Mutants of the white ABCG Transporter in Drosophila melanogaster Have Deficient Olfactory Learning and Cholesterol Homeostasis

Drosophila’s white gene encodes an ATP-binding cassette G-subfamily (ABCG) half-transporter. White is closely related to mammalian ABCG family members that function in cholesterol efflux. Mutants of white have several behavioral phenotypes that are independent of visual defects. This study characterizes a novel defect of white mutants in the acquisition of olfactory memory using the aversive olfactory conditioning paradigm. The w1118 mutants learned slower than wildtype controls, yet with additional training, they reached wildtype levels of performance. The w1118 learning phenotype is also found in the wapricot and wcoral alleles, is dominant, and is rescued by genomic white and mini-white transgenes. Reducing dietary cholesterol strongly impaired olfactory learning for wildtype controls, while w1118 mutants were resistant to this deficit. The w1118 mutants displayed higher levels of cholesterol and cholesterol esters than wildtype under this low-cholesterol diet. Increasing levels of serotonin, dopamine, or both in the white mutants significantly improved w1118 learning. However, serotonin levels were not lower in the heads of the w1118 mutants than in wildtype controls. There were also no significant differences found in synapse numbers within the w1118 brain. We propose that the w1118 learning defect may be due to inefficient biogenic amine signaling brought about by altered cholesterol homeostasis.

Transcriptional Correlates of Chronic Alcohol Neuroadaptation in Drosophila Larvae

When presented with the choice, Drosophila melanogaster females will often prefer to lay eggs on food containing a significant amount of alcohol. While, in some cases, this behavioral decision can provide a survival advantage to the developing larvae, it can also lead to developmental and cognitive problems. Alcohol consumption can affect executive functions, episodic memory, and other brain function capacities. However, in the fruit fly, the initial cognitive effects of alcohol consumption have been shown to reverse upon persistent exposure to alcohol. Using an olfactory conditioning assay where an odorant is implemented as a conditioned stimulus and paired with a heat shock as an unconditioned stimulus, a previous study has shown that when exposed to a short acute dose of alcohol, Drosophila larvae can no longer learn this association. Interestingly, upon prolonged chronic alcohol exposure, larvae seem to successfully avoid the conditioned stimulus just as well as control alcohol-naive larvae, suggestive of alcohol-induced neuroadaptations. However, the mechanisms by which Drosophila adapt to the presence of alcohol remains unknown. In this study, we explore the transcriptional correlates of neuroadaptation in Drosophila larvae exposed to chronic alcohol to understand the genetic and cellular components responsible for this adaptation. For this, we employed RNA sequencing technology to evaluate differences in gene expression in the brain of larvae chronically exposed to alcohol. Our results suggest that alcohol-induced neuroadaptations are modulated by a diverse array of synaptic genes within the larval brain through a series of epigenetic modulators.

Honey bees can store and retrieve independent memory traces after complex experiences that combine appetitive and aversive associations

Real-world experiences do often mix appetitive and aversive events. Understanding the ability of animals to extract, store and use this information is an important issue in neurobiology. We used honey bees as model to study learning and memory after a differential conditioning that combines appetitive and aversive training trials. First of all, we describe an aversive conditioning paradigm that constitutes a clear opposite of the well known appetitive olfactory conditioning of the proboscis extension response. A neutral odour is presented paired with the bitter substance quinine. Aversive memory is evidenced later as an odour-specific impairment in appetitive conditioning. Then we tested the effect of mixing appetitive and aversive conditioning trials distributed along the same training session. Differential conditioning protocols like this were used before to study the ability to discriminate odours, however they were not focused on whether appetitive and aversive memories are formed. We found that after a differential conditioning, honey bees establish independent appetitive and aversive memories that do not interfere with each other during acquisition or storage. Finally, we moved the question forward to retrieval and memory expression to evaluate what happens when appetitive and the aversive learned odours are mixed during test. Interestingly, opposite memories compete in a way that they do not cancel each other out. Honey bees showed the ability to switch from expressing appetitive to aversive memory depending on their satiation level.

Honeybee Cognition as a Tool for Scientific Engagement

Apis mellifera (honeybees) are a well-established model for the study of learning and cognition. A robust conditioning protocol, the olfactory conditioning of the proboscis extension response (PER), provides a powerful but straightforward method to examine the impact of varying stimuli on learning performance. Herein, we provide a protocol that leverages PER for classroom-based community or student engagement. Specifically, we detail how a class of high school students, as part of the Ryukyu Girls Outreach Program, examined the effects of caffeine and dopamine on learning performance in honeybees. Using a modified version of the PER conditioning protocol, they demonstrated that caffeine, but not dopamine, significantly reduced the number of trials required for a successful conditioning response. In addition to providing an engaging and educational scientific activity, it could be employed, with careful oversight, to garner considerable reliable data examining the effects of varying stimuli on honeybee learning.

CREBA and CREBB in two identified neurons gate long-term memory formation in Drosophila

Episodic events are frequently consolidated into labile memory but are not necessarily transferred to persistent long-term memory (LTM). Regulatory mechanisms leading to LTM formation are poorly understood, however, especially at the resolution of identified neurons. Here, we demonstrate enhanced LTM following aversive olfactory conditioning in Drosophila when the transcription factor cyclic AMP response element binding protein A (CREBA) is induced in just two dorsal-anterior-lateral (DAL) neurons. Our experiments show that this process is regulated by protein–gene interactions in DAL neurons: (1) crebA transcription is induced by training and repressed by crebB overexpression, (2) CREBA bidirectionally modulates LTM formation, (3) crebA overexpression enhances training-induced gene transcription, and (4) increasing membrane excitability enhances LTM formation and gene expression. These findings suggest that activity-dependent gene expression in DAL neurons during LTM formation is regulated by CREB proteins.

Nectar non-protein amino acids (NPAAs) do not change nectar palatability but enhance learning and memory in honey bees

Floral nectar is a pivotal element of the intimate relationship between plants and pollinators. Nectars are composed of a plethora of nutritionally valuable compounds but also hundreds of secondary metabolites (SMs) whose function remains elusive. Here we performed a set of behavioural experiments to study whether five ubiquitous nectar non-protein amino acids (NPAAs: β-alanine, GABA, citrulline, ornithine and taurine) interact with gustation, feeding preference, and learning and memory in Apis mellifera. We showed that foragers were unable to discriminate NPAAs from water when only accessing antennal chemo-tactile information and that freely moving bees did not exhibit innate feeding preferences for NPAAs. Also, NPAAs did not alter food consumption or longevity in caged bees over 10 days. Taken together our data suggest that natural concentrations of NPAAs did not alter nectar palatability to bees. Olfactory conditioning assays showed that honey bees were more likely to learn a scent when it signalled a sucrose reward containing either β-alanine or GABA, and that GABA enhanced specific memory retention. Conversely, when ingested two hours prior to conditioning, GABA, β-alanine, and taurine weakened bees’ acquisition performances but not specific memory retention, which was enhanced in the case of β-alanine and taurine. Neither citrulline nor ornithine affected learning and memory. NPAAs in nectars may represent a cooperative strategy adopted by plants to attract beneficial pollinators.

COVID-19 Sniffer Dog experimental training: which protocol and which implications for reliable identification?

The introduction of trained sniffer dogs for COVID-19 disease detection could be an opportunity, as previously described for other diseases. Dogs could be trained to detect volatile organic compounds (VOCs), the whiff of COVID-19 disease. Dogs involved in the study were three one male and two females from different breeds, Black German Shepherd, German Shepherd and Dutch Shepherd. The training was performed using sweat samples from COVID-19 positive apteints and from covid-19 free patients admitted at the University Hospital Campus Bio-medico of Rome. Gauze with sweat were collected in glass jar with metal top and put in metal boxes used for dog training. The dog training protocol was performed in two phase: the olfactory conditioning and the olfactory discrimintaion research. The training palnning was focused on the switch moment for the sniffer dog, the moment when the dog was able to identify VOCs specific for COVID-19 disease. At this time the dog was able to identify VOCs specific for COVID-19 disease with significant reliability, in terms of number of correct versus uncorrect (p<0.0001) reporting. In conclusion, this protocol could provide a useful tool for sniffer dogs training and their introduction in mass screening context, cheaper and faster than a conventional testing method.

Combined secondary compounds naturally found in nectars enhance honeybee cognition and survival

ABSTRACT The alkaloid caffeine and the amino acid arginine are present as secondary compounds in nectars of some flower species visited by pollinators. Each of these compounds affects honeybee appetitive behaviours by improving foraging activity and learning. While caffeine potentiates responses of mushroom body neurons involved in honeybee learning processes, arginine acts as precursor of nitric oxide, enhancing the protein synthesis involved in memory formation. Despite existing evidence on how these compounds affect honeybee cognitive ability individually, their combined effect on this is still unknown. We evaluated acquisition and memory retention in a classical olfactory conditioning procedure, in which the reward (sucrose solution) contained traces of caffeine, arginine or a mixture of the two. The results indicate that the presence of the single compounds and their most concentrated mixture increases bees' learning performance. However, memory retention, measured in the short and long term, increases significantly only in those treatments offering combinations of the two compounds in the reward. Additionally, the most concentrated mixture triggers a significant survival rate in the conditioned bees. Thus, some nectar compounds, when combined, show synergistic effects on cognitive ability and survival in an insect.

Hierarchical cross-scale analysis identifies parallel ventral striatal networks coding for dynamic and stabilized olfactory reward predictions

SUMMARYThe unbiased identification of brain circuits responsible for behavior and their local cellular computations is a challenge for neuroscience. We establish here a hierarchical cross-scale approach from behavioral modeling and fMRI in task-performing mice to cellular network dynamics to identify how reward predictions are represented in the forebrain upon olfactory conditioning. fMRI identified functional segregation in reward prediction and error computations among olfactory cortices and subcortical circuits. Among them, the olfactory tubercle contributed both to dynamic reward predictions and prediction error. In this region, cellular recordings revealed two parallel neuronal populations for prediction coding. One population produced stabilized predictions as distributed stimulus-bound transient network activity; the other evolved during anticipatory waiting and fully reflected predicted value in single-units, dynamically integrating the recent cue-specific history of uncertain outcomes. Thus, the cross-scale approach revealed regional functional differentiation among the distributed forebrain circuits with a limbic hotspot for multiple non-redundant reward prediction coding.