Genetics of cocaine and methamphetamine consumption and preference in Drosophila melanogaster.

Illicit use of psychostimulants, such as cocaine and methamphetamine, constitutes a significant public health problem. Whereas neural mechanisms that mediate the effects of these drugs are well-characterized, genetic factors that account for individual variation in susceptibility to substance abuse and addiction remain largely unknown. Drosophila melanogaster can serve as a translational model for studies on substance abuse, since flies have a dopamine transporter that can bind cocaine and methamphetamine, and exposure to these compounds elicits effects similar to those observed in people, suggesting conserved evolutionary mechanisms underlying drug responses. Here, we used the D. melanogaster Genetic Reference Panel to investigate the genetic basis for variation in psychostimulant drug consumption, to determine whether similar or distinct genetic networks underlie variation in consumption of cocaine and methamphetamine, and to assess the extent of sexual dimorphism and effect of genetic context on variation in voluntary drug consumption. Quantification of natural genetic variation in voluntary consumption, preference, and change in consumption and preference over time for cocaine and methamphetamine uncovered significant genetic variation for all traits, including sex-, exposure- and drug-specific genetic variation. Genome wide association analyses identified both shared and drug-specific candidate genes, which could be integrated in genetic interaction networks. We assessed the effects of ubiquitous RNA interference (RNAi) on consumption behaviors for 34 candidate genes: all affected at least one behavior. Finally, we utilized RNAi knockdown in the nervous system to implicate dopaminergic neurons and the mushroom bodies as part of the neural circuitry underlying experience-dependent development of drug preference.

Application of Drosophila Model Toward Understanding the Molecular Basis of Fragile X Syndrome.

Drosophila melanogaster is an ideal model to study Fragile X syndrome (FXS) as it presents us with a toolbox to identify genetic modifiers and to investigate the molecular mechanisms of FXS. Here we describe some of the methods that have been used to study FXS, ranging from reverse genetic screening using the GAL4-UAS system, to mushroom body staining and courtship behavioral assays to examine the learning and memory deficits associated with FXS.

Synaptic depression induced by postsynaptic cAMP production in the Drosophila mushroom body calyx.

KEY POINTS: Synaptic potentiation in Drosophila is observed at cholinergic synapses between antennal lobe (AL) and mushroom body (MB) neurons in the adult brain; however, depression at the AL-MB synapses has not yet been identified. By ex vivo Ca2+ imaging in an isolated cultured Drosophila brain, we found novel activity-dependent depression at the AL-MB synapses. The degree of Ca2+ responses after repetitive AL stimulation is significantly reduced in the dendritic region of MB neurons (calyx) compared with those before AL stimulation, and this reduction of Ca2+ responses remains for at least 30 min. The expression of rutabaga, which encodes Ca2+ /calmodulin-dependent adenylyl cyclase, is essential in the MB neurons for the reduction of Ca2+ responses in the calyx. Our study reveals that elevation of cAMP production in the calyx during repetitive AL stimulation induces the depression at the AL-MB synapses. ABSTRACT: Synaptic plasticity has been studied to reveal the molecular and cellular mechanisms of associative and non-associative learning. The fruit fly Drosophila melanogaster can be used to identify the molecular mechanisms of synaptic plasticity because vast genetic information or tools are available. Here, by ex vivo Ca2+ imaging of an isolated cultured Drosophila brain, we examined the novel activity-dependent synaptic depression between the projection neurons of the antennal lobe (AL) and mushroom body (MB). Ex vivo Ca2+ imaging analysis revealed that electrical stimulation of AL elicits Ca2+ responses in the dendritic (calyx) and axonal (α lobe) regions of MB neurons, and the responses are reduced after repetitive AL stimulation. Since the cAMP signalling pathway plays an important role in synaptic plasticity in invertebrates and vertebrates, we examined whether the reduction of Ca2+ responses is also regulated by the cAMP signalling pathway. The expression of rutabaga (rut), which encodes Ca2+ /calmodulin-dependent adenylyl cyclase, was essential for the reduction of Ca2+ responses in the calyx and α lobe. Furthermore, imaging analysis using a fluorescence resonance energy transfer-based cAMP indicator revealed that the cAMP level increased in the wild-type calyx during repetitive AL stimulation, whereas it decreased in rut1 mutant flies with a loss-of-function mutation of rut. Thus, our study suggests that an increase in postsynaptic cAMP level during repetitive AL stimulation contributes to the attenuation of inputs at AL-MB synapses.

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

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

Regeneration of synapses in the olfactory pathway of locusts after antennal deafferentation.

The olfactory pathway of the locust is capable of fast and precise regeneration on an anatomical level. Following deafferentation of the antenna either of young adult locusts, or of fifth instar nymphs, severed olfactory receptor neurons (ORNs) reinnervate the antennal lobe (AL) and arborize in AL microglomeruli. In the present study we tested whether these regenerated fibers establish functional synapses again. Intracellular recordings from AL projection neurons revealed that the first few odor stimulus evoked postsynaptic responses from regenerated ORNs from day 4-7 post crush on. On average, synaptic connections of regenerated afferents appeared faster in younger locusts operated as fifth instar nymphs than in adults. The proportions of response categories (excitatory vs. inhibitory) changed during regeneration, but were back to normal within 21 days. Odor-evoked oscillating extracellular local field potentials (LFP) were recorded in the mushroom body. These responses, absent after antennal nerve crush, reappeared, in a few animals as soon as 4 days post crush. Odor-induced oscillation patterns were restored within 7 days post crush. Both intra- and extracellular recordings indicate the capability of the locust olfactory system to re-establish synaptic contacts in the antennal lobe after antennal nerve lesion.

Neuron class-specific requirements for Fragile X Mental Retardation Protein in critical period development of calcium signaling in learning and memory circuitry.

Neural circuit optimization occurs through sensory activity-dependent mechanisms that refine synaptic connectivity and information processing during early-use developmental critical periods. Fragile X Mental Retardation Protein (FMRP), the gene product lost in Fragile X syndrome (FXS), acts as an activity sensor during critical period development, both as an RNA-binding translation regulator and channel-binding excitability regulator. Here, we employ a Drosophila FXS disease model to assay calcium signaling dynamics with a targeted transgenic GCaMP reporter during critical period development of the mushroom body (MB) learning/memory circuit. We find FMRP regulates depolarization-induced calcium signaling in a neuron-specific manner within this circuit, suppressing activity-dependent calcium transients in excitatory cholinergic MB input projection neurons and enhancing calcium signals in inhibitory GABAergic MB output neurons. Both changes are restricted to the developmental critical period and rectified at maturity. Importantly, conditional genetic (dfmr1) rescue of null mutants during the critical period corrects calcium signaling defects in both neuron classes, indicating a temporally restricted FMRP requirement. Likewise, conditional dfmr1 knockdown (RNAi) during the critical period replicates constitutive null mutant defects in both neuron classes, confirming cell-autonomous requirements for FMRP in developmental regulation of calcium signaling dynamics. Optogenetic stimulation during the critical period enhances depolarization-induced calcium signaling in both neuron classes, but this developmental change is eliminated in dfmr1 null mutants, indicating the activity-dependent regulation requires FMRP. These results show FMRP shapes neuron class-specific calcium signaling in excitatory vs. inhibitory neurons in developing learning/memory circuitry, and that FMRP mediates activity-dependent regulation of calcium signaling specifically during the early-use critical period.

Ubiquitin ligase HUWE1 regulates axon branching through the Wnt/ß-catenin pathway in a Drosophila model for intellectual disability.

We recently reported that duplication of the E3 ubiquitin ligase HUWE1 results in intellectual disability (ID) in male patients. However, the underlying molecular mechanism remains unknown. We used Drosophila melanogaster as a model to investigate the effect of increased HUWE1 levels on the developing nervous system. Similar to the observed levels in patients we overexpressed the HUWE1 mRNA about 2-fold in the fly. The development of the mushroom body and neuromuscular junctions were not altered, and basal neurotransmission was unaffected. These data are in agreement with normal learning and memory in the courtship conditioning paradigm. However, a disturbed branching phenotype at the axon terminals of the dorsal cluster neurons (DCN) was detected. Interestingly, overexpression of HUWE1 was found to decrease the protein levels of dishevelled (dsh) by 50%. As dsh as well as Fz2 mutant flies showed the same disturbed DCN branching phenotype, and the constitutive active homolog of ß-catenin, armadillo, could partially rescue this phenotype, our data strongly suggest that increased dosage of HUWE1 compromises the Wnt/ß-catenin pathway possibly by enhancing the degradation of dsh.

Neural circuit architecture defects in a Drosophila model of Fragile X syndrome are alleviated by minocycline treatment and genetic removal of matrix metalloproteinase.

Fragile X syndrome (FXS), caused by loss of the fragile X mental retardation 1 (FMR1) product (FMRP), is the most common cause of inherited intellectual disability and autism spectrum disorders. FXS patients suffer multiple behavioral symptoms, including hyperactivity, disrupted circadian cycles, and learning and memory deficits. Recently, a study in the mouse FXS model showed that the tetracycline derivative minocycline effectively remediates the disease state via a proposed matrix metalloproteinase (MMP) inhibition mechanism. Here, we use the well-characterized Drosophila FXS model to assess the effects of minocycline treatment on multiple neural circuit morphological defects and to investigate the MMP hypothesis. We first treat Drosophila Fmr1 (dfmr1) null animals with minocycline to assay the effects on mutant synaptic architecture in three disparate locations: the neuromuscular junction (NMJ), clock neurons in the circadian activity circuit and Kenyon cells in the mushroom body learning and memory center. We find that minocycline effectively restores normal synaptic structure in all three circuits, promising therapeutic potential for FXS treatment. We next tested the MMP hypothesis by assaying the effects of overexpressing the sole Drosophila tissue inhibitor of MMP (TIMP) in dfmr1 null mutants. We find that TIMP overexpression effectively prevents defects in the NMJ synaptic architecture in dfmr1 mutants. Moreover, co-removal of dfmr1 similarly rescues TIMP overexpression phenotypes, including cellular tracheal defects and lethality. To further test the MMP hypothesis, we generated dfmr1;mmp1 double null mutants. Null mmp1 mutants are 100% lethal and display cellular tracheal defects, but co-removal of dfmr1 allows adult viability and prevents tracheal defects. Conversely, co-removal of mmp1 ameliorates the NMJ synaptic architecture defects in dfmr1 null mutants, despite the lack of detectable difference in MMP1 expression or gelatinase activity between the single dfmr1 mutants and controls. These results support minocycline as a promising potential FXS treatment and suggest that it might act via MMP inhibition. We conclude that FMRP and TIMP pathways interact in a reciprocal, bidirectional manner.

Sleep deprivation specifically impairs short-term olfactory memory in Drosophila.

STUDY OBJECTIVES: Sleep is crucial to memory consolidation in humans and other animals; however, the effect of insufficient sleep on subsequent learning and memory remains largely elusive. DESIGN: Learning and memory after 1-day sleep deprivation (slpD) was evaluated using Pavlovian olfactory conditioning in Drosophila, and locomotor activity was measured using the Drosophila Activity Monitoring System in a 12:12 light-dark cycle. RESULTS: We found that slpD specifically impaired 1-h memory in wild type Canton-S flies, and this effect could persist for at least 2 h. However, alternative stresses (heat stress, oxidative stress, starvation, and rotation stress) did not result in a similar effect and left the flies' memory intact. Mechanistic studies demonstrated that flies with either silenced transmission of the mushroom body (MB) during slpD or down-regulated cAMP levels in the MB demonstrated no slpD-induced 1-h memory impairment. CONCLUSION: We found that slpD specifically impaired 1-h memory in Drosophila, and either silencing of MB transmission during slpD or down-regulation of the cAMP level in the MB protected the flies from slpD-induced impairment.

The Drosophila fragile X mental retardation gene regulates sleep need.

Sleep need is affected by developmental stage and neuronal plasticity, but the underlying mechanisms remain unclear. The fragile X mental retardation gene Fmr1, whose loss-of-function mutation causes the most common form of inherited mental retardation in humans, is involved in synaptogenesis and synaptic plasticity, and its expression depends on both developmental stage and waking experience. Fmr1 is highly conserved across species and Drosophila mutants carrying dFmr1 loss-of-function or gain-of-function mutations are well characterized: amorphs have overgrown dendritic trees with larger synaptic boutons, developmental defects in pruning, and enhanced neurotransmission, while hypermorphs show opposite defects, including dendritic and axonal underbranching and loss of synapse differentiation. We find here that dFmr1 amorphs are long sleepers and hypermorphs are short sleepers, while both show increased locomotor activity and shortened lifespan. Both amorphs and hypermorphs also show abnormal sleep homeostasis, with impaired waking performance and no sleep rebound after sleep deprivation. An impairment in the circadian regulation of sleep cannot account for the altered sleep phenotype of dFmr1 mutants, nor can an abnormal activation of glutamatergic metabotropic receptors. Moreover, overexpression of dFmr1 throughout the mushroom bodies is sufficient to reduce sleep. Finally, dFmr1 protein levels are modulated by both developmental stage and behavioral state, with increased expression immediately after eclosure and after prolonged wakefulness. Thus, dFmr1 expression dose-dependently affects both sleep and synapses, suggesting that changes in sleep time in dFmr1 mutants may derive from changes in synaptic physiology.

Experience improves feature extraction in Drosophila.

Previous exposure to a pattern in the visual scene can enhance subsequent recognition of that pattern in many species from honeybees to humans. However, whether previous experience with a visual feature of an object, such as color or shape, can also facilitate later recognition of that particular feature from multiple visual features is largely unknown. Visual feature extraction is the ability to select the key component from multiple visual features. Using a visual flight simulator, we designed a novel protocol for visual feature extraction to investigate the effects of previous experience on visual reinforcement learning in Drosophila. We found that, after conditioning with a visual feature of objects among combinatorial shape-color features, wild-type flies exhibited poor ability to extract the correct visual feature. However, the ability for visual feature extraction was greatly enhanced in flies trained previously with that visual feature alone. Moreover, we demonstrated that flies might possess the ability to extract the abstract category of "shape" but not a particular shape. Finally, this experience-dependent feature extraction is absent in flies with defective MBs, one of the central brain structures in Drosophila. Our results indicate that previous experience can enhance visual feature extraction in Drosophila and that MBs are required for this experience-dependent visual cognition.