Ken Honjo

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.

Nuclear DISC1 regulates CRE-mediated gene transcription and sleep homeostasis in the fruit fly

Disrupted-in-schizophrenia-1 (DISC1) is one of major susceptibility factors for a wide range of mental illnesses, including schizophrenia, bipolar disorder, major depression and autism spectrum conditions. DISC1 is located in several subcellular domains, such as the centrosome and the nucleus, and interacts with various proteins, including NudE-like (NUDEL/NDEL1) and activating transcription factor 4 (ATF4)/CREB2. Nevertheless, a role for DISC1 in vivo remains to be elucidated. Therefore, we have generated a Drosophila model for examining normal functions of DISC1 in living organisms. DISC1 transgenic flies with preferential accumulation of exogenous human DISC1 in the nucleus display disturbance in sleep homeostasis, which has been reportedly associated with CREB signaling/CRE-mediated gene transcription. Thus, in mammalian cells, we characterized nuclear DISC1, and identified a subset of nuclear DISC1 that colocalizes with the promyelocytic leukemia (PML) bodies, a nuclear compartment for gene transcription. Furthermore, we identified three functional cis-elements that regulate the nuclear localization of DISC1. We also report that DISC1 interacts with ATF4/CREB2 and a corepressor N-CoR, modulating CRE-mediated gene transcription.

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.

Optogenetic manipulation of neural circuits and behavior in Drosophila larvae

Optogenetics is a powerful tool that enables the spatiotemporal control of neuronal activity and circuits in behaving animals. Here, we describe our protocol for optical activation of neurons in Drosophila larvae. As an example, we discuss the use of optogenetics to activate larval nociceptors and nociception behaviors in the third-larval instar. We have previously shown that, using spatially defined GAL4 drivers and potent UAS (upstream activation sequence)-channelrhodopsin-2∷YFP transgenic strains developed in our laboratory, it is possible to manipulate neuronal populations in response to illumination by blue light and to test whether the activation of defined neural circuits is sufficient to shape behaviors of interest. Although we have only used the protocol described here in larval stages, the procedure can be adapted to study neurons in adult flies—with the caveat that blue light may not sufficiently penetrate the adult cuticle to stimulate neurons deep in the brain. This procedure takes 1 week to culture optogenetic flies and ∼1 h per group for the behavioral assays.

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.

Nociceptor-Enriched Genes Required for Normal Thermal Nociception

Here, we describe a targeted reverse genetic screen for thermal nociception genes in Drosophila larvae. Using laser capture microdissection and microarray analyses of nociceptive and non-nociceptive neurons, we identified 275 nociceptor-enriched genes. We then tested the function of the enriched genes with nociceptor-specific RNAi and thermal nociception assays. Tissue-specific RNAi targeted against 14 genes caused insensitive thermal nociception while targeting of 22 genes caused hypersensitive thermal nociception. Previously uncategorized genes were named for heat resistance (i.e., boilerman, fire dancer, oven mitt, trivet, thawb, and bunker gear) or heat sensitivity (firelighter, black match, eucalyptus, primacord, jet fuel, detonator, gasoline, smoke alarm, and jetboil). Insensitive nociception phenotypes were often associated with severely reduced branching of nociceptor neurites and hyperbranched dendrites were seen in two of the hypersensitive cases. Many genes that we identified are conserved in mammals.

DISC1 causes associative memory and neurodevelopmental defects in fruit flies

Originally found in a Scottish family with diverse mental disorders, the DISC1 protein has been characterized as an intracellular scaffold protein that associates with diverse binding partners in neural development. To explore its functions in a genetically tractable system, we expressed the human DISC1 in fruit flies (Drosophila melanogaster). As in mammalian neurons, DISC1 is localized to diverse subcellular domains of developing fly neurons including the nuclei, axons and dendrites. Overexpression of DISC1 impairs associative memory. Experiments with deletion/mutation constructs have revealed the importance of amino-terminal domain (46–290) for memory suppression whereas carboxyl domain (598–854) and the amino-terminal residues (1–45) including the nuclear localization signal (NLS1) are dispensable. DISC1 overexpression also causes suppression of axonal and dendritic branching of mushroom body neurons, which mediate a variety of cognitive functions in the fly brain. Analyses with deletion/mutation constructs reveal that protein domains 598–854 and 349–402 are both required for the suppression of axonal branching, while amino-terminal domains including NLS1 are dispensable. In contrast, NLS1 was required for the suppression of dendritic branching, suggesting a mechanism involving gene expression. Moreover, domain 403–596 is also required for the suppression of dendritic branching. We also show that overexpression of DISC1 suppresses glutamatergic synaptogenesis in developing neuromuscular junctions. Deletion/mutation experiments have revealed the importance of protein domains 403–596 and 349–402 for synaptic suppression, while amino-terminal domains including NLS1 are dispensable. Finally, we show that DISC1 functionally interacts with the fly homolog of Dysbindin (DTNBP1) via direct protein–protein interaction in developing synapses.

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.

Nociceptive interneurons control modular motor pathways to promote escape behavior in Drosophila

Rapid and efficient escape behaviors in response to noxious sensory stimuli are essential for protection and survival. Yet, how noxious stimuli are transformed to coordinated escape behaviors remains poorly understood. In Drosophila larvae, noxious stimuli trigger sequential body bending and corkscrew-like rolling behavior. We identified a population of interneurons in the nerve cord of Drosophila, termed Down-and-Back (DnB) neurons, that are activated by noxious heat, promote nociceptive behavior, and are required for robust escape responses to noxious stimuli. Electron microscopic circuit reconstruction shows that DnBs are targets of nociceptive and mechanosensory neurons, are directly presynaptic to pre-motor circuits, and link indirectly to Goro rolling command-like neurons. DnB activation promotes activity in Goro neurons, and coincident inactivation of Goro neurons prevents the rolling sequence but leaves intact body bending motor responses. Thus, activity from nociceptors to DnB interneurons coordinates modular elements of nociceptive escape behavior.

Visualization of DISC1-Dysbindin interaction in glutamatergic synaptic termini in fruit flies

Visualization of DISC1-Dysbindin interaction in glutamatergic synaptic termini in fruit flies