An evaluation of hedonic responses in taste-potentiated odor aversion using the analysis of licking microstructure and orofacial reactivity.

Two experiments examined the hedonic responses conditioned to odor cues in the phenomenon of taste-potentiated odor aversion. Experiment 1 analyzed the microstructure of licking behavior during voluntary consumption. A tasteless odor (amyl acetate) was delivered to rats either diluted in water or mixed with saccharin before being injected with LiCl. At test, subjects which had received the odor-taste compound during conditioning showed both lower odor consumption and lick cluster size, a result indicating an increased negative evaluation of the odor. Experiment 2 examined the orofacial reactions elicited by the odor as index of its hedonic impact. During conditioning, the rats were intraorally infused with either the odor alone or the odor-saccharin compound before being injected with LiCl. At test, they were infused with the odor and their orofacial responses video recorded. More aversive orofacial responses were elicited by the odor cue in rats that had compound conditioning, again a result indicating a strengthened negative hedonic reactivity compared to animals experiencing odor aversion conditioning alone. Taken together, these results indicate that taste-mediated potentiation of odor aversion conditioning impacts on the acquisition of conditioned hedonic reactions as well as consumption.

Neuron-glia interaction at the receptor level affects olfactory perception in adult Drosophila.

Some types of glia play an active role in neuronal signaling by modifying their activity although little is known about their role in sensory information signaling at the receptor level. In this research, we report a functional role for the glia that surround the soma of the olfactory receptor neurons (OSNs) in adult Drosophila. Specific genetic modifications have been targeted to this cell type to obtain live individuals who are tested for olfactory preference and display changes both increasing and reducing sensitivity. A closer look at the antenna by Ca2+ imaging shows that odor activates the OSNs, which subsequently produce an opposite and smaller effect in the glia that partially counterbalances neuronal activation. Therefore, these glia may play a dual role in preventing excessive activation of the OSNs at high odorant concentrations and tuning the chemosensory window for the individual according to the network structure in the receptor organ.

The Ntan1 gene is expressed in perineural glia and neurons of adult Drosophila.

The Drosophila Ntan1 gene encodes an N-terminal asparagine amidohydrolase that we show is highly conserved throughout evolution. Protein isoforms share more than 72% of similarity with their human counterparts. At the cellular level, this gene regulates the type of glial cell growth in Drosophila larvae by its different expression levels. The Drosophila Ntan1 gene has 4 transcripts that encode 2 protein isoforms. Here we describe that although this gene is expressed at all developmental stages and adult organs tested (eye, antennae and brain) there are some transcript-dependent specificities. Therefore, both quantitative and qualitative cues could account for gene function. However, widespread developmental stage and organ-dependent expression could be masking cell-specific constraints that can be explored in Drosophila by using Gal4 drivers. We report a new genetic driver within this gene, Mz317-Gal4, that recapitulates the Ntan1 gene expression pattern in adults. It shows specific expression for perineural glia in the olfactory organs but mixed expression with some neurons in the adult brain. Memory and social behavior disturbances in mice and cancer and schizophrenia in humans have been linked to the Ntan1 gene. Therefore, these new tools in Drosophila may contribute to our understanding of the cellular basis of these alterations.

Validation of an Optogenetic Approach to the Study of Olfactory Behavior in the T-Maze of Drosophila melanogaster Adults.

Optogenetics enables the alteration of neural activity using genetically targeted expression of light activated proteins for studying behavioral circuits in several species including Drosophila. The main idea behind this approach is to replace the native behavioral stimulus by the light-induced electrical activation of different points of the circuit. Therefore, its effects on subsequent steps of the circuit or on the final behavior can be analyzed. However, the use of optogenetics to dissect the receptor elements of the adult olfactory behavior presents a challenge due to one additional factor: Most odorants elicit attraction or avoidance depending on their concentration; this complicates the representative replacement of odor activation of olfactory sensory neurons (OSNs) by light. Here, we explore a dual excitation model where the subject is responding to odors while the OSNs are optogenetically activated. Thereby, we can assess if and how the olfactory behavior is modified. We measure the effects of light excitation on the response to several odorant concentrations. The dose-response curve of these flies still depends on odor concentration but with reduced sensitivity compared to olfactory stimulation alone. These results are consistent with behavioral tests performed with a background odor and suggest an additive effect of light and odor excitation on OSNs.

The Two Main Olfactory Receptor Families in Drosophila, ORs and IRs: A Comparative Approach.

Most insect species rely on the detection of olfactory cues for critical behaviors for the survival of the species, e.g., finding food, suitable mates and appropriate egg-laying sites. Although insects show a diverse array of molecular receptors dedicated to the detection of sensory cues, two main types of molecular receptors have been described as responsible for olfactory reception in Drosophila, the odorant receptors (ORs) and the ionotropic receptors (IRs). Although both receptor families share the role of being the first chemosensors in the insect olfactory system, they show distinct evolutionary origins and several distinct structural and functional characteristics. While ORs are seven-transmembrane-domain receptor proteins, IRs are related to the ionotropic glutamate receptor (iGluR) family. Both types of receptors are expressed on the olfactory sensory neurons (OSNs) of the main olfactory organ, the antenna, but they are housed in different types of sensilla, IRs in coeloconic sensilla and ORs in basiconic and trichoid sensilla. More importantly, from the functional point of view, they display different odorant specificity profiles. Research advances in the last decade have improved our understanding of the molecular basis, evolution and functional roles of these two families, but there are still controversies and unsolved key questions that remain to be answered. Here, we present an updated review on the advances of the genetic basis, evolution, structure, functional response and regulation of both types of chemosensory receptors. We use a comparative approach to highlight the similarities and differences among them. Moreover, we will discuss major open questions in the field of olfactory reception in insects. A comprehensive analysis of the structural and functional convergence and divergence of both types of receptors will help in elucidating the molecular basis of the function and regulation of chemoreception in insects.

Novel genetic approaches to behavior in Drosophila.

The study of behavior requires manipulation of the controlling neural circuits. The fruit fly, Drosophila melanogaster, is an ideal model for studying behavior because of its relatively small brain and the numerous sophisticated genetic tools that have been developed for this animal. Relatively recent technical advances allow the manipulation of a small subset of neurons with temporal resolution in flies while they are subject to behavior assays. This review briefly describes the most important genetic techniques, reagents, and approaches that are available to study and manipulate the neural circuits involved in Drosophila behavior. We also describe some examples of these genetic tools in the study of the olfactory receptor system.

Formation and function of intracardiac valve cells in the Drosophila heart.

Drosophila harbours a simple tubular heart that ensures haemolymph circulation within the body. The heart is built by a few different cell types, including cardiomyocytes that define the luminal heart channel and ostia cells that constitute openings in the heart wall allowing haemolymph to enter the heart chamber. Regulation of flow directionality within a tube, such as blood flow in arteries or insect haemolymph within the heart lumen, requires a dedicated gate, valve or flap-like structure that prevents backflow of fluids. In the Drosophila heart, intracardiac valves provide this directionality of haemolymph streaming, with one valve being present in larvae and three valves in the adult fly. Each valve is built by two specialised cardiomyocytes that exhibit a unique histology. We found that the capacity to open and close the heart lumen relies on a unique myofibrillar setting as well as on the presence of large membranous vesicles. These vesicles are of endocytic origin and probably represent unique organelles of valve cells. Moreover, we characterised the working mode of the cells in real time. Valve cells exhibit a highly flexible shape and, during each heartbeat, oscillating shape changes result in closing and opening of the heart channel. Finally, we identified a set of novel valve cell markers useful for future in-depth analyses of cell differentiation in wild-type and mutant animals.

Measuring activity in olfactory receptor neurons in Drosophila: Focus on spike amplitude.

Olfactory responses at the receptor level have been thoroughly described in Drosophila melanogaster by electrophysiological methods. Single sensilla recordings (SSRs) measure neuronal activity in intact individuals in response to odors. For sensilla that contain more than one olfactory receptor neuron (ORN), their different spontaneous spike amplitudes can distinguish each signal under resting conditions. However, activity is mainly described by spike frequency. Some reports on ORN response dynamics studied two components in the olfactory responses of ORNs: a fast component that is reflected by the spike frequency and a slow component that is observed in the LFP (local field potential, the single sensillum counterpart of the electroantennogram, EAG). However, no apparent correlation was found between the two elements. In this report, we show that odorant stimulation produces two different effects in the fast component, affecting spike frequency and spike amplitude. Spike amplitude clearly diminishes at the beginning of a response, but it recovers more slowly than spike frequency after stimulus cessation, suggesting that ORNs return to resting conditions long after they recover a normal spontaneous spike frequency. Moreover, spike amplitude recovery follows the same kinetics as the slow voltage component measured by the LFP, suggesting that both measures are connected. These results were obtained in ab2 and ab3 sensilla in response to two odors at different concentrations. Both spike amplitude and LFP kinetics depend on odorant, concentration and neuron, suggesting that like the EAG they may reflect olfactory information.

Elements of olfactory reception in adult Drosophila melanogaster.

The olfactory system of Drosophila has become an attractive and simple model to investigate olfaction because it follows the same organizational principles of vertebrates, and the results can be directly applied to other insects with economic and sanitary relevance. Here, we review the structural elements of the Drosophila olfactory reception organs at the level of the cells and molecules involved. This article is intended to reflect the structural basis underlying the functional variability of the detection of an olfactory universe composed of thousands of odors. At the genetic level, we further detail the genes and transcription factors (TF) that determine the structural variability. The fly's olfactory receptor organs are the third antennal segments and the maxillary palps, which are covered with sensory hairs called sensilla. These sensilla house the odorant receptor neurons (ORNs) that express one or few odorant receptors in a stereotyped pattern regulated by combinations of TF. Also, perireceptor events, such as odor molecules transport to their receptors, are carried out by odorant binding proteins. In addition, the rapid odorant inactivation to preclude saturation of the system occurs by biotransformation and detoxification enzymes. These additional events take place in the lymph that surrounds the ORNs. We include some data on ionotropic and metabotropic olfactory transduction, although this issue is still under debate in Drosophila.

Transcriptional basis of the acclimation to high environmental temperature at the olfactory receptor organs of Drosophila melanogaster.

BACKGROUND: Environmental temperature directly affects the concentrations of chemicals in the gas phase. Therefore, if the olfactory system does not physiologically adapt to environmental conditions, it may provide inadequate information about the distance to or direction of odor sources. Previous reports have shown at the behavioral level that temperature induces changes in olfactory sensitivity in Drosophila melanogaster. These changes are initiated in the main olfactory receptor organs, the antennae. In this work, we attempted to identify the particular genes responsible for olfactory adaptation to increasing temperatures in these organs based on current knowledge of the molecular basis of olfactory reception. RESULTS: Whole-genome transcriptional responses to transitory temperature shifts from 21-30°C were analyzed in the third antennal segments of Drosophila. More than 53% of the genome was expressed in these organs; this percentage increased slightly (55%) after heat treatment. However, the expression levels increased for 26%, decreased for 21% and remained constant for 53% of the expressed genes. Analysis of the changes produced in 389 genes related to heat response and olfactory reception, according to the current functional annotations of the Drosophila gene set, showed significant differences in 95 of these genes, which are involved in the heat response (23), perireceptor events in olfaction (50), olfactory and gustatory receptors (18) and G-proteins and transduction cascades (4). CONCLUSIONS: Gene expression was altered in response to environmental heat in the antennae of Drosophila by increasing or decreasing expression. Different acclimation patterns emerged for reception through the basiconic, trichoid and coeloconic sensilla. Changes in genes with a central role in olfactory reception, such as orco, may account for part of the acclimation reported at the behavioral level.

Environmental temperature modulates olfactory reception in Drosophila melanogaster.

Sensory systems, including the olfactory system, are able to adapt to changing environmental conditions. In nature, changes in temperature modify the volatility and concentration of odorants in the air. If the olfactory system does not adapt to these changes, it could relay wrong information about the distance to or direction of odor sources. Recent behavioral studies in Drosophila melanogaster showed olfactory acclimation to temperature. In this report, we investigated if temperature affects olfaction at the level of the receptors themselves. With this aim, we performed electroantennograms (EAGs) and single sensillum recordings (SSRs) to measure the response to several odorants in flies that had been submitted to temperature treatments. In response to all tested odorants, the amplitude of the EAGs increased in flies that had been exposed to a higher temperature and decreased after cold treatment, revealing that at least part of the reported change in olfactory perception happens at reception level. SSRs of odorant stimulated basiconic sensilla ab2 and ab3 showed some changes in the number of spikes after heat or cold treatment. However, the number and shape of spontaneous action potentials were unaffected, suggesting that the observed changes related specifically to the olfactory function of the neurons.

Expression analysis of the 3 G-protein subunits, Galpha, Gbeta, and Ggamma, in the olfactory receptor organs of adult Drosophila melanogaster.

In many species, olfactory transduction is triggered by odorant molecules that interact with olfactory receptors coupled to heterotrimeric G-proteins. The role of G-protein-linked transduction in the olfaction of Drosophila is currently under study. Here, we supply a thorough description of the expression in the olfactory receptor organs (antennae and maxillary palps) of all known Drosophila melanogaster genes that encode for G-proteins. Using RT-polymerase chain reaction, we analyzed 6 Galpha (G(s), G(i), G(q), G(o), G(f), and concertina), 3 Gbeta (G(beta5), G(beta13F), and G(beta76C)), and 2 Ggamma genes (G(gamma1) and G(gamma30A)). We found that all Galpha protein-encoding genes showed expression in both olfactory organs, but G(f) mRNA was not detected in palps. Moreover, all the Gbeta and Ggamma genes are expressed in antennae and palps, except for G(beta76C). To gain insight into the hypothesis of different G-protein subunits mediating differential signaling in olfactory receptor neurons (ORNs), we performed immunohistochemical studies to observe the expression of several Galpha and Gbeta proteins. We found that Gs, Gi, Gq, and G(beta13F) subunits displayed generalized expression in the antennal tissue, including ORNs support cells and glial cells. Finally, complete coexpression was found between Gi and Gq, which are mediators of the cyclic adenosine monophosphate and IP3 transduction cascades, respectively.

The effect of environmental temperature on olfactory perception in Drosophila melanogaster.

Olfaction provides chemical information to an animal about its environment. When environmental conditions change, individuals should be able to adequately maintain function. Temperature may influence olfaction in a double manner, as it modifies the concentrations of gaseous compounds and affects biological processes. Here, we address acclimatization to environmental temperature in the olfactory system of Drosophila melanogaster using heat and cold treatments. Because the consequences of temperature shifts persist for some time after the treatment's end, comparison of olfactory behaviors at the same temperature in treated and untreated flies allows us to infer the biological effects of temperature in olfaction. At intermediate odorant concentrations heat always generates a reduction of olfactory sensitivity, as they would be expected to compensate for the increase of volatiles in the air. Cold produces the opposite effect. These changes are observed in both sexes and in natural populations as well as in standard laboratory stocks. Short applications suffice to cause detectable olfactory perception changes, but even prolonged temperature treatments have only a transitory effect. Together, these results suggest that olfaction in Drosophila underlies acclimatization to environmental temperature. However, sensitivity changes are not immediate and may cause imperfect adjustment of olfactory function for short time periods.

Quantitative analysis of antennal mosaic generation in Drosophila melanogaster by the MARCM system.

Mosaics have been used in Drosophila to study development and to generate mutant structures when a mutant allele is homozygous lethal. New approaches of directed somatic recombination based on FRT/FLP methods, have increased mosaicism rates but likewise multiple clones in the same individual appeared more frequently. Production of single clones could be essential for developmental studies; however, for cell-autonomous gene function studies only the presence of homozygous cells for the target recessive allele is relevant. Herein, we report the number and extension of antennal mosaics generated by the MARCM system at different ages. This information is directed to obtain the appropriated mosaic type for the intended application. By applying heat shock at 10 different developmental stages from 0-12 h to 6-7 days after egg laying, more than 50% of mosaics were obtained from 5,028 adults. Single recombinant clones appeared mainly at early stages while massive recombinant areas were observed with late treatments.

The rdgB gene of Drosophila: a link between vision and olfaction.

otal (ota=olfactory trap abnormal), an X-linked mutation of Drosophila isolated by virtue of abnormal olfactory behavior, is shown to be an allele of rdgB (retinal degeneration B), a gene required for normal visual system physiology. rdgB function is shown to be necessary for olfactory response of both adult files and larvae, which have distinct olfactory systems. Electrophysiological recordings from the adult antenna indicate that rdgB is required for normal response in the peripheral olfactory system: some rdgB mutants show a delayed return to the resting potential following stimulation with ethyl acetate vapor. These results indicate that rdgB is required for both visual and olfactory physiology, and they suggest that visual and olfactory transduction may share at least one common molecular step in Drosophila.

High efficiency of a double-screening method on single P-element insertion lines to identify quantitative trait mutants in Drosophila melanogaster.

Enhancer trap P-element insertion has become a common method for generating new mutations in Drosophila melanogaster. When this method is used to isolate mutants for quantitative traits, an appropriate control must be established to define normal and mutant phenotypes. Considering that enhancer-trap lines are generated by crossing several strains, usually with no homogeneous genetic background, no clear control strain can be selected. Previous reports tried to overcome this problem by homogenizing the genetic background of the original lines. However, this is not the most common scenario, especially when functional phenotypes are studied in previously generated lines. Without such caution, is it possible to identify functional mutants among P-element insertion lines? We tested this for olfactory preference, a quantitative trait. Using as control measurement the average phenotype of 30 simultaneously generated P-element insertion lines with preferential reporter-gene expression in olfactory reception organs, we found that 25 of the lines exhibited mutant phenotypes in response to one or several of 5 tested odorants. Additional tests showed that the efficiency of the method for detecting olfactory mutations exceeded 60% even for such a small number of tested odorants. According to these results this approach greatly facilitates the identification of putative abnormal phenotypes, which must be extensively confirmed afterwards.

The Inositol 1,4,5-triphosphate kinase1 gene affects olfactory reception in Drosophila melanogaster.

The Inositol 1,4,5-triphosphate (IP3) route is one of the two main transduction cascades that mediate olfactory reception in Drosophila melanogaster. The activity of IP3 kinase1 reduces the levels of this substrate by phosphorylation into inositol 1,3,4,5-tetrakiphosphate (IP4). We show here that the gene is expressed in olfactory sensory organs as well as in the rest of the head. To evaluate in vivo the olfactory functional effects of up-regulating IP3K1, individuals with directed genetic changes at the reception level only were generated using the UAS/Gal4 method. In this report, we described the consequences in olfactory perception of overexpressing the IP3Kinase1 gene at eight different olfactory receptor-neuron subsets. Six out of the eight studied Gal-4/UAS-IP3K1 hybrids displayed abnormal behavioral responses to ethyl acetate, acetone, ethanol or propionaldehyde. Specific behavioral defects corresponded to the particular neuronal olfactory profile. These data confirm the role of the IP3kinase1 gene, and consequently the IP3 transduction cascade, in mediating olfactory information at the reception level.

Altered endogenous activation of CREB in the suprachiasmatic nucleus of mice with retinal degeneration.

The effect of cone- and rod-cell loss on the activation of transcription factor CREB (by phosphorylation at Ser133) was examined in the pacemaker of mammals, the suprachiasmatic nucleus (SCN). For this purpose, brain sections of rd/rd and wild-type C3H mice were immunolabeled with a polyclonal antibody that recognises p-CREB, i.e., the activated form of the protein. Both rd/rd and wild-type mice maintained in constant darkness showed a circadian variation of p-CREB nuclear staining: the number of immunopositive nuclear pixels at the subjective night was higher than the one observed at the subjective day. However, some differences were detected between both groups: (1) p-CREB immunolabelling in the SCN of rd/rd mice was significantly reduced throughout the 24-h cycle; (2) the time in which the activation of CREB begins to increase at the subjective night in these mice is delayed with regard to wild-type mice. When a light stimulus was given at the subjective night p-CREB immunostaining significantly increased in the SCN of both rd/rd and wild-type mice when compared to basal levels, while no significant effect was found when the stimulus was given at the subjective day. Taken together, our results suggest that despite lower levels of p-CREB, indicating that something is altered in the SCN of rd/rd mice, the main mechanisms of the clock (e.g., circadian oscillation, readjustment by light) are still fully functional in these mice. The present study supports the idea that the CREB/CRE pathway is a component of the circadian clock molecular mechanism.