Expansion microscopy in honeybee brains for high-resolution neuroanatomical analyses in social insects.

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.

Johnston's organ and its central projections in Cataglyphis desert ants.

The Johnston's organ (JO) in the insect antenna is a multisensory organ involved in several navigational tasks including wind-compass orientation, flight control, graviception, and, possibly, magnetoreception. Here we investigate the three dimensional anatomy of the JO and its neuronal projections into the brain of the desert ant Cataglyphis, a marvelous long-distance navigator. The JO of C. nodus workers consists of 40 scolopidia comprising three sensory neurons each. The numbers of scolopidia slightly vary between different sexes (female/male) and castes (worker/queen). Individual scolopidia attach to the intersegmental membrane between pedicel and flagellum of the antenna and line up in a ring-like organization. Three JO nerves project along the two antennal nerve branches into the brain. Anterograde double staining of the antennal afferents revealed that JO receptor neurons project to several distinct neuropils in the central brain. The T5 tract projects into the antennal mechanosensory and motor center (AMMC), while the T6 tract bypasses the AMMC via the saddle and forms collaterals terminating in the posterior slope (PS) (T6I), the ventral complex (T6II), and the ventrolateral protocerebrum (T6III). Double labeling of JO and ocellar afferents revealed that input from the JO and visual information from the ocelli converge in tight apposition in the PS. The general JO anatomy and its central projection patterns resemble situations in honeybees and Drosophila. The multisensory nature of the JO together with its projections to multisensory neuropils in the ant brain likely serves synchronization and calibration of different sensory modalities during the ontogeny of navigation in Cataglyphis.

Analysis of Synaptic Microcircuits in the Mushroom Bodies of the Honeybee.

Mushroom bodies (MBs) are multisensory integration centers in the insect brain involved in learning and memory formation. In the honeybee, the main sensory input region (calyx) of MBs is comparatively large and receives input from mainly olfactory and visual senses, but also from gustatory/tactile modalities. Behavioral plasticity following differential brood care, changes in sensory exposure or the formation of associative long-term memory (LTM) was shown to be associated with structural plasticity in synaptic microcircuits (microglomeruli) within olfactory and visual compartments of the MB calyx. In the same line, physiological studies have demonstrated that MB-calyx microcircuits change response properties after associative learning. The aim of this review is to provide an update and synthesis of recent research on the plasticity of microcircuits in the MB calyx of the honeybee, specifically looking at the synaptic connectivity between sensory projection neurons (PNs) and MB intrinsic neurons (Kenyon cells). We focus on the honeybee as a favorable experimental insect for studying neuronal mechanisms underlying complex social behavior, but also compare it with other insect species for certain aspects. This review concludes by highlighting open questions and promising routes for future research aimed at understanding the causal relationships between neuronal and behavioral plasticity in this charismatic social insect.

Neuroanatomical correlates of mobility: Sensory brain centres are bigger in winged than in wingless parthenogenetic pea aphid females.

Many aphid species reproduce parthenogenetically throughout most of the year, with individuals having identical genomes. Nevertheless, aphid clones display a marked polyphenism with associated behavioural differences. Pea aphids (Acyrthosiphon pisum), when crowded, produce winged individuals, which have a larger dispersal range than wingless individuals. We examined here if brain structures linked to primary sensory processing and high-order motor control change in size as a function of wing polyphenism. Using micro-computing tomography (micro-CT) scans and immunocytochemical staining with anti-synapsin antibody, we reconstructed primary visual (optic lobes) and olfactory (antennal lobes) neuropils, together with the central body of winged and wingless parthenogenetic females of A. pisum for volume measurements. Absolute neuropil volumes were generally bigger in anti-synapsin labelled brains compared to micro-CT scans. This is potentially due to differences in rearing conditions of the used aphids. Independent of the method used, however, winged females consistently had larger antennal lobes and optic lobes than wingless females in spite of a larger overall body size of wingless compared to winged females. The volume of the central body, on the other hand was not significantly different between the two morphs. The larger primary sensory centres in winged aphids might thus provide the neuronal substrate for processing different environmental information due to the increased mobility during flight.

Neuronal Plasticity in the Mushroom-Body Calyx of Bumble Bee Workers During Early Adult Development.

Division of labor among workers is a key feature of social insects and frequently characterized by an age-related transition between tasks, which is accompanied by considerable structural changes in higher brain centers. Bumble bees (Bombus terrestris), in contrast, exhibit a size-related rather than an age-related task allocation, and thus workers may already start foraging at two days of age. We ask how this early behavioral maturation and distinct size variation are represented at the neuronal level and focused our analysis on the mushroom bodies (MBs), brain centers associated with sensory integration, learning and memory. To test for structural neuronal changes related to age, light exposure, and body size, whole-mount brains of age-marked workers were dissected for synapsin immunolabeling. MB calyx volumes, densities, and absolute numbers of olfactory and visual projection neuron (PN) boutons were determined by confocal laser scanning microscopy and three-dimensional image analyses. Dark-reared bumble bee workers showed an early age-related volume increase in olfactory and visual calyx subcompartments together with a decrease in PN-bouton density during the first three days of adult life. A 12:12  h light-dark cycle did not affect structural organization of the MB calyces compared to dark-reared individuals. MB calyx volumes and bouton numbers positively correlated with body size, whereas bouton density was lower in larger workers. We conclude that, in comparison to the closely related honey bees, neuronal maturation in bumble bees is completed at a much earlier stage, suggesting a strong correlation between neuronal maturation time and lifestyle in both species.

Experience during early adulthood shapes the learning capacities and the number of synaptic boutons in the mushroom bodies of honey bees (Apis mellifera).

The honey bee mushroom bodies (MBs) are brain centers required for specific learning tasks. Here, we show that environmental conditions experienced as young adults affect the maturation of MB neuropil and performance in a MB-dependent learning task. Specifically, olfactory reversal learning was selectively impaired following early exposure to an impoverished environment lacking some of the sensory and social interactions present in the hive. In parallel, the overall number of synaptic boutons increased within the MB olfactory neuropil, whose volume remained unaffected. This suggests that experience of the rich in-hive environment promotes MB maturation and the development of MB-dependent learning capacities.

Pitfalls of using confocal-microscopy based automated quantification of synaptic complexes in honeybee mushroom bodies (response to Peng and Yang 2016).

A recent study by Peng and Yang in Scientific Reports using confocal-microscopy based automated quantification of anti-synapsin labeled microglomeruli in the mushroom bodies of honeybee brains reports potentially incorrect numbers of microglomerular densities. Whereas several previous studies using visually supervised or automated counts from confocal images and analyses of serial 3D electron-microscopy data reported consistent numbers of synaptic complexes per volume, Peng and Yang revealed extremely low numbers differing by a factor of 18 or more from those obtained in visually supervised counts, and by a factor 22-180 from numbers in two other studies using automated counts. This extreme discrepancy is especially disturbing as close comparison of raw confocal images of anti-synapsin labeled whole-mount brain preparations are highly similar across these studies. We conclude that these discrepancies may reside in potential misapplication of confocal imaging followed by erroneous use of automated image analysis software. Consequently, the reported microglomerular densities during maturation and after manipulation by insecticides require validation by application of appropriate confocal imaging methods and analyses tools that rely on skilled observers. We suggest several improvements towards more reliable or standardized automated or semi-automated synapse counts in whole mount preparations of insect brains.

Daily Thermal Fluctuations Experienced by Pupae via Rhythmic Nursing Behavior Increase Numbers of Mushroom Body Microglomeruli in the Adult Ant Brain.

Social insects control brood development by using different thermoregulatory strategies. Camponotus mus ants expose their brood to daily temperature fluctuations by translocating them inside the nest following a circadian rhythm of thermal preferences. At the middle of the photophase brood is moved to locations at 30.8°C; 8 h later, during the night, the brood is transferred back to locations at 27.5°C. We investigated whether daily thermal fluctuations experienced by developing pupae affect the neuroarchitecture in the adult brain, in particular in sensory input regions of the mushroom bodies (MB calyces). The complexity of synaptic microcircuits was estimated by quantifying MB-calyx volumes together with densities of presynaptic boutons of microglomeruli (MG) in the olfactory lip and visual collar regions. We compared young adult workers that were reared either under controlled daily thermal fluctuations of different amplitudes, or at different constant temperatures. Thermal regimes significantly affected the large (non-dense) olfactory lip region of the adult MB calyx, while changes in the dense lip and the visual collar were less evident. Thermal fluctuations mimicking the amplitudes of natural temperature fluctuations via circadian rhythmic translocation of pupae by nurses (amplitude 3.3°C) lead to higher numbers of MG in the MB calyces compared to those in pupae reared at smaller or larger thermal amplitudes (0.0, 1.5, 9.6°C), or at constant temperatures (25.4, 35.0°C). We conclude that rhythmic control of brood temperature by nursing ants optimizes brain development by increasing MG densities and numbers in specific brain areas. Resulting differences in synaptic microcircuits are expected to affect sensory processing and learning abilities in adult ants, and may also promote interindividual behavioral variability within colonies.

Age-related and light-induced plasticity in opsin gene expression and in primary and secondary visual centers of the nectar-feeding ant Camponotus rufipes.

Camponotus rufipes workers are characterized by an age-related polyethism. In the initial weeks of adult life, young workers perform tasks inside the nest before they switch to multimodal foraging tasks outside. We tested the hypothesis that this transition is accompanied by profound adaptations in the peripheral and central visual systems. Our results show that C. rufipes workers of all tested ages (between 1 and 42 days) express three genes encoding for ultraviolet (UV), blue (BL), and long-wavelength (LW1) sensitive opsins in their retina, which are likely to provide the substrate for trichromatic color vision. Expression levels of all three opsin genes increased significantly within the first two weeks of adulthood and following light exposure. Interestingly, the volumes of all three optic neuropils (lamina, medulla, and lobula) showed corresponding volume increases. Tracing of connections to higher visual centers in the mushroom bodies (MBs) revealed only one optic pathway, the anterior superior optic tract, emerging from the medulla and sending segregated input to the MB-calyx collar. The MB collar volumes and densities of synaptic complexes (microglomeruli, MGs) increased with age. Exposure to light for 4 days induced a decrease in MG densities followed by an increase after extended light exposure. This shows that plasticity in retinal opsin gene expression and structural neuroplasticity in primary and secondary visual centers comprise both "experience-independent" and "experience-dependent" elements. We conclude that both sources of plasticity in the visual system represent important components promoting optimal timing of the interior-forager transition and flexibility of age-related division of labor. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1041-1057, 2016.

Neuronal plasticity in the mushroom body calyx during adult maturation in the honeybee and possible pheromonal influences.

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.

Density of mushroom body synaptic complexes limits intraspecies brain miniaturization in highly polymorphic leaf-cutting ant workers.

Hymenoptera possess voluminous mushroom bodies (MBs), brain centres associated with sensory integration, learning and memory. The mushroom body input region (calyx) is organized in distinct synaptic complexes (microglomeruli, MG) that can be quantified to analyse body size-related phenotypic plasticity of synaptic microcircuits in these small brains. Leaf-cutting ant workers (Atta vollenweideri) exhibit an enormous size polymorphism, which makes them outstanding to investigate neuronal adaptations underlying division of labour and brain miniaturization. We particularly asked how size-related division of labour in polymorphic workers is reflected in volume and total numbers of MG in olfactory calyx subregions. Whole brains of mini, media and large workers were immunolabelled with anti-synapsin antibodies, and mushroom body volumes as well as densities and absolute numbers of MG were determined by confocal imaging and three-dimensional analyses. The total brain volume and absolute volumes of olfactory mushroom body subdivisions were positively correlated with head widths, but mini workers had significantly larger MB to total brain ratios. Interestingly, the density of olfactory MG was remarkably independent from worker size. Consequently, absolute numbers of olfactory MG still were approximately three times higher in large compared with mini workers. The results show that the maximum packing density of synaptic microcircuits may represent a species-specific limit to brain miniaturization.

Age-related plasticity in the synaptic ultrastructure of neurons in the mushroom body calyx of the adult honeybee Apis mellifera.

The mushroom bodies are high-order sensory integration centers in the insect brain. In the honeybee, their main sensory input regions are large, doubled calyces with modality-specific, distinct sensory neuropil regions. We investigated adult structural plasticity of input synapses in the microglomeruli of the olfactory lip and visual collar. Synapsin-immunolabeled whole-mount brains reveal that during the natural transition from nursing to foraging, a significant volume increase in the calycal subdivisions is accompanied by a decreased packing density of boutons from input projection neurons. To investigate the associated ultrastructural changes at pre- and postsynaptic sites of individual microglomeruli, we employed serial-section electron microscopy. In general, the membrane surface area of olfactory and visual projection neuron boutons increased significantly between 1-day-old bees and foragers. Both types of boutons formed ribbon and non-ribbon synapses. The percentage of ribbon synapses per bouton was significantly increased in the forager. At each presynaptic site the numbers of postsynaptic partners-mostly Kenyon cell dendrites-likewise increased. Ribbon as well as non-ribbon synapses formed mainly dyads in the 1-day-old bee, and triads in the forager. In the visual collar, outgrowing Kenyon cell dendrites form about 140 contacts upon a projection neuron bouton in the forager compared with only about 95 in the 1-day-old bee, resulting in an increased divergence ratio between the two stages. This difference suggests that synaptic changes in calycal microcircuits of the mushroom body during periods of altered sensory activity and experience promote behavioral plasticity underlying polyethism and social organization in honeybee colonies.

Comparison of microglomerular structures in the mushroom body calyx of neopteran insects.

Mushroom bodies (MBs) are prominent neuropils in the insect brain involved in higher order processing such as sensory integration, learning and memory, and spatial orientation. The size and general morphology of MBs are diverse across insects. In this study we comparatively investigated the microstructure of synaptic complexes (microglomeruli) in major sensory input regions of the MBs, the calyces, across various neopteran insect species. Pre- and postsynaptic compartments of microglomeruli were analyzed using anti-synapsin immunocytochemistry, f-actin-phalloidin labeling and high-resolution confocal microscopy. Our results suggest that calycal microglomeruli are present across all investigated neopteran insect species, but differences are found in the distribution of synapsin and f-actin within their pre- and postsynaptic compartments. Hymenopteran MBs contain the highest number and packing density of microglomeruli compared to all other species from the different insect orders we investigated. We conclude that the evolution of high numbers of microglomeruli in Hymenoptera may reflect an increase in synaptic microcircuits, which could enhance the computational capacities of the MBs.

Brain plasticity in Diptera and Hymenoptera.

To mediate different types of behaviour, nervous systems need to coordinate the proper operation of their neural circuits as well as short- and long-term alterations that occur within those circuits. The latter ultimately devolve upon specific changes in neuronal structures, membrane properties and synaptic connections that are all examples of plasticity. This reorganization of the adult nervous system is shaped by internal and external influences both during development and adult maturation. In adults, behavioural experience is a major driving force of neuronal plasticity studied particularly in sensory systems. The range of adaptation depends on features that are important to a particular species, and is therefore specific, so that learning is essential for foraging in honeybees, while regenerative capacities are important in hemimetabolous insects with long appendages. Experience is usually effective during a critical period in early adult life, when neural function becomes tuned to future conditions in an insect's life. Tuning occur at all levels, in synaptic circuits, neuropile volumes, and behaviour. There are many examples, and this review incorporates only a select few, mainly those from Diptera and Hymenoptera.

Synaptic organization in the adult Drosophila mushroom body calyx.

Insect mushroom bodies are critical for olfactory associative learning. We have carried out an extensive quantitative description of the synaptic organization of the calyx of adult Drosophila melanogaster, the main olfactory input region of the mushroom body. By using high-resolution confocal microscopy, electron microscopy-based three-dimensional reconstructions, and genetic labeling of the neuronal populations contributing to the calyx, we resolved the precise connections between large cholinergic boutons of antennal lobe projection neurons and the dendrites of Kenyon cells, the mushroom body intrinsic neurons. Throughout the calyx, these elements constitute synaptic complexes called microglomeruli. By single-cell labeling, we show that each Kenyon cell's claw-like dendritic specialization is highly enriched in filamentous actin, suggesting that this might be a site of plastic reorganization. In fact, Lim kinase (LimK) overexpression in the Kenyon cells modifies the shape of the microglomeruli. Confocal and electron microscopy indicate that each Kenyon cell claw enwraps a single bouton of a projection neuron. Each bouton is contacted by a number of such claw-like specializations as well as profiles of gamma-aminobutyric acid-positive neurons. The dendrites of distinct populations of Kenyon cells involved in different types of memory are partially segregated within the calyx and contribute to different subsets of microglomeruli. Our analysis suggests, though, that projection neuron boutons can contact more than one type of Kenyon cell. These findings represent an important basis for the functional analysis of the olfactory pathway, including the formation of associative olfactory memories.

Caste-specific postembryonic development of primary and secondary olfactory centers in the female honeybee brain.

Eusocial insects are characterized by division of labor among a sterile worker caste and a reproductive queen. In the honeybee both female castes are determined postembryonically by environmental factors, and queens develop substantially faster than workers. Since olfaction plays a crucial role in organizing honeybee behavior and social interactions, we compared the development of primary and secondary olfactory centers in the brain. Age-synchronized queen and worker pupae were raised in incubators at 34.5 degrees C, and their external morphology was characterized for all pupal stages. The development of olfactory synaptic neuropil was analyzed using anti-synapsin immunocytochemistry, f-actin-phalloidin labeling and confocal microscopy. In the antennal lobes of queens olfactory glomeruli formed approximately 4 days earlier than in workers. The adult number of olfactory glomeruli was in a similar range, but the total glomerular volume was slightly smaller in queens. Olfactory and visual subdivisions (lip, collar) of the mushroom-body calyx formed early, whereas the basal ring separated late. Synaptic microglomeruli in the olfactory lip were established approximately 3-4 days earlier in queens compared to workers. We propose that developmental heterochrony results in fewer synapses in olfactory centers (smaller glomeruli, fewer microglomeruli) in queens, which may result in poorer performance on olfactory learning tasks compared to workers.

Environment- and age-dependent plasticity of synaptic complexes in the mushroom bodies of honeybee queens.

Diversity in behavior plays a crucial role for the division of labor in insect societies. Social insects such as honeybees provide excellent model systems to investigate neuronal principles underlying behavioral plasticity. The two female castes, queens and workers, differ substantially in anatomy, physiology, aging and behavior. The different phenotypes are induced by environmental factors rather than genetic differences. Here we investigated environment- and age-dependent effects on the synaptic organization within higher order neuropils of the honeybee brain. Synaptic complexes (microglomeruli) in sensory-input regions of the mushroom bodies, prominent higher sensory integration centers, were analyzed quantitatively using fluorescent markers and confocal microscopy. Pre- and postsynaptic compartments of individual microglomeruli were labeled by anti-synapsin immunolabeling and f-actin detection with phalloidin in dendritic spines of mushroom-body intrinsic neurons. The results demonstrate that in queens the numbers of microglomeruli in the olfactory and visual input regions of the mushroom-body calyx are significantly lower than in workers. In queens raised in incubators, microglomeruli were affected by differences in pupal rearing temperature within the range of naturally occurring temperatures (32-36 degrees C). The highest numbers of microglomeruli developed at a lower temperature compared to workers (33.5 vs. 34.5 degrees C). We found a striking adult plasticity of microglomeruli numbers throughout the extended life-span of queens. Whereas microglomeruli in the olfactory lip increased with age ( approximately 55%), microglomeruli in the visual collar significantly decreased ( approximately 35%). We propose that developmental and adult plasticity of the synaptic circuitry in the mushroom-body calyx might underlie caste- and age-specific adaptations in behavior.

Synaptic organization in the adult honey bee brain is influenced by brood-temperature control during pupal development.

Recent studies have shown that the behavioral performance of adult honey bees is influenced by the temperature experienced during pupal development. Here we explore whether there are temperature-mediated effects on the brain. We raised pupae at different constant temperatures between 29 and 37 degrees C and performed neuroanatomical analyses of the adult brains. Analyses focused on sensory-input regions in the mushroom bodies, brain areas associated with higher-order processing such as learning and memory. Distinct synaptic complexes [microglomeruli (MG)] within the mushroom body calyces were visualized by using fluorophore-conjugated phalloidin and an antibody to synapsin. The numbers of MG were different in bees that had been raised at different temperatures, and these differences persisted after the first week of adult life. In the olfactory-input region (lip), MG numbers were highest in bees raised at the temperature normally maintained in brood cells (34.5 degrees C) and significantly decreased in bees raised at 1 degrees C below and above this norm. Interestingly, in the neighboring visual-input region (collar), MG numbers were less affected by temperature. We conclude that thermoregulatory control of brood rearing can generate area- and modality-specific effects on synaptic neuropils in the adult brain. We propose that resulting differences in the synaptic circuitry may affect neuronal plasticity and may underlie temperature-mediated effects on multimodal communication and learning.