The Digital Bee Brain: Integrating and Managing Neurons in a Common 3D Reference System

The honeybee standard brain (HSB) serves as an interactive tool for relating morphologies of bee brain neurons and provides a reference system for functional and bibliographical properties (http://www.neurobiologie.fu-berlin.de/beebrain/). The ultimate goal is to document not only the morphological network properties of neurons collected from separate brains, but also to establish a graphical user interface for a neuron-related data base. Here, we review the current methods and protocols used to incorporate neuronal reconstructions into the HSB. Our registration protocol consists of two separate steps applied to imaging data from two-channel confocal microscopy scans: (1) The reconstruction of the neuron, facilitated by an automatic extraction of the neuron's skeleton based on threshold segmentation, and (2) the semi-automatic 3D segmentation of the neuropils and their registration with the HSB. The integration of neurons in the HSB is performed by applying the transformation computed in step (2) to the reconstructed neurons of step (1). The most critical issue of this protocol in terms of user interaction time – the segmentation process – is drastically improved by the use of a model-based segmentation process. Furthermore, the underlying statistical shape models (SSM) allow the visualization and analysis of characteristic variations in large sets of bee brain data. The anatomy of neural networks composed of multiple neurons that are registered into the HSB are visualized by depicting the 3D reconstructions together with semantic information with the objective to integrate data from multiple sources (electrophysiology, imaging, immunocytochemistry, molecular biology). Ultimately, this will allow the user to specify cell types and retrieve their morphologies along with physiological characterizations

Digital, Three-dimensional Average Shaped Atlas of the Heliothis Virescens Brain with Integrated Gustatory and Olfactory Neurons

We use the moth Heliothis virescens as model organism for studying the neural network involved in chemosensory coding and learning. The constituent neurons are characterised by intracellular recordings combined with staining, resulting in a single neuron identified in each brain preparation. In order to spatially relate the neurons of different preparations a common brain framework was required. We here present an average shaped atlas of the moth brain. It is based on 11 female brain preparations, each stained with a fluorescent synaptic marker and scanned in confocal laser-scanning microscope. Brain neuropils of each preparation were manually reconstructed in the computer software Amira, followed by generating the atlas using the Iterative Shape Average Procedure. To demonstrate the application of the atlas we have registered two olfactory and two gustatory interneurons, as well as the axonal projections of gustatory receptor neurons into the atlas, visualising their spatial relationships. The olfactory interneurons, showing the typical morphology of inner-tract antennal lobe projection neurons, projected in the calyces of the mushroom body and laterally in the protocerebral lobe. The two gustatory interneurons, responding to sucrose and quinine respectively, projected in different areas of the brain. The wide projections of the quinine responding neuron included a lateral area adjacent to the projections of the olfactory interneurons. The sucrose responding neuron was confined to the suboesophageal ganglion with dendritic arborisations overlapping the axonal projections of the gustatory receptor neurons on the proboscis. By serving as a tool for the integration of neurons, the atlas offers visual access to the spatial relationship between the neurons in three dimensions, and thus facilitates the study of neuronal networks in the Heliothis virescens brain. The moth standard brain is accessible at http://www.ntnu.no/biolog/english/neuroscience/brai

Integrative Properties of the Pe1 Neuron, a Unique Mushroom Body Output Neuron

A mushroom body extrinsic neuron, the Pe1 neuron, connects the peduncle of the mushroom body (MB) with two areas of the protocerebrum in the honeybee brain, the lateral protocerebral lobe (LPL) and the ring neuropil around the α-lobe. Each side of the bee brain contains only one Pe1 neuron. Using a combination of intracellular recording and neuroanatomical techniques we analyzed its properties of integrative processing of the different sensory modalities. The Pe1 neuron responds to visual, mechanosensory, and olfactory stimuli. The responses are broadly tuned, consisting of a sustained increase of spike frequency to the onset and offset of light flashes, to horizontal and vertical movements of extended objects, to mechanical stimuli applied to the antennae or mouth parts, and to all olfactory stimuli tested (29 chemicals). These multisensory properties are reflected in its dendritic organization. Serial reconstructions of intracellularly stained Pe1 neurons using confocal microscopy reveal that the Pe1 neuron arborizes throughout all layers of MB peduncle with finger-like, vertically oriented dendrites. The peduncle of the MB is formed by the axons of Kenyon cells, whose dendritic inputs are organized in modality-specific subcompartments of the calyx region. The peduncular arborization indicates that the Pe1 neuron receives input from Kenyon cells of all calycal subcompartments. Because the Pe1 neuron changes its odor responses transiently as a consequence of olfactory learning, we hypothesize that the multimodal response properties might have a role in memory consolidation and help to establish contextual references in the long-term trace

Synaptic Spinules in the Olfactory Circuit of Drosophila melanogaster

Here we report on ultrastructural features of brain synapses in the fly Drosophila melanogaster and outline a perspective for the study of their functional significance. Images taken with the aid of focused ion beam-scanning electron microscopy (EM) at 20 nm intervals across olfactory glomerulus DA2 revealed that some synaptic boutons are penetrated by protrusions emanating from other neurons. Similar structures in the brain of mammals are known as synaptic spinules. A survey with transmission EM (TEM) disclosed that these structures are frequent throughout the antennal lobe. Detailed neuronal tracings revealed that spinules are formed by all three major types of neurons innervating glomerulus DA2 but the olfactory sensory neurons (OSNs) receive significantly more spinules than other olfactory neurons. Double-membrane vesicles (DMVs) that appear to represent material that has pinched-off from spinules are also most abundant in presynaptic boutons of OSNs. Inside the host neuron, a close association was observed between spinules, the endoplasmic reticulum (ER) and mitochondria. We propose that by releasing material into the host neuron, through a process triggered by synaptic activity and analogous to axonal pruning, synaptic spinules could function as a mechanism for synapse tagging, synaptic remodeling and neural plasticity. Future directions of experimental work to investigate this theory are proposed

RT-PCR Gels

All full RT-PCR gels for mutants and RNAi experiments, heads (Figs. 3/4) and thoraxes (no figure in publication). Eight JPG files in a ZIP container

The circuitry of olfactory projection neurons in the brain of the honeybee, Apis mellifera

In the honeybee brain, two prominent tracts - the medial and the lateral antennal lobe tract - project from the primary olfactory center, the antennal lobes, to the central brain, the mushroom bodies, and the protocerebral lobe. Intracellularly stained uniglomerular projection neurons (uPN) were reconstructed, registered to the 3D honeybee standard brain atlas, and then used to derive the spatial properties and quantitative morphology of the neurons of both tracts. We evaluated putative synaptic contacts of projection neurons using confocal microscopy. Analysis of the patterns of axon terminals revealed a domain-like innervation within the mushroom body lip neuropil. Projection neurons of the lateral tract arborized more sparsely within the lips and exhibited fewer synaptic boutons, while medial tract neurons occupied broader regions in the mushroom body calyces and the protocerebral lobe. Our data show that uPNs from the medial and lateral tract innervate both the core and the cortex of the ipsilateral mushroom body lip but differ in their innervation patterns in these regions. In the mushroombody neuropil collar we found evidence for ALT boutons suggesting the collar as a multi modal input site including olfactory input similar to lip and basal ring. In addition, our data support the conclusion drawn in previous studies that reciprocal synapses exist between projection neurons, octopaminergic-, and GABAergic cells in the mushroom body calyces. For the first time, we found evidence for connections between both tracts within the antennal lobe