Asymmetric activity of NetrinB controls laterality of the Drosophila brain.

Left-Right (LR) asymmetry of the nervous system is widespread across animals and is thought to be important for cognition and behaviour. But in contrast to visceral organ asymmetry, the genetic basis and function of brain laterality remain only poorly characterized. In this study, we performed RNAi screening to identify genes controlling brain asymmetry in Drosophila. We found that the conserved NetrinB (NetB) pathway is required for a small group of bilateral neurons to project asymmetrically into a pair of neuropils (Asymmetrical Bodies, AB) in the central brain in both sexes. While neurons project unilaterally into the right AB in wild-type flies, netB mutants show a bilateral projection phenotype and hence lose asymmetry. Developmental time course analysis reveals an initially bilateral connectivity, eventually resolving into a right asymmetrical circuit during metamorphosis, with the NetB pathway being required just prior symmetry breaking. We show using unilateral clonal analysis that netB activity is required specifically on the right side for neurons to innervate the right AB. We finally show that loss of NetB pathway activity leads to specific alteration of long-term memory, providing a functional link between asymmetrical circuitry determined by NetB and animal cognitive functions.

A sperm peptide enhances long-term memory in female Drosophila.

Can mating influence cognitive functions such as learning and memory in a permanent way? We have addressed this question using a combined behavioral and in vivo imaging approach, finding that aversive long-term memory performance strongly increases in Drosophila females in response to sperm transfer following mating. A peptide in the male sperm, the sex peptide, is known to cause marked changes in female reproductive behavior, as well as other behaviors such as dietary preference. Here, we demonstrate that this sex peptide enhances memory by acting on a single pair of serotonergic brain neurons, in which activation of the sex peptide receptor stimulates the cyclic adenosine monophosphate/protein kinase A pathway. We thus reveal a strong effect of mating on memory via the neuromodulatory action of a sperm peptide on the female brain.

Micromirror structured illumination microscope for high-speed in vivo drosophila brain imaging.

Genetic tools and especially genetically encoded fluorescent reporters have given a special place to optical microscopy in drosophila neurobiology research. In order to monitor neural networks activity, high speed and sensitive techniques, with high spatial resolution are required. Structured illumination microscopies are wide-field approaches with optical sectioning ability. Despite the large progress made with the introduction of the HiLo principle, they did not meet the criteria of speed and/or spatial resolution for drosophila brain imaging. We report on a new implementation that took advantage of micromirror matrix technology to structure the illumination. Thus, we showed that the developed instrument exhibits a spatial resolution close to that of confocal microscopy but it can record physiological responses with a speed improved by more than an order a magnitude.

Parametric and genetic analysis of Drosophila appetitive long-term memory and sugar motivation.

Distinct forms of memory can be highlighted using different training protocols. In Drosophila olfactory aversive learning, one conditioning session triggers memory formation independently of protein synthesis, while five spaced conditioning sessions lead to the formation of long-term memory (LTM), a long-lasting memory dependent on de novo protein synthesis. In contrast, one session of odour-sugar association appeared sufficient for the fly to form LTM. We designed and tuned an apparatus that facilitates repeated discriminative conditioning by alternate presentations of two odours, one being associated with sugar, as well as a new paradigm to test sugar responsiveness (SR). Our results show that both SR and short-term memory (STM) scores increase with starvation length before conditioning. The protein dependency of appetitive LTM is independent of the repetition and the spacing of training sessions, on the starvation duration and on the strength of the unconditioned stimulus. In contrast to a recent report, our test measures an abnormal SR of radish mutant flies, which might initiate their STM and LTM phenotypes. In addition, our work shows that crammer and tequila mutants, which are deficient for aversive LTM, present both an SR and an appetitive STM defect. Using the MB247-P[switch] system, we further show that tequila is required in the adult mushroom bodies for normal sugar motivation.

Localization of long-term memory within the Drosophila mushroom body.

The mushroom bodies, substructures of the Drosophila brain, are involved in olfactory learning and short-term memory, but their role in long-term memory is unknown. Here we show that the alpha-lobes-absent (ala) mutant lacks either the two vertical lobes of the mushroom body or two of the three median lobes which contain branches of vertical lobe neurons. This unique phenotype allows analysis of mushroom body function. Long-term memory required the presence of the vertical lobes but not the median lobes. Short-term memory was normal in flies without either vertical lobes or the two median lobes studied.

The Drosophila castor gene is involved in postembryonic brain development.

castor (cas) encodes a zink finger protein expressed in a subset of Drosophila embryonic neuroglioblasts where it controls neuronal differentiation. We show here that cas is expressed at larval and pupal stages in brain cell clusters where it participates in the elaboration of the adult structures. In particular using the MARCM system (mosaic analysis with a repressible cell marker), we show that cas is required postembryonically for correct axon pathfinding of the central complex (CX) and mushroom body (MB) neurons. The linotte (lio) gene encodes a transmembrane protein expressed at larval/pupal stage in a glial structure, the TIFR, and interacts with the no-bridge (nob) gene. We show that cas interacts genetically with lio and nob. These interactions do not involve direct transcription regulation but probably cellular communication processes.

Defining the role of Drosophila lateral neurons in the control of circadian rhythms in motor activity and eclosion by targeted genetic ablation and PERIOD protein overexpression.

The ventral lateral neurons (LNvs) of the Drosophila brain that express the period (per) and pigment dispersing factor (pdf) genes play a major role in the control of circadian activity rhythms. A new P-gal4 enhancer trap line is described that is mostly expressed in the LNvs This P-gal4 line was used to ablate the LNvs by using the pro-apoptosis gene bax, to stop PER protein oscillations by overexpressing per and to block synaptic transmission with the tetanus toxin light chain (TeTxLC). Genetic ablation of these clock cells leads to the loss of robust 24-h activity rhythms and reveals a phase advance in light-dark conditions as well as a weak short-period rhythm in constant darkness. This behavioural phenotype is similar to that described for disconnected1 (disco1) mutants, in which we show that the majority of the individuals have a reduced number of dorsally projecting lateral neurons which, however, fail to express PER. In both LNv-ablated and disco1 flies, PER cycles in the so-called dorsal neurons (DNs) of the superior protocerebrum, suggesting that the weak short-period rhythm could stem from these PDF-negative cells. The overexpression of per in LNs suppresses PER protein oscillations and leads to the disruption of both activity and eclosion rhythms, indicating that PER cycling in these cells is required for both of these rhythmic behaviours. Interestingly, flies overexpressing PER in the LNs do not show any weak short-period rhythms, although PER cycles in at least a fraction of the DNs, suggesting a dominant role of the LNs on the behavioural rhythms. Expression of TeTxLC in the LNvs does not impair activity rhythms, which indicates that the PDF-expressing neurons do not use synaptobrevin-dependent transmission to control these rhythms.

no-bridge and linotte act jointly at the interhemispheric junction to build up the adult central brain of Drosophila melanogaster.

The Drosophila transmembrane protein Linotte (Lio) is expressed in a glial transcient interhemispheric fibrous ring (TIFR), which was hypothesised to serve as scaffold for adult brain formation during metamorphosis. We isolated TIFR specific enhancers from the lio locus and showed that only four interhemispheric cells give rise to this complex fibrous structure. We showed that lio controls the TIFR differentiation, and confirmed the major role played by this structure in central brain metamorphosis since its destruction by apoptosis led to a pronounced adult phenotype, which included defects of lio and no-bridge (nob) mutants. lio interhemispheric expression was specifically affected in a nob(1) context, confirming that nob plays a key role in adult brain development via the TIFR.

Ciboulot regulates actin assembly during Drosophila brain metamorphosis.

A dynamic actin cytoskeleton is essential for the remodeling of cell shape during development, but the specific roles of many actin partners remain unclear. Here we characterize a novel actin binding protein, Ciboulot (Cib), which plays a major role in axonal growth during Drosophila brain metamorphosis. Loss of Cib function leads to axonal growth defects in the central brain, while overexpression of the gene during development leads to overgrown projections. The Cib protein displays strong sequence similarity to beta-thymosins but has biochemical properties like profilin: the Cib-actin complex participates in actin filament assembly exclusively at the barbed end, and Cib enhances actin-based motility in vitro. Genetic experiments show that Cib and the Drosophila profilin protein Chickadee (Chic) cooperate in central brain metamorphosis.

Identification of Drosophila mutant with memory defects after acquisition of conditioned reflex suppression of courtship.

Four lines were selected from a collection of 33 lines prepared by P insertion mutagenesis using a single-copy P-element system; the males of these four lines showed memory defects after acquisition of conditioned reflex suppression of courting. In two lines (P171 and P95), the dynamics of retention of the conditioned reflex in the repeated impregnated-female courting test were similar to those of known short-term memory mutants dnc and rut. In line P153, the dynamics were more reminiscent of the memory dynamics in a known medium-term memory mutant, amn. In line P124, the learning index was insignificant immediately after training was completed, which may indicate that this line was unable to acquire conditioned reflex suppression of courting. Determination of the positions of the P elements (P171: 48A-B; P153: 49B-C; P124: 67B-68A; P95: 77C-D) showed no correspondence with previously known mutations producing memory lesions.

Central brain postembryonic development in Drosophila: implication of genes expressed at the interhemispheric junction.

Postembryonic brain development of Drosophila has become recently a subject of intense investigations. In particular, the linotte (lio) mutants display strong structural defects in the mushroom bodies and the central complex. The Lio kinase is expressed in a glial structure at the interhemispheric junction of late larval and young pupal brain. With the aim of identifying new genes involved in the formation of adult central brain structures, 821 enhancer-trap Gal4 lines were generated and screened for late larval expression. We identified 167 lines showing expression at or near the interhemispheric junction of third-instar larval brain, an area from which the central complex differentiates. Adult brains from 104 of these 167 lines were analyzed through paraffin sections. This secondary screen allowed the recovery of five central brain mutants. Of 89 control lines showing various patterns of expression excluding the interhemispheric junction, only one anatomical mutant was isolated. These six mutations, which have been thoroughly characterized, affect the midline area of the adult brain with phenotypes of split central complex structures and/or fused mushroom body lobes. This work opens the way for further analysis of the molecular and cellular events involved in central brain reorganization during metamorphosis.

[Isolation of memory-deficient Drosophila mutants in conditioned courtship suppression paradigm]. / Vyiavlenie mutantov drozofily, proiavliaiushchikh defekty pamiati posle vyrabotki uslovnoreflektornogo podavleniia ukhazhivaniia.

Among 33 mutant stocks of Drosophila melanogaster generated by means of P-insertional mutagenesis in the system with single P element, 4 stocks have been isolated as demonstrating deficient memory in the conditioned courtship suppression paradigm. Localization of the P insertions never coincided with that of previously known mutations affecting memory.

Decreased odor avoidance after electric shock in Drosophila mutants biases learning and memory tests.

The Drosophila mutants amnesiac, dunce (dnc), and rutabaga were isolated after associative conditioning tests, during which animals were trained to associate the presence of an odor with that of electric shocks (ES). In the absence of conditioning, the odor avoidance (OA) of these mutants was shown to be normal, indicating that their poor associative conditioning performance was attributable to specific learning or memory deficits. However, I show that the OA of the mutants is greatly decreased after their exposure to ES. This effect can last for hours. These results strongly suggest that part of the defect displayed by these mutants in associative conditioning tests does not correspond to a learning or memory deficit but might arise from abnormal sensitivity to stressful stimuli. I looked at the OA after ES of two previously characterized dnc mutants. Df(1)N79f specifically decreases Dnc expression in the mushroom bodies, leading to a normal level of learning but decreased memory. Df(1)N79f mutants displayed a normal OA after ES. Df(1)N64j15 affects the entire brain expression of Dnc, leading to decreased learning and memory. Df(1)N64j15 animals showed a strong decrease of their OA after ES. Thus, the lack of Dnc "general" expression is most likely responsible for the OA defect, which would be responsible for the apparent learning defect after conditioning. In contrast, the Dnc phosphodiesterase accumulated in the mushroom bodies would be involved specifically in memory formation.

The Drosophila putative kinase linotte (derailed) prevents central brain axons from converging on a newly described interhemispheric ring.

The lio gene encodes a putative receptor tyrosine kinase, with unique motifs both in the extracellular and catalytic domains (Dura, J.-M., Préat, T., Tully, T., 1993. Identification of linotte, a new gene affecting learning and memory in Drosophila melanogaster. J. Neurogenet. 9, 1-14). We show here that a complete deletion of lio activity causes specific structural defects in the adult brain. Gal4 enhancer-trap lines used as cell markers revealed that in lio mutants central brain axons behave as if they were abnormally attracted by the midbrain area. The Lio protein is expressed in third instar larvae in a few cells at the junction of the cerebral hemispheres. These glial cells form a newly described ring structure, showing an invariable fibrous organization. In the wild-type this ring disappears at midpupation. Our results indicate that the Lio putative kinase plays a major role in the modeling of the adult brain by controlling the fate of the transient interhemispheric ring.

Abnormal mushroom body plasticity in the Drosophila memory mutant amnesiac.

In Drosophila melanogaster, adult or larval rearing conditions influence brain structure. In particular, larval density affects the number of fibers forming the mushroom bodies, a neuropil structure involved in olfactory learning. The mushroom bodies receive chemosensory inputs from the antennal lobes at the level of the calyx. In this study we report that larval density affects calyx volume measured shortly after eclosion from the pupal case. We observe that in the memory mutant amnesiac this form of experience-dependent structural plasticity is missing, whereas it is not affected in the learning mutant rutabaga and in the memory mutant radish. Independent of the plasticity effect, the calyces are on average slightly bigger than wild type in amnesiac and smaller in rutabaga flies.

The Drosophila learning and memory gene linotte encodes a putative receptor tyrosine kinase homologous to the human RYK gene product.

The linotte mutant was isolated on the basis of its learning and memory deficit. Interestingly, linotte individuals carrying a null mutation are viable, indicating that the linotte gene is not required for vital functions. We show here that the linotte gene encodes a putative receptor tyrosine kinase, homologous to the human protein RYK. These products are unique among receptor tyrosine kinases, since they possess a short extra cellular domain, and a modified intracellular catalytic domain. In particular, the subdomains directly involved in ATP binding and phosphotransfer reaction display remarkable variations. These results suggest that linotte is part of a novel signal transduction cascade involved in learning and memory.

The Suppressor of fused gene encodes a novel PEST protein involved in Drosophila segment polarity establishment.

Suppressor of fused, Su(fu), was identified as a semi-dominant suppressor of the putative serine/threonine kinase encoded by the segment polarity gene fused in Drosophila melanogaster. The amorphic Su(fu) mutation is viable, shows a maternal effect and displays no phenotype by itself. Su(fu) mutations are often found associated to karmoisin (kar) mutations but two complementation groups can be clearly identified. By using a differential hybridization screening method, we have cloned the Su(fu) region and identified chromosomal rearrangements associated with Su(fu) mutations. Two classes of cDNAs with similar developmental patterns, including a maternal contribution, are detectable in the region. Transformation experiments clearly assigned the Su(fu)+ function to one of these transcription units while the other one can be most likely assigned to the kar+ function. Surprisingly the 5' end of the kar RNA mapped within the 3' untranslated region of the Su(fu) transcribed sequence. The Su(fu) gene encodes a 53-kD protein, which contains a PEST sequence and shows no significant homologies with known proteins. Genetic analysis shows that proper development requires a fine tuning of the genetic doses of fu and Su(fu) both maternally and zygotically. These results, together with previous genetic and molecular data, suggest that fused and Suppressor of fused could act through a competitive posttraductionnal modification of a common target in the hedgehog signaling pathway.

Genetic dissection of consolidated memory in Drosophila.

Behavioral and pharmacological experiments in many animal species have suggested that memory is consolidated from an initial, disruptable form into a long-lasting, stable form within a few hours after training. We combined these traditional approaches with genetic analyses in Drosophila to show that consolidated memory of conditioned (learned) odor avoidance 1 day after extended training consisted of two genetically distinct, functionally independent memory components: anesthesia-resistant memory (ARM) and long-term memory (LTM). ARM decayed away within 4 days, was resistant to hypothermic disruption, was insensitive to the protein synthesis inhibitor cycloheximide (CXM), and was disrupted by the radish single-gene mutation. LTM showed no appreciable decay over 7 days, was sensitive to CXM, and was not disrupted by the radish mutation.

Segmental polarity in Drosophila melanogaster: genetic dissection of fused in a Suppressor of fused background reveals interaction with costal-2.

fused (fu) is a segment polarity gene that encodes a putative serine/threonine kinase. A complete suppressor of the embryonic and adult phenotypes of fu mutants, Suppressor of fused (Su(fu)), was previously described. The amorphic Su(fu) mutation is viable and displays no phenotype by itself. We have used this suppressor as a tool to perform a genetic dissection of the fu gene. Analysis of the interaction between Su(fu) and 33 fu alleles shows that they belong to three different classes. Defects due to class I fu alleles are fully suppressed by Su(fu). Class II fu alleles lead to a new segment polarity phenotype in interaction with Su(fu). This phenotype corresponds to embryonic and adult anomalies similar to those displayed by the segment polarity mutant costal-2 (cos-2). Class II alleles are recessive to class I alleles in a fu[I]/fu[II];Su(fu)/Su(fu) combination. Class 0 alleles, like class I alleles, confer a normal segmentation phenotype in interaction with Su(fu). However class II alleles are dominant over class 0 alleles in a fu[0]/fu[II];Su(fu)/Su(fu) combination. Alleles of class I and II correspond to small molecular events, which may leave part of the Fu protein intact. On the contrary, class 0 alleles correspond to large deletions. Several class I and class II fu mutations have been mapped, and three mutant alleles were sequenced. These data suggest that class I mutations affect the catalytic domain of the putative Fu kinase and leave the carboxy terminal domain intact, whereas predicted class II proteins have an abnormal carboxy terminal domain. Su(fu) enhances the cos-2 phenotype and cos-2 mutations interact with fu in a way similar to Su(fu). All together these results suggest that a close relationship might exist between fu, Su(fu) and cos-2 throughout development. We thus propose a model where the Fu+ kinase is a posterior inhibitor of Costal-2+ while Su(fu)+ is an activator of Costal-2+. The expression pattern of wingless and engrailed in fu and fu;Su(fu) embryos is in accordance with this interpretation.

Molecular organisation and expression pattern of the segment polarity gene fused of Drosophila melanogaster.

The Drosophila segment-polarity gene fused (fu) is required for pattern formation within embryonic segments and imaginal discs. We previously reported that the 5' part of the fused gene is homologous to the catalytic domain of serine/threonine kinases. We present here the sequence of the complete transcription unit, which predicts a 805 amino acid long protein. The kinase domain actually corresponds to 268 amino acids in the N-terminal part, and no known function can be attributed to the rest of the putative FUSED protein. Transcripts from the fused gene have been characterized: a unique 3.2 kb fused transcript is produced in nurse cells, in low abundance, from stage 8 of oogenesis, and persistently through the rest of oogenesis. In embryos, this transcript is evenly distributed in all embryonic cells until the extended germ band stage, after which its amount strongly decreases. Ubiquitous expression is detected later in imaginal wing and leg discs. Possible roles of the FUSED protein in signal transduction pathways required for intercellular communication at different stages of development are discussed.