Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection^1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination^4-6, herbivory of food crops⁷, and transmission of disease^8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)^10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed ''GCMR'') from neurons in the antennal lobe (AL; the insect''s primary olfactory center)^13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna''s receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques^14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.     

Distinct Projections of Two Populations of Olfactory Receptor Axons in the Antennal Lobe of the Sphinx Moth Manduca sexta

The central projections of olfactory receptor cells associatedwith two distinct types of antennal sensilla in the sphinx mothManduca sexta were revealed by anterograde staining. In bothsexes, receptor axons that arise from sexually isomorphic, type-IItrichoid sensilla (and possibly some basiconic sensilla) projectto the spheroidal glomeruli in the ipsilateral antennal lobe.Each axon terminates in one glomerulus. Axons from a limitedregion of the antenna project to glomeruli throughout the lobe,arguing against strict topographic mapping of antennal receptorcells onto the array of glomeruli. Axons of sex-pheromone-selectivereceptor cells in the male-specific type-I trichoid sensillaproject exclusively to the sexually dimorphic macroglomerularcomplex (MGC). Axons from sensilla on the dorsal surface ofthe antenna are biased toward the medial MGC and those fromventral sensilla, toward the lateral MGC. Some receptor-cellaxons branch before reaching the MGC, but their terminals arealways confined to one of the two main glomerular divisionsof the MGC, the cumulus and toroid. These findings confirm thatprimary-afferent information about pheromonal and non-pheromonalodors is segregated in the antennal lobe and suggest that thereis a functional correspondence between particular olfactoryreceptor cells and specific glomeruli. Chem. Senses 20: 313–323,1995.     

Presynaptic Inhibition of Gamma Lobe Neurons Is Required for Olfactory Learning in Drosophila

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A Model for Pheromone Discrimination in the Insect Antennal Lobe: Investigation of the Role of Neuronal Response Pattern Complexity

Based on anatomical and physiological data pertaining to severalmoth species and the cockroach, we propose a neural model forpheromone discrimination in the insect antennal lobe. The modelexploits the variety of neuronal response patterns observedin the macroglomerulus, and predicts how these complex patternsof excitation and inhibition can participate in the discriminationof the species-specific pheromone blend. The model also allowsus to investigate the relationship between the distributionof observed response patterns and the neural organization fromwhich these patterns emerge. Chem. Senses 21: 19–27, 1996.     

Interactions of Carbon Dioxide and Food Odours in Drosophila: Olfactory Hedonics and Sensory Neuron Properties

Behavioural responses of animals to volatiles in their environment are generally dependent on context. Most natural odours are mixtures of components that can each induce different behaviours when presented on their own. We have investigated how a complex of two olfactory stimuli is evaluated by Drosophila flies in a free-flying two-trap choice assay and how these stimuli are encoded in olfactory receptor neurons. We first observed that volatiles from apple cider vinegar attracted flies while carbon dioxide (CO₂) was avoided, confirming their inherent positive and negative values. In contradiction with previous results obtained from walking flies in a four-field olfactometer, in the present assay the addition of CO₂ to vinegar increased rather than decreased the attractiveness of vinegar. This effect was female-specific even though males and females responded similarly to CO₂ and vinegar on their own. To test whether the female-specific behavioural response to the mixture correlated with a sexual dimorphism at the peripheral level we recorded from olfactory receptor neurons stimulated with vinegar, CO₂ and their combination. Responses to vinegar were obtained from three neuron classes, two of them housed with the CO₂-responsive neuron in ab1 sensilla. Sensitivity of these neurons to both CO₂ and vinegar per se did not differ between males and females and responses from female neurons did not change when CO₂ and vinegar were presented simultaneously. We also found that CO₂-sensitive neurons are particularly well adapted to respond rapidly to small concentration changes irrespective of background CO₂ levels. The ability to encode temporal properties of stimulations differs considerably between CO₂- and vinegar-sensitive neurons. These properties may have important implications for in-flight navigation when rapid responses to fragmented odour plumes are crucial to locate odour sources. However, the flies’ sex-specific response to the CO₂-vinegar combination and the context-dependent hedonics most likely originate from central rather than peripheral processing.     

Olfactory Coding in Antennal Neurons of the Malaria Mosquito, Anopheles gambiae

Olfactory receptor neurons (ORNs) in the antenna of insects serve to encode odors in action potential activity conducted to the olfactory lobe of the deuterocerebrum. We performed an analysis of the electrophysiological responses of olfactory neurons in the antennae of the female malaria mosquito Anopheles gambiae s.s. and investigated the effect of blood feeding on responsiveness. Forty-four chemicals that are known to be present in human volatile emanations were used as odor stimuli. We identified 6 functional types of trichoid sensilla and 5 functional types of grooved-peg sensilla (GP) based on a hierarchical cluster analysis. Generalist ORNs, tuned to a broad range of odors, moderate specialist ORNs and 2 ORNs tuned to only one odor were identified in different sensilla types. Neurons in GP were tuned to more polar compounds including the important behavioral attractant ammonia and its synergist L-lactic acid, responses to which were found only in GP. Combinatorial coding is the most plausible principle operating in the olfactory system of this mosquito species. We document for the first time both up- and downregulation of ORN responsiveness after blood feeding. Modulation of host-seeking and oviposition behavior is associated with both qualitative and quantitative changes in the peripheral sensory system.     

Maternal-Zygotic Gene Interactions during Formation of the Dorsoventral Pattern in Drosophila Embryos

Maternal-zygotic interactions involving the three genes dorsal (dl), twist (twi) and snail (sna) are described. The results suggest that all three are involved in the process by which the dorsoventral pattern of the Drosophila embryo is established. First, the lethal embryonic mutant phenotypes are rather similar. In homozygous twi or sna embryos invagination of the ventral presumptive mesodermal cells fails to occur, and the resulting embryos are devoid of internal organs. This is very similar to the dominant phenotype described for dl; in the case of dl, however, the effect is a maternal one dependent on the mutant genotype of the female. Second, a synergistic interaction has been found whereby dominant lethality of twi- or sna-bearing zygotes is observed in embryos derived from heterozygous dl females at high temperature. The temperature sensitivity of this interaction permitted definition of a temperature-sensitive period which is probably that of dl. This was found to extend from approximately 12 hr prior to oviposition to 2–3 hr of embryogenesis. A zygotic action for the dl gene in addition to the maternal effect was revealed by the finding that extra doses of dl⁺ in the zygotes can partially rescue the dominant lethality of heterozygous twi embryos derived from heterozygous dl females. Two possible interpretations of the synergism are considered: (1) twi and sna are activated in the embryos as a result of positional signals placed in the egg as a consequence of the functioning of the dl gene during oogenesis and, thus, play a role in embryonic determination. (2) The gene products of dl⁺ and twi ⁺ (or sna⁺) combine to produce a functional molecule that is involved in the specification of dorsoventral pattern in the early embryo.     

Germ Layer Interactions in Pattern Formation of Amphibian Mesoderm during Primary Embryonic Induction

Mesodermal differentiation of dorsal marginal zone (DMZ) before and after invagination was analyzed by a series of combination experiments with different kinds of ectoderm.
Lower DMZ of early gastrula didn't show any axial-mesoderm (notochord and somitic mesoderm) but lateral mesoderm (mesenchyme, mesothelium, or blood cells) in combinant with non-competent ventral ectoderm, while combinant with competent ectoderm was found to have well-differentiated axial-mesoderm with deutero-spinocaudal neurals. The axial-mesoderms have origin in the ectoderm. Uninvaginated DMZ of middle gastrula also showed difference in mesodermal differentiation between competent and non-competent ectoderms; axial-mesoderm differentiation was much better in competent than in non-competent. The axial-mesoderm originated from the uninvaginated DMZ. Archenteron roof of late gastrula showed regional difference in mesodermal differentiation in both combinants with competent and non-competent. The present study further demonstrated that there was regionality in promoting effect of induced neurectoderm on axial-mesoderm differentiation of invaginated archenteron roof.
The present experiments suggest that the cranio-caudal and dorso-ventral axis formations of amphibian mesoderm are finally determined by sequential and reciprocal interactions between the mesodermal anlage and the overlying ectoderm. It should be also shown that lower DMZ acts to trigger a series of the sequential interactions during primary embryonic induction.     

Altered Functional Connectivity and Small-World in Mesial Temporal Lobe Epilepsy

Background

The functional architecture of the human brain has been extensively described in terms of functional connectivity networks, detected from the low–frequency coherent neuronal fluctuations that can be observed in a resting state condition. Little is known, so far, about the changes in functional connectivity and in the topological properties of functional networks, associated with different brain diseases.

Methodology/Principal Findings

In this study, we investigated alterations related to mesial temporal lobe epilepsy (mTLE), using resting state functional magnetic resonance imaging on 18 mTLE patients and 27 healthy controls. Functional connectivity among 90 cortical and subcortical regions was measured by temporal correlation. The related values were analyzed to construct a set of undirected graphs. Compared to controls, mTLE patients showed significantly increased connectivity within the medial temporal lobes, but also significantly decreased connectivity within the frontal and parietal lobes, and between frontal and parietal lobes. Our findings demonstrated that a large number of areas in the default-mode network of mTLE patients showed a significantly decreased number of connections to other regions. Furthermore, we observed altered small-world properties in patients, along with smaller degree of connectivity, increased n-to-1 connectivity, smaller absolute clustering coefficients and shorter absolute path length.

Conclusions/Significance

We suggest that the mTLE alterations observed in functional connectivity and topological properties may be used to define tentative disease markers.     

Olfactory response and resource utilization in Drosophila: interspecific comparisons

Olfactory response and resource utilization in Drosophila were compared among three domestic (D. melanogaster, D. simulans, D. immigrans) and one Australian endemic (D. lativittata) species. Olfactory response was measured in a choice type olfactometer (Fuyama, 1976). The following chemicals common in Drosophila resources were used as odourants: acetaldehyde, acetic acid, propionic acid, methanol, ethanol, n-propanol, isopropanol, n-butanol. Resource status of these chemicals was determined either from the literature or by adult longevity tests.
All species were attracted by acetaldehyde, while methanol, isopropanol and n-butanol were unattractive. Ethanol attracted all species except D. immigrans , while only D. lativittata and D. melanogaster were attracted to n-propanol, propionic acid and acetic acid
Methanol and isopropanol were not utilized as resources by any of the species, while D. melanogaster and D. lativittata showed greater utilization/tolerance of the other chemicals. Some correlation between resource utilization and olfactory response was found at the interspecific level, although not all chemicals utilized as resources are attractants. The adaptive significance of the interspecific variation in olfactory response is discussed, especially in relation to habitats selected. The results provide suggestions for habitat selection studies at the intraspecific level.     

Representation of Primary Plant Odorants in the Antennal Lobe of the Moth Heliothis virescens Using Calcium Imaging

Chemical Senses, 29,     

Genetic Analysis of Two Female-Sterile Loci Affecting Eggshell Integrity and Embryonic Pattern Formation in Drosophila Melanogaster

We have analyzed female-sterile mutations at the X-linked loci fs(1)Nas and fs(1)ph which show allele-specific effects on egg shell structure and embryonic pattern formation. The majority of mutant alleles at both loci lead to a collapsed egg phenotype. The maternal effect lethal phenotype is characterized by cuticle defects resembling those found in three autosomal mutants of the terminal class. We have analyzed the complementation behavior of various heteroallelic combinations at both loci and show that one such combination at the fs(1)Nas locus is capable of restoring normal fertility. We have investigated possible interactions between fs(1)Nas and fs(1)ph and also between the terminal allele of fs(1)Nas and various maternal effect mutations altering the anteroposterior polarity of embryos. We have isolated one new allele of fs(1)Nas which combines the locus-typical phenotypic features with novel cuticle phenotypes. Our results suggest that the products of fs(1)Nas and fs(1)ph are required for the stability of the vitelline membrane and are also involved in a morphogenetic pathway necessary for the correct differentiation of the terminal regions of the embryo. Possible mechanisms to account for the association of these two functions are discussed.     

Temporal Features of Spike Trains in the Moth Antennal Lobe Revealed by a Comparative Time-Frequency Analysis

The discrimination of complex sensory stimuli in a noisy environment is an immense computational task. Sensory systems often encode stimulus features in a spatiotemporal fashion through the complex firing patterns of individual neurons. To identify these temporal features, we have developed an analysis that allows the comparison of statistically significant features of spike trains localized over multiple scales of time-frequency resolution. Our approach provides an original way to utilize the discrete wavelet transform to process instantaneous rate functions derived from spike trains, and select relevant wavelet coefficients through statistical analysis. Our method uncovered localized features within olfactory projection neuron (PN) responses in the moth antennal lobe coding for the presence of an odor mixture and the concentration of single component odorants, but not for compound identities. We found that odor mixtures evoked earlier responses in biphasic response type PNs compared to single components, which led to differences in the instantaneous firing rate functions with their signal power spread across multiple frequency bands (ranging from 0 to 45.71 Hz) during a time window immediately preceding behavioral response latencies observed in insects. Odor concentrations were coded in excited response type PNs both in low frequency band differences (2.86 to 5.71 Hz) during the stimulus and in the odor trace after stimulus offset in low (0 to 2.86 Hz) and high (22.86 to 45.71 Hz) frequency bands. These high frequency differences in both types of PNs could have particular relevance for recruiting cellular activity in higher brain centers such as mushroom body Kenyon cells. In contrast, neurons in the specialized pheromone-responsive area of the moth antennal lobe exhibited few stimulus-dependent differences in temporal response features. These results provide interesting insights on early insect olfactory processing and introduce a novel comparative approach for spike train analysis applicable to a variety of neuronal data sets.     

Model for Transition from Waves to Synchrony in the Olfactory Lobe of Limax

A biophysical model for the interactions between bursting (B) cells and nonbursting (NB) cells in the procerebral lobe of Limax is developed and tested. Phase-sensitivity of the NB cells is exhibited due to the strong inhibition from the rhythmically bursting B cells. Electrical and chemical junctions coupled with a parameter gradient lead to sustained periodic waves in the lobe. Excitatory interactions between the NB cells, which rarely fire, lead to stimulus evoked synchrony in the lobe oscillations. A novel calcium current is suggested to explain the effects of nitric oxide (NO) on the lobe. Gap junctions are shown both experimentally and through simulations to be required for the oscillating field potentials.     

Embryonic origin and differentiation of the Drosophila heart

We have followed the normal development of the different cell types associated with the Drosophila dorsal vessel, i.e. cardioblasts, pericardial cells, alary muscles, lymph gland and ring gland, by using several tissue-specific markers and transmission electron microscopy. Precursors of pericardial cells and cardioblasts split as two longitudinal rows of cells from the lateral mesoderm of segments T2-A7 (cardiogenic region) during stage 12. The lymph gland and dorsal part of the ring gland (corpus allatum) originate from clusters of lateral mesodermal cells located in T3 and T1/dorsal ridge, respectively. Cardioblast precursors are strictly segmentally organized; each of T2-A6 gives rise to six cardioblasts. While moving dorsally during the stages leading up to dorsal closure, cardioblast precursors become flattened, polarized cells aligned in a regular longitudinal row. At dorsal closure, the leading edges of the cardioblast precursors meet their contralateral counterparts. The lumen of the dorsal vessel is formed when the trailing edges of the cardioblast precursors of either side bend around and contact each other. The amnioserosa invaginates during dorsal closure and is transiently attached to the cardioblasts; however, it does not contribute to the cells associated with the dorsal vessel and degenerates during late embryogenesis. We describe ultrastructural characteristics of cardioblast differentiation and discuss similarities between cardioblast development and capillary differentiation in vertebrates. Correspondence to: V. Hartenstein     

Olfactory coding: connecting odorant receptor expression and behavior in the Drosophila larva

The discovery of odorant receptors has significantly changed our understanding of how animals identify thousands of odorants. A recent study has shed new light on the central issue of how odor information is translated into meaningful behavior.     

Olfactory map formation in the Drosophila brain: genetic specificity and neuronal variability

The development of the Drosophila olfactory system is a striking example of how genetic programs specify a large number of different neuron types and assemble them into functional circuits. To ensure precise odorant perception, each sensory neuron has to not only select a single olfactory receptor (OR) type out of a large genomic repertoire but also segregate its synaptic connections in the brain according to the OR class identity. Specification and patterning of second-order interneurons in the olfactory brain center occur largely independent of sensory input, followed by a precise point-to-point matching of sensory and relay neurons. Here we describe recent progress in the understanding of how cell-intrinsic differentiation programs and context-dependent cellular interactions generate a stereotyped sensory map in the Drosophila brain. Recent findings revealed an astonishing morphological diversity among members of the same interneuron class, suggesting an unexpected variability in local microcircuits involved in insect sensory processing.     

Nitric Oxide Has Differential Effects on Currents in Different Subsets of Manduca sexta Antennal Lobe Neurons

Nitric oxide has been shown to regulate many biological systems including olfaction. In the moth olfactory system nitric oxide is produced in the antennal lobe in response to odor stimulation and has complex effects on the activity of both projection neurons and local interneurons. To examine the cell autonomous effects of nitric oxide on these cells, we used patch-clamp recording in conjunction with pharmacological manipulation of nitric oxide to test the hypothesis that nitric oxide differentially regulates the channel properties of these different antennal lobe neuron subsets. We found that nitric oxide caused increasing inward currents in a subset of projection neurons while the effects on local neurons were variable but consistent within identifiable morphological subtypes.