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.

Development and Evaluation of Automated Tools for Auditory-Brainstem and Middle-Auditory Evoked Potentials Waves Detection and Annotation.

Auditory evoked potentials (AEPs) are brain-derived electrical signals, following an auditory stimulus, utilised to examine any obstructions along the brain neural-pathways and to diagnose hearing impairment. The clinical evaluation of AEPs is based on the measurements of the latencies and amplitudes of waves of interest; hence, their identification is a prerequisite for AEP analysis. This process has proven to be complex, as it requires relevant clinical experience, and the existing software for this purpose has little practical use. The aim of this study was the development of two automated annotation tools for ABR (auditory brainstem response)- and AMLR (auditory middle latency response)-tests. After the acquisition of 1046 raw waveforms, appropriate pre-processing and implementation of a four-stage development process were performed, to define the appropriate logical conditions and steps for each algorithm. The tools' detection and annotation results, regarding the waves of interest, were then compared to the clinicians' manual annotation, achieving match rates of at least 93.86%, 98.51%, and 91.51% respectively, for the three ABR-waves of interest, and 93.21%, 92.25%, 83.35%, and 79.27%, respectively, for the four AMLR-waves. The application of such tools in AEP analysis is expected to assist towards an easier interpretation of these signals.

Electrophysiological differences in distinct hearing threshold level individuals with and without tinnitus distress.

Tinnitus is the perception of sound when no actual external noise is present. Tinnitus is highly prevalent, with more than 1 in 7 adults in the EU having tinnitus, and it causes negative effects on quality of life for many individuals. However, there is currently no cure for tinnitus and its pathophysiology and genesis are unknown. Auditory evoked potentials (AEPs) provide a non-invasive means by which the electrical signals evoked by the brain can be recorded, and constitute a useful indicator for the evaluation of auditory disorders such as tinnitus and hearing loss. The present study analyzed a total of 98 auditory middle evoked potential (AMLR) waveforms, a subtype of AEPs, from 49 participants with subjective tinnitus, attempting to identify differences in AMLR parameters between sufferers with and without tinnitus distress. The waveforms were divided into three categories according to the ear's hearing level, and comparisons were made between sufferers in the same hearing level category. The results of the analysis indicated some statistically significant differences in AMLR latencies and amplitudes between the compared groups. Clinical Relevance- Identification of the electro-physiological profile of subjective tinnitus sufferers based on the distress manifested by tinnitus using AMLRs.

Glossary on atmospheric electricity and its effects on biology.

There is an increasing interest to study the interactions between atmospheric electrical parameters and living organisms at multiple scales. So far, relatively few studies have been published that focus on possible biological effects of atmospheric electric and magnetic fields. To foster future work in this area of multidisciplinary research, here we present a glossary of relevant terms. Its main purpose is to facilitate the process of learning and communication among the different scientific disciplines working on this topic. While some definitions come from existing sources, other concepts have been re-defined to better reflect the existing and emerging scientific needs of this multidisciplinary and transdisciplinary area of research.

Adult Movement Defects Associated with a CORL Mutation in Drosophila Display Behavioral Plasticity.

The CORL family of CNS-specific proteins share a Smad-binding region with mammalian SnoN and c-Ski protooncogenes. In this family Drosophila CORL has two mouse and two human relatives. Roles for the mouse and human CORL proteins are largely unknown. Based on genome-wide association studies linking the human CORL proteins Fussel15 and Fussel18 with ataxia, we tested the hypothesis that dCORL mutations will cause adult movement disorders. For our initial tests, we conducted side by side studies of adults with the small deletion Df(4)dCORL and eight control strains. We found that deletion mutants exhibit three types of behavioral plasticity. First, significant climbing defects attributable to loss of dCORL are eliminated by age. Second, significant phototaxis defects due to loss of dCORL are partially ameliorated by age and are not due to faulty photoreceptors. Third, Df(4)dCORL males raised in groups have a lower courtship index than males raised as singles though this defect is not due to loss of dCORL Subsequent tests showed that the climbing and phototaxis defects were phenocpied by dCORL21B and dCORL23C two CRISPR generated mutations. Overall, the finding that adult movement defects due to loss of dCORL are subject to age-dependent plasticity suggests new hypotheses for CORL functions in flies and mammals.

Postnatal Increases in Axonal Conduction Velocity of an Identified Drosophila Interneuron Require Fast Sodium, L-Type Calcium and Shaker Potassium Channels.

During early postnatal life, speed up of signal propagation through many central and peripheral neurons has been associated with an increase in axon diameter or/and myelination. Especially in unmyelinated axons postnatal adjustments of axonal membrane conductances is potentially a third mechanism but solid evidence is lacking. Here, we show that axonal action potential (AP) conduction velocity in the Drosophila giant fiber (GF) interneuron, which is required for fast long-distance signal conduction through the escape circuit, is increased by 80% during the first day of adult life. Genetic manipulations indicate that this postnatal increase in AP conduction velocity in the unmyelinated GF axon is likely owed to adjustments of ion channel expression or properties rather than axon diameter increases. Specifically, targeted RNAi knock-down of either Para fast voltage-gated sodium, Shaker potassium (Kv1 homologue), or surprisingly, L-type like calcium channels counteracts postnatal increases in GF axonal conduction velocity. By contrast, the calcium-dependent potassium channel Slowpoke (BK) is not essential for postnatal speeding, although it also significantly increases conduction velocity. Therefore, we identified multiple ion channels that function to support fast axonal AP conduction velocity, but only a subset of these are regulated during early postnatal life to maximize conduction velocity. Despite its large diameter (∼7 µm) and postnatal regulation of multiple ionic conductances, mature GF axonal conduction velocity is still 20-60 times slower than that of vertebrate Aß sensory axons and α motoneurons, thus unraveling the limits of long-range information transfer speed through invertebrate circuits.

Longitudinal assessment of health-span and pre-death morbidity in wild type Drosophila.

The increase in human life expectancy is accompanied by age-related cognitive and motor disability, thus raising the demand for strategies toward healthy aging. This requires understanding the biology of normal aging and late-life functional phenotypes. Genetic model organisms, such as Drosophila melanogaster, can help identifying evolutionary conserved mechanisms underlying aging. Longitudinal assessment of motor performance of more than 1000 individual flies revealed age-related motor performance decline and specific late-life motor disabilities. This allows defining heath- and ill-span and scoring late-life quality of individual flies. As in mammals, including humans, onset, duration, severity, and progression dynamics of decline are heterogenic and characterized by both, progressive worsening and sudden late-life events. Flies either become increasingly incapacitated by accumulating disability over multiple days prior to death, or they escape disability until few hours prior to death. Both late-life trajectories converge into a terminal stage characterized by stereotypical signs of functional collapse and death within 3 hours. Drosophila can now be used to evaluate life prolonging manipulations in the context of late-life quality. High sugar diet increases lifespan and late-life quality, whereas lifespan prolonging antioxidant supplementation has either no, or negative effects on late-life quality, depending on base diet and gender.

Human Tau isoform-specific presynaptic deficits in a Drosophila Central Nervous System circuit.

Accumulation of normal or mutant human Tau isoforms in Central Nervous System (CNS) neurons of vertebrate and invertebrate models underlies pathologies ranging from behavioral deficits to neurodegeneration that broadly recapitulate human Tauopathies. Although some functional differences have begun to emerge, it is still largely unclear whether normal and mutant Tau isoforms induce differential effects on the synaptic physiology of CNS neurons. We use the oligosynaptic Giant Fiber System in the adult Drosophila CNS to address this question and reveal that 3R and 4R isoforms affect distinct synaptic parameters. Whereas 0N3R increased failure rate upon high frequency stimulation, 0N4R compromised stimulus conduction and response speed at a specific cholinergic synapse in an age-dependent manner. In contrast, accumulation of the R406W mutant of 0N4R induced mild, age-dependent conduction velocity defects. Because 0N4R and its mutant isoform are expressed equivalently, this demonstrates that the defects are not merely consequent of exogenous human Tau accumulation and suggests distinct functional properties of 3R and 4R isoforms in cholinergic presynapses.

CORL Expression and Function in Insulin Producing Neurons Reversibly Influences Adult Longevity in Drosophila.

CORL proteins (known as SKOR in mice, Fussel in humans and fussel in Flybase) are a family of CNS specific proteins related to Sno/Ski oncogenes. Their developmental and adult roles are largely unknown. A Drosophila CORL (dCORL) reporter gene is expressed in all Drosophila insulin-like peptide 2 (dILP2) neurons of the pars intercerebralis (PI) of the larval and adult brain. The transcription factor Drifter is also expressed in the PI in a subset of dCORL and dILP2 expressing neurons and in several non-dILP2 neurons. dCORL mutant virgin adult brains are missing all dILP2 neurons that do not also express Drifter. This phenotype is also seen when expressing dCORL-RNAi in neurosecretory cells of the PI. dCORL mutant virgin adults of both sexes have a significantly shorter lifespan than their parental strain. This longevity defect is completely reversed by mating (lifespan increases over 50% for males and females). Analyses of dCORL mutant mated adult brains revealed a complete rescue of dILP2 neurons without Drifter. Taken together, the data suggest that dCORL participates in a neural network connecting the insulin signaling pathway, longevity and mating. The conserved sequence and CNS specificity of all CORL proteins imply that this network may be operating in mammals.

Targeted Downregulation of s36 Protein Unearths its Cardinal Role in Chorion Biogenesis and Architecture during Drosophila melanogaster Oogenesis.

Drosophila chorion represents a model biological system for the in vivo study of gene activity, epithelial development, extracellular-matrix assembly and morphogenetic-patterning control. It is produced during the late stages of oogenesis by epithelial follicle cells and develops into a highly organized multi-layered structure that exhibits regional specialization and radial complexity. Among the six major proteins involved in chorion's formation, the s36 and s38 ones are synthesized first and regulated in a cell type-specific and developmental stage-dependent manner. In our study, an RNAi-mediated silencing of s36 chorionic-gene expression specifically in the follicle-cell compartment of Drosophila ovary unearths the essential, and far from redundant, role of s36 protein in patterning establishment of chorion's regional specialization and radial complexity. Without perturbing the developmental courses of follicle- and nurse-cell clusters, the absence of s36 not only promotes chorion's fragility but also induces severe structural irregularities on chorion's surface and entirely impairs fly's fertility. Moreover, we herein unveil a novel function of s36 chorionic protein in the regulation of number and morphogenetic integrity of dorsal appendages in follicles sporadically undergoing aged fly-dependent stress.

Effects of gender and age on hypothalamic-pituitary-adrenal reactivity after pharmacological challenge with low-dose 1-µg ACTH test: a prospective study in healthy adults.

OBJECTIVE: Τhe aim of this study was to observe cortisol responses to a pharmacological challenge with 1 µg ACTH as a function of age and gender. PATIENTS: Twenty-one young (13 women and 8 men; mean age: 43∙48 ± 10∙52 years) and forty elderly (20 women and 20 men; mean age: 79 ± 6∙76 years) volunteers without overt acute or chronic disease participated in this study. MEASUREMENTS: Basal serum cortisol levels were measured, and cortisol values were obtained at 30 and 60 min after administration of 1 µg ACTH. Free thyroxine, triiodothyronine, thyrotrophin, prolactin, dehydroepiandrosterone sulphate (DHEAS), insulin-like growth factor-1, follicle stimulating hormone, luteinizing hormone, oestradiol levels in women and total testosterone levels in men were also measured at baseline. RESULTS: No differences were observed between the groups of older and younger individuals in baseline plasma cortisol levels and mean cortisol responses at 30 and 60 min postchallenge. In the elderly subjects, statistically lower cortisol responses were observed in men at 30 and 60 min after 1-µg ACTH challenge (540∙17 ± 112∙28 vs 670∙11 ± 146∙49 nmol/l in women, P = 0∙003 at 30 min, and 482∙24 ± 108∙14 vs 568∙04 ± 135∙73 nmol/l in women, P = 0∙03 at 60 min). Furthermore, significant lower values in PRL, IGF-1, DHEAS and T3 were detected in older subjects when compared to the younger age group. Interestingly, a higher incidence of individuals who did not achieve normal responses to 1-µg ACTH test (>497 nmol/l) was noted in the group of elderly male subjects. CONCLUSION: This study documents a marked gender effect in the elderly, in the cortisol response levels to 1-µg ACTH stimulation. Elderly men demonstrate a decreased responsiveness compared to women in the same age range.

Constitutive activation of Ca2+/calmodulin-dependent protein kinase II during development impairs central cholinergic transmission in a circuit underlying escape behavior in Drosophila.

Development of neural circuitry relies on precise matching between correct synaptic partners and appropriate synaptic strength tuning. Adaptive developmental adjustments may emerge from activity and calcium-dependent mechanisms. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been associated with developmental synaptic plasticity, but its varied roles in different synapses and developmental stages make mechanistic generalizations difficult. In contrast, we focused on synaptic development roles of CaMKII in a defined sensory-motor circuit. Thus, different forms of CaMKII were expressed with UAS-Gal4 in distinct components of the giant fiber system, the escape circuit of Drosophila, consisting of photoreceptors, interneurons, motoneurons, and muscles. The results demonstrate that the constitutively active CaMKII-T287D impairs development of cholinergic synapses in giant fiber dendrites and thoracic motoneurons, preventing light-induced escape behavior. The locus of the defects is postsynaptic as demonstrated by selective expression of transgenes in distinct components of the circuit. Furthermore, defects among these cholinergic synapses varied in severity, while the glutamatergic neuromuscular junctions appeared unaffected, demonstrating differential effects of CaMKII misregulation on distinct synapses of the same circuit. Limiting transgene expression to adult circuits had no effects, supporting the role of misregulated kinase activity in the development of the system rather than in acutely mediating escape responses. Overexpression of wild-type transgenes did not affect circuit development and function, suggesting but not proving that the CaMKII-T287D effects are not due to ectopic expression. Therefore, regulated CaMKII autophosphorylation appears essential in central synapse development, and particular cholinergic synapses are affected differentially, although they operate via the same nicotinic receptor.

A muscle-specific p38 MAPK/Mef2/MnSOD pathway regulates stress, motor function, and life span in Drosophila.

Molecular mechanisms that concordantly regulate stress, life span, and aging remain incompletely understood. Here, we demonstrate that in Drosophila, a p38 MAP kinase (p38K)/Mef2/MnSOD pathway is a coregulator of stress and life span. Hence, overexpression of p38K extends life span in a MnSOD-dependent manner, whereas inhibition of p38K causes early lethality and precipitates age-related motor dysfunction and stress sensitivity, that is rescued through muscle-restricted (but not neuronal) add-back of p38K. Additionally, mutations in p38K are associated with increased protein carbonylation and Nrf2-dependent transcription, while adversely affecting metabolic response to hypoxia. Mechanistically, p38K modulates expression of the mitochondrial MnSOD enzyme through the transcription factor Mef2, and predictably, perturbations in MnSOD modify p38K-dependent phenotypes. Thus, our results uncover a muscle-restricted p38K-Mef2-MnSOD signaling module that influences life span and stress, distinct from the insulin/JNK/FOXO pathway. We propose that potentiating p38K might be instrumental in restoring the mitochondrial detoxification machinery and combating stress-induced aging.

Alternations of 14-3-3 θ and ß protein levels in brain during experimental sepsis.

The 14-3-3 family members play a crucial role in the determination of cell fate, exerting their antiapoptotic activity through directly interfering with the critical function of the mitochondrial core proapoptotic machinery. Dimerization of 14-3-3 is vital for the interaction with many of its client proteins and is regulated by phosphorylation. In a previous study, we observed time-dependent neuronal apoptosis during sepsis. Therefore, in the present study, we sought to evaluate the expression of 14-3-3 θ and ß isoforms in septic brain and their association with apoptosis. Sepsis was induced by a CLP model in Wistar rats that were sacrificed at predefined time points. Flow cytometric analysis showed a sepsis-induced, time-dependent alteration of 14-3-3 θ and ß isoforms in both Neun(+) and GFAP(+) cells. 14-3-3 θ was linearly correlated with apoptosis, and stratified analysis for alive and apoptotic neuronal cells demonstrated a gradual down-regulation of θ isoform in alive neurons and astrocytes. The phospho-P38 (pP38) MAP kinase levels were altered in a time-dependent manner during sepsis, presenting a peak at 6 hr post-CLP. A significant correlation between the two isoforms of 14-3-3 was observed in septic rats, with the θ isoform predominant at all time points. The hippocampus, Purkinje cells, and glia-like cells showed intense immunohistochemical reactivity for 14-3-3 θ isoform, whereas the choroid plexus showed constantly increased ß isoform expression. Our results showed that sepsis alters the expression of both 14-3-3 θ and ß isoforms in a time-, cell-, and topography-dependent manner.

The steroid hormone-regulated gene Broad Complex is required for dendritic growth of motoneurons during metamorphosis of Drosophila.

Dendrites are subject to subtle modifications as well as extensive remodeling during the assembly and maturation of neural circuits in a wide variety of organisms. During metamorphosis, Drosophila flight motoneurons MN1-MN4 undergo dendritic regression, followed by regrowth, whereas MN5 differentiates de novo (Consoulas et al. [2002] J. Neurosci. 22:4906-4917). Many cellular changes during metamorphosis are triggered and orchestrated by the steroid hormone 20-hydroxyecdysone, which initiates a cascade of coordinated gene expression. Broad Complex (BRC), a primary response gene in the ecdysone cascade, encodes a family of transcription factors (BRC-Z1-Z4) that are essential for metamorphic reorganization of the central nervous system (CNS). Using neuron-filling techniques that reveal cellular morphology with very high resolution, we tested the hypothesis that BRC is required for metamorphic development of MN1-MN5. Through a combination of loss-of-function mutant analyses, genetic mapping, and transgenic rescue experiments, we found that 2Bc function, mediated by BRC-Z3, is required selectively for motoneuron dendritic regrowth (MN1-MN4) and de novo outgrowth (MN5), as well as for soma expansion of MN5. In contrast, larval development and dendritic regression of MN1-MN4 are BRC-independent. Surprisingly, BRC proteins are not expressed in the motoneurons, suggesting that BRC-Z3 exerts its effect in a non-cell-autonomous manner. The 2Bc mutants display no gross defects in overall thoracic CNS structure, or in peripheral structures such as target muscles or sensory neurons. Candidates for mediating the effect of BRC-Z3 on dendritic growth of MN1-MN5 include their synaptic inputs and non-neuronal CNS cells that interact with them through direct contact or diffusible factors.

Evidence for cell autonomous AP1 function in regulation of Drosophila motor-neuron plasticity.

BACKGROUND: The transcription factor AP1 mediates long-term plasticity in vertebrate and invertebrate central nervous systems. Recent studies of activity-induced synaptic change indicate that AP1 can function upstream of CREB to regulate both CREB-dependent enhancement of synaptic strength as well as CREB-independent increase in bouton number at the Drosophila neuromuscular junction (NMJ). However, it is not clear from this study if AP1 functions autonomously in motor neurons to directly modulate plasticity. RESULTS: Here, we show that Fos and Jun, the two components of AP1, are abundantly expressed in motor neurons. We further combine immunohistochemical and electrophysiological analyses with use of a collection of enhancers that tightly restrict AP1 transgene expression within the nervous system to show that AP1 induction or inhibition in, but not outside of, motor neurons is necessary and sufficient for its modulation of NMJ size and strength. CONCLUSION: By arguing against the possibility that AP1 effects at the NMJ occur via a polysynaptic mechanism, these observations support a model in which AP1 directly modulates NMJ plasticity processes through a cell autonomous pathway in the motor neuron. The approach described here may serve as a useful experimental paradigm for analyzing cell autonomy of genes found to influence structure and function of Drosophila motor neurons.

Dendritic remodeling and growth of motoneurons during metamorphosis of Drosophila melanogaster.

Insect motoneurons display dramatic dendritic plasticity during metamorphosis. Many larval motoneurons survive to adulthood but undergo dendritic regression and outgrowth as they are incorporated into developing circuits. This study explores the dendritic remodeling and development of Drosophila motoneurons MN1-MN5, which innervate indirect flight muscles of the adult. MN1-MN5 are persistent larval neurons exhibiting two distinct metamorphic histories. MN1-MN4 are born in the embryo, innervate larval muscles, and undergo dendritic regression and regrowth during metamorphosis. MN5, which was identified through a combination of intracellular dye injection and retrograde staining at all stages, is also born embryonically but remains developmentally arrested until the onset of metamorphosis. In the larva, MN5 lacks dendrites, and its axon stops in the mesothoracic nerve without innervating a target muscle. It is dye coupled to the peripherally synapsing interneuron, which will become part of the giant fiber escape circuit of the adult fly. During pupal development, MN5 undergoes de novo dendritic growth and extension of its axon to innervate the developing target muscle. Its unique developmental history and identifiability make MN5 well suited for the study of dendritic growth using genetic and neurophysiological approaches.

The fate of specific motoneurons and sensory neurons of the pregenital abdominal segments inTenebrio molitor (Insecta : Coleoptera) during metamorphosis.

The anatomy and innervation of the lateral external muscle and sensory cells located in the ventral region of pregenital abdominal segments were examined at the larval and adult stages ofTenebrio molitor (Coleoptera). All seven muscles located in this region degenerate during the pupal stage, whilst only the lateral external median (lem) appears in the adult. Backfillings of the motor nerve innervating this muscle reveal that, at both larval and adult stages, it is innervated by ten neurons. Intracellular records from the muscle fibres show that two neurons are inhibitory, and at least five are excitatory. There are also two unpaired neurons. A variety of sensory organs are located in the ventral region of the larvae, whilst only campaniform sensilla are found in the adult. At both stages, the innervation pattern of the sensory nerve branches is very similar. Also, the central projections of the sensory cells occupy similar neuropilar areas. Finally, prolonged intracellular records from the lem muscle revealed that, at the larval stage, it participates only in segmental or intersegmental reflexes, whilst in the adult it has a primary expiratory role in ventilation. The results show that extensive changes occur in the number of muscles located in the ventral region of the pregenital abdominal segments, as well as in the arrangement and number of sensory neurons, in the structure of the exoskeleton, and even in the central nervous system. In contrast, only minor changes are observed in the sensory and motor nerve branches, in the sensory projections, and in the number and the location of the motoneurons innervating the lateral external median muscle.