C. Giovanni Galizia

The glomerular code for odor representation is species specific in the honeybee Apis mellifera

Odors are coded by glomerular activity patterns in the insect antennal lobe (AL) and in the mammalian olfactory bulb. We measured glomerular responses to 30 different odors in the AL of honeybees using calcium-sensitive dyes. By subsequently staining glomeruli and identifying individual glomerular outlines, we were able to compare the patterns between animals. Regardless of whether the odors were mixtures or pure substances, environmental odors or pheromones, their representations were highly conserved among individuals. Therefore, it may be possible to create a functional atlas of the AL in which particular molecular receptive ranges are attributed to each glomerulus.

Parallel Olfactory Systems in Insects: Anatomy and Function

A striking commonality across insects and vertebrates is the recurring presence of parallel olfactory subsystems, suggesting that such an organization has a highly adaptive value. Conceptually, two different categories of parallel systems must be distinguished. In one, specific sensory organs or processing streams analyze different chemical stimuli (segregate parallel systems). In the other, similar odor stimuli are processed but analyzed with respect to different features (dual parallel systems). Insects offer many examples for both categories. For example, segregate parallel systems for different chemical stimuli are realized in specialized neuronal streams for processing sex pheromones and CO(2). Dual parallel streams related to similar or overlapping odor stimuli are prominent in Hymenoptera. Here, a clear separation of sensory tracts to higher-order brain centers is present despite no apparent differences regarding the classes or categories of olfactory stimuli being processed. In this paper, we review the situation across insect species and offer hypotheses for the function and evolution of parallel olfactory systems.

Genetically Expressed Cameleon in Drosophila melanogaster Is Used to Visualize Olfactory Information in Projection Neurons

Complex external stimuli such as odorants are believed to be internally represented in the brain by spatiotemporal activity patterns of extensive neuronal ensembles. These activity patterns can be recorded by optical imaging techniques. However, optical imaging with conventional fluorescence dyes usually does not allow for resolving the activity of biologically defined groups of neurons. Therefore, specifically targeting reporter molecules to neuron populations of common genetic identity is an important goal. We report the use of the genetically encoded calcium-sensitive fluorescence protein cameleon 2.1 in the Drosophila brain. We visualized odorant-evoked intracellular calcium concentration changes in selectively labeled olfactory projection neurons both postsynaptically in the antennal lobe, the primary olfactory neuropil, and presynaptically in the mushroom body calyx, a structure involved in olfactory learning and memory. As a technical achievement, we show that calcium imaging with a genetically encoded fluorescence probe is feasible in a brain in vivo. This will allow one to combine Drosophilas advanced genetic tools with the physiological analysis of brain function. Moreover, we report for the first time optical imaging recordings in synaptic regions of the Drosophila mushroom body calyx and antennal lobe. This provides an important step for the use of Drosophila as a model system in olfaction.

The coding of odour‐intensity in the honeybee antennal lobe: local computation optimizes odour representation

We investigated strategies involved in odour intensity coding by the primary olfactory centre of insects, the antennal lobe (AL), the structural and functional analogue of the olfactory bulb. Using calcium imaging in the honeybee, we simultaneously measured the projection neuron output responses and a compound signal dominated by receptor neuron input in identified olfactory glomeruli to odours spanning seven log units of concentration. A comparison of the two processing levels indicates that the intercellular computation within the AL modulates and contrast‐enhances the primary olfactory signals. As a result the AL network optimizes the olfactory code: odour representation is improved at lower concentrations, the relative activity of olfactory glomeruli allows encoding odour quality over up to four log‐unit concentrations, and odour‐intensity is reliably represented in the overall excitation across AL.

A Command Chemical Triggers an Innate Behavior by Sequential Activation of Multiple Peptidergic Ensembles

BACKGROUND At the end of each molt, insects shed their old cuticle by performing the ecdysis sequence, an innate behavior consisting of three steps: pre-ecdysis, ecdysis, and postecdysis. Blood-borne ecdysis-triggering hormone (ETH) activates the behavioral sequence through direct actions on the central nervous system. RESULTS To elucidate neural substrates underlying the ecdysis sequence, we identified neurons expressing ETH receptors (ETHRs) in Drosophila. Distinct ensembles of ETHR neurons express numerous neuropeptides including kinin, FMRFamides, eclosion hormone (EH), crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIP), and bursicon. Real-time imaging of intracellular calcium dynamics revealed sequential activation of these ensembles after ETH action. Specifically, FMRFamide neurons are activated during pre-ecdysis; EH, CCAP, and CCAP/MIP neurons are active prior to and during ecdysis; and activity of CCAP/MIP/bursicon neurons coincides with postecdysis. Targeted ablation of specific ETHR ensembles produces behavioral deficits consistent with their proposed roles in the behavioral sequence. CONCLUSIONS Our findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles.

Odour perception in honeybees: coding information in glomerular patterns

Major advances have been made during the past two years in understanding how honeybees process olfactory input at the level of their first brain structure dealing with odours, the antennal lobe (the insect analogue of the mammalian olfactory bulb). It is now possible to map physiological responses to morphologically identified olfactory glomeruli, allowing for the creation of a functional atlas of the antennal lobe. Furthermore, the measurement of odour-evoked activity patterns has now been combined with studies of appetitive odour learning. The results show that both genetically determined components and learning-related plasticity shape olfactory processing in the antennal lobe.

Digital Atlases of the Antennal Lobe in Two Species of Tobacco Budworm Moths, the Oriental Helicoverpa assulta (Male) and the American Heliothis virescens (Male and Female)

The antennal lobe of the moth brain is the primary olfactory center processing information about pheromones and plant odors. We present here a digital atlas of the glomerular antennal lobe structures in the male of Helicoverpa assulta and the male and female of Heliothis virescens, based on synaptic antibody staining combined with confocal microscopy. The numbers of the glomeruli in the three specimens were similar, 65, 66, and 62, respectively. Whereas the male antennal lobe has a macroglomerular complex consisting of three and four units in the two species, the female lobe has two enlarged glomeruli at a corresponding position, near the entrance of the antennal nerve. Another large glomerulus, showing homology in the three specimens, is ventrally located. The small size of the heliothine moths is advantageous for confocal microscopy because the entire brain can be visualized as a single image stack. The maps are freely accessible on the internet, and the digital form of the data allows each atlas to be rotated and sectioned at any angle, providing for the identification of glomeruli in different preparations. J. Comp. Neurol. 446:123–134, 2002.

Odour coding is bilaterally symmetrical in the antennal lobes of honeybees (Apis mellifera)

The primary olfactory neuropil, the antennal lobe (AL) in insects, is organized in glomeruli. Glomerular activity patterns are believed to represent the across‐fibre pattern of the olfactory code. These patterns depend on an organized innervation from the afferent receptor cells, and interconnections of local interneurons. It is unclear how the complex organization of the AL is achieved ontogenetically. In this study, we measured the functional activity patterns elicited by stimulation with odours in the right and the left AL of the same honeybee (Apis mellifera) using optical imaging of the calcium‐sensitive dye calcium green. We show here that these patterns are bilaterally symmetrical (n = 25 bees). This symmetry holds true for all odours tested, irrespective of their role as pheromones or as environmental odours, or whether they were pure substances or complex blends (n = 13 odours). Therefore, we exclude that activity dependent mechanisms local to one AL determine the functional glomerular activity. This identity is genetically predetermined. Alternatively, if activity dependent processes are involved, bilateral connections would have to shape symmetry, or, temporal constraints could lead to identical patterns on both sides due to their common history of odour exposure.

Odor-driven attractor dynamics in the antennal lobe allow for simple and rapid olfactory pattern classification

The antennal lobe plays a central role for odor processing in insects, as demonstrated by electrophysiological and imaging experiments. Here we analyze the detailed temporal evolution of glomerular activity patterns in the antennal lobe of honeybees. We represent these spatiotemporal patterns as trajectories in a multidimensional space, where each dimension accounts for the activity of one glomerulus. Our data show that the trajectories reach odor-specific steady states (attractors) that correspond to stable activity patterns at about 1 second after stimulus onset. As revealed by a detailed mathematical investigation, the trajectories are characterized by different phases: response onset, steady-state plateau, response offset, and periods of spontaneous activity. An analysis based on support-vector machines quantifies the odor specificity of the attractors and the optimal time needed for odor discrimination. The results support the hypothesis of a spatial olfactory code in the antennal lobe and suggest a perceptron-like readout mechanism that is biologically implemented in a downstream network, such as the mushroom body.

Multiple memory traces after associative learning in the honey bee antennal lobe

We investigated the effect of associative learning on early sensory processing, by combining classical conditioning with in vivo calcium‐imaging of secondary olfactory neurons, the projection neurons (PNs) in the honey bee antennal lobe (AL). We trained bees in a differential conditioning paradigm in which one odour (A+) was paired with a reward, while another odour (B−) was presented without a reward. Two to five hours after differential conditioning, the two odour–response patterns became more different in bees that learned to discriminate between A and B, but not in bees that did not discriminate. This learning‐related change in neural odour representations can be traced back to glomerulus‐specific neural plasticity, which depended on the response profile of the glomerulus before training. (i) Glomeruli responding to A but not to B generally increased in response strength. (ii) Glomeruli responding to B but not to A did not change in response strength. (iii) Glomeruli responding to A and B decreased in response strength. (iv) Glomeruli not responding to A or B increased in response strength. The data are consistent with a neural network model of the AL, which we based on two plastic synapse types and two well‐known learning rules: associative, reinforcer‐dependent Hebbian plasticity at synapses between olfactory receptor neurons (ORNs) and PNs; and reinforcer‐independent Hebbian plasticity at synapses between local interneurons and ORNs. The observed changes strengthen the idea that odour learning optimizes odour representations, and facilitates the detection and discrimination of learned odours.