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.

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.

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.

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.

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.

Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging.

Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second.

Vitellogenin recognizes cell damage through membrane binding and shields living cells from reactive oxygen species.

Large lipid transfer proteins are involved in lipid transportation and diverse other molecular processes. These serum proteins include vitellogenins, which are egg yolk precursors and pathogen pattern recognition receptors, and apolipoprotein B, which is an anti-inflammatory cholesterol carrier. In the honey bee, vitellogenin acts as an antioxidant, and elevated vitellogenin titer is linked to prolonged life span in this animal. Here, we show that vitellogenin has cell and membrane binding activity and that it binds preferentially to dead and damaged cells. Vitellogenin binds directly to phosphatidylcholine liposomes and with higher affinity to liposomes containing phosphatidylserine, a lipid of the inner leaflet of cell membranes that is exposed in damaged cells. Vitellogenin binding to live cells, furthermore, improves cell oxidative stress tolerance. This study can shed more light on why large lipid transfer proteins have a well conserved α-helical domain, because we locate the lipid bilayer-binding ability of vitellogenin largely to this region. We suggest that recognition of cell damage and oxidation shield properties are two mechanisms that allow vitellogenin to extend honey bee life span.

Bioimpedance as an alternative tool for subjective, visual scoring of a prevalent ham quality defect.

The detection of meat quality defects can involve both subjective and objective methods. PSE-like meat is linked to a common pork defect and can be caused by rapid post-mortem damage of muscle fibers. This damage can again be linked to various factors, such as a low ultimate pH or a higher slaughter weight. PSE-like defects are characterized by discoloration, structural damage, and excessive moisture loss. However, the lack of suitable instrument-based methods makes the detection of PSE-like defects difficult, and subjective methods typically suffer from poorer reproducibility. The objective of this study was to establish how subjective visual evaluation correlates with electrical impedance spectroscopy and with traditional quality parameters. To do so, visual scoring was performed together with measurements of bioimpedance, color, and pH in two ham muscles (Adductor, Semimembranosus) for 136 animals 24-hours post-mortem. When comparing with visual scoring, Pearson correlation analysis shows the strongest correlation for bioimpedance (Py , r = -0.46, R2 = 21%), followed by pHu (r = 0.44, R2 = 19%). When using all five quality measures, i.e., Py , pHu, and CIELAB L * a * b *, the multivariate regression model had a prediction error of 0.76 for the visual scores. This was close to the error describing the subjective bias of visual scoring, more specifically the prediction error between the two observers (0.85). In all, Py showed the strongest correlation among instrument-based quality tests and alone may be used for predicting pork ham structural defects, i.e., as an instrument-based alternative for subjective, visual scoring. However, an instrument that combines Py with pH and/or L*a*b* would improve the prediction of PSE-like quality defects.

The looks of an odour--visualising neural odour response patterns in real time.

BACKGROUND: Calcium imaging in insects reveals the neural response to odours, both at the receptor level on the antenna and in the antennal lobe, the first stage of olfactory information processing in the brain. Changes of intracellular calcium concentration in response to odour presentations can be observed by employing calcium-sensitive, fluorescent dyes. The response pattern across all recorded units is characteristic for the odour. METHOD: Previously, extraction of odour response patterns from calcium imaging movies was performed offline, after the experiment. We developed software to extract and to visualise odour response patterns in real time. An adaptive algorithm in combination with an implementation for the graphics processing unit enables fast processing of movie streams. Relying on correlations between pixels in the temporal domain, the calcium imaging movie can be segmented into regions that correspond to the neural units. RESULTS: We applied our software to calcium imaging data recorded from the antennal lobe of the honeybee Apis mellifera and from the antenna of the fruit fly Drosophila melanogaster. Evaluation on reference data showed results comparable to those obtained by previous offline methods while computation time was significantly lower. Demonstrating practical applicability, we employed the software in a real-time experiment, performing segmentation of glomeruli--the functional units of the honeybee antennal lobe--and visualisation of glomerular activity patterns. CONCLUSIONS: Real-time visualisation of odour response patterns expands the experimental repertoire targeted at understanding information processing in the honeybee antennal lobe. In interactive experiments, glomeruli can be selected for manipulation based on their present or past activity, or based on their anatomical position. Apart from supporting neurobiology, the software allows for utilising the insect antenna as a chemosensor, e.g. to detect or to classify odours.

Weaker ligands can dominate an odor blend due to syntopic interactions.

Most odors in natural environments are mixtures of several compounds. Perceptually, these can blend into a new "perfume," or some components may dominate as elements of the mixture. In order to understand such mixture interactions, it is necessary to study the events at the olfactory periphery, down to the level of single-odorant receptor cells. Does a strong ligand present at a low concentration outweigh the effect of weak ligands present at high concentrations? We used the fruit fly receptor dOr22a and a banana-like odor mixture as a model system. We show that an intermediate ligand at an intermediate concentration alone elicits the neuron's blend response, despite the presence of both weaker ligands at higher concentration, and of better ligands at lower concentration in the mixture. Because all of these components, when given alone, elicited significant responses, this reveals specific mixture processing already at the periphery. By measuring complete dose-response curves we show that these mixture effects can be fully explained by a model of syntopic interaction at a single-receptor binding site. Our data have important implications for how odor mixtures are processed in general, and what preprocessing occurs before the information reaches the brain.

Aging and its modulation in a long-lived worker caste of the honey bee.

Highly social animals provide alternative aging models in which vastly different lifespan patterns are flexible, and linked to social caste. Research in these species aims to reveal how environment, including social cues, can shape the transition between short-lived and extremely long-lived phenotypes with negligible senescence. Among honey bee workers, short to intermediate lifespans are typical for summer castes, while the winter caste can live up to 10 times longer. For summer castes, experimental interventions could predictably accelerate, slow or revert functional senescence. In contrast, little is known about the partic ular conditions under which periods of negligible senescence in winter castes can be disrupted or sustained. We asked how manipulation of social environment in colonies with long-lived winter bees might alter the pace of functional senescence, measured as learning performance, as well as of cellular senescence, measured as lipofuscin accumulation. We show that behavioral senescence becomes rapidly detectable when the winter state is disrupted, and changes in social task behaviors and social environment (brood) are induced. Likewise, we found that cellular senescence was induced by such social intervention. However, cellular senescence showed marked regional differences, suggesting that particular brain regions age slower than others. Finally, by preventing post-winter colonies from brood rearing, behavioral senescence became undetectable, even after transition to the usually short-lived phenotypes had occurred. We envision that social regulation of negligible functional senescence and highly dynamic accumulation of a universal symptom of cellular aging (lipofuscin) offers rewarding perspectives to target proximate mechanisms of slowed aging.

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.

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.

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.

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.

Integrating heterogeneous odor response data into a common response model: A DoOR to the complete olfactome.

We have developed a new computational framework for merging odor response data sets from heterogeneous studies, creating a consensus metadatabase, the database of odor responses (DoOR). As a result, we obtained a functional atlas of all available odor responses in Drosophila melanogaster. Both the program and the data set are freely accessible and downloadable on the Internet (http://neuro.uni-konstanz.de/DoOR). The procedure can be adapted to other species, thus creating a family of "olfactomes" in the near future. Drosophila melanogaster was chosen because of all species this one is closest to having the complete olfactome characterized, with the highest number of deorphanized receptors available. The database guarantees long-term stability (by offering time-stamped, downloadable versions), up-to-date accuracy (by including new data sets as soon as they are published), and portability (for other species). We hope that this comprehensive repository of odor response profiles will be useful to the olfactory community and to computational neuroscientists alike.

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.

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.