Multisensory learning binds neurons into a cross-modal memory engram.

Associating multiple sensory cues with objects and experience is a fundamental brain process that improves object recognition and memory performance. However, neural mechanisms that bind sensory features during learning and augment memory expression are unknown. Here we demonstrate multisensory appetitive and aversive memory in Drosophila. Combining colours and odours improved memory performance, even when each sensory modality was tested alone. Temporal control of neuronal function revealed visually selective mushroom body Kenyon cells (KCs) to be required for enhancement of both visual and olfactory memory after multisensory training. Voltage imaging in head-fixed flies showed that multisensory learning binds activity between streams of modality-specific KCs so that unimodal sensory input generates a multimodal neuronal response. Binding occurs between regions of the olfactory and visual KC axons, which receive valence-relevant dopaminergic reinforcement, and is propagated downstream. Dopamine locally releases GABAergic inhibition to permit specific microcircuits within KC-spanning serotonergic neurons to function as an excitatory bridge between the previously 'modality-selective' KC streams. Cross-modal binding thereby expands the KCs representing the memory engram for each modality into those representing the other. This broadening of the engram improves memory performance after multisensory learning and permits a single sensory feature to retrieve the memory of the multimodal experience.

The impact of the gut microbiome on memory and sleep in Drosophila.

The gut microbiome has been proposed to influence diverse behavioral traits of animals, although the experimental evidence is limited and often contradictory. Here, we made use of the tractability of Drosophila melanogaster for both behavioral analyses and microbiome studies to test how elimination of microorganisms affects a number of behavioral traits. Relative to conventional flies (i.e. with unaltered microbiome), microbiologically sterile (axenic) flies displayed a moderate reduction in memory performance in olfactory appetitive conditioning and courtship assays. The microbiological status of the flies had a small or no effect on anxiety-like behavior (centrophobism) or circadian rhythmicity of locomotor activity, but axenic flies tended to sleep for longer and displayed reduced sleep rebound after sleep deprivation. These last two effects were robust for most tests conducted on both wild-type Canton S and w1118 strains, as well for tests using an isogenized panel of flies with mutations in the period gene, which causes altered circadian rhythmicity. Interestingly, the effect of absence of microbiota on a few behavioral features, most notably instantaneous locomotor activity speed, varied among wild-type strains. Taken together, our findings demonstrate that the microbiome can have subtle but significant effects on specific aspects of Drosophila behavior, some of which are dependent on genetic background.

The Drosophila neurogenin Tap functionally interacts with the Wnt-PCP pathway to regulate neuronal extension and guidance.

The neurogenin (Ngn) transcription factors control early neurogenesis and neurite outgrowth in mammalian cortex. In contrast to their proneural activity, their function in neurite growth is poorly understood. Drosophila has a single predicted Ngn homolog, Tap, of unknown function. Here we show that Tap is not a proneural protein in Drosophila but is required for proper axonal growth and guidance of neurons of the mushroom body, a neuropile required for associative learning and memory. Genetic and expression analyses suggest that Tap inhibits excessive axonal growth by fine regulation of the levels of the Wnt signaling adaptor protein Dishevelled.

The Drosophila homologue of the amyloid precursor protein is a conserved modulator of Wnt PCP signaling.

Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of Drosophila APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αß neurons and, in particular, is required cell-autonomously for the ß-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth.

Twists to Classical Conditioning of Adult Drosophila.

Memory has been extensively studied in Drosophila since the early 1970s. Straightforward aversive and appetitive conditioning paradigms train populations of flies to associate the pairing of one of two odors with either punishment or reward. After training, the flies show either preferential avoidance or approach behavior, to the appropriate odor, when given a choice between the two odors in a simple T-maze apparatus. These basic experimental approaches have proven useful in understanding the genetic, molecular, cellular, and neuronal network bases of various valence-specific memories in the fly brain. In addition, numerous modifications to these assays have permitted the study of a broad range of memory-related phenomena. Labile short-term avoidance and approach memories can be readily distinguished from more stable "consolidated" long-term memory equivalents. Prior or subsequent experience of the training cues, and manipulations of the flies' condition, have revealed how parallel competing memories and incompatible states can temporarily interfere with memory retrieval, providing insight into mechanisms of forgetting. Recent studies have also modified the training and testing apparatus to allow simultaneous presentation of odors and colors, providing insight into mechanisms of multisensory learning.

Classical Conditioning of Adult Drosophila.

Olfactory classical conditioning paradigms have been extensively used since the early 1970s to apply genetic approaches to the study of memory in Drosophila. Over the intervening years, advances in genetics have largely changed the focus of studies from the role of single genes in memory to investigation of memory-relevant neuronal circuits. However, the original behavioral paradigms have remained largely unaltered, besides investigators making a few useful tweaks to the training and testing apparatus and modifications to the operating procedures. In this protocol, we provide the reader with a detailed description of the manufacture and assembly of a typical T-maze apparatus, where populations of adult flies can be trained and their odor memory tested later, by giving them a binary choice between the two trained odors. We describe how variations of the training apparatus permit both aversive (odor-shock) and appetitive (odor-sugar) memories to be studied. In addition, we describe a recent modification of the apparatus and protocol that permits study of multisensory (color and odor) aversive and appetitive learning. Control assays for sensory acuity and locomotion are also included.

Prior experience conditionally inhibits the expression of new learning in Drosophila.

Prior experience of a stimulus can inhibit subsequent acquisition or expression of a learned association of that stimulus. However, the neuronal manifestations of this learning effect, named latent inhibition (LI), are poorly understood. Here, we show that prior odor exposure can produce context-dependent LI of later appetitive olfactory memory performance in Drosophila. Odor pre-exposure forms a short-lived aversive memory whose lone expression lacks context-dependence. Acquisition of odor pre-exposure memory requires aversively reinforcing dopaminergic neurons that innervate two mushroom body compartments-one group of which exhibits increasing activity with successive odor experience. Odor-specific responses of the corresponding mushroom body output neurons are suppressed, and their output is necessary for expression of both pre-exposure memory and LI of appetitive memory. Therefore, odor pre-exposure attaches negative valence to the odor itself, and LI of appetitive memory results from a temporary and context-dependent retrieval deficit imposed by competition with the parallel short-lived aversive memory.

Reduced Lateral Inhibition Impairs Olfactory Computations and Behaviors in a Drosophila Model of Fragile X Syndrome.

Fragile X syndrome (FXS) patients present neuronal alterations that lead to severe intellectual disability, but the underlying neuronal circuit mechanisms are poorly understood. An emerging hypothesis postulates that reduced GABAergic inhibition of excitatory neurons is a key component in the pathophysiology of FXS. Here, we directly test this idea in a FXS Drosophila model. We show that FXS flies exhibit strongly impaired olfactory behaviors. In line with this, olfactory representations are less odor specific due to broader response tuning of excitatory projection neurons. We find that impaired inhibitory interactions underlie reduced specificity in olfactory computations. Finally, we show that defective lateral inhibition across projection neurons is caused by weaker inhibition from GABAergic interneurons. We provide direct evidence that deficient inhibition impairs sensory computations and behavior in an in vivo model of FXS. Together with evidence of impaired inhibition in autism and Rett syndrome, these findings suggest a potentially general mechanism for intellectual disability.

A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function.

Loss of function of the FMR1 gene leads to fragile X syndrome (FXS), the most common form of intellectual disability. The loss of FMR1 function is usually caused by epigenetic silencing of the FMR1 promoter leading to expansion and subsequent methylation of a CGG repeat in the 5' untranslated region. Very few coding sequence variations have been experimentally characterized and shown to be causal to the disease. Here, we describe a novel FMR1 mutation and reveal an unexpected nuclear export function for the C-terminus of FMRP. We screened a cohort of patients with typical FXS symptoms who tested negative for CGG repeat expansion in the FMR1 locus. In one patient, we identified a guanine insertion in FMR1 exon 15. This mutation alters the open reading frame creating a short novel C-terminal sequence, followed by a stop codon. We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells. We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP. In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes.

Genetic approaches in Drosophila for the study neurodevelopmental disorders.

The fruit fly Drosophila melanogaster is one of the premier genetic model organisms used in biomedical research today owing to the extraordinary power of its genetic tool-kit. Made famous by numerous seminal discoveries of basic developmental mechanisms and behavioral genetics, the power of fruit fly genetics is becoming increasingly applied to questions directly relevant to human health. In this review we discuss how Drosophila research is applied to address major questions in neurodevelopmental disorders. This article is part of the Special Issue entitled 'Neurodevelopmental Disorders'.