Odorant-specific requirements for arrestin function in Drosophila olfaction

The ability to modulate olfactory sensitivity is necessary to detect chemical gradients and discriminate among a multitude of odor stimuli. Desensitization of odorant receptors has been postulated to occur when arrestins prevent the activation of downstream second messengers. A paucity of in vivo data on olfactory desensitization prompts use of Drosophila melanogaster genetics to investigate arrestins' role in regulating olfactory signaling pathways. Physiological analysis of peripheral olfactory sensitivity reveals decreased responsiveness to a host of chemically distinct odorants in flies deficient for arrestin1 (arr1), arrestin2 (arr2), or both. These phenotypes are manifest in odorant- and dose- dependent fashions. Additionally, mutants display altered adaptive properties under a prolonged exposure paradigm. Behaviorally, arr1 mutants are impaired in olfactory-based orientation towards attractive odor sources. As the olfactory deficits vary according to chemical identity and concentration, they indicate that a spectrum of arrestin activity is essential for odor processing depending upon the particular olfactory pathway involved. Arrestin mutant phenotypes are hypothesized to be a consequence of down-regulation of olfactory signaling to avoid cellular excitotoxicity. Importantly, phenotypic rescue of olfactory defects in arr1(1) mutants is achieved through transgenic expression of wild-type arr1. Taken together, these data clearly indicate that arrestins are required in a stimulus-specific manner for wild type olfactory function and add another level of complexity to peripheral odor coding mechanisms that ultimately impact olfactory behavior.     

A Drosophila nonvisual arrestin is required for the maintenance of olfactory sensitivity

Nonvisual arrestins are a family of multifunctional adaptor molecules that regulate the activities of diverse families of receptors including G protein-coupled receptors, frizzled, and transforming growth factor-beta receptors. These activities indicate broad roles in both physiology and development for nonvisual arrestins. Drosophila melanogaster has a single nonvisual arrestin, kurtz, which is found at high levels within the adult olfactory receptor neurons (ORNs), suggesting a role for this gene in modulating olfactory sensitivity. Using heat-induced expression of a krz cDNA through development, we rescued krz(1) lethality. The resulting adults lacked detectable levels of krz in the olfactory system. The rescued krz(1) homozygotes have an incompletely penetrant antennal structural defect that was completely rescued by the neural expression of a krz cDNA. The krz(1) loss-of-function adults without visible antennal defects displayed diminished behavioral responsiveness to both aversive and attractive odors and also demonstrated reduced olfactory receptor potentials. Both the behavioral and electrophysiological phenotypes were rescued by the targeted expression of the krz cDNA within postdevelopmental ORNs. Thus, krz is required within the nervous system for antennal development and is required later in the ORNs for the maintenance of olfactory sensitivity in Drosophila. The reduced receptor potentials in krz(1) antenna indicate that nonvisual arrestins are required for the early odor-induced signaling events within the ORNs.     

Male-male courtship behavior induced by ectopic expression of the Drosophila white gene: Role of sensory function and age

Male-male courtship behavior was recently reported to be induced in large populations of Drosophila (e.g., 600–1500 flies) by ectopic expression of the white (w) gene. Little is known about the basis of this behavior; in male-female courtship, sensory cues are believed to play an important role. Previous data are consistent with the possibility that misexpression of w causes abnormal reception or processing of sensory information. We show here that w-induced male-male courtship occurs in isolated pairs of flies. Thus the behavior does not depend on sensory cues found only among large populations of flies, or on cues produced only by a small subset of such populations. This finding enabled quantitative analysis of mechanisms that underlie the behavior. Specifically, male-male courtship does not depend on the reception of olfactory information, nor on the reception or generation of auditory cues, as determined by surgical ablation of antennae, maxillary palps, or wings. Although the rapid onset of the behavior following w induction suggested that its basis could lie in a modulation of sensory physiology, we found visual, olfactory, and gustatory function to be normal in physiological or behavioral tests. The only sensory deprivation to produce an effect on male-male courtship was testing under dim red light; the percentage of flies courting another male was reduced to one-fourth of control values. A striking age dependence of the behavior is also documented: courtship between paired male mini-w⁺ flies was not observed in tests of very young (1-day-old) flies, but occurs at high levels between the ages of 1 and 4 weeks. © 1996 John Wiley & Sons, Inc.     

Drosophila Pax‐6/eyeless is essential for normal adult brain structure and function

A role for the Pax‐6 homologue eyeless in adult Drosophila brain development and function is described. eyeless expression is detected in neurons, but not glial cells, of the mushroom bodies, the medullar cortex, the lateral horn, and the pars intercerebralis. Furthermore, severe defects in adult brain structures essential for vision, olfaction, and for the coordination of locomotion are provoked by two newly isolated mutations of Pax‐6/eyeless that result in truncated proteins. Consistent with the morphological lesions, we observe defective walking behavior for these eyeless mutants. The implications of these data for understanding postembryonic brain development and function in Drosophila are discussed. © 2001 John Wiley & Sons, Inc. J Neurobiol 46: 73–88, 2001     

Distinct roles for β‐arrestin2 and arrestin‐domain‐containing proteins in β2 adrenergic receptor trafficking

β‐arrestin 1 and 2 (also known as arrestin 2 and 3) are homologous adaptor proteins that regulate seven‐transmembrane receptor trafficking and signalling. Other proteins with predicted ‘arrestin‐like’ structural domains but lacking sequence homology have been indicated to function like β‐arrestin in receptor regulation. We demonstrate that β‐arrestin2 is the primary adaptor that rapidly binds agonist‐activated β₂ adrenergic receptors (β₂ARs) and promotes clathrin‐dependent internalization, E3 ligase Nedd4 recruitment and ubiquitin‐dependent lysosomal degradation of the receptor. The arrestin‐domain‐containing (ARRDC) proteins 2, 3 and 4 are secondary adaptors recruited to internalized β₂AR–Nedd4 complexes on endosomes and do not affect the adaptor roles of β‐arrestin2. Rather, the role of ARRDC proteins is to traffic Nedd4–β₂AR complexes to a subpopulation of early endosomes.     

Strain‐specific effects of parental age on offspring in Drosophila melanogaster

Maternal age is generally known to be negatively correlated with the lifespan of offspring in several animal models including yeast, rotifers, flies, and possibly in humans. However, several reports have shown positive effects of parental age on offspring lifespan. Thus, there was a need to investigate further the inconsistent results on the effect of parental age on lifespan. In this study, the effects of parental age on offspring fitness and lifespan were examined by using Drosophila melanogaster. The lifespan of offspring from old parents was significantly increased compared with that of the young counterparts in the Canton‐S (CS) strain but not in other D. melanogaster strains, such as Oregon‐R (OR) and w¹¹¹⁸. To find out why the lifespan is increased in the offspring from old parents in CS flies, fitness components that could modulate lifespan were examined in CS flies. Egg weight and body weight were reduced by parental aging and the offspring of old fathers or old mothers developed faster than that of the young. In addition, the offspring of old parents had increased resistance to oxidative and heat shock stresses. However, reproductive capacity, mating preference, and food intake were unaffected by parental aging. These results indicate that parental aging in CS strain D. melanogaster has beneficial effects on the lifespan and fitness of offspring. The presence of strain‐specific manner effects suggests that genetic background might be a significant factor in the parental age effect.     

Influence of the biogenic amine tyramine on ethanol‐induced behaviors in Drosophila

The biogenic amine tyramine has been implicated in drug‐induced behavior. The Drosophila inactive mutant is characterized by reduced tyramine and octopamine levels and is defective in cocaine sensitization. To test whether there is an overlap in the use of the amine neurotransmitter system in ethanol‐ and cocaine‐induced behaviors, mutant analyses were extended to the phenotypic characterization of inactive and other mutants effecting the tyramine and octopamine neurotransmitter system. The inactive mutant displays increased ethanol sensitivity and is impaired in the initial startle response upon ethanol application. Furthermore, this mutant fails to regulate its alcohol‐induced hyperactivity properly. In contrast to the defects seen after cocaine application, inactive mutants develop normal ethanol tolerance and sensitize to the locomotor activating effect of ethanol. The tyramine‐β‐hydroxylase mutant (TβH) with increased tyramine and depleted octopamine levels displays normal ethanol sensitivity, a startle repression, and hyperactivates more in response to ethanol. In addition, TβH mutants fail to develop a tolerance to the hyperactivating effect of ethanol. Ethanol‐induced sensitization does not seem to be impaired in either mutant, suggesting that tyramine is not required for this process. The comparative analysis of the phenotypes associated with inactive and TβH mutants suggests that the fine tuning of ethanol‐induced hyperactivity can be correlated with different tyramine levels. Defects in other aspects of ethanol‐induced behaviors might be due to different molecules or mechanisms. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Variations in the esterase expression pattern with respect to different light regimes in Drosophila agumbensis and Drosophila nagarholensis

Circadian clock enables organism’s to adapt under fluctuating environmental conditions by coupling of behavioral, physiological and molecular processes in a wide variety of organisms including bacteria, fungi, plants, birds and mammals. The endogenous circadian system functions to organize behavior and physiology to adapt to and anticipate environment changes in light and temperature. The present study is an attempt to understand enzyme profiles (alpha- and beta-esterases) of Drosophila agumbensis and Drosophila nagarholensis under light/dark (LD), constant dark (DD) constant light (LL), conditions over twenty generations. A polyacrylamide gel electrophoresis (7.5% – Native gel) was used to study the esterase expression patterns in two species of the montium subgroup of Drosophila. Alpha- and beta-esterase expression was significantly decreased in LL when compared to LD and DD at both the generations and species. In all the light regimes, females were found to have significantly higher level of α- and β-esterase expression than males. Flies were maintained under different light regimes showed difference in their expression patterns with respect to alpha- and beta-esterases. The present study showed that constant light conditions affect the expression of esterases in D. agumbensis and D. nagarholensis.     

dHb9 expressing larval motor neurons persist through metamorphosis to innervate adult‐specific muscle targets and function in Drosophila eclosion

The Drosophila larval nervous system is radically restructured during metamorphosis to produce adult specific neural circuits and behaviors. Genesis of new neurons, death of larval neurons and remodeling of those neurons that persistent collectively act to shape the adult nervous system. Here, we examine the fate of a subset of larval motor neurons during this restructuring process. We used a dHb9 reporter, in combination with the FLP/FRT system to individually identify abdominal motor neurons in the larval to adult transition using a combination of relative cell body location, axonal position, and muscle targets. We found that segment specific cell death of some dHb9 expressing motor neurons occurs throughout the metamorphosis period and continues into the post‐eclosion period. Many dHb9 > GFP expressing neurons however persist in the two anterior hemisegments, A1 and A2, which have segment specific muscles required for eclosion while a smaller proportion also persist in A2–A5. Consistent with a functional requirement for these neurons, ablating them during the pupal period produces defects in adult eclosion. In adults, subsequent to the execution of eclosion behaviors, the NMJs of some of these neurons were found to be dismantled and their muscle targets degenerate. Our studies demonstrate a critical continuity of some larval motor neurons into adults and reveal that multiple aspects of motor neuron remodeling and plasticity that are essential for adult motor behaviors. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1387–1416, 2016     

The Drosophila larva as a model for studying chemosensation and chemosensory learning: a review

Understanding the relationship between brain and behavior is the fundamental challenge in neuroscience. We focus on chemosensation and chemosensory learning in larval Drosophila and review what is known about its molecular and cellular bases. Detailed analyses suggest that the larval olfactory system, albeit much reduced in cell number, shares the basic architecture, both in terms of receptor gene expression and neuronal circuitry, of its adult counterpart as well as of mammals. With respect to the gustatory system, less is known in particular with respect to processing of gustatory information in the central nervous system, leaving generalizations premature. On the behavioral level, a learning paradigm for the association of odors with food reinforcement has been introduced. Capitalizing on the knowledge of the chemosensory pathways, we review the first steps to reveal the genetic and cellular bases of olfactory learning in larval Drosophila. We argue that the simplicity of the larval chemosensory system, combined with the experimental accessibility of Drosophila on the genetic, electrophysiological, cellular, and behavioral level, makes this system suitable for an integrated understanding of chemosensation and chemosensory learning.     

A species‐specific multigene family mediates differential sperm displacement in Drosophila melanogaster

Sperm competition is a postcopulatory sexual selection mechanism in species in which females mate with multiple males. Despite its evolutionary relevance in shaping male traits, the genetic mechanisms underlying sperm competition are poorly understood. A recently originated multigene family specific to Drosophila melanogaster, Sdic, is important for the outcome of sperm competition in doubly mated females, although the mechanistic nature of this phenotype remained unresolved. Here, we compared doubly mated females, second mated to either Sdic knockout or nonknockout males, and directly visualize sperm dynamics in the female reproductive tract. We found that a less effective removal of first‐to‐mate male's sperm within the female's sperm storage organs is consistent with a reduced sperm competitive ability of the Sdic knockout males. Our results highlight the role young genes can play in driving the evolution of sperm competition.     

Competing G protein‐coupled receptor kinases balance G protein and β‐arrestin signaling

Seven‐transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β‐arrestins, whose recruitment to the activated receptor is regulated by G protein‐coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal‐regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT_1AR) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)‐based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well‐established function in the desensitization of G‐protein activation, GRK2 exerts a strong negative effect on β‐arrestin‐dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2‐dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT_1AR, and HEK293 cells expressing other 7TMRs.     

Structure of ocellus photoreceptors in the ascidian Ciona intestinalis larva as revealed by an anti‐arrestin antibody

Although there have been several studies on the structure of the ocellus photoreceptors in ascidian tadpole larvae using electron microscopy, the overall structure of these photoreceptor cells, especially the projection sites of the axons, has not been revealed completely. The number of photoreceptor cells is also controversial. Here, the whole structure of the ocellus photoreceptors in the larvae of the ascidian Ciona intestinalis was revealed by using an anti‐arrestin (anti–Ci‐Arr) antibody. The cell bodies of 30 photoreceptor cells covered the right side of the ocellus pigment cell and their outer segments extended through the pigment cell into the pigment cup. The axons of the photoreceptor cells were bundled together ventro‐posteriorly in a single tract extending towards the midline. The nerve terminals diverged antero‐posteriorly at the midline of the posterior sensory vesicle (SV). The Ci‐arr gene was expressed throughout the SV at the embryonic mid‐tailbud stage and it became restricted to the neighborhood of the ocellus pigment when ocellus pigmentation occurred. At the same time, the Ci‐Arr protein was first detected, suggesting that the photoreceptor cells began to differentiate. The development of photoreceptor cells after hatching was also investigated using the anti–Ci‐Arr antibody. Three hours after hatching, the photoreceptor terminals began to ramify and then expanded. Previous behavioral analysis showed that the larvae did not respond to the step‐down of light until 2 h after hatching and then the photoresponse became robust. Accordingly, our results suggest that growth of the photoreceptor terminal is critical for the larvae to become photoresponsive. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Mutation of the central nervous system neuroblast proliferation repressor ana leads to defects in larval olfactory behavior

In the developing nervous system, interactions between glia and immature neurons or neuroblasts regulate axon pathfinding, migration, and cell division, and therefore affect structure and function. Glial control of neuroblast cell division has been documented by studies of the anachronism (ana) gene of Drosophila melanogaster. ana encodes a glycoprotein which, in the developing larval central nervous system, is secreted by glia that neighbor regulated neuroblasts. Mutations in ana lead to premature neuroblast proliferation in the larval brain. Examination of lacZ expression from an ana enhancer trap line as well as detection of the ana protein show that ana is also expressed in the larval antennal-maxillary complex (AMC) at all larval stages. As previously reported for the central nervous system, ana expression in the AMC appears to be confined to glial cells. Larval olfactory system function in ana mutants was assayed in a behavioral paradigm. When tested with the three different chemoattractants, third instar ana⁹ mutant larvae showed diminished olfactory response compared to controls. Examination of a second ana allele revealed aberrant olfactory response to ethyl acetate, demonstrating that more than one mutation in ana can give rise to abnormal larval olfactory behavior. Assays of early first instar ana⁹ mutant larvae revealed defective olfactory behavior, implying that the olfactory phenotype stems from early larval AMC and/or embryonic origins. This is consistent with proliferation analysis in the early larval AMC region which uncovered a significantly higher number of S-phase cells in ana⁹ mutants. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 199–211, 1997