3D-Reconstructions and Virtual 4D-Visualization to Study Metamorphic Brain Development in the Sphinx Moth Manduca Sexta

During metamorphosis, the transition from the larva to the adult, the insect brain undergoes considerable remodeling: new neurons are integrated while larval neurons are remodeled or eliminated. One well acknowledged model to study metamorphic brain development is the sphinx moth Manduca sexta. To further understand mechanisms involved in the metamorphic transition of the brain we generated a 3D standard brain based on selected brain areas of adult females and 3D reconstructed the same areas during defined stages of pupal development. Selected brain areas include for example mushroom bodies, central complex, antennal- and optic lobes. With this approach we eventually want to quantify developmental changes in neuropilar architecture, but also quantify changes in the neuronal complement and monitor the development of selected neuronal populations. Furthermore, we used a modeling software (Cinema 4D) to create a virtual 4D brain, morphing through its developmental stages. Thus the didactical advantages of 3D visualization are expanded to better comprehend complex processes of neuropil formation and remodeling during development. To obtain datasets of the M. sexta brain areas, we stained whole brains with an antiserum against the synaptic vesicle protein synapsin. Such labeled brains were then scanned with a confocal laser scanning microscope and selected neuropils were reconstructed with the 3D software AMIRA 4.1

The Panopticon—Assessing the Effect of Starvation on Prolonged Fly Activity and Place Preference

Animal behaviours are demonstrably governed by sensory stimulation, previous experience and internal states like hunger. With increasing hunger, priorities shift towards foraging and feeding. During foraging, flies are known to employ efficient path integration strategies. However, general long-term activity patterns for both hungry and satiated flies in conditions of foraging remain to be better understood. Similarly, little is known about how permanent contact chemosensory stimulation affects locomotion. To address these questions, we have developed a novel, simplistic fly activity tracking setup— the Panopticon. Using a 3D-printed Petri dish inset, our assay allows recording of walking behaviour, of several flies in parallel, with all arena surfaces covered by a uniform substrate layer. We tested two constellations of providing food: (i) in single patches and (ii) omnipresent within the substrate layer. Fly tracking is done with FIJI, further assessment, analysis and presentation is done with a custom-built MATLAB analysis framework. We find that starvation history leads to a long-lasting reduction in locomotion, as well as a delayed place preference for food patches which seems to be not driven by immediate hunger motivation

Sweet taste and nutrient value subdivide rewarding dopaminergic neurons in Drosophila

Dopaminergic neurons provide reward learning signals in mammals and insects [1-4]. Recent work in Drosophila has demonstrated that water-reinforcing dopaminergic neurons are different to those for nutritious sugars [5]. Here, we tested whether the sweet taste and nutrient properties of sugar reinforcement further subdivide the fly reward system. We found that dopaminergic neurons expressing the OAMB octopamine receptor [6] specifically convey the short-term reinforcing effects of sweet taste [4]. These dopaminergic neurons project to the beta\u272 and gamma4 regions of the mushroom body lobes. In contrast, nutrient-dependent long-term memory requires different dopaminergic neurons that project to the gamma5b regions, and it can be artificially reinforced by those projecting to the beta lobe and adjacent alpha1 region. Surprisingly, whereas artificial implantation and expression of short-term memory occur in satiated flies, formation and expression of artificial long-term memory require flies to be hungry. These studies suggest that short-term and long-term sugar memories have different physiological constraints. They also demonstrate further functional heterogeneity within the rewarding dopaminergic neuron population

3D Standard Brain of the Red Flour Beetle Tribolium Castaneum: A Tool to Study Metamorphic Development and Adult Plasticity

The red flour beetle Tribolium castaneum is emerging as a further standard insect model beside Drosophila. Its genome is fully sequenced and it is susceptible for genetic manipulations including RNA-interference. We use this beetle to study adult brain development and plasticity primarily with respect to the olfactory system. In the current study, we provide 3D standard brain atlases of freshly eclosed adult female and male beetles (A0). The atlases include eight paired and three unpaired neuropils including antennal lobes (ALs), optic lobe neuropils, mushroom body calyces and pedunculi, and central complex. For each of the two standard brains, we averaged brain areas of 20 individual brains. Additionally, we characterized eight selected olfactory glomeruli from 10 A0 female and male beetles respectively, which we could unequivocally recognize from individual to individual owing to their size and typical position in the ALs. In summary, comparison of the averaged neuropil volumes revealed no sexual dimorphism in any of the reconstructed neuropils in A0 Tribolium brains. Both, the female and male 3D standard brain are also used for interspecies comparisons, and, importantly, will serve as future volumetric references after genetical manipulation especially regarding metamorphic development and adult plasticity

Inhibitory muscarinic acetylcholine receptors enhance aversive olfactory conditioning in adult Drosophila

Olfactory associative learning in Drosophila is mediated by synaptic plasticity between the Kenyon cells of the mushroom body and their output neurons. Both Kenyon cells and their inputs from projection neurons are cholinergic, yet little is known about the physiological function of muscarinic acetylcholine receptors in learning in adult flies. Here, we show that aversive olfactory learning in adult flies requires type A muscarinic acetylcholine receptors (mAChR-A), particularly in the gamma subtype of Kenyon cells. mAChR-A inhibits odor responses and is localized in Kenyon cell dendrites. Moreover, mAChR-A knockdown impairs the learning-associated depression of odor responses in a mushroom body output neuron. Our results suggest that mAChR-A function in Kenyon cell dendrites is required for synaptic plasticity between Kenyon cells and their output neurons

Circuits that encode and guide alcohol-associated preference

A powerful feature of adaptive memory is its inherent flexibility. Alcohol and other addictive substances can remold neural circuits important for memory to reduce this flexibility. However, the mechanism through which pertinent circuits are selected and shaped remains unclear. We show that circuits required for alcohol-associated preference shift from population level dopaminergic activation to select dopamine neurons that predict behavioral choice in Drosophila melanogaster. During memory expression, subsets of dopamine neurons directly and indirectly modulate the activity of interconnected glutamatergic and cholinergic mushroom body output neurons (MBON). Transsynaptic tracing of neurons important for memory expression revealed a convergent center of memory consolidation within the mushroom body (MB) implicated in arousal, and a structure outside the MB implicated in integration of naive and learned responses. These findings provide a circuit framework through which dopamine neuronal activation shifts from reward delivery to cue onset, and provide insight into the maladaptive nature of memory

Neuronal mechanisms underlying innate and learned olfactory processing in Drosophila

Olfaction allows animals to adapt their behavior in response to different chemical cues in their environment. How does the brain efficiently discriminate different odors to drive appropriate behavior, and how does it flexibly assign value to odors to adjust behavior according to experience? This review traces neuronal mechanisms underlying these processes in adult Drosophila melanogaster from olfactory receptors to higher brain centers. We highlight neural circuit principles like lateral inhibition, segregation and integration of olfactory channels, temporal accumulation of sensory evidence, and compartmentalized synaptic plasticity underlying associative memory

Standard three-dimensional glomeruli of the Manduca sexta antennal lobe: a tool to study both developmental and adult neuronal plasticity.

The metamorphosing antennal lobe (AL) of the sphinx moth Manduca sexta serves as an established model system for studying neuronal development. To improve our understanding of mechanisms involved in neuronal plasticity, we have analyzed the size, shape, and localization of ten identified glomeruli at three different time points during development and in the adult, viz., (1) 13 days after pupal eclosion (P13), which reflects a time when the basic glomerular map has formed, (2) immediately after adult eclosion (A0), which represents a time when the newly formed glomeruli are uninfluenced by external odors, and (3) 4 days after adult eclosion (A4), which reflects a time when the animals have been exposed to surrounding odors. Our data from normally developing ALs of male M. sexta from P13 to A0 revealed an increase in size of all examined glomeruli of between 40% and 130%, with the strongest increases occurring in two of the three sex-specific glomeruli (cumulus, toroid). From A0 to A4, the cumulus and toroid increased significantly when correlated to AL volume, whereas the other glomeruli reached the sizes gained after A0. This study was based on antibody staining against the ubiquitous synaptic vesicle protein synaptotagmin, confocal laser scan microscopy, and the three-dimensional (3D) analysis tool AMIRA. Tissue permeability and therefore reliability of the staining quality was enhanced by using formalin/methanol fixation. The standard 3D glomeruli introduced in this study can now be used as basic tools for further examination of neuronal plasticity at the level of the identified neuropil structures, viz., the glomeruli of the AL of M. sexta.