Modulatory effects of pheromone on olfactory learning and memory in moths.

Pheromones are chemical communication signals known to elicit stereotyped behaviours and/or physiological processes in individuals of the same species, generally in relation to a specific function (e.g. mate finding in moths). However, recent research suggests that pheromones can modulate behaviours, which are not directly related to their usual function and thus potentially affect behavioural plasticity. To test this hypothesis, we studied the possible modulatory effects of pheromones on olfactory learning and memory in Agrotis ipsilon moths, which are well-established models to study sex-pheromones. To achieve this, sexually mature male moths were trained to associate an odour with either a reward (appetitive learning) or punishment (aversive learning) and olfactory memory was tested at medium- and long-term (1 h or 1.5 h, and 24 h). Our results show that male moths can learn to associate an odour with a sucrose reward, as well as a mild electric shock, and that olfactory memory persists over medium- and long-term range. Pheromones facilitated both appetitive and aversive olfactory learning: exposure to the conspecific sex-pheromone before conditioning enhanced appetitive but not aversive learning, while exposure to a sex-pheromone component of a heterospecific species (repellent) facilitated aversive but not appetitive learning. However, this effect was short-term, as medium- and long-term memory were not improved. Thus, in moths, pheromones can modulate olfactory learning and memory, indicating that they contribute to behavioural plasticity allowing optimization of the animal's behaviour under natural conditions. This might occur through an alteration of sensitization.

Interaction between cAMP and intracellular Ca(2+)-signaling pathways during odor-perception and adaptation in Drosophila.

Binding of an odorant to olfactory receptors triggers cascades of second messenger systems in olfactory receptor neurons (ORNs). Biochemical studies indicate that the transduction mechanism at ORNs is mediated by cyclic adenosine monophosphate (cAMP) and/or inositol,1,4,5-triphosphate (InsP3)-signaling pathways in an odorant-dependent manner. However, the interaction between these two second messenger systems during olfactory perception or adaptation processes is much less understood. Here, we used interfering-RNAi to disrupt the level of cAMP alone or in combination with the InsP3-signaling pathway cellular targets, InsP3 receptor (InsP3R) or ryanodine receptor (RyR) in ORNs, and quantify at ORN axon terminals in the antennal lobe, the odor-induced Ca(2+)-response. In-vivo functional bioluminescence Ca(2+)-imaging indicates that a single 5s application of an odor increased Ca(2+)-transients at ORN axon terminals. However, compared to wild-type controls, the magnitude and duration of ORN Ca(2+)-response was significantly diminished in cAMP-defective flies. In a behavioral assay, perception of odorants was defective in flies with a disrupted cAMP level suggesting that the ability of flies to correctly detect an odor depends on cAMP. Simultaneous disruption of cAMP level and InsP3R or RyR further diminished the magnitude and duration of ORN response to odorants and affected the flies' ability to detect an odor. In conclusion, this study provides functional evidence that cAMP and InsP3-signaling pathways act in synergy to mediate odor processing within the ORN axon terminals, which is encoded in the magnitude and duration of ORN response.

A microfluidic device to study neuronal and motor responses to acute chemical stimuli in zebrafish.

Zebrafish larva is a unique model for whole-brain functional imaging and to study sensory-motor integration in the vertebrate brain. To take full advantage of this system, one needs to design sensory environments that can mimic the complex spatiotemporal stimulus patterns experienced by the animal in natural conditions. We report on a novel open-ended microfluidic device that delivers pulses of chemical stimuli to agarose-restrained larvae with near-millisecond switching rate and unprecedented spatial and concentration accuracy and reproducibility. In combination with two-photon calcium imaging and recordings of tail movements, we found that stimuli of opposite hedonic values induced different circuit activity patterns. Moreover, by precisely controlling the duration of the stimulus (50-500 ms), we found that the probability of generating a gustatory-induced behavior is encoded by the number of neurons activated. This device may open new ways to dissect the neural-circuit principles underlying chemosensory perception.

In vivo functional calcium imaging of induced or spontaneous activity in the fly brain using a GFP-apoaequorin-based bioluminescent approach.

Different optical imaging techniques have been developed to study neuronal activity with the goal of deciphering the neural code underlying neurophysiological functions. Because of several constraints inherent in these techniques as well as difficulties interpreting the results, the majority of these studies have been dedicated more to sensory modalities than to the spontaneous activity of the central brain. Recently, a novel bioluminescence approach based on GFP-aequorin (GA) (GFP: Green fluorescent Protein), has been developed, allowing us to functionally record in-vivo neuronal activity. Taking advantage of the particular characteristics of GA, which does not require light excitation, we report that we can record induced and/or the spontaneous Ca(2+)-activity continuously over long periods. Targeting GA to the mushrooms-bodies (MBs), a structure implicated in learning/memory and sleep, we have shown that GA is sensitive enough to detect odor-induced Ca(2+)-activity in Kenyon cells (KCs). It has been possible to reveal two particular peaks of spontaneous activity during overnight recording in the MBs. Other peaks of spontaneous activity have been recorded in flies expressing GA pan-neurally. Similarly, expression in the glial cells has revealed that these cells exhibit a cell-autonomous Ca(2+)-activity. These results demonstrate that bioluminescence imaging is a useful tool for studying Ca(2+)-activity in neuronal and/or glial cells and for functional mapping of the neurophysiological processes in the fly brain. These findings provide a framework for investigating the biological meaning of spontaneous neuronal activity. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Presynaptic Ca2+ stores contribute to odor-induced responses in Drosophila olfactory receptor neurons.

In both vertebrates and invertebrates, olfactory receptor neurons (ORNs) respond to several odors. They also adapt to stimulus variations, and this is considered to be a simple form of non-associative learning and neuronal plasticity. Different mechanisms have been described to support neuronal and/or synaptic plasticity. For example in vertebrates, presynaptic Ca(2+) stores relying on either the ryanodine receptor (RyR) or the inositol (1,4,5)-trisphosphate receptor (InsP(3)R) have been reported to participate in synaptic transmission, in hippocampal pyramidal neurons, and in basket cell-Purkinje cell synapses. However, in invertebrates, especially in sensory neurons such as ORNs, similar mechanisms have not yet been detected. In this study, using Drosophila and taking advantage of an in vivo bioluminescence Ca(2+)-imaging technique in combination with genetic and pharmacological tools, first we show that the GFP-aequorin Ca(2+) sensor is sensitive enough to detect odor-induced responses of various durations. Second, we show that for a relatively long (5 s) odor application, odor-induced Ca(2+) responses occurring in the axon terminals of ORNs involve intracellular Ca(2+) stores. This response is decreased by specifically targeting InsP(3)R or RyR by RNAi, or application of the specific blockers thapsigargin or ryanodine, suggesting that Ca(2+) stores serve to amplify the presynaptic signal. Furthermore, we show that disrupting the intracellular Ca(2+) stores in the ORNs has functional consequences since InsP(3)R- or RyR-RNAi expressing flies were defective in olfactory behavior. Altogether, our results indicate that for long odor applications in Drosophila, the olfactory response depends on intracellular Ca(2+) stores within the axon terminals of the ORNs.

Changes of spine density and dendritic complexity in the prefrontal cortex in offspring of mothers exposed to stress during pregnancy.

Both chronic stress in adulthood and episodes of stress in the early postnatal period have been shown to interfere with neuronal development in limbic prefrontal cortical regions. The present study in rats showed for the first time that the development of layer II/III pyramidal neurons in the dorsal anterior cingulate (ACd) and orbitofrontal cortex (OFC) is significantly affected in offspring of mothers exposed to stress during pregnancy. In prenatally stressed (PS) male rat pups the ACd and OFC showed significantly lower spine densities on the apical dendrite (ACd, -20%; OFC, -25%), on basal dendrites reduced spine densities where found only in the OFC (-20% in PS males). Moreover, in both cortical areas a significant reduction of dendritic length was observed in PS males compared to control offspring, which was confined to the apical dendrites (ACd, -30%, OFC, -26%). Sholl analysis revealed that these alterations were accompanied by a significantly reduced complexity of the dendritic trees in both cortical regions. PS females displayed reductions of dendritic spine densities in the ACd and OFC on both the basal (ACd, -21%; OFC, -20%) and apical dendrites (ACd, -21%; OFC, -21%), however, in contrast to the findings in PS males, no dendritic atrophy was detected in the PS females. These findings demonstrate that gestational stress leads to significant alterations of prefrontal neuronal structure in the offspring of the stressed mothers in a sex-specific manner.