The anatomical substrates of neural nets are usually composed from reconstructions of neurons that were stained in different preparations. Realistic models of the structural relationships between neurons require a common framework. Here we present 3-D reconstructions of single projection neurons (PN) connecting the antennal lobe (AL) with the mushroom body (MB) and lateral horn, groups of intrinsic mushroom body neurons (type 5 Kenyon cells), and a single mushroom body extrinsic neuron (PE1), aiming to compose components of the olfactory pathway in the honeybee. To do so, we constructed a digital standard atlas of the bee brain. The standard atlas was created as an average-shape atlas of 22 neuropils, calculated from 20 individual immunostained whole-mount bee brains. After correction for global size and positioning differences by repeatedly applying an intensity-based nonrigid registration algorithm, a sequence of average label images was created. The results were qualitatively evaluated by generating average gray-value images corresponding to the average label images and judging the level of detail within the labeled regions. We found that the first affine registration step in the sequence results in a blurred image because of considerable local shape differences. However, already the first nonrigid iteration in the sequence corrected for most of the shape differences among individuals, resulting in images rich in internal detail. A second iteration improved on that somewhat and was selected as the standard. Registering neurons from different preparations into the standard atlas reveals 1) that the m-ACT neuron occupies the entire glomerulus (cortex and core) and overlaps with a local interneuron in the cortical layer; 2) that, in the MB calyces and the lateral horn of the protocerebral lobe, the axon terminals of two identified m-ACT neurons arborize in separate but close areas of the neuropil; and 3) that MB-intrinsic clawed Kenyon cells (type 5), with somata outside the calycal cups, project to the peduncle and lobe output system of the MB and contact (proximate) the dendritic tree of the PE1 neuron at the base of the vertical lobe. Thus the standard atlas and the procedures applied for registration serve the function of creating realistic neuroanatomical models of parts of a neural net. The Honeybee Standard Brain is accessible at www.neurobiologie.fu-berlin.de/beebrain.

In order to understand the connectivity of neuronal networks, their constituent neurons should ideally be studied in a common framework. Since morphological data from physiologically characterized and stained neurons usually arise from different individual brains, this can only be performed in a virtual standardized brain that compensates for interindividual variability. The desert locust, Schistocerca gregaria, is an insect species used widely for the analysis of olfactory and visual signal processing, endocrine functions, and neural networks controlling motor output. To provide a common multi-user platform for neural circuit analysis in the brain of this species, we have generated a standardized three-dimensional brain of this locust. Serial confocal images from whole-mount locust brains were used to reconstruct 34 neuropil areas in ten brains. For standardization, we compared two different methods: an iterative shape-averaging (ISA) procedure by using affine transformations followed by iterative nonrigid registrations, and the Virtual Insect Brain (VIB) protocol by using global and local rigid transformations followed by local nonrigid transformations. Both methods generated a standard brain, but for different applications. Whereas the VIB technique was designed to visualize anatomical variability between the input brains, the purpose of the ISA method was the opposite, i.e., to remove this variability. A novel individually labeled neuron, connecting the lobula to the midbrain and deutocerebrum, has been registered into the ISA atlas and demonstrates its usefulness and accuracy for future analysis of neural networks. The locust standard brain is accessible at http://www.3d-insectbrain.com .

In their natural environment, many insects need to identify and evaluate behaviorally relevant odorants on a rich and dynamic olfactory background. Behavioral studies have demonstrated that bees recognize learned odors within <200 ms, indicating a rapid processing of olfactory input in the sensory pathway. We studied the role of the honeybee antennal lobe network in constructing a fast and reliable code of odor identity using in vivo intracellular recordings of individual projection neurons (PNs) and local interneurons (LNs). We found a complementary ensemble code where odor identity is encoded in the spatio-temporal pattern of response latencies as well as in the pattern of activated and inactivated PN firing. This coding scheme rapidly reaches a stable representation within 50–150 ms after stimulus onset. Testing an odor mixture versus its individual compounds revealed different representations in the two morphologically distinct types of lateral- and median PNs (l- and m-PNs). Individual m-PNs mixture responses were dominated by the most effective compound (elemental representation) whereas l-PNs showed suppressed responses to the mixture but not to its individual compounds (synthetic representation). The onset of inhibition in the membrane potential of l-PNs coincided with the responses of putative inhibitory interneurons that responded significantly faster than PNs. Taken together, our results suggest that processing within the LN network of the AL is an essential component of constructing the antennal lobe population code.

Worker honeybees proceed through a sequence of tasks, passing from hive and guard duties to foraging activities. The underlying neuronal changes accompanying and possibly mediating these behavioral transitions are not well understood. We studied changes in the microglomerular organization of the mushroom bodies, a brain region involved in sensory integration, learning, and memory, during adult maturation. We visualized the MB lips' microglomerular organization by applying double labeling of presynaptic projection neuron boutons and postsynaptic Kenyon cell spines, which form microglomerular complexes. Their number and density, as well as the bouton volume, were measured using 3D-based techniques. Our results show that the number of microglomerular complexes and the bouton volumes increased during maturation, independent of environmental conditions. In contrast, manipulations of behavior and sensory experience caused a decrease in the number of microglomerular complexes, but an increase in bouton volume. This may indicate an outgrowth of synaptic connections within the MB lips during honeybee maturation. Moreover, manipulations of behavioral and sensory experience led to adaptive changes, which indicate that the microglomerular organization of the MB lips is not static and determined by maturation, but rather that their organization is plastic, enabling the brain to retain its synaptic efficacy.

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