Towards a unified vision on animal navigation.

The study of animal navigation is a complex and fertile field of research: Several questions regarding how animals relate to external stimuli, integrating them to perform their everyday movement routine, have been or are being addressed in different organisms and taxa, both from the behavioural and the neuronal activity point of view. Several invertebrate model organisms are the object of studies aimed at unravelling how they navigate and their ability to precisely return to a starting point and also how navigational information is communicated to conspecifics when precise social structures are present. Also, vertebrates are studied because of the interest in their orientation abilities while migrating, homing over impressive distances and studying exploration, orientation and space recognition. Last, research on the navigation capabilities of humans pursues a better understanding of the neural architecture involved in these processes in the remarkable effort to find answers and possible solutions to impairments, lesions and diseases. However, an 'all-inclusive' vision of navigation still appears to be in its embryonic state: A better perspective could (and should) shift from a paradigm where single research teams are centred on studying navigation in a single genus or species towards a more comprehensive evolutionary-centred view, searching systematically for behavioural analogies, and possibly for homologies in neural architecture between different taxa. In this review, we introduce examples of relevant topics in animal navigation from distinct animal groups, highlighting the similar approaches of those studies, and why, in our opinion, this research field could profit from a 'new' perspective.

Action-based attention in Drosophila melanogaster.

The mechanism of action selection is a widely shared fundamental process required by animals to interact with the environment and adapt to it. A key step in this process is the filtering of the "distracting" sensory inputs that may disturb action selection. Because it has been suggested that, in principle, action selection may also be processed by shared circuits in vertebrate and invertebrates, we wondered whether invertebrates show the ability to filter out "distracting" stimuli during a goal-directed action, as seen in vertebrates. In this experiment, action selection was studied in wild-type Drosophila melanogaster by investigating their reaction to the abrupt appearance of a visual distractor during an ongoing locomotor action directed to a visual target. We found that when the distractor was present, flies tended to shift the original trajectory toward it, thus acknowledging its presence, but they did not fully commit to it, suggesting that an inhibition process took place to continue the unfolding of the planned goal-directed action. To some extent flies appeared to take into account and represent motorically the distractor, but they did not engage in a complete change of their initial motor program in favor of the distractor. These results provide interesting insights into the selection-for-action mechanism, in a context requiring action-centered attention, that might have appeared rather early in the course of evolution. NEW & NOTEWORTHY Action selection and maintenance of a goal-directed action require animals to ignore irrelevant "distracting" stimuli that might elicit alternative motor programs. In this study we observed, in Drosophila melanogaster, a top-down mechanism inhibiting the response toward salient stimuli, to accomplish a goal-directed action. These data highlight, for the first time in an invertebrate organism, that the action-based attention shown by higher organisms, such as humans and nonhuman primates, might have an ancestral origin.

Circadian Rhythm Abnormalities in Parkinson's Disease from Humans to Flies and Back.

Clinical and research studies have suggested a link between Parkinson's disease (PD) and alterations in the circadian clock. Drosophila melanogaster may represent a useful model to study the relationship between the circadian clock and PD. Apart from the conservation of many genes, cellular mechanisms, signaling pathways, and neuronal processes, Drosophila shows an organized central nervous system and well-characterized complex behavioral phenotypes. In fact, Drosophila has been successfully used in the dissection of the circadian system and as a model for neurodegenerative disorders, including PD. Here, we describe the fly circadian and dopaminergic systems and report recent studies which indicate the presence of circadian abnormalities in some fly PD genetic models. We discuss the use of Drosophila to investigate whether, in adults, the disruption of the circadian system might be causative of brain neurodegeneration. We also consider approaches using Drosophila, which might provide new information on the link between PD and the circadian clock. As a corollary, since PD develops its symptomatology over a large part of the organism's lifespan and given the relatively short lifespan of fruit flies, we suggest that genetic models of PD could be used to perform lifelong screens for drug-modulators of general and/or circadian-related PD traits.

UCP4C mediates uncoupled respiration in larvae of Drosophila melanogaster.

Larvae of Drosophila melanogaster reared at 23°C and switched to 14°C for 1 h are 0.5°C warmer than the surrounding medium. In keeping with dissipation of energy, respiration of Drosophila melanogaster larvae cannot be decreased by the F-ATPase inhibitor oligomycin or stimulated by protonophore. Silencing of Ucp4C conferred sensitivity of respiration to oligomycin and uncoupler, and prevented larva-to-adult progression at 15°C but not 23°C. Uncoupled respiration of larval mitochondria required palmitate, was dependent on Ucp4C and was inhibited by guanosine diphosphate. UCP4C is required for development through the prepupal stages at low temperatures and may be an uncoupling protein.

Leigh syndrome in Drosophila melanogaster: morphological and biochemical characterization of Surf1 post-transcriptional silencing.

Leigh Syndrome (LS) is the most common early-onset, progressive mitochondrial encephalopathy usually leading to early death. The single most prevalent cause of LS is occurrence of mutations in the SURF1 gene, and LS(Surf1) patients show a ubiquitous and specific decrease in the activity of mitochondrial respiratory chain complex IV (cytochrome c oxidase, COX). SURF1 encodes an inner membrane mitochondrial protein involved in COX assembly. We established a Drosophila melanogaster model of LS based on the post-transcriptional silencing of CG9943, the Drosophila homolog of SURF1. Knockdown of Surf1 was induced ubiquitously in larvae and adults, which led to lethality; in the mesodermal derivatives, which led to pupal lethality; or in the central nervous system, which allowed survival. A biochemical characterization was carried out in knockdown individuals, which revealed that larvae unexpectedly displayed defects in all complexes of the mitochondrial respiratory chain and in the F-ATP synthase, while adults had a COX-selective impairment. Silencing of Surf1 expression in Drosophila S2R(+) cells led to selective loss of COX activity associated with decreased oxygen consumption and respiratory reserve. We conclude that Surf1 is essential for COX activity and mitochondrial function in D. melanogaster, thus providing a new tool that may help clarify the pathogenic mechanisms of LS.

Functional characterization of drim2, the Drosophila melanogaster homolog of the yeast mitochondrial deoxynucleotide transporter.

The CG18317 gene (drim2) is the Drosophila melanogaster homolog of the Saccharomyces cerevisiae Rim2 gene, which encodes a pyrimidine (deoxy)nucleotide carrier. Here, we tested if the drim2 gene also encodes for a deoxynucleotide transporter in the fruit fly. The protein was localized to mitochondria. Drosophila S2R(+) cells, silenced for drim2 expression, contained markedly reduced pools of both purine and pyrimidine dNTPs in mitochondria, whereas cytosolic pools were unaffected. In vivo drim2 homozygous knock-out was lethal at the larval stage, preceded by the following: (i) impaired locomotor behavior; (ii) decreased rates of oxygen consumption, and (iii) depletion of mtDNA. We conclude that the Drosophila mitochondrial carrier dRIM2 transports all DNA precursors and is essential to maintain mitochondrial function.

Evidence for a radial SNARE super-complex mediating neurotransmitter release at the Drosophila neuromuscular junction.

The SNARE proteins VAMP/synaptobrevin, SNAP-25 and syntaxin are core components of the apparatus that mediates neurotransmitter release. They form a heterotrimeric complex, and an undetermined number of SNARE complexes assemble to form a super-complex. Here, we present a radial model of this nanomachine. Experiments performed with botulinum neurotoxins led to the identification of one arginine residue in SNAP-25 and one aspartate residue in syntaxin (R206 and D253 in Drosophila melanogaster). These residues are highly conserved and predicted to play a major role in the protein-protein interactions between SNARE complexes by forming an ionic couple. Accordingly, we generated transgenic Drosophila lines expressing SNAREs mutated in these residues and performed an electrophysiological analysis of their neuromuscular junctions. Our results indicate that SNAP-25-R206 and syntaxin-D253 play a major role in neuroexocytosis and support a radial assembly of several SNARE complexes interacting via the ionic couple formed by these two residues.

Effects of oxygen concentration and pressure on Drosophila melanogaster: oxidative stress, mitochondrial activity, and survivorship.

Organisms are known to be equipped with an adaptive plasticity as the phenotype of traits in response to the imposed environmental challenges as they grow and develop. In this study, the effects of extreme changes in oxygen availability and atmospheric pressure on physiological phenotypes of Drosophila melanogaster were investigated to explore adaptation mechanisms. The changes in citrate synthase activity (CSA), lifespan, and behavioral function in different atmospheric conditions were evaluated. In the CAS test, hyperoxia significantly increased CSA; both hypoxia and hyperbaric conditions caused a significant decrease in CSA. In the survivorship test, all changed atmospheric conditions caused a significant reduction in lifespan. The lifespan reduced more after hypoxia exposure than after hyperbaria exposure. In behavioral function test, when mechanical agitation was conducted, bang-sensitive flies showed a stereotypical sequence of initial muscle spasm, paralysis, and recovery. The percentage of individuals that displayed paralysis or seizure was measured on the following day and after 2 weeks from each exposure. The majority of flies showed seizure behavior 15 days after exposure, especially after 3 h of exposure. The percentage of individuals that did not undergo paralysis or seizure and was able to move in the vial, was also tested. The number of flies that moved and raised the higher level of the vial decreased after exposure. Animal's speed decreased significantly 15 days after exposure to extreme environmental conditions. In summary, the alteration of oxygen availability and atmospheric pressure may lead to significant changes in mitochondria mass, lifespan, and behavioral function in D. melanogaster.

Optokinetic response in D. melanogaster reveals the nature of common repellent odorants.

Animals' ability to orient and navigate relies on selecting an appropriate motor response based on the perception and integration of the environmental information. This is the case, for instance, of the optokinetic response (OKR) in Drosophila melanogaster, where optic flow visual stimulation modulates head movements. Despite a large body of literature on the OKR, there is still a limited understanding, in flies, of the impact on OKR of concomitant, and potentially conflicting, inputs. To evaluate the impact of this multimodal integration, we combined in D. melanogaster, while flying in a tethered condition, the optic flow stimulation leading to OKR with the simultaneous presentation of olfactory cues, based on repellent or masking compounds typically used against noxious insect species. First, this approach allowed us to directly quantify the effect of several substances and of their concentration on the dynamics of the flies' OKR in response to moving gratings by evaluating the number of saccades and the velocity of the slow phase. Subsequently, this analysis was capable of easily revealing the actual effect, i.e. masking vs. repellent, of the compound tested. In conclusion, we show that D. melanogaster, a cost-affordable species, represents a viable option for studying the effects of several compounds on the navigational abilities of insects.

Usp14 down-regulation corrects sleep and circadian dysfunction of a Drosophila model of Parkinson's disease.

PD is a complex, multifactorial neurodegenerative disease, which occurs sporadically in aged population, with some genetically linked cases. Patients develop a very obvious locomotor phenotype, with symptoms such as bradykinesia, resting tremor, muscular rigidity, and postural instability. At the cellular level, PD pathology is characterized by the presence of intracytoplasmic neurotoxic aggregates of misfolded proteins and dysfunctional organelles, resulting from failure in mechanisms of proteostasis. Nonmotor symptoms, such as constipation and olfactory deficits, are also very common in PD. They include alteration in the circadian clock, and defects in the sleep-wake cycle, which is controlled by the clock. These non-motor symptoms precede the onset of the motor symptoms by many years, offering a window of therapeutic intervention that could delay-or even prevent-the progression of the disease. The mechanistic link between aberrant circadian rhythms and neurodegeneration in PD is not fully understood, although proposed underlying mechanisms include alterations in protein homeostasis (proteostasis), which can impact protein levels of core components of the clock. Loss of proteostasis depends on the progressive pathological decline in the proteolytic activity of two major degradative systems, the ubiquitin-proteasome and the lysosome-autophagy systems, which is exacerbated in age-dependent neurodegenerative conditions like PD. Accordingly, it is known that promoting proteasome or autophagy activity increases lifespan, and rescues the pathological phenotype of animal models of neurodegeneration, presumably by enhancing the degradation of misfolded proteins and dysfunctional organelles, which are known to accumulate in these models, and to induce intracellular damage. We can enhance proteostasis by pharmacologically inhibiting or down-regulating Usp14, a proteasome-associated deubiquitinating enzyme (DUB). In a previous work, we showed that inhibition of Usp14 enhances the activity of the ubiquitin-proteasome system (UPS), autophagy and mitophagy, and abolishes motor symptoms of two well-established fly models of PD that accumulate dysfunctional mitochondria. In this work we extended the evidence on the protective effect of Usp14 down-regulation, and investigated the beneficial effect of down-regulating Usp14 in a Pink1 Drosophila model of PD that develop circadian and sleep dysfunction. We show that down-regulation of Usp14 ameliorates sleep disturbances and circadian defects that are associated to Pink1 KO flies.

Arg206 of SNAP-25 is essential for neuroexocytosis at the Drosophila melanogaster neuromuscular junction.

An analysis of SNAP-25 isoform sequences indicates that there is a highly conserved arginine residue (198 in vertebrates, 206 in the genus Drosophila) within the C-terminal region, which is cleaved by botulinum neurotoxin A, with consequent blockade of neuroexocytosis. The possibility that it may play an important role in the function of the neuroexocytosis machinery was tested at neuromuscular junctions of Drosophila melanogaster larvae expressing SNAP-25 in which Arg206 had been replaced by alanine. Electrophysiological recordings of spontaneous and evoked neurotransmitter release under different conditions as well as testing for the assembly of the SNARE complex indicate that this residue, which is at the P(1)' position of the botulinum neurotoxin A cleavage site, plays an essential role in neuroexocytosis. Computer graphic modelling suggests that this arginine residue mediates protein-protein contacts within a rosette of SNARE complexes that assembles to mediate the fusion of synaptic vesicles with the presynaptic plasma membrane.

The impact of macroeconomic factors on suicide in 175 countries over 27 years.

BACKGROUND: Every year, more than 800,000 people die by suicide, three-quarters of which are males. Economic factors influence suicide rates, but a worldwide perspective of their impact according to age and sex is lacking. METHOD: We queried publicly available datasets on economic factors and on suicide rates stratified according to sex and age, from 1991 to 2017, for 175 countries. Thus, we analyzed approximately 21 million deaths by suicide using a multivariable regression model approach. RESULTS: Every 1% increase in global unemployment rates is associated with a 1% upsurge in male deaths by suicide (Relative risk (RR) = 1.01 [CI 95% 1.00-1.01] with respect to females) or 5000 excess male deaths. A 1% higher unemployment rate also exerts age-specific effects on suicide rates, since, among adults aged 30-59, the suicide rate is increased by 2-3%. Lastly, for every 1000 US dollar increase in the GDP per capita, suicide rates are reduced by 2% (RR = 0.98 [0.98-0.98]), corresponding to a reduction of 14,000-15,000 suicide deaths per year globally. CONCLUSIONS: Males who have lost their jobs in adulthood are those at higher risk of suicide and to whom financial support measures should be delivered in a timely manner.

A heuristic underlies the search for relief in Drosophila melanogaster.

Humans rely on multiple types of sensory information to make decisions, and strategies that shorten decision-making time by taking into account fewer but essential elements of information are preferred to strategies that require complex analyses. Such shortcuts to decision making are known as heuristics. The identification of heuristic principles in species phylogenetically distant to humans would shed light on the evolutionary origin of speed-accuracy trade-offs and offer the possibility for investigating the brain representations of such trade-offs, urgency and uncertainty. By performing experiments on spatial learning in the invertebrate Drosophila melanogaster, we show that the fly's search strategies conform to a spatial heuristic-the nearest neighbor rule-to avoid bitter taste (a negative stimulation). That is, Drosophila visits a salient location closest to its current position to stop the negative stimulation; only if this strategy proves unsuccessful does the fly use other learned associations to avoid bitter taste. Characterizing a heuristic in D. melanogaster supports the view that invertebrates can, when making choices, operate on economic principles, as well as the conclusion that heuristic decision making dates to at least 600 million years ago.

Dopamine Modulation of Drosophila Ellipsoid Body Neurons, a Nod to the Mammalian Basal Ganglia.

The central complex (CX) is a neural structure located on the midline of the insect brain that has been widely studied in the last few years. Its role in navigation and goal-oriented behaviors resembles those played by the basal ganglia in mammals. However, the neural mechanisms and the neurotransmitters involved in these processes remain unclear. Here, we exploited an in vivo bioluminescence Ca2+ imaging technique to record the activity in targeted neurons of the ellipsoid body (EB). We used different drugs to evoke excitatory Ca2+-responses, depending on the putative neurotransmitter released by their presynaptic inputs, while concomitant dopamine administration was employed to modulate those excitations. By using a genetic approach to knockdown the dopamine 1-like receptors, we showed that different dopamine modulatory effects are likely due to specific receptors expressed by the targeted population of neurons. Altogether, these results provide new data concerning how dopamine modulates and shapes the response of the ellipsoid body neurons. Moreover, they provide important insights regarding the similitude with mammals as far as the role played by dopamine in increasing and stabilizing the response of goal-related information.

DJ-1 and SOD1 Act Independently in the Protection against Anoxia in Drosophila melanogaster.

Redox homeostasis is a vital process the maintenance of which is assured by the presence of numerous antioxidant small molecules and enzymes and the alteration of which is involved in many pathologies, including several neurodegenerative disorders. Among the different enzymes involved in the antioxidant response, SOD1 and DJ-1 have both been associated with the pathogenesis of amyotrophic lateral sclerosis and Parkinson's disease, suggesting a possible interplay in their mechanism of action. Copper deficiency in the SOD1-active site has been proposed as a central determinant in SOD1-related neurodegeneration. SOD1 maturation mainly relies on the presence of the protein copper chaperone for SOD1 (CCS), but a CCS-independent alternative pathway also exists and functions under anaerobic conditions. To explore the possible involvement of DJ-1 in such a pathway in vivo, we exposed Drosophila melanogaster to anoxia and evaluated the effect of DJ-1 on fly survival and SOD1 levels, in the presence or absence of CCS. Loss of DJ-1 negatively affects the fly response to the anoxic treatment, but our data indicate that the protective activity of DJ-1 is independent of SOD1 in Drosophila, indicating that the two proteins may act in different pathways.

Lifespan and ROS levels in different Drosophila melanogaster strains after 24†h hypoxia exposure.

During recent decades, model organisms such as Drosophila melanogaster have made it possible to study the effects of different environmental oxygen conditions on lifespan and oxidative stress. However, many studies have often yielded controversial results usually assigned to variations in Drosophila genetic background and differences in study design. In this study, we compared longevity and ROS levels in young, unmated males of three laboratory wild-type lines (Canton-S, Oregon-R and Berlin-K) and one mutant line (Sod1n1) as a positive control of redox imbalance, under both normoxic and hypoxic (2% oxygen for 24 h) conditions. Lifespan was used to detect the effects of hypoxic treatment and differences were analysed by means of Kaplan-Meier survival curves and log-rank tests. Electron paramagnetic resonance spectroscopy was used to measure ROS levels and analysis of variance was used to estimate the effects of hypoxic treatment and to assess ROS differences between strains. We observed that the genetic background is a relevant factor involved in D. melanogaster longevity and ROS levels. Indeed, as expected, in normoxia Sod1n1 are the shortest-lived, while the wild-type strains, despite a longer lifespan, show some differences, with the Canton-S line displaying the lowest mortality rate. After hypoxic stress these variances are amplified, with Berlin-K flies showing the highest mortality rate and most evident reduction of lifespan. Moreover, our analysis highlighted differential effects of hypoxia on redox balance/unbalance. Canton-S flies had the lowest increase of ROS level compared to all the other strains, confirming it to be the less sensitive to hypoxic stress. Sod1n1 flies displayed the highest ROS levels in normoxia and after hypoxia. These results should be used to further standardize future Drosophila research models designed to investigate genes and pathways that may be involved in lifespan and/or ROS, as well as comparative studies on specific mutant strains.

Students' mental health problems before, during, and after COVID-19 lockdown in Italy.

The lockdown due to the coronavirus pandemic may have exacerbated mental health problems. To what degree mental health may be affected by social isolation is still poorly known. We collected prospective data on students' mental health in two instances: (i) in October and December 2019, and (ii) 6 months later, in April 2020 amidst the COVID-19 lockdown in Italy and in mid-May/June 2020, after the lifting of lockdown. A total of 358 Italian students aged 18-30 completed socio-demographic questionnaires and the Beck Depression Inventory - 2 (BDI-2), the Beck Anxiety Inventory (BAI), the Obsessive-Compulsive Inventory - Revised (OCI-R), the Eating Habits Questionnaire (EHQ), and the Eating Disorder Inventory-3 (EDI-3). We applied multiple regression models to evince any changes in the aforementioned questionnaire scores during and after lockdown with respect to the scores before lockdown. Students reported on average worse depressive symptoms during lockdown than 6 months before isolation (median increase in the BDI-2 score +2; IQR = -3, 6; ß = 0.09 ± 0.03, p = 0.005), with students without any established diagnosis of psychopathology being affected the most. The regression models predict that 86.2% (IQR = 67.9, 91.4%) of students would not experience a clinically significant worsening of symptoms, while approximately 6% of our target population could develop more severe depressive symptoms. This study supports the view that depressive symptomatology may be aggravated during lockdown, but also highlights that after the lifting of lockdown any changes quickly vanished, as the BDI-2 scores were not different from the ones reported before lockdown.

Searching for relief: Drosophila melanogaster navigation in a virtual bitter maze.

Animals use relief-based place learning to pinpoint a specific location where noxious stimuli are diminished or abolished. Here we show how the optogenetically-induced activation of bitter-sensing neurons in Drosophila melanogaster elicits pain-like behavioural responses and stimulates the search for a place where this activation is relieved. Under this "virtual" stimulation paradigm it would be feasible to test relief learning several times throughout an animal's lifespan, without the potentially damaging effects which may result from the repeated administration of "real" heat or electrical shock. Furthermore, virtual bitter taste could be used in place of virtual pain stimulation to guide conditioned place preference and study learning processes. We also propose that spatially-specific reduction of locomotor velocity may provide immediate evidence of relief-based place learning and spatial memory.

aubergine gene overexpression in somatic tissues of aubergine(sting) mutants interferes with the RNAi pathway of a yellow hairpin dsRNA in Drosophila melanogaster.

AUBERGINE (AUB) is a member of the PPD family of proteins. These proteins are implicated in RNA interference. In this article we demonstrate that the expression of the aub gene and protein increase in aub(sting) mutants. We used a genetic method to test whether aub(sting) overexpression could interfere with proper functioning of the process of RNA interference in somatic tissues of Drosophila melanogaster. This method is based on a transgenic line bearing a construct in which a fragment of the yellow (y) gene is cloned to form an inverted repeat (y-IR) under the control of the upstream activation sequence (UAS) of the yeast transcriptional activator GAL4. The UAS-y-IR transgene and the Act5C-GAL4 driver were brought together on chromosome 3 via recombination. In the resulting strain (Act5C-y-IR), transcriptional activation by GAL4 constitutively produces a dsRNA hairpin bearing cognate sequences to the yellow gene causing continuing degradation of y mRNA resulting in yellow(1) (y(1)) phenocopies. In this genetic background, the mutation of any factor involved in RNAi should repress degradation of y mRNA, restoring the wild-type phenotype. We employed this genetic approach to show that an increased amount of AUBERGINE interferes with the regular functioning of the somatic RNAi pathway.

Post-transcriptional silencing of the Drosophila homolog of human ZASP: a molecular and functional analysis.

In humans, mutations in ZASP (the gene for Z-band alternatively spliced PDZ-motif protein) are associated with dilated cardiomyopathy and left ventricular non-compaction. In particular, mutations in or around the Zasp motif seem to be related to myofibrillar myopathy. Thus, "zaspopathies" include symptoms such as Z-line disgregation, proximal and distal muscle weakness, cardiomyopathies, and peripheral neuropathies. In order to understand the role of ZASP in muscle structure and function, we have performed a molecular characterization of the Drosophila ortholog of human ZASP and a functional analysis following the post-transcriptional silencing of the Drosophila gene. Transcriptional analysis of dzasp has revealed six additional exons, with respect to the known 16, and multiple splice variants. We have produced transgenic lines harboring constructs that, through the use of the UAS/Gal4 binary system, have enabled us to drive dsRNA interference of dzasp in a tissue-specific manner. Knockdown individuals show locomotor defects associated with alterations of muscle structure and ultrastructure, consistent with a role of dzasp specifically in the maintenance of muscular integrity.