Generating parallel representations of position and identity in the olfactory system.

In Drosophila, a dedicated olfactory channel senses a male pheromone, cis-vaccenyl acetate (cVA), promoting female courtship while repelling males. Here, we show that separate cVA-processing streams extract qualitative and positional information. cVA sensory neurons respond to concentration differences in a 5-mm range around a male. Second-order projection neurons encode the angular position of a male by detecting inter-antennal differences in cVA concentration, which are amplified through contralateral inhibition. At the third circuit layer, we identify 47 cell types with diverse input-output connectivity. One population responds tonically to male flies, a second is tuned to olfactory looming, while a third integrates cVA and taste to coincidentally promote female mating. The separation of olfactory features resembles the mammalian what and where visual streams; together with multisensory integration, this enables behavioral responses appropriate to specific ethological contexts.

Beyond standard PSE testing: An exploratory study of bioimpedance as a marker for ham defects.

During post-mortem conversion from muscle to meat, diverse quality anomalies can emerge. Recent pork defects are often accompanied by deteriorating fibre structure. Here we investigate how bioimpedance response, an indicator of structural disintegration, can help in detecting quality defects. We, first, measured the relationship between standard meat quality variables (pHu, CIELAB, drip loss) and bioimpedance (BI) response. To screen for defect-biomarkers that are linked to aberrant bioimpedance and physicochemical indicators of quality decline, we performed LC-MS/MS proteomic analysis on samples, classified with a multivariate-based separation into good versus poor quality. We found that BI correlated significantly with, e.g., colour and drip loss. Proteomics revealed eleven proteins to be unique for either, good or poor ham quality groups, and maybe linked to structural degradation. In all, our data supports a wider integration of BI testing in pork quality testing to assess structural disintegration, which can render ham unsuitable for, e.g., costly curing.

The neuronal logic of how internal states control food choice.

When deciding what to eat, animals evaluate sensory information about food quality alongside multiple ongoing internal states1-10. How internal states interact to alter sensorimotor processing and shape decisions such as food choice remains poorly understood. Here we use pan-neuronal volumetric activity imaging in the brain of Drosophila melanogaster to investigate the neuronal basis of internal state-dependent nutrient appetites. We created a functional atlas of the ventral fly brain and find that metabolic state shapes sensorimotor processing across large sections of the neuropil. By contrast, reproductive state acts locally to define how sensory information is translated into feeding motor output. These two states thus synergistically modulate protein-specific food intake and food choice. Finally, using a novel computational strategy, we identify driver lines that label neurons innervating state-modulated brain regions and show that the newly identified 'borboleta' region is sufficient to direct food choice towards protein-rich food. We thus identify a generalizable principle by which distinct internal states are integrated to shape decision making and propose a strategy to uncover and functionally validate how internal states shape behaviour.

Nuclear translocation of vitellogenin in the honey bee (Apis mellifera).

Vitellogenin (Vg) is a conserved protein used by nearly all oviparous animals to produce eggs. It is also pleiotropic and performs functions in oxidative stress resistance, immunity, and, in honey bees, behavioral development of the worker caste. It has remained enigmatic how Vg affects multiple traits. Here, we asked whether Vg enters the nucleus and acts via DNA-binding. We used cell fractionation, immunohistology, and cell culture to show that a structural subunit of honey bee Vg translocates into cell nuclei. We then demonstrated Vg-DNA binding theoretically and empirically with prediction software and chromatin immunoprecipitation with sequencing (ChIP-seq), finding binding sites at genes influencing immunity and behavior. Finally, we investigated the immunological and enzymatic conditions affecting Vg cleavage and nuclear translocation and constructed a 3D structural model. Our data are the first to show Vg in the nucleus and suggest a new fundamental regulatory role for this ubiquitous protein. Supplementary information: The online version contains supplementary material available at 10.1007/s13592-022-00914-9.

Bioimpedance-based Authentication of Defrosted Versus Fresh Pork at the End of Refrigerated Shelf Life.

Correct food labeling is a legal requirement and helps consumers to make informed purchasing choices. Mislabeling defrosted meat as fresh is illegal in the EU. However, there are no standardized technologies to authenticate fresh versus defrosted meat. We address this by testing if bioimpedance-based measurements can separate defrosted meat from refrigerated-only meat at the end of shelf life, i.e., when also fresh meat shows deterioration. Pork sirloin samples from 20 pigs were first tested at 12 days postmortem ('fresh group'). This time point was chosen to represent a typical use-by date for refrigerated storage of fresh pork. Then, all samples were transferred to a -24°C freezer for 3 days and thawed for 2 days before final testing ('frozen-thawed group'). Bioimpedance analyses (BIA) were done in a frequency range of [102-106 Hz]. Weight, pH and electrode positioning were assessed to test for potential confounding effects. Statistics for treatment dependent differences were based on the established Py parameter and phase angle, which were extracted from the BI spectra. We found that using bioimpedance testing with tetrapolar electrodes, Py and phase angle allowed almost complete separation of fresh and previously frozen samples. However, within the whole sample population, there was some overlap between the spectra of fresh and frozen samples. Yet, based on Py, only one fresh sample (5% of Ntotal=20) fell in the lowest Py class with all the frozen samples. We used a multifactorial design that allowed to test the effects of potential confounding factors, such as electrode positioning and meat quality parameters. We found a relatively low explained variance for the Py parameter, indicating that confounding effects from other factors or quality defects in fresh pork may affect the detection capacity of bioimpedance-based authentication of fresh pork. Our data, therefore, suggest that reliable fresh-label authentication with bioimpedance testing should be based on testing a small number of samples to represent a specific lot of pork that is to be inspected.

Screening bioactive food compounds in honey bees suggests curcumin blocks alcohol-induced damage to longevity and DNA methylation.

Various bioactive food compounds may confer health and longevity benefits, possibly through altering or preserving the epigenome. While bioactive food compounds are widely being marketed for human consumption as 'improving health and longevity' by counteracting harmful effects of poor nutrition and lifestyle, claimed effects are often not adequately documented. Using the honey bee (Apis mellifera) as a model species, we here employed a multi-step screening approach to investigate seven compounds for effects on lifespan and DNA methylation using ELISA and whole genome bisulfite sequencing (WGBS). A positive longevity effect was detected for valproic acid, isovaleric acid, and cyanocobalamin. For curcumin, we found that lifespan shortening caused by ethanol intake, was restored when curcumin and ethanol were co-administered. Furthermore, we identified region specific DNA methylation changes as a result of ethanol intake. Ethanol specific changes in DNA methylation were fully or partially blocked in honey bees receiving ethanol and curcumin together. Ethanol-affected and curcumin-blocked differentially methylated regions covered genes involved in fertility, temperature regulation and tubulin transport. Our results demonstrate fundamental negative effects of low dose ethanol consumption on lifespan and associated DNA methylation changes and present a proof-of-principle on how longevity and DNA methylation changes can be negated by the bioactive food component curcumin. Our findings provide a fundament for further studies of curcumin in invertebrates.

Feasibility of Using Electrical Impedance Spectroscopy for Assessing Biological Cell Damage during Freezing and Thawing.

This study was performed to test bioimpedance as a tool to detect the effect of different thawing methods on meat quality to aid in the eventual creation of an electric impedance-based food quality monitoring system. The electric impedance was measured for fresh pork, thawed pork, and during quick and slow thawing. A clear difference was observed between fresh and thawed samples for both impedance parameters. Impedance was different between the fresh and the frozen-thawed samples, but there were no impedance differences between frozen-thawed samples and the ones that were frozen-thawed and then stored at +3 °C for an additional 16 h after thawing. The phase angle was also different between fresh and the frozen-thawed samples. At high frequency, there were small, but clear phase angle differences between frozen-thawed samples and the samples that were frozen-thawed and subsequently stored for more than 16 h at +3 °C. Furthermore, the deep learning model LSTM-RNN (long short-term memory recurrent neural network) was found to be a promising way to classify the different methods of thawing.

TReND in Africa: Toward a Truly Global (Neuro)science Community.

TReND is a volunteer-scientist run charity dedicated to promoting research and education on the African continent. Focusing on neuroscience, we discuss approaches to address some of the factors that currently stifle Africa's scientific development and our experience in implementing them.

Publisher Correction: An expression atlas of variant ionotropic glutamate receptors identifies a molecular basis of carbonation sensing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Monitoring Electric Impedance During Freezing and Thawing of Saline and De-ionized Water.

Physiological saline (0.9% NaCl) and deionized water were frozen in a laboratory chest freezer and impedance was monitored throughout freezing and thawing. The resistive and reactive components of electrical impedance were measured for these samples during freezing and thawing (heating) within a temperature range between 20 °C and -48 °C. The impedance of saline solution and de-ionized water increases sharply at the freezing point, similar to what is known for, e.g., complex tissues, including meat. Yet, only the saline solution impedance shows another sharp increment at a temperature between -30 and -20 °C. Changes of the electric properties after solidification suggest that the latter is linked to transformations of the ice lattice structure. We conclude that the electrical properties might serve as sensitive indicators of these phase changes.

Nutrient homeostasis - translating internal states to behavior.

Behavioral neuroscience aims to describe a causal relationship between neuronal processes and behavior. Animals' ever-changing physiological needs alter their internal states. Internal states then alter neuronal processes to adapt the behavior of the animal enabling it to meet its needs. Here, we describe nutrient-specific appetites as an attractive framework to study how internal states shape complex neuronal processes and resulting behavioral outcomes. Understanding how neurons detect nutrient states and how these are integrated at the level of neuronal circuits will provide a multilevel description of the mechanisms underlying complex feeding and foraging decisions.

Detectability of the degree of freeze damage in meat depends on analytic-tool selection.

Novel freezing solutions are constantly being developed to reduce quality loss in meat production chains. However, there is limited focus on identifying the sensitive analytical tools needed to directly validate product changes that result from potential improvements in freezing technology. To benchmark analytical tools relevant to meat research and production, we froze pork samples using traditional (-25 °C, -35 °C) and cryogenic freezing (-196 °C). Three classes of analyses were tested for their capacity to separate different freeze treatments: thaw loss testing, bioelectrical spectroscopy (nuclear magnetic resonance, microwave, bioimpedance) and low-temperature microscopy (cryo-SEM). A general effect of freeze treatment was detected with all bioelectrical methods. Yet, only cryo-SEM resolved quality differences between all freeze treatments, not only between cryogenic and traditional freezing. The detection sensitivity with cryo-SEM may be explained by testing meat directly in the frozen state without prior defrosting. We discuss advantages, shortcomings and cost factors in using analytical tools for quality monitoring in the meat sector.

Complications and technical failures are rare in knee ligament reconstruction: analyses based on 31,326 reconstructions during 10 years in Denmark.

PURPOSE: Knowledge about treatment injuries (technical failures and complications) in relation to knee ligament reconstructions is sparse. Our purpose was to describe treatment injuries to knee ligament reconstruction in Denmark during a 10-year period and to suggest initiatives to reduce the risk for treatment injuries. METHODS: Treatment injuries after knee ligament reconstructions reported to the Danish Patient Compensation Association (DPCA) 2005-2014 were analyzed and compared to information from the Danish Kneeligament Reconstruction Register and Danish National Patient Register. RESULTS: The number of knee ligament reconstructions in Denmark 2005-2014, including revisions, was 31,326. Of the 704 cases claimed to DPCA, 371 were approved as treatment injuries (1.42% of all operations). Tunnel malpositioning (135 = 0.43% of all operations), deep infection (0.27%), nerve injury (0.17%), pain (0.12%) and unrecognized combined instability (0.11%) were the most common. Patients operated with anteromedial technique for femoral tunnel placement had a lower incidence (p < 0.0001) of tunnel malpositioning compared to other techniques. Public and private hospitals had the same risk of treatment injures. Hospitals that performed the largest number of reconstructions/year had the smallest risk of a treatment injury (p < 0.001). The total compensation was 7.6 m EURO, which equals 243 EURO/performed knee ligament reconstruction. CONCLUSION: Treatment injuries as defined in this study were rare, with tunnel malpositioning being the most common. Analyses of the results indicate that it may be clinically relevant to reduce the number of treatment injures that knee ligament reconstructions are concentrated in clinics with high volume. Also, malpositioning might be reduced by routine documentation of K-wire position before tunnels are drilled. LEVEL OF EVIDENCE: III.

An expression atlas of variant ionotropic glutamate receptors identifies a molecular basis of carbonation sensing.

Through analysis of the Drosophila ionotropic receptors (IRs), a family of variant ionotropic glutamate receptors, we reveal that most IRs are expressed in peripheral neuron populations in diverse gustatory organs in larvae and adults. We characterise IR56d, which defines two anatomically-distinct neuron classes in the proboscis: one responds to carbonated solutions and fatty acids while the other represents a subset of sugar- and fatty acid-sensing cells. Mutational analysis indicates that IR56d, together with the broadly-expressed co-receptors IR25a and IR76b, is essential for physiological responses to carbonation and fatty acids, but not sugars. We further demonstrate that carbonation and fatty acids both promote IR56d-dependent attraction of flies, but through different behavioural outputs. Our work provides a toolkit for investigating taste functions of IRs, defines a subset of these receptors required for carbonation sensing, and illustrates how the gustatory system uses combinatorial expression of sensory molecules in distinct neurons to coordinate behaviour.

Metabolic enzymes in glial cells of the honeybee brain and their associations with aging, starvation and food response.

The honey bee has been extensively studied as a model for neuronal circuit and memory function and more recently has emerged as an unconventional model in biogerontology. Yet, the detailed knowledge of neuronal processing in the honey bee brain contrasts with the very sparse information available on glial cells. In other systems glial cells are involved in nutritional homeostasis, detoxification, and aging. These glial functions have been linked to metabolic enzymes, such as glutamine synthetase and glycogen phosphorylase. As a step in identifying functional roles and potential differences among honey bee glial types, we examined the spatial distribution of these enzymes and asked if enzyme abundance is associated with aging and other processes essential for survival. Using immunohistochemistry and confocal laser microscopy we demonstrate that glutamine synthetase and glycogen phosphorylase are abundant in glia but appear to co-localize with different glial sub-types. The overall spatial distribution of both enzymes was not homogenous and differed markedly between different neuropiles and also within each neuropil. Using semi-quantitative Western blotting we found that rapid aging, typically observed in shortest-lived worker bees (foragers), was associated with declining enzyme levels. Further, we found enzyme abundance changes after severe starvation stress, and that glutamine synthetase is associated with food response. Together, our data indicate that aging and nutritional physiology in bees are linked to glial specific metabolic enzymes. Enzyme specific localization patterns suggest a functional differentiation among identified glial types.

Increased survival of honeybees in the laboratory after simultaneous exposure to low doses of pesticides and bacteria.

Recent studies of honeybees and bumblebees have examined combinatory effects of different stressors, as insect pollinators are naturally exposed to multiple stressors. At the same time the potential influences of simultaneously occurring agricultural agents on insect pollinator health remain largely unknown. Due to different farming methods, and the drift of applied agents and manure, pollinators are most probably exposed to insecticides but also bacteria from organic fertilizers at the same time. We orally exposed honeybee workers to sub-lethal doses of the insecticide thiacloprid and two strains of the bacterium Enterococcus faecalis, which can occur in manure from farming animals. Our results show that under laboratory conditions the bees simultaneously exposed to the a bacterium and the pesticide thiacloprid thiacloprid had significant higher survival rates 11 days post exposure than the controls, which surprisingly showed the lowest survival. Bees that were exposed to diet containing thiacloprid showed decreased food intake. General antibacterial activity is increased by the insecticide and the bacteria, resulting in a higher immune response observed in treated individuals compared to control individuals. We thus propose that caloric restriction through behavioural and physiological adaptations may have mediated an improved survival and stress resistance in our tests. However, the decreased food consumption could in long-term also result in possible negative effects at colony level. Our study does not show an additive negative impact of sub-lethal insecticide and bacteria doses, when tested under laboratory conditions. In contrast, we report seemingly beneficial effects of simultaneous exposure of bees to agricultural agents, which might demonstrate a surprising biological capacity for coping with stressors, possibly through hormetic regulation.

Hemocyte-mediated phagocytosis differs between honey bee (Apis mellifera) worker castes.

Honey bees as other insects rely on the innate immune system for protection against diseases. The innate immune system includes the circulating hemocytes (immune cells) that clear pathogens from hemolymph (blood) by phagocytosis, nodulation or encapsulation. Honey bee hemocyte numbers have been linked to hemolymph levels of vitellogenin. Vitellogenin is a multifunctional protein with immune-supportive functions identified in a range of species, including the honey bee. Hemocyte numbers can increase via mitosis, and this recruitment process can be important for immune system function and maintenance. Here, we tested if hemocyte mediated phagocytosis differs among the physiologically different honey bee worker castes (nurses, foragers and winter bees), and study possible interactions with vitellogenin and hemocyte recruitment. To this end, we adapted phagocytosis assays, which-together with confocal microscopy and flow cytometry-allow qualitative and quantitative assessment of hemocyte performance. We found that nurses are more efficient in phagocytic uptake than both foragers and winter bees. We detected vitellogenin within the hemocytes, and found that winter bees have the highest numbers of vitellogenin-positive hemocytes. Connections between phagocytosis, hemocyte-vitellogenin and mitosis were worker caste dependent. Our results demonstrate that the phagocytic performance of immune cells differs significantly between honey bee worker castes, and support increased immune competence in nurses as compared to forager bees. Our data, moreover, provides support for roles of vitellogenin in hemocyte activity.

Take time: odor coding capacity across sensory neurons increases over time in Drosophila.

Due to the highly efficient olfactory code, olfactory sensory systems are able to reliably encode enormous numbers of olfactory stimuli. The olfactory code consists of combinatorial activation patterns across sensory neurons, thus its capacity exceeds the number of involved classes of sensory neurons by a manifold. Activation patterns are not static but vary over time, caused by the temporally complex response dynamics of the individual sensory neuron responses. We systematically analyzed the temporal dynamics of olfactory sensory neuron responses to a diverse set of odorants. We find that response dynamics depend on the combination of sensory neuron and odorant and that information about odorant identity can be extracted from the time course of the response. We also show that new response dynamics can arise when mixing two odorants. Our data show that temporal dynamics of odorant responses are able to significantly enhance the coding capacity of olfactory sensory systems.

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.