The land slug Limax performs both aversive and appetitive olfactory learning, and we investigated neurotransmitters involved in each type of learning. Slugs were conditioned by presenting a vegetable juice (appetitive conditioning) or a mixture of vegetable juice and quinidine (aversive conditioning), and the latency to reach the juice became shorter (appetitive conditioning) or longer (aversive conditioning) after conditioning. L-NAME injected either before conditioning or testing blocked the reduction in latency in appetitive conditioning but had no significant effects in aversive conditioning. 5,7-dihydroxytryptamine had no significant effects in appetitive conditioning. These results suggest different mechanisms for appetitive and aversive learning.The land mollusk Limax has a highly developed olfactory system and performs olfactory learning. Both aversive (Gelperin 1975;Sahley et al. 1981) and appetitive (Sahley et al. 1990) olfactory learning have been reported, and therefore, it is possible to compare these two forms of learning in Limax. Aversive learning occurs when an odor is associated with an aversive chemical or electrical shock, and appetitive learning occurs when an odor is associated with food. So far, aversive learning has been more extensively studied than appetitive learning. Our previous study showed using the serotonergic neurotoxin 5,7-dihydroxytryptamine (DHT), which reduces the serotonin content in the CNS to less than half, that serotonin is essential for aversive learning (Shirahata et al. 2006). However, it is not known whether appetitive learning also depends on serotonin, or whether it requires other neurotransmitters.Nitric oxide (NO) synthase is abundant in the procerebrum (PC) (Fujie et al. 2002(Fujie et al. , 2005, which is a region of the cerebral ganglion essential for olfactory learning (Kasai et al. 2006). NO applied extrinsically modulates the rhythmic activity of the local field potential (LFP) of the PC (Gelperin 1994;Gelperin et al. 2000). These observations suggest significance of NO in olfactory processing in Limax. In Helix pomatia, a related land mollusk, appetitive learning is blocked by N -nitro-L-arginine methyl ester (L-NAME), a blocker of NO synthase. In the present work, we examine using L-NAME whether NO is involved in appetitive and aversive learning in Limax. We examine dependence of the acquisition and retrieval stages on NO separately. We also examine dependence of appetitive learning on serotonin using DHT.Limax valentianus 2-4 mo old from the laboratory colony were used. The slugs were freely fed with a mixture of rat chow (Oriental Yeast), wheat starch (Wako Pure Chemical), and mixed vitamins (Oriental Yeast), until 5 d before the experiment. The slugs were kept under the light/dark cycle of 10 h/14 h, and conditioning and testing were done within 4 h from the onset of the dark period. Five days before conditioning, the slugs were moved to individual boxes floored with moistened filter paper and starved. The training and testing were made on a glass plat...

Nitric oxide (NO) modulates the dynamics of central olfactory networks and has been implicated in olfactory processing including learning. Land mollusks have a specialized olfactory lobe in the brain called the procerebral (PC) lobe. The PC lobe produces ongoing local field potential (LFP) oscillation, which is modulated by olfactory stimulation. We hypothesized that NO should be released in the PC lobe in response to olfactory stimulation, and to prove this, we applied an NO electrode to the PC lobe of the land slug Limax in an isolated tentacle-brain preparation. Olfactory stimulation applied to the olfactory epithelium transiently increased the NO concentration in the PC lobe, and this was blocked by the NO synthase inhibitor L-NAME at 3.7 mM. L-NAME at this concentration did not block the ongoing LFP oscillation, but did block the frequency increase during olfactory stimulation. Olfactory stimulation also enhanced spatial synchronicity of activity, and this response was also blocked by L-NAME. Single electrical stimulation of the superior tentacle nerve (STN) mimicked the effects of olfactory stimulation on LFP frequency and synchronicity, and both of these effects were blocked by L-NAME. L-NAME did not block synaptic transmission from the STN to the nonbursting (NB)-type PC lobe neurons, which presumably produce NO in an activity-dependent manner. Previous behavioral experiments have revealed impairment of olfactory discrimination after L-NAME injection. The recording conditions in the present work likely reproduce the in vivo brain state in those behavioral experiments. We speculate that the dynamical effects of NO released during olfactory perception underlie precise odor representation and memory formation in the brain, presumably through regulation of NB neuron activity.

Table of contentsI1 IntroductionMona Kanaan, Noreen Dadirai Mdege, Ada KedingO1 The HiSTORIC trial: a hybrid before-and-after and stepped wedge designRA Parker, N Mills, A Shah, F Strachan, C Keerie, CJ WeirO2 Stepped wedge trials with non-uniform correlation structureAndrew Forbes, Karla HemmingO3 Challenges and solutions for the operationalisation of the ENHANCE study: a pilot stepped wedge trial within a general practice settingSarah A Lawton, Emma Healey, Martyn Lewis, Elaine Nicholls, Clare Jinks, Valerie Tan, Andrew Finney, Christian D Mallen, on behalf of the ENHANCE Study TeamO4 Early lessons from the implementation of a stepped wedge trial design investigating the effectiveness of a training intervention in busy health care settings: the Thistle studyErik Lenguerrand, Graeme MacLennan, John Norrie, Siladitya Bhattacharya, Tim Draycott, on behalf of the Thistle groupO5 Sample size calculation for longitudinal cluster randomised trials: a unified framework for closed cohort and repeated cross-section designsRichard Hooper, Steven Teerenstra, Esther de Hoop, Sandra EldridgeO6 Restricted randomisation schemes for stepped-wedge studies with a cluster-level covariateAlan Girling, Monica TaljaardO7 A flexible modelling of the time trend for the analysis of stepped wedge trials: results of a simulation studyGian Luca Di Tanna, Antonio GasparriniP1 Tackling acute kidney injury – a UK stepped wedge clinical trial of hospital-level quality improvement interventionsAnna Casula, Fergus Caskey, Erik Lenguerrand, Shona Methven, Stephanie MacNeill, Margaret May, Nicholas SelbyP2 Sample size considerations for quantifying secondary bacterial transmission in a stepped wedge trial of influenza vaccineLeon Danon, Hannah Christensen, Adam Finn, Margaret MayP3 Sample size calculation for time-to-event data in stepped wedge cluster randomised trialsFumihito Takanashi, Ada Keding, Simon Crouch, Mona KanaanP4 Sample size calculations for stepped-wedge cluster randomised trials with unequal cluster sizesCaroline A. Kristunas, Karen L. Smith, Laura J. GrayP5 The design of stepped wedge trials with unequal cluster sizesJohn N.S. MatthewsP6 Promoting Recruitment using Information Management Efficiently (PRIME): a stepped wedge SWAT (study-within-a-trial)R Al-Shahi Salman, RA Parker, A Maxwell, M Dennis, A Rudd, CJ WeirP7 Implications of misspecified mixed effect models in stepped wedge trial analysis: how wrong can it be?Jennifer A Thompson, Katherine L Fielding, Calum Davey, Alexander M Aiken, James R Hargreaves, Richard J HayesS1 Stepped Wedge Designs with Multiple InterventionsVivian H Lyons, Lingyu Li, James Hughes, Ali Rowhani-RahbarS2 Analysis of the cross-sectional stepped wedge cluster randomised trialKarla Hemming, Monica Taljaard, Andrew Forbes