Jürgen Rybak

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 neurons 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.

Drosophila avoids parasitoids by sensing their semiochemicals via a dedicated olfactory circuit

Detecting danger is one of the foremost tasks for a neural system. Larval parasitoids constitute clear danger to Drosophila, as up to 80% of fly larvae become parasitized in nature. We show that Drosophila melanogaster larvae and adults avoid sites smelling of the main parasitoid enemies, Leptopilina wasps. This avoidance is mediated via a highly specific olfactory sensory neuron (OSN) type. While the larval OSN expresses the olfactory receptor Or49a and is tuned to the Leptopilina odor iridomyrmecin, the adult expresses both Or49a and Or85f and in addition detects the wasp odors actinidine and nepetalactol. The information is transferred via projection neurons to a specific part of the lateral horn known to be involved in mediating avoidance. Drosophila has thus developed a dedicated circuit to detect a life-threatening enemy based on the smell of its semiochemicals. Such an enemy-detecting olfactory circuit has earlier only been characterized in mice and nematodes.

Synaptic circuitry of identified neurons in the antennal lobe of Drosophila melanogaster

In Drosophila melanogaster olfactory sensory neurons (OSNs) establish synapses with projection neurons (PNs) and local interneurons within antennal lobe (AL) glomeruli. Substantial knowledge regarding this circuitry has been obtained by functional studies, whereas ultrastructural evidence of synaptic contacts is scarce. To fill this gap, we studied serial sections of three glomeruli using electron microscopy. Ectopic expression of a membrane‐bound peroxidase allowed us to map synaptic sites along PN dendrites. Our data prove for the first time that each of the three major types of AL neurons is both pre‐ and postsynaptic to the other two types, as previously indicated by functional studies. PN dendrites carry a large proportion of output synapses, with approximately one output per every three input synapses. Detailed reconstructions of PN dendrites showed that these synapses are distributed unevenly, with input and output sites partially segregated along a proximal–distal gradient and the thinnest branches carrying solely input synapses. Moreover, our data indicate synapse clustering, as we found evidence of dendritic tiling of PN dendrites. PN output synapses exhibited T‐shaped presynaptic densities, mostly arranged as tetrads. In contrast, output synapses from putative OSNs showed elongated presynaptic densities in which the T‐bar platform was supported by several pedestals and contacted as many as 20 postsynaptic profiles. We also discovered synaptic contacts between the putative OSNs. The average synaptic density in the glomerular neuropil was about two synapses/µm3. These results are discussed with regard to current models of olfactory glomerular microcircuits across species. J. Comp. Neurol. 524:1920–1956, 2016.

The Digital Honey Bee Brain Atlas

For a comprehensive understanding of brain function, compiling data from a range of experiments is necessary. Digital brain atlases provide useful reference systems at the interface of neuroanatomy, neurophysiology, behavioral biology and neuroinformatics. Insect brains are particularly useful because they constitute complete three-dimensional references for the integration of morphological and functional data. Image acquisition is favored by small sized brains permitting whole brain scans using confocal microscopy. Insect brain atlases thus serve different purposes, e.g. quantitative volume analyses of brain neuropils for studying closely related species, developmental processes and neuronal plasticity; documenting and storing the Gestalt and spatial relations of neurons, neural networks and neuropils; structuring large amounts of anatomical and physiological data, thus providing a repository for data sharing among researchers. This chapter focuses on the spatial relations of neurons in the honey bee brain using the Honey bee Standard Brain (HSB). The integration of neurons into the HSB requires standardized image processing, computer algorithms and protocols that aid reconstruction and visualization. A statistical shape model has been developed in order to facilitate the segmentation process. Examples from the olfactory and mechanosensory pathways in the bee brain and the organization of the mushroom bodies (MBs) are used to illustrate the implementation and strength of the HSB. An outline will be given for the use of the brain atlas to link semantic information (e.g. from physiology, biochemistry, genetics) and neuronal morphology.

The antennal lobe of Libellula depressa (Odonata, Libellulidae)

Here we describe the antennal lobe of Libellula depressa (Odonata, Libellulidae), identified on the basis of the projections of the afferent sensory neurons stemming from the antennal flagellum sensilla. Immunohistochemical neuropil staining as well as antennal backfills revealed sensory neuron terminal arborizations covering a large portion of the antennal lobe. No clear glomerular structure was identified, thus suggesting an aglomerular antennal lobe condition as previously reported in Palaeoptera. The terminal arbors of backfilled sensory neurons do, however, form spherical knots, probably representing the connections between the few afferent neurons and the antennal lobe interneurons. The reconstruction revealed that the proximal part of the antennal nerve is divided into two branches that innervate two spatially separated areas of the antennal lobe, an anterioventral lobe and a larger posteriodorsal lobe. Our data are consistent with the hypothesis that one tract of the antennal nerve of L. depressa contains olfactory sensory neurons projecting into one of the sublobes, while the other tract contains thermo-hygroreceptive neurons projecting into the other sublobe.

Drosophila FoxP Mutants Are Deficient in Operant Self-Learning

Intact function of the Forkhead Box P2 (FOXP2) gene is necessary for normal development of speech and language. This important role has recently been extended, first to other forms of vocal learning in animals and then also to other forms of motor learning. The homology in structure and in function among the FoxP gene members raises the possibility that the ancestral FoxP gene may have evolved as a crucial component of the neural circuitry mediating motor learning. Here we report that genetic manipulations of the single Drosophila orthologue, dFoxP, disrupt operant self-learning, a form of motor learning sharing several conceptually analogous features with language acquisition. Structural alterations of the dFoxP locus uncovered the role of dFoxP in operant self-learning and habit formation, as well as the dispensability of dFoxP for operant world-learning, in which no motor learning occurs. These manipulations also led to subtle alterations in the brain anatomy, including a reduced volume of the optic glomeruli. RNAi-mediated interference with dFoxP expression levels copied the behavioral phenotype of the mutant flies, even in the absence of mRNA degradation. Our results provide evidence that motor learning and language acquisition share a common ancestral trait still present in extant invertebrates, manifest in operant self-learning. This ‘deep’ homology probably traces back to before the split between vertebrate and invertebrate animals.

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 (ALs), to the central brain, the mushroom bodies (MBs), and the protocerebral lobe (PL). Intracellularly stained uniglomerular projection neurons 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 (PNs) using confocal microscopy. Analysis of the patterns of axon terminals revealed a domain-like innervation within the MB lip neuropil. PNs of the lateral tract arborized more sparsely within the lips and exhibited fewer synaptic boutons, while medial tract neurons occupied broader regions in the MB calyces and the PL. Our data show that uPNs from the medial and lateral tract innervate both the core and the cortex of the ipsilateral MB 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 PNs, octopaminergic-, and GABAergic cells in the MB calyces. For the first time, we found evidence for connections between both tracts within the AL.

Central projections of antennular chemosensory and mechanosensory afferents in the brain of the terrestrial hermit crab (Coenobita clypeatus; Coenobitidae, Anomura)

The Coenobitidae (Decapoda, Anomura, Paguroidea) is a taxon of hermit crabs that includes two genera with a fully terrestrial life style as adults. Previous studies have shown that Coenobitidae have evolved a sense of spatial odor localization that is behaviorally highly relevant. Here, we examined the central olfactory pathway of these animals by analyzing central projections of the antennular nerve of Coenobita clypeatus, combining backfilling of the nerve with dextran-coupled dye, Golgi impregnations and three-dimensional reconstruction of the primary olfactory center, the antennular lobe. The principal pattern of putative olfactory sensory afferents in C. clypeatus is in many aspects similar to what have been established for aquatic decapod crustaceans, such as the spiny lobster Panulirus argus. However, there are also obvious differences that may, or may not represent adaptations related to a terrestrial lifestyle. In C. clypeatus, the antennular lobe dominates the deutocerebrum, having more than one thousand allantoid-shaped subunits. We observed two distinct patterns of sensory neuron innervation: putative olfactory afferents from the aesthetascs either supply the cap/subcap region of the subunits or they extend through its full depth. Our data also demonstrate that any one sensory axon can supply input to several subunits. Putative chemosensory (non-aesthetasc) and mechanosensory axons represent a different pathway and innervate the lateral and median antennular neuropils. Hence, we suggest that the chemosensory input in C. clypeatus might be represented via a dual pathway: aesthetascs target the antennular lobe, and bimodal sensilla target the lateral antennular neuropil and median antennular neuropil. The present data is compared to related findings in other decapod crustaceans.

Comparative neuroanatomy of the antennal lobes of 2 homopteran species

We compared the morphology of the primary olfactory center, the antennal lobe (AL), in 2 homopteran insects, Hyalesthes obsoletus Signoret (Homoptera: Cixiidae) and Scaphoideus titanus Ball (Homoptera: Cicadomorpha). The comparison between the ALs of the 2 species is particularly interesting considering that, although both use volatile cues to locate their host plants, their feeding behavior differs considerably: specifically, H. obsoletus is a highly polyphagous species, whereas S. titanus is strictly monophagous (on grapevine). Our investigation of the AL structure using immunocytochemical staining and antennal backfills did not reveal any sexual dimorphism in either the size of the ALs or in the size of individual glomeruli for either species. Instead, the AL of H. obsoletus displayed numerous and well-delineated glomeruli (about 130 in both sexes) arranged in a multilayered structure, whereas the smaller AL of S. titanus contained fewer than 15 glomerular-like structures. This difference is likely to reflect the comparatively reduced olfactory abilities in S. titanus, probably as a consequence of the reduced number of volatiles coming from the single host plant. Instead, in H. obsoletus, the ability to distinguish among several host plants may require a more complex olfactory neuronal network.

Exploring brain connectivity in insect model systems of learning and memory

A fundamental objective in neurobiology is to understand the neuronal circuitry that underlies different aspects of behavior (sensory integration, decision making, motor control, learning, and memory formation). In invertebrates, neural circuitry is classically analyzed at the cellular level using sparse reconstruction based on single cell staining techniques (Golgi and intracellular staining) in conjunction with functional and correlative studies using immunohistology and ultrastructure analysis. These approaches led to the identification of complete circuits at the synaptic level in small invertebrates (e.g., Caenorhabditis elegans ) and in small parts of the brain (e.g., fly lamina). Advances in light microscopy techniques and the use of targeted expression of neuronal and molecular markers in transgenic animals allow more elaborate circuit mapping. High-throughput techniques in electron microscopy, genetic engineering (‘brainbow’), and three-dimensional microscopy of global brain circuitry allow the establishment of the connectome and complete wiring diagrams of dense neuropils, including synaptic connections. This chapter focuses on methods for characterizing ’microcircuits’—that is, the connectome on the synaptic level.