGCs employ both NO and serotonin in the modulation of the central neural network underlying feeding.

Role of delayed nonsynaptic neuronal plasticity in long-term associative memory.

BACKGROUND It is now well established that persistent nonsynaptic neuronal plasticity occurs after learning and, like synaptic plasticity, it can be the substrate for long-term memory. What still remains unclear, though, is how nonsynaptic plasticity contributes to the altered neural network properties on which memory depends. Understanding how nonsynaptic plasticity is translated into modified network and behavioral output therefore represents an important objective of current learning and memory research. RESULTS By using behavioral single-trial classical conditioning together with electrophysiological analysis and calcium imaging, we have explored the cellular mechanisms by which experience-induced nonsynaptic electrical changes in a neuronal soma remote from the synaptic region are translated into synaptic and circuit level effects. We show that after single-trial food-reward conditioning in the snail Lymnaea stagnalis, identified modulatory neurons that are extrinsic to the feeding network become persistently depolarized between 16 and 24 hr after training. This is delayed with respect to early memory formation but concomitant with the establishment and duration of long-term memory. The persistent nonsynaptic change is extrinsic to and maintained independently of synaptic effects occurring within the network directly responsible for the generation of feeding. Artificial membrane potential manipulation and calcium-imaging experiments suggest a novel mechanism whereby the somal depolarization of an extrinsic neuron recruits command-like intrinsic neurons of the circuit underlying the learned behavior. CONCLUSIONS We show that nonsynaptic plasticity in an extrinsic modulatory neuron encodes information that enables the expression of long-term associative memory, and we describe how this information can be translated into modified network and behavioral output.

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.

Inactivation of neuropeptide hormones (AKH I and AKH II) studied in vivo and in vitro

Abstract Timely and effective inactivation of neurohormones is necessary to ensure appropriate biological responses. We have studied the means by which the actions of insect adipokinetic hormones (AKH I: pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH 2 ) and AKH II (pGlu-Leu-Asn-Phe-Ser-Thr-Gly-Trp-NH 2 ) are terminated in the circulatory system of the desert locust, Schistocerca gregaria . When injected into locusts, radiolabelled AKH I or II are rapidly degraded and radioactive metabolites can be recovered from the haemolymph. In vitro studies using haemolymph protein extracts showed that the degradative activity is not in the circulation, suggesting that the AKH-degrading enzymes are located on the surfaces of tissues bathed by the haemolymph. This was verified by incubating AKHs with intact tissues (fat body, muscle, and Malphighian tubules) in organ culture and with membrane preparations from these tissues in vitro . Analysis of the AKH fragments by RP-HPLC and by protein sequencing indicates that inactivation is achieved by an endopeptidase which cleaves the Asn-Phe bond present in both AKHs thereby producing biologically inactive AKH fragments. The AKH fragments C-terminal to the first cleavage are subsequently metabolized by aminopeptidases present both in the circulation and on the tissue surfaces. The amino acid sequence specificity of the activating endopeptids and its sensitivity to phosphoramidon, an inhibitor of mammalian endopeptidase 24.11, indicates that circulating AKHs are inactivated by an enzyme similar to that which degrades AKH in the nervous system and supports the notion that many vertebrate and invertebrate neurohormones are inactivated by a single class of cell surface endopeptidases.