Nitric Oxide-Mediated Modulation of Central Network Dynamics during Olfactory Perception.

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

Schematic of the pathways that transmit olfactory information to the PC lobe.

<p>The PC lobe neurons (B and NB neurons) have somata in the cell mass (CM). Afferent fibers project in the terminal mass (TM) to make synapses on the NB neurons. NB neurons produce spikes that propagate afferently to activate synapses on B neurons. At the same time, spikes also propagate efferently into the internal mass (IM), where NO will be released. NO diffuses into the cell mass to depolarize B neurons, which modifies the network activity, presumably with the main effect of suppressing NB neurons.</p

Odor stimulation triggers release of NO in the PC lobe.

<p>(A) Schematic of the isolated tentacle-brain preparation used for NO measurement. NO concentration was recorded using an NO electrode placed in the PC lobe. The tentacle was connected to the brain by the superior tentacle nerve (STN). Odorant (0.001% hexanol or garlic) was applied to the olfactory epithelium. (B) Odor-evoked increase in NO concentration in the PC lobe. After application of L-NAME, the response became smaller. (C) Effect of D-NAME on odor-evoked increase in NO concentration. The response after application of D-NAME was similar to the response in normal saline. (D) Summary of NO increase in response to olfactory stimulation before and after application of L-NAME or D-NAME. Average and individual data points are shown. L-NAME significantly reduced the NO increase (**P<0.01; N = 13), while D-NAME had no significant effect (NS, not significant; N = 8).</p

Electrical stimulation of the STN evokes NO-dependent responses.

<p>(A) Schematic of the experiment. A single electrical pulse was applied to the STN from a suction electrode. (B) STN stimulation (arrow) transiently increased the frequency of LFP oscillation (top). L-NAME blocked the frequency increase (bottom). (C) Modulation of the phase lag between the apical and basal recording sites. In normal saline, the lag decreased after STN stimulation (top). In saline containing L-NAME, STN stimulation did not change the lag (bottom). (D) The amplitude of the evoked LFP immediately following the stimulation in normal saline (top) did not change after incubation with L-NAME (bottom), suggesting that fast synaptic transmission to the PC lobe is intact in the presence of L-NAME. (E) The evoked EPSP was recorded in NB neurons in normal saline (top) and L-NAME (bottom). The amplitudes of the evoked EPSP were similar under these two conditions. (F) Summary of the changes in the frequency of LFP oscillation. Average and individual data points are shown in this and subsequent graphs. The LFP oscillation increased in response to STN stimulation, and this was blocked by L-NAME (*P<0.05; N = 10). (G) Summary of the changes in the phase lag between the apical and basal recording sites. The data connected by the lines are from the same samples. The phase lag decreased following STN stimulation, and this was blocked by L-NAME (**P<0.01; N = 8). (H) Summary of the amplitude of the evoked LFP. The amplitude did not significantly change after incubation with L-NAME (NS, not significant; N = 7). (I) Summary of the amplitude of the evoked EPSP in NB neurons. The amplitude did not significantly differ between saline and L-NAME groups (N = 5 for control and N = 7 for L-NAME).</p

Odor-evoked NO release increases the frequency of the LFP oscillation in the PC lobe.

<p>(A) Ongoing LFP oscillation was recorded. During stimulation with hexanol, the LFP frequency increased (top). After application of L-NAME, the ongoing LFP oscillation was unaffected, but olfactory stimulation did not increase the LFP frequency (bottom). (B) Time course of the instantaneous frequency of the LFP oscillation in saline (left) and L-NAME (right). The dotted lines indicate the average of the pre-stimulus frequency. (C) Summary of the frequency changes by olfactory stimulation. Average and individual data are shown. L-NAME significantly reduced the frequency increase (*P<0.05; N = 7), whereas D-NAME did not significantly change the response (NS, not significant; N = 6). (D) Summary of the frequency of the resting LFP oscillation. Average and individual data points are shown. Neither L-NAME (N = 7) nor D-NAME (N = 6) significantly changed the frequency.</p

Correlation between changes in frequency and synchronicity.

<p>The decrease in phase lag was plotted against the increase in LFP frequency. Stimulation was made with hexanol at three different concentrations and EMOP at one concentration. The dotted line indicates least square fitting of all the data points. Spearman's rank correlation coefficient was 0.638 (P<0.01; N = 16).</p

Odor-evoked NO release enhances spatial synchronicity of activity in the PC lobe.

<p>(A) Schematic of the experiment. Voltage imaging was made from the PC lobe in a tentacle-brain preparation stained with Di-4-ANEPPS. Apical and basal ROIs are shown. (B) Normalized fluorescence signals from the apical and basal ROIs. Before stimulation, the apical and basal signals had a lag (top left). The lag decreased during odor stimulation (top right). In the saline containing L-NAME, olfactory stimulation did not decrease the lag (bottom). (B) Time course of the phase lag in saline (left) and L-NAME (right). The dotted lines indicate the average of the pre-stimulus phase lag. (D) Summary of the responses of phase lag to odor stimulation. Average and individual data are shown. L-NAME significantly reduced the decrease in the phase lag (*P<0.05; N = 10), whereas D-NAME did not significantly change the response (NS, not significant; N = 8).</p

Proceedings of the First International Conference on Stepped Wedge Trial Design: York, UK, 10 March 2016

I1 Introduction Mona Kanaan, Noreen Dadirai Mdege, Ada Keding O1 The HiSTORIC trial: a hybrid before-and-after and stepped wedge design RA Parker, N Mills, A Shah, F Strachan, C Keerie, CJ Weir O2 Stepped wedge trials with non-uniform correlation structure Andrew Forbes, Karla Hemming O3 Challenges and solutions for the operationalisation of the ENHANCE study: a pilot stepped wedge trial within a general practice setting Sarah A Lawton, Emma Healey, Martyn Lewis, Elaine Nicholls, Clare Jinks, Valerie Tan, Andrew Finney, Christian D Mallen, on behalf of the ENHANCE Study Team O4 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 study Erik Lenguerrand, Graeme MacLennan, John Norrie, Siladitya Bhattacharya, Tim Draycott, on behalf of the Thistle group O5 Sample size calculation for longitudinal cluster randomised trials: a unified framework for closed cohort and repeated cross-section designs Richard Hooper, Steven Teerenstra, Esther de Hoop, Sandra Eldridge O6 Restricted randomisation schemes for stepped-wedge studies with a cluster-level covariate Alan Girling, Monica Taljaard O7 A flexible modelling of the time trend for the analysis of stepped wedge trials: results of a simulation study Gian Luca Di Tanna, Antonio Gasparrini P1 Tackling acute kidney injury – a UK stepped wedge clinical trial of hospital-level quality improvement interventions Anna Casula, Fergus Caskey, Erik Lenguerrand, Shona Methven, Stephanie MacNeill, Margaret May, Nicholas Selby P2 Sample size considerations for quantifying secondary bacterial transmission in a stepped wedge trial of influenza vaccine Leon Danon, Hannah Christensen, Adam Finn, Margaret May P3 Sample size calculation for time-to-event data in stepped wedge cluster randomised trials Fumihito Takanashi, Ada Keding, Simon Crouch, Mona Kanaan P4 Sample size calculations for stepped-wedge cluster randomised trials with unequal cluster sizes Caroline A. Kristunas, Karen L. Smith, Laura J. Gray P5 The design of stepped wedge trials with unequal cluster sizes John N.S. Matthews P6 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 Weir P7 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 Hayes S1 Stepped Wedge Designs with Multiple Interventions Vivian H Lyons, Lingyu Li, James Hughes, Ali Rowhani-Rahbar S2 Analysis of the cross-sectional stepped wedge cluster randomised trial Karla Hemming, Monica Taljaard, Andrew Forbe