Fiber Number in the Mushroom Bodies of Adult Drosophila melanogaster depends on Age,Sex and Experience

The mushroom bodies are two characteristically shaped structures of the insect central brain. In Drosophila melanogaster they contain more fibers in females than in males. Within the first week of adult life the total number of fibers increases by about 15% and decreases again in flies older than 3-4 weeks. The number of mushroom body fibers is significantly reduced in flies kept under social isolation or deprived of their antennal input, but not in flies subjected to visual deprivation.     

Are the structural changes in adult Drosophila mushroom bodies memory traces? Studies on biochemical learning mutants

The pre-imaginal development of Drosophila mushroom bodies is under the influence of an unknown variable which causes populations of wild-type flies at eclosion to differ in the average number of Kenyon cell fibers. During the first week of adult life the number adjusts to an intermediate level which depends upon the experience of the flies. Under olfactory deprivation or social isolation it reaches a lower level than under favorable rearing conditions (J. Neurogenet., 1 (1984) 113-126). The biochemical learning mutants dunce and rutabaga show no experience-dependent modulation of fiber number (Fig. 2). In both strains the mushroom bodies of young adults seem to develop abnormally: in dunce a loss of about 600 fibers is observed, in rutabaga fiber number is low at eclosion and does not increase (Fig. 1a). The following model for long-term memory is proposed: in mushroom bodies outgrowth and decay of Kenyon cell fibers occur simultaneously. The fibers randomly form transient synapses onto extrinsic output neurons of the mushroom bodies and receive synapses from modulating neurons. Experience consolidates certain synapses, thus prolonging survival of the respective Kenyon cell fibers and increasing the steady state level of fiber number (Fig. 3).     

ARE THE STRUCTURAL CHANGES IN ADULT DROSOPHILA MUSHROOM BODIES MEMORY TRACES? STUDIES ON BIOCHEMICAL LEARNING MUTANTS

The pre-imaginal development of Drosophila mushroom bodies is under the influence of an unknown variable which causes populations of wild-type flies at eclosion to differ in the average number of Kenyon cell fibers. During the first week of adult life the number adjusts to an intermediate level which depends upon the experience of the flies. Under olfactory deprivation or social isolation it reaches a lower level than under favorable rearing conditions (J. Neurogenet., 1 (1984) 113–126). The biochemical learning mutants dunce and rutabaga show no experince-dependent modulation of fiber number (Fig. 2). In both strains the mushroom bodies of young adults seem to develop abnormally; in dunce a loss of aboout 600 fibers is observed, in rutabaga fiber number is low at eclosion and does not increase (Fig. 1a). The following model for long-term memory is proposed: in mushroom bodies outgrowth and decay of Kenyon cell fibers occur simultaneously. The fibers randomly form transient synapses onto extrinsic output neurons of the mushroom bodies and receive synapses from modulating neurons. Experience consolidates certain synapses, thus prolonging survival of the respective Kenyon cell fibers and increasing the steady state level of fiber number (Fig. 3).     

Fiber number in the mushroom bodies of adult Drosophila melanogaster depends on age, sex and experience

The mushroom bodies are two characteristically shaped structures of the insect central brain. In Drosophila melanogaster they contain more fibers in females than in males. Within the first week of adult life the total number of fibers increases by about 15% and decreases again in flies older than 3-4 weeks. The number of mushroom body fibers is significantly reduced in flies kept under social isolation or deprived of their antennal input, but not in flies subjected to visual deprivation.     

Fiber number in the mushroom bodies of adult Drosophila melanogaster depends on age, sex and experience

The mushroom bodies are two characteristically shaped structures of the insect central brain. In Drosophila melanogaster they contain more fibers in females than in males. Within the first week of adult life the total number of fibers increases by about 15% and decreases again in flies older than 3-4 weeks. The number of mushroom body fibers is significantly reduced in flies kept under social isolation or deprived of their antennal input, but not in flies subjected to visual deprivation.     

Are the structural changes in adult Drosophila mushroom bodies memory traces? Studies on biochemical learning mutants

The pre-imaginal development of Drosophila mushroom bodies is under the influence of an unknown variable which causes populations of wild-type flies at eclosion to differ in the average number of Kenyon cell fibers. During the first week of adult life the number adjusts to an intermediate level which depends upon the experience of the flies. Under olfactory deprivation or social isolation it reaches a lower level than under favorable rearing conditions (J. Neurogenet., 1 (1984) 113-126). The biochemical learning mutants dance and rutabaga show no experience-dependent modulation of fiber number (Fig. 2). In both strains the mushroom bodies of young adults seem to develop abnormally; in dance a loss of about 600 fibers is observed, in rutabaga fiber number is low at eclosion and does not increase (Fig. 1a). The following model for long-term memory is proposed: in mushroom bodies outgrowth and decay of Kenyon cell fibers occur simultaneously. The fibers randomly form transient synapses onto extrinsic output neurons of the mushroom bodies and receive synapses from modulating neurons. Experience consolidates certain synapses, thus prolonging survival of the respective Kenyon cell fibers and increasing the steady state level of fiber number (Fig. 3).     

Are the structural changes in adult Drosophila mushroom bodies memory traces? Studies on biochemical learning mutants

The pre-imaginal development of Drosophila mushroom bodies is under the influence of an unknown variable which causes populations of wild-type flies at eclosion to differ in the average number of Kenyon cell fibers. During the first week of adult life the number of adjusts to an intermediate level which depends upon the experience of the flies. Under olfactory deprivation or social isolation it reaches a lower level than under favorable rearing conditions (J. Neurogenet., 1 (1984) 113-126). The biochemical learning mutants dunce and rutabaga show no experience-dependent modulation of fiber number. In both strains the mushroom bodies of young adults seem to develop abnormally: in dunce a loss of about 600 fibers is observed, in rutabaga fiber number is low at eclosion and does not increase. The following model for long-term memory is proposed: in mushroom bodies outgrowth and decay of Kenyon cell fibers occur simultaneously. The fibers randomly form transient synapses onto extrinsic output neurons of the mushroom bodies and receive synapses from modulating neurons. Experience consolidates certain synapses, thus prolonging survival of the respective Kenyon cell fibers and increasing the steady state level of fiber number.     

The role of central parts of the brain in the control of sound production during courtship in Drosophila melanogaster

The question of the roles of the two main parts of the insect brain, the mushroom bodies and the central complex, in controlling motor coordination and triggering a variety of behavioral programs, including sound production, remains controversial. With the aim of improving our understanding of this question, we studied the parameters of songs used by five-day-old males during courtship for fertilized wild-type females (Canton-S, C-S) over 5-min periods at 25°C; males were of two wild-type Drosophila Melanogaster lines (Berlin and C-S). Berlin males lacking mushroom bodies because of treatment with hydroxyurea during development (chemical removal of the mushroom bodies) were used, along with two mutants with defects in the mushroom bodies (mbm ¹ and mud ¹), two mutants with defects in the central complex (ccb ^KS127 and cex ^KS181), and mutant cxb ^N71 with defects in both the mushroom bodies and the central complex. The experiments reported here showed that courtship songs in males lacking mushroom bodies were virtually identical to those of wild-type males. The main parameters of pulsatile song in mutants mbm ¹ and mud ¹ (interpulse interval and train duration) were insignificantly different from those of the songs of wild-type flies, though the stability of the pulse oscillator was the same. Flies of these lines were no different from wild-type flies in terms of courtship success (percentage of copulating pairs in 10-min tests). Conversely, the songs of mutants with defects in the central complex differed from those of wild-type males. Firstly, there was degradation of the stability of the pulse oscillator and interpulse intervals were very variable. In addition, pulses were often significantly longer and appeared multicyclic, as in the well-known cacophony mutant, while the mean train duration was significantly shorter. Males of the line cex ^KS181 usually courted very intensely, though abnormal sounds were generally emitted. Mutants cex ^KS181 and ccb ^KS127 were significantly less successful in courtship than wild-type flies. These data show that the central complex appears to play a very important role in controlling song, while the mushroom bodies are not related to this function.     

Mushroom body influence on locomotor activity and circadian rhythms in Drosophila melanogaster

Whether or not mechanisms underlying circadian locomotor rhythms and learning are related anatomically through the mushroom bodies (MBs) was investigated by monitoring behavioral rhythmicity in flies with MB lesions induced by chemical ablation and by mutations in five different genes. All flies tested were later examined histologically to assess (1) MB neuroanatomy, and (2) the condition of the putative pacemaker cells--the ventral Lateral Neurons (LN(v)s) and their terminals that project to the vicinity of the MB calyces. All groups of flies had normal rhythms except for mushroom body miniature (mbm; only in a wild-type Berlin genetic background) and mushroom body defect (mud). MB ablation had no effect on the gender-specific differences in the rhythmic activity profile that are typical of wild-type flies. However, ablated males had a slightly longer period than untreated males and were more active under constant dark conditions. LN(v)s and their arborization patterns appeared normal in MB-ablated and in most mutant flies. Activity defects of mbm flies were attributed to genetic background rather than to the mutation alone. Misrouted LN(v) projections and approximately 14% arrhythmia of mud flies were uncorrelated and attributed to pleiotropy rather than to specific effects of MB lesions. Our results imply that MBs are not involved in circadian activity rhythms but that they do have an inhibitory effect on activity levels of male flies.     

MUSHROOM BODY INFLUENCE ON LOCOMOTOR ACTIVITY AND CIRCADIAN RHYTHMS IN DROSOPHILA MELANOGASTER

Whether or not mechanisms underlying circadian locomotor rhythms and learning are related anatomically through the mushroom bodies (MBs) was investigated by monitoring behavioral rhythmicity in flies with MB lesions induced by chemical ablation and by mutations in five different genes. All flies tested were later examined histologically to assess (1) MB neuroanatomy, and (2) the condition of the putative pacemaker cells -- the ventral Lateral Neurons (LN v s) and their terminals that project to the vicinity of the MB calyces. All groups of flies had normal rhythms except for mushroom body miniature ( mbm ; only in a wild-type Berlin genetic background) and mushroom body defect ( mud ). MB ablation had no effect on the gender-specific differences in the rhythmic activity profile that are typical of wild-type flies. However, ablated males had a slightly longer period than untreated males and were more active under constant dark conditions. LN v s and their arborization patterns appeared normal in MB-ablated and in most mutant flies. Activity defects of mbm flies were attributed to genetic background rather than to the mutation alone. Misrouted LN v projections and ～14% arrhythmia of mud flies were uncorrelated and attributed to pleiotropy rather than to specific effects of MB lesions. Our results imply that MBs are not involved in circadian activity rhythms but that they do have an inhibitory effect on activity levels of male flies.     

Drosophila MAGE controls neural precursor proliferation in postembryonic neurogenesis

Necdin, a member of the MAGE family, is expressed abundantly in postmitotic neurons and is required for their differentiation and survival. In mammals, the MAGE family consists of more than 30 genes, whereas only one MAGE gene exists in the genome of nonmammalian vertebrates such as zebrafish and chicken. These nonmammalian MAGE genes are expressed in developing nervous system, and the primary structures of the encoded proteins resemble those of necdin-like MAGE proteins. Fruit fly Drosophila also carries a single necdin-like MAGE gene, which is highly expressed in neural stem cells (neuroblasts) during nervous system development. In the present study, we investigated the function of MAGE in Drosophila neurogenesis in vivo using an RNA interference (RNAi) -mediated gene knockdown system. Ubiquitous knockdown of Drosophila MAGE by double-stranded RNA injection into embryos was lethal at early stages of organogenesis. MAGE was then knocked down in developing mushroom bodies by RNAi-mediated gene silencing using the OK107-GAL4 driver. MAGE RNAi increased the population of proliferative neural precursors in larval mushroom bodies. At the pupal stage, RNAi-mediated MAGE knockdown led to a significant enlargement of the mushroom bodies as a result of increased neuronal population, presumably by accelerating the asymmetric division of neural stem cells. MAGE RNAi mushroom bodies of adult flies showed neurodegenerative changes such as vacuolation and nuclear DNA breaks, implying that supernumerary neurons undergo apoptosis during postpupal development. These results suggest that evolutionally conserved necdin-like MAGE is involved in both neural stem cell proliferation and neuronal survival during nervous system development.     

Courtship behavior induced by appetitive olfactory memory

Reinforcement signals such as food reward and noxious punishment can change diverse behaviors. This holds true in fruit flies, Drosophila melanogaster, which can be conditioned by an odor and sugar reward or electric shock punishment. Despite a wide variety of behavior modulated by learning, conditioned responses have been traditionally measured by altered odor preference in a choice, and other memory-guided behaviors have been only scarcely investigated. Here, we analyzed detailed conditioned odor responses of flies after sugar associative learning by employing a video recording and semi-automated processing pipeline. Trajectory analyses revealed that multiple behavioral components were altered along with conditioned approach to the rewarded odor. Notably, we found that lateral wing extension, a hallmark of courtship behavior of D. melanogaster, was robustly increased specifically in the presence of the rewarded odor. Strikingly, genetic disruption of the mushroom body output did not impair conditioned courtship increase, while markedly weakening conditioned odor approach. Our results highlight the complexity of conditioned responses and their distinct regulatory mechanisms that may underlie coordinated yet complex memory-guided behaviors in flies.     

Mutants with delayed cell death of the ptilinal head muscles in Drosophila.

The emergence of adult Drosophila melanogaster from the puparium is followed by the programmed degeneration of a number of muscle groups. We have isolated two X-linked mutants that delay the programmed death of at least some of these muscles. During eclosion, the fly makes use of a membranous sac, the ptilinum, which is later retracted into the head capsule. Six of the eight sets of muscles involved in the retraction then undergo degeneration. The muscle fibers initially show a gradual atrophy and then degenerate rapidly through fragmentation followed by absorption. In wild-type flies, this degeneration is obvious by 12 h after eclosion due to the loss of the birefringence of the muscles. Through mutagenesis with ethyl methanesulfonate, we isolated four mutants whose birefringence of the doomed muscles was retained even at 12 h. Mutants were genetically classified into two complementation groups; mcd-1 and mcd-2 (mcd: muscle cell death). The muscles of the mcd-1 mutants degenerate more slowly than that of the wild-type flies; the fibers enter the fragmentation step but are then not rapidly absorbed. In the mcd-2 mutants, the fibers atrophy more slowly than that in the wild-type flies and fail to undergo fragmentation. The difference in the process of the muscle death between the mcd-1 and the mcd-2 mutants suggests that at least two genes act on different steps in the process of muscle cell death.     

Mutations Affecting the cAMP Transduction Pathway Disrupt the Centrophobism Behavior

Abstract: Like vertebrates, invertebrates such as Drosophila display complex integrated behaviors that rely on locomotion for their execution. The use of genetic tools combined with sophisticated behavioral analysis has permitted researchers to investigate the brain structures implicated in those complex behaviors, such as locomotor activity. The video-tracking paradigm has allowed the study of multiple parameters of locomotor activity and has revealed that Drosophila exhibits centrophobism, a behavior related to spatial orientation. A structure/function study has demonstrated that the mushroom bodies (MBs) are implicated in this behavior. In the continuity of these former studies, we have investigated the role of the cAMP transduction pathway known to be implicated in olfactory learning and memory within the MBs. Here, we report that disturbing this pathway by using different mutants, such as dnc, rut, PKA, or amn, lead to centrophobism defect. Moreover, we found that the P[GAL4]C316 flies, used to rescue the amn mutant phenotype, like those previously reported for the learning and memory defect, are severely disturbed in centrophobism behavior. Remarkably, those flies are perfectly randomly distributed in the arena, suggesting that C316 flies carry an important mutated-gene implicated in neuronal networks required for proper spatial orientation.     

Morphometric analysis of thoracic muscles in wildtype and in bithorax Drosophila

The tergotrochanteral (TTM) "jump" muscles in the second (T2) and third (T3) thoracic segments of the fruit fly, Drosophila melanogaster, were analyzed morphologically and morphometrically in wildtype (Canton-S) and bithorax mutants (abx bx3 pbx/Df(3R)P2). In the transformed T3 segments of mutant flies, the TTMs were greatly increased in fiber number (330% of wildtype), length (141%), and volume (460%), thus manifesting both hyperplasia and hypertrophy. In contrast, TTMs in the "untransformed" T2 segments of mutant flies were both hypoplastic and hypotrophic, in that significant decreases in fiber number (93% of wildtype), length (90%), and volume (80%) were observed. Two relationships emerged from analysis of the morphometric data: 1) Although the fiber numbers and volumes of the transformed T3 TTMs in bithorax flies were greatly increased, the total combined volumes of the TTMs in T2 + T3 remained approximately the same in bithorax compared to wildtype flies. 2) The changes in TTM volumes in bithorax flies compared to those in wildtype were proportional to the relative changes in fiber numbers times the relative changes in muscle lengths. These observations suggest that the genes of the bithorax complex influence the number and the length of tubular muscles fibers of the TTMs, but do not significantly affect the mean cross-sectional areas of these fibers. Fibrillar muscle fibers, which are not found at all in T3 segments in wildtype flies, were observed in the transformed T3 segments of bithorax mutants in 11 of 18 cases (61%), but typically as wisps, not in complete muscles. We suggest that, in the T3 segment of the bithorax flies, the relative differences between the massive transformation of tubular TTMs vs. the minimal appearance of fibrillar muscles may be related, in part, to the relative availability of muscle precursors.     

The effect of isometamidium chloride onTrypanosoma vivax occurring within the insect vector (Glossina)

The effect of chemoprophylaxis on developing and matureTrypanosoma vivax inGlossina tachinoides andG. palpalis palpalis was evaluated. Newly emergedG. tachinoides andG.p. palpalis flies were infected withT. vivax by allowing them to feed on parasitaemic animals. Three experiments were conducted and in each the flies were divided into two groups. One group of infected flies was fed on animals treated 1 day previously with isometamidium (1 mg/kg body wt. in a 4% soln.) after which the surviving flies were dissected and examined for trypanosomes. The other group was fed on untreated animals. Out of a total number of 123 flies which fed on treated animals, none were found to be infected, while 51 of 127 flies which fed on untreated animals were infected. It was concluded that prophylactic treatment of animals with isometamidium would eliminateT. vivax infections from the insect vector. The potential significance of this finding in the control of trypanosomiasis in the field is discussed.     

Influence ofβ-alanine on mating and territorialism inDrosophila melanogaster

Effects of -alanine on mating behavior and aggression were studied inDrosophila melanogaster using the following competitive pairs: (1) homozygous black (b/b) flies, in which -alanine synthesis is decreased, vs. alanine is blocked vs. wild-type (e ⁺/e⁺) flies; (2) dark flies, in which -alanine incorporation is reduced, owing mainly to chromosome 3, vs. light flies collected from the same population as were the dark flies; (3) homozygous black (b/b) flies, in which -alanine synthesis is decreased, vs. -alanine-injectedb/b flies, which are phenocopies of wild-type flies. The behavior of mixed-sex groups was studied in a large, illumination-graded observation chamber containing food and in small uniformly illuminated cells also containing food. The relative competitive mating abilities of these types were measured in both experimental conditions. Uninjected black flies, but not injected ones, showed weak and unsteady gait and weak wing extension. In ebony these abnormalities were more extreme. Dark flies did not show these abnormalities. Accelerated sexual maturation was indicated in males by early onset of courtship and enhanced territorial aggression and in females by earliness of mating. Such acceleration was observed in ebony and dark flies, compared with light flies, and among -alanine-injectedb/b flies competing with uninjected black flies. Ebony males, although maturing earlier than wild-type males, were less successful than wild-type males in mating. This difference was even greater when the flies were all allowed to mature before competing. Ebony females outmated wild-type females. Dark flies outmated light flies, and -alanine-injectedb/b males outmated uninjected black males, especially in bright light. Ebony flies mated much longer than wild-type flies, and black flies mated slightly longer than injectedb/b flies. There was some spatial isolation of ebony from wild type, dark from light, and -alanine-injected from uninjectedb/b flies in the illumination-graded observation chamber. Ebony flies more than wild type concentrated near food. Flies were attracted to the current of moist inlet air. They were also attracted to deposited excrement, and males defended such deposits as a mating area, thus showing rudiments of arena behavior in which a mating area away from the oviposition site is defended. Usually, however, the defended area focused on food.This work was supported by Grant GM-18680 from the National Institutes of Health.     

Multiple Redundant Medulla Projection Neurons Mediate Color Vision in Drosophila

The receptor mechanism for color vision has been extensively studied. In contrast, the circuit(s) that transform(s) photoreceptor signals into color percepts to guide behavior remain(s) poorly characterized. Using intersectional genetics to inactivate identified subsets of neurons, we have uncovered the first-order interneurons that are functionally required for hue discrimination in Drosophila. We developed a novel aversive operant conditioning assay for intensity-independent color discrimination (true color vision) in Drosophila. Single flying flies are magnetically tethered in an arena surrounded by blue and green LEDs (light-emitting diodes). The flies’ optomotor response is used to determine the blue-green isoluminant intensity. Flies are then conditioned to discriminate between equiluminant blue or green stimuli. Wild-type flies are successfully trained in this paradigm when conditioned to avoid either blue or green. Functional color entrainment requires the function of the narrow-spectrum photoreceptors R8 and/or R7, and is within a limited range, intensity independent, suggesting that it is mediated by a color vision system. The medulla projection neurons, Tm5a/b/c and Tm20, receive direct inputs from R7 or R8 photoreceptors and indirect input from the broad-spectrum photoreceptors R1–R6 via the lamina neuron L3. Genetically inactivating these four classes of medulla projection neurons abolished color learning. However, inactivation of subsets of these neurons is insufficient to block color learning, suggesting that true color vision is mediated by multiple redundant pathways. We hypothesize that flies represent color along multiple axes at the first synapse in the fly visual system. The apparent redundancy in learned color discrimination sharply contrasts with innate ultraviolet (UV) spectral preference, which is dominated by a single pathway from the amacrine neuron Dm8 to the Tm5c projection neurons.     

Mushroom Bodies Enhance Initial Motor Activity in drosophila

Abstract: The central body (or central complex, CCX) and the mushroom bodies (MBs) are brain structures in most insect phyla that have been shown to influence aspects of locomotion. The CCX regulates motor coordination and enhances activity while MBs have, thus far, been shown to suppress motor activity levels measured over time intervals ranging from hours to weeks. In this report, we investigate MB involvement in motor behavior during the initial stages (15 minutes) of walking in Buridan's paradigm. We measured aspects of walking in flies that had MB lesions induced by mutations in six different genes and by chemical ablation. All tested flies were later examined histologically to assess MB neuroanatomy. Mutant strains with MB structural defects were generally less active in walking than wild-type flies. Most mutants in which MBs were also ablated with hydroxyurea (HU) showed additional activity decrements. Variation in measures of velocity and orientation to landmarks among wild-type and mutant flies was attributed to pleiotropy, rather than to MB lesions. We conclude that MBs upregulate activity during the initial stages of walking, but suppress activity thereafter. An MB influence on decision making has been shown in a wide range of complex behaviors. We suggest that MBs provide appropriate contextual information to motor output systems in the brain, indirectly fine tuning walking by modifying the quantity (i.e., activity) of behavior.     

The Mushroom Body of Adult Drosophila Characterized by GAL4 Drivers

Abstract: The mushroom body is required for a variety of behaviors of Drosophila melanogaster. Different types of intrinsic and extrinsic mushroom body neurons might underlie its functional diversity. There have been many GAL4 driver lines identified that prominently label the mushroom body intrinsic neurons, which are known as “Kenyon cells.” Under one constant experimental condition, we analyzed and compared the the expression patterns of 25 GAL4 drivers labeling the mushroom body. As an internet resource, we established a digital catalog indexing representative confocal data of them. Further more, we counted the number of GAL4-positive Kenyon cells in each line. We found that approximately 2,000 Kenyon cells can be genetically labeled in total. Three major Kenyon cell subtypes, the γ, α′/β′, and α/β neurons, respectively, contribute to 33, 18, and 49% of 2,000 Kenyon cells. Taken together, this study lays groundwork for functional dissection of the mushroom body.