Importance of magnetic information for neuronal plasticity in desert ants.

Many animal species rely on the Earth's magnetic field during navigation, but where in the brain magnetic information is processed is still unknown. To unravel this, we manipulated the natural magnetic field at the nest entrance of Cataglyphis desert ants and investigated how this affects relevant brain regions during early compass calibration. We found that manipulating the Earth's magnetic field has profound effects on neuronal plasticity in two sensory integration centers. Magnetic field manipulations interfere with a typical look-back behavior during learning walks of naive ants. Most importantly, structural analyses in the ants' neuronal compass (central complex) and memory centers (mushroom bodies) demonstrate that magnetic information affects neuronal plasticity during early visual learning. This suggests that magnetic information does not only serve as a compass cue for navigation but also as a global reference system crucial for spatial memory formation. We propose a neural circuit for integration of magnetic information into visual guidance networks in the ant brain. Taken together, our results provide an insight into the neural substrate for magnetic navigation in insects.

3D atlas of cerebral neuropils with previously unknown demarcations in the honey bee brain.

Honey bees (Apis mellifera) express remarkable social interactions and cognitive capabilities that have been studied extensively. In many cases, behavioral studies were accompanied by neurophysiological and neuroanatomical investigations. While most studies have focused on primary sensory neuropils, such as the optic lobes or antennal lobes, and major integration centers, such as the mushroom bodies or the central complex, many regions of the cerebrum (the central brain without the optic lobes) of the honey bee are only poorly explored so far, both anatomically and physiologically. To promote studies of these brain regions, we used anti-synapsin immunolabeling and neuronal tract tracings followed by confocal imaging and 3D reconstructions to demarcate all neuropils in the honey bee cerebrum and close this gap at the anatomical level. We demarcated 35 neuropils and 25 fiber tracts in the honey bee cerebrum, most of which have counterparts in the fly (Drosophila melanogaster) and other insect species that have been investigated so far at this level of detail. We discuss the role of cerebral neuropils in multisensory integration in the insect brain, emphasize the importance of this brain atlas for comparative studies, and highlight specific architectural features of the honey bee cerebrum.

Rotation of skylight polarization during learning walks is necessary to trigger neuronal plasticity in Cataglyphis ants.

Many animals use celestial cues for impressive navigational performances in challenging habitats. Since the position of the sun and associated skylight cues change throughout the day and season, it is crucial to correct for these changes. Cataglyphis desert ants possess a time-compensated skylight compass allowing them to navigate back to their nest using the shortest way possible. The ants have to learn the sun's daily course (solar ephemeris) during initial learning walks (LW) before foraging. This learning phase is associated with substantial structural changes in visual neuronal circuits of the ant's brain. Here, we test whether the rotation of skylight polarization during LWs is the necessary cue to induce learning-dependent rewiring in synaptic circuits in high-order integration centres of the ant brain. Our results show that structural neuronal changes in the central complex and mushroom bodies are triggered only when LWs were performed under a rotating skylight polarization pattern. By contrast, when naive ants did not perform LWs, but were exposed to skylight cues, plasticity was restricted to light spectrum-dependent changes in synaptic complexes of the lateral complex. The results identify sky-compass cues triggering learning-dependent versus -independent neuronal plasticity during the behavioural transition from interior workers to outdoor foragers.

Sex-specific and caste-specific brain adaptations related to spatial orientation in Cataglyphis ants.

Cataglyphis desert ants are charismatic central place foragers. After long-ranging foraging trips, individual workers navigate back to their nest relying mostly on visual cues. The reproductive caste faces other orientation challenges, i.e. mate finding and colony foundation. Here we compare brain structures involved in spatial orientation of Cataglyphis nodus males, gynes, and foragers by quantifying relative neuropil volumes associated with two visual pathways, and numbers and volumes of antennal lobe (AL) olfactory glomeruli. Furthermore, we determined absolute numbers of synaptic complexes in visual and olfactory regions of the mushroom bodies (MB) and a major relay station of the sky-compass pathway to the central complex (CX). Both female castes possess enlarged brain centers for sensory integration, learning, and memory, reflected in voluminous MBs containing about twice the numbers of synaptic complexes compared with males. Overall, male brains are smaller compared with both female castes, but the relative volumes of the optic lobes and CX are enlarged indicating the importance of visual guidance during innate behaviors. Male ALs contain greatly enlarged glomeruli, presumably involved in sex-pheromone detection. Adaptations at both the neuropil and synaptic levels clearly reflect differences in sex-specific and caste-specific demands for sensory processing and behavioral plasticity underlying spatial orientation.

Novel structure in the nuclei of honey bee brain neurons revealed by immunostaining.

In the course of a screen designed to produce antibodies (ABs) with affinity to proteins in the honey bee brain we found an interesting AB that detects a highly specific epitope predominantly in the nuclei of Kenyon cells (KCs). The observed staining pattern is unique, and its unfamiliarity indicates a novel previously unseen nuclear structure that does not colocalize with the cytoskeletal protein f-actin. A single rod-like assembly, 3.7-4.1 µm long, is present in each nucleus of KCs in adult brains of worker bees and drones with the strongest immuno-labelling found in foraging bees. In brains of young queens, the labelling is more sporadic, and the rod-like structure appears to be shorter (~ 2.1 µm). No immunostaining is detectable in worker larvae. In pupal stage 5 during a peak of brain development only some occasional staining was identified. Although the cellular function of this unexpected structure has not been determined, the unusual distinctiveness of the revealed pattern suggests an unknown and potentially important protein assembly. One possibility is that this nuclear assembly is part of the KCs plasticity underlying the brain maturation in adult honey bees. Because no labelling with this AB is detectable in brains of the fly Drosophila melanogaster and the ant Camponotus floridanus, we tentatively named this antibody AmBNSab (Apis mellifera Brain Neurons Specific antibody). Here we report our results to make them accessible to a broader community and invite further research to unravel the biological role of this curious nuclear structure in the honey bee central 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.

The brain of Cataglyphis ants: Neuronal organization and visual projections.

Cataglyphis ants are known for their outstanding navigational abilities. They return to their inconspicuous nest after far-reaching foraging trips using path integration, and whenever available, learn and memorize visual features of panoramic sceneries. To achieve this, the ants combine directional visual information from celestial cues and panoramic scenes with distance information from an intrinsic odometer. The largely vision-based navigation in Cataglyphis requires sophisticated neuronal networks to process the broad repertoire of visual stimuli. Although Cataglyphis ants have been subjected to many neuroethological studies, little is known about the general neuronal organization of their central brain and the visual pathways beyond major circuits. Here, we provide a comprehensive, three-dimensional neuronal map of synapse-rich neuropils in the brain of Cataglyphis nodus including major connecting fiber systems. In addition, we examined neuronal tracts underlying the processing of visual information in more detail. This study revealed a total of 33 brain neuropils and 30 neuronal fiber tracts including six distinct tracts between the optic lobes and the cerebrum. We also discuss the importance of comparative studies on insect brain architecture for a profound understanding of neuronal networks and their function.

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.

Distributed plasticity in ant visual pathways following colour learning.

Colour processing at early stages of visual pathways is a topic of intensive study both in vertebrate and invertebrate species. However, it is still unclear how colour learning and memory formation affects an insect brain in the peripheral processing stages and high-order integration centres, and whether associative colour experiences are reflected in plasticity of underlying neuronal circuits. To address this issue, we used Camponotus blandus ants as their proven colour learning and memory capabilities, precisely controllable age and experience, and already known central visual pathways offer unique access to analyse plasticity in neuronal circuits for colour vision in a miniature brain. The potential involvement of distinct neuropils-optic lobes (OLs), mushroom body (MB) input (collar) and output (vertical lobe), anterior optic tubercle (AOTU) and central complex (CX)-in associative colour experiences was assessed by quantification of volumetric and synaptic changes (MB collar) directly after colour conditioning and, 3 days later, after the establishment of long-term memory (LTM). To account for potential effects of non-associative light exposure, we compared neuronal changes in the brain of colour-naive foragers with those of foragers that had been exposed to light in a non-associative way. The results clearly show that the OLs, AOTU, and CX respond with plastic changes after colour learning and LTM formation. This suggests a complex neuronal network for colour learning and memory formation involving multiple brain levels. Such a colour-processing network probably represents an efficient design promoting fast and accurate behavioural decisions during orientation and navigation.

Opsin expression patterns coincide with photoreceptor development during pupal development in the honey bee, Apis mellifera.

BACKGROUND: The compound eyes of insects allow them to catch photons and convert the energy into electric signals. All compound eyes consist of numerous ommatidia, each comprising a fixed number of photoreceptors. Different ommatidial types are characterized by a specific set of photoreceptors differing in spectral sensitivity. In honey bees, males and females possess different ommatidial types forming distinct retinal mosaics. However, data are lacking on retinal ontogeny and the mechanisms by which the eyes are patterned. In this study, we investigated the intrinsic temporal and circadian expression patterns of the opsins that give rise to the ultraviolet, blue and green sensitive photoreceptors, as well as the morphological maturation of the retina during pupal development of honey bees. RESULTS: qPCR and histological labeling revealed that temporal opsin mRNA expression differs between sexes and correlates with rhabdom elongation during photoreceptor development. In the first half of the pupal stage, when the rhabdoms of the photoreceptors are still short, worker and (dorsal) drone retinae exhibit similar expression patterns with relatively high levels of UV (UVop) and only marginal levels of blue (BLop) and green (Lop1) opsin mRNA. In the second half of pupation, when photoreceptors and rhabdoms elongate, opsin expression in workers becomes dominated by Lop1 mRNA. In contrast, the dorsal drone eye shows high expression levels of UVop and BLop mRNA, whereas Lop1 mRNA level decreases. Interestingly, opsin expression levels increase up to 22-fold during early adult life. We also found evidence that opsin expression in adult bees is under the control of the endogenous clock. CONCLUSIONS: Our data indicate that the formation of the sex-specific retinal composition of photoreceptors takes place during the second half of the pupal development, and that opsin mRNA expression levels continue to increase in young bees, which stands in contrast to Drosophila, where the highest expression levels are found during the late pupal stage and remain constant in adults. From an evolutionary perspective, we hypothesize that the delayed retinal maturation during the early adult phase is linked to the delayed transition from indoor to outdoor activities in bees, when vision becomes important.

The Role of Celestial Compass Information in Cataglyphis Ants during Learning Walks and for Neuroplasticity in the Central Complex and Mushroom Bodies.

Central place foragers are faced with the challenge to learn the position of their nest entrance in its surroundings, in order to find their way back home every time they go out to search for food. To acquire navigational information at the beginning of their foraging career, Cataglyphis noda performs learning walks during the transition from interior worker to forager. These small loops around the nest entrance are repeatedly interrupted by strikingly accurate back turns during which the ants stop and precisely gaze back to the nest entrance-presumably to learn the landmark panorama of the nest surroundings. However, as at this point the complete navigational toolkit is not yet available, the ants are in need of a reference system for the compass component of the path integrator to align their nest entrance-directed gazes. In order to find this directional reference system, we systematically manipulated the skylight information received by ants during learning walks in their natural habitat, as it has been previously suggested that the celestial compass, as part of the path integrator, might provide such a reference system. High-speed video analyses of distinct learning walk elements revealed that even exclusion from the skylight polarization pattern, UV-light spectrum and the position of the sun did not alter the accuracy of the look back to the nest behavior. We therefore conclude that C. noda uses a different reference system to initially align their gaze directions. However, a comparison of neuroanatomical changes in the central complex and the mushroom bodies before and after learning walks revealed that exposure to UV light together with a naturally changing polarization pattern was essential to induce neuroplasticity in these high-order sensory integration centers of the ant brain. This suggests a crucial role of celestial information, in particular a changing polarization pattern, in initially calibrating the celestial compass system.

Neuronal distribution of tyramine and the tyramine receptor AmTAR1 in the honeybee brain.

Tyramine is an important neurotransmitter, neuromodulator, and neurohormone in insects. In honeybees, it is assumed to have functions in modulating sensory responsiveness and controlling motor behavior. Tyramine can bind to two characterized receptors in honeybees, both of which are coupled to intracellular cAMP pathways. How tyramine acts on neuronal, cellular and circuit levels is unclear. We investigated the spatial brain expression of the tyramine receptor AmTAR1 using a specific antibody. This antibody detects a membrane protein of the expected molecular weight in western blot analysis. In honeybee brains, it labels different structures which process sensory information. Labeling along the antennal nerve, in projections of the dorsal lobe and in the gnathal ganglion suggest that tyramine receptors are involved in modulating gustatory and tactile perception. Furthermore, the ellipsoid body of the central complex and giant synapses in the lateral complex show AmTAR1-like immunoreactivity (AmTAR1-IR), suggesting a role of this receptor in modulating sky-compass information and/or higher sensor-motor control. Additionally, intense signals derive from the mushroom bodies, higher-order integration centers for olfactory, visual, gustatory and tactile information. To investigate whether AmTAR1-expressing brain structures are in vicinity to tyramine releasing sites, a specific tyramine antibody was applied. Tyramine-like labeling was observed in AmTAR1-IR positive structures, although it was sometimes weak and we did not always find a direct match of ligand and receptor. Moreover, tyramine-like immunoreactivity was also found in brain regions without AmTAR1-IR (optic lobes, antennal lobes), indicating that other tyramine-specific receptors may be expressed there.

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.

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.

Royal jelly-like protein localization reveals differences in hypopharyngeal glands buildup and conserved expression pattern in brains of bumblebees and honeybees.

Royal jelly proteins (MRJPs) of the honeybee bear several open questions. One of them is their expression in tissues other than the hypopharyngeal glands (HGs), the site of royal jelly production. The sole MRJP-like gene of the bumblebee, Bombus terrestris (BtRJPL), represents a pre-diversification stage of the MRJP gene evolution in bees. Here we investigate the expression of BtRJPL in the HGs and the brain of bumblebees. Comparison of the HGs of bumblebees and honeybees revealed striking differences in their morphology with respect to sex- and caste-specific appearance, number of cells per acinus, and filamentous actin (F-actin) rings. At the cellular level, we found a temporary F-actin-covered meshwork in the secretory cells, which suggests a role for actin in the biogenesis of the end apparatus in HGs. Using immunohistochemical localization, we show that BtRJPL is expressed in the bumblebee brain, predominantly in the Kenyon cells of the mushroom bodies, the site of sensory integration in insects, and in the optic lobes. Our data suggest that a dual gland-brain function preceded the multiplication of MRJPs in the honeybee lineage. In the course of the honeybee evolution, HGs dramatically changed their morphology in order to serve a food-producing function.

The mushroom body of adult Drosophila characterized by GAL4 drivers.

The mushroom body is required for a variety of behaviors of Drosophila melanogaster. Different types of intrinsic and extrinsic mushroom body neurons might underlie its functional diversity. There have been many GAL4 driver lines identified that prominently label the mushroom body intrinsic neurons, which are known as "Kenyon cells." Under one constant experimental condition, we analyzed and compared the the expression patterns of 25 GAL4 drivers labeling the mushroom body. As an internet resource, we established a digital catalog indexing representative confocal data of them. Further more, we counted the number of GAL4-positive Kenyon cells in each line. We found that approximately 2,000 Kenyon cells can be genetically labeled in total. Three major Kenyon cell subtypes, the gamma, alpha'/beta', and alpha/beta neurons, respectively, contribute to 33, 18, and 49% of 2,000 Kenyon cells. Taken together, this study lays groundwork for functional dissection of the mushroom body.