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The diffraction limit of light microscopy poses a problem that is frequently faced in structural analyses of social insect brains. With the introduction of expansion microscopy (ExM), a tool became available to overcome this limitation by isotropic physical expansion of preserved specimens. Our analyses focus on synaptic microcircuits (microglomeruli, MG) in the mushroom body (MB) of social insects, high-order brain centers for sensory integration, learning, and memory. MG undergo significant structural reorganizations with age, sensory experience, and during long-term memory formation. However, the changes in subcellular architecture involved in this plasticity have only partially been accessed yet. Using the western honeybee Apis mellifera as an experimental model, we established ExM for the first time in a social insect species and applied it to investigate plasticity in synaptic microcircuits within MG of the MB calyces. Using combinations of antibody staining and neuronal tracing, we demonstrate that this technique enables quantitative and qualitative analyses of structural neuronal plasticity at high resolution in a social insect brain., (© 2023. The Author(s).)

Array Tomography (AT) is a relatively easy-to-use and yet powerful method to put molecular identity in its full ultrastructural context. Ultrathin sections are stained with fluorophores and then imaged by light and afterward by electron microscopy to obtain a correlated view of a region of interest: its ultrastructure and specific staining. By combining AT with high-pressure freezing for superior structural preservation and superresolution light microscopy, even small subcellular structures can be mapped in 3D. We established protocols for the application of superresolution AT on ultrathin plastic sections of Caenorhabditis elegans, Trypanosoma brucei, and brain tissue of Cataglyphis fortis and Apis mellifera. All steps are described in detail from sample preparation to 3D reconstruction, including species-specific modifications. We thus showcase the versatility of our protocol and give some examples for biological questions that can be answered with this technique. We offer a step-by-step recipe for superresolution AT that can be easily applied for C. elegans, T. brucei, C. fortis, and A. mellifera and adapted for other model systems., (Copyright © 2017 Elsevier Inc. All rights reserved.)

While the ability of honeybees to navigate relying on sky-compass information has been investigated in a large number of behavioral studies, the underlying neuronal system has so far received less attention. The sky-compass pathway has recently been described from its input region, the dorsal rim area (DRA) of the compound eye, to the anterior optic tubercle (AOTU). The aim of this study is to reveal the connection from the AOTU to the central complex (CX). For this purpose, we investigated the anatomy of large microglomerular synaptic complexes in the medial and lateral bulbs (MBUs/LBUs) of the lateral complex (LX). The synaptic complexes are formed by tubercle-lateral accessory lobe neuron 1 (TuLAL1) neurons of the AOTU and GABAergic tangential neurons of the central body's (CB) lower division (TL neurons). Both TuLAL1 and TL neurons strongly resemble neurons forming these complexes in other insect species. We further investigated the ultrastructure of these synaptic complexes using transmission electron microscopy. We found that single large presynaptic terminals of TuLAL1 neurons enclose many small profiles (SPs) of TL neurons. The synaptic connections between these neurons are established by two types of synapses: divergent dyads and divergent tetrads. Our data support the assumption that these complexes are a highly conserved feature in the insect brain and play an important role in reliable signal transmission within the sky-compass pathway.

Honeybees are able to solve complex learning tasks and memorize learned information for long time periods. The molecular mechanisms mediating long-term memory (LTM) in the honeybee Apis mellifera are, to a large part, still unknown. We approached this question by investigating the potential function of the calcium/calmodulin-dependent protein kinase II (CaMKII), an enzyme known as a 'molecular memory switch' in vertebrates. CaMKII is able to switch to a calcium-independent constitutively active state, providing a mechanism for a molecular memory and has further been shown to play an essential role in structural synaptic plasticity. Using a combination of knockdown by RNA interference and pharmacological manipulation, we disrupted the function of CaMKII during olfactory learning and memory formation. We found that learning, memory acquisition and mid-term memory were not affected, but all manipulations consistently resulted in an impaired LTM. Both early LTM (24 h after learning) and late LTM (72 h after learning) were significantly disrupted, indicating the necessity of CaMKII in two successive stages of LTM formation in the honeybee., (© 2015. Published by The Company of Biologists Ltd.)

Honeybee workers express a pronounced age-dependent polyethism switching from various indoor duties to foraging outside the hive. This transition is accompanied by tremendous changes in the sensory environment that sensory systems and higher brain centers have to cope with. Foraging and age have earlier been shown to be associated with volume changes in the mushroom bodies (MBs). Using age- and task-controlled bees this study provides a detailed framework of neuronal maturation processes in the MB calyx during the course of natural behavioral maturation. We show that the MB calyx volume already increases during the first week of adult life. This process is mainly driven by broadening of the Kenyon cell dendritic branching pattern and then followed by pruning of projection neuron axonal boutons during the actual transition from indoor to outdoor duties. To further investigate the flexible regulation of division of labor and its neuronal correlates in a honeybee colony, we studied the modulation of the nurse-forager transition via a chemical communication system, the primer pheromone ethyl oleate (EO). EO is found at high concentrations on foragers in contrast to nurse bees and was shown to delay the onset of foraging. In this study, EO effects on colony behavior were not as robust as expected, and we found no direct correlation between EO treatment and synaptic maturation in the MB calyx. In general, we assume that the primer pheromone EO rather acts in concert with other factors influencing the onset of foraging with its effect being highly adaptive., (© 2015 Wiley Periodicals, Inc.)

Social work force distribution in honeybee colonies critically depends on subtle adjustments of an age-related polyethism. Pheromones play a crucial role in adjusting physiological and behavioral maturation of nurse bees to foragers. In addition to primer effects of brood pheromone and queen mandibular pheromone--both were shown to influence onset of foraging--direct worker-worker interactions influence adult behavioral maturation. These interactions were narrowed down to the primer pheromone ethyl oleate, which is present at high concentrations in foragers, almost absent in young bees and was shown to delay the onset of foraging. Based on chemical analyses, physiological recordings from the antenna (electroantennograms) and the antennal lobe (calcium imaging), and behavioral assays (associative conditioning of the proboscis extension response), we present evidence that ethyl oleate is most abundant on the cuticle, received by olfactory receptors on the antenna, processed in glomeruli of the antennal lobe, and learned in olfactory centers of the brain. The results are highly suggestive that the primer pheromone ethyl oleate is transmitted and perceived between individuals via olfaction at close range.

Calcium/calmodulin-dependent protein kinase II (CaMKII) has been linked to neuronal plasticity associated with long-term potentiation as well as structural synaptic plasticity. Previous work in adult honeybees has shown that a single CaMKII gene is strongly expressed in the mushroom bodies (MBs), brain centers associated with sensory integration, and learning and memory formation. To study a potential role of CaMKII in synaptic plasticity, the cellular and subcellular distribution of activated (phosphorylated) pCaMKII protein was investigated at various life stages of the honeybee using immunocytochemistry, confocal microscopy, and western blot analyses. Whereas at pupal stages 3-4 most parts of the brain showed high levels of pCaMKII immunoreactivity, the protein was predominantly concentrated in the MBs in the adult brain. The results show that pCaMKII is present in a specific subpopulation of Kenyon cells, the noncompact cells. Within the olfactory (lip) and visual (collar) subregion of the MB calyx neuropil pCaMKII was colocalized with f-actin in postsynaptic compartments of microglomeruli, indicating that it is enriched in Kenyon cell dendritic spines. This suggests a potential role of CaMKII in Kenyon cell dendritic plasticity. Interestingly, pCaMKII protein was absent in two other types of Kenyon cells, the inner compact cells associated with the multimodal basal ring and the outer compact cells. During adult behavioral maturation from nurse bees to foragers, pCaMKII distribution remained essentially similar at the qualitative level, suggesting a potential role in dendritic plasticity of Kenyon cells throughout the entire life span of a worker bee., (Copyright © 2011 Wiley-Liss, Inc.)

The insect mushroom bodies (MBs) are paired brain centers which, like the mammalian hippocampus, have a prominent function in learning and memory. Despite convergent evidence for their crucial role in the formation and storage of associative memories, little is known about the mechanisms underlying such storage. In mammals and other species, the consolidation of stable memories is accompanied by structural plasticity involving variations in synapse number and/or size. Here, we address the question of whether the formation of olfactory long-term memory (LTM) could be associated with changes in the synaptic architecture of the MB networks. For this, we took advantage of the modular architecture of the honeybee MB neuropil, where synaptic contacts between olfactory input and MB neurons are segregated into discrete units (microglomeruli) which can be easily visualized and counted. We show that the density in microglomeruli increases as a specific olfactory LTM is formed, while the volume of the neuropil remains constant. Such variation is reproducible and is clearly correlated with memory consolidation, as it requires gene transcription. Thus stable structural synaptic rearrangements, including the growth of new synapses, seem to be a common property of insect and mammalian brain networks involved in the storage of stable memory traces.

Desert ants of the genus Cataglyphis undergo an age-related polyethism from interior workers involved in brood care and food processing to short-lived outdoor foragers with remarkable visual navigation capabilities. The quick transition from dark to light suggests that visual centers in the ant's brain express a high degree of plasticity. To investigate structural synaptic plasticity in the mushroom bodies (MBs)-sensory integration centers supposed to be involved in learning and memory-we immunolabeled and quantified pre- and postsynaptic profiles of synaptic complexes (microglomeruli, MG) in the visual (collar) and olfactory (lip) input regions of the MB calyx. The results show that a volume increase of the MB calyx during behavioral transition is associated with a decrease in MG numbers in the collar and, less pronounced, in the lip. Analysis of tubulin-positive profiles indicates that presynaptic pruning of projection neurons and dendritic expansion in intrinsic Kenyon cells are involved. Light-exposure of dark-reared ants of different age classes revealed similar effects. The results indicate that this structural synaptic plasticity in the MB calyx is primarily driven by visual experience rather than by an internal program. This is supported by the fact that dark-reared ants age-matched to foragers had MG numbers comparable to those of interior workers. Ants aged artificially for up to 1 year expressed a similar plasticity. These results suggest that the high degree of neuronal plasticity in visual input regions of the MB calyx may be an important factor related to behavior transitions associated with division of labor.