Biological aging of two innate behaviors of Drosophila melanogaster: Escape climbing versus courtship learning and memory.

Motor and cognitive aging can severely affect life quality of elderly people and burden health care systems. In search for diagnostic behavioral biomarkers, it has been suggested that walking speed can predict forms of cognitive decline, but in humans, it remains challenging to separate the effects of biological aging and lifestyle. We examined a possible association of motor and cognitive decline in Drosophila, a genetic model organism of healthy aging. Long term courtship memory is present in young male flies but absent already during mid life (4-8 weeks). By contrast, courtship learning index and short term memory (STM) are surprisingly robust and remain stable through mid (4-8 weeks) and healthy late life (>8 weeks), until courtship performance collapses suddenly at ~4.5 days prior to death. By contrast, climbing speed declines gradually during late life (>8 weeks). The collapse of courtship performance and short term memory close to the end of life occur later and progress with a different time course than the gradual late life decline in climbing speed. Thus, during healthy aging in male Drosophila, climbing and courtship motor behaviors decline differentially. Moreover, cognitive and motor performances decline at different time courses. Differential behavioral decline during aging may indicate different underlying causes, or alternatively, a common cause but different thresholds for defects in different behaviors.

Chlorogenic Acids, Acting via Calcineurin, Are the Main Compounds in Centella asiatica Extracts That Mediate Resilience to Chronic Stress in Drosophila melanogaster.

Common symptoms of depressive disorders include anhedonia, sleep problems, and reduced physical activity. Drugs used to treat depression mostly aim to increase serotonin signaling but these can have unwanted side effects. Depression has also been treated by traditional medicine using plants like Centella asiatica (CA) and this has been found to be well tolerated. However, very few controlled studies have addressed CA's protective role in depression, nor have the active compounds or mechanisms that mediate this function been identified. To address this issue, we used Drosophila melanogaster to investigate whether CA can improve depression-associated symptoms like anhedonia and decreased climbing activity. We found that a water extract of CA provides resilience to stress induced phenotypes and that this effect is primarily due to mono-caffeoylquinic acids found in CA. Furthermore, we describe that the protective function of CA is due to a synergy between chlorogenic acid and one of its isomers also present in CA. However, increasing the concentration of chlorogenic acid can overcome the requirement for the second isomer. Lastly, we found that chlorogenic acid acts via calcineurin, a multifunctional phosphatase that can regulate synaptic transmission and plasticity and is also involved in neuronal maintenance.

Withania somnifera Extracts Promote Resilience against Age-Related and Stress-Induced Behavioral Phenotypes in Drosophila melanogaster; a Possible Role of Other Compounds besides Withanolides.

Withania somnifera (WS) extracts have been used in traditional medicine for millennia to promote healthy aging and wellbeing. WS is now also widely used in Western countries as a nutritional supplement to extend healthspan and increase resilience against age-related changes, including sleep deficits and depression. Although human trials have supported beneficial effects of WS, the study designs have varied widely. Plant material is intrinsically complex, and extracts vary widely with the origin of the plant material and the extraction method. Commercial supplements can contain various other ingredients, and the characteristics of the study population can also be varied. To perform maximally controlled experiments, we used plant extracts analyzed for their composition and stability. We then tested these extracts in an inbred Drosophila line to minimize effects of the genetic background in a controlled environment. We found that a water extract of WS (WSAq) was most potent in improving physical fitness, while an ethanol extract (WSE) improved sleep in aged flies. Both extracts provided resilience against stress-induced behavioral changes. WSE contained higher levels of withanolides, which have been proposed to be active ingredients, than WSAq. Therefore, withanolides may mediate the sleep improvement, whereas so-far-unknown ingredients enriched in WSAq likely mediate the effects on fitness and stress-related behavior.

Octopamine mediates sugar relief from a chronic-stress-induced depression-like state in Drosophila.

Chronic, uncontrollable stress can result in psychiatric syndromes, including anxiety and major depressive disorder, in humans and mammalian disease models.1,2 Similarly, several days of chronic stress can induce depression-associated behavioral alteration in Drosophila accompanied by changes in biogenic amine levels in the adult brain.3-6 In our chronic stress paradigm, flies are subjected to 3 days of repetitive phases of 300 Hz vibrations combined with overcrowding and food deprivation. This treatment reduces voluntary behavioral activity, including the motivation to climb wide gaps (risk taking) and to stop for sweets (anhedonia), suggesting a depression-like state (DLS). These behavioral changes correlate with decreased serotonin release to the mushroom body (MB), a major behavioral control center in the central brain of the fly.7,8 Stressed flies are relieved from the DLS by feeding the anti-depressant serotonin precursor 5-HTP or the selective serotonin reuptake inhibitor fluoxetine. Notably, feeding sucrose to stressed flies results in elevated serotonin levels in the brain and ameliorates the DLS.3 Here, we show that this sugar relief is mediated by the neurotransmitter octopamine signaled from ventral unpaired medial neurons located in the subesophageal ganglion. The octopamine signaling of sweet sensation is transmitted to the MB via the dopaminergic PAM neurons. In addition, neuronal-silencing experiments reveal that the serotonergic dorsal paired medial (DPM) neurons innervating the MB are essential for sugar relief. Conversely, thermogenetic or optogenetic activation of DPMs can replace sweet sensation, elucidating that serotonergic signaling from DPMs takes part in positively modulating DLS-related behavioral changes.

FTD-associated mutations in Tau result in a combination of dominant and recessive phenotypes.

Although mutations in the microtubules-associated protein Tau have long been connected with several neurodegenerative diseases, the underlying molecular mechanisms causing these tauopathies are still not fully understood. Studies in various models suggested that dominant gain-of-function effects underlie the pathogenicity of these mutants; however, there is also evidence that the loss of normal physiological functions of Tau plays a role in tauopathies. Previous studies on Tau in Drosophila involved expressing the human Tau protein in the background of the endogenous Tau gene in addition to inducing high expression levels. To study Tau pathology in more physiological conditions, we recently created Drosophila knock-in models that express either wildtype human Tau (hTauWT) or disease-associated mutant hTau (hTauV337M and hTauK369I) in place of the endogenous Drosophila Tau (dTau). Analyzing these flies as homozygotes, we could therefore detect recessive effects of the mutations while identifying dominant effects in heterozygotes. Using memory, locomotion and sleep assays, we found that homozygous mutant hTau flies showed deficits already when quite young whereas in heterozygous flies, disease phenotypes developed with aging. Homozygotes also revealed an increase in microtubule diameter, suggesting that changes in the cytoskeleton underlie the axonal degeneration we observed in these flies. In contrast, heterozygous mutant hTau flies showed abnormal axonal targeting and no detectable changes in microtubules. However, we previously showed that heterozygosity for hTauV337M interfered with synaptic homeostasis in central pacemaker neurons and we now show that heterozygous hTauK369I flies have decreased levels of proteins involved in the release of synaptic vesicles. Taken together, our results demonstrate that both mutations induce a combination of dominant and recessive disease-related phenotypes that provide behavioral and molecular insights into the etiology of Tauopathies.

Integrative Biomimetics of Autonomous Hexapedal Locomotion.

Despite substantial advances in many different fields of neurorobotics in general, and biomimetic robots in particular, a key challenge is the integration of concepts: to collate and combine research on disparate and conceptually disjunct research areas in the neurosciences and engineering sciences. We claim that the development of suitable robotic integration platforms is of particular relevance to make such integration of concepts work in practice. Here, we provide an example for a hexapod robotic integration platform for autonomous locomotion. In a sequence of six focus sections dealing with aspects of intelligent, embodied motor control in insects and multipedal robots-ranging from compliant actuation, distributed proprioception and control of multiple legs, the formation of internal representations to the use of an internal body model-we introduce the walking robot HECTOR as a research platform for integrative biomimetics of hexapedal locomotion. Owing to its 18 highly sensorized, compliant actuators, light-weight exoskeleton, distributed and expandable hardware architecture, and an appropriate dynamic simulation framework, HECTOR offers many opportunities to integrate research effort across biomimetics research on actuation, sensory-motor feedback, inter-leg coordination, and cognitive abilities such as motion planning and learning of its own body size.

Drosophila Acquires a Long-Lasting Body-Size Memory from Visual Feedback.

Grasping an object or crossing a trench requires the integration of information on the operating distance of our limbs with precise distance estimation. The reach of our hands and step size of our legs are learned by the visual feedback we get during our actions. This implicit knowledge of our peripersonal space is first acquired during infancy but will be continuously updated throughout our whole life [1]. In contrast, body size of holometabolous insects does not change after metamorphosis; nevertheless, they do have to learn their body reaches at least once. The body size of Drosophila imagines can vary by about 15% depending on environmental factors like food quality and temperature [2]. To investigate how flies acquire knowledge about and memorize their body size, we studied their decisions to either refrain from or initiate climbing over gaps exceeding their body size [3]. Naive (dark-reared) flies overestimate their size and have to learn it from the parallax motion of the retinal images of objects in their environment while walking. Naive flies can be trained in a striped arena and manipulated to underestimate their size, but once consolidated, this memory seems to last for a lifetime. Consolidation of this memory is stress sensitive only in the first 2 h after training but cannot be retrieved for the next 12 h. We have identified a set of intrinsic, lateral neurons of the protocerebral bridge of the central complex [4, 5] that depend on dCREB2 transcriptional activity for long-term memory consolidation and maintenance.

Drosophila Full-Length Amyloid Precursor Protein Is Required for Visual Working Memory and Prevents Age-Related Memory Impairment.

The ß-amyloid precursor protein (APP) plays a central role in the etiology of Alzheimer's disease (AD). However, its normal physiological functions are still unclear. APP is cleaved by various secretases whereby sequential processing by the ß- and γ-secretases produces the ß-amyloid peptide that is accumulating in plaques that typify AD. In addition, this produces secreted N-terminal sAPPß fragments and the APP intracellular domain (AICD). Alternative cleavage by α-secretase results in slightly longer secreted sAPPα fragments and the identical AICD. Whereas the AICD has been connected with transcriptional regulation, sAPPα fragments have been suggested to have a neurotrophic and neuroprotective role [1]. Moreover, expression of sAPPα in APP-deficient mice could rescue their deficits in learning, spatial memory, and long-term potentiation [2]. Loss of the Drosophila APP-like (APPL) protein impairs associative olfactory memory formation and middle-term memory that can be rescued with a secreted APPL fragment [3]. We now show that APPL is also essential for visual working memory. Interestingly, this short-term memory declines rapidly with age, and this is accompanied by enhanced processing of APPL in aged flies. Furthermore, reducing secretase-mediated proteolytic processing of APPL can prevent the age-related memory loss, whereas overexpression of the secretases aggravates the aging effect. Rescue experiments confirmed that this memory requires signaling of full-length APPL and that APPL negatively regulates the neuronal-adhesion molecule Fasciclin 2. Overexpression of APPL or one of its secreted N termini results in a dominant-negative interaction with the FASII receptor. Therefore, our results show that specific memory processes require distinct APPL products.

The PKA-C3 catalytic subunit is required in two pairs of interneurons for successful mating of Drosophila.

Protein kinase A (PKA) has been shown to play a role in a plethora of cellular processes ranging from development to memory formation. Its activity is mediated by the catalytic subunits whereby many species express several paralogs. Drosophila encodes three catalytic subunits (PKA-C1-3) and whereas PKA-C1 has been well studied, the functions of the other two subunits were unknown. PKA-C3 is the orthologue of mammalian PRKX/Pkare and they are structurally more closely related to each other than to other catalytic subunits within their species. PRKX is expressed in the nervous system in mice but its function is also unknown. We now show that the loss of PKA-C3 in Drosophila causes copulation defects, though the flies are active and show no defects in other courtship behaviours. This phenotype is specifically due to the loss of PKA-C3 because PKA-C1 cannot replace PKA-C3. PKA-C3 is expressed in two pairs of interneurons that send projections to the ventro-lateral protocerebrum and the mushroom bodies and that synapse onto motor neurons in the ventral nerve cord. Rescue experiments show that expression of PKA-C3 in these interneurons is sufficient for copulation, suggesting a role in relaying information from the sensory system to motor neurons to initiate copulation.

Serotonin modulates a depression-like state in Drosophila responsive to lithium treatment.

Major depressive disorder (MDD) affects millions of patients; however, the pathophysiology is poorly understood. Rodent models have been developed using chronic mild stress or unavoidable punishment (learned helplessness) to induce features of depression, like general inactivity and anhedonia. Here we report a three-day vibration-stress protocol for Drosophila that reduces voluntary behavioural activity. As in many MDD patients, lithium-chloride treatment can suppress this depression-like state in flies. The behavioural changes correlate with reduced serotonin (5-HT) release at the mushroom body (MB) and can be relieved by feeding the antidepressant 5-hydroxy-L-tryptophan or sucrose, which results in elevated 5-HT levels in the brain. This relief is mediated by 5-HT-1A receptors in the α-/ß-lobes of the MB, whereas 5-HT-1B receptors in the γ-lobes control behavioural inactivity. The central role of serotonin in modulating stress responses in flies and mammals indicates evolutionary conserved pathways that can provide targets for treatment and strategies to induce resilience.

Motor-Skill Learning in an Insect Inspired Neuro-Computational Control System.

In nature, insects show impressive adaptation and learning capabilities. The proposed computational model takes inspiration from specific structures of the insect brain: after proposing key hypotheses on the direct involvement of the mushroom bodies (MBs) and on their neural organization, we developed a new architecture for motor learning to be applied in insect-like walking robots. The proposed model is a nonlinear control system based on spiking neurons. MBs are modeled as a nonlinear recurrent spiking neural network (SNN) with novel characteristics, able to memorize time evolutions of key parameters of the neural motor controller, so that existing motor primitives can be improved. The adopted control scheme enables the structure to efficiently cope with goal-oriented behavioral motor tasks. Here, a six-legged structure, showing a steady-state exponentially stable locomotion pattern, is exposed to the need of learning new motor skills: moving through the environment, the structure is able to modulate motor commands and implements an obstacle climbing procedure. Experimental results on a simulated hexapod robot are reported; they are obtained in a dynamic simulation environment and the robot mimicks the structures of Drosophila melanogaster.

Visual Working Memory Requires Permissive and Instructive NO/cGMP Signaling at Presynapses in the Drosophila Central Brain.

The gaseous second messenger nitric oxide (NO) has been shown to regulate memory formation by activating retrograde signaling cascades from post- to presynapse that involve cyclic guanosine monophosphate (cGMP) production to induce synaptic plasticity and transcriptional changes. In this study, we analyzed the role of NO in the formation of a visual working memory that lasts only a few seconds. This memory is encoded in a subset of ring neurons that form the ellipsoid body in the Drosophila brain. Using genetic and pharmacological manipulations, we show that NO signaling is required for cGMP-mediated CREB activation, leading to the expression of competence factors like the synaptic homer protein. Interestingly, this cell-autonomous function can also be fulfilled by hydrogen sulfide (H2S) through a converging pathway, revealing for the first time that endogenously produced H2S has a role in memory processes. Notably, the NO synthase is strictly localized to the axonal output branches of the ring neurons, and this localization seems to be necessary for a second, phasic role of NO signaling. We provide evidence for a model where NO modulates the opening of cGMP-regulated cation channels to encode a short-term memory trace. Local production of NO/cGMP in restricted branches of ring neurons seems to represent the engram for objects, and comparing signal levels between individual ring neurons is used to orient the fly during search behavior. Due to its short half-life, NO seems to be a uniquely suited second messenger to encode working memories that have to be restricted in their duration.

A screen for constituents of motor control and decision making in Drosophila reveals visual distance-estimation neurons.

Climbing over chasms larger than step size is vital to fruit flies, since foraging and mating are achieved while walking. Flies avoid futile climbing attempts by processing parallax-motion vision to estimate gap width. To identify neuronal substrates of climbing control, we screened a large collection of fly lines with temporarily inactivated neuronal populations in a novel high-throughput assay described here. The observed climbing phenotypes were classified; lines in each group are reported. Selected lines were further analysed by high-resolution video cinematography. One striking class of flies attempts to climb chasms of unsurmountable width; expression analysis guided us to C2 optic-lobe interneurons. Inactivation of C2 or the closely related C3 neurons with highly specific intersectional driver lines consistently reproduced hyperactive climbing whereas strong or weak artificial depolarization of C2/C3 neurons strongly or mildly decreased climbing frequency. Contrast-manipulation experiments support our conclusion that C2/C3 neurons are part of the distance-evaluation system.

A Fly-Inspired Mushroom Bodies Model for Sensory-Motor Control Through Sequence and Subsequence Learning.

Classification and sequence learning are relevant capabilities used by living beings to extract complex information from the environment for behavioral control. The insect world is full of examples where the presentation time of specific stimuli shapes the behavioral response. On the basis of previously developed neural models, inspired by Drosophila melanogaster, a new architecture for classification and sequence learning is here presented under the perspective of the Neural Reuse theory. Classification of relevant input stimuli is performed through resonant neurons, activated by the complex dynamics generated in a lattice of recurrent spiking neurons modeling the insect Mushroom Bodies neuropile. The network devoted to context formation is able to reconstruct the learned sequence and also to trace the subsequences present in the provided input. A sensitivity analysis to parameter variation and noise is reported. Experiments on a roving robot are reported to show the capabilities of the architecture used as a neural controller.

Modelling the insect Mushroom Bodies: Application to sequence learning.

Learning and reproducing temporal sequences is a fundamental ability used by living beings to adapt behaviour repertoire to environmental constraints. This paper is focused on the description of a model based on spiking neurons, able to learn and autonomously generate a sequence of events. The neural architecture is inspired by the insect Mushroom Bodies (MBs) that are a crucial centre for multimodal sensory integration and behaviour modulation. The sequence learning capability coexists, within the insect brain computational model, with all the other features already addressed like attention, expectation, learning classification and others. This is a clear example that a unique neural structure is able to cope concurrently with a plethora of behaviours. Simulation results and robotic experiments are reported and discussed.

Cell types and coincident synapses in the ellipsoid body of Drosophila.

Cellular ultrastructures for signal integration are unknown in any nervous system. The ellipsoid body (EB) of the Drosophila brain is thought to control locomotion upon integration of various modalities of sensory signals with the animal internal status. However, the expected excitatory and inhibitory input convergence that virtually all brain centres exhibit is not yet described in the EB. Based on the EB expression domains of genetic constructs from the choline acetyl transferase (Cha), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) genes, we identified a new set of neurons with the characteristic ring-shaped morphology (R neurons) which are presumably cholinergic, in addition to the existing GABA-expressing neurons. The R1 morphological subtype is represented in the Cha- and TH-expressing classes. In addition, using transmission electron microscopy, we identified a novel type of synapse in the EB, which exhibits the precise array of two independent active zones over the same postsynaptic dendritic domain, that we named 'agora'. This array is compatible with a coincidence detector role, and represents ~8% of all EB synapses in Drosophila. Presumably excitatory R neurons contribute to coincident synapses. Functional silencing of EB neurons by driving genetically tetanus toxin expression either reduces walking speed or alters movement orientation depending on the targeted R neuron subset, thus revealing functional specialisations in the EB for locomotion control.

A systematic nomenclature for the insect brain.

Despite the importance of the insect nervous system for functional and developmental neuroscience, descriptions of insect brains have suffered from a lack of uniform nomenclature. Ambiguous definitions of brain regions and fiber bundles have contributed to the variation of names used to describe the same structure. The lack of clearly determined neuropil boundaries has made it difficult to document precise locations of neuronal projections for connectomics study. To address such issues, a consortium of neurobiologists studying arthropod brains, the Insect Brain Name Working Group, has established the present hierarchical nomenclature system, using the brain of Drosophila melanogaster as the reference framework, while taking the brains of other taxa into careful consideration for maximum consistency and expandability. The following summarizes the consortium's nomenclature system and highlights examples of existing ambiguities and remedies for them. This nomenclature is intended to serve as a standard of reference for the study of the brain of Drosophila and other insects.

Serum response factor-mediated gene regulation in a Drosophila visual working memory.

BACKGROUND: Navigation through the environment requires a working memory for the chosen target and path integration facilitating an approach when the target becomes temporarily hidden. We have previously shown that this visual orientation memory resides in the ellipsoid body, which is part of the central complex in the Drosophila brain. Former analysis of foraging and ignorant mutants have revealed that a hierarchical PKG and RSKII kinase signaling cascade in a subset of the ellipsoid-body ring neurons is required for this type of working memory in flies. RESULTS: Here we show that mutants in the ellipsoid body open (ebo) gene, which encodes the actin-binding protein Exportin 6, exhibit excessive nuclear accumulation of actin during development and in the adult brain. ebo mutants lack the orientation memory independent of the structural defect in the ellipsoid-body neuropil, and EBO activity in any type of adult ring neurons is sufficient for orientation-memory function. Moreover, genetic interaction studies revealed that nuclear actin accumulation in ebo mutants inhibits the Drosophila coactivator myocardin-related transcription factor A (dMRTF) and therefore the transcriptional activator serum response factor (dSRF). dSRF also functions in different ring neurons, suggesting that it regulates abundance of a diffusible factor that enables a working memory in ellipsoid-body ring neurons. CONCLUSIONS: To date, SRF has only been implicated in longer forms of memory formation like synaptic long-term potentiation and depression. This study provides the first evidence that SRF-mediated gene regulation is also required for a working memory that lasts only for a few seconds.

Modeling the insect mushroom bodies: application to a delayed match-to-sample task.

Despite their small brains, insects show advanced capabilities in learning and task solving. Flies, honeybees and ants are becoming a reference point in neuroscience and a main source of inspiration for autonomous robot design issues and control algorithms. In particular, honeybees demonstrate to be able to autonomously abstract complex associations and apply them in tasks involving different sensory modalities within the insect brain. Mushroom Bodies (MBs) are worthy of primary attention for understanding memory and learning functions in insects. In fact, even if their main role regards olfactory conditioning, they are involved in many behavioral achievements and learning capabilities, as has been shown in honeybees and flies. Owing to the many neurogenetic tools, the fruit fly Drosophila became a source of information for the neuroarchitecture and biochemistry of the MBs, although the MBs of flies are by far simpler in organization than their honeybee orthologs. Electrophysiological studies, in turn, became available on the MBs of locusts and honeybees. In this paper a novel bio-inspired neural architecture is presented, which represents a generalized insect MB with the basic features taken from fruit fly neuroanatomy. By mimicking a number of different MB functions and architecture, we can replace and improve formerly used artificial neural networks. The model is a multi-layer spiking neural network where key elements of the insect brain, the antennal lobes, the lateral horn region, the MBs, and their mutual interactions are modeled. In particular, the model is based on the role of parts of the MBs named MB-lobes, where interesting processing mechanisms arise on the basis of spatio-temporal pattern formation. The introduced network is able to model learning mechanisms like olfactory conditioning seen in honeybees and flies and was found able also to perform more complex and abstract associations, like the delayed matching-to-sample tasks known only from honeybees. A biological basis of the proposed model is presented together with a detailed description of the architecture. Simulation results and remarks on the biological counterpart are also reported to demonstrate the possible applications of the designed computational model. Such neural architecture, able to autonomously learn complex associations is envisaged to be a suitable basis for an immediate implementation within an robot control architecture.

The visual orientation memory of Drosophila requires Foraging (PKG) upstream of Ignorant (RSK2) in ring neurons of the central complex.

Orientation and navigation in a complex environment requires path planning and recall to exert goal-driven behavior. Walking Drosophila flies possess a visual orientation memory for attractive targets which is localized in the central complex of the adult brain. Here we show that this type of working memory requires the cGMP-dependent protein kinase encoded by the foraging gene in just one type of ellipsoid-body ring neurons. Moreover, genetic and epistatic interaction studies provide evidence that Foraging functions upstream of the Ignorant Ribosomal-S6 Kinase 2, thus revealing a novel neuronal signaling pathway necessary for this type of memory in Drosophila.