A descending inhibitory mechanism of nociception mediated by an evolutionarily conserved neuropeptide system in Drosophila.

Nociception is a neural process that animals have developed to avoid potentially tissue-damaging stimuli. While nociception is triggered in the peripheral nervous system, its modulation by the central nervous system is a critical process in mammals, whose dysfunction has been extensively implicated in chronic pain pathogenesis. The peripheral mechanisms of nociception are largely conserved across the animal kingdom. However, it is unclear whether the brain-mediated modulation is also conserved in non-mammalian species. Here, we show that Drosophila has a descending inhibitory mechanism of nociception from the brain, mediated by the neuropeptide Drosulfakinin (DSK), a homolog of cholecystokinin (CCK) that plays an important role in the descending control of nociception in mammals. We found that mutants lacking dsk or its receptors are hypersensitive to noxious heat. Through a combination of genetic, behavioral, histological, and Ca2+ imaging analyses, we subsequently revealed neurons involved in DSK-mediated nociceptive regulation at a single-cell resolution and identified a DSKergic descending neuronal pathway that inhibits nociception. This study provides the first evidence for a descending modulatory mechanism of nociception from the brain in a non-mammalian species that is mediated by the evolutionarily conserved CCK system, raising the possibility that the descending inhibition is an ancient mechanism to regulate nociception.

Avoiding harm is fundamental for the survival of animals. Nerve cells called nociceptors can detect potential damage, such as extreme temperatures, sharp objects and certain chemicals. In humans, this detection ­ known as nociception ­ leads to signals travelling from nociceptors through the spinal cord to the brain, which perceives them as pain. Mammals such as humans and rodents can inhibit nociception by sending signals from the brain to the spinal cord to dampen pain. This top-down dampening process is believed to play a crucial role in regulating pain in mammals, and it has been implicated in the development of chronic pain. It was not known whether non-mammalian animals shared this inhibitory pathway. However, previous work had shown that fruit fly produce a molecule called Drosulfakinin, which is similar to the chemical that mammals use in the top-down signalling pathway which controls pain. To determine the role of Drosulfakinin in controlling fly nociception, Oikawa et al. manipulated its activity ­ and the activity of related genes ­ in specific neurons in the fruit fly nervous system. Without Drosulfakinin, fly larvae were more sensitive to heat exposure, suggesting that this molecule is required to inhibit nociception. Further experiments showed that Drosulfakinin is present only in the brain of fly larvae and activation of its signaling lowers the activity of neurons that transmit nociceptive signals in the insect equivalent of the spinal cord. This confirms that insect brains can dampen nociception via a top-down pathway, using a similar molecule to mammals. The findings provide an important foundation for pain studies using non-mammalian animals. The ability to manipulate nociception using genetic techniques in flies offers a powerful tool to understand the top-down process of controlling pain. This result also raises the possibility that this shared top-down inhibition mechanism may have developed over 550 million years ago, which could lead to further research into how nociception and pain regulation systems evolved.

TALE-class homeodomain transcription factors, homothorax and extradenticle, control dendritic and axonal targeting of olfactory projection neurons in the Drosophila brain.

Precise neuronal connectivity in the nervous system depends on specific axonal and dendritic targeting of individual neurons. In the Drosophila brain, olfactory projection neurons convey odor information from the antennal lobe to higher order brain centers such as the mushroom body and the lateral horn. Here, we show that Homothorax (Hth), a TALE-class homeodomain transcription factor, is expressed in many of the antennal lobe neurons including projection neurons and local interneurons. In addition, HTH is expressed in the progenitors of the olfactory projection neurons, and the activity of hth is required for the generation of the lateral but not for the anterodorsal and ventral lineages. MARCM analyses show that the hth is essential for correct dendritic targeting of projection neurons in the antennal lobe. Moreover, the activity of hth is required for axonal fasciculation, correct routing and terminal branching of the projection neurons. We also show that another TALE-class homeodomain protein, Extradenticle (Exd), is required for the dendritic and axonal development of projection neurons. Mutation of exd causes projection neuron defects that are reminiscent of the phenotypes caused by the loss of the hth activity. Double immunostaining experiments show that Hth and Exd are coexpressed in olfactory projection neurons and their progenitors, and that the expressions of Hth and Exd require the activity of each other gene. These results thus demonstrate the functional importance of the TALE-class homeodomain proteins in cell-type specification and precise wiring of the Drosophila olfactory network.

Distinctive neuronal networks and biochemical pathways for appetitive and aversive memory in Drosophila larvae.

Associative strength between conditioned stimulus (CS) and unconditioned stimulus (US) is thought to determine learning efficacy in classical conditioning. Elucidation of the neuronal mechanism that underlies the association between CS and US in the brain is thus critical to understand the principle of memory formation. With a simple brain organization, the Drosophila larva provides an attractive model system to investigate learning at the neurocircuitry level. Previously, we described a single-odor paradigm for larval associative learning using sucrose as a reward, and showed that larval appetitive memory lasts longer than 2 h. In this work, we describe behavioral and genetic characterization of larval aversive olfactory memory formed in our paradigm, and compare its stability and neurocircuitry with those of appetitive memory. Despite identical training paradigms, larval olfactory memory formed with quinine or NaCl is short-lived to be lost in 20 min. As with appetitive memory, larval aversive memory produced in this paradigm depends on intact cAMP signaling, but neither mutation of amnesiac nor suppression of CREB activity affects its kinetics. Neurocircuitry analyses suggest that aversive memory is stored before the presynaptic termini of the larval mushroom body neurons as is the case with appetitive memory. However, synaptic output of octopaminergic and dopaminergic neurons, which exhibit distinctive innervation patterns on the larval mushroom body and antennal lobe, is differentially required for the acquisition of appetitive and aversive memory, respectively. These results as a whole suggest that the genetically programmed memory circuitries might provide predisposition in the efficacy of inducing longer-lived memory components in associative learning.

Unc-51 controls active zone density and protein composition by downregulating ERK signaling.

Efficient synaptic transmission requires the apposition of neurotransmitter release sites opposite clusters of postsynaptic neurotransmitter receptors. Transmitter is released at active zones, which are composed of a large complex of proteins necessary for synaptic development and function. Many active zone proteins have been identified, but little is known of the mechanisms that ensure that each active zone receives the proper complement of proteins. Here we use a genetic analysis in Drosophila to demonstrate that the serine threonine kinase Unc-51 acts in the presynaptic motoneuron to regulate the localization of the active zone protein Bruchpilot opposite to glutamate receptors at each synapse. In the absence of Unc-51, many glutamate receptor clusters are unapposed to Bruchpilot, and ultrastructural analysis demonstrates that fewer active zones contain dense body T-bars. In addition to the presence of these aberrant synapses, there is also a decrease in the density of all synapses. This decrease in synaptic density and abnormal active zone composition is associated with impaired evoked transmitter release. Mechanistically, Unc-51 inhibits the activity of the MAP kinase ERK to promote synaptic development. In the unc-51 mutant, increased ERK activity leads to the decrease in synaptic density and the absence of Bruchpilot from many synapses. Hence, activated ERK negatively regulates synapse formation, resulting in either the absence of active zones or the formation of active zones without their proper complement of proteins. The Unc-51-dependent inhibition of ERK activity provides a potential mechanism for synapse-specific control of active zone protein composition and release probability.

A conserved nuclear receptor, Tailless, is required for efficient proliferation and prolonged maintenance of mushroom body progenitors in the Drosophila brain.

The intrinsic neurons of mushroom bodies (MBs), centers of olfactory learning in the Drosophila brain, are generated by a specific set of neuroblasts (Nbs) that are born in the embryonic stage and exhibit uninterrupted proliferation till the end of the pupal stage. Whereas MB provides a unique model to study proliferation of neural progenitors, the underlying mechanism that controls persistent activity of MB-Nbs is poorly understood. Here we show that Tailless (TLL), a conserved orphan nuclear receptor, is required for optimum proliferation activity and prolonged maintenance of MB-Nbs and ganglion mother cells (GMCs). Mutations of tll progressively impair cell cycle in MB-Nbs and cause premature loss of MB-Nbs in the early pupal stage. TLL is also expressed in MB-GMCs to prevent apoptosis and promote cell cycling. In addition, we show that ectopic expression of tll leads to brain tumors, in which Prospero, a key regulator of progenitor proliferation and differentiation, is suppressed whereas localization of molecular components involved in asymmetric Nb division is unaffected. These results as a whole uncover a distinct regulatory mechanism of self-renewal and differentiation of the MB progenitors that is different from the mechanisms found in other progenitors.

Genetic interaction of DISC1 and Neurexin in the development of fruit fly glutamatergic synapses.

Originally identified at the breakpoint of a (1;11)(q42.1; q14.3) chromosomal translocation in a Scottish family with a wide range of mental disorders, the DISC1 gene has been a focus of intensive investigations as an entry point to study the molecular mechanisms of diverse mental dysfunctions. Perturbations of the DISC1 functions lead to behavioral changes in animal models, which are relevant to psychiatric conditions in patients. In this work, we have expressed the human DISC1 gene in the fruit fly (Drosophila melanogaster) and performed a genetic screening for the mutations of psychiatric risk genes that cause modifications of DISC1 synaptic phenotypes at the neuromuscular junction. We found that DISC1 interacts with dnrx1, the Drosophila homolog of the human Neurexin (NRXN1) gene, in the development of glutamatergic synapses. While overexpression of DISC1 suppressed the total bouton area on the target muscles and stimulated active zone density in wild-type background, a partial reduction of the dnrx1 activity negated the DISC1-mediated synaptic alterations. Likewise, overexpression of DISC1 stimulated the expression of a glutamate receptor component, DGLURIIA, in wild-type background but not in the dnrx1 heterozygous background. In addition, DISC1 caused mislocalization of Discs large, the Drosophila PSD-95 homolog, in the dnrx1 heterozygous background. Analyses with a series of domain deletions have revealed the importance of axonal localization of the DISC1 protein for efficient suppression of DNRX1 in synaptic boutons. These results thus suggest an intriguing converging mechanism controlled by the interaction of DISC1 and Neurexin in the developing glutamatergic synapses.

Induction of cAMP response element-binding protein-dependent medium-term memory by appetitive gustatory reinforcement in Drosophila larvae.

The fruit fly Drosophila melanogaster has been successfully used as a model animal for the study of the genetic and molecular mechanisms of learning and memory. Although most of the Drosophila learning studies have used the adult fly, the relative complexity of its neural network hinders cellular and molecular studies at high resolution. In contrast, the Drosophila larva has a simple brain with uniquely identifiable neural networks, providing an opportunity of an attractive alternative system for elucidation of underlying mechanisms involved in learning and memory. In this paper, we describe a novel paradigm of larval associative learning with a single odor and a positive gustatory reinforcer, sucrose. Mutant analyses have suggested importance of cAMP signaling and potassium channel activities in larval learning as has been demonstrated with the adult fly. Intriguingly, larval memory produced by the appetitive conditioning lasts medium term and depends on both amnesiac and cAMP response element-binding protein (CREB). A significant part of memory was disrupted at very early phase by CREB blockade without affecting immediate learning performance. Moreover, we also show that synaptic output of larval mushroom body neurons is required for retrieval but not for acquisition and retention of the larval memory, including the CREB-dependent component.

Artificial Induction of Associative Olfactory Memory by Optogenetic and Thermogenetic Activation of Olfactory Sensory Neurons and Octopaminergic Neurons in Drosophila Larvae.

The larval brain of Drosophila melanogaster provides an excellent system for the study of the neurocircuitry mechanism of memory. Recent development of neurogenetic techniques in fruit flies enables manipulations of neuronal activities in freely behaving animals. This protocol describes detailed steps for artificial induction of olfactory associative memory in Drosophila larvae. In this protocol, the natural reward signal is substituted by thermogenetic activation of octopaminergic neurons in the brain. In parallel, the odor signal is substituted by optogenetic activation of a specific class of olfactory receptor neurons. Association of reward and odor stimuli is achieved with the concomitant application of blue light and heat that leads to activation of both sets of neurons in living transgenic larvae. Given its operational simplicity and robustness, this method could be utilized to further our knowledge on the neurocircuitry mechanism of memory in the fly brain.

Conserved cis-regulatory modules mediate complex neural expression patterns of the eyeless gene in the Drosophila brain.

The Drosophila Pax-6 homologs eyeless (ey) and twin of eyeless (toy) are expressed in the eyes and in the central nervous system (CNS). In addition to the pivotal functions in eye development, previous studies revealed that ey also plays important roles in axonal development of the mushroom bodies, centers for associative learning and memory. It has been reported that a second intron enhancer that contains several Pax-6 binding sites mainly controls the eye-specific expression, but the DNA sequences that control CNS expression are unknown. In this work, we have dissected transcriptional enhancer elements of the ey gene that are required for the CNS expression in various developmental stages. We first show that CNS expression is independent of the eye-specific enhancer of the second intron. By systematic reporter studies, we have identified several discrete DNA elements in the 5' upstream region and in the second intron that cooperatively interact to generate most of the ey expression pattern in the CNS. DNA sequence comparison between the ey genes of distant Drosophila species has identified conserved modules that might be bound by the upstream regulatory factors of the ey gene in CNS development. Furthermore, by RNA interference and mutant studies, we show that ey expression in the brain is independent of the activity of toy and ey itself whereas in the eye primordia it requires both, supporting the notion that ey and toy are regulated by parallel and independent regulatory cascades in brain development.

Early development of the Drosophila mushroom bodies, brain centres for associative learning and memory.

We have studied the formation of Drosophila mushroom bodies using enhancer detector techniques to visualize specific components of these complex intrinsic brain structures. During embryogenesis, neuronal proliferation begins in four mushroom body neuroblasts and the major axonal pathways of the mushroom bodies are pioneered. During larval development, neuronal proliferation continues and further axonal projections in the pedunculus and lobes are formed in a highly structured manner characterized by spatial heterogeneity of reporter gene expression. Enhancer detector analysis identifies many genomic locations that are specifically activated in mushroom body intrinsic neurons (Kenyon cells) during the transition from embryonic to postembryonic development and during metamorphosis.

Induction of associative olfactory memory by targeted activation of single olfactory neurons in Drosophila larvae.

It has been postulated that associative memory is formed by at least two sets of external stimuli, CS and US, that are transmitted to the memory centers by distinctive conversing pathways. However, whether associative memory can be induced by the activation of only the olfactory CS and a biogenic amine-mediated US pathways remains to be elucidated. In this study, we substituted the reward signals with dTrpA1-mediated thermogenetic activation of octopaminergic neurons and the odor signals by ChR2-mediated optical activation of a specific class of olfactory neurons. We show that targeted activation of the olfactory receptor and the octopaminergic neurons is indeed sufficient for the formation of associative olfactory memory in the larval brain. We also show that targeted stimulation of only a single type of olfactory receptor neurons is sufficient to induce olfactory memory that is indistinguishable from natural memory induced by the activation of multiple olfactory receptor neurons.

Unc-51/ATG1 controls axonal and dendritic development via kinesin-mediated vesicle transport in the Drosophila brain.

BACKGROUND: Members of the evolutionary conserved Ser/Thr kinase Unc-51 family are key regulatory proteins that control neural development in both vertebrates and invertebrates. Previous studies have suggested diverse functions for the Unc-51 protein, including axonal elongation, growth cone guidance, and synaptic vesicle transport. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we have investigated the functional significance of Unc-51-mediated vesicle transport in the development of complex brain structures in Drosophila. We show that Unc-51 preferentially accumulates in newly elongating axons of the mushroom body, a center of olfactory learning in flies. Mutations in unc-51 cause disintegration of the core of the developing mushroom body, with mislocalization of Fasciclin II (Fas II), an IgG-family cell adhesion molecule important for axonal guidance and fasciculation. In unc-51 mutants, Fas II accumulates in the cell bodies, calyx, and the proximal peduncle. Furthermore, we show that mutations in unc-51 cause aberrant overshooting of dendrites in the mushroom body and the antennal lobe. Loss of unc-51 function leads to marked accumulation of Rab5 and Golgi components, whereas the localization of dendrite-specific proteins, such as Down syndrome cell adhesion molecule (DSCAM) and No distributive disjunction (Nod), remains unaltered. Genetic analyses of kinesin light chain (Klc) and unc-51 double heterozygotes suggest the importance of kinesin-mediated membrane transport for axonal and dendritic development. Moreover, our data demonstrate that loss of Klc activity causes similar axonal and dendritic defects in mushroom body neurons, recapitulating the salient feature of the developmental abnormalities caused by unc-51 mutations. CONCLUSIONS/SIGNIFICANCE: Unc-51 plays pivotal roles in the axonal and dendritic development of the Drosophila brain. Unc-51-mediated membrane vesicle transport is important in targeted localization of guidance molecules and organelles that regulate elongation and compartmentalization of developing neurons.

Modeling schizophrenia in flies.

Schizophrenia is a debilitating mental illness that affects 1% of the population worldwide. Although its molecular etiology remains unclear, recent advances in human psychiatric genetics have identified a large number of candidate genetic risk factors involved in schizophrenia. Modeling the disease in genetically tractable animals is thus a challenging but increasingly important task. In this review, I discuss the potential problems and perspectives associated with modeling schizophrenia in fruit flies, and briefly review the recent studies analyzing the molecular and cellular functions of Disrupted-In-Schizophrenia-1 (DISC1) in transgenic flies.

Brain patterning defects caused by mutations of the twin of eyeless gene in Drosophila melanogaster.

The Drosophila Pax6 genes, eyeless (ey) and twin of eyeless (toy), are expressed in both eyes and the brain. Previous studies have demonstrated that ey plays important roles in axonal outgrowth and differentiation of mushroom bodies (MBs), which are centers for associative learning and memory in flies. However, the functional significance of toy in brain development is poorly understood. Here, we describe the expression patterns of TOY, and show that TOY expression partially overlaps with EY expression in the embryonic, larval and adult brains. Mutations of toy perturb brain neuromere formation in the embryonic stages, and result in severe deformation of the MB lobes in pharate adult brains. Moreover, we also analyzed toy functions by gain-of-function experiments, and show that overexpression of toy results in degeneration of MB lobes. Thus, our results demonstrate the importance of toy in embryonic brain patterning as well as in post-embryonic development of the major brain structures such as MBs.

UNC-51/ATG1 kinase regulates axonal transport by mediating motor-cargo assembly.

Axonal transport mediated by microtubule-dependent motors is vital for neuronal function and viability. Selective sets of cargoes, including macromolecules and organelles, are transported long range along axons to specific destinations. Despite intensive studies focusing on the motor machinery, the regulatory mechanisms that control motor-cargo assembly are not well understood. Here we show that UNC-51/ATG1 kinase regulates the interaction between synaptic vesicles and motor complexes during transport in Drosophila. UNC-51 binds UNC-76, a kinesin heavy chain (KHC) adaptor protein. Loss of unc-51 or unc-76 leads to severe axonal transport defects in which synaptic vesicles are segregated from the motor complexes and accumulate along axons. Genetic studies show that unc-51 and unc-76 functionally interact in vivo to regulate axonal transport. UNC-51 phosphorylates UNC-76 on Ser(143), and the phosphorylated UNC-76 binds Synaptotagmin-1, a synaptic vesicle protein, suggesting that motor-cargo interactions are regulated in a phosphorylation-dependent manner. In addition, defective axonal transport in unc-76 mutants is rescued by a phospho-mimetic UNC-76, but not a phospho-defective UNC-76, demonstrating the essential role of UNC-76 Ser(143) phosphorylation in axonal transport. Thus, our data provide insight into axonal transport regulation that depends on the phosphorylation of adaptor proteins.

Differential microarray analysis of Drosophila mushroom body transcripts using chemical ablation.

Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the Drosophila brain. As a way to systematically elucidate genes preferentially expressed in MBs, we have analyzed genome-wide alterations in transcript profiles associated with MB ablation by hydroxyurea. We selected 100 genes based on microarray data and examined their expression patterns in the brain by in situ hybridization. Seventy genes were found to be expressed in the posterodorsal cortex, which harbors the MB cell bodies. These genes encode proteins of diverse functions, including transcription, signaling, cell adhesion, channels, and transporters. Moreover, we have examined developmental functions of 40 of the microarray-identified genes by transgenic RNA interference; 8 genes were found to cause mild-to-strong MB defects when suppressed with a MB-Gal4 driver. These results provide important information not only on the repertoire of genes that control MB development but also on the repertoire of neural factors that may have important physiological functions in MB plasticity.

Embryonic and larval development of the Drosophila mushroom bodies: concentric layer subdivisions and the role of fasciclin II.

Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the arthropod brain. In order to understand the cellular and genetic processes that control the early development of MBs, we have performed high-resolution neuroanatomical studies of the embryonic and post-embryonic development of the Drosophila MBs. In the mid to late embryonic stages, the pioneer MB tracts extend along Fasciclin II (FAS II)-expressing cells to form the primordia for the peduncle and the medial lobe. As development proceeds, the axonal projections of the larval MBs are organized in layers surrounding a characteristic core, which harbors bundles of actin filaments. Mosaic analyses reveal sequential generation of the MB layers, in which newly produced Kenyon cells project into the core to shift to more distal layers as they undergo further differentiation. Whereas the initial extension of the embryonic MB tracts is intact, loss-of-function mutations of fas II causes abnormal formation of the larval lobes. Mosaic studies demonstrate that FAS II is intrinsically required for the formation of the coherent organization of the internal MB fascicles. Furthermore, we show that ectopic expression of FAS II in the developing MBs results in severe lobe defects, in which internal layers also are disrupted. These results uncover unexpected internal complexity of the larval MBs and demonstrate unique aspects of neural generation and axonal sorting processes during the development of the complex brain centers in the fruit fly brain.